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in english and italian
Morphophysiological Remarks
E xperimental
M edicine
R eviews
Edited by
Aldo Gerbino
Giovanni Zummo
Giuseppe Crescimanno
in english and italian
Morphophysiological Remarks
E xperimental
M edicine
R eviews
Edited by
Aldo Gerbino
Giovanni Zummo
Giuseppe Crescimanno
Dipartimento di Medicina Sperimentale
UniversitГ degli Studi di Palermo
FacoltГ di Medicina e Chirurgia
UniversitГ degli Studi di Palermo
Centro Interdipartimentale di Tecnologie della Conoscenza
Dipartimento di Medicina Sperimentale
Anatomia Umana “Emerico Luna” (via del Vespro, 129)
Istologia ed Embriologia “Arcangelo Pasqualino di Marineo” (via del Vespro, 129)
Fisiologia Umana “Giuseppe Pagano” (corso Tukory, 129)
В© Copyright
Experimental Medicine Reviews
Morphophysiological Remarks
in English and Italian
Palermo, Italy - vol 2/3, 2008/2009
Edited by
Aldo Gerbino, Full Professor of Histology and Embryology, Histology Section
Giovanni Zummo, Full Professor of Human Anatomy, Human Anatomy Section
Giuseppe Crescimanno, Full Professor of Physiology, Physiology Section
Scientific editorial office
Francesco Cappello, Laura Uzzo, Maurizio Casarrubea
Simona Corrao, Annamaria Mauro
Graphic design
Rosario Notaro
Plumelia edizioni
Collana “Ricerca”
ISBN 978-88-89876-15-2
This volume can be found on line at the following address:
Printed by
Officine Tipografiche Aiello & Provenzano, Bagheria (Palermo)
The heterogeneity of scientific papers, a member of different schools, is divided into
trasversal methodology that today spreads through way of research. This makes, on value
editorial uniformity of single capitols, some problems about harmonization. It is necessary
and perhaps useful that rise some differences about impostation, that not seem damage
editing’s quality.
The Editors
Editorial board
Everly Conway de Macario
University of Maryland Biotechnology Institute, Baltimore, MD, U.S.A.
В Felipe Cortes de Bonavides
University of Sevilla, Sevilla, Spain
В Donna E. Davies
University of Southampton, Southampton, U.K.
В Regina M. Day
Uniformed Services University of the Health Sciences, Bethesda, MD, U.S.A.
В JosГ© M. Icardo
University of Cantabria, Santander, Spain
В Alberto J. L. Macario
University of Maryland Biotechnology Institute, Baltimore, MD, U.S.A.
Abdo Jurjus
Faculty of Medicine, American University of Beirut, Beirut, Lebanon
Tomasz Konopka
Department of Oral Pathology, Wroclaw Medical University, Poland
Guest contribution
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Il sistema delle chaperonine: fisiologia e patologia]
Alberto J. L. Macario and Everly Conway de Macario
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Columbus Center, Baltimore, USA
Key words: Chaperoning system, Molecular chaperones, Heat Shock Proteins, Chaperone subpopulations,
Chaperonology, Chaperonopathies, Chaperonotherapy
Parole chiave: Sistema delle Chaperonine, Chaperoni molecolari, Proteine da Shock Termico, Chaperonopatie, Chaperonoterapie
Abstract. The concept of chaperoning system is presented here for the first time. The system encompasses
all the molecular chaperones and their anatomically and functionally related molecules and higher order
structures (multimolecular assemblies, cells, tissues). This ensemble of functionally related molecules and
structures is viewed as a physiological system whose central role is the control of protein homeostasis in
what concerns maintenance of a complete set of proteins in all fluids, cells, and tissues with the correct, native, functional conformation. The system ensures proper protein folding, re-folding, degradation, and translocation, and participates in a wide range of other molecular and cellular processes from antigen presentation to hormone receptor assembly and ligand binding, and others, that require stabilization of structure and
facilitation of intermolecular interaction. The science that studies this system is called chaperonology, which
also includes the study of disease caused by system malfunction and defective chaperones, i.e., the chaperonopathies, and their prevention and treatment using chaperone genes or proteins, i.e., chaperonotherapy.
Riassunto. Viene presentato, per la prima volta, il concetto dell’esistenza di un sistema degli chaperoni molecolari.
Questo sistema comprende tutti gli chaperoni molecolari e le molecole strutturalmente e funzionalmente correlate, tanto quanto strutture di livello superiore (complessi multimolecolari in cellule e tessuti). Questo insieme di
molecole e strutture funzionalmente correlate tra di loro viene descritto come un “sistema fisiologico” il cui ruolo
centrale è il controllo dell’omeostasi proteica, specie per ciò che concerne il mantenimento della corretta (nativa)
conformazione funzionale di proteine presenti in tutti i fluidi biologici, così come nelle cellule e nei tessuti. Questo
sistema, quindi, assicura il corretto folding delle proteine, così come il loro re-folding, ma anche la degradazione
e la traslocazione; inoltre partecipa ad un elevato numero di altre attivitГ cellulari e molecolari, che vanno dalla
presentazione antigenica all’assemblamento dei recettori ormonali, dalla formazione di complessi con ligandi ad
altre azioni che richiedono la stabilizzazione delle strutture proteiche e la facilitazione dell’interazione tra molecole.
La scienza che studia questo sistema è detta chaperonologia, e questa include anche lo studio delle malattie determinate dal suo malfunzionamento (c.d. chaperonopatie), come ad esempio per la presenza di chaperoni “difettosi”, nonché il loro utilizzo in ambito preventivo e terapeutico (c.d. chaperonoterapia), attraverso
l’espressione genica o l’utilizzo delle stesse proteine.
Note. This article contains most of the lecture by the authors entitled “Molecular chaperones and chaperonopathies in oncopathology” presented at the Symposium “New molecular targets in diagnostic oncopathology,” on the occasion of the opening of the Advanced Oncopathology Laboratory at the A.R.N.A.S. Ospedale
Civico-University of Palermo, Palermo, Italy, December 13, 2008.
Alberto J. L. Macario, Everly Conway de Macario
Since 1962 the response to stress has been studied at various levels [1]. The stressor often tested was, and is still today, a temperature elevation, heat shock. However,
there are many other stressors of various natures, chemical, physical, mechanical, biological, psychological, social, occupational, etc., which have also been investigated [2,3].
One consequence of stress that was examined from the beginning was the response of
certain genes to heat shock which resulted in the production proteins called heat-shock
proteins (Hsp). The functions of Hsp were then studied in various ways and some were
found to assist protein folding and were called molecular chaperones [1]. Work on Hsp
and chaperones has been performed over many years in many laboratories around the
world and the results have been published in a broad range of printed and electronic publications, and made available also in databases. Thus, a huge amount of data and ideas
about stress, stress response, Hsp, chaperones, and anti-stress mechanisms is disperse
in a wide variety of sources. A few years ago we started the unification of the field into a
coherent rational structure, a new scientific discipline we called chaperonology [4,5]. This
new discipline includes subspecialties such as chaperonomics, chaperonotherapy, and the
study of chaperonopathies [4-6].
In this article, we propose the unification of the molecular chaperones and Hsp molecules and the higher order structures anatomically and functionally related to these molecules into a single physiological system.
Physiology. Definitions
Chaperoning system
The chaperoning system is a newly identified physiological set of molecules and molecular teams, and pertinent cells and tissues, key to maintaining protein homeostasis
and other cellular functions. The molecular components of the system have been known
for sometime but they were not considered as members of a single physiological system
until now. Likewise, the cells containing chaperones and/or producing the chaperones
were not considered before as part of a specialized physiological system centered on
The ensemble of chaperones and closely related molecules in terms of interaction and
function and pertinent supramolecular structures (multimolecular machines, cells, tissues)
could be considered a system or an apparatus. System was chosen over apparatus because of its connotation of interconnected functions and pathways made of chaperones and
related molecules and cells and tissues, whereas apparatus has a less open-ended feel as
The Chaperoning system: physiology and pathology
it keeps the focus on the “hardware” molecules.
The system could be named in various ways, for instance the chaperone system, the
chaperonic system, or the chaperoning system. We have chosen at this time the term
chaperoning system because chaperone system may be taken to: a) designate the system
pertaining to a single chaperone from among the many that exist; b) indicate that the chaperones’ only function is to assist (to chaperone) nascent not yet folded polypeptides and
unfolded proteins to gain or regain a folded, native conformation when, in fact, chaperones
have other functions, too; and c) exclude components of the system that are not typical
chaperones but interact with them in a critical way, or that are not molecules, such as the
cells that contain and/or produce the chaperones.
The term chaperonic system was not preferred either because the spoken change
of emphasis, from the soft “…one” of chaperone to the hard “…onic” of chaperonic is
both unattractive to the ear and non-intuitive. Furthermore, chaperonic does not reflect
as well as chaperoning does the functions and interaction of the components of the
The identification of the central components of the chaperoning system, the molecular
chaperones, has originated the novel field of science named chaperonology. Chaperonology deals with the chaperoning system and with its components, molecules and higher
order structures (molecular assemblies, cells, tissues), and their abnormalities and pathologies.
The roles of chaperones
The classic concept is that chaperones assist other proteins to fold and re-fold, and
usher defective proteins toward degradation. Thus, chaperoning proteins to fold or refold
and guiding proteins toward degradation can be considered typical functions of chaperones. However, many chaperones play roles that are not typically related to protein folding
but are seemingly quite different, for example the role of Hsp70 in tumor-antigen presentation and tumor immunity [7,8]. These could be considered atypical functions of typical
chaperones. Conversely, molecules that are not typical chaperones may have functions
similar to the atypical functions of typical chaperones. For example, Hsp32 is the product
of a heat shock-inducible gene that plays a significant role in protection against oxidative
stress but has no known function in protein folding or refolding [9]. Thus, Hsp32 is a typical
heat-shock protein with a role in anti-stress mechanisms like many chaperones but it is
not a typical chaperone.
Alberto J. L. Macario, Everly Conway de Macario
Fig. 1
Subpopulations of chaperones: autochthonous vs. imported (with regard to any given cell); sessile vs. mobile
(the latter can be sedentary or nomadic); single or member of a chaperoning team with other chaperones,
co-chaperones, and co-factors, also named chaperoning machine. A chaperoning team or machine can be
a member of a chaperoning network, which is formed by various chaperoning teams and, possibly, other
molecules or molecular assemblies.
A chaperone can also form a complex with another molecule or structure (tumor antigen, cell-surface receptor, cytosolic glucocorticoid-hormone receptor, chemical compound), but in this case the complex is not
a chaperoning machine; it has other functions more or less unrelated to chaperoning. Examples: 1) Hsp70
forms complexes with tumor antigens (peptides) and cell-surface receptors; 2) Hsp90 binds glucocorticoidhormone receptor (a protein which is a transcription factor); 3) Hsp90 binds some anti-tumor compounds like
the antibiotic geldanamycin. See text for references.
Key. Circled C, molecular chaperone; 1, mobile chaperone in the cytosol; 2, chaperone inside an organelle,
such as the nucleus or mitochondrion; 3, sessile chaperone anchored to a particle (e.g., ribosome) in the
cytosol; 4 and 5, sessile chaperone anchored to the cell membrane on the cytosolic side (4) or on the outside
in the extracellular space (5); 6, mobile chaperones in the intercellular space; 7, mobile chaperone in circulation inside a vessel (blood or lymph); 8, sessile chaperone anchored to the vessel wall on the inside; 9,
mobile chaperone in the cytosol like that shown in 1, but imported from another cell. Molecular chaperones
can be found also in other locations such as cerebrospinal fluid and secretions (e.g., saliva and urine), not
shown in this figure (see Table 3)
The Chaperoning system: physiology and pathology
Molecules, cells and tissues of the chaperoning system
Chaperones are made in cells for work in the same cells in which they are made or for
export (Fig. 1). Chaperones for export are made in a cell and then travel to other locations,
inside cells or in extracellular sites, in which they will take residence and work. It can be
predicted that there are cells, and tissues or defined zones within certain tissues or organs,
specialized in the production of chaperones for export.
Chaperones are present in tissues and fluids and, as explained earlier, we call the entire
chaperone population of an organism the chaperoning system (Fig. 1). Each cell has its
own set of chaperones or subsystem that typically includes more than one chaperoning
complex or team, and teams interact forming networks inside the cell.
Pertinent timely questions
The words molecular chaperones and chaperoning system immediately suggest a number of issues and questions (Table 1). Answers to some of these questions can be found in
this article and in the bibliography.
Table 1: Questions
What are the components of the chaperoning system?
Where and when are the molecular chaperones made?
Where do the chaperones reside and work?
What do the chaperones do?
What are the diseases caused by defective chaperones?
What types of chaperonopathies are known?
How would one diagnose a chaperonopathy?
When would treatment with chaperones, i.e., chaperonotherapy, be indicated?
What types of chaperonotherapies are available?
What to do next?
Classification of molecular chaperones
A) Types of chaperones according to their size (molecular weight)
Heat-shock proteins (Hsp) senso stricto are the product of genes induced by a temperature elevation (heat shock), but the name is also applied to proteins from genes inducible
by any other stressor. Many Hsp are chaperones (for example the heat-inducible form of
Hsp70 in humans) but not all chaperones are Hsp. An example of the latter is the AlphaHemoglobin Stabilizing Protein (AHSP), which is a dedicated chaperone whose substrate is
the alpha hemoglobin chain and is encoded in a gene not known to be inducible by heat or
any other stressor [10]. Conversely, many Hsp are not chaperones, for example Hsp32 or
Alberto J. L. Macario, Everly Conway de Macario
heme oxygenase-1 (HO-1). This enzyme is the inducible isoform of heme oxygenase that
catalyzes the NADPH, O2 and cytochrome P450-reductase dependent oxidation of heme to
carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin [9]. Thus
Hsp32 is associated with the generation of biliverdin and bilirubin, potent antioxidants,
and therefore it has to be considered part of the anti-stress mechanisms of which many
molecular chaperones are important components.
Although many Hsp do not act as chaperones and, vice versa, many chaperones are
the product of genes not inducible by any stressor, the terms Hsp and chaperone have
been used as synonyms for years in a huge number of printed and electronic publications,
and in databases. It is virtually impossible at this time in the history of chaperonology
to correct these abuses in nomenclature usage. Therefore, we use here both terms as
Hsp-chaperones can be grouped according to their size in a classification that has
practical utility in research and, particularly in pathology and clinics (Table 2).
Table 2: Classification of Hsp-chaperones according to molecular weight1
Chaperone subpopulation
Other names
High MW; Hsp100
Chaperones; DnaK
Chaperonins; Cpn60
Small Hsp
sHsp; alpha-crystallins
Proteases; isomerases; AAA+ proteins (e.g.,
paraplegin, spastin]), etc.
MW (kDa)
100 or higher
34 or less
From references [2,20]
B) Types of chaperones according to their origin with regard to their place of residence
and work
Chaperones are in fixed locations inside the various cell compartments as well as in
biological fluids moving around (Fig. 1; Table 3).
Note: The following two terms refer to chaperones inside any given cell without taking
into account the cell’s organelles or membranes.
Autochthonous: a chaperone originated in the cell in which it resides. The cell of origin
and of residence is one and the same, i.e., an autochthonous chaperone resides and works
in the same cell in which it was produced, disregarding in which cell compartment the chaperone resides and works. Imported: the place of residence (a given cell) of an imported
The Chaperoning system: physiology and pathology
chaperone is not the same as that of its origin but another cell.
Table 3: Places at which chaperones reside and work
Endoplasmic reticulum
Membrane on the inside
Membrane on the outside
Intercellular space
Blood (plasma, serum)
Cerebrospinal fluid
Secretions (e.g., saliva)
C) Types of chaperones according to their ability to move and change residence
Chaperones can be classified according to their mobility (Fig. 1). Sessile: fixed, anchored to another structure (e.g., cell membrane). Mobile: not fixed, capable of moving
inside a cell (e.g., from cytosol to nucleus), or outside cells (e.g., in the blood) and change
place of residence and work. Mobile chaperones can be of two subtypes: i) Sedentary,
reside always in the same cell or cell compartment but are not fixed to any structure,
so they can move within the confines of their “home” or cellular realm; and ii) Nomadic,
change residence, i.e., the chaperone takes residence for a while in one location (e.g., a
cell or cell compartment), then in another, and so forth. Nomadic chaperones travel and
work in various successive places. Hsp60 is an example of mobile, nomadic chaperone:
it is produced in the cytosol and then translocated to the mitochondria from which it can
exit and go back to the cytosol, and even exit the cell and appear in the extracellular
space [11,12].
D) Types of chaperones according to their relation with other chaperones or other molecules
Chaperones exercise their functions alone or in associations with other molecules (Fig.
1), and they can be considered Single, i.e., a chaperone molecule that performs its role not
associated with any other chaperone, or Social. The latter form part of a Team (more precisely, Chaperoning Team), namely, a specific association of chaperones to build a chaperone machine. A Chaperoning Team includes chaperones senso stricto (e.g., Hsp70[DnaK]),
co-chaperones (e.g., Hsp40[DnaJ]), and co-factors (e.g., Nucleotide Exchange Factor [NEF]
Alberto J. L. Macario, Everly Conway de Macario
such as GrpE); all these molecules assemble into a multimolecular machine that performs
the chaperoning work. Other associations are: Network (more precisely, Chaperoning Network), which is a specific interaction between chaperone machines (e.g., Hsp70-Hsp40NEF, and Hsp60-Hsp10, and Prefoldin), or between a chaperoning team and a single chaperone. Complex designates the association of a chaperone with another molecule which
is not a chaperone, for example Hsp70 with tumor antigen [7,8], Hsp90 with glucocorticoid
receptor [13] or with geldanamycin and other inhibitors [14-16], and Hsp60 with procaspase-3 [17].
E) Other classifications of chaperones
Chaperones can be classified according to gene type into house-keeping vs. anti-stress
(constitutive vs. stress-inducible genes, respectively); and according to substrate range
into generic vs. dedicated (with many, almost any, client polypeptide vs. with a single,
specific client polypeptide or with a very restricted, specific set of client polypeptides,
Table 4: Chaperonopathies1
Mutation hereditary
Mutation sporadic
Protein coding region
Non-coding region:
promoter region (5’ UTR)
Early onset
Late onset
By mistake
AlphaA-crystallin gene, cataracts. AlphaB-crystallin gene,
desmin-related myopathy.
Hsp27 (HSPB1) dHMN/CMT22
Hsp27 (HSPB1) dHMN/CMT2
HSP70-1 (HspA1A); HSP70-2 (HspA1B); HSP70-Hom (HspA1L)
Affect longevity
HSP70-1 (HspA1A); HSP70-2 (Hsp70A1B) Affect longevity
Alpha-crystallin PTM (cataracts in diabetes)
Alpha-crystallin PTM (cataracts of senescence)
Increased: Hsp60 & Hsp10, colon, exocervix, prostate; Hsp90,
breast; HYOU-1, breast, brain
Decreased: Hsp10 and Hsp60, lung
Hsp70 necessary: breast
Prefoldin generates highly toxic Abeta oligomers rather than the
less toxic fibrils (ref. Alzheimer’s disease)
Other examples of chaperonopathies can be found in references [4,5,20,21].
Abbreviations: dHMN/CMT2, distal Hereditary Motor Neuropathy/Charcot-Marie-Tooth disease type 2; UTR, untranslated
region; PTM, post-translational modification; Hsp, heat-shock protein.
The Chaperoning system: physiology and pathology
Pathology. Disorders of protein homeostasis
Identification of chaperones and their functions made clear that the chaperoning system is a major component of anti-stress mechanisms and also plays a central role in
protein homeostasis, particularly in what concerns the maintenance of a complete set
of proteins with a native, functional conformation throughout the cell and the organism.
The realization that chaperones normally play such important roles lead to the idea that
malfunctional, defective chaperones must have a role in pathogenesis [2,18,19]. Pathological disorders in which abnormal (sick) chaperones play a pathogenetic role were called
chaperonopathies [18-21].
Protein precipitates. Here we consider protein precipitates a key indicator of protein
homeostasis abnormality or failure. As when hyperglycemia is detected one thinks of problems with insulin and the pancreas, protein precipitates ought to direct attention to chaperones, i.e., to a defect in the chaperoning system or chaperonopathy.
Chaperonopathies. The abnormalities and pathologies of the chaperones are grouped
under the newly coined name of chaperonopathies. The most common chaperonopathies are
manifestations of ageing [18]. A classification of chaperonopathies is displayed in Table 4.
Chaperonopathies by mistake or collaborationism. Pathologic conditions in which normal
chaperones are involved in pathogenetic pathways and contribute to the development of disease rather than the contrary are called chaperonopathies by mistake or collaborationism.
Significant examples of chaperonopathies by mistake are some types of cancers (Table 4).
Chaperonotherapy. Identification of chaperonopathies and other diseases (e.g., proteinmisfolding diseases) in which normal chaperones could play a beneficial role has lead to
the idea of using these molecules for treatment, namely chaperonotherapy [4,5].
Chaperonotherapy refers to the use of chaperones (genes and proteins) for treatment
of chaperonopathies, for example replacement of a structurally defective chaperone with
a normal version of it, or supplementation of a quantitative deficiency of a chaperone by
administering the pertinent chaperone as gene or protein. Chaperonotherapy can also be
used for treatment of conditions that are not chaperonopathies at all, or are not primarily
chaperonopathies (e.g., some neurodegenerative diseases) but are due primarily to abnormalities in proteins (proteinopathies) that are not chaperones.
Treatment of chaperonopathies by mistake, such as certain forms of cancer, requires antichaperone agents. When chaperones are involved in pathogenetic pathways and contribute
to the development of disease rather than the contrary they must be inhibited or eliminated.
Development of antichaperone agents is one of the most promising lines of research in
the fight against cancer.
Alberto J. L. Macario, Everly Conway de Macario
A new physiological system is presented that plays a central role in controlling protein
homeostasis at the level of molecular conformation. Proteins are major cellular and organismal components with a broad range of functions. For a cell or organism to function
correctly all its proteins in all its compartments must be at the physiological concentrations
and possess the right primary, secondary, tertiary and, when pertinent, quaternary structures. The chief function of the chaperoning system is to ensure that all proteins reach after
synthesis the mature, correct functional structure beyond the primary one, i.e., the native
conformation. The chaperoning system also monitors the native conformation of mature
proteins and restores it if these proteins tend to lose it due to stress or any other proteindenaturing factor. Furthermore, the components of the system play a variety of other roles,
many of which are not those typically assigned to chaperones (e.g., assisting nascent proteins to fold correctly or to refold if partially denatured, helping protein translocation across
membranes, or ushering damaged or unnecessary proteins toward degradation) but are diverse, including in many instances facilitation of intermolecular association and interaction
and stabilization of multimolecular assemblies. The system encompasses molecules, cells
and tissues but the central components are the molecular chaperones. System malfunction causes disturbances in protein homeostasis that lead to aggregation and formation of
protein precipitates. Failure of chaperones due to qualitative or quantitative abnormalities
cause pathological conditions named chaperonopathies. Treatment of these conditions can
be done using chaperone genes or proteins, a form of therapy called chaperonotherapy.
The concept of chaperoning system offers a new standpoint to look at molecular chaperones and their associated molecules and higher order structures and to study these
molecules and structures using fresh approaches, including those that will take into consideration interactions and interconnections between system components far apart in the
cell and in the organism. Likewise, the new concept will be instrumental to unveil aspects
not yet completely understood of known diseases. This unveiling of new features will, in
turn, provide clues on the strategies and methods that should be used for elucidating them.
Also, the chaperoning system concept, in showing new angles of pathologic disorders still
poorly characterized, will allow the investigation of these disorders using novel strategies
that should uncover details as yet hidden.
Acknowledgements. We thank Dr. Francesco Cappello for his encouragement and constructive comments
during preparation of the material for this article, and Dr. Adriana Verschoor for her critical discussion of
terminology. This is Publication No. 09-200 from the Center of Marine Biotechnology.
The Chaperoning system: physiology and pathology
[1] Haak, J. and Kregel, K.C. 1962-2077: a cell stress odyssey. Novartis Foundation Symposium 291 (London, UK, June 2007] 3-15, 2008. Discussion 15-22, 137-40.
[2] Macario, A.J.L. Heat-shock proteins and molecular chaperones: Implications for pathogenesis, diagnostics, and therapeutics. Intl J Clin Lab Res 1995; 25: 59-70.
[3] Macario, A.J.L. and Conway de Macario, E. The molecular chaperone system and other anti-stress
mechanisms in archaea. Front Biosci 2001; 6: d262-283. To see Table of Contents, Abstract, Figures,
and Tables go to:
[4] Macario, A.J.L. and Conway de Macario, E. Chaperonopathies and chaperonotherapy. FEBS Lett 2007a;
581: 3681-3688.
[5] Macario, A.J.L. and Conway de Macario, E. Chaperonopathies by defect, excess, or mistake. Ann New
York Acad Sci 2007b; 1113: 178-191.
[6] Cappello, F., Bucchieri, F., David, S., Campanella, C., Ribbene, A., Marino Gammazza, A., Ardizzone, N.,
Merendino, A., Marciano, V., Peri, G., Conway de Macario, E., Macario, A.J.L. and Zummo, G. Chaperonology: A novel research field for experimental medicine in the XXI century. Exp Med Rev 2007; 1:
[7] Calderwood, S.K., Theriault, J.R. and Gong, J. Message in a bottle: role of the 70-kDa heat shock protein
family in anti-tumor immunity. Eur J Immunol 2005; 35: 2518-2527.
[8] Murshid, A., Gong, J. and Calderwood SK. Heat-shock proteins in cancer vaccines: agents of antigen
cross-presentation. Expert Rev Vaccines 2008; 7: 1019-1030.
[9] Idriss, N.K., Blann, A.D. and Lip, G.Y. Hemoxygenase-1 in cardiovascular disease. J Am Coll Cardiol
2008; 52: 971-978.
[10] Yu, X., Kong, Y., Dore, Lc., Abdulmalik, O., Katein, A.M., Zhou, S., Choi, J.K., Gell, D., Mackay, J.P., Gow,
A.J. and Weiss, M.J. An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis. J Clin Invest 2007; 117: 1856-1865.
[11] Cappello, F., Conway de Macario, E., Marasa, L., Zummo, G. and Macario, A.J.L. Hsp60: new locations,
functions, and perspectives for cancer diagnosis and therapy. Cancer Biol Ther 2008b; 7: 801-809.
[12] Cappello, F., Di Stefano, A., Conway de Macario, E. and Macario, A.J.L. Hsp60 and Hsp10 in ageing. In:
“Heat shock proteins and whole body physiology”, edited by Alexzander A. A. Asea and Bente K. Pedersen, Springer, Heidelberg, Germany, 2009. (In press.)
[13] Sreeramulu, S., Jonker, H.R.A., Langer, T., Richter, C., Lancaster, C.R.D. and Schwalbe, H. The human
CDC37.HSP90 complex studied by heteronuclear NMR spectroscopy. J Biol Chem 2009; 284:38853896.
[14] Miyata Y. Hsp90 inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents.
Curr Pharm Des 2005; 11: 1131-1138.
[15] Stravopodis, D.J., Margaritis, L.H. and Voutsinas, G.E. Drug-mediated targeted disruption of multiple
protein activities through functional inhibition of the Hsp90 chaperone complex. Curr Med Chem 2007;
14: 3122-3138.
[16] Banerji U. Heat shock protein 90 as a drug target: some like it hot. Clin Cancer Res 2009; 15: 9-14.
[17] Campanella, C., Bucchieri, F., Ardizzone, N.M., Marino Gammazza, A., Montalbano, A., Ribbene, A., Di
Felice, V., Bellafiore, M., David, S., Rappa, F., Marasa, M., Peri, G., Farina, F., Czarnecka, A.M., Conway
de Macario, E., Macario, A.J.L., Zummo, G. and Cappello, F. Upon oxidative stress, the antiapoptotic
Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells. Eur J Histochem 2008a;
52: 221-228.
Alberto J. L. Macario, Everly Conway de Macario
[18] Macario, A.J.L. and Conway de Macario, E. Sick chaperones and ageing: A perspective. Ageing Res Rev
2002; 1: 295-311.
[19] Macario, A.J.L. and Conway de Macario, E. The pathology of anti-stress mechanisms: A new frontier.
Stress 2004; 7: 243-249.
[20] Macario, A.J.L., Grippo, T.M. and Conway de Macario, E. Genetic disorders involving molecular-chaperone genes: A perspective. Genet Med 2005a; 7: 3-12.
[21] Macario, A.J.L. and Conway de Macario, E. Sick chaperones, cellular stress and disease. New Eng J Med
2005b; 353: 1489-1501.
[22] Perng MD, Wen SF, van den IJssel P, Prescott AR, Quinlan RA. Desmin aggregate formation by R120G
alphaB-crystallin is caused by altered filament interactions and is dependent upon network status in
cells. Mol Biol Cell 2004; 15: 2335-2346.
[23] Shields, A., and Hejtmancik, J.F. Genetic origins of cataracts. Arch Ophthalmol 2007; 125: 165-173.
[24] Houlden, H., Laura, M., Wavrant-De VriГЁze, F., Blake, J., Wood, N. and Reilly, M.M. Mutations in the HSP27
(HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 71: 16601668, 2008. Comment in: Neurology 2008; 71: 1656-1657.
[25] Singh, R., KГёlvraa, S. and Rattan, S.I.S. Genetics of longevity: Special emphasis on the relevance of
hsp70 as candidate genes. Front Biosci 2007; 12: 4504-4513. To see Table of Contents, Abstract, Figures, and Tables go to:
[26] Bhattacharyya, J., Shipova, E.V., Santhoshkumar, P., Sharma, K.K. and Ortwerth, B.J. Effect of a single
AGE modification on the structure and chaperone activity of human alphaB-crystallin. Biochemistry
2007; 46: 14682-14692.
[27] Cherian M, Abraham EC. Decreased molecular chaperone property of alpha-crystallins due to posttranslational modifications. Biochem Biophys Res Commun 1995; 208: 675-679.
[28] Kumar, P.A., Kumar, M.S. and Reddy, G.B. Effect of glycation on alpha-crystallin structure and chaperonelike function. Biochem J 2007; 408: 251-258.
[29] Ozawa, K., Tsukamoto, Y., Hori, O., Kitao, Y., Yanagi, H., Stern, D.M. and Ogawa, S. Regulation of tumor
angiogenesis by oxygen-regulated protein 150, an inducible endoplasmic reticulum chaperone. Cancer
Res 2001; 61: 4206-4213.
[30] Cappello, F., Bellafiore, M., David, S., Anzalone, R. and Zummo, G. Ten kilodalton heat shock protein
(HSP10) is overexpressed during carinogenesis of large bowel and uterine exocervix. Cancer Lett
2003a; 196: 35-41.
[31] Cappello, F., Rappa, F., David, S., Anzalone, R. and Zummo, G. Immunohistochemical evaluation of PCNA,
p53, HSP60, HSP10 and MUC-2 presence and expression in prostate canrcinogenesis. Anticancer Res
2003b; 23: 1325-1332.
[32] Cappello, F., David, S., Rappa, F., Bucchieri, F., Marasa, L., Bartolotta, T.E., Farina, F. and Zummo, G. The
expression of HSP60 and HSP10 in large bowel carcinomas with lymph node metastases. BMC Cancer
2005; 5: 139. (10 pages).
[33] Czarnecka, A.M., Campanella, C., Zummo G. and Cappello, F. Heat shock protein 10 and signal transduction: a “capsula eburnea” of carcinogenesis? Cell Stress Chaperon 2006; 11: 287-294.
[34] Pick, E., Kluger, Y., Giltnane, J.M., Moeder, C., Camp, R.L.., Rimm, D.L. and Kluger, H.M. High HSP90
expression is associated with decreased survival in breast cancer. Cancer Res 2007; 67: 2932-2937.
[35] Stojadinovic, A., Hooke, J.A., Shriver, C.D., Nissan, A., Kovatich, A.J. , Kao, T-C., Ponniah, S., Peoples, G.E.,
and Moroni, M. HYOU1/Orp150 expression in breast cancer. Med Sci Monit 2007; 13 (11):BR231-239
17968289 (P,S,E,B).
[36] Cappello, F., Di Stefano, A., David, S., Rappa, F., Anzalone, R., La Rocca, G., D’Anna, S.E., Magno, F., Don-
The Chaperoning system: physiology and pathology
ner, C., Balbi, B. And Zummo, G.. Hsp60 and Hsp10 down-regulation predicts bronchial epithelial carcinogenesis in smokers with chronic obstructive pulmonary disease. Cancer 2006; 107: 2417-2424.
[37] Rohde, M., Daugard, M., Jensen, M.H. Helin, K., Nylandsted, J. and Jaattela, M. Members of the heatshock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev 2005; 19:
[38] Sakono M, Zako T, Ueda H, Yohda M, Maeda M. Formation of highly toxic soluble amyloid beta oligomers
by the molecular chaperone prefoldin. FEBS J 2008; 275: 5982-5993.
Airway cells,
epidermis, signaling
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
TGFb signaling: Roles of Smads
[La segnalazione del TGFОІ: ruoli degli Smads]
Francesca Magno*§1, Giampiero La Rocca§1, Rita Anzalone1, Simona Corrao1, Tiziana Loria1,
Melania Lo Iacono1, Gaetana Spagnolo1, Antonino Di Stefano2, Bruno Balbi2 and Giovanni
Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, UniversitГ degli Studi di Palermo,
Italy; 2Fondazione S. Maugeri, IRCCS, Veruno, NO (IT) - *Corresponding author - В§The two authors contributed
equally to this work
Key words: TGFОІ, Smads, Chronic obstructive Pulmonary Disease, Extracellular matrix
Parole chiave: TGFОІ, Smads, Broncopneumopatia Cronica Ostruttiva, Matrice extracellulare
Abstract. Transforming Growth Factor ОІ (TGFОІ) is involved in regulation of several biological and physiological processes such as stimulation and regulation of lung extracellular matrix (ECM) components. Binding
of this cytokine to specific membrane receptors induces a transduction signal mediated mainly by Smad
proteins. In the last decade many studies increased the information about the structural characteristics of
these molecules, together with their main molecular interactions. In this review we briefly summarized what
is know about these proteins, focusing on the knowledge of the key steps of TGFОІ transduction pathway.
Riassunto. Il fattore di crescita trasformante ОІ (TGFОІ) ГЁ coinvolto nella regolazione di diversi processi fisiologici, come la stimolazione e regolazione dei componenti della matrice extracellulare (ECM) polmonare.
Il legame di questa citochina a specifici recettori di membrana induce una trasduzione del segnale mediata principalmente dalle proteine Smad. Nell’ultimo decennio molti studi hanno contribuito ad aumentare
le conoscenze circa le caratteristiche strutturali di queste molecole, insieme alle loro principali interazioni
molecolari. In questa review, noi riassumiamo brevemente le informazioni circa queste proteine, focalizzando
l’attenzione sulla conoscenza dei punti chiave del processo di trasduzione del segnale mediato dal TGFβ.
1. Introduction
COPD (Chronic Obstructive Pulmonary Disease) is characterised by a slow progressive
and partially reversible airflow limitation that is associated with an abnormal inflammatory
response of the lungs to noxious particles or gases. The pathological hallmarks of COPD are
typical of the main lung diseases such as chronic bronchitis, emphysema, chronic asthma
tissue damage and fibrosis [1]. All of these pathologies are characterized by an inflammatory process with an increase of inflammatory cells and production of a large variety of
cytokines. The transforming growth factor ОІ appears to be the most important cytokine involved on stimulation and regulation of lung extracellular matrix (ECM) components. TGF-ОІ
superfamily consist of secreted growth factors involved on regulation of different cellular
Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania,
Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni
processes, such as cell growth, development, differentiation, proliferation, motility, adhesion
and apoptosis [2,3]. The mammalian TGFОІ family comprises three members: TGFОІ1, TGFОІ2
and TGFОІ3. Each of them is encoded by a distinct gene and is expressed in both a tissuespecific and developmentally-regulated fashion. Specifically, TGFОІ1 is expressed in endothelial, hematopoietic, and connective tissue cells; both TGFОІ1 and TGFОІ3 are expressed
in structures undergoing morphogenesis and mesenchymal cells, while TGFОІ2 alone is expressed in differentiating and mature epithelium [4,5]. These peptides play an important
role in the pathogenesis of different diseases, such as cancer, fibrotic and parasitic diseases
[5]. Members of this family are secreted as latent ligands by binding to their propeptide, or in
trapped form by binding to occluding factors. The active form of a TGF-ОІ cytokine is a dimer
stabilized by hydrophobic interactions, which are further strengthened by an inter-subunit
disulfide bridge in most cases. TGFОІ signal is initiated by binding to a specific pair of membrane serine/threonine kinase receptors (type I and type II receptors) and is transduced to
the nucleus by Mitogen Activate Protein Kinase (MAPKs) and, principally, by Smad proteins.
2. Basic features of Smad proteins
Eight Smad proteins are encoded in the human and mouse genome, four in Drosophila
and three in C. Elegans [6]. Only five of the mammalian Smads (R-Smads, namely Smad1,
Smad2, Smad3, Smad5 and Smad8 act as substrates for the TGFβ family’s receptors; indeed Smads1, 5, and 8 act mainly as substrates for the BMP (Bone Morphogenetic Protein)
and anti-Muellerian receptors, while Smads2 and 3 for the TGFОІ, activin, and nodal receptors. Co-Smad Smad4 serves as a common partner for all R-Smads while Smad6 and 7 are
inhibitory proteins. The name “Smad” was coined in reference to its sequence similarity
to the Sma and Mad proteins. Functional studies, together with the X-ray crystal structure
analysis, showed that these ~500 amino acids proteins consist of two conserved globular
domains (MH1 and MH2 domains) coupled by a flexible linker region rich of binding sites
for Smurf (Smad ubiquitination-related factors) ubiquitin ligase, of phosphorylation sites
for several classes of protein kinases and, in Smad4, a nuclear export signal (NES) involve
on nucleus-cytoplasmatic translocation [7]. The MH1 domain is conserved in all R-Smads
and in Co-Smad but not in inhibitory Smads; it functions as DNA-binding site containing
a ОІ-harpin structure and is stabilized by a tightly bound zinc atom [8,9]. C-terminal domain, or MH2 domain, is conserved in both Smad proteins and is involved on Smad-Smad
interaction and on R-Smad activation/phosphorylation. At limited distance a basic pocket
interacts with the phosphorylated region of the activated receptor type I (in R-Smad), or
with the phosphorylated tail of R-Smad (in case of Smad4); in the other site, a set of contiguous hydrophobic patches, the “Hydrophobic corridor”, are involved in interaction with
nucleoporins, and cytoplasmatic retention proteins [10].
TGFb signaling: Roles of Smads
3. Receptor internalitation
Binding of TGFОІ to the type I and type II receptors, induces formation of multimeric
receptors. The serine/threonine kinase receptor family in the human genome comprises
12 members, 7 type I and 5 type II receptors, all involved in TGFОІ signaling; both types
of receptors consist of about 500 amino acids, organized sequentially into an N-terminal
extracellular ligand binding domain, a transmembrane region, and a C-terminal serine/
threonine kinase domain. In contrast with BMPs, TGFОІ and Activin display a high affinity for
the type II receptors and do not interact with the isolated type I receptors [6]. TGFОІ binds
tightly to the ectodomain of the type II receptor first; this binding allows the subsequent
dimerization with the type I receptor, forming a large ligand-receptor complex involving a
ligand dimer and four receptor molecules. Lin suggested that once type II receptors phosphorylate type I receptors, they could interact at the cell surface with the cytoplasmatic
form of promyelocytic leukemia protein (cPML) that acts as a bridging factor between a
Smad Anchor for Receptor Activation (SARA) and Smad2/3. cPML promotes the transfer of
the complex formed into early endosomes, where cPML could dissociate from the complex, allowing Smad2/3 to interact with SARA and to be phosphorylated [11]. SARA is an
anchor protein which acts by presenting R-Smads to the activated type I receptor and
thereby regulating TGFОІ transduction signal. It is a multidomain protein that consist of 80
amino acids Smad-Binding Domain (SBD) and a FYVE phosholipid-binding domain that
avidly binds to phosphatidyl inositol 3-phosphate on endosomal membranes and targets
SARA preferentially to early endosomes [12]. As revealed by the X-ray crystal structure
of the Smad2–SBD complex, the SBD of SARA makes contact with the three consecutive
hydrophobic patches on the MH2 domain surface that constitute the hydrophobic corridor
[13], blocking translocation of Smad2/3 into the nucleus and formation of transcriptional
complexes; the activated TGFОІ receptor complex therefore undergoes endocytosis via two
distinct routes: by internalization via coated vesicles to early endosomes and via caveolae
to caveolin-positive vesicles for degradation [12]. However other adaptor proteins were
found to cooperate with SARA on Smad signaling, such as FYVE-containing protein, HGS
[14] Disabled-2 [15], Axin [16], and the ELF ОІ-spectrin [17].
4. Smad phosphorylation
In the basal state, Smads form homoligomers and remain in an inactive conformation through a hydrophobic interaction between the MH1 and MH2 domains. In solution,
the unphosphorylated Smad2 MH2 domain is a monomer but, once phosphorylated, both
R-Smads and Smad4 form homotrimic complexes [9,18]. As revealed by X-ray crystallographic studies, the binding of R-Smad to Smad4 forms oligomers, heterodimers (RSmad–Smad4) or heterotrimers (two R-Smads molecules plus one Smad4 molecule);
Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania,
Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni
these oligomers are stabilized by interactions within an extensive protein–protein interface
between MH2 domains plus the binding of the pSer–X–pSer motif of one MH2 domain into
the di-phosphoserine binding pocket on the adjacent MH2 domain [19]. Phosphorylation
can occur in two different sites: in the linker region sites by several kinases, including
MAPKs and CDK [20,21] and mainly on the C-terminal conserved Ser-Val-Ser (Ser-MetSer in Smad2) motif of Smad3 by the activated receptor [7,10]. Actually the cytoplasmatic
region of the type I receptor contains a canonical protein kinase domain preceded by a
regulatory region or GS domain to which the inhibitor FKBP12 binds to enforce the inactive
basal state. Phosphorylation of the GS domain by the type II receptor creates a repeated
pS-X-pS motif that serves as a docking site for both R-Smads [7]; as a result, R-Smads
decrease their affinity for SARA and the activated oligomer complex is translocated into
the nucleus where it can bind directly with DNA promoters or indirectly binding with transcription factor or co-factors. [22, 23]. However TGFОІ-independent signals can involve
target sites rich of multiple serine and threonine, both on linker region and on N-terminal
domain; indeed MAPKs bind these consensus sites and are responsable of Smad3/2 phosphorylation or Smad4 activation [24]. In vitro, ERK 1 and 2 are able to activate Smad1,
2, and 3 by phosphorylating the linker region; moreover ERK-mediated phosphorylation
of Smads 1, 2, and 3 was shown to attenuate the nuclear accumulation and signaling
activity of these proteins in response to agonists [25]. The linker region of R-Smads are
subject to phosphorylation by Cam Kinase II (Ca++- and Calmodulin-dependent kinase II)
[26], while the N-terminal and the linker region of Smad3 can be phosphorylated by CDKs
(Cyclin-dependent kinases) [27] or by GRK-2 (G protein-coupled Receptor Kinase 2) that
phosphorylates Ser197 in Smad2 [28].
5. Smad dephosphorylation
Recently phosphatases have been suggested as regulatory molecules of the TGFОІ
pathway; the interest for phosphatases started from a study in which was demonstrated
that in a cell-free system Smad2 was exported from the nucleus in a dephosphorylated
state [29]. Protein phosphatases control cellular function through cleavage of phosphate
from phosphorylated serine/threonine (pS/Ts) and tyrosine (pY) residues in proteins. The
human genome encodes about 146 phosphatases, of these. 40 are able to act on serine/
threonine residues (PS/TP). An important role on Smads activity regulation was shown for
members of the PPM subfamily namely PPM1A/PP2C-О± and PPM1B/PP2CОІ. Both PPM
phosphatases directly dephosphorylate R-Smads at the C-terminal SXS motif, but not the
phospho Ser/Thr residues in the linker region; PPM1A, is exclusively present in the nucleus,
and its activity enhances the disassembly of the activated Smad complex and facilitates
nuclear export of Smad2/3, unlike PPMB [11,30]. Elevated levels of PPM1A mitigated tran28
TGFb signaling: Roles of Smads
scriptional response to TGF-ОІ, and inhibited the anti-proliferation function of TGF-ОІ. However dephosphorylation seems to act on Smads after they end their transcriptional function
on DNA, and thereby not interfering directly with TGFОІ signaling. Recently, Wrighton and
colleagues discovered another phosphatase able to attenuate Smad signalling, the SCP1
or C-terminal domain phosphatase 1. It is a specific phosphatase that dephosphorylates
the linker region and the N-terminal domain, specifically at Thr-220 and Ser-245/250/255
in Smad2 and Thr-179 and Ser-204/Ser-S208/Ser-213 in Smad3 [31], resetting them to
their basal unphosphorylated state. The biological mechanism by which they can act on
Smad is unclear.
6. Nuclear import and export mechanism of Smads
The mechanisms of Smad nuclear import and export have been extensively studied
over the last few years, particularly for Smads 2, 3, and 4. Two recent studies showed that
Smads do not reside statically in the cytoplasm in absence of ligands or in the nucleus
upon TGFОІ stimulation; there is a spontaneous bi-directional shuttling of the R-Smads/
Smad4 complex across the nuclear envelope without TGFОІ stimulation [23] even if the
nuclear import of the oligomer complex is offset by export forces, so they cannot reach
a high concentration in the nucleus [32]. In the basal state, R-Smads are predominantly
localized in the cytoplasm, I-Smads tend to be nuclear, while Smad4 is distributed in both
the cytoplasm and the nucleus. The process of nucleocytoplasmatic shuttling can occur
with or without nuclear transport factors [23]. Some Smad proteins can undergo nuclear
import via importins; importin-О± binds importin-ОІ which directly interacts with the nuclear
pore components, namely the nucleoporins CAN/Nup214 and Nup153 [23] to negotiate
the passage of the importin-ОІ/importin-О±-cargo complex into the nucleus. However, the
nuclear translocation of Smad proteins can occur independently of importins because they
can directly interact with nucleoporins [23]. Smad2, 3, and 4 undergo nuclear import by direct interactions with nucleoporins, this process may be aided by importin-ОІ in the case of
Smad3 and 4. The intrinsic asymmetry of the nuclear pore complex and the distribution of
different nucleoporins along the span of the pore are thought to allow proteins docking on
one side of the pore to move unidirectionally to the other side and vice versa [33]. Indeed,
the direct interaction of Smad2 and 3 with nucleoporins has been shown to enable nuclear
export as well as import [23].
7. Post-translational regulation of Smads
Smad activation and transcriptional function can be regulated in both cytoplasm and
nucleus. Several types of mechanisms were involved to limit their activity: one type includes transcriptional co-repressors, such as the homeodomain protein TGIF. Binding of
Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania,
Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni
TGIF prevents the interaction of Smads with nuclear co-activators and interferes with activation of TGFОІ-target genes expression. The inhibitory Smads; Smad, 6 and 7, serve as
decoys interfering with Smad–receptor or Smad–Smad interactions; Smad7 inhibits TGFβ/
activin and BMP signaling, while Smad6 inhibits primarily the BMP pathway. Smad6 competes with Smad4 for interaction with the receptor-activated Smad1, such that inactive
Smad1–Smad6 complexes are formed [34]. Smad7 acts as a competitive inhibitor able to
bind the activated TGFОІ and BMP receptors, competing with R-Smads and prevending or
blocking their activation [35], it is involved in regulation of TGF receptor ubiquination by
SMURF ubiquitin ligase [36]. Smurf1 and Smurf2 are HECT-domain containing E3 ubiquitin
ligases that recognize R-Smads as substrates through a PPXY motif. Smurf2 can bind to
Smad7, without immediate ubiquitination and degradation, but favouring export of Smad7
to the cytoplasm. The Smurf-Smad7 complex is able to bind to the TGFОІ receptor complex
and promotes its ubiquitination, thereby down-regulating signaling; however the activity
of Smad7 is regulated at many levels: it is induced by TGFОІ, activin and BMP signaling
and by ERK [37] or inflammatory signals such as interferon-Оі, TNF-О± and interleukin-1ОІ
[38]. Activated R-Smads undergo ubiquitination and subsequent degradation. Increasing
evidence indicates that ubiquitin modification is important for regulating components of
TGFОІ signaling and can occur in both the basal and the activated state: for example, phosphorylated Smad2 is eliminated by the fast action of phosphatases and also by the slower
action of ubiquitin-dependent proteosome degradation. The susceptibility to ubiquitination
may be controlled by acetylation of the same lysine residues. Following phosphorylation,
Smad2 and 3 are translocated to the nucleus where they interact with co-activators and/or
co-repressors. Smads transcriptional activity is significantly enhanced by recruitment and
direct interaction with p300/CBP (CREB-binding protein). It is a co-activator with intrinsic
acetyltransferase activity able to transfer the acetyl group from acetyl coenzyme A to the
lysine residues in histones allowing remodeling of chromatin to a more relaxed conformation to allow transcription. Smad2, but not Smad3, can be acetylated by p300/CBP in a
ligand-dependent manner, this event involves at least three lysine residues, Lys19, Lys20,
and Lys39, that are required for efficient acetylation of Smad2. An acetylation event is required for Smad2 to mediate activin and TGFОІ signaling. Mutations of the three key lysine
residues did not alter the stability of Smad2 or its ability to form a complex with Smad4
on promoter DNA, but prevent nuclear accumulation of Smad2 and subsequent TGFОІ and
activin responses [39]. Nuclear Smad7 can be also acetylated by p300 at two N-terminal
lysines, which are also the sites of ubiquitin attachment. Acetylation prevented receptorinduced ubiquitination of Smad7 by Smurf, without affecting the subcellular distribution
of Smad7. Another Smads regulatory process is Sumoylation. In higher eukaryotes there
are three SUMO (Small-Ubiquitin-like Modifier) proteins, SUMO-1, SUMO-2 and SUMO-3,
TGFb signaling: Roles of Smads
that alter proteins function by creating a composite interaction domain [40] and regulating
protein stability and subcellular localization. Two sumoylation consensus motifs were observed in Smad4; one in MH1 domain and one in the linker region [40]. As consequence of
this regulation, however, two opposite effects were observed, an increase and a decrease
of transcriptional activity [41,42], probably as a result of potential competition between
ubiquitination and sumoylation of the same lysine residue in Smad4. Ubiquitination of RSmad/Smad4 complex can be a target of proteosomes and as a result, gene expression
is reduced. By sumoylation, ubiquitination effects are blocked, half life of the activated
complex is prolonged and gene expression is increased. SUMO modification also represses
transcriptional activity of a number of transcription factors, such as Sp3, c-Jun, c-Myb,
AP2, nuclear receptors and Elk-1, and the general co-activator p300, regulating their biological role [41].
8. Conclusions
In the last ten years the attention of scientists for Smad proteins seem to be increased.
Each piece of information contributes to better understand their possible role in maintenance and development of pathological diseases and helps scientists to understand the
molecular network of TGFОІ1-induced pathways. In this review we summarized what is
known about these molecules. This knowledge is a necessary basis in order to obtain a
clear picture of this regulatory system helps to advance future studies.
Magno Francesca, La Rocca Giampiero, Anzalone Rita, Corrao Simona, Loria Tiziana, Lo Iacono Melania,
Spagnolo G, Di Stefano A, Balbi B, Zummo Giovanni
[1] GOLD, 2006
[2] Hogan B.L.M. Bone morphogenetic proteins: Multifunctional regulators of vertebrate development.
Genes & Dev. 1996; 10: 1580-1594.
[3] Mehler, M.F, Mabie PC, Zhang D, Kessler JA. Bone morphogenetic proteins in the nervous system.
Trends Neurosci 1997; 20: 309-317.
[4] Taipale J, Saharinen J, Keski-Oja J. Extracellular matrix-associated transforming growth factor-b: role
in cancer cell growth and invasion. Adv Cancer Res 1998; 75:87-134.
[5] Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor b in human disease. N Engl J
Med 2000; 342:1350-1358.
[6] MassagueВґ J. TGF-ОІ signal transduction. Annu Rev Biochem 1998; 67, 753-791.
[7] MassaguГЁ J, Seoane J, Wotton D. Smad transcription factors. Genes and Dev 2005; 19:2783-2810.
[8] Dennler S, Itoh S, Vivien D, Ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to
critical TGFb-inducible elements in the promoter of human plasminogen activator inhibitor -type 1
gene. EMBO J 1998; 17, 3091-3100.
[9] Shi Y, Hata A, Lo RS, MassaguГЁ J, Pevletich NP. A structural basis for mutational inactivation of the
tumor suppressor Smad4. Nature 1997; 388: 87-93.
[10] Ross S, Hill CS. How the Smads regulate transcription. The International Journal of biochemistry & Cell
Biology 2008; 40: 383-408.
[11] Lin X, Duan YY, Liang Y, Su Y, Wrighton KH, Long J, Hu M, Davis CM, Wang J, Brunicardi FC, Shi Y, Chen
YG, Meng A, Feng XH. PPM1A fuction as a smad phosphatase to terminate TGFbeta signalling. Cell
2006; 125: 915-928.
[12] Di Guglielmo, GM, Le Roy C, Goodfellow, AF, Wrana, JL. Distinct endocytic pathways regulate TGF-ОІ
receptor signalling and turnover. Nat. Cell Biol. 2003; 5: 410-421.
[13] Wu G, Chen YG, Ozdamar B, Gyuricza CA, Chong PA, Wrana JL, MassaguГ© J, Shi Y. Structural basis of
Smad2 recognition by the Smad anchor for receptor activation. Science 2000; 287: 92-97.
[14] Mirura S, Takeshita T, Asao H, Kimura Y, Murata K, Sasaki Y, Hanai JI, Beppu H, Tsukazaki T, Wrana JL,
et al. Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA.
Mol Cell Biol 2000; 20, 9346-9355.
[15] Hocevar BA, Smine A, Xu XX, Howe PH. The adaptor molecule Disabled-2 links the transforming growth
factor ОІ receptors to the Smad pathway. EMBO J 2001; 20, 2789-2801.
[16] Furuhashi M, Yagi K, Yamamoto H, Furukawa Y, Shimada S, Nakamura Y, Kikuchi A, Miyazono K, Kato M.
Axin facilitates Smad3 activation in the transforming growth factor ОІ signaling pathway. Mol Cell Biol
2001; 21, 5132-5141.
[17] Tang Y, Katuri V, Dillner A, Mishra B, Deng CX, Mishra L. Disruption of transforming growth factor-ОІ
signaling in ELF О±-spectrin-deficient mice. Science 2003; 299: 574-577.
[18] Lagna G, Hata A, Hemmati-Brivanlou A, Massague J. Partnership between DPC4 and SMAD proteins in
TGF-ОІ signalling pathways. Nature 1996; 383: 832-836.
[19] Wu JW, Hu M, Chai J, Seoane J, Huse M, Li C, Rigotti DJ, Kyin S, Muir TW, Fairman R,, et al. Crystal
structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights
on Smad function in TGF-ОІ signaling. Mol Cell 2001; 8: 1277-1289.
[20] Brown JD, Di Chiara MR, Anderson KR, Gimbrone MA, Jr Topper JN. MEKK-1, a component of the stress
(stress-activated protein kinase/c-jun N-terminal kinase) pathway, can selectively activate Smad2mediated transcriptional activation in endothelial cells. J Biol Chem 1999; 274(13): 8797-8805.
TGFb signaling: Roles of Smads
[21] Shi W, Sun C, He B, Xiong W, Shi X, Yao D, Cao X. GADD34-PP1c recruited by smad7 dephosphorylates
TGF-beta type I receptor. J Cell Biol 2004; 164(2): 291-300.
[22] Xu, L., Chen, Y.G., and MassaguГ©, J. The nuclear import function of Smad2 is masked by SARA and
unmasked by TGFОІ-dependent phosphorylation. Nat Cell Biol 2000; 2: 559-562.
[23] Xu, L, Kang Y, Col S,MassaguГ©, J. Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and
Nup153 feeds TGFОІ signaling complexes in the cytoplasm and nucleus. Mol Cell 2002; 10: 271-282.
[24] Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-ОІ family signalling.
Nature 2003; 425: 577-584.
[25] Kretzschmar M, Doody J, Timokhina I MassaguГЁ J. Genes Dev 1999; 13, 804-816.
[26] Wicks SJ, Lui S, Abdel-Wahab N, Mason RM, Chantry A. Inactivation of smad-transforming growth factor
beta signaling by Ca(2+)- calmodulin-dependent protein kinase II. Mol Cell Biol 2000; 20: 8103-8111.
[27] Matsuura I, N.G. Denissova, G. Wang, D. He, J. Long, F. Liu. Cyclindependent kinases regulate the antiproliferative function of Smads. Nature 2004; 430: 226-231.
[28] Ho J, Cocolakis E, Dumas VM, Posner BI, Laporte SA, Lebrun JJ. The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction. EMBO J 2005; 3247-3258.
[29] Alonso A, Sasin J , Bottini N, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T. Proetin
tyrosine phosphatases in the human genome. Cell 2004; 117(6): 699-711.
[30] Lin X, Chen Y, Meng A, Feng X. termination of TGFb Superfamily signalling through SMAD desphosphorylation- a functional genomic view. Journal of Genetics and Genomics 2007; 34(1): 1-9.
[31] Wrighton KH, Willis D, Long J, Liu F, Lin X, Feng X.H. Small C-terminal Domain Phosphatases Dephosphorylate the Regulatory Linker Regions of Smad2 and Smad3 to Enhance Transforming Growth
Factor-ОІ Signaling. J Biol Chem 2006; 281(50): 38365-38375.
[32] Nicolas FJ, De bosscher K, Schmierer B, Hill CS. Analisys of smad nucleocytoplasmic shuttling in living
cells. J Cell Sci 2004; 177: 4113-4125.
[33] Rout, MP, Aitchison, JD, Suprapto A, Hjertaas, K, Zhao Y, Chait BT. The yeast nuclear pore complex: Composition, architecture, and transport mechanism. J Cell Biol 2000; 148: 635-651.
[34] Hata A, Lagna G, MassaguГ© J, Hemmati-Brivanlou A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes & Dev 1998; 12: 186-197.
[35] Nakao A, Afrakhte M, MorГ©n A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH,
et al. Identification of Smad7, a TGFОІ-inducible antagonist of TGF-ОІ signaling. Nature 1997; 389: 631-635.
[36] Suzuki C, Murakami G, Fukuchi M, Shimanuki T, Shikauchi Y, Imamura T, Miyazono K. Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane. J Biol Chem 2002; 277: 39919-39925.
[37] Huo YY, Hu YC, He XR, Wang Y, Song BQ, Zhou PK, Zhu MX, Li G,Wu DC. Activation of extracellular signalregulated kinase by TGF-beta1 via TbetaRII and Smad7 dependent mechanisms in human bronchial
epithelial BEP2D cells. Cell Biol Toxicol 2007; 23: 113-128.
[38] Bitzer M, Von Gersdorff G, Liang D, Dominguez-Rosales A, Beg AA, Rojkind M, Bottinger EP. A mechanism of suppression of TGF-ОІ/SMAD signaling by NF-ОІ/ RelA. Genes & Dev 2000; 14: 187-197.
[39] Tu AW, Tuo K . Acetylation of Smad2 by the Co-activator p300 Regulates Activin and Transforming
Growth Factor ОІ Response. The Journal of Biological Chemistry 2007; 29(282): 211.
[40] Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc Natl Acad Sci 2004; 101: 14373-14378.
[41] Long J, Wang G, He D, Liu F. Repression of Smad4 transcriptional activity by SUMO modification. Biochem J 2004; 379: 23-29.
[42] Chang CC, Lin DY, Fang HI, Chen RH, Shih HM. Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4. J Biol Chem 2005; 280: 10164-10173.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
New insight on immunological activation pathways of
Langerhans cells, possible tolerogenic role
[Le Cellule di Langerhans e il loro possibile ruolo tollerogenico]
Marco La Manna1, Viviana Bazan2, Aldo Gerbino3 and Alfredo Salerno1
Dipartimento di Biopatologia e Metologie Biomediche - Sezione di Patologia Generale, 2 Dipartimento di
Discipline Chirurgiche ed Oncologiche, 3 Di.Me.S. - Sezione di Istologia ed Embriologia. UniversitГ degli Studi
di Palermo (IT)
Key words: Langerhans cells, Role Tolerogenic
Parole chiave: Cellule di Langerhans, Tollerogenesi
Abstract. Langerhans cells are the prototype of antigen presenting cell, their role is to work as sentinel in
the epidermis.
Like every APC Langerhans cells act as bridge between innate and acquired immunity recognizing antigens
into the epidermis and bringing them to drying lymph node, their work is well described by the Langerhans
cell paradigm.
Recently many works designed a new and amazing role of Langerhans cells in fact they often showed tolerogenic capacity, while in many cases, they seems not necessary to promote activation of acquired immunity.
Anyway Langerhans cells remaining an interesting target for new vaccine strategies because of their localization in the epidermis and the ability to capture antigens trough different recognition pathways.
Also interesting is the possible use of langerhans cells for their tolerogenic capacity, in many experimental
models these cells opportunely addressed with appropriated cytokines appears able to down-regulate immune system response in autoimmune pathology.
Riassunto. Le cellule di Langerhans sono il prototipo delle APC (antigen presenting cells) professionali, il loro
ruolo è quello di agire da sentinella nell’epidermide.
Come ogni APC le cellule di Langerhans agiscono come ponte tra l’immunità innata e quella acquisita catturando antigeni nell’epidermide e trasportandoli nel linfonodo drenante, il loro ruolo nell’ambito del sistema
immunologico è ben descritto dal: “paradigma delle cellule di Langerhans”.
Recentemente molti lavori ha definito un nuovo interessante ruolo per le cellule di Langerhans mostrando
una loro importante attivitГ tollerogenica, mentre in molti casi sembra che queste cellule, pur avendo una
grande capacità come APC, non siano necessarie per promuovere l’attivazione della immunità adattativa.
Per la loro attivitГ di APC le cellule di Langerhans restano un interessante bersaglio per la messa a punto
di nuovi vaccini grazie alla localizzazione nell’epidermide ed alla loro capacità di catturare diversi antigeni
attraverso vari pattern di riconoscimento. Г€ inoltre di notevole interesse un loro utilizzo che sfrutti la loro
attivitГ tollerogenica, poichГ© in molti modelli sperimentali queste cellule opportunamente indirizzate con le
appropriate citochine sembrano essere in grado di sopprimere la risposta del sistema immune nel contesto
di patologie autoimmuni della cute.
Le cellule di Langerhans (LC), cellule dendritiche della linea mieloide presenti nell’epidermide, costituiscono
una importante parte del sistema immune della pelle. Sebbene scoperte da Poul Langerhans nel 1868, ci
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
sono voluti piГ№ di 100 anni prima che queste cellule fossero associate al sistema immune e riconosciute
come APC (antigen presenting cells). In particolare il gruppo di ricerca di Ralph Steiman scoprì le caratteristiche funzionali delle LC partendo dal dato che le LC presenti nell’epidermide murina, in vitro, maturavano in
cellule dendritiche con forti capacitГ immunostimolatorie.
I lavori che seguirono sullo studio delle LC; disegnarono il concetto di cellule dendritiche (DC) come APC
professionali capaci di indurre una risposta immune mediata dai linfociti T successivamente Wilson e Villadangos [4] idearono il paradigma delle LC che assegna tre funzioni chiave alle a queste cellule.
Langerhans cells “paradigm”
Langerhans cells (LC) a constituent part of the skin immune system. were discovered
by Paul Langerhans in 1868 [1], but it took more than 100 years before they were first
associated with the immune system and recognized as antigen presenting cells [2]. In particular, Ralph Steinman’s group [3] discovered the functional characteristics of LC starting
from the finding that murine epidermal LC mature into potent immunostimulatory dendritic
cells (DC) in vitro. The work that followed shaped the concept of DC as professional antigen presenting cells (APC) and inducers of T-cell immune responses largely by studying
LC, later the term �LC paradigm’ was coined by Wilson and Villadangos [4]. This concept
assigned three key functions to LC/DC [5].
First, immature LC that reside in the periphery in the steady state are highly specialized
in antigen uptake. LC represent approximately 3% of epidermal cells and form a dense
network in the epidermis where they constantly scan the environment and are ideally positioned to detect any pathogen breaching the skin barrier [6].
Second, LC transport antigen to skin draining lymph nodes (LN), which is essential
to ensure interaction with rare antigen-specific naive T cells. Stimulation by a number of
pathogen products in addition to pro-inflammatory cytokines induces LC activation [7–9].
Third, during migration the LC undergo a process of maturation in which they process
antigen acquired in the skin and present it in the context of MHC class I/II. In addition, they
dramatically upregulate their surface expression of co-stimulatory molecules and start to
produce cytokines required for proper Th1 or Th2 instruction. Thus, by the time LC arrive in
the LN, they have acquired the surface phenotype of a �functionally’ mature DC capable of
activating naive T cells and initiating an adaptive immune response specifically designed
to fight off the invading cutaneous pathogen [5].
The LC paradigm has been expanded to include LC that migrate to skin-draining LN in
the steady state in the absence of activating stimuli where they present self-peptides to T
cells [10–14]. This steady-state presentation of self-antigens has been proposed to eliminate self-reactive T cells and provide a mechanism of peripheral tolerance [15, 16].
Recently the role of LC in peripheral tolerance was deeply investigated, resulting in a
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
pivotal activity perhaps more important than usually attributed APC role
Here we wont to analyze the morphological and functional aspects of LC from their
origin to migration into lymph node to understand which mechanisms make LCs able to
accomplish their role into the immune system.
Phenotypical aspects of Langerhans cells
As immature cells, LC primary function is constantly scan the environment by extending
and retracting their dendrites to sense the environment for danger signals and capture
antigens, LC express the C-type lectin CD207 (Langerin), which represent usually the hallmark of LC [17–19]. Langerin is the major constituent of Birbeck Granules (BG), that are
a unique cytoplasmic organelle and constitute a subdomain of the endosomal recycling
compartment, perhaps being involved in antigen loading processing.
LC also express a characteristic set of cell-surface molecules, such as cutaneous lymphocyte-associated antigen (CLA), E-cadherin, the CC chemokine receptor 6 (CCR6) [2024], the invariant MHCI like CD1a receptor and epithelial cell adhesion molecule (EpCAM)
[25] which is expressed in LC, but not in other DC subsets [26], whereas langerin is also
expressed in a subset of dermal Dendritic cells (dDC) and in some CD8a1 DC in LN [25,
27, 28].
Langerin and Birbeck Granule.
Although BGs have been first observed in 1961 [29], there is still a large part of mystery
around their organization and function in the Langerhans cells. Nevertheless, BGs were
described as organelles allowing a nonclassical routing for an antigen processing
pathway [30, 31].
These distinctive rod-shaped compartments BGs may therefore correspond to a specialized membrane domain of the ERC, devoted to the loading of CD1a with glycolipids
internalized and routed to the BGs by Langerin [32].
The characterization of Langerin has provided some insight into the biogenesis of Birbeck granules. Langerin is a transmembrane type II Ca2-dependent lectin with a single carbohydrate recognition domain displaying mannose-binding specificity and an intracellular
proline-rich motif [33].
This is consistent with the observation that Ca2+ removal can cause unzipping of BGs
to various extents with inner periodical pattern disintegration [34, 35].
In freshly isolated Langerhans cells, recycling Langerin is dynamically retained in the
endosomal recycling compartment and that Birbeck granules form where Langerin accumulates.
Langerin, necessary for the formation of BG is expressed on the plasma membrane and
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
constitutively internalized into BGs, the internal membranes where it accumulates, before
returning to the plasma membrane [32, 33].
A direct role of langerin in BG formation was demonstrated from observation of BG
induction in murine fibroblasts as well as in human melanoma cell line upon langerin gene
transfection [33, 36]
Langerin extracellular region, and more precisely its carbohydrate recognition domain
(CRD), was suggested to be a key element in the BG architecture [37].
BGs also display a remarkable association with Rab11 a GTPases of the Rab family,
known to be regulators of membrane traffic between organelles. Rab11 protein has been
reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN) [38, 39]. Thus, the major intracellular pool of
Langerin is the Rab11_ BGs, which correspond to subdomains of the endosomal recycling
compartment (ERC) of LCs [32].
Langerin induces the formation of BGs in areas where it accumulates (Valladeau et
al., 2000; McDermott et al., 2004), but the cellular distribution and traffic of Langerin are
controlled by Rab11A [33-40].
The formation of BGs depends on Langerin being addressed to an appropriate Rab11Adependent membrane environment. In this environment, the coordinated action of Rab11A and Rab11A–effector complexes creates the membrane platform required for BG
formation. In the absence of this platform, the traffic of Langerin is altered and favors its
routing to lysosomal compartments where it is degraded. Because Langerin recycles actively between the endocytic system and the plasma membrane, the formation of a Rab11A
driven membrane platform must be viewed as a highly dynamic process.
Such a role, as illustrated here for Rab11A, may lead directly or indirectly to the biogenesis of a specialized compartment. BGs may therefore correspond to a specialized
membrane domain of the ERC, devoted to the loading of CD1a with glycolipids internalized
and routed to the BGs by Langerin [32].
Rab coupling protein (RCP) depletion also decreased Langerin levels and numbers of
BGs, although to a less significant extent than in the case of TfR. Rab11A–RCP complexes
couldintervene in the routing of Langerin from degradative to recycling pathways [41].
Since it was not still clear the function of Birbeck granules, two opposing theories have
been proposed.
The previous first suggests that BGs could function as alternative endocytotic structures, specific to LCs [42-46]. However, under physiological conditions, BGs appended to the
cell surface were an exceptional finding [47,48]. Moreover, there are no evidence that BGs
participate in the internalization of Langerin, one of their essential constituents. Unlike in
the physiological state, “open ended” [49] BG-like structures appended to the cell mem38
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
brane were rapidly induced by treating LCs with cytochalasin D or latrunculin A, irrespective of the presence of an anti-Langerin mAb. One possible explanation for these results
would be that BGs represent a transient endocytic intermediate that can no longer be
pinched off the cell membrane in the presence of these drugs. However, not only BG-like
structures were seen under these conditions but also a large number of coated pits, which
were labeled by gold conjugated anti-Langerin mAb, consistent with the recognized effect
of these drugs on classical receptor-mediated endocytosis [50, 51]. In addition, many of
the BG-like structures were clathrin coated, or had labeled coated pits at their cytoplasmic terminal end. These findings indicate first that Langerin endocytosis is mediated by
a classical clathrin coated pathway, regardless of whether the plasma membrane is linear
or folded, as in the characteristic zipped formation of BG-like structures and, second, that
BGs are not endocytotic structures.
The second proposed theory regarding BG function is that they could represent secretory structures formed in the Golgi area and involved in transport of cargo molecules to the
cell membrane. The disappearance of BGs from the pericentriolar area and concomitant
appearance of BG-like structures at the cell membrane after cytochalasin D or latrunculin
A treatment could signify that BGs act as transport intermediates “driving” Langerin,
and perhaps other molecules, from internal membranes to the cell surface. On arrival at
the cell surface such BGs would fuse with the plasma membrane, in a similar manner
synaptic vesicles originating from the early endosomal pathway [52].
Unzipping would follow, allowing delivery of their molecular content to the cell surface.
However, immunoelectron microscopy revealed BGs in continuity with tubular structures and also occasionally as part of early endosomal tubular networks in the pericentriolar
area, networks which became manifest after treatment with BFA. In addition, after internalization at 19.5В°C and temperature shift to 37В°C, Langerin was found both in the recycling
compartments and in pericentriolar BGs. Thus it is apparent that BGs may simply represent membrane domains of tubular recycling endosomes, the morphology of which has
been modified by the formation of molecular bridges. In this context, the elongated tubular
configuration of the recycling compartment [53] could enable two membrane leaflets to
come into close apposition, facilitating the generation of BGs. In contrast, such membrane
interaction might not occur in early sorting endosomes because of their vesicular shape.
The predominance of BGs in the ERC could also be explained if the molecules responsible for their formation transiently accumulate in this compartment during their traffic.
These last findings substantiate the hypothesis that BGs represent morphological modifications of preexisting membranes, rather than specific transport intermediates [32].
One can only speculate as to which antigen-presenting molecules Langerin might deliver bound antigen; however, on the basis of our results, CD1a is a potential target-presen39
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
ting molecule. It is demonstrated that endocytosed Langerin and CD1a molecules share a
common intracellular fate in LCs, traveling through identical intracellular compartments,
including pericentriolar BGs. Moreover, endocytosis and recycling of both molecules is arrested by LC maturation [51].
It was show that in a leprosy model infection LCs have a specialized role in the presentation of non peptide antigens to T cells, and that this function is mediated by the LCspecific pattern-recognition receptor langerin as well as the antigen-presenting molecule
CD1a, which is highly expressed by these cells [54].
Because BGs form where Langerin accumulates in the ERC and are depleted when LCs
are activated, they could in this context, serve as a loading compartment and/or a membrane reservoir for antigens before LC maturation.
Langerhans cells precursor cell subsets
The process of LC differentiation and their migration into the epidermis is not clearly
Understood It is assumed that LCs have no single origin and that local conditions within the epidermis direct theirs development. Studies of chimeric mice suggest that under
normal non inflammatory conditions, LCs are replaced entirely from a skin-resident LC
precursor pool, which could be derived from dermal CD141 cells [55]. These LC precursors
co-express Langerin and CD14, and have the potential to migrate through the dermal–
epidermal barrier to the suprabasal layer of the epidermis in response to controlled keratinocyte-derived CXCL14 and CCL20, once in epidermis, in the presence of keratinocytederived TGFb1, differentiate into immature resident
LC characterized by a weak T cell stimulatory activity [56-58].
So the final differentiation of LC precursors depends on the cytokine environment of
the epidermis (7). In fact the cutaneous cytokines granulocyte– macrophage colony-stimulating factor (GMCSF), interleukin (IL)-15, and TGF-b1 contribute to the establishment
of immature LC in the epidermis [57]
Other evidences suggest that LC are of myeloid origin and can differentiate from monocytes or CD34+ precursors [59-63].
Many studies suggested that bone marrow- derived myeloid cutaneous lymphocyte associated antigen (CLA)-expressing LC precursors travel through peripheral blood
through the dermis into the epidermis [64]. In an in vitro study it was demonstrated that
CD141CD341 PBMCs migrate across the endothelium and initiate development of immunostimulatory DCs [65].
Moreover, it was reported that the rare CD161monocyte subtype (5% to10%of peripheral blood monocytes), which is dramatically increased under inflammatory conditions,
differentiates into LCs [66].
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
It is interesting to note that no LCs of recipient origin were observed in transplanted
human hands over a period of 4.5 years [67]. These results strongly suggest that under
steady-state conditions, the replacement of human epidermal LCs from bone marrow–
derived precursors occurs very slowly.
In contrast, inflammation induces replacement of emigrated LCs, largely through a
bone marrow–derived precursor. Monocytes, MHC class II2 CD11c1 cells, and CD341 cell–
derived CD1a1 cells might give rise to development of LCs [66].
It is therefore of interest to note that while the generation of DC from monocytes requires GM-CSF and IL-4 only, the additional presence of TGFb1 in the cytokine milieu appears
to be essential for the development of LC [60, 61].
Accordingly, TGFb1-deficient mice display a severe defect in LC, but not in DC, development [68]. In vitro experiments have shown that IL-15, in cooperation with GM-CSF,
induces the differentiation of monocytes into cells that express LC markers, such as Ecadherin, CCR6 and Langerin but lacking the expression of conventional Birbeck granules
Another study reports that Activin A, a protein abundantly produced in the skin during
normal and pathological wound healing [70, 71] and inflammatory/autoimmune diseases
induces the differentiation of human CD14+monocytes in Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ cells. These langerin+ cells can be induced to differentiate by Activin A within the dermis in the absence of epidermis. In agreement with
these results, cells emigrated from skin explants could also be induced to differentiate into
Langerin+/CD1a+ cells by the presence of Activin A in vitro. These findings are compatible
with the description of CD14+, Langerin+ LC precursors located in the superficial and deep
dermis, predominantly in perivascular areas [72, 73].
Since Activin A is abundantly produced during certain inflammatory conditions, it was
proposed that this cytokine represents a new pathway, alternative to TGFb, responsible for
LC differentiation during inflammatory/autoimmune conditions [74].
Recognition of antigen
LC are able to bridge innate and adaptive immunity. They are able to interact directly
with microorganisms at the periphery to produce effector cytokines and initiate or restimulate activation of T and B lymphocytes through antigen presentation. LC express different
pattern recognition receptors such as C-type lectin receptors (CLR) langerin and Toll-like
receptors (TLR), to bind and capture pathogens. These interactions result in activation of
intracellular signalling pathways leading to LC activation and cytokine induction.
LC like other migratory DC are in an immature, highly endocytic state while sessile in
the skin/mucosa. However after pathogen binding to TLR and uptake, LC become mature
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
and migratory, down regulating their endocytic and antigen processing capacity. Mature LC
migrate to the lymph nodes where they are directly or indirectly involved in presentation of
pathogen derived antigens on MHC Class I land II or non polymorphic MHC like CD1a to T
cells, resulting in activation of antigen-specific T cells [75, 76].
LC maturation and migration from the Epidermis
LC exhibit a very slow turnover under steady-state conditions compared with other DC
subsets (including dDC), which undergo renewal at a much faster rate.
During skin inflammation, LC turnover is rapidly and markedly increased. The increased LC turnover allows the recruitment of new bone-marrow-derived LC precursors to the
epidermis, a process that requires the expression of CCR2 and CCR6 on LC precursors [25,
77, 78].
Skin contact with antigens is known to stimulate various epidermal cytokines, e.g. IL6, TNF-a, GM-CSF, and CXCL2/CXCL3 (macrophage inflammatory protein-2, MIP-2) [79],
among which IL-1b and TNF-a are essential in DC migration [80, 81]. LC leave the epidermis by a mechanism that depends on the expression of chemotactic receptors, adhesion
molecules and proteases [82-84].
Activation of Langerhans cells
Antigen recognition and subsequent activation of LC start a change in expression pattern of cytokines and surface receptors, functional to accomplish antigen presentation into
the lymph node also cytokine signalling results in altered expression of adhesion molecules, which facilitates DC migration out of the epidermis
Once activated, LC In order to leave the epidermis, need to cross the basement membrane at the dermo-epidermal junction [21, 28]. IL-1b and TNF-a play a central role in the
process of LC migration across the basement membrane [85, 86].
Upon initiation of the maturation process, LC produce IL-1b, which induces TNF-a secretion from adjacent keratinocytes [85, 86]. In brief, it is proposed that LC-derived IL-1b
performs two functions. First, LC-derived IL-1b activates the LC in an autocrine loop through
the IL-1 receptor 1 (IL-1RI). Second, IL-1b also stimulates epidermal keratinocytes (KCs) to
secrete TNF-a, which acts in a paracrine manner to facilitate DC migration through TNFRII (87).TNF-О± contributes to decreasing the attachment between LC and keratinocytes by
downregulating E-cadherin and by inhibiting the expression of CCR6, which renders LC insensitive to CCL20 produced by keratinocytes [85, 86]. TNF-a also induces the expression of
a6b1 integrin on LC, which is important for their interaction with extracellular matrix proteins
such as laminin that is present in the basement membrane of the epidermis [86, 88, 89]. The
integrin LFA-1 (leukocyte function-associated antigen-1) is also implicated in skin DC migration to LN and the LFA-1 ligand ICAM-1 is expressed by lymphatic endothelial cells [86].
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
Simultaneously, production of epidermal basement membrane degrading enzymes,
such as matrix metalloproteinases (MMPs), is upregulated in activated LC [90]. The MMP2, MMP-3, and MMP-9 have been described to facilitate in the passage of LC across the
basement membrane. Subsequently, MMPs are also essential for migration of LC and DDC
through the dermal tissue matrix, by cleavage of type IV collagen [91].
Therefore, earlier CXCR4 expression confers upon LC the capacity to migrate faster to
the dermis where the CXCR4 ligand CXCL12 is increased during inflammation. Once in the
dermis, LC complete their maturation and upregulate CCR7, allowing their final migration
to LN. CCR7 is induced only at later stages of LC maturation because its ligands CCL19 and
CCL21 are expressed in lymphatic endothelial cells that are not immediately underneath
the epidermal layer, making them not readily available for LC that are still in the epidermis
The temporal dissociation of CXCR4 and CCR7 expression would also permit LC to transiently dwell in the dermis before continuing their transit to the LN, potentially allowing
these cells to interact with and deliver antigens to resident dDC [92].
About LC stimulatory capacity, as any other DCs they are able to activated different
subsets of lymphocytes. It was seen, in different experimental models of skin pathology
an involvement of LC in priming of adaptative response in terms of CD4 or CD8 T cells
activation or B activation.
Many works report that Differentiated LC primed T cells much more efficiently than dermal DC (dDC) or monocyte-derived DC. These results led to the classical view that LC play
a prominent role in skin immunity by capturing and processing antigens in the epidermis
in order to activate T cells in the skin-draining LN [93–94].
Studies on response to viruses and gram-positive bacteria, shows that freshly purified
human LC express a restricted set of functional TLR, namely TLR2, TLR6, and TLR3, and are
directly activated by their respective ligands, while cooperation of keratinocytes and other
cell types is probably essential for response to Gram-negative bacteria and viruses in vivo.
In particular, lack of type I IFN production precludes a direct effector role of LC in viral
infections and further shows that the full development of an immune response in the skin
does not solely rely on these cells.
Of interest, activation of LC by different TLR agonists leads to specific cytokine secretion profiles. Particularly stimulation of TLR2 or TLR6 with PGN- lead to a specific IL-10
secretion, although the exact physiological relevance of this specific pattern remains to be
determined, LC may contribute to the tolerance of commensal Gram-positive bacteria that
colonize the skin of healthy individuals without causing inflammation [95].
Is interesting note that in many case reported the role played by LC is supported by
other cells subsets especially skin Keratinocites and without this cross-talk the LCs shows
very poor efficiency as APC.
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
Some studies on Mice of inbred strains C57BL_6, OT-I (39), and OT-II [96], and TAP-1deficient [97] shows that LCs isolated from the skin can cross-present exogenous soluble
and cell-associated antigen to CD8_ T cells. Furthermore, migratory LCs can take up material from keratinocytes and cross-present it.
The final outcome of cross-presentation by LCs is the induction of IFN-Оі production
and cytotoxicity in antigen-specificCD8_ T cells [98]. other study focused on in vitro LC
response to TLR ligands shown that lc from BALB/c mice pre-treated with 2,4,6-Trinitrochlorobenzene (TNCB) and 2,4,6-trinitrobenzene, are incapable of responding to TLR
ligands, as the expression of MHC class II and co-stimulatory molecules on LCs are not
augmented by the TLR-mediated signal [99]. But it was also found that CpG is capable of stimulating LCs in a certain condition, resulting in enhancement of their haptenpresenting function. This stimulatory effect was exerted only when LCs co-existed with
keratinocytes, as purified LCs mounted the CpG-induced enhancement at a lower level
than did LC–ECs.
It was also found that other TLR ligands derived from Gram-positive or negative bacteria, such as peptidoglycan and LPS, enhance the hapten-presenting ability of LCs by
augmenting of keratinocyte cytokine production [100].
Therefore, keratinocytes serve as an up-regulator of acquired immunity upon exposure
to microbes. Even if LCs are low-responsive to skin commensals, they can be stimulated
with microbial elements indirectly via keratinocytes.
Tolerogenic role of Langerhans cells
Many studies on murine experimental models, after antigens or pathogens exposure,
shows the presence of LC into draining lymph node but their role often seems to be much
more regulatory than activator and is accomplished trough the help of other cells subset.
Studies based on constitutive or transient depletion of LC in mice gave some unexpected and even contradictory findings, with reports suggesting an important role of LC in skin
immunity [100-103], whereas other studies found that LC were dispensable for inducing
skin-associated immune responses [104]. These disparate observations may be explained,
at least in part, by the variable degree of deletion of other DC subsets that also express
From work with the two Langerin–DTR–EGFP mouse strains in the setting of an acute
depletion of LC, it appears that when antigen is targeted preferentially to LC such as with
low-dose hapten, then LC appear to be stimulatory and their absence leads to diminished
CHS responses. However, when antigen is simultaneously targeted to LC and other cell
subsets like dDC such as in the setting of normal hapten doses, then LC appear largely
redundant and CHS responses are mediated by dDC. This is consistent with a model in
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
which all skin DC have similar function and the number that migrate to the CLN carrying
antigen determines the ultimate outcome [105].
More recent studies [28, 104] in which LC were selectively depleted, while other langerin1 DC subsets (including dDC) were preserved, did not show an essential role for LC in
inducing contact hypersensitivity responses to either haptens or peptide antigens [10, 15]
or in a model of skin allograft rejection [106].
Nonetheless, although LC may not be strictly required for skin immune responses in
some settings, they might still be sufficient to trigger effective protective or pathogenic
skin immune responses. Consistent with this possibility, allogeneic LC are sufficient to
trigger skin graft-versus host disease in the absence of host-derived dDC [78].
Many studies on murine experimental models, after antigens or pathogens exposure,
shows the presence of LC into draining lymph node but their role often seems to be involved in maintenance of peripheral tolerance.
Recent observations on the dynamics and functions of CD207+ cells fuel speculation
in a number of different inflammatory situations that LCs have a regulatory, rather than
stimulatory, role [107-110].
In many cases LCs rather than important in the priming of a CD4+ mediated immune
response in the sdLN, seems to lead to the development of a protective immune response
Another work described an otherwise normal mouse that constitutively lacks only epidermal LCs and used this model to demonstrate that the lack of LCs leads to an enhanced
rather than inhibited CHS response then show via cell-transfer experiments that LCs act at
the priming rather than effector phase of the response [114].
Other study on LC chimeric mice reports that PAF is involved in LC migration. However
because PAFRв€’/в€’ mice generate a CHS reaction no different from controls, these findings
support the hypothesis that LC are not involved in activating CHS. morover shows that dDC,
but notLC, are sorted from the lymph nodes of hapten sensitized mice, probably dDC can
present antigen to T cells [115].
In a murine model of parasite immunisation whith RA schistosoma larvae,
Comparison of mice vaccinated VS mice exposed to non-protective normal parasites
shows that presence of CD207+ cells in the sdLN does not correlate with the enhanced
CD4+ cell proliferation and Th1 cytokine production that is a feature of mice exposed to
the RA schistosome vaccine [112, 113, 116].
It is possible that CD207+ cells, despite expressing slightly greater amounts of MHCII,
do not have an immune priming role but regulate events in the sdLN, or crosspresentv
antigen to other APCs [98].
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
Final remarks
LC were discovered in 1868 and well characterized morphologically (i.e. Birbek granules) from1961, but still now, despite several immunological studies LC are not so well
functionally characterized especially in their regulatory role.
The understanding of the mechanisms employed by the skin to determine between
the stimulation of immune responses vs maintenance of tolerance needs further investigation.
It is still controversial whether the high plasticity, characteristic of myeloid DC, like LC,
suffice to determine between immunity or tolerance induction. In addition, whether LC
have redundant, complementary, or even opposing functions respect to other skin cells
population like DDC has not yet been clarified.
Last works support the hypothesis that, owing to their high plasticity, skin resident DC
and especially LC censor for foreign Ag but are still able to maintain the homeostasis of
the skin.
Functionally, it is probable that most of the LC and DDC remain dormant during the
steady state whereas a low number gets activated and transports self-Ag from the skin to
sDLN to present Ag in a tolerogenic pathway. In the presence of inflammatory insult, LC
and DDC may have the ability to respond to the injury and to mature into potent APC while
still being able to maintain tolerance to self-Ag.
Is interesting to note that earliest works represents LC as more modulating than activating immune response in this way is quite difficult to profile in a secure manner the LC
Surely is very important the micro-environment meaning the cross-talk with other cells
subsets, especially skin Keratinocytes, in fact cytokine production from these cells, subsequently to antigen contact, sign the destiny of LC which can so became traditionally APC,
while if there is no cross-talk with keratinocytes presentation of antigen by LC to Limphocytes is not completed by second signal and do not induce lymphocytes activation.
So the recently works from one hand reduce the classical role of LC as APC in activation
of immune response to potentials pathogens, but from the other hand attribute to LC a new
and more amazing role of cells involved in modulation of immune response and maintenance of peripheral tolerance to skin commensally bacteria.
Regardless the LC paradigm, probably this remain a valid key to explain the role of LC
in skin immunity, but surely the last and new point became perhaps the most important to
describe LC activities in the immune system.
About therapeutic use of LC more of scientific production indicate LC as possible target
for vaccine administrated subcutaneously, this due to their role of APC.
LCs, like all DCs, are cells of the innate immune system that can influence the genera46
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
tion of an adaptive T cell response through their ability to take up proteins and to process
and present them via MHC class II molecules to T cells [117].
Particularly, targeting of non peptide antigens to LCs via CD1a is a vaccine strategy that
could be used for the generation of immunity to cutaneous pathogens [118]. The location
of LCs in the epidermis allows the innate immune system to respond rapidly to microbial
invaders in the skin. But, for set up a efficient vaccine is possible controversial to target
only LC because their role of APC need the involving of other skin cells populations (Keratinocytes) which became as important as LC in activation of immune system, nevertheless
LC remain interesting as target for vaccine.
More interesting is the possible use of LC as down regulating cells against autoimmune
disease which involved skin.
As antigen-presenting cells in the epidermis, LCs control T cell–mediated allergenspecific immune responses.
Allergens can penetrate the epidermis, mucosal barriers, or both and thus induce eczema in patients with atopic disorders. The local cytokine milieu in the epidermis influences
the LC-regulated T-cell response against the encountered allergen toward silent elimination, allergy, or tolerance. The recent progress made in understanding the mechanisms by
which LCs populate the skin opens new therapeutic strategies for the modulation of immune responses in atopic diseases. Through manipulation of LC development and biology in
vitro or in vivo, new strategies for the treatment of allergic diseases could evolve.
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
Epidermic enviroment (historical himage by Von Keith R. Porter and Mary A. Bonneville, EinfГјhrung in die
Feinstruktur von Zellen und Geweben, Springer-Verlag, Berlin 1965, t.9)
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
Activation of skin immune system and different role of Langerhans cells: 1) mainly regulatory with large
amount of antigen; 2) mainly pro-inflammatory with a poor amount of antigen, in this case is pivotal the
cross-talk between Langerhans cells and keratinocytes.
Kc = keratinocites; Lc = Langerhans cells; ddc = dermal dendritic cells; Th = lymphocytes T helper; Treg =
lymphocytes T regulatory; Nt = neutrophils.
= pro-inflammatory pathways
= Regolatory pathways
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
[1] Langerhans P, Uber die Nerven der menschlichen Haut. Virchows Arch 1868; 44: 325-327.
[2] Stingl G, Katz SI, Clement L, Green I, and Shevach EM. Immunologic functions of Ia-bearing epidermal
Langerhans cells. J Immunol 1978; 121: 2005-2013.
[3] Schuler G, and Steinman RM, Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J Exp Med 1985; 161: 526-546.
[4] Wilson NS, Villadangos JA. Lymphoid organ dendritic cells: beyond the Langerhans cells paradigm.
Immunol. Cell Biol 2004; 82: 91-98.
[5] Romani N, Holzmann S, Tripp CH, Koch F, Stoitzner P. Langerhans cells - dendritic cells of the epidermis. APMIS 2003; 111:725-740.
[6] Nishibu A, Ward BR, Jester JV, Ploegh HL, Boes M, Takashima A. Behavioral responses of epidermal
Langerhans cells in situ to local pathological stimuli. J Invest Dermatol 2006; 126: 787-796.
[7] Flacher V, Bouschbacher, M, Verronese E, Massacrier C, Sisirak V, Berthier-Vergnes O, de Saint-Vis B,
et al. Human Langerhans cells express a specific TLR profile and differentially respond to viruses and
Gram-positive bacteria. J Immunol 2006; 177: 7959-7967.
[8] Cumberbatch M, Dearman RJ, Kimber I. Langerhans cells require signals from both tumour necrosis
factor-alpha and interleukin-1 beta formigration. Immunology 1997; 92: 388-395.
[9] Randolph GJ, Ochando J, Partida-Sanchez S. Migration of dendritic cell subsets and their precursors.
Annu Rev Immunol 2008; 26:293-316.
[10] Geissmann F, Dieu-Nosjean MC, Dezutter C, Valladeau J, Kayal S, Leborgne M, Brousse N, et al. Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin. J.
Exp. Med. 2002. 196: 417-430.
[11] Mayerova D, Parke EA, Bursch LS, Odumade OA, Hogquist KA. Langerhans cells activate naive selfantigen-specific CD8 T cells in the steady state. Immunity 2004; 21: 391-400.
[12] Waithman J, Allan RS, Kosaka H, Azukizawa H, Shortman K, Lutz MB, Heath WR, et al. Skin-derived
dendritic cells can mediate deletional tolerance of class I-restricted self-reactive T cells. J Immunol
2007; 179: 4535-4541.
[13] Shibaki A, Sato A, Vogel JC, Miyagawa F, Katz SI. Induction of GVHD-like skin disease by passively
transferred CD8(1) T-cell receptor transgenic T cells into keratin 14-ovalbumin transgenic mice. J Invest Dermatol 2004; 123: 109-115.
[14] Azukizawa H, Kosaka H, Sano S, Heath WR, Takahashi I, Gao XH, Sumikawa Y, et al. Induction of T-cellmediated skin disease specific for antigen transgenically expressed in keratinocytes. Eur J Immunol
2003, 33: 1879-1888.
[15] Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003; 21:
[16] Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Nat Aca Sc USA 2002; 99: 351-358.
[17] Kashihara M, Ueda M, Horiguchi Y, Furukawa F, Hanaoka M, et al. A monoclonal antibody specifically
reactive to human Langerhans cells. J Invest Dermato 1986; 87: 602-607.
[18] McDermott R, Ziylan U, Spehner D, Bausinger H, Lipsker D, et al. Birbeck granules are subdomains of
endosomal recycling compartment in human epidermal Langerhans cells, which form where Langerin
accumulates. Mol Biol Cell 2002; 13: 317-335.
[19] Valladeau J, Ravel O, Dezutter-Dambuyant C, Moore K, Kleijmeer M, et al. Langerin, a novel C-type
lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
granules. Immunity 2000; 12: 71-81.
[20] Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, et al. Cloning and characterization of a specific
receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. J Exp Med 1997; 186: 825835.
[21] Sozzani S. Dendritic cell trafficking: more than just chemokines. Cytokine Growth Factor Cytokine
Growth Factor Rev 2005; 16: 581-592.
[22] Tang A, Amagai M, Granger LG, Stanley JR, Udey MC. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature 1993; 361: 82-85.
[23] Yang D, Howard OM, Chen Q, Oppenheim JJ. Cutting edge: immature dendritic cells generated from
monocytes in the presence of TGF-beta 1 express functional C-C chemokine receptor 6. J Immunol
1999; 163: 1737-1741.
[24] Yasaka N, Furue M, Tamaki K. Expression of cutaneous lymphocyte associated antigen defined by
monoclonal antibody HECA-452 on human Langerhans cells. J Dermatol Sci 1996; 11: 19-27.
[25] Udey M C, Nagao K. Characteristics and functions of murine cutaneous dendritic cells: a synopsis of
recent developments. Mucosal Immunol 2008; 1:470-4.
[26] Borkowski TA, Nelson AJ, Farr AG, Udey MC, Expression of gp40, the murine homologue of human epithelial cell adhesion molecule (Ep-CAM), by murine dendritic cells. Eur J Immunol 1996; 26: 110-114
[27] Kaplan DH, Kissenpfennig A, Clausen BE. Insights into Langerhans cell function from Langerhans cell
ablation models. Eur J Immunol 2008; 38: 2369-2376.
[28] Bursch LS, Wang L, Igyarto B, Kissenpfennig A, Malissen B, Kaplan DH, Hogquist KA. Identification of a
novel population of Langerin1dendritic cells. J Exp Med 2007; 204: 3147-3156.
[29] Birbeck MS, Breathnach AS, Everall JD. An electron microscope study of basal melanocytes and highlevel clear cells (Langerhans cells) in vitiligo. J Invest Dermatol 1961; 37: 51-63.
[30] Hunger RE, Sieling PA, Ochoa MT, Sugaya M, Burdick AE, Rea TH, Brennan PJ, Belisle JT, Blauvelt A,
Porcelli SA, Modlin RL. Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide
antigens to T cells. J Clin Invest 2004; 113: 701-708.
[31] Hanau D, Fabre M, Schmitt DA, Stampf JL, Garaud JC, Bieber T, Grosshans E, Benezra C, Cazenave JP.
Human epidermal Langerhans cells internalize by receptor-mediated endocytosis T6 (CD1 “NA1/34”)
surface antigen. Birbeck granules are involved in the intracellular traffic of the T6 antigen ???????
1987; 89:172-177.
[32] McDermott R. et al. Birbeck granules are subdomains of endosomal recycling compartment in human
epidermal Langerhans cells, which form where Langerin accumulates. Mol Biol Cell 2002; 13: 317335.
[33] Valladeau J, Ravel O, Dezutter-Dambuyant C, Moore K, Kleijmeer M, Liu Y, Duvert-Frances V, Vincent
C, Schmitt D, Davoust J, Caux C, Lebecque S, Saeland S. Langerin, a novel C-type lectin specific to
Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity
2000; 12: 71-81.
[34] Bartosik J. Cytomembrane-derived Birbeck granules transport horseradish peroxidase to the endosomal compartment in the human Langerhans cells. J Invest Dermatol 1992; 99: 53-58.
[35] Andersson L, Bartosik J, Bendsoe N, Malmström A, Mikulowska A, Warfvinge K, Andersson A, Falk B. in
The Langerhans Cell 1988; (Thivolet, J., and Schmitt, D., Eds.) pp 185-191.
[36] McDermott R, Bausinger H, Fricker D, Spehner D, Proamer F, Lipsker D, Cazenave JP, Goud B, De La
Salle H, Salamero J, Hanau D. Reproduction of Langerin/CD207 traffic and Birbeck granule formation
in a human cell line model. J Invest Dermatol 2004; 123; 72-77.
[37] ThГ©paut M, Valladeau J, Nurisso A, Kahn R, Arnou B, Vives C, Saeland S, Ebel C, Monnier C, Dezutter-
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
Dambuyant C, Imberty A, Fieschi F. Structural Studies of Langerin and Birbeck Granule: A Macromolecular Organization Model. Biochemistry Publication Date (Web): 28 January 2009.
Wilcke M, Johannes L, Galli T, Mayau V, Goud B, Salamero J. Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the trans-Golgi
Network; The Journal of Cell Biology 2000; 151: 1207-1220.
Chavrier P, Goud B. The role of ARF and Rab GTPases in membrane transport. Curr Opin Cell Biol 1999;
11: 466-475.
McDermott, R. et al. Reproduction of Langerin/CD207 traffic and Birbeck granule formation in a human
cell line model. J Invest Dermatol 2004; 123: 72-77.
Uzan-Gafsou S, Bausinger H, Proamer F, Monier S, Lipsker D, Cazenave JP, Goud B, de la Salle H, Hanau
D, Salamero J. Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling
and Stability. Molecular Biology of the Cell 2007; 18: 3169-3179.
Tarnowski WT, Hashimoto K. Langerhans’ cell granules in histiocytosis X. The epidermal Langerhans’cell
as a macrophage. J Invest Dermatol 1967; 96; 298-304.
Hashimoto K. Langerhans cell granule. An endocytotic organelle. Arch Dermatol 1971; 104: 148-160.
Ishii M, Terao Y, Kitajima J-I, Hamada T. Sequential production of Birbeck granules through adsorptive
pinocytosis. J Invest Dermatol 1984; 82: 28-29.
Takahashi S, Hashimoto K. Derivation of Langerhans cell granules from cytomembrane. J Invest Dermatol 1985; 84: 469-471.
Bartosik J. Cytomembrane-derived Birbeck granules transport horseradish peroxidase to the endosomal compartment in the human Langerhans cells. J Invest Dermatol 1992; 99: 53-58.
Wolff K. The Langerhans cells. In: Current Problems in Dermatology, vol. 4 1972; (ed. J.W.H. Mali), Basel:
Karger, 100-103.
Stössel H, Koch F, Kämpgen E, Stöger P, Lenz A, Heufler C, Romani N, Schuler G. Disappearance of
certain acidic organelles (endosomes and Langerhans cell granules) accompanies loss of antigen
processing capacity upon culture of epidermal Langerhans cells. J Exp Med 1990; 172: 1471-1482.
Elema JD, Atmosoerodjo-Briggs JE. Langerhans cells and macrophages in eosinophilic granuloma.
An enzyme-histochemical, enzyme-cytochemical, and ultrastructural study. Cancer 1984; 54: 21742181.
Lamaze C, Fujimoto LM, Yin HL, Schmid SL. The actin cytoskeleton is required for receptor-mediated
endocytosis in mammalian cells. J Biol Chem 1997; 272: 20332-20335.
Salamero J, Bausinger H, Mommaas AM, Lipsker D, Proamer F, Cazenave J-P, Goud B, de la Salle H,
Hanau D. CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans
cells. J Invest Dermatol 2001; 116: 401-408.
Desnos C, Clift-O’Grady L, Kelly RB. Biogenesis of synaptic vesicles in vitro. J Cell Biol 1995; 130:
Hopkins CR, Gibson A, Shipman M, Strickland DK, Trowbridge IS. In migrating fibroblasts, recycling
receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma
membrane of the leading lamella. J Cell Biol 1994; 125: 1265-1274.
Miranda A, Amadeu TP, Schueler G, Alvarenga FBF, Duppre N, Ferreira H, Nery JAC, Sarno EN. Increased
Langerhans cell accumulation after mycobacterial Stimuli Histopathology 2007; 51: 649-656.
Moser B, Wolf M, Walz A, Loetscher P. Chemokines: multiple levels of leukocyte migration control.
Trends Immunol 2004; 25:75-84.
Larregina AT, Morelli AE, Spencer LA, Logar AJ, Watkins SC, Thomson AW, Falo LD Jr. Dermal-resident
CD14Гѕ cells differentiate into Langerhans cells. Nat Immunol 2001; 2: 1151-1158.
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
[57] Schaerli P, Willimann K, Ebert LM, Walz A, Moser B. Cutaneous CXCL14 targets blood precursors to
epidermalniches for Langerhans cell differentiation. Immunity 2005; 23: 331-342.
[58] Dieu-Nosjean MC, Massacrier C, Vanbervliet B, Fridman WH, Caux C. IL-10 induces CCR6 expression
during Langerhans cell development while IL-4 and IFN-gamma suppress it. J Immunol 2001; 167:
[59] Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J. GM-CSF and TNF-alpha cooperate in the
generation of dendritic Langerhans cells. Nature 1992; 360: 258-261.
[60] Gatti E, Velleca MA, Biedermann BC, Ma W, Unternaehrer J, et al. Large-scale culture and selective
maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+
progenitors. J Immunol 2000; 164: 3600-3607.
[61] Geissmann F, Prost C, Monnet JP, Dy M, Brousse N, et al. Transforming growth factor beta1, in the presence of granulocyte/macrophage colonystimulating factor and interleukin 4, induces differentiation of
human peripheral blood monocytes into dendritic Langerhans cells. J Exp Med 1998; 187: 961-966.
[62] Herbst B, Kohler G, Mackensen A, Veelken H, Kulmburg P, et al. In vitro differentiation of CD34+ hematopoietic progenitor cells toward distinct dendritic cell subsets of the birbeck granule and MIIC-positive
Langerhans cell and the interdigitating dendritic cell type. Blood 1996; 88: 2541-2548.
[63] Strobl H, Riedl E, Scheinecker C, Bello-Fernandez C, Pickl WF, et al. TGF-beta 1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors. J Immunol 1996; 157: 1499-1507.
[64] Kissenpfennig A, Malissen B. Langerhans cells-revisiting the paradigm using genetically engineered
mice (Review). Trends Immunol 2006; 27: 132-139.
[65] Ferrero E, Belloni D, Contini P, Foglieni C, Ferrero ME, Fabbri M, et al.Transendothelial migration leads
to protection from starvation-induced apoptosis in CD34+CD14+ circulating precursors: evidence for
PECAM-1 involvement through Akt/PKB activation. Blood 2003;101: 186-93.
[66] Imhof BA, Aurrand-Lions M. Adhesion mechanisms regulating the migration of monocytes. Nat Rev
Immunol 2004; 4: 432-44.
[67] Kanitakis J, Petruzzo P, Dubernard JM. Turnover of epidermal Langerhans’cells. N Engl J Med 2004;
[68] Borkowski TA, Letterio JJ, Farr AG, Udey MC. A role for endogenous transforming growth factor beta 1 in
Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal
Langerhans cells. J Exp Med 1996; 184: 2417-2422.
[69] Mohamadzadeh M, Berard F, Essert G, Chalouni C, Pulendran B, et al. Interleukin 15 skews monocyte
differentiation into dendritic cells with features of Langerhans cells. J Exp Med 2001; 194: 10131020.
[70] Hubner G, Hu Q, Smola H, Werner S. Strong induction of activin expression after injury suggests an
important role of activin in wound repair. Dev Biol 1996; 173: 490-498.
[71] Fumagalli M, Musso T, Vermi W, Scutera S, Daniele R, et al. Imbalance between activin A and follistatin
drives postburn hypertrophic scar formation in human skin. Exp Dermatol 2007; 16: 600-610.
[72] Nestle FO, Zheng XG, Thompson CB, Turka LA, Nickoloff BJ. Characterization of dermal dendritic cells
obtained from normal human skin reveals phenotypic and functionally distinctive subsets. J Immunol
1993; 151: 6535-6545.
[73] Larregina AT, Morelli AE, Spencer LA, Logar AJ, Watkins SC, et al. Dermal-resident CD14+ cells differentiate into Langerhans cells. Nat Immunol 2001; 2: 1151-1158.
[74] Musso T, Scutera S,Vermi W, Daniele R, Fornaio M, Castagnoli C, Alotto D, Ravanini M, Cambieri I, et al.
Activin A Induces Langerhans Cell Differentiation In Vitro and in Human Skin Explants. PLoS ONE 2008;
3: Issue 9 | e3271.
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
[75] Kaplan DH, Kissenpfenning A, Clausen BE. Insights into Langerhans cell function from Langerhans cell
ablation models. Eur J Immunol 2008; 38: 2369-76.
[76] Mayerova D, Parke EA, Bursch, LS, Odumade OA, Hogquist KA. Langerhans cells activate naive selfantigen-specific CD8 T cells in the steady state. Immunity 2004; 21: 391-400.
[77] Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL. et al. Langerhans cells
renew in the skin throughout life under steady-state conditions. Nat. Immunol 2002; 3: 1135-114.
[78] Merad M, Hoffmann P, Ranheim E, Slaymaker S, Manz MG, Lira SA, Charo I. et al. Depletion of host
Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host
disease. Nat Med 2004; 10: 510-517.
[79] Enk AH, Katz SI. Early molecular events in the induction phase of contact sensitivity. Proc Natl Acad Sci
USA 1992; 89: 1398-1402.
[80] Enk AH, Angeloni VL, Udey MC, Katz SI. An essential role for Langerhans cell-derived IL-1 beta in the
initiation of primary immune responses in skin. J Immunol 1993; 150: 3698-3704.
[81] Cumberbatch M, Clelland K, Dearman RJ, Kimber I. Impact of cutaneous IL-10 on resident epidermal
Langerhans’ cells and the development of polarized immune responses. J Immunol 2005: 175: 43-50.
[82] Sozzani S. Dendritic cell trafficking: more than just chemokines. Cytokine Growth Factor Rev 2005; 16:
[83] Randolph GJ, Ochando J, Partida SNS. Migration of Dendritic Cell Subsets and their Precursors. Annu
Rev Immunol 2007.
[84] Reis E, Sousa C, Stahl PD, Austyn JM. Phagocytosis of antigens by Langerhans cells in vitro. J Exp Med
1993; 178: 509-519.
[85] Villablanca EJ, Russo V, Mora JR. Dendritic cell migration and lymphocyte homing imprinting. Histol
Histopathol 2008; 23: 897-910.
[86] Randolph GJ, Angeli V, Swartz MA. Dendritic-cell trafficking to lymph nodes through lymphatic vessels.
Nat Rev Immunol 2005; 5: 617-628.
[87] Cumberbatch M, Dearman RJ, Kimber I. Langerhans cell migration in mice requires intact type I interleukin 1 receptor (IL-1RI) function. Arch Dermatol Res 1999; 291: 357-361.
[88] Ioffreda MD, Whitaker D, Murphy GF. Mast cell degranulation upregulates alpha 6 integrins on epidermal Langerhans cells. J Invest Dermatol 1993; 101: 150-154.
[89] Price AA, Cumberbatch M, Kimber I, Ager A. Alpha 6 integrins are required for Langerhans cell migration
from the epidermis. J Exp Med 1997; 186: 1725-1735.
[90] Kobayashi Y. Langerhans’ cells produce type IV collagenase (MMP-9) following epicutaneous stimulation with haptens. Immunology 1997; 90: 496-501.
[91] Ratzinger G, Stoitzner P, Ebner S et al. Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin. J Immunol 2002;
168: 4361-4371.
[92] Ouwehand K, Santegoets S, Bruynzeel D, Scheper R, de Gruijl T, Gibbs S. CXCL12 is essential for migration of activated Langerhans cells from epidermis to dermis. Eur J Immunol 2008; 38. DOI 10.1002/
[93] Schuler G,Steinman RM. Murine epidermal Langerhans cells mature into potent immunostimulatory
dendritic cells in vitro. J Exp Med 1985; 161: 526-546.
[94] Cao T, Ueno H, Glaser C, Fay JW, Palucka AK, Banchereau J. Both Langerhans cells and interstitial DC
cross-present melanoma antigens and efficiently activate antigen-specific CTL. Eur J Immunol 2007;
37: 2657-2667.
[95] Flacher V, Bouschbacher M, Verronese E, Massacrier C, Sisirak V, Berthier-Vergnes O, de Saint-Vis B, et
New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
al. Human Langerhans Cells Express a Specific TLR Profile and Differentially Respond to Viruses and
Gram-Positive Bacteria1 The Journal of Immunology 2006; 177: 7959-7967.
[96] Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in transgenic mice constructed
using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.
Immunol. Cell Biol. 1998; 76: 34-40.
[97] Van Kaer L, Ashton-Rickardt PG, Ploegh H, Tonegawa S. TAP1 mutant mice are deficient in antigen
presentation, surface class I molecules, and CD4-8+ T cells. Cell 1992; 71: 1205-1214.
[98] Stoitzner P, Tripp CH, Eberhart A, Price KM, Jung JY, Bursch L, Ronchese F, Romani N. Langerhans cells
cross-present antigen derived from skin. Proc Nat Aca Sc USA 2006; 103: 7783-7788.
[99] Mitsui H, Watanabe T, Saeki H. et al. Differential expression and function of Toll-like receptors in Langerhans cells: comparison with splenic dendritic cells. J Invest Dermatol 2004; 122: 95-102.
[100]Sugita K, Kabashima K, Atarashi K, Shimauchi T, Kobayashi M, Tokura Y. Innate immunity mediated by
epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin
Clin Exper Immunol 2006; 147: 176-183.
[101]Bennett CL, van Rijn E, Jung S, Inaba K, Steinman RM, Kapsenberg ML, Clausen BE. Inducible ablation
of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity. J Cell Biol 2005;
169: 569-576.
[102]Bennett CL, Noordegraaf M, Martina CA, Clausen BE. Langerhans cells are required for efficient presentation of topically applied hapten to T cells. J Immunol 2007; 179: 6830-6835.
[103]Stoitzner P, Green LK, Jung JY, Price KM, Tripp CH, Malissen B, Kissenpfennig A. et al. Tumor immunotherapy by epicutaneous immunization requires langerhans cells. J Immunol 2008; 180: 1991-1998.
[104]Wang L, Bursch LS, Kissenpfennig A, Malissen B, Jameson SC, Hogquist KA. Langerin expressing cells
promote skin immune responses under defined conditions. J Immunol 2008; 180: 4722-4727.
[105]Kaplan DH, Kissenpfennig A, Clausen BE. Insights into Langerhans cell function from Langerhans cell
ablation models Eur J Immunol 2008; 38: 2369-2376.
[106]Obhrai JS, Oberbarnscheidt M, Zhang N, Mueller DL, Shlomchik WD, Lakkis FG. Shlomchik, M. J. and
Kaplan, D. H., Langerhans cells are not required for efficient skin graft rejection. J Invest Dermatol
2008; 128: 1950-1955.
[107]Douillard P, Stoitzner P, Tripp CH, Clair-Moninot V, Ait-Yahia S, McLellan AD, Eggert A, Romani N, Saeland
S. Mouse lymphoid tissue contains distinct subsets of langerin/CD207 dendritic cells, only one of which
represents epidermal-derived Langerhans cells. J Invest Dermatol 2005; 125:983-994.
[108]Itano AA, McSorley SJ, Reinhardt RL, Ehst BD, Ingulli E, Rudensky AY, Jenkins MK. Distinct dendritic cell
populations sequentially present antigen to CD4 T cells and stimulate different aspects of cellmediated
immunity. Immunity 2003; 19:47-57.
[109]Kissenpfennig A, Malissen B. Langerhans cells - revisiting the paradigm using genetically engineeredmice. Trends Immunol 2006; 27:132-139.
[110]Villadangos JA, Heath WR. Life cycle, migration and antigen presenting functions of spleen and lymph
node dendritic cells: limitations of the Langerhans cells paradigm. Semin Immunol 2005; 17:262-272.
[111]Sato H, Kamiya H. Role of epidermal Langerhans’ cells in the induction of protective immunity to Schistosoma mansoni in guinea-pigs. Immunology 1995; 84:233-240.
[112]Hogg KG, Kumkate S, Mountford AP. IL-10 regulates early IL-12-mediated immune responses inducedby the radiation-attenuated schistosome vaccine. Int Immunol 2003; 15:1451-1459.
[113]Hewitson JP, Hamblin PA, Mountford AP. Immunity induced by the radiation-attenuated schistosomevaccine. Parasite Immunol 2005; 27:271-280.
[114]Kaplan DH, Jenison MC, Saeland S, Shlomchik WD, Shlomchik MJ. Epidermal Langerhans Cell-Deficient
Marco La Manna, Viviana Bazan, Aldo Gerbino, Alfredo Salerno
Mice Develop Enhanced Contact Hypersensitivity. Immunity 2005; 23: 611-620.
[115]Fukunaga A, Khaskhely NM, Sreevidya CS, Byrne SN, Ullrich SE. Dermal Dendritic Cells, and not Langerhans cells, play an essential role in inducing an immune response. J Immunol 2008; 180: 3057-3064.
[116]Kumkate S, Jenkins GR, Paveley RA, Hogg KG, Mountford AP. CD207+ Langerhans cells constitute a
minor population of skinderived antigen-presenting cells in the draining lymph node following exposure
to Schistosoma mansoni. Int J Parasitol 2007; 37: 209-220.
[117]Stingl G, Katz SI, Clement L, Green I, Shevach EM. Immunologic functions of Ia-bearing epidermal
Langerhans cells. J Immunol 1978; 121: 2005-2013.
[118]Hunger RE, Sieling PA, Ochoa MT, Sugaya M, Burdick AE, Rea TH, Brennan PJ, Belisle JT, Blauvelt A,
Porcelli SA, Modlin RL. Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide
antigens to T cells. J Clin Invest 2004; 113:701-8.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Espressione immunoistochimica della orexina A, della
orfanina FQ e della leptina nell’apparato respiratorio
del ratto
[Orexin A, Orphanin FQ, Leptin immunohistochemical expression in rat respiratory system]
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo e
Vincenzo Tessitore
Di.Me.S. – Sezione di Istologia ed Embriologia – Università degli Studi di Palermo (IT)
Parole chiave: Orexina; Orfanina FQ; Leptina; Immunoistochimica; Sistema respiratorio
Key words: Orexin; Orphanin FQ; Leptin; Immunohistochemistry; Respiratory system
Riassunto. Recenti evidenze sperimentali riferiscono sulla imprevista presenza in distretti organici periferici,
delle Orexine A e B, della Orfanina FQ e della Leptina, nuovi neuropeptidi ritenuti in origine, esclusivamente
espressi da neuroni centrali nel cui ambito svolgono un ruolo oressizzante e anoressizzante. Con riferimento anche a recenti rilievi microanalitici e farmacologici, abbiamo esteso la nostra indagine, in precedenza condotta in alcuni distretti periferici, sulla possibile espressione immunoistochimica di tali messaggeri
nell’apparato respiratorio del ratto normale. I risultati da noi ottenuti fanno rilevare che i neuro peptidi presi
in esame sono espressi, sia pure con diversa intensitГ per ciascuno di essi, da diverse strutture respiratorie
(albero tracheo-bronchiale e ghiandole annesse, bronchioli prossimo distali, bronchioli respiratori e alveoli).
In aggiunta, la immunoreattivitГ espressa da ciascun messaggero interessa in modo non uniforme i diversi
citotipi dell’epitelio mucosale bronchiolare (cellule ciliate, cellule di Clara, cellule neuroendocrine, miociti
peribronchiali). Nel distretto alveolare, soltanto i pneumociti di II tipo esprimono immunopositivitГ , di variabile
intensitГ , in relazione ai diversi neuropeptidi esaminati. I risultati ottenuti forniscono la base morfoistochimica
per ritenere che tali sostanze al di fuori della giГ nota funzione centrale oressizzante e anoressizzante, possano svolgere nuovi ruoli periferici esercitati localmente modulando diversi aspetti della funzione respiratoria
con meccanismo endocrino-paracrino, neurocrino e autocrino.
Abstract. Recent studies document that the novel neuropeptides Orexins A and B, Orphanin FQ and Leptin
considered since its first identification exclusively expressed by central neurons, may be also expressed by
different peripheral tissues (GEP system, reproductive and urinary system) like our previous immunohistochemical researches has demonstrated. Following this method, we have considered interesting to carry out
an immunohistochemical study on this neuropeptides expression and distribution in normal rat respiratory
system. Our results indicate that these substances are expressed in all respiratory structures examined, but
with different intensity. In addition, each substance is immunohistochemically expressed with different intensity in the various cytotypes of bronchiolar epithelium (Clara cells, neuroendocrine cells, ciliated epithelial
cells, peribronchiolar smooth muscle cells). In the alveoli the immunoreactivity appears in pneumocytes type
2 even though with different intensity for every tested substances. The results obtained provide a morphoistochemical basis to understand the new additional peripheral roles, apart from their already orexigenic and
anorexigenic functions, played by these regulatory peptides directly in the respiratory system with a autocrine, neurocrine, paracrine-endocrine mechanism.
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
Numerose evidenze sperimentali accumulate negli ultimi anni hanno rivelato la presenza nel polmone di un considerevole numero di neuropeptidi, alcuni dei quali sono distribuiti
in terminali assonici e agiscono pertanto da neurotrasmettitori, attivando una mediazione
peptidergica che si affianca alla giГ nota mediazione adreno-colinergica e a quella, piГ№
recente, nitrossidergica nella regolazione neurocrina delle funzioni polmonari. Altri neuropeptidi sono stati identificati in cellule neuroendocrine intraepiteliali, ove si comportano
da neurormoni che autorizzano a considerare il polmone un vero e proprio organo endocrino [1]. Alcuni dati suggeriscono inoltre che sia i peptidi neurotrasmettitoriali che quelli
neuroendocrini svolgono i loro ruoli non soltanto nella fisiologia ma anche nella patologia
polmonare flogistica e neoplastica [2,3].
Le orexine (OXA e OXB), la leptina e la orfanina FQ sono nuovi neuropeptidi ritenuti in
origine esclusivamente prodotti da neuroni del SNC, ove sono integrati in complessi circuiti dell’area ipotalamica e mesolimbica preposti alla regolazione dell’appetito e del bilancio
energetico. L’orexina e la orfanina svolgono effetti oressizzanti, la leptina (ormone della
sazietГ ) svolge effetti anoressizzanti. Tuttavia, indipendentemente dai loro effetti centrali,
grande interesse hanno suscitato, assai recentemente, alcuni effetti svolti da alcuni di essi
in distretti periferici. In proposito nostre precedenti ricerche hanno fornito documentazioni
immunoistochimiche sulla imprevista presenza di tali peptidi in alcuni distretti periferici
correlati (GEP, stomaco e ghiandole salivari) e non correlati (apparato genitourinario maschile) con la loro azione oressizzante e anoressizzante [4,5,6,7,8,9,10]. In aggiunta, ci
sembrano significativi i risultati di una recente ricerca sugli effetti stimolanti respiratori
indotti da microiniezioni di leptina nella parte respiratoria del nucleo del tratto solitario che
esprime tra l’altro alte concentrazioni di recettori leptinici [11]. Ed ancora, topi transgenici
mancanti della orexina mostrano una attenuazione della risposta ipercapnica ventilatoria
durante la veglia ma non durante il sonno; la somministrazione di un antagonista del recettore della orexina mima questa anormalitГ , rimossa al contrario da somministrazione
di orexina [12].
Con riferimento a questi imprevisti riscontri morfofunzionali, abbiamo ritenuto interessante indagare sulla possibile espressione immunoistochimica di tali neuropeptidi nelle
diverse componenti strutturali dell’apparato respiratorio nel ratto, al fine di fornire la base
morfoistochimica per la interpretazione di nuovi ruoli esplicati da tali neuropeptidi.
I risultati preliminari di tale ricerca sono stati comunicati al 32В° Congresso della SocietГ Italiana di Istochimica (Messina 2007) [13] e al 61В° Congresso Nazionale della SocietГ Italiana di Anatomia ed Istologia (Sassari 2007) [14].
Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nell’apparato respiratorio del ratto
Materiali e metodi
Sono stati impiegati 15 ratti Wistar trattati in accordo con la Convenzione di Helsinki
sull’utilizzo degli animali nella ricerca biomedica. Gli animali sono stati sacrificati previa
anestesia (50 mg/kg di Nembutal somministrato per via endoperitoneale). Sono stati
prelevati frammenti di trachea, di bronchioli prossimali e distali, di bronchioli respiratori
e di alveoli. I campioni sono stati immediatamente fissati in liquido di Bouin, inclusi in
paraffina, sezionati e infine processati per la tecnica immunoistochimica mediante il
kit EnVision+System HRP (AEC) (Dako Cytomation), utilizzando anticorpi anti-Orexina
A e anti-Orfanina FQ (Chemicon International), anti-Leptina (Santa Cruz Biotecnology). Controlli negativi sono stati realizzati su sezioni adiacenti omettendo il passaggio
dell’antisiero primario. Tutti i campioni sono stati studiati mediante fotomicroscopio
Leica DMLB.
L’epitelio pseudostratificato della mucosa tracheobronchiale appare discretamente
immunopositivo: spesso la immunoreattivitГ ГЁ addensata in forma microgranulare nel
citoplasma perinucleare (Fig.1). Gli acini sieromucosi delle ghiandole intracoriali e sottomucose mostrano discreta e parcellare immunoreattivitГ (Fig.2). In corrispondenza
della biforcazione tracheale si scorgono tronchi nervosi orfaninergici (Fig.3). L’epitelio delle diramazioni bronchiolari mostra intensa immunoreattività non uniformemente
distribuita nei vari citotipi (c. di Clara marcatamente immunoreative rispetto agli altri
epiteliociti). L’anello miocitario peribronchiolare mostra debole positività (Fig.4). Nel
distretto alveolare la rimarchevole immunopositivitГ rimane limitata ai pneumociti di II
tipo (Fig.5).
L’epitelio mucosale tracheobronchiale mostra immunoreattività sovente più intensa e
di aspetto microgranulare nell’endoplasma perinucleare e nel bordo apicale delle cellule.
Le ghiandole sieromucose tracheobronchiali mostrano discreta immunoreattività non uniformemente estesa a tutti gli adenomeri (Fig.6). Nell’epitelio bronchiolare si rileva discreta
immunoreattivitГ a carico soprattutto delle cellule neuroendocrine (Fig.7). La muscolatura
perivascolare e peribronchiale ГЁ intensamente immunopositiva (Fig.9). Parimenti reattivi
appaiono alcuni mastociti endobronchiali. Nell’interstizio peribronchiale si rilevano alcuni
piccoli neuroni gangliari fortemente immunoreattivi (Fig.8). Nel distretto alveolare alcuni
pneumociti di II tipo esibiscono debolissima immunoreattivitГ (Fig.9).
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
Modesta immunoreattività è presente nell’epitelio tracheobronchiale. L’epitelio bronchiolare appare discretamente immunopositivo con immunoreattività distribuita disomogeneamente nelle cellule di Clara e negli altri epiteliociti mucosali. La muscolatura
peribronchiale appare debolmente immunoreattiva. Nell’epitelio alveolare si rileva scarsa
immunopositivitГ a carico dei pnemociti di II tipo (Fig.10).
I risultati da noi ottenuti indicano che l’apparato respiratorio del ratto si rivela una
rimarchevole sorgente di neuropeptidi, testimoniata dalla capacitГ orfaninergica, orexinergica e leptinergica dei segmenti tracheobronchiali, bronchiolari e alveolari che si manifesta
peraltro con reattivitГ immunoistochimica di diversa intensitГ per ciascuno dei neuropeptidi presi in esame. In aggiunta, la immunoreattivitГ espressa da uno stesso neuropeptide
interessa in modo non uniforme i diversi citotipi che costituiscono i vari distretti respiratori
considerati. Così nell’epitelio mucosale bronchiale e bronchiolare le cellule di Clara, le
cellule ciliate, le cellule a muco e le cellule neuroendocrine appaiono per ciascun neuropeptide non uniformemente reattive. Nell’epitelio alveolare soltanto i pneumociti di II tipo
si mostrano apprezzabilmente immunoreattivi e con immunopositivitГ di intensitГ variabile
in relazione al tipo di neuropeptide espresso.
I nostri rilievi forniscono la base morfoistochimica per prospettare la possibilitГ che i
neuropeptidi presi in esame, al di fuori della loro giГ nota funzione oressizzante e anoressizzante centrale o periferica, ritenuta finora esclusiva, possano esplicare nuovi ruoli
agendo direttamente e localmente sulle diverse componenti immunoreattive dell’apparato
respiratorio, anche se la precisa funzione svolta da tali neuropeptidi nel polmone al presente non puГІ essere esattamente chiarita. Tuttavia il loro riscontro immunoistochimico in
diverse strutture dell’apparato respiratorio suggerisce che essi possano agire con diversi
meccanismi in maniera discriminata, controllando negli epiteliociti mucosali, con meccanismo autocrino paracrino, verosimilmente il trofismo epiteliale in epoca pre e postnatale,
la secrezione mucipara e quella del surfattante alveolare, modulando nei terminali nervosi,
con meccanismo neurocrino, la broncomotilitГ e la vasomotilitГ e pertanto la perfusione
polmonare ed infine influenzando a distanza con meccanismo neuroendocrino l’attività di
organi lontani come prospettato per altri peptidi polmonari (1).
Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nell’apparato respiratorio del ratto
Fig.1: Ratto, orfanina: discreta immunopositività Fig.2: Ratto, orfanina: parcellare immunoreattività negli adenomeri delle ghiandole sieromucose tradell’epitelio mucosale tracheale. Oc. 10x; Ob. 40x.
cheali. Oc. 10x; Ob. 20x
Fig.3 Ratto, orfanina: tronco nervoso orfaninergico Fig.4: Ratto, orfanina: intensa e disomogenea immutracheale. Oc. 10x; Ob. 20x.
noreattività dell’epitelio bronchiolare. Miociti peribronchiolari debolmente immunopositivi. Oc. 10x; Ob. 40x.
Fig.5: Ratto, orfanina: intensa immunopositivitГ nei Fig.6: Ratto, leptina: disomogenea immunoreattivitГ pneumociti di II tipo. Oc. 10x; Ob. 40x.
negli adenomeri delle ghiandole sieromucose tracheali. Oc. 10x; Ob. 20x.
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
Fig.6: Ratto, leptina: disomogenea immunoreattivitГ Fig.8: Ratto, leptina: immunopositivitГ delle cellule
negli adenomeri delle ghiandole sieromucose trache- neuroendocrine nell’epitelio mucosale bronchiolare.
ali. Oc. 10x; Ob. 20x.
Oc. 10x; Ob. 40x.
Fig.9: Ratto, leptina: neuroni gangliari leptinergici Fig.10: Ratto, leptina: miociti perivascolari e perinell’interstizio peribronchiolare. Oc. 10x; Ob. 63x.
bronchiolari intensamente immunoreattivi. Debole
immunopositivitГ dei pneumociti di II tipo. Oc. 10x;
Espressione immunoistochimica della orexina A, della orfanina FQ e della leptina nell’apparato respiratorio del ratto
[1] Cutz E. Neuro-endocrine (APUD-type cells of the lung), in: “Ultrastucture of endocrine cells and tissues”
Motta PM (Eds), Chapter 13, 1984.
[2] Vernooy JH, Drummen NE, Suylen RJ, Cloots RH, Möller GM, Bracke KR, Zuyderduyn S, Dentener MA,
Brusselle GG, Hiemstra PS, Wouters EF. Enhanced pulmonary leptin expression in patients with severe
COPD and asymptomatic smokers. Thorax. 2009 Jan; 64(1):26-32. Epub 2008 Oct 3.
[3] Havemann K. et al. “Peptide hormones in lung cancer”. Springer-Verlag 1985.
[4] Tessitore V, Uzzo ML, Bonaventura G, Spatola GF. Immunohistochemical expression of Orexin A and
Orexin Type 2 receptors in the rat gastrointestinal apparatus and endocrine pancreas. Eur J Histochem
2005; vol. 49 Suppl.1, p. 19.
[5] Bonaventura G, Tessitore V, Uzzo ML, Spatola GF. Immunohistochemical distribution of orphanin FQ in the
rat gastrointestinal apparatus and pancreas. Ital J Anat Embryol 2005; vol. 110 Suppl.1, Fasc3, p. 262.
[6] Tessitore V. et al. Immunohistochemical studies on leptin, ghrelin, orexin, orphanin FQ and endocannabinoids expression in the stomach of lean and obese (fa/fa) zucker rats. First world congress on
therapies against obesity. Parigi 18-19 maggio 2006.
[7] Uzzo ML, Spatola GF, Mandracchia R, Buscemi M, Farina Lipari E, Lipari D, Valentino B, Gerbino A. Orphanin FQ and motility in seminal vesicles. Ital J Anat Embryol 2006; Vol.111, Suppl. 2 [3] 267.
[8] Uzzo ML, Martorana A, Cappello F, Farina Lipari E, Valentino B, Lipari D, Aragona F, Buscemi M, Gerbino
A. Immunohistochemical expression of orphanin FQ, atrial natriuretic peptide, and oxytocin in normal
human seminal vesicles. Eur J Histochem 2007; 51: Suppl. 2,30.
[9] Bonaventura G. Rilievi immunoistochimici della orfanina nell’apparato genitourinario del ratto. Exper
Medicine Rev (A Gerbino, G Zummo, G Crescimanno Eds), Plumelia, Palermo 2008; Vol.1 271-277.
[10] Bonaventura G, Cucco D, Leone A, Mandracchia R, Spatola GF, Tessitore V, Uzzo ML. The role of new
neurocrine and neuroendocrine messengers on the regulation of the function of major salivary glands
in some mammals. an immunohistochemical study. Ital J Anat Embryol 2008; Vol.113, Suppl. 1 [2] 40.
[11] Inyushkin AN, Inyushkina EM, Merkulova NA. Respiratory responses to microinjections of leptin into the
solitary tract nucleus. Neurosci Behav Physiol 2009; Mar; 39(3):231-40. Epub 2009 Feb 21.
[12] Kuwaki T. Orexinergic modulatio n of breathing across vigilance states. Respir Physiol Neurobiol 2008;
Dec 10;164(1-2):204-12.
[13] Bonaventura G, Cucco D, Spatola GF, Uzzo ML, Tessitore V. Espressione immunoistochimica di alcuni
neuropeptidi e del recettore CB1 nel polmone di ratto. nota I: bronchioli e alveoli. Eur J Histochem 2007;
vol. 51 (2), p. 24.
[14] Bonaventura G, Cucco D, Spatola G., Uzzo M.L, Tessitore V. Immunohistochemical expression of some
neuropeptides and CB1 cannabinoid receptor in rat lung. Note II: Trachea and Bronchi. Ital J Anat 2007;
Embryol. Vol.112, Suppl. 1 [2] 44.
Heart, circulation
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
trabecular meshwork FIBROSIS
[La non compattazione ventricolare sinistra isolata, “stroke” e fibrosi trabecolare]
Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana D’Angelo, Loredana Sutera, Emiliano
Maresi**, Giuseppina Novo, Aldo Gerbino* and Salvatore Novo
Department of Cardiology, *Department of Experimental Medicine, Section of Histology and Embryology;
**Department of Legal Medicine; University of Palermo (IT)
Key words: Non-compaction, Stroke, Fibrosis, Recesses
Parole chiave: Non-compaction, Stroke, Fibrosi, Recessi
Abstract. Left ventricular non-compaction is a myocardium disease characterized by an increased and
excessively prominent ventricular trabecular meshwork, interrupted by deep wall recesses. Although the
presence of intertrabecular recesses undoubtedly is a favourable condition for thrombus formation, the real
prevalence of this event in non-compacted hearts is still unknown. We assessed prevalence of strokes and
echocardiographic findings of thrombi inside the left ventricle in a continuous series of 229 patients, and
we detected that only 5 of them (2%) had a history of ischemic stroke or thrombus. Also, in macroscopic
heart specimen in cases of left ventricular myocardium non-compaction shows many prominent trabeculae
separated by recesses: by observing the wall meshwork, it is possible to notice fibrosis striae on the external
borders of every trabecula. Peritrabecular fibrosis is due to the stress induced by blood on the left ventricular
wall, which is further enhanced by turbulent flow into the wall recesses. If blood stasis into the left ventricle
wall recesses is not presents it is reasonable to infer that non-compaction in itself does not predispose to
the endoventricular thrombosis.
Riassunto. La “non compattazione ventricolare sinistra” è una malattia caratterizzata dalla presenza di
un’ipertrabecolatura della parete del ventricolo sinistro. Sebbene la presenza di trabecole e recessi, indubbiamente è una condizione favorevole per la formazione di trombi, la reale prevalenza di eventi emboligeni
cerebrali ГЁ ancora sconosciuta. Abbiamo valutato la prevalenza di ictus in una serie continua di 229 pazienti
affetti da non compattazione, e abbiamo rilevato che solo 5 di loro (2%) avevano una storia di ictus ischemico.
Inoltre, nelle sezioni anatomo-patologiche macroscopiche di miocardio ventricolare sinistro non compattato mostrano molti eminenti trabeculae separati da recessi ГЁ possibile notare la presenza di fibrosi nelle
trabecole più esterne. Verosimilmente tale fibrosi è dovuta allo stress indotto dal sangue sulla parete ventricolare sinistra, stress determinato da un flusso turbolento del sangue all’interno delle trabecole. È nostra
opinione quindi che se ГЁ presente un flusso turbolento non sarГ presente stasi, pertanto non sarГ presente
un rischio aumentato di trombosi in questa malattia.
Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana D’angelo, Loredana Sutera,
Emiliano Maresi, Giuseppina Novo, Aldo Gerbino, Salvatore Novo
Figura 1
Left ventricular non-compaction is a myocardium disease characterized by an increased and excessively prominent ventricular trabecular meshwork, interrupted by deep
wall recesses. Although the presence of intertrabecular recesses undoubtedly is a favourable condition for thrombus formation, the real prevalence of this event in non-compacted
hearts is still unknown [1-4]. With regard to this matter, Sahin S. et al [1] reported an unusual
case of cerebral cardioembolism in a 33-year-old man due to ventricular non-compaction,
diagnosed by magnetic resonance imaging [1]. As the same Authors write, only 1 adult case of
stroke among patients affected by non-compaction has been documented so far.
Overall, just 3 isolated cases of thrombus formation due to non-compaction of the left ventricular myocardium were described in the literature [2-3].
In 2004 Stollberger and Finesterer reported in a review a low prevalence of ventricular
thrombus findings and embolic events in patient populations with non-compacted myocardium [4]. These data were confirmed by the same Authors in a retrospective analysis of 62
patients [5].
We assessed prevalence of strokes and echocardiographic findings of thrombi inside
the left ventricle in a continuous series of 229 patients, and we detected that only 5 of them
(2%) had a history of ischemic stroke or thrombus. On the basis of the opposing results
Isolated left venticular non-compaction, Stroke and Trabecular Meshwork fibrosis
gathered so far, it is not still clear if the presence of intertrabecular recesses can lead to
thrombi formation, with possible thromboembolic consequences [1-10].
Observations and Conclusions
So, a further clarification is needed: the Figure 1 shows a macroscopic heart specimen
in a case of left ventricular myocardium non-compaction, in which many prominent trabeculae separated by recesses of varying depth are visible.
By observing the wall meshwork, it is possible to notice fibrosis striae on the external
borders of every trabecula. Peritrabecular fibrosis is due to the stress induced by blood on
the left ventricular wall, which is further enhanced by turbulent flow into the wall recesses.
Therefore fibrosis ultimately proves the presence of a turbulence flow pattern within the
trabecular meshwork.
According to the theory correlating myocardium non-compaction with endoventricular
thrombus formation, blood stasis into the left ventricle wall recesses is likely to be the
pathophysiologic link between these events by favouring coagulation activation. But the
presence of peritrabecular fibrosis refutes this theory and the assumption on which it is
Therefore, it is reasonable to infer that non-compaction in itself does not predispose to
the endoventricular thrombosis.
Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana D’angelo, Loredana Sutera,
Emiliano Maresi, Giuseppina Novo, Aldo Gerbino, Salvatore Novo
[1] Sahin S, Sekban A, Ayalp S, Karsidag S. An Unusual Cause of Cardioembolic Stroke: Isolated Left Ventricular Noncompaction. Neurologist 2008; 14(2):125-127.
[2] Ritter M, Oechslin E, Sutsch G, Attenhofer C, Schneider J, Jenni R. Isolated noncompaction of the myocardium in adults. Mayo Clin Proc 1997;72:26-31.
[3] Hascelik S, Yalnizoglu D, Kafali G, Celiker A,Cila A, Topcu M, Gurgey A. Stroke owing to noncompaction
of myocardium. J Child Neurol 2003;18:437-9.
[4] Stollberger C, Finsterer J. Thrombi in left ventricular hypertrabeculation/noncompaction - Review of the
literature. Acta Cardiol 2004;59:341-4.
[5] Stollberger C, Finsterer J. Left ventricular hypertrabeculation/noncompaction and stroke or embolism.
Cardiology 2005;103:68-72.
[6] Fazio G, Pipitone S, Iacona MA, Marchì S, Mongiovì M, Zito R, Sutera L, Novo G, Novo S. Evaluation of diastolic function by the Tissue doppler in children affected by non-compaction. Int J Cardiol
[7] Fazio G, Sutera L, Corrado G, Novo S. The chronic heart failure is not so frequent in non-compaction.
Eur Heart J 2007; 28:1269.
[8] Fazio G, Corrado G, Pizzuto C, Zachara E, Rapezzi C, Sulafa AK, Sutera L, Stollberger C, Sormani L, Finsterer J, Benatar A, Di Gesaro G, Novo G, Cavusoglu Y, Baumhakel M, Drago F, Carerj S, Pipitone S, Novo
S. Supraventricular arrhythmias in non-compaction of left ventricle: Is this a frequent complication? Int
J Cardiol 2007.
[9] Corrado G, Zachara E, Rapezzi C, Sulafa AK, Sutera L, Pizzuto C, Stollberger C, Sormani L, Finsterer J,
Benatar A, Di Gesaro G, Cascio C, Cangemi D, Cavusoglu Y, Baumhakel M, Drago F, Carerj S, Pipitone
S, Novo S. Ventricular tachycardia in non-compaction of left ventricle: is this a frequent complication?
Pacing Clin Electrophysiol 2007; 30:544-6.
[10] Fazio G, Sutera L, Vernuccio F, Fazio M, Vernuccio D, Pizzuto C, Di Gesaro G, Cascio C, Novo S. Heart
failure and cardiomyopathies: a case report. G Ital Cardiol 2007; 8:129-32.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
The Chromogranin A-derived peptides: structural and
functional features in heart biology
[Peptidi derivati dalla cromogramina A: caratteristiche funzionali e strutturali relative alla
biologia del cuore]
Valentina Di Felice*, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella
Marino Gammazza, Francesco Cappello and Giovanni Zummo
Human Anatomy Section “E. Luna”, Department of Experimental Medicine, University of Palermo (IT)
Key words: Vasostatin I, Vasostatin II, Natural peptides, Neuroendocrine tissue, Heart physiology
Parole chiave: Vasostatina I, Vasostatina II, Peptidi naturali, Tessuto neuroendocrino, Fisiologia del cuore
Abstract Chromogranin A (CGA) is a 48 kDa acidic protein co-stored in secretory granules of several endocrine and neuroendocrine cells with other compounds and it is co-released with them by exocytosis. Either
inside the granules or in extracellular fluid or into circulation, CGA is cleaved by tissue-specific proteolytic
enzymes into smaller peptides with different biological activities. High levels of plasma CGA have been correlated to endocrine and neuroendocrine tumours, renal failure and heart failure and it has been recently
demonstrated that CGA is produced not only by the diffuse neuroendocrine cells but also by cells of different
embryologic origins. Moreover, new effects of CGA-derived peptides come out every day.
The main CGA-derived peptides correlated to heart biology are Pancreastatin (PST) and Vasostatins (VSs).
PST is a 49-amino-acid peptide, corresponding to bovine CGA248-293 and human CGA240-288, able to stimulate
secretion in neonatal rat atrial cells. Vasostatin I (VS-I) and Vasostatin II (VS-II) correspond to the N-terminal
fragments CGA1-76 and CGA1-113 respectively, and seem to be the best cardioregulatory peptides in mammals,
since they counteract the ОІ-adrenergic-mediated positive inotropism in vertebrate hearts. These findings let
us suppose that CGA and CGA-derived peptides may play an autoregolatory role in heart secretion.
This review would be a summary of the possible relationships among CGA and CGA-derived peptides and
heart physiology, biology and pathology.
Riassunto La cromogranina A (CGA) ГЁ una proteina acida di 48 kDa conservata nei granuli di secrezione di
diverse cellule endocrine e neuroendocrine insieme ad altre sostanze e viene rilasciata insieme ad esse per
esocitosi. Sia all’interno dei granuli o nel fluido extracellulare o nella circolazione, la CGA viene tagliata da
enzimi tessuto-specifici in peptidi piГ№ piccoli che possiedono svariate attivitГ biologiche. Alti livelli plasmatici
di CGA sono stati correlati a tumori endocrini e neuroendocrini, ad alterazioni renali e cardiache ed ГЁ stato
recentemente dimostrato cha la CGA viene prodotto non solo dalle cellule del sistema neuroendocrino diffuso ma anche da cellule di diversa derivazione embriologica. Inoltre, vengono scoperti ogni giorno nuovi
effetti di peptidi derivati dalla CGA.
I principali peptidi derivati dalla CGA, correlati con la biologia del cuore, sono la Pancreastatina (PST) e le
Vasostatine (VSs). La PST ГЁ un peptide di 49 aa, corrispondente alla CGA248-293 bovina e alla CGA240-288
umana, capace di stimolare la secrezione nelle cellule striali di ratto neonato. La Vasostatina I (VS-I) e la
Vasostatina II (VS-II) corrispondono ai frammenti N-terminali CGA1-76 e CGA1-113 rispettivamente, e sembrano essere i migliori peptidi cardioregolatori nei mammiferi, dal momento che sono in grado di contrastare
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso,
Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo
l’inotropismo positivo mediato dai recettori b-adrenergici nei cuori dei vertebrati. Queste scoperte ci lasciano
supporre che la CGA e i peptidi da essa derivati possano svolgere un ruolo regolatore nella secrezione cardiaca.
Questa “review” rappresenta un riassunto delle possibili relazioni esistenti tra la CGA ed i suoi peptidi, e la
fisiologia, la biologia e la patologia del cuore.
Chromogranin A (CGA) is an acidic secretory protein detected in several endocrine and
neuroendocrine tissues, co-stored and co-secreted with catecholamines. CGA undergoes tissue-specific proteolytic events, and it gives rise to several derived peptides with a wide range
of functions and tissue targets, acting in an autocrine, paracrine and endocrine manner.
High levels of CGA have been detected, also, in pathological conditions regarding the
neuroendocrine, the renal and the cardiac system, postulating its possible involvement in
the patho-physiology of these diseases.
The purpose of this mini-review is to correlate the old and new knowledge about CGA
and its derived-peptides with heart biology and physio-pathology, suggesting a modulation
and cardiac-protective role for these molecules.
CGA and CGA derived peptides: structural and functional features
CGA is a 48 kDa acidic water-soluble protein, identified for the first time by K. Helle in
1966 in the secretory granules of bovine adrenal medullary chromaffin cells, and successively detected also in other endocrine and nervous tissues. It represents the 40% of the
soluble proteins of the granules, is co-stored with other granule secretory molecules such
as catecholamines, enzymes, neurotransmitters, calcium, and is co-released by exocytosis
upon splancnic nerve acetylcholine stimulation. Only after twenty years from its discovery,
two distinct groups obtained for the first time the primary sequence of bovine CGA (see
Figure1) [3, 11]. The analysis of the primary sequence shows many important features: a
signal sequence, composed of the first 18 amino acid residues of the primary pro-peptide
transcript, targeting it to rough endoplasmic reticulum membrane for maturation; numerous polyglutamic acid clusters in several regions of the molecule conferring a net negative
charge, and an isoelectric point at about 5; two cysteine residues at positions 17 and 38
forming a disulphide bridge important for many CGA activities; an RGD sequences at the
residues 43-45, probably implicated in adhesive processes between the cells and the extracellular matrix; several sites of post-translational modifications, such as O-glicosilation,
phosphorilation and sulphation; twelve cleavage sites recognized by endopeptidase enzymes, five of them situated in the N-terminus domain and the others in the C-terminus.
CGA seems to have a double function: intracellulary, it binds both calcium and cate72
The Chromogranin A-derived peptides: structural and functional features in heart biology
cholamines and it regulates the granule biogenesis acting as an “on/off switch”; extracellulary, it acts as pro-hormone. CGA is important as a whole molecule, but now they are
becoming very important the several smaller peptides derived from this secretory protein,
which showed to have different autocrine, paracrine or endocrine biological functions. In
fact, CGA can be degraded by specific proteases (such as Prohormone convertases) either
inside the granules, outsides in the extracellular environment or in the blood stream. This
process and the fragments derived have a tissue- and specie-specific pattern. The peptides derived from the cleavage of bovine CGA and their functions are listed in Table 1.
Vasostatins correlation with the heart: old and new findings
CGA and its derived fragments have been detected also in the cardiac tissue. Recently,
Glattard and colleagues [8] investigated the pattern of N-terminal CGA derived peptides
in the cardiac tissue, revealing the presence of several VS-I- and VS-II-containing CGA
Vasostatin-I (VS-I) and Vasostatin-II (VS-II) derive from the cleavage at the first two pair of
basic amino acids of the N-terminal domain of CGA, respectively K77-K78 and K114-R115
[15], giving rise to two peptides corresponding to the fragments CGA1-76 and CGA1-113 [7].
The prediction of the secondary structure of VS-I, the most abundant natural N-terminal
CGA fragment, is compatible with an О±-helical orientation and shows three anphipatic
domains: two in the N-terminal region, including the fragment CGA1-16 and the fragment
CGA17-38 containing the cysteine-bridged loop, and the third in the C-terminus comprehending the fragment CGA47-66. Moreover, the latest was well characterized and named chromofungin for its capability to cross the fungi membranes blocking their growing.
These fragments were named “Vasostatins (VSs)” because of their first role discovered:
an inhibition of the vasoconstriction, evoked by the potent vasoconstrictor ET-1, on isolated
segments of human internal thoracic artery and saphenous vein [1].
Subsequent evidences suggested a regulatory role for VSs acting via autocrine, paracrine or endocrine mechanisms in function of the cell/tissue target and the local peptide
concentration [10]. VSs, in fact, promote the adhesion of fibroblasts and smooth muscle
cells on extracellular matrix, exert autocrine inhibition of parathyroid hormone secretion in
parathyroid cells, have an antimicrobial activity and, finally, have an effect on heart [20].
It has been well established that VSs exert a general negative effect on the physiology
of eel, frog and rat hearts [2,6]. VSs and several other smaller CGA-N-terminal fragments
(i.e.CGA7-57 , CGA1-40 , CGA4-16 CGA47-66), in fact, have been tested on isolated and perfused
working hearts both in basal and isoproterenol (ISO)-stimulated conditions mimicking a
ОІ-adrenergic response. They acted modulating negatively the hearts inotropism on basal
condition and counteracting the positive inotropism stimulated by the ОІ-adrenergic agonist
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso,
Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo
ISO. This effect seems to be due to the fragment containing the disulfide-bridge loop, because the N- terminal fragments CGA4-16 and CGA47-66 are able to counteract the ISO-stimulated inotropy only at higher concentration than the peptide CGA1-40 with the intact loop [19].
Moreover, VS-II shows a lower inotropic potency than VS-I, probably because of a different
steric conformation. Both in eel, frog and rat hearts VS cardio suppressive effect depends
on extracellular calcium influx, as previously showed by Aardal and Helle [1] in saphenous
vein, and potassium channels. Furthermore, only in eel heart the intact endothelium is fundamental for VS activities. In fact, while in frog heart a treatment with Triton X-100 has no
effect on VS activities, in eel heart it abolishes the VS-mediated inotropic effect.
Moreover, recent studies clarified the role of the cytoskeleton in VS signal, both in eel
and frog hearts, being involved in several cellular functions, such as, for instance, the
modulation of some ion channels (i.e. Calcium channels or Sodium channels) [14]. Using
specific cytoskeleton inhibitors in concentrations which did not affect the functional integrity of the contractile cardiac machinery, it has been demonstrated a total block of the VS
negative inotropism. Thus the cytoskeleton integrity plays a crucial role in the VS action.
Nevertheless, VS interaction with the cells has not been discovered yet and the pathway
or pathways affected by VSs have not been well characterised yet, neither in mammals nor
in other vertebrates. The most recent studies, in fact, deal with the insight of the possible
cell-interaction way and with the characterization of a precise cellular pathway triggered
or affected by VSs.
A classical and specific receptor for VSs has not been discovered yet, thus two possible
different hypotheses of interaction with the cells could be taken into account [20]. The
extracellular hypothesis proposes an interaction either with the membrane phospholipids
or with some membrane proteins, for instance integrins, and a subsequent interference
with the function of cellular effectors (I.E ion channels, receptors, enzymes). Blois and
colleagues using a Langmuir film balance apparatus at 37В°C demonstrated, in vitro, an
interaction between VS-I at low concentration with membrane phospholipids in physiological conditions. The electrostatic and hydrophobic interaction affected the film fluidity [4].
On the other hand, we studied the effects of VS-I on 3D-cultured primary cardiomyocytes
establishing its influence in extracellular matrix-cell interactions affecting the localisation
of intracellular proteins such as endothelial nitric oxide synthase (eNOS) and HSP 90 [20].
The intracellular hypothesis suggests an internalisation of VS-I and its action inside the
cell, as it has been demonstrated using rhodamine-labelled CGA 47-70 on smooth muscle layer of rat posterior cerebral artery [13]. Nevertheless, further studies have to be performed
to clarify which hypothesis is correct, or whether both could be considered as combined
interaction ways.
Another aim of the last studies was to detect the pathways involved in VS action.
The Chromogranin A-derived peptides: structural and functional features in heart biology
Figure 1. Schematic rapresentation of the main features of bovine CGA structure. (S-S) Disulphide bridge; (P)
Phosphorylation; (G) Glycosilation; (t) Cleavage site.
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso,
Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo
Figure 2. Hypothetical sites of action and pathways involving Vasostatin-I. (AC) Adenylyn Cyclate; (TnI) Troponin I; (TnC) Troponin C.
The Chromogranin A-derived peptides: structural and functional features in heart biology
Previously works carried out in eel and frog heart preparations, showed important differences in the pathways affected by VS-I, due to species-specific features. The implication of endocardial endothelium (EE) and the Nitric Oxide (NO)-cGMP signal transduction
pathway seems to be essential in mediating VS effects only in eel heart [6, 18]. In fact,
it has been demonstrated that the use of either NOS or soluble guanylate cyclase (GC) or
cGMP-activated protein kinase (PKG) (an important NO target) inhibitors abolishes completely the effects of VS-I. The negative VS-inotropism can be explained considering that the
NOS-dependent NO production increases the level of cGMP which activates PKG. PKG, on
one hand, phosphorilates the troponin I and causes the decrease of the affinity of troponin
C for calcium, regulating negatively the cardiac contraction; on the other hand, it phosphorilates the alpha-subunit of the Gi/0 protein, which negatively affects adenylyn cyclase,
decreases the cAMP levels and finally inhibits the L-type Ca current. Lastly, both pathways
elicit the negative regulation of the cardiac contraction. Recently, Cappello and colleagues
[16] studied, for the first time, the pathways affected by VS-I in mammalian heart, using as
a model the rat heart. Using specific inhibitors, they demonstrated the involvements of the
same NOS-NO-cGMP-PKG system as in the eel heart.
Gallo and co-workers [12] detected whether anti-adrenergic effects of VS-I occurs directly on cardiomyocytes or on other cell types of the cardiac tissue. They demonstrated
that the synthesis of NO induced by VS-I takes places not in cardiomyocytes but in EE and
it depends on Phosphoinositide-3 kinase (PI3K) activation.
Role of CGA and CGA derived peptides in heart physio-pathology
High serum levels of CGA have been correlated to several kind of neuroendocrine
tumours for long time [9]. Recently high levels of CGA have been also reported in patients with heart failure and myocardial infarction [5]. Ceconi and co-workers correlated
the increase of serum CGA with the chronic heart failure (CHF) [5], which is a syndrome
characterized by neuroendocrine activation, in which the serum levels of catecholamines,
natriuretic peptides and several peptides signalling system increase. These work demonstrated for the first time that serum CGA levels increased in patients with CHF and it was
related to the clinical severity of the syndrome. The increase in CGA was found already in
the early stages of the failure and it was a predictive factor of mortality. Moreover, it has
been speculated that the high CGA levels may represent the response of the organism to
counteract the effects of an excessive neuroendocrine activation. On the other hand, being
CGA a component of the neuroendocrine system, the increase of its levels could be just the
consequence of the over stimulation of the neuroendocrine system [5].
It has been well established that CGA either into the neuroendocrine vescicles or in the
blood stream undergoes proteolytic events, giving rise to several CGA derived peptides
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso,
Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo
showing autocrine and paracrine effects. Moreover, it has been demonstrated that the
cardiac tissue, in both in normal and pathological condition, is able to produce CGA [17].
Thus, CGA and its derived peptides could play an auto-endocrine regulation on the heart,
counteracting an excessive adrenergic stimulation in normal condition and especially in
pathological condition correlated with their high plasma levels. In this view CGA shows a
role in myocardial protection. Recently, it has been, in fact, proposed a preconditioninglike effect of VS-I, if given before ischemia/reperfusion and it has been demonstrated a
reduction of the infarct size after the administration of VS-I before I/R. Furthermore, it has
been established the involvement of, at least, two different pathways: the NO-cGMP-PKG
pathway and the adenosine-pathway. Indeed, the inhibition of eNOS blocked totally this
effect and on the other hand the inhibition of A1-receptors reduced it about 50% [16].
Little is still known about the role of CGA and its derived peptides in physio-pathology
of the heart and further studies are necessary. However, these molecules, acting as neurormones, could play a central role in the cardiac physio-pathology and could represent the
main target of the new pharmacological therapy.
We thank the University of Palermo, Italy, for a doctoral scholarship to Antonella Montalbano and the Ministero della UniversitГ e della Ricerca, for financial support (MURST ex 60% Prof. Giovanni Zummo).
List of abbreviations
Chromogranin A
VS-I =
Vasostatin I
Vasostatin II
Tumor necrosis factor О±
ET-1 =
Atrial Natriuretic Factor
Phospholipase C
Inositol 1,4,5 triphosphate
Protein Kinase C
eNOS =
Endothelial nitric oxide synthase
Endocardial endothelium
Nitric oxide
Guanylate cyclase
The Chromogranin A-derived peptides: structural and functional features in heart biology
cGMP-activated protein kinase
Phosphoinositide-3 kinase
Chronic Heart Failure
Table I. Bovine CGA derived peptides and their main function
Vasostatin I (CGA1-76)
Vasostatin II (CGA1-113)
(rat-ОІgranin (CGA1-128))
Chromofungin (CGA47-66)
Chromostatin (CGA124-143)
Chromacin (CGA173-194)
Inhibition vasoconstriction
Promotion of adhesion
Inhibition parathyroid hormone secretion
Antimicrobial effects
Negative inotropism
Antimicrobial effects
Inhibition chromaffin cells secretion
Antimicrobial effects
Pancreastatin (CGA248-293)
Inhibition of glucose-stimulated insulin secretion
Take part in lipid and glucose metabolism
WE-14 (CGA316-331)
No Data
Parastatin (CGA347-419)
Inhibition of parathormone and CGA secretion from parathyroid cells
Catestatin (CGA344-364)
Inhibition of catecholamine secretion; promotion histamine release in mast cells
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso,
Antonella Marino Gammazza, Francesco Cappello, Giovanni Zummo
[1] Aardal S, et al. Vasostatins, comprising the N-terminal domain of chromogranin A, suppress tension in
isolated human blood vessel segments. Journal of Neuroendocrinology 1993; 5(4): 405-412.
[2] Angelone T, et al. The emerging cardio-inhibitory role of the Hippocampal Cholinergic Neurostimulating
Peptide (HCNP). Journal Pharmacological Experimental Terapeutics 2006; in press.
[3] Benedum U M, et al. The primary structure of bovine chromogranin A: a representative of a class of
acidic secretory proteins common to a variety of peptidergic cells. Embo J 1986; 5(7): 1495-1502.
[4] Blois A, et al. Interactions of chromogranin-A-derived Vasostatins and monolayer of phosphatydilserine, phosphatidilcholine, phosphatydilethanolamine. Regulatory Peptides (accepted).
[5] Ceconi C, et al. Chromogranin A in heart failure; a novel neurohumoral factor and a predictor for mortality. Eur Heart J 2002; 23(12): 967-974.
[6] Corti A, et al. Chromogranin A N-terminal fragments vasostatin-1 and the synthetic CGA 7-57 peptide act
as cardiostatins on the isolated working frog heart. Gen Comp Endocrinol 2004; 136(2): 217-224.
[7] Corti A, et al. Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) - evidence of dimer-monomer equilibria. Eur J Biochem 1997; 248(3): 692-699.
[8] Glattard E, et al. Characterization of natural vasostatin-containing peptides in rat heart. Febs J 2006;
273(14): 3311-3321.
[9] Gratton J P, et al. Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1
complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from
caveolin-1. J Biol Chem 2000; 275(29): 22268-22272.
[10] Helle KB, Metz-Boutigue M-H, and Aunis D. Chromogranin A as a Calcium-Binding Precursor for a
Multitude of Regulatory Peptides for the Immune, Endocrine and Metabolic Systems. Curr Med Chem Immun, Endoc & Metab Agents 2001; 1: 119-140.
[11] Iacangelo A, et al. Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine
tissues. Nature 1986; 323(6083): 82-86.
[12] Malan D, et al. Microtubules mobility affects the modulation of L-type I(Ca) by muscarinic and betaadrenergic agonists in guinea-pig cardiac myocytes. J Mol Cell Cardiol 2003; 35(2): p. 195-206.
[13] Mandala M, et al. Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery.
Regul Pept 2005; 124(1-3): 73-80.
[14] Mazza R, et al. Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts. Regul Pept 2007; 138(2-3): 145-151.
[15] Metz-Boutigue M H, et al. Intracellular and extracellular processing of chromogranin A. Determination
of cleavage sites. Eur J Biochem 1993; 217(1): 247-257.
[16] Pagliaro P, et al., Protection against ischemia/reperfusion injures by vasostatin-1 in the isolated rat
heart, in “Physiological Society Meeting”. 2005: University of Oxford, UK.
[17] Pieroni M, et al. Myocardial production of chromogranin A in human heart: a new regulatory peptide of
cardiac function. Eur Heart J 2007; 28(9): 1117-1127.
[18] Tota B, et al. Vasostatins and Negative Inotropy in Vertebrate Hearts. Curr Med Chem – Immun, Endoc
& Metab Agents 2004; 4: 195-201.
[19] Tota B, et al. Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart. Regul Pept 2003; 114(2-3): 123-130.
[20] Tota B, et al. New biological aspects of Chromogranin A-derived peptides: Focus on vasostatins. Comparative Biochemistry and Physiology (Part A) 2007; 147: 11-18.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
New insights into the role of matrix metalloproteinases
in heart angiogenesis induced by exercise
[Ruolo delle MMPs di matrice nell’angiogenesi del cuore indotta dall’esercizio fisico]
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
Dipartimento di Medicina Sperimentale, sezione di Anatomia Umana “E. Luna”, Università degli Studi di
Palermo (IT)
Key words: Angiogenesis, Exercise, Heart, MMPs
Parole chiave: Angiogenesi, Esercizio fisico, Cuore, MMPs
Abstract. Angiogenesis induced by exercise has been observed in both cardiac and skeletal muscle and
plays a fundamental role in maintaining tissue function adequate to the increase in metabolic requests.
Mechanical and haemodynamic forces are strong starters of angiogenic process via regulation of secondary mediators such as vascular endothelial growth factor (VEGF). A crucial step of vessel sprouting is the
degradation of the basement membrane and remodelling of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs). It has long been accepted that MMPs are involved in the angiogenesis, but the exact
mechanisms are not well characterized. Cryptic fragments and neo-epitopes released by proteolysis of basement membrane components by MMPs can promote or block capillary growth. MMPs are also involved in
the release and activation of growth factors embedded into ECM such as VEGF. In turn, it has been showed in
vitro that VEGF can stimulate endothelial and smooth muscle cells to produce MMPs suggesting a reciprocal
relationship between VEGF and MMPs. The expression patterns of MMPs are temporally dissociated by those
of VEGF in vivo: MMPs and VEGF seem to be independently regulated and involved respectively in sprout
formation and capillary proliferation. Little is known about the regulation of MMPs by exercise and the most
studies were carried out in the skeletal muscle. The increase in MMP activity is an early and critical feature
of angiogenesis in skeletal muscle when initiated by mechanical stimuli which lead to breakage of the basement membrane but not by stimuli such as shear stress that act via the luminal surface of vessels. Although
MMPs seem to play a important role in sprouting angiogenesis induced by exercise, in our knowledge their
function in exercised hearts has not been still tested. It is known that elevated levels of tissue inhibitors of
the MMPs (TIMPs) may contribute to the accumulation of collagen content in the infarcted heart leading to
myocardial fibrosis. Therefore, a positive modulation of MMP system by exercise in heart failure-related
myocardial remodelling could lead to a ECM with a less fibrous component and more angiogenic sprouting
in order to preserve the cardiac function.
Riassunto. L’angiogenesi è stata osservata nel miocardio e nel muscolo scheletrico in risposta all’esercizio
fisico e riveste un ruolo fondamentale nel mantenere l’attività del tessuto adeguata all’aumento delle richieste metaboliche. Le forze meccaniche ed emodinamiche sono potenti iniziatori del processo angiogenico
mediante la regolazione di mediatori secondari, come il fattore di crescita vascolare endoteliale (VEGF). Una
fase cruciale per la formazione di nuovi capillari ГЁ la degradazione della membrana basale e il rimodellamento della matrice extracellulare (ECM) da parte delle metalloproteasi di matrice (MMPs). Sebbene sia
noto da tempo che le MMPs sono coinvolte nell’angiogenesi, gli esatti meccanismi non sono stati ancora
ben caratterizzati. Frammenti criptici e neo-epitopi rilasciati mediante la proteolisi dei componenti della
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
membrana basale da parte delle MMPs possono promuovere o inibire la crescita capillare. Le MMPs sono
anche coinvolte nel rilascio e nell’attivazione di fattori di crescita intrappolati nella ECM così come il VEGF.
A sua volta, ГЁ stato dimostrato in vitro che il VEGF puГІ stimolare le cellule endoteliali e le cellule muscolari
lisce a produrre le MMPs suggerendo una relazione reciproca tra il VEGF e le MMPs. I profili di espressione
delle MMPs sono temporalmente dissociati da quelli delle MMPs in vivo: le MMPs e il VEGF sembrano essere regolati indipendentemente e coinvolti rispettivamente nella gemmazione del nuovo vaso e nella proliferazione capillare. Poco è noto circa la regolazione delle MMPs da parte dell’esercizio fisico e la maggior
parte degli studi sono stati condotti sul muscolo scheletrico. L’incremento dell’attività delle MMPs è una
caratteristica cruciale e precoce dell’angiogenesi nel muscolo scheletrico qualora innescata da stimoli meccanici che portano alla rottura della membrana basale; tuttavia non da stimoli quali le forze da taglio che
agiscono attraverso la superficie del lume dei vasi. Sebbene le MMPs sembrino rivestire un ruolo importante
nell’angiogenesi indotta dall’esercizio fisico, nell’ambito della nostra conoscenza la loro funzione nei cuori
allenati non è stata ancora studiata. È noto che livelli elevati degli inibitori tissutali delle MMPs (TIMPS) possano contribuire all’accumulo del contenuto di collagene nei cuori infartuati portando a fibrosi del miocardio.
Pertanto, una modulazione positiva del sistema delle MMPs da parte dell’esercizio nel rimodellamento del
miocardio con insufficienza cardiaca potrebbe condurre ad una ECM con una minore componente fibrosa e
con una maggiore angiogenesi tale da garantire la funzionalitГ del miocardio.
Angiogenesis is the formation of new blood vessels from existing ones. A fundamental
step of this process is the degradation of the basement membrane and remodelling of the
extracellular matrix (ECM) in order to allow endothelial cells to migrate into the extravascular space where they proliferate and form abluminal sprouts which become functional
on connection to other segments of capillary network. Subsequently, the sprouts acquire
newly formed basement membrane and become surrounded by pericytes. For the any components of matrix, there are a large number of enzymes controlling stroma turnover. One
important group are the matrix metalloproteinases (MMPs) that regulate the degradation
of components of the ECM in conjunction with specific inhibitors (TIMPs) whose net effects
determine the structure of the ECM at any particular point in time [1]. This process of ECM
degradation need to be highly controlled and coordinated because unguarded dissolution
of the matrix would result in a loss of the integrity and function of the microvasculature.
Angiogenesis process occurs in the healthy adult organism in specific situations including endocrine changes such as in the female reproductive organs, hemodynamic and mechanical stress and humoral stimuli in response to exercise [2]. Capillary growth induced
by exercise has been observed in both cardiac and skeletal muscle and it results to be
essential for maintaining tissue function adequate to increased metabolic demand [3]. The
present review points to the regulation of MMPs by exercise in the cardiac angiogenesis
and potential implications that regular and prolonged exercise could have on the heart
failure-related ECM turnover.
New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise
Description of ECM components in cardiac tissue
The myocardial ECM is a dynamic structure whose component proteins are maintained
by a finely controlled homeostatic balance between deposition and degradation. Within the
myocardium, myocytes are linked by an intricate lattice of ECM proteins which provide structural integrity to the tissue and a means for optimal vectoral transmission of force. Although
myocytes occupy two thirds of the tissue volume, 70% of cells within the myocardium are
fibroblasts [4]. The primary role of cardiac fibroblasts (CFs) appears to be the synthesis of
ECM components and regulation of their turnover by the secretion and activation of proteolytic enzymes such as the MMPs. The lattice surrounding the myocytes includes a complex
network of structural proteins (collagen and elastic fibers) and adhesive proteins (fibronectin, laminin) within a hydrated proteoglycan and glycosaminoglycan-rich milieu [5].
Fibrillar collagens are the most abundant of the ECM proteins and at least 28 distinct
collagen types are expressed in vertebrates. Within the myocardium, collagen types I, III,
IV, V, VI and VIII have been identified [6]. The fibrillar collagen types I and III coexist to form
the collagenous network of the myocardium comprising 85% and 11% of the collagen in
healthy mammalian hearts respectively [7]. Type I collagen fibrils have an extremely high
tensile strength and in combination with the giant sarcomeric muscle protein, titin, largely
determine the stiffness of the myocardium [8]. Type III collagen is more distensible than
type I by comparison and is argued to maintain the structural integrity of the network.
Other less abundant collagen types of the myocardium are types IV, V, VI and VIII. Type IV
is expressed by myocytes and endothelial cells and does not form fibrils but instead assembles into an open network within basement membranes [7]. Type V coexists with type
IV collagen in the basement membrane whilst both types V and VI collagens co-localize
with collagen I and III fibrils in the interstitium. Type V appears to play contrasting roles to
other collagens, as it is thought to inhibit endothelial cell adhesion and proliferation [9].
Type VIII collagen expression is predominant during development, yet is detectable at very
low levels in the adult myocardium and is likely to play a role in the morphogenesis of cardiac valves [10]. To date, a full understanding of the roles of these less abundant collagens
in the heart remains to be realized. Collagenous matrices exhibit high levels of resistance
to both tensile forces and proteolytic degradation. These features are enhanced by the
process of cross-linking which provides a final step in the maturation of collagen fibrils.
Robinson et al. (1988) have identified elastic fibers associated with the coiled perimysial fibres of the collagen fibers axis and suggested that the elastic fibers counteract
stretch in the myocardium and are responsible for passive recoil [11].
Fibronectin, laminin and collagen type IV not only constitute the structural support of the
basement membranes, but are also involved in signalling and coupling the myocyte to the
collagenous ECM scaffold [12].
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
Involvement of MMPs in the capillary growth
MMPs, also called matrixins, are a family of zinc-containing endopeptidases that are
capable of degrading various components of the ECM. There are two main types of MMPs:
those that are secreted into the extracellular space and those that are membrane bound.
The secreted MMPs are synthesized as inactive pro-enzymes and are activated by cleavage of the regulatory peptide sequence. This cleavage determines a conformational change
and exposure of the catalytic domain to ECM substrate. Activation of MMPs is achieved by
serine proteases, such as plasmin, and other MMPs. The membrane-type MMPs, or MTMMPs, undergo intracellular activation and are proteolytically active once inserted into the
cell membrane. MT-MMPs constitute an important pathway for activation of other MMPs
within the ECM [13]. Another important control point of MMP activity is through the presence of tissue inhibitors of the MMPs, called TIMPs. Four different TIMP species have been
identified and bind to activated MMPs in a 1:1 stoichiometric ratio [14]. By contrast, MTMMPs do not appear to bind and be regulated by TIMPs.
It has long been accepted that MMPs play an important role in angiogenesis, but the exact mechanisms are not well characterized. Recently, cryptic fragments and neo-epitopes
released by proteolysis of basement membrane components result to be involved in the
promoting or blocking angiogenesis (Table 1). For instance, both MMP-2 and MMP-9 expose a cryptic epitope within collagen IV that promotes angiogenesis. Fragments generated from NC1 domain of collagen IV, VIII, XV and XVIII can be released by cleavage involving MMPs and act as anti-angiogenic factors. Other components of basement membrane,
such as laminin-5 and fibronectin, also harbor cryptic sites that are release by MMP activity (MMP-2, -3, -12, -13 and -14) and fragments generated can induce cell migration [15].
However, the mechanisms responsible for the release of all these fragments have not been
clearly elucidated.
MMPs are also involved in the release and activation of growth factors and cytokines embedded into ECM [1]. Vascular endothelial growth factor (VEGF) and transforming
growth factor-ОІ (TGF-ОІ) represent two examples of such factors that are stored within
the ECM and can be released by MMP proteolysis increasing their bioavailability. VEGF is
involved in the process of angiogenesis because it enhances vascular permeability and
promotes new vessel growth. Two different tumor models implicate MMP-9 in the release
of VEGF from the ECM. In the insulinoma model, release of VEGF by MMP-9 is associated
with an angiogenic switch, which promotes tumor progression [16]. In an ovarian carcinoma model, activated MMP-9 and, to lesser extent, MMP-2 are involved in increased
bioavailability of VEGF in culture [15]. Even though these are tumor models, it is highly
likely that similar mechanisms for the release of VEGF, leading to increased angiogenesis,
occur in non-pathological conditions. Furthermore, VEGF can stimulate endothelial and
New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise
smooth muscle cells to produce MMPs in vitro to facilitate migration and tube formation.
All this suggests that a reciprocal relationship between VEGF and MMPs in different cell
types in vitro exists. In vivo, much less is known about interactions between MMP activities and VEGF expression during angiogenesis, where temporal patterns of expression may
differ from in vitro. Rivilis et al. (2002) showed that the increase in MMP preceded VEGF in
response to skeletal muscle stretch whereas with stimulation, it lagged behind VEGF [17].
Moreover, during treatment with prazosin vasodilator, VEGF was increased very early on
when MMP expression was actually slightly decreased. MMPs are unlikely to be important
for endothelial proliferation since this proceeded unaltered in stimulated muscles treated
with a MMP inhibitor whereas sprout formation was suppressed [18]. VEGF, on the other
hand, might play a more prominent role in capillary proliferation as increases in its expression were dissociated temporally from those of MMPs. Thus, these factors appears to be
regulated independently in vivo.
Cardiac angiogenesis in response to exercise: role of MMPs
In the literature there are controversial data about heart angiogenesis in response to
exercise. It has been shown that various variables such as the age of the animals, kind
and intensity of exercise may affect the coronary capillary growth. It is clear, however,
mechanical and haemodynamic forces are strong starters of angiogenic process. For
istance, the increased myocardial stroke volume and contractility induced by dobutamine
mechanically can vary the capillary stretch and spreading throughout the cardiac cycle.
In the same way, the increase in the coronary blood flow, stroke volume and contractility
induced by exercise training may increase the shear stress, wall tension, capillary stretch
and compression forces on vessels [19]. It has been reported that these stimuli can initiate
angiogenesis process via regulation of secondary mediators such as VEGF. Indeed, several
authors found that VEGF is up-regulated by ventricle wall stretching and increased dyastolic pressure [20,21].
Angiogenesis induced by exercise training appears to occur by vessel sprouting rather
than intussusception process. Angiogenic sprouting consists into branching out of activated endothelial cells from an existing capillary, extending through the degraded surrounding
matrix to form a cordlike structure [2].
Little is known about the regulation of MMPs by exercise and most studies were carried out in the skeletal muscle. In electrically stimulated skeletal muscles, total and active MMP-2 protein increased after 7 days coinciding with the appearance of angiogenic
sprouts [18]. Both stretched and stimulated, but not vasodilator treated, muscles also
showed increased expression of mRNA and protein for MMP-2 and MT1-MMP [17,18].
Moreover, inhibition of MMP activity prevented the growth of new capillaries in stimulated
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
muscles; instead it did not affect the capillary-associated cell proliferation. These findings
point to increased MMP activity as an early and critical feature of angiogenesis in skeletal
muscle when initiated by mechanical stimuli which lead to breakage of the basement
membrane but not by stimuli such as shear stress that act via the luminal surface of
vessels. In addition, capillary-associated cell proliferation seems to precede and occurs
independently of endothelial cell sprout formation [18]. In stretched muscles, lengthening of capillaries by changes in sarcomere length might be the main stimulus whereas in
stimulated muscles, the repetitive shortening and relaxation of myocytes, and the 2-fold
increase in capillary pressure and hence wall tension could act in combination. Moreover,
cyclic strain applied to rat microvascular endothelial cells led to time-dependent upregulation of MT1-MMP mRNA and protein indicating the importance of physical tensile forces in
the regulation of extracellular matrix degrading factors [19]. Although MMPs seem to play
a important role in sprouting angiogenesis induced by exercise, in our knowledge their
function in exercised hearts has not been still tested. Verzola et. al. studied the effect of
acute swimming training with anaerobic component on MMP activity and myosin heavy
chain gene expression in the rat myocardium. They found an increase in expression and
activity of MMP-2 72, 96 and 120 hours after the beginning of exercise training suggesting
precocious heart remodeling. This modification was associated with myocardium hypertrophy induced by training protocol suggesting that increased MMP activity is necessary to
allow tissue growth in order to support the enhanced metabolic requests during exercise.
However, vascular remodelling that might have contributed to the significant increase in
heart weight has not been examined in this study [22]. In our previous work, we found
that mouse heart hypertrophy induced by endurance training was significantly due to an
increase in the capillary growth rather than increased myocardiocyte mass [23]. Moreover,
we showed that both expression of VEGF receptors and MMP-2 and MMP-9 activity were
involved in the angiogenic process of exercised mouse hearts and were differently regulated by intensity and time of training [24].
Exercise training is an important complementary intervention for heart health
The benefits of exercise in terms of reduced risk of coronary disease and mortality are
widely recognized. There is no doubt that the direct effects of repeated bouts of exercise
on the myocardium – improved contractile function, increased end-diastolic dimensions,
resting bradycardia, induction of protective heat shock proteins, altered energy substrate
utilization and improved anti-oxidant capacity [25] – contribute significantly to the enhanced ventricular performance of the trained heart. Moreover, it is known that physical
training programs are beneficial to the heart because chronic exercise increases myocardial vascularity and protects the myocardium against ischemic insult [26]. This highlights
New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise
the significance of coronary vascular growth and remodelling as intrinsic to the protective
benefits of exercise. Brown et al. (1994) showed a increase in the capillary density in pigs
hearts with a region of infarction about 15% of the left ventricular free wall during induced
bradycardia. After 4 weeks, capillary density was significantly reduced in the border zone
of the infarct compared with that in undamaged left ventricle of the same hearts or control
sham-operated pig hearts. When infarcted pig hearts were electrically paced to bradycardia for 4 weeks, maintaining a basal heart rate of 60–80 beats min-1 compared with around
110 beats min-1 in unpaced infarcted or normal conscious pigs, capillary densities in both
the border zone and the undamaged left ventricle were significantly greater. This enlargement of the capillary bed most likely enabled more homogeneous tissue perfusion, contributing to the improvement in cardiac function of paced infracted hearts during inotropic
challenge. Proliferation of capillaries after pacing was confirmed in non-infarcted hearts by
an increase in proportion of those labelled by a cell proliferation marker [27].
In agreement with what has been said the knowledge of the mechanisms of vascular
growth in response to exercise may be used to target specific portions of the coronary
vascular tree for therapeutic purposes. A positive modulation of MMP system by exercise in
heart failure-related myocardial remodelling could be an important complementary intervention. It is generally accepted that ECM and myocardial maladaptive is a unifying feature
in the progression to heart failure [28]. For this reason an exercise-related reduction of
TIMPs expression may improve the balance between MMPs and TIMPs and enhance the
proteolytic activity, thus decrease the collagen accumulation in post-infarcted myocardium
[29]. Indeed, Webb et al. (2006) demonstrated that TIMP-1 levels were higher at day 1
post-infarction and remained substantially elevated through day 180 in patient with myocardial infarct. The elevated TIMP-1 level may contribute to the accumulation of collagen
content in the infarcted heart leading to myocardial fibrosis [30]. Moreover, a ECM with
a less fibrous component could improve sprouting angiogenesis in response to exercise,
thereby preserve the cardiac function.
Little is known about the signals that promote vascular growth and remodelling in the
heart in response to exercise. It is still necessary to study as hearts that are trained and/
or affected by angiogenic stimuli such as haemodynamic and mechanical stresses (typical of physical activity) may improve angiogenesis. Furthermore, although MMP system
appears to be involved in one of essential step of angiogenesis process, there are no data
about their function in cardiac angiogenesis induced by exercise training. For this reason,
it could be interesting to study the expression and activity of cardiac MMPs and TIMPs and
their regulation by exercise. Moreover, at the present it is not possible to prescribe with
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
adequate precision the kind, duration and intensity of exercise that may promote vascular growth. Therefore, developing the knowledge about the regulation of MMP system by
exercise might allow to draw up specific training protocols for improving performance of
healthy and diseased hearts.
Table 1. Cryptic basement membrane fragments involved in angiogenesis
ECM protein
Collagen IV
Cryptic epitope
Collagen IV
NC1 a1, arresten
NC1 a 2, canstatin
NC1 a 3, tumstatin
NC1 a 6
Collagen VIII
NC1 a1, vastatin
Collagen XV
NC1, restin
Collagen XVIII
NC1, endostatin
C-term, endorepellin
Modified from Mott JD and Werb Z 2004 [15]
New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise
[1] McCawley L, Matrisian L. Matrix metalloproteinases: they’re not just for matrix anymore! Curr Opin Cell
Biol 2001; 13:534-40.
[2] Bloor CM. Angiogenesis during exercise and training. Angiogenesis 2005; 8: 263-271.
[3] Hudlicka O, Brown MD, Egginton S.: Angiogenesis in skeletal and cardiac muscle. Physiol Rev 1992;
72: 369-417.
[4] Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, McFadden A, Rice M, Borg TK. Organization
of fibroblasts in the heart. Dev Dyn 2004; 230: 787-794.
[5] Manabe I, Shindo T, Nagai R. Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy. Circ Res 2002; 91: 1103-1113.
[6] Kitamura M, Shimizu M, Ino H, Okeie K, Yamaguchi M, Fujino N, Mabuchi H, Nakanishi I. Collagen
remodeling and cardiac dysfunction in patients with hypertrophic cardiomyopathy: the significance of
type III and VI collagens. Clin Cardiol 2001; 24: 325-329.
[7] Linehan KA, Seymour AM, Williams PE. Semiquantitative analysis of collagen types in the hypertrophied
left ventricle. J Anat 2001; 198: 83-92.
[8] Linke WA, Popov VI, Pollack GH. Passive and active tension in single cardiac myofibrils. Biophys J 1994;
[9] Fukuda K, Koshihara Y, Oda H, Ohyama M, Ooyama T. Type V collagen selectively inhibits human endothelial cell proliferation. Biochem Biophys Res Commun 1988; 151: 1060-1068.
[10] Iruela-Arispe ML, Sage EH. Expression of type VIII collagen during morphogenesis of the chicken and
mouse heart. Dev Biol 1991; 144: 107-118.
[11] Robinson TF, Geraci MA, Sonnenblick EH, Factor SM. Coiled perimysial fibers of papillary muscle in rat
heart: morphology, distribution, and changes in configuration. Circ Res 1988; 63:577-592.
[12] Tryggvason K.: The laminin family. Curr Opin Cell Biol 1993; 5: 877-882.
[13] Knauper V, Murphy G. Membrane-type matrix metalloproteinases and cell surface-associated activation cascades for matrix metalloproteinases. In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases.
San Diego, Calif: Academic Press 1998; 199–218.
[14] Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and
function. Biochim Biophys Acta 2000; 1477(1-2): 267-83.
[15] Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004;
16(5): 558-64.
[16] Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z,
Hanahan D. Matrix metalloproteinase 9 triggers the angiogenic switch during carcinogenesis. Nat Cell
Biol 2000; 2: 737-744.
[17] Rivilis I, Milkiewicz M, Boyd P, Goldstein J, Brown MD, Egginton S, Hansen FM, Hudlicka O, Haas TL.
Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis. Am J Physiol Heart Circ Physiol 2002; 283(4):H1430-8.
[18] Haas TL, Milkiewicz M, Davis SJ, Zhou AL, Egginton S, Brown MD, Madri JA, Hudlicka O. Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle. Am J Physiol
2000; 279:H1540-47.
[19] Brown MD, Hudlicka O. Modulation of physiological angiogenesis in skeletal muscle by mechanical
forces: involvement of VEGF and metalloproteinases. Angiogenesis 2003; 6(1):1-14.
[20] Li J, Hampton T, Morgan JP, Simons M. Stretch-induced VEGF expression in the heart. J Clin Invest
1997; 1:100(1):18-24.
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
[21] Zheng W, Seftor EA, Meininger CJ, Hendrix MJ, Tomanek RJ. Mechanisms of coronary angiogenesis in
response to stretch: role of VEGF and TGF-beta. Am J Physiol Heart Circ Physiol 2001; 280(2):H909-17.
[22] Verzola RM, Mesquita RA, Peviani S, Ramos OH, Moriscot AS, Perez SE, Selistre-de-AraГєjo HS. Early remodeling of rat cardiac muscle induced by swimming training. Braz J Med Biol Res 2006; 39(5):621-7.
[23] Bellafiore M, Sivverini G, Palumbo D, Macaluso F, Bianco A, Palma A, Farina F. Increased cx43 and angiogenesis in exercised mouse hearts. Int J Sports Med 2007; 28(9):749-55.
[24] Bellafiore M, Battaglia G., Bianco A, Palma A, Farina F. Molecular factor involved in the angiogenesis
process of exercised mouse hearts. “13th Annual Congress of the European College of Sport Science”;
Estoril, Portugal. 2008 (9-12 july); 13:532.
[25] Powers SK, Lennon SL, Quindry J, Mehta JL. Exercise and cardioprotection. Curr Opin Cardiol 2002;
[26] Scheuer J. Effects of physical training on myocardial vascularity and perfusion. Circulation 1982;
[27] Brown MD, Davies MK, Hudlicka O, Townsend P. Long term bradycardia by electrical pacing: a new
method for studying heart rate reduction. Cardiovasc Res 1994; 28(12):1774-9.
[28] Graham HK, Horn M, Trafford AW. Extracellular matrix profiles in the progression to heart failure. Acta
Physiol (Oxf). European Young Physiologists Symposium Keynote Lecture-Bratislava 2007. 2008;
[29] Xu X, Wan W, Ji L, Lao S, Powers AS, Zhao W, Erikson JM, Zhang JQ. Exercise training combined
with angiotensin II receptor blockade limits post-infarct ventricular remodelling in rats. Cardiovasc Res
2008; 78(3):523-32.
[30] Webb CS, Bonnema DD, Ahmed SH, Leonardi AH, McClure CD, Clark LL, Stroud RE, Corn WC, Finklea
L, Zile MR, Spinale FG. Specific temporal profile of matrix metalloproteinase release occurs in patients
after myocardial infarction: relation to left ventricular remodeling. Circulation 2006; 114(10):1020-7.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Capillaroscopia orale: nostra esperienza]
Francesco Carini1, Giuseppe Alessandro Scardina3, Gianfranco Cocorullo4, Pierluigi Varia1,
Chiara Lo Piccolo1, Angelo Leone2, Pietro Messina3 and Vincenzo Valenza1
Department of Experimental Medicine, Section of Human Anatomy; 2Department of Experimental Medicine,
Section of Histology and Embryology; 3Department of Dental Stomatological Sciences, Section of Dental
Medicine; 4Department of General and Urgent Surgery, University of Palermo (IT)
Key words: Capillaroscopy, Vasculitis, Oral mucositis
Parole chiave: Capillaroscopia, Vasculite, Mucosite orale
Abstract. Capillaroscopy has now definitely become an important and indispensable exam in the area of
diagnosing vasculopathies and in particular micro angiopathies. The type of damages connected to these
anatomy pathologies regarding the actual micro vascular unit work in a functional pathogenic of an extreme
importance, in conditioning the course and the eventual aggravation of micro angiopathies properly called
vasculity and also of macroangiopathies. In all, this underlines the great importance achieved by using
certain instrumental exams which complements the clinical research, exactly because of the valid elements
which have been given for a complete and precise morphological valuation for the surrounding circulation
conditions. Such techniques, like the laser Doppler or the pletismography do not let us express a prognostic
verdict on eventual risks with tropic damages, which are instead valued directly on realistic times with the
aid of oral capillaroscopy, of whose use has permitted new morphological and functional acquisitions.
The unharmful, no-invocatory and easily repeatable test and its moderate cost, also allows us to monitor and
up-date the future state of peripheral vasculopathies, as well as the efficiency of the medicines which have
possibly been used.
Riassunto. La capillaroscopia è entrata a pieno diritto tra gli esami diagnostici indispensabili nell’ambito
delle vasculopatie e in particolare nelle microangiopatie. Le lesioni anatomo-patologiche a carico dell’unità microvascolare svolgono una funzione patogenetica estremamente importante, se non addirittura fondamentale, nel condizionare il decorso e l’eventuale aggravamento sia di microangiopatie propriamente dette,
vasculiti, sia di macroangiopatie.
Tutto ciò sottolinea il grande valore assunto da alcuni esami strumentali quale complemento all’indagine
clinica, proprio perché capaci di fornire elementi validi per una più completa e minuziosa valutazione morfologica e funzionale delle condizioni circolatorie distrettuali. Varie tecniche, come il laser doppler o la pletismografia non ci consentono di esprimere un giudizio prognostico sull’eventuale rischio di lesioni trofiche, che
sono invece valutate direttamente in vivo e in tempo reale con l’ausilio della capillaroscopia orale, il cui uso
ha permesso nuove acquisizioni morfologiche e funzionali. La non invasività , l’innocuità , l’agevole ripetibilità dell’esame, unite ai costi relativamente contenuti consentono inoltre di monitorare nel tempo l’andamento
delle vasculopatie periferiche, nonché l’efficacia dei farmaci eventualmente impiegati.
Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo,
Angelo Leone, Piero Messina and Vincenzo Valenza
Morfological parametres
The experiment research was effectuated with a preference on the bottom lip
and the capillaries are visible like curves
in the skin papilla. A normal person has
parallel curvatures, formed in mainly upside U, a regular size, without a large dilatation; either way they occupy in a homogeneous density in their reserved space.
The morphology of the curves, the
number and size of the vases, the color,
Morfological parameters in a capillaroscopic image
the visualization of the vein system, of
micro-haemorrhage and the together of intravascular permit a diagnosis and to have a
major number of information on the condition of the microcirculation in functional arteriopathies such as diabetes and in a less specified way, in artery-sclerosis. The use of capillaroscopy has though a precise electivity in allowing a rapid diagnosis of dermato sclerosis
in the presence of a pre existing functional arteriopathy. To also keep in mind is that an
aneurismatic dystrophy in the extreme flexible part of the capillaries, mega capillaries,
stasis, hemorrhages aggregated or prodigious with the mega capillaries, to the point of an
ischemic skin disorder with a capillary deduction, there is a possibility of verifying a such
picture of the various states in a patient with a dermato-sclerosis.
The valuation of these morphologic parameters is complex and needs an attentive
analysis with expert training. It is for this exact reason and we are obligated, that to value,
according to certain objective parameters that we can distinguish between morphologies
and functional .
Between the morphologic parameters we can distinguish: visibility, morphology of the
ansae, an orientation of the ansae, the density of the capillary, the length of the curves in
the veins and their diameters.
Functional parameters
A fundamental innovative characteristic regarding capillaroscopy is the dynamic study
in vivo of the circulation. An abnormal biochemical solicitation, turns into a functional breakdown, to a complete penalization of the contracting elements of the area pre-capillary, in
an excessive or longer spasm of the post-capillary veins.
The insufficient microcirculation that it becomes into is characterized by a progressive
slowing down till stopping for a hyper aggregability of the elements in the blood.
The conventional capillary permits one to distinguish either a continuous flow or the
Oral Capillaroscopy: our experience
ribbon-shaped flow, a granulose flow being present a micro aggregation of erythrocytes,
an intermittent characterized by a visualization of plasmatic spaces visibly empty. The
Video Capillary using fluorescent techniques represent a superior realistic of the morphologic phase of the study of capillaroscopy; in fact, in a conventional stereo-microscope,
even though being able to receive quite important aspects of the capillary flow, we cannot
quantify and neither value the real grade of the capillary’s permeability. This last comment
could also be expressed, in suggestive terms, by the grade of an interstice edema. However, it can be distinguished that an ordinary or conventional capillaroscopy, useful in the
valuation of the morphological parameters, and that of a dynamic one, aimed towards a
functional research, which either can or not use fluorescent tracers. These permit an even
more precise valuation in the way that the microcirculation unit replies to pharmacological
or physical stimulations, being in a good state of health or in a bad one.
A capillaroscopic exam and a bioptic exam
In spite of the vast quantity of contributions in the world’s literature offered by conventional and dynamic, the conceptual scheme regarding the skin’s and mucosa’s micro
circulation have not changed all that much: clinging to a par cellular and limited vision of
the capillaries morph structure of certain skin and mucosa areas. The characteristic “hairpin” disposition started off a large study on morphological data, in an attempt to correlate
structural modifications of the ascending and descending ansae or of the apex of the
ansae, or of their number and of an optimum visualization, to pathologic situations of the
micro circulation of the body in general. Much of this data has contributed effectively to a
much better comprehension and a codification of certain queries diagnostic differentials,
with a particular attention to certain syndromes them being organic or functional, also
for micro angiopathies, such as diabetes, rheumatism, sclera dermas and hypertension.
The very rapid evolution of the microcirculation acknowledgment request a conceptual
correction during its course, by going back to morphological and structural acquisitions,
but with an even more dynamic impression than usual. With this to have a better knowledge of the most important aspects on in vivo circulation and not just because of the
bi-optical findings. To answer the question if or not more capillary-copy-cal images are
really un-replaceable for a correct diagnosis, the answer is negative; we will attenuate,
in fact, even them being more aggressive, that will give us a certified diagnosis regarding
the parameters and relation, as in an histological; the figure of an anatomy pathologist
would not be less in terms of diagnosis. Diagnosis damages to the microcirculation, with
the use of capillaroscopy orally, in a practical way, can be made apart from the fact of a
bi-optical exam; and it’s for this reason that capillaroscopy is generally classified as in vivo
and not in vitro in contradiction with the the researches on the bioptical statistics, as a
Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo,
Angelo Leone, Piero Messina and Vincenzo Valenza
complementary and supportive research with the clinical diagnosis. To also have a proper
characteristic which gives us secure information and immediately, on the dynamic of the
blood flow in the artery system regarding the whole of the organism and gives us data on
the organic and functions of the vase illness at the moment, because of presence or absence of aggregated erythrocytes, takes us to a significant and of a great merit diagnosis
regarding the capillaroscopy, to assume one integrant and un-replaceable fact which is
prognostic. The experiences reached with this technique outlines, not needing help, but
an operative proposal based on objective data ,difficult to refute, that the re-activity of the
micro-vascular tissue system has towards the endogens, especially with the pharmacologic, which is represented as the most reliable for the hoped for solution, of the prognostic
query of the micro vascular. The term re-activity its underlined that the whole complex of
circumstances them being the function-anatomy that invests as a whole these so called
micro vascular-tissues, whose energetic motor is represented by the modality functions
of the small arteries, from the arteries pre-capillary and from the muscular components of
the dispositive of the end arteries and of the blocking end arterial volume regulators and
the speed of the endo capillary.
The capillaries function structured passively, in the sense that accessibility flow, the
velocity of transfer of the elements represented in the blood, the actual emetic mass, essentially depend on the systolic potency of the small arteries and arterials .
Our experience
30 patients under examination ; medium age В±DS = 55,61В±2,55; range = 45-65). A
capillaroscopy examination was effectuated on the mucosa of the lower lip near the attaching part of the tongue to the pavement of the mouth; this area was chosen because of the
notable transparent mucosa and for a simpler application of the technique, the feeler, even
if small, would have had difficulty entering in different areas. The patients were not chosen
by chance; in fact these subjects had previously been through a bi-optical operation, in the
same lip area, for pathologies in that area: to study properly analogies and differences of
the microcircuit on two methods of diagnostic research, classic and un replaceable, the
histology exam and an innovative and complementary, the video capillaroscopy with an
optical tracker and with a polarized light. The methodic of video imaging biomicroscopy
was carried out thanks to the utilization of the capillaroscopy with acknowledgements:
in fact during the capillaroscopy the temperature of the room was kept between 21В° and
24В°, and the patient in resting conditions without smoking and or haven drunk alcohol for
at least an hour before the test. The choice of the objective was easily taken, because in
the examination of the micro circulation of the mucosa it is advisable to use contact objectives. The usage of these objectives could be compromised by the scarce consistency of
Oral Capillaroscopy: our experience
the examination surface, for which focusing is very precarious; for an altogether vision,
after having set the illumination intensity, that then was calibrated for all the other exams,
a 50x was then applied. With this enlargement the capillaries are distinguished thanks the
red colour of blood. To examine adequately the morphological aspects of a district micro
circulation, which permits the identification of the regional microvascular, a 200x was
then used which let us explore point to point all the morph structural characteristic of the
capillaroscopy field. Having a diameter of 7,5 they appear to the observer as small points
with a variable velocity in the single capillaries; appreciated though is the phenomenon
of aggregations erythrocytes and the capillaries or of the smaller veins in whose flow is
slowed down o momentarily still. The video capillaroscopy is constituted: a body central
which contains a source of light of cold halogen from a lamp of 100w which comes from
a device that automatically controls the luminous intensity; a tracker with optical fibers,
which consists of a flexible cable measuring 2 meters which presents an optical-video
terminal, which also contains a micro colored television camera and with a support for the
dwelling of different objectives. Furthermore for the digitizing of the histological images
we used LEICA IM10000 attached directly to the microscope, which gave us the possibility, once calibrated, the exact measurements, in fact, setting both the resolution and the
enlargement, the histological image presented itself inside a crowded network scales and
borders, within which it was permitted the correct recognition of the dimension of any cell,
including the micro circulation.
Valued capillaroscopic parameters: density of the capillaries (d-c); width of capillaries
(LC); diameter of ansae efferent; diameter of ansae afferent (DAA).
Valued histological parameter: capillaries density (d-c); width of capillaries (LC); presence hyper keratosis (IP).
All of this at the end is to correlate affinity between the two different methods of research
which altogether are different from each other, this demonstrates an major attend ability of
the capillaroscopy data, by an accurate means of images digitalized so differently.
Experimentation iconographic confrontation
Capillary examination
Using the media of 4 analyzed parameters we have :d-c 0 14 x mm2; LC media 0 11 В±
0.8 Вµ DAA Media 0 5 В± 2,90 Вµ; DAE media =6 В± 2,10 Вµ
Histological examination
The results of our observation is evidence that there are perfect analogies by the observation of the micro circulation in vivo and in vitro, with a small range error which turns out
Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo,
Angelo Leone, Piero Messina and Vincenzo Valenza
to be around a В±0.3 Вµ and also regarding the capillary density we can see a variation of В±
2 capillaries per mm2. There have also been found general values observed in the capillaroscopy studies of these years; or where the cap. density (d-c), determines the number
of ansae present per surface unit shines between 12 and 16 capillary per mm2, and also
the diameter of the afferent curvature is always smaller than the efferent one for a small
micron: or rather is the first one really 4-8 Вµ, the second around 7-10 Вµ it has also been
confirmed that the width of the capillary is around 9-14 Вµ.
All the capillaroscopy techniques and in particular the Video Capillaroscopy using an
Optic Feeler and with Polarized Light offer scientific and practical starting points on a clinical plan for a global valuation of the damages to the microcirculation with a widespread
ubiquity; it’s principal virtue consists in the possibility of valuing the anatomy-topographic
differences to the entity of the microcirculation�s damage and it continues today and will so
in the future to value this damage as it is that what represents the most original innovation:
the possibility if monitoring the damage in each accessible body part, following, in subsequent phases, the evolution being positive or negative and associating with capillaroscopic
a clinical and symptomatologic frame.
Oral Capillaroscopy: our experience
[1] Hettema ME, Zhang D, Stienstra Y, Oomen PN, Smit AJ, Kallenberg CG, Bootsma H. Decreased capillary permeability and capillary density in patients with systemic sclerosis using large-window sodium
fluorescein videodensitometry of the ankle. Rheumatology, 2008; 47(9): 1409-12.
[2] Cortes S, Clemente-Coelho P. Nailfold capillaroscopy in the evaluation of Raynaud’s phenomenon and
undifferentiated connective tissue disease. Acta Reumatol Port 2008 Apr-Jun; 33(2): 203-9.
[3] Dynnik OB, MostovoI�SE, BerezovskiI�VA. Biophysical methods in assessment of the skin microcirculation
system. Fiziol Zh 2008; 54(2): 100-8.
[4] Ingegnoli F, Boracchi P, Gualtierotti R, Lubatti C, Meani L, Zahalkova L, Zeni S, Fantini F. Prognostic
model based on nailfold capillaroscopy for identifying Raynaud’s phenomenon patients at high risk for
the development of a scleroderma spectrum disorder. Arthritis Rheum 2008 Jul; 58(7): 2174-82.
[5] Aschwanden M, Daikeler T, Jaeger KA, Thalhammer C, Gratwohl A, Matucci-Cerinic M, Tyndall A. Rapid
improvement of nailfold capillaroscopy after intense immunosuppression for systemic sclerosis and
mixed connective tissue disease. Ann Rheum Dis 2008 Jul; 67(7): 1057-9.
[6] Allegra C, Antignani PL, Schachter I, Koverech A, Messano M, Virmani A. Propionyl-L-carnitine in lerichefontaine stage II peripheral arterial obstructive disease. Ann Vasc Surg 2008 Jul-Aug; 22(4): 552-8.
[7] Mosca M, Tani C, Bombardieri S. A case of undifferentiated connective tissue disease: is it a distinct
clinical entity? Nat Clin Pract Rheumatol 2008 Jun; 4(6): 328-32.
[8] Scardina GA, Messina P. Modifications of interdental papilla microcirculation: a possible cause of periodontal disease in Hashimoto’s thyroiditis? Ann Anat 2008; 190(3): 258-63.
[9] Herrick A. Diagnosis and management of scleroderma peripheral vascular disease. Rheum Dis Clin
North Am 2008 Feb;34(1): 89-114.
[10] Micali G, Nardone B, Scuderi A, Lacarrubba F. Videodermatoscopy enhances the diagnostic capability of
palmar and/or plantar psoriasis. Am J Clin Dermatol 2008; 9(2): 119-22.
[11] Kim SH, Kim HO, Jeong YG, Lee SY, Yoo WH, Choi TH, Lee SI. The diagnostic accuracy of power Doppler
ultrasonography for differentiating secondary from primary Raynaud’s phenomenon in undifferentiated
connective tissue disease. Clin Rheumatol 2008 Jun; 27(6): 783-6.
[12] Christen-Zaech S, Seshadri R, Sundberg J, Paller AS, Pachman LM. Persistent association of nailfold
capillaroscopy changes and skin involvement over thirty-six months with duration of untreated disease
in patients with juvenile dermatomyositis. Arthritis Rheum 2008 Feb; 58(2): 571-6.
[13] Rouster-Stevens KA, Gursahaney A, Ngai KL, Daru JA, Pachman LM. Pharmacokinetic study of oral
prednisolone compared with intravenous methylprednisolone in patients with juvenile dermatomyositis. Arthritis Rheum 2008 Feb 15; 59(2): 222-6.
[14] Settergren M, Pernow J, Brismar K, Jörneskog G, Kalani M. Endothelin-A receptor blockade increases
nutritive skin capillary circulation in patients with type 2 diabetes and microangiopathy. J Vasc Res
2008; 45(4): 295-302.
[15] Szabo N, Csiki Z, Szanto A, Danko K, Szodoray P, Zeher M. Functional and morphological evaluation
of hand microcirculation with nailfold capillaroscopy and laser Doppler imaging in [15]Raynaud’s and
Sjögren’s syndrome and poly/dermatomyositis. Scand J Rheumatol 2008 Jan-Feb; 37(1): 23-9.
[16] Lu W, Wang D, Liu L, Xiong J, He Q. Nail fold capillary observation in replanted severed fingers. Microsurgery 2008; 28(1): 13-6.
[17] Dondorp AM, Ince C, Charunwatthana P, Hanson J, van Kuijen A, Faiz MA, Rahman MR, Hasan M, Bin
Yunus E, Ghose A, Ruangveerayut R, Limmathurotsakul D, Mathura K, White NJ, Day NP. Direct in vivo
assessment of microcirculatory dysfunction in severe falciparum malaria. J Infect Dis 2008 Jan 1;
Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo,
Angelo Leone, Piero Messina and Vincenzo Valenza
197(1): 79-84.
[18] Scardina G.A., Pisano T., Carini F., Valenza V., Messina P. Burning Mouth sindrome: evaluation of “in
vivo” microcirculation. Journal of American Dentistry 2008,139, 940-946.
[19] Aguilar Peralta GR, ArГ©valo Gardoqui J, Llamas MacГ­as FJ, Navarro Ceja VH, Mendoza Cisneros SA,
MartГ­nez MacГ­as CG. Clinical and capillaroscopic evaluation in the treatment of chronic venous insufficiency with Ruscus aculeatus, hesperidin methylchalcone and ascorbic acid in venous insufficiency
treatment of ambulatory patients. Int Angiol 2007 Dec; 26(4): 378-84.
[20] Hudson M, Taillefer S, Steele R, Dunne J, Johnson SR, Jones N, Mathieu JP, Baron M. Improving the
sensitivity of the American College of Rheumatology classification criteria for systemic sclerosis. Clin
Exp Rheumatol 2007 Sep-Oct; 25(5): 754-7.
[21] Scardina GA., Cacioppo A, Carini F, Ruggeri A, Valenza V, Messina P. Periodontal morphological microcirculation in oral lichen planus. It J Anat Embryol 2007; 12(4): 281-292.
[22] Scardina GA, Carini F, P. Messina. Capillaroscopic and histological examination of the human labial
mucosa: a new non invasive methodology compared with the histological exam. Gazz Med Ital-Arch Sci
2005; 164: 113-8.
[23] Scardina GA, Messina P, Carini F, Leone A. Capsaicin stimulation and neurogenic experimentaly induced
infiammation of the human labial mucosa. Dent Med Probl 2005; 42: 31-6.
[24] Scardina GA, Carini F, Messina P. Vasodilatation of human gengiva and neurogenic inflammation. Clinical Hemorheology and Microcirculation. 2005; 32(4): 279-85.
[25] Scardina GA, Carini F, Leone A, Valenza V, FucГ G, Messina P. Panoramic periodontal capillaroscopy: a
new diagnostic method. Dent Med Probl 2004; 3: 449-453.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Abdominal aortic aneurysm]
Guido Bajardi1, Felice Pecoraro2, Annamaria Mauro3, Umberto Marcello Bracale1
Cattedra di Chirurgia vascolare - 2Scuola di specializzazione in Chirurgia vascolare - 3Di.Me.S. Sezione di
Istologia ed Embriologia, UniversitГ degli Studi di Palermo (IT)
Parole chiave: Aneurisma dell’Aorta Addominale, AAA, ACE, IL, MMPs, PAF, TIMP-2, Aterosclerosi, EVAR
Key words: Abdominal Aortic Aneurysm, ACE, IL, MMPs, PAF, TIMP-2, Atherosclerosis, EVAR
Riassunto. Nelle ultime due decadi si è registrato un incremento dell’incidenza dell’aneurisma dell’aorta
addominale ( AAA) a causa di una maggiore aspettativa di vita nella popolazione, il fumo di sigaretta ed
ai programmi di screening. Nei Paesi sviluppati l’AAA è responsabile dell’1.3% di tutte le morti negli uomini di età compresa tra 65 ed 85 anni. Sebbene l’AAA venga descritto come una massa pulsante nella
regione dell’epicondrio od epigastrio, di solito resta misconosciuta fino alla rottura. L’AAA è comunemente
definito quale incremento di 1·5 volte del diametro rispetto al normale. L’AAA è stato considerato come
manifestazione tardiva del processo aterosclerotico. Studi recenti mostrano che, almeno parzialmente, il
processo dilatativo differisce da quello ostruttivo: l’AAA è caratterizzato da un processo degenerativo trans
murale localizzato in un segmento arterioso specifico. I principali fattori di rischio per l’AAA sono il fumo, il
sesso maschile, l’ipertensione, la broncopatia cronica ostruttiva, l’iperlipemia e la lostoria familiare di malattia. Anche l’infiammazione è stata coinvolta come uno dei principali protagonisti nel processo aneurismatico.
L’aorta normale è un condotto fibro-elastico compliante, resistente e duraturo. Le principali caratteristiche
di un’aorta normale sono legate all’elastina presente nella media ed al collagene presente nell’avventizia.
Nello sviluppo ed evoluzione dell’AAA sia le fibre di elastina che di collagene vanno incontro ad un rimaneggiamento indirizzato verso la proteolisi. La riduzione delle cellule muscolari lisce è coinvolta nella formazione
dell’AAA per quanto riguarda il rimodellamento vascolare. Anche la presenza costante del trombo endoluminale nell’AAA viene associato a condizioni di ipossia locale ed incremento dello stress parietale . soalmnete
il 30-40% degli AA viene diagnosticato all’esame obiettivo. L’eco-color-doppler è la metodica diagnostica più
semplice ed economica per la valutazione della presenza dell’AAA ed è usata convenzionalmente nei programmi di screening. La tomografia computerizzata è l’esame con la migliore accuratezza e viene utilizzata
nel periodo preoperatorio od in soggetti sintomatici. I programmi di screening riducono significativamente la
mortalità per AAA ( 43% negli uomini con età tra 65 e 74 anni). Il tasso di crescita per AAA di piccole dimensioni (≤ 5.5 cm) è di 3.6-3.2 mm per anno ed incrementa al crescere dell’aneurisma.
Solitamente gli AAA sono asintomatici fino alla rottura. Questa ГЁ caratterizzata da un improvviso dolore,
shock, e presenza di massa pulsante. Il grado di shock dipende dalla localizzazione della rottura sulla parete
aorica: una rottura antero-laterale è associata ad un’emorragia massiva in peritoneo. Differentemente una
rottura posteriore puГІ essere tamponata nello spazio retro peritoneale e permettere una riparazione urgente
dell’AAA. Una rottura tamponata è sempre seguita da una rottura maggiore nel giro di poche ore.
La decisione di trattare in elezione un paziente dipende dal rischio di rottura rispetto al rischio operatorio
nel contesto dell’aspettativa di vita del paziente. Il diametro massimo di 5.5 cm è il limite per l’indicazione
al trattamento; questo ГЁ inferiore di 5 mm nelle donne. Altri parametri da considerare nella riparazione sono
la crescita rapida dell’AAA (≥1 cm/anno o ≥ 0.5 cm/6 mesi), la presenza di sintomi e la conformazione ana-
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
tomica (tortuositГ ed asimmetria).
Il tasso di mortalità per la riparazione elettiva dell’AAA varia da 1.1% ad 8.0%. le ultime decadi hanno visto
una riduzione della mortalità legata alla riparazione elettiva dell’AAA; tale tendenza però non si è registrata
in quelli rotti. Il tasso di mortalitГ per AAA rotti varia dal 30% al 70% per i pazienti che giungono vivi in
ospedale. Il trattamento medico viene usualmente proposto ai pazienti in regime followup. Sono in corso
studi per la valutazione dell’efficacia di doxicilina, roxithromicina e delle statine. Il trattamento chirurgico
tradizionale consiste in una aneurismectomia ed innesto di una protesi in Dacron. L’intervento chirurgico può
essere complicato da problemi cardiaci, polmonari, renali ed emorragici. Il trattamento endovascolare (EVAR)
ГЁ una metodica piГ№ recente che prevede il posizionamento di una endoprotesi dalle arterie feomrali evitando
l’apertura dell’addome. Questo trattamento seppur meno invasivo può essere complicato da endoleaks,
endotension migrazione della protesi ed infezione delle ferite inguinali. Diversi trials randomizzati sono stati
realizzati con l’intenzione di verificare quale sia il trattamento migliore. Attualmente questi studi hanno mostrato una mortalità perioperatoria più bassa dopo EVAR con risultati a lungo termina ancora da valutare.
Alcuni aspetti come la prevenzione primaria con programmi di screening nella popolazione a rischio e
l’identificazione di biomarkers predittivi di rottura dovrebbero essere investigati. Anche le indicazione al trattamento open ed EVAR dovrebbero essere valutate specialmente in quei pazienti con AAA rotto.
Abstract. Last two decades have seen an increased incidence of abdominal aortic aneurysm (AAA) because
of higher life-expectancy, smoking and screening program. In developing countries the AAA is responsible of
1.3% of all death in men between 65 and 85 years. Even if AAA has been defined as a pulsating mass in hypochondriac or epigastric region it usually remain asymptomatic until rupture. For AAA definition it has been
proposed as a criterion that the infrarenal diameter should be 1В·5 times the expected normal diameter.
AAA have been usually considered as a late manifestation of atherosclerotic disease. Recent studies show
that aneurysm process differ, at least in part, from arterial obstructive disease: AAA is characterized by a
transmural degenerative process usually localized in a specific arterial segment. Tobacco smoking, masculine sex, hypertension, chronic obstructive bronchopneumopathy, hyperlipemiaand family history are the
most important AAA risk factor. Also inflammation have been involved in AAA process as one of the most
important aspect.
Normal aorta is a compliant, resistant and durable fibro-elastic conduit. The most important characteristics
of normal aorta are linked to elastin in the tunica media and collagen in the tunica adventitia. In AAA development and evolution both elastin and collagen fibers turnover are pendent to proteolysis. The smooth muscle
cells reduction play a relevant role in vessel remodeling and consequent AAA formation. Also the constant
presence of thrombus into the AAA has been linked to local hypoxia and increased wall stress.
Only 30-40% of all AAA is diagnosed on physical examination. Echo-color-doppler is the simplest and cheapest diagnostic method to evaluate the presence of AAA and is conventionally used in screening programs.
Computed tomography is the exam with the best accuracy and it is used in the preoperative period or in
symptomatic patients. Screening programs significantly reduce AAA related death (43% in men between 65
and 74 years). Growth rate for small AAA (≤ 5.5 cm) is 3.6-3.2 mm/years and increase with AAA dimension.
Usually AAA are asymptomatic until rupture. AAA rupture is characterized by sudden pain, shock and pulsating mass. Shock degree is dependent on the aortic wall rupture portion: an antero-lateral rupture is associated to a massive hemorrhage in peritoneal space. Differently a posterior rupture could be tamponade by the
retroperitoneal space and give the opportunity for an urgent treatment of AAA. A tamponade rupture is always
followed by a major rupture in hours.
The decision to electively operate a patient depend on the risk rupture versus the risk related to operation
in the context of overall life expectancy. Maximum diameter of 5.5 cm remains the gold standard as cut-off
Gli aneurismi dell’aorta addominale
in the operation decision; this limit is 5 mm inferior in women. Other parameters involved in the decision to
operate are rapid growth (≥1 cm/year or ≥ 0.5 cm/6 months) , symptoms presence and anatomic details
(tortuosity and asymmetry).
Mortality rate for elective AAA repair ranges between 1.1% and 8.0%. AAA management in the last decade
have decreased elective aneurysm-related deaths differently this trend has not been registered in ruptured
AAA. Mortality rate reported for ruptured AAA ranges between 30% and 70% for those who reach hospital
alive. Medical treatment is usually administered for those patients who have AAA in the surveillance range.
Further studies are required to evaluate the real action and activity of doxycyline, roxithromycin and statin therapy. Surgical treatment consists of an open surgery approach or an endovascular aneurysm repair
(EVAR). The open surgical treatment contemplates an aneuryectomy and Dacron interposition graft. The main
complications of the open surgery are cardiac, pulmonary, renal and hemorrhagic. A totally laparoscopic
surgical repair is a feasible option. EVAR treatment is a relatively more recent option that consists in the
endovascular position of the prosthesis from femoral arteries avoiding abdominal incision and general anesthesia. Most frequent complications after EVAR are endoleaks, endotension, device migration and wound
infection. To evaluate the superiority of the two surgical treatment many randomized control trials have
been proposed. Actually these studies show no survival improvement for AAA repair less than 5.5 cm; lower
perioperative death after EVAR with unknown long-term results; and no better results in patient considered
unfit for open surgery.
Some aspects like primary prevention with screening programs in population at risk and the identification
of biomarkers predictive of rupture should be investigated. Also better indication to open or EVAR treatment
should evaluated especially in those patients with ruptured AAA.
L’Aneurisma dell’Aorta Addominale (AAA) rappresenta una malattia cronica degenerativa con severe implicazioni per la vita. Si ritiene che gli AAA possano essere causati da una
forma di danno della parete aortica a cui si sovrappone la presenza di fattori degenerativi
legati all’età , l’emodinamica, fattori di rischio sistemici e genetici anche se la natura o la
sequenza precisa di questi eventi non ГЁ nota.
Nei Paesi sviluppati l’AAA è responsabile dell’1.3% della mortalità tra gli uomini di età compresa tra 65 ed 85 anni. Nelle ultime due decadi si è assistito ad un incremento dell’incidenza dell’AAA causato dalla maggiore aspettativa di vita nella popolazione, l’incremento del numero dei soggetti fumatori ed anche per l’introduzione di programmi di screening
ed il miglioramento delle apparecchiature diagnostiche1. L’AAA rappresenta la tredicesima
causa di morte negli Stati Uniti2 ed ГЁ piГ№ frequente nei soggetti con storia di fumo [3].
Sebbene in alcuni casi l’AAA possa presentarsi con una sintomatologia vaga,caratterizzata
da dolore dorsale od addominale, la maggior parte degli AAA restano asintomatici fino alla
rottura3 ed è generalmente riferito come una massa pulsante localizzata al’ipocondrio sinistro od all’epigastrio [4].
Nel 1991, la Society for Vascular Surgery e l’International Society for Cardiovascular
Surgery Ad Hoc Committee on Standards in Reporting hanno proposto come criterio di
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
definizione dell’AAA un’aorta sottorenale di almeno 1.5 volte rispetto al diametro normale5.
L’AAA può presentarsi in una variante infiammatoria caratterizzata dalla presenza di una
spessa parete, una marcata fibrosi perianeurismatica e retroperitoneale e dense adesioni
che coinvolgono le strutture adiacenti [6].
Strategia di ricerca e criteri di selezione.
La ricerca ГЁ stata condotta su MEDLINE per la letteratura recente con le parole chiave
�Abdominal’, �Aortic’, �Aneurysm’, �Trial’, �Rupture’, �EVAR’ and �open repair’. Queste sono
state utilizzate singolarmente ed in associazione. Sono state selezionate principalmente
pubblicazioni degli ultimi 5 anni e quelle maggiormente referenziate, anche se antecedenti
Ogni anno circa 300.000 persone negli Stati Uniti improvvisamente muoiono senza una
diagnosi certa, di queste il 4-5% per una rottura misconosciuta di un AAA [7]. Nel Regno
Unito la rottura dell’AAA causa circa 8.000 morti ogni anno [8]. Il tasso di mortalità globale
per i pazienti con AAA rotto è compresa tra il 65% e l’ 85% [3,8], inoltre circa la metà delle
morti attribuite alla rottura di AAA avviene prima che il paziente raggiunga la sala operatoria [9,10]. Per prevenire la rottura negli Stati Uniti ogni anno vengono eseguiti circa 40.000
trattamenti in elezione degli AAA asintomatici. Comunque queste procedure causano circa
1500 morti [11].
L’AAA risulta essere più frequente negli uomini che nelle donne con un rapporto maschi/femmine che varia da 3/1 ad 8/1 [12-16]. La settima decade di vita è la più colpita
presentandosi raramente prima dei 50 anni [15-19]. Ciononostante, visto che il fumo rappresenta uno dei principali fattori di rischio per l’AAA e visto l’incremento sia delle donne
fumatrici che dei giovani fumatori, le caratteristiche epidemiologiche legate all’età ed al
sesso dell’AAA probabilmente cambieranno in futuro [14,15].
Etiologia e fattori di rischio
Anche se la reale causa della formazione dell’AAA rimane sconosciuta, questo è stato
comunemente considerato una manifestazione terminale della patologia aterosclerotica
[20]. Recenti studi clinici e di laboratorio indicano che il meccanismo patogenetico alla
base degli AAA differisce, almeno in parte, da quello responsabile della patologia ostruttiva
[21,22]. Infatti l’anatomia patologica degli AAA è caratterizzata da un processo degenerativo transmurale generalmente localizzato in uno specifico segmento arterioso, mentre
la patologia ostruttiva è una malattia diffusa dell’intima che coinvolge i vasi lungo tutto
l’albero arterioso. Defawe et al. sono giunti alle stesse considerazioni analizzando i livelli
di due inibitori fisiologici delle proteasi (il Tissue inhibitor of metalloproteinase [TIMP-2] ed
Gli aneurismi dell’aorta addominale
il Plasminogen Activator Inhibitor-1[PAI-1]) nel contesto della parete vasale responsabili di
una diversa evoluzione verso una patologia dilatativa od ostruttiva [23].
Solo una limitata percentuale di AAA risulta essere in relazione a cause specifiche come
traumi, infezioni (brucellosi, salmonellosi, tubercolosi), malattie infiammatorie (Behçet eTakayasu) [24,25] e malattie del tessuto connettivo (Sindrome di Marfan, Ehlers-Danlos tipo
IV) [26].
Dal momento che non tutti i pazienti con aterosclerosi sviluppano un AAA, ulteriori
fattori sono coinvolti nello sviluppo dell’aneurisma. Tra questi il fumo è responsabile dello
sviluppo e della progressione aneurismatica [16,27-29] determinando nei soggetti fumatori una prevalenza di circa 4 volte superiore 3. Altri fattori di rischio coinvolti sono il sesso
maschile, l’età , l’ipertensione, la bronco pneumopatia cronica ostruttiva, l’iperlipemia e
la storia familiare della malattia [14-16]. La componente genetica , secondo Shibamura
H. et al, potrebbe contribuire al 70% o più nella genesi dell’AAA [31]: i loci coinvolti nello
sviluppo sono stati individuati sui cromosomi 19q13 e 4q31 [32,33], che includono i geni
della risposta immune e dell’apoptosi [32].
Anche la tendenza familiare dell’AAA non è dovuta esclusivamente ad un’unica causa:
il background genetico gioca un ruolo principale in associazione con fattori esterni [34].
L’associazione tra antigeni dell’ HLA di I [35] e di II [36] Classe è stata rilevata sia negli
AAA di tipo infiammatorio che non infiammatorio [37], dando supporto all’ipotesi di una
patogenesi comune immuno-mediata modulata da fattori HLA -indipendenti.
Oltre al PAI-1 ed al TIMP-2 sono numerosi i geni e le molecole chiamate in causa nel
processo di formazione dell’AAA: l’Angiotensin Converting Enzyme (ACE); l’Interleukina(IL)
10; le metallo proteinasi (MMPs 2,7,9,12); la methylenetetrahydrofolate reductase (MTHFR) ed il platelet activating factor (PAF) [38-45].
L’aneurisma dell’aorta addominale (AAA) è legato, tra gli altri fattori identificati, ad una
eccessiva degradazione della matrice extracellulare (ECM) che rende la parete del vaso
meno resistente alla dilatazione.
Si ritiene che questa de-regolazione della degradazione della ECM sia dovuta all’aumento dell’espressione e dell’attività delle metalloproteinasi di matrice (MMPs), un gruppo
di enzimi collettivamente capaci di degradare praticamente qualsiasi componente della
ECM [133].
Vari studi legano lo sviluppo dell’aneurisma dell’aorta addominale all’overespressione
di diverse metalloproteinasi di matrice tra cui ricordiamo le MMP-1, -2, -3, -7, -8, -9, -12
e -13 [128,141,142].
Nella patogenesi della AAA si pensa sia coinvolta soprattutto la degradazione del collagene e delle fibre elastiche, per questo motivo, tra le varie MMPs, i primi candidati ad
essere indagati come possibili responsabili della patologia sono state la MMP-9 e la MMP103
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
2, enzimi degradanti collagene ed elastina. GiГ da molti anni negli aneurismi aortici addominali ГЁ nota la presenza di livelli aumentati di MMP-9 [138] ed MMP-2 [129]. Altri studi
riportano che la MMP-2 ГЁ la principale MMP espressa nei piccoli aneurismi mentre la
MMP-9 ГЁ la principale espressa negli aneurismi di grandi dimensioni [131].
A sostegno di questi dati, esperimenti di delezione genica hanno mostrato che topi
knock-out, privati del gene della MMP-9 o della MMP-2, non sviluppavano aneurismi
dell’aorta addominale neanche dopo induzione con cloruro di calcio, al contrario di quanto
avveniva in topi normali [135]. Inoltre la doxyciclina, un inibitore delle MMPs ad ampio spettro ha dimostrato in molti studi su modelli animali di ridurre l’incidenza di AAA [136,127].
L’overespressione della MMP-9 in particolare è stata spesso associata con una maggiore probabilità di rottura dell’aneurisma e con lo sviluppo di aneurismi di maggiori dimensioni [140,144].
Г€ stata anche studiata la possibile associazione tra polimorfismi dei geni per le diverse
MMPs e sviluppo dell’AAA, ed in alcuni casi si è visto che determinate varianti geniche
aumentavano la predisposizione allo sviluppo della patologia [132,143].
Per questi motivi le MMPs possono essere dei buoni target per lo sviluppo di terapie per
la prevenzione e la cura dell’AAA e alcuni tentativi e trial clinici sono già in corso [134].
Gli inibitori tissutali delle metalloproteinasi di matrice (TIMPs) regolano l’attività delle
MMPs e possono influenzare l’equilibrio tra deposizione e degradazione della matrice extracellulare, ne consegue che anche l’espressione di queste molecole è stata ampiamente investigata come potenziale target di terapie contro l’aneurisma aortico addominale
Anche il processo infiammatorio è oggetto di studio nella genesi dell’AAA per la presenza di cellule infiammatorie mononucleate nel tessuto aneurismatico che potrebbero
intervenire nell’apoptosi delle cellule muscolari lisce (SMC) [46-50] e nella produzione di
citochine pro infiammatorie (tumour necrosis factor-a [TNF-a], interferon-g [IFN-g], IL- 5
ed IL-6) [39].
Recenti studi avanzano l’ipotesi che la formazione dell’AAA sia la conseguenza di una
alterata e persistente rispostaauto- immune [38,51] all’interno della parete aortica [50,52
53,54]. CiГІ ГЁ supportato dal riscontro istologico di follicoli simil-linfoidi organizzati nel
contesto della parete aortica [39] la cui presenza fa ipotizzare anche un’etiologia infettiva
[55] oltre che autoimmune (verso peptidi derivanti dalla degradazione dell’elastina o lipoproteine ossidate a bassa densità ) [56].
L’aorta normale è un condotto fibroelastico i cui requisiti funzionali nei confronti della
deformazione al continuo stress emodinamico sono la compliance (elasticitГ ), la resistenza
Gli aneurismi dell’aorta addominale
e la durata. Queste particolari necessità vengono soddisfatte dalle proprietà biomeccaniche dell’elastina e del collagene, proteine fibrillari di matrice che si trovano nella tonaca
media ed avventizia dell’aorta. Uno dei principali e precoci riscontri istologici del tessuto
aneurismatico è la frammentazione delle fibre elastiche in associazione con la ridotta concentrazione dell’elastina durante la crescita aneurismatica fino al momento della rottura
[57,58]. Il tessuto avventiziale, in cui il collagene ГЁ predominante, ГЁ responsabile della
resistenza aortica in assenza dell’elastina contenuta nella media:la degradazione del collagene potrebbe rappresentare la causa finale di rottura [58]. Nell’AAA è stato descritto
un incremento del turnover del collagene [59]: uno sbilanciamento tra la degradazione del
collagene e la sua sintesi potrebbe creare la condizione catabolica che porta alla rottura
[60]. Le cellule muscolari lisce della media ed i fibroblasti avventiziali producono le proteasi responsabili dell’alterazione dell’elastina e del collagene [61].
La parete aortica aneurismatica ГЁ caratterizzata da uno spesso strato di infiltrato di
monociti, linfociti B, Plasmacellule e Linfociti T, indice di un processo infiammatorio cronico
concomitante [62-65]. Pertanto l’immunità potrebbe giocare un ruolo anche nello sviluppo dell’AAA [66,67], con la produzione di enzimi proteolitici dell’elastina e del collagene
[68-71] e conseguente sbilanciamento verso la proteolisi [23,72]. Un evento chiave nello
sviluppo dell’AAA, è la riduzione delle densità delle SMC della media elastica [73] che partecipano nel rimodellamento della parete vascolare [74].
Lo sviluppo dell’AAA è anche associato alla presenza del trombo murale nella maggioranza dei pazienti. Diversamente dalla patologia ostruttiva, il flusso ematico è mantenuto
per il continuo rimodellamento delle componenti del trombo.. Sebbene il trombo possa
ridurre significativamente lo stress della parete aneurismatica, il suo incremento continuo
di spessore potrebbe condurre ad una condizione di ipossia locale nello strato interno della media e conseguente induzione della neovascolarizzazione ed infiammazione mediale
[75,76]. Anche la relazione che il trombo murale possa avere con la quota figurata del
sangue e con i fattori della coagulazione dovrebbe essere investigato.
Solamente il 30-40% degli AAA viene riconosciuta all’esame obiettivo a seconda della
dimensione [77] e di eventuale obesitГ addominale [78]. La sensibilitГ della palpazione
bimanuale dell’area sopraombelicale incrementa con il diametro della lesione: 61% per
AAA di diametro tra 3.0 e 3.9 cm, 69% per quelli tra 4.0 e 4.9, e del 76-82% per quelli
superiori a 5 cm [78].
La radiografia standard addominale puГІ incidentalmente essere diagnostica se sono
presenti delle calcificazioni nella parete aortica che permettono la visualizzazione della
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
L’EcoColorDoppler (ECD) è la metodica diagnostica più semplice ed economica che
può misurare l’aorta in tutte le sue dimensioni con un’accuratezza di 3 mm [79]. L’ECD è
ampiamente utilizzato non solo per la diagnosi iniziale ed il follow-up, ma anche per studi
o programmi di screening.
Quando il diametro dell’aneurisma è tale da richiederel’intervento chirurgico, la Tomografia Computerizzata (TC) rappresenta lì indagine più idonea per determinare la tipologia
di trattamento ed eventuali anomalie che possono essere di notevole impedimento durante
l’accesso chirurgico al “colletto” (vena cava sinistra, vena renale sinistra posteriore) [80].
L’esame TC può anche visualizzare la presenza di sangue all’interno del trombo come
segno predittivo di rottura imminente [81]. In caso di AAA di tipo infiammatorio, la TC
evidenzia un importante spessore della parete e la presenza di fibrosi para-aortica [6]. Lo
stravaso del mezzo di contrasto è diagnostico per rottura dell’AAA. Attraverso la diagnostica tridimensionale, la TC spirale e l’angio-TC possono fornire ulteriori dettagli anatomici,
fondamentali in caso di programmazioni di procedure endovascolari [82].
La Risonanza Magnetica (RM), combinata con l’angio-RM, è un’ulteriore metodica da
considerare soprattutto per l’utilizzo di mezzo di contrasto non nefrotossico, a differenza
dell’angiografia tradizionale, che può portare ad insufficienza renale ed embolizzazione
distale. L’utilizzo dell’angiografia è attualmente indicato in caso di posizionamento di dispositivi endovasclolari
Lo screening su uomini con età tra 65 e 74 anni riduce significativamente la mortalità correlata all’AAA [2]. Tradizionalmente, il diametro è stato utilizzato quale principale segno
nella progressione dell’AAA. L’esame ECD addominale è stato dimostrato quale metodica
diagnostica accurata e riproducibile per studi di screening per patologia aneurismatica
[83]. La prevalenza di AAA asintomatici negli uomini e donne con etГ superiore a 60 anni ГЁ
compresa tra il 4% e l’8% e tra lo 0.5% e l’1.5%, rispettivamente [84].
L’US Preventive Services Task Force ha concluso che l’ECD può ridurre la mortalità del
43% negli uomini con etГ compresa tra 65 e 75 anni [2]. Nel piГ№ grande studio di screening su popolazione statunitense il 90% degli AAA identificati era di dimensione inferiore a
5.5 cm di diametro [85]. La crescita media per piccoli AAA (≤5.5 cm di diametro) è di circa
2.6-3.2mm/anno, e tale tendenza incrementa al crescere del diametro [86,87]. L’espansione dell’AAA in 1743 pazienti con follow-up a 7 anni è stata significativamente associata al
diametro di partenza. Differentemente non ГЁ stata riscontrata associazione per etГ o sesso.
Il fumo di sigaretta ГЁ stato associato con un incremento addizionale di 0.4 mm per anno.
Gli aneurismi dell’aorta addominale
Presentazione Clinica
Gli aneurismi non rotti sono generalmente asintomatici nella maggioranza dei pazienti. Sono essenzialmente diagnosticati incidentalmente durante un attento esame clinico,
specialmente in pazienti visitati per patologia coronarica, periferica o cerebrovascolare
nonchГ© durante screening su popolazione [10].
La rottura dell’AAA è fortemente correlata alla triade dolore improvviso (al centro
dell’addome o al fianco con possibilità di irradiazione allo scroto), shock e presenza di
massa addominale pulsante. L’appendicite e la colecistite acuta possono determinare una
sintomatologia simile alla rottura dell’AAA [88]. Il grado di shock varia a seconda della localizzazione, della dimensione e del tempo intercorso dalla rottura. La rottura della parete
anterolaterale nella cavitГ peritoneale ГЁ usualmente un evento drammatico associato a
morte repentina. La maggioranza dei pazienti che giungono in ospedale vivi presentano,
invece, una rottura della parete posterolaterale nello spazio retro peritoneale; una rottura
tamponata determina una perdita ematica limitata. Tale evento è sistematicamente seguito da una rottura maggiore entro poche ore. L’evoluzione bifasica della rottura dell’AAA
enfatizza l’importanza del periodo che intercorre tra le due rotture, che dovrebbe essere
utilizzato per il trasferimento e l’intervento urgente . Solo raramente l’AAA si rompe spontaneamente nel duodeno [89]: la fistola aorto-duodenale si realizza di solito dopo un precedente intervento di riparazione per AAA [88,90]. Anche la rottura dell’AAA in vena cava è
un evento che puГІ realizzarsi con quadro di edema apparente agli arti inferiori [91].
Indicazioni per il trattamento
L’indicazione al trattamento viene posta in considerazione delle dimensioni dell’AAA,
del rischio peri-operatorio e dell’aspettativa di vita del paziente. [92,93] In una review di
32 articoli di Norman et al. ha rilevato che la sopravvivenza a 5 anni dei pazienti sottoposti
ad intervento per AAA rispetto alla popolazione generale ГЁ del 70% [94].
La chiave ГЁ naturalmente come incorporare le comorbiditГ del paziente rispetto
all’aspettativa di vita [15,87]. Il diametro massimo dell’AAA resta la miglior metodica per la
stima del rischio di rottura: l’United Kingdom Small Aneurysm Trial (UK SAT) e l’Aneurysm
Detection and Management study (ADAM) suggeriscono quale limite per l’intervento di
riparazione aneurismatica un diametro di 5.5 cm negli uomini [87,96] e 5 mm meno nelle
donne [97]. Il rischio di rottura aneurismatica ГЁ significativo quando il diametro trasverso ГЁ superiore a 5.5 cm ed incrementa esponenzialmente al crescere della dimensione
Anche se il diametro aneurismatico ГЁ il principale indice per la rottura, in taluni pazienti
puГІ fallire: alcuni AAA vanno incontro a rottura anche a dimensioni inferiori [92]. Infatti ad
un anno il rischio ГЁ del 9.4% per AAA tra 5.5 e 5.9 cm, 10.2% per AAA tra 6.0 e 6.9 cm. e
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
del 32.5% per AAA superiori a 7.0 cm ed un tasso del 25.7% a 6 mesi per diametri superiori ad 8 cm [98].
La metodica �finite element analysis of AAA wall stress’ che usa ricostruzioni tridimensionali TC si è rilevata migliore sia nel differenziare gli AAA sintomatici prossimi alla rottura
che nel predirne il rischio di rottura [93].
La crescita rapida di AAA (≥ 1 cm/annuo o ≥ 0.5 cm in 6 mesi) e l’insorgenza di sintomi
sono fattori indipendenti per l’indicazione all’intervento. Alcuni criteri anatomici possono
incrementare il rischio di rottura come l’asimmetria e la tortuosità vasale [97,100]. Aneurismi eccentrici, sacciformi o con blisters parietali hanno un rischio di rottura maggiore
La familiarità rappresenta un fattore di rischio per l’AAA con un tasso di rottura precoce
4 volte superiore rispetto a casi sporadici [101].
Vi è stata ed è presente una costante ricerca per l’identificazione, di altri fattori che
possano predire il rischio di rottura. Tre questi la MMP-9, che ГЁ stata direttamente correlata nella degradazione proteolitica della matrice extracellulare della parete aortica [102],
potrebbe essere un utile marker misurandone i livelli plasmatici [103].
Dati preliminari ottenuti alla tomografia ad emissione di positroni per l’AAA hanno mostrato un maggiore captazione focale di 18-fluorodeoxyglucose (18F-FDG) nel contesto
della parete aortica in pazienti con AAA di grandi dimensioni, ad espansione rapida o sintomatici. La captazione di18F-FDG rappresenta l’immagine funzionale dell’infiltrato infiammatorio e quindi potenziale tecnica non invasiva per l’identificazione di AAA instabili in fase
di rottura [104].
La mortalità correlata alla riparazione dell’AAA varia in relazione all’ospedale ed alla
equipe operatoria [88-105]. La mortalitГ media a 30 giorni risulta essere compresa tra
l’1.1% e l’8.0% nel trattamento in elezione [88,106-109] e dovrebbe essere contenuta al
di sotto del 2% nella maggioranza degli ospedali [108,109]. Nelle ultime decadi il miglioramento dei risultati nel trattamento elettivo non ГЁ stato seguito da risultati sovrapponibili
nel trattamento per rottura, con una mortalitГ tra il 30% ed il 70% [3,10]. E, se considerata
la mortalità combinata alla rottura, durante il trasferimento, all’ammissione ospedaliera ed
all’intervento, solamente il 18% dei pazienti con AAA rotto sopravvive. Prance et al hanno
suggerito cinque fattori di rischio preoperatori per la stima della mortalità legata all’AAA:
(1) etГ superiore a 76 anni; (2) livelli di creatininemia superiori a 190 Вµmol/L; (3) emoglobina inferiore a 8g/dl; (4) perdita di coscienza; (5) ECG con evidenza di ischemia. Nel loro
studio il tasso di mortalitГ fu del 100% quando il paziente presentava tre o piГ№ fattori di rischio decrescendo al 48%, 28% e 18% se i fattori di rischio presenti erano rispettivamente
due, uno o nessuno [110]. Attualmente particolare importanza viene conferita alla �emostasi ipotensiva’, che consiste nell’infusione di liquidi limitatamente al raggiungimento di
Gli aneurismi dell’aorta addominale
valori pressori sistolici di 90 mmHg, ed al riconoscimento della sindrome compartimentale
addominale specialmente in relazione al trattamento endovascolare [88,111].
Trattamento medico
La diagnosi di AAA ГЁ solitamente occasionale in corso di indagini per altre patologie o
durante programmi di screening. Alla diagnosi il 90% di questi AAA risulta essere all’interno
della zona di sorveglianza [84]. In tale periodo viene usualmente somministrata una terapia
medica adiuvante. In particolare la terapia con statina ha dimostrato un effetto biologico
pleiotropico inclusa la riduzione dei livelli di proteina C-reattiva circolante,che potrebbe
essere rilevante nella patogenesi dell’AAA [112]. Ad oggi, comunque, non vi sono evidenze
sufficienti per raccomandare la terapia con statina alla diagnosi di AAA. La somministrazione del propanololo è di beneficio controverso nell’AAA. Evidenze di Livello A dall’American College of Cardiology/ American Heart Association clinical practice guidelines indicano
che il propanololo non inibisce la crescita aneurismatica [113]. Dati provenienti da studi
su animali dimostrano che l’utilizzo degli ACE-inibitori o del losartan riducono il tasso di
espansione aneurismatica [84]. L’utilizzo di roxitromicina, in caso di riscontro sierico di
infezione da Chlamydia pneumoniae, e delle tetracicline ГЁ stato associato ad una riduzione
del tasso di espansione aneurismatica [114,115]. Un’interessante target terapeutico nella farmacoterapia degli aneurismi potrebbe essere la risposta infiammatoria interferendo
con la via delle MMPs. Anti-infiammatori non steroidei, come l’indometacina, sono stati in
grado di inibire la crescita aneurismatica in modelli animali [116]. Risultati da studi clinici
hanno suggerito che la doxiciclina previene la crescita aneurismatica negli uomini [117].
Ciononostante sono necessari ulteriori studi per chiarire il ruolo potenziale della doxicilina,
della roxitromicina e della terapia con statine nella progressione della patologia aneurismatica [88].
Trattamento chirurgico
Per la riparazione chirurgica dell’AAA sono disponibili due forme di trattamento. La riparazione chirurgica �open’ viene utilizzata ampiamente da più di 50 anni. Più recentemente,
la riparazione endovascolare aneurismatica (EVAR) è stata sviluppata per offrire un’alternativa meno invasiva e sta superando in frequenza di utilizzo la metodica �open’ [11].
Il primo trattamento chirurgico dell’AAA fu realizzato da Charles Dubost il 29 Marzo
1951 mediante un’aneurismectomia ed interposizione di homograft [118]. Il trattamento
odierno deriva da Creech, del gruppo di DeBakey, che ha pubbliacto nel 1966 una semplificazione dell’intervento mediante un’endoaneurismectomia ed interposizione protesica
divenendo quello ad oggi piГ№ utilizzato [119].
Durante il trattamento chirurgico, l’addome è aggredito, di norma, mediante una lunga
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
incisione mediana. Un approccio retro peritoneale viene raccomandato per pazienti con
patologia polmonare cronica ostruttiva. Gli svantaggi di tale approccio includono sia l’impossibilità all’esplorazione del contenuto intraperitoneale che la difficoltà d’accesso all’arteria iliaca destra. Una volta aperta la cavità addominale deve essere identificato il colletto
aneurismatico per permettere il controllo dello stesso. In caso di colletto soprarenale od infrarenale potrebbe essere necessario posizionare una clamp al di sopra delle arterie renali.
Le arterie iliache vengono controllate in maniera analoga. In taluni casi si rende necessario
il repertare l’arteria mesenterica inferiore per il suo successivo reimpianto nella protesi
aortica mediante patch di Carrell al fine di garantire un flusso soddisfacente al sigma ed al
retto. Comunemente vengono utilizzate protesi in Dacron a �zero porosità ’ rese impermeabili dall’impregnazione con collagene, albumina o gelatina [88]. L’anastomosi prossimale
ГЁ del tipo termino-terminale e la distale puГІ essere realizzata alla biforcazione aortica, alla
biforcazione iliaca o sull’arteria femorale comune a seconda dell’estensione aneurismatica
ed alla pervietГ delle arterie iliache esterne. Viene posta molta cura nel mantenere almeno
un’arteria iliaca interna pervia ed nel diagnosticare preoperatoriamente una potenziale
ischemia colica sinistra o del retto. Talvolta può rendersi necessario l’impianto di un corto
�side arm graft’ per riperfondere un’arteria iliaca interna. Negli uomini sessualmente attivi,
la raccomandazione è di non dissecare la parete aortica sinistra e l’arteria iliaca comune
sinistra. Le principali complicanze dopo trattamento chirurgico �open’ dell’AAA sono quelle cardiache, polmonari, renali ed emorragiche [120]. La paraplegia postoperatoria, una
complicanza rilevante nella chirurgia per aneurismi toraco-addominali, ГЁ infrequente negli
AAA. L’incidenza della paraplegia dopo trattamento EVAR o chirurgico viene riportata tra lo
0.21%121 e lo 0.25-0.9% rispettivamente [122].
Coggia et al hanno dimostrato, in uno studio preliminare, la fattibilitГ della riparazione
chirurgia per via totalmente laparoscopica. Anche se tale tecnica risulta meno invasiva e
riduce il trauma chirurgica, sono richieste piГ№ esperienza e miglioramento delle conoscenze per assicurare un beneficio reale di questa tecnica rispetto alla riparazione tradizionale
Il trattamento EVAR, introdotto per la prima volta da Parodi nel 1991 [124], prevede il
posizionamento di un’endoprotesi (stentgraft) all’interno dell’AAA e il suo fissaggio sulla
parete sana dell’aorta e delle iliache. Non è necessaria una laparotomia, bensì la protesi
endovascolare viene impiantata attraverso piccole incisioni femorali. Dall’epoca del primo
impiego di un’endoprotesi, l’uso delle tecniche endovascolari per gli AAA ha avuto larga
diffusione e ad oggi sono stati impiantati migliaia di dispositivi in tutto il mondo. La tecnica
ГЁ stata ampiamente studiata in diversi trial randomizzati, multicentrici, in Europa (DREAM,
EVAR) e negli USA (OVER) che ne hanno dimostrato la fattibilità e l’efficacia [125]. Non
tutti i pazienti portatori di AAA possono essere sottoposti ad un intervento endovascolare,
Gli aneurismi dell’aorta addominale
ma sono necessarie caratteristiche anatomiche specifiche. La TC spirale e l’angioTC sono
metodiche indispensabili per la scelta del piГ№ idoneo stentgraft. Lo stengraft ГЁ composto,
quindi, da tessuto e stents di metallo giГ premontati su un sistema di rilascio. Sotto guida
fluoroscopia, il sistema ГЁ inserito attraverso le arterie femorali su guide angiografiche fino
al corretto posizionamento dello stentgraft al di sopra ed al di sotto del segmento aneurismatico. Il rilascio dello stentgraft in corretta posizione esclude il flusso ematico dalla sacca aneurismatica con conseguenteeliminazione della pressione sulla parete. La principale
complicanza dopo EVAR è l’endoleak ossia l’incompleta esclusione dell’aneurisma dalla
circolazione. Vi è la possibilità che l’endoleak possa risolversi spontaneamente, ma una
parte di essi persistono e sono associati ad un rischio tardivo di rottura. Altre complicanze
sono rappresentate dall’endotension, dalla migrazione del device e dalle infezioni delle ferite inguinali [88]. Le controindicazioni all’EVAR consistono in aspetti anatomo-morfologici
attentamente valutati alla TC preoperatoria..
Sono stati pubblicati diversi trials controllati randomizzati (RCTs) per la valutazione del
migliore trattamento dell’AAA. Lederle et al. hanno recentemente rivisto la letteratura analizzando solo RCTs per riparazione versus sorveglianza di AAA di piccole dimensioni, trattamento chirurgico �open’ versus EVAR e trattamento EVAR versus controllo per pazienti non
candidabili a riparazione �open’. Nel gruppo riparazione versus sorveglianza di AAA di piccole dimensioni né l’ADAM né l’UKSAT trial hanno raggiunto una differenza statisticamente
significativa per mortalità legata a tutte le cause (rischio relativo [RR] 1.1 [ 95% da 0.771.32]) o per mortalità legata all’AAA (RR 0.78 [95% CI; 0.56-1.10]). In entrambi gli RCTs le
differenze della qualitГ di vita rilevate furono modeste tendendo a favorire, ad un anno, la
riparazione chirurgica, sebbene la maggioranza degli AAA monitorati erano asintomatici.
Nel gruppo trattamento chirurgico �open’ versus EVAR il Dutch Randomized Endovascular
Aneurysm Management trial (DREAM), l’Eindhoven Stent Prothesis for Aneurysm Study
(ESPAS), l’Endovascular Aneurysm Repair trial (EVAR)-1 ed il Montreal trial hanno rilevato
che il trattamento EVAR riduce la mortalitГ legata a tutte le cause a 30 giorni rispetto al
trattamento chirurgico �open’ (RR 1.6% vs. 4.8%; 0.33 [95%CI, 0.17-0.64]). La differenza
nella mortalità precoce però scompare entro 2 anni nel DERAM e nell’EVAR-1trial, risultando in nessuna differenza statisticamente significativa nella mortalità a medio termine. GLI
RCTs pubblicati hanno mostrato una ridotta mortalitГ operatoria e un miglioramento nella
qualitГ di vita precoce dopo trattamento EVAR ma non hanno rilevato alcuna differenza
statisticamente significativa in termini di qualitГ di vita o mortalitГ relata a tutte le cause
nel medio termine. Nel gruppo trattamento EVAR versus controllo per pazienti non candidabili a riparazione tradizionale solamente l’EVAR-2 trial è stato considerato. Lo studio non
ha riportato alcuna differenza statisticamente significativa per quanto riguarda la mortalitГ per tutte le cause. Il trend fu rilevato favorevole per i soggetti sottoposti ad osservazione
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
Fig 1: Foto intraoperatoria: AAA infiammatorio.
Fig 2. TC di AAA infiammatorio con caratteristica
�cuff’ perianeurismatica.
Fig 3. Foto intraoperatoria: voluminoso trombo endo- Fig 4. AAA di grandi dimensioni che deforma il profilo
della parete addominale.
Fig 5. TC addominale di AAA di grandi dimensioni che Fig 6. AAA rotto: voluminoso ematoma retro ed intradeforma il profilo della parete addominale.
Gli aneurismi dell’aorta addominale
Fig 7. Foto intraoperatoria: AAA di grandi dimensio- Fig 8. Protesi in Dacron retta 20mm aorto-aortica.
niisolato con posizionamento di clamps ed apertura
della parete.
Fig 9. Protesi in Dacron biforcata 18-9mm aortobisiliaco.
Fig 10. Rx addome: controllo EVAR.
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
(hazard ratio [HR] 1.21 [95% CI, 0.87-1.69]). Non ГЁ stata rilevata inoltre, alcuna differenza
statisticamente significativa nella mortalitГ per AAA (HR 1.01 [95% CI; 0.55-1.84]). Le
conclusioni degli Autori furono che la sopravvivenza non migliora operando AAA inferiori
a 5.5 cm; il trattamento EVAR ГЁ associato ad una minore mortalitГ operatoria ma con
risultati a lungo termine sconosciuti rispetto al trattamento chirurgico �open’ e l’EVAR non
ha mostrato miglioramenti in pazienti che hanno controindicazioni mediche al trattamento
chirurgico �open’ [126].
La sopravvivenza non ha mostrato miglioramenti per riparazioni di AAA inferiori a 5.5
cm; l’EVAR è associata a minore mortalità operatoria, re interventi più frequenti e risultati a
lungo termine sconosciuti rispetto al trattamento chirurgico �open’. L’EVAR non ha mostrato
miglioramenti in termini di sopravvivenza in pazienti che presentano controindicazioni mediche al trattamento chirurgico �open’. I risultati a lungo termine degli RCTs che valutano
il trattamento chirurgico �open’ rispetto all’EVAR sono necessari e si attendono. Per gli
uomini un valore appropriato di AAA per considerare l’intervento è un diametro di 5,5 cm
e tale valore deve essere ridotto a 5 cm per le donne. Gli AAA inferiori a tale dimensioni
dovrebbero essere sorvegliati mediante esame ECD con un intervallo di 6 mesi e corretti
chirurgicamente nel caso in cui dovessero raggiungere le dimensioni appropriate oppure
se la crescita durante l’intervallo tra due esami sia superiore a 0.5 cm o insorgano dei
sintomi legati all’AAA.
Le ultime 5 decadi sono state caratterizzate da un progressivo miglioramento nella
diagnosi, terapia e prognosi nel trattamento dell’AAA. Sicuramente alcuni aspetti dovranno
essere approfonditi da future ricerche riferendoci in particolare alla prevenzione primaria mediante programmi di screening, al riconoscimento della popolazione a rischio ed
all’identificazione di eventuali biomarcatori predittivi di rottura. Si auspica, inoltre, una
migliore definizione delle indicazioni al trattamento �open’ e d EVAR al fine di migliorare i
risultati specialmente nei pazienti con AAA rotto.
Gli aneurismi dell’aorta addominale
[1] Sakalihasan N, Limet R, Defawe OD. Abdominal aortic aneurysm. Lancet 2005; 365:1577–89.
[2] Fleming C, Whitlock EP, Beil TL, Lederle FA. Screening for abdominal aortic aneurysm: a best-evidence
systematic review for the U.S. Preventive Services Task Force. Ann Intern Med 2005; 142: 203-11.
[3] Kniemeyer HW, Kessler T, Reber PU, Ris HB, Hakki H, Widmer MK. Treatment of ruptured abdominal
aortic aneurysm, a permanent challenge or a waste of resources? Prediction of outcome using a multiorgan-dysfunction score. Eur J Vasc Endovasc Surg 2000; 19: 190–96.
[4] Gray H. “Gray’s Anatomy: Descriptive and Surgical”. 15th ed. London: Chancellor Press; 1994. p. 526.
[5] Johnston KW, Rutherford RB, Tilson MD, Shah DM, Hollier L, Stanley JC. Suggested standards for reporting on arterial aneurysms. Subcommittee on Reporting Standards for Arterial Aneurysms, Ad Hoc
Committee on Reporting Standards, Society for Vascular Surgery and North American Chapter, International Society for Cardiovascular Surgery. J Vasc Surg 1991; 13: 452–58.
[6] Tang T, Boyle JR, Dixon AK, Varty K. Inflammatory abdominal aortic aneurysms. Eur J Vasc Endovasc
Surg 2005; 29: 353-62.
[7] Kent KC, Zwolak RM, Jaff MR, Hollenbeck ST, Thompson RW, Schermerhorn ML, et al; Society for Vascular Surgery; American Association of Vascular Surgery; Society for Vascular Medicine and Biology.
Screening for abdominal aortic aneurysm: a consensus statement. J Vasc Surg 2004 Jan;39(1): 267-9.
[8] Thompson MM. Controlling the expansion of abdominal aortic aneurysms. Br J Surg 2003; 90: 897–
[9] Scott RA, Ashton HA, Kay DN. Abdominal aortic aneurysm in 4237 screened patients: prevalence, development and management over 6 years. Br J Surg 1991; 78: 1122–25.
[10] Wilmink TB, Quick CR, Hubbard CS, Day NE. The influence of screening on the incidence of ruptured
abdominal aortic aneurysms. J Vasc Surg 1999; 30: 203–08.
[11] Nowygrod R, Egorova N, Greco G, Anderson P, Gelijns A, Moskowitz A, et al. Trends, complications, and
mortality in peripheral vascular surgery. J Vasc Surg 2006; 43: 205-16.
[12] Lederle FA, Johnson GR, Wilson SE, et al. The aneurysm detection and management study screening
program: validation cohort and final results. Arch Intern Med 2000; 160: 1425–30.
[13] Lindholt JS, Vammen S, Juul S, Fasting H, Henneberg EW. Optimal interval screening and surveillance
of abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2000; 20: 369–73.
[14] Lederle FA, Johnson GR, Wilson SE. Abdominal aortic aneurysm in women. J Vasc Surg 2001; 34:
[15] Singh K, Bonaa KH, Jacobsen BK, Bjork L, Solberg S. Prevalence of and risk factors for abdominal aortic
aneurysms in a population-based study: the Tromso Study. Am J Epidemiol 2001; 154: 236–44.
[16] Vardulaki KA, Walker NM, Day NE, Duffy SW, Ashton HA, Scott RA. Quantifying the risks of hypertension,
age, sex and smoking in patients with abdominal aortic aneurysm. Br J Surg 2000; 87: 195–200.
[17] Huber TS, Wang JG, Derrow AE, Dame DA, Ozaki CK, Zelenock GB, et al. Experience in the United States
with intact abdominal aortic aneurysm repair. J Vasc Surg 2001;33:304-10; discussion 310-1.
[18] Johnston KW. Influence of sex on the results of abdominal aorticaneurysm repair. Canadian Society for
Vascular Surgery Aneurysm Study Group. J Vasc Surg 1994;20:914-23; discussion 923-6.
[19] Steickmeier B. Epidemiology of arotic disease: Aneurysm, dissection, occlusion. Radiologe 2001;41:62432.
[20] Reed D, Reed C, Stemmermann G, Hayashi T. Are aortic aneurysms caused by atherosclerosis? Circulation 1992; 85: 205–11.
[21] Tilson MD. Aortic aneurysms and atherosclerosis. Circulation 1992; 85: 378–79.
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
[22] Xu C, Zarins CK, Glagov S. Aneurysmal and occlusive atherosclerosis of the human abdominal aorta. J
Vasc Surg 2001; 33: 91–96.
[23] Defawe OD, Colige A, Lambert CA, Munaut C, Delvenne P, LapiГЁre CM, et al. TIMP-2 and PAI-1 mRNA
levels are lower in aneurysmal as compared to athero-occlusive abdominal aortas. Cardiovasc Res
2003; 60: 205-13.
[24] Erentu V, Bozbua N, Omerolu SN, Ardal H, Eren E, GГјГ§lГј M, et al. Rupture of abdominal aortic aneurysms
in Behçet’s disease. Ann Vasc Surg 2003; 17: 682–85.
[25] Matsumura K, Hirano T, Takeda K, Matsuda A, Nakagawa T, Yamaguchi N, et al. Incidence of aneurysms
in Takayasu’s arteritis. Angiology 1991; 42: 308–15.
[26] Towbin JA, Casey B, Belmont J. The molecular basis of vascular disorders. Am J Hum Genet 1999; 64:
[27] Brown LC, Powell JT. Risk factors for aneurysm rupture in patients kept under ultrasound surveillance.
UK Small Aneurysm Trial Participants. Ann Surg 1999; 230: 289–96; discussion 296–97.
[28] Blanchard JF, Armenian HK, Friesen PP. Risk factors for abdominal aortic aneurysm: results of a casecontrol study. Am J Epidemiol 2000; 151: 575–83.
[29] Brady AR, Thompson SG, Fowkes FG, Greenhalgh RM, Powell JT. Abdominal aortic aneurysm expansion:
risk factors and time intervals for surveillance. Circulation 2004; 110: 16–21.
[30] Gillum RF. Epidemiology of aortic aneurysm in the United States. J Clin Epidemiol 1995; 48: 1289–98.
[31] Powell JT, Greenhalgh RM. Multifactorial inheritance of abdominal aortic aneurysm. Eur J Vasc Surg
1987; 1: 29–31.
[32] Shibamura H, Olson JM, van Vlijmen-Van Keulen C, Buxbaum SG, Dudek DM, Tromp G, et al. Genome
scan for familial abdominal aortic aneurysm using sex and family history as covariates suggests genetic heterogeneity and identifies linkage to chromosome 19q13. Circulation 2004;109: 2103–2108.
[33] Van Vlijmen-Van Keulen CJ, Rauwerda JA, Pals G. Genome-wide linkage in three Dutch families maps
a locus for abdominal aortic aneurysms to chromosome 19q13. 3. Eur J Vasc Endovasc Surg 2005:
30; 29–35.
[34] Powell JT. Familial clustering of abdominal aortic aneurysm-smoke signals, but no culprit genes. Br J
Surg 2003; 90: 1173–74.
[35] Sugimoto T, Sada M, Miyamoto T, Yao H. Genetic analysis on HLA loci in Japanese patients with abdominal aortic aneurysm. Eur J Vasc Endovasc Surg 2003; 26: 215–218.
[36] Rasmussen TE, Hallett JW Jr, Tazelaar HD, Miller VM, Schulte S, O’Fallon WM, et al. Human leukocyte
antigen class II immune response genes, female gender, and cigarette smoking as risk and modulating
factors in abdominal aortic aneurysms. J Vasc Surg 2002; 35: 988–993.
[37] Rasmussen TE, Hallett JW Jr, Schulte S, Harmsen WS, O’Fallon WM, Weyand CM. Genetic similarity in
inflammatory and degenerative abdominal aortic aneurysms: a study of human leukocyte antigen class
II disease risk genes. J Vasc Surg 2001; 34: 84–89.
[38] Juvonen J, Surcel HM, Satta J, Teppo AM, Bloigu A, Syrjälä H, et al. Elevated circulating levels of inflammatory cytokines in patients with abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 1997; 17:
[39] Forester ND, Cruickshank SM, Scott DJ, Carding SR. Functional characterization of T cells in abdominal
aortic aneurysms. Immunology 2005; 115: 262–270
[40] Lucarini L, Sticchi E, Sofi F, Pratesi G, Pratesi C, Pulli R, Gensini GF, Abbate R, Pepe G, Fatini C. ACE and
TGFBR1 genes interact in influencing the susceptibility to abdominal aortic aneurysm. Atherosclerosis
2009 Jan;202(1):205-10.
[41] Wallinder J, Bergqvist D, Henriksson AE. Proinflammatory and anti-inflammatory cytokine balance in
Gli aneurismi dell’aorta addominale
patients with abdominal aortic aneurysm and the impact of aneurysm size. Vasc Endovascular Surg
2009 Jun-Jul;43(3):258-61.
Reeps C, Pelisek J, Seidl S, Schuster T, Zimmermann A, Kuehnl A, Eckstein HH. Inflammatory infiltrates and neovessels are relevant sources of MMPs in abdominal aortic aneurysm wall. Pathobiology
Thompson AR, Drenos F, Hafez H, Humphries SE. Candidate gene association studies in abdominal aortic aneurysm disease: a review and meta-analysis. Eur J Vasc Endovasc Surg 2008 Jan;35(1):19-30.
Sandford RM, Bown MJ, London NJ, Sayers RD. The genetic basis of abdominal aortic aneurysms: a
review. Eur J Vasc Endovasc Surg 2007 Apr;33(4):381-90.
Unno N, Nakamura T, Mitsuoka H, Uchiyama T, Yamamoto N, Saito T, Sugatani J, Miwa M, Nakamura
S. Association of a G994-T missense mutation in the plasma platelet-activating factor acetylhydrolase
gene with risk of abdominal aortic aneurysm in Japanese Ann Surg 2002 Feb;235(2):297-302.
Koch AE, Haines GK, Rizzo RJ, Radosevich JA, Pope RM, Robinson PG, et al. Human abdominal aortic aneurysms. Immunophenotypic analysis suggesting an immune-mediated response. Am J Pathol
1990; 137: 1199–1213.
Brophy CM, Reilly JM, Smith GJ, Tilson MD.The role of inflammation in nonspecific abdominal aortic
aneurysm disease. Ann Vasc Surg 1991; 5: 229–233.
Galle C, SchandenГ© L, Stordeur P, Peignois Y, Ferreira J, Wautrecht JC, et al. Predominance of type 1
CD4+ T cells in human abdominal aortic aneurysm. Clin Exp Immunol 2005; 142: 519-527.
Ocana E, BohГіrquez JC, PГ©rez-Requena J, Brieva JA, RodrГ­guez C. Characterisation of T and B lymphocytes infiltrating abdominal aortic aneurysms. Atherosclerosis 2003; 170: 39-48.
Yen HC, Lee FY, Chau LY. Analysis of the T cell receptor VОІ repertoire in human aortic aneurysms.
Atherosclerosis 1997;135: 29–36.
Forester ND, Cruickshank SM, Scott DJ, Carding SR. Increased natural killer cell activity in patients with
an abdominal aortic aneurysm. Br J Surg 2006; 93: 46–54.
Kuivaniemi H, Platsoucas CD, Tilson MD 3rd. Aortic aneurysms: an immune disease with a strong genetic component. Circulation 2008; 117: 242–252.
Bobryshev YV, Lord RS. Vascular-associated lymphoid tissue (VALT) involvement in aortic aneurysm.
Atherosclerosis 2001; 154: 15–21.
Caligiuri G, Rossignol P, Julia P, Groyer E, Mouradian D, Urbain D, et al. Reduced immunoregulatory
CD31+ T cells in patients with atherosclerotic abdominal aortic aneurysm. Arterioscler Thromb Vasc
Biol 2006; 26: 618–623.
Ozsvath KJ, Hirose H, Xia S, Tilson MD. Molecular mimicry in human aortic aneurysmal diseases. Ann
N Y Acad Sci 2006; 800: 288–293.
Platsoucas CD, Lu S, Nwaneshiudu I, Solomides C, Agelan A, Ntaoula N, et al. Abdominal aortic aneurysm is a specific antigen-driven T cell disease. Ann N Y Acad Sci 2006; 1085: 224–235
Sakalihasan N, Heyeres A, Nusgens BV, Limet R, Lapiere CM. Modifications of the extracellular matrix of
aneurysmal abdominal aortas as a function of their size. Eur J Vasc Surg 1993; 7: 633–37.
Dobrin PB, Mrkvicka R. Failure of elastin or collagen as possible critical connective tissue alterations
underlying aneurismal dilatation. Cardiovasc Surg 1994; 2: 484–88.
Satta J, Juvonen T, Haukipuro K, Juvonen M, Kairaluoma MI. Increased turnover of collagen in abdominal aortic aneurysms, demonstrated by measuring the concentration of the aminoterminal propeptide
of type III procollagen in peripheral and aortal blood samples. J Vasc Surg 1995; 22: 155–60.
Huffman MD, Curci JA, Moore G, Kerns DB, Starcher BC, Thompson RW. Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms. Surgery
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
2000; 128: 429–38.
[61] Herron GS, Unemori E, Wong M, Rapp JH, Hibbs MH, Stoney RJ. Connective tissue proteinases and inhibitors in abdominal aortic aneurysms: involvement of the vasa vasorum in the pathogenesis of aortic
aneurysms. Arterioscler Thromb 1991; 11: 1667–77.
[62] Shah PK. Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological
paradigm in aortic aneurysm. Circulation 1997; 96: 2115–17.
[63] Newman KM, Jean-Claude J, Li H, Ramey WG, Tilson MD. Cytokines that activate proteolysis are increased in abdominal aortic aneurysms. Circulation 1994; 90: II224–27.
[64] Walton LJ, Franklin IJ, Bayston T, Brown LC, Greenhalgh RM, Taylor GW, et al. Inhibition of prostaglandin
E2 synthesis in abdominal aortic aneurysms: implications for smooth muscle cell viability, inflammatory
processes, and the expansion of abdominal aortic aneurysms. Circulation 1999; 100: 48–54.
[65] Hance KA, Tataria M, Ziporin SJ, Lee JK, Thompson RW. Monocyte chemotactic activity in human abdominal aortic aneurysms: role of elastin degradation peptides and the 67-kD cell surface elastin
receptor. J Vasc Surg 2002; 35: 254–61.
[66] Hirose H, Tilson MD. Abdominal aortic aneurysm as an autoimmune disease. Ann N Y Acad Sci 2001;
947: 416–18.
[67] Curci JA, Thompson RW. Adaptive cellular immunity in aortic aneurysms: cause, consequence, or context? J Clin Invest 2004; 114: 168–71.
[68] Carrell TW, Burnand KG, Wells GM, Clements JM, Smith A. Stromelysin-1 (matrix metalloproteinase-3)
and tissue inhibitor of metalloproteinase-3 are overexpressed in the wall of abdominal aortic aneurysms. Circulation 2002; 105: 477–82.
[69] Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in
concert to produce aortic aneurysms. J Clin Invest 2002; 110: 625–32.
[70] Tromp G, Gatalica Z, Skunca M, Berguer R, Siegel T, Kline RA, et al. Elevated expression of matrix metalloproteinase-13 in abdominal aortic aneurysms. Ann Vasc Surg 2004; 18: 414–20.
[71] Eriksson P, Jones KG, Brown LC, Greenhalgh RM, Hamsten A, Powell JT. Genetic approach to the role of
cysteine proteases in the expansion of abdominal aortic aneurysms. Br J Surg 2004; 91: 86–89.
[72] Knox JB, Sukhova GK, Whittemore AD, Libby P. Evidence for altered balance between matrix metalloproteinases and their inhibitors in human aortic diseases. Circulation 1997; 95: 205–12.
[73] Lopez-Candales A, Holmes DR, Liao S, Scott MJ, Wickline SA, Thompson RW. Decreased vascular
smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms. Am J Pathol
1997; 150: 993–1007.
[74] Allaire E, Muscatelli-Groux B, Mandet C, Guinault AM, Bruneval P, Desgranges P, et al. Paracrine effect
of vascular smooth muscle cells in the prevention of aortic aneurysm formation. J Vasc Surg 2002; 36:
[75] Lindholt JS, Jorgensen B, Fasting H, Henneberg EW. Plasma levels of plasmin-antiplasmin-complexes
are predictive for small abdominal aortic aneurysms expanding to operationrecommendable sizes. J
Vasc Surg 2001; 34: 611–15.
[76] Vorp DA, Lee PC, Wang DH, Makaroun MS, Nemoto EM, Ogawa S, et al. Association of intraluminal
thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening. J Vasc Surg 2001; 34:
[77] Kiev J, Eckhardt A, Kerstein MD. Reliability and accuracy of physical examination in detection of abdominal aortic aneurysms. Vasc Endovasc Surg 1997;31:143.
[78] Fink HA, Lederle FA, Roth CS, Bowles CA, Nelson DB, Haas MA. The accuracy of physical examination
to detect abdominal aortic aneurysm. Arch Intern Med 2000;160:833-6.
Gli aneurismi dell’aorta addominale
[79] Quill DS, Colgan MP, Sumner DS. Ultrasonic screening for the detection of abdominal aortic aneurysms.
Surg Clin North Am 1989; 69: 713–20.
[80] Bajardi G, Vitale G, Mirabella D, Bracale UM. Retroaortic left renal vein and inflammatory abdominal
aortic aneurysm. Gen Thorac Cardiovasc Surg (2010) 58: in press.
[81] Arita T, Matsunaga N, Takano K, Nagaoka S, Nakamura H, Katayama S, et al. Abdominal aortic aneurysm: rupture associated with the high-attenuating crescent sign. Radiology 1997; 204: 765–68.
[82] Sprouse LR 2nd, Meier GH 3rd, Parent FN, DeMasi RJ, Stokes GK, LeSar CJ, et al. Is three-dimensional
computed tomography reconstruction justified before endovascular aortic aneurysm repair? J Vasc
Surg 2004; 40: 443–47.
[83] Wilmink AB, Forshaw M, Quick CR, Hubbard CS, Day NE. Accuracy of serial screening for abdominal
aortic aneurysms by ultrasound. J Med Screen. 2002; 9: 125–127.
[84] Baxter TB, Terrin MC, Dalman RL. Medical Management of Small Abdominal Aortic Aneurysms. Circulation 2008; 117: 1883-1889
[85] Lederle FA, Johnson GR, Wilson SE, Chute EP, Littooy FN, Bandyk D, et al. Aneurysm Detection and Management (ADAM) Veterans Affairs Cooperative Study Group. Prevalence and associations of abdominal
aortic aneurysm detected through screening. Ann Intern Med 1997; 126:441-449.
[86] United Kingdom Small Aneurysm Trial Participants. Long-term outcomes of immediate repair compared
with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002; 346: 1445-1452.
[87] Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, et al. Immediate repair compared
with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002; 346: 1437-1444.
[88] Chaikof EL, Brewster DC, Dalman RL , Makaroun MS, Illig KA, Sicard GA, et al. The care of patients with
an abdominal aortic aneurysm: The Society for Vascular Surgery practice guidelines. J Vasc Surg 2009
Oct; 50(4 Suppl): S2-49.
[89] Parry DJ, Waterworth A, Kessel D, Robertson I, Berridge DC, Scott DJ. Endovascular repair of an inflammatory abdominal aortic aneurysm complicated by aortoduodenal fistulation with an unusual presentation. J Vasc Surg 2001; 33: 874-79.
[90] Lemos DW, Raffetto JD, Moore TC, Menzoian JO. Primary aortoduodenal fistula: a case report and
review of the literature. J Vasc Surg 2003; 37: 686-89
[91] Davis PM, Gloviczki P, Cherry KJ Jr, Toomey BJ, Stanson AW, Bower TC, et al. Aorto-caval and ilio-iliac
arteriovenous fistulae. Am J Surg 1998; 176: 115–18.
[92] Fillinger M. Who should we operate on and how do we decide: predicting rupture and survival in patients with aortic aneurysm. Semin Vasc Surg 2007; 20:121-127.
[93] Fillinger MF, Marra SP, Raghavan ML, Kennedy FE. Prediction of rupture risk in abdominal aortic aneurysm during observation: wall stress versus diameter. J Vasc Surg 2003Вё37: 724-732.
[94] Norman PE, Semmens JB, Lawrence-Brown MM. Long-term relative survival following urgery for
abdominal aortic aneurysm: a review. Cardiovasc Surg 2001; 9: 219–24.
[95] Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;
352: 1649-1655.
[96] Fillinger MF, Racusin J, Baker RK, Cronenwett JL, Teutelink A, Schermerhorn ML, et al. Anatomic characteristics of ruptured abdominal aortic aneurysm on conventional CT scans: implications for rupture
risk. J Vasc Surg 2004; 39: 1243-1252.
[97] Lederle FA, Johnson GR, Wilson SE, Ballard DJ, Jordan WD Jr, Blebea J, et al. Rupture rate of large
abdominal aortic aneurysms in patients refusing or unfit for elective repair. JAMA 2002; 287: 29682972.
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
[98] Endovascular aneurysm repair and outcome in patients unfit for open repair of abdominal aortic aneurysm (EVAR trial 2): Randomised controlled trial. Lancet 2005; 365: 2187-2192.
[99] Brewster DC, Cronenwett JL, Hallett JW Jr, Johnston KW, Krupski WC, Matsumura JS, et al. Guidelines
for the treatment of abdominal aortic aneurysms. Report of a subcommittee of the Joint Council of the
AmericanAssociation for Vascular Surgery and Society for Vascular Surgery. J Vasc Surg 2003; 37:
[100]Tanquilut EM, Veith FJ, Ohki T, Lipsitz EC, Shaw PM, Suggs WD, et al. Nonoperative management with
selective delayed surgery for large abdominal aortic aneurysms in patients at high risk. J Vasc Surg
2002; 36:41-46.
[101]Italian Society of Vascular and Endovascular Surgery (SICVE). Linee Guida per la Diagnosi ed il Trattamento degli Aneurismi Dell’aorta Addominale 2009.
[102]Sakalihasan N, Delvenne P, Nusgens BV, Limet R, Lapiere CM. Activated forms of MMP2 and MMP9 in
abdominal aortic aneurysms. J Vasc Surg 1996; 24: 127–33.
[103]Lindholt JS, Vammen S, Fasting H, Henneberg EW, Heickendorff L. The plasma level of matrix metalloproteinase 9 may predict the natural history of small abdominal aortic aneurysms: a preliminary study.
Eur J Vasc Endovasc Surg 2000; 20: 281–85.
[104]Sakalihasan N, Van Damme H, Gomez P, Rigo P, Lapiere CM, Nusgens B, et al. Positron emission tomography (PET) evaluation of abdominal aortic aneurysm (AAA). Eur J Vasc Endovasc Surg 2002; 23:
[105]Dueck AD, Kucey DS, Johnston KW, Alter D, Laupacis A. Longterm survival and temporal trends in patient and surgeon factors after elective and ruptured abdominal aortic aneurysm surgery. J Vasc Surg
2004; 39: 1261–67.
[106]Kieffer E, Koskas F, Dewailly J, Gouny P. Mortalite peroperatoire de la chirurgie elective de l’anevrysme
de l’aorte abdominale: etude multicentrique de l’AURC. In: Kieffer E, ed. “Les aneurysmes de l’aorte
abdominal sous-renale”. Paris, France: AERCV, 1990: 235–243.
[107]Brady AR, Fowkes FG, Greenhalgh RM, Powell JT, Ruckley CV, Thompson SG. Risk factors for postoperative death following elective surgical repair of abdominal aortic aneurysm: results from the UK Small
Aneurysm Trial. Br J Surg 2000; 87: 742–49.
[108]Elkouri S, Gloviczki P, McKusick MA, Panneton JM, Andrews J, Bower TC, et al. Perioperative complications and early outcome after endovascular and open surgical repair of abdominal aortic aneurysms. J
Vasc Surg 2004; 39: 497–505.
[109]Hertzer NR, Mascha EJ, Karafa MT, O’Hara PJ, Krajewski LP, Beven EG. Open infrarenal abdominal
aortic aneurysm repair: the Cleveland Clinic experience from 1989 to 1998. J Vasc Surg 2002; 35:
[110]Prance SE, Wilson YG, Cosgrove CM, Walker AJ, Wilkins DC, Ashley S. Ruptured abdominal aortic aneurysms: selecting patients for surgery. Eur J Vasc Endovasc Surg 1999; 17: 129–32.
[111]Bajardi G, Pecoraro F, Mirabella D, Bracale UM, Bellisi MG. Abdominal Compartment Syndrome (ACS)
After Surgical Abdominal Aortic Aneurysm (AAA) Repair. Ann Ital Chir 2009 in press.
[112]Gotto AM Jr. Role of C-reactive protein in coronary risk reduction: focus on primary prevention. Am J
Cardiol 2007; 99: 718-725.
[113]Gibbons RJ, Smith S, Antman E. American College of Cardiology/ American Heart Association clinical
practice guidelines: part I: where do they come from? Circulation. 2003; 107: 2979-2986.
[114]Lindholt JS, Ashton HA, Scott RA. Indicators of infection with Chlamydia pneumoniae are associated
with expansion of abdominal aortic aneurysms. J Vasc Surg 2001;34:212–215.
[115]Mosorin M, Juvonen J, Biancari F, Satta J, Surcel HM, Leinonen M, et al. Use of doxycycline to decrease
Gli aneurismi dell’aorta addominale
the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebocontrolled pilot
study. J Vasc Surg 2001;34:606–610.
[116]Holmes DR, Petrinec D, Wester W, Thompson RW, Reilly JM. Indomethacin prevents elastase-induced
abdominal aortic aneurysms in the rat. J Surg Res 1996; 63: 305–09
[117]Baxter BT, Pearce WH, Waltke EA, Littooy FN, Hallett JW Jr, Kent KC, et al. Prolonged administration of
doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective
(Phase II) multicenter study. J Vasc Surg 2002; 36: 1–12.
[118]Dubost C, Allary M, Oeconomos N. A’ propos du traitment des anevrysmes de l’aorte. Ablation de
l’Anevrysme. Retabblissemment de la continuitè par greffe d’aorte humain conservèe. Men Acad Chir
1951; 77: 381-3.
[119]Creech O Jr. Endo-aneurysmorrhaphy and treatment of aortic aneurysm. Ann Surg 1966 Dec; 164(6):
[120]Rutherford RB, editor. “Vascular Surgery”. 6th ed. Philadelphia Elsevier Saunders; 2005. p. 1431.
[121]Berg P, Kaufmann D, van Marrewijk CJ, Buth J. Spinal cord ischaemia after stent-graft treatment for
infra-renal abdominal aortic aneurysms: analysis of the Eurostar database. Eur J Vasc Endovasc Surg
2001; 22: 342–47.
[122]Picone AL, Green RM, Ricotta JR, May AG, DeWeese JA. Spinal cord ischemia following operations on
the abdominal aorta. J Vasc Surg 1986; 3: 94–103.
[123]Coggia M, Javerliat I, Di Centa I, Colacchio G, Cerceau P, Kitzis M, et al. Total laparoscopic infrarenal
aortic aneurysm repair: preliminary results. J Vasc Surg 2004; 40: 448–54.
[124]Parodi JC, Palmaz JC, Barone HD. Transfemoral intraluminal graft implantation for abdominal aortic
aneurysms. Ann Vasc Surg 1991; 5: 491–99.
[125]Lederle FA, Freischlag JA, Kyriakides TC, Padberg FT Jr, Matsumura JS, Kohler TR, et al; Open Versus
Endovascular Repair (OVER) Veterans Affairs Cooperative Study Group. Outcomes following endovascular vs open repair of abdominal aortic aneurysm: a randomized trial. JAMA 2009 Oct 14; 302(14):
[126]Lederle FA, Kane RL, MacDonald R, Wilt TJ. Systematic Review: Repair of Unruptured Abdominal Aortic
Aneurysm. Ann Intern Med 2007; 146: 735-741.
[127]Bartoli MA, Parodi FE, Chu J, Pagano MB, Mao D, Baxter BT, Buckley C, Ennis TL, Thompson RW. Localized administration of doxycycline suppresses aortic dilatation in an experimental mouse model of
abdominal aortic aneurysm. Ann Vasc Surg 2006 Mar; 20(2): 228-36.
[128]Cho BS, Roelofs KJ, Ford JW, Henke PK, Upchurch GR Jr. Decreased collagen and increased matrix
metalloproteinase-13 in experimental abdominal aortic aneurysms in males compared with females.
Surgery 2009 Sep 18.
[129]Davis V, Persidskaia R, Baca-Regen L, Itoh Y, Nagase H, Persidsky Y, Ghorpade A, Baxter BT. Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms.
Arterioscler Thromb Vasc Biol 1998 Oct; 18(10): 1625-33.
[130]Eskandari MK, Vijungco JD, Flores A, Borensztajn J, Shively V, Pearce WH. Enhanced abdominal aortic
aneurysm in TIMP-1-deficient mice. J Surg Res 2005 Feb; 123(2): 289-93.
[131]Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalgh RM, Powell JT. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 1995 Aug;
15(8): 1145-51.
[132]Jones GT, Phillips VL, Harris EL, Rossaak JI, van Rij AM. Functional matrix metalloproteinase-9 polymorphism (C-1562T) associated with abdominal aortic aneurysm. J Vasc Surg 2003 Dec; 38(6): 1363-7.
[133]Kadoglou NP, Liapis CD. Matrix metalloproteinases: contribution to pathogenesis, diagnosis, surveillan-
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
ce and treatment of abdominal aortic aneurysms. Curr Med Res Opin 2004 Apr; 20(4): 419-32.
[134]Lindeman JH, Abdul-Hussien H, van Bockel JH, Wolterbeek R, Kleemann R. Clinical trial of doxycycline
for matrix metalloproteinase-9 inhibition in patients with an abdominal aneurysm: doxycycline selectively depletes aortic wall neutrophils and cytotoxic T cells. Circulation 2009 Apr 28; 119(16): 2209-16.
[135]Longo GM, Xiong W, Greiner TC, Zhao Y, Fiotti N, Baxter BT. Matrix metalloproteinases 2 and 9 work in
concert to produce aortic aneurysms. J Clin Invest 2002 Sep; 110(5): 625-32.
[136]Manning MW, Cassis LA, Daugherty A. Differential effects of doxycycline, a broad-spectrum matrix
metalloproteinase inhibitor, on angiotensin II-induced atherosclerosis and abdominal aortic aneurysms.
Arterioscler Thromb Vasc Biol 2003 Mar 1; 23(3): 483-8.
[137]Mateos-CГЎceres PJ, LГіpez-FarrГ© AJ, Morata PC, Ramos-Mozo P, Macaya C, Serrano FJ, MoГ±ux G.
Pravastatin increases the expression of the tissue inhibitor of matrix metalloproteinase-1 and the oncogene Bax in human aortic abdominal aneurysms. Can J Physiol Pharmacol. 2008 Jul;86(7):431-7.
[138]McMillan WD, Pearce WH. Increased plasma levels of metalloproteinase-9 are associated with abdominal aortic aneurysms. J Vasc Surg 1999 Jan; 29(1): 122-7.
[139]Nishimura K, Ikebuchi M, Kanaoka Y, Ohgi S, Ueta E, Nanba E, Ito H. Relationships between matrix metalloproteinases and tissue inhibitor of metalloproteinases in the wall of abdominal aortic aneurysms.
Int Angiol 2003 Sep; 22(3): 229-38.
[140]Petersen E, Gineitis A, WГҐgberg F, Angquist KA. Activity of matrix metalloproteinase-2 and -9 in abdominal aortic aneurysms. Relation to size and rupture. Eur J Vasc Endovasc Surg 2000 Nov; 20(5):
[141]Reeps C, Pelisek J, Seidl S, Schuster T, Zimmermann A, Kuehnl A, Eckstein HH. Inflammatory infiltrates
and neovessels are relevant sources of MMPs in abdominal aortic aneurysm wall. Pathobiology 2009;
76(5): 243-52.
[142]Rizas KD, Ippagunta N, Tilson MD 3rd. Immune cells and molecular mediators in the pathogenesis of
the abdominal aortic aneurysm. Cardiol Rev 2009 Sep-Oct; 17(5): 201-10.
[143]Smallwood L, Allcock R, van Bockxmeer F, Warrington N, Palmer LJ, Iacopetta B, Golledge J, Norman
PE. Polymorphisms of the matrix metalloproteinase 9 gene and abdominal aortic aneurysm. Br J Surg
2008 Oct; 95(10): 1239-44.
[144]Wilson WR, Anderton M, Choke EC, Dawson J, Loftus IM, Thompson MM. Elevated plasma MMP1 and
MMP9 are associated with abdominal aortic aneurysm rupture. Eur J Vasc Endovasc Surg 2008 May;
35(5): 580-4.
Cellular stress
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Proteine da shock termico: fattori chiave nella patogenesi e terapia dell’artrite reumatoide]
Luana Lipari
Department of Experimental Medicine - Section of Histology and Embryology “Arcangelo Pasqualino di Marineo” - School of Medicine. University of Palermo (IT)
Key words: Heat shock proteins, Rheumatoid arthritis, Experimental arthritis, Molecular chaperones, Pathogenesis, Risk factors
Parole chiave: Proteine da shock termico, Artrite reumatoide, Artrite sperimentale, Chaperonine, Patogenesi,
Fattori di rischio
Abstract. The pathogenesis of rheumatoid arthritis (RA) is very complex, the cell types involved have been
characterized: monocyte/macrophages, T and B cells, synoviocytes and endothelial cells. Chronicity and destructive potential are characteristic features of the inflammatory response in the synovial membrane, it has
proposed that an exogenous antigen, likely an infectious organism, targets the synovia and elicits a chronic
immune response. A major effort has been made to determine which cytokines and inflammatory mediators
are produced at the site of disease. Tissue residing and infiltrating cells secrete proinflammatory cytokines
in situ, which likely have a role in ampliflyng and maintaining the inflammation. Heat shock proteins (HSPs)
are a group of proteins that are present in all cells, they are induced when a cell undergoes various types
of environmental stresses like heat, cold and oxygen deprivation but HSPs are also present in cells under
perfectly conditions. Heat shock proteins trigger immune response through activities that occur both inside
the cell and outside the cell, they play an important role in protein folding and assembly in the cells, defective
HSPs can contribute to the pathogenesis of a number of disease, known as chaperonopathies. Bacterial HSPs
are recognized by the immune system and thus may provide a missing link between an immune response
to infection and autoimmunity, infact, the overwhelming presence of bacterial HSPs on the mucosal surfaces
of the gastrointestinal tract, the expression of self-HSP at sites of inflammation and the cross-reactivity
between bacterial and self-HSP, self-HSP reactive T cells are normally controlled by the immune system.
The generation of self-HSP specific T cells may even be essential for the maintenance of self-tolerance in a
healthy individual.
Riassunto. La patogenesi dell’ artrite reumatoide (AR) è molto complessa, le cellule coinvolte nella patogenesi
sono: monociti/macrofagi, linfociti T e B, sinoviociti e cellule endoteliali. Le caratteristiche principali dell’AR
sono la cronicitГ e la totale distruzione della principale sede verso la quale si rivolge la risposta infiammatoria
ossia: la membrana sinoviale; si presuppone che un antigene esogeno, verosimilmente un agente di natura
infettiva, s’insedi in corrispondenza della membrana sinoviale ed eliciti l’attivazione della risposta infiammatoria verso la stessa. A seguito della risposta infiammatoria vengono prodotte in situ numerose citochine proinfiammatorie in grado di richiamare dal torrente ematico altre cellule pro-infiammatorie amplificando in tal
modo e perpetuando la risposta infiammatoria quindi il danno a carico della membrana sinoviale. Le proteine
da shock termico (HSPs) sono un gruppo di proteine presenti in tutti i tipi cellulari, la loro espressione viene
indotta a seguito dell’esposizione a stress di varia natura come l’elevate o basse temperature e la privazione
Luana Lipari
di O2 sebbene possono esser presenti anche in condizioni fisiologiche. Le HSPs attivano la risposta immune
e partecipano ai processi di assemblaggio e ripiegamento di proteine all’interno della cellula stessa, pertanto
possono contribuire alla patogenesi di numerose patologie note come chaperonopatie. Le HSPs batteriche
sono riconosciute dal sistema immune ed attivando la risposta immune fungono da legame tra risposta
immune attivata nei confronti di un agente di natura infettiva e l’autoimmunità , infatti l’overespressione di
HSPs batteriche sulla mucosa del tratto gastrointestinale, l’espressione di HSP-self nel sito d’infiammazione
determina una cross-reattività tra HSPs batteriche e linfociti T diretti contro HSPs self innescando ma soprattutto amplificando la risposta immune nei confronti di strutture self dell’organismo stesso.
Rheumatoid Arthritis: what’s known on pathogenesis
Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease that affects 0.51% of the population and causes progressive joint destruction that leads to the restriction
of activities of daily living and deterioration of quality of life. The inflammatory process is
characterized by infiltration of inflammatory cells into the joints, leading to the proliferation
of fibroblast-like synoviocytes and the destruction of cartilage and bone. In synovial tissue
of patients with RA, the infiltrating cells consist of macrophages, T cells, B cells, plasma
cells, neutrophilis, mast cells, dendritic cells and natural killer cells. Migration of leukocytes into the synovium is a regulated multistep process involving interactions between
leukocytes and endothelial cells and cellular adhesion molecules, as well as between leukocytes, chemokines and chemokine receptors. Chemokines are small, chemoattractant
cytokines that play key roles in the accumulation of inflammatory cells at the site of inflammation [8].
Among risk factors: genetic and environmental agents
Although the pathogenesis of RA has not yet been fully elucidated, it is considered to be
a complex disease that is influenced by both genetic and environmental factors. Genetic
influences that contribute to RA susceptibility have been demonstrated in both studies of
twins and families, as well as in genome-wide linkage scans. These studies estimated that
genetic factors are responsible for 50-60% of the risk of developing RA and that environmental factors may explain the remaining risk. This quantification was made by a classical
approach to separating the quantitative influence of genetic factors in RA with nationwide
studies of twins with RA [7].
Several infectious agents have been reported to be risk factors for RA, including human
parvovirus B19 (B19), Epstein-Barr virus, retroviruses, alphaviruses, hepatitis B virus, Mycobacterium tuberculosis, Escherichia coli, Proteus mirabilis and Mycoplasma. Moreover, a
large number of epidemiological studies have demonstrated an association between cigarette smoking and the development of RA. In particular, smoking is known to be associated
with rheumatoid factor positive and anti peptide C citrullinate positive, and to interact
Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis
with the HLA-DRB1 shared epitope alleles. In addition, some studies have suggested that
smoking also influences RA disease severity, although this remains controversial. While
the mechanisms responsible for the influence of smoking in RA are not clear, some studies have shown an association between RA and the toxic compounds found in cigarette
smoke, such as nicotine, 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD) and reactive oxygen
species. It was found that the mRNA and protein levels of aryl hydrocarbone receptor (AhR)
were higher in RA synovial tissue than in osteoarthritis tissue, and that TCDD upregulated
the expression of IL-1ОІ, IL-6 and IL-8 through binding to AhR, with this effect transmitted
via the nuclear factor-kB and extracellular signal-regulated kinase signalling cascades. In
addition, AhR expression in synovial cells was upregulated by TNFО± [30].
The increased risk of RA in women has lead to evaluation of the role of sex hormones
in disease susceptibility. It is well known that the levels of male sex hormones, particularly
testosterone, are lower in men who have RA. By contrast, the levels of female sex hormones do not differ significantly between RA patients and control individuals. It has been
reported that bone erosion occurs more frequently in men than in women, and that arthritis
tends to occur earlier in men. Furthermore, male gender is correlated with a higher risk of
bone erosions and an accelerated disease course, while female gender is correlated with
structural consequences of joint destruction. Joint surgery is performed more frequently
in female RA patients than in male patients. In addition, gender influences the risk, as well
as the pattern, of organ involvement in RA. Nodules, and rheumatoid lung disease are typical manifestations in men, whereas women typically develop sicca syndrome. Exogenous
hormones have also been reported to influence RA disease risk [5].
It is widely accepted that RA frequently remits during pregnancy. Although the mechanism for this is unclear, it is reported that the amelioration of RA during pregnancy is associated with a disparity in HLA class II antigens between mother and fetus. These studies
suggested that the maternal immune response to paternal HLA antigens may play a role
in pregnancy-induced RA remission. By contrast, several studies have suggested that nulliparous women have an increased during the postpartum period, particularly after the first
pregnancy. Subsequent investigations demonstrated that much of this increased risk is
associated with breastfeeding, and that women who breastfeed after their first pregnancy
are at the greatest risk of developing RA. It is also suggested that the association among
breastfeeding, pregnancy and RA may be related to either increased prolactin levels or an
abnormal, pro-inflammatory response to prolactin [4]. Recently, it has been showed that
the amelioration of disease activity in the third trimester corresponded to the increased
number of regulatory T cells that induced a pronounced anti-inflammatory cytokine milieu.
These data suggests that pregnancy leads to a beneficial effect of regulatory T cells on RA
disease activity [12].
Luana Lipari
Few reports have addressed the influence of diet on RA development and progression.
The addition of omega-3 fatty acids to the diets of RA patients has been associated with
improvement in RA. Furthermore, diets high in eicosapentaenoic acid have a favourable
effect on the clinical outcome of RA. Although the association between diet and RA onset
is unclear, it is accepted that such fatty acids compete with arachidonic acids, the latter of
which are involved in inflammation. Some reports have demonstrated the influence of vitamins on RA, for example greater intake of vitamin D, primarily from fish and fish products,
has been associated with a lower risk of RA. In addition, the vitamin K, which is primarily
derived from vegetables and legumes could inhibit the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis [29].
Experimental arthritis as a research model
Animal models of arthritis play an important role in unravelling mechanisms of chronic
inflammation in rheumatoid synovial tissue. They are used extensively to study disease
pathogenesis and to apply new treatment strategies for rheumatoid arthritis and juvenile
idiopathic arthritis (JIA). However, regardless of their limitations and drawbacks, experimental arthritis models can provide useful information on both pathogenesis and novel venues
for therapy. A good example is the ongoing study of the most commonly used models of
experimental arthritis. Its clinical and pathological features mimic several human chronic
arthritic diseases, including RA, reactive arthritis and JIA. It can be used as a model to study
other aspects of chronic arthritis such as pain, neuro-endocrine dysbalance, cartilage degeneration and even physical therapy. Nevertheless, the model is probably best known for
its role in the study of the immune pathogenesis of chronic synovial best known for its role
in the study of the immune pathogenesis of chronic synovial inflammation and exploring
novel immune interventions in arthritis. Over the years, studies on the role of T cell recognition in autoimmune diseases have been performed [1]. As a consequence of these studies,
novel venues for possible immune intervention with Heat Shock Proteins (HSPs) in human
chronic arthritis are currently being explored, as we are going to discuss later.
Adjuvant arthritis: description of histopathological steps
Adjuvant arthritis (AA) is one of the most widely used experimental arthritis models. The
first classical model of AA has described in 1956 by a single intracutaneous injection with
heat-killed mycobacteria incomplete Freund’s adjuvant (CFA). The presence of mycobacteria in CFA suggested an infectious cause of the polyarthritis; however, anti-inflammatory
drugs, but not antibiotics, are successful in attenuating disease severity [23]. AA is induced
by intradermal or footpad injection of heat-killed mycobacterial species, preferably in a fine
suspension in a mineral or vegetable oil. The disease is restricted to susceptible rodents,
Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis
mostly certain rat strains, such as Lewis, Buffalo, Sprague-Dawly and Wistar rats. Interestingly, arthritis can also be induced in humans through immunization with mycobacterial
antigens. The pathogenesis of this BCG-induced reactive arthritis in man is still largely unknown, but the disease appears T cell driven, presumably with an important pathogenetic
role for both CD4+ and CD8+ lymphocytes. Some data suggest that, as in AA, HSPs may
play a triggering role [22]. Following AA induction with CFA, rats not only develop arthritis
but also tract and a loss in body weight that starts 24-48 hours before the clinical onset
of arthritis. AA is a symmetric polyarthritis, affecting primarily the peripheral joints and
quite frequently the spine. The affected joints are red, swollen and painful. The onset of
overt clinical arthritis is seen 10-14 days following the induction of AA with CFA. The first
histopathological signs of arthritis, an accumulation of mononuclear cells in connective
tissues adjacent to periostal surfaces, are already manifest 6 days after disease induction.
Approximately 10 days after disease induction, the first radiological signs of inflammation
become visible: localized osteoporosis, with erosive lesions and periostal reaction. The
synovial infiltrate leads to pannus formation, resulting in cartilage deformation and severe
destruction of the joint [21].
In the early stages of overt disease, the histopathological abnormalities of AA are characterized by the infiltration of mononuclear cells into the synovial tissue. This cell infiltrate
consists primarily of lymphocytes, but also plasma cells and at later stages neutrophils
are present. Lymphocytes play a central role in triggering AA. It was shown that adoptive
transfer of the disease is possible with the injection of primed lymph node cells or thoracic
duct lymphocytes from diseased animals into recipients rats without co-immunization with
mycobacteria or any triggering antigen. During the past, it was shown that the induction
of AA is T cell driven, it was generated a T cell line, A2, directed at M. tubercolosis, that is
capable of transferring AA to irradiated recipient rats. The same T cell line is capable of
vaccinating non-irradiated rats against subsequent AA induction with CFA immunization.
Thus a microbial agent may pull the trigger and initiate a self-perpetuating autoimmune
process in a susceptible individual [3].
Heat shock proteins: players of rheumatoid arthritis pathogenesis
HSPs are highly conserved cellular proteins. They play an important role in protein
folding and assembly in the cells, as so called molecular chaperones. Based on size, four
main groups of HSPs can be distinguished; Hsp90, Hsp70, Hsp60 and the small-HSPs.
HSPs can be considered immunologic important proteins [14,15,17,26]. Defective HSPs
can contribute to the pathogenesis of a number of diseases, now known as chaperonopathies [13,16,18].
First of all, HSPs are called stress proteins because their production increases when a
Luana Lipari
cell is confronted with an environmental stressor, such as a rise in temperature, hypoxia,
irradiation, viral infection or exposure to toxic chemicals. Because of this, self-HSP expression is up-regulated at sites of cell stress, such as in damaged or inflamed tissues,
irrespective of the cause of damage. In inflamed synovial tissue from both rats with AA and
from patients with either RA or JIA, an increased expression of self-Hsp60 can be found in
synovial lining cells [25].
Secondly, HSPs are found in cells of all eukaryotes and prokaryotes and are remarkably conserved during evolution. Human Hsp60 has 40-50% identical amino acid residues
and an additional 20% conservative replacements in comparison to mycobacterial Hsp65,
GroEL [6].
Thirdly, despite the high degree of homology among HSPs of different species, they
have been found to be strong immunogens. Bacterial HSPs are easily recognized by the
immune system, which in turn can lead to cross recognition of self-HSP, and thus may
provide a missing link between an immune response to infection and autoimmunity [11].
However, in the healthy immune system this is not the case. Despite, or maybe as a consequence of, the overwhelming presence of bacterial HSPs on the mucosal surfaces of the
gastrointestinal tract, the expression of self-HSP at sites of inflammation, e.g. during an
infection, and the cross-reactivity between bacterial and self-HSP, self-HSP reactive T cells
are normally controlled by the immune system. Over the years, evidence has mounted that
the generation of self-HSP specific T cells may even be essential for the maintenance of
self-tolerance in a healthy individual. From that point of view, self-HSP-specific T cells may
effectively play a key regulatory role in the control of potential danger situations, such as
inflammation [8].
A new approach for therapy of rheumatoid arthritis: the chaperonotherapy
Following the initial discovery that the T cell epitope of the arthritogenic T cell clone A2b
in AA is a Hsp60-derived sequence, several attempts have been made to induce arthritis
with Hsp60, or peptides derived from Hsp60. To date, all these attempts have been unsuccessful, even when the arthritogenic epitope itself or self-Hsp60 was used. Intraperitoneal
immunization with the 180-188 Mhsp65 epitope of A2b induces a specific immune response towards both the 180-188 peptide and whole M. tuberculosis [27]. However, such
immunization fails to induce arthritis, but instead can prevent the subsequent induction of
AA. Conversely, as immunization with MHsp65 protects in AA and in practically all forms of
experimental arthritis. Since that initial discovery, many approaches using Hsp60 or Hsp60
derived peptides have been successfully applied to induce protection in AA. These approaches included, among others, oral and nasal tolerance induction and DNA vaccination
[9, 10]. Bacterial HSPs epitopes are present to the mucosal surface of the gastrointestinal
Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis
tract. This can lead to the expansion of tolerized T cells, specific for these bacterial epitopes,
but capable of cross-reacting with conserved self-hsp epitopes. Such a mechanism can
be functionally fully comparable with the induction of regulatory T cells in oral tolerance.
Subsequently, when cells are confronted with stress, e.g., during joint inflammation, selfHSP-reactive T cells [24]. These tolerized self-HSP-reactive cells, induced by the bacterial
flora of the intestinal tract, may be part of the regulatory network of the healthy immune
system controlling inflammation [20]. There is evidence from experimental arthritis models
that supports this concept.
Heat shock proteins have received broad interest over recent years. Initially, they were
found to be dominantly immunogenic microbial antigens, then the raised presence of immunity to HSPs in virtually all conditions of inflammation including autoimmune diseases,
transplantation rejection and atherosclerosis has emphasized the critical significance of
immunity to HSP inflammatory.
The observations that HSPs can be released and that they can directly or indirectly elicit
potent immunoregulatory activities give a new perspective on the roles of HSP and antiHSP reactivity in autoimmunity, transplantation, vascular disease and other conditions.
Indeed, HSPs have unique arthritis-protective properties that may play a role in determining the disease outcome, not only in experimental arthritis, but also in patients. [2, 28].
In conclusion, the interaction of Hsps with the immune system, and the potential use of
chaperones as therapeutic agents, i.e. the chaperonotherapy [19], is a putative new field
of research for next years.
Luana Lipari
[1] Berner B, Akca D, Jung T, Muller GA, Reuss-Borst MA. Analysis of Th1 and Th2 cytokines expressing
CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry. J Rheumatolol 2000; 27:1128-35.
[2] Chen G, Hao J, Xi Y, Wang W, Wang Z, Li N, Li W. The therapeutic Effect of Vasoactive Intestinal Peptide
on Experimental Arthritis is associated with CD4 CD25 T Regulatory Cells. Scand J Immunol 2008;
[3] Cope AP. T cells in rheumatoid arthritis. Arthritis Res Ther 2008;10(Suppl 1):S1. Epub 2008 Oct 15.
[4] Cutolo M. Sex hormone adjuvant therapy in rheumatoid arthritis. Rheum Dis Clin North Am 2000;
[5] Gerosa M, De Angelis V, Riboldi P, Meroni PL. Rheumatoid arthritis: a female challenge. Womens Health
(Lond Engl) 2008; 4:195-201.
[6] Hromadnikova I, Nguyen TT, Zlacka D, Sedlackova L, Popelka S, Veigl D, Pech J, Vavrincova P, Sosna A
Expression of heat shock protein receptors on fibroblast-like synovial cells derived from rheumatoid
arthritis-affected joints. Rheumatol Int 2008; 28:837-44 Epub 2008 Jan 30.
[7] Imboden JB. The Immunopathogenesis of Rheumatoid Arthritis. Annu Rev Pathol. Epub 2008 Oct 27.
[8] Iwamoto T, Okamoto H, Toyama Y and Momohara S. Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients. FEBS Journal Epub 2008.
[9] Kamphuis S, Kuis W, de Jager W, Teklenburg G, Massa M, Gordon G, Boerhof M, Rijkers GT, Uiterwaal
CS, Otten HG, Sette A, Albani S, Prakken BJ. Tolerogenic immune responses to novel T-cell epitopes
from heat-shock protein 60 in juvenile idiopathic arthritis. Lancet 2005; 366:50-6.
[10] Kaufmann SH, Ladel CH, Flesch IE. T cells and cytokines in intracellular bacterial infections: experience
with Mycobacterium bovis BCG. Ciba Found Symp 1995; 195:132-32; discussion: 132-6.
[11] Kim HR, Kim EY, Cerny J, Mougdil KD. Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis. J Immunol 2006;
[12] Kobayashi S, Momohara S, Kamatani N, Okamoto H. Molecular aspects of rheumatoid arthritis: role of
environmental factors. FEBS Journal Epub 2008.
[13] Macario AJL. Heat-shock proteins and molecular chaperones: Implications for pathogenesis, diagnostics, and therapeutics. Intl J Clin Lab Res 1995; 25:59-70.
[14] Macario AJL, Lange M, Ahring BK, Conway de Macario E. Stress genes and proteins in the Archaea.
Microbiol Mol Biol Reviews 1999; 63:923-967.
[15] Macario AJL, Conway de Macario E. The molecular chaperone system and other anti-stress mechanisms in archaea. Front Biosci 2001; 6:d262-283.
[16] Macario AJL, Conway de Macario E. Sick chaperones and ageing: A perspective. Ageing Res Rev 2002;
[17] Macario AJL, Conway de Macario E. The pathology of anti-stress mechanisms: A new frontier. Stress
2004; 7:243-249.
[18] Macario AJL, Conway de Macario E. Sick chaperones, cellular stress, and disease. N Engl J Med 2005;
[19] Macario AJL, Conway de Macario E. Chaperonopathies and chaperonotherapy. FEBS Letters 2007;
[20] Massa M, Passlia M, Manzoni SM, Campanelli R, Ciardelli L, Yung GP, Kamphuis S, Pistorio A, Meli V,
Sette A, Prakken B, Martini A, Albani S. Differential recognition of heat-shock protein dnaJ-derived
epitopes by effector and Treg cells leads to modulation of inflammation in juvenile idiopathic arthritis.
Heat shock proteins: key players in pathogenesis and therapy of rheumatoid arthritis
Arthritis Rheum 2007; 56:1648-57.
[21] Mirshfiey A, Mohsenzadegan M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 2008; 7:195-202.
[22] Moudgil KD. Diversification of response to hsp65 during the course of autoimmune arthritis is regulatory rather than pathogenic. Immunol Rev 1998; 164:175-84.
[23] Prakken B, Kuis W, van Eden W, Albani S. Heat shock proteins in juvenile idiopathic arthritis: keys for
understanding remitting arthritis and candidate antigens for immune therapy. Curr Rheumatol Rep
2002; 4:466-73.
[24] Prakken BJ, Roord S, Ronaghy A, Wauben M, Albani S, van Eden W. Heat shock protein 60 and adjuvant
arthritis: a model for T cell regulation in human arthritis. Springer Semin Immunopathol 2003; 25:47-63.
[25] Puga Yung GL, Le TD, Roord S, Prakken B, Albani S. Heat shock proteins (HSP) for immunotherapy of
rheumatoid arthritis (RA). Inflamm Res 2003; 52:443-51.
[26] Sedlackova L, Nguyen TT, Zlacka D, Sosna A, Hromadnikova D. Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells. Autoimmunity Epub. 2008 Jun 12.
[27] Sheikhi A, Nazarian M, Khadem-Al-Melleh A, Nasab NM, Esmaeilzadeh A, Yahaghi N, Sheikhi R. In vitro
effects of Mycobacterial bovis BCG-lysate and its derived heat shock proteins on cytokines secretion
by blood mononuclear cells of rheumatoid arthritis patients in comparison with healthy controls. Int
Immunopharmacol 2008; 8:887-92. Epub 2008 Mar 7.
[28] Sherer Y, Gerli R, Bocci EB, Gilburd B, VaBistoni O, Shoenfeld Y. Heat-shock protein 65 autoantibodies
are differently associated with early atherosclerosis in rheumatoid arthritis and in healthy subjects. Ann
N Y Acad Sci 2007; 1108:408-13.
[29] Strietholt S, Maurer B, Peters MA, Pap T, Gay S. Epigenetic modifications in rheumatoid arthritis. Arthritis Res Ther 2008; 10:219 Epub 2008 Oct 10.
[30] Weyand CM, Goronzy JJ. Pathogenesis of rheumatoid arthritis. Med Clin North Am 1997; 81:29-55.
Brain, behavior
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Effetti dell’Apomorfina nella reazione di orientamento nel ratto]
Maurizio Casarrubea, Filippina Sorbera and Giuseppe Crescimanno
Department of Experimental Medicine, Human Physiology Section, University of Palermo (IT)
Key words: Dopamine, A9 and A10 neurons, Nigrostriatal Dopaminergic Neurons, Mesolimbic-Mesocortical
Dopaminergic Neurons, Head turning, Sensorimotor integration.
Parole chiave: Dopamina, Neuroni A9 e A10, Neuroni Dopaminergici Nigrostriatale, Neuroni Dopaminergici
Mesolimbico-Mesocorticali, Rotazione del capo, Integrazione sensitivomotoria.
Abstract. Apomorphine, a mixed D1 and D2 dopaminergic agonist, provokes rapid modifications of A9 and A10
neuronal firing. Aim of the research was to study the possible relationship between rapid apomorphine-induced
modifications of midbrain dopaminergic neuron activity and rapid modifications of a motor response triggered by
sensory stimuli (sensorimotor integration). With the aid of a videoanalysis system, effects of apomorphine (0.1,
0.5, 1.0, 2.0 mg/kg IP) on latency and duration of head orienting movement, induced by acoustic stimulation, were
examined. During trials lasting 20 min, in a control group of animals, receiving only acoustic stimulation, latency
and duration of head turning movement were respectively 184.21 + 48.30 ms and 257.82 + 49.19 ms. On vehicle
+ acoustic administered animals, no significant modifications of head turning parameters occurred. Following apomorphine administration, ANOVA test revealed significant decrease of head turning latency and duration. Post-hoc
comparisons, between drug and corresponding control conditions, showed that a) the two larger doses provoked
significant effects only on head turning latency; b) the slight increase, observed with doses of 0.1 and 0.5 mg/
kg, was no significant. An apomorphine activity on pre- or postsynaptic receptors of nigrostriatal and mesolimbicmesocortical dopaminergic neurons could explain the effects; hence, a change on neurotransmitter release, leading
to an inhibition or facilitation of dopamine-mediated responses to environmental stimuli, may be hypothesized.
Riassunto. L’apomorfina, un agonista dopaminergico sia D1 che D2, induce rapide modificazioni dell’attività dei neuroni A9 e A10. Scopo della ricerca è stato di studiare la possibile relazione tra le rapide modificazioni
dell’attività neuronale indotte dall’apomorfina e le rapide modificazioni di una risposta motoria innescata da
una stimolazione sensoriale (integrazione sensitivo-motoria). Per mezzo di un sistema di videoanalisi, sono stati
esaminati gli effetti della somministrazione di apomorfina (0.1, 0.5, 1.0, 2.0 mg/kg IP) sulla latenza e durata del
movimento di orientamento della testa (HT), indotto da stimolazione acustica. Durante sedute della durata di 20
minuti, in un gruppo di animali controllo che riceveva solo stimoli acustici, la latenza e la durata dell’HT erano
rispettivamente 184.21 + 48.30 ms e 257.82 + 49.19 ms. Animali cui era somministrata soltanto la soluzione
veicolo e ai quali erano erogati gli stimoli acustici, non esibivano significative variazioni dei parametri dell’HT.
In seguito alla somministrazione di apomorfina, il test di significatività ANOVA mostrava significativi decrementi sia della latenza che della durata dell’HT. La post-hoc analisi dimostrava che: a) le due dosi maggiori
di apomorfina provocavano effetti significativi solo sulla latenza dell’HT; b) solo un piccolo e non significativo
effetto si osservava con le dosi di 0.1 e 0.5 mg/kg. Gli effetti osservati possono essere spiegati con un’azione
dell’apomorfina sui recettori presinaptici dei neuroni dopaminergici nigrostriatali e mesolimbico-mesocorticali.
In conseguenza di ciò, è possibile ipotizzare che una modificazione dell’entità del rilascio di neurotrasmettitore,
possa condurre ad una alterazione delle risposte agli stimoli ambientali mediate dalla dopamina.
Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno
Presentation of novel auditory stimuli elicits a motor response which has been referred to as the orienting reaction. Modifications of this response have been considered as
sensitive and reliable indexes of changes in attentive condition and sensory information
processing speed [1-3].
Different neuronal systems, in which a dopaminergic (DA) activity has been demonstrated, seem to play a role in orientational capacities [4-8]. Among them, A9 neurons in pars
compacta of the substantia nigra and A10 group neurons of ventral tegmental area (VTA)
show an increased neuronal firing in response to sensory stimuli [9-15]. Moreover, lesions
of these systems result in characteristic sensorimotor impairments and attention disorders
with affected animals unable to perform a correct orientation toward incoming sensory
stimuli [8,16-18].
It has been previously demonstrated that the rapid increase of neuronal firing, observed
on A9 and A10 DA neurons, following sulpiride administration, is correlated to rapid modifications of specific behavioral activities needing sensorimotor integration [19]. Moreover,
in freely moving rats, acute apomorphine (APO), a mixed D1 and D2 DA agonist directly
binding to dopamine receptors [20], has been proved to provoke dose-dependent rapid
changes of A9 and A10 neuronal firing [21].
Possible behavioral correlates of this last finding have not been fully examined.
Aim of the present research, was to investigate possible relationships between rapid
APO-induced modifications of midbrain DA neuron activity and rapid modifications of a
motor response triggered by sensory stimuli (sensorimotor integration).
To this purpose, after acute administration of different APO doses, the acoustically-evoked head turning (HT) movement was studied in rats by means of a videoanalysis system
allowing a subtle frame-by frame scanning.
Since HT is a component of the orienting reaction toward environmental stimuli, i.e. a
response needing sensorimotor integration, HT latency and duration can be considered as
useful parameters to evaluate possible rapidly-induced changes on sensorimotor processing speed [1,22,3].
Materials and methods
The study was performed on male wistar rats (250 - 300 g), housed in separate cages
(room temperature 21В°C), with free access to food and water. Experimental sessions were
carried out during light phase (from 0700 to 1900 h) of day-night cycle.
Effects of Apomorphine on Orienting Reaction in the Rat
experiments were carried out in a soundproof room, maintained at constant illumination
and temperature. Illuminated and ventilated perspex boxes (30 x 30 x 30 cm) were used
as observation cages. Two loud-speakers placed within the box walls (one on the left wall
and the other on the right one), exactly in the centre and 5 cm from the bottom, allowed to
induce an orienting reaction whenever the acoustic stimulus occurred. Loudspeakers were
controlled by an acoustic stimulator (Coulbourn), whose output switched on a LED, placed
in the front of the cage, indicating, on the monitor, stimulus start and duration. Stimuli (300
Hz, 2 s) were randomly delivered as for spatial localization and intertrial interval (from 1 to
5 min; a mean of six stimuli in each trial) to avoid habituation; their intensity was checked
not to induce avoidance responses.
Behavioral analysis
Six groups of animals, each composed of 10 rats, were tested during one session only.
One group received acoustic stimulation, one was stimulated and received vehicle solution
IP, and four groups were stimulated and administered different doses of APO (0.1, 0.5, 1.0,
and 2.0 mg/kg IP). Animal’s habituation to novel environment was performed twenty-four
hrs before trials and consisted of ten min exploration of the observation box, without delivering any acoustic stimuli.
Experiments, lasting 20 min, were recorded on videotapes by a videocamera apparatus
and analyzed via a videocassette recorder.
Motor sequence was measured on playback in slow motion and frame-by-frame with
a temporal resolution of 40 ms. HT latency and duration were calculated analyzing initial
and final frame. HT initial time was considered to occur the frame preceding the start
of head-turning movement following acoustic stimulation, and HT final time the frame
preceding the end of head movement. Interval between onset of acoustic stimulus and
beginning of head movement indicated HT latency, and interval between HT initial and final
time indicated HT duration. Experiments were conducted in accordance with the European
Communities Council Directive (86/609/EEC) regarding care and use of animals for experimental procedures.
Apomorphine hydrochloride (Sigma Chemical Co., St. Louis, MO) was dissolved in saline
and injected intraperitoneally at concentration of 1.0 mg/ml at a dose of 0.1, 0.5, 1.0 and
2.0 mg/kg. Control group received same volume of vehicle calculated at the 1.0 mg/kg
Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno
Statistical analysis
All values are expressed as means + S.D. Statistical analysis was performed using one
way analysis of variance (ANOVA) followed by the Newman-Keuls (N-K) post-hoc test for
multiple comparisons. Values of p < 0.05 were considered significant. Unpaired Student’s
t-test (two-tailed) was used when comparison was made between vehicle and only acoustically treated group.
Acoustic stimulation provoked in the rat a head orienting movement toward stimulus
source. Animal position, relatively to the active loudspeaker, varied throughout trials along
a 360В° degrees spectrum, depending on a) animal standing area in the box when the randomized acoustic stimulus occurred, and b) interstimulus animal activity. Frame-by-frame
analysis of responses revealed a sequence of fixed motor acts: auricle erection, coordinated eye deviation, neck and head torsion, with drug beginning to take effect (i.e. when
animals showed first behavioral change) 1-2 min post injection. Likely because of rapid
metabolism of the compound [23], the specific behavioral effects, we investigated, lasted
no longer than 20 min.
Two specific parameters of motor sequence were analyzed: HT latency and duration.
As to latency, the mean + S.D. was 184.21 + 48.30 ms and to duration 257.82 + 49.19
ms. Results, observed in animals only delivered acoustic stimulation, were not significantly
modified by vehicle administration (mean + S.D. 181.15 + 50.66 ms for latency, p<0.892
unpaired Student’s t-test, and 261.30 + 46.48 ms for duration, p<0.873 unpaired Student’s t-test). Moreover, to study whether rat response may change depending on different
frequencies (100 to 500 Hz, steps of 100 Hz) and durations (1, 2, 3 sec) of acoustic stimulus, control tests were carried out for each variation of parameters and for each dose
of APO (two animals for each control test). Only slight and non significant modifications of
HT latency and duration were observed. However, following acoustic stimuli at frequency
of 500 Hz, coupled to all train durations, animals did not orient their heads, but exhibited a
jerk interfering with the orienting response.
When the drug began to be effective, low doses induced whisker movements, occasionally-occurred sequences of head oscillations and the typical snout-to-surface (of the box
walls) contact. Larger doses also provoked licking, gnawing, sniffing, and rare climbing
episodes. Neither retching, nor other signs of nausea or general discomfort were observed. Acoustically-evoked HT movements, occurring during sequences of oscillatory head
movements, were not taken into consideration. On the other hand, a careful interstimulus
analysis allowed to discriminate behavioral modifications if depending or not on acoustic
Effects of Apomorphine on Orienting Reaction in the Rat
ANOVA test showed that APO IP administration evoked a significant decrease of HT
latency when compared to vehicle injected animals. As for HT latencies, means + S.D. for
each dosage were 188.61 + 43.82 ms, 191.36 + 45.67 ms, 125.40 + 33.78 ms, 115.52
+ 37.12 ms respectively, F(4,49)=7.46 p<0.0001 (fig. 1). As for HT durations, means +
S.D. for each dosage were 282.56 + 38.17 ms, 274.45 + 46.86 ms, 243.29 + 43.90 ms,
231.44 + 41.63 ms respectively, F(4,49)=2.38, p<0.066 (fig. 1). Newman-Keuls post hoc
comparisons between drug and corresponding control conditions (vehicle injected animals) revealed that the two larger doses of APO (1.0 and 2.0 mg /kg) produced significant
decrease of HT latency (p<0.05), whereas they failed to induce significant modifications of
HT duration. As for APO at 0.1 and 0.5 mg/kg, post-hoc analysis showed that both doses
failed to induce significant changes.
Results of present research clearly indicate that APO provokes significant modifications
of the orienting movement induced by an acoustic stimulation. Specifically, a decrease of
HT latency was observed. Moreover, post-hoc analysis revealed one main, significant effect, i.e. a decrease of HT latency with the two larger doses which, on the other hand, failed
to induce significant modifications on HT durations. A slight, non significant increases of
both HT parameters resulted from post-hoc analysis of APO effects with the lower doses.
These findings give rise to some interesting questions: 1) which central mechanisms
are influenced by APO and, 2) which APO properties could explain the effects.
1) Nigrostriatal and mesocorticolimbic DA systems have been demonstrated to be closely involved in orienting behavior and in regulation of attentive condition [4-6,11,13,24,25].
Accordingly, changes in A9 and A10 neuronal activity, e.g. trains of burst firing, were observed in response to different sensory stimuli and during orientation [9-15]. Lower doses
of APO inhibited A9 and A10 DA neuron firing, on the contrary larger doses increased it
[21]. On the other hand, a slowed reaction capacity was observed in rats treated with the
DA antagonist chlorpromazine [26] or О±-flupenthyxol [27]. Our results suggest a possible
correlation between rapid APO-induced modifications of A9 and A10 neuronal firing and
rapid modifications of specific parameters of a sensory-triggered motor response (sensorimotor integration).
Since HT, which follows an acoustic stimulation, can be considered as a component of
the orienting reaction toward environmental stimuli, changes of HT latency might depend
on APO influence on DA systems regulating sensorimotor integration and attention shift
[28]. These functions have been suggested to be under the control of both mesolimbicmesocortical system, where DA neurons of VTA change their activity after presentation of
different kinds of sensory stimuli [11,29,15,25] and of neostriatum, which shares these
Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno
properties [9,7,12,14], and also receives input from brain areas associated with sensory as
well as motor processing [30]. Moreover, dopamine receptor agonists, like bromocriptine
and APO, induced an acute recovery of function from severe neglect produced by unilateral
destruction of posterior parietal cortex in rats. This effect seems to be due to an activity
on a DA system underlying directed attention and involving medial agranular cortex and
its connection to the neo-striatum [31]. In addition, nigrostriatal dopamine system has
long been associated with control of motor and postural mechanisms [12]; hence, APOinduced modifications of HT duration may arise from a specific influence on neurons of
this system.
2) As to specific drug properties, implicated in the observed effects, it has been previously demonstrated that APO administration resulted in a significant restoration of
orientation to all modalities of stimuli [17]. Further interesting remarks are suggested
by the slight increase of HT latency and duration, when doses of 0.1 and 0.5 mg/kg
were administered. If on the one hand, this effect resembles inhibition of operant motor
performance obtained in rats trained to press force-sensitive beams [24], on the other
hand, these delayed or weaker motor responses may be explained taking into account
that dopamine autoreceptors are more sensitive to the direct-acting dopamine agonist
APO than postsynaptic receptors are [32]. In parallel studies, different effects were obtained. In fact, APO caused dose-related modifications of lever-press responding for reward
in an instrumental motor task, i.e., a general increase was observed both when stimuli
were paired to conditioned reinforcers, and when they were not [33]. Moreover, single i.v.
APO injection to freely moving rats has been reported to decrease A9 DA neuron activity
[21,9]. Thus, effects of lower APO doses on DA neuronal activity and on orienting parameters may be due to selective activation of autoreceptors with a decrease of dopamine
release and synthesis [34-36]. On the contrary, since larger doses have been proved to
influence postsynaptic receptors, inducing specific behavioral effects [37,38], an increased APO-induced DA activity might provoke a facilitation of dopamine-mediated response
to environmental stimuli, thus resulting a decrease of response latency and a facilitation
of orienting shift. In addition, APO stimulates both D1 and D2 receptors and this activity
has been mimicked in experiments where intracerebral or systemic injections of D1 and
D2 agonists were carried out provoking enhancement of the rewarding effect of brain stimulation [39]. These evidences suggest that modifications of HT parameters could not be
exclusively dependent on pre-or-postsynaptic drug activity, but also on a specific D1 and/
or D2 mediation [40].
In conclusion, drugs affecting DA activity can be effectively used to identify nature,
timing and duration of different stages of sensorimotor integration, a function often damaged in diseases arising from an altered DA transmission.
Effects of Apomorphine on Orienting Reaction in the Rat
Effects of different doses of apomorphine (0.1, 0.5, 1.0, 2.0 mg/kg IP, hatched columns) and vehicle solution
(blank columns) on HT latency and duration. Each bar shows the mean + S.D. of results obtained from the
analysis of 10 animals, p < 0.0001 for latency and p < 0.066 for duration (ANOVA). * Newman-Keuls significant (О± = 0.05) post-hoc comparison.
Maurizio Casarrubea, Filippina Sorbera, Giuseppe Crescimanno
[1] Muir JL. Attention and stimulus processing in the rat. Cogn Brain Res 1996; 3: 215-25.
[2] Siddle D, Stephenson D, Spinks JA. Elicitation and habituation of the orienting response. In: Siddle, D.,
(ed.), Orienting and habituation: Perspectives in human research. John Wiley & Sons, New York, 1983;
pp. 109-82.
[3] Sokolov EN. Neuronal models and the orienting reflex. In: Brazier, M.A.B., (Ed.), The central nervous
system and behaviour. Josiah Macy Jr Foundation, New York, 1960; pp. 187-276.
[4] Bos van den R, Charria-Ortiz GA, Bergmans AC, Cools AR. Evidence that dopamine in the nucleus accumbens is involved in the ability of rats to switch to cue-directed behaviors. Brain Res. 1991; 42:107-14.
[5] Conde H, Benita M, Dormont JF, Schmied A, Cadoret A. Control of reaction time performance involves
the striatum. J Physiol (Paris) 1981; 77: 97-105.
[6] Crescimanno G, Sorbera F, Emmi A, Amato G. Inhibitory effect of A10 dopaminergic neurons of the
ventral tegmental area on the orienting response evoked by acoustic stimulation in the cat. Brain Res
Bull 1998; 45: 61-65.
[7] Keller RW jr, Stricker EM, Zigmond MJ. Environmental stimuli but not homeostatic challenges produce
apparent increases in dopaminergic activity in the striatum: an analysis by in vivo voltammetry. Brain
Res 1983; 279: 159-70.
[8] Le Moal M, Simon H. Mesocorticolimbic dopaminergic network: functional and regulatory role. Physiol
Rev 1991; 71: 155-233.
[9] Freeman AS, Meltzer LT, Bunney BS. Firing properties of substantia nigra dopaminergic neurons in
freely moving rats. Life Sci 1985; 36: 1983-94.
[10] Horvitz JC, Stewart T, Jacobs BL. Burst activity of ventral tegmental dopamine neurons is elicited by
sensory stimuli in the awake cat. Brain Res 1997; 759: 251-58.
[11] Kosobud A E K, Harris GC, Chapin JK. Behavioral association of neuronal activity in the ventral tegmental area of the rat. J Neurosci 1994; 14: 7117-29.
[12] Overton PG, Clark D. Burst firing in midbrain dopaminergic neurons. Brain Res Rev 1998; 25: 312-34.
[13] Schultz W, Romo R. Dopamine neurons of the monkey midbrain: contingencies of responses to stimuli
eliciting behavioral reactions. J Neurophysiol 1990; 63: 607-24.
[14] Sesack SR, Bunney BS. Central dopaminergic systems: neurophysiology and electrophysiological pharmacology. In: Henn, F.A., De Lisi, L.E., (Eds.), Handbook of schizophrenia Vol. 2: “Neurochemistry and
Neuropharmacology of Schizophrenia”. Elsevier Science Publishers, New York, 1987; pp. 149-78.
[15] West CHK, Michael RP. Responses of units in the mesolimbic system to olfactory and somatosensory
stimuli: modulation of sensory input by ventral tegmental stimulation. Brain Res 1990; 532: 307-16.
[16] Marshall JF, Berrios N, Sawyer S. Neostriatal dopamine and sensory inattention. J Comp Neurol 1980;
94: 833-46.
[17] Marshall JF, Gotthelf T. Sensory inattention in rats with 6-hydroxydopamine-induced degeneration of
ascending dopaminergic neurons: apomorphine-induced reversal of deficits. Exp Neurol 1979; 65:
[18] Robbins TW, Everitt BJ. Functions of dopamine in the dorsal and ventral striatum. Semin Neurosci 1992;
4: 119-27.
[19] Crescimanno G, Mannino M, Casarrubea M, Amato G. 2000. Effects of sulpiride on the orienting movement evoked by acoustic stimulation in the rat. Pharmacol Biochem Behav 2000; 66: 747-50.
[20] Corrodi H, Fuxe K, Hokfelt T. Effect of ergot drugs on central catecholamine neurons: evidence for a
stimulation of central dopamine neurons. J Pharm Pharmacol 1973; 25: 409-12.
Effects of Apomorphine on Orienting Reaction in the Rat
[21] Freeman AS, Bunney BS. Activity of A9 and A10 dopaminergic neurons in unrestrained rats: further
characterization and effects of apomorphine and cholecystokinin. Brain Res 1987; 405: 46-55.
[22] Ostry DJ. Execution-time movement control. In Stelmach G.E., Requin J., (Eds.), Tutorials in motor
behavior. North Holland Publishing Company, Amsterdam, 1980; pp. 457-68.
[23] Davis M, Aghajanian GK. Effects of apomorphine and haloperidol on the acoustic startle response in
rats. Psychopharmacology 1976; 47: 217-23.
[24] Xiaoqing L, Strecker RE, Brener JM. Low doses of apomorphine suppress operant motor performance
in rats. Pharmacol Biochem Behav 1996; 53: 335-40.
[25] Zhang H, Kiyatkin EA, Stein EA. Behavioral and pharmacological modulation of ventral tegmental dendritic dopamine release. Brain Res 1994; 656: 59-70.
[26] Spirduso WW, Abraham LD, Wolf MD. Effects of chlorpromazine on escape and avoidance responses: a
closer look. Pharmacol Biochem Behav 1980; 14: 433-38.
[27] Amalrich M, Koob GF. Depletion of dopamine in the caudate nucleus but not nucleus accumbens impairs reaction time performance in rats. J Neurosci 1987; 7: 2129-34.
[28] Yee BK, Russig H, Feldon J. Apomorphine-induced prepulse inhibition disruption is associated with a paradoxical enhancement of prepulse stimulus reactivity. Neuropsychopharmacology 2004; 29: 240-48.
[29] Ljungberg T, Apicella P, Schultz W. Responses of monkey dopamine neurons during learning of behavioral reactions. J Neurophysiol 1992; 67: 145-63.
[30] Pycock CJ, Phillipson OT. A neuroanatomical and neuropharmacological analysis of basal ganglia output. In: Iversen, L.L., Iversen, S.D., Snyder, S.H., (Eds.), Handbook of Psychopharmacology vol. 18,
“Drugs, Neurotransmitters and Behavior”. Plenum Press, New York, 1984; pp. 191-278.
[31] Corwin JV, Burcham KJ, Hix GI. Apomorphine produces an acute dose-dependent therapeutic effect
on neglect produced by unilateral destruction of the posterior parietal cortex in rats. Behav Brain Res
1996; 79: 41-49.
[32] Skirboll LR, Grace AA, Bunney BS. Dopamine auto- and postsynaptic receptors: electrophysiological
evidence for different sensitivity to dopamine agonists. Science, 1979; 206: 80-2.
[33] Robbins TW, Watson BA, Gaskin M, Ennis C. Contrasting interactions of pipradrol, d-amphetamine,
cocaine, cocaine analogues, apomorphine and other drugs with conditioned reinforcement. Psychopharmacology 1983; 80: 113-19.
[34] Aghajanian GK, Bunney BS. Pre- and postsynaptic feedback mechanisms in central dopaminergic neurons. In: Seeman, P., Brown, J.M. (Eds.), Frontiers of neurology and neuroscience research. University of
Toronto Press, Toronto, 1974; pp. 4-11.
[35] Di Chiara G, Corsini GU, Mereu GP, Tissari A, Gessa GL. Self inhibitory dopamine receptors: Their role in
the biochemical and behavioral effects of low doses of apomorphine. In: Roberts, P.J., (Ed.) Advances
in biochemical psychopharmacology. Raven Press, New York, 1978; pp. 275-84.
[36] Seeman P, Van Tol HHM. Dopamine receptor pharmacology. Trends Pharmacol Sci. 1994; 15: 264-69.
[37] Bunney BS. Neuroleptics: electrophysiological pharmacology. In: Coyle, J.T., Enna, S.J. (Eds.), Neuroleptics: Neurochemical, Behavioral, and Clinical perspectives. Raven Press, New York, 1983; pp. 157-81.
[38] Xiaoqing L, Strecker RE, Brener JM. Low doses of apomorphine suppress operant motor performance
in rats. Pharmacol Biochem Behav 1996; 53: 335-40.
[39] Ranaldi R, Beninger RJ. The effects of systemic and intracerebral injections of D1 and D2 agonists on
brain stimulation reward. Brain Res 1994; 651: 283-92.
[40] Pezze MA, Dalley JW, Robbins TW. Differential roles of dopamine D1 and D2 receptors in the nucleus
accumbens in attentional performance on the five-choice serial reaction time task. Neuropsychopharmacology 2007; 32: 273-83.
Clinical Embryology
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
The balance of Hsps expression during pregnancy: the
role of placenta
[Il bilancio dell’espressione delle HSPs nella gravidanza: il ruolo della placenta]
Simona Corrao1#, Giampiero La Rocca1#, Rita Anzalone1, Francesca Magno1, Tiziana Loria1,
Melania Lo Iacono1, Everly Conway de Macario2, Alberto JL Macario2, Francesco Cappello1*
and Felicia Farina1
#The two authors contributed equally to this work. 1Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, UniversitГ degli Studi di Palermo (IT); 2University of Maryland Biotechnology Institute, Center of
Marine Biotechnology, Baltimore MD (USA). *Corresponding author
Key words: Placenta, Placental Pathologies, Pregnancy, Heat Shock Proteins, Chaperones, Immune Regulation, Immune Tolerance
Parole chiave: Placenta, Patologie Placentari, Gravidanza, Proteine da Shock Termico, Chaperoni, Regolazione Immunitaria, Tolleranza Immunitaria
Abstract. The outcome of pregnancy depends on a series of events that start from embryo implantation and
maintenance and end, under normal conditions, with a full-term delivery, passing through those events that
underlie correct embryo development. Placenta has a key role in all of these processes. Placenta constitutes
a highly differentiated organ of extra-embryonic origin with a structure characterized by a successive series
of membranes: amnion, chorion, trophoblast of chorionic villi, and decidua, through which mother/foetus
metabolic exchanges occur. Chorionic villi and respective capillaries are chiefly responsible for metabolic
exchange. Any deviation in placental structures causes foetus damage, resulting in miscarriage, or other adverse effects. In pregnancy maintenance, inhibition of the immune response is essential to prevent a reaction
against the semi-allogeneic embryo. Here we present the placenta’s components of the chaperoning system,
including heat-shock proteins (Hsps) and chaperones. Hsps, many of which are chaperones, are involved in a
pivotal manner during placentation, embryogenesis, and immune response regulation to allow foetal growth.
Hence, a better understanding of the various aspects of these proteins (synthesis, modifications, structure,
gene regulation, and function), and of those cellular processes associated with Hsps in the placenta is of
primary importance for the recognition and treatment of placental pathologies.
Riassunto. La riuscita di una gravidanza è legata ad una serie di eventi che iniziano dall’impianto e mantenimento dell’embrione e che si concludono, in condizioni normali, con il parto a termine, passando attraverso
quei meccanismi che sono alla base del corretto sviluppo embrionale. In tutti questi processi, la placenta
svolge un ruolo fondamentale. La placenta ГЁ un organo altamente differenziato di origine extra-embrionale;
la sua struttura è caratterizzata da una serie di membrane: l’amnios, il corion, il trofoblasto dei villi coriali e la
decidua, attraverso cui avvengono gli scambi metabolici madre/feto. I villi coriali ed i relativi capillari sono i
principali responsabili degli scambi metabolici. Ogni modificazione della struttura placentare si puГІ riflettere
in un danno al feto, con effetti avversi che possono culminare nell’aborto. Nel mantenimento dello stato di
gravidanza è essenziale l’inibizione della risposta immunitaria nei confronti del embrione semi-allogeneico.
In questo articolo, noi presentiamo le componenti placentari del sistema chaperonico, comprendente pro-
Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono,
Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina
teine da shock termico (Hsps) e chaperoni. Le Hsps, molte delle quali sono chaperoni, sono coinvolte in modo
cruciale durante la placentazione, l’embriogenesi e la regolazione della risposta immune, per permettere la
crescita fetale. Quindi, una migliore comprensione delle caratteristiche biomolecolari di tali proteine (sintesi,
modificazioni, struttura, regolazione genica e funzione) e dei meccanismi cellulari ad esse associati nella
placenta potrebbero assumere una importanza fondamentale nel riconoscimento e nel trattamento delle
patologie placentari.
Morphology of placenta and physiological implications
Placenta is a component of extra-embryonic annexes. It is a highly differentiated organ
in which reside the mechanisms that create the conditions for embryo implantation and
correct growth of the foetus, i.e. respiration, nourishment, and metabolic exchanges. Placenta also has a controlled and complex endocrine activity.
Placental structure varies throughout pregnancy. The trophoblast is the first layer that is
formed during transition from morula to blastocyst stage. The next layer is the hypoblast,
which originates inside the trophoblast and beneath the inner cell mass of the blastocyst.
Finally, the chorion originates from the trophoblast that is backed by mesoderm [1, and refs.
therein]. The serial organization of human placenta membranes at term from the side of the
foetus to the side of the uterus consists of amnion, chorion, trophoblast of chorionic villi, and
decidua, (Fig.1) [2]. During placentation, vascularization is the most important event that induces the formation of continuous capillaries, allowing metabolic exchanges at the mother/
foetus interface. Maintenance of the structure of the capillary endothelium is necessary for
correct vascular permeability [3]. Foetal blood is separated from the maternal blood by three tissue layers that constitute the placental barrier: foetal capillary endothelium, stromal
connective tissue of the villus, and chorionic epithelium of the villus (or trophoblast). The
trophoblast is constituted of two layers, the cytotrophoblast (inner proliferative zone) and
the syncytiotrophoblast (outer terminally differentiated epithelium) (Fig.1).
All of the placental structures, especially the villous tree, are involved in the finely regulated functional interaction between mother and foetus. Full-term pregnancy depends on
a globally good condition of placental components. Pre-term birth is a common cause of
perinatal morbidity and death, often related to infections or utero-placental vascular disease [4]. The thickness of the villous epithelium depends on correct proliferation of cytotrophoblast cells and their recruitment into syncytiotrophoblast. Since the villous epithelium
is involved in metabolic exchanges, any deviation of its thickness beyond the physiological
range will affect diffusive conductance. Alterations in placental barrier permeability due,
for example, to chronic inflammation and/or immune complex formation, will contribute to
the development of pregnancy-related disorders such as pre-eclampsia [5,6] and intrauterine growth restriction (IUGR) [6,7,8].
The balance of Hsp expression during pregnancy: the role of placenta
Fig.1: Organization of human placenta at term. A, amnion; B, chorion; C, villous tree; D, foetal capillary; E,
syncytiotrophoblast; F, cytotrophoblast; G, decidua. Other structures such as umbilical vein (H) and arteries (I),
and maternal arteries (J) and veins (K) are also shown. Oxygen and nutrients exchanges between maternal
oxygenated blood and foetal deoxygenated blood occur in the intervillous space (L).
Placenta as a secretory organ: hormones and Hsps and their involvement in placental development and pathogenesis
In addition to its fundamental role in connecting anatomically and physiologically mother and foetus, the placenta has an important endocrine activity during pregnancy that
allows embryo implantation. In the early stages of pregnancy, the placenta produces si151
Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono,
Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina
gnificant amounts of progesterone and estrogens, substituting and influencing the ovary’s
activity. The placental endocrine activity includes the release of Chorionic Gonadotrophin
(hCG); Placental Lactogen (hPL); growth factors such as Epidermal Growth Factor (EGF),
Insulin-like Growth Factor (IGF)-I and -II, and Platelet-derived Growth Factor (PDGF); and
chemokines and cytokines [9]. Placenta also releases Vascular Endothelial Growth Factor
(VEGF) [10]. These hormones act together modifying the uterine structure and creating the
correct environment to ensure embryo survival and growth.
In addition to being a part of the endocrine system, placenta also contributes to the
chaperoning system. For instance, the decidua has also a role in the establishment of pregnancy possibly with the participation of Hsps as suggested by the fact that during the first
trimester of pregnancy, both the decidua and placenta produce a series of Hsps [11,12].
Hsps are highly conserved cellular proteins, many of which are the products of stressinducible genes present in all organisms (from bacteria to man) and many of which are
chaperones. These proteins along with other molecules are part of the chaperoning system
whose main function is the maintenance of protein homeostasis [see article by Macario
and Conway de Macario in this volume]. Hsps and chaperones are major components of
the anti-stress mechanisms that are activated in all cell types that undergo endogenous or
exogenous stress, and prevent and correct molecular damage [13]. Participation of Hsps
in various steps of reproduction has been reported, including spermatogenesis and oogenesis [14], probably because of their roles in the maintenance of a high cell-proliferation
rate and in the maturation of newly synthesised proteins as they are chaperones. The trophoblast cells of blastocyst proliferate rapidly during implantation, differentiating into inner
cytotrophoblast and outer syncytiotrophoblast, all of which are accompanied by a series of
invasion mechanisms on endometrial connective tissue [rev. in 9]. Possible roles of Hsps in
protein synthesis and cell-damage prevention at these stages are likely, so these proteins
would be key players in foetal development and in the outcome of pregnancy. Hsp27 is
actively transcribed during trophoblast differentiation, during which acts as a regulator of
actin filaments structure and dynamics. Moreover, Hsp27 expression levels are related to
expression of molecules involved in trophoblast invasion and motility such as MMP2 and
iNOS [15]. Hsp27 interacts with isoform beta (ERb) of the estrogen receptor, acting as a
co-repressor. Since ERb is over-expressed in vascular injury, Hsp27 could also be involved
in vascular disease [16, and refs. therein]. An open question is whether Hsp27 has a role
in vascular disease-related pre-term birth.
Other Hsps, such as Hsp90 and Hsp60, were investigated in order to determine their
involvement in stress-related pre-term labour. The levels of these two Hsps were unchanged during the first trimester of pregnancy and also did not change following the onset
of labour [17]. An immunohistochemical investigation was carried out on the levels of
The balance of Hsp expression during pregnancy: the role of placenta
Hsp70 and Hsp90 in chorionic villi of women who experienced first trimester miscarriage
compared to the levels in the placentas of women who had full-term, normal delivery [18].
Both, Hsp70 and Hsp90 (Hsp90A and Hsp90B isoforms) were found increased in the cases
with miscarriage compared to full-term placentas. These findings suggest participation of
the Hsps investigated in the miscarriage mechanisms and/or reflect the stressful situation
inherent to pregnancy and miscarriage. An association between over-expression of Hsp70
and endothelial activation and immune response in placental vascular disease has been
reported [19]. Hsp70 might interact with proteins involved in cell cycle modulation in embryo development and thus play a role in the whole process of pregnancy at the placental,
foetal, and maternal levels [rev. in 20].
Since a successful pregnancy depends on first cleavage steps of the zygote and, therefore, on embryo development and cell differentiation, and because of ethical problems
in carrying out studies on human embryos, most of the information about involvement of
Hsps in embryogenesis comes from studies in animal models. For instance, in an in vitro
mouse model, using antibodies specific for Hsp60, Hsp70, and Hsp90, an impairment of
embryo development was shown after 3, 5, and 7 days respectively [21].
Hsp70 has also been found extracellularly and thereby participates, along with other
Hsps, in the regulation of the immune system [22,23]. Microbial Hsp60 and Hsp70, which
can originate from bacteria in genital tract infections and penetrate into circulation before
or during pregnancy, stimulate the immune system to produce antibodies. These antibodies can cross-react with the human Hsps, inducing autoimmunity, pro-inflammatory responses and, probably, pre-term delivery [24] or other adverse pregnancy outcomes [25].
In women with unexplained recurrent spontaneous abortions, elevated levels of the Th1
cytokines (IFNОі and TNFО±) were observed, compared to women with a normal pregnancy;
the latter showed, instead, higher Th2 cytokines production than the other women [26]. All
these observations reveal that a very interesting aspect of placental physiopathology is its
involvement in the regulation of the immune system. The foetus possesses a half paternal
chromosomal inheritance and, therefore, represents a homologue graft against which the
maternal immune system might react.
Pregnancy establishment seems to be tightly related to Hsps release. An important role
is probably performed by Hsp10 during the first stages of pregnancy. In fact, Hsp10 was
previously known as Early Pregnancy Factor (EPF) because of its release during the first
stages of development, within 24h after oocyte fertilization and continuing until, at least,
up to the first half of gestation [27]. EPF was first purified and characterized from various
sources, including human pregnancy sera [28], and human platelets, showing a molecular
weight of 10,843 Da and an amino-acid sequence approximately 70% identical to that of
the rat mitochondrial chaperonin 10 (Cpn10), the homologue of human Hsp10 [29]. Further
Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono,
Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina
analyses demonstrated that the EPF gene coding sequence has high similarity with the
sequence of the human Hsp10 gene [30]. EPF is thought to be the extracellular form of
Hsp10 [31,32].
Current knowledge about EPF and its participation in the suppression of the immune
system and, thus, in facilitating the establishment of pregnancy, lead us to hypothesize
that Hsp10 plays a crucial role in various aspects of the embryos’ life and beyond. In fact,
the downregulation effect of Hsp10 on immune cells was also seen in ovarian cancer [33].
Marked inhibition was observed of the expression of the signal-transducing zeta chain
associated with the T cell receptor (TCR), known as CD3-zeta [33]. Also, it was observed
that during pregnancy a T-cell signaling modulation by pregnancy-associated exosomes
(which have been isolated from the blood of pregnant women) [34], which suggests that
the exosomes might be involved in the mechanism of Hsp10/EPF release. Likely, EPF is
also involved in embryonic development, as seen in in vivo and in vitro mouse models [35].
These observations regarding EPF suggest that Hsp10 could play key roles in cell differentiation during placentation and, consequently, any modifications in its expression levels
could have an impact on the progression of pregnancy.
Conclusions and perspectives
The complex mechanisms that take part in all the events starting from oocyte fertilization and leading to a full-term gestation are finely regulated by an intricate network
of hormones, proteins, and immune response-related molecules. The balance between
endogenous and exogenous stresses can influence placental functions, leading to damage
that could have and impact on embryo fate. The role of the chaperoning system, Hsps in
particular, in normal and pathological placenta is not yet fully understood. Further studies
about the functions of Hsps and chaperones should be carried out in order to give a definitive response to as yet unanswered questions such as, how do Hsps participate in the
interaction between mother and foetus through the placenta? And, what kind of pathways
are induced or blocked by Hsps in pregnancy-related pathological conditions? These and
other similar questions represent a stimulating challenge to basic and applied scientists
alike who are interested in the chaperoning system and its role in health and disease.
The balance of Hsp expression during pregnancy: the role of placenta
[1] Enders AC, Blankenship TN. Comparative placental structure. Adv Drug Deliv Rev 1999; 38:3-15.
[2] Benirschke K. Remarkable placenta. Clin Anat 1998; 11:194-205.
[3] Firth JA and Leach L. Structure and permeability in human placental capillaries- a review. Trophobl Res
1997; 10:205-213.
[4] Baker AM, Braun JM, Salafia CM, Herring AH, Daniels J, Rankins N, Thorp JM. Risk factors for uteroplacental vascular compromise and inflammation. Am J Obstet Gynecol 2008; 199:256.e1-256.e9.
[5] Mayhew TM, Leach L. Aspect of structure and function in human placenta. Curr Obstet Gynaecol 1998;
[6] Mayhew TM, Mangani R, Ohadike C, Wijsekara J, Baker PN. The placenta in pre-eclampsia and in intrauterine growth restriction: studies on exchange surface areas, diffusion distances and villous membrane diffusive conductances. Placenta 2007; 28:233-238.
[7] Chaddha V, Viero S, Huppertz B, Kingdom J. Developmental biology of the placenta and the origins of
placental insufficiency. Semin Fet Neonat Med 2004; 9:357-369.
[8] Huppertz B, Burton G, Cross JC, Kingdom JCP. Placental morphology: from molecule to mother-a dedication to Peter Kaufmann-a review. Placenta 2006; 27:S3-S8.
[9] Gude NM, Roberts CT, Kalionis B, King RJ. Growth and function of the normal placenta. Thromb Res
2004; 114:397-407.
[10] Kingdom J, Huppertz B, Seaword G, Kaufmann P. Development of the placental villous tree and its
consequences for foetal growth. Eur J Obstet Gynecol Reprod Biol 2000; 92:35-43.
[11] Shah M, Stanek J, Handwerger S. Differential localization of heat shock proteins 90, 70, 60 and 27 in
human decidua and placenta during pregnancy. Histochem J 1998; 30:509-518.
[12] Ron A, Birkenfeld A. Stress proteins in the human endometrium and decidua. Hum Reprod 1987; 2:277280.
[13] Macario AJL, Conway de Macario E. The pathology of anti-stress mechanisms: a new frontier. Stress
2004; 7:243-249.
[14] Neuer A, Spandorfer SD, Giraldo P, Dieterle S, Rosenwaks Z, Witkin SS. The role of heat shock proteins
in reproduction. Hum Reprod Update 2000; 6:149-159.
[15] Matalon ST, Drucker L, Fishman A, Ornoy A, Lishner M. The role of heat shock protein 27 in extravillous
trophoblast differentiation. J Cell Biochem 2008; 103:719-729.
[16] Miller H, Poon S, Hippert B, Rayner K, Chen Y-X, O’Brien ER. Modulation of estrogen signaling by the novel
interaction of Heat shock Protein 27, a biomarker for atherosclerosis, and Estrogen Receptor ОІ. Mechanistic insight into the vascular effects of estrogen. Artherioscler Thromb Vasc Biol 2005; 25:e10-e14.
[17] Divers MJ, Bulmer JN, Miller D, Lilford NJ. Placental heat shock proteins: no immunohistochemical evidence for a differential stress response in pre-term labour. Gynecol Obstet Invest 1995; 40:236-243.
[18] Sotiriou S, Liatsos K, Ladopoulos I, Arvantis DL. A comparison of heat shock proteins (HSP) 70 and 90
on chorionic villi of human placenta in normal pregnancies and missed miscarriages. Clin Exp Obstet
Gynecol 2004; 31:185-190.
[19] Liu Y, Li N, You L, Liu X, Li H, Wang X. HSP70 is associated with endothelial activation in placental vascular diseases. Mol Med 2008; 14:561-566.
[20] Luft JC and Dix DJ. Hsp70 expression and function during embryogenesis. Cell Stress Chaperones
1999; 4:162-170.
[21] Neuer A, Mele C, Liu HC, Rosenwaks Z, Witkin SS. Monoclonal antibodies to mammalian heat shock
proteins impair mouse embryo development in vitro. Hum Reprod 1998; 13:987-990.
Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono,
Everly Conway de Macario, Alberto JL Macario, Francesco Cappello, Felicia Farina
[22] Pockley AG, Muthana M, Calderwood SK. The dual immunoregulatory roles of stress proteins. Trends
Biochem Sci 2008; 33:71-79.
[23] Cappello F, Conway de Macario E., Marasa L, Zummo G, Macario AJL. Hsp60: new locations, functions,
and perspectives for cancer diagnosis and therapy. Cancer Biol Ther 2008; 7:801-809.
[24] Ziegert M, Witkin SS, Sziller I, Alexander H, Brylla E, Hortig W. Heat Shock Proteins and Heat Shock
Protein-antibody complexes in placental tissues. Infect Dis Obstet Gynecol 1999; 7:180-185.
[25] Witkin SS. Immunity to Heat Shock Proteins and pregnancy outcome. Infect Dis Obstet Gynecol 1999;
[26] Raghupathy R, Makhseed M, Azizis F, Hassan N, Al-Azemi M, Al-Shamali E. Maternal Th1- and Th2-type
reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous
abortions. Cell Immunol 1999; 196:122-130.
[27] Morton H, Rolfe BE, Cavanagh AC. Ovum factor and early pregnancy factor. Curr Top Dev Biol 1987;
[28] Mehta AR, Eessalu TE, Aggarwal BB. Purification and characterization of Early Pregnancy Factor from
human pregnancy sera. J Biol Chem 1989; 264:2266-2271.
[29] Cavanagh AC, Morton H. The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10. Eur J Biochem 1994; 222:551-560.
[30] Summers KM, Murphy RM, Webb GC, Peters GB, Morton H, Cassady AI, Cavanagh AC. The human early
pregnancy factor/chaperonin 10 gene family. Biochem Mol Med 1996; 58:52-58.
[31] Cavanagh AC. Identification of early pregnancy factor as chaperonin 10: implications for understanding
its role. Rev Reprod 1996; 1:28-32.
[32] Morton H. Early pregnancy factor: an extracellular chaperonin 10 homologue. Immunol Cell Biol 1998;
[33] Akyol S, Gercel-Taylor C, Reynolds LC, Taylor DD. HSP10 in ovarian cancer: expression and suppression
of T-cell signalling. Gynecol Oncol 2006; 101:481-486.
[34] Taylor DD, Akyol S and Garcel-Taylor C. Pregnancy-associated exosomes and their modulaton of T cell
signaling. J Immunol 2006; 176:1534-1542.
[35] Athanasas-Platsis S, Corcoran CM, Kaye PL, Cavanagh AC, Morton H. Early Pregnancy Factor is required
at two important stages of embryonic development in the mouse. Am J Reprod Immunol 2000; 43:223233.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Expression of cytokeratins in human umbilical cord at
[Espressione delle citocheratine nel cordone ombelicale umano a termine]
Annamaria Mauro, Maria Buscemi and Aldo Gerbino
Departiment of Experimental Medicine, Section of Histology and Embryology, School of Medicine, University
of Palermo (IT).
Key words: Human umbilical cord, Cytokeratin, Immunohistochemistry, RT-PCR
Parole chiave: Cordone ombelicale umano, Citocheratine, Immunoistochimica, RT-PCR
Abstract. Background. Cytokeratins are the proteins that costitute intermediate filaments. In the cytoplasm,
keratin filaments extend from the nuclear membrane to the inner side of the plasma membrane. This link is
important for the structural organization of the cytoplasm and for many cellular processes such as mitosis,
cell movement, cell differentiation and adhesion. The pattern of expression of cytokeratins is frequently organ
or tissue specific and depends mainly on the type of epithelium, the level of differentiation and the stage of
The expression of cytokeratins in umbilical cord has been investigated by many groups, but these previous
studies gave only fragmentary informations: each single study focused only on few types of cytokeratins and
usually concerned only their expression at protein level.
Aim. With this paper we tryed to make light on the pattern of expression of a wide range of cytokeratins in
human umbilical cord both at protein and at RNA level.
Results. Through immunohistochemistry we detected in the amniotic epithelium of human umbilical cord
at term immunoreactivity for cytokeratin clone AE1/AE3 (Pankeratins) antibody, clone MNF116 (antibody
against cytokeratins 1, 5, 10 and 14) and clone 34ОІE12 (antibody against cytokeratins 5, 6, 8, 17 and 19).
RT-PCR analysis revealed gene expression for cytokeratin 1, 5, 6, 8, 10, 14 and 17.
Riassunto. Premessa. Le citocheratine sono le proteine costituenti dei filamenti intermedi. Nel citoplasma
i filamenti di cheratine si estendono dal nucleolemma fino alla faccia interna della membrana plasmatica;
questa connessione è importante per l’organizzazione strutturale del citoplasma e per molti altri processi
cellulari come la mitosi, il differenziamento cellulare e l’adesione. Il pattern di espressione delle citocheratine
ГЁ spesso organo-specifico o tessuto-specifico e dipende principalmente dal tipo di epitelio, dallo stadio di
differenziamento e dallo stadio di sviluppo.
L’espressione delle citocheratine nel cordone ombelicale è stata studiata da diversi gruppi, ma tali precedenti
studi hanno dato soltanto informazioni frammentarie; ogni singolo studio si concentrava soltanto su pochi tipi
di citocheratine ed in genere riguardava solo la loro espressione a livello proteico.
Obiettivo. Con questo lavoro abbiamo cercato di far luce sul pattern di espressione di un vasto gruppo di
citocheratine nel cordone ombelicale umano sia a livello proteico che di RNA.
Risultati. Per immunohistochimica abbiamo rilevato nell’epitelio amniotico del cordone ombelicale umano
a termine immunoreattivitГ per AE1/AE3 (anticorpo contro pancheratine), per il clone MNF116 (anticorpo
contro le citocheratine 1, 5, 10 e 14) e per il clone 34ОІE12 (anticorpo contro le citocheratine 5, 6, 8, 17 e 19).
L’analisi per RT-PCR ha rivelato espressione genica per le citocheratine 1, 5, 6, 8, 10, 14 e 17.
Annamaria Mauro, Maria Buscemi, Aldo Gerbino
Cytokeratins (CKs) are the proteins that build intermediate filaments. These filaments,
along with actin microfilaments and microtubules, constitute the cytoskeleton of epithelial
cells and their primary function is to provide mechanical strength to cells.
The classification of cytokeratins (CK1-CK20) is based on the catalogue of Moll et
al. [1].
There are two types of cytokeratins: the low weight, acidic type I cytokeratins and the
high weight, basic or neutral, type II cytokeratins.
The high molecular weight cytokeratins comprise subtypes CK1 to CK8. The low molecular weight cytokeratins comprise subtypes CK9 to CK20.
Cytokeratins are made of a central О±-helix rich domain and non-О±-helical N- and C- terminal domains. The О±-helical domain comprises four segments in which a seven-residue
pattern is repeated. Into this repeated pattern, the first and fourth residues are hydrophobic
and the charged residues show alternate positive and negative polarity, resulting in the
polar residues being located on one side of the helix. This central domain of the chain provides the molecular alignment in the keratin structure and makes the chains form coiled
dimers in solution.
The end-domain sequences of cytokeratin chains contain in both sides of the rod domain the subdomains V1 and V2. The subdomains V1 and V2 contain residues enriched
by glycines (Gly) and/or serines (Ser), the former providing the cytokeratin chain a strong
insoluble character and facilitating the interaction with other molecules.
Cytokeratins are usually found in tetramers comprising two type I cytokeratins and two
type II cytokeratins. Two dimers of cytokeratin associate into a keratin tetramer by antiparallel binding (Fig.1). This cytokeratin tetramer is considered to be the main building
block of the cytokeratin chain. By head-to-tail linking of the cytokeratin tetramers, the
protofilaments are originated, which in turn intertwine in pairs to form protofibrils. Four
protofibrils give place to one cytokeratin filament.
In the cytoplasm, the keratin filaments form a complex network which extends from the
surface of the nucleus to the cell membrane.
This association between the plasma membrane and the nuclear surface provides important
implications for the organization of the cytoplasm and cellular communication mechanisms. Apart from the relatively static function
provided in terms of supporting the nucleus
and providing tensile strength to the cell, the
Fig.1: Graphic representation of a cytocheratins
cytokeratin networks undergo rapid depolyme- tetramer.
Expression of cytokeratins in human umbilical cord at term
rization which is important for many cellular processes such as mitosis, cell movement and
Cytokeratins also interact with desmosomes and hemidesmosomes, thus collaborating
to cell-cell adhesion and basal cell-underlying connective tissue connection.
The expression of cytokeratins is frequently organ or tissue specific.
The subsets of cytokeratins which an epithelial cell expresses depends mainly on the
type of epithelium, the moment in the course of terminal differentiation and the stage of
development. Thus this specific cytokeratin fingerprint allows the classification of all epithelia upon their cytokeratin expression profile. Furthermore this applies also to the malignant counterparts of the epithelia (carcinomas). The main clinical implication is that the
study of the cytokeratin profile by immunohistochemistry techniques is a tool of immense
value widely used for tumor diagnosis and characterization in surgical pathology.
Cytokeratin 1 (CK1) is specifically expressed in the spinous and granular layers of the
epidermis, usually associated with cytokeratin 10. Mutations in these genes have been
associated with bullous congenital ichthyosiform erythroderma.
Cytokeratin 5 (CK5) is specifically expressed in the basal layer of the epidermis and is
usually found as a heterotetramer with two keratin 14 molecules. Mutations in these genes
have been associated with a complex of diseases termed epidermolysis bullosa simplex.
Cytokeratin 6 (CK6) is expressed with family members CK16 and/or CK17 in the filiform
papillae of the tongue, the stratified epithelial lining of oral mucosa and esophagus, the
outer root sheath of hair follicles, and the glandular epithelia. Mutations in these genes
have been associated with pachyonychia congenita.
Cytokeratin 8 (CK8), together with cytokeratin 18, was first identified in liver and as
marker of mouse embryonal carcinoma (EC) and embryonic stem (ES) cell differentiation.
Investigation on early mouse embryos confirmed that the differentiation of the inner cell
mass of mouse blastocysts to trophoblast derivatives and extra-embryonic endoderm parallels the induction and accumulation of CK8 and CK18 [2].
Cytokeratin 10 (CK10) is an acidic (type I) cytokeratin. It is synthesized in the suprabasal cell layers of the human epidermis as well as in several epithelial tumours, squamous
metaplasias and transformed keratinocytes [3]. Cytokeratins 10 is closely related to CK11
and they are mostly coexpressed with the basic cytokeratin 1.
Cytokeratin 14 (CK14) is an acidic (type I) cytokeratin, it is usually coexpressed with
cytokeratin 5.
Cytokeratin 17 (CK17) is involved in the formation and maintenance of various skin
appendages, specifically in determining shape and orientation of hair. CK17 is also considered a marker of basal cell differentiation in complex epithelia and recognizes cervical
stem cells [4].
Annamaria Mauro, Maria Buscemi, Aldo Gerbino
The expression of CK17 protein has been found increased in cancerous tissues compared with normal tissues in cervical squamous cell carcinoma, laryngeal squamous cell
carcinoma, oesophageal squamous cell carcinoma and lung cancers [5].
The different epithelia of the human body express cytokeratins which are not only characteristic of the type of epithelium, but also related to the degree of differentiation.
The following pattern of expression of cytokeratins is usually found in the different
types of epithelia. One-layered epithelium, generally referred to as “simple epithelium”
primarily expresses cytokeratins 8 and 18 and often cytokeratins 7, 19 and 20.
Stratified epithelia are subdivided into the keratinizing, the non-keratinizing and
transitional type. The keratinizing and non-keratinizing epithelia have a basal cell layer
which invariably expresses cytokeratins 5 and 14. The suprabasal cell layers express
cytokeratins 4 and 13 in noncornifying epithelia and cytokeratins 1 and 10 in cornifying
epithelia. The transitional epithelium of the urinary bladder expresses cytokeratins 4 and
13 together with the cytokeratins of the “simple” epithelia (CK7, 8, 18, 19 and 20).
In combined epithelia, morphologically characterized by a basal cell layer and a layer
of columnar cells both in contact with the basement membrane, the basal cells express
cytokeratins 5, 14 and 17, while the luminal cells can express different combinations of
cytokeratins 7, 8, 18 and 19.
Squamous epithelia with a high cell turnover express cytokeratins 6 and 16.
CKs can be expressed also in non-epithelial cells; in a few mesenchymal cells such as
certain smooth muscle cells (particularly vascular wall and myometrium), myofibroblasts,
arachnoidal cells, fibroblastic reticulum cells of lymph nodes and rarely endothelial cells,
as well as cells of the umbilical cord and foetal myocardium, CK8 and CK18 have been
identifyed in addition to the original mesenchymal intermediate filaments (vimentin, desmin).
CKs can be expressed in non-neoplastic epithelial lesions. Upon various kinds of damage, epithelial cells may change their pattern of CK expression. The changes may indicate
an immature CK pattern of regenerative cells or a metaplastic process which sometimes
is not morphologically evident [1].
Carcinomas generally express CK patterns, which at least in part represent the pattern
of the putative cells of origin. The consequent profile, which reflects both epithelial type
and differentiation status, may therefore be useful in tumour diagnosis. Certain carcinomas
may lose the ability to express one or more CKs found in the putative tissue of origin while
other types of carcinoma may switch on certain CKs. Non-epithelial tumours are usually
CK negative. However, in a number of mesenchymal tumours CK positivity, usually faint or
focal and limited to CK8, CK18 and CK19, has been demonstrated [1].
Recent publications have classified breast cancers on the basis of expression of cytok160
Expression of cytokeratins in human umbilical cord at term
eratin 5 and 17 at the RNA and protein levels, and demonstrated the importance of these
markers in defining sporadic tumours with bad prognosis; expression of basal cytokeratins
(CK5, 6, 14, 17) is correlated with adverse clinical outcome for breast cancer [6,7,8].
In the field of diagnostic medicine the antibodies against cytokeratins are usefull in
the distinction of epithelial malignancies from sarcomas and lymphomas of various types.
Cytokeratin subtype expression patterns are however also being used in the distinction of
different types of epithelial malignancies.
Umbilical cord is the structure that connects the fetus to the placenta. Umbilical cord
at term is made of three vessels (two arteries arranged in coils around a vein) surrounded
by the Wharton’s jelly, a mucous connective tissue rich of water, described as a threedimensional spongy network of interlacing collagen fibers, bundles of glycoprotein microfibrils and an interfibrillar soluble phase [9] which are responsible for its elasticity and its
mechanical properties [10].
The epithelium of the human umbilical cord at term is composed of between one and
five layers of flattened cells, nuclei and recognizable organelles are absent from some of
the superficial cells and their cytoplasm contains a loose meshwork of filamentous material. The plasma membrane is thickened.
Wharton’s jelly cells resemble mesenchymal fibroblasts. Ultrastructural studies indicate
that their intrinsic properties are also similar to smooth muscle cells [11] and they are
therefore considered as myofibroblasts.
The endothelium of the umbilical vessels is made of a single layer of endothelial cells
containing cisternae of endoplasmic reticulum, mitochondria and Golgi apparatus. Large
accumulations of glycogen and Weibel & Palade bodies are present [12].
The expression of cytokeratins in umbilical cord has been investigated by many groups;
Bader et al. [13] and Bea et al. [14] showed the presence of low levels of cytokeratin 8, 18
and 19 in smooth muscle cells of the blood vessel walls of the human umbilical cord, while
Mizoguchi et al. [15] investigated through immunohistochemistry the presence of several
cytokeratins in the umbilical epithelium. Mahdi et al. [16] found CK1 expression in human
umbilical vein endothelial cells (HUVEC).
In literature, previous studies gave only fragmentary informations regarding cytokeratins expression in human umbilical cord; the single studies focused only on few types
of Cytokeratins and usually concerned only their expression at protein level (immunohistochemical or immunoblotting investigations). With this paper we want to make light on
the pattern of expression of a wide range of cytokeratins in human umbilical cord both
at the protein level through immunohistochemistry and at RNA level through RT-PCR
Annamaria Mauro, Maria Buscemi, Aldo Gerbino
Materials and Methods
The analysis was performed on human umbilical cords at term obtained from caesarian
delivery. The umbilical cords were provided by the Gynecologic and Obstetric clinic of the
University “Paolo Giaccone” in Palermo.
Human umbilical cords were collected in physiological solution. One section (1 cm
thick) was dissected from each umbilical cord and fixed in formalin solution. After fixation
the tissue was dehydrated in a graded series of alcohols, cleared in xylene and paraffin
embedded. Sections of 7 Ојm were cut on a Leica microtome RM2145, dryed overnight at
37В°C and then stored at R.T. until use. On the day of the experiment slides were dewaxed
in xylene and rehydrated in a graded series of alcohols. Slides were then transferred into
distilled water for 5 min. Samples were subjected to enzymatic pre-treatment with pepsin
(Dako, code S3002) for 10 min at 37В°C.
The immunohistochemistry was performed using the “DakoCytomation EnVision + System-HRP (AEC)” kit from Dako, following the manufacturer’s instructions. Briefly: Sections
were covered with the “Peroxidase block” reagent and incubated 5 min at room temperature. The samples were rinsed once in PBS buffer pH 7.2. The sections were covered with
the antibody solution and incubated at 4В°C O/N. Mouse anti human cytokeratin clone AE1/
AE3 antibody (Dako; 1:50 dilution), mouse anti human cytokeratin clone 34ОІE12 antibody
(Dako; 1:50 dilution) and mouse anti human cytokeratin clone MNF116 antibody (Dako; 1:
50 dilution) were used. The antibodies were diluted in a 0.1% BSA solution.
Samples were rinsed twice in PBS pH 7.2 and then incubated with the “Peroxidase
Labelled Polymer” reagent for 30 min. Samples were rinsed twice in PBS pH 7.2, then incubated with the “Substrate-Chromogen” reagent and immediately observed under a light
microscope; the reaction was carryed on until the stainig appeared (2-10 min). Reaction
was stopped rinsing the slides in distilled water. Negative control sample was treated in
the same way but omitting primary antibody. Slides were coverslipped using the “DakoCytomation Faramount Aqueous Mounting Medium”.
The specimens were observed under a Leica DM1000 light microscope.
Total RNA extraction
Samples of human umbilical cord were frozen in liquid nytrogen immediately after
delivery and stored at -80В°C until use. Total RNA extraction was accomplished using the
“illustra RNAspin Mini Kit” (Amersham Biosciences, Milan) following the manufacturer’s in162
Expression of cytokeratins in human umbilical cord at term
structions. RNA yield was evaluated spectrophotometrically (A260/A280) and RNA aliquots
were stored at -80В°C until use.
Reverse-Transcription (RT) Polymerase Chain Reaction (PCR)
For RT reaction 2 Ојg RNA was used. To avoid DNA contamination in the RNA samples
DNAse digestion was performed using “AMPD1 kit” (SIGMA).
RT reaction was performed using the “Enhanced avian HS RT-PCR kit” (SIGMA) following
the manufacturer’s instructions. Briefly: 1μl random nonamers and 1 μl anchored oligo
(dT)23 were added to the DNAse digestion product and incubated at 70В°C for 10 min to denature the sample. Then 2 Ојl 10x Buffer, 1 Ојl deoxynucleotide mix, 1 Ојl RNAse inhibitor, 1 Ојl
Enhanced AMV Reverse Transcriptase enzyme and DEPC water were added to the sample.
The RT reaction was performed in 20 Ојl total volume at 42В°C for 50 min, followed by 95В°C
5 min to inactivate the enzyme. The PCR was performed using the “PCR enzyme Selection
Kit- High specificity” (Invitrogen) following the manufacturer’s instructions. Briefly: 2 μl of
template DNA, 0.5 Ојl of the Primers mix (200 nM final concentration) and 22.5 Ојl of the
“Platinum Super Mix” were mixed together. The reaction was cycled for 94°C 3 min, then 40
cycles of 94В°C 60 sec, 56В°C 60 sec, 72В°C 60 sec, with a final extension at 72В°C 10 minutes.
GAPDH gene was used as internal positive control. The PCR products were visualized on 2%
agarose gels stained with ethidium bromide. Primers sequences are showed in table 1.
Table 1: Names, sequences and amplification product size of the PCR primers used in this work.
Primer name
GAPDH human forward
GAPDH human reverse
Cytokeratin-1 forward
Cytokeratin-1 reverse
Cytokeratin-5 forward
Cytokeratin-5 reverse
Cytokeratin-6 forward
Cytokeratin-6 reverse
Cytokeratin-8 forward
Cytokeratin-8 reverse
Cytokeratin-10 forward
Cytokeratin-10 reverse
Cytokeratin-14 forward
Cytokeratin-14 reverse
Cytokeratin-17 forward
Cytokeratin-17 reverse
Primer nucleotide sequence
agg agg tgg acg tgg tag tg
gag ggc aga cag gac cat aa
caa gcg tac cac tgc tga ga
tca tac tgg gcc ttg acc tc
ggg ttt cag tgc caa ctc ag
ata gct gcc tcc ggc tct
aca tcg aga tcg cca cct ac
aga cac cag ctt ccc atc ac
tcg cta ctg tgt gca gct ct
cgg aac ttc cct ctc ctt ct
gac cat tga gga cct gag ga
ggc tct caa tct gca tct cc
cag ttc acc tcc tcc agc tc
tca cct cca gct cag tgt tg
Product size
238 bp
220 bp
252 bp
226 bp
272 bp
185 bp
224 bp
325 bp
Annamaria Mauro, Maria Buscemi, Aldo Gerbino
AE1/AE3 antibody reacts with the basic, high molecular weight cytokeratins 1, 2, 3, 4,
5, 6, 7 and 8, and with the acidic low molecular weight cytokeratins 10, 13, 14, 15, 16
and 19. The immunoreactivity for this antibody is localized in the amniotic epithelium of
human umbilical cord (Fig.2,3). The fibroblasts of Wharton’s jelly didn’t react with AE1/AE3
antibody so that we belive they don’t express any type of cytokeratin. Endothelial cells and
vessels walls do not express cytokeratins.
Cytokeratin antibody, clone 34ОІE12, recognizes cytokeratin 1, 5, 10 and 14. Immunohistochemistry showed the expression of these types of cytokeratins (or at least one of
them) in the cells of the umbilical epithelium (Fig.4,5), while all the other regions of the
umbilical cord didn’t react with the antibody.
MNF116 antibody recognizes cytokeratin 5, 6, 8, 17 and 19. The immunoreactivity for
this antibody is localized only in the
cells of the umbilical epithelium (Fig.6,
7) while all the other cells of the umbilical cord seems not to express these
types of cytokeratins.
The transcriptional expression of
the genes encoding for these proteins
was investigated through Reverse
Transcription-Polymerase Chain ReCK1
action (RT-PCR). The analysis revealed
the expression of cytokeratins 1, 5, 6, Fig. 8: Representative picture of RT-PCR analysis performed
on human umbilical cord. Lane 1: 100bp DNA ladder; lane 2:
8, 10, 14 and 17 (Fig.8).
Cytokeratin 8 and 14 were detec- cytokeratin-1 amplification product (220 bp); lane 3: cytokeratin-5 amplification product (252 bp); lane 4: cytokeratin-6 amted in the totality (100%) of umbilical plification product (226 bp); lane 5: cytokeratin-8 amplification
cords tested, CK 5 in the 83% of the product (272 bp); lane 6: cytokeratin-10 amplification product
samples and CKs 1, 6, 10 and 17 in (185 bp); lane 7: cytokeratin-14 amplification product (224
bp); lane 8: cytokeratin-17 amplification product (325 bp).
the 66% of the samples.
The immunohistochemical expression of all the three cytokeratin’s antibodies used in
this work is limited to the amniotic epithelium of human umbilical cord. The immunoreactivity for all these three antibodies is intense and clear and appears to be specific with the
positive cells stained with dark brown granules and no background reactivity.
The RT-PCR analysis confirmed the immunohistochemical data showing the transcriptional expression of all the cytokeratins testeded CK-1, 5, 6, 8, 10, 14 and 17.
Expression of cytokeratins in human umbilical cord at term
Fig.2: AE1/AE3 immunoreaction in the amniotic epi- Fig.3: AE1/AE3 immunoreaction in amniotic epithethelium of human umbilical cord (40X).
lium (100X).
Fig.4: Cytokeratin antibody, clone 34ОІE12, immuno- Fig.5: Cytokeratin antibody, clone 34ОІE12, immunoreactivity in amniotic epithelium (40X).
reactivity in amniotic epithelium (100X).
Fig.6: Cytokeratin antibody, clone MNF116, immuno- Fig.7: Cytokeratin antibody, clone MNF116, immunoreactivity in amniotic epithelium (40X).
reactivity in amniotic epithelium (100X).
Annamaria Mauro, Maria Buscemi, Aldo Gerbino
[1] Moll R, Franke WW, Schiller DL, Geiger B, Krepler R. The catalog of human cytokeratins: pattern of
expression in normal epithelia, tumors and cultured cells. Cell 1982 Nov; 31(1):11-34.
[2] Maurer J, Nelson B, CeceГ±a G, Bajpai R, Mercola M, Terskikh A, Oshima RG. Contrasting expression of
keratins in mouse and human embryonic stem cells. PLoS ONE 2008; 3(10):e3451. [3] Meinhold-Heerlein I, Brandstetter T, Kommoss F, Bettendorf H, Hagedorn M, Bauknecht T. Localization
of cytokeratin 10 mRNA in human epidermis using nonradioactive in situ hybridization as a routine
method. Arch Dermatol Res 1998 May; 290(5):286-8.
[4] Martens JE, Arends J, Van der Linden PJ, De Boer BA, Helmerhorst TJ. Cytokeratin 17 and p63 are
markers of the HPV target cell, the cervical stem cell. Anticancer Res 2004; 24: 771–775.
[5] Wei K-J, Zhang L, Yang X, Zhong L-P, Zhou X-J, Pan H-Y, Li J, Chen W-T, Zhang Z-Y. Overexpression of
cytokeratin 17 protein in oral squamous cell carcinoma in vitro and in vivo. Oral Diseases 2009; (15):
[6] Gusterson BA, Ross DT, Heath VJ, Stein T. Basal cytokeratins and their relationship to the cellular origin
and functional classification of breast cancer. Breast Cancer Res 2005; 7(4): 143–148.
[7] Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT, Perou CM. Phenotypic evaluation
of the basal-like subtype of invasive breast carcinoma. Mod Pathol 2006 Feb; 19(2):264-71.
[8] Chen MH, Yip GW, Tse GM, Moriya T, Lui PC, Zin ML, Bay BH, Tan PH. Expression of basal keratins and
vimentin in breast cancers of young women correlates with adverse pathologic parameters. Mod Pathol
2008 Oct; 21(10):1183-91. Epub 2008 Jun 6.
[9] Meyer FA Wharton’s Jelly of the Umbilical cord; in: Extracellular Matrix. Tissue Function, W.D. Comper,
ed Harwood Academic Publishers, Amsterdam 1996. Vol. 1, 443-456.
[10] Sobolewski K, Bankokski E, Chyczewski L, Jaworski S. Collagen and glycosaminoglycans of Wharton’s
jelly. Biol Neonate 1997; 71(1):11-21.
[11] Can A, Karahuseyinoglu S. Concise review: Human umbilical cord stroma with regard to the source of
fetus-derived stem cells. Stem cells 2007 Nov; 25(11):2886-95.
[12] Parry EW, Abramovich DR. The ultrastructure of human umbilical vessel endothelium from early pregnancy to full term. J Anat 1972 Jan; 111(Pt 1):29-42.
[13] Bader BL, Jahn L, Franke WW. Low level expression of cytokeratins 8, 18 and 19 in vascular smooth
muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the
chromosomal locus containing the cytokeratin 19 gene. Eur J Cell Biol 1988 Dec; 47(2):300-19.
[14] Bea F, Bär H, Watson L, Blessing E, Kübler W, Kreuzer J, Jahn L. Cardiac alpha-actin in smooth muscle
cells: detection in umbilical cord vessels and in atherosclerotic lesions. Basic Res Cardiol 2000 Apr;
[15] Mizoguchi M, Ikeda S, Suga Y, Ogawa H. Expression of cytokeratins and cornified cell envelope-associated proteins in umbilical cord epithelium: a comparative study of the umbilical cord, amniotic epithelia
and foetal skin. J Invest Dermatol 2000 Jul; 115(1):133-4.
[16] Mahdi F, Shariat-Madar Z, Todd RF 3rd, Figueroa CD, Schmaier AH. Expression and colocalization of
cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells. Blood 2001 Apr;
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
The evolutionary course of Vertebrate foot, from Fish
to Man
[Il corso evolutivo del piede dei Vertebrati, dal pesce all’uomo]
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello and Elvira Farina Lipari
Department of Experimental Medicine, University of Palermo (IT)
Key words: Humans, Anthropoids, Primates, Reptiles, Amphibians
Parole chiave: Uomo, Antropoidi, Primati, Rettili, Anfibi
Abstract. The evolutionary course of Vertebrates foot starts from the posterior (or pelvic) fins of aquatic
Vertebrates (Fishes) and from these it follows a long way through Amphibians, Reptilians, archaic Mammals,
Primates and modern Man. So, a functional analogy can be seen between the Fish fins and the Vertebrate
limbs. During the evolution, the foot becomes “a mechanic complex” with longitudinal and transverse arches
united to constitute a “structural unity” that plays a role of supporting the body weight and discharging it to
the ground while allowing a walking movement. In this review we do an excursus of the evolution of vertebrate foot, from fish to Man.
Riassunto. Il corso evolutivo del piede dei Vertebrati inizia dalle pinne posteriori (o pelviche) di vertebrati
acquatici (Pesci) e da questo esso segue un lungo percorso attraverso gli Anfibi, Rettili, Mammiferi arcaici,
Primati e Uomo moderno. Pertanto, una analogia funzionale puГІ essere osservata tra le pinne dei pesci e gli
arti dei Vertebrati. Durant l’evoluzione, il piede diviene “un complesso meccanico” con archi longitudinali e
trasversali uniti a costituire una “unità strutturale” che svolge un ruolo di supporto al corpo per scaricare il
peso sulla terra durante il cammino. In questa review noi facciamo un excursus della evoluzione del piede
dei Vertebrati, dai pesci all’uomo.
1. The fish fins
Fishes appear in the middle Paleozoic era, in a period included between 450 and 330
million years ago. Among the most advanced pluricellular primordial organisms, they are
endowed with lengthen body, head, tail, nervous system, an impair skin fold and two pair
From the impair skin fold derive the impair fins: the dorsal, tail and anal fins; from the
pair folds derive the pectoral and pelvic fins (Fig.1a). These fins were originally unarmed,
successively armed with contractile and skeletal elements; consequently they take on
the role of body appendix able to grant the wavering tail movement, making it stable and
orienting its direction [4,9]. In particular, Fish fins framework, initially made of cartilaginous
tissue formed by little and flexible laminae and sequentially by bone tissue, allows fish mo-
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari
vements into the aquatic habitat without
having, however, any supporting role for
the body weight.
Besides we must signal that, while
we can certainly recognize a functional
correspondence or analogy between pair
Fish fins and terrestrial Vertebrate limbs,
it is not demonstrable the homology or
morphological correspondence [7].
2. The amphibian foot
Amphibian foot of late Paleozoic era,
included between about 330 and 240
million years ago, is considered the progenitor of Vertebrate foot (Fig.1b). Already in this era we can recognize a general
organization of pentadactyl base (fivetoed) and the three constitutive parts are
outlined yet: a proximal (tarsus), a middle
(metatarsus) and a distal one (phalanges).
In the tarsus of Amphibian foot there
are eleven bones (Fig.1c) ordered in two
transverse rows, a proximal one and a
distal one. The proximal row is made by
three (variable) bones, the tibial (ti), the
intermedium (i) and the fibular (fi) one
which is in relationship with tibia (T) and
fibula (F); the distal row has five tarsal
bones (T1-T5) each of them in relationship with the correspondent metatarsal
bone. Between the proximal row and the
distal one there are mainly three central
bones, a proximal central (cp) and two
distal central (cd) ones. Besides, there
are two inconstant adding members: one
along the medial margin, the prehallux
a) Fins in a Fish belonging to the Palaeozoic era
(between 450 and 330 million years ago). b) Pentadactyl basic organization of Primitive Amphibian
foot of the late Palaeozoic era (between 330 and 240
million years ago). Its anatomical parts were already defined (tarsus, metatarsus and phalanges). c)
Tarsal bones of Amphibian foot with two additional
inconstant members, the prehallux ray (Ph) and the
postminimus ray (Pm). (T: tibia, F: fibula, Ti: tibial, i:
intermedium, fi: fibular, cp: central proximal, cd: central distals, T1-T5: metatarsals); from Morton 1948,
modified. d) Metatarsal bones (M1-M5) and phalanges* of Amphibian foot toes from the first to the fifth;
digital formula: 2,3,4,5,4.
The evolutionary course of Vertebrate foot, from Fish to Man
ray (Ph) and the other one along the lateral margin, the postminimum ray (Pm).
Concerning metatarsal bones and phalanges (Fig.1d), there are five metatarsal bones
and in the five toes there are totally eighteen phalanges with a digital formula, 2,3,4,5,4,
characteristic in Amphibian foot, where the first toe is constituted of two phalanges, the
second of three, the third of four, the fourth of five and the fifth of four. The primitive Amphibian pentadactyl foot is wholly constituted of thirty-four skeletal segments.
In Amphibians, bones composing foot show an incomplete ossification, and its longitudinal axis seems to be located on the same plan of the leg one. So Amphibian foot, being
partially able to support the body weight against the terrestrial force of gravity, allows
these partially terrestrial Vertebrates a certain raising of the animal centre of gravity from
the ground, permitting the crawling movement of the body on the ground. In this precarious
static condition, the load imbalance on the foot lateral margin determines the increasing
number of lateral toes phalanges, and consequently the digital formula 2,3,4,5,4 [7].
3. The reptilian foot
In the late Paleozoic era and in Mesozoic era, during the Reptiles age, which is included
between 240 and 85 million years ago, by reaching correct connection between proportion and sturdiness, foot, being able to raise the animal centre of gravity, takes the role of
organ suitable for translating it on the ground: bones that constitute it, lined in an anteriorposterior axis direction, as for a longitudinal axis that form an angle with the leg axis, allow
foot movements of dorsal-plantar flexion in the talocrural articulation.
Making a comparison between the Amphibian skeletal foot components and the Reptilian one, in the latter while we find a complete mineralization of the bones which constitute
it for bearing charges, we register a numerical reduction in the tarsus bones from eleven to
seven, because of the fusion or reabsorbing of some of them [17].
In particular (Fig.2a):
The intermedium (i) melts with central proximal (cp) in order to compose the talus;
The fibular (fi) increases remarkably in its dimensions and volume, constituting the
heel, disposing under the talus and entering in contact with the ground;
The tibial (ti) progressively reabsorbs;
The two central distal (cd and cd) melt together in order to compose the navicular;
About the five tarsal bones, T1, T2 and T3 form respectively the medial cuneiform, the
intermedium and the lateral while T4 and T5 melt together in order to compose the cuboid,
which is the bone with the main volume in this row;
The components of prehallux and postminimun ray, if present, progressively reabsorb;
The five metatarsal bones remain as they were;
The toes bones, phalanges, ordered in a linear succession, modify (Fig.2b) the digital
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari
formula of Amphibian foot (2,3,4,5,4)
into a 2,3,3,3,3 formula, where the
first toe is composed by two phalanges, proximal and distal, and the other
four toes by thre phalanges, proximal,
middle and distal.
This digital formula is successively
found again in Mammal and Primate
foot. Totally, Reptile foot, compared to
Amphibian one, presents a great reduction, (about 25%), of skeletal segments; from thirty-four bones in Amphibian foot to twenty-six in Reptilian
one; besides these ones, we must consider even the sesamoid bones, which
are present along the tendons path of
the long flexor muscles of foot toes.
4. The mammal foot
The archaic Mammals settled on
earth during the end of Cretaceous
period of Mesozoic era and during the
Eocene and Oligocene periods of Cenozoic era, between about 85 and 35 million years ago. These Mammals, whose
ancestry were actually represented by
Therapsid group of “mammals-like”
reptiles, during the Triassic period of
Mesozoic era, were present on earth
after the Jurassic mammals extinction
These Mammal foot registers: the
complete and definitive disappearance
of marginal toes; the acquisition by the
talus of a pulley, the trochlea, which inserts into wedge-shaped articular socket of the ankle joint and of a head for
a) Reptilian foot tarsal bones in the Mesozoic era
(between 240 e 85 million years ago). We observe:
fusion between the intermedium (i) and central proximal (cp) bones, which form the talus; increment of the
fibular (fi) dimension which forms the calcaneus; fusion between two central distals (cd+cd) which forms
the navicular; tarsal bones T1,T2,T3 which form the
cuneiform medial, the intermedium and the lateral;
fusion of the tarsal bones T4 and T5 which form the
cuboid; progressive tibial (ti) reabsorption and prehallux ray (ph) (from Stark 1955, modified). b) Metatarsal
bones (M1-M5) and phalanges* of Reptilian foot toes
from first to the fifth; digital formula: 2,3,3,3,3.
The evolutionary course of Vertebrate foot, from Fish to Man
the talocalcaneonavicular joint; the definitive positioning of the heel under the talus with
the plantar flexor muscles which insert on the heel; the steady union of tarsal bones with
each other; the sturdiness and divergence of first metatarsal; the prehensile characteristic
of hallux, which is developed and abducted; the long toes with the formula 2,3,3,3,3; the
foot longitudinal axis which involves the talus, the heel, the lateral cuneiform, the three
metatarsal and the third toe, that is the centre of the lever action of dynamic foot (Fig.3a).
With the complete acquisition of all these morphostructural elements, foot is already
conformed for his primitive role of gripping organ which allows the arboreal phase in these
Mammals. Archaic Mammal foot, briefly, presents all the components of Reptilian foot that
for this reason can be placed in the centre of the Vertebrate foot evolutionary course. Once
that Mammals defined the essential and fundamental skeletal structures, a great variety of
modifications took place in thoracic and pelvic limbs in toto.
In Modern Mammals, appeared less than 35 million years ago, during the Miocene,
Pliocene and Pleistocene period of Cenozoic era, proper evolutionary lines defined in conFig.3:
a) Foot skeleton of archaic Mammals, settled on earth
during the end of Cretaceous period, between 85 and
35 million years ago. We observe: divergence of the
marginal toes; presence of trochlea and talus head;
divergence of the first metatarsal and hallux; long toes,
with formula 2,3,3,3,3; foot longitudinal axis passing
through the third ray. b) During the evolutionary course
from the Monkeys to Man, we observe the formation
of the longitudinal medial arch; the heel gains the sustentaculum tali and the functional axis of the third ray
in Monkey moves medially in Ape, between the first
and the second ray; instead in modern Man it involves
the second ray. c) The gradual changes of metatarsal
bones torsion, symbolized by the oblique lines and the
arrows, is clear on foot of Monkey, Ape and Man (from
Morton 1948, modified). d) The arboreal Monkeys foot:
angulated leverage axis. The semiterrestrial Apes foot:
partial correction of the leverage axis. Lengthening of
the first metatarsal bone causes a reduction in the angle BEF; adduction of the bone to D’ further reduces the angle to BEF’. The terrestrial Human foot: the leverage axis forms a straight line AB. A-B: The
functional axis, tarsal and metatarsal portion; A-F: The angulated leverage axis, tarsal and metatarsal
portion; BEF and BEF’: The angle; C-D and C-D’: Line between the heads of metatarsal I and II which act
as the fulcrum; E-F: Line of metatarsal portion of leverage axis perpendicular to C-D; G-H: The direction
of body movement. (From Morton 1948, modified).
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari
nection with the animal way of life, the role they are intended to have and the habitat.
Limbs can be transformed, for example, in flight organ (Bats), in running organ (Equidae),
paddles for swimming (Cetaceans), in limbs with gripping function thanks to the opposition
of thumb and hallux with the other fingers or toes, as we can see in Primates and Man. A
particular interest is in the evolutionary course variations of the extreme distal of thoracic
and pelvic limbs relative to the number of toes, which are pair (in artiodactyls) or impair
(in perissodactyls).
In the extreme distal of Horse limb we can find that only the third toe supports the body
weight and lean on the ground with the distal phalange endowed with a hoof, in Ruminants
there are the third and the fourth toes, in Swine there are the third and the fourth toes but
also the second and fifth ones, that however do not lean on the ground, in Hippopotamus
there are four toes while in Marsupials there are five toes.
5. The primate foot
Mammal order is represented by Primates which include Monkeys and Anthropoids
Apes (Chimpanzee, Gibbon, Orang, Gorilla). Anthropoids apes are considered the most advanced existent Primates and they use thoracic and pelvic limbs for moving among the trees [15], the so called “brachiating”; these Primates during the Miocene period of Cenozoic
era, were preceded by a primitive stock of quadrupedal Primates and, in a very long space
of time, they underwent a fundamental modification in their posture that changed from horizontal to vertical for a bigger development of superior limbs, for the accentuate shoulder
mobility and for the pelvic limbs moving in a direct continuity with the trunk [8,10].
We usually believe that between pronogrades Anthropoids and ortogrades Hominids
and between these ones and Modern Man there are still some connections, unknown by
our knowledge and interpretation, which could explain and clarify particular aspects about
form and actual structure [1].
During the Cenozoic era, probably before the beginning of Miocene period, about 40
million years ago, Man inherited form unknown arboreal bipeds progenitors, a foot endowed with a short and mobile hallux [6]. So, Primate and Man foot derives from a unique
ancestor, the Australopithecus, whose structures had not reach the definitive and peculiar
organization [16]. This ancestor reaches the terrestrial bipedism with a gradual transfer
of locomotory functions on pelvic limbs, with the heel leaning on the ground and with the
reduction of foot grasping function on the ground and his assuming lever functions for
lifting and pushing body against the force of gravity.
For defining and achieving the evolutionary course towards human foot, we need the
intervention of further modifications regarding: the constitution of the medial longitudinal
arch; the moving of foot functional axis between the first and the second ray; the toes
The evolutionary course of Vertebrate foot, from Fish to Man
reduction in length; the torsion of lateral metatarsal bones; the first metatarsal bone divergence.
The medial longitudinal arch takes form during the evolutionary course between Monkeys and Anthropoids apes, the heel medial surface, besides, is endowed with a very
important bracket shaped relief, the sustentaculum tali, and foot functional axis moves
medially from the third ray, involving in Man the second ray (Fig.3b). The reduced fingers
length is necessary because they have not a role as grasping for climbing anymore. The
medial torsion of lateral metatarsal bones and their flexion, that converge towards the first
metatarsal, can be easy recognized (Fig.3c) in Chimpanzee foot, it is indicated by the oblique lines and arrows, and it characterizes the foot role as an arboreal organ; in terrestrial
Gorilla we can notice that the metatarsal bones torsion is remarkable reduced, instead, in
Man, the torsion is practically disappeared and metatarsal bones plantar turned.
The first metatarsal divergence is evident looking at the Chimpanzee, Gorilla and Man
feet (Fig.3d). In the Cimpanzee arboreal foot, it is clear the first metatarsal divergence and
shortness: between the metatarsal anterior part of lever axis and the foot functional axis
there is an angle included; the direction of body on ward translation movement is realized
on the metatarsal part of lever axis, thanks to a movement of foot external rotation, from
which it derives the characteristic walking of these Monkeys; in the terrestrial Gorilla foot,
the lengthening and the partial adduction towards the lever axis of first metatarsal determines a reduction of the abduction angle, in Man foot, the complete adduction of first metatarsal determines the disappearing of the abduction angle, and the lever axis coincides
with the functional axis which goes the second ray [7].
The finger length, the lateral metatarsal bones torsion, the first metatarsal bone divergence then represents the morphostructural conditions right for an arboreal bipedism, but
not for the terrestrial bipedism of Human foot.
6. The human foot
The skeletal elements of Human foot (Fig.4) are twenty-eight and are represented by
seven bones at tarsal level (talus, heel, navicular, medial, intermedium and lateral cuneiform, cuboid); five bones at metatarsal level; fourteen phalanges at toe level (two phalanges, a proximal and a distal one, for the first toe, three phalanges, a proximal, a middle
and a distal one, for the second, third, four and five toe); there are also two other canonical
bones: the sesamoid hallux bones [14].
In the embryonic and foetal life, during the human foot ontogenesis, bones that composed it, at the beginning, present a linear series disposition on a sole plane, as is in the
hand; successively, following a series of genetic messages, it occurs the so called “podalic
winding” where the medial longitudinal arch rises, wrapping around the lateral longitudinal
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari
arch, making an helicoidally structure known as plantar vault; in the wake of this “podalic
winding”, the skeletal elements of hindfoot overlaps, while in the forefoot and midfoot they
dispose on the same plane [2].
Human foot is therefore made up by a dome-shaped bone system (fig.4) with the dorsum looking upward, posteriorly convex and anteriorly flattened; the sole is concave and
comparable to a tripod resting on three points: posteriorly, the calcanear tuberosity, anteriorly and medially the head of the first metatarsal, anteriorly and laterally the head of the
five metatarsal.
Actually, load share that burdens on the forefoot uses for leaning against the ground
six points so represented: two points on the two sesamoids of first metatarsal head, four
points, one for each ones, on the second, third, fourth, fifth metatarsal heads [12].
Each foot, from an architectonic point of view, is actually represented by the curvilinear
section of a half-vault, which basis is represented by the lateral border of the foot and the
summit by the medial border; a complete dome is formed only when the two medial borders of the foot are approached together.
Therefore Human foot can be defined as a complex mechanical construction with a
Human foot bones seen from the dorsal surface (a) and from the medial margin (b,c). We can observe
a 90В° angle between the leg and the foot at talocrural region (c); it is also evident the almost complete
disappearing of grasping functions.
The evolutionary course of Vertebrate foot, from Fish to Man
curvilinear axis with the medial longitudinal arch, more important than the lateral one, is
higher and with a strong resilience; a lateral longitudinal arch, on the contrary, is much
flatter and rigid in contact with the ground. The transverse arches of the foot run from side
to side; they are numerous and they link the two longitudinal arches. Mainly we describe
two main transverse arches: a posterior one lying the midfoot and the forefoot, at a Lisfranc
line, and an anterior one in the forefoot, at a metatarsophalangeal line [12].
The longitudinal and transversal arches, complete and fixed together by strong ligaments, especially plantar, constitute a real “structural unity” that has two important function: to support the body weight and to serve as a lever to propel the body forward in
walking and running.
7. Exegeses and views
Greater anatomic differences distinguish the terminal segment of the lower limbs of
Homo sapiens from the corresponding segment of the upper limb than in another Primates
or Mammals. The anatomy of the foot is more characteristic of the human species than the
anatomy of the hand; the erect bipedal habit account for these differences.
The evolutionary course of Vertebrate foot, from Fishes to Man, reach its finish line
through a series of successive improving valuable thanks to the following objective data
(Fig. 4): foot and leg rotation of 180В° with the first ray is medially arranged, on the same
plane of the other toes; horizontal direction and 90В° position compared to leg in order to
amplify the supporting surface; completely vault-like morphological order with a plantar
niche turning down in a way to consent an adequate protection for its contents against
the mechanical stresses; foot bones volume and dimension, particularly at hindfoot level,
where talus and heel must support the body weight and convey it to the ground; adequate and perfect inserting of astragalic trochlea into the wedge-shaped articular socket of
the ankle joint for constituting together a powerful lever able to support and lift the body
weight against the gravity, also allowing step pushing, riding, jumping [13].
During Human foot evolutionary period, at the forefoot level, we assist to the almost
complete disappearing of grasping function (Fig.4) which shows through:
atrophy of toe phalanges, which are shorter, more little and less mobile than the other
Primates ones; hallux, that usually is the longest, only supports the foot static functions
and the dynamical ones transmitted during the locomotion;
first ray reduced mobility and loss of the other toes opposing function;
hallux position on the plane of other toes with a flexor surface plantar-like turned, as for
the other lateral toes, to constitute a unique complex suitable for its propulsive function;
sturdiness of metatarsal bones, which are more mobile at head level and endowed with
a larger possibility of executing movements of dorsal-plantar bending and limited move175
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello, Elvira Farina Lipari
ment of abduction and adduction compared to the axis toe represented in human foot by
the second toe, partially fixed.
8. Conclusions
Did the evolutionary process of Vertebrate foot effectively complete and exhaust its
course? Would it not be possible to have more transformations in diversified or modified
aspects under an organizing point of view, during the time? It is well-known that, through
chemical-physical factors, it is possible to introduce changes inside the genetic molecule
and that genetic mutation can realize, without any logic concatenation, even naturally and
not only experimentally.
The actual Human modus vivendi is under everyone’s attention; with the correct exceptions, we don’t know or only partially know the pleasure in using the inferior limbs; yet
terrestrial bipedism was the great conquest that diversify us from the other Primates! It
becomes more and more strong an invitation to walk, even because this activity is good
for health, it is useful to take weight under control, reduces risk in cardiovascular disease
Even if we share this problematic, we express our perplexity and our worry because,
in a very long time, according to the vertebrate evolutionary course rules, it can intervene
modifications in the organizing order, in the lower limb and, in particular, in the foot not in
a positive, but in a negative sense, or even in a regressive one, in the wake of a gradual
process of atrophy ex non usu. In this perspective, only posterity in the future and after
several decennials, could verify the rightness of our not really optimistic forecasts.
The evolutionary course of Vertebrate foot, from Fish to Man
[1] Bruno G. Origine e perfezionamento del piede umano. Riv Biol 1958; 50:297-310.
[2] Gravante G, Pomara F, Russo G, Amato G, Cappello F, Ridola C. Plantar pressure distribution analysis
in normal weight young women and men with normal and claw feet. A cross-sectional study. Clin Anat
[3] Harcourt-Smith WEH, Aiello LC. Fossils, feet and the evolution of human bipedal locomotion. J Anat
[4] Hayama S, Nakatsukasa M, Kunimatsu Y. Monkey performance: the development of bipedalism in
trained Japanese Monkey. Acta Anat Nippon 1992;67:169-185.
[5] Leghissa S. “Anatomia comparata dei Vertebrati”. Tinarelli, Bologna, 1960.
[6] McHenry H, Jones AL. Hallucial convergence in hominids. J Human Evol 2006; 50:534-539.
[7] Morton DJ. “The human foot”. Columbia University Press, New York, 1948.
[8] Nakatsukasa M. Acquisition of bipedalism: the Miocene hominoid record and modern analogues for
bipedal protohominids. J Anat 2004; 204:385-402.
[9] Nakatsukasa M, Hayama S. Skeletal responses to bipedalism in macaques: with emphasis on cortical
bone distribution of the femur. Cour Forsch-Inst Senckenberg 2004; 243:35-45.
[10] Richmond BG, Jungers Wl. Orrorin tugenensis femoral morphology and the evolution of human bipedalism. Science 2008; 319:1599-1601.
[11] Ridola C, Cappello F, Palma A. Anatomy of the foot. Med Sport 2002; 55:229-240.
[12] Ridola C, Palma A. Functional anatomy and imaging of the foot. It. J Anat Embryol 2005; 106:85-98.
[13] Ridola CG, Cappello F, Lipari D, MarcianГІ V, Palma A. Clinical anatomy of joints of the foot. Med Sport
2006; 59:497-508.
[14] Ridola CG, Cappello F, MarcianГІ V, Farina-Lipari E, Palma A. The synovial joints of the human foot. It J
Anat Embryol 2007; 112:61-80.
[15] Sellers WI, Cain GM, Wang W, Crompton RH. Stride lenght, speed and energy costs in walking of Australopithecus Afarensis: using evolutionary robotics to predict locomotionof early human ancestors. J R
Soc Interface 2005; 2:431-441.
[16] Senut B, Pickford M, Gommery D, Mein P, Cheboi K, Coppens Y. First hominid from the Miocene (Lukeino
Formation, Kenya). C R Acad Sci 2001; Paris 332:137-144.
[17] Stark D. ”Embryology”. Stuttgart, Thieme, 1955.
[18] “Terminologia Anatomica”, International Anatomical Terminology. FCAT, Stuttgart, Thieme, 1998.
Gastrointestinal tract
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Orexina e Orfanina FQ nuovi neuropeptidi regolatori
delle funzioni del Sistema Gastroenteropancreatico.
Uno studio immunoistochimico
[Orexin and orphanin FQ in GEP system. An immunohistochemical study]
Giuseppe Bonaventura, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore
Di.Me.S. - Sezione di Istologia ed Embriologia - UniversitГ degli Studi di Palermo (IT)
Parole chiave: Orexina, Orfanina FQ, Sistema gastroenteropancreatico, Immunoistochimica
Key Words: Orexin, Orphanin FQ, GEP system, Immunohistochemistry
Riassunto. La orexina A (OXA) e B (OXB) chiamate pure ipocretina 1 e 2 e la orfanina FQ sono membri di una
famiglia di neuropeptidi ritenuti in origine esclusivamente prodotti da neuroni dell’area ipotalamica posterolaterale ove regolano l’assunzione alimentare e il peso corporeo attivando specifici recettori e svolgendo
un ruolo oressizzante. In aggiunta la orfanina FQ che presenta somiglianze strutturali con i neuropeptidi
oppioidi (enkefaline-endorfine) ГЁ stata implicata nella nocicezione. PiГ№ di recente, evidenze sperimentali
fanno ritenere che tali neuropeptidi svolgano i loro effetti al di fuori del S.N.C. in distretti periferici correlati o non correlati con l’equilibrio nutrizionale. In riferimento a tali acquisizioni registrate al di fuori del
SNC abbiamo svolto indagini immunoistochimiche sulla espressione dei neuropeptidi summenzionati nel
Sistema Gastroenteropancreatico del ratto normale. I nostri risultati forniscono documentazioni immunoistochimiche che autorizzano ad ammettere che sia la Orexina A con il suo recettore tipo 2 che la orfanina FQ
possono essere espresse perifericamente da molti citotipi costitutivi del Sistema GEP. In aggiunta, i nostri
reperti forniscono la base morfoistochimica per l’interpretazione del ruolo funzionale svolto dall’orexina e
dall’orfanina in tali distretti suggerendo la possibilità che esse possano esercitare un’azione diretta di tipo
periferico sulle strutture summenzionate esplicando nuovi ruoli con meccanismi neurocrini, endocrini e
Abstract. The orexin A (OXA) and B (OXB) ipocretin 1 and 2 also called and the orphanin FQ are members of a
family of neuropeptides originally considered to be exclusively produced by hypothalamic neurons in the posterolateral region, where regulate food intake and body weight by activating specific receptors, and playing
a orexigenic role. In addition, the orphanin FQ presenting structural similarities with the opioid neuropeptides
(enkefalin-endorphins) has been implicated in nociception. More recently experimental evidences suggest
that these neuropeptides play their effects outside the CNS in peripheral districts related or not related to
the nutritional balance. In reference to these acquisitions verified outside the CNS, we investigated the immunohistochemical expression of these neuropeptides in the Gastroenteropancreatic System of normal rats.
Our immunohistochemical results provide the documentation that authorizes to admit that the orexin A with
its receptor type 2 and the orphanin FQ can be expressed by several peripheral citotypes of the GEP system.
In addition, our findings provide the morphoistochemical basis for the functional interpretation of the role
undertaken by orexin and orphanin in these districts and suggest the possibility that they may bring a direct
peripheral action on these structures playing new roles probably with neurocrine, endocrine and autocrineparacrine mechanism.
Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
L’orexina A (OXA) e B (OXB), chiamate pure ipocretina 1 e 2 e la orfanina FQ sono membri
di una famiglia di neuropeptidi ritenuti in origine esclusivamente prodotti da neuroni centrali dell’area ipotalamica posterolaterale ove regolano l’assunzione alimentare e il peso
corporeo attivando specifici recettori e svolgendo un ruolo oressizzante. In aggiunta l’orfanina FQ che presenta somiglianze strutturali con i neuropeptidi oppioidi (enkefaline-endorfine) è stata implicata nella nocicezione, donde la denominazione alternativa di nocicettina
ad essa attribuita. Successive precisazioni hanno chiarito che la orfanina ГЁ estesamente
distribuita nel S.N.C. [1] per cui ГЁ stato ritenuto che essa sia implicata non soltanto nei
complessi e discussi fenomeni nocicettivi ma anche nei fenomeni di apprendimento e di
memoria, nell’ansia e nella epilessia.
PiГ№ di recente aldilГ di tali neuroeffetti centrali, evidenze sperimentali fanno ritenere
che tali neuropeptidi svolgano i loro effetti al di fuori del S.N.C. in distretti periferici correlati
o non correlati con l’equilibrio nutrizionale [2-12]. Tutto ciò ovviamente dischiude dal punto
di vista clinico-pratico nuove prospettive farmacoterapeutiche.
In riferimento a tali acquisizioni registrate al di fuori del SNC abbiamo svolto indagini
immunoistochimiche sulla espressione dei neuropeptidi summenzionati nel Sistema Gastroenteropancreatico del ratto normale.
I risultati preliminari di tale ricerca sono stati comunicati al 31В° Congresso della SocietГ Italiana di Istochimica (Pisa 2005) e al 59В° Congresso della SocietГ Italiana di Anatomia e
Istologia (Sorrento, 2005) [13,14].
Materiali e metodi
Sono stati impiegati 10 ratti Wistar trattati in accordo con la Convenzione di Helsinki
sull’utilizzo degli animali nella ricerca biomedica. Gli animali sono stati sacrificati previa
anestesia (50 mg/kg di Nembutal somministrato per via endoperitoneale). Sono stati prelevati lo stomaco, l’intestino tenue e il pancreas. I campioni sono stati immediatamente
fissati in liquido di Bouin ed inclusi in paraffina. I campioni sono stati sezionati, processati
per la tecnica immunoistochimica e svelati mediante kit di rivelazione Envision + System
HRP (AEC) (DAKO Citomation), utilizzando anticorpi policlonali anti Orexina A (Chemicon
AB3098), anti Orexina-2 Receptor (Chemicon AB3094) data l’indisponibilità commerciale
di un anticorpo per il recettore tipo 1 ed un anticorpo policlonale anti Orfanina FQ (Chemicon AB5515). Per la identificazione delle cellule principali gastriche sono stati impiegati il
blue di toluidina e la ematossilina ferrica. Contemporaneamente i controlli negativi sono
stati realizzati su sezioni adiacenti processate in base allo stesso protocollo omettendo il
passaggio dell’antisiero primario. Tutti i campioni sono stati studiati mediante fotomicroscopio Leica DMLB.
Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico.
Uno studio immunoistochimico
Nello stomaco
Orexina A: intensa immunopositivitГ si evidenzia in cellule endocrine incastonate nel
contesto degli adenomeri tubulari delle ghiandole della regione del fondo gastrico, le
cui cellule parietali e zimogeniche mostrano una attivitГ orexinergica immunoistochimicamente assai debole (Fig.1-2). Nessuna immunopositivitГ ГЁ rilevabile a carico delle
cellule principali. Nella regione antropilorica la immunopositivitГ per la orexina A ГЁ piГ№
evidente ed estesa essendo piГ№ abbondanti le cellule endocrine presenti soprattutto
nella porzione mediobasale delle ghiandole tubulari ramificate. Sia nella regione del
fondo gastrico che nella regione antropilorica il rilievo immunoistochimico del recettore
OX2R ГЁ assai scarso e limitato ad alcuni citotipi endocrini presenti soprattutto nella
regione mediobasale delle ghiandole. Discreta immunopositivitГ al recettore tipo 2 mostrano alcune cellule del corion mucosale che attorniano i fondi ghiandolari al limite con
la sottomucosa.
Fig.1: Stomaco di ratto – regione antrale: intensa
immunoreattivitГ OXA delle cellule endocrine (frecce
nere); debole immunoreattivitГ a carico delle cellule
parietali (frecce azzurre) - Ob. 63x
Fig.2: Stomaco di ratto – regione del corpo: intensa
immunoreattivitГ orexinergica di cellule endocrine
(frecce); scarsa immunoreattivitГ OXA in numerose
cellule parietali - Ob. 63x
Fig.3: ImmunoreattivitГ N/OFQ delle cellule endocrine Fig.4: ImmunoreattivitГ N/OFQ delle cellule principali
(freccie) e delle cellule principali delle ghiandole della delle ghiandole della regione fundica (freccie verdi) e immunonegativitГ delle cellule parietali (freccie
regione fundica - 40x
nere) - 40x
Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
Orfanina FQ: nella regione antropilorica intensa immunopositivitГ si rileva in isolate cellule endocrine presenti negli adenomeri tubulari (Fig.3). Sia nella regione antropilorica che
nella regione fundica discreta immunoreattivitГ ГЁ evidenziabile suggestivamente a carico
delle cellule principali (Fig.4) identificate in sezioni adiacenti a quelle trattate immunoistochimicamente con il blu di toluidina e con la ematossilina ferrica. Le cellule parietali sono
immunoistochimicamente negative (Fig.4). In alcune sezioni si scorgono pure fini fibre
orfaninergiche tra gli adenomeri o tra i citotipi secernenti.
Fig.5: Intestino tenue di ratto: immunopositività ore- Fig.6: Intestino tenue di ratto: intensa immunoespressione dell’OX2R
xinergica degli enterociti mucosali. Nel corion villare degli enterociti mucosali dei villi. Assai marcata immunoespressionumerosi citotipi immunopositivi - Ob. 40x
ne dell’OX2R in grosse cellule dello stroma villoso - Ob. 20x
Fig.7: Stomaco di ratto – regione antrale: immunoe- Fig.8: Immunoreattività N/OFQ negli enterociti villaspressione OXA in piccoli neuroni submucosali ente- ri (freccie nere); cellule caliciformi immunonegative
rici e negli epiteliociti degli adenomeri – Ob. 40x
(freccie verdi) - Ob. 20x
Fig.9: Pancreas endocrino di ratto: marcata reattivitГ Fig.10: Intensa immunoreattivitГ N/OFQ pancreatica
orexinergica dei citotipi endocrini - Ob. 40x
nei citotipi insulari e negli acini - Ob. 20x
Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
Nell’ intestino tenue (duodeno, digiuno-ileo)
Orexina A: rilevante immunopositivitГ orexinergica ГЁ estesa uniformemente al citoplasma degli enterociti mucosali (Fig.5). Consistente immunoreattivitГ ГЁ rilevabile anche a carico delle cellule secernenti delle cripte di Galeazzi-LieberkГјhn concentrata soprattutto nel
loro settore apicale. Nell’asse stromale dei villi e nel corion perighiandolare si rinvengono
numerosi citotipi, verosimilmente appartenenti al Sistema Immune Secretorio (linfociti-macrofagi) che esibiscono assai intensa immunopositivitГ (Fig.5). Cellule endocrine scaglionate
tra gli enterociti mucosali tra le cellule esocrine delle cripte di galeazzi esibiscono intensa
immunoreattivitГ . La espressione immunoistochimica del recettore OX2R ГЁ vistosa da parte
degli enterociti e delle cellule secernenti esocrine dei segmenti intestinali prese in esame.
Nell’asse stromale dei villi si rinvengono alcuni grossi citotipi che esprimono intensamente
il recettore tipo 2 (Fig.6).
Sia nell’ambito gastrico che enterico una numerosa subpopolazione di neuroni del plesso mioenterico di Auerbach e del plesso sottomucoso di Meissner esibiscono discreta immunoreattività (Fig.7).
Orfanina: intensa positività per la orfanina si rileva uniformememnte estesa nel citoplasma medioapicale degli enterociti mucosali assorbenti (Fig.8 freccia nera). Le cellule caliciformi e l’asse dei villi risultano immunoistochimicamente negativi (Fig.8 freccia verde).
Isolate cellule endocrine esprimono intensa immunoreattivitГ .
Nel pancreas:
Orexina A: intensa immunopositività è estesa uniformemente a tutti i citotipi dell’isola langheransiana (Fig.9). Il recettore OX2R è espresso in maniera meno rilevante in tutti i citotipi
Orfanina: tutti i citotipi insulari esprimono assai intensamente immunoreattivitГ orfaninergica. Nel pancreas esocrino le cellule acinose esprimono discreta immunoreattivitГ orfaninergica di aspetto fioccoso soprattutto concentrata nella porzione citoplasmatica
medioapicale (Fig.10). Nel connettivo stromale si rileva rimarchevole immunopositivitГ a
carico di tronchicini nervosi.
I nostri risultati forniscono la documentazione immunoistochimica che autorizza ad
ammettere che sia l’Orexina A con il suo recettore tipo 2 che la Orfanina FQ neuropeptidi
ritenuti in origine prodotti esclusivamente nei neuroni centrali possono essere espressi
perifericamente anche da alcuni citotipi costitutivi del Sistema GEP. Questa doppia sede di
nascita cerebrale e gastroenteropancreatica rafforza il concetto che per questi messag185
Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del Sistema Gastroenteropancreatico.
Uno studio immunoistochimico
geri, così come accade per altri neuropeptidi, possa esistere un asse correlativo cervellointestino (brain-gut axis). In aggiunta, i nostri reperti forniscono la base morfoistochimica per l’interpretazione del ruolo funzionale esplicato dall’orexina e dall’orfanina in tali
distretti suggerendo la possibilità che esse possano esercitare un’azione diretta di tipo
periferico sulle strutture summenzionate eplicando nuovi ruoli verosimilmente svolti con
diversi meccanismi: neurocrino, suggerito dalla immunopositivitГ dei neuroni orexinergici
e orfaninergici del sistema enterico, con il quale esse potrebbero controllare l’attività motoria gastroenterica e la vasomotilità ; endocrino suggerito dalla loro espressione immunoistochimica in cellule endocrine gastroenteropancreatiche con un possibile conseguente
controllo sulla secrezione acida gastrica intestinale e pancreatica modulando il senso
dell’appetito e l’omeostasi glicemica; autocrino-paracrino suggerito dalla loro espressione
immunoistochimica nelle cellule adelomorfe gastriche, negli enterociti, nelle cellule degli
adenomeri intestinali e nei citotipi esocrini pancreatici, tramite il quale questi neuropeptidi
potrebbero essere implicati nel controllo della funzione secretoria gastrica ed intestinale,
dell’assorbimento dei nutrienti, del trasporto ionico ed infine della funzione secernente
esocrina del pancreas.
Giuseppe Bonaventura, Giovanni Franco Spatola, Maria Laura Uzzo, Vincenzo Tessitore
[1] Neal CR Jr et al.: Localization of orphanin FQ (nociceptin) peptide and messenger RNA in Central Nervous System of the rat. J Comp Neurol 1999; 406:503.
[2] Bonaventura G. Rilievi immunoistochimici della orfanina nell’apparato genitourinario del ratto. Exper
Med Rev (eds A Gerbino, G Zummo, G Crescimanno) “Plumelia” Bagheria, Palermo 2008; Vol.1, pp.271277.
[3] Takahashi N. et al.: Stimulation of gastric acid secretion by centrally administrered Orexin-A in conscious rats. Biochem Biophys Res Comm 1999; 254:623-627.
[4] Kirchgessner A, Liu M. Orexin synthesis and response in the gut. Neuron 1999, 24:941-951.
[5] Krzystof W et al. Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies. Life
Sciences 2000; 66 (5):449-454.
[6] Naslund E. et al.: Localizzation and effects of orexin on fasting motility in the rat duodenum. Am J
Physiol Gastointest Liver Physiol 2002; 282:G470-G479.
[7] Yazdani A, et al. Functional significance of a newly discovered neuropeptide, orphanin FQ, in rat gastrointestinal motility. Gastroenterology 1999; 116(1):108-17.
[8] Osinski MA, Brown DR. Orphanin FQ/nociceptin: a novel neuromodulator of gastrointestinal function?
Peptides 2000; 21:999.
[9] Giuliani S. et al. Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur J Pharmacol 1997; 333:177-179.
[10] Fischer A, et al. Nociceptin-induced inhibition of tachykinergic neurotransmission in guinea pig bronchus. J Pharmacol Exp Ther 1998; 285:902-907.
[11] Shah S, et al. Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway. Br
J Pharmacol 1998; 125:510-516.
[12] Giuliani S, et al. The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats. Br J
Pharmacol 1998; 124:156-672.
[13] Bonaventura G, et al. Immunohistochemical distribution of orphanin FQ in the rat gastrointestinal apparatus and pancreas. It J Anat Embryol 2005; 110/Suppl.1 (3):262.
[14] Tessitore V, et al. Immunohistochemical expression of Orexin A and Orexin Type 2 receptors in the rat
gastrointestinal apparatus and endocrine pancreas. EJH 2005;49 (Suppl.1):19.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Effects of cronical treatment with a CB1 receptor angatonist (Rimonabant) on pancreatic
apoptosis of obese and diabetic Zucker rats. An immunohistochemical study]
Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola e
Maria Laura Uzzo
Di.Me.S. - Sezione di Istologia ed Embriologia - UniversitГ degli Studi di Palermo (IT)
Parole chiave: CB1, Rimonabant, Apoptosi, Pancreas, Ratti Zucker
Key words: CB1, Rimonabant, Apoptosi, Pancreas, Zucker rats
Riassunto. Recenti ricerche indicano che vi ГЁ una iperattivazzione del sistema endocannabinoide (EC) in ratti
e umani obesi. In una precedente pubblicazione abbiamo dimostrato che nei ratti Zucker obesi e diabetici
vi ГЁ un danno apoptotico nelle cellule B degli isolotti pancreatici. Inoltre, il recettore CB1 ГЁ iperespresso da
parte di tutti i citotipi endocrini in confronto con gli analoghi citotipi dei ratti normali in cui l’immunoreattività del CB1 è limitata alle cellule A. Inoltre, altri AA. hanno dimostrato che l’attivazione del recettore CB1 per i
cannabinoidi induce una intolleranza al glucosio nei ratti. Gli studi clinici in umani obesi trattati con un antagonista del recettore CB1 dei cannabinoidi (Rimonabant) hanno mostrato una riduzione del peso corporeo
e il miglioramento della resistenza insulinica. Con riferimento a quanto dimostrato abbiamo studiato gli effetti
del trattamento cronico con Rimonabant sulla apoptosi delle cellule B nel pancreas endocrino di ratti Zucker
obesi e diabetici (ZDF) utilizzando come indicatore immunoistochimico di apoptosi la Caspase-9 e valutando
la presenza di nNOS / iNOS implicati nel danno cellulare. I nostri risultati dimostrano che il trattamento con
Rimonabant riduce significativamente l’incidenza di apoptosi nelle isole di ratti ZDF rispetto a quelle dei ratti
normali non-diabetici. Questi risultati forniscono una base morfoistochimica a supporto dell’uso di questo
farmaco nella sindrome metabolica associata con l’obesità .
Abstract. Recent reports suggest that there is an overactivation of the endocannabinoid system (EC) in obese
humans and rats. In a previous publication we have shown that in obese and diabetic Zucker rats the pancreatic islets show B-cells apoptotic damage. In addition the CB1 receptor is overexpressed by all endocrine cells
in comparison with normal rat islets in which CB1 immunoreactivity is restricted to A-cells. Also, other AA. have
demonstrated that activation of cannabinoid CB1 receptor induce glucose intolerance in rats. Clinical trials in
obese men with a cannabinoid CB1 receptor antagonist (Rimonabant) have shown a reduction of body weight
and improvement of insulin resistance. With references to findings we have studied the effects of chronic
treatment with Rimonabant on B-cells apoptosis in endocrine pancreas of the Zucker diabetic fatty rats (ZDF)
underlining immunohistochemically Caspase-9 as an indicator of apoptosis and nNOS/iNOS activities implicated in cellular damage. Our results show that Rimonabant treatment significantly decreases the apoptotic
incidence in the islets of ZDF rats compared with those of non-diabetic normal rats. These findings provide a
morphohistochemical basis to support the use of this drug in metabolic syndrome associated with obesity.
Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo
Recenti evidenze sperimentali suggeriscono che gli endocannabinoidi (EC) regolano
l’appetito e il peso corporeo rappresentando uno dei più importanti sistemi oressizzanti
del nostro organismo. Tale effetto si esplica sia con un meccanismo di azione ritenuto in
un primo tempo esclusivamente centrale sia anche con un meccanismo periferico piГ№ di
recente acquisito, entrambi svolti per attivazione del recettore CB1 espresso sia da neuroni
dell’ipotalamo e del sistema limbico (sistema endocannabinoide centrale) sia da cellule
di organi periferici (muscolo, fegato, organo adiposo, sistema gastroenteropancreatico,
ghiandole salivari) (sistema endocannabinoide periferico) [1]. Nuove evidenze sperimentali
indicano che gli EC modulano le funzioni del pancreas esocrino ed endocrino, nuovo target
periferico nell’azione degli EC nel controllo dell’equilibrio nutrizionale [2,3] tramite interazione con i recettori pancreatici CB1 e CB2. La identificazione recettoriale pancreatica fa
supporre anzitutto che gli endocannabinoidi esercitano un controllo diretto sul pancreas
con meccanismo autocrino o paracrino-endocrino e dischiude implicitamente nuove prospettive terapeutiche nell’ambito della patofisiologia pancreatica esocrina ed endocrina
con l’impiego di farmaci che interferiscono in modo vario con i suddetti recettori.
Nel pancreas esocrino, molte strutture (cellule acinari, cellule duttali, nervi) esprimono
immunoistochimicamente nel ratto e nei soggetti normali sia il recettore CB1 che il recettore CB2 [2], tuttavia i risultati degli studi sull’esatto ruolo svolto in situazioni patofisiologiche dagli EC in questo distretto, sono ancora frammentari e per alcuni aspetti controversi.
Ad esempio la attivazione nell’uomo dei recettori CB1 e CB2 con bassi dosaggi di agonisti
dei cannabinoidi attenua la risposta infiammatoria nella pancreatite acuta, mentre alte
dosi la incrementano [4]. Per contro l’antagonista AM251 del recettore CB1 prolunga la
sopravvivenza del ratto affetto da severa pancreatite acuta [5].
Per ciГІ che riguarda i rapporti tra EC e pancreas endocrino la recente letteratura fornisce rilievi non univoci per quanto attiene la identificazione dei citotipi endocrini capaci di
esprimere i recettori CB1 e CB2 tali dispareri sono verosimilmente asccrivibili sia all’impiego
di modelli sperimentali diversi di volta in volta, sia all’adozione di tecniche non similari. In
nostre precedenti ricerche abbiamo per la prima volta fornito documentazioni immunoistochimiche in vivo e nel ratto normale relative all’espressione del CB1 nelle cellule A (Fig.1);
utilizzando ratti Zucker obesi ed iperfagici la immunoreattivitГ per il CB1 si estende a tutti i
citotipi insulari interessando non soltanto i citotipi A, come avviene nel ratto normale (Fig.2)
[3]. Jian PicГІ e coll hanno identificato in vitro nel topo la presenza del recettore CB1 nei citotipi A e del recettore CB2 nei citotipi A e B [6]. BermГ№dez-Silva e coll hanno documentato
nell’uomo immunofluorescenza per il CB1 nelle cellule A [7]. William G. Tharp e coll hanno
rilevato nei ratti Zucker l’espressione del CB1 colocalizzato con la somatostatina nelle cellule
D [8]. Matias e coll hanno dimostrato in vitro la presenza di endocannabinoidi nelle cellule
Effetti del trattamento cronico con un antagonista del recettore cb1 (rimonabant)
sul pancreas apoptotico di ratti zucker obesi e diabetici. Uno studio immunoistochimico
B del ratto (rat insulinoma RIN-m5F ОІ-cells) e dei topi DIO [9]. Starowicz e coll nei topi DIO
hanno documentato l’espressione immunoistochimica del CB1 in cellule A insieme agli enzimi di sintesi dei cannabinoidi e di enzimi degradanti cannabinoidi nelle cellule B [10]. È
stato infine dimostrato che in animali sottoposti a diete iperlipidiche e nell’obesità umana il
sistema endocannabinoide ГЁ iperattivato e che nei vari targets centrali e periferici il CB1 ГЁ
iperespresso [3,9-12]. Questi risultati fanno ritenere che gli EC esplichino un ruolo cruciale
nell’obesità , per cui per il suo trattamento è stato recentemente proposto l’impiego di un
antagonista del CB1. È stato pure documentato che l’attivazione del recettore CB1 induce
intolleranza al glucosio e diminuzione della secrezione insulinica da parte delle beta cellule
il che suggerisce un ruolo di controllo degli EC sulla omeostasi glicemica [13,14]. L’insieme
di questi dati ha stimolato a proporre nell’obesità e nelle comorbilità associate l’impiego di
un antagonista del CB1 il Rimonabant, che in numerosi trials clinici ha prodotto in pazienti
obesi e diabetici non soltanto una significativa riduzione del peso corporeo ma anche un
aumento della insulinoresistenza e del profilo glicemico [15-18]. Con riferimento a quanto
sopra, abbiamo studiato l’effetto di un trattamento cronico con Rimonabant sulla apoptosi
Fig.1: Pancreas endocrino ratto normale: CB1 immuno- Fig.2: Pancreas endocrino ratto Zucker: CB1 immureattivitГ sostanzialmente limitata alle cellule A - 40x noreattivitГ omninsulare - 40x
Fig.3: Pancreas esocrino ratto Zucker non trattato: Fig.4: Pancreas esocrino ratto Zucker trattato: nointensa immunoreattività caspasica nei nuclei delle tevole riduzione dell’attività caspasica nucleare del
cellule acinose - 20x
comparto acinoso - 20x
Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo
delle beta cellule nel pancreas endocrino di ratti Zucker obesi e diabetici (ZDF) valutando immunoistochimicamente la Caspasi 9 come indicatore dell’apoptosi e le attività enzimatiche
NOS implicate spesso nel danno cellulare attraverso una iperproduzione di NO.
Materiali e metodi
Sono stati utilizzati ratti Zucker obesi e diabetici (ZDF) della Harlan Italy S.r.l. sottoposti
a dieta tipo Harlan Tekland, Rodent diet # 8640. Gli animali sono stati divisi in due gruppi.
Al primo gruppo ГЁ stato somministrato tramite sondino gastrico Rimonabant (10mg pro
Kg/pro die per 28 giorni). Il secondo gruppo riceveva dosi orali di Carbossimetilcellulosa
come veicolo (5ml proKG/pro die per 28 giorni). Di ciascun animale ГЁ stato controllato il
peso corporeo e su ciascuno ГЁ stato effettuato un prelievo ematico per il monitoraggio dei
valori glicemici sia il 1В° giorno di trattamento che il 28В° giorno. Tutti gli animali sono stati
sacrificati allo scadere dei 28 giorni. I campioni di pancreas prelevati sono stati fissati in
liquido di Bouin e inclusi in paraffina. Alcune sezioni seriate ottenute, dello spessore di 8
Fig.5: Pancreas endocrino ratto Zucker non trattato: Fig.6: Pancreas endocrino ratto Zucker trattato: signifiisola apoptotica; immunoreattivitГ caspasica dei ci- cativa riduzione dei fenomeni apoptotici con immunototipi residui - 20x
reattivitГ caspasica limitata a qualche citotipo - 40x
Fig.7: Pancreas endocrino ratto Zucker non trattato: Fig.8: Pancreas endocrino ratto Zucker trattato: immunoisola apoptotica; intensa immunoreattivitГ nNOS nei reattivitГ nNOS estesa come di norma a molti citotipi del
parenchima insulare sostanzialmente non apoptotico - 40x
citotipi residui - 40x
Effetti del trattamento cronico con un antagonista del recettore cb1 (rimonabant)
sul pancreas apoptotico di ratti zucker obesi e diabetici. Uno studio immunoistochimico
Ој, sono state colorate con ematossilina eosina per lo studio morfologico e altre sono state
processate con un anticorpo anti Caspase-9 (Sigma) and anti nNOS/iNOS (Transduction
Laboratories) by Envision + System HRP (AEC) (DAKO Citomation). Tutte le sezioni sono
state osservate mediante microscopio Leica DM1000.
Risultati e conclusioni
Pancreas Esocrino: Nel comparto acinoso dei ratti non trattati le cellule acinari e duttali rivelano intensa attivitГ caspasica nucleare in molti citotipi (Fig. 3). Negli animali trattati i citotipi
esocrini non esibiscono immuno reattivitГ caspasica nucleare e rivelano solo scarsa reattivitГ citoplasmatica (Fig.4). In entrambi i gruppi la immunoreattivitГ iNOS e nNOS ГЁ assai debole.
Pancreas Endocrino: Nei ratti non trattati si evidenzia una destrutturazione dell’architettura insulare all’esame morfologico con EE. Nei citotipi residui la immunoreattività caspasica nucleare è assai intensa denunciando un elevato grado di apoptosi (Fig.5). La
immunoreattivitГ nNOS ГЁ intensa e limitata a quei citotipi endocrini che sfuggono ai fenomeni apoptotici (Fig.7), la iNOS ГЁ assai poco evidente. Nei ratti trattati le isole non presentano sostanzialmente, allo studio morfologico, fenomeni apoptotici come dimostra la
immunoreattività caspasica limitata a pochi citotipi endocrini (Fig.6). La immunoreattività nNOS è discretamente estesa come di norma ai citotipi dell’isola (Fig.8), la iNOS è sostanzialmente assente.
I risultati da noi ottenuti dimostrano che la somministrazione di un antagonista del CB1
(Rimonabant) puГІ modificare la funzione esocrina pancreatica per cui gli EC modulandola
direttamente possono essere implicati, quindi, nel meccanismo fisiopatologico delle malattie esocrine del pancreas [2]. Per ciГІ che attiene il pancreas endocrino i nostri reperti
fanno ritenere che il Rimonabant esercita un freno sulla comparsa della destrutturazione
apoptotica a carico delle isole di Langherans migliorando l’insulite data la deflessione
nei ratti trattati della immunoreattivitГ caspasica insulare, da noi considerata un indice
proapoptotico. La somministrazione di un antagonista del CB1 blocca secondo la nostra
interpretazione una sequenza di eventi che nel ratto Zucker non trattato esitano nella alterazione insulare e nel conseguente dissesto glicemico; infatti la documentata iperattivazione del CB1 svincolata dal freno che nel ratto Zucker la leptina non puГІ esercitare come di
norma causa una iperfagia incontrollata e protratta con conseguente lipotossicitГ insulare
e distruzione delle beta cellule. Il Rimonabant con la sua azione antagonista sul CB1 insulare garantisce la integritГ morfofunzionale delle beta cellule e la normale insulinopoiesi.
Il Rimonabant è capace quindi di svolgere sul piano clinico non soltanto un effetto anoressizzante ma anche metabolico sulle comorbidità associate all’obesità (diabete) Data la
sovrapponibilitГ dei reperti immunoistochimici attinenti alla NOS nei due gruppi di animali
non sembra che essa sia coinvolta nel determinismo della destrutturazione apoptotica.
Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo
[1] Cota D, Woods SC. The role of the endocannabinoid system in the regulation of energy homeostasis.
Curr Opin Endocr Diab 2005; 12: 338-351.
[2] Izzo AA, Camilleri M. Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and
clinical aspects. Gut 2008; 57(8):1140-55. Epub 2008 Apr 8.
[3] Tessitore V, et al. Immunohistochemical study and densitometric comparision on CB1 receptor expression in the gastrointestinal tract and pancreas of obese (fa/fa) and lean Zucker rats. It J Anat Embryol
2006; 111 (Suppl 12):256.
[4] Michalski CW, et al. Cannabinoids ameliorate pain and reduce disease pathology in cerulein-induced
acute pancreatitis. Gastroenterology 2007; 132(5):1968-78. Epub 2007 Feb 21.
[5] Matsuda K, et al. The cannabinoid 1 receptor antagonist, AM251, prolongs the survival of rats with
severe acute pancreatitis. Tohoku J Exp Med 2005; 207(2):99-107.
[6] Juan-PicГІ P, et al. Cannabinoid receptors regulate Ca 2+ signals and insulin secretion in pancreatic
ОІ-cell. Cell Calcium 2006; 39:155-162.
[7] BermГ№dez-Silva FJ, et al. Presence of functional cannabinoid receptors in human endocrine pancreas.
Diabetologia 2008; 51:476-487.
[8] Tharp WG, et al. The cannabinoid CB1 receptor is expressed in pancreatic Оґ-cells. Biochem Biophys
Res Comm 2008; 372:595-600.
[9] Matias I, et al. Regulation, function, and dysregulation of endocannabinoids in models of adipose and
ОІ-pancreatic cells and in obesity and hyperglycemia. J Clin Endocrinol Metab 2006; 91(8): 31713180.
[10] Starowicz KM, et al. Endocannabinoid dysregulation in the pancreas and adipose tissue of mice fed
with a high-fat diet. Obesity 2008; 16:553-565.
[11] Di Marzo V, et al. The role of endocannabinoids in the regulation of gastric emptying: alterations in mice
fed a high-fat diet. Br Journ of Pharm 2008; 153:1272-1280.
[12] Engeli S, et al. Activation of the peripheral endocannabinoid system in human obesity. Diabetes 2005;
[13] BermГ№dez-Silva FJ, et al. Activation of cannabinoid CB1 receptors induces glucose intolerance in rats
Eur J of Pharm 2006; 531:282-284.
[14] Masanori N, Toshihiko Y. Cannabinoids inhibit insulin secretion and cytosolic Ca 2+ oscillation in islet
ОІ-cells via CB1 receptors. Regulatory Peptides 2008; 145:49-53.
[15] Ravinet Trillou C, et al. CB1 cannabinoid receptor knockout in mice leads to leanness, resistance to dietinduced obesity and enhanced leptin sensitivity. Int J Obes Relat Metab Disord 2004; 28(4):640-8.
[16] Ravinet Trillou C, et al. Anti-obesity effect of SR141716, a CB1 receptor antagonist, in diet-induced
obese mice. Am J Physiol Regul Integr Comp Physiol 2003; 284(2):R345-53. Epub 2002 Oct 24.
[17] Vickers SP, et al. Preferential effects of the cannabinoid CB1 receptor antagonist, SR 141716, on food
intake and body weight gain of obese (fa/fa) compared to lean Zucker rats. Psychopharmacology (Berl).
2003; 167(1):103-11. Epub 2003 Mar 11.
[18] Hildebrandt AL, et al. Antiobesity effects of chronic cannabinoid CB1 receptor antagonist treatment in
diet-induced obese mice. Eur J Pharmacol 2003; 462(1-3):125-32.
Tumor process
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari
e clinici
[Heredofamilial and pediatric GISTs: biomolecular and clinical aspects]
Antonio Russo1, Sergio Rizzo1, Daniele Fanale1, Valentina CalГІ1, Nello Grassi2, Gianni Pantuso2, Luigi Sandonato2, Michele Frazzetta3, Giuseppe Cicero1, Massimo Cajozzo4, Aldo Gerbino5, Giuseppe Badalamenti1, Nicola Gebbia1 e Viviana Bazan1
Sezione di Oncologia Medica, 2Sezione di Oncologia Chirurgica e 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4UnitГ di Chirurgia Toracica; 5Sezione di Istologia ed Embriologia,
Di.Me.S., UniversitГ degli Studi di Palermo (IT)
Parole chiave: Tumori stromali gastrointestinali; Mutazioni attivanti; KIT; PDGFRA; Carney-Stratakis; Neurofibromatosi
Key words: Gastrointestinal stromal tumors; Germline mutations; KIT; PDGFRA; Carney-Stratakis; Neurofibromatosis
Riassunto. I tumori stromali gastrointestinali (GISTs) sono rare neoplasie sporadiche che insorgono prevalentemente in etГ avanzata; tuttavia sono stati riportati casi di aggregazione familiare e pediatrici. Tali casi
hanno caratteristiche clinicopatologiche e molecolari distinte da quelli sporadici. Infatti nei GISTs pediatrici
mancano le mutazioni attivanti in KIT e PDGFRA comunemente riscontrabili negli adulti. Le analisi dei profili di espressione genica dei GISTs pediatrici mostrano caratteristiche peculiari e suggeriscono un’origine
comune rispetto ai GISTs degli adulti. La review tratta inoltre i GISTs familiari con mutazioni germinali nei
geni KIT e PDGFRA e le forme che non presentano queste mutazioni come la triade di Carney, la sindrome di
Carney-Stratakis, e la neurofibromatosi di tipo 1.
Abstract. Gastrointestinal stromal tumors (GISTs) are rare sporadic tumors that typically occur late in life;
however, there also are reports of familial and pediatric cases. These rare subsets of GISTs have clinicopathologic and molecular features distinct from their sporadic counterparts. Most pediatric GISTs lack
the gain-of-function mutation in KIT or PDGFRA commonly found in adult cases. Gene expression profiling
studies of pediatric GISTs show distinct molecular signatures, suggesting a unique origin as compared with
adult GISTs. The review discusses also familial GISTs with germline KIT or PDGFRA genes mutations and
forms that don’t harbour mutations in these genes such as Carney triad, Carney-Stratakis syndrome, and
neurofibromatosis type 1.
I tumori stromali gastrointestinali (GISTs) sono tumori rari la cui l’incidenza è stata chiarita di recente, dopo la ridefinizione dei criteri diagnostici in seguito al riconoscimento del
caratteristico profilo immunoistochimico di queste neoplasie.
Questi tumori inizialmente erano infatti considerati eccezionali, con una incidenza in
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
passato stimata a circa 1,5 casi per milione di abitanti all’anno, ma oggi non sono più tali
dato che la reale incidenza di queste neoplasie è 10 volte superiore a quanto precedentemente ipotizzato e pari a circa 1000 casi all’anno in Italia e a circa 4500-5000 casi negli
I GIST rappresentano meno dell’1% di tutte le neoplasie maligne, ma sono i più comuni
tumori mesenchimali del tratto gastroenterico (0,1-3 % in questa sede).
Si presentano in piГ№ del 99% dei casi in forma sporadica ma esiste una minima percentuale di GIST eredo-familiari caratterizzati da mutazioni germinali in diversi geni di
suscettibilitГ . In particolare si distinguono forme familiari quando nella stessa famiglia
piГ№ soggetti sono affetti dallo stesso tipo di tumore e forme ereditarie quando si riesce a
dimostrare che la predisposizione allo sviluppo della neoplasia ГЁ trasmessa geneticamente
dai genitori ai figli.
Aspetti biomolecolari dei GIST: c-KIT e PDGFRA.
La genetica molecolare non solo ha svelato il meccanismo coinvolto nella genesi dei
GIST, ma, anche, ci ha consegnato un marcatore immunofenotipico il c-KIT.
La maggior parte dei GIST (90-95%) mostrano mutazioni attivanti di c-KIT con immunopositivitГ KIT/CD117 pari circa al 95%. Tale marcatore consente quindi nel 90-95% dei
casi di produrre una diagnosi molto affidabile. c-KIT ГЁ un oncogene che mappa sul cromosoma 4q12 ed ГЁ costituito da 21 esoni codificanti. Nel gene sono presenti 4 hot spot di
mutazione che cadono in ordine di frequenza negli esoni 11, 9, 13 e 17.
KIT ГЁ una glicoproteina transmembrana di 145KD con attivitГ tirosin-chinasica che ГЁ
un’attività enzimatica indispensabile per lo sviluppo (differenziamento) e la crescita delle
cellule (proliferazione). In condizioni non patologiche il recettore c-KIT ГЁ una molecola che
ha attività TK cioè è un “interruttore cellulare” che se attivato in seguito all’interazione del
proprio ligando (SCF) con la porzione extracellulare del recettore attraverso processi di
dimerizzazione innesca un pathway intracellulare cioГЁ avvia una cascata di segnali il cui
fine è l’aumento della proliferazione, il differenziamento e l’inibizione dell’apoptosi.
Entrando piГ№ in dettaglio, il legame di una molecola di SCF con ciascuno dei monomeri
c-KIT dà luogo alla formazione di un c-KIT dimero. L’interazione di due recettori adiacenti
(omodimero) ГЁ mediata proprio da due molecole di SCF. La omodimerizzazione comporta
cambiamenti strutturali dei recettori con attivazione della tirosinchinasi intracellulare che
catalizza anzitutto la auto-fosforilazione a livello di specifici residui tirosinici, che antagonizza l’auto-inibizione ed è necessaria per la trasduzione a valle del segnale conseguente
all’interazione del ligando col recettore.
Tra i pathways attivati ci sono: PI3-Kinasi/AKT, RAS/RAF/MAPK e JAK/STAT.
Nella situazione patologica la maggior parte delle mutazioni di questo gene innescano
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
una omodimerizzazione spontanea che, in definitiva, provoca un’autoattivazione con un
vantaggio proliferativo e un effetto anti-apoptotico.
Sappiamo che le mutazioni del gene c-KITvengono riconosciute come l’evento patogenetico principale (90% dei casi), ma non rappresentano l’unico evento patogenetico. Infatti
esistono GIST che non presentano mutazioni in c-KIT, ma, con rapporto mutualmente esclusivo, nel gene PDGFR-alfa (fattore di crescita di derivazione piastrinica) codificante per una
molecola con attivitГ tirosin-kinasica con funzione sostanzialmente analoga a c-KIT.
Strutturalmente il recettore ГЁ costituito da tre porzioni principali:
1. Una porzione extracellulare contenente i siti di legame con il ligando, costituita da 5
domini Ig-simili.
In questa regione si riconoscono:
-- il dominio di legame col ligando, vale a dire quella parte che lega specificatamente
il ligando o fattore di crescita costituita da 4 domini Ig-simili;
-- il dominio di dimerizzazione che è alla base dell’attivazione del recettore.
2. Una porzione transmembranaria
3. Una porzione intracellulare contenente l’attività tirosin-chinasica, suddivisa in due domini:
-- il dominio iuxtamembrana o loop auto-inibitorio (codificato dall’esone 11 e capace
di auto-inibire l’attività chinasica in assenza dello specifico ligando) sito al di sotto
della membrana cellulare
-- il dominio chinasico (catalitico) che ГЁ la parte attiva del recettore cioГЁ la parte che ha
la proprietà biochimica di trasferire un gruppo fosfato dall’ATP ad un certo substrato.
In particolare il dominio TK1contiene il sito di legame per l’ATP mentre il dominio
TK2 contiene l’attività fosfotransferasica.
Hot spot di mutazioni di c-KIT e PDGFRA.
Più dell’90% dei GIST presentano mutazioni attivanti a livello del gene c-KIT (in circa il
90-95% dei casi) o meno frequentemente del gene PDGFRA (circa il 5-10% dei casi).
In KIT sono stati identificati quattro hot-spots a livello degli esoni 9, 11, 13 e 17.
Esiste una gerarchia nelle mutazioni dei GIST.
L’esone maggiormente interessato è l’esone 11, in circa il 70-80% dei casi di GIST, che
codifica per il “loop auto-inibitorio”. Questo dominio esercita l’azione auto-inibitoria nei
confronti della tirosin-chinasi del recettore.
L’esone 9, risulta mutato nel 10-20% dei GIST. L’esone 9 codifica per il quinto dominio
simil-immunoglobulinico della regione extracellulare del recettore.
Meno frequenti (1-5%) sono le mutazioni puntiformi a carico degli esoni 13 e 17 che
codificano per i domini tirosin-chinasici della porzione intracitoplasmatica del recettore.
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
Il gene PDGFRA codifica per il recettore del fattore di crescita di derivazione piastrinica e condivide con c-KIT circa il 35% della sequenza aminoacidica. Anche questa ГЁ una
molecola con attività tirosin-chinasica. Il pathway che segue all’attivazione del recettore è
sostanzialmente analogo a quello di c-KIT.
Modello di progressione tumorale dei GIST
Le mutazioni attivanti in c-KIT o in PDGFRA si osservano pressochè nell’intera popolazione cellulare dei GIST, mentre le alterazioni cromosomiche si osservano solo in una percentuale di cellule neoplastiche. Ciò suggerisce che le mutazioni di c-KIT rappresentano
un’evento precoce, presumibilmente, aventi un ruolo di iniziazione della trasformazione
neoplastica, mentre le successive alterazioni citogenetiche sono coinvolte nella progressione neoplastica.
I GIST a basso gradohanno spesso un cariotipo normale o presentano la perdita del
braccio corto del cromosoma 1p o la perdita del cromosoma 14 o 22 (50% dei casi).
I GIST ad alto grado sono spesso accompagnati da perdita da almeno 3 delezioni cromosomiche (1p, 9p,11p o 22p).
I GIST metastatici sono caratterizzati da perdita nel cromosoma 9p associata a guadagno in 5q, 8q, 17q e 20q. Inoltre presentano LOH di c-KIT e diminuzione dell’espressione
di TP53 e MSI.
Bisogna, tuttavia, sottolineare che questa sequenza di eventi rappresenta una semplificazione e che non tutti i GIST acquisiscono alterazioni genetiche nell’ordine riportato.
Gist pediatrici
Sono definiti pediatrici i GIST diagnosticati entro il diciottesimo anno d’età . I GIST pediatrici finora descritti rappresentano tra l’1 e il 2% di tutti i casi di GIST, e quindi in età giovanile sono tumori raramente diagnosticati.
La ricerca di base sulla biologia dei GIST ГЁ stata ostacolata dalla raritГ della malattia e
dalla mancanza di modelli sperimentali. Nonostante tali limitazioni, le conoscenze acquisite
dallo studio dei GIST negli adulti e dei tipi tumorali ad essi correlati (per es. paragangliomi),
oltre alla nascita di nuove metodiche (per es. tecniche di microarrays) hanno permesso
significativi passi in avanti nella comprensione delle rare forme pediatriche. Questi tumori
possono avere una differente patogenesi rispetto ai GIST degli adulti, poichГ© possono mancare le mutazioni nei geni KIT o PDGFRA (sono cioГЁ GIST wild-type). Questo dato potrebbe
suggerire che siano attivi all’interno delle cellule altri meccanismi di attivazione di KIT o
pathways oncogeniche non correlati ad esso. Nella maggior parte dei GIST pediatrici esaminati, non ГЁ stata riscontrata alcuna anomalia citogenetica o alterazioni degli esoni 9, 11 o
13 di KIT. Tra i pochi GIST pediatrici sottoposti ad analisi mutazionale e riportati in letteratu200
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
ra, solo nell’11% dei casi è stata evidenziata mutazioni nei geni KIT o PDGFRA, equamente
distribuite. Questo dato ГЁ differente da quello osservato nei GIST adulti sporadici in cui le
mutazioni in KIT sono 10 volte piГ№ comuni delle mutazioni in PDGFRA. Nelle forme pediatriche tali mutazioni sono identificate prevalentemente nei maschi. Infatti, ГЁ stato riportato
un solo caso di paziente di sesso femminile con una mutazione in PDGFRA. Probabilmente
queste mutazioni rappresentano degli eventi molecolari casuali. Si potrebbe ipotizzare la
presenza di mutazioni localizzate in altri esoni di KIT e PDGFRA, differenti da quelle riscontrate negli adulti, ma quando ГЁ stato effettuato il sequenziamento diretto del gene non sono
state identificate nuove mutazioni in KIT. Nonostante non ci siano generalmente mutazioni
a carico di KIT nelle forme pediatriche di GIST, tale gene risulta spesso espresso ed il suo
pathway attivato. In relazione all’esiguo numero di casi ad oggi identificati, le alterazioni
genetiche alla base dell’insorgenza di un GIST pediatrico sono comunque poco note e forse
sono implicate anche alterazioni epigenetiche o mutazioni ipomorfiche (in porzioni genomiche che regolano il gene coinvolto). Infatti, il grado di attivazione misurato dalla fosforilazione della corrispondente proteina in tali pazienti risulta essere simile a quello riscontrato
nei GIST adulti KIT-mutati. Inoltre, risultano attivati anche i geni a valle di KIT: MAPK, Akt, S6
e mTOR . Questi dati avvalorano l’ipotesi che KIT ed i geni a valle di esso possano essere
importanti target terapeutici anche nei pazienti pediatrici.
La maggior parte dei GIST pediatrici che non presenta mutazioni in KIT o PDGFRA ГЁ
caratterizzata da pochi riarrangiamenti cromosomici su larga scala. Effettuando esperimenti di microarrays mediante SNP analysis, ГЁ stato osservato che la quasi totalitГ dei
GIST pediatrici wild type mostrano preservazione dell’eterozigosità e copy number diploide
lungo l’intero genoma. Questo dato concorda con precedenti valutazioni di GIST in età pediatrica mediante citogenetica classica, che hanno evidenziato cariotipi diploidi. Mediante
successiva analisi CGH (ibridazione genomica comparativa), il 60% dei 13 GIST wild type
pediatrici non mostrava alterazioni cromosomiche. Nel restante 40% di tumori con variazioni cromosomiche, è stato osservato che l’anomalia più comunemente identificata era la
delezione 1q, riscontrata in 3 casi.
I profili di espressione genica dei GIST pediatrici sono differenti da quelli dei GIST adulti
wild type e sono caratterizzati dall’incrementata espressione dei seguenti geni: BAALC, IGF1R, FGF4, CRLF1, PLAG1 e NELL1. Inoltre, in uno studio del 2005, Prakash S. et al. hanno
analizzato l’espressione genica di campioni tumorali di GIST provenienti da bambini, giovani adulti (età inferiore ai 30 anni) e adulti, trovando un’alta espressione dei geni PHKA1,
FZD2, NLGN4, IGF1R e ANK3 nei primi due gruppi di pazienti. I GIST che si sviluppano in
giovani adulti, con caratteristiche simili a quelle osservate nelle forme pediatriche, mostrano profili di espressione genica piГ№ simili a quelli dei GIST pediatrici wild type. Tuttavia, gli
esperimenti di gene expression descritti sono stati effettuati usando un numero esiguo di
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
campioni tumorali e i risultati non sono stati confermati in seguito a causa della difficoltà di ottenere campioni di tessuto disponibili per l’analisi dell’RNA.
L’espressione di IGF1R (“insulin-like growth factor 1 receptor”) è 5 volte più alta nei
GIST wild type pediatrici che in quelli wild type degli adulti. IGF1 e il suo recettore (IGF1R)
giocano un ruolo chiave nella crescita e proliferazione cellulare e nello sviluppo. L’analisi
dell’espressione proteica di IGF1R nei GIST, mediante western blotting e immunoistochimica, mostra che tale proteina è espressa in tutti i GIST, ma che i suoi livelli di espressione
sono molto piГ№ alti nel GIST wild type . IGF1R ГЁ attivato in molti GIST, ma i suoi livelli di
attivazione non sono correlati alla sua espressione. Г€ stato osservato, inoltre, che, durante
la stimolazione con IGF1, l’inibizione di IGF1R da parte di piccole molecole riduce la proliferazione di una linea cellulare di GIST KIT-mutata. Questi dati supportano il ruolo di IGF1R
come potenziale target terapeutico nei GIST pediatrici.
Come nei GIST degli adulti, le manifestazioni cliniche piГ№ frequenti al momento della
diagnosi comprendono il sanguinamento gastro-intestinale e l’anemia ad esso correlata e
talvolta ГЁ presente una massa addominale palpabile, voluminosa e tale da determinare compressione intestinale. Ai GIST insorti in etГ pediatrica possono associarsi alcuni tumori, e cioГЁ
l’osteosarcoma e il neuroblastoma. Circa il 10% dei GIST pediatrici si presentano nel contesto della triade o della diade di Carney (vedi più oltre), di conseguenza altre caratteristiche
suggestive di questo complesso, in particolare l’iperpigmentazione e la sindrome da inappropriata secrezione di catecolamine associata ai paragangliomi, devono essere attentamente
ricercate al momento della valutazione iniziale di un paziente pediatrico affetto da GIST.
L’istotipo più comune è a cellule epitelioidi o a morfologia mista fusata/epitelioide. Il
decorso clinico, in genere indolente, è tuttavia meno prevedibile rispetto all’adulto, anche
prendendo in considerazione i fattori prognostici comunemente considerati (dimensioni,
indice mitotico e sede del tumore). Altra caratteristica comune è la multifocalità nel contesto della regione anatomica sede di insorgenza che condiziona l’insorgenza di recidive
locali dopo diversi anni dall’intervento chirurgico.
Prima dell’introduzione dei farmaci a bersaglio molecolare (in primo luogo Imatinib),
nella terapia dei GIST la chirurgia rappresentava l’unico trattamento possibile e costituisce
a tutt’oggi lo standard quando il tumore è radicalmente resecabile. Nonostante la dissezione linfonodale non sia raccomandata negli adulti, nei GIST pediatrici, per l’incidenza di
metastasi linfonodali relativamente elevata è prevista l’asportazione della stazioni linfonodali sospette al momento dell’intervento chirurgico. L’intervallo tra i controlli successivi
all’intervento chirurgico dovrebbe prevedere, oltre all’esame obiettivo, il ricorso periodico
ad esami di imaging quali ecografie, TC, RM e PET, che dovrebbero essere effettuati per i
primi 2 anni ogni 3 mesi e ogni 6 mesi nei due anni successivi, infine annualmente. come
del resto negli adulti.
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
Prima dell’introduzione dell’Imatinib mesilato, l’intervento chirurgico con intento radicale veniva proposto anche a pazienti con malattia localmente avanzata ovvero non resecabile in maniera completa oppure metastatica. Tuttavia i dati disponibili evidenziano che
la resezione completa di lesioni secondarie non si accompagna a guarigione e l’ulteriore
ripresa di malattia si verifica nella quasi totalitГ dei casi (90-100%). Nei GIST pediatrici il
quadro ГЁ sovrapponibile .
La chirurgia estesa a scopo di debulking, cui si ricorreva in passato, in mancanza di altre terapie mediche efficaci, non appare piГ№ una opzione praticabile in epoca di Imatinib.
I GIST, analogamente ai tumori mesenchimali, sono neoplasie scarsamente responsive
alla chemioterapia. Studi randomizzati con antracicline, taxani, ifosfamide ed altri agenti
variamente combinati hanno registrato tassi di risposta inferiori al 10%.
L’Imatinib mesilato ha permesso di ottenere risultati decisamente migliori rispetto ad
ogni tipo di chemioterapia o altro trattamento. La dose ottimale di Imatinib nei pazienti
pediatrici, come terapia iniziale, varia da 230 a 400 mg/die e puГІ essere aumentata a 600800mg/die allorchГЁ la malattia ГЁ in progressione; in alternativa possono essere utilizzati
Sunitinib o Nilotinib.
Gist eredo-familiari
I tumori stromali gastrointestinali (GIST) eredo-familiari rappresentano complessivamente meno dell’1% di tutti i GIST e sono caratterizzati da mutazioni ereditate in diversi
geni di suscettibilitГ . Li possiamo distinguere in GIST con mutazioni che interessano KIT e
PDGFRA, che è l’evento patogenetico principale in oltre il 90% dei GIST sporadici, e GIST
con mutazioni su altri geni.
GIST eredo-familiari con mutazioni di KIT e PDGFRA
Le eventuali mutazioni germinali attivanti i geni KIT e PDGFRA vengono trasmesse con
modalitГ autosomica dominante e sono state riportate in un numero esiguo di famiglie. Le
manifestazioni cliniche negli individui portatori di mutazioni germinali del gene KIT variano in relazione all’esone coinvolto. La, che codifica per il dominio iuxtamembranario del
recettore di membrana KIT, rappresenta un evento fondamentale e molto precoce nella
oncogenesi dei GIST.
Quando ereditata per via germinale comporta iperplasia delle cellule interstiziali di Cajal
(ICC) che evolve precocemente verso la formazione di GIST multipli; comporta inoltre mastocitosi ed iperpigmentazione della cute, specialmente a livello perineale, ascellare, delle
mani e della faccia (urticaria pigmentosa). La presenza di disordini della pigmentazione e
la mastocitosi sono giustificate dal fatto che KIT ГЁ coinvolto anche nella melanogenesi e
nello sviluppo delle mastcellule.
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
L’associazione di GIST ed iperplasia generalizzata delle ICC supporta l’origine di questi
tumori da cellule staminali mesenchimali CD34+ lungo la via di differenziazione delle ICC
(Fig.1). L’attivazione di KIT è sufficiente per determinare una iperplasia delle ICC (stimolo
proliferativo alle cellule progenitrici dei GIST), ma sono necessari altri eventi oncogenici
per generare un GIST.
I domini tirosin-chinasici del recettore comportano invece GIST multipli dello stomaco e
dell’intestino tenue in assenza di evidenti disordini cutanei.
Anche le non comportanopigmentazione della pelle o mastocitosi ma i GIST possono
essere associati ad anomalie nella peristalsi esofagea.
L’età media di insorgenza di questo gruppo di GIST eredo-familiari è 46 anni, di poco
inferiore all’età media di insorgenza dei GIST sporadici dell’adulto (circa 50 anni) e fuori
dal range indicato per i GIST pediatrici. Le loro fasi iniziali appaiono biologicamente simili
a quelle del GIST sporadico, con altrettanto simili meccanismi di progressione citogenetica
e comparabili profili di espressione genica. Le mutazioni germinali in KIT e PDGFRA sono
prevalentemente simili a quelle trovate nei GIST sporadici. Tuttavia, in due famiglie con
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
GIST ereditario sono state riscontrate due mutazioni mai viste nel GIST sporadico, rispettivamente in KIT e PDGFRA (Asp419del e Tyr555Cys). Inoltre, un recente studio ha riportato
il caso di un paziente che ha sviluppato lipomi e GIST e che presentava una mutazione
germinale (Asp561Val) in PDGFRA. La Tabella 1 riassume quanto esposto evidenziando
anche le principali correlazioni genotipo-fenotipo di questo gruppo eredo-familiare.
Tabella 1- GIST eredo-familiari con mutazioni di KIT e/o PDGRFA:
Principali correlazioni genotipo-fenotipo
Esone 11 KIT (V559A, delV559, W557R)
Urticaria pigmentosa; mastocitosi
Esone 13 KIT (K642E)
Nessuna pigmentazione della pelle o mastocitosi
Esone 17 KIT (D820Y)
Nessuna pigmentazione della pelle o mastocitosi; anomalie nella peristalsi esofagea
GIST eredo-familiari con mutazioni su altri geni:
Il complesso di Carney (da nome di un noto anatomo-patologo della Majo Clinic che ha
dedicato la propria attivitГ allo studio di queste patologie) comprende la Triade di Carney,
descritta per la prima volta nel 1977, e la Diade di Carney/Stratakis.
La Triade di Carney ГЁ una sindrome ad insorgenza giovanile (etГ media circa 20 anni)
caratterizzata da GIST gastrici multipli ad insorgenza multifocale che si presentano in associazione ad altri tumori: paragangliomi extra-surrenalici e condromi polmonari. Un attento follow-up di questi pazienti ha mostrato un aumentato rischio anche di adenomi
Nell’85% dei casi è colpito il sesso femminile con decorso in genere lento ed indolente
(80% dei pazienti vivi a 20 anni); le recidive locali, così come le metastasi epatiche linfonodali e peritoneali sono poco frequenti. I geni KIT, PDGFRA e SDH risultano wild type.
La Diade di Carney-Stratakis è caratterizzata da GIST gastrici e paragangliomi multipli. L’età media di comparsa è 19 anni ed alcuni pazienti possono presentare, insieme al
GIST, anemia ed emorragia gastrointestinale in assenza di evidenza di paraganglioma. Nella maggior parte dei pazienti con sindrome di Carney-Stratakis non si riscontra la presenza
di mutazioni in KIT e PDGFRA, bensì mutazioni germinali o delezioni nei geni SDH B, C o D
che codificano per le subunitГ B, C e D della succinato deidrogenasi (SDH, complesso mitocondriale II). La predisposizione allo sviluppo di questi tumori ГЁ ereditata come carattere
autosomico dominante a penetranza incompleta.
La succinato deidrogenasi ГЁ un sistema enzimatico che ha un duplice ruolo nel ciclo
di Krebs e nella catena di trasporto degli elettroni nei mitocondri. La SDH ГЁ costituita da 4
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
subunitГ codificate dai geni SDHA, SDHB, SDHC e SDHD, formati rispettivamente da 15, 8,
6 e 4 esoni. I primi due geni, localizzati nel cromosoma 5p15 e nel cromosoma 1p35, codificano per le due subunitГ catalitiche, mentre gli ultimi due codificano per le due subunitГ idrofiliche transmembrana.
Mutazioni germinali nel gene tumor-suppressor SDH erano state precedentemente
associate al paraganglioma ereditario (PGL1) e al feocromocitoma e dovrebbero essere
ricercate in pazienti con GIST familiare e paragangliomi.
La Tabella 2 riassume le principali caratteristiche del Complesso di Carney
È stata riportata un’aumentata incidenza (circa 200 volte) di GIST in pazienti con Neurofibromatosi di tipo 1. La NF1 è una tra le più comuni malattie autosomiche dominanti,
e colpisce nel mondo 1 su 2500–3000 nati, con una prevalenza di circa 1 su 4000–5000
individui nella popolazione generale. Il tratto viene trasmesso in maniera autosomica dominante, con una penetranza del 100% a espressivitГ variabile in quanto esistono anche
forme segmentali o a mosaico. Il background genetico dei pazienti influenza invece la
manifestazione dei fenotipi correlati.
Il gene NF1 codifica per una proteina, detta neurofibromina, di circa 220-250 kDa, con
funzione di proteina inducente la funzione GTPasica (GAP) delle proteine della famiglia Ras.
La sua azione comporta la stimolazione dell’attività GTPasica di Ras, con la conseguente
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
conversione del complesso mitogenico Ras-GTP nella forma inattiva Ras-GDP. Pertanto la
perdita di questa proteina determina la permanente attivazione di Ras, condizione tipica di
molti tumori sporadici in cui mutazioni attivanti di Ras ne eliminano l’attività catalitica.
A supporto di questa funzione, ГЁ stato osservato che linee cellulari umane e murine
prive del gene NF1 (NF1-null) che presentano un fenotipo iperproliferativo possono essere
riportate alla normalità con l’inibizione dell’attività di Ras.
La proteina Ras ГЁ anche implicata nelle vie di trasduzione del segnale attivate da c-KIT,
ed infatti i pazienti con NF1 spesso presentano GIST multifocali nell’intestino tenue, più
raramente nello stomaco. L’incidenza di GIST in questo sottogruppo di pazienti è circa il
5-25%. Anche questi insorgono in assenza di mutazioni in KIT e PDGFRA, prevalentemente
nel sesso femminile e hanno un decorso clinico indolente.
Gist eredo-familiari e principi di counselling oncogenetico
L’identificazione dei geni di suscettibilità , coinvolti nelle varie sindromi tumorali ereditarie ha fornito la base molecolare dei test genetici, che permettono di individuare tra gli
individui a rischio quelli portatori della mutazione germline, considerati “ad alto rischio” di
sviluppare uno specifico tumore.
Questa nuova acquisizione della genetica molecolare ha evidenziato la necessitГ di impiegare il counselling oncogenetico per venire incontro alle esigenze di tutti quei soggetti,
affetti da tumore o sani, che desiderano comprendere il rischio di ricorrenza di una malattia genetica all’interno della famiglia e programmare una adeguata gestione clinica.
Il numero di laboratori di ricerca e di professionisti del settore che si dedicano al counselling oncogenetico sta crescendo enormemente, visto l’aumento della richiesta da parte di
oncologi e altri medici a cui pervengono individui con forti fattori di rischio d’insorgenza di
tumori eredo-familiari, che vengono indirizzati alla consulenza genetica.
Il primo fattore di rischio fra tutti che deve far pensare ad un tumore ereditario è la precoce età d’insorgenza, anche in assenza di anamnesi familiare positiva. Nel caso dei GIST
eredo-familiari, l’età a cui si fa riferimento è circa 45 anni.
La presenza dello stesso tipo di tumore in diversi membri da un solo lato (paterno o
materno) della famiglia o l’associazione di vari tumori dovuti a mutazioni di KIT, PDGFRA e
SDH (per esempio paragangliomi e condromi) costituiscono fattori di rischio molto forti per
identificare famiglie candidate al counselling genetico.
Il counselling oncogenetico (CGO) ГЁ un atto medico, rivolto alla persona nella sua globalitГ . Si svolge presso centri specializzati e prevede un approccio multidisciplinare ed
integrato tra diverse figure professionali, quali il medico oncologo, il genetista e lo psicologo; ciascuno di questi specialisti svolge un ruolo ben determinato ed interviene a
livelli differenti delle varie fasi di cui il counselling si compone (fase pre-test, fase del test
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
genetico, fase post-test, follow-up). La multidisciplinarietГ tiene conto dei diversi aspetti
e dei diversi bisogni della persona a rischio di cancro ereditario. Gli individui a rischio di
sviluppare una neoplasia vengono sottoposti all’analisi della storia personale e familiare
attraverso la ricostruzione dell’albero genealogico nella fase pre-test.
Le principali caratteristiche della storia personale che suggeriscono la natura ereditaria di un GIST comprendono la diagnosi di GIST in etГ precoce, GIST primitivi multipli, o
diagnosi di GIST e un altro tumore (ad esempio paragangliomi, adenomi, condromi, neurofibromi).
La ricostruzione della storia familiare del soggetto (probando), rappresenta un momento fondamentale del processo, in quanto la dettagliata descrizione dell’albero genealogico consente agli specialisti di effettuare una diagnosi più corretta, prevedere la prognosi
piГ№ accuratamente e, quindi, consigliare il consultante sulle decisioni da intraprendere.
Г€ necessario raccogliere per almeno tre generazioni le informazioni personali e cliniche
dei parenti di primo (figli, fratelli, genitori), secondo (nonni, zii, nipoti) e terzo grado (cugini, prozii) del probando in modo da facilitare l’identificazione di pattern ereditari presenti
eventualmente nella famiglia.
Per la corretta costruzione di un “pedigree” è necessario raccogliere il maggior numero
di informazioni possibile, considerando: a) sia il ramo paterno che materno; b) la paternitГ ,
la consanguineità , l’uso di tecnologie di fecondazione assistita.
Г€ importante raccogliere informazioni sia sui familiari affetti da patologie tumorali che
sui familiari non affetti. Per ogni familiare affetto da tumore ГЁ necessario valutare: a) il tipo
e la sede del tumore primitivo; b) l’età d’insorgenza alla diagnosi del tumore primitivo;
c) l’età attuale se l’individuo è ancora in vita o l’età del decesso e la causa della morte;
d) l’esposizione a carcinogeni (per es., uso di tabacco, esposizione a radiazioni); d) altri
problemi di salute significativi (per es., presenza di malattie genetiche note che possono
predisporre allo sviluppo di tumori).
Per ogni familiare non affetto da tumore è necessario esaminare: a) l’età attuale o l’età del decesso; b) se deceduto, la causa del decesso; c) gli interventi chirurgici che possono
aver ridotto il rischio di insorgenza del cancro; d) se routinariamente sottoposti a screening
per la diagnosi precoce di tumore; e) l’esposizione a carcinogeni; f) altre patologie.
In particolare, le caratteristiche che possono suggerire la predisposizione genetica
all’insorgenza di un GIST comprendono la presenza di:
-- due o piГ№ parenti con familiaritГ di I grado con GIST
-- un membro della famiglia che ha avuto diagnosi di GIST e un altro con diagnosi di un
altro tumore primitivo raro
-- un membro della famiglia con GIST che ha una storia personale o familiare delle tipiche
manifestazioni cutanee, multifocalitГ o risulta portatore di mutazioni nel gene NF1.
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
Nell’immagine di seguito riportata (Fig.3) viene illustrato l’albero genealogico di una
famiglia affetta da Diade di Carney-Stratakis, caratterizzato dalla verticalità della trasmissione ereditaria per 4 generazioni e dall’associazione dei casi di GIST con paragangliomi.
Da notare infine la precoce etГ di insorgenza della malattia rispetto ai casi sporadici.
Dopo la valutazione della storia personale e familiare segue la fase del test genetico.
L’identificazione di una mutazione genetica può consentire, grazie alle correlazioni genotipo-fenotipo associate alla sindrome, di inquadrare le manifestazioni cliniche nell’ambito
delle sindromi precedentemente descritte. La fase post-test prevede la consegna del referto, e nei portatori di mutazione, il follow up allo scopo di adottare programmi di sorveglianza individualizzati.
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta,
Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti, Nicola Gebbia, Viviana Bazan
[1] Agaram NP, Laquaglia MP, Ustun B, et al. Molecular characterization of pediatric gastrointestinal stromal
tumors. Clin Cancer Res 2008; 14:3204-15.
[2] Bolland M, Benn D, Croxson M, et al. Gastrointestinal stromal tumour in succinate dehydrogenase subunit B mutation-associated familial phaeochromocytoma/paraganglioma. ANZ J Surg 2006; 76:763-4.
[3] Braconi C, Bracci R, Bearzi I, et al. Insulin-like growth factor (IGF) 1 and 2 help to predict disease outcome in GIST patients. Ann Oncol 2008; 19:1293-8.
[4] Carballo M, Roig I, Aguilar F, et al. Novel c-KIT germline mutation in a family with gastrointestinal
stromal tumors and cutaneous hyperpigmentation. Am J Med Genet A 2005; 132:361-4.
[5] Carney JA, Stratakis CA. Familial paraganglioma and gastric stromal sarcoma: a new syndrome distinct
from the Carney triad. Am J Med Genet 2002; 108:132-9.
[6] Chiarugi M, Galatioto C, Lippolis P, Zocco G, Seccia M. Gastrointestinal stromal tumour of the duodenum
in childhood: a rare case report. BMC Cancer 2007; 7:79.
[7] Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors. J Clin Oncol 2004;
[8] Cypriano MS, Jenkins JJ, Pappo AS, Rao BN, Daw NC. Pediatric gastrointestinal stromal tumors and
leiomyosarcoma. Cancer 2004; 101:39-50.
[9] Hartmann K, Wardelmann E, Ma Y, et al. Novel germline mutation of KIT associated with familial gastrointestinal stromal tumors and mastocytosis. Gastroenterology 2005; 129:1042-6.
[10] Isozaki K, Terris B, Belghiti J, Schiffmann S, Hirota S, Vanderwinden JM. Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors. Am J Pathol 2000;
[11] Janeway KA, Liegl B, Harlow A, et al. Pediatric KIT wild-type and platelet-derived growth factor receptor
alpha-wild-type gastrointestinal stromal tumors share KIT activation but not mechanisms of genetic
progression with adult gastrointestinal stromal tumors. Cancer Res 2007; 67:9084-8.
[12] Kuroiwa M, Hiwatari M, Hirato J, et al. Advanced-stage gastrointestinal stromal tumor treated with
imatinib in a 12-year-old girl with a unique mutation of PDGFRA. J Pediatr Surg 2005; 40:1798-801.
[13] Li FP, Fletcher JA, Heinrich MC, et al. Familial gastrointestinal stromal tumor syndrome: phenotypic and
molecular features in a kindred. J Clin Oncol 2005; 23:2735-43.
[14] McWhinney SR, Pasini B, Stratakis CA. Familial gastrointestinal stromal tumors and germ-line mutations. N Engl J Med 2007; 357:1054-6.
[15] Miettinen M, Lasota J. Gastrointestinal stromal tumors: review on morphology, molecular pathology,
prognosis, and differential diagnosis. Arch Pathol Lab Med 2006; 130:1466-78.
[16] Miettinen M, Lasota J, Sobin LH. Gastrointestinal stromal tumors of the stomach in children and young
adults: a clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases with longterm follow-up and review of the literature. Am J Surg Pathol 2005; 29:1373-81.
[17] O’Riain C, Corless CL, Heinrich 17. Nishida T, Hirota S, Taniguchi M, et al. Familial gastrointestinal
stromal tumours with germline mutation of the KIT gene. Nat Genet 1998; 19:323-4.
[18] Pasini B, McWhinney SR, Bei T, et al. Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits
SDHB, SDHC, and SDHD. Eur J Hum Genet 2008; 16:79-88.
[19] Prakash S, Sarran L, Socci N, et al. Gastrointestinal stromal tumors in children and young adults: a
clinicopathologic, molecular, and genomic study of 15 cases and review of the literature. J Pediatr
Hematol Oncol 2005; 27:179-87.
GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
[20] Price VE, Zielenska M, Chilton-MacNeill S, Smith CR, Pappo AS. Clinical and molecular characteristics of
pediatric gastrointestinal stromal tumors (GIST). Pediatr Blood Cancer 2005; 45:20-4.
[21] Rubin BP, Antonescu CR, Scott-Browne JP, et al. A knock-in mouse model of gastrointestinal stromal
tumor harboring kit K641E. Cancer Res 2005; 65:6631-9.
[22] Sommer G, Agosti V, Ehlers I, et al. Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase. Proc Natl Acad Sci U S A 2003; 100:6706-11.
[23] Tarn C, Rink L, Merkel E, et al. Insulin-like growth factor 1 receptor is a potential therapeutic target for
gastrointestinal stromal tumors. Proc Natl Acad Sci U S A 2008; 105:8387-92.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
[L’impatto del carico emotivo e del distress del caregiver sull’adattamento alla malattia del
paziente affetto da carcinoma della mammella]
Gianluca Lo Coco1, Elena Foddai1,2, Salvatore Gullo1, Maria Viviana Cicero1, Giovanna Manna1, Francesca Paola Guadagna1,2, Viviana Bazan2, Maria Buscemi4, Massimo Cajozzo3, Giuseppe Bronte2, Sergio Rizzo2 and Antonio Russo2
Department of Psychology; 2Section of Medical Oncology, Department of Surgery and Oncology, 3Unit of
Toracic Surgery, 4Histology and Embryology Section, Di.Me.S., UniversitГ di Palermo (IT)
Key words: Breast cancer, Caregiver, Distress, Adjustment
Parole chiave: Carcinoma della mammella, Caregiver, Distress
Abstract. Introduction: Breast cancer involves continually stressful situation caused by the threat of the
illness for both patient and caregiver. Impairment of coping and support processes can result in this context.
The aim of the present study was to obtain further knowledge regarding the relationship between the breast
cancer patient and the caregiver’s psychological distress. Methods: Participants were recruited in an outpatient oncology clinic. Measures included Impact of Events Scale (IES), Family Strain Questionnaire (FSQ),
Mental Adjustment to Cancer (MAC) and Profile of Mood States (POMS). Patients completed a measure of
psychological adjustment to their cancer and caregivers underwent a measure of caregiver strain. Results:
A sample of 56 patients with breast cancer and their respective caregivers was included in the study. Mean
age of patients was 56 (range: 30-75). The IES-Avoidance scores of patients were significantly correlated
with the IES-Intrusion of caregivers (p < .05), the POMS-Anxiety (p < .01), Depression (p < .01) and Hostility (p < .01) scores of their caregivers. The POMS-Confusion scores of caregivers were correlated with the
IES-A scores of patients. Conclusions: Our findings indicated that the caregiver is as likely to experience
psychological distress as the cancer patient, and lends weight to the assertion that both members of the
dyad experience similar levels of distress. Moreover, we found that the hopelessness and the sense of
the uncontrollability of cancer in the patients are predicted by the vigour and the emotional burden of the
Riassunto. Introduzione: Il carcinoma della mammella frequentemente genera situazioni stressanti legate
alla gravità della malattia, nel paziente così come nel caregiver. In questo contesto possono verificarsi alterazioni nel coping e nel processo di supporto. Lo scopo del presente studio è quello di investigare la relazione
tra il distress psicologico del paziente affetto da carcinoma della mammella e il suo caregiver. Metodi: I partecipanti sono stati reclutati in un DH di oncologia. Come questionari sono stati somministrati l’Impact of Events
Scale (IES), il Family Strain Questionnaire (FSQ), il Mental Adjustment to Cancer (MAC) e il Profile of Mood
States (POMS). I pazienti hanno completato un questionario sull’adattamento psicologico alla loro malattia
mentre al caregiver ГЁ stato proposto un questionario relativo al proprio malessere. Risultati: Il campione
comprendeva 56 pazienti con carcinoma della mammella e i loro rispettivi caregiver. L’età media dei pazienti
è stata di 56 anni (range: 30-75). Il punteggio dell’IES-evitamento dei pazienti è risultato significativamente
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna,
Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo
correlato con l’IES-pensieri intrusivi dei caregiver (p < .05), con i punteggi del POMS-Ansia (p < .01), Depressione (p < .01) e Ostilità (p < .01) dei loro caregiver. I punteggi del POMS-Confusione dei caregiver è risultato
correlato con i punteggi dell’IES-Evitamento dei pazienti. Conclusioni: I nostri risultati suggeriscono che il
caregiver ha una probabilitГ di sperimentare un distress psicologico proporzionale a quella del paziente, e
avvalora la tesi secondo la quale entrambi i membri della diade sperimentano livelli simili di distress. Inoltre,
abbiamo riscontrato che il senso di disperazione e di incontrollabilitГ della malattia da parte del paziente affetto da carcinoma puГІ essere predetto dal vigore e dal carico emotivo del caregiver.
The experience of cancer may heavily depend on patients’ interpersonal context and,
notably, their primary caregivers. Caregivers of cancer patients may take an active role
in key decisions concerning treatment options and provide emotional and instrumental
support to the patient [1]. There is a general consensus that breast cancer poses a major
stressor for patients as well as caregivers and that patients and partners are involved in
each other’s coping and support processes [2]. Significantly, an appropriate adjustment to
cancer depends on the totality of the cognitive, emotional and behavioural responses to the
diagnosis on the part of both the patient and the significant other.
Most studies concerning breast cancer has reported that this life-threatening illness
produces high stress in both patients and families, and a number of studies have found
a significant correlation between distress in patients and partners [3]. Moreover Hodges
[4], in a meta-analytic investigation conducted on 21 independent samples regarding the
relationship between the psychological distress of various types of cancer patients and
their caregivers confirm the positive association between patients’ and caregivers’ psychological distress. Most studies of couples in which the woman has cancer have focused on breast cancer patients. Several studies indicates that the psychological distress
of breast cancer patients is higher than that of their spouses [5,6], while others found that
spouses present more psychosocial problems than patients [7,8]. Northouse [9] described
husbands’ level of distress as similar to that of their wives and higher, up to 18 months
after diagnosis, than that in non-patient populations. A recent meta-analysis has indicated
that differences in distress within couples coping with cancer could clearly be ascribed to
gender: women were found to report more distress than men, regardless of whether they
were the individuals with cancer or the partners [10].
Several studies have documented that the primary caregiver of the cancer patient
is in a continually stressful situation caused by the threat of the illness and, simultaneously, by the assumption of the role of main informal social support resource for the patient in the illness process. Previous research has consistently found that a good marital
relationship predicts better emotional adaptation to breast cancer [11,12]. In contrast,
social constraints and unresponsiveness of family members in talking about the illness
The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
have been related to poor mental health among cancer patients [13]. Emotional distress
and depression in the caregiver can have important consequences for the diagnosed
patient and can negatively affect his/her adjustment and how the couple copes with
challenges [14,15].
Overall, it appears that perceived reactions of the patient’s caregiver represent an important influence on the woman’s response to the diagnosis and treatment of this disease
[16]. Caregivers are at the center of the support network for the patient, and it is therefore
extremely important to reach a detailed appreciation of their needs and experiences. Given
this, it seems important to know more about how caregiver reactions relate to the patient’s
emotional well-being, whether the effect is an adverse one or a beneficial one. The aims
of this study were to determine the correspondence between the psychological distress
of cancer patients and their caregivers, and ascertain the nature of this relationship. The
following specific questions were raised.
Research Question 1: Is there a significant positive correlation between breast cancer
patient and caregiver psychological distress?
Research Question 2: Is the distress of the caregiver a predictor of the patient’s poor
adjustment to breast cancer ?
Patients and methods
For enrolment participants should be new and returning cancer patients seen at the
medical oncology clinic at the University of Palermo (Italy), with their caregivers. As caregiver we considered a person indicated by the patient as the “primary informal caregiver.”
Patients were approached in the medical oncology clinic before or after their treatment
appointments and asked to participate in the study. If a patient was interested, the research assistant explained the informed consent form to the patient and asked for the patient’s permission to contact his/her caregiver to request his/her participation. Exclusion
criteria for patients were: prior history of psychiatric hospitalization (3 participants were
excluded), and major concurrent disease (6 participants were excluded). Approximately
85% of the patients contacted who met eligibility criteria agreed to participate. Patients
and caregivers who agreed to participate were separately interviewed before leaving the
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna,
Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo
Impact of Events Scale (IES) [17]. Psychological distress was assessed using a shortened version of the Impact of Events Scale. This scale has been seen to offer a reliable
assessment of intrusive thoughts related to stressful events and conscious avoidance of
feelings and ideas about the events [18]. The shortened version of the scale includes eight
items (four for each subscale). Patients and caregivers respond to statements about the
frequency (0 = not at all, 1 = rarely, 3 = sometimes, 5 = often) in the prior 7 days of intrusive
thoughts related to the patient’s cancer (e.g. “I thought about the diagnosis when I didn’t
mean to”) and to the frequency of avoidance behaviors (e.g. �I kept away from reminders of
the cancer’). In the present study, Cronbach’s alphas for patients for the Intrusion and the
Avoidance scale were .72 and .89, respectively, and .73 and .82 for caregivers.
Family Strain Questionnaire (FSQ) [19]. The FSQ consists of a semi-structured interview
and questionnaire that can be administered in a total of about 20 min. The semi-structured
interview was focused on the caregiver’s knowledge of/beliefs about the disease and the
way in which it is managed. The questionnaire consists of 35 dichotomous items (yes/no)
covering five factors: Emotional Burden (EB); Problem of Social Involvement (SI); The Need
for Knowledge of the Disease (KD); Quality of Family Relationships (FR); Thoughts about
Death (TD). Nine non-factorial dichotomous items concern the caregiver’s attribution of
problems relating to social stigma. As the �yes’ answers are attributed a score of 1, the
higher the score for each area, the greater the problems involved, with the exception of
satisfaction of family relationships (in which a high score indicates good relationships).
The FSQ has been validated in Italy and has good psychometric properties. In this study
Cronbach’s alpha ranged from .72 (TD) to .87 (EB).
Mental Adjustment to Cancer (MAC) [20]. The MAC is a 40 items scale which identifies
four behavioral styles of coping: fighting spirit, fatalism, helplessness/hopelessness, and
anxious preoccupation. Patients’ responses were rated on a 4-point scale. Fighting Spirit
(FS): identifies the patient’s tendency to see cancer as a challenge and to take an active role
in therapy and recovery, and to adopt an optimistic attitude. Helplessness (H): measures
cognitive schema and corresponding behaviors characterized by a sense of the uncontrollability of cancer. Stoic Acceptance/Fatalism (F): which indicates resignation and passive
acceptance of the illness and the therapy, without personal involvement. Anxious Preoccupation (AP): evaluates anxiety and diffuse worry about cancer and its possible recurrence,
associated with excessive search for reassurance and information. In the present study,
internal consistency (Cronbach’s alpha) of the subscales of MAC were: FS=.76, H=.69,
AP=.67, F=.62, respectively.
The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
Profile of Mood States (POMS) [21]. Patients and caregivers were asked to rate the
Profile of Mood States (POMS), a 65-items measure designed to assess subjective mood
states, including positive and negative affects. Responses were on a four-point scale, ranging from “Not at all like this” to “Very much like this”. The POMS yields a score for total
mood disturbance based on six subscales: Anxiety (A), Depression (D), Hostility (H), Confusion (C), Vigor (V), and Fatigue (F). In this study, all the alpha coefficients for the six POMS
scales ranged from .84 (C) to .95 (D) (patients); the alpha coefficients ranged from .76 (F)
to .89 (A) for caregivers.
Statistical analyses
Means and standard deviations were computed for demographic and medical information as well as for psychosocial variables. Paired-sample t tests were conducted to
compare mean differences between patients and caregivers scores in IES and POMS.
Pearson correlation coefficients were calculated to examine the bivariate associations
among study variables. Six separate stepwise regressions were constructed to examine
the role of family strain (FSQ) and mood states (POMS) of caregivers in predicting the
patient adjustment to cancer (IES-I, -A; MAC-FS, -H/H, -AP, F). The equations had 2
steps: in step 1, we entered the age of the patient and caregiver as control variables, in
step 2 we entered the independent variables with a stepwise procedure. The stepwise
regression procedure describes how much more each independent or predictor variable has contributed to the prediction from the preceding predictor variables, and it is
generally used in exploratory procedures. In this study, we adopted a more restrictive
level of .01.
Fifty-six patient-caregiver couples participated.
Demographic and medical characteristics of the sample are presented in Table 1. Patients had a mean age of 56 (range: 30-75). Eighty percent were married, and sixty-six
percent had at least a high school diploma, and in terms of occupation, sixty-seven percent were unemployed. Eighty-five percent underwent surgical treatment or surgery and
chemotherapy. Caregivers had a mean age of 48.8 (range: from 23 to 75), and 55.5%
were female. Fifty-five percent were the patients’ spouses, twenty-seven percent were the
children, and eighteen percent had other relationships (parents, sisters). Only 44% were
employed caregivers.
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna,
Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo
Table 1. Demographic and medical characteristics
Demographic and medical characteristics
Mean age
single (or separated/widowed)
less than high school
high school or more
Employment status
Type of cancer
colon and other
II and III
Range time since diagnosis
surgery or surgery + chemio
(N = 56)
(N = 56)
56,02 (11,36)
48,85 (13,92)
1 - 10 months
A first step in the analysis was the comparison between patients and caregivers in
terms of psychological distress (IES & POMS). Only the IES-Avoidant subscale was higher
in cancer patients than caregivers (t = 2.02 p < .05). Table 2 shows zero-order correlations for the observed variables. The IES-Avoidance scores of patients were significantly
correlated with the IES-Intrusion of caregivers (p < .05), the POMS-Anxiety (p < .01), De218
The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
pression (p < .01), and Hostility (p < .01), scores of their caregivers. The IES-Intrusion of
patients were correlated with the FSQ-Emotional Burden and KD scores of their caregivers
(p < .05; p < .01, respectively). The FSQ-Emotional Burden of caregivers was correlated
with the MAC-Helplessness (p < .05). The POMS-Confusion scores of caregivers were correlated with the IES-A scores of patients.
Table 2. Zero-order correlations (Pearson’s r) between patients and caregivers
IES Intr
,034 ,231
,025 ,214
-,064 ,188
-,080 -,129
,131 ,180
-,013 ,148
,128 ,174
,335* ,105
-,048 -,073
,160 ,218
-,161 ,170
,087 ,254
,102 ,407(**)
,178 -,288(*)
-,105 -,398**
,065 ,189 ,056 -,114 ,108
,054 ,150 -,006 -,060 ,100
,062 ,147 ,039 -,094 ,048
-,182 -,196 -,212 -,155 -,076
-,013 ,195 -,060 -,084 ,057
-,081 ,063 -,078 -,063 ,015
,294* ,179 ,408** -,003 ,208
,112 ,184 ,123 -,095 ,232
-,180 ,022 -,164 -,058 -,082
,296* ,230 ,232 ,189 ,243
,057 ,070 ,077 -,045 -,020
,016 ,058 -,058 ,083 ,046
* Correlation is significant at the 0.05 level.
With regard to the regression analyses (see the statistical analyses section), the first
block of equations (see Table 3) was conducted on the IES Intrusion and Avoidance scores
of patients. The first final model was significant (F = 3.255, p < .01), and revealed that the
FSQ-KD (ОІ = .40, p < .001) was the only predictor of IES-Intrusion of patients. The second
equation showed that age of the patient had quite a significant effect (p < .05). A second
block of multiple regression analyses was conducted on the MAC scores of patients as the
dependent variables. In the final model both the POMS-Vigour and the age of patient were
predictors of the MAC-Helplessness/Hopelessness (ОІ = -.36, p < .01; ОІ = .42, p < .01,
respectively). The FSQ-EB showed a low effect on MAC Helplessness/Hopelessness (p <
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna,
Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo
Table 3. Hierarchical multiple-regression analyses predicting patient’s adjustment to cancer
I – IES Intrusion
I – IES Avoidance
Age of patients
II - MAC Helplessness/Hopelessness
Age of patients
- .36
Adj R2
p of R2 change
Only significant variables and those approaching significance are included in the table. The Table displays
the standardised regression coefficients (ОІ), the significance of R squared increment provided at each step
(p of R2 change), and the overall Adjusted R squared (Adj R2).
IES Intrusion and Avoidance = subscales of Impact Event Scale; MAC Helplessness/Hopelessness = subscale
from Mental Adjustment to Cancer; FSQ KD, EB = knowledge of disease, emotional burden, subscales of
Family Strain Questionnaire; POMS – C = confusion, subscale of Profile of Mood States.
In this study, the aim was to test if there exists a positive relationship between breast
cancer patient and caregiver psychological distress, and if the caregiver’s distress may be a
predictor of the patient’s poor adjustment to cancer. Our findings confirm a significant positive relationship between patient and caregiver psychological distress. Caregiver distress of
(POMS-Confusion, Hostility, Depression, and Anxiety) is positively correlated with the patient’s
avoidance of feelings and ideas about the cancer (IES-AV). Interestingly, the Vigor (POMS) of
the caregiver is positively correlated with the Vigor and Fighting Spirit, and negatively with the
Fatigue and the Helplessness of the patient. These findings seem to indicate that the caregiver is as likely to experience psychological distress as the cancer patient, and lends weight
to the assertion that both members of the dyad experience similar levels of distress [3]. In
our study, only the IES-Avoidant scale is higher in breast cancer patients than their caregivers. A cursory review of the literature regarding distress in breast cancer patients and their
caregivers suggests that it is not cumulative and that it is beset by quite contradictory claims.
Studies have reported that patients are as distressed as caregivers, that patients with cancer
are more distressed than their partners, that caregivers are more distressed than patients.
Our findings are consistent with previous research which has shown that the difference
between patient and caregiver psychological distress appears to vary over time [4], and that
The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
from the 3-month assessment period onwards, no significant difference in distress between
patient and caregiver levels exist. One critical issue in the interpretation of findings regarding
distress in patients and caregivers appears to be gender. Studies that find greater distress
among partners may have confounded the patient–partner role with gender.
Moreover, the findings of the multiple regression analyses show that different aspects of
the caregiver’s psychological distress may predict the adjustment to breast cancer and distress of the cancer patient. The need of hear the disease (FSQ-KD) of the caregiver predicts
the patient’s intrusive thoughts related to cancer (IES-Intrusion). The hopelessness and the
sense of the uncontrollability of cancer in the patient are predicted by the vigour and the
emotional burden of the caregiver. These findings seem to suggest the association between
the psychological distress of caregiver and patient, and they might well be interpreted as
support for the notion that patient-caregiver pairs react as an interdependent emotional system. These findings are also consistent with previous research on women with breast cancer,
which shows that the family caregiver’s vulnerability is heightened because he or she finds
himself/herself in a double and conflictive role. As the primary supporter, he/she must assume new roles in the household and provide tangible as well as emotional support; and at
the same time must cope with the distress emanating from the significance of the patient’s
diagnosis in terms of suffering and the threat to life [22]. Further research will be necessary
to investigate how the distress of caregivers may influence the patient’s distress. Moreover,
some important variables, such as gender and role, need to be taken into account, in order
to study the relationship between the breast cancer patient and his/her caregiver. A recent
meta-analysis [4] which covered 21 studies of distress in people with cancer and their caregivers outlined that while these caregivers were mainly partners, they also included siblings,
children, and close friends. In our sample, only 55% of the primary caregivers indicated
by patients are the spouses. The available literature suggests caution about assuming that
partners of people with breast cancer have substantial caregiving responsibilities, in terms
of providing assistance with medical management or the activities of daily life. Rather than
being accepted uncritically as a synonym for partners of people with cancer, use of the term
“caregiver” requires justification with explicit reference to instrumental caregiving tasks or
demands of home management or functional limitations of the person with cancer. Finally,
attention needs to be directed toward factors other than breast cancer as direct influences of
distress in these couples and to mediators and moderators of the cancer experience.
A limitation of this study was the modest size and the heterogeneous characteristics of
both the samples of patient and caregiver. Results should therefore be considered tentative and exploratory until confirmed by further research studies. Despite these limitations
on generalizability, our findings seem to offer useful information in the attempt to reach a
clearer understanding concerning the experiences of breast cancer patients.
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca Paola Guadagna,
Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo, Antonio Russo
[1] Pistrang N, Barker C. The partner relationship in psychological response to breast cancer. Soc Sci Med
1995; 40: 789-797.
[2] Manne S, Ostroff J, Sherman M, et al. Couples’ support-related communication, psychological distress,
and relationship satisfaction among women with early stage breast cancer. J Consult Clin Psychol
2004; 72: 660-670.
[3] Manne SL. Cancer in the marital context: A review of the literature. Cancer Invest 1998; 16(3):188-202.
[4] Hodges LJ, Humphries GM, MacFarlane G. A meta-analytic investigation of the relationship between the
psychological distress of cancer patients and their caregivers. Soc Sci Med 2005; 60:1-12.
[5] Ben-Zur H, Gilbar O, Lev S. Coping with breast cancer: patient, spouse and dyad models. Psychosom
Med 2001; 63:32–39.
[6] Hoskins CN. Adjustment to breast cancer in couples. Psychol Rep 1995; 77:435-454.
[7] Goodyear-Smith F, Buetow S. Power issues in the doctor-patient relationship. Health Care Analysis
2001; 9: 449-462.
[8] Yeo W, Kwan WA, Teo PML, et al. Psychosocial impact of breast cancer surgeries in Chinese patients
and their spouses. Psychooncology 2004; 13:1 32-139.
[9] Northouse L. Sharing the cancer experience: Husbands of women with initial and recurrent breast
cancer. In Baider L, Cooper C L, & Kaplan De-Nour A. (ed), “Cancer and the family”. Wiley, New York
1996, pp 304-317.
[10] Hagedoorn M, Sanderman R, Bolks HN, et al. “Distress in Couples Coping With Cancer: A Meta-Analysis
and Critical Review of Role and Gender Effects”. Psychological bulletin, 2008; 134 (1): 1-30.
[11] Skerrett K. Couple adjustment to the experience of breast cancer. Fam Syst Health 1998;16, 281-298.
[12] Wimberly SR, Carver CS, Laurenceau J, et al. Perceived Partner Reactions to Diagnosis and Treatment of Breast
Cancer: Impact on Psychosocial and Psychosexual Adjustment. J Consult Clin Psychol 2005; 73, 300-311.
[13] Lepore SJ, Helgeson VS. Social constraints, intrusive thoughts and mental health after prostate cancer.
J Soc Clin Psychol 1998; 17: 89-106.
[14] Lewis FM, Woods NF, Hough EE, et al. The family’s functioning with chronic illness in the mother: The
partner’s perspective. Soc Sci Med 1989; 29: 1261-1269.
[15] Walker BL. Adjustment of husbands and wives to breast cancer. Cancer Pract 1997; 5: 92-98.
[16] Holmberg SK, Scott LL, Alexy W, et al. Relationship issues of women with breast cancer. Cancer Nurs
2001; 24: 53-60.
[17] Zilberg NJ, Weiss DS, Horowitz MJ. Impact of Event Scale: a cross validation study and some empirical
evidence supporting a conceptual model of stress response syndromes. J Consult Clin Psychol 1982;
50: 407-414.
[18] Horowitz MJ, Wilner NR, Alvarez W. Impact of Event Scale. A measure of subjective stress. Psychosom
Med 1979; 41: 209-218.
[19] Rossi Ferrario S, Baiardi P, Zotti AM. Update on the Family Strain Questionnaire: A tool for the general
screening of caregiving-related problems. Qual Life Res 2004, 13: 1425-1434.
[20] Watson M, Greer S, Young J, et al. Development of a questionnaire measure of adjustment to cancer:
the MAC scale. Psychol Med 1988; 18: 203-209.
[21] McNair D, Lorr M, Droppleman LF. “Profile of Mood States” (POMS) (2nd ed.). Educational and Industrial
Testing Service, San Diego 1981.
[22] Spencer SM, Carver CS, Price AA. Psychological and social factors in adaptation. In: Holland J (ed.),
“Psycho-Oncology”. Oxford University Press: New York 211-222, 1998.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Oncogenetic counselling: biomolecular and clinical implications]
Antonio Russo1, Sergio Rizzo1, Valentina CalГІ1, Loredana Bruno1, Laura La Paglia1, Nello
Grassi2, Gianni Pantuso2, Michele Frazzetta3, Massimo Cajozzo3, Domenico Guarneri4, Giuseppe Lo Dico4, Maria Buscemi5, Nicola Gebbia1 e Viviana Bazan1
Sezione di Oncologia Medica e Centro di Riferimento Regionale per la Caratterizzazione e lo Screening
Genetico dei Tumori Ereditari, Dipartimento di Discipline Chirurgiche ed Oncologiche; 2Sezione di Oncologia
Chirurgica, Dipartimento di Discipline Chirurgiche ed Oncologiche; 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4Ginecologia ed Ostetricia, Dipartimento Materno Infantile;
Sezione di Istologia ed Embriologia, Di.Me.S., UniversitГ degli Studi di Palermo (IT)
Key words: Genetic counselling, Hereditary breast cancer, Ovarian cancer, Genetic testing
Parole chiave: Consulenza genetica, Cancro ereditario della mammella, Cancro dell’ovaio, Test genetici
Abstract. Oncogenetic counselling is a multistep process. The aim is to evaluate the risk that the client
carries a heritable or germline mutation in a cancer susceptibility gene. The process begins by collecting information about the client’s personal medical history and family history to assess heritable cancer risk. Then
the multidisciplinary team, including a oncologist, a psychologist and a geneticist, should acquire informed
consent for genetic testing when applicable, and propose options for medical management with regard to
the risk category.
Riassunto. Il “counselling oncogenetico” è un processo di comunicazione multistep finalizzato alla valutazione del rischio di essere portatore di una mutazione germinale in un gene di suscettibilità per tumore
ereditario. Tale processo ГЁ suddiviso in varie fasi partire dalla raccolta delle informazioni relative alla storia
personale e familiare al fine di stabilire il rischio di neoplasia ereditaria. Il team multidisciplinare, composto
da un oncologo, da uno psicologo e da un genetista, se il paziente è candidato all’esecuzione del test genetico deve acquisire il consenso informato all’analisi molecolare e proporre le appropriate opzione per la
gestione clinica in funzione del rischio.
Il counselling oncogenetico (Consulenza Genetica Oncologica - CGO) ГЁ un processo di
comunicazione multistep e multidisciplinare che consente di individuare:
- soggetti “ad alto rischio” di essere portatori di mutazioni germinali nei geni di suscettibilità coinvolti nelle varie sindromi tumorali ereditarie, che sono quindi candidati al test
genetico (membri di famiglie con nota predisposizione ai tumori ereditari o individui sani
con una forte storia familiare per determinati tumori o con altre caratteristiche suggestive
di sindromi ereditarie di predisposizione al cancro);
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
- soggetti “a basso rischio” per i quali non sarà necessario sottoporsi al test.
Le nuove acquisizioni ottenute nel campo della genetica molecolare hanno consentito
di identificare la predisposizione genetica allo sviluppo di specifiche neoplasie e hanno
evidenziato la necessitГ di impiegare la CGO per venire incontro alle esigenze di tutti quei
soggetti affetti da tumore o sani che desiderano comprendere il rischio di una malattia
genetica all’interno della famiglia.
Negli ultimi quindici anni, tali acquisizioni hanno permesso ai membri delle famiglie a
rischio in base alla loro etГ o alla loro condizione patologica di intraprendere strategie di
chemioprevenzione e trattamenti medici e chirurgici adeguati.
Il numero dei Centri di ricerca e di professionisti del settore che si dedicano alla CGO sta
crescendo enormemente, di pari passo all’aumentata richiesta di oncologi e clinici, a cui
pervengono individui a rischio d’insorgenza di tumori eredo-familiari che devono essere
indirizzati alla CGO. La principale terminologia utilizzata nella CGO ГЁ riportata in Tabella 1.
Tabella 1. Principale terminologia utilizzata nella consulenza oncogenetica.
Albero Genealogico:
rappresentazione grafica, secondo le relazioni di parentela, di tutti gli individui che compongono più generazioni di una stessa famiglia. Esso fornisce indicazioni sull’eventuale modalità di trasmissione ereditaria della malattia.
ГЁ una delle possibili forme o varianti di un gene. Per ogni gene (in un determinato locus sul
cromosoma) esistono due alleli, ereditati dal padre e della madre. Quando i due alleli sono
uguali, l’individuo è definito omozigote per quel specifico tratto; se gli alleli sono diversi,
l’individuo è definito eterozigote.
Cellula germinale (ga- spermatozoi e ovuli.
Cellula somatica:
tutte le cellule del corpo eccetto quelle germinali.
EreditarietГ autosomi- la presenza di una singola copia del gene mutato (eterozigosi) ГЁ sufficiente per il manifeca dominante:
starsi della patologia. Sono colpiti entrambi i sessi ed ГЁ trasmessa da entrambi i sessi. Il
figlio dell’unione di un affetto con un non-affetto ha il 50% di probabilità di essere affetto.
EreditarietГ autosomi- ГЁ necessario che entrambe le copie del gene sui due cromosomi omologhi di derivazione
ca recessiva:
paterna e materna siano mutate per il manifestarsi della patologia. Gli individui affetti di solito
sono figli di individui non affetti. I genitori degli individui affetti di solito sono portatori asintomatici. Sono colpiti entrambi i sessi. Ciascun figlio ha la probabilitГ di essere affetto del 25%.
quando in un locus sono presenti due differenti alleli nei due cromosomi omologhi.
insieme dei caratteri che l’individuo manifesta, frutto dell’interazione tra genotipo e fattori
DNA microsatellite:
sequenze di DNA molto semplici (2-5 paia di basi) ripetute (tandem) anche molte volte, disperse nel genoma, e situate il piГ№ delle volte al di fuori delle porzioni codificanti di un gene.
La loro funzione non ГЁ ancora del tutto nota e la frequenza di ripetizione di queste sequenze
puГІ essere utilizzata come markers di instabilitГ genomica.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
segmento genico rappresentato nell’RNA messaggero dopo il processo di eliminazione dagli
introni (maturazione, vedi Splicing). I singoli esoni possono contenere DNA codificante e/o
DNA non codificante (sequenze non tradotte).
FamiliaritГ :
presenza, nell’albero genealogico di più familiari affetti da neoplasia.
insieme dei caratteri che l’individuo manifesta, frutto dell’interazione tra genotipo e fattori
Gene di suscettibilitГ :
gene la cui alterazione patogena ГЁ alla base di una sindrome ereditaria di predisposizione
ai tumori.
ГЁ la costituzione genetica di un individuo, data dalla totalitГ dei geni presenti nel suo genoma.
InstabilitГ dei DNA mi- fenomeno caratteristico di alcune cellule tumorali in cui, durante la replicazione del DNA, il
crosatelliti (MSI):
numero di copie ripetute dei DNA microsatelliti ГЁ soggetto a cambiamenti casuali.
sito di una specifica sequenza di DNA (frequentemente un gene o un marcatore) sul cromosoma.
Mutazione founder:
specifica mutazione patogena presente solo o prevalentemente in alcune popolazioni o
gruppi etnici.
quando in un locus ГЁ presente lo stesso allele nei due cromosomi omologhi.
capacitГ di una variante genica di manifestarsi fenotipicamente quando presente.
esistenza di almeno due varianti alleliche non patologiche in un locus di un gene, ciascuna
delle quali presente in una popolazione con una frequenza maggiore dell’1%. Il polimorfismo dovuto alla variazione di un singolo nucleotide è denominato SNP (Single Nucleotide
ГЁ il primo membro affetto di una famiglia a rischio di tumore ereditario ad essere studiato. In molti
casi ГЁ il membro piГ№ giovane affetto da neoplasia a cui viene proposto il test genetico per la ricerca
dell’alterazione del gene di suscettibilità . In caso di esito positivo, a quest’ultima può far seguito
l’indagine in altri membri della stessa famiglia, ammalati o sani. Viene anche indicato come “caso
indice” (index case) ovvero propositus/proposita (rispettivamente maschio/femmina).
comprendono inserzioni, delezioni o duplicazioni di ampi frammenti genomici, non rilevabili
con le metodiche comunemente usate per l’identificazione delle mutazioni puntiformi, e di
significato in genere patogeno.
processo di maturazione del trascritto primario di RNA messaggero che prevede l’eliminazione degli introni. Lo splicing alternativo è il processo attraverso il quale, mediante un
diverso arrangiamento degli esoni durante la maturazione dell’RNA messaggero, da uno
stesso gene possono derivare diverse proteine.
Tumore Ereditario:
neoplasia caratterizzata dalla presenza di alterazioni note in geni di suscettibilitГ , che si
trasmettono ai discendenti.
Tumore Familiare:
aggregazione di piГ№ casi di neoplasia nella stessa famiglia, spesso a precoce insorgenza
che puГІ suggerire la presenza di alterazioni in geni di suscettibilitГ , e quindi un tumore
ereditario, anche se le alterazioni stesse non vengono riconosciute. PuГІ anche dipendere
dal fatto che i membri di una stessa famiglia sono spesso esposti agli stessi fattori di rischio
ambientali o relativi agli stili di vita.
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
A. Fasi del Programma di Counselling oncogenetico
La consulenza ГЁ un processo di accertamento e comunicazione offerto al probando e si
articola in diverse fasi ben distinte tra loro. La CGO ГЁ un atto medico che si svolge presso
centri competenti e prevede un approccio multidisciplinare ed integrato tra diverse figure
professionali, quali il genetista, il medico oncologo e lo psico-oncologo; ciascuno di questi
specialisti svolge un ruolo ben determinato ed interviene a livelli differenti delle varie fasi di
cui il counselling si compone. La multidisciplinarietГ tiene conto dei diversi aspetti e dei diversi bisogni della persona a rischio di cancro ereditario. Gli individui a rischio di sviluppare
una neoplasia di tipo ereditario vengono sottoposti prima all’analisi della storia personale
e successivamente a quella della storia familiare attraverso la ricostruzione dell’albero
genealogico e successivamente alla valutazione del rischio personale.
Fondamentalmente il programma di consulenza prevede quattro fasi: fase pre-test, test
genetico, fase post-test e follow-up.
1. Fase pre-test
Questa prima fase ГЁ molto importante per fronteggiare i problemi clinici, psicologici ed
etici che insorgono durante tutto il processo di consulenza. La fase pre-test solitamente
prevede un primo momento in cui il probando e/o i suoi familiari vengono informati sui
concetti di base (genetici, epidemiologici, clinici) dei tumori eredo-familiari e sui metodi
diagnostici e preventivi esistenti; quindi si procede all’esame della storia personale e alla
costruzione dell’albero genealogico, raccogliendo informazioni sulla storia personale e familiare del probando. Si procede quindi alla stima del rischio di insorgenza di un tumore
attraverso l’anamnesi della famiglia e/o con programmi statistici di calcolo del rischio. La
raccolta del consenso informato è l’altro elemento fondamentale di questa fase.
a. Raccolta della storia personale e familiare
Г€ un momento fondamentale, in cui vengono raccolte tutte le informazioni necessarie,
che possono aiutare lo specialista a far luce sulla reale origine genetica della malattia.
La raccolta dei dati clinici deve comprendere la storia personale del soggetto a rischio (probando) vale a dire l’età attuale e gli altri fattori di rischio. Il primo fattore di
rischio fra tutti è la precoce età d’insorgenza di un tumore anche in assenza di storia
familiare. Nel caso dei tumori della mammella, l’età a cui si fa riferimento è circa 45 anni,
invece per quelli colorettali l’età considerata è circa 50 anni.
Nell’analisi della storia personale bisogna tenere conto che esistono varie patologie
ereditarie che presentano più frequentemente particolari istotipi (fenotipo “triple negative”
del BC nei carrier di mutazioni del gene BRCA1, l’istotipo midollare del carcinoma della
tiroide, l’istotipo diffuso del carcinoma gastrico).
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
Anche la presenza di adenomi multipli, amartomi o polipi del colon in etГ giovanile sono
indicativi di sindromi ereditarie.
Un attenzione particolare deve essere posta nella valutazione della storia personale
quando viene diagnosticato un tumore raro, quale ad esempio il carcinoma alla mammella
in un maschio, il carcinoma midollare della tiroide o il retinoblastoma, che hanno spesso
un’origine ereditaria.
Inoltre ГЁ necessaria la valutazione di caratteristiche suggestive di sindromi ereditarie di
predisposizione al cancro. Gli esempi piГ№ classici sono i disordini della tiroide nella sindrome di Cowden, le cheratocisti odontogeniche nella sindrome di Gorlin, i tumori desmoidi, le
anomalie dentarie della FAP, i nevi displastici nel melanoma familiare.
Allo stesso modo ГЁ importante valutare quei soggetti affetti da piГ№ tumori primitivi.
La ricostruzione della storia familiare del probando, rappresenta un momento fondamentale del processo, in quanto la dettagliata descrizione dell’albero genealogico consente agli
specialisti di effettuare una diagnosi piГ№ corretta, prevedere una prognosi piГ№ accurata e,
quindi, consigliare il probando sulle decisioni da intraprendere. Г€ necessario raccogliere per
almeno tre generazioni le informazioni personali e cliniche dei parenti di primo (figli, fratelli,
genitori), secondo (nonni, zii, nipoti) e terzo grado (cugini, prozii) del probando in modo da
facilitare l’identificazione di pattern ereditari presenti eventualmente nella famiglia.
La presenza dello stesso tipo di tumore in diversi membri da un solo lato della famiglia
o l’associazione di tumori dovuti a mutazioni in un singolo gene, come quelli di mammella/
ovaio e prostata o quelli colon/utero, ovaio, stomaco e apparato urogenitale, costituiscono
altri criteri utilizzati per selezionare le famiglie candidate per il counselling genetico.
Per la corretta costruzione di un albero genealogico o di un “pedigree” è necessario,
quindi, raccogliere il maggior numero di informazioni possibile, considerando: a) sia il ramo
paterno che materno; b) la paternità , la consanguineità , l’uso di tecnologie di fecondazione
assistita; c) il gruppo etnico di appartenenza.
Un ruolo molto importante è determinato dalla conoscenza del gruppo etnico di appartenenza dell’individuo in esame. Infatti, la conoscenza del gruppo etnico può influenzare le
decisioni riguardanti il test genetico perchГ© ГЁ noto che mutazioni specifiche (le cosiddette
“founder mutations”) possono verificarsi con maggiore frequenza in popolazioni selezionate. Per esempio, come detto in precedenza, i soggetti ebrei Ashkenazi hanno un rischio
alto di essere portatori di 3 mutazioni founder dei geni BRCA1/2 (185delAG, 5382insC del
gene BRCA1 e 6174delT del gene BRCA2) e della mutazione I1307K del gene APC.
Inoltre, ГЁ importante raccogliere informazioni sia sui familiari affetti da patologie tumorali che sui familiari non affetti. Per ogni familiare affetto da tumore ГЁ necessario valutare:
a) il tipo e la sede del tumore primitivo; b) l’età d’insorgenza alla diagnosi del tumore
primitivo; c) l’età attuale se l’individuo è ancora in vita o l’età del decesso e la causa della
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
morte; d) l’esposizione a carcinogeni (per es., uso di tabacco, esposizione a radiazioni,
esposizione all’uso di terapie ormonali per lunghi periodi di tempo o comunque senza
adeguate interruzioni); d) altri problemi di salute significativi (per es., presenza di malattie
genetiche note che possono predisporre allo sviluppo di tumori).
Per ogni familiare non affetto da tumore è necessario esaminare: a) l’età attuale o l’età del decesso; b) se deceduto, la causa del decesso; c) gli interventi chirurgici che riducono
il rischio di insorgenza del cancro; d) se routinariamente sottoposti a screening per la diagnosi precoce di tumore; e) ogni lesione preneoplastica tipica della sindrome in questione;
f) l’esposizione a carcinogeni; g) altri problemi di salute significativi.
Gli aspetti più salienti di un “pedigree” che fanno supporre l’insorgenza di un tumore
ereditario sono: la precoce etГ di insorgenza, la presenza dello stesso tipo di tumore in un
gruppo ristretto di parenti; una inusuale etГ di insorgenza del tumore; due o piГ№ tumori primitivi in uno stesso familiare; evidenza di una ereditarietГ autosomica dominante; presenza di tumori bilaterali; presenza di tumori associati a note sindromi familiari, caratteristiche
fisiche associate a note sindromi ereditarie e naturalmente la presenza di una mutazione
di un gene di suscettibilitГ nella famiglia (Fig. 1).
b. Verifica della storia familiare.
Dato che le sindromi ereditarie di predisposizione al cancro vengono identificate fondamentalmente mediante l’attenta analisi della storia familiare, è necessario che le informazioni ottenute durante la costruzione del pedigree siano molto accurate. Purtroppo spesso
ciГІ non si verifica perchГ© il probando non ricorda bene o ha informazioni incomplete o inesatte. Pertanto, ГЁ buona norma cercare sempre di verificare quanto riportato dal probando
visionando i referti anatomo-patologici, i certificati
di morte e/o i registri tumori.
In figura 2 ГЁ mostrato
l’esempio di un pedigree
costruito considerando solamente quanto riportato
verbalmente dal probando
e quello reale modificato
dopo aver consultato i referti anatomo-patologici.
Come si puГІ notare, il proFigura 1: Albero genealogico di una famiglia probabilmente affetta da
bando ricordava che nella
sindrome tumorale ereditaria.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
propria famiglia vi erano stati giГ dei casi di tumore, ma i suoi ricordi erano estremamente
imprecisi. Solamente dopo aver consultato i referti anatomo-patologici lo specialista ha
potuto ricostruire in modo esatto l’albero genealogico del probando, dal quale si evinceva
la possibile presenza di una sindrome ereditaria mammella/ovaio.
c. DinamicitГ della storia familiare.
Un altro aspetto da considerare ГЁ la dinamicitГ della storia familiare, poichГ© questa non
è statica, ma varia col trascorrere del tempo. È quindi necessario aggiornare periodicamente l’albero genealogico inserendo ad esempio le eventuali nascite, i decessi, l’insorgenza di lesioni preneoplastiche, di nuovi tumori e le ricadute.
Ogni volta che viene acquisita una nuova informazione al pedigree ГЁ opportuno riconsiderare la significativitГ della storia familiare, dal momento che queste nuove informazioni
possono influire sulla valutazione del rischio, sui programmi di sorveglianza, e sulle raccomandazioni cliniche fornite al probando (Fig. 3).
d. Identificazione e comunicazione del rischio
L’obiettivo principale di questa fase è quello di fornire ai probandi informazioni accurate
Figura 2: Verifica della storia familiare. Albero genealogico costruito tenendo conto di quanto riportato dal
paziente (a) e dello stesso realizzato dopo aver consultato i referti anatomo-patologici (b)
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
sul proprio rischio di sviluppare un tumore. La CGO ГЁ offerta ad individui che sono ad alto e
medio rischio di tumore ereditario e/o familiare; i soggetti a basso rischio sono tutti coloro
che sviluppano un carcinoma in etГ avanzata e non hanno storia familiare di tumore.
Nel caso dei tumori eredo-familiari di mammella/ovaio, gli individui a medio rischio (a
basso rischio ГЁ la popolazione in genere) sono i soggetti con parenti di I grado che hanno
avuto diagnosi di tumore alla mammella sotto i 50 anni di etГ oppure soggetti con parenti
di I o II grado dallo stesso lato della famiglia che hanno avuto diagnosi di tumore alla mammella o all’ovaio sempre sotto i 50 anni di età .
La definizione del rischio di essere portatori di mutazione si basa su differenti sistemi
di valutazione. I sistemi empirici, basati essenzialmente sul numero di casi di tumore nella
storia familiare o sull’età alla diagnosi, possono essere utilmente impiegati per selezionare
le famiglie da sottoporre a CGO. Chiaramente i modelli matematici sono piГ№ affidabili dei
sistemi empirici in quanto riescono effettivamente a fornire una stima del rischio di essere
portatori di mutazione in un gene di suscettibilitГ .
Se per esempio prendiamo in esame la sindrome HBOC, in questi casi vengono utilizzati
due metodi analitici per il calcolo del rischio medio di sviluppare un carcinoma della mammella: a) il modello di Claus, che si basa sulle informazioni relative alla storia familiare di
I grado della stessa linea parentale, ma prende in considerazione unicamente i carcinomi
mammari presenti e b) il modello di Gail, che tiene conto sia dei fattori ormonali, a cui ГЁ
stata soggetta la donna nel corso della vita, che i fattori di familiaritГ solo dei parenti di
primo grado affetti con esclusione della storia familiare paterna. Ma il modello di Gail non
risulta uno strumento adeguato alla valutazione del rischio
di sindrome ereditaria, perchГ©
tarato per stimare la probabilità d’insorgenza di BC nella
popolazione generale.
Gli individui ad alto rischio
sono quei soggetti che fanno
parte di famiglie, che presentano precoce etГ di insorgenza
di carcinoma di mammella ed
ovaio, o bilateralitГ dei tumori, associazione di tumore alla
mammella e all’ovaio e presenza di carcinomi mammari
Figura 3: DinamicitГ della storia familiare. Albero genealogico della
maschili. Tali soggetti vengono
stessa famiglia dopo follow up di due anni.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
sottoposti durante la consulenza ad alcuni modelli matematici piГ№ specifici, quali BRCAPRO
e Couch model che estendono le informazioni ottenute a livello familiare, tenendo conto
anche dei casi di carcinoma ovarico presenti e danno un calcolo matematico di probabilitГ di essere portatore di mutazione nei geni di suscettibilitГ BRCA1 e BRCA2 (Tabella 2).
Tabella 2. Modelli di calcolo del rischio di carcinoma della mammella
per la popolazione generale e per i soggetti ad alto rischio
EtГ del menarca
EtГ del primo parto
Numero dei parenti di I grado affetti
Numero di precedenti biopsie mammarie
Presenza di iperplasie atipiche
EtГ alla diagnosi
Numero dei parenti di I grado affetti da BC
EtГ alla diagnosi
Parenti di I e II grado affetti da BC o CO
EtГ del paziente
EtГ di morte del parente non affetto
Quando viene utilizzato il BRCA-PRO software il test genetico viene proposto dal genetista quando la probabilitГ a priori di essere portatori di mutazione ГЁ almeno del 10%. Prima
di comunicare il rischio al probando ГЁ importante valutare il suo grado di comprensione e
di percezione del rischio. Lo specialista deve sempre considerare che il probando di frequente non ha molta dimestichezza con i concetti di probabilitГ statistica, di rischio relativo
e assoluto, di proporzione, per cui deve trovare il metodo migliore (mediante numeri, parole
o grafici) per comunicare al probando la stima del rischio assicurandosi che quest’ultimo
gli abbia dato un giusto peso. Infatti, il probando spesso tende a sovrastimare la propria
probabilitГ di ammalarsi soprattutto se in famiglia ci sono stati altri casi di tumore. Di conseguenza, la percezione personale del rischio non sempre corrisponde al valore oggettivo
del rischio statistico calcolato.
La scelta del metodo di comunicazione del rischio ГЁ fondamentale perchГ© una stessa informazione data usando metodi differenti puГІ essere recepita in maniera diversa.
Anche in questo caso ГЁ importante il sostegno offerto dallo psicologo in sede di consulenza.
Una volta effettuata la stima del rischio dovrГ essere ottenuto dal probando il consenso
ad effettuare il test genetico. Questa decisione deve essere presa liberamente dal probando, informato circa i vantaggi ed i limiti di un test genetico.
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
e. Consenso informato
Ottenere il consenso informato scritto prima della fase di testing ГЁ necessario per chiarire: a) i rischi, i benefici, i limiti e le alternative al test genetico; b) il significato di un
risultato positivo, negativo, indeterminato o con varianti di sequenza di significato clinico
sconosciuto (inconclusivo); c) le possibili ripercussioni di un simile risultato non soltanto a
livello personale e familiare, ma anche sociale.
Le informazioni che il consenso informato deve contenere riguardano le opzioni di stima del rischio di insorgenza di un tumore alternative al test genetico, la possibilitГ che il
test sia indeterminato o inconclusivo.
Spesso vi ГЁ la possibilitГ di disagio psicologico personale e familiare o la possibilitГ di
discriminazioni nel settore lavorativo e assicurativo.
Г€importante che si evinca la volontГ di comunicare il risultato del test ad altri membri
della famiglia. Il probando dovrГ inoltre accettare il potenziale uso futuro (per es. per nuove
tecniche) del materiale a disposizione e gli eventuali costi del test.
B. Fase del test genetico
I test molecolari disponibili sono molteplici, con tempi di indagine differenti e con diversi limiti in rapporto alla differente sensibilitГ e specificitГ .
L’eterogeneità genetica delle sindromi rende la scelta del test molto difficile. Differenti
sindromi possono essere accompagnate dallo sviluppo di un particolare tipo di tumore;
così, per esempio, i tumori ereditari del colon possono essere conseguenza di poliposi adenomatosa familiare (FAP), cancro colorettale ereditario non poliposico (HNPCC), sindrome
di Peutz-Jegher (PJS), Juvenile polyposis syndrome (JPS), ognuno con una diversa base
genetica; oppure diversi geni possono essere responsabili della stessa sindrome (HNPCC
puГІ essere dovuta ad alterazioni in uno dei 6 geni del mismatch repair).
Anche l’eterogeneità allelica può condizionare il test genetico. Differenti mutazioni nello
stesso gene possono conferire un diverso rischio o essere associate a un differente fenotipo. Se, per esempio, in linea generale le mutazioni di APC sono associate a centinaia/
migliaia di polipi colici e alla FAP classica, alcune mutazioni specifiche possono essere
responsabili di forme intermedie con un minor numero di polipi e un piГ№ basso rischio di
sviluppare il cancro al colon-retto, o predisporre ad alterazioni a carico della retina invece
che allo sviluppo di tumori desmoidi.
Pertanto, la selezione di un appropriato test genetico per un dato individuo richiede
profonde conoscenze cliniche e molecolari ed il rapido aggiornamento delle tecniche di
screening genetico che subiscono continuamente aggiornamenti e/o miglioramenti.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
Figura 4: Struttura dei geni BRCA
1. Alterazioni genetiche nelle principali Sindromi di predisposizione ereditaria
Carcinoma ereditario della mammella e/o dell’ovaio (HBOC).
Le mutazioni dei geni BRCA non ricadono all’interno di “hot spots”, ma sono uniformemente distribuite lungo l’intero gene (Fig. 4).
Le mutazioni identificate nei geni BRCA a partire dalla loro scoperta e le rispettive
frequenze vengono catalogate in un registro internazionale denominato Breast Cancer Information Core Database (BIC; http// Nel database BIC sono
state riportate oltre 600 mutazioni patologiche sia per BRCA1 che per BRCA2. Spesso si
osserva una prevalenza di alcune di esse in rapporto all’area geografica di appartenenza
del portatore (mutazioni “founder” o ricorrenti, Tabella 3).
Tabella 3. Mutazioni “founder” dei geni BRCA in diverse popolazioni
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
• Finlandia
• Francia
• Islanda
• Italia
- Calabria
- Sardegna
- Sicilia
• Norvegia
3347delAG 1135insA
• USA/Israele
(ebrei Ashkenazi)
9345 +1G>A
7708 C>T
8555 T>G
6174delT and 995delG
Il 70-80% delle alterazioni genetiche sono patogene e comportano la formazione di una
proteina tronca di dimensione piГ№ piccola. Si tratta principalmente di mutazioni frameshift,
nonsense, piccole inserzioni e delezioni e in minima parte missense.
I riarrangiamenti genomici (grosse duplicazioni e delezioni) costituiscono il 3-5% delle
alterazioni genetiche patogene e sono principalmente a carico di BRCA1. Oltre il 40% del
gene BRCA1 ГЁ costituito da sequenze piccole sequenze ripetitive localizzate nelle regioni
introni che denominate Alu, che giustificano l’elevata percentuale di riarrangiamenti genomici che si verificano in questo gene.
Dal punto di vista funzionale, oltre alle alterazioni genetiche patologiche si distinguono
varianti di sequenza di significato sconosciuto e mutazioni puntiformi sinonime (Tabella 4).
Tabella 4. Mutazioni puntiformi
-- Frame-shift (mutazione con slittamento del modulo di lettura): modifica della sequenza di un gene per inserzione/delezione di un singolo nucleotide.
-- Sinonima (“silente”): sostituzione di un singolo nucleotide che determina un codone
diverso ma codificante per lo stesso amminoacido.
-- “Nonsense”: sostituzione non sinonima di un singolo nucleotide che produce un
codone di stop e quindi la formazione di una proteina tronca.
-- “Missense” (di senso errato): sostituzione non sinonima di un singolo nucleotide
con cambiamento di un aminoacido. Le mutazioni “missense” possono essere clas-
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
sificate in conservative (sostituzione di un amminoacido con un altro chimicamente
simile) o non conservative (sostituzione di un amminoacido con un altro che ha una
catena laterale completamente diversa).
Normale sequenza
Sostituzione AВ®T
Delezione di T
Inserzione di T
N.B. Le mutazioni frame-shift sono sempre patogene, così come le nonsense. Le mutazioni missense possono non
comportare la compromissione della funzione della proteina e quindi non essere patogene. Le sinonime sono ovviamente
sempre prive di significato patologico.
B. Carcinoma ereditario del colon retto
Sindrome di Lynch (HNPCC)
Il 70% dei soggetti portatori di mutazione nelle famiglie affette da HNPCC hanno mutazioni germinali nei geni hMSH2 e hMLH1. Altri geni quali hPMS1, hPMS2 e hMSH6 sono
coinvolti in misura minore, ma anche le loro mutazioni manifestano alta penetranza. Tali
geni sono oncosoppressori ed appartengono al gruppo dei geni “caretakers” coinvolti nei
processi di riparazione del DNA ed in particolare degli errati accoppiamenti dei nucleotidi
durante la fase di replicazione “mismatch”, impedendo così la trasmissione di alterazioni
geniche. La perdita della eterozigosi nella cellula somatica del tessuto bersaglio, che nel
caso di hMSH2 è la cripta del colon dove si trovano le cellule epiteliali attivamente proliferanti, comporta la genesi del “fenotipo ipermutabile” la cui espressione è l’instabilità delle
sequenze dei microsatelliti nel DNA (MSI) e l’accumulo delle mutazioni necessarie alla
trasformazione neoplastica in rapida sequenza.
L’instabilità genomica a livello microsatellitare è un evento precoce nella rapida progressione adenoma-carcinoma dell’HNPCC mentre non si ritrova frequentemente nei carcinomi colorettali sporadici e non è necessariamente associata alla perdita di eterozigosi
a livello dei geni coinvolti nel “mismatch repair” (MMR). Questi sono localizzati in 3 diversi
cromosomi e codificano per proteine che agiscono sottoforma di eterodimeri che lavorano
in sequenza per riconoscere e riparare i “mismatch” del DNA.
Poliposi adenomatosa familiare (FAP)
Il gene di suscettibilità della FAP è l’APC, localizzato sul cromosoma 5 (5q21). Tale gene
codifica per una proteina citoplasmatica che riveste un ruolo chiave nella regolazione di
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
apoptosi, ciclo cellulare, interazione ed adesione intercellulare, processi di migrazione e
metastatizzazione. La maggior parte delle mutazioni identificate nel gene APC portano alla
formazione di proteine tronche. Sino al 30% dei pazienti presentano mutazioni “de novo”.
Le mutazioni del gene APC sono presenti a livello somatico in circa l’80% dei tumori
colorettali sporadici, e, a livello germinale in piГ№ del 70% delle famiglie con FAP ed in circa
il 25% delle famiglie con la cosiddetta “attenuated FAP” (AFAP). In particolare nei soggetti
con AFAP, le mutazioni si localizzano preferenzialmente nella porzione 3’-terminale del
gene APC e negli esoni soggetti a splicing alternativo. In alcuni pazienti con FAP o AFAP ГЁ
stata riscontrata evidenza di mosaicismo somatico, ovvero la presenza di mutazione solo
in alcune sottopopolazioni cellulari.
In altre forme di AFAP vengono ereditate in maniera autosomica recessiva mutazioni del
gene MYH codificante per una glicosilasi coinvolta nel meccanismo di riparazione dei danni
al DNA a singola elica, denominato base excision repair (BER). Le mutazioni bialleliche di
questo gene sono presenti in circa il 25-30% dei soggetti AFAP ed in una minima frazione
di soggetti con FAP. L’eventuale consanguineità tra i membri della famiglia che rende conto
di alcune mutazioni bialleliche, potrebbe falsare il test genetico.
Le correlazioni genotipo-fenotipo delle alterazioni del gene APC sono state descritte
ampiamente e spiegano l’eterogeneità clinica dei pazienti. Ad esempio, la forma aggressiva di FAP con esordio precoce, caratterizzata da un numero molto alto di adenomi, è
associata con mutazioni nei codoni compresi tra il 1250 e il 1464 (in particolare nel codone
1309), invece l’AFAP è associata con mutazioni all’estremità 5’ o 3’ del gene APC e negli
esoni soggetti a splicing alternativo.
2. Test di screening
a) Materiale biologico
Il DNA genomico viene estratto o da sangue o da tessuto.
Sangue. L’estrazione del DNA richiede la lisi dei linfociti e la digestione delle proteine
della membrana plasmatica e degli istoni , attraverso tamponi contenenti sodio dodecilsolfato (SDS) e l’enzima Proteinasi K, con conseguente rilascio del DNA in essi contenuto. Segue una fase di purificazione e concentrazione del DNA mediante precipitazione in
soluzione alcolica. Accanto a tale metodica ГЁ possibile disporre di kit commerciali basati
sul principio della cromatografia per filtrazione che accorciano i tempi di estrazione e purificazione.
Tessuto. Il DNA genomico può essere estratto da prelievo bioptico o chirurgico di tessuto neoplastico fresco, congelato o fissato in formalina ed incluso in paraffina. L’utilizzo di
tessuti paraffinati fornisce DNA di qualitГ inferiore rispetto a quella del tessuto fresco.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
Il DNA estratto viene siglato con un codice nel rispetto della privacy e conservato a 4В°C,
per poter essere utilizzato nel percorso diagnostico molecolare che prevede successivamente una metodica di amplificazione del DNA. Se opportunamente conservato il DNA puГІ
essere utilizzato anche a distanza di tempo (diversi anni) per ulteriori indagini.
b) Amplificazione del DNA
La reazione di polimerizzazione a catena (PCR) ГЁ una tecnica estremamente sensibile
che consente in vitro una amplificazione rapida, su base enzimatica (DNA polimerasi), di
uno specifica sequenza di DNA. Per ogni reazione di PCR viene utilizzato un controllo negativo, in cui il DNA stampo viene sostituito con acqua.
c) Metodiche per lo screaning mutazionale
Una volta amplificato il DNA, si ha a disposizione materiale sufficiente per la ricerca e
l’identificazione delle mutazioni genetiche, per le quali possono essere utilizzati metodi
indiretti e diretti.
I metodi indiretti, quali ad esempio la dHPLC (denaturing High Performance Liquid
Chromatography o cromatografia liquida denaturante), rivelano la presenza di alterazioni
del DNA e la loro localizzazione, ma non sono in grado di caratterizzarle. Viceversa i metodi
diretti quali ad esempio il sequenziamento permettono di identificare e caratterizzare le
mutazioni nei principali geni di suscettibilitГ .
Principali metodi indiretti. dHPLC (denaturing High Performance Liquid Chromatography o cromatografia liquida denaturante). Г€ una cromatografia a scambio ionico in condizioni denaturanti che rappresenta una metodica di pre-screening genetico.
Essa permette di analizzare in poco tempo un elevato numero di pazienti e di visualizzare la presenza di mutazioni geniche puntiformi. Tra i vantaggi si ha l’automatizzazione
della tecnica, l’elevata sensibilità (> 98%), alta accuratezza, elevato rendimento, rapida
velocità d’analisi ed i costi ridotti.
Nella figura 5a sono riportati i cromatogrammi (profili di eluizione) che identificano
rispettivamente un profilo wild type, un mutato ed un polimorfismo. Come si evince dal
grafico il picco che identifica la mutazione ГЁ distinguibile da quello relativo al polimorfismo.
Il limite di questa tecnica consiste nel fatto che, pur identificando la presenza di una mutazione del gene interessato, non consente di specificare di quale mutazione si tratti (per
esempio, nelle mutazioni puntiformi non chiarisce quali basi azotate siano interessate).
Nella Sindrome di Lynch, vengono eseguiti preventivamente dei test di screening quali
l’analisi dell’MSI e l’immunoistochimica (IHC). Tali analisi necessitano del tessuto tumorale
del membro più giovane all’interno della famiglia e consentono di discriminare quale dei
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
Figura 5: Mutazione nell’esone 7 del gene BRCA2 (V211I)
geni MMR studiare evitando analisi mutazionali inutili in termini di costi e di tempo. L’IHC
fornisce perГІ solo indicazioni sulle alterazioni geniche (mutazioni puntiformi o riarrangiamenti genomici) che determinano la formazione di proteine tronche non funzionali. La
possibilitГ che alcune mutazioni missense non determinino la mancata espressione delle
proteine, riduce l’utilità diagnostica di tale metodica.
L’analisi della MSI prevede l’identificazione di specifiche sequenze dei microsatelliti nel
DNA della linea somatica tumorale (markers microsatellitari) mediante l’utilizzo di primers
marcati con fluorocromi che si appaiano alla sequenza bersaglio per dare inizio alla sintesi
del filamento complementare e la loro amplificazione mediante PCR. I prodotti di amplificazione vengono separati, secondo le loro dimensioni (espresse in coppie di basi) mediante
elettroforesi capillare in un sequenziatore automatico, e queste dimensioni paragonate a
quelle dei frammenti contenuti nello standard. Tale metodo ha una sensibilitГ pari al 93%
per i portatori di mutazione nei geni MMR, ma, paragonato all’IHC, non offre indicazioni su
altri geni che potrebbero essere alterati.
Si procede, quindi, all’analisi mutazionale dei geni MMR. Se viene rilevata una determinata mutazione in un membro affetto, si effettuerà il test sugli altri membri non affetti. Se
invece non ГЁ stata identificata alcuna mutazione nel membro affetto, non ГЁ indicato il test
sugli altri membri della famiglia in quanto risultati falsi negativi possono essere dovuti alla
presenza di altri geni MMR sconosciuti.
Nelle famiglie con una storia familiare molto sospetta, inoltre, ГЁ consigliato comunque
lo screening mutazionale qualunque sia il risultato dell’analisi microsatellitare.
Principali metodi diretti. Sequenziamento automatico diretto (Direct Sequencing DS). A seguito dell’analisi dHPLC, nei campioni che mostrano un profilo di cromatogramma
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
alterato rispetto al profilo wild type viene effettuato il DS.
In questi casi il DS permette di identificare e caratterizzare il tipo di mutazione puntiforme con una sensibilità di circa il 100%. Questa metodica prevede l’utilizzo di dideossinucleotidi marcati con un fluorocromo diverso per ciascun nucleotide, usati come terminatori,
cioГЁ come ultimo nucleotide, durante la polimerizzazione della catena di DNA. I frammenti
ottenuti a seguito della polimerizzazione, vengono separati mediante elettroforesi capillare
e quindi colpiti da un raggio laser, che eccita i vari fluorocromi. La conseguente emissione
di fluorescenza viene raccolta da una cella fotoelettrica e riportata in un grafico denominato elettroferogramma, composto da una sequenza di picchi colorati corrispondenti alla
sequenza dei nucleotidi diversamente marcati.
Nella figura 5b sono riportati un elettroferogramma tipico di un wild type ed uno in cui
ГЁ possibile osservare il doppio picco che indica la presenza di una mutazione puntiforme
del tipo missense (cambio del nucleotide 859 da G ad A) con effetto patogeno a carico del
gene BRCA2. A causa di questa mutazione, infatti, l’aminoacido isoleucina sostituisce la
valina con la formazione di una proteina non piГ№ funzionale.
Le alterazioni genetiche alla base delle sindromi ereditarie di predisposizione possono
comprendere riarrangiamenti genomici come nel caso del gene BRCA1 in cui questi si formano, per ricombinazione omologa, a causa dell’alta frequenza di sequenze ripetute come
quelle Alu nelle regioni introniche. I riarrangiamenti genomici sono costituiti da grosse
delezioni-duplicazioni e vengono ricercati nei casi risultati negativi con le tecniche comunemente usate per lo screening delle mutazioni puntiformi (dHPLC e DS). Tali alterazioni
possono essere identificate con il saggio MLPA, acronimo di Multiple Ligation-dependent
Probe Amplification. Dal momento che i riarrangiamenti genomici comportano variazioni
del numero di copie degli esoni del gene di suscettibilità , l’MLPA consente di identificare
queste variazioni quantitative che potranno essere meglio caratterizzate successivamente
mediante sequenziamento.
In particolare si utilizzano sonde oligonucleotidiche che si appaiono alle sequenze adiacenti gli esoni codificanti il gene. Quindi i prodotti di legame vengono amplificati e separati,
mediante elettroforesi capillare, secondo la loro lunghezza.
Nella figura 6 ГЁ riportato un elettroferogramma relativo al gene BRCA2 (a) tipico di un
wild type (controllo) ed uno (b) in cui è possibile osservare in corrispondenza dell’esone 10
un picco marcatamente più basso che indica una delezione dell’esone 10.
Negli ultimi anni sono state sviluppate metodiche di sequenziamento capaci di produrre
in tempo reale notevoli quantitГ di sequenze di lunghezza inferiore rispetto al sequenziamento classico ma ad un costo minore e soprattutto ad una velocitГ maggiore.
Il pirosequenziamento è una di queste tecniche: esso prevede la rilevazione del pirofosfato rilasciato dalla DNA polimerasi dopo l’incorporazione di uno o più nucleotidi. Il pirofo239
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
sfato inorganico viene trasformato in ATP (sulfurilasi) e queste molecole vengono utilizzate
dalla luciferasi per ossidare la luciferina e produrre un segnale luminoso che viene rilevato
da un’apposita camera fotosensibile (CCD). L’intensità di segnale è proporzionale al numero di nucleotidi incorporati e viene tradotta in un grafico (fluorogramma) in cui ad ogni base
corrisponde un picco di colore specifico.
Tale metodica è particolarmente indicata per la ricerca di mutazioni “founder” in specifiche popolazioni o per l’analisi dei DNA microsatelliti a partire da frammenti di DNA di
lunghezza limitata (circa 100 nucleotidi).
3. Fase post-test
Anche tale fase del counselling ГЁ di estrema importanza, poichГ© prevede la comunicazione del risultato del test e delle sue implicazioni. Prevede una discussione sulle misure
di sorveglianza, di prevenzione e/o cure esistenti, sui possibili trattamenti chirurgici profilattici. Inoltre viene discussa la possibilitГ di offrire il test genetico anche ad altri membri
della famiglia che pur essendo asintomatici potrebbero essere a rischio di sviluppare una
Figura 6: Elettroferogramma relativo ad analisi con MLPA del gene BRCA2. La freccia indica l’esone 10. (a)
In alto gli esoni del gene BRCA2 wild type (controllo). (b) In basso, in corrispondenza dell’esone 10 un picco
marcatamente più basso che indica una delezione dell’esone 10.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
1. Comunicazione del risultato del test e delle sue implicazioni
Il test genetico viene effettuato allo scopo di migliorare la stima del rischio tumorale.
Tale rischio ГЁ dato dalla ricostruzione della storia personale e familiare del probando e dal
risultato del test genetico stesso. La stima è importante per proporre ai soggetti “a rischio”
e alle loro famiglie un appropriato piano di sorveglianza diverso da quello proposto alla
popolazione generale.
Nonostante la scoperta di nuove tecniche e lo sviluppo delle piГ№ moderne tecnologie,
non tutte le mutazioni possono essere identificabili; inoltre anche se il test dovesse risultare positivo esso indica la probabilitГ ma non la certezza del 100% che il portatore sviluppi
la patologia tumorale.
Г€ importante che il genetista comunichi al probando il risultato del test utilizzando un
linguaggio semplice e molto diretto, che eviti di aumentare lo stato d’ansia del probando.
Prima di procedere, è opportuno che lo specialista chieda un secondo consenso informato per accertarsi che il probando sia ancora disposto a conoscere l’esito del proprio test,
avendo considerato a fondo il possibile impatto che il risultato potrebbe avere sulla propria
famiglia, sulla vita professionale e sul suo stato emotivo. A volte, infatti, puГІ succedere
che durante la fase di testing (la cui durata ГЁ variabile in base al tipo di test effettuato) il
probando cambi idea e non voglia più conoscere l’esito del test. In questo caso, il probando potrà comunque scegliere di sottoporsi periodicamente a specifici controlli clinici e, in
futuro, conoscere il risultato del proprio test qualora lo desideri. Anche in questa fase, dal
punto di vista psicologico vengono toccati elementi fondamentali nell’equilibrio individuale,
per cui la presenza dello psico-oncologo puГІ supportare gli stati ansia del probando per i
propri familiari e per il proprio futuro.
2. Risultato del test genetico
Il risultato del test genetico puГІ essere positivo, negativo, indeterminato o inconclusivo
(Fig. 7).
a) L’esito è positivo quando si identifica una alterazione genetica patogena a cui si può
associare un rischio alto di sviluppare una neoplasia (nel caso in cui il probando sia sano)
oppure di sviluppare un secondo tumore (nel caso in cui il probando sia un individuo affetto
in precedenza dalla neoplasia, ma possibilmente guarito). In caso di risultato positivo dovrГ essere adottata una strategia multidisciplinare di sorveglianza intensiva o altre procedure
di riduzione del rischio.
b) L’esito di un test genetico effettuato a scopo predittivo in un individuo non affetto,
cioè quando una alterazione responsabile di predisposizione genetica è già stata identificata in un familiare di primo grado, è negativo se l’individuo non l’ha ereditata. Ciò implica
che il soggetto ГЁ sfuggito al rischio neoplastico su base genetica e presenta un rischio di
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
sviluppare la patologia pari a quello della popolazione generale. In questo caso le raccomandazioni per lo screening ricalcano quelle della popolazione generale.
c) L’esito è indeterminato quando nel probando non viene identificata alcuna variante
di sequenza o riarrangiamento genico. Tale risultato puГІ dipendere da diversi fattori:
• Può trattarsi di un falso negativo: nella famiglia è presente una alterazione genica
non riscontrabile con le tecniche comunemente impiegate (es. dHPLC, sequenziamento, MLPA).
• L’aggregazione familiare potrebbe non essere attribuibile ai geni di suscettibilità studiati, (es. BRCA1/BRCA2 in caso di carcinoma della mammella) ma essere dovuta
a altri geni ad alta, media o bassa penetranza.
• Fenocopia cioè un fenotipo causato da fattori ambientali che mimano nel risultato un
carattere geneticamente determinato (Fig. 8).
• Il consenso al test potrebbe essere stato rifiutato per ragioni personali.
In caso di risultato indeterminato il rischio di tumore si basa sulla storia personale e
familiare e devono essere proposti programmi di sorveglianza personalizzati.
d) Il test ГЁ definito inconclusivo se viene identificata una variante di sequenza di significato clinico ancora non conosciuto (variants of uncertain significance o VUS), escludendo
i polimorfismi cioГЁ le varianti alleliche non patologiche in un locus di un gene.
Nella maggior parte dei casi tale esito ГЁ dovuto ad una mutazione missense nella regione codificante del gene che comporta una sostituzione aminoacidica il cui effetto sulla
funzione della proteina non ГЁ prevedibile, ma possono anche essere presenti inserzioni/
delezioni in frame nelle porzioni introniche o nelle regioni 5’ UTR o 3’ UTR.
Le VUS possono essere “nuove” cioè non precedentemente riportate in letteratura, ovvero già “note” ma con significato ancora incerto.
Anche in questo caso sulla base della storia personale e familiare viene quantificato il
rischio individuale e pianificate le strategie di sorveglianza.
3. Implicazioni del risultato del test per i familiari
Un test genetico non coinvolge solamente chi ad esso si sottopone, ma anche i membri
della sua famiglia. Lo specialista ГЁ tenuto a rispettare il segreto professionale e in nessun
caso puГІ contattare direttamente i familiari del probando e/o comunicare il risultato del
test se il probando stesso non ha fornito il suo consenso. Alla fine verrà consegnata al probando una relazione scritta contenente l’esito del test e gli eventuali suggerimenti clinici in
rapporto al rischio individuale.
Tra i rischi previsti per il test genetico vi ГЁ lo stress psicologico a cui viene sottoposto
il probando e gli eventuali cambiamenti che possono avvenire nella dinamica familiare.
Ciò poiché l’idea di una patologia potenzialmente ereditaria non viene ovviamente ben
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
Figura 7. Fasi del Counselling Oncogenetico. a) Test genetico diagnostico. b) Test genetico predittivo.
CI: Consenso Informato
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
Figura 13: Esempio di fenocopia. Nel probando con carcinoma della mammella a 35 anni non viene identificata la mutazione 633delC di BRCA1 responsabile della stessa neoplasia negli altri familiari. Pertanto il
probando ГЁ una fenocopia.
accettata dal probando e/o dai suoi familiari che talvolta possono vederla come un fattore
discriminante nella societГ .
Allo scopo di minimizzare questi rischi, ГЁ stata introdotta la figura dello psico-oncologo
a coadiuvare il ruolo del genetista e del medico oncologo.
Implicazioni psicologiche del test genetico
La presenza dello psico-oncologo all’interno del CGO è un modo nuovo ed efficace per
rispondere ai bisogni del probando aiutandolo a maturare con consapevolezza le scelte
necessarie a mantenere la propria qualitГ di vita.
Si tratta di scelte a forte valenza emozionale in quanto legate a tematiche quali salute,
malattia, procreazione, morte, futuro, etc. che richiedono da parte del singolo, della coppia
o di gruppi familiari una assoluta autonomia decisionale. La comunicazione “non direttiva”
fa si che le scelte decisionali siano ad esclusivo appannaggio del soggetto e che il processo di decision making sia incentrato sui suoi reali bisogni e valori, così da essere il solo
responsabile di sГЁ stesso e del proprio cambiamento.
Lo psico-oncologo, tramite una comunicazione chiara ed aperta, prende in carico la
persona, dГ informazioni sulle diverse e complesse implicazioni psicologiche della patologia, in modo da aiutarla a preservare la propria integritГ psichica, a ridefinire le proprie
aspettative rispetto al nuovo percorso da affrontare e a perseguire tutti quegli obiettivi
rilevanti per la propria salute.
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
Un “disordine di natura genetica” lacera l’individuo perchè va a colpire il modo in cui
percepisce se stesso, il suo senso di identitГ personale, il senso di appartenenza familiare,
il desiderio interno di stabilitГ , di controllo ed il comune senso di onnipotenza; ГЁ comunque una ferita narcisistica che fa emergere sentimenti di rabbia, vergona, sensi di colpa e
Gli aspetti maggiormente critici sono: percezione del rischio, motivazioni speranze e
paure prima del test genetico, vissuti psicoemozionali ad esso legati, modalitГ di adattamento al risultato, implicazioni psicosociali della comunicazione ai familiari; tutti questi
elementi possono provocare ansia, depressione e paura ed ГЁ per questo che il CGO ГЁ
un momento importante che serve sia per monitorare le modalitГ con le quali i familiari
hanno vissuto le tappe della malattia del proprio congiunto sia per intervenire nella nuova
Il malessere che può accompagnare il test genetico diagnostico è legato all’incertezza
circa la natura ereditaria della propria patologia e alla paura di avere trasmesso l’alterazione genetica ai figli ed è in questo delicato momento che il supporto psicologico si
rivela particolarmente utile. È compito di tutta l’equipe, ma in particolar modo dello psicooncologo fornire tutti gli strumenti psicologici utili al paziente per la gestione dello stress
che può derivare dal nuovo stato di “malato di rischio”.
La comunicazione del rischio genetico comporta importanti cambiamenti nel modo in
cui l’individuo percepisce se stesso, maggiormente quando l’esito del test risulta indeterminato o inconclusivo. Entrambi i risultati possono dar luogo ad un disagio psicoemotivo
perchГЁ diventa piГ№ difficoltoso poter programmare un percorso di sorveglianza. Questo
stato di incertezza può generare ansia e determinare l’erronea percezione del proprio rischio con sovra- o sottostima del reale rischio di potersi ammalare.
4. Follow-up
Il programma di counselling genetico non termina con la comunicazione del risultato
del test al probando. Infatti, il consulente genetico dovrГ essere disponibile per rispondere
ad ulteriori domande insorte nel probando dopo aver conosciuto il proprio esito, ma anche successivamente. SarГ inoltre necessario monitorare nel tempo il probando a rischio
ed i suoi familiari poichГ© la storia familiare ГЁ continuamente in evoluzione. Infine, tenuto
conto della rapida evoluzione della ricerca medica, i probandi devono essere incoraggiati
a ricontattare periodicamente il consulente per ricevere le piГ№ recenti informazioni circa i
progressi in campo preventivo e terapeutico di questo settore.
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni Pantuso, Michele Frazzetta,
Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi, Nicola Gebbia e Viviana Bazan
[1] Augello C, Bruno L, Bazan V, Calo V, Agnese V, Corsale S, Cascio S, Gargano G, Terrasi M, Barbera F, Fricano S, Adamo B, Valerio MR, Colucci G, Sumarcz E, Russo A. Y179C, F486L and N550H are BRCA1 variants that may be associated with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo
Oncologico dell’Italia Meridionale) prospective study. Ann Oncol 2006;17 Suppl 7:vii30-33.
[2] CalГІ V, Agnese V, Gargano G, Corsale S, Gregorio V, Cascio S, Cammareri P, Bruno L, Augello C, Gullo A,
Sisto PS, Badalamenti G, Valerio MR, Napoli L, Gebbia N, Bazan V, Russo A. A new germline mutation in
BRCA1 gene in a sicilian family with ovarian cancer. Breast Cancer Res Treat 2006;96:97-100.
[3] DeVita VT, Lawrence TS, Rosenberg SA. DeVita, Hellman, and Rosenberg’s Cancer: Principles & Practice
of Oncology, 8th edition. Philadelphia, Wolters Kluwer/Lippincott Williams & Wilkins, 2008.
[4] Falchetti M, Lupi R, Rizzolo P, Ceccarelli K, Zanna I, CalГІ V, Tommasi S, Masala G, Paradiso A, Gulino A,
Giannini G, Russo A, Palli D, Ottini L. BRCA1/BRCA2 rearrangements and CHEK2 common mutations are
infrequent in Italian male breast cancer cases. Breast Cancer Res Treat 2008;110:161-167.
[5] Ferla R, Calo V, Cascio S, Rinaldi G, Badalamenti G, Carreca I, Surmacz E, Colucci G, Bazan V, Russo A.
Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol 2007;18 Suppl 6:vi93-98.
[6] Hendriks YM, de Jong AE, Morreau H, Tops CM, Vasen HF, Wijnen JT, Breuning MH, Brocker-Vriends AH.
Diagnostic approach and management of Lynch syndrome (hereditary nonpolyposis colorectal carcinoma): a guide for clinicians. CA Cancer J Clin 2006;56:213-225.
[7] Jorde LB, Carey JC, Bamshad MJ. Medical Genetics, 4th edition. Philadelphia, Mosby/Elsevier, 2009.
[8] La Paglia L, Lauge A, Weber J, Champ J, Cavaciuti E, Russo A, Viovy JL, Stoppa-Lyonnet D. ATM germline mutations in women with familial breast cancer and a relative with haematological malignancy.
Breast Cancer Res Treat 2009.
[9] Lynch HT, Lynch JF, Lynch PM, Attard T. Hereditary colorectal cancer syndromes: molecular genetics,
genetic counseling, diagnosis and management. Fam Cancer 2008;7:27-39.
[10] Ottini L, Palli D, Rizzo S, Federico M, Bazan V, Russo A. Male breast cancer. Crit Rev Oncol Hematol
[11] Russo A, Agnese V, Rizzo S, La Paglia L, Bazan V. The genetics of breast cancer: application in clinical
practice. In: Giordano A, Normanno N (Eds.). Breast Cancer in the Post-Genomic Era. New York, Humana
Press, 2009, pp. 39-54.
[12] Russo A, CalГІ V, Agnese V, Bruno L, Corsale S, Augello C, Gargano G, Barbera F, Cascio S, Intrivici C,
Rinaldi G, Gulotta G, Macaluso M, Surmacz E, Giordano A, Gebbia N, Bazan V. BRCA1 genetic testing in
106 breast and ovarian cancer families from Southern Italy (Sicily): a mutation analyses. Breast Cancer
Res Treat 2007;105:267-276.
[13] Russo A, CalГІ V, Augello C, Bruno L, Agnese V, Schiro V, Barbera F, Cascio S, Foddai E, Badalamenti G,
Intrivici C, Cajozzo M, Gulotta G, Surmacz E, Colucci G, Gebbia N, Bazan V. 4843delC of the BRCA1 gene
is a possible founder mutation in Southern Italy (Sicily). Ann Oncol 2007;18 Suppl 6:vi99-102.
[14] Russo A, CalГІ V, Bruno L, Rizzo S, Bazan V, Di Fede G. Hereditary ovarian cancer. Crit Rev Oncol Hematol
[15] Russo A, CalГІ V, Bruno L, Schiro V, Agnese V, Cascio S, Foddai E, Fanale D, Rizzo S, Di Gaudio F, Gulotta
E, Surmacz E, Di Fede G, Bazan V. Is BRCA1-5083del19, identified in breast cancer patients of Sicilian
origin, a Calabrian founder mutation? Breast Cancer Res Treat 2009;113:67-70.
[16] Russo A, Migliavacca M, Zanna I, Macaluso M, Gebbia N, Bazan V. Caretakers and Gatekeepers. In:
Encyclopedia of the Human Genoma. New York, Wiley, 2003.13.
[17] Russo A, Zanna I, Tubiolo C, Migliavacca M, Bazan V, Latteri MA, Tomasino RM, Gebbia N. Hereditary
La consulenza genetica in oncologia: implicazioni biomolecolari e cliniche
common cancers: molecular and clinical genetics. Anticancer Res 2000;20:4841-4851.
[18] Schneider K. Counseling About Cancer: Strategies for Genetic Counseling, 2nd edition. New York, WileyLiss, 2002.
[19] Smith A, Dougall A, Posluszny D, Somers T, Rubinstein W, Baum A. Psychological distress and quality of
life associated with genetic testing for breast cancer risk. J Psycho- Oncology 2008;17:767-773.
[20] Stormorken AT, Bowitz-Lothe IM, Noren T, Kure E, Aase S, Wijnen J, Apold J, Heimdal K, Moller P. Immunohistochemistry identifies carriers of mismatch repair gene defects causing hereditary nonpolyposis
colorectal cancer. J Clin Oncol 2005;23:4705-4712.
[21] Veschi S, Aceto G, Scioletti AP, Gatta V, Palka G, Cama A, Mariani-Costantini R, Battista P, Calo V, Barbera
F, Bazan V, Russo A, Stuppia L. High prevalence of BRCA1 deletions in BRCAPRO-positive patients with
high carrier probability. Ann Oncol 2007;18 Suppl 6:vi86-92.
[22] Vogelstein B, Kinzler K. The Genetic Basis of Human Cancer, 2nd edition. New York, Mc-Graw-Hill,
[23] Walsh T, Casadei S, Coats KH, Swisher E, Stray SM, Higgins J, Roach KC, Mandell J, Lee MK, Ciernikova
S, Foretova L, Soucek P, King MC. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. Jama 2006;295:1379-1388.
[24] Welcsh PL, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
I tumori della mammella e/o dell’ovaio di tipo eredofamiliare: strategie di prevenzione nelle donne ad alto
[Heredo-familial breast and/or ovarian cancers: prevention strategies in high risk women]
Sergio Rizzo1, Giuseppe Bronte1, Valentina CalГІ1, Loredana Bruno1, Nello Grassi2, Gianni
Pantuso2, Luigi Sandonato2, Michele Frazzetta3, Giuseppe Cicero1, Girolamo Guarneri4, Giuseppe Lo Dico4, Massimo Cajozzo3, Nino Dispensa5, Aldo Gerbino6, Nicola Gebbia1, Viviana
Bazan1 e Antonio Russo1
Sezione di Oncologia Medica e Centro di Riferimento Regionale per la caratterizzazione e lo Screening
Genetico dei Tumori Ereditari, Dipartimento di Discipline Chirurgiche ed Oncologiche; 2Sezione di Oncologia
Chirurgica, Dipartimento di Discipline Chirurgiche ed Oncologiche; 3Sezione di Chirurgia Generale, Dipartimento di Discipline Chirurgiche ed Oncologiche; 4Ginecologia ed Ostetricia, Dipartimento Materno Infantile;
Sezione di Urologia, Dipartimento di Medicina Interna, Malattie Cardiovascolari e Nefrourologiche; 6Sezione
di Istologia ed Embriologia, Di.Me.S., UniversitГ degli Studi di Palermo (IT)
Parole chiave: Fattori di rischio, Carcinoma ereditario della mammella e Sidrome da Carcinoma ovarico,
Screening, Prevenzione
Key words: Risk factors, Hereditary Breast and Ovarian Cancer Syndrome, Screening, Prevention
Riassunto. Numerosi fattori sono stati coinvolti nell’insorgenza del carcinoma mammario e dell’ovaio tra cui
l’età , fattori ormonali e riproduttivi come l’età e il numero di gravidanze e l’utilizzo di contraccettivi orali e, in
minor misura, fattori ambientali e stili di vita.
Le possibilitГ per la gestione clinica delle pazienti considerate ad alto rischio di sviluppare carcinoma della
mammella e/o dell’ovaio includono chirurgia profilattica (es. mastectomia bilaterale profilattica o ovarosalpingectomia bilaterale), chemioprevenzione (principalmente tamoxifene e raloxifene ma anche, più recentemente, inibitori dell’aromatasi nelle donne ad alto rischio in postmenopausa), cambiamenti nelle abitudini
di vita e sorveglianza intensiva attraverso l’esame clinico periodico, la Risonanza Magnetica Mammaria, la
Mammografia talvolta in associazione all’Ecografia e, per la prevenzione del carcinoma ovarico, lo screening
combinato utilizzando il dosaggio del CA125 e l’ecografia transvaginale.
Abstract. Several factors should be taken into account to estimate the risk for breast and ovarian cancer
development such as age, reproductive and hormonal factors including parity and oral contraceptive use,
strong family history of breast and ovarian cancer, and – to a lesser extend – environmental and lifestyle
Management options for the women at high-risk for breast and ovarian cancer development include prophylactic surgery (i.e. bilateral mastectomy and bilateral ovarosalpingectomy), chemoprevention (using mainly
tamoxifen, but also raloxifen, and - recently - aromatase inhibitors in high-risk post-menopausal women),
lifestyle changes and intensive surveillance which includes regular clinical examination, regular Magnetic
Resonance Imaging, Mammography potentially in association with breast Ultrasonography, and ovarian cancer surveillance with transvaginal ultrasound and CA125 combined.
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
Incidenza dei carcinomi della mammella e dell’ovaio
Il carcinoma della mammella ГЁ frequente nelle popolazioni del mondo occidentale soprattutto in donne in post-menopausa. In Europa rappresenta il 22% dei tumori maligni
femminili e colpisce il 27% delle donne affette da tumori. In Italia circa una donna su 11
si ammala di tumore della mammella entro i 70 anni; il nostro Paese, con una stima di
circa 40.000 nuovi casi annui, si pone di poco al di sotto dei valori medi europei, con una
incidenza del 30% inferiore nelle regioni del Sud rispetto alle regioni del Nord.
Nel mondo occidentale il carcinoma dell’ovaio rappresenta una delle principali malattie
ginecologiche ad alta mortalitГ , la quarta causa di morte per tumore nelle donne, con una
incidenza di circa 192.000 nuovi casi ogni anno.
Fattori di rischio per i carcinomi della mammella e dell’ovaio
Carcinoma della mammella
Ad oggi i fattori eziologici del carcinoma mammario sono in gran parte sconosciuti.
Diversi studi epidemiologici hanno identificato alcuni fattori che possono aumentare il
rischio di sviluppare un tumore della mammella.
• Fattori Familiari e Genetici: la presenza di un familiare di primo grado con carcinoma mammario è associata ad un rischio doppio di sviluppare la patologia nel corso della
vita rispetto alla popolazione generale. La percentuale di rischio aumenta se i familiari
affetti da carcinoma della mammella sono due o piГ№ (rischio fino al 40%). Si ritiene che
circa il 10% di tutti i casi di tumore della mammella siano attribuibili a fattori genetici, in
particolare a mutazioni all’interno dei geni di suscettibilità per questa neoplasia (BRCA1 e
BRCA2) che conferiscono un rischio cumulativo (all’età di 80 anni) di sviluppare il carcinoma della mammella del 56-85% (Fig. 1).
• Età : le diagnosi di carcinoma della mammella prima dei 30 anni sono rare, di contro
la frequenza di casi aumenta nel periodo della menopausa e successivamente. PiГ№ del
50% delle neoplasie alla mammella ГЁ infatti diagnosticato in donne di oltre 65 anni.
• Fattori Riproduttivi: le donne di età inferiore a 21 anni che hanno già sostenuto
una gravidanza sviluppano un tumore al seno meno frequentemente rispetto a quelle che
hanno il primo figlio dopo i 30 anni. Il rischio di insorgenza di un carcinoma mammario ГЁ
dunque inversamente correlato con l’età della prima gravidanza. Si stima inoltre che esso
ГЁ anche inversamente correlato al numero di gravidanze portate a termine. Sembra esserci
poi una relazione diretta e lineare tra il numero di mesi di allattamento al seno ed il rischio
relativo di insorgenza del tumore. Tra gli altri fattori che potrebbero influenzare l’insorgenza di un carcinoma della mammella rientrano anche l’età di comparsa di mestruazioni e
la menopausa: la prima mestruazione (menarca) precoce e la menopausa tardiva sembra250
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
no predisporre maggiormente all’insorgenza della neoplasia, infatti corrispondono ad una
maggiore durata della vita riproduttiva ed una più lunga esposizione dell’epitelio ghiandolare agli stimoli proliferativi degli estrogeni ovarici.
• Fattori Ormonali: gli ormoni sessuali hanno un ruolo fondamentale nello sviluppo
e nella progressione del carcinoma mammario. Evidenze suggeriscono che pazienti che
assumono una terapia sostitutiva ormonale per prevenire i tipici disturbi della menopausa
abbiano un incrementato rischio di neoplasia mammaria, ma solo per assunzioni prolungate (8-10 anni). Si ritiene dunque che per i primi anni di trattamento i benefici derivanti dalla terapia sostitutiva ormonale, tra cui la possibile riduzione di problemi cardiaci e
dell’osteoporosi, compensino il rischio, per quanto aumentato, di sviluppare un tumore del
seno. L’uso dei contraccettivi orali, soprattutto in caso di assunzioni prolungate con inizio
in etГ molto giovane, puГІ incrementare il rischio di insorgenza di tumore della mammella
e ridurre quello di tumore dell’ovaio.
• Fattori Dietetici: è stato rilevato che soggetti che hanno una dieta basata su farine
eccessivamente raffinate e su alimenti di origine animale, come quella prevalente nei Paesi occidentali, sono maggiormente suscettibili allo sviluppo del tumore della mammella;
inoltre, è stato evidenziato un legame tra l’obesità ed una maggiore suscettibilità a sviluppare un tumore al seno, sulla base del fatto che la principale fonte di estrogeni circolanti
nella donna in post-menopausa ГЁ rappresentata dal tessuto adiposo.
Carcinoma della mammella
Figura 1. Rischio cumulativo di carcinoma della mammella e dell’ovaio all’età di 80 anni
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
• Radiazioni: sono rari i casi di carcinomi mammari causati da radiazioni. Sono considerate pericolose le radiazioni ionizzanti (raggi X) assunte con la radioterapia per la cura di
tumori vicini alla mammella, ad esempio tiroide e linfomi; mentre, sono considerate meno
dannose le radiazioni assorbite durante le mammografie o altri esami diagnostici.
Carcinoma dell’ovaio
• Fattori Familiari e Genetici: Per il carcinoma ovarico l’unico fattore di rischio realmente significativo sembra, e una vasta rete bibliografica lo sottolinea (1-27), la storia familiare di questa malattia. Il rischio nel corso della vita di sviluppare un carcinoma ovarico
aumenta dall’1.6% nella popolazione generale al 4% quando un parente di I grado è affetto
da carcinoma ovarico e al 7% in presenza di due parenti di I grado affetti.
Il rischio di neoplasia familiare diminuisce con l’incremento dell’età del familiare di I
grado che ha sviluppato la malattia.
Mutazioni nei geni BRCA predispongono anche al carcinoma ovarico con un rischio cumulativo pari al 20% per i portatori di mutazione nel gene BRCA2 e al 50% per i portatori
di mutazione nel gene BRCA1 (Fig. 1).
• Fattori Riproduttivi: le donne che non hanno mai avuto una gravidanza sono più
a rischio di sviluppare il carcinoma dell’ovaio. Anche un numero più basso di gravidanze
correla con un aumentato rischio di ammalarsi di questa patologia, rispetto alle donne che
hanno avuto un maggior numero di gravidanze.
• Fattori Ormonali: aumento del rischio è associato ai farmaci che inducono ovulazione incessante mediante stimolazione gonadotropinica continua, usati nel trattamento
della sterilitГ . Questa associazione ГЁ stata spiegata con il verificarsi ad ogni ovulazione
di microtraumi a carico dell’epitelio celomatico, che contribuirebbero allo sviluppo della
neoplasia. Anche l’utilizzo in post-menopausa di terapie ormonali sostitutive è capace di
aumentarne il rischio. Al contrario le donne che fanno uso di contraccettivi orali presentano
un rischio inferiore di questa neoplasia.
• Fattori Dietetici: una dieta particolarmente ricca di grassi saturi e alcol e povera di
frutta e verdura sarebbe associata con l’aumento del rischio.
Carcinoma ereditario della mammella e/o dell’ovaio (HBOC)
Principali geni di suscettibilitГ Sebbene il carcinoma della mammella sia piГ№ frequentemente sporadico, circa il 5-10%
dei casi sono ereditari. Meno della metГ di questi sono associati a mutazioni germinali
in geni di suscettibilitГ noti che conferiscono un alto rischio (BRCA1, BRCA2, TP53) o un
rischio moderato (CHEK2, PTEN, ATM ed altri) di sviluppare la malattia nell’arco della vita.
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
In oltre il 60% dei casi di carcinoma della mammella eredo-familiare sembrano essere
coinvolte mutazioni in geni di suscettibilitГ ad alta penetranza non ancora identificati o
polimorfismi in piГ№ loci a bassa penetranza, ciascuno dei quali conferisce un rischio di
carcinoma della mammella lieve-moderato (suscettibilitГ poligenica). I due principali geni
di suscettibilitГ sono Brca1 e BRCA2, entrambi geni oncosoppressori caretakers coinvolti
nella riparazione del danno al DNA, le cui alterazioni determinano un’instabilità genetica
che favorisce la comparsa di ulteriori mutazioni in altri geni importanti nel controllo del
ciclo cellulare. Le mutazioni di questi geni sono ad alta penetranza e vengono trasmesse
con modalitГ autosomica dominante (Fig. 2).
Ma nella maggior parte dei casi la suscettibilitГ al carcinoma della mammella ГЁ soggetta a meccanismi poligenici, cioГЁ a combinazioni additive o moltiplicative di piГ№ varianti alleliche a bassa penetranza. Gli studi di associazione genomica che consentono di
identificare caratteristiche genetiche comuni (GWA) relativi al carcinoma della mammella
hanno consentito di identificare 8 varianti alleliche a bassa penetranza (FGFR2, TNCR9/
Tox3, MAP3K1, LSP1, H19, 8q, 2q35, ECHDC/RNF) che giustificano appena il 5% dei casi
di carcinoma mammario eredo-familiare.
Figura 2. Carcinoma ereditario della mammella e/o dell’ovaio (HBOC): principali geni di suscettibilità 253
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
Sindromi ereditarie
Le famiglie i cui membri sono portatori di mutazioni Brca1/BRCA2 possono presentare
nell’albero genealogico esclusivamente casi di carcinoma della mammella e si parla allora
di Carcinoma Ereditario della Mammella (Hereditary Breast Cancer, HBC), casi di carcinoma
della mammella e carcinoma dell’ovaio e si parla di Carcinoma Ereditario della Mammella
e dell’Ovaio (Hereditary Breast and Ovarian Cancer, HBOC) ovvero, meno frequentemente,
soltanto casi di carcinoma dell’ovaio e si parla di Carcinoma Ereditario dell’Ovaio (Hereditary Ovarian Cancer, HOC) (Fig. 3).
In particolare HBC, HOC e HBOC rappresentano lo spettro di una stessa patologia genetica con manifestazioni fenotipiche differenti.
I criteri utilizzati per la selezione dei pazienti candidati alla consulenza oncogenetica
e al test genetico allo scopo di identificare la sindrome di predisposizione al carcinoma
ereditario della mammella e/o dell’ovaio includono una precoce età di insorgenza di carcinoma della mammella e/o carcinoma dell’ovaio, carcinoma della mammella bilaterale,
carcinoma della mammella e/o carcinoma dell’ovaio nella stessa persona a qualsiasi età e carcinoma della mammella maschile (Male Breast Cancer, carcinoma della mammella
maschile) (Fig. 4).
Esistono casi di carcinoma ovarico familiare nell’ambito di altre sindomi ereditarie che
Figura 3. Sindromi ereditarie di predisposizione al carcinoma della mammella e/o dell’ovaio
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
Figura 4. Profili familiari dei portatori di mutazione nei geni BRCA
coinvolgono il carcinoma del colon, in tal caso si parla di sindrome di Lynch II. I geni di
suscettibilità coinvolti sono quelli del sistema del “mismatch repair” (geni MMR).
Rischio cumulativo
Le mutazioni nel gene BRCA1 conferiscono nelle donne portatrici un rischio compreso
tra il 50 e l’85% di sviluppare un carcinoma della mammella nell’arco della loro vita, un
rischio del 40-60% di sviluppare un carcinoma della mammella controlaterale e un rischio
compreso tra il 15 e il 45% di sviluppare un carcinoma dell’ovaio o un carcinoma tubarico.
Anche le mutazioni nel gene BRCA2 conferiscono nelle donne portatrici un rischio compreso tra il 50 e l’85% di sviluppare un carcinoma della mammella e un rischio minore
(20-30%) di sviluppare un carcinoma dell’ovaio.
Alcuni ritengono che mutazioni nel gene BRCA1 nei soggetti maschili non comportino
rischi di ammalarsi di CM ma piuttosto di carcinoma della prostata o del colon. Gli uomini
che risultano portatori di mutazione del gene BRCA2 possono sviluppare un cancro alla
mammella nel corso della vita con un rischio del 6%. Le diverse casistiche mostrano inoltre un leggero incremento (6-14%) del rischio di sviluppare carcinomi della prostata, del
colon e del pancreas.
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
I dati in letteratura suggeriscono che il rischio di sviluppare un carcinoma della mammella in donne portatrici di mutazioni Brca1/BRCA2 sia del 10-20% a 40 anni di etГ , del
30-50% a 50 anni e del 40-85% a 70 anni, rispetto a un rischio nella popolazione generale
rispettivamente dello 0,5%, 2% e 8%.
Le mutazioni in BRCA1 sono associate ad un rischio di carcinoma dell’ovaio nel corso
della vita stimato tra il 15% e il 45%, in confronto al rischio dell’1.6% nella popolazione
generale. Il rischio cumulativo di carcinoma dell’ovaio in famiglie che risultano portatrici di
mutazione nel gene BRCA2 risulta compreso tra 0.4% e il 27%.
Strategie di prevenzione nei gruppi ad alto rischio (Figg. 5,6)
a. Chirurgia profilattica. Rappresenta la misura piГ№ efficace di prevenzione primaria.
La mastectomia profilattica bilaterale con rimozione completa del parenchima mammario, dell’areola e del capezzolo, con o senza asportazione del rivestimento cutaneo delle
mammelle (total o skin-sparing), dovrebbe quasi azzerare il rischio di Carcinoma o Cancro
della Mammella (BC) ma, essendo una procedura drastica e traumatica, va considerata
esclusivamente in donne portatrici di mutazioni BRCA1/BRCA2 o in altri geni di suscettibilità che conferiscono un alto rischio di BC; può essere proposta inoltre a donne con anamnesi di carcinoma lobulare in situ poiché anch’esse ad alto rischio di carcinoma invasivo nella
mammella inizialmente interessata e in quella controlaterale. In mancanza di diagnosi
Figura 5. Strategie di prevenzione per BC in portatori di mutazioni BRCA e/o famiglie ad alto rischio
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
istopatologica di BC nel tessuto asportato, non ГЁ indicata linfadenectomia ascellare.
La mastectomia sottocutanea (nipple-sparing) consente di risparmiare l’areola e il capezzolo, con migliori risultati estetici, e dovrebbe ridurre il rischio di BC di almeno il 90%.
Tale rischio non puГІ essere del tutto eliminato a causa della possibilitГ di insorgenza del BC
a livello dei dotti galattofori del capezzolo, in prossimità dell’areola e nelle isole di parenchima mammario risparmiate dall’intervento chirurgico. È stata anche impiegata l’asportazione del parenchima mammario e del capezzolo e non dell’areola (areola-sparing) ma
manca un follow-up adeguato a supporto di questa procedura.
Nonostante il rischio di Carcinoma Ovarico o Cancro dell’Ovaio (OC) sia decisamente
inferiore rispetto al rischio di BC, la ovaro-salpingectomia bilaterale profilattica dovrebbe essere proposta a tutte le donne portatrici di mutazioni BRCA1/BRCA2 oppure
ad alto rischio, in perimenopausa o al completamento del programma familiare, perchГЁ
dimezza il rischio di BC, nonostante la maggior parte dei tumori in portatori di mutazione
BRCA1 non esprimano recettori ormonali; nei confronti del carcinoma ovarico consente
una riduzione del rischio di almeno il 95%, anche se tale rischio non puГІ essere com-
Figura 6
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
Figure 7a - 7b
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
pletamente eliminato per la possibile comparsa di carcinomi primitivi peritoneali, per
la comune origine embriologica del peritoneo e dell’epitelio di rivestimento ovarico. Le
salpingi possono rappresentare un sito elettivo di tumore nei portatori di mutazione BRCA
ed ГЁ quindi indicata la contemporanea isterectomia per evitare la possibile insorgenza in
corrispondenza dell’impianto delle tube nell’utero.
b. Chemioprevenzione. La possibilitГ di ridurre il rischio cumulativo di sviluppare un
tumore da parte di un soggetto predisposto mediante terapia ormonale, senza l’obbligo di
ricorrere alla demolizione chirurgica è, data l’età delle pazienti interessate, un obiettivo di
grande interesse. Tuttavia occorre considerare che i BC in portatori di mutazioni in BRCA1
sono per la maggior parte ER negativi (>80%) mentre i BC in portatori di mutazioni in
BRCA2 sono prevalentemente ER positivi (75-80%). Di conseguenza, i portatori di mutazioni in BRCA2 potrebbero essere il gruppo che beneficia della chemioprevenzione con ormonoterapia; al contrario nei portatori di mutazione BRCA1, il ruolo ГЁ discutibile (Fig. 7a,7b).
Nonostante l’approccio con SERMs (Selective Estrogen Receptor Modulators) come Tamoxifene e Raloxifene sia stato ampiamente studiato nei soggetti ad alto rischio familiare
in pre- e post-menopausa (vedi NCCN Breast Cancer Risk Reduction Guidelines 2009), il
loro utilizzo è comunque gravato da un’aumentata incidenza di tumori e sanguinamenti
Figura 8
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
endometriali così come di eventi tromboembolici.
Un retinoide (Fenretide) ha mostrato di ridurre significativamente il rischio di recidiva
locale nelle donne in pre-menopausa con anamnesi di BC in stadio I, indipendentemente
dallo stato recettoriale del tumore primitivo. Tale risultato rappresenta la base degli studi di
prevenzione con Fenretide in corso nelle donne giovani ad alto rischio.
Sempre nell’ambito di studi clinici controllati, è in corso la valutazione dell’efficacia
preventiva degli inibitori dell’aromatasi (anastrozolo ed exemestane) nelle donne in postmenopausa ad alto rischio (Fig. 8).
c. Cambiamento dello stile di vita. Circa la possibilitГ di influire sui fattori di rischio
non-genetici, poiché la produzione periferica di estrogeni aumenta con l’aumentare dell’età ed è correlata al grado di obesità , sono indicati la riduzione del peso, dell’assunzione di
grassi e del consumo di alcool e l’incremento dell’esercizio fisico. Tuttavia la valutazione
dei singoli interventi ГЁ difficile ed occorrono tempi lunghi per definire se le modifiche dello
stile di vita possono davvero modificare il rischio.
È stata riportata un’associazione tra la Terapia Ormonale Sostitutiva (TOS) in postmenopausa e lo sviluppo di cancro ovarico, anche se la potenza statistica della maggior parte
degli studi condotti a questo riguardo è assai limitata. All’interno di una casistica inglese di ampie proporzioni, le donne classificate come utilizzatrici correnti (30%), pregresse
(20%) o non utilizzatrici (50%) di TOS (intesa come assunzione di estrogeni con o senza
progestinici) venivano seguite per lo sviluppo di carcinoma ovarico. Le donne utilizzatrici
di TOS avevano un’incidenza significativamente maggiore di cancro ovarico rispetto alle
non utilizzatrici (RR 1.20). Non ГЁ stato osservato alcun aumento significativo nelle donne
appartenenti alla categoria delle utilizzatrici pregresse e nemmeno nelle donne che avevano utilizzato la TOS per un periodo inferiore ai 5 anni. Le utilizzatrici di TOS presentavano
inoltre un aumento significativo del rischio di morte per carcinoma ovarico rispetto alle non
utilizzatrici (RR 1.23). Di conseguenza, la terapia ormonale sostitutiva dovrebbe essere
prescritta soltanto per brevi periodi.
Il possibile effetto protettivo dell’assunzione protratta (6 anni) di contraccettivi orali
nei confronti dell’ OC (riduzione del rischio del 60%) sembra bilanciato da un ulteriore
incremento di rischio di BC tra le portatrici di mutazioni BRCA1/BRCA2 (RR = 1.2), di conseguenza l’utilizzo di contraccettivi orali non può essere raccomandato tra le opzioni per
ridurre il rischio di carcinoma ovarico nelle pazienti a rischio.
d. Sorveglianza senologica. Le metodiche di sorveglianza convenzionalmente utilizzate
nello screening per il BC nella popolazione generale comprendono autopalpazione, esame
clinico della mammella, ecografia e mammografia. Soltanto la mammografia ha mostrato
di ridurre la mortalitГ per BC del 20-35 % nelle donne di etГ 50-69 anni e di meno del 20%
nelle donne di etГ 40-49 anni. Per delineare una strategia integrata di sorveglianza inten260
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
siva da adottare nelle donne ad alto rischio di BC occorre valutare la precoce età di insorgenza (premenopausa), l’elevato rischio conseguente all’esposizione a radiazioni ionizzanti
(associato alla possibile compromissione della capacitГ di riparazione dei danni cellulari
radioindotti nei portatori di mutazione BRCA), la minore sensibilitГ della mammografia nei
seni “densi”, l’alta velocità di crescita nei tumori BRCA che condiziona il maggior rischio di
carcinomi d’intervallo, l’elevata percentuale di positività linfonodale alla diagnosi.
Nei soggetti ad alto rischio ГЁ quindi opportuno adottare programmi di sorveglianza con
esordio precoce (25 anni) e brevi intervalli (6 mesi) tra due controlli successivi, prediligendo tecniche di imaging poco dipendenti dalle variazioni della densitГ ghiandolare.
Tabella 1. Linee guida sulla sorveglianza nelle donne ad alto rischio e BRCA1/2 mut.
(Forza Operativa Nazionale sul Carcinoma Mammario)
Auto-esame della
Esame clinico
EtГ > 25 в†’ ogni 6 mesi
(National Comprehensive Cancer
EtГ > 18 в†’ ogni mese
(dopo training adeguato)
EtГ > 25 в†’ ogni 6 mesi
EtГ > 30 в†’ ogni 12 mesi - Dose
ghiandolare media complessiva (per
mammelle standard) <4 mGy (= solo
Mammografia (Mx)
EtГ 35-50 в†’ ogni 12 mesi
EtГ > 25 в†’ ogni 12 mesi
Dose ghiandolare media complessiva
(per mammella standard) <8 mGy
Eta > 50 в†’ ogni 12 mesi
Dose standard
EtГ 25-35 в†’ ogni 6 mesi
della mammella
EtГ 35-50 в†’ ogni 12 mesi (ogni 6
mesi se seni”densi” alla Mx)
Solo a giudizio del Radiologo
EtГ > 50 в†’ a giudizio del Radiologo
Risonanza Magnetica EtГ > 25 в†’ ogni 12 mesi
Ecografia TransvagiEtГ > 35 в†’ ogni 12 mesi
nale + CA-125
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
Numerosi studi hanno valutato l’impiego della risonanza magnetica (RM) con mezzo di
contrasto paramagnetico nella sorveglianza delle donne ad alto rischio, concludendo che
questa tecnica ГЁ la piГ№ sensibile ad ogni etГ per la diagnosi del BC (70-96%). Tuttavia tale
metodica risulta sensibilmente meno specifica rispetto alla mammografia e può comportare la ripetizione dell’esame prima del successivo passaggio di screening e agobiopsie
non necessarie. Nonostante non sia stata ancora dimostrata una riduzione della mortalitГ mediante lo screening con RM nelle donne portatrici di mutazioni BRCA1/BRCA2 e ad
alto rischio, le neoplasie così diagnosticate sono però mediamente di minori dimensioni e
meno frequentemente associate a coinvolgimento linfonodale.
Il protocollo di sorveglianza proposto nel Maggio 2008 dalla Forza Operativa Nazionale
per il Carcinoma della Mammella (FONCAM) ГЁ illustrato nella tabella (confronto con NCCN)
(Tabella 1).
e. Sorveglianza ginecologica. Una strategia di screening multimodale basata sulla
combinazione di ecografia pelvica e dosaggio del CA125 sierico, non indicata nella popolazione generale, ГЁ suggerita nei pazienti ad alto rischio per la mutazione dei geni BRCA1/
BRCA2, con l’indicazione a ripetere semestralmente tali indagini.
Implicazioni terapeutiche
La maggior parte dei BC con mutazioni germinali o sporadiche di BRCA1, specialmente
quelli diagnosticati al di sotto dei 50 anni, presentano le caratteristiche immunoistochimiche del fenotipo triplo-negativo e, per quanto riguarda l’espressione genica, appartengono
alla classe molecolare basal-like. Nei tumori sporadici basal-like i livelli della proteina
BRCA1 possono essere ridotti anche per fenomeni di tipo epigenetico, quali la metilazione
del promotore del gene e il silenziamento dovuto alla espressione di repressori di BRCA1,
ad esempio il fattore ID4. L’inattivazione delle proteine BRCA causerebbe una maggiore
sensibilitГ delle cellule neoplastiche agli agenti citotossici che producono addotti al DNA
come il cisplatino; viceversa proteine BRCA1 funzionalmente attive sarebbero necessarie
affinchГЁ gli agenti antimicrotubuli come i taxani possano esercitare la loro azione citocida.
Inoltre la selezione dei pazienti con neoplasia mammaria basal-like basata sullo stato mutazionale di BRCA1 potrebbe portare all’utilizzo di molecole target dirette contro l’enzima
poli-ADPribosio-polimerasi (PARP). Le cellule tumorali che presentano la mutazione del
gene BRCA dipendono dall’attività di questo enzima per la riparazione del DNA danneggiato
e quindi per la sopravvivenza. Con la nuova classe degli inibitori di PARP il bersaglio ГЁ costituito dalla riparazione del DNA danneggiato. Vari agenti (raggi UV), radiazioni ionizzanti,
sostanze chimiche, chemiotertici come i derivati del platino, ecc.) possono produrre danni
al DNA, con rottura della singola elica o di entrambi. Nel primo caso il proceso di riparazione è detto base excision repair ed è controlato dall’enzima PARP. Le cellule tumorali, anche
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
a causa della rapida proliferazione, hanno difetti riparativi e molteplici danni da instabilità genetica. in caso di danno an un singolo filamento, PARP si lega ai punti di rottura, formando catene di poli-ADPribosio: così vengono recluatati specifici enzimi di riparazione.
La mancata riparazione del singolo filamento, come conseguenza dell’esposizione ad un
inibitore di PARP, determina una rottura della doppia elica nella fase S del ciclo cellulare.
Le cellule con mutazioni di BRCA1/BRCA2 presentano difetti di riparazione delle rotture
nella doppia elica. Di conseguenza il double stand break conseguente all’inibizione di PARP
ed eventualmente occorso quando la cellula passa in fase S, non puГІ essere riparato.
In effetti, le cellue con mutazioni o in attivazione di BRCA1/BRCA2 si mostrano particolarmente sensibili all’effetto cicotida degli inibitori di PARP e in alcuni studi clinici di fase
II, gli inibitori di PARP hanno mostrato risultati incoraggianti in pazienti affette da neoplasie
mammarie ed ovariche con mutazioni di BRCA1/BRCA2.
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe
Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan, Antonio Russo
[1] Augello C, Bruno L, Bazan V, et al. Y179C, F486L and N550H are BRCA1 variants that may be associated
with breast cancer in a Sicilian family: results of a 5-year GOIM (Gruppo Oncologico dell’Italia Meridionale) prospective study. Ann Oncol 2006; 17 Suppl 7:vii30-3.
[2] CalГІ V, Agnese V, Gargano G, et al. A new germline mutation in BRCA1 gene in a sicilian family with
ovarian cancer. Breast Cancer Res Treat 2006; 96:97-100.
[3] Chung DC, Yoon SS, Lauwers GY, et al. Case records of the Massachusetts General Hospital. Case 222007. A woman with a family history of gastric and breast cancer. N Engl J Med 2007; 357:283-91.
[4] DeVita VT, Lawrence TS, Rosenberg SA. DeVita, Hellman, and Rosenberg’s Cancer: Principles & Practice
of Oncology, 8th edition. Philadelphia, Wolters Kluwer/Lippincott Williams & Wilkins, 2008.
[5] Easton DF, Pooley KA, Dunning AM, et al. Genome-wide association study identifies novel breast cancer
susceptibility loci. Nature 2007; 447:1087-93.
[6] Falchetti M, Lupi R, Rizzolo P, et al. BRCA1/BRCA2 rearrangements and CHEK2 common mutations are
infrequent in Italian male breast cancer cases. Breast Cancer Res Treat 2008; 110:161-7.
[7] Ferla R, CalГІ V, Cascio S, et al. Founder mutations in BRCA1 and BRCA2 genes. Ann Oncol 2007; 18
Suppl 6:vi93-8.
[8] Guillem JG, Wood WC, Moley JF, et al. ASCO/SSO review of current role of risk-reducing surgery in common hereditary cancer syndromes. Ann Surg Oncol 2006; 13:1296-321.
[9] Hendriks YM, de Jong AE, Morreau H, et al. Diagnostic approach and management of Lynch syndrome
(hereditary nonpolyposis colorectal carcinoma): a guide for clinicians. CA Cancer J Clin 2006; 56:21325.
[10] Jorde LB, Carey JC, Bamshad MJ. Medical Genetics, 4th edition. Philadelphia, Mosby/Elsevier, 2009.
[11] La Paglia L, Lauge A, Weber J, et al. ATM germline mutations in women with familial breast cancer and
a relative with haematological malignancy. Breast Cancer Res Treat 2009.
[12] Lynch HT, Lynch JF, Lynch PM, et al. Hereditary colorectal cancer syndromes: molecular genetics, genetic counseling, diagnosis and management. Fam Cancer 2008; 7:27-39.
[13] Lynch HT, Shaw TG, Lynch JF. Inherited predisposition to cancer: a historical overview. Am J Med Genet
C Semin Med Genet 2004; 129C:5-22.
[14] Ottini L, Palli D, Rizzo S, et al. Male breast cancer. Crit Rev Oncol Hematol 2009.
[15] Russo A, Agnese V, Rizzo S, La Paglia L, Bazan V. The genetics of breast cancer: application in clinical practice. In: Giordano A, Normanno N (Eds.). “Breast Cancer in the Post-Genomic Era”. New York,
Humana Press, 2009, pp. 39-54.
[16] Russo A, CalГІ V, Agnese V, et al. BRCA1 genetic testing in 106 breast and ovarian cancer families from
Southern Italy (Sicily): a mutation analyses. Breast Cancer Res Treat 2007; 105:267-76.
[17] Russo A, CalГІ V, Augello C, et al. 4843delC of the BRCA1 gene is a possible founder mutation in Southern Italy (Sicily). Ann Oncol 2007; 18 Suppl 6:vi99-102.
[18] Russo A, CalГІ V, Bruno L, et al. Hereditary ovarian cancer. Crit Rev Oncol Hematol 2009; 69:28-44.
[19] Russo A, CalГІ V, Bruno L, et al. Is BRCA1-5083del19, identified in breast cancer patients of Sicilian
origin, a Calabrian founder mutation? Breast Cancer Res Treat 2009; 113:67-70.
[20] Russo A, Migliavacca M, Zanna I, Macaluso M, Gebbia N, Bazan V. Caretakers and Gatekeepers. In:
“Encyclopedia of the Human Genoma”. New York, Wiley, 2003.
[21] Russo A, Zanna I, Tubiolo C, et al. Hereditary common cancers: molecular and clinical genetics. Anticancer Res 2000; 20:4841-51.
[22] Schneider K. Counseling About Cancer: Strategies for Genetic Counseling, 2nd edition. New York, Wiley-
I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
Liss, 2002.
[23] Stormorken AT, Bowitz-Lothe IM, Noren T, et al. Immunohistochemistry identifies carriers of mismatch
repair gene defects causing hereditary nonpolyposis colorectal cancer. J Clin Oncol 2005; 23:470512.
[24] Veschi S, Aceto G, Scioletti AP, et al. High prevalence of BRCA1 deletions in BRCAPRO-positive patients
with high carrier probability. Ann Oncol 2007; 18 Suppl 6:vi86-92.
[25] Vogelstein B, Kinzler K. “The Genetic Basis of Human Cancer”, 2nd edition. New York, Mc-Graw-Hill,
[26] Walsh T, Casadei S, Coats KH, et al. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in
families at high risk of breast cancer. Jama 2006; 295:1379-88.
[27] Welcsh PL, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet
2001; 10:705-13.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Central giant cell of the jaw]
Silvia Tortorici1, Maria Lidia Buzzanca1, Francesco Burruano1, Aldo Gerbino2 e Maria Buscemi2
Dipartimento di Scienze Stomatologiche “G. Messina” Sezione di Chirurgia Speciale Odontostomatologica;
Dipartimento di Medicina Sperimentale Sezione di Istologia ed Embriologia “Arcangelo Pasqualino di Marineo”, Facoltà di Medicina e Chirurgia, Università degli Stuti di Palermo (IT)
Parole chiave: CGCG, Ossa mascellari, Osteoclasta; kB (RANK)
Key words: CGCG, Jaws, Osteoclast, kB (RANK)
Riassunto. Il granuloma centrale a cellule giganti (CGCG), ГЁ una lesione fibro-ossea. La maggiore frequenza
ГЁ osservata nella mandibola e principalmente in bambini ed in giovani adulti, piГ№ spesso in femmine che in
maschi. Questa lesione, la cui patogenesi rimane ancora oscura, si presenta come un’area di radiotrasparenza uni o multiloculare con trabecolature ossee.
Lo studio istologico mostra cellule giganti, cellule fibroblastiche, focolai emorragici e tessuto osteoide. In
base alle caratteristiche cliniche, radiologiche ed istologiche, il CGCG può essere classificato come “nonaggressivo” o “aggressivo”; l’analisi istomorfometrica delle forme aggressive, mostra un aumento di volume
delle cellule giganti e incremento dell’attività mitotica. Invece l’immunoistochimica (Ki-67 e p53) non mostra
differenze significative. Da un punto di vista clinico i pazienti affetti da forme aggressive riferiscono intenso
dolore e, all’esame obiettivo, si evidenziano: tumefazione, riassorbimento degli apici radicolari, perforazione
della corticale e/o ricorrenza della lesione dopo asportazione chirurgica. Le caratteristiche cliniche e istomorfometriche possono quindi essere considerate indicatori certi per la differenziazione fra CGCG aggressivo e
non aggressivo, per il trattamento terapeutico e il follow-up. L’asportazione chirurgica ed un adeguato followup rappresentano il trattamento più opportuno nei pazienti affetti da CGCG.
Abstract. Central giant cell granuloma (CGCG), is a fibro-osseous lesion. The majority of lesions are observed
in the mandible and mainly in children and young adults, more often in females than in males. This lesion,
which the pathogenesis still remains obscure, appears as radiolucency, well or ill-defined, uni or multilocular
with trabeculations coursing through the lesion. Histologic study shows giant cells, fibroblastic cells, and
foci of hemorrhage and osteoid tissue. On the basis of clinical, radiological and histologic features, central
giant cell granulomas can be classified as “non-aggressive” or “aggressive”; the histomorphometric analysis
prove a significant increase in large giant cells, fractional surface area, and mitotic activity in aggressive
CGCG lesions. Immunohistologic investigation (Ki-67 and p53 stain) reveal no significant differences. The
patients with aggressive lesions show aggressive growth, pain, massive swelling, root resorption, cortical
perforation, and/or recurrence. Clinical and histomorphometric features may be reliable indicators for the
differentiation between aggressive and nonaggressive CGCG. This should be accounted for to improve the
individual planning of the treatment and follow-up. Alternative treatments are worthy of consideration, although surgical excision remains the treatment of choice.
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi
In accordo col World Health Organization (W.H.O.) il granuloma centrale a cellule giganti
(CGCG) ГЁ una lesione endo-ossea benigna costituita da tessuto cellulare fibroso nel cui
contesto si trovano focolai multipli emorragici, cellule giganti multinucleate e trabecole di
osso spugnoso. Г€ una lesione ossea non frequente che ha una percentuale di presentazione di meno del 7% rispetto alle altre lesioni ossee benigne dei mascellari [1].
Fu Jaffe [2] che, nel 1953, descrisse questa patologia come entità nosologica a sé stante e la definì col termine di “granuloma riparativo a cellule giganti” (GRCG) per distinguerla
dai tumori a cellule giganti che insorgevano nelle epifisi delle ossa lunghe; l’autore sosteneva che i GRCG dei mascellari non erano delle neoplasie ma costituivano una reazione
riparativa locale conseguente a cause traumatiche di varia natura. Il granuloma centrale a
cellule giganti (CGCG) ГЁ considerato una lesione osteolitica di variabile dimensione e con
tendenza alla progressione la cui natura ed etiologia sono ancora controverse [3].
Il CGCG interessa quasi esclusivamente le ossa mascellari; raro ГЁ il suo riscontro nella restante parte dello scheletro cranio-facciale. In letteratura vengono riportati casi che
interessano la base del cranio, l’osso sfenoidale, l’osso temporale, i seni paranasali ed
etmoidali, l’osso occipitale, l’orbita; tali localizzazioni sono infrequenti e meritano una particolare attenzione viste le diverse modalità di approccio chirurgico che caratterizzano le
varie forme.
Nonostante la quasi esclusiva localizzazione alle ossa del massiccio facciale, il CGCG
puГІ interessare anche le ossa extragnatiche e, in particolar modo, le ossa brevi di mani e
piedi; rarissima ГЁ invece la presentazione nelle ossa lunghe.
Nell’ambito dei mascellari il CGCG è considerevolmente più frequente nella mandibola rispetto al mascellare superiore con una percentuale che raggiunge il 70% circa, soprattutto nell’area molare-premolare, estendendosi anche alla branca montante. Il condilo
mandibolare rappresenta una sede piuttosto rara. Al mascellare superiore sono descritti
casi in zona alveolare e al palato duro.
Il CGCG dei mascellari interessa un ampio range di etГ con una prevalenza nella seconda decade di vita; si presenta quindi in adolescenti e giovani adulti approssimativamente
nel 48% dei casi, mentre, nel 60% dei casi, le lesioni sono evidenziate prima dei 30 di etГ .
Rara ГЁ la manifestazione in etГ infantile.
Il sesso femminile ГЁ interessato nel 60% dei casi circa e predomina, con un rapporto di
quasi 3 ad 1, nel gruppo di etГ tra gli 11 ed i 30 anni [4].
Sono riportati in letteratura casi di lesioni giganto-cellulari in soggetti affetti da sindromi
quali Cherubismo, Sindrome di Noonan, Sindrome oculare ectodermica, sindrome descritta
per la prima volta nel 1993, in soggetti con fibromatosi di tipo-1 e con iperparatiroidismo
primario e feocromocitoma [5].
Il granuloma centrale a cellule giganti dei mascellari
La patogenesi e la natura del CGCG dei mascellari sono incerte. Secondo alcuni autori
[2] la lesione puГІ essere correlata ad un trauma, ad un processo riparativo o ad un difetto
di sviluppo; secondo altri ad una cisti aneurismatica o ad una cisti traumatica [6].
Le cellule multinucleate esibiscono delle caratteristiche fenotipiche degli osteoclasti
quali: positivitГ alla vitronectina (VNR) e alla fosfatasi acida tartrato-resistente (TRAP), risposte inibitorie alla calcitonina e capacitГ di determinare riassorbimento lacunare osseo.
Le cellule mononucleate rappresentano invece una eterogenea popolazione di macrofagi
e di cellule simili ai fibroblasti. Incerto ГЁ il ruolo che queste cellule fibroblastiche giocano
nell’indurre la formazione degli osteoclasti e il riassorbimento osseo nel CGCG.
Recente ГЁ la identificazione dei fattori cellulari ed umorali che inducono la formazione
degli osteoclasti; essa richiede un’interazione tra le cellule stromali che esprimono il recettore attivatore del ligando del fattore nucleare kB (RANKL), e il recettore attivatore del fattore nucleare kB (RANK) espressione dei fagociti mononucleati precursori degli osteoclasti,
trovati sia in circolo sia nei tessuti. Come simili fattori agiscano nell’indurre la formazione
di cellule giganti osteoclastiche è ancora sconosciuto [7,8]. Queste ricerche sono importanti non solo per la determinazione della patogenesi ma anche per la valutazione dell’uso
di strategie terapeutiche non chirurgiche, come l’uso di farmaci che inibiscono la formazione e la funzionalità degli osteoclasti, per esempio la calcitonina, e per il trattamento delle
lesioni osteolitiche ricorrenti.
Fig. 1 - Neoformazione a superficie irregolare, di consistenza duro-elastica, ricoperta da mucosa tesa che ne fa trasparire il colore rossastro; la massa ha dislocato posteriormente un elemento dentario.
Fig.2 - La sezione istologica mostra numerose cellule di grandi dimensioni a contorni netti ma irregolari, contenenti
molteplici nuclei. Tali elementi sono separati da stroma fusocellulare. Sono presenti stravasi emorragici interstiziali.
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi
Aspetti clinici
Г€ largamente diffuso il concetto che le lesioni a cellule giganti mostrano un comportamento clinico alquanto variabile; sebbene la maggior parte delle lesioni sia costituita
da processi circoscritti a lenta crescita e che rispondono bene al semplice courettage,
un certo numero di queste patologie sono caratterizzate da un atteggiamento aggressivo
caratterizzato da dolori, parestesie, riassorbimento degli apici radicolari, da distruzione
ossea, e tendenza alla recidiva dopo trattamento. L’incidenza delle recidive varia dall’11
al 49% [9].
Nella maggior parte dei casi, c’è una prima fase di latenza in cui la sintomatologia è
muta e sono esami radiografici occasionali che evidenziano, nel contesto del mascellare
colpito, un’area di rarefazione; a volte è l’elemento dentario che presenta mobilità e, una
volta estratto, l’alveolo rimane beante e si va riempiendo di un tessuto rossastro, di aspetto
cerebroide, molle elastico e indolente alla palpazione; il CGCG può condurre ad un’espansione della corticale in maniera proporzionale alla sua crescita determinando mobilità e
spostamento dei denti.
Nella fase di deformazione compare una tumefazione evidente, e la concomitante erosione della corticale ossea esteriorizza una massa neoformata, ricoperta da mucosa tesa,
ora normale ora iperemica, a consistenza duro-elastica; gli elementi dentari vengono spostati e si mobilizzano fino a cadere spontaneamente.
La compressione di un tronco nervoso o una reazione infiammatoria possono dar luogo
a sintomatologia piГ№ o meno intensa. La neoformazione puГІ raggiungere dimensioni notevoli. La sintomatologia assume, quindi, caratteristiche diverse secondo la localizzazione e
l’entità della neoformazione.
Poiché la manifestazione clinica di alcune di queste lesioni è una lieve reazione riparativa, oggi sono più comunemente accettati la designazione di “granuloma a cellule giganti”
o il più evasivo termine di “lesione giganto-cellulare”. È ancora oggetto di speculazione se
il granuloma a cellule giganti delle ossa mascellari sia un tumore; secondo alcuni autori
rappresenterebbe infatti la variante benigna del tumore a cellule giganti (osteoclastoma).
Tuttavia, l’accettazione del termine granuloma a cellule giganti, indica come non si tratti di
una neoplasia ma di un’iperplasia.
Sebbene il granuloma a cellule giganti possa qualche volta presentare un accrescimento rapido e sia localmente distruttivo, il suo decorso ГЁ comunque benigno rispetto ai tumori
a cellule giganti che hanno un comportamento locale più invasivo e tendono a metastatizzare nonostante l’aspetto istologico benigno.
I tumori a cellule giganti si localizzano frequentemente a livello delle ossa lunghe ed
alcune volte puГІ risultare molto difficile distinguerli dal granuloma a cellule giganti, sia dal
comportamento clinico sia dal quadro istologico. Diversamente dal granuloma a cellule
Il granuloma centrale a cellule giganti dei mascellari
giganti, i tumori a cellule giganti tendono a non avere predilezione per uno dei due sessi,
ed a colpire pazienti piГ№ anziani. Inducono di solito, dolore e presentano un rapido accrescimento determinando, nel 10% dei casi, una frattura patologica che rappresenta il primo
segno clinico. Anche i CGCG delle ossa mascellari con comportamento “aggressivo” oppure con un aspetto istologico simile ai tumori a cellule giganti, sono molto meno distruttivi
dei tumori a cellule giganti delle ossa lunghe (TGC) che hanno una percentuale di recidiva
del 60% [10].
All’ispezione il CGCG si presenta come una tumefazione rotondeggiante od ovoidale, a
superficie liscia ovvero irregolarmente mammellonata; il volume varia da quello di un cece
a quello di una grossa noce, raramente puГІ raggiungere diametri maggiori.
Alla palpazione la consistenza va dal molliccio al duro elastico.
Aspetti radiografici
Da un punto di vista radiografico il CGCG si presenta come un’area di radiotrasparenza
uniloculare o multiloculare. Non sempre i limiti con il tessuto osseo sono netti, a volte la
radiotrasparenza sfuma verso i tessuti circostanti e non si riesce a delimitarne i contorni.
Le forme localizzate al mascellare superiore si presentano sotto forma di molteplici aree
osteolitiche a margini irregolari; nella mandibola invece il quadro radiografico ГЁ piГ№ netto,
simil cistico.
L’aspetto radiografico varia con le dimensioni delle lesioni. Le lesioni piccole di solito si
presentano come aree di radiotrasparenza uniloculare e prive di setti ossei interni. Le lesioni
di grandi dimensioni invece appaiono come aree di radiotrasparenza multiloculare [11].
Kaffe et al. [12] osservarono che, su 80 casi esaminati, il 51% delle lesioni erano multiloculari, il 44% uniloculari, il 5% non erano loculari, e il 68% delle lesioni multiloculari
interessavano la mandibola. Essi evidenziarono anche che le lesioni uniloculari avevano
una dimensione media di 4.05 cm, mentre le lesioni multiloculari di 7.38 cm. Essi stabilirono, quindi, una correlazione statisticamente significativa tra le dimensioni delle lesioni e
la loro modalitГ di presentazione.
Whitaker e Waldron [13], analogamente, trovarono che il 61% delle lesioni diagnosticate come CGCG erano multiloculari mentre il 39% erano uniloculari.
Cohen e Hertzanu [14] stabilirono inoltre una correlazione tra la localizzazione delle
lesioni e la loro locularità . Essi evidenziarono, infatti, che l’80% delle lesioni multiloculari
erano mandibolari, il 75% delle lesioni uniloculari interessavano la mascella.
Osservarono che le dimensioni medie delle lesioni uniloculari erano di 23.75 mm e di
quelle multiloculari di 53.00 mm .
Vi era quindi una differenza statisticamente significativa tra le dimensioni delle lesioni
uniloculari e quelle multiloculari. Questi studi sono stati avvalorati recentemente da lavori
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi
effettuati da Gungormus M. et al. [15].
L’esame radiografico fornirebbe delle indicazioni anche sul grado di maturazione della
neoformazione: sembra che il CGCG più maturo abbia un’attività osteoformatrice evidenziata dalla presenza di trabecole ossee; il CGCG non ancora maturo mostra, invece, una
zona di osteocondensazione reattiva da parte del tessuto circostante e si presenta uniloculare [15].
Comunque l’immagine radiografica non è patognomonica.
Numerose sono, infatti, le patologie che mostrano aree di osteorarefazione quali:
• Cisti odontogena
• Cisti non odontogene
• Tumori odontogeni a matrice differenziata
• Forme localizzate del mieloma multiplo o plasmocitoma solitario
• Reticolosarcoma primitivo dell’osso o linfoma primitivo dell’osso
• Carcinoma metastatico a partenza da parotide, retto, prostata, mammella, polmone
• Condroma nella sua forma mista
• Osteoma osteoide
• Osteosarcoma
• Condrosarcoma
• Emangioma centrale dei mascellari
• Lesioni similtumorali: il tumore melanotico neuroectodermico dell’infanzia, il granuloma eosinofilo, la displasia fibrosa (forma monostotica), il sarcoma di Ewing
• Osteiti rarefacenti
• Malattia di Paget
• Malattia di Recklinghausen
Poiché il quadro radiologico non è patognomonico, fondamentale è il ruolo dell’indagine
citologica per agoaspirazione e dell’indagine istologica per un’esatta diagnosi e per la
programmazione di un appropriato approccio terapeutico.
Macroscopicamente il CGCG si presenta come una massa rotondeggiante o ovoidale, a
superficie liscia o mammellonata con aspetto cerebroide, di colore rosso o rosso-brunastro; la consistenza va dal molliccio al duro-elastico. La mancanza di una capsula fa sì che
tale neoformazione non sia ben delimitata rispetto all’osso circostante.
Il quadro microscopico classico (Fig. 1,2) mostra che il CGCG ГЁ costituito da tessuto
mesenchimale. Gli elementi cellulari sono essenzialmente rappresentati da fibroblasti e
macrofagi immersi in uno stroma costituito da scarse fibre connettive, vasi e cellule giganti; possono riscontrarsi anche leucociti, linfociti, plasmacellule ed eritrociti, espressione di
episodi flogistici sovrapposti.
Il granuloma centrale a cellule giganti dei mascellari
Le indagini ultrastrutturali al microscopio elettronico evidenziano che le cellule stromali
piГ№ rappresentative sono i fibroblasti, elementi allungati ed ovali; il loro citoplasma ГЁ abbondante con reticolo endoplasmatico rugoso, alcune cisterne dilatate, parecchie chiazze
di reticolo endoplasmatico liscio, numerosi poliribosomi liberi ed un esteso complesso di
Golgi. Sono presenti inoltre cellule fagocitiche mononucleari, con superficie irregolare per
la presenza di microvilli che possono dar luogo ad interdigitazione fra cellule contigue creando un quadro ritenuto tipico. Questi macrofagi contengono parecchie inclusioni anche a
tipo fosfolipidico. Il tessuto vascolare, sempre rappresentato, puГІ essere tanto abbondante
da conferire aspetto angiomatoso; sono presenti stravasi emorragici interstiziali depositi di
emosiderina e zone di necrosi.
Le cellule giganti sono gli elementi caratteristici della lesione; di forma rotondeggiante
od ovalare, sono caratterizzate da una membrana cellulare irregolare per la presenza di
microvilli ed invaginazioni. Le dimensioni sono varie e comunque di diametro compreso
fra 10 e 150 micron. Il citoplasma ГЁ basofilo alla periferia ed acidofilo al centro, il numero
dei nuclei ГЁ variabile e di solito non supera il numero di 20 essendo rari (6%) i casi con
un numero maggiore. Le cellule giganti si localizzano vicino ai vasi sanguigni, a volte si
dispongono a manicotto attorno ad essi o sporgono all’interno del lume vascolare; quando
una cellula gigante sporge di poco nel lume vasale, ГЁ sempre ricoperta da un sottile strato
di cellule endoteliali, quando sporge ampiamente tale continuitГ endoteliale ГЁ interrotta. Il
citoplasma contiene numerosi mitocondri ed un reticolo endoplasmatico rugoso discretamente rappresentato. L’apparato di Golgi presenta vescicole ectasiche. I nuclei mostrano
un’abbondanza in acidi nucleinici; ciò denuncia una notevole attività metabolica.
L’aspetto istologico del CGCG risulta molto simile a quello riscontrato nell’iperparatiroidismo. Le cellule giganti mostrano delle risposte molto simili agli osteoclasti per quanto
riguarda l’utilizzo della calcitonina e dell’immunoistochimica .
La natura e il significato delle cellule giganti sono stati indagati mediante indagini morfologiche ultrastrutturali, citometriche per stabilire l’attività nucleinica; indagini immunoistochimiche in cui si esamina l’espressione di p53, MDM2, Ki-67, PCNA; tali studi convergono verso la considerazione che queste cellule sono osteoclasto-simili, e che hanno come
precursori i monociti macrofagi [16].
Poiché l’evoluzione clinica di questa patologia è varia, alcuni autori hanno cercato di
evidenziare delle correlazioni fra i quadri microscopici e il comportamento clinico delle
Su 142 casi di CGCG dei mascellari Whitaker et al. [13] osservarono che la maggior
parte delle lesioni era asintomatica e relativamente innocua; tuttavia alcune mostravano
un decorso clinico piГ№ aggressivo caratterizzato da riassorbimento radicolare, dolore o
parestesia e perforazione corticale. In tutti i casi la percentuale di recidiva andava dall’11
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi
al 49%. Gli autori supposero che quei quadri istologici caratterizzati da una regolare distribuzione di cellule giganti e dalla presenza di figure mitotiche riconoscibili rappresentavano processi neoplastici a crescita autonoma, invece, lГ dove le cellule giganti erano
irregolarmente distribuite e dove erano presenti non poche figure mitotiche, si trattava di
un processo reattivo. La presenza di tessuto osteoide alla periferia di lesioni non recidivanti
suggeriva che queste erano state oggetto di un courettage piГ№ profondo o che un processo
osteogenico reattivo le aveva delimitate.
In realtГ non sono ancora ben chiari i rapporti tra CGCG e tumori gigantocellulari.
Diagnosi differenziale
Г€ opportuno menzionare altre lesioni delle ossa mascellari caratterizzate dalla presenza
di cellule multinucleate simil-osteoclastiche; attualmente si riserva l’appellativo di neoformazioni “a cellule giganti” solo a quei quadri patologici dove l’espressione istologica
fondamentale della lesione ГЁ costituita dalle suddette cellule. In tale gruppo troviamo:
• Il tumore bruno
• I tumori a cellule giganti
• Il granuloma a cellule giganti periferico
• La cisti ossea aneurismatica e la displasia ossea fibrosa
• Il cherubismo
Terapia - note conclusive
Il trattamento delle lesioni giganto-cellulari dei mascellari ГЁ sempre chirurgico.
Il courettage ГЁ la prima scelta terapeutica e di solito ГЁ risolutiva. Vista la mancanza di
capsula a volte residuano piccoli frammenti di tessuto che di solito non necessitano di
ulteriore terapia e qualche volta vanno incontro a risoluzione spontanea. Altre volte invece
queste formazioni tendono ad assumere un decorso locale piГ№ aggressivo se non sono
completamente rimosse; le recidive necessitano di ulteriori interventi di courettage.
Le lesioni ossee a cellule giganti localizzate al massiccio facciale comprendono una
serie di quadri molto diversi fra loro per etiologia e comportamento clinico.
La somiglianza istologica, clinica e radiologica di tali lesioni rende necessaria un’attenta valutazione prima di intraprendere un qualsiasi atto terapeutico.
La piГ№ frequente delle lesioni giganto cellulari dei mascellari ГЁ rappresentata dal CGCG
che, pur presentando invasività locale e rapido accrescimento ha un comportamento clinico benigno. Contrapposto troviamo il “tumore osseo a cellule giganti” che ha un comportamento clinico più aggressivo; in realtà dalla letteratura si evince come sia difficile una
diagnosi differenziale ben definita tra le due forme. Alcuni autori [3,4,13] hanno distinto
le lesioni giganto cellulari in forme non aggressive e forme aggressive in base all’aspetto
Il granuloma centrale a cellule giganti dei mascellari
clinico, biologico ed istologico. Le lesioni non aggressive richiedono solo un’asportazione
della neoformazione, insieme ad un accurato courettage della cavitГ , in quanto in questa
categoria è molto rara l’insorgenza di recidive.
Nelle lesioni aggressive il semplice courettage non ГЁ spesso sufficiente e la terapia piГ№
adeguata consiste invece nella resezione in blocco del segmento osseo interessato dalla
lesione, con immediata ricostruzione.
Alcune neoformazioni richiedono un approccio diverso; nonostante la scelta dell’intervento dipenda primariamente dall’aspetto clinico della lesione, occorre considerare la sede
di interessamento delle ossa mascellari e l’età del paziente.
In presenza di una forma aggressiva, se richiesta una resezione in blocco accompagnata alla perdita di strutture importanti, puГІ essere preferibile trattare tale lesione come
non aggressiva e praticare un semplice courettage accettando il rischio di una recidiva e
riservando ad un secondo tempo eventualmente la chirurgia resettiva.
Nel mascellare superiore, a prescindere dall’aggressività della lesione, il courettage
puГІ costituire un ottimo compromesso come trattamento poichГ© le caratteristiche anatomiche di questa struttura ne rendono maggiormente complessa, rispetto alla mandibola, la
ricostruzione dopo l’intervento di osteotomia [19].
Recenti studi propongono terapie alternative a quella chirurgica nel management del
CGCG. Tali terapie si basano sulle caratteristiche biologiche di alcuni tipi di lesioni giganto
cellulari. Sono state sperimentate iniezioni di calcitonina, iniezioni intralesionali di steroidi,
e iniezioni subcutanee di alfa-interferone in soggetti con lesioni multiple, recidivanti o
particolarmente aggressive [20-23]. In realtГ i meccanismi di azione non sono ancora definiti ed i risultati non sono uniformi anche perchГ© limitati a pochi casi, pertanto la terapia
chirurgica in atto rimane quella elettiva.
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino, Maria Buscemi
[1] Kramer IRH, Pindborg JJ, Shear M. Histological Typing of Odontogenic Tumors. In: WHO International
Histological Classification of Tumours, 2nd edn. Berlin, Germany, Springer-Verlag 1992.
[2] Jaffe HL. Giant cell reparative granuloma, traumatic bone cyst and fibrous (fibro-osseus) dysplasia of
the jaw bones. Oral Surg Oral Med Oral Pathol 1953; 6: 159-75.
[3] Sapp P, Eversole CR, Wysocki GP. Contemporary oral pathology. St Louis: CV Mosby Co; 1997.
[4] Waldrom CA, Shafer WG. The giant cell reparative granuloma of the jaws: an analysis of 38 cases. Am
J Clin Pathol 1966; 45: 437-47.
[5] Dahlin DC. Giant cell tumors of bone: highlights of 407 cases. AJR 1985; 144: 955-60.
[6] Chiba I, Teh BG, Iizuka T, Fukuda H. Conversion of a Traumatic Bone Cyst Into Central Giant Cell Granuloma: Implications for Pathogenesis - A case report. J Oral Maxillofac Surg 2002; 60: 222-225.
[7] Liu B, Yu SF, Li TJ. Multinucleated giant cells in various forms of giant cell containing lesions of the jaws
express features of osteoclasts. J Oral Pathol Med 2003; 32(6): 367-75.
[8] Itonaga I, Hussein I, Kudo O, Sabokbar A, Watt-Smith S, Ferguson D, Athanasou NA. Cellular mechanisms of osteoclast formation and lacunar resorption in giant cell granuloma of the jaw. J Oral Pathol
Oral Med 2003; 32: 224-31.
[9] Regezi JA, Sciubba JJ. Oral pathology: clinical pathologic correlations, 3rd edn. Philadelphia: WB Sauders 1993; 368-370.
[10] Andersen L, Fejerskov O, Philipsen HP. Oral giant cell granulomas. A clinical and histological study of
129 new cases. Acta Pathol Microbiol Scand [ A] 1973; 81(5): 606-16.
[11] Stavopoulos F, Katz J. Central giant cell granulomas: a systematic review of the radiographic characteristics with the addition of 20 new cases. Dentomaxillofac Radiol 2002; 31(4): 213-7.
[12] Kaffe I, Ardekian L, Taicher S, Littner MM, Buchner A. Radiologic features of central giant cell granuloma
of the jaws. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996; 81: 720-6.
[13] Whitaker SB, Waldron CA. Central giant cell lesion of the jaws: A clinical, radiological, and histopathologic study. Oral Surg Oral Med Oral Pathol 1993; 75(2): 199-208.
[14] Cohen MA, Hertzanu Y. Radiologic features, including those seen with computed tomography, of central
giant cell granuloma of the jaws. Oral Surg Oral Med Oral Pathol 1988; 65(2): 255-61.
[15] Gungormus M, Akgul M. Central Giant cell Granuloma of the Jaws: A Clinical and Radiologic Study. The
Journal of Contemporary Dental Practice 2003; 4(3), 1-8.
[16] Souza PE, Mesquita RA, Gomez RS. Evaluation of p53, PCNA, Ki-67, MDM2 and AgNOR in oral peripheral and central giant cell lesions. Oral Dis 2000; 6 (1): 35-9.
[17] Chiba I, Teh BG, Iizuka T, Fukuda H. Conversion of a Traumatic Bone Cyst Into Central Giant Cell Granuloma: Implications for Pathogenesis - A case report. J Oral Maxillofac Surg 2002; 60: 222-225.
[18] Sapp P, Eversole CR, Wysocki GP. Contemporary oral pathology. St Louis: CV Mosby Co; 1997.
[19] Cannistra C, Fadda T, Guerrieri L, Vero S, Della Rocca C, Iannetti G. Central giant cell granuloma of the palate:
unusual localization in a five year old child. Bull Group Int Rech Sci Stomatol Odontol 1999; 41 (4): 112-4.
[20] Kaban LB, Mulliken JB, Ezekowitz A. Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alpha-2a. Pediatrics 1999; 103: 1145-419.
[21] O’Regan EM, Gibb DH, Odell EW. Rapid growth of giant cell granuloma in pregnancy treated with calcitonin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 92(5): 532-8.
[22] Pogrel MA. Calcitonin therapy for central giant cell granuloma. J Oral Maxillofac Surg 2003; 61(6): 649-53.
[23] Khafif A, Krempl G, Medina JE. Treatment of giant cell granuloma of the maxilla with intralesional injection of steroids. Head Neck 2000; 22(8): 822-5.
Physical activity
Clinical applications
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Spettroscopia di risonanza magnetica: principi ed applicazioni cliniche]
Giulia Bivona1, Maria Buscemi2, Aldo Gerbino2 and Marcello Ciaccio1
Chair of Clinical Biochemistry, 2Chair of Histology and Embryology (Di.Me.S.), School of Medicine, University
of Palermo (IT)
Key words: Magnetic Resonance Spectroscopy, Neurodegeneration, Mitochondrial Diseases
Parole chiave: Spettroscopia di Risonanza Magnetica, Neurodegenerazione, Malattie Mitocondriali
Abstract. While Magnetic Resonance Imaging (MRI) provides anatomic images and morphometric characterization of disease, magnetic resonance spectroscopy (MRS) provides metabolite/biochemical information
about tissues non-invasively in vivo. MRS has been used clinically for more than two decades in the investigation of neurological dysfunction and in the evaluation of the prostate gland (1H MRS) and muscle tissue
(31P MRS).
in vivo proton MRS allows the presence of certain metabolites in brain tissue which present clinical importance, including: N-acetyl aspartate (Naa), Creatine (Cr), Choline (Cho) and Myoinositol (mI). Other metabolites that can be detected via proton MRS include lactate and amino acids. This technique is useful whenever
biochemical or metabolic assessment may be necessary, such as in differential diagnosis of focal brain
lesions (neoplastic and non-neoplastic diseases); diagnosis of neurodegenerative diseases; assessment of
epilepsy; biochemical alterations in hepatic encephalopathies and inborn errors of metabolism and hypoxic
P MRS can be used to investigate energy metabolism in several tissues, but muscle is in many ways an
ideal tissue for MRS studies; 31P MRS spectra metabolites from muscle contain the following peaks: О±, ОІ
and Оі ATP, phosphocreatine (PCr) inorganic phosphates (Pi); phosphomonoesters (PME) and phosphodiesters
(PDE). The technique provides information about intracellular pH and intracellular magnesium concentration.
P MRS can be used for diagnostic purposes in metabolic myopathies and mitochondrial diseases, in which
such an information can be obtained to correctly identify the pathology.
Riassunto. La Spettroscopia di Risonanza Magnetica del Protone, differentemente rispetto all’Imaging Morfologico Standard, consente di ottenere informazioni di natura biochimica e metabolica sul distretto indagato,
che puГІ essere rappresentato dal Sistema Nervoso Centrale, dal muscolo scheletrico o dalla ghiandola
Mediante Spettroscopia del protone è possibile valutare i seguenti metaboliti indicativi dello stato metabolico- funzionale del Sistema Nervoso Centrale: N- acetil aspartato, Creatina, Colina e Mioinositolo; altri metaboliti rilevabili includono il Lattato ed alcuni aminoacidi. L’indagine biochimica può essere molto utile ai fini
della diagnosi differenziale fra patologie neurologiche di natura neoplastica e non neoplastica, nella diagnosi
delle patologie neurodegenerative e nelle epilessie, nelle encefalopatie epatica ed ipossico- ischemica. La
spettroscopia del fosforo è utile nell’indagine metabolico-funzionale di molti tessuti fra i quali in particolare
emerge in termini di applicazioni cliniche quello muscolo-scheletrico. I metaboliti rintracciabili sono i seg-
Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio
uenti: О±, ОІ e Оі ATP, fosfocreatina (PCr) fosfato inorganico (Pi); fosfomonoesteri (PME) e fosfodiesteri (PDE).
È possibile inoltre, contestualmente all’indagine, ottenere delle informazioni riguardanti le concentrazioni
intracellulari di pH e di Magnesio. Le applicazioni principali in biomedicina della tecnica includono la diagnosi
ed il monitoraggio delle malattie mitocondriali e delle miopatie metaboliche.
The first in vivo magnetic resonance spectroscopy was performed at the beginning
of the 1980s and was done using resonance of the phosphorus nucleus (31P), thereby
revealing the energy metabolism of the tissue cells studied. However, this method had
the disadvantages of low magnetic sensitivity, low concentration of 31P atoms and also
inadequate spatial resolution in focal brain lesions with small and mild dimensions, and it
has been replaced by proton (hydrogen) spectroscopy, especially in brain studies. Proton
(1H) resonance is nowadays the method most frequently used in neurospectroscopy, because hydrogen is the most abundant atom in the human body and its nucleus emits the
most intense radiofrequency signal, when in an external magnetic field, in relation to other
nuclei. Moreover, proton magnetic resonance spectroscopy is more quickly accomplished
and easily interpreted [1].
While Magnetic Resonance Imaging (MRI) provides anatomic images and morphometric characterization of disease, magnetic resonance spectroscopy (MRS) provides metabolite/biochemical information about tissues non-invasively in vivo. MRS has been used
clinically for more than two decades in the investigation of neurological dysfunction and in
the evaluation of the prostate gland (1H MRS, figures 1-2-3) and muscle tissue (31P MRS,
figures 4-5). The main difference between standard MRI and MRS is that the frequency of
the MR signal is used to encode different types of information: while MRI uses high spatial
resolution to generate anatomical images, MRS, based on the principle of chemical shift,
provides chemical information about the tissues presented as line spectra. This technique
has provided a useful bridge between metabolism and the anatomic and physiological
studies available from MRI, and represents a versatile diagnostic tool in biomedicine. Biochemical informations in vivo are provided by MRS selecting volumes of interest (voxel or
VOI) on the standard imaging sequence and the peaks on the spectra obtained correspond
with various metabolites which are typical for every tissue examined and may be identified
There are two methods of magnetic resonance spectroscopy: single voxel and multivoxel, with or without spectroscopic imaging. Single voxel proton magnetic resonance
spectroscopy provides a rapid biochemical profile of a localized volume within a region of
interest that may be determined, especially in brain studies. Spectroscopic imaging provides biochemical information about multiple, small and contiguous volumes focalized on a
particular region of interest that may allow the mapping of metabolic tissue distribution. By
Magnetic Resonance Spectroscopy: Principles and Clinical Applications
using this method, the data obtained may be manipulated by computer and superimposed
on the image of an abnormality, thereby illustrating the distribution of such metabolites
within that area.
In vivo proton MRS allows the presence of certain metabolites in brain tissue to be
detected if the minimum concentrations are between 0.5 and 1.0 mM. Some of these
present clinical importance, such as: N-acetyl aspartate (Naa), which is a neuronal marker that is present in neuron bodies and axons and indicates their density and viability;
the Naa peak in proton spectroscopy will be decreased whenever there is neuronal loss,
such as in glioma, ischemia and degenerative diseases; Creatine (Cr) is a marker of the
aerobic energy metabolism, its peak is practically constant and may be used as a control
value in relation to other metabolites. Choline (Cho), which is a constituent molecule of the
phospholipid metabolism of cell membranes and reflects membrane turnover. Increased
choline indicates greater membrane synthesis and cell proliferation. Its concentration is
normally greatly increased in cases of brain neoplastic expansible processes. Lactate is
not commonly detected in proton spectroscopy of brain tissue: its presence indicates a
pathological condition with regard to the final products of anaerobic metabolism. Lactate
can be identified in cysts, hypoxic/ischemic tissues and some neoplasms. Lipids are usually not detected by proton spectroscopy, either. In pathological situations in which necrosis occurs, such as in malignant neoplasms and inflammatory/ infectious processes, there
is an accentuated lipid peak, signifying cell membrane degradation; Myoinositol, which is
considered to be a glial function marker, generally presents reduction in hepatic encephalopathy and elevation in Alzheimer’s disease. Other metabolites that can be detected via
proton MRS include the following amino acids: alanine, acetates and succinates [2-3].
Every metabolite has a normal concentration that generates a pattern of peaks that is the
same from person to person unless there is an underlying pathology. Diagnosis with MRS
can therefore be made by either comparing the numeric values of metabolite concentrations or by recognizing abnormal patterns of peaks in the spectra (Figg. 2-3).
Proton magnetic resonance spectroscopy of the brain is useful whenever biochemical
or metabolic assessment may be necessary, such as in differential diagnosis of focal brain
lesions (neoplastic and non-neoplastic diseases); diagnosis of dementia and other degenerative diseases; follow-up radiation therapy for patients with brain neoplasms; demyelinating diseases such as multiple sclerosis and leukodystrophy; diagnosis and prognosis
of brain ischemic and traumatic lesions; assessment of epilepsy; biochemical alterations
in hepatic encephalopathies; and neuropediatric affections such as brain tumors, inborn
errors of metabolism and hypoxic encephalopathy [2-4].
P MRS can be used to investigate energy metabolism in several tissues, but muscle
is in many ways an ideal tissue for MRS studies because it can be stressed in the magnet
Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio
with exercise while the biochemical response is monitored. 31P MRS records signals from
high energy phosphate compounds which are central to energy metabolism in vivo. Phosphorus MR spectra from muscle contain seven resonances: three arise from the phosphate
groups of adenosine triphosphate (О±, ОІ and Оі ATP), one from phosphocreatine (PCr) and
one from inorganic phosphates (Pi); two additional smaller resonances can be observed
from phosphomonoesters (PME) and phosphodiesters (PDE). The spectral distance between Pi and PCr provides information about intracellular pH while the chemical shift of ATP
can be used to calculate intracellular magnesium concentration. The concentration of the
metabolically active adenosine diphosphate (ADP), which is important in the regulation of
rates of mitochondrial ATP synthesis, does not produce a signal visible in the spectra, but
can be calculated indirectly using the creatine kinase (CK) equilibrium equation. One of
the most important features of 31P MRS is its ability to monitor time-dependent changes of
metabolites non invasively. In normal exercising muscle, one can observe in the MR spectra
a PCr decrease stoechiometrically linked to Pi accumulation, while the ATP signal remains
unchanged at least when exercise intensity is light or moderate. During recovery, [PCr]
gradually increases, [Pi] and [ADP] decrease, and pH returns to its rest level (Figg. 4-5) .
P MRS can be used for diagnostic purposes in metabolic myopathies and mitochondrial diseases, in which such an information can be obtained to correctly identify the pathology. Particularly regarding on mitochondrial diseases, the clinical spectrum of these
disorders is large, due in part to their genetic and biochemical heterogeneity [5]. A majority
of these diseases include neurological symptoms besides muscle symptoms and can be
suspected when an increase in serum of lactate and pyruvate is found, even at rest. The
proportion of mutant versus wild-type mitochondrial DNA determines the biochemical phenotype; however, the link between the biochemical phenotype and the clinical phenotype
is not always clear making the diagnosis difficult. Defects of respiratory chain enzymes
are the most prevalent types of mitochondrial diseases and their common endpoint is
a deficit in ATP production leading to a decreased exercise tolerance and a shared pattern of detectable abnormalities. Among syndromic mitochondrial diseases at least two
encephalomyopathies are greatly known and studied: Leber Hereditary Optic Neuropathy (LHON) and Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like
episodes (MELAS); LHON is characterized by visual loss resulting from retinal ganglion
cell degeneration; despite the important role of respiratory chain deficiency and oxidative
stress induced by mtDNA point mutations affecting complex I, excitotoxic injury has been
postulated as a concurrent pathogenic factor. MELAS syndrome is the most common syndromic manifestation of mitochondrial encephalomyopathies that occur in all age groups.
The clinical course of MELAS is slowly progressive with stepwise deterioration; the severity
of lactic acidosis correlates with the severity of neurologic deficit.
Magnetic Resonance Spectroscopy: Principles and Clinical Applications
Fig 1. 1HMRS multivoxel spectra of prostate gland.
Fig 2. 1HMRS voxel localization and correspondent spectrum.
Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio
Fig 3. Differences between 1HMRS spectra peaks and correspondent metabolite concentrations.
Fig 4. 31 PMRS spectrum obtained during rest.
Fig 5. 31 PMRS spectrum obtained during exercise and recovery after exercise.
Magnetic Resonance Spectroscopy: Principles and Clinical Applications
Deficits in carbohydrate metabolism can present either with exercise-induced symptoms
such as painful cramps, contractures, and rhabdomyolysis or with progressive weakness.
Muscles with impaired glycolysis or glycogenolysis lack the ability to produce lactic acid
during exercise, deficiency of glicogenolytic and glycolytic enzymes leads to impaired anaerobic synthesis of ATP in association with reduced production of lactate; the insufficient
production of reducing equivalents (nicotinamide adenine dinucleotide, NADH) and pyruvate during aerobic exercise can also result in impaired mitochondrial ATP production. This
represents the biochemical basis for a distinctive muscle 31PMRS pattern in glycogenolytic
enzyme defects, characterized by a rapid depletion of PCr, appearance of cytosolic alkalosis or reduced acidification during muscle exercise and slow rate of PCr resynthesis
during recovery after exercise. One of the most studied myopathy in this group is the
myophosphorylase deficiency, known as McArdle disease, in which 31P MRS findings is PCr
rapid depletion and paradoxical increase in muscle pH, due to proton consumption by PCr
hydrolysis during exercise [6].
Giulia Bivona, Aldo Gerbino, Maria Buscemi, Marcello Ciaccio
[1] Van der Knaap MS, Van der Grond J, Van Rijen PC, Faber JA, Valk J, Willemse K. Age-dependent changes in localized proton and phosphorus MR spectroscopy of the brain. Radiology 1990; 176: 509-15.
[2] Lin A, Ross BD, Harris K, Wong W. Efficacy of Proton Magnetic Resonance Spectroscopy in Neurological
Diagnosis and Neurotherapeutic Decision Making. NeuroRx 2005; 2: 197-214.
[3] Kantarci K. 1H Magnetic Resonance Spectroscopy in Dementia. Br J Radiol, 2007; 80(2): S146-52.
[4] Soares DP, Law M. Magnetic resonance spectroscopy of the brain: review of metabolites and clinical
applications. Clinical Radiology 2009; 64: 12-21.
[5] Kemp GJ, Meyerspeer M, Moser E. Absolute quantification of phosphorus metabolite concentrations in
human muscle in vivo by 31P MRS: a quantitative review. NMR Biomed 2007; 20(6): 555-65.
[6] Prompers JJ, Jeneson JA, Drost MR, Oomens CC, Strijkers GJ, Nicolay K. Dynamic MRS and MRI of
skeletal muscle function and biomechanics. NMR Biomed 2006; 19(7): 927-53.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Immunoistochimica e analisi d’immagine multi spettrale
[Immunohistochemistry and multispectral image analisys]
Giovanni Francesco Spatola
Di.Me.S. Sezione di Istologia ed Embriologia – Facoltà di Medicina - Università degli Studi di Palermo (IT)
Parole chiave: Analisi d’immagine, Immagini multispettrali, Immunoistochimica
Key words: Image analisys, Multispectral, Immunohistochemistry
Sommario. L’uso di metodiche d’analisi d’immagine ha permesso di rendere più affidabili e facilmente riproducibili i risultati ottenuti mediante tecniche di immunoistochimica (IHC). La maggiore diffusione e la semplificazione dei software che permettono uno studio colorimetrico ha fatto sì che tali metodiche siano alla portata di
tutti e ha permesso di standardizzare la tecnica mediante dei sistemi di “score” affidabili. Inoltre, l’introduzione
negli ultimi anni di metodiche di acquisizione d’immagine mediante sistemi multispettrali ha affinato ulteriormente tali tecniche permettendo di evitare artefatti e svincolare la valutazione dell’operatore dai dati oggettivi.
Abstract. The use of methods of image analysis has allowed us to make more reliable and reproducible
results obtained by immunohistochemistry (IHC). Wider use and simplification of software allowing a colorimetric study has meant that these methods are available to everyone and made it possible to standardize
the technique by reliable systems score. Moreover, the introduction in recent years of multispectral image
acquisition systems methods has further refined these techniques allowing to avoid artifacts and release the
evaluation of the data by the observer.
La tecnica immunistochimica viene impiegata sia nel campo della ricerca per evidenziare antigeni specifici sia dagli anatomo patologi per evidenziare marker tipici di svariate
patologie. Sin dalla sua messa a punto essa ha sofferto di alcune gravi limitazioni intrinseche all’esecuzione della tecnica stessa. Se i passaggi che precedono la valutazione dei
risultati possono comunque essere standardizzati [1,2], molto piГ№ difficile ГЁ ottenere una
ripetitivitГ e una corretta valutazione dei dati ottenuti. Infatti la lettura di un preparato IHC
dipende strettamente dall’operatore e dalla sua esperienza. Vi sono molteplici fattori che
influenzano l’osservazione a partire dal tipo di microscopio, alla temperatura di colore della
luce utilizzata, al tipo di colorante usato per la rivelazione, alla capacità visiva dell’operatore stesso ed ultimo, ma non ultimo per importanza, dalla non linearità ottica delle reazioni.
Difatti se per basse concentrazioni di antigene si puГІ affermare che ad un aumento della
concentrazione aumenta anche l’intensità della colorazione questo dato si perde a maggiori concentrazioni di antigene. Pertanto la nostra percezione ottica non permette una
valutazione corretta della effettiva reattivitГ .
Giovanni Francesco Spatola
Al fine di superare questi limiti sopra esposti negli ultimi anni si ГЁ cercato di standardizzare al massimo la tecnica IHC sia nelle fasi di preparazione, che nella vera e propria
reazione e nella successiva lettura dei dati [1,2]. A tal fine un importante aiuto ГЁ stato
fornito dai sistemi di imaging digitale che da circa 15 anni vengono affiancati da software
sempre più perfezionati di analisi d’immagine. L’analisi d’immagine è una metodica informatica che ci permette mediante software più o meno dedicati di avere una valutazione
sia del numero di cellule positive sia dell’intensità della reazione intesa come parametri
colorimetrici derivanti da un immagine digitale.
Analisi d’immagine
I maggiori problemi legati alla valutazione delle reazioni immunoistochimiche dipendono, come precedentemente sottolineato, da diversi parametri prevalentemente soggettivi.
Quello che per un operatore ГЁ una positivitГ del test per un altro puГІ essere invece assolutamente negativo e ciГІ dipende dalla percezione personale dei colori, dalle condizioni di
osservazione, dalla propria capacitГ visiva etc.
Bisogna, inoltre, tenere conto del fatto che le reazioni IHC non hanno una resa totalmente lineare dal punto di vista cromogenico. Esse difatti risultano effettivamente lineari a
bassa concentrazione dell’anticorpo nella cellula ma se la sostanza ricercata risulta essere
presente in maggiori concentrazioni la risposta cromogenica risulta essere non lineare
ossia dopo una certa soglia (per altro molto bassa) non risulta piГ№ possibile discriminare
in maniera attendibile la quantità di sostanza presente all’interno della cellula. Diverso è il
caso della immunofluorescenza in quanto essa tende a dare una risposta quasi del tutto
lineare ossia a maggiore concentrazione della sostanza corrisponde un effettivo aumento
della fluorescenza. Per tale motivo oggi si tende ad utilizzare sempre piГ№ questa ultima
A partire dai primi anni novanta l’uso di immagini digitali in microscopia si è sempre
piГ№ diffuso e ha permesso di ottenere un sempre crescente numero di informazioni dai
campioni studiati. Chiaramente in sistemi pioneristici come i primi metodi basati sull’acquisizione di immagini video e non fotografiche la bassa risoluzione dei frames acquisiti
non permetteva di potere effettuare valutazioni colorimetriche accurate. Nonostante ciГІ
l’introduzione di alcuni software come il Lucia M della Nikon sono da considerare come
una vera rivoluzione nel campo della valutazione IHC in quanto hanno permesso di semplificare sia il conteggio di cellule positive sia la valutazione dei risultati introducendo il
concetto di quantificazione colorimetrica. Tale tipo di valutazione permetteva di avere, sia
pure in maniera approssimativa, una idea della quantità di sostanza presente all’interno
di vari citotipi. A causa della bassa risoluzione si era convenuto che bisognava utilizzare
immagini di tipo binario (bianco e nero a 2 bit) oppure immagini in scala di grigio che
Immunoistochimica e analisi d’immagine multi spettrale
Fig.1 Profili CMYK per i piГ№ comuni cromogeni
risultano essere piГ№ ricche di informazioni in quanto a 8 bit o 256 toni di grigio. Г€ importante considerare che il nostro occhio riesce a distinguere circa 70 toni di grigio quindi
un uso di immagini a 256 toni necessita di un software che effettui una valutazione corretta dell’intensità di reazione. L’uso del b&w evitava le differenze legate ai diversi tipi di
cromogeni usati e permetteva una maggiore standardizzazione delle tecniche di analisi
d’immagine [3,4].
Per ovviare alla predetta non linearità delle reazioni IHC e alla impossibilità di valutazione oggettiva del singolo operatore (frasi del tipo “diffusamente reattivo”, oppure “molto
presente” o ancora “scarsamente immunoreattivo” risultano essere assolutamente inutili
e imprecise) e al fine di individuare correttamente la presenza e la distribuzione di una
sostanza risulta sempre più di fondamentale importanza l’uso di un sistema di analisi
d’immagine che provveda a quantificare mediante uno score la reattività permettendoci di
rispondere alle domande base che sono:
• La reazione è positiva in rapporto ai controlli ?
• Se è positiva che intensità presenta e questa è correlata alla quantità di antigene ?
• Dove è localizzata la reattività ?
L’evoluzione dei sistemi di acquisizione di immagine ha modificato alcuni parametri
per cui se fino a qualche anno fa risultava indispensabile utilizzare le immagini binarie o
al massimo ad 8 bit, come prima chiarito, oggi la maggiore risoluzione permette anche un
uso di immagini a colori.
Le immagini digitali a colori si basano su alcuni parametri fondamentali ed in particolare su degli istogrammi legati alla combinazione dei colori di base siano essi RGB (rosso,
giallo, blu) oppure CMYK (ciano, magenta, giallo, e nero). I profili di questi istogrammi
variano in maniera sensibile in base al colore che andiamo a prendere in considerazione.
In particolare si ГЁ visto [5] che il profilo CMYK ГЁ quello che maggiormente ci permette una
valutazione progressiva ed in particolare il canale del giallo è indipendente dall’osservatore
e applicabile a diversi tipi di cromogeno in quanto sensibile a minime variazioni di intensitГ 291
Giovanni Francesco Spatola
IHC con una scala lineare che permette di ottenere un corretto score riproducibile con
facilitГ . Teniamo conto che ogni canale viene suddiviso in 250 toni quindi basta associare
secondo una progressione lineare a un determinato valore un punteggio variabile da 1 a
7, dove 1 corrisponde all’assenza di colore o 0 e 7 al massimo valore ottenibile in base al
cromogeno, ossia 250 (Fig.1).
Parallelamente all’evoluzione dei sistemi di acquisizione di immagini la semplificazione
dei software di analisi ha permesso una diffusione della metodica che risulta oggi alla
portata di chiunque abbia un minimo di esperienza nell’uso di comuni programmi di fotoritocco come ad esempio Photoshop [6,7] che nelle sue ultime versioni implementa anche
un menù di analisi d’immagine dove con pochi semplici comandi è possibile effettuare una
serie di rilevazioni che vanno dal conteggio di cellule positive fino alla quantificazione di
valori colorimetrici a cui assegnare uno score [4,8,9].
Analisi d’immagine multi spettrale
Recentemente alla valutazione basata su uno score derivante dal profilo CMYK si ГЁ
affiancata la metodica di acquisizione di immagini multi spettrali. Tale metodica, utilizzata inizialmente per migliorare la qualitГ di immagini acquisite dai satelliti e applicata in
diversi campi di studio, dalla valutazione geologica del suolo sino a studi di archeologia
dove le immagini multispettrali permettono di valutare la presenza di insediamenti nel
sottosuolo, si basa sulla possibilitГ mediante particolari espedienti tecnici di superare i
limiti di visione dell’occhio umano che ha una percezione del colore dipendente dalla luce
e dalle condizioni di osservazione. In fisica uno stimolo colore ГЁ definito univocamente
dall’”intensità ” che esso assume nello spettro di luce visibile. Pertanto ad ogni colore si
deve associare una “curva spettrale” costruita in base all’energia irradiata dalle singole
lunghezze d’onda [10]. Per evidenziare tali curve è necessario acquisire delle immagini
che tengano conto di quella che in fisica viene definita come “riflettanza” ossia la proporzione di luce incidente che una data superficie è in grado di riflettere. Un sistema di
imaging multi spettrale pertanto permette di determinare la riflettanza nello spettro del
visibile e di ricostruire fedelmente i colori della stessa immagine. I sistemi multi spettrali
utilizzano particolari filtri ottici sensibili alle diverse lunghezze d’onda nello spettro del
visibile e successivamente dei sistemi software capaci di ricostruire le curve di riflettanza
in ogni punto della scena restituendoci una immagine che puГІ essere studiata nelle sue
varie componenti colorimetriche.
In pratica i sistemi di analisi multi spettrali sono composti da un sistema di filtri che ci
permettono, guardando attraverso uno di essi settato per una certa lunghezza d’onda nel
range visibile, di distinguere il singolo colore (Fig.2). Un “cubo” di dati spettrali è ottenuto
per selezione di una lunghezza d’onda dopo l’altra, acquisendo un’immagine per ciascuna
Immunoistochimica e analisi d’immagine multi spettrale
Fig. 2 Curve spettrali e filtri
di essa e ricostruendola mediante appositi software. La possibilitГ di acquisire immagini a
diverse lunghezze d’onda fa si che si possa anche sottrarre alcune di esse dall’immagine
finale e quindi mettere in evidenza soltanto quelle lunghezze d’onda specifiche per un
determinato cromogeno o fluorocromo. Le applicazioni risultano essere quindi molteplici. In particolare nel caso di immagini in fluorescenza la possibilitГ di separare le varie
lunghezze d’onda permette di eliminare tutta o in massima parte l’autofluorescenza dei
tessuti con indubbio vantaggio per la valutazione di risultati legati ad una determinata
reattività specifica. Nell’immunoistochimica classica l’uso di tale metodica permette di
isolare ed evidenziare, mediante una elaborazione definita pseudo fluorescenza (immagini
trasformate in negativo e ben contrastate), la presenza di un determinato cromogeno, la
sua localizzazione e successivamente anche la sua intensitГ (Fig.3-4-5). Inoltre ГЁ possibile
separare ed isolare diversi cromogeni utilizzati per valutare una colocalizzazione di antigeni diversi all’interno della stessa cellula o dello stesso tessuto [11,12,13]. Successivamente alla fase di acquisizione applicando una separazione selettiva dei vari spettri mediante
una eliminazione progressiva di quelli che non corrispondono ai cromogeni in esame ГЁ
possibile anche qui trasformare l’immagine secondo il profilo CMYK e valutare il canale del
giallo ottenendo come giГ precedentemente esposto una curva colorimetrica lineare a cui
applicare uno score per la valutazione dell’intensità della reazione e quindi indirettamente
quantificare la presenza del nostro antigene.
Giovanni Francesco Spatola
La metodica IHC ancora oggi risulta essere utile a visualizzare la presenza all’interno
di cellule di determinati antigeni. Associare le metodiche di analisi d’immagine e di analisi
multi spettrale permette inoltre di ottenere risultati riproducibili e separati dalle limitazioni
oggettive determinate dai vari osservatori. La possibilitГ inoltre di quantificare mediante
uno score la quantitГ di antigene presente nel nostro campo microscopico ci permette in
associazione a metodiche di biologia molecolare di avere una visione più completa e sicuramente più corrispondente alla realtà rispetto all’utilizzo delle singole metodiche.
Immunoistochimica e analisi d’immagine multi spettrale
Fig.3 Acquisizione multi spettrale
- positivitГ leptinergica (cromogeno AEC) in alcuni epiteliociti
duttali – immagine multispettrale
- Ob. 10x
Fig.4 Acquisizione multi spettrale - positivitГ MMP9 (cromogeno
AEC) in endoteliociti di aneurisma
aortico dilatativo umano – immagine multispettrale - Ob. 40x
Fig.5 Confronto tra microscopia classica (A) e immagine multi spettrale (B) - positivitГ MMP9 (cromogeno
AEC) in cellule di polpa dentale – immagine multispettrale - Ob. 40x
Giovanni Francesco Spatola
[1] Walker RA. Quantification of immunohistochemistry issues concerning methods, utility and semiquantitative assessment. I; Histopathology 2006; 49: 406-410.
[2] Taylor CR, Levenson RM, Quantification of immunohistochemistry issues concerning methods, utility
and semiquantitative assessment. II; Histopathology 2006; 49: 411-424.
[3] Oberholzer M, Ostreicher M, Christen H, BrГјhlmann M. Methods in quantitative image analysis. Histochem Cell Biol 1996; May 105(5):333-55.
[4] Kaczmarek E et al, Techniques of image analysis for quantitative immunohistochemistry. Annales Academiae Medicae Bialostocensis Vol. 49, 2004; Suppl. 1, Proceedings.
[5] Nhu-An Pham et al, Quantitative image analysis of immunohistochemical stains using a CMYK color
model. Diagnostic Pathology 2007; 2:8.
[6] Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Application of photoshop-based image
analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem
1997; 45(11): 1559-65.
[7] Kirkeby S, Thomsen CE. Quantitative immunohistochemistry of fluorescence labelled probes using lowcost software. J Immunol Methods 2005; Jun;301(1-2):102-13.
[8] Skaland I, Ovestad I, Janssen EA, Klos J, Kjellevold KH, Helliesen T, Baak JP. Digital image analysis
improves the quality of subjective HER-2 expression scoring in breast cancer. Appl Immunohistochem
Mol Morphol 2008; Mar;16(2):185-90.
[9] Skaland I, Г�vestad I, Janssen EA, Klos J, Kjellevold KH, Helliesen T, Baak JP. Comparing subjective and
digital image analysis HER2/neu expression scores with conventional and modified FISH scores in
breast cancer. J Clin Pathol 2008; Jan;61(1):68-71. Epub 2007 Apr 5.
[10] G. Novati et al, Acquisizione di immagini multispettrali - 2003.
[11] Gavrilovic M, Wählby C. Quantification of colocalization and cross-talk based on spectral angles. J
Microsc 2009; Jun; 234(3):311-24.
[12] Jaskolski F, Mulle C, Manzoni OJ. An automated method to quantify and visualize colocalized fluorescent signals. J Neurosci Methods 2005; Jul 15;146(1):42-9.
[13] Landmann L, Marbet P. Colocalization analysis yields superior results after image restoration. Microsc
Res Tech 2004; Jun 1;64(2):103-12.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Effetti dell’allenamento specifico sui livelli ormonali nelle atlete]
Arcangelo Benigno, Attilio Licciardi and Giuseppe Morici
Department of Experimental Medicine (Di.Me.S), University of Palermo (IT)
Key words: Exercise, Menstrual disorders, Soccer, Gonadotropins, Training
Parole chiave: Esercizio, Alterazioni mestruali, Calcio, Gonadotropine, Allenamento
Abstract. Intensity of exercise, specific type and amount of training, reduced caloric intake, low body mass
index (BMI) or body fat may contribute to cause various forms of reproductive dysfunctions including oligomenorrhea, amenorrhea and luteal phase defects. To explore the possible relationships between hormone
levels and specific training programmes we determined plasma levels of ОІ-endorphin, prolactin (PRL), follicle
stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in 15 female soccer players (SP) of the
same team (6 eumenorrheic, 9 oligomenorrheic) at rest and after an individualized role-related training session (IT), and in 10 sedentary eumenorrheic controls (C) at rest. At rest, FSH, LH and E2 levels were lower, and
ОІ-endorphin level higher, in SP than in C. ОІ-endorphin level was unrelated to hormone levels in either SP or C.
IT increased hormone concentrations, with higher values of E2 and LH in eu- compared to oligomenorrheic SP.
ОІ-endorphin increased less in eu- than in oligomenorrheic SP. In the entire SP group after IT, we found that a
higher increase in ОІ-endorphin resulted in a lower change in LH (r= -0.59, p<0.05) and E2 (r= -0.58, p<0.05).
As for positional roles, most forwards were eumenorrheic. Conversely, all midfielders were anovulatory oligomenorrheic, and their LH response to IT correlated with the ОІ-endorphin response (r=-0.99, p=0.01). Our
data suggest an interaction between specific training, hormone levels and menstrual status in female SP.
Riassunto. L’intensità dell’esercizio, la specificità e la quantità dell’allenamento, la riduzione dell’apporto
calorico, un basso indice di massa corporea (IMC) o una ridotta percentuale di grasso corporeo possono
contribuire a causare diverse forme di disfunzioni della riproduzione come oligomenorrea, amenorrea e
difetti della fase luteinica. Per esaminare la possibile relazione tra livelli ormonali e programmi di allenamento
specifici abbiamo determinato i livelli plasmatici di: ОІ-endorfina, prolattina (PRL), ormone follicolostimolante
(FSH), ormone luteinizzante (LH) e estradiolo (E2) in 15 giocatrici di calcio (SP) della stessa squadra (6 eumenorroiche, 9 oligomenorroiche) a riposo e dopo una seduta di allenamento personalizzata per il ruolo (IT).
Dieci donne sedentarie eumenorroiche a riposo (C) sono state studiate come controlli. A riposo, i livelli di
FSH, LH e E2 erano piГ№ bassi, ed i livelli di ОІ-endorfina piГ№ alti, nelle SP che nei C; i livelli di ОІ-endorfina non
erano correlati con i livelli ormonali nelle SP o nei C. L’IT incrementava le concentrazioni ormonali, con valori
più alti di E2 e LH nelle SP eu- rispetto alle oligomenorroiche. L’incremento di β-endorfina era inferiore nelle
SP eu- che nelle oligomenorroiche. Nell’intero gruppo delle SP dopo IT, l’incremento della β-endorfina era
inversamente correlato alle variazioni di E2 (r=-0.58, p<0.05) e di LH (r=-0.59, p<0.05). In considerazione
dei ruoli, la maggior parte delle attaccanti erano eumenorroiche. Viceversa, tutte le centrocampiste erano
oligomenorroiche anovulari e la risposta all’IT dell’ormone LH correlava con la risposta della β-endorfina (r=0.99, p<0.01). I nostri dati suggeriscono un’interazione tra allenamento specifico, livelli ormonali e funzione
mestruale nelle calciatrici.
Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici
Paragraph 1: Intensity of exercise, specific type (endurance vs. power) and amount of
training [1-4], reduced caloric intake, low body mass index (BMI) or body fat [4, 5] may
partially contribute to cause various forms of menstrual cycle disorders (MCDs). Evidence
accumulated to date indicates that low energy availability plays a causal role in the development of exercise induced amenorrhea [5]. Disordered eating in combination with high
energy expenditure from exercise can lead to energy deprivation [6].
Paragraph 2: The most frequent alterations of the menstrual cycle in female athletes
are delayed puberty if agonistic training begins before puberty, and hypothalamic chronic
oligo-/amenorrhea if agonistic training begins in post-menarcheal age [7-9]. The trainingassociated changes in the menstrual cycle are reversible, with return to eumenorrhea upon
cessation of training [10].
Paragraph 3: The aetiology of exercise-associated menstrual disorders is not completely understood. Plasma levels of endogenous opioid hormones like ОІ-endorphin, prolactin
(PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) are
believed to reflect an alteration in the hypothalamic control of gonadotropin release [1113]. The pulsatility of gonadotropins is also reduced in athletes with anovulatory oligomenorrhea [14,15]. The decrease of estrogenic hormone concentrations is proportional
to training intensity [16,17], and plasma levels of ОІ-endorphin increase after short-term
anaerobic exercise [18]. However, there is a lack of information regarding the effects of
exhaustive aerobic endurance exercise and of aerobic-anaerobic alternated sports, like
soccer, on sex steroids and ОІ-endorphin [19,20].
Paragraph 4:Soccer players need a high aerobic power in order to sustain a high workrate for 90 minutes. But, according to their position in the team, they need high anaerobic
or explosive power, too. The greatest running distance is covered by midfielders, while the
frequency of jumps is greater among forwards than fullbacks or midfielders. Therefore,
the alactacid anaerobic power of forwards should be higher than that of players in other
positions. For these reasons, training programmes for soccer players have been developed
in accordance with their positional roles in the team [21].
Paragraph 5: The aim of this study in eumenorrheic and anovulatory oligomenorrheic
female soccer players was to determine the acute effects of an individualized role-related
training session on plasmatic levels of ОІ-endorphin, PRL and gonadotropins.
Paragraph 6: Fifteen female soccer players (SP) from the same team, and ten healthy
Effects of specific training on hormone levels in female athletes
sedentary women of similar age (C) were recruited for the study. All experiments were in
agreement with the ethical standards of the local University Ethical Committee. All subjects
gave written informed consent to the study. No use of tobacco, alcohol, cannabis or other
drugs was allowed by the study protocol. No subject was on oral contraceptives or other
exogenous hormones, or had a history of eating disorders (anorexia, bulimia).
Paragraph 7: The anthropometric characteristics of SP and C were similar (age:
20.4В±6.4 yr in SP, 20.6В±3.4 yr in C; body weight: 54.0В±7.9 kg in SP, 59.3В±9.7 kg in C;
height: 157.2В±5.5 cm in SP, 157.5В±6.2 cm in C; percentage of body fat: 26.5В±4.1% in SP,
27.5В±6.7% in C). All C women had cyclic menstrual cycles (11-13 menses/year) and their
menarcheal age was 12.5В±1.1 yr. The SP group comprised 6 eumenorrheic (11-13 menses/year), and 9 oligomenorrheic (5-7 menses/year) women. In the SP group, menarcheal
age was 13.4В±1.0 yr, slightly higher than in C (p<0.05 by unpaired t-test), but similar in
eu- (13.7В±1.1 yr) and oligomenorrheic SP (13.2В±0.9 yr). The oligomenorrheic SP were
studied on the 3rd day of the cycle to assess basal gonadotropin pattern, and by basal body
temperature to ascertain oligomenorrheic anovulation, leading to the diagnosis of stressrelated hypothalamic chronic anovulation [22].
Paragraph 8: All SP had been playing agonistic soccer for at least 6 years. The team
included 4 forwards, 4 midfielders and 7 fullbacks. Each training session lasted approximately 2 hrs, and included skills, tactics and physical conditioning exercises. Their training
programme (4 sessions/week) was individualized according to positional roles in the team:
most work carried out by midfielders and fullbacks was to develop aerobic capacity, whereas training in forwards aimed at improving strength and alactacid anaerobic capacity
and power.
Paragraph 9: Data in SP were collected between 4 and 6 p.m. towards the end of a
14-week training period (23). Data in controls were collected in the same month and hour
of the day. The percentage of body fat was determined by using a bioelectrical impedance
apparatus (Human - IM Scan, DS MediGroup, Milan, Italy).
Paragraph 10: In all subjects, blood sampling was scheduled during the follicular phase of the menstrual cycle between days 10 and 14 (to emphasize the possible different
conditions between eumenorrheic and anovulatory oligomenorrheic groups). In SP, blood
samples were obtained 29 В± 4 min before, and 28 В± 5 min after the training session. During the training session, water drinking was allowed at pleasure.
Paragraph 11: Blood was drawn from the subject’s antecubital vein into sterile tubes
containing EDTA (7,2 mg/5 ml) at 4•C, and centrifuged for 15 min at 2000 rpm. Aliquots of
plasma were frozen and kept at - 20•C in polycarbonate tubes and subsequently analysed
Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici
in duplicate for ОІ-endorphin (Incaster RIA Kit, Stillwater, Minnesota, USA), LH and FSH (Cis
Bio-International RIA Kit, Gif-Sur-Yvette Cedex, France), and E2 and PRL (Biodata RIA Kit,
Rome, Italy).
Paragraph 12: Results are presented as means В± standard deviation (SD). Data from
SP and C, and from eumenorrheic and anovulatory oligomenorrheic SP, were compared
by two-tailed unpaired t-test. Statistical evaluation in the SP group according to positional
roles was by analysis of variance, with Bonferroni multiple-range test. Association between
variables was assessed by simple linear regression. The level of significance was set at
Resting conditions
Paragraph 13: Table 1 reports plasma hormone concentrations at rest in SP and C.
Compared to C, both eu- and oligomenorrheic SP showed decreased plasma levels of LH
and E2. Both subgroups showed increased plasma level of ОІв€’EP, while plasma FSH was
significantly lower than normal in oligomenorrheic SP only. Eumenorrheic SP showed significantly lower plasma PRL values than C. As expected, there was a difference between
eu- and oligomenorrheic SP at rest regarding the plasma level of E2, which was significantly lower in the latter than in the former group (p<0.005 by unpaired t-test).
Paragraph 14: No significant relationship was found at rest between plasma hormones
and PRL or ОІ-endorphin concentration in any group (C, all SP, eu- or oligomenorrheic SP).
Table 1. - Hormones values at rest in controls and soccer players
Sedentary controls (n=10)
Soccer players
Eumenorrheic (n=6)
Oligomenorrheic (n=9)
Values are meanВ± S.D.
* indicates p<0.05 between soccer players and controls
Effects of specific training on hormone levels in female athletes
Fig. 1. Summary data of hormonal levels at rest (empty bars) and after exercise (hatched bars) in eumenorrheic and anovulatory oligomenorrheic SP (upper and lower panel, respectively).
* indicates significant difference between resting and post-exercise values.
Fig. 2. Hormonal responses to exercise in eumenorrheic (empty bars) and oligomenorrheic (filled bars) SP
expressed as percentage change from baseline.
* indicates significant difference between eumenorrheic and oligomenorrheic SP.
Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici
fullbacks, eumenorrheic (n=3)
fullbacks, oligomenorrheic (n=4)
midfielders, oligomenorrheic (n=4)
forwards, eumenorrheic (n=3)
forwards, oligomenorrheic (n=1)
Fig. 3. Analysis of exercise-associated average hormonal changes according to positional role in the team
and to menstrual status.
Effects of specific training on hormone levels in female athletes
Response to exercise according to menstrual status
paragraph 15: Post-exercise values of all hormones and ОІ-endorphin were higher than
pre-exercise values in both eu- and oligomenorrheic SP (Figure 1), but the E2 level after
exercise was much higher in eumenorrheic SP.
Paragraph 16: Figure 2 illustrates exercise-induced changes in hormonal levels expressed as percentage increase over baseline: the three-fold increase in plasma LH found
in eumenorrheic SP was blunted in oligomenorrheic SP (p=0.001 by unpaired t-test),
while ОІ-endorphin showed a slightly larger increase in oligo- than in eumenorrheic SP
(p<0.05, Figure 2). The increase in FSH, PRL and E2 were not statistically different in the
two groups.
Paragraph 17: In the whole SP group (eu- + oligomenorrheic SP), exercise-induced
changes in ОІ-endorphin correlated inversely with changes in LH (r=-0.59, p<0.05) and
E2 (r=-0.58, p<0.05). No significant relationship was found between exercise-induced
changes in ОІ-endorphin and PRL or FSH, either in the whole SP group or in the two subgroups.
Response to exercise according to positional role in the team
Paragraph 18: Both 3 out of 4 forwards and 3 out of 7 fullbacks were eumenorrheic. All
4 midfielders were oligomenorrheic. The average trends in hormonal responses to exercise in SP, according to both the menstrual status and the role in the team are reported in
figure 3. ОІ-endorphin increased similarly after exercise irrespective of positional roles or
menstrual status. No significant difference among positional roles was evident for any hormone. Eumenorrheic SP showed a significant increase in plasma LH and E2 after exercise,
which was blunted in oligomenorrheic SP (Figure 3). The LH response to exercise correlated negatively to the ОІ-endorphin response in midfielders (r=-0.99, p=0.01).
Paragraph 19: Exercise-induced menstrual irregularities appear related to the intensity and duration of training [1,2]. Menstrual disturbances, such as hypothalamic chronic
anovulation, have been associated with significant weight loss, decreased body fat, stress,
intensity of athletic training, disordered eating in combination with high energy expenditure from exercise [6,14,24]. Still, the mechanism by which strenuous exercise might lead to
decreased reproductive function has not been elucidated [22].
Paragraph 20: High plasma levels of ОІ-endorphin have been shown to decrease the basal levels of LH and FSH. ОІ-endorphin has been implicated in the regulation of circulating
gonadotropin-releasing hormone (GnRH) from the median eminence of the hypothalamus.
Furthermore, the pulsatile secretion of GnRH may be inhibited by the increased level of
Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici
ОІ-endorphin, which binds to norepinephrine receptors in the hypothalamus leading to the
suppression of LH and FSH release. These hormonal changes result in hypothalamic chronic anovulation [14,25,26].
Paragraph 21: In our sample, ОІ-endorphin at rest was higher, and FSH, LH, and E2 lower,
in SP than in controls. In addition, a similar percentage of body fat in SP and controls ruled
out any possible effect of altered body composition on hormonal levels at rest. Our data do
not support an exclusive role of ОІ-endorphin in reproductive hormone regulation, since no
significant difference in ОІ-endorphin was found between eu- and oligomenorrheic SP at
rest. These data suggest that other factors, including the individual sensitivity to hormones
and possibly the type of training, may modulate the relationship between ОІ-endorphin and
reproductive hormone levels.
Paragraph 22: The effect of acute strenuous exercise on plasma hormone concentrations
has also been the object of much controversy. Serum FSH, LH and E2 concentrations were
reported to be increased, decreased or unchanged with both acute and chronic exercise
[14]. Our study provides evidence that when SP undergo intense exercise, FSH, LH, E2 and
PRL all increase significantly. Furthermore, despite different basal levels, the percentage
increase over baseline after exercise was not different for PRL, FSH and E2 in eu- and oligomenorrheic SP. However, anovulatory oligomenorrheic SP displayed higher ОІ-endorphin
and lower LH increase after the training session, in agreement with the known reduced variation of gonadotropin secretion in response to different stress in this group. In the whole
SP sample, we found that a higher exercise-induced increase in ОІ-endorphin resulted in
a lower increase in LH. These data suggest that the acute exercise-induced alterations in
ОІ-endorphin and LH are tightly associated.
Paragraph 23: As the possible role of PRL is concerned, lower resting levels of PRL were
found in eu- than in oligomenorrheic SP. Since PRL was not increased over baseline in
oligomenorrheic SP, our data suggest a minor role of PRL in causing chronic hypothalamic
anovulation in athletes. In addition, the PRL response to exercise was similar in eu- and
oligomenorrheic SP.
Paragraph 24: Little information is presently available regarding the effects of aerobic
exhaustive endurance exercise and aerobic-anaerobic alternated sports like soccer on sex
steroids and ОІ-endorphin [27,28]. Ideally, the evaluation of the long-term effects of training
on reproductive hormone concentrations and menstrual cycle would require a long-term
follow-up of a large sample of women along many seasons of training and agonistic practice. In our study, we compared the FSH, LH, E2, PRL and ОІ-endorphin responses to exercise in forwards, midfielders and fullbacks. Although no statistical difference in hormonal
levels was found among positional roles in the team, one interesting observation was that
all midfielders in our study were affected by hypothalamic chronic anovulation. A possible
Effects of specific training on hormone levels in female athletes
explanation for this particular endocrine pattern is that, as already shown in long-distance
runners [12], chronic aerobic stress may modify the pulsatile secretion of GnRH and determine hypothalamic chronic anovulation. Conversely, forwards who perform an intermittent
anaerobic stress, were mostly eumenorrheic. Therefore, positional roles seem to affect
the endocrine pattern possibly through different effects of role-specific training. Midfielders and fullbacks perform a more constant and continuous physical aerobic-anaerobic
alternated exercise than forwards, who perform a strenuous and fast but discontinuous
anaerobic exercise with several pauses. The hypothesis of a modulating effect of the type
of exercise is supported by the findings of correlation analysis in fullbacks indicating that
exercise-induced changes in LH are linked to changes in ОІ-endorphin.
Paragraph 25: In conclusion, a low LH or E2 response to exercise in female soccer
players was associated with a large increase in ОІ-endorphin concentration. On the other
hand, intense exercise increased reproductive hormones and ОІ-endorphin concentrations
in both eumenorrheic and anovulatory oligomenorrheic athletes. It is possible that anovulatory oligomenorrheic soccer players are more susceptible to exercise-related increase
in ОІ-endorphin concentration. The large post-stress increase in hormone concentrations
noted in eumenorrheic SP supports that the integrity of hypothamic-pituitary-gonadal axis
is necessary for a ready response to exercise-induced stress.
Paragraph 26: Our results in soccer players confirm the known effects of chronic exercise on menstrual irregularities. Because no significant differences in body composition were found between soccer players and controls, the data suggests that the type of
training (aerobic versus aerobic/anaerobic) can significantly affect the menstrual status
of women involved in competitive sports. Soccer appears as a very useful model to study
exercise-associated menstrual dysfunction, because of the marked differences among
training programmes according to positional roles. The size of soccer teams (at least 15
subjects) may allow assessing the effects of different training patterns in a relatively homogeneous context (i.e same trainer and frequency of training sessions, etc). Longitudinal
studies are warranted to better define soccer-induced menstrual abnormalities and the
impact of progressive specific training according to positional roles in the team.
The Authors gratefully thank: Dr. Rosario Giuliano, Specialist in Sport Medicine for his help in data collection
and analysis.
Arcangelo Benigno, Attilio Licciardi, Giuseppe Morici
[1] Bonen A. Recreational exercise does not impair menstrual cycle: a prospective study. Int J Sports Med
1992; 13: 100-120.
[2] Lehmann M, Knizia K, Gastmann U. Influence of 6 week, 6 days per week, training on pituitary function
in recreational athletes. Br J Sports Med 1993; 27: 186-192.
[3] Warren MP, Perlroth NE. The effects of intense exercise on the female reproductive System. J Endocrinol 2001; 170: 3-11.
[4] Castelo-Branco C, Reina F, Montivero AD, ColodrГІn M, Vanrell JA. Influence of highintensity training
and of dietetic and anthropometric on menstrual cycle disorders in ballet dancers. Gynecol Endocrinol
2006; 22 (1): 31-35.
[5] Williams NI, Helmreich DL, Parfitt DB, Caston-Balderrama A, Cameron JL. Evidence for a causal role
of low energy availability in the induction of menstrual cycle disturbances during strenuous exercise
training. J Clin Endocrinol Metab 2001; 86 (11): 5184-5193.
[6] Zanker C, Hind K. The effect of energy balance on endocrine function and bone health in youth. Med
Sport Sci 2007; 51: 81-101.
[7] Constantini NW. Clinical consequences of athletic amenorrhoea. Sport Med 1994; 17: 213-223.
[8] Ohsawa S, Ji CY, Kasai N. Age at menarche and comparison of the growth performance of pre and post
menarcheal girls in China. Am J Hum Biol 1997; 9: 205-212.
[9] Little NG, Day JAP, Steinke L. Relationship of physical performance to maturation in perimenarcheal
girls. Am J Hum Biol 1997; 9: 163-171.
[10] Loucks AB. Effects of exercise training on the menstrual cycle: existence and mechanism. Med Sci
Sports Exerc 1990; 22: 275-280.
[11] Li CH. ОІ-endorphin: a pituitary peptide with potent morphine-like reactivity. Arch Biochem Biophys
1977; 183: 595-660.
[12] Loucks AB, Mortola JF, Girton L, Yen SSC. Alteration of the hypothalamic-pituitary-ovarian and the
hypothalamic-pituitary-adrenal axes in athletic women. J Clin Endocrinol Metab 1989; 68: 402-410.
[13] Lefebvre P, Bringen J, Orsetti A. Sport et axe gonadotrope fГ©minin. Science & Sports 1997; 12: 19-25.
[14] Arena B, Mafulli N, Mafulli F, Morleo MA. Reproductive hormones and menstrual changes with exercise
in female athletes. Sports Med 1995; 19: 278-287.
[15] Russell JB, Mitchell DE, Musey PI, Collins DC. Collins DC. The role of ОІ-endorphin and catechol estrogens on the hypothalamic-pituitary axis in female athletes. Fertil Steril 1984; 42: 690-695.
[16] Fry RW, Morton AR, Garcia-Webb P. Biological responses to overload training in endurance sports. Eur
J Appl Physiol 1992; 64: 335-344.
[17] Lehmann M, Gastmann U, Petersen KG. Defined increase in training volume versus intensity in experienced middle-and long-distance runners. Br J Sports Med 1992; 26: 233-242.
[18] Schwarz L, Kindermann W. Changes in ОІ-endorphin levels in response to aerobic and anaerobic exercise. Sports Med 1992; 13: 25-36.
[19] Fellmann N. Hormonal and plasma volume alterations following endurance exercise. A brief review.
Sports Med 1992; 13: 37-49.
[20] Kraemer RR, Acevedo EO, Dzewaltowski D, Kilgore L, Kraemer GR, Castracane VD. Effects of lowvolume resistive exercise on beta-endorphin and cortisol concentrations. Int J Sports Med 1996; 17:
[21] Di Salvo V, Pigozzi F. Physical training of football players based on their positional roles in the team.
Effects on performance-related factors. J Sports Med Phys Fitness 1998; 38: 294-7.
Effects of specific training on hormone levels in female athletes
[22] Chen EC, Brzyski RG. Exercise and reproductive dysfunction. Fertil Steril 1999; 71:1-6.
[23] Dent RRM, Guilleminault C, Albert LH, Posner BI, Cox BM, Goldstein A. Diurnal rhythm of plasma immunoreactive ОІ-endorphin and its relationship to sleep stages and plasma rhythms of cortisol and
prolactin. J Clin Endocrinol Metab 1982; 52: 942-7.
[24] Bonen A. Exercise-induced menstrual cycle changes: a functional temporary adaptation to metabolic
stress. Sports Med 1994; 17: 373-392.
[25] De Cree C. Endogenous opioid peptides in the control of normal menstrual cycle and their possible role
in athletic menstrual irregulation. Obstet Gynecol 1989; 44: 720-728.
[26] Loucks AB, Heath EM, Law T. Dietary restriction reduces luteinizing hormone (LH) pulse frequency, during waking hours and increase LH pulse amplitude during sleep in young menstruating women. J Clin
Endocrinol Metab 1994; 78: 910-915.
[27] Hakkinen K, Pakarinen A. Acute hormonal responses to heavy resistance exercise in men and women
at different age. Int J Sports Med 1995; 16: 507-513.
[28] Raynaud E, Mannier JF, Brun JF. Biochimie et hormonologie de l’exercise submaximal: standardisation
d’un test d’effort chez le sportif. Science & Sports 1997; 12: 72-74.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Effetti positivi dell’allenamento d’endurance leggero in topi adulti mdx]
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno and Attilio Licciardi
Department of Experimental Medicine (Di.Me.S), University of Palermo (IT)
Key words: Training, Strength, Degree of fatigue, Body weight, Muscular dystrophy
Parole chiave: Allenamento, Forza, Grado di fatica, Peso corporeo, Distrofia muscolare
Abstract. High intensity exercise induces muscle damage in mdx mice; however, the effects of low intensity training on the contractile properties of mdx mice muscles are still controversial. The aim of this study
was to examine whether low intensity endurance training had beneficial effects on strength, fatigue and
body weight in adult mdx mice. Wild-type (C57BL/10) and mdx mice (C57BL/10Sc-Dmdmdx/J) ran daily on
a motorized Rota-Rod under standard laboratory conditions (temperature: 20В° C, humidity: 50%). Exercise
bouts were performed daily five times per week for a total time period of 4 weeks at progressively increasing
loads. To study the recovery effects, mice were also observed after two weeks of cessation of training. At
week 3, sedentary mdx mice (MDX-SD) showed a lower grip strength fatigue (-2.8 В± 33.5 %) compared to
both exercised mdx mice (MDX-EX) (23.2 В± 13.6 %, p<0.05) and exercised wild-type mice (WT-EX) (30.3 В±
18.4 %, p<0.05). Conversely, after recovery MDX-SD group showed a grip strength fatigue (39.9 В± 7.3 %)
significantly greater than MDX-EX (11.1 В± 1.1 %, p<0.05), WT-EX (19.2 В± 15.5 %, p<0.05) and sedentary
wild-type mice (WT-SD) groups (6.9 В± 15.2 %, p<0.05). In conclusion, low intensity endurance training in
mdx mice induced physiological adaptations resulting in a trend to lower body weight gain and in a positive
effect on the degree of fatigue. These preliminary data provided evidence for beneficial effects of low intensity endurance training in adult mdx mice.
Riassunto: L’esercizio molto intenso determina danno muscolare nei topi mdx; comunque, gli effetti di un
allenamento di bassa intensitГ sulle proprietГ contrattili dei muscoli di topi mdx sono ancora controversi.
Lo scopo di questo studio è stato quello di osservare se l’allenamento di resistenza leggero avesse effetti
positivi sulla forza, la fatica e il peso corporeo in topi mdx adulti. Topi allenati wild-type (C57BL/10) e mdx
(C57BL/10Sc-Dmdmdx/J) correvano giornalmente su un Rota-Rod motorizzato in condizioni di laboratorio
standard ( temperatura 20В°C, umiditГ 50%). Gli allenamenti sono stati effettuati giornalmente cinque volte la
settimana per un periodo totale di 4 settimane aumentando il carico in modo progressivo. Per osservare gli
effetti del recupero, i topi sono stati studiati dopo due settimane di cessazione dell’allenamento. Alla terza
settimana di allenamento, il gruppo dei topi mdx sedentari (MDX-SD) mostrava una ridotta fatica al grip
strength (-2.8 В± 33.5 %) sia rispetto al gruppo dei topi mdx allenati (MDX-EX) (23.2 В± 13.6 %, p<0.05) che
al gruppo dei topi wild-type allenati (WT-EX) (30.3 В± 18.4 %, p<0.05). Invece, dopo il recupero, il gruppo
MDX-SD mostrava una maggiore fatica al grip strength (39.9 В± 7.3 %) rispetto ai gruppi MDX-EX (11.1 В± 1.1
%, p<0.05), WT-EX (19.2 ± 15.5 %, p<0.05) e wild-type sedentari (WT-SD) (6.9 ± 15.2 %, p<0.05). In conclusione, l’allenamento di resistenza leggero ha indotto nei topi mdx adattamenti fisiologici quali una tendenza
ad un ridotto accrescimento ponderale e un effetto positivo sul grado di fatica. Questi dati preliminari hanno
evidenziato degli effetti benefici dell’allenamento di resistenza leggero nel topo adulto mdx.
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi
Paragraph 1: Dystrophin-deficient (mdx) mice are the classical models for Duchenne
muscular dystrophy (DMD). The mdx mice have a mutated dystrophin gene and are therefore genetically valid models of DMD. Although these animals have been useful for genetic
and pharmacologic treatment manipulations nevertheless they have not been considered
an ideal clinical model [1].
Paragraph 2: Young mdx mice (3-4 weeeks of age) show muscle weakness and appear
more susceptible to fatigue (increased myofibre necrosis) than control mice, in mdx adult
mice (over 8 weeks of age) this acute phase decreases significantly to a chronic low level of
damage, <10% muscle affected, which persists throughout life but is further decreased by
1 year of age [2]. The onset of progressive replacement of muscle by fibrous connective tissue is reported in limb muscle of mdx mice from 10-13 weeks with extensive fibrosis from
16 to 20 months of age [3]. No obvious clinical weakness is apparent until 18 months.
Paragraph 3: As DMD results in progressive skeletal muscle atrophy and weakness,
appropriate exercise may be a suitable therapy. A number of studies (Table 1) have shown
that muscle from mdx mice adapts differently to exercise. These discrepancies in the literature may be explained by differences in experimental procedures: duration, intensity
and type of exercise, variations in age of mdx mice, different environmental conditions. However, whether exercise therapy is beneficial or detrimental to dystrophic skeletal muscle
needs to be further documented.
Paragraph 4: Moreover, Connolly et al. showed that mdx mice at 8-10 weeks of age
weighed significantly more than age-matched normal mice. This excess weight-gain was
related to chronic under-activity in mdx mice [4].
Paragraph 5: The aim of this study was to examine whether regular low intensity endurance training had beneficial effects on strength, fatigue and body weight in adult mdx
Table 1. Studies on the effects of different training protocols in mdx mice
Author, year [ref]
Starting age
Exercise Time Weeks of
(min per day) training
Main results
per week
Granchelli JA et al.
2000 [5]
12 m/min
19 % decrease in strength
Kaczor JJ et al.
2007 [6]
9 m/min
Decreased lipid and protein
oxidative damage
Beneficial effects of low intensity endurance training in adult mdx mice
De Luca A et al.
2003 [7]
12 m/min
Muscular damage. Mdx mice
significantly weaker than
EE et al., 1994 [8]
5 km/day (approximately)
No deleterious effects on
soleus muscle function
Call JA et al.
2008 [9]
1-2 km/day
Increased running distance
Hayes A and Williams
DA, 1996 [10]
Hayes A and Williams
DA, 1998 [11]
until exhaustion
Faist V et al.
2001 [12]
4 (young)
and 16
Wineinger MA et al.
1998 [13]
Force output improvement of
4 km/day at soleus muscle and greater
1.6 km/h (ap- fatigue resistance of soleus
proximately) muscle and of the extensor
digitorum longus
No detrimental effects
480 m/day
Mitochondrial oxygen consumption was significantly
lower in skeletal muscle of
exercised young mdx mice
Beneficial effects on both
the old mdx mice and their
controls. 9% reduction in the
fatigability of the extensor
digitorum longus of the old
mdx mice after the exercise
Paragraph 6: Experiments were carried out in our laboratory (temperature: 20В°C, humidity: 50%) using 31 male dystrophic mdx mice (C57BL/10ScSn-Dmdmdx/J from Jackson
Laboratories) and 35 male wild type mice (C57BL/10ScSn from Harlan Italy). All mice were
housed under a 12h day-night cycle and allowed access to food and water ad libitum. Experiments were carried out in agreement with the European Communities Counsil Directive
(86/609/EEC) regarding care and use of animals for experimental procedures.
Exercise training protocol
Paragraph 7: At 10 weeks of age mdx mice were randomly assigned to sedentary
(n=17; MDX-SD) or trained (n=14; MDX-EX) groups. At 14 weeks of age wild type mice
were randomly assigned to sedentary (n=19; WT-SD) or trained (n=16; WT-EX) groups
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi
too. Preliminarily all mice underwent a 2-week period of acclimatization trial at very low
Paragraph 8: To avoid the confounding effects of the accelerated necrotic stage of the
disease present in young mdx mice (3-4 weeks of age) and the hormone effects on skeletal muscle typical of pre-adolescence and puberty in mice (5-7 weeks of age) [1] we tested
the effects of low intensity endurance training in adult mice (> 8 weeks of age).
Paragraph 9: Training was performed in the same room where the mice were housed
using a motorized rotor (Rota-Rod; Ugo Basile, Biological Research Apparatus, Comerio
Varese, Italy). Mice ran 5 days/week for 4 weeks at progressively increasing loads. In order
to study the recovery effects, mice were housed for more 2 weeks after completion of the
exercise training protocol described above (Table 2).
Paragraph 10: Wild-type and mdx mice were weighed at the start time of exercise
training protocol and after 2, 3 and 4 weeks of training. Mice were still weighed after the
recovery period.
Forelimb strength and fatigue
Paragraph 11: All mice groups were examined for forelimb strength at 1, 3 and 4 weeks
of training and after recovery. Briefly, the mouse was placed on a metal grid attached to a
force transducer (Grip strength meter; Ugo Basile, Biological Research Apparatus, Comerio
Varese, Italy), and pulled gently from the base of the tail until release. Because the increase
in strength paralleled somatic growth in normal animals, we analyzed the strength of the
mdx and controls as a function of weight. Each mouse underwent five trials and peak grip
strength was normalized for body weight (force/BW) and recorded as the average of the
three best efforts divided by the mouse’s body weight. Grip strength fatigue was calculated
by comparing the first two pulls to the last two pulls [4]. The decrement between pulls 1 +
2 and pulls 4 + 5 gives a measure of fatigue. In the formula (4 + 5)/(1 + 2), animals with
no fatigue have a value of 1. For convenience, the value resulted from the formula above
was subtracted from the value of “1” so that an animal without fatigue has a calculated
fatigue value of 0, and an animal that can only complete three pulls, because completely
fatigued, has a value of “1”. The fatigue was expressed as percentage so that a value of
0% fatigue would be assigned to mice without fatigue and a value of 100% fatigue to mice
with complete fatigue.
Statistical analysis
Paragraph 12: Results are reported as mean В± SD. Analysis of variance (ANOVA) and
Fisher’s PLDS correction for post hoc comparison were used to test for differences between sedentary wild type (WT-SD), trained wild type (WT-EX), sedentary dystrophic mice
(MDX-SD) and trained dystrophic mice (MDX-EX) at different time points. Time trends in
Beneficial effects of low intensity endurance training in adult mdx mice
each group were assessed by paired t-test (Statview 5.0.1; SAS Institute, Inc., Cary, NC).
Significance was at p<0.05.
Table 2. Training protocol on the rotarod apparatus.
Session time
Body weight and exercise
Paragraph 13: As shown in Figure 1, at the beginning of exercise training protocol, body
weight in MDX-EX (26.2 В± 2.2 g, p<0.05) and MDX-SD (24.8 В± 1.9 g, p<0.001) was lower
compared to WT-EX (27.9 В± 1.8 g) and WT-SD (28.0 В± 2.0 g). After 2 weeks of training,
body weight was lower in MDX-SD (26.8 В± 2.0 g) than in WT-SD (28.8 В± 2.3 g, p<0.05).
After 3 and 4 weeks of training and after recovery no significant differences were found
between groups; however, in MDX-EX body weight tended to be lower than in MDX-SD.
Forelimb strength
Paragraph 14: After 1, 3 and 4 weeks of training and after recovery there was no significant difference in forelimb strength (FS) between all groups. The time course of forelimb
strength was analysed for each group of mice. The main variations were found between
week 1 and 3, and reported as follows. In MDX-EX, FS was 63.9 В± 14.3 g at week 1 and
51.6 В± 8.3 g at week 3 (p<0.05); in WT-EX, FS was 64.2 В± 15.7 g at week 1 and 49.5 В±
16.6 g at week 3 (p<0.0001); in WT-SD, FS was 61.2 В± 13.5 g at week 1 and 47.4 В± 9.8 g
at week 3 (p<0.05); in MDX-SD, FS did not change at any time point.
Forelimb strength normalized for weight
Paragraph 15: Forelimb strength normalized for body weight (FSW, gram strength per
gram of body weight) did not show significant difference at any time point of the training
protocol and after recovery between all groups. The time course of FSW was analysed for
each group of mice. The main variations were found between week 1 and 3, and reported
as follows. In MDX-EX, FSW was 2.40 В± 0.61 g/g at week 1 and 1.88 В± 0.34 g/g at week
3 (p<0.05); in WT-EX, FSW was 2.29 В± 0.57 g/g at week 1 and 1.76 В± 0.57 g/g at week 3
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi
(p<0.0001); in WT-SD, FSW was 2.15 В± 0.46 g/g at week 1 and 1.64 В± 0.28 g/g at week
3 (p<0.05). In MDX-SD, FSW did not change at any time point.
Paragraph 16: Grip strength fatigue is reported in Figure 2. At week 3, MDX-SD showed
a lower grip strength fatigue (-2.8 В± 33.5 %) than both MDX-EX (23.2 В± 13.6 %, p<0.05)
and WT-EX (30.3 В± 18.4 %, p<0.05). Conversely, after recovery, grip strength fatigue was
significantly greater in MDX-SD compared to all groups. In particular, MDX-SD showed a
greater fatigue (39.9 В± 7.3 %) than MDX-EX (11.1 В± 1.1 %, p<0.05), WT-EX (19.2 В± 15.5
%, p<0.05) and WT-SD (6.9 В± 15.2 %, p<0.05) (Fig. 3). In addition, the time course of fatigue was analyzed for each group of mice. A reduced grip strength fatigue was observed
in MDX-EX after recovery compared with week 3 (11.1 В± 1.1 % vs 23.2 В± 13.6 %, respectively; p<0.05).
Paragraph 17: The main finding of this study is that forced low intensity endurance
training, without any corticosteroid and/or nutritional therapies may have beneficial effects
in adult mdx mice.
Paragraph 18: It has been previously reported that voluntary endurance exercise in
mdx mice induced greater force-generating capacity of soleus muscles and more fatigue
resistance in extensor digitorum longus [10,13]. Other studies showed that voluntary wheel running produced no effects on soleus or extensor digitorum longus muscle function
in mdx mice [8,14]. In a recent study Landisch and co-workers [15] hypothesized that a
“voluntary, endurance type of exercise training would improve mdx mouse muscle to the
same extent that exercise benefited muscle from healthy control mice”. In contrast, high
intensity forced treadmill running, especially with episodes of eccentric contractions, induced or accelerate muscle damage in mdx mice [7,16,17,18].
Paragraph 19: Moreover, an excess of weight-gain and obesity in mdx mice at 8-10
weeks of age was related to their chronic under-activity [4].
Paragraph 20: However, it is not currently known whether exercise therapy in mdx mice
is beneficial or detrimental to dystrophic skeletal muscle. Muscle from mdx mice adapts
differently to exercise, depending on the exercise protocol and age. At present, exercise
parameters (type, duration, intensity, frequency) need to be defined.
Paragraph 21: To fill this gap, we have trained adult mdx mice according to a low intensity endurance protocol to determine whether beneficial physiological adaptation may
be induced. Moreover, we tested the effects of low intensity endurance training in adult
mice (> 8 weeks of age) to avoid the confounding effects of acute skeletal muscle necrosis
present in young mdx mice (3-4 weeks of age) and the hormone effects on skeletal muscle
Beneficial effects of low intensity endurance training in adult mdx mice
typical of pre-adolescence and puberty in mice (5-7 weeks of age).
Paragraph 22: The novel finding of our study in mdx mice is that the low intensity endurance training protocol proposed had not detrimental effects on forelimb strength. Indeed,
in exercised mdx mice we found: a) lower grip-strength fatigue after recovery compared
to sedentary mdx mice; b) a trend toward an increased forelimb strength after 4 weeks of
low intensity endurance training; c) a body weight similar to that of exercised wild-type
mice with no evidence of obesity.
Paragraph 23: In conclusion, our preliminary data suggest that low intensity endurance
training had beneficial effects on skeletal muscle of adult mdx mice. Therefore, remediation of skeletal muscle weakness and fatigue changes may represent a physiologic adaptive response to specific training in mdx mice. These results may be relevant for therapeutic
exercise in patients with Duchenne Muscular Dystrophy but further studies are necessary
to determine the intensity and/or duration of exercise in humans. В 315
Beneficial effects of low intensity endurance training in adult mdx mice
Figure 1. Body weight changes in sedentary and exercised mdx and wild type mice during training and recovery periods. Data are expressed as means. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice;
WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice.
Figure 2. Grip strength fatigue in sedentary and exercised mdx and wild type mice during training and recovery periods. Data are expressed as means. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice;
WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice.
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi
Figure 3. Fatigue of forelimb strength in sedentary and exercised mdx and wild type mice after 3 weeks
of training (upper panel; * p<0.05 vs MDX-EX and WT-EX) and after recovery (lower panel; * p<0.05 vs all
groups). Data are expressed as means В± SD. MDX-SD: sedentary mdx mice; MDX-EX: exercised mdx mice;
WT-SD: sedentary wild type mice; WT-EX: exercised wild type mice.
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno, Attilio Licciardi
[1] Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol
Dis 2008 Jul; 31(1):1-19.
[2] McGeachie JK, Grounds MD, Partridge TA, Morgan JE. Age-related changes in replication of myogenic
cells in mdx mice: quantitative autoradiographic studies. J Neurol Sci 1993; 119:169-79.
[3] Lefaucheur JP, Pastoret C, Sebille A. Phenotype of dystrophinopathy in old mdx mice. Anatom Rec
1995; 242:70-76.
[4] Connolly AM, Keeling RM, Mehta S, Pestronk A, Sanes JR. Three mouse models of muscular dystrophy:
the natural hystory of strength and fatigue in dystrophin-, dystrophin/utrophin-, and laminin О± 2-deficient mice. Neuromuscul Disord 2001; 11:703-12.
[5] Granchelli JA, Pollina C, Hudecki MS. Pre-clinical screening of drugs using the mdx mouse. Neuromuscul Disord 2000; 10 (4-5):235-9.
[6] Kaczor JJ, Hall JE, Payne E, Tarnopolsky MA. Low intensity training decreases markers of oxidative
stress in skeletal muscle of mdx mice. Free Radic Biol Med 2007; 43 (1):145-54.
[7] De Luca A, Pierno S, Liantonio A, Cetrone M, Camerino C, Fraysse B, Mirabella M, Servidei S, Ruegg UT,
Conte Camerino D. Enhanced dystrophic progression in mdx mice by exercise and beneficial effects of
taurine and insulin-like growth factor-1. J Pharmacol Exp Ther 2003; 304 (1):453-63.
[8] Dupont-Versteegden EE, McCarter RJ, Katz MS. Voluntary exercise decreases progression of muscular
dystrophy in diaphragm of mdx mice. J Appl Physiol, 1994; 77 (4):1736-41.
[9] Call JA, Voelker KA, Wolff AV, McMillan RP, Evans NP, Hulver MW, Talmadge RJ, Grange RW. Endurance
capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green
tea extract. J Appl Physiol 2008; 105 (3):923-32.
[10] Hayes A, Williams DA. Beneficial effects of voluntary wheel running on the properties of dystrophic
mouse muscle. J Appl Physiol 1996; 80 (2):670-9.
[11] Hayes A , Williams DA. Contractile function and low-intensity exercise effects of old dystrophic ( mdx)
mice. Am J Physiol 1998; 274 (4 Pt 1):C1138-44.
[12] Faist V, Konig J, Hoger H, Elmadfa I. Decreased mitochondrial oxygen consumption and antioxidant
enzyme activities in skeletal muscle of dystrophic mice after low-intensity exercise. Ann Nutr Metab
2001; 45 (2):58-66.
[13] Wineinger MA, Abresch RT, Walsh SA, Carter GT. Effects of aging and voluntary exercise on the function
of dystrophic muscle from mdx mice. Am J Phys Med Rehabil 1998; 77 (1):20-7.
[14] Carter GT, Wineinger MA, Walsh SA, Horasek SJ, Abresch RT, Fowler WM Jr. Effect of voluntary wheelrunning exercise on muscles of the mdx mouse. Neuromusc Disord 1995; 5 (4):323-32.
[15] Landisch RM, Kosir AM, Nelson SA, Baltgalvis KA, Lowe DA. Adaptive and nonadaptive responses to
voluntary wheel running by mdx mice. Muscle Nerve 2008; 38:1290-303.
[16] Vilquin JT, Brussee V, Asselin I, Kinoshita I, Gingras M, Tremblay JP. Evidence of mdx mouse skeletal
muscle fragility in vivo by eccentric running exercise. Muscle Nerve 1998; 21:567-76.
[17] Ridgley JA, Pinniger GJ, Hamer PW, Grounds MD. The physiological effects of IGF-1 (class 1:Ea transgene) over-expression on exercise-induced damage and adaptation in dystrophic muscles of mdx mice.
Pflugers Arch 2008. DOI 10.1007/s00424-008-0568-4.
[18] Payne ET, Yasuda N, Bourgeois JM, Devries MC, Rodriguez MC, Yousuf J, Tarnopolsky MA. Nutritional
therapy improves function and complements corticosteroid intervention in mdx mice. Muscle Nerve
2006; 33:66-77.
Invited article
and report
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Epigenetics of galectin-3- a novel regulatory mechanism
of its transcription during carcinogenesis
[Epigenetica della Galectina-3. Un nuovo meccanismo regolatorio della sua trascrizione]
Hafiz Ahmed
University of Maryland, Biotechnology Institute, Baltimore (USA)
Key words: Epigenetics, Galectin-3, Carcinogenesis, Promoter methylation, Prostate cancer
Parole chiave: Epigenetica, Galectina-3, Carcinogenesi, Promoter della metilazione, Carcinoma prostatico
Abstract. Protein-carbohydrate interactions play significant role in modulating cell-cell and cell-extracellular
matrix interactions, which, in turn, mediate various biological processes such as growth regulation, immune
function, cancer metastasis, and apoptosis. Galectin-3, a member of a b-galactoside-binding protein family, is found multifunctional and is involved in normal growth development as well as cancer progression
and metastasis, but the detailed mechanisms of its functions or its transcriptional regulations are not well
understood. Besides, several regulatory elements such as GC box, CRE motif, AP-1 site, and NF-ОєB sites,
the promoter of galectin-3 gene contains several CpG islands that can be methylated during tumorigenesis
leading to the gene silencing. This review discusses the galectin-3 epigenetics, which represents a novel
regulatory mechanism of its transcription.
Riassunto. Le interazioni proteine-carboidrato giocano un ruolo significativo nella modulazione delle interazioni cellula-cellula e cellula-matrice extracellulare, che, in definitiva, mediano i vari processi biologici
quali la regolazione della crescita, la funzione immunitaria, la metastatizzazione tumorale e l’apoptosi. La
galectina-3, un membro della famiglia delle proteine leganti il b-galactoside, ГЁ una molecola multifunzionale
ed è coinvolta nei processi fisiologici di sviluppo così come nella progrressione tumorale e nella metastatizzazione, ma i meccanismi dettagliati delle sue funzioni o delle sue regolazioni trascrizionali non sono stati
ancora ben compresi. Oltre a diversi elementi regolatori come il box CG, il motivo CRE, il sito per AP-1 e i siti
per NF-kB, il promotore del gene per la galectina-3 contiene diverse isole CpG che possono essere mutilate
durante la tomorigenesi, portando al silenziamento del gene. Questa review tratta l’epigenetica della galectina-3, che rappresenta un nuovo meccanismo regolatorio della sua trascrizione.
Interactions between cells, and between cells and the extracellular matrix (ECM) are
pivotal for proper cellular function. In recent years, protein-carbohydrate interactions have
been considered as very important for modulation cell-cell and cell-ECM interactions (Fig.
1), which, in turn, mediate various biological processes such as cell activation, growth
regulation, cancer metastasis, and apoptosis. Thus, the identification of carbohydratebinding proteins (lectins) and their partners (carbohydrate ligands), and the detailed un321
Hafiz Ahmed
derstanding of the molecular mechanisms and downstream effects of these protein-carbohydrate interactions are subjects of current intense research. Galectins (gal), a family
of b-galactoside-binding proteins, are involved in growth development as well as cancer
progression and metastasis [1-5]. However, the detailed mechanisms of these functions
remain largely unknown. Of the fifteen members of the galectin family identified so far,
gal1, 2, 5, 7, 10, 11, 13, 14, and 15 are examples of the “proto” type galectins (one carbohydrate-recognition domain [CRD] per subunit), while Gal4, 6, 8, 9, and 12 are “tandemrepeat” type galectins, which contain two CRDs [6]. Gal3 is the only representative of the
“chimera” galectin type, containing one CRD connected to a collagen-like sequence rich
in proline and glycine [7].
Gal3, previously known as Mac-2, L-29, L-31, L-34, IgE binding-protein, CBP35, and
CBP30, is a very interesting multifunctional molecule and probably the most studied
member of the galectin family [2,8,9]. Gal3 plays an important role in normal development and tumorigenesis through regulating cell proliferation, apoptosis, cell adhesion,
invasion, angiogenesis and metastasis by binding to the cell surface b-galactose-containing glycoconjugates or glycolipids. Gal3 may exert its multiple biological roles intracellularly within the nucleus or the cytoplasm, or after its secretion, at the cell surface
and/or the extracellular space, mediating interactions between cells and the extracellular
matrix [3,4,6]. Its expression in normal and tumor tissues and regulation of its expression
including the newly discovered epigenetic regulation in some tumor tissues have been
discussed in this review.
Gal3 expression in normal tissues- role in growth development
Gal3 is developmentally regulated and expressed in many tissues of adults [10,11].
During mouse embryogenesis, gal3 first appears at fourth day of gestation in the trophectoderm of blastocyst, followed by its expression in the notochord cells between 8.5 and
11.5 days of gestation [10]. In later stages of mouse development, gal3 is expressed in the
cartilage, ribs, facial bones, the suprabasal layer of epidermis, the endodermal lining of the
bladder, larynx and oesophagus [9]. In adult, gal3 is mainly expressed in the epithelial cells
such as small intestine, colon, cornea, kidney, lung, thymus, breast, and prostate [9]. The
expression of gal3 is also detected in ductal cells of salivary glands, pancreas, kidney, and
eye and in intrahepatic bile ducts [9]. Regarding cell type, gal3 expression is observed in
fibroblasts, chondrocytes and osteoblasts, osteoclasts, keratinocytes, Schwann cells and
gastric mucosa, endothelial cells, and also immune related cells such as neutrophils, eosinophils, basophils and mast cells, Langerhans cells, dendritic cells, as well as monocytes
and macrophages from different tissues [3,9,12].
Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
Gal3 expression (increased or decreased) in tumors- role in tumor progression
and metastasis
Gal3 mediates homotypic and heterotypic aggregation and promotes tumor cells endothelial interactions, angiogenesis, and tumor metastasis. It was shown that cell surface
gal3 mediates homotypic cell adhesion by binding to soluble complementary glycoconjugates [13]. Interactions of metastatic cancer cells with vasculatory endothelium are critical
during early stages of cancer metastasis. Recent studies demonstrate that the endothelium gal3 participates in docking of cancer cells including breast and prostate cancers on
capillary endothelium by specifically interacting with cancer cells-associated ThomsenFriedenreich disaccharide (Gal b1,3GalNAc) [14]. Intracelluar gal3 in cancer cells confers
anti-apoptotic activity. For example, gal3 acts as a specific binding partner of activated
K-Ras and this interaction promotes strong K-Ras activation of PI3K (phosphoinositide
3-kinase) [15]. Gal3 is the only member of the galectin family that contains the NWGR antideath domain of the Bcl-2 family. Bcl-2 translocation to the mitochondrial membrane leads
to anti-apoptosis activity resulting from blocking cytochrome c release [8]. In human breast
carcinoma BT547 cells, gal3 can also inhibit cytochrome c release thereby protecting cells
against nitric oxide-induced apoptosis [8]. Moreover, gal3 binds Bcl-2 protein in vitro to
form heterodimers and thus behaves as a mitochondria-associated apoptotic regulator in
the cytoplasm. Interestingly, synexin (annexin 7, a Ca2+- and phospholipid-binding protein),
is required for gal3 prevention of mitochondrial damage [8]. Extracellular gal3 secreted
from tumor cells induces apoptosis of cancer-infiltrating T-cells suggesting its role in the
immune escape mechanism during tumor progression [16].
Expression of gal3 is observed in many tumors. However, the intensity of the gal3 expression in tumors depends on the type of tumor, its invasiveness and metastatic potential
[17,18]. For example, increased expression of gal3 is observed in colon, head and neck,
gastric, endometrial, thyroid, liver, bladder cancers and breast carcinomas [17,18]. Gal3
plays an important role in tumor progression and metastasis in many cancers such as
breast cancer, colon cancer, and human brain tumors [2,4,8,12,17,18]. Gal3 transfected
human breast cancer cells BT549, which is gal3 null, after intrasplenic injection, formed
metastatic colonies in the liver, while gal3 null BT549 cells did not [19]. In colorectal cancer,
gal3 expression in the primary lesions of the cancer was significantly increased compared
to the normal tissue, correlating with the progression of clinical stage, liver metastasis,
venous invasion, and lymph node metastasis [17,18]. The expression of the endogenous
gal3 also correlates with the malignant potential and metastasis of tumors in the central
nervous system. Change in cellular localization of gal3 is observed during progression of
various cancers. Down-regulation of gal3 expression has been demonstrated in colorectal
cancer, with increased cytoplasmic expression of gal3 at more advanced stages [17,18].
Hafiz Ahmed
In tongue cancer, nuclear gal3 is decreased, but cytoplasmic gal3 is increased during progression from normal to cancer [17,18].
The decreased expression of galectin-3 is known so far in prostate [20-22], kidney [23],
and pituitary cancers [24]. Recent data by us and others indicated that decreased expression of gal3 in pituitary and prostate tumors is, in part, due to its gal3 promoter methylation
[22,24,25, please see described later].
Transcriptional regulation of gal3 expression
Although a large body of data about gal3 expression is available in the literature, the
mechanisms of regulation of gal3 expression are not well understood. However, the expression of gal3 depends on cell type, external stimuli and environmental conditions and
involves numerous transcription factors and signaling pathways. Gal3 expression may serve as differentiation marker for certain cell types. For example, the differentiation of the
human monocytes or promyelocytic cell line HL-60 to macrophage-like cells induced by
phorbol ester is accompanied by increased expression of gal3 [26]. Gal3 expression is upregulated in phagocytic macrophages and thus considered as a “macrophage activation
marker” [9]. Gal3 expression is also elevated in microglia and macrophages activated by
phagocytosis of myelin or when exposed to granulocyte-macrophage colony-stimulating
factor [9]. In contrast, activation of human monocots by lipopolysaccharide and interferon-Оі
is accompanied by decrease of gal3 expression [9]. The reduced expression of gal3 was
also observed in monocytic THP-1 cells treated with non-steroidal or corticosteroidal antiinflammatory drugs [9]. Interestingly, gal3 expression is absent or barely detected in the
resting lymphocytes, but the activated B and T cells induce gal3 expression [9].
In the promoter region of the gal3 gene, several regulatory elements such as five putative Sp1 binding sites (GC boxes), five cAMP-dependent response element (CRE) motifs,
four AP-1- and one AP-4-like sites, two NF-ОєB-like sites, one sis-inducible element (SIE)
and a consensus basic helix–loop–helix (bHLH) core sequence are found [27]. The presence of multiple GC box motifs for binding ubiquitous expressed Sp1 transcription factor is
a characteristic of constitutively expressed “housekeeping” genes. The activation of the
Sp1 binding transcription factor is responsible for gal3 induction by Tat protein of HIV [9].
The SIE that binds sisinducible factors was suggested to be a possible candidate for the
growth-induced activation of gal3 gene expression, caused by the addition of serum. The
presence of CRE and NF-ОєB-like site in the gal3 promoter suggests that the activation of
gal3 expression could be regulated through the signaling pathways involving the cAMPresponse element-binding protein (CREB) or the NF-ОєB transcription factor. The CREB/ATF
and the NF-ОєB/Rel transcription factors pathways may be involved in the regulation of
Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
gal3 expression by the Tax protein during HTLV-I infection of T cells [9]. The involvement of
the NF-ОєB transcription factor in regulation of gal3 expression, as well as the Jun protein,
a component of AP-1 transcription factor has recently been confirmed [9]. The regulation
of gal3 expression through the NF-ОєB transcription factor was shown to be mediated by
nucling, a novel apoptosis-associated protein, which interferes with NF-ОєB via the nuclear
translocation process of NF-ОєB/p65, thus inhibiting galectin-3 expression on both protein
and mRNA level [9]. In skeletal tissues, the regulation of gal3 expression is mediated by
the transcription factor Runx2 [9]. Very recently, gal3 expression is found to be regulated
in pituitary and prostate tumors by methylation of CpG islands in promoter region [22, 24,
25], which is described later in a greater detail.
Epigenetic events- DNA methylation in the promoter results in transcriptional
Epigenetic mechanisms such as DNA methylation and histone modification play essential roles in many molecular and cellular alterations associated with the development
and progression of cancer [28]. DNA methylation (the addition of a methyl group to the
carbon-5 position of cytosine residues) refers to the covalent binding of a methyl group
specifically to the dinucleotide CpG (Fig. 2). This is catalyzed by a family of enzymes, the
DNA methyltransferases (DNMTs) [29]. Two types of DNA methylation alterations have
been demonstrated in human cancers. The first refers to global hypomethylation where
the genomes of cancer cells show decreased methylation compared to normal cells [30].
This hypomethylation is primarily due to the loss of methylation in repetitive elements and
other non-transcribed regions of the genome, which results in genomic instability. The
second type of methylation alteration in cancer cells is the methylation of CpG islands that
lie in promoter regions of tumor suppressor and other regulatory genes that are normally
unmethylated. The promoter regions of these genes are inactivated by methylation and
their gene expression silenced. This is referred to as gene hypermethylation. It is thought
that DNA methylation alters chromosome structure and defines regions for transcriptional
regulation. Clusters of CpG sites are found dispersed around the genome and are referred
to as CpG islands [31]. These islands are found in the promoter region of about 60% of
genes, in exons and introns, and in repetitive elements. In normal cells, most CpG islands
in the promoter regions are unmethylated whereas CpG islands in intronic regions and
repetitive sequences are heavily methylated, perhaps to help the cell identify regions for
gene transcription. The suppression of transcription by DNA methylation is probably due
to direct inhibition of transcription factor binding [32]. However, some transcription factors
are insensitive to methylation [32], and many gene promoters do not have CpG sites within
their recognition sequences. Recently, a more generally applicable mechanism by which
Hafiz Ahmed
DNA methylation can maintain transcriptional repression has been proposed. A family of
proteins known as methylbinding domain (MBD) proteins specifically binds to DNA containing methylated CpG sites [33]. At least three of the five known members of this family
(MeCP2, MBD2 and MBD3) have been shown to be associated with large protein complexes containing histone deacetylase (HDAC1 and HDAC2) and chromatin-remodeling (Sin3a
and mi-2) activities [34]. A large number of genes (such as DNA repair and tumor suppressor genes) have been found hypermethylated in cancer. These genes are correlated with
pathological grade or clinical stage, and thought to contribute to initiation and progression
of the disease [35].
Expression of gal3 gene is transcriptionally regulated by DNA methylation.
Gal3 was shown highly expressed in androgen independent PC-3 and DU-145 cells,
but weakly expressed in androgen dependent LNCaP cells [25]. Treatment of LNCaP cells
with azacytidine (DNA methyltransferase inhibitor) showed restored expression of gal3
indicating that the promoter methylation is responsible for gal3 gene silencing [25] (Fig. 3).
We have also demonstrated DNA methylation on the gal3 promoter in LNCaP cells. For this
purpose, DNA was treated with sodium bisulfite and subsequently amplified by PCR [25,
36] with primer pairs located outside the CpG sites. This method allows precise analysis of
methylation in a selected region by converting all non-methylated cytosines (C) into uracil
(U), while methylated cytosines remain unchanged. All cytosines of CpG sites (about 50)
in the gal3 promoter from LNCaP DNA were methylated in the 384 bp PCR product, but
no gal3 promoter methylation was observed in azacytidine-treated LNCaP. Similarly, no
methylation in the gal3 promoter from either PC-3 or DU-145 DNA was observed [22, 25].
Gal3 silencing by promoter methylation is not specific to prostate cancer cells, rather it is a
common mechanism for gal3 gene silencing in cancer, as treatment of two gal3 null breast
cancer cell lines BT549 [25] and SKBR3 [24] with azacytidine induced expression of gal3.
Expression of gal1 was also shown to be regulated by promoter methylation [37]. A small
genomic region of approximately 100 base pairs surrounding the transcriptional start site
(-50/+50) accounts for most transcriptional activity of gal1 [37].
Decreased expression of gal3 in pituitary and prostate tumors is associated with
increased methylation in the gal3 promoter.
Expression of gal3 in conjunction with its promoter methylation has been recently been
investigated in pituitary [24] and prostate [22, 25] tumors. Among pituitary tumors, mainly
in follicle-stimulating hormone/luteinizing hormone-producing (38%) and null cell (57%)
adenomas, the gal3 promoter was found to be methylated and silenced [24]. Interestingly,
the prolactin- and adrenocorticotropic hormone-producing tumors were unmethylated and
Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
expressed gal3 [24]. We investigated gal3 expression in normal, benign prostatic hyperplasia (BPH), and various stages of prostate adenocarcinoma [22]. Gal3 was found strongly
expressed in normal, BPH, and high-grade prostatic intraepithelial neoplasia (HGPIN, a
precursor lesion to development of invasive prostatic adenocarcinoma) tissues in both the
nucleus and the cytoplasm. However, gal3 showed decreasing immunopositivity during
stage evolution. Moreover, localization of gal3 is interesting during stage evolution. In particular, stage I tumors showed a strong immunopositivity both in nucleus and cytoplasm,
while in more advanced stages immunostaining was less intense and localized mainly in
cytoplasm, with rare, occasional nucleus positivity. However, two stage I specimens (out of
10) showed little or no gal3 immunopositivity [22]. Similar observation on gal3 expression
in prostate tumor was made by others [20,38]. The role of cytoplasmic and nuclear gal3 in
cancer progression was examined by specifically expressing gal3 in either cytoplasm or
nucleus of LNCaP, a gal3-negative human prostate cancer cell line [18]. Cytoplasmic gal3
was anti-apoptotic and promoted tumor growth and angiogenesis. Interestingly, nuclear
gal3 affected these parameters in an opposite fashion with an overall antitumoral activity [18]. The role of decreased expression of gal3 in early stage of prostate tumor is not
known. However, in normal cells gal3 is believed to interact to the members of Nkx homeodomain family [18], especially to a prostate tumor suppressor, Nkx3.1 [39] and decrease
the expression of the cancer phenotype [18]. Therefore, the silencing of gal3 gene during
the development of prostate tumor could be necessary to suppress the influence of Nkx3.1
gene and thereby to help tumor cells to proliferate.
Since gal3 expression is decreased in stage evolution of prostate adenocarcinoma as
evidenced from our studies [22] and others [20], we hypothesized that DNA isolated from
early stages of prostate cancer would be methylated in the gal3 promoter region. Results revealed that the gal3 promoter from multiple specimens of stage II tumor is heavily
methylated throughout its entire length, but that from multiple specimens of stage III and
IV tumor is lightly methylated. Whereas gal3 promoter in stage III showed few methylation
sites, mostly between -199 to -252 nt, the gal3 promoter from stage IV tumor specimens
was methylated between -112 to -227 nt. In stage I prostate cancer, however, both light
and heavy methylation is evident in the gal3 promoter. In multiple normal prostate and BPH
samples, the gal3 promoter was almost unmethylated. Overall, results indicated that the
decreased expression of gal3 in tumor prostate is associated with the hypermethylation of
its promoter.
Concluding remarks
Recent studies by us and others have shown that the expression of gal3 in prostate
and breast cancer cell lines is regulated by the DNA methylation in its promoter and is
Hafiz Ahmed
negatively correlated [22,24,25]. Moreover, decreased expression of gal3 in pituitary and
prostate tumors was found associated with hypermethylation in the gal3 promoter [22,24].
The gal3 promoter showed almost complete methylation in all CpG sites in early stages
of prostate cancer, but is lightly methylated in later stages. The methylation pattern of the
gal3 promoter at various stages of prostate cancer, and in particular its complete methylation in early stages, is unique, because in other genes such as GSTP1, CpG methylation
correlates positively with tumor grade and stage (i.e. low methylation in early stages and
high methylation in late stages) [40]. However, the degree of gal3 promoter methylation
and its expression at various stages of prostate cancer cannot be correlated from this
study [22]. This is because the tumor tissue is heterogeneous and so DNA population extracted from the tumor tissue may not necessarily represent single tumor stage. To answer
this question, however, further studies are needed from the micro-dissected tissues such
as those obtained from laser capture micro-dissection approach.
I apologize to the authors whose works are not cited due to the limited space. The work
carried out in the author’s laboratory has been supported by the UMBI Presidential Proof of
Concept Award and the National Institute of Health Grant RO3 CA133935-01.
Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
Fig. 1 - Schematic representation of lectin-mediated cell-cell and cell-extracellular matrix (ECM)
interactions. Galectins mediate interactions between cells or between cells and ECM by binding to laminin,
integrin and other cell surface glycoconjugates A representative of each type of galectins, proto (gal1), chimera (gal3), and tandem-repeat (gal8) is shown.
Fig. 2 - DNA methylation catalyzed by DNA methyltransferase. DNA methyltransferase transfers methyl
group from S-adenosyl methionine (SAM-CH3) to cytosine yielding S-adenosyl homocysteine (SAH) and
Hafiz Ahmed
Fig. 3 - Simplified cartoon showing gal3 transcription by unmethylated promoter (A) and gal3 gene
silencing by the methylated promoter (B). A. In normal prostate and pituitary tissues, gal3 promoter is
unmethylated and accessible to binding to the transcription factors such as AP-1 and Sp-1 for stimulation
of gal3 transcription. B. In prostate and pituitary tumors, gal3 promoter is methylated and therefore bound
by the methyl binding proteins (MBD) and histone deacetylase (HDAC). Thus the methylated promoter is not
accessible to binding to the transcription factors and inactive.
Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
[1] Ahmed H, Du SJ, O’Leary N, Vasta GR. Biochemical and molecular characterization of galectins from
zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis. Glycobiology. 2004; 14:219-32.
[2] Nakahara S, Raz A. Regulation of cancer-related gene expression by galectin-3 and the molecular
mechanism of its nuclear import pathway. Cancer Metastasis Rev 2007; 26:605-10.
[3] Rabinovich GA, Liu FT, Hirashima M, Anderson A. An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer.
Scand J Immunol 2007; 66:143-58.
[4] Nakahara S, Raz A. Biological modulation by lectins and their ligands in tumor progression and metastasis. Anticancer Agents Med Chem 2008; 8:22-36.
[5] Vasta GR.Roles of galectins in infection. Nat Rev Microbiol 2009; 7:424-38.
[6] Elola MT, Wolfenstein-Todel C, Troncoso MF, Vasta GR, Rabinovich GA. Galectins: matricellular glycanbinding proteins linking cell adhesion, migration, and survival. Cell Mol Life Sci 2007; 64:1679-700.
[7] Gong HC, Honjo Y, Nangia-Makker P, Hogan V, Mazurak N, Bresalier RS, Raz A. The NH2 terminus
of galectin-3 governs cellular compartmentalization and functions in cancer cells, Cancer Res 1999;
[8] Nakahara S, Oka N, Raz A. On the role of galectin-3 in cancer apoptosis. Apoptosis 2005; 10: 267-75.
[9] Dumic J, Dabelic S, Flögel M. Galectin-3: an open-ended story. Biochim Biophys Acta 2006; 1760:61635.
[10] Colnot C, Ripoche MA, Scaerou F, Foulis D, Poirier F. Galectins in mouse embryogenesis. Biochem Soc
Trans 1996; 24:141-6.
[11] Poirier F. Roles of galectins in vivo. Biochem Soc Symp 2002; 69:95-103.
[12] Liu FT, Rabinovich GA. Galectins as modulators of tumour progression. Nat Rev Cancer 2005; 5:2941.
[13] Inohara, H., et al., Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion. Cancer Res 1996; 56:4530-4.
[14] Glinsky VV, Glinsky GV, Rittenhouse-Olson K, Huflejt ME, Glinskii OV, Deutscher SL, Quinn TP. The role of
Thomsen-Friedenreich antigen in adhesion of human breast and prostate cancer cells to the endothelium. Cancer Res 2001; 61:4851-4857.
[15] Elad-Sfadia G, Haklai R, Balan E, Kloog Y. Galectin-3 augments K-Ras activation and triggers a Ras
signal that attenuates ERK but not phosphoinositide 3-kinase activity. J Biol Chem 2004; 279:3492230.
[16] Fukumori T, Takenaka Y, Yoshii T, Kim HR, Hogan V, Inohara H, Kagawa S, Raz A. CD29 and CD7 mediate
galectin-3-induced type II T-cell apoptosis. Cancer Res 2003; 63:8302-11.
[17] Danguy A, Camby I, Kiss R. Galectins and cancer. Biochim Biophys Acta 2002; 1572:285-93.
[18] Califice S, Castronovo V, Van Den BrГ»le F. Galectin-3 and cancer. In: J Oncol 2004; 25:983-92.
[19] Song YK, Billiar TR, Lee YJ. Role of galectin-3 in breast cancer metastasis: involvement of nitric oxide.
Am J Pathol 2002; 160:1069-75.
[20] Pacis RA, Pilat MJ, Pienta KJ, Wojno K, Raz A, Hogan V, Cooper CR. Decreased galectin-3 expression in
prostate cancer. Prostate 2000; 44:118-23.
[21] Merseburger AS, Kramer MW, Hennenlotter J, Simon P, Knapp J, Hartmann JT, Stenzl A, Serth J, Kuczyk
MA. Involvement of decreased Galectin-3 expression in the pathogenesis and progression of prostate
cancer. Prostate 2008; 68:72-7.
Hafiz Ahmed
[22] Ahmed H, Cappello F, Rodolico V, Vasta GR. Evidence of heavy methylation in the galectin-3 promoter in
early stages of prostate adenocarcinoma: Development and validation of a methylated marker for early
diagnosis of prostate cancer. Translational Oncology 2009 (In press).
[23] Merseburger AS, Kramer MW, Hennenlotter J, Serth J, Kruck S, Gracia A, Stenzl A, Kuczyk MA. Loss
of galectin-3 expression correlates with clear cell renal carcinoma progression and reduced survival.
World J Urol 2008; 26:637-42.
[24] Ruebel KH, Jin L, Qian X, Scheithauer BW, Kovacs K, Nakamura N, Zhang H, Raz A, Lloyd RV. Effects of
DNA methylation on galectin-3 expression in pituitary tumors. Cancer Res 2005; 65:1136-40.
[25] Ahmed H, Banerjee PB, Vasta GR. Differential expression of galectins in normal, benign and malignant
prostate epithelial cells: Silencing of galectin-3 expression in prostate cancer by its promoter methylation. Biochem Biophys Res Commun 2007; 358: 241-6.
[26] Abedin MJ, Kashio Y, Seki M, Nakamura K, Hirashima M. Potential roles of galectins in myeloid differentiation into three different lineages. J Leukoc Biol 2003; 73:650-6.
[27] Kadrofske MM, Openo KP, Wang JL. The human LGALS3 (galectin-3) gene: determination of the gene
structure and functional characterization of the promoter. Arch Biochem Biophys 1998; 349:7-20.
[28] McKenna ES, Roberts CW. Epigenetics and cancer without genomic instability. Cell Cycle 2009; 8:23-6.
[29] Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de
novo methylation and mammalian development. Cell 1999; 99:247-57.
[30] Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 2002; 21:5400-13.
[31] Cross SH, and Bird AP. CpG islands and genes. Curr Opin Genet Dev 1995; 5:309-14.
[32] Tate PH, Bird AP. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin
Genet Dev 1993; 3:226-31.
[33] Hendrich B, Bird A. Identification and characterization of a family of mammalian methyl-CpG binding
proteins. Mol Cell Biol 1998; 18:6538-47.
[34] Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, Bird A. Transcriptional repression
by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998;
[35] Das PM, Singhal R. DNA methylation and cancer. J Clin Oncol 2004; 22:4632-42.
[36] Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, and Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.
Proc Natl Acad Sci US. 1992; 89:1827-31.
[37] Salvatore P, Benvenuto G, Caporaso M, Bruni CB, Chiariotti L. High resolution methylation analysis of the
galectin-1 gene promoter region in expressing and nonexpressing tissues. FEBS Lett 1998; 421:152-8.
[38] van den BrГ»le FA, Waltregny D, Liu FT, Castronovo V. Alteration of the cytoplasmic/nuclear expression
pattern of galectin-3 correlates with prostate carcinoma progression. Int J Cancer 2000; 89:361-7.
[39] Kim MJ, Bhatia-Gaur R, Banach-Petrosky WA, Desai N, Wang Y, Hayward SW, Cunha GR, Cardiff RD,
Shen MM, Abate-Shen C. Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis.
Cancer Res 2002; 62:2999-3004.
[40] JerГіnimo C, Henrique R, Hoque MO, Mambo E, Ribeiro FR, Varzim G, Oliveira J, Teixeira MR, Lopes C,
and Sidransky D. A quantitative promoter methylation profile of prostate cancer. Clin Cancer Res 2004;
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare
[1859: Naturalism. Motifs for the Evolution and Cellular Anatomy]
Aldo Gerbino
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia “Arcangelo Pasqualino di Marineo”, Facoltà di Medicina e Chirurgia, Università degli Studi di Palermo (IT)
Parole chiave: Evoluzione, Radiolari, Peduncolo, Flagello, FC, LCA
Key words: Evolution, Radiolarians, Peduncle, Flagellum, First Cell, Last Common Ancestor
Relazione su invito per: “Incontri di Morfologia Funzionale”, Dipartimento di Anatomia Umana, Farmacologia e Scienze Medico Forensi di Parma, nell’ambito del Forum: “La morfologia
delle costituzioni nella tradizione anatomica dello Studium di Parma: Lorenzo Tenchini (18521906), Cesare Lombroso (1835-1909)”; a cura di Roberto Toni, Marco Vitale e Guglielmo Masotti (Parma, 25 marzo 2009 Aula Canuto-Ospedale Maggiore. Facoltà di Medicina e Chirurgia, Università degli Studi di Parma; Società di Medicina e Scienze Naturali di Parma).
27 dicembre 1831. Il brigantino �Beagle’, armato di dieci cannoni, salpa da Devonport
al comando del capitano Fitzroy; a bordo, un giovane intellettuale d’eccezione, Charles
Darwin, ГЁ pronto ad intraprendere un viaggio che avrГ la durata di ben cinque anni (1836).
Un lustro per un’incubazione e un fermento speculativo che lo trasformeranno, da curioso
osservatore della natura, in uno scienziato sorretto da una pertinace idea del mondo, portatore, al fine, di un metodo, d’una rivoluzionaria poetica della natura. Il 13 aprile del 1834,
il �Beagle’, ancorato alla foce del Santa Cruz, fiume a 100 Km a Sud di Porto San Giuliano,
in Patagonia, segna la dimensione assoluta della sua ricerca estrema, in un territorio compreso tra valli di basalto ed una inquietante omogeneitГ geologica, ma, allo stesso tempo,
stimolatrice di suggerimenti speculativi (Fig. 1). CiГІ che in natura appare, a prima vista,
poco significativo, nasconde molto spesso tra le sue pieghe un mondo variegato, tutto da
rivelare e decifrare, in cui ГЁ possibile avviare nuove comparazioni, focalizzare insospettati
comportamenti. Una regione che, se in apparenza si mostra «completamente priva d’interesse», disegna invece un vivido scenario metafisico. Darwin annoterà nel suo �Diario’, tra
i tanti aspetti etologici che lo attraggono, quello del cannibalismo: В«Per quanto povera sia
sotto certi aspetti, la Patagonia puГІ vantare il maggior numero di piccoli roditori di quello
che possa farlo qualsiasi altra parte del mondo. Parecchie specie di topi sono caratterizzate esternamente da orecchie grandi e sottili e da una bellissima pelliccia. Questi animali
Aldo Gerbino
Fig. 1. Charles Darwin. Il �Beagle’ ancorato alla foce del Santa Cruz. L’Origine della specie per elezione naturale, pubblicata a Londra nel 1859, nella sua prima traduzione italiana del 1864.
abbondano fra i boschetti nelle valli, dove per mesi di seguito non possono assaggiare una
sola goccia d’acqua, tranne la rugiada. Sembrano tutti cannibali perché non appena un
topo era preso in una delle mie trappole, veniva divorato dagli altri.» [1] Scorrendo l’enorme mole di notizie raccolte in questo esaltante viaggio, possono di continuo essere percepiti le compenetrazioni esistenti tra �generale’ e �particolare’, tra ambiente e animali che lo
popolano, tra animali e piante, tra macro e microstrutture, tutti nell’accezione posta nella
celebre frase di Goethe: В«Il generale e il particolare coincidono; il particolare ГЁ il generale
che appare sotto condizioni diverse» [2]. Un’espressione che, per camminamenti diversi,
ritrova le sue coincidenze nel naturalista inglese, per quell’essere portata, sottolinea Giuseppe Zamboni scrivendo sulle �riflessioni’ goethiane, «a stabilire leggi organiche nella vita
dell’uomo e della natura», tendendo, così, a «sollevarsi dall’individuale e particolare all’idea
e alla regolaВ» [3]. Anche Darwin troverГ la sua regola, quella regola che detiene il governo
del mondo, portatrice di una palmare potenzialità trasformativa: l’“evoluzione”.
1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare
Fig. 2. a. Ernst Haeckel e l’acquerello raffigurante lo Stretto di Messina. b. Tavola XXXIII del Die Radiolarien
Tale strettissimo vincolo in cui il particolare è visto quale modello dell’interezza biologica
e in cui la cellula (l’unicellulare) si esprime in forma di marchio arcaico, evolutivamente
disposto alla mutazione, lo ritroviamo nella misura culturale di un intellettuale e morfologo
d’eccezione: Ernst Haeckel (1835-1919), l’autore del celebre Die Radiolarien (1862), della
notissima Natürliche Schöpfungsgeschichte (“La storia della creazione naturale”, 1868) la
quale, con le sue ben dodici ristampe, rappresenta l’opera che chiama a raccolta «tutte le letture sulla teoria dell’evoluzione in generale e quelle di Darwin, Goethe e Lamark
in particolare, sull’applicazione di queste teorie all’origine dell’uomo e ad altre questioni
fondamentali della scienza» [4], contribuendo, così, alla comprensione e conoscenza della
ricerca darwiniana. È del 1874 la monografia sulle �spugne calcaree’ per le quali postula
come «l’evoluzione ontogenetica ricapitola la filogenesi», e, infine, nel 1870, la stampa della
laboriosa Antrhopogenie oder Entwickelungsgeschichte des Menschen (“Antropogenia o la
storia dello sviluppo del genere umano”). Haeckel si era formato alla università di Wurzburg
seguendo il chimico analitico Scherer, l’istologo Rudolf Albert von Kölliker (1817-1905), il
patologo Rudolf Ludwig K. Virchow (1821-1902), ma, pur studiando medicina, dichiara con
convinzione di vedere «l’anatomia esclusivamente dal punto di vista della storia naturale dell’uomo, non dal punto di vista medico!». L’anatomia cellulare pervade la mente di
Haeckel interrelando le sue conoscenze biologiche e fisiologiche con i dati mineralogici
di Christian Samuel Weiss (1780-1856), colui il quale definì, in maniera inoppugnabile, la
simmetria dei cristalli. Allora, i radiolari non sono soltanto organismi unicellulari che vivono
nelle grandi vertigini dei mari sin dal periodo precambriano, ma, come intuito da Haeckel,
Aldo Gerbino
Fig. 3. Dendrogrammi di linee filetiche. Da Martino Rizzotti, 1998 (mod.).
rappresentano un’estetica, una �cristallografia organica’, una filosofia, una scienza ed anche
un’arte, tanto da imprimere nell’immaginario artistico di René Binet la possibilità di ricreare
forme e contenuti ideali nelle versioni architettoniche destinate alla �Esposizione’ parigina
del 1900; una estetica che trovò fertile substrato creativo nel tessuto espressivo dello Jugendstil [5, 6]. Con Haeckel sopravvivono le Nereidi dello Stretto di Messina, l’antica Zancle,
per quelle migliaia di forme in migrazione capaci di alimentare il sospiro del mondo, l’anima
stessa delle nostre ombre. Dello stretto messinese esiste un lontano acquerello che ci narra
delle sue ricerche operate in questo luogo del mito. Stimolato dal pittore Hermann Allmers
(1821-1902), il suo percorso scientifico sui Radiolari restituisce, proprio attraverso i capillari
disegni, la meraviglia degli “Sphaerozoum” nelle varietà �italicum’, �spinosum’, �ovodimare’,
�punctatum’, o quelle della “Aulacantha scolymantha”, e della “Thalassicolla Zanclea”, o del
“Dictyophimus Tripus”, e, ancora, delle eleganti geometrie delle “Heliosphaere” (�inermis’,
�tenuissima’, �actinota’, �echinoides’, �elegans’) oppure delle stellari iridescenze che circondano il pulviscolo costituito dalle catenarie di “Dictyocha Messanensis” [7] (Fig.2, a, b) .
1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare
Fig. 4. a. Cellula di lievito. b. Flagello batterico (Gram-). Da Vera Bianchi, Pietro Amodeo, 1983 (mod.).
È di estremo interesse comprendere, nell’ecosistema cellulare, come i vari compartimenti, embricati nell’ambiente cellulare, entrino nella sostanza del processo filogenetico in
rapporto sinergico, ora attraverso il comparto endocellulare (�locus genico’), ora attraverso
quello extracellulare. Sappiamo, inoltre, quale grande importanza rivestano, ad esempio, le
parti mobili delle cellule; cioГЁ quelle specializzazioni che sembrano essere alla base delle
primitive tappe filogenetiche, capostipiti per nuovi filomeri, attori nella confezione futura
di una cronospecie. CiГІ in virtГ№ dello sviluppo di quelle cellule evolutive identificate come
First Cell (FC, prima cellula capace di traduzione), ed LCA (Last Common Ancestor o Cenancestor), l’ultimo progenitore comune dell’attuale biosfera (3,5→1,8 mld anni; Fig. 3). Un
esempio di compartecipazione biologica tra le varie classi di cellule (procarioti ed eucarioti)
può essere alimentato dalla “cellula del lievito” (eucariotica/ancestrale), tradizionalmente
ascritta ai funghi ascomiceti o lieviti (saccaromiceti). Durante la sua divisione, da corpiccioli
posti in prossimitГ del nucleolemma, si formano microtubuli che entrano in rapporto con i
cromatidi. In essa coesistono la �parete’ polisaccaridica di stampo procariotico e gli organelli
citoplasmatici, una novitГ evoluzionistica (mitocondri, complesso del Golgi, reticolo endoplasmatico granulare), inoltre: un distinto compartimento nucleare delimitato da una membrana (costituita da due membranelle unitarie di 2x6 nm con una intercapedine di 20 nm)
attraversata da pori, mentre, nella cariolinfa, sono presenti alcuni cromosomi, un nucleolo e
delle vescicole con materiale di riserva. La “Ochromonas danica” (una cloroficea delle alghe
verdi, eucariota) mostra, giГ organizzato, un flagello ed un corpuscolo basale; inoltre, un
cloroplasto (marchio delle cellule vegetali) con uno stigma a forma di sferule, e, al di sotto,
Aldo Gerbino
Fig. 5. Ipotesi del peduncolo in un eucariote unicellulare. Da Martino Rizzotti, 1998 (mod.).
un fotorecettore. L’ “Euglena gracilis” (un’altra alga verde eucariota, fornita di flagelli simmetrici) offre all’osservazione cloroplasti multipli ricchissimi di clorofilla, mitocondri, e varie
figure organulari. Quadri biologici, questi, interpretati attraverso fenomeni endosimbiontici e
inseriti in un ventaglio polifiletico dove coesistono, pur avendo avuto origine da uno stesso
philum, forme arcaiche e forme evolutivamente avanzate. Anche gli apparati mobili cellulari
mostrano similitudini e divergenze sul piano della complessitГ molecolare tra procarioti ed
eucarioti. Nelle procellule (batteri) troviamo un meccano-enzima formato da 6 subunitГ (giГ presente in LCA), innestato in una sorta di motore rotante destinato a imprimere al flagello
un movimento passivo. Nelle eucellule (dalla rodoficee alle cellule dei primati) le macchine
proteiche, legate alla genesi del movimento, mostrano complessi di actina e miosina o di
tubulina e dineina, e, nel ciglio vibratile della cellula eucariotica, troviamo un codificato apparato mobile espresso in una articolazione biologica in cui agiscono 600 proteine (Fig. 4, a,
b) [8]. Appare particolarmente interessante, quanto suggestiva, nella genesi del movimento,
l’ipotesi del peduncolo [9], in cui il “sistema mobile ciglio” sembra, infatti, trarre origine da
1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare
un �peduncolo’ sorretto dai microtubuli presenti nell’eucariote unicellulare (per specializzazione del citoscheletro); oppure, come è d’altre fonti sperimentali sostenuto, le componenti
microtubulari verrebbero assunte, per simbiosi extracellulare, da altri microrganismi (Spirocheta) [10]. La formazione originaria di un peduncolo emesso da un corpo cellulare, sospinto da necessitГ alimentari, organizzerebbe i microtubuli disponendoli in una architettura
ordinata nella esemplare ultrastruttura dell’assonema (9+2), consentendo un’interazione
con bracci di una dineina primitiva, proteina ad alta attivitГ ATPasica (Fig. 5).
Sollevarsi da un substrato, con una rete di microtubuli allineati in parallelo, consentirebbe
alle cellule ancestrali di acquisire una maggiore flessibilitГ ed autonomia dal substrato originario, e, quindi, essere oggetto di una probabile selezione filogeneticamente piГ№ vantaggiosa.
Un vantaggio che potrebbe essere non esclusivamente ricondotto alla individualitГ cellulare
ma, secondo la “regola di Hamilton”, al gene “altruistico” (1964), a quei geni comuni, già osservati da J. B. S. Haldane [11] nel 1955, condivisi da una molteplicità di familiari, e che
verrebbero altruisticamente preservati durante il processo evolutivo. Durante tale processo
si assiste, comunque, ad un incremento della complessità (in senso �epigenetico’: domini degli aggregati biologici agenti nella categoria spazio/tempo) rispetto alla �semplicità ’ operativa
genica; la definizione di cellula, allora, letta piГ№ comunemente come un sistema dualistico
(geni/proteine: genotipo/fenotipo), dovrebbe essere interpretata, secondo i suggerimenti di
Barbieri, anche come entitГ triadica (geni/codemaker/proteine: genotipo/fenotipo/ribotipo),
dove il �codemaker’ (il «fabbricante di codici organici») è caratterizzato dal ribotipo, cioè dal
sistema delle ribonucleoproteine impegnato a leggere e costruire i materiali proteici [12].
Dopo i tempi prebiotici, caratterizzati da una evoluzione della materia inanimata (evoluzione
conformazionale), l’evoluzione adattativa (darwiniana) trova una sua specificità con la costruzione di linee filetiche adattabili alle varie esigenze ambientali le quali, allontanandosi
sempre piГ№ dalla condizione di immobilitГ e fissitГ propria della materia inanimata [13],
conserverebbero nel grande bagaglio della memoria chimica progetti che saranno ritrovati
nella loro futura biologia. Le cellule riprodurranno e rifletteranno forme e significati di transizione, traducendo, trasformando e facendo proprie nuove orme �reali’ di appartenenza. I
cambiamenti evolutivi identificano nuovi filomeri e aggregano citotipi in quell’ammodernato
“darwinismo di Edelman” [14] che inevitabilmente conduce ad una revisione epistemologica
del concetto di �forma’ [15], per una morfologia cellulare, specchio primario della corporeità futura, sollecitata, soprattutto nella sua fase di sviluppo, nel delicato momento della aggregazione e della selezione cellulare. Ciò è quanto fu notato anche da Haeckel, quando, nel
lontanissimo settembre del 1869, al largo dell’isola norvegese di Gisoe, descrivendo il minuscolo flagellato dalla forma sferica, la Magosphera planula, caratterizzò il suo plastico ciclo
vitale, ponendolo quale anello di transizione tra la fase unicellulare e quella pluricellulare
[16]. Dai protozoi ai metazoi; dunque, verso le scandite bioarchitetture delle “città cellulari”.
Aldo Gerbino
[1] Charles Darwin. Viaggio di un naturalista intorno al mondo (Trad. di M. Magistretti). Firenze, Giunti-Martello, 1982.
[2] Wolfgang Goethe. Aus den maximen und reflexionen ein brevier (Trad. It., “Breviario dalle Massime e
Riflessioni”). Firenze, Fussi, 1950.
[3] Giuseppe Zamboni, in Goethe, op. cit., p. 11.
[4] Olaf Breidbach, in: Ernst Haeckel, The Radiolarian Atlas of 1862. London, Prestel, 2005.
[5] Sylvain Ageorges. Sur les traces des Expositions universelles, Paris, Parigramme, 2007.
[6] AA.VV. Paris 1900. Paris, MusГ©e Carnavalet, 2001.
[7] Aldo Gerbino. 1859: Naturalism. Motivs for the Evolution and Cellular Anatomy. В«Acta Biomed.В», in
[8] Vera Bianchi, Pietro Amodeo (a cura). Anatomia della cellula. Milano, В«Le ScienzeВ» (quaderni), 1983.
[9] Martino Rizzotti. Prime tappe dell’evoluzione cellulare. Bologna, Decibel/Zanichelli, 1998.
[10] Lynn Margulis. Archaeal-eubacterial mergers in the origin of Eukarya. В«Proc. Natl. Acad. Sci.В», USA,
93:1071-1073, 1996; Id.: Symbiosis in cell evolution. New York, Freeman, 1993.
[11] William Donald Hamilton. The collected papers of W.D. Hamilton, Narrow Road of Gene.New York, LH
Freeman, 1996.
[12] Marcello Barbieri. I codici organici: metafore o realtГ ? В«Systema NaturaeВ», 5: 99-113, 2003.
[13] Nikolay P. Kolomiytsev, Nadezhda Ya. Poddubnaya. L’origine della vita come risultato del cambiamento
del meccanismo evolutivo. В«Biology ForumВ», 100: 11-26, 2007.
[14] Gerald M. Edelman. Topobiology, an Introduction to Molecular Embryology. New York, В«Basic BooksВ»,
1988; Id.: Topobiology. В«Scientific AmericanВ», May 76-88, 1989; Id.: CAMs and Igs: Cell Adhesion and
the Evolutionary Origins of Immunity. В«Immunol. Rev.В», 100: 9-43, 1987b.
[15] Roberto Toni, Epistemologia della forma corporea. В«Systema Naturae, Annali di Biologia TeoricaВ», 2:
200-333, 1999a; Id.: Topobiology: Epistemological Implication of an Ontic Theory in Biomorphology.
В«EpistemologiaВ», XXVII: 83-106, 2004.
[16] Andrew Reynolds, Norbert Hülsmann. Ernst Haeckel’s Discovery of Magosphera planula: A Vestige of
Metazoan Origins? В«History and Philosophy of the Life SciencesВ», 30: Вѕ, 2008.
Proceedings & Meetings
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Ambiente staminale e prospettive di transdifferenziamento verso il fenotipo osteoblastico
[Staminal environment and transdifferentiation perspectives towards the osteoblastic phenotype]
Annamaria Mauro, Luana Lipari, Maria Buscemi e Aldo Gerbino
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia “Arcangelo Pasqualino di Marineo”, Facoltà di Medicina e Chirurgia, Università degli Studi di Palermo (IT)
Key words: MSCs, Transdiffentiation, Wharton Jelly
Parole chiave: Cellule Staminali Mesenchimali, Transdifferenziazione, Gelatina del Wharton
Abstract. In the last years there is a growing interest in the use of adult stem cells for the treatment of many
pathologies. Our group has focused its attention on the study of “mature mucous tissue” and on the stem
cells that can be isolated from it; Mesenchimal Stem Cells (MSCs) derived from the Wharton’s jelly of umbilical cord and stem cells from dental pulp.
The isolation of these types of cells is obtained through Fluorescence Activated Cell Sorter (FACS) targeting
specific stem cell markers (surface antigens).
The differentiation towards the phenotypes of interest is induced exposing the stem cells to specific differentiation media; in particular the osteogenic differentiation is induced with a medium containing Dexamethasone, Ascorbic acid and ОІ-Glycerophosphate, while the condrogenic differentiation can be induced with
a medium containing Transforming Growth Factor (TGF), Dexamethasone, Ascorbic acid, Sodium Pyruvate,
Insulin, Transferrin and Linoleic acid.
Negli ultimi anni la ricerca e la medicina stanno tentando sempre di più di avvalersi dell’uso delle cellule
staminali come strumento per la cura di molte patologie. Le cellule staminali adulte o somatiche suscitano
un particolare interesse in quanto piГ№ facili da reperire
e sottoposte a minori restrizioni di carattere legislativo
o etico. Una caratteristica delle cellule staminali adulte
di fondamentale importanza per le applicazioni cliniche, è la loro “plasticità ” o capacità di “transdifferenziare”, cioè la possibilità di differenziarsi in tipi cellulari
caratteristici di altri tessuti.
Il nostro gruppo ha da diversi anni rivolto la sua attenzione allo studio del tessuto mucoso maturo e delle
cellule staminali che da esso possono essere isolate:
Annamaria Mauro, Luana Lipari, Maria Buscemi, Aldo Gerbino
cellule staminali da polpa dentale e cellule staminali mesenchimali (MSCs) derivate dalla
gelatina di Wharton del cordone ombelicale.
L’isolamento delle cellule staminali dalla gelatina di Wharton viene ottenuta con la citofluorimetria di flusso (FACS) utilizzando markers specifici per cellule staminali mesenchimali (CD105, CD44 e CD73) [1]. Il differenziamento in senso condrogenico viene indotto
con un mezzo contenente: TGF (transforming growth factor), 100 nM dexamethasone, acido ascorbico, sodio piruvato, insulina, trasferrina e acido linoleico. Il differenziamento in
senso osteogenico viene indotto con un mezzo contente 100 nM dexamethasone, acido
ascorbico e 10 mM glicerofosfato per 4 settimane [1], oppure con l’esposizione alla Bone
Morphogenetic Protein 2 (BMP2) [2].
Per confermare il potenziale osteogenico delle cellule sottoposte ad induzione vengono ricercati alcuni markers specifici come la fosfatasi alcalina, sialoproteina ossea, il
collagene tipo I, l’osteocalcina e l’osteopontina. Viene valutata anche la risposta a fattori
osteoinduttivi come l’ormone paratiroideo, IGF1, BMP2 e TGF-β1.
Da qualche tempo cellule staminali sono state isolate anche dalla polpa dentale, ciГІ ГЁ
di grande interesse in quanto il rapporto cellule staminali/massa di questo tessuto ГЁ uno
dei più elevati ed inoltre sia il livello proliferativo di queste cellule che la loro “capacità di
transdifferenziamento” sono ragguardevoli. Le cellule staminali estratte dalla polpa dentale vengono separate mediante FACS in base all’espressione di specifici markers staminali
quale ad es. STRO-1. Il differenziamento in senso osteogenico viene indotto utilizzando
dexamethasone, acido ascorbico e beta-glicerofosfato, mentre il differenziamento in senso
condrogenico con acido ascorbico, sodio piruvato e TGF-ОІ [3].
Le MSCs sono state utilizzate in vari trattamenti: difetti ossei in modelli animali sperimentali e in piccoli gruppi di pazienti. I fattori cruciali nel controllo della proliferazione
e differenziazione non sono del tutto noti. Tra di essi il TGF-ОІ che media le funzioni di
osteoblasti/osteoclasti e la LTBP che media l’accumulo di TGF-β nell’osso e l’accumulo di
matrice extracellulare [4]. Topi knockout per LTBP-3 mostravano una ridotta proliferazione
cellulare e una ridotta differenziazione osteogenica.
Referenze bibliografiche
Karahuseyinoglu S et al. Stem Cells. 2007 Feb; 25(2):319-31.
Hou T et al. Tissue Eng (Part A). 2009 Feb 18.
Lindroos B et al. Biochem Biophys Res Commun. 2008 Apr 4;368(2):329-35.
Koli et al. Bone. 43(2008) 679-688.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
[Cause della rinite allergica e dell’asma]
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone and Vincenzo Bellia
Dipartimento di Medicina, Pneumologia, Fisiologia e Nutrizione Umana (DIMPEFINU), University of Palermo,
Palermo (IT)
Key words: Allergic Rhinitis, Asthma
Parole chiave: Rinite Allergica, Asma
Abstract. Many allergic diseases may involve the respiratory tract: the most common are rhinitis and asthma.
These are linked one to the other, according to the concept of “one airway, one disease”. These two diseases
are common worldwide and there is evidence that their prevalence has been increasing in the last decades.
Several studies have clearly shown that modifications of the pattern of microbial exposure of children associated with social and economic development represent a critical factor underlying the rising severity and
prevalence of atopic disorders. These observations are the basis for the so-called “hygiene hypothesis”, that
could be a reasonable explanation for the allergy epidemics. The purpose of the present review is to sum up
how allergy is linked to the development of rhinitis and asthma.
Riassunto. Numerose malattie allergiche possono coinvolgere
l’apparato respiratorio: le più comuni sono l’asma e la rinite allergica, che sono anche legate l’una all’altra secondo il concetto “una
via aerea, un’unica malattia”. Queste due malattie sono molto comuni
in tutto il mondo e la loro prevalenza ГЁ aumentata negli ultimi anni.
Numerosi studi hanno chiaramente dimostrato che le modificazioni
nel tipo di esposizione microbica dei bambini associato con lo sviluppo socio-economico rappresenta un fattore critico che sta alla base
dell’aumento di gravità e di prevalenza delle malattie atopiche. Queste
osservazioni sono il fondamento della cosiddetta “ipotesi igienica”,
che potrebbe essere una ragionevole spiegazione dell’epidemia allergica. Lo scopo della presente revisione è quello di riassumere come
l’allergia sia legata allo sviluppo della rinite e dell’asma.
Allergic rhinitis is defined as a symptomatic disorder
of the nose, induced after allergen exposure, by an IgEmediated inflammation of the nasal membranes [1]. It is
characterized by symptoms of nasal congestion, rhinorrhea, sneezing, and itching, and it is often associated with
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia
ocular symptoms [2,3]. The first description of “hay fever” was formulate by John Bostock
[4] who observed a patients suffered form allergy with both rhinitis and asthmatic manifestations. He wrote: “About the beginning or middle of June in every year… a sensation of
heat and fulness is experienced in the eyes... to this succeeds irritation of the nose producing sneezing… to the sneezings are added a further sensation of tightness of the chest,
and a difficulty of breathing”
On the basis of the timing of exposure allergic rhinitis was previously distinguished
into seasonal, perennial, and occupational. This subdivision is not entirely satisfactory. A
novel classification of allergic rhinitis based on duration: subdivides into “intermittent” and
“persistent” disease. Depending on severity of symptoms and quality of life impairment,
the disease is further subdivided into “mild” or “moderate-severe” forms,.
The counterpart of rhinitis at the level of intrapulmonary airways is asthma: it is defined
as a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role [5]. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and
coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable, airflow obstruction of airways that is often reversible
either spontaneously or with treatment.
Rhinitis and asthma are pathogenetically linked one to the other, suggesting the concept
of “one airway, one disease” [1]. Indeed, many cross-sectional studies have demonstrated
that rhinitis and asthma occur commonly together. Moreover, patients with allergic rhinitis
and no clinical evidence of asthma frequently exhibit bronchial hyperresponsiveness to
bronchoconstrictor agents such as methacholine or histamine. For these reasons, some
investigators have postulated that bronchial hyperreactivity may represent an intermediate phase along the route leading from nasal allergy to symptomatic asthma [6]. It is well
demonstrated not only that the rhinitis impacts on asthma but also that the severity of the
two conditions are correlated [7,8].
Allergic rhinitis is a common disease worldwide, affecting at least 10 to 25 % of the
population and its prevalence is increasing. Although it is not usually a severe disease, it
affects health-related quality of life, and it is a risk factor for asthma [1].
Asthma is a worldwide problem as well, with an estimated population of 300 million
affected individuals [9]. Asthma is the third leading cause of hospitalization among persons
under 18 years of age in the United States [12]. There is evidence that asthma prevalence
has been increasing in the last decades in some countries, including Italy [10,11]. According
to the Centers for Disease Control and Prevention, the prevalence of asthma among U.S.
Causes of allergic rhinitis and asthma
children increased from 3.6% in 1980 to 5.8% in 2003. There is widespread concern about
such a rising in prevalence in developed world: in these countries the economic and humanitarian effects of asthma are probably greater and the prevalence is also rising [13].
Analyses of the cost of asthma lead to conclude that the burden of the disease depends
on on the effectiveness of prevention and early therapy of exacerbations since emergency
treatment is far more expensive than regular treatment [5]. In Italy, it has been calculated
that direct costs account for 47.3% of the total economic burden of the disease, medications being the largest component [14]. In 2001 direct medical costs for asthma were
above € 800.000.000, i.e. 1% of the entire Italian health care system expenses, asthma
medications being responsible for € 650.000.000 [15].
A number of risk factors are associated with the development of asthma, including (but
not limited to) sensitisation to aeroallergens, maternal diet during pregnancy and/or lactation,
pollutants (particularly environmental tobacco smoke), microbes and their products, and psychosocial factors. However, evidence for effectiveness of avoidance measures in preventing
asthma is lacking in many cases. Factors that influence the development and the expression
of asthma include host factors (which are primarily genetic, i.e. atopy) [16] and environmental factors (including viruses) [17], allergens and occupational exposures [18]. However, the
mechanisms that influence the development and expression of asthma are complex.
Epidemiological studies [19-25] have clearly shown that modifications of the pattern of
microbial exposure of children associated with Westernisation represent a critical factor
underlying the rising severity and prevalence of atopic disorders: the so-called “hygiene
hypothesis” will be discussed below. This mechanism is a reasonable explanation for the
“allergy epidemics” [13].
Although allergy is closely linked to asthma, a large proportion of asthmatics are not allergic. Therefore, the mechanisms reviewed hereafter may be not valid for such patients.
Asthma pathogenesis: allergic inflammation in asthma
Airway inflammation in asthma is a multicellular process involving mainly eosinophils,
neutrophils, lymphocytes and mast cells, with eosinophilic infiltration being the most peculiar feature [26]. The inflammatory process involves central airways and spreads distally
to include the small airways [27,28].
A fundamental feature of asthma associated with allergic sensitisation is the ability of
the airways to recognize common environmental allergens and to generate a Th2 cytokine
response to them. IgE antibodies have been linked to the severity of asthma [29] and the
early and late phase responses of the airway to allergens.
To initiate the synthesis of IgE, inhaled allergens must encounter dendritic cells situated in the airway epithelium and submucosa and extending their processes to the airway
surface [30,31]. These dendritic cells uptake and process the inhaled allergens and then
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia
they migrate to draining lymph nodes, where they present processed antigen to T and B
cells [32]. Interactions among these cells in presence of specific cytokines (such as interleukin-4 or IL-13) and other mediators prompt B cells to the production of IgE antibodies.
After their release by B cells, IgE antibodies bind to high-affinity IgE receptors on the surface of tissue mast cells or peripheral-blood basophils [32]. The interaction of allergen with
receptor-bound IgE molecules causes activation of the cell and the release of preformed
and newly generated mediators (such as IL-4 or IL-13). In addition uptake of allergen is
enhanced by IgE bound to high affinity receptors on dendritic cells that facilitate allergen
internalisation [33]. These mechanism contribute to amplify the synthesis of IgE.
Several data demonstrate that about 40% of the Western population is atopic and have
elevated IgE antibodies to common environmental allergens, only about 7% express their
atopy in the form of asthma [34]. On this basis, it has been proposed that the immune
response to allergens is regulated at the surface of the airways: this mechanism should
explain why only a small part of patients with atopy show a specific expression in the
conducting airways [34].
The role of the type 2 T helper (Th2) cell-mediated immune response against “innocuous” environmental antigens (allergens) in the immunopathogenesis of allergic atopy is
well documented. It has been proposed that the immune system of the newborn infant is
skewed toward Th2 cells and needs timely and appropriate environmental stimuli to create
a balanced immune response. in this contest, infections have been identified as factors
that enhance Th1-mediated responses and that are associated with a reduced incidence
of allergy and asthma [32]. Several data suggest that allergen-specific T-cell response can
be shifted from Th2 to Th1 by microbial products that favour the production of IL-12 and
interferons by cells of the innate immunity, thus strongly supporting the concept that many
bacterial and viral infections that stimulate dendritic cells and natural killer cells may not
only promote Th1 polarization but also inhibit the development of Th2 cells. The effect of
reduced microbial stimulation on the Th2вЃ„Th1 balance (missing immune deviation) should
lead to the over expression of Th2 cells-mediated immune and in turn it should favour the
development of allergy [35].
The link between the Th1/Th2 balance and allergic diseases is mediated in part by IgE:
Th2 cells, by secreting IL-4 and IL-13, promote immunoglobulin class-switch recombination to IgE [36].
Hygiene hypothesis
As it has been presented above, the hygiene hypothesis links epidemiological observations on the rising in prevalence of atopic disorders associated with the Westernisation
[21-24] to experimental evidence of Th2вЃ„Th1 imbalance due to reduced microbial stimula348
Causes of allergic rhinitis and asthma
tion (37-40). In addition, the importance of reduced activity of T regulatory cells (reduced
immune suppression) has been emphasized [41-43].
In discussing the hygiene hypothesis at least three aspects can be considered: i) bacterial, viral and parasites infections in early life; ii) exposure to microbial compounds, such
as endotoxins, during childhood; iii) interaction of the first two conditions with the innate
and adaptive immune response [44].
Bacterial, viral and parasites infections in early life: child’s increased exposure to
viral infections through day care or contact with other children can influence the phenotypic expression. In cross-sectional surveys recurrent respiratory tract infections during
the first 3 years of life have been shown to be negatively associated with atopy among
asthmatic children at school age (45, 46). In a study involving 1035 children followed since
birth as a part of the Tucson Children’s Respiratory Study, exposure of young children to
older children at home or to other children at day care protects against the development of
asthma and frequent wheezing later in childhood [47]: the editorial article of this paper had
a provocative and appealing title: “please, sneeze on my child” [48].
There is good evidence to suggest that in endemic areas, such as in Africa or Latin
America, parasitic infections are strongly inversely related to the development of atopy
[41,49]. The helminthic infections favour a Th2 type of immune response and this condition
should protected from allergic diseases [50].
Attention should be paid in generalising these observations. Indeed, among respiratory
viruses, some might exert more deleterious effects than others and infections of the lower
respiratory tract early in life have been identified as risk factors for persistent wheeze
and asthma [51,52]. In this perspective, two viruses have been associated with asthma
and wheeze in a large number of studies: rhinovirus and respiratory syncytial virus (RSV).
Rhinovirus has been detected in 80% of nasal aspirates of school-age asthmatic children
within 4 days after parents reported episodes of wheezing [53]. A birth cohort of high-risk
infants (Childhood Origins of ASThma [COAST] Study) has extended this work to younger
age groups and confirmed that also in infants and toddlers up to the age of 3 years rhinovirus was the main isolate from nasal lavage specimens taken during symptomatic
periods [54]. Infantile rhinovirus illnesses were the ones most significantly associated with
the prevalence of wheezing in the third year of life. In older children viral infections might
interact with airway inflammation in several ways. Damage to the airway epithelium and
consequently enhanced absorption of aeroallergens might lead to increased airway inflammation (55). Furthermore, induction of pro-inflammatory cytokines (IL-6, IL-1b, and TNFa), chemokines (IL-8), adhesion molecules and leukotrienes enhance cellular recruitment,
activation and inflammatory responses [56, 57].
Other data suggest that the site of infection could be important [58]. In the prospective
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia
Multicentre Allergy Study (MAS) cohort, that involved 1314 children followed from birth to
the age of 7 years, it was found that repeated viral infections of the upper respiratory tract
early in life may reduce the risk of developing asthma up to school age, whereas repeated
lower respiratory tract infections in the first 3 years of life showed a positive association
with wheeze up to the age of 7 years [58].
Exposure to microbial compounds during childhood: a large number of studies has
documented that children raised in farms have a lower prevalence of hay fever and atopic
sensitisation in childhood, as well as in adulthood, whereas effects on asthma are more
consistent for the atopic than the non-atopic phenotype [23,24,59,60]. Higher concentrations of bacterial products were found in the mattresses of farm children compared with
concentrations found in the mattresses of non-farm children [25,61] and higher levels of
fungal exposures were found in these environments [22]. Timing of the exposure played a
crucial role. In fact, the protection was strongest when the exposures occurred in the first
years of life compared with in later years [62].
A paradox has been observed in the hygiene hypothesis in US inner cities, where asthma is rising, despite unclean living conditions. Indeed, asthma prevalence is higher for
those living in USA urban versus rural communities, but it is not significantly influenced by
ethnicity or poverty [63]. However, for hygiene hypothesis considerations, unclean innercity living conditions might not be the equivalent of farm and rural living. In this context, endotoxin levels in homes of low-income metropolitan Denver families were much lower than
from farm homes or rural homes and similar to those of non–low-income families [64].
Finally, it might be considered that the role of infections could be different in relation
to the timing of the exposure: early exposure might prevent subsequent allergic disease
development, whereas exposure worsens asthma once it is established.
Atopic sensitization to common aeroallergens, especially perennial inhalant allergens,
is an important risk factor associated with asthma [38]. Several types of aeroallergens are
particularly important in relation to asthma, including house dust mites, animal allergens
(including cockroaches) and molds.
A Cochrane analysis questioned the effectiveness of house dust mite avoidance for the
treatment of established asthma [65]. Moreover, there is no evidence that anti-house dust
mite measures prevent the onset of asthma [66-68]. The relationship between exposure
and sensitization to allergens from companion animals is not clear, and there are insufficient data to recommend for, or against, the presence of a pet in the house unless the child
has become sensitized to the pet species [38,69-71]. Exposure to cockroach allergen in
Causes of allergic rhinitis and asthma
the living quarters is associated with the development of sensitization, and this, in turn,
is associated with an increased risk of developing asthma [72]. Sensitization to alternaria
is a risk factor not only for the development of asthma in children, but also for its severity
[73,74]. Alternaria is usually considered an outdoor aeroallergen, but outdoor and indoor
concentrations may be similar [75].
On the basis of epidemiological and experimental studies the “hygiene hypothesis” has
been developed and has received much attention. According to this hypothesis, the development of asthma is due in part to a lack of exposure to infections (viruses, bacteria and
parasites) and microbial products early in life. However, to date, the hygiene hypothesis
argument for the prevention of allergy and asthma has not been developed strongly enough to warrant any course of action for either prevention or therapy.
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia
[1] Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, Zuberbier T, Baena-Cagnani CE, Canonica GW, van Weel C, et al. Allergic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy 2008;63 Suppl 86:8-160.
[2] Bousquet J, Van Cauwenberge P, Khaltaev N. Allergic rhinitis and its impact on asthma. J Allergy Clin
Immunol 2001;108:S147-334.
[3] Bush RK. Etiopathogenesis and management of perennial allergic rhinitis: a state-of-the-art review.
Treat Respir Med 2004;3:45-57.
[4] Bostock J. Case of a periodical affection of the eyes and chest. Med. Chir. Trans. 1819;10:161-5.
[5] NHLBI/WHO. Global Initiative for Asthma (GINA). Workshop Report: Global Strategy for Asthma Management and Prevention. NIH 2002:Publication No 02-3659. Revised 2008.
[6] Corren J. The impact of allergic rhinitis on bronchial asthma. J Allergy Clin Immunol 1998;101:S352-6.
[7] Togias A. Rhinitis and asthma: evidence for respiratory system integration. J Allergy Clin Immunol
2003;111:1171-83; quiz 1184.
[8] Togias AG. Systemic immunologic and inflammatory aspects of allergic rhinitis. J Allergy Clin Immunol
[9] Masoli M, Fabian D, Holt S, Beasley R. 2004. Global Burden of Asthma Report; Developed for the Global
Initiative for Asthma (GINA). Available from; accessed on July 2009.
[10] Viegi G, Pedreschi M, Baldacci S, Chiaffi L, Pistelli F, Modena P, Vellutini M, Di Pede F, Carrozzi L. Prevalence rates of respiratory symptoms and diseases in general population samples of North and Central
Italy. Int J Tuberc Lung Dis 1999;3:1034-42.
[11] de Marco R, Poli A, Ferrari M, Accordini S, Giammanco G, Bugiani M, Villani S, Ponzio M, Bono R, Carrozzi L, Cavallini R, Cazzoletti L, Dallari R, Ginesu F, Lauriola P, Mandrioli P, Perfetti L, Pignato S, Pirina
P, Struzzo P. The impact of climate and traffic-related NO2 on the prevalence of asthma and allergic
rhinitis in Italy. Clin Exp Allergy 2002;32:1405-12.
[12] National Center for Health Statistics. 2009. Health, United States, 2008 (With Chartbook). Hyattsville,
MD. Available at; Accessed July 2009.
[13] Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 2006;355:2226-35.
[14] Accordini S, Bugiani M, Arossa W, Gerzeli S, Marinoni A, Olivieri M, Pirina P, Carrozzi L, Dallari R, De Togni
A, de Marco R. Poor control increases the economic cost of asthma. A multicentre population-based
study. Int Arch Allergy Immunol 2006;141:189-98.
[15] de Marco R, Accordini S, Cazzoletti L, Marinoni A. Il carico individuale, sociale ed economico dell’asma.
In: de Marco R, Bugiani M, Cerveri I, editors. L’Epidemiologia dell’asma in Italia. Pisa: EDI-AIPO; 2001.
[16] Cookson W. The alliance of genes and environment in asthma and allergy. Nature 1999;402:B5-11.
[17] Stein RT, Sherrill D, Morgan WJ, Holberg CJ, Halonen M, Taussig LM, Wright AL, Martinez FD. Respiratory
syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 1999;354:541-5.
[18] Venables KM, Chan-Yeung M. Occupational asthma. Lancet 1997;349:1465-9.
[19] Strachan DP. Hay fever, hygiene, and household size. Bmj 1989;299:1259-60.
[20] Alm JS, Swartz J, Lilja G, Scheynius A, Pershagen G. Atopy in children of families with an anthroposophic lifestyle. Lancet 1999;353:1485-8.
[21] Matricardi PM, Rosmini F, Riondino S, Fortini M, Ferrigno L, Rapicetta M, Bonini S. Exposure to foodborne and orofecal microbes versus airborne viruses in relation to atopy and allergic asthma: epidemiological study. Bmj 2000;320:412-7.
[22] Schram-Bijkerk D, Doekes G, Douwes J, Boeve M, Riedler J, Ublagger E, von Mutius E, Benz MR, Pers-
Causes of allergic rhinitis and asthma
hagen G, van Hage M, Scheynius A, Braun-Fahrlander C, Waser M, Brunekreef B. Bacterial and fungal
agents in house dust and wheeze in children: the PARSIFAL study. Clin Exp Allergy 2005;35:1272-8.
Eduard W, Douwes J, Omenaas E, Heederik D. Do farming exposures cause or prevent asthma? Results
from a study of adult Norwegian farmers. Thorax 2004;59:381-6.
Elliott L, Yeatts K, Loomis D. Ecological associations between asthma prevalence and potential exposure to farming. Eur Respir J 2004;24:938-41.
Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, Maisch S, Carr D, Gerlach F, Bufe A,
Lauener RP, Schierl R, Renz H, Nowak D, von Mutius E. Environmental exposure to endotoxin and its
relation to asthma in school-age children. N Engl J Med 2002;347:869-77.
Kay AB. The role of eosinophils in the pathogenesis of asthma. Trends Mol Med 2005;11:148-52.
Tulic MK, Christodoulopoulos P, Hamid Q. Small airway inflammation in asthma. Respir Res 2001;2:333-9.
Kraft M, Martin RJ, Wilson S, Djukanovic R, Holgate ST. Lymphocyte and eosinophil influx into alveolar
tissue in nocturnal asthma. Am J Respir Crit Care Med 1999;159:228-34.
Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels
and skin-test reactivity to allergens. N Engl J Med 1989;320:271-7.
von Garnier C, Filgueira L, Wikstrom M, Smith M, Thomas JA, Strickland DH, Holt PG, Stumbles PA.
Anatomical location determines the distribution and function of dendritic cells and other APCs in the
respiratory tract. J Immunol 2005;175:1609-18.
Hammad H, Lambrecht BN. Recent progress in the biology of airway dendritic cells and implications for
understanding the regulation of asthmatic inflammation. J Allergy Clin Immunol 2006;118:331-6.
Busse WW, Lemanske RF, Jr. Asthma. N Engl J Med 2001;344:350-62.
Kitamura K, Takeda K, Koya T, Miyahara N, Kodama T, Dakhama A, Takai T, Hirano A, Tanimoto M, Harada
M, Gelfand EW. Critical role of the Fc receptor gamma-chain on APCs in the development of allergeninduced airway hyperresponsiveness and inflammation. J Immunol 2007;178:480-8.
Beasley R, Pekkanen J, Pearce N. Has the role of atopy in the development of asthma been over-emphasized? Pediatr Pulmonol 2001;Suppl 23:149-50.
Romagnani S. The increased prevalence of allergy and the hygiene hypothesis: missing immune deviation, reduced immune suppression, or both? Immunology 2004;112:352-63.
Geha RS, Jabara HH, Brodeur SR. The regulation of immunoglobulin E class-switch recombination. Nat
Rev Immunol 2003;3:721-32.
Martinez FD, Holt PG. Role of microbial burden in aetiology of allergy and asthma. Lancet 1999;354
Suppl 2:SII12-5.
Platts-Mills T, Vaughan J, Squillace S, Woodfolk J, Sporik R. Sensitisation, asthma, and a modified
Th2 response in children exposed to cat allergen: a population-based cross-sectional study. Lancet
Romagnani S. Regulation of the development of type 2 T-helper cells in allergy. Curr Opin Immunol
AA.VV. Immunologic influences on allergy and the TH1/TH2 balance. J Allergy Clin Immunol
Yazdanbakhsh M, Kremsner PG, van Ree R. Allergy, parasites, and the hygiene hypothesis. Science
Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med
Wills-Karp M, Santeliz J, Karp CL. The germless theory of allergic disease: revisiting the hygiene hypothesis. Nat Rev Immunol 2001;1:69-75.
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone, Vincenzo Bellia
[44] Schaub B, Lauener R, von Mutius E. The many faces of the hygiene hypothesis. J Allergy Clin Immunol
2006;117:969-77; quiz 978.
[45] von Mutius E, Illi S, Hirsch T, Leupold W, Keil U, Weiland SK. Frequency of infections and risk of asthma,
atopy and airway hyperresponsiveness in children. Eur Respir J 1999;14:4-11.
[46] Calvani M, Jr., Alessandri C, Bonci E. Fever episodes in early life and the development of atopy in children with asthma. Eur Respir J 2002;20:391-6.
[47] Ball TM, Castro-Rodriguez JA, Griffith KA, Holberg CJ, Martinez FD, Wright AL. Siblings, day-care attendance, and the risk of asthma and wheezing during childhood. N Engl J Med 2000;343:538-43.
[48] Christiansen SC. Day care, siblings, and asthma--please, sneeze on my child. N Engl J Med
[49] Cooper PJ. Intestinal worms and human allergy. Parasite Immunol 2004;26:455-67.
[50] Yazdanbakhsh M, van den Biggelaar A, Maizels RM. Th2 responses without atopy: immunoregulation in
chronic helminth infections and reduced allergic disease. Trends Immunol 2001;22:372-7.
[51] Arshad SH, Kurukulaaratchy RJ, Fenn M, Matthews S. Early life risk factors for current wheeze, asthma,
and bronchial hyperresponsiveness at 10 years of age. Chest 2005;127:502-8.
[52] Nafstad P, Brunekreef B, Skrondal A, Nystad W. Early respiratory infections, asthma, and allergy: 10year follow-up of the Oslo Birth Cohort. Pediatrics 2005;116:e255-62.
[53] Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, Symington P, O’Toole S, Myint
SH, Tyrrell DA, et al. Community study of role of viral infections in exacerbations of asthma in 9-11 year
old children. Bmj 1995;310:1225-9.
[54] Lemanske RF, Jr., Jackson DJ, Gangnon RE, Evans MD, Li Z, Shult PA, Kirk CJ, Reisdorf E, Roberg KA,
Anderson EL, Carlson-Dakes KT, Adler KJ, Gilbertson-White S, Pappas TE, Dasilva DF, Tisler CJ, Gern
JE. Rhinovirus illnesses during infancy predict subsequent childhood wheezing. J Allergy Clin Immunol
[55] Sakamoto M, Ida S, Takishima T. Effect of influenza virus infection on allergic sensitization to aerosolized ovalbumin in mice. J Immunol 1984;132:2614-7.
[56] Schroth MK, Grimm E, Frindt P, Galagan DM, Konno SI, Love R, Gern JE. Rhinovirus replication causes
RANTES production in primary bronchial epithelial cells. Am J Respir Cell Mol Biol 1999;20:1220-8.
[57] Garofalo RP, Hintz KH, Hill V, Patti J, Ogra PL, Welliver RC, Sr. A comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus. J Med Virol
[58] Illi S, von Mutius E, Lau S, Bergmann R, Niggemann B, Sommerfeld C, Wahn U. Early childhood infectious
diseases and the development of asthma up to school age: a birth cohort study. Bmj 2001;322:390-5.
[59] Ernst P, Cormier Y. Relative scarcity of asthma and atopy among rural adolescents raised on a farm. Am
J Respir Crit Care Med 2000;161:1563-6.
[60] Leynaert B, Neukirch C, Jarvis D, Chinn S, Burney P, Neukirch F. Does living on a farm during childhood protect against asthma, allergic rhinitis, and atopy in adulthood? Am J Respir Crit Care Med
[61] van Strien RT, Engel R, Holst O, Bufe A, Eder W, Waser M, Braun-Fahrlander C, Riedler J, Nowak D, von
Mutius E. Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid
in mattress dust, and its association with respiratory health. J Allergy Clin Immunol 2004;113:860-7.
[62] Riedler J, Braun-Fahrlander C, Eder W, Schreuer M, Waser M, Maisch S, Carr D, Schierl R, Nowak D, von
Mutius E. Exposure to farming in early life and development of asthma and allergy: a cross-sectional
survey. Lancet 2001;358:1129-33.
[63] Aligne CA, Auinger P, Byrd RS, Weitzman M. Risk factors for pediatric asthma. Contributions of poverty,
Causes of allergic rhinitis and asthma
race, and urban residence. Am J Respir Crit Care Med 2000;162:873-7.
[64] Gereda JE, Klinnert MD, Price MR, Leung DY, Liu AH. Metropolitan home living conditions associated
with indoor endotoxin levels. J Allergy Clin Immunol 2001;107:790-6.
[65] Gotzsche PC, Johansen HK. House dust mite control measures for asthma. Cochrane Database Syst
Rev 2008:CD001187.
[66] Corver K, Kerkhof M, Brussee JE, Brunekreef B, van Strien RT, Vos AP, Smit HA, Gerritsen J, Neijens HJ,
de Jongste JC. House dust mite allergen reduction and allergy at 4 yr: follow up of the PIAMA-study.
Pediatr Allergy Immunol 2006;17:329-36.
[67] Marks GB, Mihrshahi S, Kemp AS, Tovey ER, Webb K, Almqvist C, Ampon RD, Crisafulli D, Belousova
EG, Mellis CM, Peat JK, Leeder SR. Prevention of asthma during the first 5 years of life: a randomized
controlled trial. J Allergy Clin Immunol 2006;118:53-61.
[68] Woodcock A, Lowe LA, Murray CS, Simpson BM, Pipis SD, Kissen P, Simpson A, Custovic A. Early life
environmental control: effect on symptoms, sensitization, and lung function at age 3 years. Am J Respir
Crit Care Med 2004;170:433-9.
[69] Bufford JD, Gern JE. Early exposure to pets: good or bad? Curr Allergy Asthma Rep 2007;7:375-82.
[70] Ownby DR, Johnson CC, Peterson EL. Exposure to dogs and cats in the first year of life and risk of
allergic sensitization at 6 to 7 years of age. Jama 2002;288:963-72.
[71] Platts-Mills JA, Custis NJ, Woodfolk JA, Platts-Mills TA. Airborne endotoxin in homes with domestic
animals: implications for cat-specific tolerance. J Allergy Clin Immunol 2005;116:384-9.
[72] Morgan WJ, Crain EF, Gruchalla RS, O’Connor GT, Kattan M, Evans R, 3rd, Stout J, Malindzak G, Smartt
E, Plaut M, Walter M, Vaughn B, Mitchell H. Results of a home-based environmental intervention among
urban children with asthma. N Engl J Med 2004;351:1068-80.
[73] O’Hollaren MT, Yunginger JW, Offord KP, Somers MJ, O’Connell EJ, Ballard DJ, Sachs MI. Exposure to
an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma.
N Engl J Med 1991;324:359-63.
[74] Salo PM, Arbes SJ, Jr., Sever M, Jaramillo R, Cohn RD, London SJ, Zeldin DC. Exposure to Alternaria
alternata in US homes is associated with asthma symptoms. J Allergy Clin Immunol 2006;118:892-8.
[75] O’Connor G T, Walter M, Mitchell H, Kattan M, Morgan WJ, Gruchalla RS, Pongracic JA, Smartt E, Stout
JW, Evans R, Crain EF, Burge HA. Airborne fungi in the homes of children with asthma in low-income
urban communities: The Inner-City Asthma Study. J Allergy Clin Immunol 2004;114:599-606.
Experimental Medicine Reviews (Eds: A. Gerbino, G. Zummo, G. Crescimanno)
В© Plumelia Ricerca (ISBN 978-88-89876-15-2) - Vol. 2/3 - 2008/2009
Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries
[Celiachia: Una diffusa patologia emergente nei Paesi del Mediterraneo]
Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello
Department of Pediatrics; UniversitГ degli Studi di Palermo (IT)
Key words: Celiac Disease, Gluten Enteropathy, Upper Endoscopy, Histological Features, Gluten Free Diet
Parole Chiave: Celiachia, Enteropatia da Glutine, EGDS, Istologia, Dieta aglutinata
Abstract. Celiac Disease is up to date the most well known example of autoimmune disease. It is largely
widespread all over the world and its incidence of 0,5-1% is similar in different countries. Gluten intake
is necessary for triggering disease in genetically predisposed subjects. Genetic factors and environmental
ones are embedded in determining gluten intolerance. Diagnosis of celiac disease is combined and based
on clinical features, serology, histology and genetics. In this review we consider clinical ways of presentation, several serological markers available at the moment, endoscopical patterns, and histological features
following duodenal biopsy. Up to date Gluten Free Diet remains the only safe and effective therapy provided
that is well conducted and long-life.
Riassunto. Ad oggi la celiachia è l’esempio più conosciuto di patologia
autoimmunitaria. Essa ГЁ largamente diffusa in tutto il mondo e la sua
incidenza si aggira tra 0,5 ed 1% in tutti i paesi. L’introito di glutine è
necessario per innescare la patologia in soggetti geneticamente predisposti. Fattori genetici ed ambientali si embricano nel determinare
l’intolleranza al glutine. La diagnosi di malattia celiaca è combinata e si
basa su segni clinici, markers sierologici, aspetti endoscopici e quadri
istologici successivi alla biopsia duodenale. Ad oggi la dieta priva di glutine è l’unica terapia codificata: efficace e scevra di effetti collaterali.
Celiac Disease (CD) is a permanent, gluten dependent,
autoimmune enteropathy. Ingested gluten is responsible
of intestinal damage triggered by T lymphocytes and magnified by tissue trans-glutaminase and pro-inflammatory
cytokines. Starting in the gut the physio-pathological process may extend involving other organs. Clinical features
and mucosal intestinal damage are reversible provided
that patients start a gluten free diet (GFD) [1,2].
Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello
CD incidence is increased in the last decade. Some years ago several reports showed
different incidence rates also among near countries. This finding was understood as an
epidemiological paradox. Nowadays the incidence of disease is about 1:100-1:180, also
in people coming from developing countries, thanks to higher sensitivity and specificity of
new serological screening tests such as Anti Endomysium Antibodies (AEA) and anti human
trans glutaminase (hTTG) antibodies [3-6].
In Mediterranean countries the larger consumption of wheat and other gluten containing cereals (barley, rye) contribute to increase incidence of disease. In particular, modern
grain cultivars are esaploid ones, such as Triticum Aestivum, in which gluten derived proteins may exceed 50%. Gluten derived proteins are useful to make dough, bread and other
baker products because of their glue properties.
This is the reason why polyploid wheat cultivars were selected across centuries [3,7,8].
Amongst the world affected population Mossi people, living in Burkina Faso, seem to be
free from CD in spite of gluten presence in their diet regimen. On the other side Saharawi
people, living in Tindourf oasis in West Algeria, have the highest incidence of disease in
the world reaching 5.6%.
In fact long lasting endogamic tradition is responsible of enormous diffusion of CD
predisposing genes, first of all DQ2. The association with large gluten consumption in
their daily diet rich of cous-cous and other wheat derived foodstuffs also contribute to CD
development [9].
In determining CD the above mentioned environmental factor is embedded with genetic
ones [10].
HLA related and HLA unrelated genes confer susceptibility to disease. 90% of celiac
affected people carry II Class HLA heterodimer DQ2 and almost the remaining CD patients
who, are DQ2 negative, carry DQ8 aplotype. Only a small amount of CD patients is both
DQ2 and DQ8 negative [7,11,12]. In fact non-HLA genes also matter in determining predisposition to develop CD [10,13-15]. Antigen Presenting Cells of intestinal Lamina Propria,
containing DQ2 or DQ8 aplotype, interact with TCR of CD4+ cells triggering a Th1 inflammatory response.
Gliadin peptides rich of prolyn, lisin and glutamine in the lamina propria undergo a
transamination process by TTG conferring them a negative electrical charge. This kind of
biochemical change is responsible of increased interaction between HLA DQ2 aplotype
present in APC and the TCR of CD4+ lamina propria cells. Enterocytes themselves act as
APC towards CD8+ intra epithelial lymphocytes. IL-15 produced by NK cells and entero358
Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries
cytes amplifies innate immune response contributing to tissue destruction [15-21].
Cooperation between T and B cells results in antibodies production against Gliadin
(AGA), Endomysium (AEA) and Tissue Transglutaminase (TTG) [22].
Clinical features
Clinical features in CD may be distinct in typical and atypical ones. The first are directly
gut related and constitute the classical presentation way of disease in younger children. In
deed failure to thrive, abdominal distension, chronic diarrhea, vomiting, bloating, malabsorption with fat in stools, represent the clinical features of overt forms of CD [2,3,10].
The atypical ones are more common in the elder child and in adulthood and consist of
extra intestinal features giving proof of a systemic disease [23,24]. (Table 1).
CD is often associated to other clinical conditions such as Type 1 Mellitus Diabetes,
thyroditis, IgA deficiency or genetic syndromes like Down’s Syndrome or William’s Syndrome [25]. These syndromes confer to affected individuals an additional, sometimes HLA
independent, risk of doing CD.
In these last years risk of malignancy in CD and in particularly of enteropathy associated T Lymphoma, was undersized. In fact it seems more related to Refractory Celiac Sprue,
that probably is a pre-neoplastic pathological condition different than CD [16].
Up to date serology consists of three different kind of antibodies against Gliadin, Endomysium and Transglutaminase respectively. AGA are those with best sensitivity and
specificity under two years of age, reflecting the first immunological response towards
heterologous proteins of wheat gliadin.
They are the first kind of antibodies detected in serum samples of celiac patients. In
early 90’s AEA were also available by Indirect Immuno-Fluorescence method conferring to
serologic suspicion more reliability also for the follow up of the disease. In fact this new
serologic finding allowed the ESPGHAN committee to revise the diagnostic criteria for CD,
reducing the number of duodenal biopsies needed to diagnosis, from three (Interlaken
1970) to only one [26]. In 1997 the last and more accurate category of serologic antibodies was discovered by Dieterich e Coll. and provided a new useful tool for screening and
monitoring the disease without undergoing duodenal biopsy: anti TTG antibodies in deed
are the highest specific and sensitive class of serologic markers for CD suspected diagnosis [4]. First line generation of TTG substrate was extracted by guinea pig while the more
recent ones by human recombinant TTG. Also for AEA the first used substrate was monkey
esophagus in recent times substituted by human umbilical cord. All kind of serologic an359
Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello
tibodies may be performed using IgA or IgG circulating classes. IgG response ought to be
considered in IgA deficient patients.
It is the most common way to perform duodenal biopsy for diagnostic purpose. Upper
endoscopy allows multiple bioptic samples and the direct visualization of duodenum giving
to the performer possibility of choosing bioptic location. The endoscopic pattern of duodenal damage may be largely variable going from absolutely normal to several grades of
endoscopical damage: scalloping, hyperemia, decreased number and height of Kerckring
folds, vascular reticule evidence [27].
Histological damage in CD consists of three different periods. The infiltration phase
characterized by intraepithelial lymphocytes (IEL) infiltration, the hyperplastic phase with
decrease of villous/crypt ratio and the destructive one consisting of villous atrophy and
mucosal flattening.
The early period of CD is linked to intraepithelial infiltration by CD3+ CD8+ lymphocytes.
More than 25 IEL per cent enterocytes are suggestive of CD in its infiltrative form. Diagnosis may be possible only with immunohistochemical staining for CD3+ cells.
The hyperplastic phase is the result of over expressed bowel cellular mitosis at the
crypt level associated to reduced villous height (Vh/CrD ratio).
Total mucosal atrophy is typical of the last destructive phase.
Up to date diagnosis of CD rests upon this histological findings of increased intra epithelial lymphocyte infiltration, decreased villous/crypt ratio and mucosal flattening according to Marsh-Oberhuber stadiation [28,29].
Diagnosis of CD depends on clinical, serological, genetic, endoscopic, and histological
Anyway duodenal biopsy remains the gold standard. Long life Gluten Free Diet is at the
moment the only effective and safe therapy although new ones are under experimental
evaluation [30]. Bacterial endo-peptidase, able to digest gluten peptide may be, in the next
future, useful to detoxify gliadin for celiac intolerant subjects. In the early diagnosis of CD,
especially in the younger child, a combined GFD-lactose free diet may be useful in relieving
abdominal symptoms due to secondary lactase deficiency.
Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries
Table 1
Classic Form
Atypical Form
Chronic Diarrhea
Iron Deficiency Anaemia
Failure to Thrive
Short Height
Stop of the growth
Herpetiform Dermatitis
Muscular Atrophy
Recurrent Miscarriage
Recurrent Vomiting
Autoimmune Hepatitis
Cryptogenic Hypertransaminasemia
Autoimmune Thyroiditis
Enamel defects
Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello
[1] Fasano A, Catassi C. Current Approaches to Diagnosis and treatment of Celiac Disease. An Evolving
Spectrum. Gastroenterology 2001; 120:636-651.
[2] Green P, Jabri B: Coeliac Disease. The Lancet 2003; 36(9381):383-91.
[3] Catassi C, Fasano A, Fornaroli F et al. La celiachia: un mondo in esplorazione. Minerva Pediatr 2003;
[4] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 1997 Jul;3(7):797-801.
[5] Westerlund A, Ankelo M, Simell S, Ilonen J, Knip M, Simell O, Hinkkanen AE. Affinity maturation of
immunoglobulin A anti tissue transglutaminase autoantibodies during development of coeliac disease.
Clin Exp Immunol. 2007 May;148(2):230-40.
[6] Caputo I, Barone MV, Martucciello S, Lepretti M, Esposito C. Tissue transglutaminase in celiac disease:
role of autoantibodies. Amino Acids. 2009 Apr;36(4):693-9.
[7] Accomando S, Cataldo F. The Global village of Celiac Disease. Digestive and liver Disease 2004;
[8] Accomando S, Pellitteri V, Corsello G. Biochemical Markers in Celiac Disease. Frontiers in Bioscience.
In Press.
[9] Catassi C, Ratsch IM, Gandolfi L, Pratesi R, Fabiani E, El Asmar R, et al. Why is coeliac disease endemic
in the people of the Sahara? Lancet 1999; 354:647-8.
[10] Bensussan NC, Cellier C, Heyman M et al. Coeliac Disease: An Update on fact and Questions on the 10
International Symposium on Coeliac Disease. Journal of Pediatric Gastroenterology and Nutrition Oct
2003; 37; 412-421.
[11] Sollid LM. Molecular basis of celiac disease. Annu Rev Immunol 2000; 18: 53– 81.
[12] Spurkland A, Sollid LM, Polanco I, Vartdal F, Thorsby E. HLA-DR and -DQ genotypes of celiac disease
patients serologically typed to be non-DR3 or non-DR5/7. Hum Immunol 1992; 35: 188– 192.
[13] Greco L, Romino R, Coto I et al. The first large population based twin study of coeliac disease. Gut 2002;
50: 624– 628.
[14] Petronzelli F, Bonamico M, Ferrante P et al. Genetic contribution of the HLA region to the familial clustering of coeliac disease. Ann Hum Genet 1997; 61: 307– 317.
[15] Maiuri L, Ciacci G, Ricciardelli I et al. Association between innate response to gliadin and activation of
pathogenic T cells in Celiac Disease. Lancet 2003; 362: 30-37.
[16] Verkarre V, Romana SP, Cellier C et al. Recurrent Partial Trisomy 1922-944 in Clonal Intrepithelial Lymphocyties in Refractory Celiac Sprue. Gastroenterology 2003; 125:10-16.
[17] Mazzarella G, Stefanile R, Camarca A, Giliberti P, Cosentini E, Marano C, Iaquinto G, Giardullo N, Auricchio
S, Sette A, Troncone R, Gianfrani C. Gliadin activates HLA class I-restricted CD8+ T cells in celiac disease
intestinal mucosa and induces the enterocyte apoptosis. Gastroenterology. 2008 Apr;134(4):1017-27.
[18] Bernardo D, Garrote JA, Allegretti Y, LeГіn A, GГіmez E, Bermejo-Martin JF, Calvo C, Riestra S, FernГЎndezSalazar L, Blanco-QuirГіs A, Chirdo F, Arranz E. Higher constitutive IL15R alpha expression and lower
IL-15 response threshold in coeliac disease patients. Clin Exp Immunol. 2008 Oct;154(1):64-73.
[19] Benahmed M, Meresse B, Arnulf B, Barbe U, Mention JJ, Verkarre V, Allez M, Cellier C, Hermine O,
Cerf-Bensussan N. Inhibition of TGF-beta signaling by IL-15: a new role for IL-15 in the loss of immune
homeostasis in celiac disease. Gastroenterology. 2007 Mar;132(3):994-1008.
[20] Ebert EC. IL-15 converts human intestinal intraepithelial lymphocytes to CD94 producers of IFN-gamma
and IL-10, the latter promoting Fas ligand-mediated cytotoxicity. Immunology. 2005 May;115(1):118-26.
Celiac Disease: a Widespread Emerging Condition in Mediterranean Countries
[21] Tang F, Chen Z, Ciszewski C, Setty M, Solus J, Tretiakova M, Ebert E, Han J, Lin A, Guandalini S, Groh
V, Spies T, Green P, Jabri B. Cytosolic PLA2 is required for CTL-mediated immunopathology of celiac
disease via NKG2D and IL-15. J Exp Med. 2009 Mar 16;206(3):707-19.
[22] HJ Ellis, Pollock EL, Engel W et al. Investigastion of the putative immunodominant T cell epitopes in
coeliac disease. Gut 2003; 52:212-217.
[23] Catassi C, Fabiani E. The spectrum of coeliac disease in children. Baillière’s Clin Gastroenterol 1997;
[24] Corazza GR, Gasbarrini G. Celiac disease in adults. Baillière’s Clin Gastroenterol 1995; 9:329-50.
[25] Ventura A, Neri E, Ughi C, Leopaldi A, Citta A, Not T. Gluten-dependent diabetes-related and thyroidrelated autoantibodies in patients with celiac disease. J Pediatr 2000; 137:263-5.
[26] Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition: Revised
criteria for diagnosis of coeliac disease. Arch Dis Child 1990; 65, 909–11.
[27] Ravelli AM, Tobanelli P, Minelli L et al. Endoscpic features of celiac disease in children. Gastrointest
Endosc 2001; 54: 736-742.
[28] Marsh MN. Gluten, major histocompatibility complex and the small intestine. A molecular and immunobiologic approach to the spectrum of glutensensitivity (�celiac sprue’). Gastroenterology 1992;
[29] Oberhuber G et al. The histopathology of celiac disease: time for a standardized report scheme for
pathologists. European Journal of Gastroenterology and hepatology 1999; 11 (10) 1185-1194.
[30] Accomando S, Albino C. Celiac disease: new implications in an old condition. Capsula Eburnea 2006;
1:1 1-5.
Analitical index
and Contributors
Analitical index and Contributors
Abdominal Aortic Aneurysm, 99
Accomando S., 357
Ahmed Hafiz., 321
Allergic Rhinitis, 345
Angiogenesis, 81
Anphibians, 167
Anthropoids, 167
Anzalone R., 25, 149
Asthma, 345
Atherosclerosis, 99
Badalamenti G., 197
Bajardi G., 99
Baldi B., 25
Basile I., 67
Battaglia G., 81
Battaglia S., 345
Bazan V., 35, 197, 213, 223, 249
Bellafiore M., 81
Bellia V., 345
Benigno A., 297, 309
Bivona G., 281
Bonaventura G., 57, 181, 189
Bracale UM., 99
Breast cancer, 213, 223, 249
Bronte G., 213, 249
Bruno L., 223, 249
Burruano F., 269
Buscemi M., 157, 213, 223, 269, 281, 343
Buzzanca ML., 269
Caiozzo M., 197, 213, 223, 249
CalГІ V., 197, 213, 249
Capillaroscopy, 91
Cappello F., 71, 149, 167
Carini F., 91
Casarrubea M., 137
CB1 receptor, 189
Celiac disease, 357
Central giant cell granuloma, 269
Chaperoning system, 9
Chimenti L., 309
Chronic obstructive pulmonary disease, 25
Ciaccio M., 281
Cicero G., 197, 249
Cicero MV., 213
Cocorullo G., 91
Conway de Macario E., 9, 149
Corrao S., 25, 149
Corsello G., 357
Crescimanno G., 137
Cucco D., 57, 181
Cytokeratins, 157
D’Angelo L., 67
De Luca A., 71
Di Felice V., 71
Di Stefano A., 25
Dispensa N., 249
Dopamin, 137
Evolution, 333
Fanale D., 197
Farina F., 81, 149
Fazio G., 67
Fibrosis, 67
First Cell, 333
Foddai E., 213
Frazzetta M., 197, 223, 249
Galectin-3, 321
Gastrointestinal stromal tumors, 197
Gebbia N., 197, 223, 249
Genetic counselling, 223
GEP system, 181
Gerbino A., 35, 67, 157, 197, 249, 269, 281, 333
Grassi N., 197, 223, 249
Guadagna FP., 213
Guarneri D., 223
Guarneri G., 249
Gullo S., 213
Heat shock proteins, 9, 125
Human umbilical cord, 157
Image analysis, 289
La Manna M., 35
La Paglia L., 223
La Rocca G., 25, 149
Langerhans cells, 35
Last Common Cell, 333
Leone A., 91
Leptin, 57
Licciardi A., 297, 309
Lipari E.F., 167
Lipari L., 125, 343
Lo Coco G., 213
Lo Dico G., 223, 249
Lo Iacono M., 25, 149
Lo Piccolo C., 91
Loria T., 25, 149
Lunetta M., 67
Macaluso F., 71
Macario AJL., 9, 149
Magnetic resonance spectroscopy, 281
Magno F., 25, 149
Manna G., 213
MarcianГІ V., 167
Maresi E., 67
Marino Gammazza A., 71
Matrix Metalloproteinases, 99
Mauro A., 99, 157, 343
Mesenchimal Stem Cells, 343
Mesolimbic-mesocortical dopaminergic neurons, 137
Messina P., 91
Montalbano A., 71
Morici G., 297, 309
Muscular dystrophy, 309
Naturalism, 333
Nigrostriatal dopaminergic neurons, 137
Non-compaction, 67
Novo G., 67
Novo S., 67
Orexin, 57, 181
Orphanin, 57, 181
Ovarian cancer, 223, 249
Paglino G., 345
Pantuso G., 197, 223, 249
Pecoraro F., 99
Peduncle, 333
Pellitteri V., 357
Placenta, 149
Primates, 167
Prostate cancer, 321
Radiolarians, 333
Reptiles, 167
Rheumatoid arthritis, 125
Ridola CG., 167
Rimonabant, 189
Rizzo S., 197, 213, 223, 249
Role tolerogenic, 35
Russo A., 197, 213, 223, 249
Salerno A., 35
Sandonato L., 197, 249
Scardina GA., 91
Scichilone N., 345
Smads, 25
Sorbera F., 137
Spagnolo G., 25
Spatola GF., 57, 181, 189, 289
Sutera L., 67
Tessitore V., 57, 181, 189
Tortrici S., 269
Training, 297, 309
Transdifferentiation, 343
Uzzo ML., 57, 181, 189
Valenza V., 91
Varia P., 91
Vasostatin I, II, 71
Wharton’s Jelly, 157, 343
Zummo G., 25, 71
Guest contribution
Alberto J. L. Macario and Everly Conway de Macario
23 Airway cells,epidermis, signaling
25TGFb signaling: Roles of Smads
Francesca Magno, Giampiero La Rocca, Rita Anzalone, Simona Corrao, Tiziana Loria, Melania Lo Iacono, Gaetana Spagnolo, Antonino Di Stefano, Bruno Balbi and Giovanni Zummo
35 New insight on immunological activation pathways of Langerhans cells, possible tolerogenic role
Marco La Manna, Viviana Bazan, Aldo Gerbino and Alfredo Salerno
57 Espressione immunoistochimica della orexina A, della orfanina FQ e
della leptina nell’apparato respiratorio del ratto
Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo
65 Heart, circulation
67ISOLATED Left ventricular NON-COMPACTION, STROKE AND trabecular meshwork FIBROSIS
Giovanni Fazio, Ivana Basile, Monica Lunetta, Luciana D’Angelo, Loredana Sutera, Emiliano Maresi,
Giuseppina Novo, Aldo Gerbino and Salvatore Novo
71The Chromogranin A-derived peptides: structural and functional features in heart biology
Valentina Di Felice, Antonella Montalbano, Angela De Luca, Filippo Macaluso, Antonella Marino Gammazza, Francesco Cappello and Giovanni Zummo
81 New insights into the role of matrix metalloproteinases in heart angiogenesis induced by exercise
Giuseppe Battaglia, Marianna Bellafiore, Felicia Farina
Francesco Carini, Giuseppe Alessandro Scardina, Gianfranco Cocorullo, Pierluigi Varia, Chiara Lo Piccolo, Angelo Leone, Pietro Messina and Vincenzo Valenza
Guido Bajardi, Felice Pecoraro, Annamaria Mauro, Umberto Marcello Bracale
123 Cellular stress
Luana Lipari
135 Brain, behavior
Maurizio Casarrubea, Filippina Sorbera and Giuseppe Crescimanno
147 Organogenesis, Philogenesis, Clinical Embryology
149 The balance of Hsps expression during pregnancy: the role of placenta
Simona Corrao, Giampiero La Rocca, Rita Anzalone, Francesca Magno, Tiziana Loria, Melania Lo Iacono, Everly Conway de Macario, Alberto JL Macario, Francesco Cappello and Felicia Farina
157 Expression of cytokeratins in human umbilical cord at term
Annamaria Mauro, Maria Buscemi and Aldo Gerbino
167The evolutionary course of Vertebrate foot, from Fish to Man
Carlo G. Ridola, Vito MarcianГІ, Francesco Cappello and Elvira Farina Lipari
179 Gastrointestinal tract
181Orexina e Orfanina FQ nuovi neuropeptidi regolatori delle funzioni del
Sistema Gastroenteropancreatico. Uno studio immunoistochimico
Giuseppe Bonaventura, Giovanni Francesco Spatola, Maria Laura Uzzo e Vincenzo Tessitore
Vincenzo Tessitore, Giuseppe Bonaventura, Daniela Cucco, Giovanni Francesco Spatola e Maria Laura Uzzo
195 Tumor process
197 GISTs Eredo-familiari e pediatrici: aspetti biomolecolari e clinici
Antonio Russo, Sergio Rizzo, Daniele Fanale, Valentina CalГІ, Nello Grassi, Gianni Pantuso, Luigi Sandonato, Michele Frazzetta, Giuseppe Cicero, Massimo Cajozzo, Aldo Gerbino, Giuseppe Badalamenti,
Nicola Gebbia e Viviana Bazan
213The impact on the patient’s adjustment to breast cancer of the caregiver’s burden and distress
Gianluca Lo Coco, Elena Foddai, Salvatore Gullo, Maria Viviana Cicero, Giovanna Manna, Francesca
Paola Guadagna, Viviana Bazan, Maria Buscemi, Massimo Cajozzo, Giuseppe Bronte, Sergio Rizzo and
Antonio Russo
Antonio Russo, Sergio Rizzo, Valentina CalГІ, Loredana Bruno, Laura La Paglia, Nello Grassi, Gianni
Pantuso, Michele Frazzetta, Massimo Cajozzo, Domenico Guarneri, Giuseppe Lo Dico, Maria Buscemi,
Nicola Gebbia e Viviana Bazan
249I tumori della mammella e/o dell’ovaio di tipo eredo-familiare: strategie di prevenzione nelle donne ad alto rischio
Sergio Rizzo, Giuseppe Bronte, Valentina CalГІ, Loredana Bruno, Nello Grassi, Gianni Pantuso, Luigi
Sandonato, Michele Frazzetta, Giuseppe Cicero, Girolamo Guarneri, Giuseppe Lo Dico, Massimo Cajozzo, Nino Dispensa, Aldo Gerbino, Nicola Gebbia, Viviana Bazan e Antonio Russo
267 Oral
Silvia Tortorici, Maria Lidia Buzzanca, Francesco Burruano, Aldo Gerbino e Maria Buscemi
279 Methodology, Physical activity, Clinical applications
Giulia Bivona, Maria Buscemi, Aldo Gerbino and Marcello Ciaccio
289Immunoistochimica e analisi d’immagine multi spettrale
Giovanni Francesco Spatola
Arcangelo Benigno, Attilio Licciardi and Giuseppe Morici
Giuseppe Morici, Filippo Macaluso, Laura Chimenti, Arcangelo Benigno and Attilio Licciardi
319 Invited article and report
321 Epigenetics of galectin-3- a novel regulatory mechanism of its transcription during carcinogenesis
Hafiz Ahmed
333 1859: Naturalismo. Motivi evoluzionistici e anatomia cellulare
Aldo Gerbino
341 Proceedings & Meetings
343Ambiente staminale e prospettive di transdifferenziamento verso il
fenotipo osteoblastico
Annamaria Mauro, Luana Lipari, Maria Buscemi e Aldo Gerbino
Salvatore Battaglia, Giuseppe Paglino, Nicola Scichilone and Vincenzo Bellia
357 Celiac Disease: a Widespread Emerging Condition in Mediterranean
Salvatore Accomando, Veronica Pellitteri e Giovanni Corsello
365 Analitical index and Contributors
Finito di stampare
nel mese di dicembre 2009
presso le
Officine Tipografiche Aiello & Provenzano
Bagheria, Palermo