Helsinki University Biomedical Dissertations No. 79
Lymphatic vessels in health and disease:
Role of the VEGF-C/VEGFR-3 pathway and the
transcription factor FOXC2
Terhi Kärpänen
Molecular/Cancer Biology Laboratory
Haartman Institute and Biomedicum Helsinki
&
Division of Biochemistry
Department of Biological and Environmental Sciences
Faculty of Biosciences
University of Helsinki
Finland
Academic dissertation
To be publicly discussed, with the permission of the Faculty of Biosciences of the
University of Helsinki, in the lecture hall 2, Biomedicum Helsinki, Haartmaninkatu 8,
Helsinki on June 30, 2006, at 12 o’clock noon
Helsinki 2006
Supervised by
Kari Alitalo, M.D., Ph.D.
Research Professor of the Finnish Academy of Sciences
Molecular/Cancer Biology Laboratory
Haartman Institute and Biomedicum Helsinki
University of Helsinki
Finland
Reviewed by
Juha Partanen, Ph.D.
Docent
Institute of Biotechnology
University of Helsinki
Finland
and
Marko Salmi, M.D., Ph.D.
Docent
MediCity Research Laboratory
University of Turku
Finland
Thesis opponent
Ralf Adams, Ph.D.
Cancer Research UK
London Research Institute
Lincoln’s Inn Fields Laboratories
London
UK
ISBN: 952-10-3230-8 (nid.)
ISBN: 952-10-3231-6 (PDF)
ISSN: 1457-8433
http://ethesis.helsinki.fi
Yliopistopaino
Helsinki 2006
2
Tietä käyden tien on vanki,
vapaa on vain umpihanki
Aaro Hellaakoski
3
CONTENTS
ABBREVIATIONS__________________________________________________________ 6
ORIGINAL PUBLICATIONS _________________________________________________ 7
ABSTRACT _______________________________________________________________ 8
REVIEW OF THE LITERATURE _____________________________________________ 9
1. Structure and function of the two vascular systems __________________________________________ 9
1.1
The blood vascular system _______________________________________________________ 9
1.2
The lymphatic vascular system ___________________________________________________ 10
2. Development of the two vascular systems _________________________________________________
2.1
Vasculogenesis _______________________________________________________________
2.2
Angiogenesis and arteriogenesis__________________________________________________
2.3
Lymphangiogenesis ___________________________________________________________
2.4
Angiogenesis and lymphangiogenesis in adult tissues _________________________________
11
12
12
13
14
3. Molecular regulation of vascular development _____________________________________________
3.1
Vascular endothelial growth factors _______________________________________________
3.2
Vascular endothelial growth factor receptors ________________________________________
3.3
Molecules that modulate VEGFR signalling_________________________________________
3.4
Molecules that regulate remodelling and maturation of blood and lymphatic vessels __________
3.5
Transcription factors implicated in the development of lymphatic vasculature_______________
3.6
Molecular specification of lymphatic endothelial identity ______________________________
3.7
Role of VEGFs outside the cardiovascular and hematopoietic systems ____________________
14
15
21
24
29
35
36
38
4. Blood vessels in pathological conditions ___________________________________________________ 40
4.1
Tumor angiogenesis and its inhibition _____________________________________________ 40
5. Lymphatic vessels in pathological conditions ______________________________________________
5.1
Tumor lymphangiogenesis and metastasis __________________________________________
5.2
Lymphedema ________________________________________________________________
5.3
Lymphangiectasia and lymphatic neoplasms ________________________________________
5.4
Inflammatory diseases _________________________________________________________
5.5
Prolymphangiogenenic therapy __________________________________________________
41
41
44
47
47
48
AIMS OF THE STUDY _____________________________________________________ 50
MATERIALS AND METHODS ______________________________________________ 51
RESULTS AND DISCUSSION_______________________________________________ 55
1. The carboxyterminal silk-like propeptide enhances lymphangiogenic potential of VEGF-C (I) ______ 55
2. NP2 is involved in VEGF-C and VEGF-D induced VEGFR-3 signalling (II) _____________________ 56
3. VEGF-C mediated tumor lymphangiogenesis is inhibited by blocking VEGFR-3 signalling (III) ____ 58
3.1
Possible mechanisms of VEGF-C enhanced lymphatic metastasis ________________________ 60
4. Postnatal maturation renders lymphatic vessels independent of ligand-induced
VEGFR-3 signalling (IV)______________________________________________________________ 62
5. Foxc2 regulates the interactions of the lymphatic endothelium with vascular mural cells and is
required for the morphogenesis of lymphatic valves (V)_____________________________________ 64
CONCLUDING REMARKS _________________________________________________ 68
ACKNOWLEDGEMENTS __________________________________________________ 69
REFERENCES ___________________________________________________________ 70
5
ABBREVIATIONS
AAV
Ad
Ang
BEC
BM
CAM
CT
CUB
E
EC
ECM
EEA-1
EGF
ES cell
FIGF
Flk1
Flt1
Flt4
FOXC2
HA
HDMVEC
HIF-1
HSPG
Ig
K14
kDa
KDR
LD
LEC
LYVE-1
MFH-1
mRNA
NP
P
PAE
PC
PCNA
PDGF
PDGFR
PDZ
PECAM-1
PG
PI3K
PlGF
Prox1
SCID
SEMA
SLC
SMA
SMC
Tek
TGF
Tie
VE-cadherin
VEGF
VEGFR
Vezf1
VHD
VHL
VPF
VRF
VRP
Adeno associated virus
Adenovirus
Angiopoietin
Blood vascular endothelial cell
Basement membrane
Chorioallantoic membrane
Carboxyterminus
Protein domain; the name is derived from the first three proteins the domain was found in
(Complement subcomponents C1r/C1s, sea urchin EGF-like protein, bone morphogenic protein 1)
Embryonic day
Endothelial cell
Extracellular matrix
Early endosome antigen 1
Epidermal growth factor
Embryonic stem cell
c-fos induced growth factor (VEGF-D)
Fetal liver kinase 1 (mouse VEGFR-2)
Fms-like tyrosine kinase 1 (VEGFR-1)
Fms-like tyrosine kinase 4 (VEGFR-3)
Forkhead box C2
Hyaluronan
Human dermal microvascular endothelial cell
Hypoxia-inducible factor 1
Heparan sulphate proteoglycan
Immunoglobulin
Keratin-14
Kilodalton
Kinase insert domain containing receptor (human VEGFR-2)
Lymphedema-distichiasis
Lymphatic endothelial cell
Lymphatic endothelial hyaluronan receptor 1
Mesenchyme forkhead 1 (FOXC2)
Messenger ribonucleic acid
Neuropilin
Postnatal day
Porcine aortic endothelial
Pericyte
Proliferating cell nuclear antigen
Platelet derived growth factor
PDGF receptor
Protein domain; the name is derived from the first three proteins the domain was found in (PSD95,
Dlg, ZO1)
Platelet endothelial adhesion molecule 1
Proteoglycan
Phosphatidylinositol 3’-kinase
Placenta growth factor
prospero-related homeobox protein 1
Severe combined immunodeficiency
Semaphorin
Secondary lymphoid chemokine
Smooth muscle α-actin
Smooth muscle cell
Tunica interna endothelial cell kinase (Tie-2)
Transforming growth factor
Tyrosine kinase with Ig and EGF homology domains (Tie-1)
Vascular endothelial cadherin
Vascular endothelial growth factor
Vascular endothelial growth factor receptor
Vascular endothelial zinc finger 1
VEGF homology domain
Von Hippel Lindau protein
Vascular permeability factor (VEGF)
VEGF related factor (VEGF-B)
VEGF related protein (VEGF-C)
6
ORIGINAL PUBLICATIONS
This thesis is based on the following original articles, which are referred to in the text by their
Roman numerals:
I
Karpanen, T. and Alitalo, K. The silk-like carboxyterminal propeptide regulates the
lymphangiogenic activity of VEGF-C. Manuscript in preparation (2006).
II
Karpanen, T.*, Heckman, C.A.*, Keskitalo, S., Jeltsch, M., Ollila, H., Neufeld, G.,
Tamagnone, L. and Alitalo, K. Functional interaction of VEGF-C and VEGF-D with
neuropilin receptors. The FASEB Journal, in press (2006).
III
Karpanen, T.*, Egeblad, M.*, Karkkainen, M. J., Kubo, H., Ylä-Herttuala, S.,
Jäättelä, M. and Alitalo, K. Vascular endothelial growth factor C promotes tumor
lymphangiogenesis and intralymphatic tumor growth. Cancer Research 61, 1786-1790
(2001).
IV
Karpanen, T., Wirzenius, M., Mäkinen, T., Veikkola, T., Haisma, H.J., Achen M.G.,
Stacker, S.A., Pytowski, B., Ylä-Herttuala, S. and Alitalo, K. Lymphangiogenic
growth factor responsiveness is modulated by postnatal lymphatic vessel maturation.
The American Journal of Pathology, in press (2006).
V
Petrova, T.V.*, Karpanen, T.*, Norrmén, C., Mellor, R., Tamakoshi., T., Finegold,
D., Ferrell, R., Kerjaschki, D., Mortimer, P., Ylä-Herttuala, S., Miura, N. and Alitalo,
K. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular
failure in lymphedema distichiasis. Nature Medicine 10, 974-981 (2004).
* equal contribution
7
ABSTRACT
The circulatory system consists of two vessel types, which act in concert but significantly
differ from each other in several structural and functional aspects as well as in mechanisms
governing their development. The blood vasculature transports oxygen, nutrients and cells to
tissues whereas the lymphatic vessels collect extravasated fluid, macromolecules and cells of
the immune system and return them back to the blood circulation. Understanding the
molecular mechanisms behind the developmental and functional regulation of the lymphatic
system long lagged behind that of the blood vasculature. Identification of several markers
specific for the lymphatic endothelium, and the discovery of key factors controlling the
development and function of the lymphatic vessels have greatly facilitated research in
lymphatic biology over the past few years. Recognition of the crucial importance of
lymphatic vessels in certain pathological conditions, most importantly in tumor metastasis,
lymphedema and inflammation, has increased interest in this vessel type, for so long
overshadowed by its blood vascular cousin.
VEGF-C (Vascular Endothelial Growth Factor C) and its receptor VEGFR-3 are essential for
the development and maintenance of embryonic lymphatic vasculature. Furthermore, VEGFC has been shown to be upregulated in many tumors and its expression found to positively
correlate with lymphatic metastasis. Mutations in the transcription factor FOXC2 result in
lymphedema-distichiasis (LD), which suggests a role for FOXC2 in the regulation of
lymphatic development or function. This study was undertaken to obtain more information
about the role of the VEGF-C/VEGFR-3 pathway and FOXC2 in regulating lymphatic
development, growth, function and survival in physiological as well as in pathological
conditions. We found that the silk-like carboxyterminal propeptide is not necessary for the
lymphangiogenic activity of VEGF-C, but enhances it, and that the aminoterminal propeptide
mediates binding of VEGF-C to the neuropilin-2 coreceptor, which we suggest to be involved
in VEGF-C signalling via VEGFR-3. Furthermore, we found that overexpression of VEGF-C
increases tumor lymphangiogenesis and intralymphatic tumor growth, both of which could be
inhibited by a soluble form of VEGFR-3. These results suggest that blocking VEGFR-3
signalling could be used for prevention of lymphatic tumor metastasis. This might prove to be
a safe treatment method for human cancer patients, since inhibition of VEGFR-3 activity had
no effect on the normal lymphatic vasculature in adult mice, though it did lead to regression
of lymphatic vessels in the postnatal period. Interestingly, in contrast to VEGF-C, which
induces lymphangiogenesis already during embryonic development, we found that the related
VEGF-D promotes lymphatic vessel growth only after birth. These results suggest, that the
lymphatic vasculature undergoes postnatal maturation, which renders it independent of ligand
induced VEGFR-3 signalling for survival but responsive to VEGF-D for growth. Finally, we
show that FOXC2 is necessary for the later stages of lymphatic development by regulating the
morphogenesis of lymphatic valves, as well as interactions of the lymphatic endothelium with
vascular mural cells, in which it cooperates with VEGFR-3. Furthermore, our study indicates
that the absence of lymphatic valves, abnormal association of lymphatic capillaries with
mural cells and an increased amount of basement membrane underlie the pathogenesis of LD.
These findings have given new insight into the mechanisms of normal lymphatic
development, as well as into the pathogenesis of diseases involving the lymphatic vasculature.
They also reveal new therapeutic targets for the prevention and treatment of tumor metastasis
and lymphatic vascular failure in certain forms of lymphedema. Several interesting questions
were posed that still need to be addressed. Most importantly, the mechanism of VEGF-C
promoted tumor metastasis and the molecular nature of the postnatal lymphatic vessel
maturation remain to be elucidated.
8
REVIEW OF THE LITERATURE
1. Structure and function of the two vascular systems
In vertebrates oxygen, nutrients, hormones and cells of the immune system are delivered to
and waste products removed from the tissues by blood vessels. These form a closed,
hierarchically ordered circulatory system, where the heart pumps oxygenated blood via large
arteries and arterioles, designed for fast flow and low permeability, into capillaries, which are
specialized for bidirectional exchange of gases and metabolites between the blood and the
surrounding tissue. Postcapillary venules, which are the site for leukocyte extravasation to the
interstitium, and veins return the deoxygenated blood from the systemic circulation back to
the heart, from where it is circulated to the lungs for reoxygenation. Pressure inside the blood
vascular system causes plasma to leak out from the capillaries into the interstitial space. The
lymphatic vessels, which start blind ended in the tissues, serve for recovery and unidirectional
transport of some of the extravasated fluid and macromolecules from the periphery back to
the blood circulation. In addition to their drainage function, the lymphatic vessels are crucial
for the absorption of long-chain dietary triglycerides and lipophilic compounds in the
intestine. Furthermore, the lymphatic vasculature, which together with the lymphoid organs
comprises the lymphatic system, plays an essential role in body’s immune defence, by
transporting antigens and antigen presenting cells from the interstitium to be displayed for B
and T cells in the lymph nodes.
1.1
The blood vascular system
The lumen of blood vessels is lined by a monolayer of blood vascular endothelial cells. In
most vessels these are firmly attached to each other by tight junctions, which form a barrier
that regulates paracellular permeability to plasma solutes and leukocytes as well as maintains
cell polarity to apical and basolateral surfaces (reviewed in Liebner et al., 2006). In addition
to tight junctions, adherens junctions serve to further regulate vessel permeability, and are
important for contact inhibition of growth whereas gap junctions are cell to cell
communication structures providing a passage for exchange of small molecule solutes
between neighbouring endothelial cells (reviewed in Dejana, 2004). These junctional
complexes are numerous and well developed in arteries and in the blood vessels of organs
with strictly controlled permeability, such as the brain, but are less well organized in
postcapillary venules and in the specialized capillaries of organs with a high rate cell and
particle trafficking, such as the liver, spleen and bone marrow. These organs contain vessels
characterized by a discontinuous, also referred to as porous or sinusoidal, endothelium
containing pores or gaps that allow an unrestricted exchange of cells and macromolecules
between the vessel lumen and the interstitium (reviewed in Risau et al., 1998; Bazzoni &
Dejana, 2004). Another type of specialized blood vessels are the fenestrated capillaries, which
are adapted for a high secretion rate, filtration or absorption of fluid and macromolecules and
found in endocrine organs, such as adrenal and thyroid glands, as well as in the choroid
plexus, kidney and small intestine. The endothelial cells of the fenestrated endothelium are
characterized by the presence of fenestrae, clusters of circular transcellular openings or pores
containing a diaphragm (reviewed in Risau, 1998).
9
On their basolateral side the blood vascular endothelial cells are seated on a basement
membrane (BM), a thin sheet of extracellular matrix (ECM) primarily composed of the type
IV and XVIII collagens, laminin, fibronectin and heparan sulphate proteoglycans (HSPGs).
The composition and integrity of the BM varies depending on the vessel type and the tissue
environment. Proper assembly of the BM is necessary for vessel stability (Thyboll et al.,
2002). Only the discontinuous blood endothelium is devoid of a BM.
The endothelium of blood vessels is further supported and stabilized by a rather
heterogeneous group of vascular mural cells, the two major classes of which are pericytes
(PCs) and vascular smooth muscle cells (SMCs). PCs surround newly formed blood vessels,
capillaries and postcapillary venules. They are embedded within the endothelial BM, make
close contacts with the endothelium and extend numerous protrusions around the endothelial
cells (Figure 1). Vascular SMCs reside on the abluminal side of the endothelial BM and form
one or more cell layers around the bigger vessels. The walls of big arteries, designed to
withstand high pressure, are composed of three distinct zones. The inner coat, tunica intima,
consists of the endothelial cell layer, BM, ECM proteins, mesenchymal cells and a few layers
of SMCs. The middle coat, tunica media or the smooth muscle cell layer, is separated from
the intima by the internal elastic lamina, and is composed of oriented, contractile SMCs,
elastic fibers, collagen and proteoglygans. The outermost coat, tunica adventitia or the
connective tissue layer, is separated from the media by the external elastic lamina and consists
of fibroblasts, SMCs and mast cells in a matrix of collagen and PGs. The adventitia is
penetrated by nerve fibers, blood capillaries and lymphatic vessels, which also partly protrude
into the media.
1.2
The lymphatic vascular system
In contrast to the blood vasculature, the lymphatic vasculature does not form a closed
circulatory system. Instead, the initial lymphatic capillaries form a network in the tissues.
They drain into the precollecting lymphatic vessels, which transport the lymph into successive
sets of lymph nodes. Lymph from the intestinal, hepatic and lumbar regions is collected into
the cisterna chyli. Finally, the collecting lymphatic vessels drain into the thoracic duct or,
from the upper right quadrant of the human body, into the right lymphatic duct. These are
then emptied into the venous circulation at the junctions of left or right internal jugular veins
and subclavian veins, respectively. A unique bicuspid valve prevents venous reflux into the
lymphatic ducts (reviewed in Aalami et al., 2000). Avascular tissues like the epidermis,
cartilage and cornea, as well as some vascularized tissues like the brain, bone marrow and
retina do not contain lymphatic vessels (reviewed in Oliver & Detmar, 2002).
Compared to blood vessels, lymphatic vessels are thin walled and have a relatively wide
lumen. The endothelial cells of initial lymphatic capillaries lack tight junctions. Instead, the
neighbouring lymphatic endothelial cells are partly overlapping, forming valve like openings,
which allow easy access for fluid, macromolecules and cells into the lymphatic lumen
(Figure 1). The lymphatic capillaries completely lack mural cells and have no or only an
incomplete, fragmented BM (Leak & Burke, 1966). The endothelial cells of lymphatic
capillaries are attached to the surrounding ECM by elastic fibres, the so called anchoring
filaments, which maintain the patency of lymphatic vessels, and during increased tissue
pressure or inflammation pull the endothelial cells apart, opening the gaps between adjacent
10
cells thus further facilitating the uptake of fluid and macromolecules (Leak & Burke, 1968;
reviewed in Casley-Smith, 1980; Witte et al., 1997). In the absence of pressure or
contractility of surrounding SMCs, the alternating compression and dilation of lymphatic
capillaries by respiratory movement, contraction of skeletal muscles and the pulsation of the
adjacent arteries help to propel the lymph forward. The precollecting and collecting lymphatic
vessels have a BM and are surrounded by a SMC layer with intrinsic contractile activity,
which promotes lymph flow. They also contain valves to prevent retrograde flow of the
lymph.
The endothelial cells of blood and lymphatic vessels (BECs and LECs, respectively) represent
two related yet distinct cell lineages. Techniques for isolation of primary LECs and BECs
have greatly facilitated lymphatic research over the past few years, for example by enabling
identification of the molecular differences between these two endothelial cell types
(Kriehuber et al., 2001; Makinen et al., 2001b; Podgrabinska et al., 2002; Hirakawa et al.,
2003). Interestingly, in a mixed culture both cell types make preferentially homotypic
interactions.
Figure 1: Structure of the lymphatic vessels. The lymphatic capillaries start blind ended in tissues. Their
endothelial cells form overlapping valve-like junctions and are attached to the surrounding stroma by elastic
fibres called anchoring filaments (green), which in conditions of increased tissue pressure pull the lymphatic
endothelial cells apart, opening gaps between them to facilitate access of fluid, macromolecules and cells to the
lymphatic lumen. In contrast, the endothelial cells of blood vessels form tight and adherence junctions. The
lymphatic capillaries lack pericytes and have no or only a discontinuous basement membrane (grey fragments).
The intermediate and collecting lymphatic vessels are surrounded by a smooth muscle cell layer (blue), have a
basement membrane (grey) and, like veins, contain valves that prevent lymph backflow. The valve regions are
devoid of smooth muscle cells. In contrast, blood vessels have a continuous basement membrane (grey) and are
surrounded by pericytes and smooth muscle cells (blue), which form one or multiple layers increasing in
thickness with the vessel size. Figure modified from (Jones et al., 2001a; Alitalo et al., 2005).
2. Development of the two vascular systems
The cardiovascular system is the first organ to start functioning during embryogenesis and its
proper development is crucial for the survival and growth of the embryo. Blood vessels are
required to meet the metabolic requirements of the tissues, but the endothelial cells also
11
provide instructive morphogenetic cues to promote organ formation and patterning (Lammert
et al., 2001; Matsumoto et al., 2001; Mattot et al., 2002; Eremina et al., 2003; Lammert et al.,
2003; Maynard et al., 2003; Bjarnegard et al., 2004; Yoshitomi & Zaret, 2004). In turn,
organs influence the endothelial cells and blood vessels to adopt tissue specific features, like
fenestrations or the blood-brain barrier (reviewed in Nikolova & Lammert, 2003; Coultas et
al., 2005). This reflects the interdependent development and intimate association of the blood
vessels and their cognate organs.
2.1
Vasculogenesis
During embryonic development the first blood vessels evolve by vasculogenesis, formation of
an immature, uniform network of endothelial tubes by de novo differentiation of mesodermal
precursor cells called angioblasts. Vasculogenesis starts in the extra-embryonic tissues by
assembly of cell clusters known as blood islands. The cells in the center of the blood islands
develop into hematopoietic stem cells, whereas the outer cells differentiate into angioblasts
and further to endothelial cells. These then form a primitive capillary plexus consisting of
homogenously sized and shaped endothelial channels. Except for some specific regions,
vasculogenesis in the embryo proper occurs without the concomitant differentiation of
hematopoietic cells. The dorsal aorta, cardinal vein and endocardium are formed directly by
aggregating angioblasts, whereas other vessels in the embryo arise from the intra-embryonic
capillary plexus (reviewed in Risau & Flamme, 1995; Flamme et al., 1997; Eichmann et al.,
2000; Luttun et al., 2002a; Ferguson et al., 2005). The close association of the nascent
endothelial and hematopoietic cells in the blood islands has suggested that both cell lineages
are derived from common progenitors, the hemangioblasts. Progenitors that can give rise to
both endothelial and hematopoietic cells have been isolated from embryonic stem (ES) cell
cultures and gastrulating mouse embryos, which provides evidence for the existence of
hemangioblasts (Choi et al., 1998; Chung et al., 2002; Huber et al., 2004). It cannot be
excluded though that these represent a more primitive progenitor with a broader potential to
differentiate into multiple mesodermal lineages including vascular SMCs (Yamashita et al.,
2000; Ema et al., 2003).
2.2
Angiogenesis and arteriogenesis
The uniform plexus of primitive endothelial channels undergoes extensive remodelling in
order to form a hierarchically ordered network of larger and smaller vessels. This remodelling
requires both regression, or pruning, of vessels and angiogenesis, a process that includes
sprouting, branching, splitting, intercalation and fusion of pre-existing vessels. These
processes adjust the vascular density to meet the metabolic demand of the tissue (reviewed in
Risau, 1997; Carmeliet, 2000). The vascular network is created with remarkable precision and
reproducibility with respect to the branching pattern and hierarchy of differently sized arteries
and veins (reviewed in Weinstein, 1999). The final outcome is determined by a combination
of genetic programming and extrinsic influences, such as hypoxia and haemodynamic flow (le
Noble et al., 2004; Ramirez-Bergeron et al., 2004; reviewed in Coultas et al., 2005).
Arteriogenesis refers to maturation of the blood vessels and includes the deposition of BM
components as well as recruitment and organization of supporting vascular mural cells, PCs
and SMCs, into the vessel wall (reviewed in Carmeliet, 2000).
12
2.3
Lymphangiogenesis
The lymphatic vessels arise after the cardiovascular system is established and fully functional.
Two theories exist for the origin of the lymphatic vessels, both of which date back to the
beginning of the 20th century and have gained support from data obtained with the more
recent methods of molecular and cellular biology. The prevailing one postulates a venous
origin for the lymphatic endothelial cells (Sabin, 1902, 1904, 1909). The lymphatic
development starts around midgestation, when a distinct subpopulation of endothelial cells on
one side of the anterior cardinal vein commit to a lymphatic lineage, and subsequently sprout
and migrate to form primary lymph sacs (van der Putte, 1975; reviewed in Oliver & Detmar,
2002; Oliver, 2004). Subsequently, in addition to these two jugular ones several other lymph
sacs are formed in different regions of the embryo. The peripheral lymphatic vasculature is
then generated by centrifugal sprouting of lymphatic vessels from these lymph sacs, followed
by a merging of the separate lymphatic capillary networks, and remodelling and maturation of
the lymphatic vasculature (Figure 2).
Another theory suggests that lymphatic endothelial cells are derived from mesenchymal
progenitor cells (Huntington & McClure, 1908; Kampmeier, 1912). The existence of
lymphatic endothelial cell precursors, lymphangioblasts, has been shown in birds and
amphibians, but their existence in mammals is still controversial (Schneider et al., 1999;
Papoutsi et al., 2001; Ny et al., 2005). Thus, in addition to dedifferentiation of LECs from
venous endothelial cells and subsequent sprouting lymphangiogenesis, differentiation from
mesenchymal precursor cells may contribute to the formation of the lymphatic vasculature
during embryogenesis.
Figure 2: Development of the lymphatic vasculature. Upon stimulation by as yet unidentified signal(s) a
subset of venous endothelial cells becomes committed to the lymphatic endothelial fate. These differentiating
lymphatic endothelial cells express Prox1, LYVE1 and VEGFR-3. Stimulated by VEGF-C, which is secreted by
the adjacent tissue, they migrate and proliferate to form primary lymph sacs, from which the lymphatic vessels
sprout further. The initial lymphatic network (green) becomes separated from the blood vessels (orange) and
undergoes remodelling and maturation to create a lymphatic vasculature consisting of a lymphatic capillary
network, which lacks pericytes, and of collecting lymphatic vessels, which contain valves (yellow) and are
associated with smooth muscle cells (red). Molecules involved at these later stages of the lymphatic
development include ephrinB2, NP2, Ang2, podoplanin, integrin α9 and the transcription factors FOXC2, Net,
Sox18 and Vezf1 (see text and Table I for references). Figure modified from (Alitalo et al., 2005).
13
2.4
Angiogenesis and lymphangiogenesis in adult tissues
In adults, endothelial cells are normally in a quiescent state, but can be induced by a variety of
stimuli. New blood vessel growth is required in several physiological processes, such as
wound healing and the female reproductive cycle, including ovulation, menstruation and
placental growth. As during development, blood vessel growth in the adult is tightly
controlled, as sustained neovascularization and excessive or insufficient angiogenesis
contribute to the development of several pathological conditions (reviewed in Carmeliet,
2003).
Angiogenesis and arteriogenesis, sprouting of new blood vessels from pre-existing ones and
their subsequent maturation, are regarded as the primary methods of neovascularization in the
adult. Collateral growth, the expansion of pre-existing vessels forming collateral bridges
between arterial networks, represents an additional mechanism for blood vessel formation in
the adult, especially in ischemic settings. It has recently been suggested that bone marrow
derived cells can be incorporated into nascent blood vessels, in a process referred to as
postnatal vasculogenesis (reviewed in Carmeliet & Luttun, 2001; Kopp et al., 2006). The
relative contribution of these putative endothelial progenitor cells (EPCs) to adult
neovascularization, as well as their exact identity, is still unclear, and currently a hot topic of
research in the field of angiogenesis.
Lymphatic vessel growth appears to follow that of the blood vessels also in the adult, for
example during wound healing and in inflammation (Paavonen et al., 2000; Baluk et al.,
2005). New lymphatic vessels are probably required to drain the fluid and macromolecules
that leak out from the newly formed immature blood capillaries, and to regulate the
inflammatory responses. As in the case of blood vessels, new lymphatic vessels are generally
thought to grow primarily by sprouting from existing ones. Although the existence of bone
marrow derived or circulating putative progenitors capable of differentiating into lymphatic
endothelial cells has been suggested, the currently available data are insufficient for definitive
conclusions about their contribution to neolymphangiogenesis in the adult. Endothelial cells
of host origin in the newly formed lymphatic vessels of inflamed kidney transplants were
explained to be derived from bone marrow precursors (Kerjaschki et al., 2006) and in
inflamed mouse cornea macrophages were suggested to transdifferentiate to lymphatic
endothelial cells (Maruyama et al., 2005). In other studies bone marrow derived cells were
found to contribute to perivascular mural cells but not detectably to the blood or lymphatic
endothelium (He et al., 2004b; Rajantie et al., 2004). Interestingly, whereas new blood
vessels rapidly regress after the withdrawal of the stimulus, newly formed lymphatic vessels
persist longer (Baluk et al., 2005).
3. Molecular regulation of vascular development
Development of the blood and lymphatic vascular systems are multistep processes that are
tightly regulated by a proper balance between a variety of stimulatory and inhibitory factors
from several families. Some of these act directly on endothelial cells, others act indirectly by
stimulating associated cells such as macrophages or stromal cells. In the following, only the
key regulators of blood and lymphatic vessel development and those of direct importance to
the studies presented in this thesis are discussed in detail.
14
3.1
Vascular endothelial growth factors
Vascular endothelial growth factors (VEGFs) belong structurally to the VEGF/PDGF
(platelet-derived growth factor) family of growth factors characterized by eight conserved
cysteine residues in their VEGF homology domain (VHD). Although currently comprising
five mammalian members, VEGF and VEGF-C appear to be the only essential factors of the
VEGF family, the former for vasculogenesis and angiogenesis and the latter for embryonic
lymphangiogenesis. The other members, PlGF, VEGF-B and VEGF-D, are likely to play
modifying roles under specific conditions, such as pathological angiogenesis.
The existence of a VEGF receptor homolog and several VEGF homologs in simple
invertebrates, which lack a closed vascular system, suggests that this family of growth factors
appeared very early in the evolution of multicellular organisms, and mediated more
primordial developmental functions like cell migration (Duchek et al., 2001; Heino et al.,
2001; Cho et al., 2002; Ishimaru et al., 2004). In Drosophila, the VEGFR homolog (PVR) is
expressed in the blood cells, the so called hemocytes, and is required for hemocyte migration,
stimulated by the three VEGF homologs expressed along the hemocyte migration routes.
3.1.1
VEGF
Vascular endothelial growth factor (VEGF), also referred to as vascular permeability factor
(VPF), has since its identification less than two decades ago been recognized as one of the
most important regulators of endothelial cell physiology mediating both developmental,
physiological and pathological angiogenesis as well as inducing vascular permeability
(Senger et al., 1983; Keck et al., 1989; Leung et al., 1989; Ferrara et al., 1998). It promotes
angiogenesis by stimulating vascular endothelial cell proliferation and migration, and is
required for the survival of endothelial cells in newly formed blood vessels (Connolly et al.,
1989; Ferrara & Henzel, 1989; Alon et al., 1995; Benjamin et al., 1999). Although the
endothelium of mature blood vessels in adults was believed not to require VEGF for
maintenance, recent evidence indicates that at least the fenestrated capillaries of endocrine
glands are dependent on continuous VEGF signalling (Gerber et al., 1999a; Kamba et al.,
2006). Deletion of even a single Vegf allele leads to embryonic lethality due to abnormal
formation of blood islands and blood vessels, which are even more impaired in embryos
lacking both Vegf alleles, demonstrating a remarkably strict dose-dependence of early blood
vessel development for VEGF (Carmeliet et al., 1996; Ferrara et al., 1996). The essential role
of regulated VEGF signalling during development is further demonstrated by the early
embryonic lethality of mice with moderate overexpression of Vegf from its endogenous locus
(Miquerol et al., 2000). Furthermore, Vegf deficient ES cells have a dramatically reduced
ability to form tumors in nude mice, indicating the importance of VEGF in pathological
angiogenesis (Ferrara et al., 1996). It has also been suggested that VEGF induces migration
but not proliferation of vascular SMCs, at least in vitro (Grosskreutz et al., 1999).
VEGF is an antiparallel dimer, covalently linked by two disulfide bonds with receptor binding
sites at each pole (Muller et al., 1997; Wiesmann et al., 1997). Human VEGF exists as at least
eight proangiogenic isoforms, generated by alternative splicing of a single gene (reviewed in
Takahashi & Shibuya, 2005). These differ in their association to heparan sulphate rich ECM
and cell surface HSPGs, receptor binding characteristics and expression pattern, which
influences their potential to affect endothelial cell proliferation and migration. VEGF121 does
15
not bind heparin and is freely diffusible in the extracellular space, whereas VEGF189 strongly
binds heparin and is completely sequestered in the ECM and at the cell surface. The
predominant isoform, VEGF165, has a moderate affinity to heparin and thus to HSPGs in the
ECM and cell surface (Park et al., 1993). The less frequent isoforms of human VEGF include
VEGF145, VEGF148, VEGF162, VEGF183 and VEGF206. The respective mouse isoforms are one
amino acid shorter each (Takahashi & Shibuya, 2005). All VEGF isoforms bind to and
activate both VEGFR-1 and VEGFR-2, although with different potential (Figure 3).
Furthermore, neuropilins (NP) act as isoform specific VEGF receptors (discussed in more
detail in section 3.3.1). Additionally, an endogenous inhibitory form of VEGF, VEGF165b, that
binds to VEGFR-2 but does not activate it or its downstream signalling pathways, has been
described (Woolard et al., 2004). The ECM bound isoforms can be released as bioactive
fragments through cleavage by plasmin, urokinase or matrix metalloproteinases at their
carboxyterminus (Park et al., 1993; Plouet et al., 1997; Lee et al., 2005). Although the
VEGF165 isoform plays the most central role in vascular development, the less prevalent
isoforms also have distinct roles in vascular patterning and especially arterial development
(Stalmans et al., 2002). Mice lacking heparin-binding Vegf isoforms die within two weeks of
birth, due to impaired myocardial angiogenesis and ischemic cardiomyopathy (Carmeliet et
al., 1999b). These mice also exhibit a specific decrease in capillary branch formation and
impairement of the directed extension of endothelial cell filopodia, leading to an increase in
capillary lumen size of the existing vessels, which incorporate the nascent endothelial cells
(Ruhrberg et al., 2002). Opposite defects, excess endothelial filopodia and abnormally thin
capillary branches at ectopic sites, were found in mice expressing only a heparin binding form
of Vegf (Ruhrberg et al., 2002). This suggests that the specific localization of VEGF isoforms
in the extracellular space provides spatially restricted stimulatory cues that polarize and guide
sprouting endothelial cells to initiate vascular branch formation.
VEGF has a ubiquitous tissue distribution, and is produced by a variety of cell types,
indicating its role as a paracrine factor for endothelial cells (Breier et al., 1992; Ferrara,
1999). VEGF gene expression is regulated by a variety of stimuli such as growth factors,
hormones, transformation, p53 mutation, tumor promoters and nitric oxide, the most
important probably being hypoxia (Takahashi & Shibuya, 2005). The key mediator of
hypoxic responses is the transcriptional activator HIF-1 (hypoxia inducible factor 1)
consisting of two constitutively expressed subunits, HIF-1α and HIF-1β. Under normoxic
conditions HIF-1α is rapidly targeted for degradation by the ubiquitin E3 ligase complex
containing the tumor suppressor VHL (von Hippel-Lindau protein). The interaction of VHL
protein with HIF-1α is dependent on hydroxylation of two specific proline residues in HIF-1α
by a prolyl-4-hydroxylase, which requires oxygen and iron for its activity. Under hypoxia, the
prolyl hydroxylation of HIF-1α is suppressed, leading to decreased ubiquitination and thus
stabilization and increased levels of HIF-1α (Ivan et al., 2001; Jaakkola et al., 2001).
Furthermore, oxygen dependent asparaginyl hydroxylation of HIF-1α by FIH (factor
inactivating HIF) inactivates its transcriptional activity (Lando et al., 2002). In addition to the
tight transcriptional control, VEGF is also regulated at the posttranscriptional level as well as
at the level of mRNA stability (Takahashi & Shibuya, 2005).
When highly overexpressed in the skin, as well as during wound healing and in some
experimental tumors, VEGF also induces lymphangiogenesis (Nagy et al., 2002; Hong et al.,
2004b; Hirakawa et al., 2005), whereas in some other experimental settings, like in the
16
differentiated chick chorioallantoic membrane, and in a mouse insulinoma tumor model,
VEGF specifically promotes the growth of blood vessels (Oh et al., 1997; Gannon et al.,
2002). Although VEGFR-2 is expressed in some, mainly collecting and activated, lymphatic
vessels (Saaristo et al., 2002b; Hong et al., 2004b; Hirakawa et al., 2005), at least some of the
lymphatic effects of VEGF might be secondary, due to the induction of vessel permeability
and recruitment of inflammatory cells that produce lymphangiogenic growth factors
(Cursiefen et al., 2004; Baluk et al., 2005).
Figure 3: Growth factors of the VEGF family, their binding to VEGF receptor tyrosine kinases and
signalling pathways induced. Circle, immunoglobulin homology domain; SS, disulfide bond. Positions of the
tyrosine residues are indicated in black and the identified tyrosine phosphorylation sites by numbers in the
intracellular domains of the receptors. R after the number indicates that the use of the phosphorylation site is
regulated, depending on the angiogenic state of the endothelial cell (VEGFR-2), on the particular ligand (PlGF
but not VEGF for VEGFR-1) or heterodimerization (marked VEGFR-3 sites are phosphorylated only in the
VEGFR-3 homodimer but not in a VEGFR-2/VEGFR-3 heterodimer). Binding of signalling molecules to certain
phosphorylation sites (boxed numbers) initiates signalling cascades, which lead to specific biological responses
(boxes). The mode of initiation of signalling cascades marked with dashed arrows is unclear. JM,
juxtamembrane domain; TK1 and TK2, tyrosine kinase domain; KI, kinase insert; CT, carboxyterminus; HPC,
hematopoietic progenitor cell; EC, endothelial cell; DAG, diacylglycerol; eNOS, endothelial nitric oxide
synthase; FAK, focal adhesion kinase; HSP27, heat-shock protein-27; MAPK, mitogen-activated protein kinase;
MEK, MAPK and ERK kinase; PI3K, phosphatidylinositol 3’-kinase; PKC, protein kinase C; PLCγ,
phospholipase Cγ; TSAd, T cell specific adaptor. The putative unknown pathway for activation of VEGFR-3 is
indicated by a question mark (see Results and Discussion, paragraph 4). Figure modified from (Olsson et al.,
2006).
17
3.1.2
PlGF
PlGF (Placenta Growth Factor) exists as four alternatively spliced isoforms, PlGF-1 to PlGF4, which are most highly expressed in the placenta and upregulated in tumors. All PlGF
isoforms bind to VEGFR-1, but not to the major angiogenic signal transducer VEGFR-2, and
the heparin binding forms PlGF-2 and PlGF-4 also interact with neuropilins (Takahashi &
Shibuya, 2005). PlGF can also form heterodimers with VEGF (DiSalvo et al., 1995; Cao et
al., 1996). It has been reported that PlGF induces angiogenesis in vivo and that it is
chemotactic and mitogenic for endothelial cells in vitro (Ziche et al., 1997). Furthermore,
PlGF stimulates the migration of monocytes (Clauss et al., 1996). Plgf deficiency does not
affect embryonic blood vessel development in mice, but reduces pathological angiogenesis,
permeability and collateral growth during ischemia, inflammation, wound healing and in
cancer (Carmeliet et al., 2001). It has been suggested that PlGF mediates its effects in
pathological angiogenesis by mobilizing bone marrow derived endothelial precursor cells and
by potentiating the effects of VEGF (Carmeliet et al., 2001). In line with these results, it has
been suggested that PlGF has therapeutic potential in ischemic conditions without side effects
associated with VEGF, such as edema (Luttun et al., 2002b).
3.1.3
VEGF-B
VEGF-B, also known as VEGF related factor (VRF), exists as two isoforms generated by
alternative splicing, the heparin binding form VEGF-B167 and the hydrophobic, proteolytically
processable VEGF-B186 (Olofsson et al., 1996a; Olofsson et al., 1996b). Both forms bind to
VEGFR-1 and to NP1 (Olofsson et al., 1998; Makinen et al., 1999). Recently, the crystal
structure of VEGF-B has suggested a structural basis for this receptor specificity (Iyer et al.,
2006). VEGF-B has a wide tissue distribution, but is most abundant in the heart and skeletal
muscle (Aase et al., 1999; Olofsson et al., 1999). Although studies on PlGF have
demonstrated the role of VEGFR-1 in pathological conditions, the involvement of VEGF-B in
developmental or pathological angiogenesis is not clear. Vegfb-/- mice are viable and fertile,
but whereas one group has reported smaller hearts, dysfunctional coronary arteries and an
impaired recovery from experimentally induced myocardial ischemia, others have discovered
only a subtle cardiac phenotype in the form of an atrial conduction abnormality, characterized
by a prolonged PQ interval (Bellomo et al., 2000; Aase et al., 2001). VEGF-B has also been
implicated in pathological vascular remodelling in inflammatory arthritis, and in protecting
the brain from ischemic injury (Mould et al., 2003; Sun et al., 2004).
3.1.4
VEGF-C
VEGF-C (or VEGF-related protein, VRP) is produced as a preproprotein consisting of a
signal peptide, an aminoterminal propeptide with no homology to known proteins, VEGF
homology domain (VHD) and a carboxyterminal propeptide (CT) with cysteine-rich repeats
homologous to the Balbiani ring 3 domain of silk proteins (Joukov et al., 1996; Lee et al.,
1996). Instead of having splice variants, the receptor binding pattern and affinity and thus the
biological activity of VEGF-C is regulated by stepwise proteolytic processing (Figure 4).
Upon proteolytic cleavage the affinity of VEGF-C towards VEGFR-3 increases and the fully
processed, mature form of VEGF-C also binds to and activates VEGFR-2 (Joukov et al.,
1997). Plasmin is able to cleave both propeptides and the proprotein convertases furin, PC5
and PC7 are capable of cleaving the carboxyterminal propeptide of VEGF-C, at least in vitro
18
(McColl et al., 2003; Siegfried et al., 2003). The first step, cleavage of the carboxyterminal
propeptide, primarily occurs before, but is not prerequisite for, secretion. Beyond that not
much is known about the regulation of the proteolytic processing of VEGF-C.
A paracrine expression pattern between VEGF-C and VEGFR-3 during embryogenesis at the
sites of first lymphatic sprouting suggests a role for VEGF-C in the regulation of
lymphangiogenesis (Kaipainen et al., 1995; Kukk et al., 1996). Indeed, VEGF-C has been
shown to specifically induce growth of lymphatic but not blood vessels both in the skin of
transgenic mice and in differentiated chicken chorioallantoic membrane (CAM; Jeltsch et al.,
1997; Oh et al., 1997). Although not required for lymphatic endothelial commitment, VEGFC is essential for the initial sprouting and directed migration as well as for the subsequent
survival of lymphatic endothelial cells (Karkkainen et al., 2004). Deletion of both Vegfc
alleles in mice results in embryonic lethality, and heterozygous Vegfc+/- mice display defects
in lymphatic vascular development, indicating the importance of tightly regulated VEGF-C
concentration (Karkkainen et al., 2004). Although dispensable for blood vascular
development in mice, VEGF-C was found to be required for angiogenesis in both zebrafish
and in Xenopus tadpoles (Ober et al., 2004; Ny et al., 2005). At higher concentrations and
under specific conditions, like in the ischemic limb, or in mouse cornea and undifferentiated
CAM, which do not contain lymphatic vessels, VEGF-C is also able to induce the growth of
blood vessels (Cao et al., 1998; Witzenbichler et al., 1998; Rissanen et al., 2003) and to
increase vascular permeability (Joukov et al., 1997). In vitro, VEGF-C has been shown to act
synergistically with VEGF to induce tube formation of bovine aortic endothelial cells (Pepper
et al., 1998), and to promote proliferation and migration of both lymphatic and blood vascular
endothelial cells (Joukov et al., 1997; Makinen et al., 2001b). The VEGF-C induced effects
are probably mediated by VEGFR-2 on the blood vascular endothelium and primarily by
VEGFR-3 on the lymphatic endothelium, although the expression of VEGFR-2 on lymphatic
vessels might contribute to mitogenic signalling (Veikkola et al., 2001; Saaristo et al.,
2002b). Thus the degree of proteolytic processing of the VEGF-C precursor might dictate its
angiogenic versus lymphangiogenic potential.
VEGF-C is most abundant in the heart and lung and is produced primarily by mesenchymal
cells and vascular SMCs (Kukk et al., 1996; Karkkainen et al., 2004). VEGF-C expression
does not seem to be regulated by hypoxia (Enholm et al., 1997), but its expression is
increased in response to proinflammatory cytokines, suggesting a role in inflammatory
responses (Ristimaki et al., 1998).
3.1.5
VEGF-D
VEGF-D, also known as c-Fos-induced growth factor (FIGF), has a homologous domain
structure and undergoes proteolytic processing in a similar manner to VEGF-C (Orlandini et
al., 1996; Achen et al., 1998; Stacker et al., 1999). Like VEGF-C, fully processed human
VEGF-D binds to and activates both VEGFR-2 and VEGFR-3 (Achen et al., 1998; Stacker et
al., 1999). Interestingly, unlike its human counterpart, mouse VEGF-D is a specific ligand of
VEGFR-3, unable to bind to VEGFR-2 (Baldwin et al., 2001a). Furthermore, alternative
splicing generates two isoforms of mouse, but not human, VEGF-D, which differ in their
carboxyterminal domain in respect to length and glycosylation pattern (Baldwin et al.,
2001b). VEGF-D has a wide tissue distribution, being most abundant in the lung
(Avantaggiato et al., 1998; Farnebo et al., 1999). VEGF-D is mitogenic for endothelial cells
19
(Achen et al., 1998). Although reported to be angiogenic in rabbit cornea and muscle, VEGFD rather specifically induces lymphangiogenesis in some other systems, such as mouse skin
(Marconcini et al., 1999; Veikkola et al., 2001; Rissanen et al., 2003). VEGF-D is
dispensable for embryonic development of both blood vascular and lymphatic systems, the
only defect caused by deletion of Vegfd in mice being a slight reduction in the number of
lymphatic vessels around the lung bronchioles (Baldwin et al., 2005).
Figure 4: Structure and proteolytic processing of VEGF-C. VEGF-C is produced as a 61 kDa prepropeptide
consisting of a signal peptide (SP, gradient), aminoterminal propeptide (NT, striped box), VEGF homology
domain (VHD, white box) and a carboxyterminal propeptide (CT, grey box). Removal of the signal peptide
generates a 58 kDa VEGF-C propeptide, which forms antiparallel homodimers. After proteolytic cleavage the
carboxyterminus remains covalently attached to the aminoterminus of the other peptide by a disulfide bond (SS). This first cleavage occurs predominantly intracellularly, but some cells secrete also the unprocessed
propeptide to a variable extent. Only after cleavage of the aminoterminus, which occurs extracellularly or at the
cell surface, both propeptides are released and the mature VEGF-C is thus a noncovalent (dotted line)
antiparallel dimer of 20 or 21 kDa peptides. Additionally, several alternatively processed minor forms exist. The
primary cleavage sites are marked with arrowheads and the identified proteases are indicated. The
experimentally verified and potential sites for N-linked glycosylation are marked with bold or plain N,
respectively, and the cysteine residues in the VHD with black ovals, whereas the cysteine residues in CT are
omitted for clarity. Numbers indicate the apparent molecular mass of the corresponding polypeptides in kDa.
Proteolytic processing increases the affinity of VEGF-C towards VEGFR-3 and the fully processed form can
bind to and activate also VEGFR-2. Figure modified from (Joukov et al., 1997).
20
3.1.6
VEGF-E and snake venom VEGF
Several viruses, known to induce vascular lesions, carry VEGF homologues in their genomes.
These are collectively referred to as VEGF-E. Most of them are specific ligands for VEGFR2, and some also bind NP1. Many VEGF-E forms have been shown to stimulate endothelial
cell proliferation and angiogenesis, and to induce vascular permeability, although to variable
extents (Lyttle et al., 1994; Ogawa et al., 1998; Meyer et al., 1999; Wise et al., 1999; Kiba et
al., 2003; Wise et al., 2003).
Snakes utilize venom specific VEGF-like molecules to induce hypotension for the
enhancement of toxicity in envenomination. Snake venom VEGFs show considerable
variation in their potential to induce vessel permeability versus angiogenesis, reflecting their
binding profiles to VEGFR-1 and/or VEGFR-2 (Komori et al., 1999; Gasmi et al., 2000;
Junqueira de Azevedo et al., 2001; Gasmi et al., 2002; Takahashi et al., 2004).
The unique receptor binding patterns of these VEGF-like molecules make them convenient
research tools for dissecting VEGFR-1 versus VEGFR-2 mediated signalling effects.
3.2
Vascular endothelial growth factor receptors
VEGFs mediate their signals primarily through their high affinity cell surface receptor
tyrosine kinases VEGFR-1, VEGFR-2 and VEGFR-3. All VEGFRs share a common structure
characterized by an extracellular region consisting of seven immunoglobulin (Ig) homology
domains, a transmembrane domain and an intracellular tyrosine kinase domain split by a
kinase insert, followed by a carboxyterminal region (Figure 3). The second Ig homology
domain, eventually in combination with the first or the third one, is responsible for ligand
binding (Jeltsch et al., 2006). Ligand binding causes homo- or heterodimerization of the
VEGFRs and transphosphorylation of tyrosine residues within their cytoplasmic domains,
which induces the kinase activity of the receptor and creates docking sites for cytoplasmic
downstream signal transduction molecules, finally leading to cellular responses.
3.2.1
VEGFR-1
VEGFR-1, also known as Flt1 (Fms-like tyrosine kinase 1), is expressed as a membrane
bound receptor on blood vascular endothelial cells, PCs, monocytes/macrophages, dendritic
cells, hematopoietic stem cells, trophoblasts and osteoclasts and also exists as a soluble form
(sVEGFR-1, reviewed in Shibuya, 2001; see also chapter 3.7). Although possessing only a
weak kinase activity, partly owing to a repressor sequence in its juxtamembrane domain
(Waltenberger et al., 1994; Seetharam et al., 1995; Gille et al., 2000), VEGFR-1 has been
shown to be involved in migration of monocytes/macrophages (Barleon et al., 1996; Clauss et
al., 1996; Sawano et al., 2001; Hattori et al., 2002; Shibuya & Claesson-Welsh, 2006). Vegfr1
deficient mice die at E8.5 due to disorganized blood vessels caused by increased
hemangioblast commitment and endothelial cell overgrowth, which suggested that VEGFR-1
functions as a negative regulator of angiogenesis during embryonic development, possibly
owing to its high affinity for VEGF (Fong et al., 1995; Fong et al., 1999). Deletion of the
Vegfr1 tyrosine kinase domain allows normal developmental angiogenesis, further suggesting
a role for VEGFR-1 as an inert decoy receptor regulating the bioavailability of VEGF
(Hiratsuka et al., 1998). Membrane fixation, although not tyrosine kinase activity, of
21
VEGFR–1 was subsequently found to be important for its function, suggesting that VEGFR-1
might be required for recruitment of VEGF to the cell surface (Hiratsuka et al., 2005). Recent
studies have revealed a more active signalling role for VEGFR-1 tyrosine kinase, both in
macrophage migration and in pathological angiogenesis (Hiratsuka et al., 1998; Hiratsuka et
al., 2001). Conversely, inhibition of VEGFR-1 suppresses pathological neovascularization
and inflammation, which was attributed to a reduced mobilization of bone marrow derived
myeloid progenitors into the peripheral blood and impaired infiltration of leukocytes into
inflamed tissues (Luttun et al., 2002b). VEGFR-1 seems to have some positive signalling of
its own when stimulated by PlGF, but not by VEGF (Autiero et al., 2003). This is surprising
in light of the fact that crystallization of PlGF and VEGF in combination with the second Iglike domain of VEGFR-1 have revealed that these two ligands interact with the same binding
interface of VEGFR-1 in a very similar manner (Wiesmann et al., 1997; Iyer et al., 2001;
Christinger et al., 2004).
VEGFR-1 has been suggested to positively modulate VEGFR-2 activity, for example during
pathological conditions, possibly through heterodimerization of the two receptors or
attenuation of intracellular negative regulatory pathways like those involving phosphotyrosine
phosphatases (Carmeliet et al., 2001; Huang et al., 2001; Autiero et al., 2003). On the other
hand, several groups have reported that VEGFR-1 negatively regulates VEGFR-2 function by
still unknown mechanisms, which might involve heterodimerization or PI3K dependent
interference with VEGFR-2 downstream signalling in addition to the strong ligand trapping
effect (Rahimi et al., 2000; Zeng et al., 2001; Roberts et al., 2004). It remains to be elucidated
whether these apparently opposing effects of VEGFR-1 on VEGFR-2 activity operate under
different conditions or in different endothelial cell types (reviewed in Olsson et al., 2006).
VEGFR-1 is upregulated by hypoxia through HIF-1 (Gerber et al., 1997). Despite the many
potential interactions of VEGFR-1 with intracellular signalling molecules including PI3K,
PLCγ and Grb2, signalling events downstream of VEGFR-1 are unknown (Olsson et al.,
2006). VEGFR-1 expression has so far not been detected in lymphatic vessels (Hirakawa et
al., 2003; Hong et al., 2004b).
3.2.2
VEGFR-2
VEGFR-2, also known as Flk1 (fetal liver kinase 1) in mice and as KDR (kinase insert
domain containing receptor) in humans, is the earliest marker for hemangioblasts, the
precursors of both endothelial and hematopoietic cells (Millauer et al., 1993; Kabrun et al.,
1997). Deletion of Vegfr2 in mice leads to embryonic lethality at E9 due to lack of both
hematopoietic and endothelial cells, indicating an essential role for VEGFR-2 in the
differentation of endothelial cell progenitors (Shalaby et al., 1995). Furthermore, VEGFR-2
seems to be required for the migration of cells to the correct locations to form blood islands,
from the posterior primitive streak to the yolk sac and, possibly, to the intra-embryonic sites
of early hematopoiesis (Shalaby et al., 1997). Owing to its strong tyrosine kinase activity,
VEGFR-2 is regarded as the major positive signal transducer in angiogenesis, mediating
proliferation, migration and survival signals in endothelial cells (reviewed in Shibuya &
Claesson-Welsh, 2006). In endothelial cells VEGFR-2 seems to activate the
Raf1/MEK/MAPK cascade, leading to DNA synthesis and cell proliferation, independent of
Ras through the PLCγ-PKC pathway (Takahashi et al., 1999b; Takahashi et al., 2001). In
22
addition, VEGFR-2 activates PI3-kinase using the adapter molecule Shb to regulate focal
adhesions for the induction of cell migration (Holmqvist et al., 2004) and to activate AKT for
promoting cell survival (Gerber et al., 1998) and for phosphorylation of endothelial nitric
oxide synthase, which regulates vessel permeability (Dimmeler et al., 1999; Fulton et al.,
1999). In tumor vessels, the T cell specific adapter, TSAd, is employed to mediate migration
(Matsumoto et al., 2005). The identified tyrosine phosphorylation sites and known
downstream signalling pathways of VEGFR-2 are schematically illustrated in Figure 3. Mice
expressing Vegfr2 mutated at the binding site of PLCγ and Shb, tyrosine 1173, display
vascular defects resembling those of Vegfr2-/- mice, indicating the essential function of this
tyrosine residue (Sakurai et al., 2005). Attenuation of VEGFR-2 signalling does not seem to
involve the c-Cbl mediated receptor ubiquitination and turnover, but rather seems to occur by
degradation primed by activation of nonclassical PKCs by a mechanism that requires two
carboxyterminal serine residues of VEGFR-2 but not ectodomain shedding (Singh et al.,
2005).
VEGFR-2 expression declines during later stages of vascular development, but becomes
upregulated in physiological and pathological angiogenesis in adults. In addition, VEGFR-2
expression has been shown to be high in the endothelial tip cells, which through their
filopodia sense the VEGF concentration gradient to guide vascular sprouting, and lower in the
proliferating stalk cells (Gerhardt et al., 2003). Furthermore, VEGFR-2 is expressed in
hematopoietic stem cells (Ziegler et al., 1999). Non-endothelial expression of VEGFR-2 has
been observed in neuronal cells, osteoblasts, pancreatic duct cells, retinal progenitor cells and
megakaryocytes (reviewed in Shibuya & Claesson-Welsh, 2006; see chapter 3.7). A soluble
form of VEGFR-2 has been detected, but its generation, whether by proteolytic cleavage or
alternative splicing, is unclear (Ebos et al., 2004).
3.2.3
VEGFR-3
VEGFR-3, also known as Flt4, has a distinct structural feature as compared to other VEGFRs
in that it is proteolytically cleaved in its fifth Ig homology domain, and a disulfide bridge
keeps the aminoterminal part connected with the rest of the molecule (Pajusola et al., 1993).
Additionally, in humans, alternative splicing into the long terminal repeat of a retrovirus
integrated between the last two exons generates two VEGFR-3 isoforms, the shorter one
lacking 65 amino acid residues in its carboxyterminus (Pajusola et al., 1993; Hughes, 2001).
VEGFR-3 was shown to be able to heterodimerize with VEGFR-2, which leads to differential
carboxyterminal phosphorylation and potentially differential signal transduction properties of
VEGFR-3 than in the homodimeric configuration (Dixelius et al., 2003).
During the early stages of vascular development, before the evolvement of lymphatic vessels,
VEGFR-3 is expressed in the endothelium of the blood vasculature. Although not required for
vasculogenesis, VEGFR-3 is essential for the remodelling and maturation of the primary
vascular plexus and mice deficient of Vegfr3 die at E9.5 due to a cardiovascular failure
(Dumont et al., 1998). Later in embryogenesis expression of VEGFR-3 decreases in the blood
vessel endothelium, first in the arteries and then in the veins, finally becoming restricted
primarily to lymphatic endothelial cells (Kaipainen et al., 1995). VEGFR-3 mediates the
sprouting and migration of differentiated lymphatic endothelial cells from the veins, and is
required for the survival and maintenance of lymphatic vessels during embryonic
development (Makinen et al., 2001a; Karkkainen et al., 2004). VEGFR-3 mediated signals
23
alone are sufficient for inducing the growth of lymphatic vessels, as a VEGF-C mutant,
VEGF-C156S, which is unable to bind VEGFR-2, is capable of stimulating
lymphangiogenesis (Joukov et al., 1998; Veikkola et al., 2001). In addition to stimulating cell
migration, VEGFR-3 induces PKC dependent activation of the p42/p45 MAPK signalling
cascade and PI3-kinase dependent Akt phosphorylation to mediate growth and survival
signals in isolated lymphatic endothelial cells (Makinen et al., 2001b). In adults, VEGFR-3 is
also expressed in fenestrated blood vessels and in monocytes/macrophages (Partanen et al.,
2000; Skobe et al., 2001a; Schoppmann et al., 2002). Furthermore, VEGFR-3 expression
becomes upregulated in the angiogenic blood vessels of tumors (Partanen et al., 1999; Valtola
et al., 1999). Recently, VEGFR-3 expression was also reported in osteoblasts and in neural
progenitor cells (Le Bras et al., 2006; Orlandini et al., 2006; see chapter 3.7).
3.3
Molecules that modulate VEGFR signalling
VEGFs and their specific tyrosine kinase receptors directly interact with several cell surface
molecules of various classes. These interactions significantly modify the signalling activities
of VEGFRs. A good example of such interaction is the formation of a mechanosensory
complex comprising of VEGFR-2, PECAM-1 and VE-cadherin, which acts upstream of
integrin activation to confer responsiveness to shear stress (Tzima et al., 2005). In addition,
coordinated crosstalk between VEGFR and other signalling pathways is crucial for providing
specificity.
3.3.1
Neuropilins
Neuropilin-1 (NP1) and NP2 are transmembrane non-tyrosine kinase glycoproteins (Takagi et
al., 1991; Kolodkin et al., 1997). They were originally found to be expressed in the tips of
growing axons and to function as high-affinity receptors for class III semaphorins (SEMA3),
secreted molecules signalling repulsive axon guidance (Takagi et al., 1995; Kawakami et al.,
1996; Chen et al., 1997; He & Tessier-Lavigne, 1997; Kolodkin et al., 1997). The
extracellular part of neuropilins consists of two complement binding (CUB) domains (a1 and
a2 domains) involved in SEMA3 binding, two coagulation factor V/VIII homology domains
(b1 and b2 domains) involved in interactions with SEMA3 and VEGF, and a meprin (MAM
or c) domain thought to be important for neuropilin dimerization and for the interaction of
neuropilins with other receptors (Figure 5). As deletion of the short intracellular domain of
NP1 did not affect SEMA3A induced growth cone collapse, neuropilins were thought not to
have a signalling capacity of their own, but to interact with other cell surface receptors, in the
case of neural cells with members of the plexin family, for signal transduction (Nakamura et
al., 1998; Takahashi et al., 1999a; Tamagnone et al., 1999). Later, a chimeric receptor
consisting of the extracellular domain of epidermal growth factor receptor fused to the
transmembrane and intracellular domains of NP1 was shown to be capable of inducing ligand
stimulated endothelial cell migration, but not proliferation, suggesting that neuropilins do
possess a limited intrinsic signalling activity (Wang et al., 2003). This signalling is probably
mediated by the last three evolutionary conserved amino acids of the intracellular domain of
NP1, which interact with a PDZ domain containing neuropilin interacting protein NIP (Cai &
Reed, 1999).
24
Neuropilins are also expressed in the vascular system, where NP1 is detected mainly in the
endothelium of arteries, whereas NP2 is found in the endothelial cells of lymphatic vessels,
and at low levels in veins (Herzog et al., 2001; Moyon et al., 2001; Yuan et al., 2002). In
chick embryos the expression of NP1 and NP2 is segregated to separate blood islands and to
the future arterial and venous parts of the primitive capillary plexus already before the onset
of blood flow, and therefore represents one of the earliest markers in the determination of
arterial and venous identity (Herzog et al., 2005). Overexpression or inactivation of Np1 in
mice leads to embryonic death due to developmental defects in the nervous and vascular
systems (Kitsukawa et al., 1995; Kitsukawa et al., 1997; Kawasaki et al., 1999). The vascular
defects in Np1 deficient mouse embryos include impairment in neural vascularization,
improper development and organization of the large vessels and the branchial arches, and
disorganized and insufficient development of vascular networks in the yolk sac (Kawasaki et
al., 1999). Np2 mutant mice on the other hand are viable, but in addition to having mild
neuronal defects they are born without small lymphatic vessels and capillaries, while the
blood vasculature and larger lymphatic vessels develop normally (Chen et al., 2000; Giger et
al., 2000; Yuan et al., 2002). Postnatally the small lymphatic vessels and capillaries also grow
without NP2. Although not essential for development of the blood vasculature, NP2 has been
shown to be involved in retinal neovascularization, suggesting a role in pathological
angiogenesis (Shen et al., 2004). Mice lacking both Np1 and Np2 die earlier and suffer from
more severe vascular defects than either of the single knockouts, suggesting that both
neuropilins are necessary for normal blood vascular development, but have partly overlapping
functions (Takashima et al., 2002).
In addition to binding class III semaphorins, neuropilins also interact with specific splice
variants of several VEGF family members. NP1 binds VEGF165, VEGF-B167, VEGF-B186,
PlGF-2 and VEGF-C (Migdal et al., 1998; Soker et al., 1998; Makinen et al., 1999; Ober et
al., 2004), whereas NP2 binds VEGF165, VEGF145, PlGF-2 and VEGF-C (Gluzman-Poltorak et
al., 2000; Karkkainen et al., 2001). NP1 has been shown to enhance the interaction of
VEGF165 with VEGFR-2 and to promote VEGF165 induced endothelial cell proliferation and
migration (Soker et al., 1998; Soker et al., 2002). Although the vascular defects in Np1 and
Np2 deficient mice were originally thought to reflect disruption in Vegf signalling, recent
evidence indicates that also semaphorin induced signalling is crucial for vascular
morphogenesis (Feiner et al., 2001; Gu et al., 2003; Serini et al., 2003; Gitler et al., 2004).
Semaphorins may also signal to endothelial cells without the requirement of neuropilins.
SEMA3E acts as a repulsive cue for endothelial cells expressing its receptor plexinD1 and
this signalling system controls endothelial cell positioning and patterning of the developing
vasculature (Torres-Vazquez et al., 2004; Gu et al., 2005).
Both NP1 and NP2 have been shown to form complexes with VEGFR-1 (Fuh et al., 2000;
Gluzman-Poltorak et al., 2001). The interaction of NP1 with VEGFR-1 might be required for
SEMA3A induced repulsion of neural progenitor cells (Bagnard et al., 2001). NP1 also forms
a complex with VEGFR-2. VEGF165 induced association of NP1 and VEGFR-2 occurs not
only on the surface of endothelial cells, in which case the complexes are internalized, but also
in a juxtacrine manner via association of endothelial VEGFR-2 with soluble or tumor cell
NP1 (Soker et al., 2002). It has been suggested that NP1 has a receptor-clustering role instead
of just behaving as an affinity-converting subunit in the complex between NP1 and VEGFR-2
(Whitaker et al., 2001). The interaction of NP1 with VEGFR-2 could at least partly account
25
for the NP1 dependent potentiation of VEGF165 induced endothelial cell migration (Soker et
al., 1998). Adding to the complexity, plexins may also associate with VEGFRs, as shown for
plexinA1 and VEGFR-2 (Toyofuku et al., 2004).
Figure 5: Structure of neuropilins and their interaction with ligands of the VEGF and semaphorin
families as well as with other cell surface receptors. The extracellular part of neuropilins NP1 and NP2 consist
of two CUB-domains (a1 and a2 domains, boxes with rounded corners), two coagulation factor V/VIII
homology domains (b1 and b2 domains, ovals) and a MAM domain (c, octagon). The VEGF and semaphorin
ligands of neuropilins are shown (small font size indicates low binding affinity). It has been suggested that NP1
could interact through ionic bonding with heparin binding sites of several other proteins as well (West et al.,
2005). NP1 can form complexes with VEGFR-1 and VEGFR-2, whereas NP2 has been reported to form
complexes with VEGFR-1. Interaction of NP2 with VEGFR-2 and VEGFR-3 is discussed in the Results and
Discussion, paragraph 2. The involvement of plexins in these signalling complexes is still unclear. In addition,
NP1 has been shown to interact with cell adhesion molecule L1 (Castellani et al., 2002), and plexins with the
receptor tyrosine kinases VEGFR-2, off-track kinase (OTK), ErbB2 and the hepatocyte growth factor/scatterfactor receptor Met (reviewed in Kruger et al., 2005).
Neuropilins and Semaphorins in tumorigenesis
NP1 is widely expressed by epithelial cells and its expression is detected in several carcinoma
cell lines and in carcinomas of a variety of organs, while NP2 is expressed predominantly by
neuronal tumors and by melanomas (reviewed in Bielenberg et al., 2006; Guttmann-Raviv et
al., 2006).
Several soluble neuropilin (sNP) forms are produced by alternative splicing (Rossignol et al.,
2000; Cackowski et al., 2004). Whereas monomeric sNP1 inhibits VEGF165 function by
acting as a ligand trap, dimerized sNP1 potentiates VEGF165 induced VEGFR-2
phosphorylation and promotes angiogenesis (Yamada et al., 2001). Accordingly, a naturally
26
occuring monomeric sNP1 lacking the MAM domain was shown to function as an inhibitor of
tumor angiogenesis and tumor progression (Gagnon et al., 2000). Conversely, NP1 expressed
by prostate or colon carcinoma tumor cells enhances tumor angiogenesis and tumor
progression, probably by in trans potentiating VEGF165/VEGFR-2 signalling on endothelial
cells or by enhancing storage and bioavailability of VEGF165 within the tumor
microenvironment (Miao et al., 2000; Parikh et al., 2004). VEGF165, but not VEGF121, was
also reported to be an autocrine survival factor for VEGFR-2 negative breast cancer cells,
suggesting a role for NP1 in cell survival (Bachelder et al., 2001).
VEGF165 and SEMA3A compete for an overlapping binding site in NP1 (Gu et al., 2002).
Thus SEMA3A inhibits VEGF165 induced migration of endothelial cells and both
developmental and in vitro angiogenesis (Miao et al., 1999; Bates et al., 2003; Serini et al.,
2003). SEMA3A also inhibits migration and spreading of NP1 and plexinA1 expressing
tumor cells (Bachelder et al., 2003). SEMA3F has also been shown to inhibit tumorigenesis,
both by directly controlling the adhesive and migrational behavior of NP1 or NP2 expressing
tumor cells and by suppressing tumor angiogenesis (Xiang et al., 2002; Nasarre et al., 2003;
Bielenberg et al., 2004; Kessler et al., 2004). SEMA3F has been shown to be chemorepulsive
for lymphatic and blood endothelial cells, and to inhibit EC proliferation, adhesion and
migration (Serini et al., 2003; Bielenberg et al., 2004; Kessler et al., 2004). The chromosomal
region containing SEMA3F is commonly deleted in lung cancer and its expression is often
lost in metastatic cancer cells (reviewed in Bielenberg et al., 2006). The inhibition of
adhesion and migration of both tumor cells and endothelial cells suggests a unique tumor
suppressor function for SEMA3F, which may therefore possess therapeutic potential. These
results also indicate a dual role for neuropilins in the promotion of angiogenesis through their
VEGF ligands, and in the inhibition of angiogenesis through their SEMA ligands.
3.3.2
Heparan sulphate proteoglycans
Several receptor tyrosine kinases are known to require HSPGs as coreceptors for efficient
signal transduction. The longer isoforms of VEGF as well as PlGF-2 and VEGF-B167 contain
heparin-binding domains that mediate their interaction with HSPGs in the ECM and on the
cell surface (Gitay-Goren et al., 1992; Houck et al., 1992; Hauser & Weich, 1993; Olofsson
et al., 1996a). VEGFR-1 and VEGFR-2 also contain a heparin-binding site in their
extracellular domain (Cohen et al., 1995; Dougher et al., 1997). It has been suggested that
whereas binding of VEGF to VEGFR-2 is enhanced by heparin, heparin might inhibit binding
of VEGF to VEGFR-1 (Terman et al., 1994). More recently, it was shown that heparin
enhances signalling by VEGF165, but not by VEGF121, through VEGFR-2 (Ashikari-Hada et
al., 2005). Furthermore, HSPGs expressed in trans by perivascular SMCs fully support the
formation of functional VEGFR complexes on endothelial cells, which leads to prolonged and
enhanced signalling properties in response to VEGF165 (Jakobsson et al., 2006). Cell surface
HSPGs or heparin might induce conformational changes in the receptor or the ligand, which
facilitates their interaction. Alternatively, heparin or HSPGs might mediate formation of
higher order receptor complexes (Yayon et al., 1991). HSPGs could also be needed for
localizing the ligand to the cell surface and increasing its avidity to the receptor. Another
aspect to the importance of the interaction of VEGFs with HSPGs is that anchoring of the
heparin binding VEGF isoforms to the ECM and cell surface creates the growth factor
gradients required for proper vessel guidance (Carmeliet et al., 1999b; Ruhrberg et al., 2002).
27
3.3.3
Integrins
Integrins are heterodimeric non-tyrosine kinase transmembrane receptors that mediate cell
adhesion to the ECM and signalling from the ECM to the cell. In coordination with growth
factor receptor signalling, integrin transduced signals are central to a variety of cellular
processes such as migration, proliferation, differentiation and apoptosis (reviewed in Eliceiri,
2001).
Several integrins, most importantly α vβ3 and αvβ5 as well as the collagen and laminin
receptors α1β1 and α2β1 and the fibronectin receptor α5β1, have been assigned important roles
in VEGF-induced angiogenesis (Friedlander et al., 1995; Senger et al., 1996; Senger et al.,
1997; Byzova et al., 2000; Kim et al., 2000). Integrin α vβ3, which binds to the arginineglycine-aspartic acid (RGD) peptide motif, present in ECM proteins such as fibronectin,
vitronectin, fibrinogen, osteopontin and thrombospondin, is expressed in angiogenic but not
in quiescent blood vessels. It has been shown to directly interact with VEGFR-2 and its
ligation enhances VEGFR-2 kinase activity and mitogenic signalling (Soldi et al., 1999;
Borges et al., 2000). Disruption of αvβ3 or αvβ5 ligation either by blocking antibodies or by
cyclic RGD peptides inhibits angiogenesis and tumor growth (Brooks et al., 1994a; Brooks et
al., 1994b; Brooks et al., 1995; Drake et al., 1995; Hammes et al., 1996). Surprisingly, mice
lacking β3 or both β3 and β5 integrins display enhanced pathological angiogenesis, and reveal
that αvβ3 and αvβ5 integrins are not necessary for vascular development or neovascularization
(Reynolds et al., 2002). This controversy has been explained by the upregulation of VEGFR2 expression in the absence of β3 and β 5 integrins, either due to removal of the negative
regulators or to molecular compensation, or with pro-apoptotic signalling by unligated
integrins, but the exact mechanisms by which these αv integrins are involved in the regulation
of angiogenesis remains obscure (discussed in Cheresh & Stupack, 2002; Hynes, 2002;
Sheppard, 2002; Hodivala-Dilke et al., 2003). Mice deficient of all α v integrins die around
midgestation due to hemorrhages caused by extensive vasculogenesis and angiogenesis
(Bader et al., 1998).
The integrin α1 chain, a subunit of a receptor for collagen and laminin, and α9, a subunit of a
receptor for osteopontin and tenascin, are expressed at higher levels in LECs than in BECs
(Petrova et al., 2002). Interestingly, mice deficient in integrin α9β1 are incompatible with
postnatal life and display chylothorax, which suggest an underlying function for integrin α9β1
in lymphatic development or function (Huang et al., 2000).
3.3.4
VE-cadherin
Vascular endothelial (VE-) cadherin is an endothelial cell specific transmembrane adhesion
molecule located in adherens junctions. Inactivation of VE-cadherin or truncation of its βcatenin binding cytosolic domain in mice leads to impaired remodelling and maturation of the
primary capillary plexus due to endothelial cell apoptosis, causing embryonic lethality at E9.5
(Carmeliet et al., 1999a). VE-cadherin was shown to inhibit endothelial cell apoptosis by
forming a complex with VEGFR-2, β-catenin and PI3-kinase, which leads to activation of
Akt and increased expression of the anti-apoptotic protein Bcl2 (Carmeliet et al., 1999a). On
the other hand, VE-cadherin is involved in contact inhibition of endothelial cell growth by
negatively regulating VEGFR-2 activity (Caveda et al., 1996; Rahimi & Kazlauskas, 1999). It
has been suggested that this is due to dephosphorylation of VEGFR-2 by junctional
28
phosphatases at the cell-cell contacts in complex with VE-cadherin and β-catenin (Grazia
Lampugnani et al., 2003). VE-cadherin was also shown to bind the negative regulator of Src
family kinases, Csk (Baumeister et al., 2005).
3.4
Molecules that regulate remodelling and maturation of blood and lymphatic
vessels
The primary capillary plexus has to undergo extensive remodelling of the homogenous
endothelial channels to form a highly branched hierarchical vascular network consisting of
arteries, arterioles, capillaries, venules and veins. The branching pattern of the vascular
system is highly specified and its precise wiring requires a coordinated set of guidance cues
from many different classes of molecules. The distinction between arteries and veins,
previously assumed to be a late event in development, and to reflect functional and
physiological differences, seems to be genetically determined before the onset of circulation.
Despite this, the overall architecture of the vascular tree still has considerable plasticity
during early embryonic development, and can adapt to alterations in perfusion and oxygen
content by changing the arterial or venous identity both functionally, morphologically and
genetically, indicating the importance of hemodynamic forces in shaping the vascular pattern
(Moyon et al., 2001; le Noble et al., 2004).
As the blood and lymphatic vasculatures extend to many different tissues throughout the body
in a manner similar to the nervous system, it is not surprising that the guided growth and
branching of vessels shares several molecular cues with that of the nerve fibers. Endothelial
tip cells, which extend long filopodia to explore their microenvironment in order to guide
blood vessel sprouting, closely resemble the growth cones that navigate the pathfinding of
axons (Gerhardt et al., 2003). In addition to semaphorins and ephrins, which are discussed in
more detail, the common guidance molecules used by blood vessels and neurons include
Netrins and Slits, which mediate predominantly repulsive or inhibitory signals through their
Unc and Robo receptors, respectively (reviewed in Eichmann et al., 2005a; Eichmann et al.,
2005b; Klagsbrun & Eichmann, 2005). Peripheral nerves are also thought to provide a
template that determines the organotypic blood vessel branching pattern and arterial
differentiation, at least in the skin, via local secretion of VEGF (Mukouyama et al., 2002).
In addition to remodelling of the vascular pattern, maturation of blood vessels involves
deposition of the BM and recruitment of non-endothelial cells to stabilize the vessel wall.
Vascular mural cells, PCs and SMCs, are a rather heterogenous group of cells in respect to
their origins, morphology and molecular characteristics. PCs, found in small blood vessels
and capillaries as well as in newly formed blood vessels, are embedded within the vascular
BM and make close contacts with the endothelial cells. Vascular SMCs, which constitute one
or multiple layers to the wall of larger arteries and veins, are separated from the endothelium
by its BM and rich ECM. Vascular mural cells, especially PCs, are of critical importance to
vessel maturation and stabilization. Recruitment of mural cells leads to a quiescent state of the
endothelium, decreases vessel permeability, brings structural support and provides elasticity
for the vessel wall. In the absence of PCs, endothelial cells overproliferate and fail to form
mature inter-endothelial junctions. Furthermore, the luminal surface of the endothelium
becomes highly irregular with multiple folds, which is likely to impair blood flow (reviewed
in von Tell et al., 2006).
29
Generally used molecular markers of vascular mural cells include smooth muscle α-actin
(SMA), desmin, NG2, PDGFR-β, aminopeptidases A and N and RGS5, but none of these
markers is absolutely specific for vascular mural cells and none of them recognizes all
PCs/SMCs (reviewed in Armulik et al., 2005). Vascular mural cells are generally of
mesodermal or, in the head region, of neural crest origin, although transdifferentiation from
other cell types and even from bone marrow progenitors has recently been suggested
(Rajantie et al., 2004). In addition, vascular mural cells present considerable plasticity and
can even give rise to other types of mesenchymal cells.
Although less understood than blood vessel remodelling, the primordial lymphatic vascular
plexus also undergoes extensive remodelling and maturation (reviewed in Karpanen &
Makinen, 2006). This process continues in the postnatal period and forms a superficial
lymphatic capillary network by sprouting, and deeper collecting lymphatic vessels by
deposition of BM, recruitment of SMCs and development of lymphatic valves (Makinen et
al., 2005). Like sprouting blood vessel endothelium, sprouting lymphatic endothelial cells
also form filopodial extensions with subsequent elongation (He et al., 2005; Makinen et al.,
2005). Lymphatic capillaries lack PCs, whereas the collecting lymphatic vessels have a SMC
layer. Lymphatic vessels also lack many BM components and express a different set of
adhesion molecules than blood vessels, which may partially account for their different
interaction with mural cells.
3.4.1
Establishment of arterio-venous identity
VEGF has a specific role in artery development, but less is known about the signals
governing vein formation (Lawson et al., 2002; Mukouyama et al., 2002; Stalmans et al.,
2002). In Zebrafish, it has been shown that VEGFR-2 and VEGFR-3 cooperate during artery
morphogenesis but not differentiation (Covassin et al., 2006).
Notch
A number of genes involved in the Notch signalling pathway are specifically expressed in
arteries, where its activation is required to suppress the venous fate and to allow arterial
differentiation (Zhong et al., 2000; Lawson et al., 2001; Zhong et al., 2001; Lawson et al.,
2002; Duarte et al., 2004; Fischer et al., 2004; Gale et al., 2004; Krebs et al., 2004). In
addition, Notch3 has been shown to be necessary for specifying the arterial characteristics of
vascular SMCs (Domenga et al., 2004).
COUP-TFII
The orphan nuclear receptor COUP-TFII is specifically expressed in the venous but not in the
arterial endothelium. Deletion of COUP-TFII in endothelial cells enables veins to acquire
arterial characteristics and its ectopic expression in endothelial cells suppresses the expression
of NP1 and other arterial markers, suggesting that COUP-TFII is part of an active pathway
promoting venous identity (You et al., 2005).
Ephrins and Eph-receptors
Eph receptor tyrosine kinases and their cell surface bound ephrin ligands are important
regulators of embryonic patterning and tissue morphogenesis. In the nervous system they
30
control axon guidance as well as cell migration and adhesion primarily by providing repulsive
cues. In addition to acting as ligands for EphB receptors, upon ligation the transmembrane
ephrinBs are capable of actively transducing signals through their conserved cytoplasmic
domain to their host cell, referred to as reverse signalling (Bruckner & Klein, 1998). Two
ephrinB ligands and three EphB receptors are expressed in the vascular system, where they
are required for early vascular remodelling by regulating capillary sprouting and endothelial
mesenchymal interactions as well as by defining arterio-venous boundaries (Adams et al.,
1999). EphrinB2 is highly expressed in the arterial endothelium from the onset of
angiogenesis, whereas one of its receptors, EphB4, is found predominantly in veins (Wang et
al., 1998). Mice deficient in ephrinB2 or EphB4 die around midgestation due to defects in
remodelling of the uniform primary capillary plexus into a hierarchical vascular network of
arteries and veins (Wang et al., 1998; Adams et al., 1999; Gerety et al., 1999). Reverse
signalling downstream of ephrinB2 and its expression in endothelial cells is required for
vascular remodelling, as mice lacking the cytoplasmic tail of ephrinB2 or with an endothelial
cell specific deletion of ephrinB2 phenocopy the vascular defects of the conventional
ephrinB2 deficient mice (Adams et al., 2001; Gerety & Anderson, 2002). During postnatal
neovascularization, EphB4 acts as a negative regulator of blood vessel branching and reduces
vessel permeability by activating the Ang1/Tie2 system (Erber et al., 2006).
In addition to arterial endothelium, ephrinB2 is expressed in the vascular mural cells of
arteries and larger veins (Wang et al., 1998; Adams et al., 1999; Gale et al., 2001; Shin et al.,
2001; Makinen et al., 2005). A proportion of double mutant mice deficient in both EphB2,
expressed in vascular mural cells, and EphB3, found in veins and aortic arches, exhibit a
phenotype similar to mice deficient of ephrinB2 signalling, suggesting a requirement of
ephrin-Eph signalling between endothelial and surrounding mural cells (Adams et al., 1999).
The critical requirement of ephrinB2 for the proper association of vascular mural cells with
blood vessels was revealed by vascular mural cell specific deletion of ephrinB2 in mice (Foo
et al., 2006). These mice die shortly after birth due to respiratory failure, and display edema
and extensive haemorrhaging. Although the number of PCs/SMCs around the vasculature was
normal, these cells displayed an abnormally rounded morphology, and made only loose
contacts with the endothelium, leading to incomplete coverage of microvessels. The ephrinB2
deficient SMCs were defective in spreading and focal adhesion formation, and showed
increased but nonpolarized motility in culture (Foo et al., 2006). Interestingly, deletion of
ephrinB2 in SMCs also lead to abnormal migration of SMCs to lymphatic capillaries (Foo et
al., 2006).
In the lymphatic vasculature, ephrinB2 is expressed in collecting lymphatic vessels, whereas
EphB4 was detected both in collecting lymphatic vessels and in lymphatic capillaries
(Makinen et al., 2005). PDZ-dependent signalling of ephrinB2 was found to be dispensable
for blood vascular development, but crucial for the postnatal remodelling of the lymphatic
vasculature into a hierarchically organized lymphatic vessel network consisting of lymphatic
capillaries and collecting lymphatic vessels (Makinen et al., 2005). Mice expressing
ephrinB2, which lacks its carboxyterminal binding site for PDZ-domain containing proteins,
display hyperplasia of the collecting lymphatic vessels and defective formation of luminal
valves as well as disturbed sprouting of lymphatic endothelial cells, leading to blunt-ended
protrusions, indicating a requirement for ephrinB2 reverse signalling in the elongation and
guidance of sprouting lymphatic endothelial cells (Makinen et al., 2005). In addition, the
31
specification of collecting versus capillary lymphatic vessel identity fails in these mice as
demonstrated by persistant LYVE-1 expression in all lymphatic vessels, and by abnormal
recruitment of SMCs into lymphatic capillaries (Makinen et al., 2005).
3.4.2
Separation of the lymphatic and vascular systems
Lymph is returned to the bloodstream mainly through the thoracic and the right lymphatic
ducts, which make a connection to the veins at the junctions of left or right internal jugular
and subclavian veins, respectively. Additional lymphatico-venous communications exist at
least in the kidney, adrenal gland and liver, but are thought to be non-functional, except when
the intralymphatic pressure increases (reviewed in Aalami et al., 2000). Lymphatico-venous
anastamoses can be observed in lymphedema, chylous ascites and chylothorax (Aalami et al.,
2000).
Mice deficient in the intracellular signalling molecules Syk or Slp76, expressed almost
exclusively in hematopoietic but not in endothelial cells, display arterio-venous shunts and
abnormal lymphatico-venous connections, leading to blood filled lymphatic vessels. This
suggests that circulating hematopoietic cells are involved in controlling the separation of the
lymphatic and blood vascular systems (Abtahian et al., 2003).
3.4.3
Stabilization of vessels by mural cell recruitment
Reciprocal communication between the endothelium and the mesenchyme is important for the
proper development of blood and lymphatic vessels. Mesenchymal cells signal endothelial
cells via angiopoietins, which bind to their endothelial specific Tie receptors. Endothelial
cells recruit PCs and vascular SMCs through the platelet derived growth factor (PDGF)
signalling pathway. TGF-β signalling is crucially involved in many aspects of angiogenesis
and vessel maturation. In addition to these ligand/receptor systems discussed in more detail
below and to the EphB-ephrinB signalling presented in chapter 3.4.1, some other signalling
pathways are also known to contribute to the proper mural cell coverage of blood vessels.
These include sphingosine-1-phosphate signalling via its G-protein coupled receptor S1P1
(Edg1), which leads to redistribution of N-cadherin in endothelial cells to facilitate
interactions with mural cells (Paik et al., 2004).
Angiopoietins and Tie-receptors
Tie1 and Tie2, also known as Tie and Tek respectively, comprise, along with VEGFRs,
another endothelial cell specific receptor tyrosine kinase family. Although dispensable for
vasculogenesis, both receptors are essential in later stages of vascular development for vessel
remodelling, maturation and stabilization (Puri et al., 1999). Tie1 is essential for the
establishment of endothelial cell integrity, and deletion of Tie1 in mice leads to edema and
hemorrhages (Puri et al., 1995; Sato et al., 1995). Tie1 is still an orphan receptor, but was
recently found to be phosphorylated upon stimulation with the angiopoietins Ang1 and Ang4
(Saharinen et al., 2005).
Tie2 has currently three known angiopoietin ligands. Whereas Ang1 obligately activates Tie2
signalling, Ang2 can activate Tie2 on some cells but act as a competitive antagonist in others
(Davis et al., 1997; Maisonpierre et al., 1997; Teichert-Kuliszewska et al., 2001; Mochizuki
et al., 2002). In addition, mouse Ang3 and its human orthologue Ang4 are agonists of Tie2,
32
but mouse Ang3 has a strong activity only on the endothelial cells of its own species
(Valenzuela et al., 1999; Lee et al., 2004). Tie2 or Ang1 deficient mice die around
midgestation due to cardiovascular defects, including insufficient angiogenesis, defective
maintenance of the primary capillary plexus and detachment of PCs from blood vessels
(Dumont et al., 1994; Sato et al., 1995; Suri et al., 1996). Conversely, transgenic
overexpression of Ang1 leads to an increased number of enlarged, leakage resistant blood
vessels (Suri et al., 1998; Thurston et al., 1999). During the postnatal period Ang1 induces
vessel enlargement, especially of the veins, by promoting endothelial cell proliferation in the
absence of sprouting (Thurston et al., 2005). In adults Ang1 protects the vasculature against
plasma leakage without changing the vessel morphology (Thurston et al., 2000). In humans
an activating mutation in TIE2 causes venous malformations associated with abnormal
vascular SMC coverage (Vikkula et al., 1996).
These genetic loss and gain of function studies have indicated an essential role for the Ang1
induced Tie2 signalling in vessel maturation. Ang1 is expressed by PCs and SMCs and
provides paracrine signals by activating its endothelial cell specific Tie2 receptor to mediate
vessel stabilization by promoting interactions of endothelial cells with the surrounding ECM
and mesenchymal cells. Ang2 on the other hand is expressed mainly by endothelial cells,
where it is stored in specialized secretory vesicles and can be rapidly released upon
stimulation (Fiedler et al., 2004). Ang2 is thought to antagonize the constitutive vessel
stabilizing effects of Ang1 and to provide autocrine vessel destabilizing signals by inhibiting
Tie2 signalling. This leads to PC detachment and disrupted interactions of endothelial cells
with ECM, making the endothelial cells more responsive to a variety of stimuli. In the
presence of VEGF, this leads to induction of vessel sprouting and angiogenesis, whereas in
the absence of VEGF the endothelial cells undergo apoptosis and vessels regress. This is
further supported by the fact that in the adult Ang2 is highly expressed only at sites of active
angiogenesis, such as placenta, ovary and tumors (Maisonpierre et al., 1997; Goldman-Wohl
et al., 2000; Etoh et al., 2001). Furthermore, both Tie1 and Tie2 can activate signalling
pathways that promote cell survival and are suggested to be required for the survival of
endothelial cells in vivo (Partanen et al., 1996; Hayes et al., 1999; Papapetropoulos et al.,
2000; Jones et al., 2001b; Kontos et al., 2002). Recently, Ang2 was suggested to play a role
as an autocrine regulator of endothelial cell inflammatory responses (Fiedler et al., 2006b).
Both Tie1 and Tie2 are expressed by lymphatic endothelial cells (Saharinen et al., 2005).
Mice deficient in Ang2 reveal that Ang2 is dispensable for embryonic development, but
required for postnatal vascular remodelling (Gale et al., 2002). Ang2 deficient mice also
display generalized lymphatic dysfunction caused by disorganized collecting lymphatic
vessels with poorly associated SMCs and irregularly patterned hypoplastic lymphatic
capillary network (Gale et al., 2002). Interestingly, the lymphatic defects, but not the blood
vascular ones, are rescued by Ang1, suggesting that unlike in the blood vasculature Ang2 acts
as a Tie2 agonist in the lymphatic endothelium (Gale et al., 2002). Ang1 has also been shown
to induce growth of lymphatic vessels in adult tissues (Morisada et al., 2005; Tammela et al.,
2005).
33
PDGFs and PDGFRs
Platelet derived growth factors (PDGFs) -A, -B, -C and -D, signalling through their PDGF
receptors -α and -β, are regarded as mitogens and chemotactic agents for several cell types of
mesodermal and neuro-ectodermal origin with major functions during development (reviewed
in Betsholtz, 2004). Genetic studies in mice have revealed a critical role for the PDGFB/PDGFR-β signalling pathway in the recruitment of PCs to newly formed blood vessels.
Endothelial cells at sites of active angiogenesis, especially the migratory tip cells at the
leading edge of angiogenic sprouts, secrete PDGF-B, which induces proliferation and
migration of vascular mural cells that express its receptor PDGFR-β (reviewed in Armulik et
al., 2005). Deletion of either Pdgfb or Pdgfrb in mice leads to perinatal death caused by
vascular and renal dysfunction (Levéen, 1994; Soriano, 1994; Lindahl et al., 1998). The
primary cause of the vascular abnormalities is pronounced reduction in PC recruitment and
coverage of microvessels (Lindahl et al., 1997; Crosby et al., 1998; Hellstrom et al., 1999).
Loss of PCs leads to secondary consequences in the endothelium, which include endothelial
hyperplasia and abnormal junctions, as well as compensatory upregulation of VEGF causing
vascular leakage and hemorrhage (Hellstrom et al., 2001). Although PCs are initially induced
in the absence of PDGF-B signalling, they fail to expand and spread along the microvessels
(Hellstrom et al., 1999). Recently, PDGF-B induced migration, but not proliferation, of
vascular SMCs was shown to be inhibited by the neuronal pathfinding molecule Slit2 (Liu et
al., 2006).
The importance of endothelial PDGF-B has been confirmed with an endothelial cell specific
PDGF-B knockout (Enge et al., 2002; Bjarnegard et al., 2004). Retention of PDGF-B to the
microvessel endothelium and the surrounding extracellular space by way of the HSPG
binding motif is essential for proper recruitment and organization of PCs to the vessel wall
(Lindblom et al., 2003). These mouse mutants as well as signalling defective Pdgfrb mutants
confirm that up to 90% PC reduction is still compatible with basic vascular functions and
postnatal life although with persistant pathological changes (Tallquist et al., 2003).
Interestingly, one study has suggested that PDGF-B would promote tumor metastasis by
inducing tumor lymphangiogenesis (Cao et al., 2004).
TGF-β
The members of the TGF-β superfamily are among the most versatile carriers of growth and
differentiation signals affecting several aspects of development. The TGF-β signalling
machinery is also of critical importance for vascular development and function by regulating
EC proliferation and migration, promoting production of ECM and proteases, inducing
differentiation of mesodermal cells into vascular mural cells and contributing to arteriovenous specialization. In the vasculature, at least the neural crest derived SMCs of pharyngeal
arch arteries express TGF-β2 and TGF-β3, whereas TGF-β1 is expressed by the endothelium
(Gittenberger-de Groot et al., 2006). TGF-β family members can be either stimulators or
inhibitors of angiogenesis, probably because TGF-β binding to TGF-β type II receptor can
activate either the endothelial cell specific TGF-β type I receptor ALK1, which via Smad1/5
stimulates endothelial cell proliferation and migration, or the more broadly expressed ALK5
with opposing effects, signalling differentiation via Smad2/3 (Goumans et al., 2002; Lebrin et
34
al., 2005). ENDOGLIN is a coreceptor for TGF-β family members on endothelial cells, and
promotes ALK1 signalling, thereby shifting the TGF-β response towards proliferation (Lebrin
et al., 2004). In humans, loss of function mutations in ENDOGLIN or ALK1 cause hereditary
hemorrhagic telangiectasia type 1 or 2, respectively, diseases characterized by vascular
malformations, arterio-venous shunts and recurrent hemorrhages (McAllister et al., 1994;
Johnson et al., 1996). Mice lacking Endoglin die around midgestation due to defective
vascular remodelling and poor SMC coverage of blood vessels, whereas mice deficient for
alk1 develop serious arterio-venous malformations resulting from fusion of major arteries and
veins (Li et al., 1999; Urness et al., 2000). Similarly, deletion of Tgfβ1, Alk5, TGF-β receptor
type II or Smad5 in mice leads to serious vascular defects (Dickson et al., 1995; Oshima et
al., 1996; Yang et al., 1999; Larsson et al., 2001). The primary defect upon disruption of
TGF-β signalling is likely to occur in the endothelium, but in addition to structural changes,
for example in the BM, defective signalling from endothelial to mural cells, partly via TGF-β
itself, may also contribute to secondary defects in the differentiation and recruitment of
vascular mural cells (Carvalho et al., 2004; reviewed in Armulik et al., 2005).
3.5
Transcription factors implicated in the development of lymphatic vasculature
In addition to PROX1 and FOXC2 discussed below, also the transcription factors Net, SOX18
and Vezf1 have been implicated in lymphatic development (see Table I).
3.5.1
PROX1
In the vascular system the expression of the homeobox transcription factor PROX1 is specific
for lymphatic vessels. In mice, the first Prox1 positive endothelial cells are detected as a
restricted subpopulation on one side of the anterior cardinal vein at E9.5 and soon thereafter
these cells start budding from the vein and migrating in a polarized manner, eventually
forming lymph sacs (Wigle & Oliver, 1999). By E14.5, the initial lymphatic vessels have
started sprouting from the lymph sacs and a day later form a superficial capillary plexus.
Prox1 deficient embryos completely lack lymph sacs and lymphatic vessels. The endothelial
cells initially bud and sprout from the cardinal vein, although in an unpolarized manner, but
their migration is soon arrested. These cells fail to express lymphatic endothelial markers and
instead retain their blood vascular endothelial phenotype (Wigle & Oliver, 1999; Wigle et al.,
2002). Overexpression of PROX1 in human blood vascular endothelial cells suppresses the
expression of several genes specific for the blood vascular endothelium and upregulates
lymphatic endothelial cell specific gene expression, which further suggests a function for
PROX1 as a master control gene determining lymphatic endothelial cell fate (Hong et al.,
2002; Petrova et al., 2002). In addition, PROX1 upregulates cyclin E1 and E2, which suggests
that it also induces cell proliferation (Petrova et al., 2002). The signals leading to polarized
expression of PROX1 in lymphatic endothelial cells are not known. The population of
Prox1+/- mice surviving until adulthood in an outbred background as well as mice with an
endothelial specific deletion of Prox1 develop chylous ascites and adult-onset obesity,
indicating a link between impaired lymph drainage and tissue adiposity (Harvey et al., 2005).
Recently, FGFR-3 was identified as a direct target of PROX1 in LECs and thus suggested a
role in lymphatic vessel development (Shin et al., 2006). However, Fgfr3 deficient mice do
35
not display any obvious defects in lymphatic development or function (Tatiana Petrova,
personal communication).
3.5.2
FOXC2
FOXC2, also known as Mesenchyme Fork Head 1 (MFH-1), is a member of the forkhead or
“winged-helix” family of transcription factors, which in mammals currently comprises over
30 members with important roles in embryonic patterning and morphogenesis. Foxc2 is
widely expressed in the embryonic mesenchyme and in tissues derived from it, such as
kidney, bones, cartilage, endocardium and blood vessels (Miura et al., 1993; Kaestner et al.,
1996). Deletion of Foxc2 leads to embryonic or perinatal lethality due to interrupted aortic
arch and skeletal defects (Iida et al., 1997; Winnier et al., 1997). In the adult, Foxc2 is also
expressed in adipose cells, where it seems to counteract diet induced insulin resistance and to
protect against type 2 diabetes (Cederberg et al., 2001).
Linkage of several primary lymphedema syndromes with mutations in the FOXC2 gene
suggested a role for this transcription factor in the development of the lymphatic vasculature
(see chapter 5.2.1). As will be discussed in the Results and Discussion, paragraph 5, Foxc2 is
expressed in the developing lymphatic vessels as well as in lymphatic valves in adults, and is
involved in regulating the lymphatic capillary phenotype and morphogenesis of lymphatic
valves (V, Dagenais et al., 2004).
3.6
Molecular specification of lymphatic endothelial identity
Traditionally, the lymphatic vessels have been differentiated from blood vessels by
ultrastructural differences. These include the relatively wide lumen, presence of anchoring
filaments, discontinuous or absent BM, lack of PCs and loose overlapping or interdigitated
intercellular junctions in the lymphatic endothelium. At the molecular level, as compared to
blood vascular endothelium lymphatic capillaries are often devoid of the BM proteins
collagen IV, fibronectin and laminin, contain desmoplakin I and II in their specialized
junctional complexes, complexus adhaerentes, possess high 5’-nucleotidase and low alkaline
phosphatase enzymatic activity and lack the surface protein PAL-E. The more recently
identified markers specific for the lymphatic endothelium within the vasculature, such as the
surface proteins VEGFR-3, LYVE-1 and podoplanin, the transcription factor PROX1 and the
chemokine SLC, have greatly facilitated lymphatic research over the last decade (reviewed in
Sleeman et al., 2001). In addition, mannose receptor 1, involved in leukocyte trafficking, and
chemokine receptor D6, involved in the post-inflammatory clearance of β-chemokines and
thus in resolving the inflammatory response, are specific markers for a subset of lymphatic
vessels (Irjala et al., 2001; Nibbs et al., 2001; Jamieson et al., 2005).
3.6.1
Lymphatic endothelial hyaluronan receptor 1 (LYVE-1)
LYVE-1, lymphatic endothelial hyaluronan receptor 1, is one of the most specific and widely
used lymphatic endothelial markers (Banerji et al., 1999; Prevo et al., 2001). It is expressed
early on in the differentiating lymphatic endothelial cells, but in adult its expression in
collecting lymphatic vessels decreases and remains high only in lymphatic capillaries
(Makinen et al., 2005). In addition to LECs, it is also expressed in macrophages and in liver
sinusoids (Mouta Carreira et al., 2001). The exact function of LYVE-1 is currently unknown
36
but it may be involved in hyaluronan transport and turn-over or in leukocyte trafficking
(Prevo et al., 2001; Jackson, 2004). Mice lacking LYVE-1 have normal lymphatic vessels
(reviewed in Alitalo et al., 2005).
3.6.2
Podoplanin
Podoplanin is a small, mucin-type transmembrane glycoprotein expressed in osteoblasts and
in various epithelial cells like podocytes, keratinocytes and those of the choroid plexus and
lung alveoles (Rishi et al., 1995; Wetterwald et al., 1996; Breiteneder-Geleff et al., 1997).
Within the vascular system, podoplanin is predominantly expressed in the lymphatic
endothelium, where its expression starts around E11 and remains high both in collecting
lymphatic vessels and in lymphatic capillaries also in the adult (Breiteneder-Geleff et al.,
1999; Schacht et al., 2003). Podoplanin deficient mice die at birth due to respiratory failure
and have defects in lymphatic but not in blood vessel formation and patterning, leading to
diminished lymphatic transport and congenital lymphedema (Schacht et al., 2003). In cultured
endothelial cells podoplanin promotes cell adhesion, migration and tube formation (Schacht et
al., 2003). Podoplanin is also upregulated in the invasive front of many human carcinomas
and its expression promotes tumor cell invasion in the absence of epithelial-mesenchymal
transition (Wicki et al., 2006).
3.6.3
Secondary lymphoid chemokine (SLC)
The chemokine CCL21, also known as secondary lymphoid chemokine (SLC), is expressed in
high endothelial venules and T cell areas of lymph nodes and Peyer’s patches as well as in
lymphatic endothelium of multiple organs (Gunn et al., 1998). It is involved in lymphocyte
homing to secondary lymphoid organs (Gunn et al., 1999). Interestingly, as discussed below,
it has also been implicated in the metastasis of tumor cells expressing its receptor, CCR7
(Muller et al., 2001; Wiley et al., 2001).
3.6.4
Lymphatic endothelial heterogeneity
In contrast to the acknowledged molecular heterogeneity of blood vessels in distinct organs,
reflecting their structural, functional and microenvironmental specificities, little is known
about the heterogeneity of the lymphatic endothelium in different types of vessels and in
different organs. Given the multiple and specialized tasks of different lymphatic vessels, like
the absorption of dietary lipids by the central lacteals of the intestinal villi, the transport of
immune cells by the afferent lymphatic vessels and the exposure of lymphatic capillaries to
high hydrostatic pressure in the extremities, it is reasonable to presume that such differences
exist. VEGFR-2 and ephrinB2 are found predominantly in collecting lymphatic vessels,
whereas VEGFR-3 and NP2 are more specifically expressed in lymphatic capillaries (Saaristo
et al., 2002b; Yuan et al., 2002; Veikkola et al., 2003; Makinen et al., 2005). Although
LYVE-1 is expressed in all lymphatic endothelia during early development, it appears in adult
tissues primarily in the lymphatic capillaries (Makinen et al., 2005). Lymphatic endothelial
cell heterogeneity was also observed in lymphatic vessel structures induced to differentiate in
murine embryonic stem cell cultures aggregated to form embryoid bodies (Kreuger et al.,
2006). Furthermore, tumor lymphatic vessels are likely to express specific markers different
from normal lymphatic vessels (Laakkonen et al., 2002; Laakkonen et al., 2004; Fiedler et al.,
2006a).
37
3.7
Role of VEGFs outside the cardiovascular and hematopoietic systems
Given the anatomical resemblance of the vascular and nervous systems, and the well
recognized involvement of axonal pathfinding molecules in the guidance of vessel branching,
it is not surprising that VEGFs and VEGFRs also have a role in mediating migration,
proliferation and survival of neural cells. VEGF is likely to affect development of the nervous
system and to provide neuroprotection both as a direct neurotrophic agent and indirectly by
stimulating endothelial cells to provide adequate blood perfusion and to secrete neurotrophic
factors (Sondell et al., 1999; Jin et al., 2000; Sondell et al., 2000; Oosthuyse et al., 2001;
Haigh et al., 2003; Rosenstein & Krum, 2004; Schwarz et al., 2004; Storkebaum &
Carmeliet, 2004; Storkebaum et al., 2005). Although the expression of both VEGFR-1 and
VEGFR-2 has been reported in neural cells, the direct effects of VEGF on these cells might
be at least partly mediated by NP1. Neural progenitor cells in both mouse and Xenopus laevis
embryos express VEGFR-3 and their proliferation was reported to be severely reduced in the
absence of VEGF-C (Le Bras et al., 2006). Furthermore, Vegfc deficient mouse embryos
displayed a selective loss of oligodendrocyte precursor cells in the embryonic optic nerve (Le
Bras et al., 2006). A neuroprotective function has also been suggested for VEGF-B (Nag et
al., 2002; Sun et al., 2004, 2006).
All the VEGFRs are also expressed in osteoblasts and in addition to regulating bone
formation indirectly by inducing vascularization, VEGF might also directly induce osteoblast
differentiation and migration (Gerber et al., 1999b; Deckers et al., 2000; Nakagawa et al.,
2000; Maes et al., 2002; Zelzer et al., 2002; Maes et al., 2004; Zelzer et al., 2004; Orlandini
et al., 2006). Recently, VEGF-D signalling through VEGFR-3 has been suggested to be
critical for osteoblast maturation, functioning as a downstream effector of VEGF in
osteogenesis (Orlandini et al., 2006).
In addition, VEGFR-2 may have a role in maturation of β-cells from pancreatic duct cells
(Oberg et al., 1994). Furthermore, overexpression of VEGF in the testis leads to infertility,
possibly at least partly due to targeting several nonendothelial cell types expressing VEGFR-1
and VEGFR-2 in this tissue (Ergun et al., 1997; Korpelainen et al., 1998).
38
Table I: Genes identified to be involved in lymphatic vascular development and maturation based on
mouse mutant studies. Modified from (Alitalo et al., 2005).
Gene
Lymphatic phenotype
Lethality
Reference
Ang2
-/-
Defects in the function and
patterning
Perinatal-P14 (129/J)
>90% survival (c57bl/6j)
(Gale et al., 2002)
(Fiedler et al., 2006b)
EphrinB2
ΔV/ΔV
Absence of valves, defective
remodelling, ectopic mural cells
ΔV/ΔV
(Makinen et al., 2005)
-/-
Absence of valves, abnormal
patterning, ectopic mural cells
+/Hyperplasia (also lymph nodes)
-/-
E12.5-perinatal
IV
+/-
normal
(Kriederman et al., 2003)
-/-
-/-
perinatal
normal
(Huang et al., 2000)
Foxc2
Integrin α9
Lymphedema, chylothorax
+/ΔV
+/-
Net
mut/mut
perinatal
normal
mut/mut
Lymphangiectasia, chylothorax
+/mut
Transient absence of lymphatic
capillaries
mut/mut
p85 subunit
of PI3K
-/-
-/-
Podoplanin
-/-
Np2
Prox1
Slp76
Sox18
mut/mut
perinatal
normal
Chylous ascites
+/mut
+/-
postnatal or survive
normal
(Ayadi et al., 2001)
(Yuan et al., 2002)
postnatal or survive
normal
(Fruman et al., 2000)
perinatal
normal
(Schacht et al., 2003)
defects in lymphatic function,
lymphangiectasia, lymphedema
-/-
-/-
No differentiation of LECs
Defects in the lymphatic function,
adult onset obesity
-/-
+/-
+/-
E14.5
perinatal (in most
backgrounds)
(Wigle & Oliver, 1999;
Wigle et al., 2002; Harvey
et al., 2005)
-/-
Failure to separate blood and
lymphatic vasculatures, chylous ascites
-/-
(Abtahian et al., 2003)
mut/mut
mut/mut
Edema, chylous ascites
+/-
+/-
perinatal
normal
+/mut
(Pennisi et al., 2000)
Failure to separate blood and
lymphatic vasculatures, chylous ascites
-/-
Trisomy 16
Nucheal edema, abnormal size and
structure of jugular lymph sacs
E16-E20
(Gittenberger-De Groot et
al., 2004)
Vegfc
-/-
-/-
(Karkkainen et al., 2004)
Syk
-/-
perinatal
normal
+/-
Vegfr3
No lymph sacs or lymphatic vessels
Hypoplasia, chylous ascites
+/mut
+/-
+/-
perinatal
normal
E17-E19
perinatal or normal
mut/mut
Hypoplasia, chylous ascites
+/mut
Vezf1
+/-
Lymphatic hypervascularization and
edema in the jugular region
(incompletely penetrant and transient
phenotype)
-/+/-
E10
perinatal or normal
E9.5-E16.5
normal
(Abtahian et al., 2003)
(Karkkainen et al., 2001)
(Kuhnert et al., 2005)
ΔV: Deletion of a valine residue in the conserved carboxyterminus leading to an inability to bind PDZ-domain
containing proteins
mut
: inactivating mutation (Net and Np2, gene targeted; Sox18, spontaneous; Vegfr3, chemically induced)
39
4. Blood vessels in pathological conditions
Excessive or insufficient angiogenesis is centrally involved in several pathological conditions
including diabetic retinopathy, psoriasis, rheumatoid arthritis, cardiovascular diseases and
cancer (reviewed in Carmeliet & Jain, 2000; Carmeliet, 2003). The ability to induce or inhibit
blood vessel growth in a controlled manner would thus provide an additional strategy for the
management and treatment of such diseases.
Although successful in animal models, clinical trials testing the proangiogenic potential of
VEGF or FGF to promote revascularization of ischemic tissues have failed to show a
conclusive clinical benefit (reviewed in Simons, 2005). Perhaps a combination therapy
promoting both vessel growth and maturation or factors like stabilized HIF-1α might prove to
be more potent choices than individual angiogenic factors (Pajusola et al., 2005).
4.1
Tumor angiogenesis and its inhibition
It has long been recognized that tumors depend on the growth of new blood vessels to provide
the increasing tumor mass with sufficient oxygen and nutrients (reviewed in Folkman, 1996).
Therefore inhibition of tumor angiogenesis, especially by interfering with VEGF/VEGFR-2
interaction or by inhibition of VEGFR-2 signal transduction, is regarded as an efficient
method of inhibiting tumor growth, and after success in mouse models, several such agents
are already in clinical use or in trials (reviewed in Ferrara et al., 2003). Inhibition of tumor
angiogenesis is considered an attractive alternative to inhibition of tumor growth, since it can
act on any tumor type, endothelial cells are easily accessible, they are genomically stable and
thus do not develop resistance, and normal tissues are minimally affected since quiescent
blood vessels have been thought to be independent of VEGF signalling. Furthermore, there is
increasing evidence that some tumor cells might express VEGFRs and inhibition of the
VEGF/VEGFR autocrine loop could thus directly inhibit the growth of tumor cells (Vieira et
al., 2005; Wey et al., 2005). The clinical use of Bevacizumab (Avastin), a VEGF specific
neutralizing antibody, in the treatment of colorectal cancer has shown that inhibition of tumor
angiogenesis by interfering with the VEGF signalling alone is not able to eradicate the tumor.
Many types of angiogenic factors are secreted by stromal cells, such as infiltrated
macrophages and inflammatory cells, in addition to the tumor cells. Thus, complete inhibition
of tumor angiogenesis might require inhibition of several other angiogenic pathways in
addition to VEGF. On the other hand, antiangiogenic therapy could prove to be efficient when
combined with other means of treatment. Bevacizumab has been found to improve the
efficacy of chemotherapeutic agents in colorectal cancer (reviewed in Hurwitz, 2004; Hurwitz
et al., 2004). This might be due in part to normalization of leaky, malfunctioning, mosaic
tumor blood vessels, which improves the transport of chemotherapeutic agents and lowers the
interstitial pressure (Jain et al., 2006).
The endothelium of tumor blood vessels selectively expresses certain molecules not found in
normal blood vasculature, which allows identification of peptides specifically homing to
tumors. Such peptides could be used for targeted delivery of chemotherapeutic drugs into the
tumor, which could increase efficacy and diminish side effects in other tissues (Arap et al.,
1998). Furthermore, recruitment of endothelial precursor cells has been suggested to be
necessary for tumor angiogenesis, thus providing an additional target for inhibition of blood
vessel growth into the tumor (Lyden et al., 2001).
40
5. Lymphatic vessels in pathological conditions
5.1
Tumor lymphangiogenesis and metastasis
Although the importance of angiogenesis for tumor progression is well recognized, it is still
rather unclear if lymphatic vessels have an active or a mere passive role in tumorigenesis
(reviewed in Folkman, 1995; Carmeliet & Jain, 2000). Fatality of cancer is seldom caused by
the primary tumor but rather by the metastatic spread of malignant cells to distant organs.
Although tumor cell dissemination can occur by a variety of mechanisms, including local
tissue invasion, direct seeding of body cavities or surfaces and spread through the blood
vascular system, several clinical and pathological observations have suggested that for many
types of solid human tumors the most common pathway of initial metastasis is through the
lymphatic system to regional lymph nodes (reviewed in Sleeman, 2000; Stacker et al.,
2002b). From there, further dissemination may occur to distant organs either through the
lymphatic system draining into the venous circulation or directly via the blood vasculature.
The spread of tumor cells to regional lymph nodes via the lymphatic vasculature has long
been used as an important prognostic marker of tumor aggressiveness and as an indicator for
therapeutic choices (reviewed in Alitalo & Carmeliet, 2002; Pepper et al., 2003). Lymphatic
vessels containing clusters of tumor cells are frequently observed in the periphery of
malignant tumors (Deutsch et al., 1992; Cann et al., 1995; reviewed in Witte et al., 1997).
However, the existence of functional lymphatic vessels within human cancers and the ability
of tumors to actively stimulate lymphangiogenesis has been controversial (reviewed in Jain,
1989; Padera et al., 2004).
Intratumoral lymphatic vessels have been detected in some human cancers such as
melanomas and head and neck carcinomas, and in a few cases these were reported to be
proliferating (Beasley et al., 2002; Dadras et al., 2003; Maula et al., 2003; Straume et al.,
2003; Gombos et al., 2005). Although in some cancers the presence of lymphatic vessels
inside the tumor was reported to positively correlate with lymph node metastasis
(Schoppmann et al., 2001; Beasley et al., 2002; Hall et al., 2003; Kuroyama et al., 2005;
Nakamura et al., 2006), intratumoral lymphatic vessels might not be a prominent feature and
are probably not required for lymphatic metastasis (Wong et al., 2005). However, many types
of solid human tumors express the lymphangiogenic growth factor VEGF-C and several
clinicopathological studies have reported a positive correlation of its expression with
lymphatic invasion, lymph node and distant metastasis and poor patient survival, but not
necessarily with the density of tumor associated lymphatic vessels (Table II and references
therein). On the other hand, the relationship of VEGF-D with lymph node metastasis is much
more unclear. Whereas some tumors show upregulation of VEGF-D and a positive correlation
between its expression and lymphatic metastasis, in some cases no correlation or even a
negative correlation was reported (Table II and references therein). VEGF-D was found to be
downregulated in most human tumors in a broad cancer array (Pirjo Laakkonen and Kari
Alitalo, unpublished observation). Furthermore, the lymphatic endothelial specific receptor
VEGFR-3 is upregulated in the blood vessels of many tumor types, and it was suggested to be
involved in tumor angiogenesis or the integrity of tumor blood vessels (Partanen et al., 1999;
Valtola et al., 1999; Kubo et al., 2000; Clarijs et al., 2002).
41
Table II: Correlation of VEGF-C or VEGF-D expression in primary human tumors with lymphatic
involvement, tumor metastasis and patient survival. Modified from (Stacker et al., 2002a; Pepper et al.,
2003; He et al., 2004a).
Tumor
Relation of VEGF-C expression with tumor
parameters
Reference
Breast cancer
Positive correlation with LVI, DFSa,b
Not significant but clear trend for DFSb
No correlation with VI, MVD, LNMb
Positive correlation with LNM
(Kinoshita et al., 2001)
(Yang et al., 2002)
(Gunningham et al., 2000)
(Kurebayashi et al., 1999)
Cervical cancer
Positive correlation with LNM, poor prognosisa,b
(Hashimoto et al., 2001)
(Ueda et al., 2001)
(Van Trappen et al., 2002)
(Gombos et al., 2005)
Positive correlation with LVI, LNM, DFS b
Colorectal cancer
Positive correlation with LNM, LVI, invasion deptha,b
Positive correlation with LVI, LNM, poor histological
grade, depth of invasion, venous invasion, liver
metastasis and Duke's stageb
No correlation with LNMa,b
(Akagi et al., 2000)
(Kaio et al., 2003)
(Kawakami et al., 2003a)
(Furudoi et al., 2002)
(George et al., 2001)
Endometrial
carcinoma
Positive correlation with LVI, LNM, DFS, vascular
invasion, depth of invasionb
(Hirai et al., 2001)
Esophageal cancer
Positive correlation with LVI, LNM, depth of invasion,
tumor stage, venous invasionb
(Kitadai et al., 2001)
Gallbladder cancer
Positive correlation with LVI, LNM, poor outcome, but
not with MVDb
(Nakashima et al., 2003)
Gastric cancer
Positive correlation with LVI, LNM, venous invasion,
tumor infiltrating patterns, poor prognosisa
Positive correlation with LVI, venous invasion (VEGF-C
expression had a significant unfavorable impact on
prognosis of patients with negative staining for VEGF)b
Positive correlation with LVI, LNM in undifferentiated
adenocarcinomas but not in those of differentiated
tumorsb
Positive correlation with LVI, LNM, MVD, depth of
tumor invasionb
Positive correlation with LVI, no correlation with LNMb
(Yonemura et al., 1999)
Head and neck
squamous cell
carcinoma
Positive correlation with LNM (increased expression of
VEGF-C in tumors versus normal epithelium)a
No correlation of VEGF-C with intratumoral lymphatic
vessel proliferation
(O-Charoenrat et al., 2001)
Lung
adenocarcinoma
Positive correlation of a low VEGF-D:VEGF-C ratio
with LVI, tendency for higher VEGF-C levels in the
node-positive group & ratio of VEGF-D:VEGF-C
significantly lower in the node-positive groupb
(Niki et al., 2000)
Melanoma
Increased LVD associated with improved patient 5-year
survivalb
(Straume et al., 2003)
42
(Ichikura et al., 2001)
(Ishikawa et al., 2003)
(Amioka et al., 2002)
(Kabashima et al., 2001)
(Beasley et al., 2002)
Neuroblastoma
No correlation with LNMa
(Komuro et al., 2001)
Non-small cell
lung cancer
(NSCLC)
Positive correlation with LVD, no correlation between
LVD and LNMb
Positive correlation with LNMb
Positive correlation with LVI, LNM, DFSb
Positive correlation with DFSb
(Ohta et al., 1999)
Oral cancer
Positive correlation with LNM and pathological grade,
but not with clinical grades in squamous carcinomasb
Positive correlation with LVDa
(Wen et al., 2001)
Ovarian carcinoma
Positive correlation with LNM, peritoneal metastasis,
DFSb
(Yokoyama et al., 2003)
Pancreatic cancer
Positive correlation with LVI, LNM, not with DFSb,c
(Tang et al., 2001)
Prostate cancer
Higher expression of VEGF-C in lymph node-positive
than in lymph node-negative groupd
(Tsurusaki et al., 1999)
Thyroid tumors
Increased expression of VEGF-C in lymph node invasive
tumorsa,b
Positive correlation with LNMb
(Bunone et al., 1999)
Tumor
Relation of VEGF-D expression with tumor
parameters
Reference
Breast cancer
Positive correlation with LNM, poor DFS and overall
survivalb
No correlation with LNM, but with inflammatory
response
(Nakamura et al., 2003)
VEGF-D expression downregulated in tumors with LVIa
No correlation with LNM, VEGF-D expression
downregulated in polyps and in tumorsa,b
Positive correlation with LVI, LNM, an independent
prognostic factor for both disease-free and overall
survivalb
(Kawakami et al., 2003b)
(George et al., 2001)
Gastric cancer
Positive correlation with LVI, LNM in undifferentiated
early gastric cancersb
(Ishikawa et al., 2003)
Head and neck
squamous cell
carcinoma
Decreased expression of VEGF-D in tumors versus
normal epitheliuma
(O-Charoenrat et al., 2001)
Lung
adenocarcinoma
Tendency for lower VEGF-D levels in the node-positive
group, none of the 11/60 tumors with high VEGF-D
levels metastasized to lymph nodesb
(Niki et al., 2000)
Ovarian carcinoma
Positive correlation with LNM and peritoneal metastasis
& VEGF-D was an independent prognostic indicator of
poor survivalb
(Yokoyama et al., 2003)
Colorectal cancer
(Ohta et al., 2000)
(Kajita et al., 2001)
(Arinaga et al., 2003)
(Yu et al., 2002)
(Tanaka et al., 2002)
(Kurebayashi et al., 1999)
(White et al., 2002)
LVI, lymphatic vessel invasion; VI, vascular invasion; LNM, lymph node metastasis; LVD, lymphatic vessel
density; MVD, microvascular density; DFS, poor disease-free survival
a
mRNA expression detected by reverse transcription polymerase chain reaction
b
Protein detected by immunohistochemistry
c
mRNA expression detected by Northern blot analysis
d
mRNA expression detected by in situ hybridization
43
Recently, the relationship of VEGF-C and VEGF-D with tumorigenesis has been a focus of
intensive experimental research, and both growth factors have been indicated to increase
tumor lymphangiogenesis and lymphatic metastasis in several xenotransplantation and
transgenic animal models. Furthermore, tumor lymphangiogenesis and lymphatic metastasis
were inhibited by blocking the VEGFR-3 signalling pathway (reviewed in Achen et al.,
2005). These studies are presented and the possible mechanisms for VEGF-C induced
increase in tumor metastasis are discussed in more detail in the Results and Discussion,
paragraph 3.
Tumor metastasis is not a random event but an extremely complex, highly organized and
tissue-specific process of multiple sequential steps involving many different classes of
molecules (reviewed in Gassmann et al., 2004; Christofori, 2006; DiMeo & Kuperwasser,
2006). To metastasize, tumor cells have to detach from the surrounding tumor mass and
ECM, migrate, intravasate into blood or lymphatic vessels, survive in the circulation, adhere
in the target organ microvasculature and extravasate, invade and, finally, to be able to escape
immune surveillance, grow and induce angiogenesis in their destination (reviewed in
Yeatman & Nicolson, 1993; Fidler, 2003). The nature of the molecular cues guiding certain
tumor types at different stages to metastasize to specific target organs are starting to be
elucidated. The vascular endothelium has specific molecular characteristics in each tissue,
which facilitates tumor cell adhesion and homing to selected organs (reviewed in Ruoslahti &
Rajotte, 2000; Ruoslahti, 2004). Interestingly, the chemokine receptors CCR7 and CXCR4
are highly expressed by human breast cancer cells whereas their respective ligands, secondary
lymphoid chemokine (SLC/CCL21) and CXCL12 are produced by the lymphatic endothelium
in lymph nodes, bone marrow, lung and liver, which represent the first destinations of breast
cancer metastasis (Gunn et al., 1998; Muller et al., 2001). Inhibition of the CXCL12/CXCR4
interaction significantly reduces breast cancer metastasis to the lymph nodes and the lung
(Muller et al., 2001). Similarly, expression of CCR7 in murine melanoma cells promoted
metastasis to lymph nodes, and this was blocked by neutralizing SLC/CCL21 antibodies
(Wiley et al., 2001). These kinds of molecular cues could represent a more general
mechanism of guiding tumor cell homing to specific destinations and might provide a
potential therapeutic target for inhibition of tumor progression to lethal metastatic disease
(reviewed in Zlotnik, 2004). Another recently suggested and interesting mechanism dictating
organ specific tumor spread is that bone marrow derived VEGFR-1 positive hematopoietic
progenitors establish a premetastatic niche at tumor specific sites prior to tumor cell arrival
(Kaplan et al., 2005). The early stages of tumor cell growth in the target organ are also
affected by a supportive microenvironment providing paracrine growth stimuli.
5.2
Lymphedema
Dysfunction of the lymphatic vasculature and impairment of lymphatic drainage causes
interstitial accumulation of proteins and associated fluid, which leads to a chronic,
progressive swelling of the extremities, a condition called lymphedema. Lymphedema can be
either hereditary or with unidentifiable etiology, in which case it is called primary
lymphedema, or a consequence of an earlier disease, trauma, surgery or radiotherapy, in
which case it is referred to as secondary or acquired lymphedema. Common complications of
lymphedema include progressive dermal fibrosis, accumulation of adipose and connective
44
tissue, impaired wound healing, decreased immune defence and thus increased susceptibility
to infections and subsequent cellulitis (reviewed in Rockson, 2001). In rare cases,
lymphedema can also place the patient at risk for developing lymphangiosarcoma. Although
seldom life threatening, lymphedema is a disabling and disfiguring condition severely
affecting the quality of life. At the moment no curative treatment exists, traditional
management of the disease includes manual lymphatic drainage by physiotherapy, massage
and external compressions (reviewed in Witte et al., 2001).
Whereas lymphedema normally affects the body extremities, chylous ascites and chylothorax
are rare conditions caused by lymph extravasation leading to accumulation of high-fat
containing fluid (chyle) in the abdomen or thorax as a result of trauma or abnormal
development of lymphatic vessels.
5.2.1
Primary lymphedema
Although the pattern of inheritance for the forms of primary lymphedema with a linkage to an
identified genomic locus is generally autosomal dominant, the penetrance is in some cases
reduced or variable, suggesting an oligogenic condition or substantial contribution by
modifier genes and/or environmental factors (Holberg et al., 2001; Brice et al., 2005).
Furthermore, the classification of lymphedema syndromes by phenotypic features does not
directly reflect the underlying genetic failure (Finegold et al., 2001). Primary lymphedemas
are estimated to affect approximately one in six thousand people, with a sex ratio of about one
male to three females (Dale, 1985).
Hereditary Lymphedema Type I
Hereditary Lymphedema Type I, also called Milroy’s disease or primary congenital
lymphedema (MIM 153100), is an early onset form of lymphedema, which becomes apparent
at birth and affects primarily the legs and feet. Lymphangiography has revealed that the
lymphatic vessels of these patients are absent or extremely hypoplastic in the affected but not
in the unaffected areas (Bollinger et al., 1983; Brice et al., 2005). Inheritance of this disease,
at least in some families, has been linked to the VEGFR-3 locus in chromosome 5q35.3
(Ferrell et al., 1998; Witte et al., 1998; Evans et al., 1999; Evans et al., 2003). Most of the
disease associated alleles contain missense mutations encoding tyrosine-kinase-negative
VEGFR-3 proteins with extended cellular half-lives, which function as dominant negative
receptors diminishing downstream signalling (Irrthum et al., 2000; Karkkainen et al., 2000).
Hereditary Lymphedema Type II
Hereditary Lymphedema Type II, also called Meige disease or lymphedema praecox (MIM
153200), is a late onset form of lymphedema, commonly detected around puberty. At least in
one family, the inheritance of this disease was linked to an inactivating mutation in the
FOXC2 gene (Finegold et al., 2001).
Lymphedema syndromes linked to inactivating mutations in the FOXC2 gene
Lymphedema-distichiasis syndrome (LD, MIM 153400), lymphedema and ptosis (MIM
153000) as well as lymphedema and yellow nail syndrome (MIM 153300) are multisymptom
disorders characterized by lymphedema of the limbs, with pubertal or variable age of onset,
45
associated with a variety of congenital abnormalities. Individuals with LD have an extra row
of eyelashes, which irritates the cornea, and lymphedema affects their legs and feet but not the
arms (Figure 6). Other associated complications may include cardiac defects, cleft palate,
spinal extradural cysts, varicose veins and photophobia (Fang et al., 2000). Unlike in
congenital lymphedema, where lymphatic vessels are aplastic or hypoplastic, LD is associated
with a normal or even an increased number of lymphatic vessels, which however display a
decreased uptake and reflux of a tracer (Brice et al., 2002). These syndromes show an
autosomal dominant mode of inheritance, and in several families inheritance has been linked
to mutations in the FOXC2 gene (Mangion et al., 1999; Bell et al., 2000; Fang et al., 2000;
Bell et al., 2001; Erickson et al., 2001; Finegold et al., 2001; Bahuau et al., 2002; Brice et al.,
2002; Traboulsi et al., 2002; Brooks et al., 2003). Most of these mutations are small
insertions or deletions leading to a frameshift causing a premature, inactivating truncation of
the transcription factor, but two missense single nucleotide substitutions have also been
identified. In two families, despite linkage to the FOXC2 locus, no mutations in the coding
region of FOXC2 were found, suggesting a promoter mutation (Sholto-Douglas-Vernon et al.,
2005). Reflecting the developmental role of FOXC2 in multiple tissues, a recent study
reported renal disease and diabetes mellitus in combination with LD caused by a FOXC2
mutation (Yildirim-Toruner et al., 2004).
Other syndromes involving lymphedema with identified genetic linkages
An unusual association of lymphedema with hypotrichosis and telangiectasia (MIM 607823)
was recently reported and linked to mutations in the gene encoding for the transcription factor
SOX18 (Glade et al., 2001; Irrthum et al., 2003). Furthermore, the locus for cholestasislymphedema syndrome, also known as Aagenaes syndrome, (MIM 214900) has been mapped
to chromosome 15q, but the gene involved is still unknown (Bull et al., 2000). The locus
responsible for the variable symptoms of Turner syndrome, frequently associated with
lymphedema, has been mapped to region Xp11.2-p22.1, which interestingly harbors also the
VEGF-D gene (Rocchigiani et al., 1998; Zinn et al., 1998).
Figure 6: Lymphedemadistichiasis. Lymphedemadistichiasis is manifested by
late onset lymphedema
affecting the legs and feet (A)
and by distichiasis, an
additional row of eyelashes (B,
arrows). Images by courtesy of
Prof. Peter Mortimer and Dr.
Russell Mellor.
46
5.2.2
Secondary lymphedema
Post-surgical edema, especially after mastectomy, represents the primary form of
lymphedema in industrialized countries. Its incidence, in approximately 6 to 30% of operated
patients, is increased by post-operative radiotherapy, but its etiology and pathophysiology are
still not fully understood, and appear to be multifactorial (reviewed in Petrek & Heelan, 1998;
Pain & Purushotham, 2000; Rockson, 2001). Some of the susceptibility may have a genetic
basis.
Worldwide the most common cause of lymphedema is filariasis, currently affecting over 120
million people, mostly in tropical areas. It is caused by mosquito-transmitted infection with
the parasitic nematodes Wuchereria bancrofti, Brugia malayi or Brugia timori. These
parasites live and reproduce in the lymphatic system, causing a massive dilation of lymphatic
vessels and finally a complete and permanent disruption of lymphatic transport, which leads
to a predisposition to long-term recurrent bacterial infections and a condition called
elephantiasis (reviewed in Dreyer et al., 2000). Although several microfilaricidal agents are
currently in use, these drugs are only partially effective against the long-lived adult worms
and thus require long-term application for up to 20 years. Recently, tetracyclines are used to
target the endosymbiotic bacteria, Wolbachia, which results in filarial growth retardation and
infertility.
5.3
Lymphangiectasia and lymphatic neoplasms
Lymphangiectasia, local saccular dilation of lymphatic vessels, is visible as translucent
vesicles. It is mostly associated with postsurgical lymphedema but can also occur as a
consequense of other local lymphatic damage. Lymphangioma, benign lymphatic
malformation, represents similar symptoms as lymphangiectasia but has a genetic, mostly
inherited, cause. Lymphangiosarcomas, malignant lymphatic tumors, are very rare and occur
mostly as a consequence of long lasting lymphedema.
Kaposi sarcoma, characterized by nodules of spindle shaped tumor cells with a prominent
vasculature, is associated with infection by Kaposi sarcoma herpesvirus/human herpesvirus 8
(KSHV/HHV-8). Kaposi sarcoma spindle cells are suggested to be of endothelial origin and
express both blood and lymphatic endothelial markers (Jussila et al., 1998). One of KSHV
envelope glycoproteins interacts with and activates VEGFR-3 and integrin α 3β1, resulting in
increased endothelial cell growth and migration (Zhang et al., 2005b). The transcriptional
profile of Kaposi sarcoma cells closely resembles that of lymphatic endothelial cells and
furthermore, infection of BECs in vitro with KSHV was shown to result in the expression of
several LEC specific genes (Hong et al., 2004a; Wang et al., 2004).
5.4
Inflammatory diseases
In addition to decreasing edema caused by inflammation, lymphatic vessels are actively
involved in the regulation of inflammatory responses by transporting leukocytes from the site
of inflammation to secondary lymphoid organs. Although less characterized than the
molecular mechanisms controlling the extravasation of leukocytes from postcapillary venules
into tissues, several molecules have recently been implicated in the process of their
47
recirculation through the lymphatic system. The chemokine receptor CCR7 is essential for
migration of dendritic cells into afferent lymphatic vessels, which express its ligand
SLC/CCL21 (Ohl et al., 2004). Mannose receptor 1 and common lymphatic endothelial and
vascular endothelial receptor 1 (CLEVER-1), both expressed by the lymphatic endothelium,
were found to control lymphocyte traffic in lymphatic vessels (Irjala et al., 2001; Salmi et al.,
2004).
VEGF-C is upregulated by proinflammatory cytokines, suggesting it stimulates lymphatic
vessel growth during inflammation (Ristimaki et al., 1998). This upregulation presumably
occurs through NF-κB, which is an important transcription factor for signal transduction of
proinflammatory factors and has a putative binding site in the VEGF-C promoter (Chilov et
al., 1997). Interestingly, NF-κB was shown to be constitutively active in lymphatic vessels
(Saban et al., 2004).
Inflammatory cells, especially macrophages, express VEGFRs and are thus attracted by
angiogenic and lymphangiogenic signals. They also stimulate further lymphangiogenesis by
themselves secreting VEGF-C and VEGF-D (Mimura et al., 2001; Skobe et al., 2001a;
Schoppmann et al., 2002; Cursiefen et al., 2004; Hamrah et al., 2004). Furthermore, recent
reports suggest that macrophages might also contribute to lymphangiogenesis by
transdifferentiating into lymphatic endothelial cells and incorporating into the lymphatic
endothelium (Kerjaschki, 2005; Maruyama et al., 2005; Kerjaschki et al., 2006).
Proliferation of lymphatic vessels has been reported in human kidney transplants undergoing
rejection, in a mouse model of chronic airway inflammation and in cornea models of
inflammatory neovascularization (Hamrah et al., 2003; Chen et al., 2004; Cursiefen et al.,
2004; Kerjaschki et al., 2004; Baluk et al., 2005). In kidney transplants, lymphatic vessels
contribute to the export of the lymphocyte rich inflammatory infiltrate, but are also involved
in the maintenance of the detrimental alloreactive immune response by attracting CCR7positive immune cells by producing SLC/CCL21 (Kerjaschki et al., 2004). Infection of the
mouse airway epithelia by Mycoplasma pulmonis results in massive lymphangiogenesis
induced by VEGF-C and VEGF-D producing inflammatory cells. Inhibition of
lymphangiogenesis by a VEGFR-3 decoy leads to severe mucosal edema, consistent with the
importance of lymphatic vessels as an exit route for the immune cells and fluid (Baluk et al.,
2005). Interestingly, the new lymphatic vessels were found to regress much later than newly
formed blood vessels after the inflammation (Baluk et al., 2005).
5.5
Prolymphangiogenic therapy
Stimulation of lymphatic growth could prove to be advantageous for the treatment of those
forms of primary and secondary lymphedema characterized by lymphatic aplasia or
hypoplasia or even to control edema under some inflammatory conditions. Adenoviral gene
transfer of VEGF-C into normal adult mouse skin leads to a strong lymphangiogenic
response, promising an effective treatment for lymphedema (Enholm et al., 2001). Indeed,
growth of functional lymphatic vessels has been successfully achieved with VEGF-C gene
therapy in the Chy-mouse model of congenital lymphedema. Like patients with Milroy’s
disease, these mice harbour an inactivating mutation in one of their VEGFR-3 alleles
allowing some wild type VEGFR-3 activity, which could be enhanced by overexpression
VEGF-C (Karkkainen et al., 2001). VEGF-C gene transfer was also used to reconstruct a
48
functional lymphatic vessel network after surgical incision, suggesting important implications
for the prevention and treatment of surgically induced secondary lymphedema (Saaristo et al.,
2004).
When stimulating growth of new lymphatic vessels several concerns should be taken into
account. This kind of treatment should be local to avoid a possible risk of increasing tumor
lymphangiogenesis and metastasis. This might exclude the use of prolymphangiogenic
therapy for the treatment of postsurgical lymphedema in cancer patients. The systemic effect
of VEGF-C is expected to be minimal, as the half-live of VEGF-C in blood circulation is very
short (Veikkola et al., 2001). Furthermore, prolymphangiogenic therapy should be specific
without angiogenic or vascular permeability side effects. Whereas VEGF-C induces blood
vessel enlargement and leakiness, the VEGFR-3 specific form of VEGF-C, VEGF-C156S,
was shown to stimulate lymphangiogenesis with minimal blood vascular side effects (Saaristo
et al., 2002a; Saaristo et al., 2002b). Also, the stability of the newly formed lymphatic vessels
and their sufficiency to decrease edema remain critical issues to be assessed. So far, VEGF-C
and VEGF-D have been the factors of choice for preclinical prolymphangiogenic studies.
Knowledge of the biological function of their propeptides would be important for decreasing
side effects (I). Furthermore, rational design of recombinant growth factors may lead to even
better prolymphangiogenic properties (Jeltsch et al., 2006).
Despite several safety and ethical issues of concern, gene therapy by means of recombinant
viruses is attractive for the treatment of monogenic diseases, and has already been applied in
humans (Wagner et al., 1999; Kay et al., 2000). Development of delivery systems capable of
efficient gene transfer to a specific tissue without causing pathogenic side effects is a key
factor for improving the success of gene therapy (reviewed in Verma & Weitzman, 2005).
Gene therapy with adeno and adeno associated viruses has successfully been used in animal
models to stimulate or inhibit angiogenesis and lymphangiogenesis (reviewed in YlaHerttuala & Alitalo, 2003). Targeting of gene delivery into endothelial cells by modifying the
surface adhesion molecules of the viral particles or allowing expression only in endothelial
cells under the control of specific, and possibly inducible, promoters are future challenges in
the field.
49
AIMS OF THE STUDY
This study was undertaken to elucidate the role of the VEGF-C/VEGFR-3 pathway and to
define the function of the transcription factor FOXC2 in the regulation of lymphatic vessel
development, growth and survival under normal as well as pathological conditions.
To achieve these goals we specifically studied:
I
The biological function of the carboxyterminal silk-like propeptide of VEGF-C
II
Involvement of NP2 in VEGF-C and VEGF-D signalling via VEGFR-3
III
The role of the VEGF-C/VEGFR-3 pathway in tumor growth, vascularization and
metastasis
IV
Dependence of the normal lymphatic vasculature on VEGFR-3 signalling
V
The mechanisms by which Foxc2 regulates the development of the lymphatic
vasculature
50
MATERIALS AND METHODS
A list of the materials and methods used is given in the form of tables. The materials and
methods are described in more detail in the original publications.
Cell line
Description
Source or
reference
Used in
293T
Human embryonic kidney fibroblast cell line
expressing SV40 T antigen
I, II, IV, V
BEC
Primary blood vascular endothelial cells
CAEC
HDMVEC
HepG2
LEC
Primary human coronary artery endothelial cells
Primary human dermal microvascular endothelial cells
Human kidney epithelial cell line
Primary lymphatic endothelial cells
MCF-7
S1 subclone of human MCF-7 breast carcinoma cell
line
Porcine aortic endothelial cells stably transfected to
express human NP2 and VEGFR-3
Porcine aortic endothelial cells stably transfected to
express human NP2
Porcine aortic endothelial cell line
American Type
Culture
Collection
(ATCC)
(Makinen et al.,
2001b)
Promo Cell
Promo Cell
ATCC
(Makinen et al.,
2001b)
ATCC
II
II
II
II
Dr. Lena
Claesson-Welsh
(Pajusola et al.,
1994)
Invitrogen
Promo Cell
II
NP2/R3-PAE
NP2-PAE
PAE
R3-PAE
S2
SaVEC
Porcine aortic endothelial cells stably transfected to
express human VEGFR-3
Drosophila Schneider S2 cells
Primary human saphenous vein endothelial cells
II
V
II, V
IV, V
II, V
III
II
II, IV
V
Recombinant
Protein
Description
Source or
reference
Used in
VEGF165, human
Produced in Sf21 cells using baculovirus expression
system
Human VEGF-C consisting of the amino acid residues
103-215 with a hexahistidine tag, produced in S2 cells
Human VEGF-D consisting of the amino acid residues
93-201 with a hexahistidine tag, produced in Sf21 cells
Fusion protein containing the first five Ig homology
domains of human VEGFR-1 fused to the Fc region of
hIgG
Fusion protein containing the first three Ig homology
domains of human VEGFR-2 fused to the Fc region of
hIgG
Fusion protein containing the first three Ig homology
domains of human VEGFR-3 fused to the Fc region of
hIgG
R&D Systems
II
II
II
R&D Systems
II
(Makinen et al.,
2001a)
II, IV
(Uutela et al.,
2004)
II
(Makinen et al.,
2001a)
I, II, IV
VEGF-C, mature
human
VEGF-D, mature
human
VEGFR-1-Ig
VEGFR-2-Ig
VEGFR-3-Ig
51
Antigen
Antibody
Source or reference
Used in
Collagen IV,
mouse
EEA-1
Endoglin, mouse
Rabbit polyclonal
Cosmo Bio
V
Rabbit polyclonal
Rat monoclonal
II
V
FOXC2, human
FOXC2, human
Foxc2, mouse
Laminin, mouse
Lyve-1, mouse
Goat polyclonal
Mouse monoclonal
Rat monoclonal
Rabbit polyclonal
Rabbit polyclonal
Abcam
Pharmingen/BD
Biosciences
Abcam
(Yang et al., 2000)
(Furumoto et al., 1999)
Dr. Karl Tryggvason
V, (Prevo et al., 2001)
NG2, mouse
NP2, human
PCNA
Pecam-1, mouse
Rabbit polyclonal
Rabbit polyclonal (H-300)
Mouse monoclonal, biotinylated
Rat monoclonal
V
II
III
I, III, IV, V
Podoplanin, human
Rabbit polyclonal
Podoplanin, mouse
Syrian hamster monoclonal
PROX1, human
SMA, human
V5 epitope of
paramyxovirus
VEGF-C, human
VEGF-C, human
VEGF-D, human
VEGFR-2, human
VEGFR-2, human
Vegfr-2, mouse
VEGFR-3, human
Rabbit polyclonal
Mouse monoclonal, Cy-3 conjugated
Mouse monoclonal
(Ozerdem et al., 2001)
Santa Cruz
Zymed Laboratories
Pharmingen/BD
Biosciences
(Breiteneder-Geleff et al.,
1999)
Developmental Studies
Hybridoma Bank,
University of Iowa
(Karkkainen et al., 2004)
Sigma
Invitrogen
Goat polyclonal, affinity purified
Rabbit polyclonal antiserum 882
Goat polyclonal, affinity purified
Rabbit polyclonal (RS-2)
Rat monoclonal (1121B)
Rat monoclonal
Mouse monoclonal clones 9D9F9 and
2E11D11
Rabbit polyclonal (C-20)
Goat polyclonal, biotinylated
Rat monoclonal (AFL4)
Rat monoclonal (mF4-31C1)
Rabbit polyclonal
R&D Systems
(Joukov et al., 1997)
R&D Systems
(Waltenberger et al., 1994)
(Zhu et al., 2003)
(Kubo et al., 2000)
(Jussila et al., 1998)
I, II
III, IV
II
II
II
IV
II
Santa Cruz
R&D Systems
(Kubo et al., 2000)
(Pytowski et al., 2005)
DAKO
II
I, IV, V
III, IV
IV
V
Recombinant
Adenovirus
Description
Source or reference
Used in
AdLacZ
AdFOXC2
AdVEGF-C
AdVEGFR-2-Ig
Encodes β-galactosidase
Encodes human FOXC2
Encodes full length human VEGF-C
Encodes a fusion protein containing the first
three Ig homology domains of human
VEGFR-2 fused to the Fc region of hIgG
Encodes a fusion protein containing the first
three Ig homology domains of human
VEGFR-3 fused to the Fc region of hIgG
(Laitinen et al., 1998)
V
(Enholm et al., 2001)
Dr. Hidde J. Haisma
III, IV, V
V
IV
IV
III
III, IV
VEGFR-3, human
Vegfr-3, mouse
Vegfr-3, mouse
Vegfr-3, mouse
vWF, human
AdVEGFR-3-Ig
52
V
V
V
III
I, III, IV, V
V
V
V
V
I
Plasmid
Description
Source or
reference
Used in
6xFOXC2/pTAL-Luc
A reporter plasmid containing six FOXC2 binding sites
in front of the sequences coding for the firefly luciferase
The sequences coding for the carboxyterminal amino
acids 237-501 (264), 338-501 (163) or 414-501 (87) of
FOXC2 cloned in frame with those coding for the Gal4
DNA binding site
FOXC2 mutants A, B, C, F or K in pAMC vector for
testing transactivation of the luciferase reporter gene
Wild type FOXC2 in pAMC vector for testing
transactivation of the luciferase reporter gene
Encodes the a1a2, b1b2, a1a2b1b2 or c domain of
human NP1 fused to the Fc region of hIgG
V
V
V
V
V
V
V
V
II
II
Encodes human NP1 extracellular domain fused to the
Fc region of hIgG
Encodes the a1a2, b1b2, a1a2b1b2 or c domain of
human NP2 fused to the Fc region of hIgG
II
II
II
II
Encodes human NP2 (a22) form
Encodes human NP2 extracellular domain fused to the
Fc region of hIgG
PCI-neo vector (Promega) with an aminoterminal myctag
A luciferase reporter plasmid with five Gal4 binding
sites
A vector that contains the sequences coding for the Gal4
DNA binding domain
Renilla luciferase reporter with herpes simplex virus
thymidine kinase promoter
Reporter plasmid coding for the firefly luciferase
Encodes human SEMA3F with carboxyterminal myc
and 6 x His tags
Encodes human VEGF165
Encodes human VEGF165
II
(Karkkainen et
al., 2001)
(Tiainen et al.,
1999)
Promega
II
II
Clontech
V
Promega
V
Clontech
(Kessler et al.,
2004)
III
(Olofsson et
al., 1998)
I
V
II
I, II
II
I, II
II
II
III
(Veikkola et
al., 2001)
III
II
I, II, III
II
II
II
III
III
FOXC2 CT/pM1
FOXC2 mut/pAMC
FOXC2 WT/pAMC
NP1(a1a2)/spIg
NP1(b1b2)/spIg
NP1(a1a2b1b2)/spIg
NP1(c)/spIg
NP1/spIg
NP2(a1a2)/spIg
NP2(b1b2)/spIg
NP2(a1a2b1b2)/spIg
NP2(c)/spIg
NP2/pcDNA3.1z
NP2/spIg
pAMC
pG5-luc
pM1
pRL-TK
pTAL-Luc
SEMA3F/pcDNA3.1
VEGF165/pEBS7
VEGF165/pSG5
VEGF-C CT/pMosaic
VEGF-CΔC/pMosaic
VEGFCΔNΔC/pAdCMV
VEGF-CΔN/pMosaic
VEGF-C/pEBS7
VEGF-D
/pcDNA3.1z+
VEGFR-1-Ig/pEBS7
VEGFR3/pcDNA3.1hygro
VEGFR-3-Ig/pEBS7
Encodes the carboxyterminus of human VEGF-C with
Igκ signal peptide and a V5 tag
Encodes human VEGF-CΔC with Igκ signal peptide
Encodes human VEGF-CΔNΔC with endogenous signal
peptide
Encodes human VEGF-CΔN with Igκ signal peptide
Encodes full length human VEGF-C
Encodes full length human VEGF-D
Encodes a fusion protein containing the first five Ig
homology domains of human VEGFR-1 fused to the Fc
region of hIgG
Encodes human VEGFR-3 (long form)
Encodes a fusion protein containing the first three Ig
homology domains of human VEGFR-3 fused to the Fc
region of hIgG
53
V
V
III
II
I
III
Method
Used in
Adenoviral transduction of cells or mice
Cell culture
DNA subcloning
ELISA (enzyme-linked immunosorbent assay)
FACS (fluorescence activated cell sorting)
Generation of transgenic mice
Immunofluorescence
Immunohistochemistry
Immunoprecipitation
Ligand binding assays
Metabolic labelling
Microlymphography (Evans blue dye and fluorescent dextran)
Northern Blotting
PCR
Preparation of mouse tissues
Quantitation of lymphatic vessel number and area
Recombinant production and purification of proteins
RNA extraction
RT-PCR (quantitative or non-quantitative)
Southern blotting
Transfection of cells
Tumor implantation in mice
Western blotting
X-gal staining of tissues
III, IV
I, II, III, IV, V
I, II, III
III, IV
IV
I
I, II, IV, V
I, III, IV, V
I, II, III, IV
I, II, IV
I, II, III, IV
I, III, IV, V
I, V
I, II, III, IV, V
I, III, IV, V
I, III, IV, V
II, III, IV
I, V
I, IV
I, V
I, II, III, IV, V
III
I, II
IV, V
54
RESULTS AND DISCUSSION
1. The carboxyterminal silk-like propeptide enhances lymphangiogenic potential of
VEGF-C (I)
The unique cysteine-rich carboxyterminal propeptide of VEGF-C has high homology to silk
proteins. It is evolutionally conserved and found also in the VEGF homologs of Drosophila,
which suggests that it has an important function. Its removal by proteolytic processing
regulates the affinity and specificity of VEGF-C to VEGFR-2 and VEGFR-3, but beyond that
its biological significance is unknown. To get further insight to its role in vivo, we generated
transgenic mice, which express either VEGF-C lacking the carboxyterminal propeptide
(VEGF-CΔC) or which express the carboxyterminal propeptide (CT) alone in the skin under
the transcriptional control of the keratin-14 (K14) promoter. VEGF-CΔC was capable of
inducing hyperplasia of the lymphatic capillary network in the skin of the transgenic mice,
indicating that the CT is not required for the lymphangiogenic activity of VEGF-C.
Surprisingly, expression of the CT separately severely reduced the number but increased the
size of the lymphatic capillaries. The double transgenic mice displayed increased lymphatic
hyperplasia when compared to the K14-VEGF-CΔC mice, suggesting that the CT enhances
the lymphangiogenic potential of VEGF-C. In vitro, CT coprecipitated along with VEGFCΔC, which binds to VEGFR-3, suggesting that CT is able to complement VEGF-CΔC when
expressed in the same cells.
The heparin-binding carboxyterminal domain of VEGF has been shown to be critical for its
mitogenic activity (Keyt et al., 1996). In addition, the proper concentration gradient and
biodistribution of the heparin-binding VEGF isoforms in the ECM is required for initiation
and guidance of vascular branch formation (Ruhrberg et al., 2002; Stalmans et al., 2002).
Similarly, retention of PDGF-B by its carboxyterminal HSPG binding domain to microvessel
endothelium is essential for proper recruitment and organization of PCs (Lindblom et al.,
2003). Accurately localized depots of PDGF-B might help PCs to migrate along the
abluminal surface of the microvessel wall, similar to the requirement of ECM associated
VEGF isoforms for endothelial cell migration along astrocytes in the postnatal retina
(reviewed in Armulik et al., 2005). We found that the CT is associated with ECM when
expressed in 293T cells. Thus, a possible mechanism for the enhancement of the
lymphangiogenic activity of VEGF-C by its silk-like CT is that the CT anchors VEGF-C to
ECM thus creating a proper concentration gradient for guided sprouting of lymphatic
endothelial cells. Given its unique structure, the CT could also form a matrix or a
“prelymphatic channel” for adhesion and migration of lymphatic endothelial cells, which
express a rather different set of adhesion molecules as compared to blood vessel endothelial
cells. Alternatively, binding to cell surface HSPGs could increase the avidity of VEGF-C to
VEGFR-3 and to NP2. Conversely, an excess of the carboxyterminal propeptide in the skin of
K14-CT mice might sequester endogenous VEGF-C too tightly to the matrix where it is not
available for receptor binding or, alternatively, it could block the matrix binding sites from
the endogenous VEGF-C, which then leads to a reduced number of lymphatic capillaries.
In summary, although the mechanism remains to be elucidated, our results indicate that the
carboxyterminal silk-like propeptide of VEGF-C is not absolutely required for, but does
potentiate the lymphangiogenic activity of VEGF-C.
55
2. NP2 is involved in VEGF-C and VEGF-D induced VEGFR-3 signalling (II)
NP2 participates in axon guidance during development of the nervous system. It is also
expressed in the lymphatic and venous endothelium and genetic evidence has implicated a
transient role for NP2 in the development of the lymphatic vasculature (Yuan et al., 2002). In
order to elucidate how NP2 is involved in lymphatic endothelial cell biology, we studied its
interaction with the primary regulators of lymphangiogenic signalling, VEGFR-3 and its
ligands VEGF-C and VEGF-D.
With in vitro binding assays using dimeric soluble neuropilin fusion proteins we found that
both VEGF-C and VEGF-D interact with NP2. Whereas both the partially processed and the
mature form of VEGF-C were able to interact with NP2 without heparin, only the partially
processed form of VEGF-D bound to NP2 in a heparin dependent manner. The partially
processed but not the mature forms of both growth factors also interacted with NP1 in the
presence of heparin. Although the mature form of VEGF-C could bind to NP2, the
aminoterminal propeptide of VEGF-C greatly enhanced the interaction of VEGF-C with NP2,
and was the major mediator of NP1 binding, whereas the carboxyterminal propeptide of
VEGF-C seemed to be dispensable for interaction with neuropilins. The binding site for
VEGF and VEGF-C was mapped to the b1b2 domain of neuropilins. SEMA3F was able to
compete with VEGF-C in binding to both neuropilins, indicating overlapping binding sites.
Upon stimulation of human dermal microvascular endothelial cells (HDMVECs), VEGF-C
and VEGF-D were cointernalized along with NP2 to endocytic vesicles, which demonstrates
that the interaction of these lymphangiogenic vascular endothelial growth factors with NP2
occurs also in the cellular context. Coimmunoprecipitation assays revealed that NP2 also
interacts with VEGFR-3. Furthermore, NP2 and VEGFR-3 were cointernalized to early
endosome antigen 1 (EEA-1) positive endosomal vesicles upon stimulation of LECs with
VEGF-C or VEGF-D. These results indicate, that NP2 is directly associated in an active
signalling complex together with VEGFR-3 and its ligands and suggests a possible
mechanism of how NP2 is involved in the development of the lymphatic vasculature. As
neuropilins are primarily involved in mediating cell guidance and migration, one can
speculate that NP2 would modulate VEGF-C and VEGF-D induced VEGFR-3 mediated
lymphatic endothelial cell migration, and possibly also proliferation.
Receptor internalization upon activation by a specific ligand often leads to termination of
signalling either by degradation of the complex in lysosomes or by recycling the dissociated
receptor back to the plasma membrane. Recently, receptor endocytosis has been found to
control the intensity, duration and nature of cell signalling in a more versatile manner
(reviewed in Miaczynska et al., 2004; Le Roy & Wrana, 2005). Several growth factor
receptors, such as the receptors for TGF-β, epidermal growth factor (EGF) and nerve growth
factor (NGF), were found to maintain their signalling activity in the intracellular compartment
(Le Roy & Wrana, 2005). Furthermore, receptor internalization is a mechanism to localize
receptor tyrosine kinase signalling to facilitate guided migration (Jekely et al., 2005).
Interestingly, NP2 is not internalized upon VEGF-C stimulation in PAE cells expressing only
NP2, but is cointernalized along with VEGFR-3 in cells expressing both NP2 and VEGFR-3,
suggesting that VEGF-C induced NP2 internalization is VEGFR-3 dependent. On the other
hand, VEGFR-3 can be internalized without NP2. This suggests, that although VEGFR-3 is
capable of active signalling on its own, NP2 might modulate VEGFR-3 signalling by
56
polarizing it for directed migration or controlling the signalling intensity, duration or
downstream effector interactions.
Interestingly, although VEGF165 is able to bind NP2, we did not observe internalization of
NP2 in HDMVECs upon stimulation with VEGF165. Nor could we detect
coimmunoprecipitation of NP2 along with VEGFR-2, suggesting that in the vascular system
NP2 might interact specifically with and modulate the signalling of VEGFR-3. This is
supported by the fact that blood vascular development was unaffected in NP2 mutant mice
(Yuan et al., 2002; Shen et al., 2004). If NP2 was important for modulation of VEGF
signalling via VEGFR-2, major blood vascular defects would be expected upon inactivation
of NP2, yet these were restricted to changes in pathological angiogenesis (Shen et al., 2004).
Recently, NP2 was shown to enhance the biological responses of human endothelial cells
induced by VEGF or VEGF-C (Favier et al., 2006). In contrast to our observations in primary
human blood vascular endothelial cells, this study reports an interaction of NP2 with VEGFR2 in transfected 293T cells, and suggests that this interaction could at least partly be
responsible for the NP2 mediated potentiation of VEGF and VEGF-C induced endothelial cell
survival and migration in endothelial cells. On the other hand, VEGF-C and VEGF-D also
bind to NP1 and have angiogenic effects possibly mediated through VEGFR-2, suggesting
that they could use NP1 as a coreceptor when interacting with VEGFR-2.
The fact that the binding of SEMA3F to NP2 could not be competed by VEGF165 has lead to
the suggestion that SEMA3F and VEGF165 bind to separate sites in NP2 (Gluzman-Poltorak et
al., 2001). In our experiments the binding of SEMA3F to neuropilins was also not inhibited
by VEGF-C, whereas VEGF-C binding to neuropilins was competed by increasing
concentrations of SEMA3F. This result indicates at least partially overlapping binding sites
for VEGF-C and SEMA3F in NP2, and suggests that the affinity of SEMA3F for neuropilins
is higher than that of VEGF-C. With the exception of VEGF-B186, the neuropilin binding of
the VEGF family members is mediated by their heparin binding domains (Soker et al., 1998;
Makinen et al., 1999; Gluzman-Poltorak et al., 2000; Mamluk et al., 2002). Full-length
VEGF-C binds to heparin only weakly whereas mature VEGF-C does not interact with
heparin (our unpublished observations). Although the mature form of VEGF-C is able to bind
to NP2, we located the major mediator of the neuropilin binding to the aminoterminal
propeptide of VEGF-C. This was further confirmed by the neuropilin binding ability of a
chimeric protein consisting of the amino- and carboxyterminal propeptides of VEGF-C fused
to the growth factor domain of VEGF, which by itself is unable to interact with neuropilins
(our unpublished observations). The interaction of VEGF-C and VEGF-D thus represents a
unique mechanism for neuropilin binding. As in the case of other VEGF family members,
heparin still potentiates the interaction of VEGF-C and VEGF-D with both NP1 and NP2,
possibly by altering the conformation of neuropilins.
Taken together, our results suggest that NP2 is actively involved in the regulation of
lymphatic development by modifying VEGF-C and VEGF-D induced VEGFR-3 signalling.
Whether this occurs by localizing VEGFR-3 signalling for directed cell migration or
adhesion, by controlling the tyrosine kinase activity of VEGFR-3, by modulating its
interactions with downstream signalling molecules or with the cytoskeleton, by simply
increasing the avidity of VEGF-C and VEGF-D to VEGFR-3, or by some other mechanisms
remains to be elucidated.
57
3. VEGF-C mediated tumor lymphangiogenesis is inhibited by blocking VEGFR-3
signalling (III)
VEGF-C is highly expressed in many types of human tumors and its expression positively
correlates with vascular invasion, lymph node and distant metastasis and poor patient survival
(reviewed in Stacker et al., 2002a; He et al., 2004a). Furthermore, VEGFR-3 is upregulated in
the blood vessels of several tumor types and its expression has been suggested to contribute to
tumor angiogenesis (Partanen et al., 1999; Valtola et al., 1999; Clarijs et al., 2002). To study
the possible effects of VEGF-C on tumor growth, vascularization and metastasis we
overexpressed full length human VEGF-C or the ligand binding domain of VEGFR-3 as a
dimeric soluble immunoglobulin-fusion protein (VEGFR-3-Ig) in MCF-7 human breast
carcinoma cells. The expression of these proteins did not affect the growth rate of MCF-7
cells in vitro. When implanted into the mammary fat pads of SCID mice, overexpression of
VEGF-C significantly accelerated the growth of MCF-7 tumors, but had no detectable effect
on the density of tumor blood vessels. Instead, VEGF-C strongly promoted the growth of
tumor associated lymphatic vessels, which in the tumor periphery were commonly infiltrated
by tumor cells. However, the increase in tumor growth caused by VEGF-C was not as
dramatic as that caused by overexpression of VEGF165, which also increased tumor
angiogenesis approximately two-fold. In contrast, the VEGF165 expressing and control tumors
only occasionally contained lymphatic vessels. The effects of VEGF-C, the slightly increased
tumor growth rate, tumor lymphangiogenesis and intralymphatic tumor growth, were
inhibited by the soluble VEGFR-3-Ig fusion protein expressed either by the tumor cells or by
an intravenously injected recombinant adenovirus.
These results strongly suggest that VEGF-C promotes tumor metastasis via the lymphatic
system by inducing growth of tumor associated lymphatic vessels and by promoting tumor
cell invasion into and growth inside the lymphatic vasculature. Furthermore, our results
indicate that tumor spread via the lymphatic system can be inhibited by blocking the VEGFC/VEGFR-3 signalling pathway (Figure 7).
Several experimental tumor studies using either xenotransplantation or transgenic mouse
models have since shown that overexpression of the lymphangiogenic growth factors VEGFC or VEGF-D induces not only tumor lymphangiogenesis but also increases lymphatic
metastasis (Mandriota et al., 2001; Skobe et al., 2001b; Stacker et al., 2001; Yanai et al.,
2001; Padera et al., 2002; Kawakami et al., 2005). The efficacy of the soluble VEGFR-3-Ig in
blocking tumor lymphangiogenesis and lymphatic metastasis was later confirmed in several
studies employing either ectopically or endogenously VEGF-C expressing tumor models (He
et al., 2002; Krishnan et al., 2003; He et al., 2005; Lin et al., 2005). Furthermore, tumor
lymphangiogenesis and lymphatic metastasis were also inhibited by neutralizing VEGFR-3
antibodies (Shimizu et al., 2004; Roberts et al., 2006), small interfering RNA-mediated
downregulation of VEGF-C (Chen et al., 2005) and by neutralizing VEGF-D antibodies
(Stacker et al., 2001), all interfering with the ligand induced VEGFR-3 pathway.
The prevailing belief that lymphatic vessels could not grow inside tumors because of the high
interstitial pressure was partly developed due to the lack of reliable lymphatic specific
markers. In our study, the staining of the lymphatic endothelial cells for proliferating cell
nuclear antigen (PCNA) reveals that the lymphatic vessels in the periphery of the MCF-7
tumors are indeed actively growing. On the other hand, the intratumoral lymphatic vessels
58
detected in our study and in several other xenotransplantation tumor models (Skobe et al.,
2001b; Stacker et al., 2001) might well be pre-existing ones entrapped or co-opted by the
growing tumor mass. This view is supported by the lack of intratumoral lymphatic vessels
despite massive VEGF-C induced lymphangiogenesis on the tumor periphery in a transgenic
model of pancreatic cancer (Mandriota et al., 2001).
Figure 7: VEGF-C and VEGF-D induced tumor lymphangiogenesis and lymphatic metastasis can be
inhibited by blocking the VEGFR-3 signalling pathway. VEGF-C and VEGF-D secreted by tumor cells,
infiltrated inflammatory cells or other stromal cells stimulate the growth of tumor associated lymphatic vessels
(yellow), and may in some tumor types also contribute to tumor angiogenesis (red vessels). Tumor cells easily
access the lumen of lymphatic vessels either by active or passive mechanisms, grow inside the lymphatic vessels
and spread through the lymphatic vasculature to draining lymph nodes and from there via the blood or lymphatic
vessels further to distant organs. Blocking the VEGFR-3 signalling pathway by a variety of approaches can be
used to inhibit tumor lymphangiogenesis and lymphatic metastasis (top). Figure modified from (Karpanen &
Alitalo, 2001).
In some studies VEGF-C or VEGF-D were found to contribute also to tumor angiogenesis
(Skobe et al., 2001a; Stacker et al., 2001; Padera et al., 2002), whereas in our study and in
several others VEGF-C had no effect on the density of the tumor blood vessels (Mandriota et
al., 2001; Skobe et al., 2001b). The angiogenic effect of VEGF-C or VEGF-D could be
explained by expression of VEGFR-3 in tumor blood vessels, by the efficient proteolytic
processing of VEGF-C to the mature form able to signal also through VEGFR-2, by efficient
recruitment of macrophages or by stimulation of other stromal cells to secrete additional
angiogenic factors in some tumor models but not in others. We did not detect any significant
expression of VEGFR-3 in the blood vessels of MCF-7 tumors (unpublished observations, see
also Mattila et al., 2002). In addition, most of VEGF-C secreted by the transfected MCF-7
cells in vitro, and by MCF-7 tumors in vivo (Mattila et al., 2002), was only partially
59
processed, thus making the involvement of VEGF-C induced VEGFR-2 signalling in this
tumor model less probable. The increased tumor growth of VEGF-C expressing MCF-7
tumors as compared to control MCF-7 tumors despite the lack of increased blood vessel
density or autocrine growth stimulation could be explained by the increased amount of
functional lymphatic vessels as shown by the Evan’s blue microlymphangiography and thus
better drainage, which might lead to decreased interstitial pressure and better blood perfusion
in the VEGF-C overexpressing tumors.
We did not detect metastasis of the VEGF-C expressing MCF-7 cells, whereas in the study of
Mattila et al. (2002) VEGF-C induced micrometastases of the poorly invasive MCF-7 tumors
were observed in the regional lymph nodes. This can be explained by difference in the
observation times (6-12 weeks in the study by Mattila et al. as compared to 2-3 weeks in our
study), different mouse model (nude vs. SCID mice) and tumor implantation site (5th
mammary fat pads bilaterally in the study by Mattila et al. and 2nd mammary fat pad
unilaterally in our study).
In conclusion, our study demonstrated that VEGF-C strongly induces growth of tumor
associated lymphatic vessels, and suggested that the observed intralymphatic tumor growth
promotes tumor metastasis to regional lymph nodes and perhaps even to more distant organs.
Furthermore, our results indicate that tumor lymphangiogenesis and lymphatic metastasis can
be inhibited by interfering with the ligand induced VEGFR-3 pathway.
3.1
Possible mechanisms of VEGF-C enhanced lymphatic metastasis
Although the metastatic dissemination of tumor cells to regional lymph nodes is a common
feature of many human cancers and has long been used as an important prognostic marker and
an indicator for therapeutic choices, its mechanisms are still poorly understood. It is not clear
whether lymphatic metastasis requires active tumor associated lymphangiogenesis or if the
pre-existing lymphatic vasculature is sufficient for tumor spread. Furthermore, it is not known
whether the entry of malignant cells into the lymphatic vasculature is an active or a passive
process. VEGF-C may promote lymphatic metastasis in several ways.
First, VEGF-C stimulates the growth of new tumor associated lymphatic vessels and the
enlargement of the existing lymphatic channels draining the tumor, thus increasing the contact
area of malignant cells with the lymphatic vasculature (II, Mandriota et al., 2001; Skobe et
al., 2001a; Skobe et al., 2001b; Stacker et al., 2001; Yanai et al., 2001; He et al., 2002;
Padera et al., 2002; He et al., 2005; Kawakami et al., 2005). The observation that the nearby
lymphatic endothelium extensively forms filopodia towards VEGF-C producing tumor cells
suggested that tumor emboli are actively entrapped inside the sprouting lymphatic vessels (He
et al., 2005; reviewed in Alitalo et al., 2005). Nevertheless, a mere contact with the lymphatic
endothelium is not sufficient for metastatic spread, but the tumor cells need to possess some
additional intrinsic invasive abilities to metastasize as was shown by the lack of lymphatic
metastasis despite increased tumor lymphangiogenesis by overexpression of VEGF-C in a
nonmetastatic lung cancer model (He et al., 2002). The fact that inhibition of tumor
metastasis by VEGFR-3-Ig blocks tumor lymphangiogenesis, but does not affect the preexisting lymphatic vessels suggests, that newly formed tumor associated lymphatic vessels
might indeed be necessary for lymphatic metastasis (He et al., 2002; Krishnan et al., 2003; He
et al., 2005; Lin et al., 2005). On the other hand, some studies have shown that
60
lymphangiogenesis is unnecessary and the pre-existing lymphatic vessels are sufficient for
lymph node metastasis (Wong et al., 2005). Thus, VEGF-C induced tumor
lymphangiogenesis and the increase of the lymphatic surface area in contact with tumor cells
can be seen as elimination of one rate-limiting step of the metastatic process.
Second, as VEGF-C is able to induce vascular permeability, it could also promote metastasis
by increasing the interstitial pressure inside the tumor, which might be an important
determinant driving tumor cell seeding both into blood and lymphatic vessels. The increased
number of tumor lymphatic vessels does not necessarily alleviate the increase of interstitial
pressure, especially when most intratumoral lymphatic vessels are thought to be dysfunctional
(Padera et al., 2002; Isaka et al., 2004).
Third, VEGF-C might activate the lymphatic endothelium to facilitate tumor cell entry into
the lymphatic vasculature by promoting molecular interactions between tumor cells and
lymphatic endothelial cells. This could be mediated by secretion of paracrine factors such as
proteases and chemotactic agents by the lymphatic endothelium, which induce detachment,
migration or invasion of tumor cells or alteration of the functional properties of the lymphatic
endothelium to facilitate adhesion and intravasation of malignant cells into lymphatic vessels.
Activation of the lymphatic endothelium via the VEGFR-3 pathway is supported by the
observation that inhibition of VEGFR-3 signalling more effectively suppresses lymph node
and distant metastasis than inhibition of VEGFR-2, although either treatment potently
suppresses tumor lymphangiogenesis, and blockade of VEGFR-2 more effectively inhibits
tumor angiogenesis and growth (Roberts et al., 2006). Furthermore, in several
clinicopathological studies high levels of VEGF-C correlate with lymphatic vessel invasion
and lymph node metastasis, but are not necessarily associated with increased lymphatic vessel
density (Table II). Alternatively, VEGF-C might activate the lymphatic endothelium to
promote survival of malignant cells in the lymphatic system.
Fourth, VEGF-C secreted by the primary tumor might also affect the transport, extravasation
and/or growth of malignant cells in the sentinel lymph nodes. Although metastasis to distant
organs is thought to occur via the blood circulation, it might often require the initial spread of
tumor cells through the lymphatic vessels to lymph nodes, from where they metastasize
further either via the blood or the lymphatic vasculature. This view is supported by the fact
that in some experimental studies VEGF-C promotes metastasis not only to lymph nodes but
also to the lung, and in some cases lung metastasis can also be inhibited by VEGFR-3-Ig
(Krishnan et al., 2003). Furthermore, some studies have reported expression of VEGFR-3 by
the tumor cells, allowing autocrine growth stimulation (Van Trappen et al., 2003; Su et al.,
2006).
Finally, SEMA3F, through binding to NP2 expressed by tumor cells, suppresses tumor
metastasis by inhibiting tumor cell adhesion and chemotaxis (reviewed in Bielenberg et al.,
2006; Guttmann-Raviv et al., 2006). As we have shown in the study discussed above, VEGFC and SEMA3F compete for the same binding site in NP2 (II). Thus it is tempting to
speculate that one mechanism by which VEGF-C could enhance tumor metastasis is to
counteract the inhibitory activity of SEMA3F.
The mechanism by which VEGF-C promotes tumor metastasis remains a crucial issue to be
resolved in order to identify the most potential targets for the development of an effective
therapy for the prevention of tumor spread.
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4. Postnatal maturation renders lymphatic vessels independent of ligand-induced
VEGFR-3 signalling (IV)
Spread of cancer cells from the primary tumor to distant organs is the major cause of
mortality of cancer patients. The primary route of metastasis is through the lymphatic vessels
first to sentinel lymph nodes and then via the lymphatic or the blood vasculature further to
distant organs. In our studies described above we have shown that inhibition of ligandinduced VEGFR-3 signalling by the soluble VEGFR-3-Ig fusion protein effectively blocks
tumor lymphangiogenesis and intralymphatic tumor growth (III). Inhibition of VEGFR-3
signalling by the soluble VEGFR-3 decoy was later shown also to inhibit lymphatic
metastasis (He et al., 2002; He et al., 2005; Lin et al., 2005). Other potential means of
blocking tumor cell dissemination via the lymphatic vasculature by targeting the VEGFC/VEGF-D/VEGFR-3 lymphangiogenic pathway include neutralizing antibodies towards
VEGFR-3 or its ligands (Kubo et al., 2000; Stacker et al., 2001; Pytowski et al., 2005;
Roberts et al., 2006), inhibitory peptides blocking interaction of VEGFR-3 with its ligands,
small molecules inhibiting the tyrosine kinase activity of VEGFR-3 or its downstream
effectors or inhibition of the proteolytic enzymes involved in the processing of VEGF-C and
VEGF-D. To be able to employ the inhibition of VEGF-C/VEGF-D/VEGFR-3 signalling
cascade for the prevention of lymphatic metastasis in cancer patients it is important to know
how inhibition of ligand-induced VEGFR-3 signalling affects the normal lymphatic
vasculature.
To study this we transduced mice of different ages with a recombinant adenovirus encoding
the soluble VEGFR-3-Ig fusion protein. The inhibition of the ligand induced VEGFR-3
pathway lead to regression of lymphatic capillaries and medium sized lymphatic vessels in
mice under two weeks of age, while the collecting lymphatic vessels and the blood
vasculature remained unaffected. If the mice were transduced with the recombinant
adenovirus at the age of two weeks or later, inhibition of ligand-induced VEGFR-3 signalling
had no effect on the lymphatic vasculature. Partial regression of the lymphatic capillaries was
also observed in response to intraperitoneal injections with recombinant VEGFR-3-Ig
proteins and blocking antibodies against VEGFR-3, indicating that the regression was not a
side effect of the adenovirus. These results demonstrate that lymphatic vessels are dependent
on VEGFR-3 until two weeks of age, after which they become independent of ligand-induced
VEGFR-3 signalling. This strongly suggests that lymphatic vessels undergo postnatal
maturation, in a similar manner to what has previously been observed for blood vessels.
In nude mice transduced with the recombinant VEGFR-3-Ig adenovirus at the age of seven
days, the lymphatic capillaries initially regressed, but started to regenerate by the age of four
weeks despite neutralizing levels of VEGFR-3-Ig in the serum. This suggests that a VEGFR-3
ligand-independent mechanism of lymphatic growth must exist after the postnatal period of
two weeks. Factors that could contribute to this VEGF-C/VEGF-D/VEGFR-3 independent
regrowth of lymphatic vessels include VEGF (Nagy et al., 2002; Cursiefen et al., 2004;
Bjorndahl et al., 2005b; Hirakawa et al., 2005), hepatocyte growth factor (HGF, Kajiya et al.,
2005), insulin-like growth factor 1 (IGF-1, Bjorndahl et al., 2005a) and possibly
angiopoietins (Gale et al., 2002; Morisada et al., 2005; Tammela et al., 2005) and fibroblast
growth factor 2 (FGF-2, Kubo et al., 2002; Chang et al., 2004), which have all been shown to
induce lymphangiogenesis, either in a VEGFR-3 independent or dependent manner. Although
62
apparently not required any more, the VEGF-C and VEGF-D induced pathway is still
functional after this stage (Enholm et al., 2001; Karkkainen et al., 2001; Saaristo et al.,
2002b; Rissanen et al., 2003).
Whereas VEGF-C is critically required during embryogenesis (Karkkainen et al., 2004),
VEGF-D appears to be dispensable for lymphatic development (Baldwin et al., 2005). On the
other hand, both VEGF-C and VEGF-D are lymphangiogenic in the adult (Veikkola et al.,
2001). To obtain further insight into the postnatal lymphatic maturation we studied the
relative abilities of these two known VEGFR-3 ligands to induce lymphangiogenesis at
different developmental stages. Interestingly, we did not detect any increase in the number or
area of lymphatic vessels in transgenic mice expressing VEGF-D in the skin under the
keratin-14 promoter at E14.5, at a stage when the VEGF-C transgene expressed under the
same promoter had already induced a clear increase in the number and area of lymphatic
capillaries. The first signs of lymphangiogenesis in the K14-VEGF-D mice were apparent one
day after birth and a week later the lymphatic vessels were even more hyperplastic in the
K14-VEGF-D than in the K14-VEGF-C mice. This suggests that the postnatal lymphatic
vessel maturation is involved with as yet unidentified changes in the lymphatic endothelium
required for the lymphangiogenic activity of VEGF-D. These changes might overlay those
required for the VEGFR-3 ligand independent survival and growth of lymphatic vessels after
the postnatal period.
The molecular nature of the postnatal lymphatic vessel maturation remains an interesting
question to be resolved. A good candidate are signals provided by the ECM through the
integrins. The first clues of the involvement of the ECM in the survival and growth of the
lymphatic endothelium came from the observation that LECs can survive and proliferate
without VEGFR-3 ligands on fibronectin, but not on uncoated tissue culture plates (Makinen
et al., 2001b). Indeed, it was later shown that integrin β1, when in contact with collagen or
fibronectin, can directly interact with and induce the tyrosine phosphorylation of VEGFR-3 as
well as to stimulate migration of endothelial cells to some extent even in the absence of a
cognate ligand (Wang et al., 2001). Integrin α5β1 was shown to significantly participate in the
activation of VEGFR-3 by VEGF-C156S and its downstream PI3 kinase/Akt signalling
pathway, which is essential for fibronectin-mediated LEC survival and proliferation (Zhang et
al., 2005a). Integrin α 9β1 deficient mice die 6-12 days after birth due to chylothorax, which
suggest an underlying function for integrin α9β1 in lymphatic development or function (Huang
et al., 2000). The exact cellular and molecular mechanism of this is unknown, but might start
to be elucidated by the observation that integrin α 9β1 directly binds VEGF-C and VEGF-D,
and that EC adhesion to and migration on VEGF-C and VEGF-D is dependent on integrin
α9β1 (Vlahakis et al., 2005). Furthermore, initial experiments indicate that VEGF-C/VEGF-D
double knockout embryos have a normal blood vasculature and thus do not reproduce the
VEGFR-3 knockout phenotype (Paula Haiko et al., unpublished observation). This suggests,
that VEGFR-3 must have either an additional, as yet unidentified ligand or an alternative
pathway of activation. This unknown mechanism of VEGFR-3 stimulation might be required
for embryonic blood vessel remodelling and later for lymphatic vessel maturation, but can not
substitute for VEGF-C in embryonic development and early postnatal survival of lymphatic
vessels.
63
Postnatal regeneration of lymphatic capillaries has also been observed in mice heterozygous
for Vegfc (Karkkainen et al., 2004), in Chy mice that are heterozygous for an inactivating
mutation in the Vegfr3 gene (Karkkainen et al., 2001), in transgenic mice expressing VEGFR3-Ig in the skin (Makinen et al., 2001a) and in Np2 deficient mice (Yuan et al., 2002). The
cellular origin of postnatal lymphatic regrowth is not known. In the AdVEGFR-3-Ig treated
mice it seems that the lymphatic remnants function as the origin of lymphatic sprouting.
Similarly, the larger lymphatic vessels in several of the above mentioned mouse models could
serve the same function. Other possibilities include transdifferentiation of the venous to
lymphatic endothelial cells or, more unlikely, a de novo differentiation from other cell types
(Maruyama et al., 2005) or mobilization and differentiation of putative lymphatic progenitor
cells (Kerjaschki et al., 2006).
Blood vascular endothelial cells are dependent on VEGF signalling for survival during the
early postnatal period, but this susceptibility is greatly decreased after the fourth postnatal
week (Gerber et al., 1999a; Baffert et al., 2004). Acquisition of PC coverage was thought to
be the major contributor to the loss of VEGF dependency during maturation of blood vessels
(Alon et al., 1995; Benjamin et al., 1998). However, inhibition of VEGF signalling was
shown to induce regression of PC covered, fenestrated blood vessels in several organs in
adults (Kamba et al., 2006), indicating that PCs cannot be the only factor for blood vessel
maturation. It remains to be determined whether the maturation of blood and lymphatic
vessels involves common molecular and cellular mechanisms.
In summary, our results suggest that during the first few weeks after birth the lymphatic
vessels in mice undergo maturation, with changes that render them independent of ligandinduced VEGFR-3 signalling, but on the other hand responsive to VEGF-D. The molecular
mechanism of this lymphatic maturation remains to be elucidated. Importantly, our results
indicate that inhibition of the VEGF-C/VEGF-D/VEGFR-3 pathway should be a safe method
for preventing tumor metastasis in adults, as it does not affect the normal lymphatic
vasculature at this stage. However, it is still important to find out how the inhibition of
VEGFR-3 signalling affects VEGFR-3 positive fenestrated blood vessels of the endocrine
organs or newly formed lymphatic vessels in the adult.
5. Foxc2 regulates the interactions of the lymphatic endothelium with vascular mural
cells and is required for the morphogenesis of lymphatic valves (V)
To get insight into the mechanism of lymphatic vascular failure in lymphedema-distichiasis
(LD), which is caused by inactivating mutations in the FOXC2 gene, and unlike most other
forms of lymphedema is not associated with hypoplasia of the lymphatic vessels, we studied
the lymphatic phenotype of Foxc2 gene targeted mice. Foxc2 deficient mice have previously
been reported to die pre- or perinatally due to cardiac, aortic arch, skeletal and genitourinary
defects (Iida et al., 1997; Winnier et al., 1997). In contrast to recently published observations
(Kriederman et al., 2003), we could not detect any functional or structural defects in the
lymphatic vasculature of adult Foxc2+/- mice, whereas all of them displayed distichiasis, a
double row of eyelashes. This controversy could be explained by variability in the genetic
background, or by differences in the analysis methods. In Foxc2-/- embryos early lymphatic
development seemed to occur normally. But at later stages of embryonic development the
lymphatic vessels of Foxc2 deficient embryos appeared irregularly patterned, were closely
64
associated with an abnormally high amount of PCs/SMCs and had an increased deposition of
the BM protein collagen IV. Additionally, the collecting lymphatic vessels of Foxc2-/embryos lacked lymphatic valves, causing abnormal retrograde flow of a fluorescent tracer
from the collecting lymphatics into the lymphatic capillaries. Furthermore, abnormally
patterned lymphatic vessels with an increased amount of closely associated PCs/SMCs were
also observed in mice double heterozygous for Foxc2 and Vegfr3, suggesting cooperation
between these two pathways. Interestingly, an increased amount of smooth muscle actin
positive perivascular cells was closely associated with the lymphatic capillaries also in the
skin of affected feet, but not in the unaffected arms of human LD patients. This difference
between the lymphatic capillaries in the affected and unaffected areas of LD patients suggests
a substantial contribution by additional factors such as hydrostatic pressure on top of the
genetic predisposition to the onset of lymphedema. These results indicate that Foxc2 is not
required for early lymphatic development, but is essential at later stages by regulating
morphogenesis of lymphatic valves as well as controlling the interactions of lymphatic
endothelial cells with vascular mural cells and deposition of BM components.
The structural abnormalities caused by FOXC2 insufficiency, the abnormal lymphatic
vascular wall structure and valve defects, are likely to underlie the functional failure of
lymphatic vessels in LD patients. As demonstrated with the fluorescent tracer, the lack or
malformation of valves leads to retrograde flow of the lymph from the collecting lymphatic
vessels to lymphatic capillaries. Additionally, the entry of protein rich fluid from the
interstitial space into the lymphatic capillaries might be hindered by the abnormal presence of
vascular mural cells and increased deposition of BM in the lymphatic capillary wall, making
it less permeable. Furthermore, the uneven lumen size and uncoordinated contractility of the
ectopic mural cells might impair lymph flow within the lymphatic capillaries.
During embryonic development we observed Foxc2 expression in all lymphatic vessels,
whereas the skin blood vessels and their surrounding SMCs, in contrast to the endothelium
and SMCs of the big arteries and veins, had no or only little Foxc2 expression. This suggests,
that the observed abnormal patterning of lymphatic vessels associated with increased
recruitment of PCs/SMCs, as well as valve agenesis in the lymphatic vessels of Foxc2-/embryos represent lymphatic endothelial cell autonomous defects and not a failure of
PCs/SMCs. This is further supported by the presence of similar structural abnormalities in the
lymphatic capillaries of Foxc2 heterozygous embryos with diminished Vegfr3 signalling in
lymphatic endothelial cells. In the adult, high expression of Foxc2 persists in the lymphatic
valves but is downregulated in lymphatic capillaries, suggesting a role for Foxc2 in the
maintenance of valve function. Interestingly, the valve regions of colleting lymphatics are
devoid of SMCs.
Our findings demonstrate, that Foxc2 and Vegfr3 cooperate to establish a PC/SMC free
lymphatic capillary network. The fact that Vegfr3 mRNA or protein were not reduced in the
endothelial cells of Foxc2-/- embryos, whereas Foxc2 mRNA was downregulated in the
endocardium of Vegfr3-/- embryos, suggests a model where Foxc2 acts downstream of Vegfr3.
Vegfr3 signalling might also control Foxc2 transcriptional activity in addition to its
expression. Foxc2 seems not to be involved in Vegfr3 mediated lymphatic endothelial cell
proliferation, but rather for negative regulation of SMC recruitment. Interestingly, Vegfr3
expression is downregulated in collecting lymphatic vessels, which contain a SMC layer, and
65
contact with SMCs results in decreased VEGFR-3 expression in cultured endothelial cells
(Saaristo et al., 2002b; Veikkola et al., 2003).
The lymphatic endothelial cells of Foxc2 deficient mice were found to express abnormally
high amounts of Pdgfb and endoglin. Furthermore, in cultured human lymphatic endothelial
cells, overexpression of FOXC2 was shown to downregulate expression of PDGF-B and
collagen IV. This suggests, that FOXC2 inhibits the interactions of lymphatic endothelial
cells with vascular mural cells at least partly by directly or indirectly downregulating the
expression of endoglin and PDGF-B, which have both been implicated in the recruitment
PCs/SMCs to blood vessels, and of collagen IV, which as a BM component might also
regulate adhesion of vascular mural cells. These findings may reveal new targets for
prevention of lymphatic vascular failure in LD.
Abnormal interactions of lymphatic vessels with PCs/SMCs have also been reported in mice
lacking Ang2, in which the collecting lymphatic vessels have poorly associated SMCs (Gale
et al., 2002), as well as in mice expressing an ephrinB2 mutant deficient in PDZ signalling
(Makinen et al., 2005) and in mice lacking ephrinB2 expression in PCs/SMCs (Foo et al.,
2006), in which lymphatic capillaries have ectopic coverage by PCs/SMCs similar to the
lymphatic capillaries in Foxc2 deficient embryos. Whether an interaction between the FOXC2
pathway and ephrinB2 exists remains to be elucidated. Interestingly, it has been shown that
ephrinB can be phosphorylated by activated Tie2 or PDGF receptors (Bruckner et al., 1997;
Adams et al., 1999).
Figure 8: Inactivating mutations in the gene coding for the transcription factor FOXC2 lead to
dysfunctional lymphatic vasculature due to abnormal coverage of lymphatic capillaries by
pericytes/smooth muscle cells and lack of valves in collecting lymphatic vessels of lymphedema-distichiasis
patients. A. In healthy individuals shown at the left lymphatic capillaries (green) lack pericytes, whereas
collecting lymphatic vessels are covered by smooth muscle cells (orange) and contain valves (white). In
lymphedema-distichiasis patients the lymphatic capillaries are ectopically covered by mural cells and the valves
of the collecting lymphatic vessels are absent (shown at right). The blood vessels are shown in red. Drawing by
Paula Saarinen. B and C. Ectopic coverage of LYVE-1 positive lymphatic capillaries (green) by smooth muscle
α-actin positive PCs/SMCs (red) in the skin of Foxc2 deficient (B) but not in wild type (C) mouse embryos.
66
Recently, mouse embryos compound mutant for Foxc2 and the closely related Foxc1 were
reported to exhibit defects in the early sprouting of lymphatic endothelial cells from the
cardinal vein (Seo et al., 2006). This is most likely attributed to a reduced Vegfc expression in
the mesenchymal cells surrounding the cardinal vein, which coexpress these two Foxc
transcription factors. In addition, the Foxc1/Foxc2 compound mutant embryos displayed
arterio-venous malformations and lack of induction of arterial markers, in which process these
Foxc transcription factors were placed upstream of the Notch signalling pathway (Seo et al.,
2006).
Venous insufficiency and incompetent venous valves are a common feature in lymphedemadistichiasis. Recently, mutations in FOXC2 were linked with varicose veins in otherwise
healthy individuals (Ng et al., 2005). As FOXC2 is expressed in venous valves, our results
might prove also to be important in understanding the mechanisms underlying venous
insufficiency, which is a common complication affecting almost half of the adult population.
In conclusion, our results have provided new insights into the mechanisms of lymphatic
development and the morphogenesis of vascular valves (Figure 8). They might also provide
possible new targets for the prevention and rational treatment not only of lymphedemadistichiasis but also of other forms of lymphedema and the more common venous
insufficiency, which traditionally can be treated only by massage, physiotherapy and external
compressions.
67
CONCLUDING REMARKS
Although the structure and main function of the lymphatic vessels was revealed in the
beginning of the last century, only the past decade has brought lymphatic research to the
molecular era. VEGFR-3 was identified as the first lymphatic specific marker, and it was
found to mediate proliferation, migration and survival signals for lymphatic endothelial cells.
The known ligands of VEGFR-3, VEGF-C, which is necessary for formation and
maintenance of lymphatic vasculature during embryogenesis, and VEGF-D, which is not
required during embryonic development, are rather specific inducers of lymphangiogenesis.
The studies presented here shed light on the function of the unique propeptides of VEGF-C,
and identify NP2 as a potential modifier of VEGF-C and VEGF-D signalling through
VEGFR-3. Furthermore, they reveal VEGF-C as a strong inducer of tumor
lymphangiogenesis, and suggest a role for VEGF-C in tumor metastasis, which could be
inhibited by blocking VEGFR-3 signalling. Moreover, ligand induced VEGFR-3 signalling
was found not to be required for the survival of established adult lymphatic vasculature,
suggesting that VEGFR-3 blockage is a safe method for inhibiting lymphatic tumor
metastasis. On the other hand, ligand induced VEGFR-3 signalling was observed to be
necessary for the survival of lymphatic vessels until the postnatal maturation of the lymphatic
vasculature. Interestingly, this maturation is associated with changes that also render the
lymphatic vessels responsive for VEGF-D. Finally, our work places the transcription factor
FOXC2 downstream of VEGFR-3 signalling, and shows that it is necessary for the
morphogenesis of lymphatic valves as well as for inhibition of the interaction of the
lymphatic endothelial cells with vascular mural cells.
These data have produced new insights into the development and pathogenesis of lymphatic
vessels. They have also revealed potential new targets for specific prevention and treatment of
life threatening tumor metastasis and severely disabling lymphedema-distichiasis, two human
diseases involving lymphatic vessels. Furthermore, given the association of FOXC2 with
varicose veins, a common disease affecting the majority of the adult human population, the
possibilities for human therapy become even wider.
Identification of the molecular basis underlying postnatal lymphatic maturation as well as the
nature of the VEGFR-3 ligand independent growth of lymphatic vessels remain a challenge
for lymphatic research of the future. Furthermore, understanding the mechanisms behind
tumor metastasis are crucial for development of effective and targeted treatments for
preventing the spread of tumor cells, the major cause of cancer mortality. The recent
improvements in the methods for isolation and culture of lymphatic endothelial cells, genome
wide transcriptional profiling as well as spatially and temporally controlled gene targeting in
mice greatly facilitate the characterisation of new molecular regulators and modifiers of
lymphatic development.
68
ACKNOWLEDGEMENTS
This work was carried out at the Molecular/Cancer Biology Laboratory, University of Helsinki during the years
1998-2006. I wish to express my gratitude to all the fellow researchers and employees in Haartman Institute and
Biomedicum Helsinki for creating a pleasant working environment, and Eero Saksela and Olli Jänne for
excellent working facilities.
I am most grateful to my supervisor Kari Alitalo for giving me the chance to work in his group of excellent and
dedicated scientists. It has been a privilege to learn and to experience the fascinating world of science at the front
line of lymphatic research. His inexhaustible ideas, countless international connections and interesting research
topics have provided me with opportunities beyond compare.
I am deeply indebted to all my co-authors and collaborators without whom this work would never have been
possible. I especially owe thanks to Tanya for motivating me with her inspiring enthusiasm for science, even at
the most frustrating times, and for all the scientific and nonscientific discussions, which have greatly helped me
over the years. I sincerely thank Caroline, Mikala, Taija, Salla, Maria and Camilla for fruitful collaboration,
stimulating discussions and many joyful moments in and out of work. I will also remember with delight the
sailing lessons with Caroline, hiking with Taija as well as most of the nicknames created by Salla.
I am thankful to Tomi Mäkelä, Aija Kaitera and Terhi Kulmala for the opportunities provided by the Helsinki
Biomedical Graduate School, as well as Jorma Keski-Oja and Petri Salven for thesis follow up and interest in my
work. I also thank Juha Partanen and Marko Salmi for their constructive comments on this manuscript on a very
tight schedule.
I kindly thank all the former and present members of the M/CBL for their help and support, for an enjoyable
atmosphere and for the numerous memorable times in and out of the lab. I would like to express my special
thanks to Pipsa, Pirjo and Paula for helpful discussions, refreshing chats and also for all that friendly teasing. I
also want to thank Michael for keeping my German active and my computer running, Nana for her friendship
since the early times, Birgitta for her never ending enthusiasm and for introducing me to the lab at the start, as
well as Kristiina, Anne and Marko for always believing that this thesis would become a reality one day. Antti,
Jarkko, Juho, Katri, Kostya, Marja, Tuomas and Wolfgang are acknowledged for their friendly attitude and
advice, and Hanna for training me to teach and for doing the practical work while I was writing.
I am deeply grateful to Tapio for his kind help in all the practical matters, Alun, Angela, Kaisa, Mari, Niina,
Paula, Pipsa, Riikka, Sanna and Tanja for their friendly and skilful assistance, which has been invaluable for this
work, Marja-Leena, Mia and Tarja for secretarial help, Seija, Merja and Marjatta for providing clean
instruments, as well as to the animal facility staff for gentle care of the mice. Alun is also greatly acknowledged
for kindly revising the language of this thesis and many other manuscripts.
I wish to express my warmest thanks to Samu for his friendship, help and encouragement, for sharing the lunch
breaks with me and for teaching me to see beauty also in the urban world.
I kindly thank all the people who have taught, tutored and worked with me in Helsinki, in München and in
Innsbruck, especially Thomas for guiding my early steps on a scientific career.
I warmly thank my friends, especially Eeva and Petri, for all the skiing, skating, canoeing, cycling and hiking
tours without which I would never have had the power to carry out this work, as well as Ingvild, Roger, Walter
and Vidar for teaching me the secrets of surviving in the wild. My sincere thanks belong to Tommi for our
shared years, Mine for cheering me up with her positive attitude and Marja, Nina and Taina for always being
there for me ever since our shared years at school and for reminding me that the dreams of youth are still worth
believing in, even at the times when the world seemed to turn upside down.
My heartfelt thanks belong to my mother Terttu, my father Heikki, the best sisters in the world Taija and Tarja
as well as their chosen ones René and Andy for their caring, encouragement and support during my whole life.
I have been financially supported by personal grants from the Ida Montin Foundation, Ella and Georg Ehrnrooth
Foundation, Finnish Cultural Foundation, Finnish-Norwegian Foundation for Medical Research, Emil Aaltonen
Foundation, Finnish Cancer Organizations, Research and Science Foundation of Farmos, Paulo Foundation,
Aarne Koskelo Foundation, Intrumentarium Science Fund, Biomedicum Helsinki Foundation, Finnish
Foundation for Cardiovascular Research and K. Albin Johansson Foundation, which are sincerely
acknowledged.
Helsinki, June 2006
69
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