Originalarbeit
© Schattauer 2012
Microscopic analysis of lymphatic
vessels in primary lymphedematous
skin
X. Wu1; R. Li2; N. Liu1
1Lymphology Center of Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital, Shanghai
Jiao Tong University School of Medicine, Shanghai 200011, China; 2Department of Osthopaedcs, the Second Hospital
of Fuzhou, Fujian 35000, China
Keywords
Microscopic, Lymphatic vessels, Primary lymphedema
Summary
Background: Although skin lymphatic system
is implicated in primary lymphedema, the
morphology and distribution of lymphatic
vessels in the affected skin of the lower extremity of this disease are less described and
understood. Our aim was to characterize the
structural and distributional features of lymphatic vessels in the affected skin of the lower
extremity of primary lymphedema patients
and pinpoint their role in this disease.
Methods: Skin biopsies of lower limb of 14 patients with primary lymphedema and 10 agematched controls were performed for immunohistochemistry microscopic studies using
podoplanin (a lymphatic marker) antibodies.
All lymphatic vessels present in each section
were counted and inner luminal diameter was
measured by software.
Results: The total density and inner luminal diameter of lymphatic vessels in the affected
skin of the lower extremity of primary lymphedema patients were greater compared with
controls (p=0.004, p=0.014, respectively).
However, in the superficial 300 μm band of
dermis, the density of lymphatic vessels in primary lymphedema was lower with respect to
controls (p=0.00). In contrast, in deep dermis
Korrespondenzadresse
Ningfei Liu
Lymphology Center of the Department of Plastic and
Reconstructive Surgery Shanghai 9th People’s Hospital
Shanghai Jiao Tong University School of Medicine
Shanghai, 200011, China
Tel. +86–21–23271699 X 5734
Fax +86–21–53078128
E-Mail: [email protected]
(>300 μm), the density of lymphatic vessels in
primary lymphedema was significantly higher
compared with controls (p=0.00).
Conclusion: The affected skin of the lower extremity of primary lymphedema patients is
characterized by hyperplasia and dilation of
lymphatic vasculature mainly located in deep
dermis, which might have a pathogenic role in
the evolution and in the clinical manifestations
of the disease.
Schlüsselwörter
mikroskopisch, lymphatische Gefäße, primäres
Lymphödem
Zusammenfassung
Hintergrund: Obwohl das Lymphsystem der
Haut bei den primären Lymphödemen einbezogen ist, werden die Morphologie und die Anordnung der Lymphgefäße in der betroffenen
Hautregion der unteren Extremität bei dieser
Krankheit wenig beschrieben und verstanden.
Unser Ziel war es, die strukturellen und räumlichen Eigenschaften der Lymphgefäße in der betroffenen Hautregion der unteren Extremität
bei Patienten mit primären Lymphödemen zu
charakterisieren und ihre Rolle bei dieser
Krankheit herauszuarbeiten.
Methoden: Es wurden Hautbiopsien der unteren Extremität von 14 Patienten mit primärem
Lymphödem sowie von 10 altersmäßig ent-
sprechenden Kontrollen entnommen und immunhistochemisch sowie mikroskopischeuntersucht unter Verwendung von podoplanin-Antikörpern (einem lymphatischen Marker). Die Lymphgefäße jedes Abschnittes wurden gezählt und der innere luminale Durchmesser bestimmt.
Ergebnisse: Die Gesamtdichte und der innere
luminale Durchmesser der Lymphgefäße in
der betroffenen Haut der untereren Extremität der Patienten mit primärem Lymphödem waren verglichen mit Kontrollen größer
(p=0,004 bzw. p=0,014 ). Im oberen Bereich
der Lederhaut bis 300 μm war die Dichte der
Lymphgefäße der Lymphödem-Patienten
niedriger im Vergleich zu den Kontrollen
(p=0,00). Demgegenüber war die Dichte der
Lymphgefäße in der tiefen Lederhaut
(>300 μm) bei primärem Lymphödem höher
als bei den Kontrollen (p=0,00).
Zusammenfassung: Die betroffene Haut der
unteren Extremität der Primärlymphödem-Patienten wird gekennzeichnet durch die Hyperplasie und Dilatation von Lymphvasculaturen
hauptsächlich in der tiefen Lederhaut, was auf
eine pathogene Rolle bei der Entstehung und
der klinischen Manifestation der Krankheit
hindeuten könnte.
Mikroskopische Analyse von Lymphgefäßen in primär lymphödematöser Haut
Phlebologie 2012; 41: 13–17
received: July 24, 2011
accepted: January 20, 2012
Phlebologie 1/2012
Downloaded from www.phlebologieonline.de on 2017-06-16 | IP: 88.99.165.207
For personal or educational use only. No other uses without permission. All rights reserved.
13
14
X. Wu: Microscopic analysis of lymphatic vessels in primary lymphedematous skin
Lymphedema is caused by impairment of the
lymphatic vessels and insufficient lymphatic
function, leading to abnormal accumulation
of protein-rich fluids and subsequent chronic
inflammation and definitive fibrosis (1).
Lymphedema is classified into a primary and
a secondary form according to the underlying causes. In contrast to secondary lymphedema that develops after lymphatic failure due to trauma, surgery, radiotherapy or
parasite infection, primary lymphedema is
associated with developmental abnormalities of the lymphatic system that result
from genetic mutations. To date, several
underlying genes including VEGFR3,
FOXC2, SOX18, CCBE1, NEMO, GJC2 (2–7)
have been identified and linked to different
phenotypes of primary lymphedema, providing valuable insight into the molecular
mechanisms regulating the development
and function of the lymphatic vasculature.
Clinically, primary lymphedema, most
often affecting the lower limbs (8), is a disfiguring and disabling disease. The diagnosis of lymphedema is currently dependent on medical history and physical examination. Although treatments including
physiotherapy, compression garments, liposuction, and occasionally surgery (9, 10)
could reduce the swelling, no curable treatment for primary lymphedema is available.
A transport failure of the cutaneous
lymphatic vessels due to gene mutations
causes primary lymphedema, accompanied by thickening of the skin, deposition of adipose tissue, and dermal fibrosis
of the affected limbs. The investigation of
lymphatic vessels has been long hampered
by the lack of lymphatic endothelial cell
markers.
The discovery of podoplanin (11), a most selective marker of skin lymphatic vessels (12,
13), has provided possibility for histological
study of skin lymphatic vessels.
Control
(n=10)
Age (years), range
26 (11–54)
27 (13–62)
Male/female
7/7
6/4
Sampling region:
Left/right lower limb
4/10
5/5
Control
a
Total vessel density
Inner luminal diameter
7.5 (5–14)
b
Superficial 300 μm dermis
Deep dermis (>300 μm)
a Vessels/mm2,
mm2;
Tab. 1
Summary of clinical
data.
Morphometric data of lymphatic vessels.
Measure
d
16.51 (12.34–21.88)
c
Materials and methods
Patients
In the present study we evaluated the distribution and structure of lymphatic vessels
identified with podoplanin in the affected
skin of the lower extremity of primary
lymphedema patients compared to controls. The aim was to characterize the structural and distributional features of lymphatic vessels in the affected skin of the
lower extremity of primary lymphedema
patients and pinpoint their role in the disease.
Studies on skin lymphatic vessels of patients affected by primary lymphedema are
few. Previously, indirect lymphography
with Iotasul demonstrated hyperplasia of
the dermal precollectors present in lymphedema praecox and lymphedema tardum
(14). Recently, studies using fluorescent
Primary lymphedema
(n=14)
Tab. 2
microlymphangiography (FML) showed
more extensive dye-filled lymphatic capillary network and microlymphatic hypertension and higher filling velocity in primary lymphedema compared with normals (15). Despite these evidences, to date,
there are no microscopic studies on lymphatic vessels in primary lymphedematous
skin which includes lymphatic capillaries
and precollectors (16).
14(7–20)
3.5 (2–10)
b μm;
Lymphedema
P
14.5 (6–21)
0.004
20.68 (16.89–33.84)
0.014
2.0 (0–7)
0
20 (14–33)
0
Median (minimum to maximum),
Median (minimum to maximum), c Vessels/
d
2
Median (minimum to maximum), Vessels/mm ; Median (minimum to maximum)
Biopsies of affected skin were obtained
from the lower limbs of 14 patients affected
by primary lymphedema diagnosed by a
clinician experienced in the field. All patients were staged III based on the International Society of Lymphology (ISL) staging system (17). The main clinical data are
summarized in 씰Table 1. Site-matched
normal skin samples were obtained from
10 sex- and age-matched individuals with
no clinical evidence of lymphedema or history of edema. The study was approved by
Shanghai 9th people’s Hospital Ethics
Committee. Each participant gave informed, written consent.
Immunohistochemistry
Samples were fixed in 10 % formalin and
embedded in paraffin. Before immunohistochemical labeling, sections were rehydrated via xylene and ethanol and placed
for 30 minutes in 0.1 % Trypsin Solution
(Sigma, St. Louis, MO, USA) for antigen retrieval. Endogenous peroxidase activity
was quenched with 3 % H2O2 for 10 minutes in the dark, and unspecific binding
sites were blocked for 30 minutes with
phosphate buffered saline containing 10 %
goat serum. Labeling was performed by the
use of the mouse anti-human monoclonal
antibody podoplanin (Angiobio). Lymphatic vessels were stained by incubating
sections overnight at 4°-refrigerator with
podoplanin diluted 1:100 in phosphatebuffered saline containing 0.5 % bovine
serum albumin (hereafter referred to as
buffer), followed by 30-minute incubation
Phlebologie 1/2012
© Schattauer 2012
Downloaded from www.phlebologieonline.de on 2017-06-16 | IP: 88.99.165.207
For personal or educational use only. No other uses without permission. All rights reserved.
X. Wu: Microscopic analysis of lymphatic vessels in primary lymphedematous skin
with HRP-goat anti-mouse IgG (H+L) secondary antibody (DAKO) diluted 1:50 in
buffer. The reaction was revealed with
3,3′-diaminobenzidine Substrate Kit according to manufacturer’s instructions
(VECTOR). The slides were viewed and
images were captured using light microscope (Nikon Japan).
Morphometric analysis
The total area of dermis was obtained from
the total section area computed with a 20×
objective after subtracting the area occupied by hypodermic fat. The whole area of
each section was then examined with a 20×
objective and all the lymphatic (stained in
brown by podoplanin) vessels in each section were counted. Total area and number,
and inner luminal diameter were measured
using the morphometric software “ImagePro Plus (Media Cybernetics USA)” by
Nikon. Vessel density was evaluated as the
total number of lymphatic vessels in the
total cross-sectional area of the dermis. Podoplanin positive lymphatic vessels were
counted in the superficial 300 μm band of
dermis and deep dermis outside 300 μm
(<300 μm, >300 μm, etc.) and the density
in each band was calculated.
Statistical analysis
Data are expressed as the median (minimum to maximum). The statistical significance of the difference between each group
was evaluated using the Wilcoxon test, and
the comparison between groups was analyzed by the Wilcoxon test statistical analysis software (SPSS 16.0, SPSS Inc. South
Wacker Drive, Chicago USA). P <0.05 was
considered statistically significant.
Results
Fig. 1
Histological sections
of skin stained with
antibodies to podoplanin. (a) Control
limb. (b) Primary
lymphedema limb.
(c) The deep part of
affected skin of primary lymphedema
foot. Lymphatic
vessels, indicated by
arrows, are positive
for podoplanin
(brown).
brosis, elongation of the dermal papillae,
irregularity of the epidermal/dermal junction (씰Fig. 1).
Immunohistochemistry
General morphology
Under light microscopy, there were many
tissue changes in the affected skin of the
lower extremity of primary lymphedema
patients with respect to controls: increased
thickness of the dermis with pronouced fri-
Lymphatic vessels were consistently and intensely stained by podoplanin antibody in the
affected skin of primary lymphedema patients and controls. Cross-reactivity was only
found in some epithelial cells. Most of the
lymphatic vessels had a patent lumen delin-
eated by a tortuous and irregular profile. In
addition, lymphatic vessels were composed of
a single layer of endothelial cells which were
stained in dark brown by the immunohistochemical reaction (씰Fig. 1).
Morphometric evaluation
In primary lymphedema, the total density
and inner luminal diameter of lymphatic
vessels were significantly higher than in
controls (p=0.004 vs p=0.014, 씰Tab. 2). In
© Schattauer 2012
Phlebologie 1/2012
Downloaded from www.phlebologieonline.de on 2017-06-16 | IP: 88.99.165.207
For personal or educational use only. No other uses without permission. All rights reserved.
15
16
X. Wu: Microscopic analysis of lymphatic vessels in primary lymphedematous skin
Fig. 2 Lymphatic vessel densities, inner luminal diameter, and distribution in lower limb skin in controls and patients with primary lymphedema. (a) Vessels total density. (b) Inner luminal diameter of lymphatic vessels. (c) Vessel density in the superficial 300 μm band of dermis. (d) Vessel density in the deep
dermis (>300 μm). The medians are represented by horizontal lines. Lymphatic densities and diameter
in the primary lymphedema group were significantly increased relative to the control group. In the
superficial dermis, the lymphatic density was significantly higher in primary lymphedema with respect
to controls, whereas in deep dermis (>300 μm) the lymphatic density in the primary lymphedema group
tended to be lower than in controls. For statistical tests, see Table 2.
the superficial 300 μm band of dermis, the
density of lymphatic vessels in primary
lymphedema was lower with respect to
controls. In contrast, in deep dermis
(>300 μm), the density of lymphatic vessels
in primary lymphedema was significantly
higher compared with controls (p=0.00).
Podoplanin positive lymphatic vessels were
seen in the whole dermis with the highest
density in the most superficial 300 μm
band of dermis in controls, whereas lymphatic vessels were mainly located in deep
dermis (>300 μm) in primary lymphedema
(p=0.00, 씰Fig. 2).
Discussion
Although studies of the last decades have
revealed the importance of lymphatic
vessels for the pathogenesis of primary
lymphedema, only microlymphography
and indirect lymphography studies focuses on lymphatic vessels in the affected
skin of the lower extremity of this disease
(14, 15).
The present immunohistochemical
study is, to the best of our knowledge, the
first detailed morphometric analysis of
lymphatic vessels in the limb skin of patients affected by primary lymphedema.
Our results showed that the morphology
and distribution of lymphatic vessels in the
skin lesions of primary lymphedema patients differ from that in the normal skin.
Firstly, skin remodeling was evident in the
affected skin of primary lymphedema patients in comparison to controls. Increased
dermis thickness and fibrosis were observed in primary lymphedema. The dermal/epidermal junction became irregular
and the dermal papillae elongated.
Secondly, a significant increase (data not
shown) of the inner luminal diameter of
lymphatic vessels and total density was observed in the dermis of primary lymphedema patients compared with controls. Since
skin lymphatic vessels tend to be collapsed
flat, dilation of lymphatic vessels in the
present study could be due to compensa-
tory mechanism for the reduction in
number of functional lymphatic vessels.
In addition, enlarged lymphatic vessels
may also suggest the presence of a certain
degree of lymphostasis possibly as a result
of the overall reduced capacity of lymph
drainage caused by the dysfunctional lymphatic vessels. Increased total lymphatic
density in the skin of primary lymphedema
patients indicated that lymphangiogenesis
might occur despite the mutated genes that
controlled the lymphatic development.
Possible explanations behind lymphangiogenesis in the affected skin of primary
lymphedema patients include:
● The underlying gene did not have an adverse impact on proliferation and budding of lymphatic endothelial cells.
● Chronic lymphedema is usually accompanied by inflammation that stimulates
lymphangiogenesis (18, 19).
These findings indicated such phenotype
of primary lymphedema is, to some extent,
ascribed to a profound functional failure of
initial lymphatics rather than hypoplasia of
it. Several potential mechanisms might be
responsible for dysfunction of lymphatic
vessels. For example, microlymphatic hypertension might have an adverse impact
on the capacity for fluid or cellular uptake
of lymphatic capillaries (15).
In addition, it is not excluded that during progression of lymphedema, chronic fibrosis can either directly or indirectly damage anchoring filaments which regulate the
absorption capacity of lymphatic capillaries (20).
Finally, fibrosis might contribute to
lymphatic dysfunction by promoting a direct lymphatic endothelial cell-mesenchymal cell transdifferentiation (21). Further
studies are required to clarify the mechanism of dysfunction.
Interestingly, an increase in lymphatic
density in the primary lymphedema foot
was most evident in the deep dermis
(>300 μm), whereas density in the superficial 300 μm band of dermis was significantly reduced. Several possible causes
could account for this scenario. Firstly,
lymph vessels in dermal papillae might be
occluded from the outside by tough, fibrotic connective tissues. Whereas abundant expression of VEGFC by skin append-
Phlebologie 1/2012
© Schattauer 2012
Downloaded from www.phlebologieonline.de on 2017-06-16 | IP: 88.99.165.207
For personal or educational use only. No other uses without permission. All rights reserved.
X. Wu: Microscopic analysis of lymphatic vessels in primary lymphedematous skin
ages that could be preserved during dermal
fibrosis (22, 23), might be responsible for
lymphangiogenesis in deep dermis. Additionally, dermal fibrosis might move lymphatic vessels down during the progression
of lymphedema.
Regardless of the mechanism, the dysfunction and abnormal distribution of
lymphatic vessels could have a pathogenic
role in the evolution of the disease. A transport failure of the cutaneous lymphatic
vessels leads to stagnation of protein-rich
fluids and macromolecules in the interstitial space, resulting in edema at first and
subsequent skin remodeling that leads to
redistribution of skin lymphatic vessels.
Conclusion
We showed, for the first time, that histollogically abnormal morphology and distribution of lymphatic vessels are characteristic for the affected skin of primary
lymphedema. This observation could facilitate a better understanding of the pathogenic mechanisms of the disease. In addition, our findings also provide a new guideline for modulating primary lymphedema
and have potential prognostic implications
as patients with enlarged lymphatic capillaries may respond better to complex physical therapy than those with aplasia of
micro-lymphatic vessels (24).
Acknowledgements
The study is funded by the Shanghai
Science and Technology Committee
(grant no. 09410706400, 10411964100,
10440711000).
References
1. Rockson SG. Lymphedema. Am J Med 2001; 110:
288–295.
2. Verstraeten VL, Holnthoner W, van Steensel MA et
al. Functional analysis of FLT4 mutations associated
with Nonne-Milroy lymphedema. J Invest Dermatol 2009; 129: 509–512.
3. Petrova TV, Karpanen T, Norrmén C et al. Defective
valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med 2004; 10: 974–981.
4. Irrthum A, Devriendt K, Chitayat D et al. Mutations
in the transcription factor gene SOX18 underlie recessive and dominant forms of hypotrichosislymphedema-telangiectasia. Am J Hum Genet
2003; 72: 1470–1478.
5. Alders M, Hogan BM, Gjini E et al. Mutations in
CCBE1 cause generalized lymph vessel dysplasia in
humans. Nat Genet 2009; 41: 1272–1274.
6. Saban MR, Mémet S, Jackson DG et al. Visualization of lymphatic vessels through NF-kappaB activity. Blood 2004; 104: 3228–3230.
7. Ferrell RE, Baty CJ, Kimak MA et al. GJC2 missense
mutations cause human lymphedema. Am J Hum
Genet. 2010; 86: 943–948.
8. Schook CC, Mulliken JB, Fishman SJ et al. Primary
lymphedema: clinical features and management in
138 pediatric patients. Plast Reconstr Surg 2011;
127: 2419–2131.
9. Radhakrishnan K, Rockson SG. The clinical spectrum of lymphatic disease. Ann N Y Acad Sci 2008;
1131: 155–184.
10. Warren AG, Brorson H, Borud LJ, Slavin SA. Lymphedema: a comprehensive review. Ann Plast Surg
2007; 59: 464–472.
11. Breiteneder-Geleff S, Soleiman A et al. Angiosarcomas express mixed endothelial phenotypes of blood
and lymphatic capillaries: podoplanin as a specific
marker for lymphatic endothelium. Am J Pathol
1999; 154: 385–394.
12. Baluk P, Fuxe J, Hashizume H et al. Functionally
specialized junctions between endothelial cells of
lymphatic vessels. J Exp Med 2007; 204: 2349–2362.
13. Kahn HJ, Marks A. A new monoclonal antibody,
D2–40, for detection of lymphatic invasion in primary tumors. Lab Invest 2002; 82: 1255–1257.
14. Partsch H, Urbanek A, Wenzel-Hora B. The dermal
lymphatics in lymphoedema visualized by indirect
lymphography. Br J Dermatol 1984; 110: 431–438.
15. Bollinger A, Amann-Vesti BR. Fluorescence microlymphography: diagnostic potential in lymphedema and basis for the measurement of lymphatic
pressure and flow velocity. Lymphology 2007; 40:
52–62.
16. Berens V, Rautenfeld D, Lubach I. New techniques
of demonstrating lymph vessels in skin biopsy
specimens and intact skin with the scanning electron microscope. Ariii DenmUol Kes 1987; 279:
J27–34.
17. The diagnosis and treatment of peripheral lymphedema. 2009 Consensus document of the International Society of Lymphology. Lymphology 2009;
42: 51–60.
18. Xu H, Edwards J, Banerji S, Prevo R, Jackson DG,
Athanasou NA. Distribution of lymphatic vessels in
normal and arthritic human synovial tissues. Ann
Rheum Dis 2003; 62: 1227–1229.
19. Kajiya K, Detmar M. An important role of lymphatic vessels in the control of UVB-induced edema
formation and inflammation. J Invest Dermatol
2006; 126: 920–922.
20. Suami H, Pan WR, Taylor GI. Changes in the lymph
structure of the upper limb after axillary dissection:
radiographic and anatomical study in a human cadaver. Plast Reconstr Surg 2007; 120: 982–991.
21. Clavin NW, Avraham T, Fernandez J et al. TGFbeta1 is a negative regulator of lymphatic regeneration during wound repair. Am J Physiol Heart Circ
Physiol 2008; 295: H2113–2127.
22. Ibba-Manneschi L. Niissalo S. Milia A.F. Variation
of neuronal nitric oxide synthase in systemic sclerosis skin. Arthritis Rheum 2006; 54: 202–213.
23. Rossi A, Sozio F, Sestini P et al. Lymphatic and blood
vessels in scleroderma skin, a morphometric analysis. D Hum Pathol 2010; 41: 366–374.
24. Foldi M, E Foldi. Die komplexe physikalische Entstauungs-Therapie des Lymphödems. Phlebol
Proktol 1984; 13: 79.
© Schattauer 2012
Phlebologie 1/2012
Downloaded from www.phlebologieonline.de on 2017-06-16 | IP: 88.99.165.207
For personal or educational use only. No other uses without permission. All rights reserved.
17