Expression pattern of VEGFR-1, -2, -3 and D2-40 protein in the skin of patients with systemic sclerosis

ARTICLE

ejd.2011.1284

Auteur(s) : Nobuyo HIGASHI-KUWATA, Takamitsu MAKINO, Yuji INOUE, Hironobu IHN ihn-der@kumamoto-u.ac

Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

Reprints.: H. IHN

Systemic sclerosis (SSc) is a generalized connective tissue disorder [1-3] characterized by vascular signs and symptoms (e.g., Raynaud's phenomenon, fingertip ulcers and gangrene) due to endothelial damage [4, 5]. Vascular changes precede the development of skin fibrosis and lead to vessel wall intimal proliferation and obliteration, and decreased capillary density due to both inflammatory/immune processes and ischemia-reperfusion damage [6, 7]. It has been reported that despite the reduced capillary density, there is a lack of new vessel formation in the skin of patients with SSc [8].

Recent studies have provided evidence that the formation of new vessels in postnatal life does not result solely from the sprouting of pre-existing vessels (angiogenesis) but also involves the recruitment of bone marrow-derived progenitors that are the precursors of endothelial cells (vasculogenesis) [9, 10]. As such, there have been many studies examining angiogenesis [11, 12] and vasculogenesis [13, 14] in SSc. However, detailed in situ examination of microcirculatory changes including the lymphatic circulation in the skin of patients with SSc has not been performed [15].

Among the angiogenic growth factors, vascular endothelial growth factor (VEGF) has been identified as a key mediator of angiogenesis [16]. VEGF induces differentiation, proliferation and migration of endothelial cells that contribute to the formation of vessels through both angiogenesis and vascular remodelling. VEGF exerts its biological functions by binding to the tyrosine kinase receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and VEGFR-3 (Flt-4) [17, 18]. In addition, it has been reported that circulating endothelial cells, which are thought to contribute to vasculogenesis, also express VEGFR-2 [19].

The blood vascular and the lymphatic system play complementary roles in tissue perfusion and fluid reabsorption. Despite its critical role in mediating tissue fluid homeostasis and the immune response, the lymphatic system has received less attention than the vascular system in research into SSc. Developmentally, it is known that lymphatic vessels arise by sprouting from veins, suggesting a close tie between the two systems [20].

Therefore, the aim of our study was to examine the expression patterns of VEGFR-1, -2, -3 and the lymphatic endothelial cell marker D2-40 in the skin of patients with SSc.

Patients and methods

Patients

The study was approved by the Ethics Committee of Kumamoto University. Nine patients with systemic sclerosis (SSc) with a mean age of 60.3 ± 9.4 years (mean ± SD) were recruited and subdivided into those with diffuse cutaneous disease (dcSSc) and limited cutaneous disease (lcSSc) as defined by the LeRoy criteria. Four (two males and two females) patients had dcSSc while the others (one male and four females) had lcSSc. Disease duration varied from 4 to 108 months (table 1). Patients were from southern Japan and visited the dermatology outpatient clinic of Kumamoto University Hospital. Skin biopsy specimens were obtained from the forearm of patients with written informed consent. Biopsy specimens of normal skin from six adults were used as controls.

Table 1 Summary of patients.

Antibodies

The following antibodies were used: anti-VEGFR-1 (dilution 1:100, rabbit anti-human IgG, Abcam, Cambridge, UK), anti-VEGFR-2 (dilution 1:100, mouse anti-human IgG1, clone A-3, Santa Cruz Biotechnology, CA, USA), anti-VEGFR-3 (dilution 1:100, mouse anti-human IgG, clone C-20, Santa Cruz Biotechnology), anti-D2-40 (mouse anti-human IgG1, Nichirei Bioscience, Tokyo, Japan), anti-CD68 (dilution 1:300; mouse anti-human IgG1, clone KP1; DakoCytomation, Carpinteria, CA) and anti-Von Willebrand Factor (dilution 1:50, mouse anti-human IgG, clone F8/86, DakoCytomation, Glostrup, Denmark).

Immunohistochemistry

Skin biopsy samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Four-micrometre sections were prepared. After sections were deparaffinized in xylene and rehydrated in a graded ethanol series, antigens were retrieved by incubation with trypsin (Invitrogen, NY, USA) for 30 min for the detection of CD68. For the detection of VEGFR-1, VEGER-2, VEGFR-3 and D2-40, antigen retrieval was performed by incubation with citrate buffer at pH 6 for 5 min in a microwave oven. Endogenous peroxidase activity was inhibited, after which sections were incubated with 5% normal goat serum for 20 min and then reacted with the antibodies (anti CD68, VEGFR-1, VEGFR-2, VEGFR-3 or D2-40 antibody) at 4 °C for 6 hr. Excess antibody was washed out with phosphate-buffered saline and samples were incubated with horseradish peroxidase-labelled goat anti-mouse antibody (Nichirei) for 60 min. The reaction was visualized using the 3,3′-diaminobenzidine substrate system (Dojin, Kumamoto, Japan). Slides were lightly counterstained with Mayer's haematoxylin and examined under a light microscope (Olympus BX50, Tokyo, Japan).

Quantitative analysis of staining

The staining intensity was graded as follows: no staining (-), weak staining (+/-), distinct staining (+), intense staining (++), very intense staining (++ +). Based on the graded intensity, the staining was scored as follows: 0 for -, 1 for +/-, 2 for +, 3 for ++ and 4 for ++ + . We also measured the area of lymphatic lumen using the WinROOF image processing software (Mitani Corp., Tokyo, Japan) for Windows.

Statistical analysis

Statistical analysis was carried out using the Mann-Whitney U test or Wilcoxon signed-ranks test for comparison of means. The Spearman's rank correlation coefficient was used to examine the relationship between two continuous variables. P-values < 0.05 were considered statistically significant. All data are expressed as mean ± standard deviation (SD).

Results

Qualitative analysis of VEGFR-1, -2, -3 and D2-40 staining

VEGFR-1

Staining for VEGFR-1 was negative in all patients and controls tested in this study. It was not possible to analyse these results statistically. The VEGFR-1 antibody was recommended by the manufacturer for use on formaldehyde-fixed/paraffin wax-embedded tissue, and antigen retrieval methods did not result in any improvement in the staining. However, positive sample controls (placenta, data not shown) confirmed the sensitivity of the staining.

VEGFR-2

Weak to intense staining of VEGFR-2 was found in capillaries adjacent to epithelia in all nine patients (figure 1A). Capillaries not adjacent to epithelia and larger vessels – such as arterioles, venules, arteries and veins – were negative or only weakly stained for VEGFR-2. Furthermore, non-vascular VEGFR-2 staining was observed in several cells in six of nine patients, some of which were confirmed to express CD68 protein by using the technique of serial sectioning (figure 2). In controls, only a few endothelial cells were positive for VEGFR-2 and the other cells were negative (figure 1B).

VEGFR-3

Similar to VEGFR-2, larger vessels such as arterioles, venules, arteries and veins were negative. Weak to intense staining of VEGFR-3 was found in capillaries adjacent to epithelia and lymphatic vessels situated around blood vessels in three of nine patients (figure 1C). In controls, only a few endothelial cells were positive for VEGFR-3 and the other cells were negative (figure 1D).

D2-40

Very intense staining of D2-40 was found only in lymphatic endothelial cells in all nine patients and controls as well (figure 3). An example of the VEGFR and D2-40 staining pattern in a skin sample from a patient with lcSSc and control is shown in table 2.

Table 2 An example of the staining pattern of VEGFR and D2-40 in a patient with systemic sclerosis and normal control.

Quantitative analysis of VEGFR-2, -3 and D2-40 staining

VEGFR-2 and VEGFR-3

There was significantly greater staining intensity of VEGFR-2 in capillaries in the papillary layer in each dcSSc group (1.8 ± 0.9), lcSSc group (2.4 ± 0.5), and combined SSc (dcSSc and lcSSc) group (2.1 ± 0.7) compared with that in controls (0.5 ± 0.5) (P < 0.05) (table 3). The intensity of VEGFR-3 staining of capillaries in the papillary layer did not show a significant difference compared with that in controls (data not shown). There was also a significantly greater intensity of VEGFR-3 staining in lymphatic vessels in the dcSSc group (1.1 ± 0.6), lcSSc group (1.2 ± 0.5), and combined SSc group (1.1 ± 0.8) compared with that in controls (0.25 ± 0.5) (P < 0.05) (table 3). There was no significant difference between the dcSSc and lcSSc groups in regards to VEGFR-2 and VEGFR-3 staining (data not shown).

Table 3 Result of the immunohistochemical staining scores of capillaries in the papillary layer (VEGFR-2) and lymphatics (VEGFR-3 and D2-40).

Data are expressed as mean ± SD, P < 0.05.

D2-40

The intensity of D2-40 staining of lymphatic vessels in the SSc group did not show a significant difference compared with that in controls (table 3). Lymphatic vessels were differentiated from blood vessels by staining with anti-human Von Willebrand Factor antibody (data not shown).

The area of lymphatic lumen per 1,000 μm² in SSc patients was significantly greater in the dcSSc group (20.2 ± 1.4 μm²), lcSSc group (35.7 ± 2.5 μm²) and combined SSc group (22.5 ± 1.5 μm²) compared to that in healthy controls (4.9 ± 1.2 μm², P < 0.05) (figure 4). However, there was no significant difference between the dcSSc group (20.2 ± 1.4 μm²) and lcSSc group (35.7 ± 2.5 μm²).

Discussion

Our study revealed three major findings. The intensity of VEGFR-2 and VEGFR-3 staining in the skin of patients with dcSSc or lcSSc was significantly higher than that in healthy controls. Non-vascular VEGFR-2 staining was observed in some mononuclear cells in the skin of patients with dcSSc or lcSSc. The lumen area of lymphatic vessels in the skin of patients with either dcSSc or lcSSc was significantly larger than that in healthy controls.

In earlier studies, expression of VEGFR-1 and VEGFR-2 was found to be upregulated in endothelial cells of SSc patients [15, 16]. Increased VEGFR-3 expression in lymphatic vessels and ectopically expressed VEGFR-3 in blood vessels has been reported as well [17].Our results regarding increased vascular VEGFR-2 and lymphatic VEGFR3 expression are in agreement with those from other studies. However, it was not possible to detect the expression of VEGFR-1 in our study. Furthermore, the expression of VEGFR-2 in the skin of patients with SSc was not restricted to endothelial cells and non-vascular VEGFR-2 staining was clearly observed. Regarding the discrepancy of the VEGF-R1 staining, it might be caused by the difference of the antibody source and staining method we used. Each anti-VEGFR-1 rabbit antibody purchased from Santa Cruz Biotechnology [15] or DakoCytomation [16] was used in previous studies whereas anti-VEGFR-1 rabbit antibody was purchased from Abcam and used in the present study. Moreover, TechMate Horizon staining robot programmed for the biotin-streptavidin protocol was utilized for staining in one of previous studies [15]. On the contrary, we performed a standard manual staining as described in the materials and methods.

The intensity of D2-40 expression on lymphatic vessels was equally high in both SSc and controls, and was not significantly different between the two groups, whereas the lumen area of lymphatic vessels per 1,000 μm² in SSc patients was significantly greater than that of controls. As we have previously reported [21] and we also confirmed in the specimens (data not shown), the number of lymphatic vessels was reduced in the skin of SSc patients. Therefore the dilation of lymphatic vessels might be interpreted as a compensatory mechanism for the reduction in their number. The contribution of the endothelin/NO system on SSc vascular pathogenesis has been reported [22] and it has been also reported that endothelin-1 directly stimulates lymphatic endothelial cells [23].

However, the exact cellular mechanism of lymphatic endothelial cells for vessel dilation in SSc patients has not been addressed. Although there was no significant difference in the intensity of D2-40 staining between patients with SSc and controls, it was slightly higher than that in the control group. Moreover, the expression of VEGFR-3 – a member of a receptor tyrosine kinase family specific for lymphatic endothelial cells – was significantly higher in the skin of patients with SSc, indicating a reactive change on lymphatic vessels in the skin of patients with SSc. The vascular and lymphatic systems play complementary roles in tissue perfusion and fluid reabsorption. We speculate that a disturbance in tissue fluid homeostasis and immune response caused by dysregulation of the lymphatic system may influence the uncontrolled expression of VEGF and its receptors in SSc [16].

In light of previously reported evidence and the results of this study, we conclude that the increased expression of VEGFR-2 and VEGFR-3 and the dilated lymphatic vessels may play a role in the pathogenesis of systemic sclerosis. As this study was descriptive and the involvement of dilated lymphatic vessels in the pathogenesis of SSc is speculative, further studies are needed to provide a better understanding of the pathogenesis of this disease, including an analysis of the in situ contribution of the vascular and lymphatic systems.

Disclosure

Acknowledgements: The authors sincerely thank Dr. Faith Chengetayi Muchemwa for her valuable discussions and critical reading of this manuscript. We also thank Chiemi Shiotsu and Junko Suzuki for their technical assistance with histopathology. Financial support: none. Conflict of interest: none.

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