Background Levels of VEGF (vascular endothelial growth factor) are significantly elevated in systemic sclerosis (SSc) patients even before the development of skin fibrosis.
Objectives To analyze the effects of VEGF on vasculopathy and skin fibrosis in different animal models of SSc.
Methods Density and wall thickness of dermal microvessels as well as (hypo-)dermal thickness were assessed by histology using HE and Masson’s trichrome staining and immunohistochemistry using anti-vWF and anti-α-SMA antibodies. In the model of bleomycin-induced skin fibrosis, homo- (+/+) (n=8) and heterozygous (+/–) (n=9) VEGF transgenic (tg) mice and wildtype (wt) mice (n=6/9) were treated with bleomycin and compared to saline treated controls. Additionally, TSK (tight skin)1/VEGF+/+ mice were generated (n=4). For statistical analysis, GraphPad Prism software was employed. Parametric non-related data were expressed as mean±SEM, nonparametric non-related data as median(Q1,Q3). P-values <0.05 were considered statistically significant.
Results In unchallenged VEGF tg mice, the number of dermal microvessels spontaneously increased, which was most pronounced in VEGF+/– mice (mean±SEM saline treated VEGF+/– 21.5±1.3 vs. VEGF+/+ 15.3±3.1 vs. 5.2±1.1/HPF in wt mice; p<0.05). Similar effects were observed for vessel wall thickness (saline treated VEGF+/– 4.2±0.2 vs. VEGF+/+ 3.5±0.3 vs. 1.9±0.2μm in wt mice; p<0.05), thus suggesting that in vivo, intermediate doses of VEGF are pro-angiogenic, whereas high doses lead to decreased angiogenesis. Strikingly, VEGF+/+, but not VEGF+/– tg mice spontaneously developed skin fibrosis (increase of skin thickness by 1.8±0.2 fold compared to wt mice; p<0.05), indicating a dose-dependent pro-fibrotic effect of VEGF in vivo. Challenge with bleomycin enhanced the effects of VEGF on vasculopathy. VEGF+/– compared to VEGF+/+ tg and wt mice showed the most pronounced increase in both the number of microvessels (31.8±1.6 vs. 19.1±1.4 vs. 10.2±0.9/HPF; p<0.05) and vessel wall thickness (4.0±0.2 vs. 3.3±0.1 vs. 2.3±0.2μm; p≤0.05). Additionally, bleomycin further increased skin thickness in VEGF+/+ and induced skin fibrosis in VEGF+/– tg mice (2.3±0.08 and 2.3±0.07 fold; p<0.05). Bleomycin challenged wt mice showed the same increase in skin thickness (1.7±0.02 fold; p<0.05) as VEGF+/+ tg mice without bleomycin challenge. In conclusion, pro-inflammatory conditions aggravated the effects of VEGF. In the non-inflammatory TSK1 mice, VEGF had similar effects. Whereas TSK1 mice showed a reduced number of dermal microvessels compared to wt mice (median(Q1,Q3) 2.0(1,2) vs. 5.5(3,6)/HPF; p<0.05), in VEGF+/+/TSK1 mice, microvessel density (16.5(11,25) vs. 2.0(1,2)/HPF in TSK1 mice; p<0.05) and vessel wall thickness increased (4.9(4,8) vs. 2.9(3,4)μm in TSK1 mice; p<0.05) which was paralleled by an increase in hypodermal thickness (694.5±15.1 vs. 456.0±20.6 in TSK1 vs. 102.9±5.7μm in wt mice; p<0.05).
Conclusions These in vivo data on VEGF tg mice and two SSc models provide a novel link between the vasculopathy and onset of fibrosis in SSc. High levels of VEGF lead to a decrease of angiogenesis. Notably, these high levels of VEGF have potent profibrotic effects and induce dermal fibrosis without additional stimuli.
Disclosure of Interest B. Maurer: None Declared, A. Akhmetshina: None Declared, R. Gay: None Declared, G. Schett: None Declared, B. Michel: None Declared, S. Gay: None Declared, J. Distler Shareholder of: stock owner of 4D Science, Grant/Research support from: Boehringer Ingelheim, Celgene, Bayer Pharma, Actelion, Pfizer, Ergonex, BMS, JB Therapeutics, Anaphore, Inc, Sanofi-Aventis, Novartis, Array Biopharma and Active Biotec in the area of potential treatments of scleroderma, Consultant for: Boehringer Ingelheim, Celgene, Bayer Pharma, Actelion, Pfizer, Ergonex, BMS, JB Therapeutics, Anaphore, Inc, Sanofi-Aventis, Novartis, Array Biopharma and Active Biotec in the area of potential treatments of scleroderma, O. Distler Grant/Research support from: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac in the area of potential treatments of scleroderma and its complications. Received lecture honoraria from Actelion, Pfizer, Encysive and Ergonex., Consultant for: Actelion, Pfizer, Ergonex, BMS, Sanofi-Aventis, United BioSource Corporation, medac in the area of potential treatments of scleroderma and its complications. Received lecture honoraria from Actelion, Pfizer, Encysive and Ergonex.
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