Overexpression of VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, leads to insufficient angiogenesis in patients with systemic sclerosis
Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, fibrosis, and autoimmunity that affects the skin and internal organs. Although in SSc there is a lack of sufficient angiogenic response to chronic tissue ischemia culminating in the loss of capillary vessels, the expression of vascular endothelial growth factor-A (VEGF) has paradoxically been shown to be upregulated in SSc skin and circulation. However, previous studies in the field did not distinguish between the proangiogenic VEGF165 and antiangiogenic VEGF165b isoforms that are generated by alternative splicing in the terminal exon of VEGF pre-RNA. In the present study, we investigated whether VEGF isoform expression could be altered in skin and circulation of SSc patients. Using RT-PCR, quantitative real-time PCR, Western blotting, immunohistochemistry and confocal microscopy, we could show that the VEGF165b splice variant was selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF165b expression correlated with increased expression of profibrotic transforming growth factor-β1 (TGF-β1) and serine/arginine protein 55 (SRp55) splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. ELISA on plasma samples revealed that circulating levels of VEGF165b were significantly higher in SSc patients than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF165b than healthy MVECs (H-MVECs). TGF-β1 upregulated the expression of VEGF165b and SRp55 in both SSc- and H-MVECs. In SSc-MVECs, VEGF receptor-2 (VEGFR-2) was overexpressed, but its phosphorylation and ERK1/2 downstream signaling were impaired. Recombinant human VEGF165b and SSc-MVEC–conditioned medium inhibited VEGF165-mediated VEGFR-2 phosphorylation, ERK1/2 activation and capillary morphogenesis on Matrigel in H-MVECs. The addition of anti-VEGF165b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC–conditioned medium. Capillary morphogenesis was severely impaired in SSc-MVECs and could be ameliorated by treatment with recombinant VEGF165 and anti-VEGF165b blocking antibodies. In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia. The combination of proangiogenic VEGF165 administration and VEGF165b neutralization might represent a potential therapeutic strategy to promote effective angiogenesis and capillary regeneration in SSc.
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