Overexpression of VEGF 165 b, an Inhibitory Splice Variant of Vascular Endothelial Growth Factor, Leads to Insufficient Angiogenesis in Patients With Systemic Sclerosis

Abstract

Rationale: Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF 165 and antiangiogenic VEGF 165 b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA. Objective: We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc. Methods and Results: Here, we show that the endogenous antiangiogenic VEGF 165 b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF 165 b expression correlated with increased expression of profibrotic transforming growth factor-β1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF 165 b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF 165 b than healthy MVECs. Transforming growth factor-β1 upregulated the expression of VEGF 165 b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF 165 b and SSc-MVEC–conditioned medium inhibited VEGF 165 -mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF 165 b 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 VEGF 165 and anti-VEGF 165 b blocking antibodies. Conclusions: In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.