The Different Effects of VEGFA121 and VEGFA165 on Regulating Angiogenesis Depend on Phosphorylation Sites of VEGFR2

The effects of VEGFA isoforms on the vascular permeability and structure are still unclear. In this study, we found that VEGFA121 and VEGFA165, 2 isoforms of VEGFA, exerted the opposing effects of antiangiogenesis and proangiogenesis on regulating vascular endothelia cells proliferation and tube formation. The 2 isoforms affected the protein expression of Ras-related protein 1-GTPase-activating protein 1 (Rap1GAP) and thrombospondin 1, 2 important signal molecules of Rap1GAP/thrombospondin 1 signal pathway in primary human umbilical vein endothelial cells by regulating 2 different phosphorylating sites of VEGFR2, Tyr(1175) and Tyr(1214). We also found that VEGFA121 and VEGFA165 regulating angiogenesis was related to their regulating VEGFR2 and Rap1GAP/thrombospondin 1 signal pathway with the technology of RNA intervening the gene expression of VEGFR2 and Rap1GAP. Meanwhile, 2 inhibitors of VEGFR2, cabozantinib malate and ZM 323881 HCl (ZM), were used to investigate the relationship among VEGFA(121 and 165), VEGFR2, and angiogenesis. It was demonstrated that cabozantinib malate blocked VEGFA121 and VEGFA165 binding to VEGFR2 and inhibited angiogenesis by specifically binding to VEGFR2 rather than changing VEGFR2 phosphorylation or regulating the expression of VEGFR2. However, ZM antagonized the effect of VEGFA on angiogenesis by specifically reversing the phosphorylation induced by VEGFA121 and VEGFA165. The experiments in vivo also demonstrated that obvious abnormality of VEGFA121 and VEGFA165 presented in the serum of ulcerative colitis (UC) rats compared with that of the normal rats. ZM could promote the repairation of the injuries of the vessels and tissues of colonic mucosa of UC rats and caused mild inflammation in colonic mucosa of normal rats. On the contrary, cabozantinib malate caused injury of vessels and inflammation in the colonic mucosa of normal rats and aggravated the injuries of the vessels and inflammation in the colonic mucosa of UC rats. Hence, our data indicated that the activation of different phosphorylation sites of VEGFR2 leaded to VEGFA121 and VEGFA165 exerting opposing effects on angiogenesis, and it might be an underlying pathogenesis of UC and a potential target for UC treatment.