Shiying Wang, Boyun Sun, Jianye Yuan, Wanjun Zhang, Ping Tao, Jiang Lin, Hongyi Hu
*Department of Gastroenterology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China; and †Institute of Digestive Diseases, China-Canada Center of Research for Digestive Diseases (ccCRDD), Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Inflamm Bowel Dis. 2017 Apr;23(4):603-616. doi: 10.1097/MIB.0000000000001055.
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.
血管内皮生长因子A（VEGFA）异构体对血管通透性和结构的影响仍不清楚。在本研究中，我们发现VEGFA的两种异构体VEGFA121和VEGFA165在调节血管内皮细胞增殖和管腔形成方面发挥了抗血管生成和促血管生成的相反作用。这两种异构体通过调节VEGFR2的两个不同磷酸化位点Tyr(1175)和Tyr(1214)，影响了原代人脐静脉内皮细胞中Ras相关蛋白1 - GTP酶激活蛋白1（Rap1GAP）和血小板反应蛋白1这两个Rap1GAP/血小板反应蛋白1信号通路重要信号分子的蛋白表达。我们还通过RNA干扰VEGFR2和Rap1GAP基因表达的技术发现，VEGFA121和VEGFA165调节血管生成与其调节VEGFR2和Rap1GAP/血小板反应蛋白1信号通路有关。同时，使用两种VEGFR2抑制剂，苹果酸卡博替尼和盐酸ZM 323881（ZM），来研究VEGFA（121和165）、VEGFR2与血管生成之间的关系。结果表明，苹果酸卡博替尼通过特异性结合VEGFR2而非改变VEGFR2磷酸化或调节VEGFR2表达，阻断VEGFA121和VEGFA165与VEGFR2的结合并抑制血管生成。然而，ZM通过特异性逆转VEGFA121和VEGFA165诱导的磷酸化来拮抗VEGFA对血管生成的作用。体内实验还表明，与正常大鼠相比，溃疡性结肠炎（UC）大鼠血清中VEGFA121和VEGFA165存在明显异常。ZM可促进UC大鼠结肠黏膜血管和组织损伤的修复，并在正常大鼠结肠黏膜中引起轻度炎症。相反，苹果酸卡博替尼在正常大鼠结肠黏膜中导致血管损伤和炎症，并加重UC大鼠结肠黏膜血管损伤和炎症。因此，我们的数据表明，VEGFR2不同磷酸化位点的激活导致VEGFA121和VEGFA165在血管生成上发挥相反作用，这可能是UC的潜在发病机制和UC治疗的潜在靶点。
