Du Y, Chen Q, Huang L, Wang S, Yin X, Zhou L, Ye Z, Ren X, Cai Y, Ding X, Ouyang H, Li X, Ju R
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Chengdu Aier Eye Hospital, Chengdu, 610041, China.
Curr Mol Med. 2018;18(5):273-286. doi: 10.2174/1566524018666181004115304.
Whereas retinal pigment epithelial (RPE) cells are known to secrete VEGF-A and VEGFR2, the functions of the autocrine VEGF signaling remain unclear. Meanwhile, anti-VEGF therapies have been applied routinely to treat ocular vascular diseases.
The aim of this study was to determine the functions of the VEGF signaling in RPE cells and evaluate the consequences of its interruption.
The genes involved in the VEGF and Hippo signal pathways were knocked down with siRNAs in both ARPE-19 cell line and human primary RPE cells via transient transfection whereas overexpression of VEGFR2 was mediated via adenovirus transduction. Expression of the epithelial-mesenchymal transition (EMT) markers and the downstream genes of YAP were determined by real-time PCR and Western Blot analysis. Immunofluorescence staining was utilized to determine gene expression in tissue and mouse samples.
Knockdown of VEGFR2 results in epithelial-mesenchymal transition in vitro and in vivo. Overexpression of VEGFR2 suppresses TGF β-mediated EMT in RPE cells. Loss of VEGF-C rather than VEGF-A induces EMT. Mechanistically, the VEGFR2 ablation-induced EMT in RPE cells is mediated by activation of YAP, an effector of the Hippo pathway. Finally, the immunohistochemical analysis of VEGFR2 and YAP in human proliferative vitreoretinopathy (PVR) membranes indicates a tendency of an inverse correlation between VEGFR2-positive and YAP-positive cells.
Our results disclose unexpected novel roles of VEGFR2 and VEGF-C in the process of EMT of RPE cells and in the Hippo pathway. The data shown here demonstrated that VEGFR2 and VEGF-C are important to maintain the normal physiological state of RPE cells.
已知视网膜色素上皮（RPE）细胞可分泌血管内皮生长因子A（VEGF-A）和血管内皮生长因子受体2（VEGFR2），但自分泌VEGF信号传导的功能仍不清楚。同时，抗VEGF疗法已被常规应用于治疗眼部血管疾病。
本研究旨在确定VEGF信号在RPE细胞中的功能，并评估其阻断的后果。
通过瞬时转染，利用小干扰RNA（siRNA）在ARPE-19细胞系和人原代RPE细胞中敲低VEGF和Hippo信号通路相关基因，而通过腺病毒转导介导VEGFR2的过表达。通过实时聚合酶链反应（PCR）和蛋白质免疫印迹分析确定上皮-间质转化（EMT）标志物和Yes相关蛋白（YAP）下游基因的表达。利用免疫荧光染色确定组织和小鼠样本中的基因表达。
敲低VEGFR2在体外和体内均导致上皮-间质转化。VEGFR2的过表达抑制RPE细胞中转化生长因子β（TGFβ）介导的EMT。VEGF-C而非VEGF-A的缺失诱导EMT。机制上，RPE细胞中VEGFR2缺失诱导的EMT由Hippo通路的效应器YAP的激活介导。最后，对人增殖性玻璃体视网膜病变（PVR）膜中VEGFR2和YAP的免疫组织化学分析表明，VEGFR2阳性细胞和YAP阳性细胞之间存在负相关趋势。
我们的结果揭示了VEGFR2和VEGF-C在RPE细胞EMT过程和Hippo通路中意想不到的新作用。此处显示的数据表明，VEGFR2和VEGF-C对维持RPE细胞的正常生理状态很重要。
