VEGFR2 and VEGF-C Suppresses the Epithelial-Mesenchymal Transition Via YAP in Retinal Pigment Epithelial Cells

Abstract

Background: 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.

Objective: The aim of this study was to determine the functions of the VEGF signaling in RPE cells and evaluate the consequences of its interruption.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Hippo pathway; Retinal pigment epithelial cell; VEGFR2; epithelial-mesenchymal transition; eye; vascular endothelial growth factor; yes-associated protein..