Physical disruption of cell–cell contact induces VEGF expression in RPE cells

PURPOSE: To investigate the role of RPE cell–cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). METHODS: Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell–cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, β-catenin, and claudin-1 in RPE cultures. RESULTS: Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, β-catenin, and claudin-1 successfully localized to cell–cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, β-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell–cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell–cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. CONCLUSIONS: VEGF expression increases after physical disruption of RPE cell–cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in the cytoplasm of RPE cells.