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Farhad Farjood, Elizabeth Vargis; Effect of Intracellular Junction Formation on VEGF Expression in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1054.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been implicated in choroidal neovascularization associated with age-related macular degeneration (AMD). In this work, we investigated the role of cell-cell contact and intracellular junction formation on VEGF expression in cultures of a commercially available human retinal pigment epithelial (RPE) cell line (ARPE-19) and primary human RPE (hRPE) cells.
Two in vitro methods, micropatterning and scratching, were used to control the physical dissociation of RPE cell-cell contact. Micropatterning was achieved using a stencil patterning method. Scratching was performed by scoring monolayers of RPE cells using a pipette tip. Extracellular VEGF expression was assessed using an enzyme-linked immunosorbent assay (ELISA) kit for VEGF. Immunocytochemistry (ICC) was performed to visualize the differential expression of VEGF and a tight junction protein, zonula occludens-1 (ZO-1), in micropatterned and scratched RPE cultures.
Micropatterning RPE cells on different size circular patterns produced varying concentrations of cells with lost cell-cell junctions. Increasing the concentration of cells with fewer intracellular junctions increased extracellular VEGF secretion from both ARPE-19 and hRPE cells. Higher expression of VEGF protein by cells on the edges of patterned and scratched RPE layers was confirmed by ICC. Moreover, removing intracellular junctions by micropatterning and scratching resulted in dislocalization of zonula occludens-1 (ZO-1) from intracellular junctions to the nucleus. The loss of intracellular junction formation and accumulation of ZO-1 in the nucleus correlated with increased VEGF expression.
VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with both the loss of intracellualr junctions and the localization of ZO-1 in the nucleus of RPE cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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