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Magali Saint-Geniez, Carrie Spencer, Stephanie Abend, Kevin McHugh; Synergistic interaction between endothelial cells and retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2014;55(13):383.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have demonstrated the critical interdependence of choroidal endothelial cells (EC) and retinal pigment epithelial (RPE) cells. For example, we recently showed that production of soluble VEGF isoforms by RPE is necessary for choriocapillaris survival. Here we analyze the reciprocal interaction between RPE and EC using a long-term coculture system and determine the effect of RPE-EC interaction on cell behavior and matrix deposition.
ARPE-19 and HUVECs were seeded on opposite sides of polyester transwells and cultured for up to 4 weeks in low serum conditions.Cell viability was quantified using a trypan blue assay. Cell morphology was evaluated by H&E staining, SEM and immunohistochemistry. RPE barrier function was determined by recording of the transepithelial resistance and localization of ZO-1. RPE phagocytic function was examined by performing an 18-hour fluorescent bead assay. Gene expression analysis was performed on both RPE and ECs by qPCR. Quantification of extracellular matrix deposition was performed on acellular transwells stained for collagen IV, fibronectin, and fibrillin. EC angiogenic activity was quantified in a 2D tube-formation assay.
Coculture with RPE sustains primary human EC survival and proliferation in low serum for up to 4 weeks. Presence of ECs significantly improves RPE barrier and phagocytic functions with no effect on RPE survival or morphology. Gene expression analysis reveals that coculture with ECs strongly induces RPE expression of matrix- and visual cycle-associated genes such as RPE65 and CRALBP. Presence of ECs leads to the accumulation of BrM-like material on the RPE basal side as measured by electron microscopy and immunohistological analysis. Expression of PEDF and Thrombospondin-1 by RPE was also significantly upregulated in co-culture condition, suggesting that RPE acquired an anti-angiogenic status. Respectively, gene expression and tube formation studies confirmed that co-culture with RPE significantly decreased the angiogenic phenotype of ECs.
This study demonstrates that long-term RPE-EC coculture improves RPE differentiation and deposition of a BrM-like matrix while inhibiting the ECs angiogenic potential and further emphasizes the importance of RPE-EC interaction in the maintenance of retinal homeostasis.
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