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Stephanie Abend, Kevin McHugh, Patricia A. D'Amore, Magali Saint-Geniez; Effect of Endothelial Cells on RPE Differentiation and Matrix Deposition. Invest. Ophthalmol. Vis. Sci. 2011;52(14):879.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have described 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. In addition, developmental studies have suggested that formation of Bruch’s membrane (BrM) depends on the presence of both choroidal EC and RPE. To examine the reciprocal interaction between RPE and EC, we developed a long-term coculture system and determined the effect of RPE-EC interactions on cell behavior and matrix deposition.
ARPE-19, a line of human RPE cells, and primary human umbilical vein EC, HUVEC, were seeded on the opposite sides of polyester transwells (0.4 µm pores) at 1.7x105 cells/cm2 and 5x104 cells/cm2, respectively and cultured for up to 4 weeks in EBM-2, 1% FCS. Cell viability was quantified using trypan blue. Transepithelial resistance was monitored with an epithelial voltohmmeter. Cell morphology was evaluated by ZO-1 immunohistochemistry. Matrix deposition and expression of relevant genes were determined by qPCR and western blot analysis. Matrix accumulation was visualized by TEM analysis.
Coculture with RPE sustained EC survival and proliferation in low serum for up to 2 weeks, a trophic effect that appeared to be contact dependent. Presence of ECs significantly improved RPE barrier function with no effect on RPE survival or morphology. Analysis of gene expression revealed that coculture with ECs strongly induced RPE expression of matrix-associated proteins such as collagen I, elastin, decorin and lysyl oxydase. Expression of cellular retinaldehyde-binding protein by RPE was also significantly increased in presence of EC. TEM analysis revealed that presence of ECs led to the accumulation of BrM-like material on the basal side of RPE.
RPE differentiation and deposition of a BrM-like matrix are influenced by long-term coculture with EC, further emphasizing the importance of RPE-EC interactions in retinal homeostasis.
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