Choroid endothelial cells regulate RPE tight junctions through modulation of Bruch′s membrane assembly

Abstract

Purpose : A hallmark of terminal retinal differentiation is the coordinated maturation of RPE, Bruch′s membrane (BM) and choroid vasculature. Recent studies demonstrate that endothelial cells (ECs) play key instructive roles in the differentiation and maintenance of parenchymal cells. We tested the hypothesis that choroid ECs regulate terminal differentiation of RPE and the outer blood-retina barrier by modulating BM assembly, which in turn triggers remodeling of RPE tight junctions (TJs).

Methods : We co-cultured in Transwell chambers human fetal RPE (hfRPE) and either human umbilical vein ECs or mouse choroid ECs isolated by in vivo labeling of VE-cadherin and flow cytometry cell sorting. We assessed the effect of ECs on hfRPE TJs by measuring transepithelial electrical resistance (TER) and occludin subcellular localization by cell surface biotinylation and quantitative microscopy. We studied extracellular matrix (ECM) deposition by hfRPE by scanning electron microscopy and immunofluorescence assays. Lysyl oxidase activity, which catalyzes collagen and elastin crosslinking, was inhibited in EC-conditioned media by β-aminopropionitrile (BAPN) treatment. We carried out RNAseq analyses of choroid ECs from developing (P5) and adult (P30) mouse retinas. We evaluated newborn mice treated for 30 days with BAPN for visual function by optomotor response and for BM maturation by transmission electron microscopy.

Results : EC-conditioned media induced a specific increase in hfRPE TER and the accumulation of occludin along hfRPE TJs. These effects were significantly impaired by BAPN treatment and EC lysyl oxidase-like 2 (LOXL2) knockdown. ECs enhanced the appearance of bundled collagen I-positive fibers in the ECM deposited by hfRPE, and increased the levels of active β1 integrin at the hfRPE basal plasma membrane. RNAseq analyses showed that several ECM-related genes were differentially expressed in developing versus adult choroid ECs. In particular, LOXL2 was significantly more expressed in P5 than in P30 choroid ECs. In vivo, BAPN treatment during mouse terminal retinal differentiation induced defects in BM structure and visual function.

Conclusions : Our results suggest that the regulation of BM composition and structure by choroid ECs is key for the maturation of RPE TJs and the establishment of the outer blood-retina barrier, essential for proper vision.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.