Crosstalk between RPE and choroid endothelium regulates RPE tight junctions

Abstract

Purpose: The retina is irrigated by two blood circulations, the choroid and the retinal blood vessels. The retinal pigment epithelium (RPE) performs a key role as a perm-selective barrier between the neural retina and the fenestrated choroid capillaries. RPE tight junctions (TJs) are a key component of this blood-retinal barrier. Recent studies have demonstrated that endothelial cells (ECs) display organ-specific transcriptomes and play key instructive roles in the differentiation and maintenance of parenchymal cells. Here, we isolated mouse choroid ECs (ChECs) and retinal ECs to high purity, analyzed their transcriptome relative to each other and to other body ECs and studied the regulation of RPE TJs by ChECs.

Methods: We performed co-culture experiments on Transwell inserts using human umbilical vein ECs expressing the adenoviral protein E4 (VeraVecs), which are able to survive in the absence of endothelial factors or serum, and human fetal RPE (hfRPE). The effect of VeraVecs on hfRPE transepithelial electrical resistance (TER) was assessed. Expression levels of occludin and different claudins in hfRPE were analyzed by real time PCR. The effect of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 on hfRPE TER and claudin levels was also evaluated. We isolated mouse ChECs by in vivo EC labeling and flow cytometry cell sorting. ChEC conditioned medium was used in hfRPE wound healing assays. We performed RNAseq analysis of mouse native ECs extracted from choroid, neural retina, heart, liver and lung.

Results: VeraVec and ChEC-conditioned media specifically induced an increase in hfRPE TER during both de novo TJ formation and TJ recovery after disruption by TNFα treatment, and a decrease in hfRPE claudin-2 mRNA levels. Bioinformatic analyses showed that ChECs presented a significantly high expression of a set of genes involved in development and wound healing, including the EGFR ligand HBEGF. Recombinant HBEGF increased hfRPE TER, and EGFR tyrosine kinase inhibition reduced EC-mediated changes in hfRPE TER and claudin-2 levels. Wound healing assays showed that ChEC conditioned media improved hfRPE wound closure.

Conclusions: Our results suggest a role of ChECs in the formation and maintenance of the outer blood-retinal barrier by modulating RPE TJ function, and in repair processes after RPE injury. Thus, ChECs emerge as a potential new therapeutic tool to treat RPE-related vision pathologies.