Characterization of exosomes liberated from damaged RPE: implication in new blood vessel formation due to overexpression of VEGF receptor 1 and 2

Abstract

Purpose: Oxidative stress induces activation of VEGF in RPE cells, causing choroidal neovascularization in a number of blinding diseases. RPE cells also liberate exosomes, small membrane vesicles, which may influence the fate of neighboring cells. We hypothesized that RPE-derived exosomes are relevant in new blood vessel formation after ethanol-induced oxidative damage.

Methods: Exosomes were purified from ARPE-19 cells by sequential centrifugation and ultracentrifugation after a 24 h treatment with different ethanol concentrations (40 and 80 mM). Exosomes were observed by electron microscopy and quantified by flow cytometry using an anti-CD9 antibody. The protein content of VEGFR-1 and -2 were analyzed by Western blot. Positive VEGFR-1 and -2 exosome populations were observed by flow cytometry. qPCR was conducted to assess the mRNA content of VEGFRs. Neovascularization assays were performed after applying control or treated RPE-derived exosomes into endothelial cell cultures (HUVEC).

Results: Exosomes were observed in extracellular medium of both control and ethanol-treated cells, exhibiting the typical morphology and size (30-100 nm). It was observed that cells treated with 80 mM ethanol released a larger number of exosomes (448,248 ± 26,541 per µL) than those untreated (201,031 ± 21,264 per µL). VEGF receptors were expressed in the membrane of exosomes originated from both, treated and untreated cells. VEGFR-1-positive exosomes increased significantly when cells were previously treated with 80 mM ethanol (103,392 ± 4,792 per µL vs. control: 60,000 ± 5,042 per µL). A similar effect was noted when the expression of VEGFR-2 was studied (control: 677 ± 33 per µL vs. 80mM ethanol: 3,162 ± 243 per µL). Moreover, exosomes from 80mM ethanol-treated cells enclosed extra cargo of VEGFR-1 mRNA (328% more than control) and VEGFR-2 mRNA (194% more than control). Angiogenesis assays confirmed that endothelial cells (HUVEC) hold an enhanced tube formation capacity when they are exposed to exosomes derived from ethanol-treated RPE cells.

Conclusions: Oxidative damage initiated by ethanol induces the release of increased amounts of exosomes in ARPE-19 cells. These “pathologic” exosomes exhibit greater concentrations of membrane-bound VEGFR-1 and -2, which might contribute to the formation of new vessels in retinal disorders.