Downregulation of miR-205-5p in ARPE-19 promotes angiogenesis in HUVEC cell

Abstract

Purpose : Accumulating evidence proposes that dysregulation of miRNAs is closely associated with the initiation or progression of diabetic retinopathy (DR). Several studies have pointed miRNAs as potential regulators of ocular angiogenesis. It has been proved that miR-205-5p inhibits vessel formation, targeting VEGFA. This miRNA also travels in extracelular vesicles (EVs) playing a role in paracrine communication. The present work aimed to identify the role of mir-205-5p in angiogenesis processes.

Methods : HUVEC cells were seeded at a density of 3,0x10⁴cells/cm² in a Matrigel, then treated with ARPE-19 cells media for 5 hours. 150 μl of medium were incorporated to each well. The different conditions were determined by the conditioned medium used; control media, control media and miR-205-5p mimic, control media and N-acetylcysteine, control media with N-acetylcysteine and miR-205-5p mimic, 35 mM glucose media from, 35 mM glucose media and miR-205-5p mimic , 35 mM glucose media and N-acetylcysteine and 35 mM glucose media with N-acetylcysteine and miR-205-5p mimic. sEVs from ARPE-19 cell media were isolated using the Total Exosome Isolation Reagent from cell culture media (Thermo Fisher Scientific, Waltham, USA).Pictures were taken with Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan). Total tube length from images were quantified by Image J using the Angiogenesis Analyzer plugin. Values are expressed as mean ± SEM (n= 3). The p-value was obtained by ANOVA one way; *p < 0.05 and **p < 0.01.

Results : The HUVEC cells were used for a functional angiogenesis assay to confirm the involvement of miR-205-5p, VEGFA and glucose exposure in vasculogenic regulation. The vasculogenic processes in HUVEC cells treated with control media were not modify. However, the vasculogenic processes increase after 35 mM glucose exposure (202.4 ± 28.7). This effect was reverse due to the administration of N-Acetylcysteine (127.9 ± 12.24), mimic miR-205-5p (11.7 ± 12.57) and combination of both (113.1 ± 15.53). Only the EVs of medium from ARPE-19 cells treated with glucose 35 mM were able to increase angiogenic process in HUVEC cells (133.7 ± 7.4).

Conclusions : Decrease of miR-205-5p from damaged RPE cells resulted in an increased growth of vessels. When the level of miR-205-5p returned to physiological levels, through the addition of a mimic, neovascularization was also normalized.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.