Regulation of vasculogenic processes by miR-302a-3p encapsulated in small extracellular vesicles

Abstract

Purpose : Environmental cell conditions can promote extracellular vesicles (EVs) release. EVs are nanovesicles produced by most human cells containing numerous products including mRNA, microRNA (miRNAs), proteins and signaling molecules that have a crucial role in cell-to-cell communication. miRNAs are noncoding RNAs about 22 nucleotides long. They are considered post-transcriptional regulators of gene expression that influence the expression of hundreds of genes simultaneously. They are involved with up and downregulation is associated with many pathologies. Diabetic retinopathy (DR), age-related macular disease (AMD) and cancers are characterized by neovascularization processes. Oxidative damage increases the EVs release regulating angiogenesis. It has been proved that miR-302a-3p inhibits vessel formation targeting VEGFA via zinc finger protein 91 (ZFP91) and hypoxia induced factor 1α (HIF-1 α).

Methods : Our objective was to demonstrate that oxidative stress stimulates EVs release, down regulating miR-302a-3p and increasing angiogenesis. We used arising Retinal Pigment Epithelial cell line (ARPE-19) treated with 600 μM H₂0₂ or 4mM N-Acetylcysteine (NAC). Then, Human Vein Endothelial Cells (HUVEC) were grown indifferent conditions with or without EVs from control media, H₂O₂ or NAC media collecting EVs and isolating miRNA in order to confirm the results.

Results : H₂O₂ and H₂O₂-induced EVs exposure increases reactive oxygen levels (ROS) levels and decreases cell viability in ARPE-19 overexpressing VEGFA mRNA levels, those changes were reverted with NAC. miR-302a-3p mimic exposure decreased H₂O₂ induced VEGFA mRNA expression similarly to NAC addition. It is known that miR-302a-3p decreases vascular processes. Control-released EVs significantly downregulated the new vessels formation. More EVs were released after miR-302a-3p mimic addition and it was correlated with a stronger angiogenesis inhibition, showing a relationship between them. The use of miR-302a-3p inhibitor abolishes the effect. On the other hand, the miR-302a-3p mimic inhibitor restored VEGFA mRNA expression to control levels.

Conclusions : Oxidative stress promotes EVs release with proangiogenic properties. Those are inhibited by miR-302a-3p and promoted by ROS. This opens a new strategy against oxidative- induced angiogenesis by promoting miR-302a-3p as VEGFA mRNA repressor.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.