miRNA profile of human retinal endothelial cells in starvation or angiogenic stimulation with and without VEGF inhibitors

Abstract

Purpose : MicroRNAs (miRNAs) are short, single-stranded ribonucleic acids (RNA). Mature miRNAs are about 20-22 nucleotides long, form a subset of non-coding RNAs and attenuate mRNA translation. They are involved in fine-tuning of diverse biological processes including angiogenesis. Retinal microvascular endothelial cells (RMVECs) are instrumental in retinal angiogenesis. The aim of this study was to decipher the miRNA profile of RMVECs with and without angiogenic stimulation and to determine changes upon VEGF inhibition.

Methods : We analyzed miRNA expression profiles of human RMVECs under 5 different conditions: (i) 24h of starvation, (ii) 24h of angiogenic stimulation (AS), (iii - v) AS for 12h followed by 12h of incubation with one of the VEGF inhibitors aflibercept, bevacizumab or ranibizumab. Cells were harvested and miRNA expression was determined using next-generation sequencing (Illumina). Following data analysis, the significantly (p≤0.05) and relevantly (>2fold) changed miRNAs were validated by qPCR. The confirmed miRNA modulations, as well as unregulated miRNAs that were very highly expressed were further studied by gene ontology-based enrichment analysis (GO) to determine their potential effect on intracellular signaling pathways.

Results : In all groups, miR-21-5p made up 40% of the RMVEC miRNome. Other abundantly expressed miRNAs were miR-29a-3p, miR-100-5p and miR-126-5p/3p. These were equally expressed (<2fold change) in all groups and might represent a RMVEC specific miRNA signature. Ten miRNAs with lower expression rates were significantly different in starvation and AS. Four of them (miR-335-5p/3p and miR-139-5p/3p) were validated by qPCR. GO analysis linked these miRNAs to regulation of angiogenesis, cell migration, apoptotic signaling and hypoxia response. Interestingly, supplementation of VEGF inhibitors did not alter the previously induced angiogenic miRNA expression profile within the short time frame of our experiments.

Conclusions : While the majority of miRNAs remained stable, we identified a regulated subset of miRNAs in RMVECs exposed to angiogenic stimulation. Addition of VEGF inhibitors to these angiogenically activated RMVECs for 12h did not alter their miRNA expression profile. These findings suggest that VEGF inhibition alone does not have acute effects on miRNA expression profiles of activated RMVECs.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.