Abstract 668: Identification And Functional Characterization Of Novel MicroRNAs Targeting Epsins For Angiogenesis And Vascular Remodeling

Abstract

Objective: Epsin is evolutionarily conserved protein that involved in clathrin-mediated endocytosis. Our previous studies showed Epsins regulate VEGF signaling, which is critical for angiogenesis, and Epsins can also augment inflammatory signaling in endothelial cells (EC). However, how Epsins are regulated during inflammation mediated angiogenesis is not clear. We hypothesize that post-transcriptional regulation of Epsins by miRNA are critical for inflammation-induced angiogenesis. In this study, we aim to identify novel miRNAs that can bind to Epsin1/2 genes for regulation of inflammation mediated angiogenesis. Approach and Results: In vitro, we found Epsin1/2 are significantly induced by TNFa and oxidized LDL in mouse aortic endothelial cells (MAECs). To screen miRNAs that can regulate Epsin1/2 expressions, we performed small RNA-sequencing for the control and treated MAECs samples, and identified differentially expressed miRNAs that are predicted to bind to Epsin1/2. We identified 78 differentially expressed (DE) known miRNA and 464 DE novel miRNAs. Among them, 12 known miRNAs and 69 novel miRNAs were predicted to bind Epsin1/2. We validated 2 known miR (miR-6390 and miR-6539) and 3 novel miRNA candidates (miR105462, miR39474 and miR67284) by in vitro miRNA mimics experiments, and found that they all can robustly suppress Epsin1/2 expressions. We then generated miR105462-flox mouse lines by homologous recombination using CRISPR/Cas9. We cross the miR105462-flox mice with Cdh5-Cre ERT2 to generate inducible EC-specific deletion by tamoxifen (miR105562-iEKO). Interestingly, neonatally induced endothelial-specific loss of miR105462 lead to reduced microvascular density and fewer tip cells in the retina of postnatal day6 pups. Additionally, higher Epn1 expressions in retinal EC were detected in EC-specific miR105462 deficient mice. Conclusion: We identified novel miRNA that can regulate Epsin expression in EC. Using novel genetic models, we identified angiogenic defects in vivo when miR105462 is conditionally deleted in EC, implicating critical function elicit by these novel miRNAs. Future studies will be directed at defining the novel miRNA-dependent molecular mechanism for the angiogenesis.