Abstract 12544: A miRNA Cassette for Reprogramming Vascular Smooth Muscle Cells Into Endothelial Cells

Abstract

Introduction: Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) exhibit unique miRNA profiles, which regulate their overall function. However, whether differentially expressed miRNAs can be used for transcriptional reprogramming of SMCs into ECs is unknown. MiRNA profiling of SMCs and ECs revealed differential regulation of miR-143-3p, miR-145-3p, miR-146a-5p, and miR-181b-5p as a set of candidates for transdifferentiation. Methods: Human coronary SMCs were transfected with miR-143-3p and miR-145-3p inhibitors alongside miR-146a-5p and miR-181b-5p mimics and selectively expanded. Induced endothelial cells (iECs) were then assessed for their transcriptional, protein expression, and functional similarities to ECs (Fig. 1). Results: Principal component and gene set enrichment analyses determined that iECs are transcriptionally clustered closer to ECs than SMCs in whole transcriptome profiling and selected EC and SMC gene sets. Top pathways included ‘blood vessel morphogenesis’, ‘regulation of angiogenesis’ and ‘Notch, VEGF, integrin signaling’. Transcriptome data was validated via PCR and Western blot analyses as iECs exhibited robust upregulation in EC markers CD31, VE-Cadherin, and VEGFR2 and downregulation of SMC markers MHY11, Vimentin, and αSMA. The similarity of iECs to ECs was functional as iECs demonstrated oxLDL and Ac-LDL uptake, tube formation in Matrigel™ assays, eNOS phosphorylation in response to VEGF, and iEC migratory and cell growth properties were similar to human umbilical vein ECs in the presence of VEGF. A hindlimb ischemia model validated iEC similarity to ECs as injected iECs restored blood flow and significantly reduced limb necrosis similar to ECs. Conclusion: A miRNA cassette consisting of miR-143-3p and miR-145-3p inhibitors and miR-146a-5p and miR-181b-5p mimics transformed SMCs into endothelial-like cells and show promise as a potential novel regenerative strategy for endothelial cells.