Abstract 11323: Role of Mir-6236 on Skeletal Muscle Angiogenesis

Abstract

Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is characterized by impaired blood flow to the lower extremities. There are no medical treatments available and in severe cases, PAD may lead to critical limb ischemia requiring amputation. The pathophysiology of PAD is characterized by marked changes in gene expression and reduced angiogenesis. Although many studies have investigated the molecular mechanisms involved in PAD, less is known about the role of microRNAs (miRNA) in the pathogenesis of the disease. MiRNAs are important regulators of gene expression that bind to specific target mRNA and repress their expression. Using a mouse model of PAD, we investigated miRNAs induced by ischemia using RNAseq analysis of ischemic and non-ischemic hind limb tissue. Our data showed 77 miRNAs were upregulated and 40 miRNAs were downregulated in ischemic mouse hind limbs versus non-ischemic controls. Among these miRNAs, miR-6236 was significantly upregulated suggesting a possible role in post ischemic adaptation. We validated miR-6236 expression by qPCR (Ischemic 2.04±0.39 vs Non-Ischemic 1.0±0.11; n=5-6; p<0.05). In vitro, using primary mouse microvascular endothelial cells (MVECs), ischemia induced miR-6236 expression in a time dependent manner up to 72 hrs. At the same time, ischemia caused decrease in proliferation (Ischemia (Isch) 0.12±0.03 vs Normoxia (Norm) 0.23±0.02; n=4-6; p<0.05), migration (Isch 46.7±0.52 vs Norm 72.02±2.74; n=6; p<0.05) and tube formation (Isch 31.5±3.58 vs Norm 82.12±6.5; n=8; p<0.05), and increased apoptosis (Isch 0.94±0.05 vs Norm 0.59±0.02; n=4-5; p<0.05). Treatment of mouse MVECs with miR-6236 inhibitor (miRINH, 100 nM) prior to ischemia decreased miR-6236 expression by 50% and improved proliferation (miRINH + Isch 0.21±0.01 vs Isch 0.12±0.03; n= 4; p<0.05), migration (miRINH + Isch 63.71±2.08 vs Isch 46.7±0.52; n=6; p<0.05), tube formation (miRINH + Isch 50±5.92 vs Isch 31.5±3.58; n=8; p<0.05) and decreased ischemia-induced apoptosis (miRINH + Isch 0.64±0.04 vs Isch 0.94±0.05; n=4-5; p<0.05). We conclude that miR-6236 serves as a critical regulator of endothelial cell function in ischemic skeletal muscle and can be a promising target for therapeutic skeletal muscle angiogenesis.