miRNA-6236 Regulation of Postischemic Skeletal Muscle Angiogenesis

Background: Peripheral arterial disease affects >200 million people worldwide and is characterized by impaired blood flow to the lower extremities. There are no effective medical treatments available. Using the mouse hind-limb ischemia model and miRNA sequencing, we identified a novel miRNA, miR-6236, whose expression significantly elevated in ischemic mouse limbs compared with nonischemic limbs. The role of miR-6236 in general or in postischemic angiogenesis is not known. Here we describe its role using in vivo and in vitro models of peripheral arterial disease. Methods and Results: In primary mouse and human endothelial cells, we studied the effect of simulated ischemia on miR-6236 expression and assessed its role in cell viability, apoptosis, migration, and tube formation during ischemia. Furthermore, we developed miR-6236 null mice and tested its role in postischemic perfusion recovery using the hind-limb ischemia model. Lastly, using bioinformatics and gene expression analysis, we identified putative angiogenic miR-6236 targets. In vitro simulated ischemia-enhanced miR-6236 expression in mouse and human endothelial cells, whereas its inhibition improved viability, migration, tube formation, and reduced apoptosis. In vivo ischemic mouse skeletal muscle tissue showed higher miR-6236 expression compared with nonischemic muscles. Loss of miR-6236 improved impaired postischemic perfusion recovery and poor angiogenesis associated with streptozotocin-induced diabetes in mice. Six of the 8 miR-6236 predicted angiogenic target mRNAs showed expression consistent with regulation by miR-6236 in ischemic skeletal muscle. Conclusions: Our results show for the first time that miR-6236 plays a key role in regulating postischemic perfusion recovery and angiogenesis.