miRNA-6236 Regulation of Postischemic Skeletal Muscle Angiogenesis

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.

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.

Our results show for the first time that miR-6236 plays a key role in regulating postischemic perfusion recovery and angiogenesis.