MicroRNA-93 Controls Perfusion Recovery After Hindlimb Ischemia by Modulating Expression of Multiple Genes in the Cell Cycle Pathway

We used C57BL/6J and BALB/cJ mice to examine microRNA expression in ischemic hindlimb muscles at a time point when the 2 strains had similar recovery to identify microRNAs that are differentially regulated. We used computational techniques to identify and quantitative real-time Background —MicroRNAs are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease. Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hindlimb ischemia between inbred mouse strains to identify key microRNAs involved in perfusion recovery from hindlimb ischemia. Methods and Results —From comparative microRNA profiling between inbred mouse strains that display profound differences in their extent of perfusion recovery after hindlimb ischemia, we found that the mouse strain with higher levels of microRNA-93 (miR-93) in hindlimb muscle before ischemia and the greater ability to upregulate miR-93 in response to ischemia had better perfusion recovery. In vitro, overexpression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells and enhanced proliferation in both cell types. In addition, miR-93 overexpression enhanced endothelial cell tube formation. In vivo, miR-93 overexpression enhanced capillary density and perfusion recovery from hindlimb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in vitro and in vivo modulation of miR-93 resulted in alterations in the expression of >1 cell cycle pathway gene in 2 different cell types. Conclusions —Our data indicate that miR-93 enhances perfusion recovery from hindlimb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue. ( Circulation . 2013;127:1818-1828.) washed with PBS and mounted with Vectashield mounting medium (Vector Lab, Burlingame, CA). Secondary antibody only without primary antibody was used as negative control to assess non-specific binding. Three representative pictures from each section were taken under 400X magnification, using Olympus BX51 high-magnification microscope. Total number of CD31 positive spots/field and total number of muscle fiber/field were counted, and capillaries expressed as CD31 positive spots/muscle fiber.