LAUSR

Background: miR-499, a muscle specific-miR, is enhanced in diabetic heart and has been suggested to be a therapeutic target for cardiovascular disease in diabetes. Recent studies provided the evidence that overexpressing miR-499 in ECs inhibits the capillary tube networks. Here, we tested the hypothesis that under diabetic conditions, myocyte-derived exosomes transfer miR-499 to EC/EPCs thereby impairing their angiogenic properties. Methods and Results: miR-499 expression is abundant and enhanced in heart, skeletal muscle, cardiomyocytes and skeletal muscle cells (SKMCs) of db/db mice. We observed that miR-499 level was also increased in diabetic EC/EPCs. In vitro, high D-glucose (25 mM) increased miR-499 level in SKMCs but not in ECs, suggesting that enhanced miR-499 in diabetic EC/EPCs is not regulated by hyperglycemia. To study whether SKMC-derived exosomes transfer miR-499 from SKMCs to EC/EPCs, we first examined the miR-499 levels in diabetic SKMC-derived exosomes and plasma-derived exosomes from db/db mice. We observed that miR-499 levels were greater in diabetic SKMC- and plasma-derived exosomes. Furthermore, by co-culture, we found that diabetic SKMCs increased miR-499 levels in human microvascular endothelial cells (ECs) and impaired EC tube formation and migration which was blocked by exosome inhibitor GW4869 (GW). We also observed that GW partially rescued diabetic SKMC- and plasma-derived exosome-mediated impairment in tube formation and migration of ECs. Overexpression of miR-499 in ECs decreased angiogenic factors and impaired tube formation/migration. Furthermore, in a hind-limb ischemia model of db/db mice, GW treatment improved ischemic hindlimb blood perfusion and angiogenesis. Our study suggests that diabetes-enhanced miR-499 in myocytes can be transferred to ECs via myocyte-derived exosomes thus impairs EC/EPC function. Mechanistically, Sex-determining region Y-box 6 (SOX6), one of the validated target of miR-499, was significantly decreased in diabetic EC/EPCs. Knockdown of SOX6 impairs tube formation and migration of ECs. Conclusions: Our results suggest that exosomal transfer of muscle specific miR-499 to EC/EPCs impairs angiogenesis and ischemic tissue injury repair in diabetes via suppression of SOX6.