Abstract 11333: Muscle Specific MicroRNA-499-5p Impairs Angiogenesis in Ischemic Hindlimb of Diabetic Micehindlimb of Diabetic Mice

Abstract

Introduction: Limb ischemia is one of the most prevalent cardiovascular complications in diabetic patients. The underlying mechanisms remain incompletely understood. Previously, we have reported that microvascular endothelial cells (MVECs) play an important role in angiogenesis. Recent study demonstrated that skeletal muscle cells (SKMCs) regulate angiogenesis process via paracrine signals; level of miR-499, a muscle specific miR, was enhanced in diabetic hearts/cardiomyocytes and overexpression of miR-499 impaired MVEC function. We observed that level of miR-499-5p, but not -3p, was increased in skeletal muscle cells (SKMCs), MVECs and SKMC-derived extracellular vehicles (SKMC-EVs) in db/db mice. Hypothesis: SKMC-EVs-derived miR-499-5p impairs MVEC function in diabetes. Methods: MVECs and SKMCs, and SKMC-EVs were isolated from 8-10-week male db/+ and db/db mice. Ischemic hind limb (IHL) surgery was conducted in 8-10-week male wildtype C57BL/6J, diabetic db/db and non-diabetic db/+ mice. Results: Level of miR-499-5p was increased in SKMCs and MVECs from db/db mice. Overexpression of miR-499-5p impaired tube formation and migratory activity of MVECs. Intramuscular injection of anti-miR-499-5p lentivirus ameliorated blood perfusion and capillary density in IHL of db/db mice. Mechanistically, miR-499-5p level was increased in diabetic SKMC-EVs. Inhibition of EV formation/section by GW4869 ameliorated diabetic SKMCs-enhanced miR-499-5p level in MVECs. Local injection of diabetic SKMC-EVs impaired blood perfusion and capillary density in C57BL/6J mice. Moreover, diabetic SKMC-EVs-impaired tube formation and migratory activity of MVECs was ameliorated by silencing of miR-499-5p in the diabetic SKMCs ameliorated. Level of sex-determining region Y-box 6 (SOX6), the most attractive gene targeted by miR-499-5p, was decreased in MVECs of db/db mice. Finally, silencing of SOX6 impaired tube formation, migration and pro-angiogenic factor secretion from MVECs. Conclusions: miR-499-5p impairs angiogenic property of MVECs in diabetic mice via SKMC-EV/miR-499-5p/SOX6/proangiogenic factors cascades. miR-499-5p and SOX6 may be novel targets for prevention/therapeutics of ischemic limb injury in diabetic patients.