Abstract P2065: Ischemic Injury Aggravated Muscle-specific Mir-499-5p-impaired Angiogenic Property Of Endothelial Cell In Hindlimb Of Diabetic Db/db Mice: Role Of Small Extracellular Vesicles

Abstract

Background: Recently, skeletal muscle cells (SKMCs) have been reported to be critical for regulation of EC function in limbs. miR-499, a muscle specific microRNA (miR), was found to be modulated in diabetes and ischemic injury. Here, we studied the role of miR-499 in EC dysfunction in diabetic ischemic limb injury. Methods: ECs and SKMCs were isolated from ischemic or non-ischemic hindlimb (IHL) of male db/+ and db/db mice. Serum- and SKMC-derived small extracellular vesicles/exosomes (EV/Exo) were isolated by gradient ultracentrifugation. Ischemic hindlimb (IHL) surgery was conducted by unilateral ligation of formal artery. Results: miR-499-5p level was increased in SKMCs and ECs of db/db mice which was synergistically increased by ischemic injury. Overexpression of miR-499-5p impaired tube formation and migratory activity of ECs. Intramuscular injection of anti-miR-499-5p lentivirus improved blood prefusion and neovascularization in IHL of db/db mice. Mechanistically, miR-499-5p level was enhanced in serum- and SKMC-derived EV/Exo from db/db mice which was synergistically increased by ischemic injury. Diabetic SKMC-EV/Exo impaired blood perfusion in wildtype mice. Anti-miR-499-5p rescued diabetic SKMC-EV/Exo-impaired EC function. Co-culture of diabetic SKMCs with wildtype ECs increased miR-499-5p expression in ECs which was inhibited by EV/Exo inhibitor GW4869. Sex-determining region Y-box 6 (SOX6), the most attractive gene targeted by miR-499-5p, was decreased in ECs from db/db mice which was synergistically reduced by ischemic injury. SOX6 siRNA impaired pro-angiogenic factor secretion and function of ECs. Anti-miR-499-5p significantly enhanced SOX6 level in SKMs from IHL of db/db mice. Finally, overexpression of SOX6 by transduction of lentivirus improved EC function of db/db mice. Conclusions: Enhanced miR-499-5p expression in ECs of SKMs from hindlimb of db/db mice is synergistically increased by ischemic injury. EV/Exo transfer miR-499-5p from SKMCs to ECs. miR-499-5p impairs angiogenic property of EC via, at least partially, SOX6/proangiogenic factors axis. miR-499-5p may be a novel target for treatment of critical limb ischemia in diabetic patients.