Abstract Body: Background: Emerging evidence suggest that skeletal muscle cells (SKMC) play critical roles in the regulation of angiogenesis in diabetic critical limb ischemia with defective angiogenesis response. However, the molecular mechanisms linking skeletal muscle to impaired angiogenic property of endothelial cells (EC) remain unidentified. The current study attempted to clarify whether and how muscle-specific miR-499-5p may impair EC function in ischemic limb in diabetes.
Methods: Hind limb ischemia was established by unilateral ligation of left femoral artery in 8- to 10-week-old, male db/+ and db/db mouse. ECs and SKMCs were isolated from sham or ischemic hind limbs (IHL). SKMC-derived small extracellular vesicles (SKMC-sEVs) were isolated from the culture medium of SKMCs.
Results: miR-499-5p level was markedly increased in SKMCs and surprisingly in ECs from hindlimb of db/db mice. Ischemic injury significantly aggravated diabetes-enhanced miR-499-5p level in ECs from IHL of db/db mice. Tube formation and/or migratory activity was reduced in ECs from IHL of db/db mice and miR-499-5p overexpressing human microvascular endothelial cells (HMVECs). Intramuscular injection of lentiviral-anti-miR-499-5p improved blood prefusion and angiogenesis in IHL of db/db mice. Mechanistically, we found that skeletal muscle-derived SKMC-sEV carried higher level of miR-499-5p in db/db mice and transferred miR-499-5p to ECs. Further, Intramuscular injection of diabetic SKMC-sEVs repressed IHL recovery in wildtype mice. Blocking sEV biosynthesis/release by intraperitoneal injection of GW4869 markedly improved angiogenesis/neovascularization and blood perfusion in IHL of db/db mice. We identified that sex-determining region Y-box 6 (SOX6) is direct downstream target of miR-499-5p. Silencing of SOX6 suppressed release of proangiogenic factors from ECs. Targeting miR-499-5p significantly enhanced SOX6 level in ECs from IHL of db/db mice. Finally, overexpression of SOX6 improved angiogenic property of ECs from IHL of db/db mice.
Conclusions: SKMC-sEV-mediated transfer of myo-miR-499-5p and subsequent suppression of SOX6 plays a critical role in diabetes-impaired angiogenesis in IHL of db/db mice. Targeting miR-499-5p-mediated pathogenic communication between SKMCs and ECs may be a novel avenue for therapeutics of critical limb ischemia in patients with diabetes.