Extracellular vesicles derived from hypoxia-preconditioned bone marrow mesenchymal stem cells ameliorate lower limb ischemia by delivering miR-34c

Hypoxic mesenchymal stem cell-derived extracellular vesicles (EVs) have been suggested as a promising therapy for various diseases. This study aims to determine the effect of EVs derived from bone marrow mesenchymal stem cells (BMMSCs) under hypoxia on lower limb ischemia and the underlying mechanism. Human BMMSCs were subjected to hypoxia or normoxia followed by the isolation of EVs. Nanoparticle trafficking analysis (NTA), transmission electron microscopy (TEM), and Western Blotting using corresponding markers were performed to confirm the EVs. The EVs from BMMSCs under hypoxia condition (Hyp-EVs) or normoxia condition (Nor-EVs) were subjected to hindlimb ischemia (HI) mice. MiR-34c expression in BMMSCs and BMMSC-EVs was detected. The role of miR-34c in regulating M2 macrophage polarization, as well as the target of miR-34c, were explored. HI mice with Hyp-EV treatment, as compared to the Nor-EV or the PBS group, had better blood flow and higher capillary density. MiR-34c expression was increased in BMMSCs, BMMSC-EVs, and the adductor muscle of HI mice. Hyp-EVs promoted the M2 macrophage polarization and anti-inflammatory cytokine production, and enhanced the blood flow and capillary density in HI mice, while the knockdown of miR-34c partly reversed these effects. PTEN is a target of miR-34c, and the PTEN silencing facilitated M2 macrophage polarization, whereas the inhibition of AKT signaling partly abolished the effect. Hyp-EVs promoted M2 macrophage polarization by delivering miR-34c via PTEN/AKT pathway, which could be a promising therapeutic strategy to ameliorate lower limb ischemia.