Macrophage M2 polarization induced by exosomes from adipose-derived stem cells contributes to the exosomal proangiogenic effect on mouse ischemic hindlimb

BACKGROUND: M2 macrophages and exosomes from adipose-derived stem cells (ASCs) are both reported to promote angiogenesis. However, the possible synergistic effects between exogenous exosomes and endogenous M2 macrophages are poorly understood. METHODS: Exosomes were isolated from conditioned medium of normoxic and hypoxic ASCs using the combined techniques of ultrafiltration and size-exclusion chromatography and were identified with nanoparticle tracking analysis and immunoblotting for exosomal markers. Macrophages were collected from the mouse peritoneal cavity. M1 and M2 macrophages were detected by immunoblotting for the intracellular markers inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) and by flow cytometry for the surface markers F4/80, CD86, and CD206. Murine models of Matrigel plug and hindlimb ischemia were employed as in vivo angiogenic assays. RESULTS: When M1 macrophages were treated with exosomes from normoxic ASCs (Nor/Exo), and particularly from hypoxic ASCs (Hyp/Exo), the expression of the M1 marker iNOS decreased, and the M2 marker Arg-1 increased in a time- and dose-dependent manner. Additionally, a decrease in the M1 surface marker CD86 and an increase in the M2 surface marker CD206 were observed, which suggested that M1 macrophages were polarized to an M2-like phenotype. Conditioned medium from these M2-like macrophages presented lower levels of proinflammatory cytokines and higher levels of proangiogenic factors and promoted endothelial cell proliferation, migration, and tube formation. Furthermore, M2 polarization and angiogenesis were induced upon the administration of exosomes in mouse Matrigel plug and hindlimb ischemia (HLI) models. Interestingly, these exosomal effects were attenuated by using a colony stimulating factor 1 receptor (CSF-1R) inhibitor, BLZ945, in vitro and in vivo. Downregulation of microRNA-21 (miR-21) in hypoxic ASCs reduced the exosomal effects on M2 polarization, Akt phosphorylation, and CSF-1 secretion. A similar reduction in exosomal activity was also observed when exosomes were administered along with BLZ945. CONCLUSION: Our findings provide evidence that exosomes from ASCs polarize macrophages toward an M2-like phenotype, which further enhances the exosomal proangiogenic effects. Exosomal delivery of miR-21 and positive feedback of secreted CSF-1 may be involved in macrophage polarization.