Extracellular vesicles from young-mouse plasma promote wound healing in aged mice and are associated with M2-like macrophage polarization

Background Wound healing is a challenging problem, especially in aging patients, where wound healing becomes more difficult. Young-mouse plasma EV-enriched preparations (young EVs) have anti-aging properties. Thus, the purpose of this study was to examine potential pathways and the impact of young EVs on wound healing. Methods Plasma EVs were isolated from young (2-month-old) and aged (20-month-old) male C57BL/6J mice and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. In vitro, macrophage senescence was induced with LPS (100 ng/mL, 24 h), and the effects of young EVs (15 μg/mL) on senescence markers and macrophage polarization were assessed. In vivo, 24 male C57BL/6J mice (6 young, 18 aged) were used. Two full-thickness circular wounds (6 mm) were created on the back of each mouse. Aged mice received subcutaneous injections of PBS, aged EVs, or young EVs (15 μg protein per wound, approximately 2 × 10 9 particles) at four sites around the wound margin once daily for 3 consecutive days. Wound closure was measured on days 0, 3, and 7. Angiogenesis, collagen synthesis, and macrophage polarization in wound tissue were evaluated on day 7. The involvement of the SIRT1-AMPK pathway was examined using the AMPK inhibitor compound C. Results Isolated EVs exhibited a circular vesicle morphology with a mean diameter of approximately 100 nm and were enriched for CD9, CD63, and TSG101. In LPS-induced macrophages, young EVs reduced the expression of senescence markers and decreased the proportion of SA-β-gal-positive cells (young EVs vs. LPS: 15.3% ± 3.1% vs. 36.7% ± 4.5%, mean ± SD, P < 0.001). Young EVs also decreased M1 markers (iNOS and CD86) and increased M2 markers (Arg-1 and CD206), while reducing TNF-α and IL-1β and increasing IL-10. In aged mice, young EV treatment accelerated wound closure on day 7 compared with PBS (wound closure rate: 0.36% ± 0.2% vs. 0.75% ± 0.8%, mean ± SD, P < 0.001), increased CD31-positive vessel density, and upregulated α-SMA, collagen I, and collagen III expression. Young EVs also increased the proportion of M2 macrophages (CD206 + F4/80 + ) and decreased M1 macrophages (CD86 + F4/80 + ) in wound tissue. Mechanistically, young EVs activated the SIRT1-AMPK pathway, and the AMPK inhibitor compound C reversed the effects of young EVs on macrophage senescence and polarization. Conclusions Young EVs could promote wound healing in senescent individuals by increasing M2 polarization and inhibiting cellular senescence through activation of the SIRT1-AMPK pathway. Accordingly, young EVs are a promising approach to the treatment of wound healing.