Identification of Angiogenic Cargoes in Human Fibroblasts-Derived Extracellular Vesicles and Induction of Wound Healing

A complete redevelopment of the skin remains a challenge in the management of acute and chronic wounds. Recently, the application of extracellular vesicles (EVs) for soft tissue wound healing has received much attention. As fibroblasts are fundamental cells for soft tissues and skin, we investigate the proangiogenic factors in human normal fibroblast-derived EVs (hNF-EVs) and their effects on wound healing. Normal fibroblasts were isolated from human skin tissues and characterized by immunofluorescence (IF) and Western blotting (WB). hNF-EVs were isolated by ultracentrifugation and characterized using transmission electron microscopy and WB. The proangiogenic cargos in hNF-EVs were identified by a TaqMan assay and a protein array. Other in vitro assays, including internalization assays, cell counting kit-8 analysis, scratch wound assays, WBs, and tube formation assays were conducted to assess the effects of hNF-EVs on fibroblasts and endothelial cells. A novel scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue was applied onto full-thickness skin wounds in mice. The wound healing therapeutical effect of hNF-EVs was assessed by calculating the rate of wound closure and through histological analysis. Isolated hNF was confirmed by verifying the expression of the fibroblast markers vimentin, αSMA, Hsp70, and S100A4. Isolated hNF-EVs showed intact EVs with round morphology, enriched in CD81 and CD63, and devoid of the cell markers GM130, Calnexin, and Cytochrome C. Our TaqMan assay showed that hNF-EVs were enriched in miR130a and miR210, and protein arrays showed enriched levels of the proangiogenic proteins’ vascular endothelial growth factor (VEGF)-D and CXCL8. Next, we found that the internalization of hNF-EVs into hNF increased the proliferation and migration of hNF, in addition to increasing the expression of bFGF, MMP2, and αSMA. The internalization of hNF-EVs into the endothelial cells increased their proliferation and tube formation. A scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue accelerated the wound healing rate in full-thickness skin wounds in mice, and the treatments increased the cellular density, deposition, and maturation of collagens in the wounds. Moreover, the scaffold-free noninvasive delivery of hNF-EVs with or without fibrin glue increased the VEGF and CD31 expression in the wounds, indicating that hNF-EVs have an angiogenic ability to achieve complete skin regeneration. These findings open up for new treatment strategies to be developed for wound healing. Further, we offer a new approach to the efficient, scaffold-free noninvasive delivery of hNF-EVs to wounds.