Hypoxia-Preconditioned Adipose Stem Cell Exosomes Promote Adipose Graft Vascular Regeneration via miRNA-126

Background and Objective Adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASCs-Exos) possess angiogenic potential, which can enhance the retention rate of fat grafts. Hypoxic preconditioning can augment their functionality. However, the optimal conditions for hypoxic preconditioning and the specific mechanisms by which it exerts its effects are not well defined. This study aimed to screen for the optimal hypoxic culture conditions for ASCs and to preliminarily elucidate the mechanisms by which they exert their effects. Methods Flow cytometry and TUNEL fluorescence staining were employed to determine the optimal oxygen concentration and culture duration for hypoxic preconditioning of ASCs-Exos. Subsequently, a mixture of human adipose tissue and hypoxic preconditioned ASCs-Exos was implanted into the subcutaneous fat of nude mice. At 4 and 12 weeks post-transplantation, the fat graft specimens were weighed, and hematoxylin and eosin (H&E) staining and immunohistochemistry were performed to examine the samples. The expression of CD31 was assessed to analyze the formation of neovascularization. The expression of miRNA in Exos was then detected, and the influence of ASCs-Exos derived miRNA-126 on the proliferative capacity of neovascularization was verified through an EDU fluorescence assay. Results ASCs-Exos exhibited optimal performance when cultured under an oxygen concentration of 5% for 24 h. After transplantation of human adipose tissue mixed with hypoxic preconditioned ASCs-Exos for 4 to 12 weeks, the weight of fat grafts and the number of viable adipocytes in the hypoxic ASCs-Exos group were significantly higher than those in the control group (P < 0.05), with better adipocyte integrity and enhanced neovascularization capacity. Detection of miRNA derived from hypoxic preconditioned ASCs-Exos revealed that miRNA-126 demonstrated robust angiogenic and proliferative capabilities in both in vitro and in vivo experiments. Conclusions In summary, hypoxic preconditioning of ASCs-Exos at 5% oxygen for 24 h significantly enhances their angiogenic properties, thereby improving the viability and retention of fat grafts. The study elucidates miRNA-126 as a critical mediator of neovascularization and adipocyte survival, offering a promising avenue for enhancing the efficacy of ASCs-Exos in regenerative medicine. No Level Assigned This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.