SFL-3D-Cultured Adipose Mesenchymal Cell-Derived Extracellular Vesicles Promote Diabetic Wound Healing by Alleviating Microvascular Endothelial Senescence via PI3K/AKT/mTOR/4EBP1-Mediated Cap-Dependent Translation

Abstract Background Diabetic foot ulcers (DFUs) are severe complications of diabetes, and treatment options for DFUs are limited. Current research on impaired angiogenesis in DFUs predominantly relies on generic endothelial cells, which inadequately reflect the pathophysiological microenvironment of the diabetic microvasculature. In contrast, this study focused specifically on human dermal microvascular endothelial cell (HDMEC) senescence as a central mechanism in DFU progression. We used a self-feeder layer 3D (SFL-3D) culture system to reprogram adipose-derived mesenchymal stem cells (ADSCs) to prepare functionally enhanced three-dimensional adipose stem cells (tdASCs) and their extracellular vesicles (tdASC-EVs) to mitigate HDMEC senescence and improve diabetic healing. Methods EVs were isolated from SFL-3D-induced tdASCs and characterized. In vitro, high-glucose-induced human dermal microvascular endothelial cells were treated with tdASC-EVs, after which senescence, oxidative stress, mitochondrial function, and angiogenesis were assessed. Pathway-specific inhibitors and a phosphorylation-mimetic 4EBP1 plasmid were used to validate the mechanistic involvement of the PI3K/AKT/mTOR/4EBP1 axis and cap-dependent translation via m7GTP pull-down. In vivo, a fat graft model was used to assess the stem cell function of tdASCs, whereas a diabetic wound model was used to evaluate the therapeutic efficacy of tdASC-EVs. Results tdASC-EVs significantly attenuated high-glucose-induced HDMEC senescence, oxidative stress, and mitochondrial dysfunction while increasing tube formation. Mechanistically, tdASC-EVs activated PI3K/AKT/mTOR signalling, increased 4EBP1 phosphorylation, and promoted eIF4E–eIF4G assembly. In diabetic mice, tdASC-EVs accelerated wound closure and increased microvascular density, which correlated with reduced p16 expression and increased CD31 and p-4EBP1 expression. In the Bama miniature pig diabetic large animal model, tdASC-EVs exhibited markedly superior therapeutic effects compared with those induced by conventional ADSC-EVs, as evidenced by significantly faster wound closure, greater neovascularization density, narrower scar width, and more orderly collagen deposition. Conclusions tdASC-EVs ameliorated diabetic wound healing by targeting HDMEC senescence through PI3K/AKT/mTOR/4EBP1-mediated cap-dependent translational activation. The preclinical large animal data further confirmed the superior efficacy of tdASC-EVs over ADSC-EVs, highlighting the therapeutic potential of SFL-3D-modified EVs for DFU treatment.