Sustained FTO-Enriched 3D-ADSC-Derived Large Extracellular Vesicles Promote Angiogenesis and Wound Healing by Upregulating PPARγ

Extracellular vesicles (EVs) have shown considerable promise as cell-free therapeutics for wound repair. During EVs isolation, however, the large extracellular vesicles (LEVs) fraction is usually discarded, despite its larger vesicle size and abundant cargo. The biological function and mechanism of 3D-cultured adipose-derived stem cell-derived LEVs (3D-ADSC-LEVs) in wound repair remain unclear. Here, ADSC-derived LEVs were obtained using a 3D culture system and incorporated into hyaluronic acid methacrylate (HAMA) hydrogel for sustained local delivery. Compared with sEVs, LEVs more effectively protected H2O2-injured human umbilical vein endothelial cells, enhancing proliferation, migration, tube formation, and VEGF/CD31 expression. In a mouse full-thickness wound model, LEVs@HAMA accelerated wound closure, re-epithelialization, collagen deposition, and angiogenesis. Transcriptomic analysis identified activation of peroxisome proliferator-activated receptor (PPAR) signaling in LEVs-treated endothelial cells, while proteomic analysis revealed enrichment of fat mass and obesity-associated protein (FTO) in 3D-ADSC-LEVs. Further validation showed that LEVs restored FTO, PPARγ, VEGF, and CD31 expression under oxidative stress, whereas pharmacological FTO inhibition and siRNA-mediated PPARγ knockdown attenuated LEV-induced VEGF/CD31 recovery and tube formation. These findings indicate that FTO-enriched 3D-ADSC-LEVs promote angiogenic wound repair through the PPARγ-VEGF/CD31 axis and support LEVs@HAMA as a promising cell-free strategy for tissue regeneration.