Oxidative Stress Alters Angiogenic and Antimicrobial Content of Extracellular Vesicles and Improves Flap Survival

Extracellular vesicles (EVs) secreted from adipose-derived mesenchymal stem cells (ADSCs) (ADSC-EVs) improve flap survival after ischemia–reperfusion injury. Exposure of parent ADSCs to oxidative stress has been shown to enhance this effect, but mechanisms are unclear. We aimed to determine whether angiogenesis-promoting protein and microRNA (miRNA) content is altered in EVs after preconditioning with hydrogen peroxide (H(2)O(2) ADSC-EVs) and whether H(2)O(2) ADSC-EVs can increase viability of random pattern skin flaps. METHODS: EVs secreted by human ADSCs were isolated after culture in EV-depleted medium ± H(2)O(2). Nanoparticle tracking analysis determined size and concentration of purified EVs. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. ADSC-EVs, H(2)O(2) ADSC-EVs, or vehicle were injected into random pattern skin flaps of BALB/c mice (4–5 mice per group). Viable and necrotic areas were measured on day 7, and tissues underwent histologic analysis. RESULTS: Angiogenic and antimicrobial protein content of EVs was altered with H(2)O(2) preconditioning. Functional enrichment analysis identified constitutive photomorphogenesis 9 signalosome (known to direct vascular endothelial growth factor production) as the major enriched Gene Ontology term unique to H(2)O(2) ADSC-EVs. Two miRNAs were increased, and 12 (including 10 antiangiogenic miRNAs) were reduced in H(2)O(2) ADSC-EVs. Enhanced viability (P < 0.05) of flaps treated with H(2)O(2) ADSC-EVs compared with vehicle corresponded to increased capillary density in the H(2)O(2) group (P < 0.001). CONCLUSION: Altered protein and miRNA content in ADSC-EVs after H(2)O(2) pretreatment likely contributes to enhanced therapeutic effects on flap survival observed in preclinical models.