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

Unlabelled: 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 (HO ADSC-EVs) and whether HO ADSC-EVs can increase viability of random pattern skin flaps.

Methods: EVs secreted by human ADSCs were isolated after culture in EV-depleted medium ± HO. 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, HO 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 HO 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 HO ADSC-EVs. Two miRNAs were increased, and 12 (including 10 antiangiogenic miRNAs) were reduced in HO ADSC-EVs. Enhanced viability ( < 0.05) of flaps treated with HO ADSC-EVs compared with vehicle corresponded to increased capillary density in the HO group ( < 0.001).

Conclusion: Altered protein and miRNA content in ADSC-EVs after HO pretreatment likely contributes to enhanced therapeutic effects on flap survival observed in preclinical models.