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

Flap necrosis is a serious complication following reconstructive surgery for soft tissue coverage, with treatment at times requiring a return to the operating room or prolonged wound care.1 Inadequate neovascularization and insufficient production of pro-angiogenic mediators are likely culprits for the poor healing. Injection of adipose-derived mesenchymal stem cells (ADSCs) into skin flaps has been shown by several investigators to increase flap survival and angiogenesis in various skin flap models.2–5 Recently, extracellular vesicles (EVs) released from ADSCs as a cell-free alternative were also found to improve flap viability after ischemia–reperfusion injury.6 Bai et al determined that exposure of parent ADSCs to oxidative stress via a low concentration of hydrogen peroxide (H2O2) enhanced the angiogenic effects of the EVs, but the mechanisms by which this occurs remain unclear.7 The beneficial effects of mesenchymal stem cell (MSC)-based therapies have been proposed to be mediated predominantly by paracrine activity, to include soluble factors and EVs secreted from MSCs.8–11 EVs are composed of different types of vesicles including exosomes (40–200 nm), which are of endosomal origin, and microvesicles (150–1,000 nm), which directly bud from the cell membrane.8–11 Because current isolation methods cannot physically separate exosomes from small microvesicles, the term “EVs” in this manuscript refers to vesicles in the size range of exosomes. MSCs have been shown to release EVs that can selectively package and transfer proteins, nucleic acids including microRNAs (miRNAs), and lipids to injured tissues which are capable of modulating gene expression and the biological activity of recipient cells.8–11 MiRNAs are an important class of highly conserved, small (~22 nucleotide), nonprotein-coding RNAs that regulate gene expression via degradation or translational repression of their targeted mRNA transcripts.12 They are significant modulators of numerous fundamental physiological and cellular processes including angiogenesis, and their dysregulation is implicated in several diseases.12,13 Results from several studies indicate that angiogenesis-related miRNAs and proteins are the main components in EVs to exert their pro-angiogenic function (reviewed in Bian et al)14 ADSC-EVs are enriched with specific protein and miRNA cargo molecules that simultaneously activate their related signal pathway to regulate the expression of angiogenic factors in endothelial cells.13–17 EV cargo can change in response to different microenvironments, for example, EVs secreted under hypoxic conditions have been shown to contain increased expression of platelet-derived growth factor, epidermal-derived growth factor, fibroblast growth factor, and nuclear factor kappaB signaling pathway proteins and pro-angiogenic miRNAs including miR-126, miR-130a, and miR-210.18,19 The identity of which components of the EV proteome and miRNome responsible for the increased skin flap viability observed after H2O2 preconditioning of parent ADSCs has not been determined.7 Therefore, the aims of this study were to (1) use unbiased proteomics and next-generation sequencing approaches to comprehensively characterize the protein and miRNA content of EVs released from human ADSCs under normal culture conditions (control ADSC-EVs) and from ADSCs preconditioned with H2O2 (H2O2 ADSC-EVs) and (2) determine whether H2O2-induced changes in EV composition can increase viability of random pattern skin flaps in an established mouse model.