Physiological oxygen conditions enhance the angiogenic properties of extracellular vesicles from human mesenchymal stem cells

Abstract Background Following an ischemic injury to the brain, the induction of angiogenesis is critical to neurological recovery. The angiogenic benefits of mesenchymal stem cells (MSCs) have been attributed at least in part to the actions of extracellular vesicles (EVs) that they secrete. EVs are membrane-bound vesicles that contain various angiogenic biomolecules capable of eliciting therapeutic responses and are of relevance in cerebral applications due to their ability to cross the blood–brain barrier (BBB). Though MSCs are commonly cultured under oxygen levels present in injected air, when MSCs are cultured under physiologically relevant oxygen conditions (2–9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. There is a need to determine the effects of MSC-EVs in models of cerebral angiogenesis and whether those from MSCs cultured under physiological oxygen provide greater functional effects. Methods Human adipose-derived MSCs were grown in clinically relevant serum-free medium and exposed to either headspace oxygen concentrations of 18.4% O2 (normoxic) or 3% O2 (physioxic). EVs were isolated from MSC cultures by differential ultracentrifugation and characterized by their size, concentration of EV specific markers, and their angiogenic protein content. Their functional angiogenic effects were evaluated in vitro by their induction of cerebral microvascular endothelial cell (CMEC) proliferation, tube formation, and angiogenic and tight junction gene expressions. Results Compared to normoxic conditions, culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance. Conclusions EVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function.