Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro

Abstract

Mesenchymal stem/stromal cells (MSCs) display a variety of therapeutically relevant effects, such as the induction of angiogenesis, particularly under hypoxic conditions. It is generally recognized that MSCs exert their effects by secretion of paracrine factors and by stimulation of host cells. Furthermore, there is increasing evidence that some therapeutically relevant effects of MSCs are mediated by MSC-derived extracellular vesicles (EVs). Since our current knowledge on MSC-derived EVs released under hypoxic conditions is very limited, we aimed to characterize MSC-derived EVs from normoxic vs. hypoxic conditions (5% O2). Adipose-derived MSCs were grown under normoxic and hypoxic conditions, and EVs were analyzed by flow cytometry using lactadherin as a marker for EVs exposing phosphatidylserine, CD63 and CD81 as EV markers, as well as CD73 and CD90 as MSC surface markers. Particle concentration and size distribution were measured by nanoparticle tracking analysis (NTA), and the EV surface antigen signature was characterized using bead-based multiplex flow cytometry. Furthermore, we evaluated the potential of MSC-derived EVs obtained under hypoxic conditions to support angiogenesis using an in vitro assay with an hTERT-immortalized human umbilical vein endothelial cell (HUVEC) line. Proliferation and viability of MSCs were increased under hypoxic conditions. EV concentration, size, and surface signature did not differ significantly between normoxic and hypoxic conditions, with the exception of CD44, which was significantly upregulated on normoxic EVs. EVs from hypoxic conditions exhibited increased tube formation as compared to normoxic EVs or to the corresponding supernatants from both groups, indicating that tube formation is facilitated by EVs rather than by soluble factors. In conclusion, hypoxia conditioned MSC-derived EVs appear to be functionally more potent than normoxic MSC-derived EVs regarding the induction of angiogenesis.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…EVs are produced constitutively or upon activation, due to inflammation, hypoxia, oxidative stress, shear stress, senescence, cell death, exposure to bacterial endo/exotoxins, uremia, etc. [10][11][12][13][14][15]. The cargo varies depending on type and differentiation of the parent cell, microenvironmental variables, and agents that triggers EV release.…”

Biology and therapeutic potential of mesenchymal stem cell‐derived exosomes

“…Furthermore, the biogenesis and secretion of exosomes released from MSC also depend on external stimuli. For instance, MSC undergoing hypoxia or inflammation could influence the biomolecule packaging process into exosomes and affect their functional properties, such as the hypoxia‐conditioned MSC‐derived exosomes exerting more angiogenic activity than normoxic exosomes 12 . The Wnt and mTOR pathways are considered as “master regulators” involved in the enhancement of self‐renewal in MSC by increasing β‐catenin expression, and are also required for exosome secretion 13‐15 .…”

Current Knowledge and Future Perspectives on Mesenchymal Stem Cell-Derived Exosomes as a New Therapeutic Agent

“…Co-transplantation of exosomes with a fat graft resulted in increased expression of EGF, fibroblast growth factor, angiopoietin-1, and angiopoietin receptor (Tie-1) proteins [110]. It was observed that exosomes from hypoxia-primed MSCs versus MSCs cultured under normoxic conditions were more easily taken up by HUVECs; the uptaken exosomes promoted vascular endothelial growth factor (VEGF) expression and protein kinase A signaling pathway activation, which resulted in improved angiogenesis by HUVECs [111,112]. Exosomes from hypoxia-primed MSCs have also been shown to promote myocardial repair.…”