Abstract 180: Blood Outgrowth Endothelial Cell-derived Exosomes Mediate Therapeutic Neovascularization

Background: Blood outgrowth endothelial cells (BOECs) mediate therapeutic neovascularization in experimental models. We hypothesized that BOECs promote angiogenesis via secretion of exosomes, extracellular vesicles with an important role in cell-to-cell communication. Methods: We derived exosomes from BOECs of patients with ischemic heart failure (HF with LVEF <35%) and age-matched controls (CON) in normoxia and 1% O 2 hypoxia by differential ultracentrifugation and used nano tracking (NTA) and immunoblot analysis to identify size, number, and surface markers. We characterized protein and miRNA content by proteomic LC-MS-Orbitrap analysis of trypsin-digested exosomes and validated selected peptides using ELISA. We measured expression of pro-angiogenic miRNAs and tested in vitro angiogenic potential of HUVECs in the presence or absence of exosomes using matrigel 2D-tube formation and 3D-spheroid sprouting assays. We compared in vivo angiogenesis using a wound healing model in nude mice. Results: NTA showed higher exosome concentrations in hypoxia compared to normoxia (14.38±0.23x10E8 vs 12.05±0.23x10E8 particles/ml, n=7, P<0.001) with robust expression of exosome markers TSG101 and Flotilin-1. Compared to serum stimulation (positive control), BOEC-derived exosomes similarly enhanced 2D vascular network growth and 3D spheroid sprouting angiogenesis whereas absence of serum or exosomes did not (P<0.001). Bioinformatic proteome analysis identified 249 shared proteins in HF and CON, and 72 proteins exclusively present in HF with abundant expression of matricellular proteins and angiogenic growth factors. Exosomes derived in hypoxia contained significantly higher levels of pro-angiogenic miR-210-3p, miR-210-5p and miR-21-3p (P<0.05 vs normoxia, n=6). Closure of the skin wound after 4 and 5 days was significantly better in animals treated with BOEC-derived exosomes and accompanied with greater density of CD31-positive neovessels. Conclusion: BOEC-derived exosomes effectively induce vascular network formation and sprouting angiogenesis and stimulate wound healing in vivo. The identified protein and miRNA content in BOEC-derived exosomes may constitute a promising allogenic approach for therapeutic neovascularization.