Abstract P244: Hypoxic Stimulation of Vasoreparative Functions in Human CD34 + cells is Mediated by Angiotensin Converting Enzyme-2 and Mas Receptor

Abstract

CD34 + stem/progenitor cells have the propensity of re-endothelialization and vascular regeneration. Angiotensin Converting Enzyme-2 (ACE2) generates Angiotensin (Ang)-(1-7), which produces vasoprotection by acting on Mas receptor (MasR). Hypoxic preconditioning has been shown to stimulate vascular repair-relevant functions of CD34 + cells, which was impaired in MasR-deficient HSPCs. The current study tested the hypothesis that hypoxic stimulation of CD34 + cell functions are mediated by ACE2 and MasR. CD34 + cells were isolated from mononuclear cells (MNCs) derived from healthy volunteers (n=46). Cells were exposed to normoxia (20% O 2 ) or hypoxia (1% O 2 ). Protein and mRNA expressions of ACE2 and MasR were determined. ACE2 activity was determined by using an enzyme-specific fluorogenic substrate, and a selective inhibitor, MLN4760. CD34 + cells were transduced with lentiviral particles carrying ACE2- or MasR- or scramble-3’-UTR (Scr) fused downstream to firefly luciferase reporter (flr) gene. Co-transfection luciferase assay was performed using MicroRNA, miR-421 or miR-143 with ACE-2 or MasR luciferase construct, respectively. Luciferase activity was determined by using luciferase assay kit. Hypoxia stimulated mRNA and protein expressions of ACE2 and MasR in CD34 + cells (54.9±4.8% and 51.6±14.7%, respectively, higher than normoxia, n=5, P<0.05) but not in MNCs. No changes in ACE or AT1 receptor expressions were observed. Effects of hypoxia were blocked by 2-methoxyestradiol (0.5μM), an inhibitor of hypoxia-inducible factor-1α (n=5, P<0.01). Luciferase activity was increased by hypoxia in cells expressing ACE2- or MasR-FLR (258±23% and 214±19%, respectively, of Scr, n=6, P<0.001). Effect of hypoxia is recapitulated by vascular endothelial growth factor (100 nM) (n=5, P<0.01 vs untreated) or stromal-derived factor-1α (100 nM) under normoxia (n=5, P<0.05 vs untreated). Axitinib (30 nM), a nonspecific inhibitor of VEGFR, or AMD3100 (10 μM), a specific CXCR4 inhibitor, decreased the luciferase activity in ACE2- or MasR-FLR expressing cells (n=4, P<0.01). Collectively, these observations provide compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34 + cells, which contribute to their vasoreparative functions.