Hypoxic Stimulation of Vasoreparative Functions in Human CD34 + Cells is Mediated by Angiotensin Converting Enzyme‐2 and Mas Receptor

CD34 + hematopoietic stem/progenitor cells (HSPCs) have the propensity of re‐endothelialization and vascular regeneration. Angiotensin Converting Enzyme‐2 (ACE2) generates the heptapeptide, Angiotensin (Ang)‐(1‐7), which produces vasoprotective effects by acting on Mas receptor (MasR). We have previously reported that hypoxic preconditioning robustly stimulated vascular repair‐relevant functions of CD34 + cells, which was impaired in MasR‐deficient murine HSPCs. The current study tested the hypothesis that hypoxic stimulation of HSPC functions are mediated by ACE2 and MasR. CD34 + cells were isolated from mononuclear cells (MNCs) derived from healthy volunteers (n=46). Exposure to normoxia (20% O 2 ) or hypoxia (1% O 2 ) was accomplished by using a hypoxia chamber (BioSpherix). Protein and mRNA expressions of ACE2 and MasR were determined in cells by western blotting or by flow cytometry and real‐time PCR, respectively. ACE2 activity was determined in cell‐lysates by using an enzyme‐specific fluorogenic substrate, 7‐Mca‐YVADAPK (Dnp) and a selective inhibitor, MLN4760. CD34 + cells were transduced with lentiviral particles (LV) carrying either ACE2‐, MasR‐ or scramble‐3′‐UTR fused downstream to firefly luciferase reporter gene. Luciferase activity was determined using a luminometer. 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 change in angiotensin converting enzyme (ACE) or angiotensin type 1 (AT1) receptor expression was found in cells exposed to hypoxia compared to normoxia. Accordingly, ACE2 activity was increased in the CD34 + cell lysates (n=6, P<0.05). In the presence of 2‐methoxyestradiol (0.5 μM), an inhibitor of hypoxia‐inducible factor‐1α (HIF‐1α), hypoxic upregulation of ACE2 or MasR was not observed (n=5, P<0.01 vs untreated). Luciferase activity was increased by hypoxia, compared to normoxia, in either ACE2‐ or MasR‐luciferase expressing cells (258±23% and 214±19%, respectively, higher than scramble, n=6, P<0.001). Co‐transfection experiments using ACE2 and MasR specific miRNA (miR‐421 or miR‐143 for ACE2 and MasR, respectively) confirmed that the observed luciferase activity in both normoxic and hypoxic condition was dependent on ACE2 or MasR, respectively. The effects of hypoxia were mimicked by stimulation with hypoxia‐regulated factors, Vascular Endothelial Growth Factor (VEGF) (100 nM) (n=5, P<0.01 vs untreated) and stromal‐derived factor‐1α (SDF) (100 nM) in normoxic conditions (n=5, P<0.05 vs untreated). In the presence of axitinib (30 nM), a nonspecific inhibitor of VEGFR, or AMD3100 (10 μM), a specific CXCR4 inhibitor, luciferase activity was decreased in ACE2‐ or MasR‐luciferase expressing cells (n=4, P<0.01) confirming transcriptional regulation of ACE2 and MasR by VEGF or SDF. Collectively, these results provide compelling evidence for the hypoxic upregulation of ACE2 and MasR in CD34 + cells, which largely contribute to the revascularization of ischemic areas following vascular injury. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .