Abstract 4916: Hepatocyte Growth Factor Mediates Proangiogenic Effects by Activating the NADPH Oxidase Nox2
Hepatocyte growth factor (HGF) is a potent pro-angiogenic stimulus. We determined the effect of HGF on the mobilization of endothelial progenitor cells (EPCs) and endothelial aortic ring outgrowth as a model for ex vivo angiogenesis. As growth factor signaling frequently involves reactive oxygen species (ROS), we analyzed the effects of HGF on ROS formation and identified their enzymatic sources and the contribution of ROS to HGF signaling. In vivo application of HGF in mice induced the mobilization of EPCs (identified by FACS analysis as sca-1+, flk-1+, lineage negative mononuclear cells). Ex vivo, HGF stimulated endothelial cell outgrowth and increased the H2O2 formation of EPCs and the peroxynitrite formation in HUVEC as determined by peroxidase-catalyzed amplex red oxidation and DCHF-DA oxidation, respectively. This difference in ROS formation between EPCs and HUVECs was a consequence of the low expression of endothelial NO synthase (eNOS) in EPCs: In HUVECs, HGF increased the phosphorylation of eNOS at Ser1177, which increases the catalytic activity of the enzyme. As NADPH oxidases of the Nox family are important sources of ROS, their contribution to the signaling of HGF was determined. Down-regulation or genetic deletion of Nox2 prevented the HGF-stimulated ROS formation. As compared to wild type controls, the HGF-induced endothelial outgrowth was significantly reduced in aortic rings from Nox2 deficient (Nox2y/-) mice. Moreover, in vivo, the HGF-induced EPC mobilization was absent in Nox2y/- mice. Accordingly, HGF induced colony forming unit (CFU) formation in EPCs of WT but not Nox2y/- mice. ROS formation was essential for HGF-induced signaling: In HUVEC, HGF stimulated the phosphorylation of Jak2 and STAT3 and these effects were absent after depletion of the NADPH oxidase Nox2. We conclude that HGF-signaling critically involves the NADPH oxidase Nox2 and that ROS-formation by this enzyme contributes to the pro-angiogenic effects of HGF by stimulating EPC mobilization and endothelial activity.