Abstract 773: The NADPH Oxidase Nox2 Is Required For Hypoxia-induced Mobilization Of Endothelial Progenitor Cells
Hypoxia plays a major role in the regulation of vascular homeostasis and provides a potent stimulus for angiogenesis. Hypoxia also affects reactive-oxygen species (ROS) production and signalling. Endothelial progenitor cells (EPC) contribute to angiogenesis but the impact of hypoxia and ROS on mobilization of these cells is unclear. We studied the role of hypoxia for EPC mobilization and the contribution of the NADPH oxidase Nox2, the most important source of ROS in endothelial cells, in this process. Murine EPCs, defined as lineage negative and sca-1+/flk-1+, were measured by FACS from peripheral blood. Hypoxia (10%, 48hours) induced mobilization of EPC in wild-type mice but not in NADPH oxidase knockout mice (Nox2y/−). Colony forming unit (CFU) assays on methylcellulose were used as an index for repair capacity. In vivo hypoxia induced a marked increase in CFUs from blood of WT but not from blood of Nox2y/− mice. Through the activation of HIF-1 alpha, hypoxia induces the formation of vascular endothelial growth factor (VEGF) and erythropoietin (EPO), which both might contribute to EPC mobilization. Despite the difference in mobilization, EPO production in response to hypoxia was less pronounced in WT than in Nox2y/− mice. Interestingly, also the EPO-induced mobilization of EPCs was greater in Nox2y/− as compared to WT mice. These data suggest that EPO is not responsible for the difference in EPC mobilization between WT and Nox2y/− mice in response to hypoxia. In contrast to EPO, quantitative RT-PCR revealed that VEGF was differentially regulated between WT and Nox2y/− mice: Hypoxia induced a marked induction of VEGF A, B, C as well as of the VEGF receptor 2 in WT but not in Nox2y/− mice. Moreover, unlike WT cells, VEGF-induced migration was completely absent in endothelial cells cultured from Nox2y/− mice. In contrast to these observations in endothelial cells, VEGF-induced invasion of bone marrow cells using a boyden chamber assay was similar in both mice strains. We conclude that Nox2 is involved in hypoxia-induced EPC mobilization. This effect seems to be mediated by differences in VEGF expression and signalling and by the responsiveness of bone marrow endothelial cells involved in the process of ECP mobilization of EPCs.