Endothelial progenitor cells (EPC) promote vascular repair but the impact of systemic hypoxia on EPC function is unclear. Hypoxia is a strong stimulus for angiogenesis and affects reactive-oxygen species (ROS) production and signalling. We studied the role of hypoxia for EPC mobilization and vascular repair and the contribution of the NADPH oxidase Nox2, the most important source of ROS in endothelial cells, in this process.

Murine EPCs were defined as linage negative and sca-1/flk-1 positive and measured by FACS from whole blood. Nox2 is the predominant NADPH oxidase in these cells as analysed by quantitative RT-PCR. Vascular repair was induced by the carotid artery wire and electro injury model. Hypoxia (10%, 96hours) induced mobilisation of EPC and improved vascular repair after injury in wild-type mice but not in NADPH oxidase knockout mice (Nox2y/-). Hypoxia induces the formation of erythropoietin (EPO), which might contribute to EPC mobilization. EPO production in response to hypoxia however was enhanced in Nox2y/- as compared to WT mice. Importantly, EPO, which induced pronounced EPC mobilization in WT mice, failed to do so in Nox2-/- mice. These data suggest that EPO signalling is defective in Nox2y/- mice. Indeed, in EPCs from WT-mice, EPO induced ROS production and proliferation and both responses were missing in EPCs from Nox2y/- mice as well as in Nox2 antisense oligonucleotide transfected human EPCs. The activation of the downstream signaling of the EPO-receptor was reduced as revealed by luciferase reportergene assay. As vascular repair by EPCs includes the homing onto the area of damage we used a in vitro model were EPCs were allowed to attach onto a monolayer of endothelial- or smooth muscle cells. Attachement of EPCs was attenuated in the absence of Nox2 as well as after treatment with radical scavengers or inhibitors of ROS-production.

We conclude that Nox2-derived ROS are required for EPO-signalling in stem cells and that hypoxia-induced EPC mobilisation and vascular repair requires the NADPH oxidase Nox2.