Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization involves not only angiogenesis but also mobilization of EPCs from BM and their recruitment to the ischemic sites. We demonstrated that reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in reparative angiogenesis induced by hindlimb ischemia. However, role of Nox2-derived ROS in BM and EPC function in postnatal neovascularization is unknown. Here we show that Nox2 is the most highly expressed Nox enzymes in mouse BM cells (BMCs) and EPCs. Hindlimb ischemia of mice significantly increases Nox2 mRNA expression (2.3-fold) and ROS production (7.2-fold) in BMCs at 3 days after surgery, which is associated with an increase in number of EPC-like c-kit+Flk-1+ cells in peripheral blood (3.9-fold). Nox2-deficient mice show impairment of ischemia-induced flow recovery (68% inhibition) and significant reduction of ROS levels in BM (98% decrease) and EPC mobilization, as assessed by EPC culture assay (76% decrease) and FACS analysis of c-kit+Flk-1+ cells (33% decrease). Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization. Conversely, WT mice transplanted with Nox2-deficient BM show significant decrease of flow recovery (41% decrease) and capillary density (24% decrease) compared to WT-BM transplanted control. Intravenous infusion of WT-BM-mononuclear cells (MNCs), but not Nox2-deficient MNCs, into WT mice at 1 day after hindlimb ischemia significantly promotes neovascularization (37% increase). Infusion of fluorescent dye-labeled WT- and Nox2-deficient BMCs reveals that homing capacity of Nox2-deficient BMCs in ischemic border zone is significantly reduced (52% decrease). In vitro, VEGF-induced EPC migration (48% decrease) and BMCs invasion (68% decrease) are significantly inhibited in Nox2-deficient cells. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing and angiogenic capacity of EPCs, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for ischemic cardiovascular diseases.
The role of bone marrow-derived endothelial progenitor cells and angiogenic responses in chronic obstructive pulmonary disease