Abstract-Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated through mobilization of EPCs from BM and their recruitment to the ischemic sites. Reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in postnatal neovascularization; however, their role in BM and EPC function is unknown. Here we show that hindlimb ischemia of mice significantly increases Nox2 expression and ROS production in BMmononuclear cells (BMCs), which is associated with an increase in circulating EPC-like cells. Mice lacking Nox2 show reduction of ischemia-induced flow recovery, ROS levels in BMCs, as well as EPC mobilization from BM. Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization, whereas WT mice transplanted with Nox2-deficient BM show reduced flow recovery and capillary density compared to WT-BM transplanted control. Intravenous infusion of WT-and Nox2-deficient BMCs into WT mice reveals that neovascularization and homing capacity are impaired in Nox2-deficient BMCs in vivo. In vitro, Nox2-deficient c-kit ϩ Lin Ϫ BM stem/progenitor cells show impaired chemotaxis and invasion as well as polarization of actins in response to stromal derived factor (SDF), which is associated with blunted SDF-1-mediated phosphorylation of Akt. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing, and angiogenic capacity of EPCs and BM stem/progenitor cells, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for promoting neovascularization in ischemic cardiovascular diseases. (Circ Res. 2008;103:212-220.)

Abstract-Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo. (Circ Res. 2008;102:1182-1191.)Key Words: protein tyrosine phosphatase 1B Ⅲ vascular endothelial growth factor Ⅲ endothelial cell Ⅲ cell-cell adhesions Ⅲ angiogenesis