Vascular endothelial cells (ECs) are responsible for post-ischemic angiogenesis, a process that is regulated by reactive oxygen species. Recent studies indicate that endothelial Nox4 based NADPH oxidase may have a key role. This study examines the role of endothelial Nox4 in ischemia-induced angiogenesis and explores the potential mechanisms involved. Mouse lines overexpressing human Nox4 wild type (EWT) or its dominant negative form P437H (EDN) specifically in the endothelium were used. Non-transgenic littermate mice (NTg) were used as controls. Following hind limb ischemia, blood flow recovery was enhanced in EWT and was impaired in EDN compared with NTg. The critical angiogenesis regulating genes vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS) and transforming growth factor beta 1 (TGF beta 1) were upregulated in EWT both in the ischemic muscle and in heart ECs, while TGF beta 1 was downregulated in EDN ECs. In EC, both VEGFA and TGF beta 1 stimulated EC proliferation, migration, and capillary-like network formation in EWT but failed to do so in EDN. Application of TGF beta 1 increased both VEGFR2 and eNOS expression levels, whereas blocking TGF beta 1 or addition of catalase inhibited the phosphorylation of VEGFR2 and eNOS, indicating H2O2 and TGF beta 1 signaling downstream of Nox4 is critical to maintain EC angiogenic functions. Use of cell specific transgenic mice with both upregulation and downregulation of endothelial Nox4 indicate several mechanisms linked to Nox4 play a role in angiogenesis. Endothelial Nox4 regulates ischemia-induced angiogenesis, likely through H2O2- and TGF beta 1-mediated activation of cell signaling pathways essential for endothelial function. (C) 2014 Elsevier B.V. All rights reserved.