Reactive oxygen species contribute to angiogenesis as they promote migration, proliferation and tube-formation of endothelial cells. Particular in endothelial cells, the NADPH oxidase Nox4, which is a source of H₂O₂, is abundantly expressed. We generated a Nox4-knockout mouse (Nox4^-/-) and investigated the contribution of Nox4 to peripheral angiogenesis in response to voluntary exercise and ischemia after vascular occlusion. Although the running distance was similar between wildtype (WT) and Nox4^-/--mice, exercise induced skeletal muscle capilarization was observed only in WT- but not Nox4^-/--mice. In disease conditions as in the hindlimb ischemia model blood flow recovery in Nox4^-/--mice was significantly attenuated when compared to WT-animals. Accordingly depletion of H₂O₂ by injection of PEG-catalase also impaired the blood flow recovery in WT-mice. Importantly, genetic deletion of Nox1 or Nox2 did not affect blood flow recovery.

In vitro Nox4-deficient lung endothelial cells (LECs) exhibited a reduced capacity to form tubes on matrigel. Low amounts of H₂O₂ selectively rescued this phenotype, whereas PEG-Catalase impaired tube formation in WT-LECs. In vitro migration of WT-LEC was strongly induced by serum from WT-mice but not Nox4^-/--mice with hind limb ischemia. Analyses of the cytokine composition of the different sera revealed that VEGF level was significantly lower in the serum of Nox4^-/--mice when compared to those of WT-mice.

Conclusion: Nox4 is essentially involved in exercise- and hypoxia-induced angiogenesis in murine skeletal muscles. H₂O₂ produced by Nox4 is crucial for basal tube formation and Nox4-dependent VEGF expression is required for proper angiogenic responses in vivo.