A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4′-hydroxy-3′methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal–regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.

Rationale:: The function of Nox4, a source of vascular H ₂ O ₂ , is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. Objective:: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4 ^−/− mice. Methods and Results:: Nox4 was highly expressed in the endothelium and contributed to H ₂ O ₂ formation. Nox4 ^−/− mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4 ^−/− lung endothelial cells (LECs) was attenuated and restored by low concentrations of H ₂ O _2, whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. Conclusion:: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

Aging is considered to be the major risk factor for the development of atherosclerosis and, therefore, for coronary artery disease. Apart from age-associated remodeling of the vascular wall, which includes luminal enlargement, intimal and medial thickening, and increased vascular stiffness, endothelial function declines with age. This is most obvious from the attenuation of endothelium-dependent dilator responses, which is a consequence of the alteration in the expression and/or activity of the endothelial NO synthase, upregulation of the inducible NO synthase, and increased formation of reactive oxygen species. Aging is also associated with a reduction in the regenerative capacity of the endothelium and endothelial senescence, which is characterized by an increased rate of endothelial cell apoptosis.

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O₂^̇̄), Nox4 produces hydrogen peroxide (H₂O₂). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H₂O₂ versus L-012 and cytochrome c for O₂^̇̄. The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H₂O₂ to O₂^̇̄ generation while preserving expression and intracellular localization. In the presence of an NO donor, the O₂^̇̄-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O₂^̇̄. In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H₂O₂ but increased O₂^̇̄ formation by Nox4 without affecting Nox1-dependent O₂^̇̄ formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H₂O₂. Mutation of this but not of four other conserved histidines also switched Nox4 from H₂O₂ to O₂^̇̄ formation. Thus, H₂O₂ formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O₂^̇̄ egress and/or provide a source for protons, allowing dismutation to form H₂O₂.

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.