The Nox family NADPH oxidases are reactive oxygen species (ROS)-generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE^−/−) mice. Wild-type (C57Bl6/J), ApoE^−/−, and Nox2^−/y/ApoE^−/−mice were maintained on a high-fat (21%) diet from 5 wk of age until they were 12 or 19 wk old. Mice were euthanized and their aortas removed for measurement of Nox2 expression (Western blot analysis and immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to N^ω-nitro-l-arginine), and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE^−/−mice before the appearance of lesions, and this was associated with elevated ROS levels. Within developing plaques, macrophages were also a prominent source of Nox2. The absence of Nox2 in Nox2^−/y/ApoE^−/−double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 wk of age. However, an en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2^−/y/ApoE^−/−versus ApoE^−/−mice, and this was associated with a marked decrease in aortic ROS production and an increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability, and early lesion development in ApoE^−/−mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis.

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2^−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8⁺ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2^−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2^−/y mice. The numbers of influenza-specific CD8+D^bNP₃₆₆+ and D^bPA₂₂₄+ T cells in the BALF and spleen were comparable in WT and Nox2^−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner. Influenza A virus pandemics are imminent and with emerging anti-viral resistance highlight an ongoing, urgent need for novel generic pharmacological strategies. Ideally these strategies should reduce both viral replication and lung inflammation, irrespective of the infecting strain by modulating the host immune response. An important paradigm strongly suggests that the lung damage arising from not only influenza A viruses but other pathogens including, but not restricted to, SARS, parainfluenza viruses, human respiratory syncytial virus and Streptococcus pneumoniae consists of an excessive host response characterised by a rapid, influx of inflammatory cells into the lungs leading to excessive reactive oxygen species (ROS) production. Our study demonstrates that the primary enzymatic source of inflammatory cell ROS, Nox2-containing NADPH oxidase, promotes airways inflammation to low and high pathogenicity influenza A virus infection and impedes with the host's ability to clear the virus. Thus, Nox2 inhibitors could be considered individually or in combination with current antiviral strategies for control of future influenza A virus pandemics.

Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here we investigate the regulation of NADPH oxidase activity by nitric oxide (NO). Human cultured microvascular endothelial cells (HMEC-1) were treated with the NO donors, diethylenetriamine (DETA)-NONOate, S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 0.5-24 h. Superoxide production was measured by lucigenin chemiluminescence and dihydroethidium fluorescence, while NADPH oxidase subunit expression was measured via Western blotting. S-nitrosylation was assessed using the 2,3-diaminonapthalene (DAN) assay, and via immunoblotting with an anti-nitrosocysteine antibody. Specific siRNA reduced Nox2 and Nox4 protein expression and markedly decreased superoxide production in HMEC-1. DETA-NONOate (10-300 micromol/L) suppressed superoxide production in HMEC-1 in a concentration- and time-dependent manner, which was not entirely attributable to stoichiometric reaction with NO, for the effect was observed more than 6 h after removing DETA-NONOate from solution. Similarly, sustained attenuation of superoxide production was achieved with SNP (10-100 micromol/L) and SNAP (10-100 micromol/L). The suppressive effect of NO was not dependent on (1) the sGC/cGMP/PKG pathway, (2) peroxynitrite-formation, (3) reduced protein expression of NADPH oxidase subunits or (4) dissociation of NADPH oxidase subunits. Treatment with NO caused S-nitrosylation of the crucial organizer subunit p47phox, and de-nitrosylation with UV light restored superoxide production. NO causes sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells by S-nitrosylation of p47phox. These findings highlight a novel approach by which vascular oxidative stress might be suppressed by NO donors.

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.