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.

NADPH oxidases are important sources of vascular superoxide, which has been linked to the pathogenesis of atherosclerosis. Previously we demonstrated that the Nox4 subunit of NADPH oxidase is a critical catalytic component for superoxide production in quiescent vascular smooth muscle cells. In this study we sought to determine the role of Nox4 in superoxide production in human aortic smooth muscle cells (AoSMC) and embryonic kidney (HEK293) cells under proinflammatory conditions. Incubation with tumor necrosis factor-α (TNF-α, 10 ng/ml) for 12h increased superoxide production in both cell types, whereas angiotensin II, platelet-derived growth factor or interleukin-1β had little effects. Superoxide production was completely abolished by the NADPH oxidase inhibitors diphenyline iodonium and apocynin, but not by inhibitors of xanthine oxidase, nitric oxide synthase or mitochondrial electron transport. TNF-α upregulated the expression of Nox4 in AoSMC at both message and protein levels, while Nox1 and Nox2 were unchanged. In contrast, upregulation of Nox2 appeared to mediate the enhanced superoxide production by TNF-α in HEK293 cells. We suggest that Nox4 may be involved in increased superoxide generation in vascular smooth muscle cells under proinflammatory conditions.

The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNFα) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2′,7′-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NFκB) was measured by luciferase activity assay. TNFα stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5–1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNFα-induced NFκB promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNFα whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNFα-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNFα-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNFα-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NFκB pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.

Purpose: Corneal injury that occurs after burning with alkali initiates wound-healing processes, including inflammation, neovascularization, and fibrosis. Excessive reactions to injury can reduce corneal transparency and thereby compromise vision. The NADPH oxidase (Nox) enzyme complex is known to be involved in cell signaling for wound-healing angiogenesis, but its role in corneal neovascularization has been little studied. Methods: The center corneas of wild-type and Nox4 knockout (KO) mice were injured with 3 µL 1 M NaOH, while the contralateral corneas remained untouched. On day 7, mRNA expression levels of NADPH oxidase isoforms, the proangiogenic factors VEGF-A and TGFβ1, and proinflammatory genes ICAM-1 and VCAM-1 were determined. Corneal neovascularization and fibrosis were visualized using PECAM-1 antibody and picrosirius red staining, respectively, on the same day. Results: Expressions of both Nox2 and Nox4 gene isoforms as well as the above genes were markedly increased in the injured corneas at 7 days. Injured corneas showed neovascularization and fibrosis as well as an increase in clinical opacity score. All responses stimulated by alkali burn were abrogated in Nox4 KO mice. Conclusions: Nox4 could be a new target to treat pathologic corneal wound-healing responses and such targeting might prevent blindness caused by burn injuries.

Objective Reactive oxygen species including superoxide have been shown to promote atherogenesis. We previously showed that a major source of superoxide, the NADPH oxidase system, is upregulated in the intima and adventitia during remodelling induced by periarterial collars in rabbits. We have now examined the action of the NADPH oxidase inhibitor apocynin, given via the adventitia, on the neointima formation and endothelial function in this model. Methods Perivascular collars were implanted around the common carotid arteries of male NZW rabbits for 14 days to induce intimal thickening. The periarterial space of one collar was filled with apocynin (1 mM) while the contralateral collar with the vehicle (0.1% DMSO). Results After 14 days, local treatment with apocynin via the adventitia, reduced superoxide generation. In addition, apocynin significantly reduced neointima formation and proliferation of cells in both the neointima and adventitia. Moreover, NO-dependent vasorelaxation to acetylcholine, which is normally impaired in collared arteries, was improved, and apocynin suppressed the endothelial expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1. Conclusions NADPH oxidase is implicated in vascular remodelling and superoxide-stimulated cell proliferation in the neointima contributes to intimal hyperplasia in this collar model. Targeting NADPH oxidase via adventitial drug delivery not only reduces superoxide generation, but also normalises endothelial cell function. Targeting the primary source of NADPH oxidase-derived superoxide is an effective approach to prevent deleterious arterial remodelling, providing a rationale for designing more efficacious and selective inhibitors of vascular NADPH oxidase as potential therapeutics for human vascular disease.