Superoxide anion (O 2 ؊ ) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O 2 ؊ is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O 2 ؊ production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O 2 ؊ -generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22 phox , gp91 phox , p47 phox , and p67 phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O 2 ؊ production. Furthermore, immunodepletion of p67 phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67 phox . Further study into the regulation of this adventitial source of O 2 ؊ is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.In recent years, reactive oxygen species (ROS) such as superoxide anion (O 2 Ϫ ) have been shown to play a number of roles in the body. Phagocytic cells produce ROS as a primary host defense mechanism (1), whereas other cells utilize ROS as intracellular second messengers for a wide range of cellular functions. For example, ROS participate in Ras-mediated mitogenic signaling in fibroblasts as well as in leukocyte apoptosis (2, 3).The phagocyte NADPH oxidase or respiratory burst oxidase is the best characterized ROS-generating system and is a multicomponent enzyme complex that catalyzes the oneelectron reduction of oxygen to O 2 Ϫ . Its components include the two membrane-spanning polypeptides, p22 phox and gp91 phox , which comprise flavocytochrome b 558 , and three cytoplasmic polypeptides, p40 phox , p47 phox , and p67 phox (4-6). Additionally, the cytosolic guanine nucleotide-binding protein Rac2 is required for oxidase activation (7). Exposure of the cell to a variety of agonists induces the association of the cytosolic with the membrane-associated components and causes activation of the normally dormant oxidase (4-6).Several groups have shown that NAD(P)H oxidase(s) exist in nonphagocytic cells including carotid body (8), mesangial cells (9), vascular smooth muscle cells (10, 11), endothelial cells (12, 13), and fibroblasts (14). The NAD(P)H oxidase systems in these cells have not yet been well characterized, and even the substrate specificity of these oxidase(s) with regard to NADH and NADPH is still not clear (10)(11)(12)15). In vascular smooth muscle cells of the bovine pulmonary artery and rat aorta, an NADH oxidase has been described, and in the rat aorta these cells express...

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even in environmental toxicity. The complexity of this family’s effects on cellular processes stems from the fact that there are 7 members, each with unique tissue distribution, cellular localization and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophillic fatty acids has impact on many redox sensitive pathologies, and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. The following review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburgh’s Vascular Medicine Institute and Department of Pharmacology and Chemical Biology, and encompasses further interaction and discussion among the presenters.

In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2 and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In the present study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B loop of Nox2, would inhibit ROS production by Nox2-, but not by Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O2•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance (EPR) confirmed that Nox2ds inhibits O2•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production in either Nox1 or Nox4 oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2 oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

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NADPH oxidases (Nox) are established as major sources of reactive oxygen species (ROS). Over the past two decades, Nox-derived ROS have emerged as pivotal in the development of myriad diseases involving oxidative stress. In contrast, Nox are also involved in signaling mechanisms necessary for normal cell function. The study of these enzymes in physiological and pathophysiological conditions is made considerably more complex by the discovery of 7 isoforms: Nox1 through 5 as well as Duox1 and 2, each with its own specific cytosolic components, regulatory control mechanisms, subcellular localization and/or tissue distribution. A clear understanding of the role individual isoforms play in a given system is hindered by the lack of isoform-specific inhibitors. In animal models, knockdown or knockout methodologies are providing definitive answers to perplexing questions of the complex interplay of multiple Nox isoforms in cell and tissue signaling. However, the complex structures and interactions of these heteromeric isozymes predict pleiotropic actions of the Nox subunits and thus suppression of these proteins is almost certain to have untoward effects. Thus, as both therapies and pharmacological tools, molecule-based inhibitors continue to prove extremely useful and rational in design. Unfortunately, many of the available inhibitors have proven non-specific, falling into the category of scavengers or inhibitors of more than one source of ROS. Here, we will review some of the efforts that have been undertaken to develop specific inhibitors of NADPH oxidase over the past decade, from the peptidic inhibitor Nox2ds-tat to more recent small molecule inhibitors that have emerged from high-throughput screening campaigns.

Significance: Numerous studies in animal models and human subjects corroborate that elevated levels of reactive oxygen species (ROS) play a pivotal role in the progression of multiple diseases. As a major source of ROS in many organ systems, the NADPH oxidase (Nox)

The regulation of platelets by oxidants is critical for vascular health and may explain thrombotic complications in diseases such as diabetes and dementia, but remains poorly understood. Here, we describe a novel technique combining electron paramagnetic resonance spectroscopy and turbidimetry, which has been utilized to monitor simultaneously platelet activation and oxygen radical generation. This technique has been used to investigate the redox-dependence of human and mouse platelets. Using selective peptide inhibitors of NADPH oxidases (NOXs) on human platelets and genetically modified mouse platelets (NOX1 −/− or NOX2 −/− ), we discovered that: 1) intracellular but not extracellular superoxide anion generated by NOX is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent activation; 3) NOX1 is the main source of oxygen radicals in response to collagen, while NOX2 is critical for activation by thrombin; 4) two platelet modulators, namely oxidized low density lipoproteins (oxLDL) and amyloid peptide β (Aβ), require activation of both NOX1 and NOX2 to pre-activate platelets. This study provides new insights into the redox dependence of platelet activation. It suggests the possibility of selectively inhibiting platelet agonists by targeting either NOX1 (for collagen) or NOX2 (for thrombin). Selective inhibition of either NOX1 or NOX2 impairs the potentiatory effect of tested platelet modulators (oxLDL and Aβ), but does not completely abolish platelet hemostatic function. This information offers new opportunities for the development of disease-specific antiplatelet drugs with limited bleeding side effects by selectively targeting one NOX isoenzyme.