We previously reported enhanced expression of the p67^phoxand gp91^phoxcomponents of NAD(P)H oxidase in angiotensin (Ang) II–induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II–induced O₂⁻production, we designed a chimeric peptide that inhibits p47^phoxassociation with gp91^phoxin NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91^phox(known to interact with p47^phox). As a control, we constructed a chimera containingtatand a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tatdecreased O₂⁻levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O₂⁻-generating enzyme xanthine oxidase or potassium superoxide–generated O₂⁻. We infused vehicle, Ang II (0.75 mg · kg⁻¹· d⁻¹), Ang II+gp91ds-tat(10 mg · kg⁻¹· d⁻¹), or Ang II+scramb-tatintraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tatbut was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II–induced O₂⁻production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47^phoxand gp91^phox(or its homologues) can block O₂⁻production and attenuate blood pressure elevation in mice.

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.

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.

Although NAD(P)H oxidase-derived superoxide (O₂⁻) is increased during the development of angiotensin II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O₂⁻. Here we compare vascular levels of O₂⁻and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after 7 days of ANG II infusion (750 μg · kg⁻¹· day⁻¹ip) in C57B1/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O₂⁻in the thoracic aorta as measured using lucigenin (25 μM)-enhanced chemiluminescence. Both p67^phoxand gp91^phoxwere detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT₁receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O₂⁻are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.

The redox signaling network in mammals has garnered enormous interest and taken on major biological significance in recent years as the scope of NADPH oxidases (NOXs) as regulators of physiological signaling and cellular degeneration has grown exponentially. All NOX isoforms have in common the capacity to generate reactive oxygen species (ROS) superoxide anion (O₂^•−) and/or hydrogen peroxide (H₂O₂). A baseline, normal level of ROS formation supports a wide range of processes under physiological conditions. A disruption in redox balance caused by either the suppression or “super” induction of NOX off balance with antioxidant systems is associated with myriad diseases and cell/tissue damage. Over the past two to three decades, our understanding of NOXs has progressed from almost entirely a phagocyte-, antimicrobial-centered perspective to that of a family of enzymes that is vital to broad cellular function and organismal homeostasis. It is becoming increasingly evident that highly regulated, targeted oxidative protein modifications are elicited in a spatiotemporal manner and initiated at cell membranes in humans by seven NOX isoforms [NOXs 1, 2, 3, 4, 5 and dual oxidases (DUOXs) 1 and 2]. In a sense, this renders NOX-ROS signaling akin to that of other second messenger systems involving localized Ca²⁺ dynamics and tyrosine kinase transactivation. Accordingly, the study of ROS compartmentalization in subcellular organelles has been shown to be crucial to elucidating their role in cell phenotype modulation under physiological and pathophysiological conditions. The NOXs are as distinct in their distribution and activation as they are in their cellular functions, ranging from host defense, second messenger posttranslational modifications (PTMs) to transcriptional, epigenetic, and (de)differentiating effects. This review integrates past knowledge in the field with new focus areas on the leading edge of NOX-centered ROS signaling, including how a new wave of structural information provides insights for NOX biology and targeted therapies.