Nox Inhibition Research Articles

Aminolevulinic Acid Attenuates Increases in Pulmonary Arterial Superoxide Elicited By Organoid Culture with Endothelin‐1 and Exposure of Mice to Chronic Hypoxia Through Mechanisms Potentially Involving Protoporphyrin IX‐Elicited Stimulation of Soluble Guanylate Cyclase and cGMP Protein Kinase Signaling

Our studies have detected that treatment of mice with the heme biosynthesis precursor δ‐aminolevulinic acid (ALA, 50mg/kg/day) prevents the development of pulmonary hypertension (PH) in mice exposed to 21 days of hypoxia (10% O2). Since ALA may promote the accumulation of protoporphyrin IX, a direct activator of soluble guanylate cyclase, we investigated if the known beneficial effects of cGMP or inhibiting the actions of endothelin‐1 (ET) and Nox2 in treating PH could originate from an attenuation of ET‐elicited pulmonary arterial superoxide generation by ALA & cGMP. Organoid culture of bovine pulmonary arteries (PA) with ET (10 μM, 24hr) increased the detection of superoxide by 5uM lucigenin. This was attenuated by stimulation of protein kinase G (PKG) activation in the presence of 0.1mM ALA, 0.1mM 8‐bromo‐cGMP or 0.1mM dihydroxyepiandrosterone, or by Nox inhibition (0.1mM apocynin), respectively. ALA also activated PKG and lowered PA superoxide in mice exposed to 21D hypoxia.

Apocynin treatment attenuated middle cerebral artery remodeling in life‐long obesity in Sprague‐Dawley rats

Metabolic syndrome is a disorder that includes abdominal obesity and hypertension, and these increase stroke risk. Diet‐induced obesity and hypertension lead to remodeling of the middle cerebral artery (MCA) through a mechanism dependent on superoxide anion (O2•−). In the vasculature, most O2•− is generated by the enzyme NADPH‐oxidase (Nox). We hypothesized that Nox inhibition with apocynin (Apo) would attenuate obesity‐induced MCA remodeling in rats. Three‐week old male Sprague‐Dawley rats fed a high‐fat diet (HF) for 17 weeks were treated with Apo (HF+Apo, n=7) or vehicle (HF, n=12). Rats fed normal chow were controls (NC, n=11). MCAs were mounted on a pressure myograph to assess passive structure. Data are mean ± SEM, NC vs HF vs HF+Apo. Rats fed HF had higher visceral fat:body weight (1.42±0.13, 3.92±0.19, 4.27±0.45, p<0.05). Apo prevented the HF‐induced increase in mean arterial pressure (126±3, 146±6, 125±3mmHg, p<0.01). At 80mmHg intralumenal pressure, Apo increased MCA lumen diameter (266±6.6, 245±8.3, 280±8.3μm, p<0.05), decreased wall thickness (20.2±1.5, 33.0±2.8, 18.0±1.2μm, p<0.05) and wall‐to‐lumen ratio (0.08±0.01, 0.14±0.01, 0.07±0.01, p<0.05). HF±Apo had no effect on distensibility (56.9±5.6, 48.8±4.6, 50.3±4.3). These data suggest that Apo prevented the increase in blood pressure in rats fed HF. This might be responsible for the beneficial effects of Apo in the MCA structure.

Cyclooxygenase‐2 inhibition prevent angiotensin II‐induced oxidative stress but not hypertension in humans

Angiotensin II (AngII) is known as an important oxidative stress mediator in hypertension. There is controversy whether cyclooxygenase‐2 (COX‐2) specific inhibitors would be associated with elevations or decrease in blood pressure (BP) despite evidences showing decrease in ROS generation under COX‐2 inhibition. The aim of this study was to determine in healthy humans whether the COX‐2 selective inhibitor celecoxib has antioxidant and hypertensive protective effects against AngII. BP and selected plasma oxidative stress markers (8OHdG; AOPP), peroxynitrite activity (nitrotyrosine), endothelin‐1 (ET1) and nitric oxide metabolism (NOx) were measured at baseline and in response to a graded infusion of AngII before and after 14 days of celecoxib in 14 healthy subjects in high‐salt balance. In response to AngII challenge, all markers of oxidative stress were increased and there was blunting of NOx. Though no effect was observed in terms of BP response to AngII challenge, celecoxib significantly attenuated the increase in oxidative stress as observed by a reduction in the rise of 8OHdG, nitrotyrosine, aldosterone and the NOx inhibition induced by AngII infusion. In conclusion, exposure to AngII increases oxidative stress in humans, though this AngII‐mediated upregulation is attenuated with COX‐2 inhibition. Supported by Alberta Innovates Health Solutions and Canadian Institutes for Health Research.

β2‐Adrenoceptors, NADPH oxidase, ROS and p38 MAPK: another ‘radical’ road to heart failure?

Persistent activation of the cardiac β-adrenergic system may contribute to the pathogenesis of congestive heart failure. Both β₁- and β₂-adrenoceptors are known to mediate these noxious effects, yet the β₁-adrenoceptor-PKA axis has received greater attention with less information available on β₂-adrenoceptor driven pathways. In the present issue, Xu and colleagues provide new evidence, showing that β₂-adrenoceptor over-expression leads to increased reactive oxygen species (ROS) emission, mainly caused by up-regulation of reduced nicotinamide adenine dinucleotide phosphate oxidase (Nox) 2 and 4. Increase in ROS levels is accompanied by p38 mitogen-activated protein kinase activation, fibrosis, apoptosis and cardiac dysfunction. Both Nox inhibition and administration of the antioxidant N-acetyl cysteine prevent these adverse effects. Interestingly, antioxidant treatment also prevents the increase in Nox expression, suggesting that β₂-adrenoceptor stimulation triggers a vicious cycle eventually amplified by both Nox isoforms. The possible existence of a circuitry to enhance ROS signalling and detrimental consequences on myocardial remodelling are also discussed, in light of the recent description of intracellular localization of Nox4.

Initial evidence linking synaptic superoxide production with poor short-term memory in aged mice

Unregulated production of reactive oxygen species (ROS) is a marker of cellular and organismal aging linked to cognitive decline in humans and rodents. The sources of elevated ROS contributing to cognitive decline are unknown. Because NADPH oxidase (Nox) inhibition may prevent memory decline with age, we hypothesized that Nox and not mitochondrial sources of synaptic ROS production are linked to individual variance in cognitive performance in aged mice. Young (8 months) and aged (26 months) mice were tested in the novel object recognition task (NORT). Mitochondrial and Nox ROS production was assayed in isolated synaptosomes using spin trapping electron paramagnetic resonance (EPR) spectroscopy. Aged mice exhibited variance in NORT performance, with some performing similar to young mice while others exhibited poorer short-term memory. EPR studies indicated that Nox rather than mitochondria was the major ROS source at the synapse, and Nox-induced but not mitochondrial-induced ROS levels correlated with NORT performance in aged mice. Our findings support the hypothesis that variance in Nox-specific synaptic ROS production may predict short-term memory deficits with age.

Nox inhibition in isolated rat mesenteric arteries via plasmid electroporation

Collateral growth (CG) is impaired with age. Previously, we have demonstrated that antioxidants, including a NAD(P)H oxidase (Nox) inhibitor, can reverse age‐related impairment in both flow‐mediated nitric oxide (NO) production and CG. The goal of the present study was to determine the optimal conditions for electroporation‐mediated delivery of siRNA to suppress Nox expression in rat mesenteric arteries. A plasmid containing shRNA to the Nox subunit p22phox, along with GFP DNA, was delivered into the arterial wall via square wave electroporation. We have designed a 4mm wide electroporation chamber to allow for both isolated fragment and in vivo electroporation of a 10–15 mm long mesenteric vascular bundle with minimal vector volume and mesenteric tissue manipulation. The results show optimal electroporation conditions of isolated vessels at 125V (312.5V/cm) with 8 pulses of 10msec duration as shown by comparative GFP fluorescence at 48hrs post electroporation. Superoxide production was assessed by monitoring DHE staining of the vessel fragments. Future studies will be focused on optimizing in vivo electroporation conditions and determining the Nox isoforms responsible for the observed flow‐mediated impaired collateral response. Sponsored by NIH HL092012‐S1.

Small-Molecule NOX Inhibitors: ROS-Generating NADPH Oxidases as Therapeutic Targets

NOX NADPH oxidases are electron-transporting membrane enzymes whose primary function is the generation of reactive oxygen species (ROS). ROS produced by NOX enzymes show a variety of biologic functions, such as microbial killing, blood pressure regulation, and otoconia formation. Strong evidence suggests that NOX enzymes are major contributors to oxidative damage in pathologic conditions. Blocking the undesirable actions of NOX enzymes, therefore, is a therapeutic strategy for treating oxidative stress-related pathologies, such as ischemia/reperfusion tissue injury, and neurodegenerative and metabolic diseases. Most currently available NOX inhibitors have low selectivity, potency, and bioavailability, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. This review has two main purposes. First, we describe a systematic approach that we believe should be followed in the search for truly selective NOX inhibitors. Second, we present a critical review of small-molecule NOX inhibitors described over the last two decades, including recently published patents from the pharmaceutical industry. Structures, activities, and in vitro/in vivo specificity of these NOX inhibitors are discussed. We conclude that NOX inhibition is a pertinent and promising novel pharmacologic concept, but that major efforts will be necessary to develop specific NOX inhibitors suited for clinical application.

RETRACTED: Apocynin-induced nitric oxide production confers antioxidant protection in maize leaves

The effect of apocynin on nitric oxide (NO) synthesis and oxidative stress was studied in corn (Zea mays) seedlings. After treatment with 100 microM apocynin, strongly increased amounts of NO were detected in the leaves. This NO production was reduced by more than 70% by N(G)-nitro-l-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, but there was no reduction in NO production when apocynin was applied in combination with diphenylene iodonium (a plant NOX inhibitor). When maize seedlings were UV-B-irradiated, cellular damage occurred and reactive oxygen species (ROS) were found widely distributed in chloroplasts and mesophyll cells. Pre-treatment with apocynin and coinciding NO accumulation prevented this damage. However, the protective effect was averted by L-NAME application. Leaf discs placed in 1M H(2)O(2) for 24h showed a reduction in chlorophyll content that could also be avoided by apocynin treatment. Our results show that apocynin induces the accumulation of NO in leaves of maize seedlings through a NOS-like activity, a mechanism alternative to NOX inhibition, and confers an augmented tolerance to different types of abiotic oxidative stress. Indeed, we propose the use of apocynin as an alternative approach to study NO functionality in plants.

Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: Effects of nitrosothiol exposure

Mechanisms regulating NADPH oxidase remain open and include the redox chaperone protein disulfide isomerase (PDI). Here, we further investigated PDI effects on vascular NADPH oxidase. VSMC transfected with wild-type PDI (wt-PDI) or PDI mutated in all four redox cysteines (mut-PDI) enhanced (2.5-fold) basal cellular ROS production and membrane NADPH oxidase activity, with 3-fold increase in Nox1, but not Nox4 mRNA. However, further ROS production, NADPH oxidase activity and Nox1 mRNA increase triggered by angiotensin-II (AngII) were totally lost with PDI overexpression, suggesting preemptive Nox1 activation in such cells. PDI overexpression increased Nox4 mRNA after AngII stimulus, although without parallel ROS increase. We also show that Nox inhibition by the nitric oxide donor GSNO is independent of PDI. PDI silencing decreased specifically Nox1 mRNA and protein, confirming that PDI may regulate Nox1 at transcriptional level in VSMC. Such data further strengthen the role of PDI as novel NADPH oxidase regulator.

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase-Dependent Activation of Phosphoinositide 3-Kinase and p38 Mitogen-Activated Protein Kinase Signal Pathways Is Required for Lipopolysaccharide-Induced Microglial Phagocytosis

The importance of microglial reactive oxygen species (ROS) signaling in neuroinflammatory processes has been well demonstrated; however, relatively little is known regarding the related mechanisms underlying these processes. Here, we show that ROS-dependent signal pathways that govern microglial phagocytosis are highly dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activation. Specifically, phagocytosis was greatly reduced by both antioxidant and Nox inhibitor treatments in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Additionally, there was a marked reduction in intracellular ROS content. These results suggest that Nox is the main ROS source for LPS-induced microglial phagocytosis. More decisive evidence for the involvement of ROS in phagocytosis was obtained from an examination of phosphatidyl inositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK) signal pathway activation under reduced ROS levels. These two kinases were activated by LPS treatment and inhibited by ROS neutralization and Nox inhibition. We conclude that microglial phagocytosis requires ROS-dependent PI3-K and p38 MAPK activation and that Nox-derived ROS functions as an upstream regulator of both PI3-K and p38 MAPK. These findings will provide a fundamental basis for a therapeutic modality in inflammation-mediated neurodiseases.

Inhibition of odd oxygen production in the carbon bond four and generic reaction set mechanisms

Abstract Outdoor smog chamber experiments have been conducted by researchers in Australia at lower initial NOx and VOC-to-NOx ratios than those chamber experiments which were used to formulate photochemical reaction mechanisms such as the Carbon Bond Four (CB4). When the Australians simulated their experiments with CB4, they found that CB4 underpredicted ozone production at low VOC-to-NOx ratios, i.e. in the NOx-rich region of the ozone isopleth diagram above the ridge line. CB4 predicts a significant NOx inhibition in this region whereas the empirical General Reaction Set (GRS) mechanism, which was developed by the Australian researchers to fit their data, does not. In this study, a mass balance and process analysis technique is used to explain the origins of the NOx -inhibition in CB4 simulations. The causes are determined to be in the inorganic reactions of the CB4, which are believed to be both complete and well-known. The primary cause was a strong negative feedback of new radical production from the photolysis of ozone which occurred because of the longer delay needed to oxidize the higher initial NO. The CB4 mechanism could still be incorrect if it is missing a strong radical source, for example, in the poorly understood aromatics chemistry. The GRS, which lacks representation of the inorganic processes responsible for NOx-inhibition, may be achieving its predictive accuracy by compensating errors. If this is true, then the GRS would be unsatisfactory for ambient air use. Additional smog chamber experiments at low VOC-to-NOx ratios and better characterization of the Australian chambers are needed to resolve the discrepancy in CB4 and GRS predictions.