Nox Activity Research Articles

Promotion effect of SO42−/Fe2O3 modified MnOx catalysts for simultaneous control of NO and CVOCs

To improve the simultaneous removal of NOx and CVOCs catalytic activity for MnOx catalysts, SO42−/Fe2O3 modified catalysts were prepared by the coprecipitation method. Compared with pure MnOx, the activity of the SO42–0.10Fe–MnOx catalysts was significantly improved, with >90% NO conversion and CB oxidation. The synergistic effect between SO42−/Fe2O3 and Mn can lead to higher BET, Mn4+/Mn3+ and oxygen vacancy contents, which can provide large number of acid sites. In addition, the characterization results showed that SO42−/Fe2O3 modification led to the generation of more Fe2+/Fe3+ species and chemisorbed oxygen species, and the reduction performance of the catalyst can be influenced by the redox cycle: Fe2+ + Mn4+ ↔ Fe3+ + Mn3+. Therefore, the enhancement of redox performance and abundant surface acidic sites are the main reasons for the improvement of synergistic catalytic activity over SO42–0.10Fe–MnOx catalysts.

Glycolysis and the Pentose Phosphate Pathway Promote LPS-Induced NOX2 Oxidase- and IFN-β-Dependent Inflammation in Macrophages.

Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-β expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-β response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-β increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.

Regulation of Neutrophil NADPH Oxidase, NOX2: A Crucial Effector in Neutrophil Phenotype and Function.

Reactive oxygen species (ROS), produced by the phagocyte NADPH oxidase, NOX2, are involved in many leukocyte functions. An excessive or inappropriate ROS production can lead to oxidative stress and tissue damage. On the other hand, an absence of ROS production due to a lack of a functional NADPH oxidase is associated with recurrent infections as well as inflammation disorders. Thus, it is clear that the enzyme NADPH oxidase must be tightly regulated. The NOX2 complex bears both membrane and cytosolic subunits. The membrane subunits constitute the flavocytochrome b 558, consisting of gp91phox (Nox2) and p22phox subunits. The cytosolic subunits form a complex in resting cells and are made of three subunits (p47phox, p40phox, p67phox). Upon leukocyte stimulation, the cytosolic subunits and the small GTPase Rac assemble with the flavocytochrome b 558 in order to make a functional complex. Depending on the stimulus, the NADPH oxidase can assemble either at the phagosomal membrane or at the plasma membrane. Many studies have explored NOX2 activation; however, how this activation is sustained and regulated is still not completely clear. Here we review the multiple roles of NOX2 in neutrophil functions, with a focus on description of its components and their assembly mechanisms. We then explain the role of energy metabolism and phosphoinositides in regulating NADPH oxidase activity. In particular, we discuss: 1) the link between metabolic pathways and NOX2 activity regulation through neutrophil activation and the level of released ROS, and 2) the role of membrane phosphoinositides in controlling the duration of NOX2 activity.

Effects of the preparation method of Cu⁃SAPO⁃34 catalysts in de-NOx SCR by propylene

Cu-SAPO-34 catalysts prepared by incipient wet impregnation (IWI), liquid ion-exchange (LIE) and one-pot (OP) hydrothermal methods were applied in selective catalytic reduction of NO with propylene (C3H6-SCR). Under diesel engine exhaust conditions containing 10% O2 and 5% H2O, Cu-SAPO-34(OP) obtained ~95% NOx conversion and ~95% N2 yield at 250–300 °C. Compared the IWI and LIE samples, the Cu-SAPO-34(OP) sample had abundant Lewis acidic sites, the largest specific surface area, and the most isolated Cu2+ species, thus it exhibited the best de-NOx performance. In contrast, more inactive CuO species formed on the surface of the LIE and IWI samples. The isolated Cu2+ ions in the 8MR rings have high reducibility, and are supposed to be the main active sites of Cu-SAPO-34 catalysts in the C3H6-SCR reaction. In situ DRIFTS suggests that isolated copper ions are favourable for the adsorption and activation of NOx and C3H6, thus enhancing the formation of isocyanate (-NCO) and -CN species, which act as key intermediates and further produce N2. This study can be helpful to provide some references for the design of HC-SCR catalyst and application of diesel NOx abatement.

Hepatocyte Specific gp130 Signalling Underlies APAP Induced Liver Injury.

N-acetyl-p-aminophenol (APAP)-induced liver damage is associated with upregulation of Interleukin-11 (IL11), which is thought to stimulate IL6ST (gp130)-mediated STAT3 activity in hepatocytes, as a compensatory response. However, recent studies have found IL11/IL11RA/gp130 signaling to be hepatotoxic. To investigate further the role of IL11 and gp130 in APAP liver injury, we generated two new mouse strains with conditional knockout (CKO) of either Il11 (CKOIl11) or gp130 (CKOgp130) in adult hepatocytes. Following APAP, as compared to controls, CKOgp130 mice had lesser liver damage with lower serum Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST), greatly reduced serum IL11 levels (90% lower), and lesser centrilobular necrosis. Livers from APAP-injured CKOgp130 mice had lesser ERK, JNK, NOX4 activation and increased markers of regeneration (PCNA, Cyclin D1, Ki67). Experiments were repeated in CKOIl11 mice that, as compared to wild-type mice, had lower APAP-induced ALT/AST, reduced centrilobular necrosis and undetectable IL11 in serum. As seen with CKOgp130 mice, APAP-treated CKOIl11 mice had lesser ERK/JNK/NOX4 activation and greater features of regeneration. Both CKOgp130 and CKOIl11 mice had normal APAP metabolism. After APAP, CKOgp130 and CKOIl11 mice had reduced Il6, Ccl2, Ccl5, Il1β, and Tnfα expression. These studies exclude IL11 upregulation as compensatory and establish autocrine, self-amplifying, gp130-dependent IL11 secretion from damaged hepatocytes as toxic and anti-regenerative.

Metformin mitigates gas explosion‑induced blast lung injuries through AMPK‑mediated energy metabolism and NOX2‑related oxidation pathway in rats

Gas explosions are a recurrent event in coal mining that cause severe pulmonary damage due to shock waves, and there is currently no effective targeted treatment. To illustrate the mechanism of gas explosion-induced lung injury and to explore strategies for blast lung injury (BLI) treatment, the present study used a BLI rat model and supplementation with metformin (MET), an AMP-activated protein kinase (AMPK) activator, at a dose of 10 mg/kg body weight by intraperitoneal injection. Protein expression levels were detected by western blotting. Significantly decreased expression of phosphorylated (p)-AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and metabolic activity were observed in the BLI group compared with those in the control group. However, the mitochondrial stability, metabolic activity and expression of p-AMPK and PGC1α were elevated following MET treatment. These results suggested that MET could attenuate gas explosion-induced BLI by improving mitochondrial homeostasis. Meanwhile, high expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX2) and low expression of catalase (CAT) were observed in the BLI group. The expression levels of NOX2 and CAT were restored in the BLI + MET group relative to changes in the BLI group, and the accumulation of oxidative stress was successfully reversed following MET treatment. Overall, these findings revealed that MET could alleviate BLI by activating the AMPK/PGC1α pathway and inhibiting oxidative stress caused by NOX2 activation.

Activation of the MAC1-ERK1/2-NOX2 Pathway Is Required for LPS-Induced Sustaining Reactive Microgliosis, Chronic Neuroinflammation and Neurodegeneration.

Recent studies suggest that improper resolution of acute neuroinflammation may lead to long-lasting low-grade chronic neuroinflammation and drive progressive neurodegeneration. However, the molecular mechanism underlying the transition from acute to chronic neuroinflammation remains unclear. The main purpose of this study was to search for potential pathways mediating LPS-elicited chronic neuroinflammation and resultant neurodegeneration. Using microglia cultures prepared from C57BL/6J, MAC1-deficient, and MyD88-deficient mice, the initial study showed that activation of TLR-4 is not sufficient for maintaining chronic neuroinflammation despite its essential role in LPS-initiated acute neuroinflammation. Opposite to TLR-4, our studies showed significantly reduced intensity of chronic neuroinflammation, oxidative stress, and progressive loss of nigral dopaminergic neurons in MAC1-deficient neuron/glial cultures or mice stimulated with LPS. Mechanistic studies revealed the essential role ERK1/2 activation in chronic neuroinflammation-elicited neurodegeneration, which was demonstrated by using an ERK1/2 inhibitor in neuron-glial cultures. Taken together, we propose a key role of the MAC1-NOX2-ERK1/2 signaling pathway in the initiation and maintenance of low-grade chronic neuroinflammation. Continuing ERK1/2 phosphorylation and NOX2 activation form a vicious feedforward cycle in microglia to maintain the low-grade neuroinflammation and drive neurodegeneration.

Ordered Mesoporous MnAlOx Oxides Dominated by Calcination Temperature for the Selective Catalytic Reduction of NOx with NH3 at Low Temperature

Manganese alumina composited oxides (MnAlOx) catalysts with ordered mesoporous structure prepared by evaporation-induced self-assembly (EISA) method was designed for the selective catalytic reduction (SCR) of NOx with NH3 at low temperature. The effect of calcination temperature of MnAlOx catalysts was investigated systematically, and it was correlated with SCR activity. Results showed that with an increase in calcination temperature, the SCR activity of MnAlOx catalysts increased. When the calcination temperature was raised up to 800 °C, the NOx conversion was more than 90% in the operation temperature range of 150~240 °C. Through various characterization analysis, it was found that MnAlOx-800 °C catalysts possessed enhanced redox capacities as the higher content of Mn4+/(Mn3+ + Mn4+). Moreover, the improved redox properties could contribute to a higher NOx adsorption and activation ability, which lead to higher SCR performance of MnAlOx-800 °C catalysts. In situ DRIFTs revealed that the adsorbed NO2 and bidentate nitrate are the reactive intermediate species, and NH3 species bonded to Lewis acid sites taken part in SCR progress. The SCR progress predominantly followed E–R mechanism, while L–H mechanism also takes effect to a certain degree.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n= 15) and normozoospermic (n= 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

PILATES METHOD IMPROVES PHYSICAL CAPACITY AND ANTI-OXIDATIVE SYSTEM IN ELDERLY WOMEN

Objective: The decline in physical capacity in humans occurs as a consequence of ageing. It is well established that oxidative mechanisms play a role in ageing process, leading to oxidative accumulation in tissue damage. Regular physical exercise can minimize the physiological changes that occur during aging and also contribute to improving the individual’s health and well-being. The aim of the present study is to verify the effects of resistance physical training on oxidative damage and physical capacity in elderly women who practice Pilates (P) or Strength Training (ST). Design and method: Elderly volunteer were evaluated and divided into P (68 ± 4 years age) or ST (66 ± 3 years age) groups (n = 20 per group), according to individual choice for the modality. The training protocol (moderate intensity) lasted 12 weeks, with 2 sessions of 60 minutes per week. All volunteers underwent dual-energy x-ray absorptiometry (DXA) and handgrip strength (HGS) for physical and functional assessment. Blood sample was collected for the analysis of oxidative stress markers (TBARS, Carbonilated proteins, NOX and SOD activities and Frap). Results: The results show that resistance exercises in P and ST modalities are effective in inducing changes in physical composition (body weight, fat percentage and bone density did not change). On the other hand, ST was effective in reducing fat mass in upper and lower limbs and increasing HGS (+40%), while P increased lean mass in limbs and trunk. Pilates method was more efficient in increasing muscle mass throughout the body, while strength training was more efficient in allowing fat loss. Finally, Pilates method was effective in increasing SOD activity (+20%), reducing protein oxidation (17%). On the other hand, strength training implemented an increase in the antioxidant system at the end of the ROS production cascade, which seems not to have been effective in reducing lipid oxidation (Frap increased 37%, while TBARS and NOX maintained 50% and 30% higher compared to Pilates, respectively). Conclusions: The results allow us to conclude that Pilates method, for a period of 12 weeks, is effective in bringing anti-oxidative capacity and in the quality of life of sedentary elderly women.

Yin and Yang of NADPH Oxidases in Myocardial Ischemia-Reperfusion.

Oxidative stress is critically involved in the pathophysiology of myocardial ischemic-reperfusion (I/R) injury. NADPH oxidase (Nox) 2 and 4, major sources of reactive oxygen species (ROS) in cardiomyocytes, are upregulated in response to I/R. Suppression of Nox-derived ROS prevents mitochondrial dysfunction and endoplasmic reticulum (ER) stress, leading to attenuation of myocardial I/R injury. However, minimal levels of ROS by either Nox2 or Nox4 are required for energy metabolism during I/R in the heart, preserving hypoxia-inducible factor-1α (HIF-1α) and peroxisome proliferator-activated receptor-α (PPARα) levels. Furthermore, extreme suppression of Nox activity induces reductive stress, leading to paradoxical increases in ROS levels. Nox4 has distinct roles in organelles such as mitochondria, ER, and ER-mitochondria contact sites (MAMs). Mitochondrial Nox4 exerts a detrimental effect, causing ROS-induced mitochondrial dysfunction during I/R, whereas Nox4 in the ER and MAMs is potentially protective against I/R injury through regulation of autophagy and MAM function, respectively. Although Nox isoforms are potential therapeutic targets for I/R injury, to maximize the effect of intervention, it is likely important to optimize the ROS level and selectively inhibit Nox4 in mitochondria. Here, we discuss the ‘Yin and Yang’ functions of Nox isoforms during myocardial I/R.

Targeting NOX2 via p47/phox-p22/phox Inhibition with Novel Triproline Mimetics.

On the basis of the knowledge that the proline-rich hot spot PPPRPP region of P(151)PSNPPPRPP(160), an oligopeptide derived from the cytosolic portion of p22phox (p22), binds to the single functional bis-SH3 domain of the regulatory protein p47phox (p47), we designed a mimetic of the tripeptide PPP based on NMR and X-ray crystallographic data for the p22(151-161) peptide PPSNPPPRPPA with a peptide construct. Incorporation of the synthetic pseudo-triproline mimetic Pro-Pro-Cyp in a molecule derived from molecular modeling studies led to only a 7-fold diminution in activity in a surface plasmon resonance assay relative to the same molecule containing the natural Pro-Pro-Pro tripeptide. The alternative sequence corresponding to a Pro-Cyp-Pro insertion was inactive. This is a first example of the use of a triproline mimetic to interfere with the formation of the p47-p22 complex, which is critical for the activation of NOX, leading to the production of reactive oxygen species as superoxide anions.

The Antileukemic and Anti-Prostatic Effect of Aeroplysinin-1 Is Mediated through ROS-Induced Apoptosis via NOX Activation and Inhibition of HIF-1a Activity.

Aeroplysinin-1 is a brominated isoxazoline alkaloid that has exhibited a potent antitumor cell effect in previous reports. We evaluated the cytotoxicity of aeroplysinin-1 against leukemia and prostate cancer cells in vitro. This marine alkaloid inhibited the cell proliferation of leukemia Molt-4, K562 cells, and prostate cancer cells Du145 and PC-3 with IC50 values of 0.12 ± 0.002, 0.54 ± 0.085, 0.58 ± 0.109 and 0.33 ± 0.042 µM, respectively, as shown by the MTT assay. Furthermore, in the non-malignant cells, CCD966SK and NR8383, its IC50 values were 1.54 ± 0.138 and 6.77 ± 0.190 μM, respectively. In a cell-free system, the thermal shift assay and Western blot assay verified the binding affinity of aeroplysinin-1 to Hsp90 and Topo IIα, which inhibited their activity. Flow cytometry analysis showed that the cytotoxic effect of aeroplysinin-1 is mediated through mitochondria-dependent apoptosis induced by reactive oxygen species (ROS). ROS interrupted the cellular oxidative balance by activating NOX and inhibiting HIF-1α and HO-1 expression. Pretreatment with N-acetylcysteine (NAC) reduced Apl-1-induced mitochondria-dependent apoptosis and preserved the expression of NOX, HO-1, and HIF-1a. Our findings indicated that aeroplysinin-1 targeted leukemia and prostate cancer cells through multiple pathways, suggesting its potential application as an anti-leukemia and prostate cancer drug lead.

MP07-17 MECHANISMS INVOLVED IN NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE (NOX)-DERIVED REACTIVE OXYGEN SPECIES (ROS) MODULATION OF MUSCLE FUNCTION IN HUMAN BLADDERS.

You have accessJournal of UrologyCME1 May 2022MP07-17 MECHANISMS INVOLVED IN NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE (NOX)-DERIVED REACTIVE OXYGEN SPECIES (ROS) MODULATION OF MUSCLE FUNCTION IN HUMAN BLADDERS. Nagat Frara, Dania Giaddui, Alan S. Braverman, Mary F. Barbe, Changhao Wu, and Michael R. Ruggieri Nagat FraraNagat Frara More articles by this author , Dania GiadduiDania Giaddui More articles by this author , Alan S. BravermanAlan S. Braverman More articles by this author , Mary F. BarbeMary F. Barbe More articles by this author , Changhao WuChanghao Wu More articles by this author , and Michael R. RuggieriMichael R. Ruggieri More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002529.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Generation of reactive oxygen species (ROS) by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is a highly regulated process that constitutes one of the main redox signaling components. Excessive ROS production may cause oxidative damage to tissue and organ. Although, the role of redox signaling in different pathological processes has been of intense interest, its importance in bladder pathology remains unexplored. This aim was to use ex vivo muscle strip contractility studies to further explore the physiological role of ROS/Nox in regulating bladder function in humans. METHODS: Mucosa-denuded bladder muscle strips were obtained from human organ transplant donors; three males and two females were mounted in muscle baths. The maximal contractile responses to 120 mM potassium chloride were measured. Trains of electrical field stimulation (EFS) of 1 ms pulse duration, 12 V, 8 Hz at 90 s intervals were applied to each strip for about 20 minutes. Then, subsets of strips were incubated with ROS/Nox agonists or antagonists for 20 minutes in continued trains of EFS. Next, same subsets of strips that were treated with antagonists were retreated with agonists and their responses were detected. All responses are expressed in milli Newtons (mN) or as percentages of control. RESULTS: In male and female human bladders, exogenous application of ROS (hydrogen peroxide; H2O2), at the physiological concentration of 100μM, enhanced EFS-induced smooth muscle strip contractions. Apocynin (100μM), the inhibitor of ROS generating enzyme Nox, attenuated intrinsic muscle strip activity. However, treatment with H2O2 brings the EFS-induced muscle activity back to control level. Application of a key Nox activator and a pro-inflammatory peptide angiotensin II (1μM) augmented EFS-induced smooth muscle contractions. Treatment with AT1 receptor specific antagonist ZD7155 (1μM) inhibited EFS-induced activity, while retreatment with angiotensin II after ZD7155 blockade restores muscle activity to control level. CONCLUSIONS: The enhancement of EFS-evoked contractions by H2O2 and the inhibition of these contractions by the Nox inhibitor apocynin demonstrates the functional relevance of ROS in regulating human bladder smooth muscle activity and suggests that endogenous Nox-derived ROS regulates smooth muscle function. The augmentation of contractions by angiotensin II suggests that activation of Nox via a receptor by its ligand can also enhance smooth muscle activity and that the effect of angiotensin II is mediated by AT1, which was further supported by the inhibitory effect of the selective antagonist ZD7155. Collectively, these data provide evidence for the functional significance of Nox-derived ROS in human bladder and that ROS can modulate bladder function without exogenous stimuli. Since, inflammation is an important mechanism associated with oxidative damage, the effects of angiotensin II on bladder smooth muscle function may have significant pathologic implications. Source of Funding: NIA 1R01AG049321-01A1 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e122 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nagat Frara More articles by this author Dania Giaddui More articles by this author Alan S. Braverman More articles by this author Mary F. Barbe More articles by this author Changhao Wu More articles by this author Michael R. Ruggieri More articles by this author Expand All Advertisement PDF DownloadLoading ...

PFKFB3 Mediated Changes in ROS/NO Balance Contribute to Endothelial Dysfunction in Obesity

Obesity is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS). The biological actions of NO can be compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that is mainly generated by NADPH oxidases (NOXs). However, the signals coordinating the increased superoxide production leading to loss of NO with altered metabolism remain unknown.We have found that the expression of 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐biphosphatase 3 (PFKFB3), which is a key glycolytic regulatory enzyme, is significantly increased in endothelial cells of obese animals. Overexpression of PFKFB3 within the endothelium of blood vessels is accompanied by the loss of vascular relaxation and in decreased NO production as a result of increased eNOS phosphorylation on the inhibitory T495 residue. PFKFB3 overexpression also blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation and activation of eNOS at S1177. While PFKFB3 exerted negative effects on NO signaling, it increased the activity and mRNA levels of NOX1, a major contributor to endothelial dysfunction. Further, we have also found that pharmacological inhibition of NOX1 attenuates the decreased vasodilator responses in PFKFB3 overexpressing aortic rings.These results demonstrate a novel functional relationship between endothelial metabolism, ROS and NO production that may contribute to endothelial dysfunction in obesity.

Lysine Deacetylases and Demethylases: Early Epigenetic Regulators of Intermittent Hypoxia induced Sympathetic Activation and Blood Pressure

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT‐IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation leading to persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Lysine modifications of histones is another major epigenetic mechanism associated with gene regulation. Interplay between lysine deacetylases (HDACs) and demethylases (KDMs) differentially regulate gene expression. Lysine acetylation of histone opens up the chromatin thereby facilitating binding of transcription factors. On the other hand, KDMs regulate transcription by demethylating histone lysines which activate gene expression. Here we report that exposure of rat pheochromocytoma (PC)‐12 cells to IH in vitro exhibit reduced HDAC5 and increased KDM6B enzyme activity leading to increased HIF‐1α stability and transcriptional activity. Treatment of rats with Trichostatin A (TSA), an inhibitor of HDACs increased HIF‐1 dependent NADPH oxidase (NOX)‐4 transcription, in adrenal medullae (AM), resulting in elevated plasma catecholamines and blood pressure. Treating IH exposed rats with GSKJ4 an inhibitor of KDMs blocked HIF‐1 dependent transcriptional activation of NOX4, as well as absence of elevated plasma catecholamines and hypertension. These findings indicate a hitherto uncharacterized role of HDACs and KDMs as early epigenetic regulators in IH‐augmented sympathetic nerve activation and hypertension in rodent models of IH.

Inhibition of Peroxiredoxin 6 PLA2 Activity Decreases Oxidative Stress and the Severity of Acute Lung Injury in the Mouse Cecal Ligation and Puncture Model

IntroductionALI is associated with bacterial infection (sepsis), either as an initiating factor or as a secondary complication. Activation of polymorphonuclear leucocytes (PMN) recruited into the lungs causes the release of free radicals and reactive oxygen species (ROS) that are responsible for many of the clinical manifestations of ALI. Since NADPH oxidase (NOX2) is the major source of ROS (i.e., superoxide anion) in PMN, NOX2 blockade has been proposed for the treatment of Acute Lung Injury (ALI). We reasoned that NOX2 blockers would be counter‐productive in ALI associated with bacterial infection by interfering with the bactericidal activity of PMN.HypothesisPhospholipase A2 inhibitory peptide (PIP‐2) that inhibits NOX2 activation (by preventing activation of Rac, a necessary NOX2 co‐factor) would inhibit the bactericidal activity of PMN and thereby limit clearance of bacteria from the lung; we posited that this effect of PIP‐2 could be overcome by the administration of appropriate antibiotics.MethodsUsing the cecal ligation and puncture (CLP) model of ALI associated with sepsis, we investigated the effect of inhibiting NOX2derived ROS production by administration of PIP‐2.ResultsThe number of colony‐forming bacteria in lungs and peritoneal fluid of mice with CLP approximately doubled at 18 h after intravenous treatment with 2 µg PIP‐2 /gram body weight (wt). Treatment with 10 µg PIP‐2/g body wt resulted in 100% mortality within 18 hr. Antibiotic treatment abolished both the increase in lung bacteria with low dose PIP‐2 and the increased mortality with high dose PIP‐2. Treatment with PIP‐2 in the presence of antibiotics resulted in significantly improved lung histology, decreased PMN infiltration, decreased lung fluid accumulation, and decreased oxidative lung injury compared to antibiotics alone.ConclusionAdministration of PIP‐2 provides partial protection against lung injury in a model of ALI due to bacterial infection while concurrent antibiotic treatment abolishes the deleterious effects of PIP‐2 on lung bacterial clearance. These results suggest that addition of PIP‐2 to the antibiotic regimen would be beneficial for treatment of ALI associated with bacterial infection.

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification, apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2•-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47phox (aa 1–286), p67phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.

Reversal of NADPH Oxidase-Dependent Early Oxidative and Inflammatory Responses in Chronic Obstructive Pulmonary Disease by Puerarin.

In the present study, we investigated effects of Puerarin on the early oxidative and inflammatory responses in the lung triggered by acute cigarette smoking (ACS). C57BL/6 mice were exposed to ACS for 1 hr in the presence or absence of Puerarin and harvested at 2, 6, and 24 hours. ACS induced significant increases in superoxide production in mouse lungs at 2 and 6 hours; and superoxide production was also elevated in a time and concentration dependent manner in cigarette smoke extract (CSE) stimulated human small airway epithelial cells (HSAECs), which was dose-dependently abrogated by Puerarin. ACS exposure upregulated NOX1, NOX2, and NOX4 protein expression in mouse lungs. Likewise, NOX1 and NOX4 were upregulated in CSE-stimulated HSAECs. These responses were significantly or completely attenuated by Puerarin. ACS induced significant infiltrations of neutrophils and macrophages in mouse lung parenchyma and BAL fluid, which were completely or significantly abrogated by Puerarin, so was the activation of the NF-кB pathway and the upregulation in inflammatory mediators including TNF-α, KC (murine homolog of IL-8), COX-2, IL-6 and MCP-1. Nuclear translocation of p65, IL-8 secretion, and upregulation of COX-2 in CSE stimulated HSAECs were also markedly attenuated by Puerarin. Moreover, ACS or CSE stimulated upregulation in reactive oxygen species (ROS) production and expression of inflammatory mediators were alleviated by ROS scavenger TEMPO in vivo and vitro, with no synergy combining with Puerarin, indicating that the effects of Puerarin are redox-sensitive following activation of NOX. In summary, our data for the first time demonstrate that Puerarin robustly attenuates NOX isoform-dependent ROS production and inflammatory activation in ACS exposed mice and CSE treated HSAECs, indicating that Puerarin might be used as a robust therapeutic agent for early or early stage COPD.