NADPH Oxidase-induced Oxidative Stress Research Articles

Elevated expression of toll-like receptor 4 is associated with NADPH oxidase-induced oxidative stress in B cells of children with autism

Autism spectrum disorder (ASD) is a childhood disorder with neurodevelopmental dysfunction which manifests as impairment in social behavior and communication skills. B cells play an important role in immune dysfunction where toll-like receptor 4 (TLR4) may contribute through oxidative inflammatory process. TLR4 related signaling and oxidative stress have been reported in the periphery of ASD subjects, however it has not been evaluated in peripheral B cells of ASD subjects and compared with typically developing control (TDC) children. This study evaluated TLR4 expression and related signaling [Bruton’s tyrosine kinase (BTK), spleen tyrosine kinase (SYK), NF-kB, NADPH oxidase (NOX2), nitrotyrosine, superoxide dismutase (SOD)] in ASD and TDC subjects. Current investigation in B cells shows that ASD subjects have increased TLR4 expression and oxidative stress as exhibited by upregulated NOX2 and nitrotyrosine expression as compared to TDC subjects. B cell relevant pathways, BTK/SYK/NF-kB were also upregulated in B cells of ASD group. Treatment with TLR4 agonist, LPS led to upregulation of NOX2 and nitrotyrosine in B cells of ASD whereas it had no significant effect on TDC subjects. Treatment with NF-kB inhibitor caused inhibition of LPS-induced upregulation of NOX2 and nitrotyrosine in B cells of ASD. Therefore, current investigation proposes the notion that TLR4 expression is elevated in B cells which is associated with increased NF-kB signaling and oxidant stress in ASD subjects. In short, peripheral B cells could contribute to systemic oxidative inflammation and contribute to the immune dysfunction in ASD.

Myeloid-specific dopamine D2 receptor signalling controls inflammation in acute pancreatitis via inhibiting M1 macrophage.

Macrophage infiltration and activation is a critical step during acute pancreatitis (AP). We have shown that pancreas-specific D2 receptor signalling protects against AP severity. As it is unclear to what extent myeloid-specific D2 receptor mediates AP, we investigated the role of myeloid-specific D2 receptor signalling in AP. Using wild-type and LysM+/cre D2 fl/fl mice, AP was induced by l-arginine, caerulein and LPS. Murine bone marrow-derived macrophages and human peripheral blood mononuclear cells (PBMCs) were isolated, cultured and then induced to M1 phenotype. AP severity was assessed by measurements of serum amylase and lipase and histological grading. Macrophage phenotype was assessed by flow cytometry and qRT-PCR. NADPH oxidase-induced oxidative stress and NF-κB and NLRP3 inflammasome signalling pathways were also evaluated. We found that dopaminergic system was activated and dopamine reduced inflammatory cytokine expression in M1-polarized macrophages from human PBMCs. Dopaminergic synthesis was also activated, but D2 receptor expression was down-regulated in M1-polarized macrophages from murine bone marrows. During AP, myeloid-specific D2 receptor deletion worsened pancreatic injury, systematic inflammation and promoted macrophages to M1 phenotype. Furthermore, M1 macrophages from LysM+/cre D2 fl/fl mice exhibited increased NADPH oxidase-induced oxidative stress and enhanced NF-κB and NLRP3 inflammasome activation. D2 receptor activation inhibited M1 macrophage polarization, oxidative stress-induced NF-κB and NLRP3 inflammasome activation. Our data for the first time showed that myeloid-specific D2 receptor signalling controls pancreatic injury and systemic inflammation via inhibiting M1 macrophage, suggesting D2 receptor activation might serve as therapeutic target for AP.

Recurrent insulin-induced hypoglycemia induces AngII and COX2 leading to renal (pro)renin receptor expression and oxidative stress

Background: Recurrent insulin-induced hypoglycemia (RIIH) is an avoidable consequence in the therapeutic management of diabetes mellitus. RIIH has been implicated in causing hypertension through an increase in renal and systemic AngII production.Objective: The present study was performed to assess the hypothesis that chronic insulin treatment enhances AngII and COX2 formation which in turn increases (pro) renin receptor (PRR) expression and NADPH oxidase-mediated oxidative stress, leading to renal and cardiac injury.Methods: The present studies were conducted in Male Sprague Dawley rats treated with daily subcutaneous injections of 7u/kg insulin or saline for 14 days. On the 14th day, surgery was performed for treatment infusion (captopril 12mg/kg, NS398 0.3mg/kg or vehicle), and renal interstitial fluid sample and urine collections for biomarker measurements. At the end of the experiments, kidneys and hearts were harvested to evaluate PRR and NOX2 (NADPH oxidase subunit) expression and oxidative stress.Results: We found that RIIH enhanced AngII and COX2 activity, leading to renal PRR expression and NADPH oxidase-induced oxidative stress in the heart and kidney. 8-isoprostane was evaluated as a renal biomarker of oxidative stress, which was induced in insulin treated animals and modulated by captopril and NS398. In addition, there was a slight increase in NGAL, a urinary biomarker of acute kidney injury (AKI), in insulin treated animals when compared to control.Conclusion: These results demonstrate that RIIH induces renal PRR expression and oxidative stress through increasing AngII and COX2 in the heart and kidney, leading to end-organ damage.

Role of NADPH Oxidase in Metabolic Disease-Related Renal Injury: An Update.

Metabolic syndrome has been linked to an increased risk of chronic kidney disease. The underlying pathogenesis of metabolic disease-related renal injury remains obscure. Accumulating evidence has shown that NADPH oxidase is a major source of intrarenal oxidative stress and is upregulated by metabolic factors leading to overproduction of ROS in podocytes, endothelial cells, and mesangial cells in glomeruli, which is closely associated with the initiation and progression of glomerular diseases. This review focuses on the role of NADPH oxidase-induced oxidative stress in the pathogenesis of metabolic disease-related renal injury. Understanding of the mechanism may help find potential therapeutic strategies.

Pigment Epithelium–Derived Factor Improves Metabolic Derangements and Ameliorates Dysregulation of Adipocytokines in Obese Type 2 Diabetic Rats

Oxidative stress and inflammation in the adipose tissues contribute to the metabolic syndrome. Pigment epithelium-derived factor (PEDF) inhibits vascular inflammation through its anti-oxidative properties. However, it remains unclear whether PEDF could suppress adipocyte inflammation. We investigated the effects of long-term administration or suppression of PEDF on adipocyte inflammation and metabolic derangements in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes with insulin resistance. Circulating and adipose tissue PEDF levels were increased as OLETF rats became more obese and insulin resistant. Long-term administration of PEDF improves metabolic parameters, ameliorates dysregulation of adipocytokines, and suppresses NADPH oxidase-induced oxidative stress and macrophage infiltration in the adipose tissues of OLETF rats, whereas these variables are exacerbated by the knockdown of PEDF by administering siRNAs. Our study suggests that PEDF could improve metabolic derangements by suppressing the inflammatory and oxidative reactions in adipose tissues of OLETF rats. PEDF levels may be elevated as a countersystem against obesity-related metabolic derangements.

Chapter Ten - Neuroimmune Basis of Alcoholic Brain Damage

Alcohol-induced brain damage likely contributes to the dysfunctional poor decisions associated with alcohol dependence. Human alcoholics have a global loss of brain volume that is most severe in the frontal cortex. Neuroimmune gene induction by binge drinking increases neurodegeneration through increased oxidative stress, particularly NADPH oxidase-induced oxidative stress. In addition, HMGB1-TLR4 and innate immune NF-κB target genes are increased leading to persistent and sensitized neuroimmune responses to ethanol and other agents that release HMGB1 or directly stimulate TLR receptors and/or NMDA receptors. Neuroimmune signaling and glutamate excitotoxicity are linked to alcoholic neurodegeneration. Models of adolescent alcohol abuse lead to significant frontal cortical degeneration and show the most severe loss of hippocampal neurogenesis. Adolescence is a period of high risk for ethanol-induced neurodegeneration and alterations in brain structure, gene expression, and maturation of adult phenotypes. Together, these findings support the hypothesis that adolescence is a period of risk for persistent and long-lasting increases in brain neuroimmune gene expression that promote persistent and long-term increases in alcohol consumption, neuroimmune gene induction, and neurodegeneration that we find associated with alcohol use disorders.

NADPH oxidase: short-term foe, long-term friend

in this issue of Journal of Applied Physiology , Nottin and colleagues ([9][1]) in their manuscript “β-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger,” examine the involvement of both β-

β-Adrenergic receptors desensitization is not involved in exercise-induced cardiac fatigue: NADPH oxidase-induced oxidative stress as a new trigger

Prolonged strenuous exercise (PSE) induces transient left ventricular (LV) dysfunction. Previous studies suggest that β-adrenergic pathway desensitization could be involved in this phenomenon, but it remains to be confirmed. Moreover, other underlying mechanisms involving oxidative stress have been recently proposed. The present study aimed to evaluate the involvement of both the β-adrenergic pathway and NADPH oxidase (Nox) enzyme-induced oxidative stress in myocardial dysfunction in rats following PSE. Rats were divided into 4 groups: controls (Ctrl), 4-h exercised on treadmill (PSE), and 2 groups in which Nox enzyme was inhibited with apocynin treatment (Ctrl APO and PSE APO, respectively). We evaluated cardiac function in vivo and ex vivo during basal conditions and isoproterenol stress. GSH/GSSG ratio, cardiac troponin I (cTnI) release, and lipid peroxidation (MDA) were evaluated. PSE induced a decrease in LV developed pressure, intrinsic myocardial contractility, and relaxation associated with an increase in plasma cTnI release. Our in vivo and ex vivo results demonstrated no differences in myocardial response to isoproterenol and of effective dose 50 between control and PSE rats. Interestingly, the LV dysfunction was reversed by apocynin treatment. Moreover, apocynin prevented cellular oxidation [GSH/GSSG ratio: PSE APO rats vs. PSE rats in arbitrary units (au): 1.98 ± 0.07 vs. 1.35 ± 0.10; P < 0.001]. However, no differences in MDA were observed between groups. These data suggest that myocardial dysfunction observed after PSE was not due to β-adrenergic receptor desensitization but could be due to a signaling oxidative stress from the Nox enzyme.

Oxidative Modification of Mitochondrial Integrity and Nerve Fiber Density in the Ischemic Overactive Bladder

To our knowledge the mechanism of neurodegeneration in the overactive bladder remains unknown. We examined mitochondrial integrity and searched for markers of oxidative neural injury in the ischemic overactive bladder. A rabbit model of overactive bladder was developed by inducing moderate pelvic ischemia. After 16 weeks cystometrograms and blood flow recordings from overactive bladders were compared with those in age matched controls. Bladder tissues were processed to assess oxidative products, oxidative stress sensitive genes and nerve fiber density using enzyme immunoassay, quantitative real-time polymerase chain reaction and immunohistochemical staining, respectively. Tissue ultrastructure was examined by transmission electron microscopy. Ischemia increased spontaneous bladder contractions and led to cyclic ischemia-reperfusion. Tissue levels of oxidative and nitrosative products, and oxidative stress sensitive genes encoding superoxide dismutase and aldose reductase were up-regulated in the overactive bladder. Transmission electron microscopy of overactive bladder tissues showed mitochondria with distinctive morphological features, characterized by swollen membranes, decreased granules, a total loss of granules and sporadic membrane damage. These changes were associated with sporadic loss of epithelial mucosal membrane, twisted smooth muscle cells, diffused vacuolization and marked neurodegeneration. Our findings suggest free radical mediated ultrastructural damage and neurodegeneration in the overactive bladder. Overactivity associated mitochondrial stress may have a central role in epithelial damage, smooth muscle cell injury and neurodegeneration. Superoxide dismutase and aldose reductase up-regulation in the overactive bladder imply intrinsic defensive reaction against free radicals that apparently fails to prevent oxidative damage and neurodegeneration. Therapeutic strategies targeting basic mitochondrial processes such as energy metabolism or free radical generation may help better manage wall degeneration and neuropathy in the overactive bladder.

Antioxidant SOD Mimetic Prevents NADPH Oxidase-Induced Oxidative Stress and Renal Damage in the Early Stage of Experimental Diabetes and Hypertension

Aims: The presence of hypertension increases renal oxidative stress by increasing NADPH oxidase-dependent superoxide production and by decreasing antioxidant defense in the early stage of experimental diabetes mellitus (DM). In the present study, we investigated whether the administration of an antioxidant mimetic of the superoxide dismutase (SOD) (tempol) corrects the oxidative imbalance and oxidative stress-induced renal injury in the presence of DM and hypertension. Methods: DM was induced in spontaneously hypertensive rats (SHR) by streptozotocin at 4 weeks of age. The diabetic rats either did or did not receive tempol for 20 days. Oxidative-stress parameters and indices of renal injury were evaluated. Results: Tempol reestablished the imbalance in redox status induced by DM. It elevated the expression of renal antioxidant extracellular SOD, p < 0.0001; decreased (p = 0.049) the production of renal NADPH-dependent superoxide production, and diminished (p = 0.016) a marker of oxidative stress-induced DNA damage, 8-hydroxy-2′-deoxyguanosine. Reduction of oxidative stress markers was associated with reduction in renal damage parameters associated with DN. DM-induced albuminuria and elevation in renal expression of collagen IV were reduced to the level observed in control rats. Conclusion: We conclude that an imbalance in renal redox status is associated with markers of renal injury in the early stage of DM and hypertension. Antioxidant treatment reestablished the redox status and prevented oxidative stress-induced renal damage.

Abstract 669: ET A Receptor Activation and ET B Receptor Deficiency Reduce Circulating Endothelial Progenitor Cells in Salt-Sensitive Hypertension

Background: Circulating endothelial progenitor cells (EPCs) are reduced in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. DOCA-salt and ET B receptor deficiency rats (ET B −/ −) have salt-sensitive hypertension, characterized by increased ET-1 and oxidative stress levels. We hypothesized that ET A receptor activation and ET B receptor deficiency reduce circulating EPCs in adult male DOCA-salt rats. Methods and Results: Systolic blood pressure (SBP) was higher in DOCA-salt (4-wk regimen) vs. Sham rats (199.3 ± 4.8 vs. 133.4 ± 4.6 mmHg, n =8 –15, p&lt;.05), which was significantly reduced by in vivo treatment with ET A antagonist ABT-627 (5 mg/kg/d) or NADPH oxidase inhibitor apocynin (1.5 mmol/L) (167.8 ± 6.8 and 156.9 ± 16.6 mmHg, respectively). Flow cytometry showed that circulating CD34- and Flk-1-positive EPCs were significantly lower in DOCA vs. Sham rats (31.5 ± 0.2 vs. 18.3 ± 0.8% and 21.1 ± 0.9 vs. 16.3 ± 0.7%, respectively, n=7– 8, p&lt;.05). Dil-acLDL and isolectin double-stainings also showed reduced EPCs in DOCA vs. Sham rats (4.5 ± 0.4 vs. 10.2 ± 0.3 cells/hpf, n=5–15, p&lt;.01), which was rescued by in vivo treatment with ABT-627 (10.1 ± 1.7) or apocynin (8.9 ± 0.8). Real-time RT-PCR and fluorescence confocal microscopy showed that ET A and ET B receptor mRNA and proteins were present in EPCs of SD rats. Intracellular ROS level was increased by &gt;50% in EPCs of DOCA vs. Sham rats (DCF fluorescence, n = 9 –11, p&lt;.05). Apoptosis was increased by &gt;80% in EPCs of DOCA vs. Sham rats, which was reversed by ABT-627 or apocynin (TUNEL assay, n=5– 6, p&lt;.05). EPCs were significantly lower in ET B −/ − vs. ET B +/+ rats (6.3 ± 0.6 cells/hpf, n =7– 8), paralleled with higher SBP (telemetry 151 ± 1.2 vs. 129 ± 0.9 mmHg, n = 6), plasma ET-1 level (5.2 ± 0.3 vs. 3.6 ± 0.1 pg/ml, n = 5–12), and suppressed telomerase activity (354.6 ± 105.5 vs. 83.3 ± 26.2%, n = 3) (all p&lt;.05). ET-1 treatment (10 nM, 48 hrs) also reduced EPCs of normal rats (6.9 ± 0.1 vs. 10.3 ± 0.3 cells/hpf, n = 5– 6, p&lt;.05), which was rescued by ABT-627 (9.9 ± 0.3), but not by ET B antagonist BQ788 (7.6 ± 0.4). Conclusion: ET A activation and ET B deficiency reduce circulating EPCs in DOCA-salt and ET B −/ − rats, in part, due to NADPH oxidase-induced oxidative stress, apoptosis, and telomerase inactivation.

Oxidative Stress after Subarachnoid Hemorrhage in gp91phox Knockout Mice

Oxidative stress largely contributes to early brain injury after subarachnoid hemorrhage (SAH). One of the major sources of reactive oxygen species is NADPH oxidase, upregulated after SAH. We hypothesized that NADPH oxidase-induced oxidative stress plays a major causative role in early brain injury after SAH. Using gp91phox knockout (ko) and wild-type (wt) mice, we studied early brain injury in the endovascular perforation model of SAH. Mortality rate, cerebral edema, oxidative stress, and superoxide production were measured at 24 h after SAH. Neurological evaluation was done at 23 h after SAH surgery. Genotyping confirmed the existence of a nonfunctional gp91phox gene in the ko mice. CBF measurements did not show differences in SAH-induced acute ischemia between ko and wt mice. SAH caused a significant increase of water content in the ipsilateral hemisphere as well as an increase of Malondialdehyde (MDA) levels and superoxide production. There were no significant differences in post-SAH mortality rate, brain water content and the intensity of the oxidative stress between knockout and wild type groups of mice. Our results suggest that gp91phox is not critically important to the early brain injury after SAH. An adaptive compensatory mechanism for free radical production in knockout mice is discussed.

Angiotensin II, oxidative stress, and extracellular matrix degradation during transition to LV failure in rats with hypertension

Angiotensin II (Ang-II) plays pivotal roles in the progression of left ventricular (LV) remodeling in diseased hearts; it remains to be elucidated how Ang-II links to degradation of the extracellular matrix (ECM). Using hypertensive Dahl salt-sensitive rats that show the distinctive transition from concentric LV hypertrophy to LV remodeling, we chronically treated them with an angiotensin type-1 receptor blocker (telmisartan 5 mg/kg/day, ARB group) or vehicle (0.5% CMC, CHF group). During the process of LV remodeling, we assessed, (1) in-vivo LV shape and function; (2) animal survival; (3) amounts of ECM in LV using a scanning electron microscope (SEM); (4) mRNA (by real time RT-PCR) and protein (by immunoblotting) levels in LV of NADPH oxidase, glutathione peroxidase-1 (GPX-1), and matrix metalloproteinase (MMP)-2, -9, and -13; (5) immunohistochemical staining of myocardial 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine; (6) nuclear factor kappa-B (NFκB) protein levels in the nuclear extract; and (7) endogenous activities of MMP-2 and -9 by an antibody capture method. Compared with CHF, ARB group showed an improvement of survival and preserved LV shape and function, and ECM density in SEM that was accompanied by decreases in oxidative stress-mediated protein degenerations, activities of GPX-1, NADPH oxidase, NFκB, and MMP-2, -9, and -13. Local activation of Ang-II in hypertrophic LV triggers MMP-mediated ECM degradation, namely LV remodeling, at least in part, through NADPH oxidase-induced oxidative stress and the subsequent NFκB activation.

NADPH Oxidase-dependent Oxidation and Externalization of Phosphatidylserine during Apoptosis in Me2SO-differentiated HL-60 Cells: ROLE IN PHAGOCYTIC CLEARANCE

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.