Reduced NADPH Oxidase Activity Research Articles

Paricalcitol prevents renal tubular injury induced by ischemia-reperfusion: Role of oxidative stress, inflammation and AT1R

The vitamin D receptor (VDR) is associated with antioxidative and anti-inflammatory effects and modulation of the renin-angiotensin-aldosterone system. This study evaluated whether VDR agonist paricalcitol protects renal ischemia-reperfusion (IR) induced tubular injury in rats by evaluating: 1) ATP-dependent tubular Na+ transport; 2) renal redox signaling; 3) renal content of proinflammatory cytokines TNF-α and IL-6; and 4) renal content of renin and angiotensin II receptor type 1 (AT1R). Paricalcitol prevented IR-induced tubular injury, evidenced by the prevention of histopathological changes and renal fibrosis with preservation of the activity of ATP-dependent Na+ transporters in the renal cortex. Paricalcitol decreased renal oxidative stress by reducing NADPH oxidase activity and increasing catalase. Paricalcitol also decreased the renal content of TNF-α, IL-6, and AT1R. The NADPH oxidase inhibitor apocynin did not present additive protection to paricalcitol-induced effects. The protective effects of paricalcitol on tubular injury induced by renal IR may dependent on the modulation of redox and proinflammatory signaling and renal angiotensin II/AT1R signaling.

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

Resposta Vascular da Triiodotironina sobre Anéis de Aortas Isoladas: Contribuição de Mecanismos Redox

Abstract Background Vascular dysfunction constitutes the etiology of many diseases, such as myocardial infarction and hypertension, with the disruption of redox homeostasis playing a role in the imbalance of the vasomotor control mechanism. Our group previously has shown that thyroid hormones exert protective effects on the aortic tissue of infarcted rats by improving angiogenesis signaling. Objective Investigate the role of triiodothyronine (T3) on vascular response, exploring its effects on isolated aortas and whether there is an involvement of vascular redox mechanisms. Methods Isolated aortic rings (intact- and denuded-endothelium) precontracted with phenylephrine were incubated with T3 (10-8, 10-7, 10-6, 10-5, and 10-4 M), and tension was recorded using a force-displacement transducer coupled with an acquisition system. To assess the involvement of oxidative stress, aortic rings were preincubated with T3 and subsequently submitted to an in vitro reactive oxygen species (ROS) generation system. The level of significance adopted in the statistical analysis was 5%. Results T3 (10-4 M) promoted vasorelaxation of phenylephrine precontracted aortic rings in both intact- and denuded-endothelium conditions. Aortic rings preincubated in the presence of T3 (10-4 M) also showed decreased vasoconstriction elicited by phenylephrine (1 µM) in intact-endothelium preparations. Moreover, T3 (10-4 M) vasorelaxation effect persisted in aortic rings preincubated with NG-nitro-L-arginine methylester (L-NAME, 10 µM), a nonspecific NO synthase (NOS) inhibitor. Finally, T3 (10-4 M) exhibited, in vitro, an antioxidant role by reducing NADPH oxidase activity and increasing SOD activity in the aorta’s homogenates. Conclusion T3 exerts dependent- and independent-endothelium vasodilation effects, which may be related to its role in maintaining redox homeostasis.

Vascular Response of Triiodothyronine on Isolated Aortic Rings: Contribution of Redox Mechanisms.

Vascular dysfunction constitutes the etiology of many diseases, such as myocardial infarction and hypertension, with the disruption of redox homeostasis playing a role in the imbalance of the vasomotor control mechanism. Our group previously has shown that thyroid hormones exert protective effects on the aortic tissue of infarcted rats by improving angiogenesis signaling. Investigate the role of triiodothyronine (T3) on vascular response, exploring its effects on isolated aortas and whether there is an involvement of vascular redox mechanisms. Isolated aortic rings (intact- and denuded-endothelium) precontracted with phenylephrine were incubated with T3 (10-8, 10-7, 10-6, 10-5, and 10-4 M), and tension was recorded using a force-displacement transducer coupled with an acquisition system. To assess the involvement of oxidative stress, aortic rings were preincubated with T3 and subsequently submitted to an in vitro reactive oxygen species (ROS) generation system. The level of significance adopted in the statistical analysis was 5%. T3 (10-4 M) promoted vasorelaxation of phenylephrine precontracted aortic rings in both intact- and denuded-endothelium conditions. Aortic rings preincubated in the presence of T3 (10-4 M) also showed decreased vasoconstriction elicited by phenylephrine (1 µM) in intact-endothelium preparations. Moreover, T3 (10-4 M) vasorelaxation effect persisted in aortic rings preincubated with NG-nitro-L-arginine methylester (L-NAME, 10 µM), a nonspecific NO synthase (NOS) inhibitor. Finally, T3 (10-4 M) exhibited, in vitro, an antioxidant role by reducing NADPH oxidase activity and increasing SOD activity in the aorta's homogenates. T3 exerts dependent- and independent-endothelium vasodilation effects, which may be related to its role in maintaining redox homeostasis.

Trimetazidine Reduces Cardiac Fibrosis in Rats by Inhibiting NOX2-Mediated Endothelial-to-Mesenchymal Transition.

PurposeEndothelial-to-mesenchymal transition (EndMT) is an important mechanism underlying cardiac fibrosis. The anti-ischemic drug trimetazidine (TMZ) is reportedly useful in ventricular remodeling and associated with NADPH oxidase (NOX) 2. This study aimed to investigate the possible effect of TMZ on cardiac fibrosis exerted via the inhibition of NOX2-mediated EndMT.MethodsA cardiac fibrosis model was established in Sprague-Dawley rats through a subcutaneous injection of isoproterenol (ISO, 5 mg/kg/d). Echocardiographic parameters, myocardial fibrosis, NOX2 expression and EndMT were assessed. An in vitro model of EndMT was developed using human umbilical vein endothelial cells (HUVECs) via treatment with transforming growth factor-β (TGF-β) at 10 ng/mL for 24 h. HUVECs were administrated with TMZ or TMZ and lentivirus, the expression of EndMT and related proteins was observed by wound healing assay, immunoblotting, and immunofluorescence.ResultsRats injected with ISO exhibited severe interstitial cardiac fibrosis and perivascular fibrosis, decreased left ventricular ejection fraction, and increased NOX activity. TMZ treatment mitigated cardiac fibrosis, ameliorated left ventricular dysfunction, and reduced NOX activity. In addition, TMZ effectively inhibited EndMT in ISO-treated rat hearts and TGF-β-treated HUVECs, as manifested by increased CD31 expression, decreased α-SMA expression, and suppressed cell migration. Compared with the control group, the expression of NOX2, nuclear factor-κB (NF-κB), and Snail was increased in vivo and in vitro but decreased with TMZ treatment. Furthermore, the overexpression of NOX2 by lentivirus abolished the protective effects of TMZ on TGF-β-induced EndMT.ConclusionTMZ may ameliorate EndMT and ISO-induced cardiac fibrosis through the NOX2/NF-κB/Snail pathway. The findings of the study may provide new insights into the potential role of TMZ in the pathophysiology of cardiac fibrosis.

Effects of canagliflozin on human myocardial redox signalling: clinical implications.

AimsRecent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans.Methods and resultsStudy 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide () sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM.ConclusionsWe demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin.

Prolyl oligopeptidase inhibition reduces oxidative stress via reducing NADPH oxidase activity by activating protein phosphatase 2A

Oxidative stress (OS) is a common toxic feature in various neurodegenerative diseases. Therefore, reducing OS could provide a potential approach to achieve neuroprotection. Prolyl oligopeptidase (PREP) is a serine protease that is linked to neurodegeneration, as endogenous PREP inhibits autophagy and induces the accumulation of detrimental protein aggregates. As such, inhibition of PREP by a small-molecular inhibitor has provided neuroprotection in preclinical models of neurodegenerative diseases. In addition, PREP inhibition has been shown to reduce production of reactive oxygen species (ROS) and the absence of PREP blocks stress-induced ROS production. However, the mechanism behind PREP-related ROS regulation is not known. As we recently discovered PREP's physiological role as a protein phosphatase 2A (PP2A) regulator, we wanted to characterize PREP inhibition as an approach to reduce OS. We studied the impact of a PREP inhibitor, KYP-2047, on hydrogen peroxide and ferrous chloride induced ROS production and on cellular antioxidant response in HEK-293 and SH-SY5Y cells. In addition, we used HEK-293 and SH-SY5Y PREP knock-out cells to validate the role of PREP on stress-induced ROS production. We were able to show that absence of PREP almost entirely blocks the stress-induced ROS production in both cell lines. Reduced ROS production and smaller antioxidant response was also seen in both cell lines after PREP inhibition by 10 μM KYP-2047. Our results also revealed that the OS reducing mechanism of PREP inhibition is related to reduced activation of ROS producing NADPH oxidase through enhanced PP2A activation. In conclusion, our results suggest that PREP inhibition could also provide neuroprotection by reducing OS, thus broadening the scope of its beneficial effects on neurodegeneration.

Probiotic Bifidobacterium breve prevents DOCA-salt hypertension.

Many probiotics that affect gut microbial ecology have been shown to produce beneficial effects on renin-angiotensin-dependent rodent models and human hypertension. We hypothesized that Bifidobacterium breve CECT7263 (BFM) would attenuate hypertension in deoxycorticosterone acetate (DOCA)-salt rats, a renin-independent model of hypertension. Rats were randomly divided into five groups: control, DOCA-salt, treated DOCA-salt-BFM, treated DOCA-salt-butyrate, and treated DOCA-salt-acetate, for 5weeks. BFM prevented the increase in systolic blood pressure, cardiac weight, and renal damage induced by DOCA-salt. BFM increased acetate-producing bacterial population and gut acetate levels, improved colonic integrity, normalized endotoxemia, plasma trimethylamine (TMA) levels, and restored the Th17 and Treg content in mesenteric lymph nodes and aorta. Furthermore, BFM improved nitric oxide-dependent vasorelaxation induced by acetylcholine in aortic rings and reduced NADPH oxidase activity in DOCA-salt animals. These protective effects were mimicked by acetate, but not by butyrate supplementation. These data demonstrate that BFM induces changes in gut microbiota linked with attenuation of endothelial dysfunction and increase in blood pressure in this low-renin form of hypertension. These beneficial effects seem to be mediated by increased acetate and reduced TMA production by gut microbiota, thus, improving gut integrity and restoring Th17/Tregs polarization and endotoxemia.

Changes to the gut microbiota induced by losartan contributes to its antihypertensive effects.

Hypertension is associated with gut dysbiosis. Here we have evaluated the effects of the angiotensin receptor antagonist losartan on gut microbiota in spontaneously hypertensive rats (SHR) to assess their contribution to its antihypertensive effects. Twenty-week-old Wistar Kyoto rats (WKY) and SHR were treated with losartan for 5 weeks (SHR-losartan). Faecal microbiota transplantation (FMT) was performed from donor SHR-losartan group to recipient untreated-SHR. Blood pressure (BP) was measured using tail-cuff plethysmography. Composition of the gut microbiota was assessed by amplification of the V3-V4 region of 16S rRNA gene. T cells were analysed in gut/aorta by flow cytometry. Faeces from SHR showed gut dysbiosis, characterised by higher Firmicutes/Bacteroidetes ratios, lower acetate- and higher lactate-producing bacteria, and lower levels of strict anaerobic bacteria, effects which were restored to normal by losartan. Improvement of gut dysbiosis was linked to higher colonic integrity and lower sympathetic drive in the gut. In contrast, hydralazine reduced BP, but it neither restored gut dysbiosis nor colonic integrity. FMT from SHR-losartan to SHR reduced BP, improved the aortic endothelium-dependent relaxation to ACh, and reduced NADPH oxidase activity. These vascular changes were accompanied by both increased Treg and decreased Th17 cell populations in the vascular wall. In SHR, losartan treatment reduced gut dysbiosis and sympathetic drive in the gut, thus improving gut integrity. The changes induced by losartan in gut microbiota contributed, in part, to protecting the vasculature and reducing BP, possibly by modulating the immune system in the gut.

INTERPRETATION OF DHR RESULTS FOR CHRONIC GRANULOMATOUS DISEASE (CGD): IMPROVING DIAGNOSTIC ACCURACY

Introduction Very-early-onset inflammatory bowel disease is associated with an increased rate of immune dysregulation. A 14-year-old male with Crohn's and recurrent liver abscesses had 2 dihydrorhodamine (DHR) oxidative burst and phorbol myristate acetate (PMA) induced assays reported as normal. Case Description The patient had gastrointestinal symptoms since infancy and was diagnosed with Crohn's disease at 6 years. He had recurrent sinopulmonary infections and 2 liver abscesses. Two prior DHR assays were reported as normal based on percentage of neutrophils demonstrating oxidative burst (84%, 73%), without reporting NADPH oxidase (NOX) activity measured by mean fluorescence intensity (MFI). Due to high suspicion for chronic granulomatous disease (CGD), a third oxidative burst was obtained from another reference laboratory. It revealed significantly reduced NOX activity in neutrophils. Flow cytometry for NADPH-oxidase specific proteins showed normal expression of gp91phox, p22phox, p67phox and p40phox and absent p47phox. The patient had a heterozygous NCF1 delta GT mutation and a wild-type second allele. He underwent successful hematopoietic stem cell transplantation from a fully matched unrelated donor. Discussion DHR assay is the standard diagnostic test for CGD. However, complete interpretation requires both MFI and percentage of positive cells, and variability exists in how DHR is performed and reported by different reference laboratories. This patient's diagnosis and treatment were delayed by a lack of recognition of abnormal test results. It is essential for ordering physicians to understand the test, including performance characteristics, analysis and reporting by laboratories. Further, specific protein flow can be used to confirm an abnormal DHR result enabling rapid genetic triage. Very-early-onset inflammatory bowel disease is associated with an increased rate of immune dysregulation. A 14-year-old male with Crohn's and recurrent liver abscesses had 2 dihydrorhodamine (DHR) oxidative burst and phorbol myristate acetate (PMA) induced assays reported as normal. The patient had gastrointestinal symptoms since infancy and was diagnosed with Crohn's disease at 6 years. He had recurrent sinopulmonary infections and 2 liver abscesses. Two prior DHR assays were reported as normal based on percentage of neutrophils demonstrating oxidative burst (84%, 73%), without reporting NADPH oxidase (NOX) activity measured by mean fluorescence intensity (MFI). Due to high suspicion for chronic granulomatous disease (CGD), a third oxidative burst was obtained from another reference laboratory. It revealed significantly reduced NOX activity in neutrophils. Flow cytometry for NADPH-oxidase specific proteins showed normal expression of gp91phox, p22phox, p67phox and p40phox and absent p47phox. The patient had a heterozygous NCF1 delta GT mutation and a wild-type second allele. He underwent successful hematopoietic stem cell transplantation from a fully matched unrelated donor. DHR assay is the standard diagnostic test for CGD. However, complete interpretation requires both MFI and percentage of positive cells, and variability exists in how DHR is performed and reported by different reference laboratories. This patient's diagnosis and treatment were delayed by a lack of recognition of abnormal test results. It is essential for ordering physicians to understand the test, including performance characteristics, analysis and reporting by laboratories. Further, specific protein flow can be used to confirm an abnormal DHR result enabling rapid genetic triage.

Full-length cDNA cloning, characterization of catalase from Ulva prolifera and antioxidant response to diphenyliodonium

Here, we present a method for the acquisition of the catalase gene, among the most important enzymes involved in the elimination of reactive oxygen species, from Ulva prolifera, and investigate the relationship of NADPH oxidase, salicylic acid, and high-temperature stress on U. prolifera. The full-length complementary DNA (cDNA) of the catalase gene from the green alga U. prolifera (UpCAT) was cloned by polymerase chain reaction using rapid amplification of cDNA ends. The activity of superoxide dismutase, catalase, NADPH oxidase, and phenylalanine ammonia-lyase and the expression level of UpCAT and glutathione reductase (GR) were investigated following treatment with the NADPH oxidase inhibitor diphenyliodonium and salicylic acid at high temperature. The UpCAT cDNA sequence consisted of 1919 bp containing a 5′-untranslated region of 133 bp, a 3′-untranslated region of 307 bp with a polyA tail, and an open reading frame of 1479 bp encoding a polypeptide of 492 amino acids with molecular weight of 56.88 kDa and theoretical pI of 6.658. The deduced amino acid sequence of UpCAT had significant homology to catalases from other plants. The UpCAT protein sequence contained a proximal heme-ligand signature of 344RIFSYPDTQ352 and a proximal active-site signature of 54FDRERIPERVVHARGAAA71. Additionally, three key residues correlating to catalytic activity, His65, Asn139, and Tyr348, were in the UpCAT sequence. Phylogenetic-tree analysis showed that U. prolifera grouped with Ulva fasciata, Chlamydomonas reinhardtii, Haematococcus pluvialis, and Volvox carteri. Furthermore, we observed that under the high-temperature treatment condition, diphenyliodonium treatment (0.01–0.5 mg L−1) reduced NADPH oxidase activity over the entire time, reduced superoxide dismutase, catalase, and phenylalanine ammonia-lyase activity at the early time and induced the expression of UpCAT. Additionally, the expression level of glutathione reductase was suppressed after DPI (0.5 mg L−1) treatment. Analysis of the UpCAT gene substantially advanced our understanding of green-lineage evolution. The results indicated that NADPH oxidase is involved in the process of salicylic acid-induced alleviation of high-temperature stress; however, the specific mechanism requires further study.

Haploinsufficiency of NADPH Oxidase Subunit Neutrophil Cytosolic Factor 2 Is Sufficient to Accelerate Full-Blown Lupus in NZM 2328 Mice.

We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE). We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus. Clinical disease, serology, and immunopathology were evaluated. NCF-2-null mice on the B6 background were susceptible to Aspergillus fumigatus pneumonia characteristic of chronic granulomatous disease, but did not develop systemic lupus disease. In contrast, NCF-2-null and even NCF-2-haploinsufficient mice on the NZM 2328 background developed accelerated full-blown lupus with significantly accelerated lupus kidney disease. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments, increased expression of type I interferon-responsive genes, and generation of neutrophil extracellular traps, which were observed even in the absence of NADPH oxidase activity. Just as patients with chronic granulomatous disease who lack NADPH oxidase rarely develop SLE, NCF-2-null mice on a nonautoimmune background were susceptible to a chronic granulomatous disease-like opportunistic infection but did not develop lupus. In contrast, on a lupus-prone background, even haploinsufficiency of NCF-2 accelerated the development of full-blown lupus disease. This establishes an interaction between reduced oxidase activity and other lupus-predisposing genes, paralleling human SLE-associated variants predicted to have only reduced NADPH oxidase activity.

Thioredoxin attenuates oxidized low-density lipoprotein induced oxidative stress in human umbilical vein endothelial cells by reducing NADPH oxidase activity

Oxidative stress is recognized as one of the most important contributing factors to the development of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) can induce vascular reactive oxygen species (ROS) production, trigger endothelial dysfunction and initiate the progression of atherosclerosis. Previous studies have demonstrated that thioredoxin-1 (Trx) is one of the key regulators of intracellular redox, which is pivotal in atherogenesis. However, the regulation mechanism is still unclear. In this study, we investigated the effects of Trx1 on NADPH oxidase in human umbilical vein endothelial cells (HUVECs), whose ROS level is mainly produced by NADPH oxidase, especially Nox4 isoform. Our data demonstrated that Trx decreased NADPH oxidase activity, ROS production and ICAM-1 expression in ox-LDL treated HUVECs. Genetic gain-of-function and loss-of-function studies showed that Trx1 suppressed ox-LDL-induced Nox4 and p22phox expression. A co-immunoprecipitation assay indicated that Trx1 decreased Nox4-p22phox complex level during ox-LDL stimulation. Transient transfection of Nox4 and p22phox significantly increased intracellular ROS generation, which could be blocked by Trx overexpression. In addition, Trx overexpression also prevented ox-LDL-induced Nox2 and Rac1 protein levels. These results suggest that Trx suppresses NADPH oxidase activity in vascular endothelia under pathological conditions and may prevent the initiation of atherosclerosis by attenuating exceeding ROS production.

Ferulic acid, a bioactive component of rice bran, improves oxidative stress and mitochondrial biogenesis and dynamics in mice and in human mononuclear cells

The aim of the study was to characterize the vascular effects of rice bran enzymatic extract (RBEE). ApoE−/− mice were fed a high-fat/cholesterol diet (HFD) or HFD supplemented with 5% RBEE for 21 weeks. RBEE prevented development of atherosclerotic plaques and oxidative stress in mouse aorta as well as the down-regulation of markers of mitochondrial biogenesis. Analysis of the bioactive components identified ferulic acid (FA) as responsible component. In healthy human volunteers, FA intake reduced NADPH oxidase activity, superoxide release, apoptosis and necrosis in peripheral blood mononuclear cells. Differentiation and proliferation of endothelial progenitor cells were improved. In summary, the study identifies FA as a major active component of rice bran, which improves expression of mitochondrial biogenesis and dynamics markers and reduces oxidative stress in a mouse model of vascular damage as well as in endothelial cells and human mononuclear cells.

Rac1 Pharmacological Inhibition Rescues Human Endothelial Dysfunction.

BackgroundEndothelial dysfunction contributes significantly to the development of vascular diseases. However, a therapy able to reduce this derangement still needs to be identified. We evaluated the effects of pharmacological inhibition of Rac1, a small GTPase protein promoting oxidative stress, in human endothelial dysfunction.Methods and ResultsWe performed vascular reactivity studies to test the effects of NSC23766, a Rac1 inhibitor, on endothelium‐dependent vasorelaxation of saphenous vein segments collected from 85 subjects who had undergone surgery for venous insufficiency and from 11 patients who had undergone peripheral vascular surgery. The endothelium‐dependent vasorelaxation of the varicose segments of saphenous veins collected from patients with venous insufficiency was markedly impaired and was also significantly lower than that observed in control nonvaricose vein tracts from the same veins. Rac1 activity, reactive oxygen species levels, and reduced nicotine adenine dinucleotide phosphate (NADPH) oxidase activity were significantly increased in varicose veins, and NSC23766 was able to significantly improve endothelium‐dependent vasorelaxation of dysfunctional saphenous vein portions in a nitric oxide–dependent manner. These effects were paralleled by a significant reduction of NADPH oxidase activity and activation of endothelial nitric oxide synthase. Finally, we further corroborated this data by demonstrating that Rac1 inhibition significantly improves venous endothelial function and reduces NADPH oxidase activity in saphenous vein grafts harvested from patients with vascular diseases undergoing peripheral bypass surgery.ConclusionsRac1 pharmacological inhibition rescues endothelial function and reduces oxidative stress in dysfunctional veins. Rac1 inhibition may represent a potential therapeutic intervention to reduce human endothelial dysfunction and subsequently vascular diseases in various clinical settings.

Nitrite-mediated reduction of macrophage NADPH oxidase activity is dependent on xanthine oxidoreductase-derived nitric oxide but independent of S-nitrosation

BackgroundInorganic nitrite has shown beneficial effects in cardiovascular and metabolic diseases partly via attenuation of NADPH-oxidase (NOX)-mediated oxidative stress. However, the exact mechanisms are still unclear. Here we investigated the role of S-nitrosation or altered expression of NOX subunits, and the role of xanthine oxidoreductase (XOR) in nitrite-derived nitric oxide (NO) production. MethodsMouse macrophages were activated with LPS in the presence or absence of nitrite. NOX activity was measured by lucigenin-dependent chemiluminescence. Gene and protein expression of NOX2 subunits and XOR were investigated using qPCR and Western Blot. S-nitrosation of Nox2 and p22phox was studied with a Biotin Switch assay. Uric acid levels in cell culture medium were analyzed as a measure of XOR activity, and NO production was assessed by DAF-FM fluorescence. ResultsNOX activity in activated macrophages was significantly reduced by nitrite. Reduced NOX activity was not attributed to decreased NOX gene expression. However, protein levels of p47phox and p67phox subunits were reduced by nitrite in activated macrophages. Protein expression of Nox2 and p22phox was not influenced by this treatment and neither was their S-nitrosation status. Increased uric acid levels after nitrite and diminished NO production during XOR-inhibition with febuxostat suggest that XOR is more active during nitrite-treatment of activated macrophages and plays an important role in the bioactivation of nitrite. ConclusionsOur findings contribute to the mechanistic understanding about the therapeutic effects associated with nitrite supplementation in many diseases. We show that nitrite-mediated inhibition of NOX activity cannot be explained by S-nitrosation of the NOX enzyme, but that changes in NOX2 expression and XOR function may contribute.

Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., impedes oxalate/calcium oxalate induced pathways of oxidative stress in male wistar rats

BackgroundOxalate and/or calcium oxalate, is known to induce free radical production, subsequently leading to renal epithelial injury. Oxidative stress and mitochondrial dysfunction have emerged as new targets for managing oxalate induced renal injury. HypothesisPlant products and antioxidants have gained tremendous attention in the prevention of lithiatic disease. Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., has shown free radical scavenging, antioxidant activity and has been reported to interfere in signaling pathways leading to inflammation and apoptosis. In this study, the potential role of rottlerin, in rats exposed to hyperoxaluric environment was explored. MethodsHyperoxaluria was induced by administering 0.4% ethylene glycol and 1% ammonium chloride in drinking water to male wistar rats for 9 days. Rottlerin was administered intraperitoneally at 1mg/kg/day along with the hyperoxaluric agent. Prophylactic efficacy of rottlerin to diminish hyperoxaluria induced renal dysfunctionality and crystal load was examined along with its effect on free radicals generating pathways in hyperoxaluric rats. Results0.4% ethylene glycol and 1% ammonium chloride led to induction of hyperoxaluria, oxiadtive stress and mitochondrial damage in rats. Rottlerin treatment reduced NADPH oxidase activity, prevented mitochondrial dysfunction and maintained antioxidant environment. It also refurbished renal functioning, tissue integrity and diminished urinary crystal load in hyperoxaluric rats treated with rottlerin. ConclusionsThus, the present investigation suggests that rottlerin evidently reduced hyperoxaluric consequences and the probable mechanism of action of this drug could be attributed to its ability to quench free radicals by itself and interrupting signaling pathways involved in pathogenesis of stone formation.

Nitric oxide generation by the organic nitrate NDBP attenuates oxidative stress and angiotensin II‐mediated hypertension

NO deficiency and oxidative stress are crucially involved in the development or progression of cardiovascular disease, including hypertension and stroke. We have previously demonstrated that acute treatment with the newly discovered organic nitrate, 2-nitrate-1,3-dibuthoxypropan (NDBP), is associated with NO-like effects in the vasculature. This study aimed to further characterize the mechanism(s) and to elucidate the therapeutic potential in a model of hypertension and oxidative stress. A combination of ex vivo, in vitro and in vivo approaches was used to assess the effects of NDBP on vascular reactivity, NO release, NADPH oxidase activity and in a model of hypertension. Ex vivo vascular studies demonstrated NDBP-mediated vasorelaxation in mesenteric resistance arteries, which was devoid of tolerance. In vitro studies using liver and kidney homogenates revealed dose-dependent and sustained NO generation by NDBP, which was attenuated by the xanthine oxidase inhibitor febuxostat. In addition, NDBP reduced NADPH oxidase activity in the liver and prevented angiotensin II-induced activation of NADPH oxidase in the kidney. In vivo studies showed that NDBP halted the progression of hypertension in mice with chronic angiotensin II infusion. This was associated with attenuated cardiac hypertrophy, and reduced NADPH oxidase-derived oxidative stress and fibrosis in the kidney and heart. The novel organic nitrate NDBP halts the progression of angiotensin II-mediated hypertension. Mechanistically, our findings suggest that NDBP treatment is associated with sustained NO release and attenuated activity of NADPH oxidase, which to some extent requires functional xanthine oxidase.

Cluster Differentiating 36 (CD36) Deficiency Attenuates Obesity-Associated Oxidative Stress in the Heart.

RationaleObesity is often associated with a state of oxidative stress and increased lipid deposition in the heart. More importantly, obesity increases lipid influx into the heart and induces excessive production of reactive oxygen species (ROS) leading to cell toxicity and metabolic dysfunction. Cluster differentiating 36 (CD36) protein is highly expressed in the heart and regulates lipid utilization but its role in obesity-associated oxidative stress is still not clear.ObjectiveThe aim of this study was to determine the impact of CD36 deficiency on cardiac steatosis, oxidative stress and lipotoxicity associated with obesity.Methods and ResultsStudies were conducted in control (Lean), obese leptin-deficient (Lepob/ob) and leptin-CD36 double null (Lepob/obCD36-/-) mice. Compared to lean mice, cardiac steatosis, and fatty acid (FA) uptake and oxidation were increased in Lepob/ob mice, while glucose uptake and oxidation was reduced. Moreover, insulin resistance, oxidative stress markers and NADPH oxidase-dependent ROS production were markedly enhanced. This was associated with the induction of NADPH oxidase expression, and increased membrane-associated p47phox, p67phox and protein kinase C. Silencing CD36 in Lepob/ob mice prevented cardiac steatosis, increased insulin sensitivity and glucose utilization, but reduced FA uptake and oxidation. Moreover, CD36 deficiency reduced NADPH oxidase activity and decreased NADPH oxidase-dependent ROS production. In isolated cardiomyocytes, CD36 deficiency reduced palmitate-induced ROS production and normalized NADPH oxidase activity.ConclusionsCD36 deficiency prevented obesity-associated cardiac steatosis and insulin resistance, and reduced NADPH oxidase-dependent ROS production. The study demonstrates that CD36 regulates NADPH oxidase activity and mediates FA-induced oxidative stress.

Abstract P085: Angiotensin II Type 2-receptor Agonist C21 Reduces Proteinuria and Oxidative Stress in Kidney of High-salt Fed Obese Zucker Rats

Oxidative and nitrosative stress have been implicated in hypertension and kidney diseases. Recently we reported blood pressure reducing effect of AT2-receptor agonist C21 in high sodium-diet (HSD)-fed obese Zucker rats. In the present study we investigated C21-mediated reno-protection against HSD-related oxidative and nitrosative stress in obese Zucker rats. Obese rats (13 week) were fed normal sodium-diet (NSD) or HSD 4%, for 14 days, with/without C21, delivered subcutaneously via osmotic pump, 1 mg/kg/day. Compared with NSD controls, HSD-fed rats exhibited reduced eGFR (15.2±2.3 vs. 43.5±9.9 μL/min) and increased proteinuria (0.18±0.02 vs. 0.12±0.02 μg/min) and albuminuria (0.19±0.03 vs. 0.12±0.02 μg/min), which were associated with decreased cortical expression of the protein recycling receptors, megalin (0.25±0.02 vs. 0.47±0.07) and cubilin (0.77±0.16 vs. 1.27±0.14). C21 improved eGFR (27.4±4.9 μL/min) and reduced proteinuria (0.11±0.02 μg/min) and albuminuria (0.08±0.01 μg/min) without any effects on megalin or cubilin. Cortical NADPH oxidase (NOX) activity in HSD-fed rats was increased by 3-fold (0.37±0.07 vs. 0.13±0.04 ΔRLU/μg protein/min), while enzymatic defense (catalase/superoxide dismutase activity ratio) was unchanged. Urinary excretion of H2O2 (41.7±4.7 vs. 23.0±2.1 μM/mg creatinine) and 8-isoprostanes (7.9±1.6 vs. 1.4±0.2 pg/μg creatinine), indices of oxidative stress, were elevated in HSD-fed rats. C21 reduced NOX activity (0.11±0.04 ΔRLU/μg protein/min) as well as the excretion of H2O2 (30.5±3.0 μM/mg creatinine) and 8-Isoprostanes (4.1±1.0 pg/μg creatinine) in HSD-fed rats. Similarly, C21 also reduced carbonyls (5.5±1.4 vs. 10.5±0.9 μM/mg protein), an indicator of protein modification caused by oxidative stress. 3-nitrotyrosine, an indicator of nitrosative stress, remained unaltered in HSD and/or C21 groups. C21-treatment of HSD-fed rats improved plasma nitrites (22.1±2.2 vs. 15.3±0.5 μM/L), which could have been due to reduced oxidative stress in this group. Together results indicate that AT2R agonist exerts antioxidant effects, possibly by reducing NOX activity and by rescuing NO levels, which potentially contributes to the reduction in salt-induced proteinuria/nephropathy in obesity.