Adenine Dinucleotide Phosphate Oxidase Activity Research Articles

Activation of NADPH oxidase mediates mitochondrial oxidative stress and atrial remodeling in diabetic rabbits

AimsThe mechanisms of atrial fibrillation (AF) in diabetes mellitus (DM) involve a complex interplay between increased oxidative stress, mitochondrial dysfunction and atrial remodeling. In this study, we examined the effects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation on mitochondrial oxidative stress and atrial remodeling in a rabbit model of diabetes mellitus (DM). Main methodsHealthy rabbits were selected and randomly divided into control, diabetic and apocynin administration group. Parameters of echocardiography, atrial electrophysiology, oxidative stress and mitochondrial function were compared between the different groups. Key findingsCompared to the control group, the DM group showed higher activity of NADPH oxidase, increased oxidative stress, larger left atrial diameter, a reduction in atrial mean conduction velocity. These findings were associated with increased interstitial fibrosis of the atria and higher atrial fibrillation (AF) inducibility. Moreover, atrial ultrastructure and mitochondrial function such as the mitochondrial respiratory control rate (RCR) were impaired. NADPH oxidase inhibition using the pharmacological agent apocynin improved these changes. SignificanceNADPH oxidase activity plays an important role in mitochondrial oxidative stress, which is associated with AF inducibility by promoting adverse atrial remodeling. The NADPH oxidase inhibitor apocynin can prevent these pathological changes and may be a potential drug for AF treatment.

Preventive Effect of Canstatin against Ventricular Arrhythmia Induced by Ischemia/Reperfusion Injury: A Pilot Study.

Ventricular arrhythmia induced by ischemia/reperfusion (I/R) injury is a clinical problem in reperfusion therapies for acute myocardial infarction. Ca2+ overload through reactive oxygen species (ROS) production is a major cause for I/R-induced arrhythmia. We previously demonstrated that canstatin, a C-terminal fragment of type IV collagen α2 chain, regulated Ca2+ handling in rat heart. In this study, we aimed to clarify the effects of canstatin on I/R-induced ventricular arrhythmia in rats. Male Wistar rats were subjected to I/R injury by ligating the left anterior descending artery followed by reperfusion. Ventricular arrhythmia (ventricular tachycardia and ventricular fibrillation) was recorded by electrocardiogram. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and ROS production in neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen glucose deprivation/reperfusion (OGD/R) were measured by lucigenin assay and 2′,7′-dichlorodihydrofluorescein diacetate staining, respectively. The H2O2-induced intracellular Ca2+ ([Ca2+]i) rise in NRCMs was measured by a fluorescent Ca2+ indicator. Canstatin (20 µg/kg) inhibited I/R-induced ventricular arrhythmia in rats. Canstatin (250 ng/mL) inhibited OGD/R-induced NOX activation and ROS production and suppressed the H2O2-induced [Ca2+]i rise in NRCMs. We for the first time demonstrated that canstatin exerts a preventive effect against I/R-induced ventricular arrhythmia, perhaps in part through the suppression of ROS production and the subsequent [Ca2+]i rise.

Epigallocatechin Gallate Reduces Homocysteine-Caused Oxidative Damages through Modulation SIRT1/AMPK Pathway in Endothelial Cells.

Elevated plasma concentration of total homocysteine is a pathological condition that causes vascular endothelial injury and subsequently leads to the progression of endothelial apoptosis in atherosclerosis. Epigallocatechin gallate (EGCG), a well-known anti-oxidant in green tea, has been reported with benefits on metabolic and cardiovascular diseases. This study aimed to explore that EGCG ameliorates homocysteine-induced endothelial cell apoptosis through enhancing the sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK) survival signaling pathway. Human umbilical endothelial cells were treated with homocysteine in the presence or absence of EGCG. We found that EGCG significantly increased the activities of SIRT1 and AMPK. EGCG diminished homocysteine-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation by inhibiting protein kinase C activation as well as reactive oxygen species (ROS) generation and recovered the activity of the endogenous antioxidant enzyme, superoxidase dismutase (SOD). Besides, EGCG also restores homocysteine-mediated dephosphorylation of Akt and decreases endothelial NO synthase (eNOS) expression. Furthermore, EGCG ameliorates homocysteine-activated pro-apoptotic events. The present study shows that EGCG prevents homocysteine-induced endothelial cell apoptosis via enhancing SIRT1/AMPK as well as Akt/eNOS signaling pathways. Results from this study indicated that EGCG might have some benefits for hyperhomocysteinemia.

Probucol decreases homocysteine-stimulated CRP production in rat aortic smooth muscle cells via regulating HO-1/NADPH oxidase/ROS/p38 pathway.

The elevated homocysteine level is an independent risk factor for atherosclerosis, which is characterized as a chronic inflammatory disease associated with oxidative stress. We have confirmed that homocysteine can stimulate the production of C-reactive protein (CRP) in rat aortic smooth muscle cells (RASMCs). In the present study, we investigated the role of probucol in homocysteine-induced CRP expression in cultured RASMCs and high-methionine-diet-induced hyperhomocysteinemic rats. The results showed that probucol decreased homocysteine-induced CRP mRNA and protein expression in RASMCs in a concentration-dependent manner. In addition, the animal experiment showed that probucol not only inhibited CRP expression in the vessel wall but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further investigations revealed that probucol markedly increased heme oxygenase-1 activity, suppressed nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, diminished superoxide anion generation, and decreased p38 phosphorylation in RASMCs and hyperhomocysteinemic rat aorta. These data demonstrate that probucol can inhibit homocysteine-induced CRP generation by interfering with the NADPH oxidase/p38 signal pathway in RASMCs, which will provide new evidence for the anti-inflammatory and anti-atherosclerotic effects of probucol.

Different Effects of Metformin and A769662 on Sodium Iodate-Induced Cytotoxicity in Retinal Pigment Epithelial Cells: Distinct Actions on Mitochondrial Fission and Respiration.

Oxidative stress-associated retinal pigment epithelium (RPE) cell death is critically implicated in the pathogenesis of visual dysfunction and blindness of retinal degenerative diseases. Sodium iodate (NaIO3) is an oxidative retinotoxin and causes RPE damage. Previously, we found that NaIO3 can induce human ARPE-19 cell death via inducing mitochondrial fission and mitochondrial dysfunction. Although metformin has been demonstrated to benefit several diseases possibly via AMP-activated protein kinase (AMPK) activation, it remains unknown how AMPK affects retinopathy in NaIO3 model. Therefore, in this study, we compared the effects of metformin and AMPK activator A769662 on NaIO3-induced cellular stress and toxicity. We found that A769662 can protect cells against NaIO3-induced cytotoxicity, while metformin exerts an enhancement in cell death. The mitochondrial reactive oxygen species (ROS) production as well as mitochondrial membrane potential loss induced by NaIO3 were not altered by both agents. In addition, NaIO3-induced cytosolic ROS production, possibly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and counteracting cell death, was not altered by A769662 and metformin. Notably, NaIO3-induced mitochondrial fission and inhibition of mitochondrial respiration for ATP turnover were reversed by A769662 but not by metformin. In agreement with the changes on mitochondrial morphology, the ERK-Akt signal axis dependent Drp-1 phosphorylation at S616 (an index of mitochondrial fission) under NaIO3 treatment was blocked by A769662, but not by metformin. In summary, NaIO3-induced cell death in ARPE cells primarily comes from mitochondrial dysfunction due to dramatic fission and inhibition of mitochondrial respiration. AMPK activation can exert a protection by restoring mitochondrial respiration and inhibition of ERK/Akt/Drp-1 phosphorylation, leading to a reduction in mitochondrial fission. However, inhibition of respiratory complex I by metformin might deteriorate mitochondrial dysfunction and cell death under NaIO3 stress.

Docosahexaenoic acid inhibits ethanol/palmitoleic acid-induced necroptosis in AR42J cells.

Fatty acid ethyl esters (FAEEs), non-oxidative metabolites of ethanol, are the main causative agents of severe acute pancreatitis resulting from alcohol abuse. Pancreatic acinar cells exposed to ethanol in combination with the fatty acid palmitoleic acid (EtOH/POA) display increased levels of palmitoleic acid ethyl ester and cell death. Oxidative stress and acinar cell necroptosis are implicated in the pathology of severe acute pancreatitis. Docosahexaenoic acid (DHA) serves as a powerful anti-oxidant that reduces pancreatic inflammation and improves the outcomes of patients with acute pancreatitis. We investigated whether treatment of EtOH/POA, as an in vitro model of alcoholic pancreatitis, increases reactive oxygen species (ROS), necroptosis-regulating proteins, and cell death by increasing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and intracellular calcium. Also, we investigated whether DHA inhibits EtOH/POA-induced alterations in pancreatic acinar AR42J cells. As a result, EtOH/POA increased intracellular and mitochondrial ROS levels, NADPH oxidase activity, necroptosis-regulating proteins, and cell death, which was inhibited by NADPH oxidase inhibitor apocynin, the Ca2+ chelator BAPTA, and DHA. However, DHA did not reduce EtOH/POA-induced increases in Ca2+ oscillation or levels in AR42J cells. Furthermore, EtOH/POA induced mitochondrial dysfunction by reducing mitochondrial membrane polarization and hence, adenosine triphosphate (ATP) production. DHA treatment attenuated EtOH/POA-induced mitochondrial dysfunction. In conclusion, DHA inhibits EtOH/POA-induced necroptosis by suppressing NADPH oxidase activity, reducing ROS levels, preventing mitochondrial dysfunction, and inhibiting activation of necroptosis-regulating proteins in AR42J cells.

Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants-identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

Silver-nanoparticle and abscisic acid modulate sub1A quantitative trait loci functioning towards submergence tolerance in rice (Oryza sativa L.)

An experiment was conducted to explore the silver-nanoparticles (Ag-NPs) and abscisic acid (ABA) to elicit sub1A quantitative trait loci (QTL) responses under submergence stress. Rice genotypes cv. Swarna and Swarna Sub1 were pre-treated with 100 mg ml−1Ag-NP [Ag-NP(+)] and 10 μM ABA [ABA(+)] at 7-day-old seedlings for 1 day in two separate sets and subsequent transfer to complete submergence for 5 days. sub1A QTL regulatory APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) expression was mitigated for both genotypes under submergence; however, retrieved with Ag-NP(+) and ABA(+) variably. ABA(+) had recorded affirmative responses than Ag-NP(+) in net assimilation rate, relative growth rate, chlorophyll content, and metal accumulation under stress. cv. Swarna Sub1 under submergence had better recovery of proline and relative water content than others. Phenolics, flavonoids, carotenoids, and lycopene content were more influenced by ABA(+) than Ag-NP(+) to recover under submergence. The development of superoxide and hydrogen peroxide had a positive and fair correlation with nicotinamide adenine dinucleotide phosphate oxidase (EC 1.6.3.1) activity under submergence. cv. Swarna Sub1 responded better under submergence through invertase (EC 3.2.1.26), starch phosphorylase (EC 2.4.1.1) and sucrose synthase (EC 2.4.1.13) activity more with ABA(+) than Ag-NP(+). The functioning of sub1A QTL for anaerobic metabolism was improved in cv. Swarna Sub1 than cv. Swarna with the support of polymorphic expression of alcohol dehydrogenase (EC 1.1.1.1). Finally, given results may arise the prospect for mitigation submergence stress by Ag-NP(+) and ABA(+) through modulation of cellular responses affiliated to sub1A QTL.

Interactive effects of mercuric oxide nanoparticles and future climate CO2 on maize plant

So far, the phytotoxic hazards of nano-sized mercuric oxide (HgO-NPs) are not investigated. Herein, the phytotoxicity of fully characterized HgO-NPs (100 mg/kg soil), prepared by coprecipitation method, on maize grown under ambient (aCO2, 410 ppm) and elevated CO2 (eCO2, 620 ppm) was investigated. Regardless of CO2 concentration, HgO-NPs treatment increased Hg levels in maize organs. HgO-NPs induced severe oxidative stress in aCO2 grown plants as indicated by reduced growth and photosynthesis and accumulation of reactive oxygen species (ROS), through photorespiration and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities, and lipid and protein oxidation products. Although HgO-NPs increased molecular (polyphenols, flavonoids, tocopherols) and enzymatic (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, glutathione peroxidase) antioxidants in shoots of aCO2 plants, but this failed to fight the eruption of increased ROS. On contrary, eCO2 treatment mitigated the HgO-NPs impact by promoting photosynthesis and reducing the Hg-induced ROS production. Moreover, eCO2 promoted ROS detoxification via molecular antioxidants overproduction, enhanced superoxide dismutase, catalase and peroxidases activities, and modulation of reduced ascorbate/oxidized ascorbate and reduced glutathione/oxidized glutathione homeostasis. The combined HgO-NPs + eCO2 treatment also enhanced the glutathione-S-transferase activity. This study suggests that HgO-NPs cause severe phytotoxic hazards and this effect will be less detrimental under future CO2 climate.

ELABELA attenuates deoxycorticosterone acetate/salt-induced hypertension and renal injury by inhibition of NADPH oxidase/ROS/NLRP3 inflammasome pathway

ELABELA (ELA), a 32-residue hormone peptide abundantly expressed in adult kidneys, has been identified as a novel endogenous ligand for APJ/Apelin receptor. The aim of this study was to investigate the role of ELA in deoxycorticosterone acetate (DOCA)/salt-induced hypertension and further explore the underlying mechanism. In DOCA/salt-treated rats, the mRNA level of ELA greatly decreased in the renal medulla. Next, overexpression of ELA in the kidney was found to attenuate DOCA/salt-induced hypertension and renal injury, including lower blood pressure, reversed glomerular morphological damage, decreased blood urea nitrogen (BUN), and blocked the accumulation of fibrotic markers. Mechanistically, ELA overexpression inhibited renal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and subsequent reactive oxygen species (ROS) production, thus resulted in the blockade of formation and activation of Nod-like receptor protein 3 (NLRP3) inflammasome. The inhibitory effects of ELA on Aldosterone-stimulated NADPH oxidase/ROS/NLRP3 inflammasome pathway were confirmed in the human renal tubular cells. Furthermore, our in vivo and in vitro results showed that the deficiency of the apelin receptor APJ did not influence the antihypertensive effect and blockage to NADPH oxidase/ROS/NLRP3 pathway of ELA. Moreover, in heterozygous ELA knockout mice (ELA+/−), the ELA deficiency remarkably accelerated the onset of DOCA/salt-induced hypertension. Our data demonstrate that ELA prevents DOCA/salt-induced hypertension by inhibiting NADPH oxidase/ROS/NLRP3 pathway in the kidney, which is APJ independent. Pharmacological targeting of ELA may serve as a novel therapeutic strategy for the treatment of hypertensive kidney disease.

Metabolic adaptability in liver and gastrocnemius muscle of mice following subacute lead toxicity.

Lead (Pb) is one of several environmental pollutants that adversely affect human health by producing toxicity at the tissue level. The aim of the study was to understand the effects of Pb on the metabolic profiles of liver and gastrocnemius muscle of mice in relation to carbohydrate and fat metabolisms. Swiss albino mice were chosen and divided into two groups, control and Pb-treated. The Pb-treated animals were exposed to Pb at a dose of 5 mg/kg body weight for 30 days orally, which resulted in hypoglycemia, glycosuria, and increased glycogenolysis in the liver and gastrocnemius muscle of treated mice. Pyruvic acid, the end product of glycolysis decreased in muscular tissue and increased in the liver. Additionally, the activity of G-6Pase was depressed in the liver, whereas lactate dehydrogenase activity was increased in skeletal muscle only. An adaptive mechanism was initiated by stimulating glycogenolytic and retarding glycolytic activity in the liver and also by alteration of liver and muscle pyruvate dehydrogenase activity along with increased activity of malate dehydrogenase in skeletal muscle. There was enhancement of succinate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase activities in the studied tissues. Interestingly, cholesterol, high-density lipoprotein, and low-density lipoprotein levels were elevated, whereas those of triglycerides were decreased in Pb-treated mice serum. The activities of fatty acid synthase and glyceraldehyde 3 phosphate dehydrogenase were depressed in Pb-treated mice livers. Pb also significantly altered the morphological features of the liver, skeletal muscle, and pancreas. These data suggested that subacute Pb exposure was responsible for metabolic modulation in an adaptive fashion in the liver and skeletal muscle of mice.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. Methods: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b₅₅₈ were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. Results: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. Conclusions: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.

Systemic Redox Imbalance in Patients with Chronic Granulomatous Disease.

The aim of our study was to evaluate redox status, enzymatic and non-enzymatic antioxidant barriers, oxidative damage of proteins, lipids and DNA, as well as concentration of coenzyme Q10 and vitamins A and E in patients with chronic granulomatous disease (CGD). The study was performed on fifteen Caucasian individuals (median age 24 years and seven months) diagnosed with CGD. The mutation in the NCF1 gene was confirmed in ten patients, and in the CYBB gene in five patients. We demonstrated high levels of total oxidant status (TOS) and oxidative stress index (OSI), lipids (↑8-isoprostanes (8-isoP), ↑4-hydroxynonenal (4-HNE)), proteins (↑advanced oxidation protein products (AOPP)) and DNA (↑8-hydroxy-2’-deoxyguanosine (8-OHdG)) oxidation products in CGD individuals as compared to sex- and age-matched healthy controls. We showed enhanced serum enzymatic activity of catalase (CAT) and superoxide dismutase-1 (SOD) and significantly decreased coenzyme Q10 concentration. Our study confirmed redox disturbances and increased oxidative damage in CGD patients, and indicated the need to compare redox imbalance depending on the type of mutation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The question regarding effectiveness of antioxidant therapy in patients with CGD is open, and the need to establish guidelines in this area remains to be addressed.

Deterioration of cognitive function after transient cerebral ischemia with amyloid-\u03b2 infusion\u2014possible amelioration of cognitive function by AT2 receptor activation

BackgroundTo promote understanding of the pathogenesis of cognitive impairment or dementia, we explored the potential interaction between transient cerebral ischemia and amyloid-β (Aβ) infusion in mediating cognitive decline and examined the possible ameliorative effect of angiotensin II type 2 (AT2) receptor activation in vascular smooth muscle cells (VSMC) on this cognitive deficit.MethodsAdult male wild-type mice (WT) and mice with VSMC-specific AT2 receptor overexpression (smAT2) were subjected to intracerebroventricular (ICV) injection of Aβ1-40. Transient cerebral ischemia was induced by 15 min of bilateral common carotid artery occlusion (BCCAO) 24 h after Aβ injection.ResultsAβ injection in WT induced a cognitive decline, whereas BCCAO did not cause a significant cognitive deficit. In contrast, WT with BCCAO following Aβ injection exhibited more marked cognitive decline compared to Aβ injection alone, in concert with increases in superoxide anion production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and expression of p22phox, p40phox, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1β in the hippocampus, and upregulation of RAGE (receptor for advanced glycation end product), an Aβ transporter. BCCAO following Aβ injection further enhanced neuronal pyknosis in the hippocampus, compared with BCCAO or Aβ injection alone. In contrast, smAT2 did not show a cognitive decline, increase in oxidative stress, inflammation, and RAGE level or neuronal pyknosis, which were induced by BCCAO with/without Aβ injection in WT.ConclusionsTransient cerebral ischemia might worsen Aβ infusion-mediated cognitive decline and vice versa, with possible involvement of amplified oxidative stress and inflammation and impairment of the RAGE-mediated Aβ clearance system, contributing to exaggerated neuronal degeneration. AT2 receptor activation in VSMC could play an inhibitory role in this cognitive deficit.

Exercise training delays renal disorders with decreasing oxidative stress and increasing production of 20-hydroxyeicosatetraenoic acid in Dahl salt-sensitive rats

Exercise training has antihypertensive and renoprotective effects in humans and rats. However, the effects of exercise training on renal disorders that occur with salt-sensitive hypertension remains unclear. The study aim was to investigate the effects and mechanisms of exercise training on renal function in a rat model of salt-sensitive hypertension. Six-week-old male Dahl salt-sensitive rats were divided into normal-salt (0.6% NaCl) diet, high-salt (8% NaCl) diet, and high-salt diet with exercise training groups. The high-salt diet with exercise training group underwent daily treadmill running for 8 weeks. The high-salt diet induced severe hypertension and renal dysfunction. Exercise training significantly improved high-salt diet-induced urinary protein, albumin, and L-type fatty acid-binding protein excretion, and glomerulosclerosis but not renal interstitial fibrosis without changing blood pressure. Exercise training significantly attenuated high-salt diet-induced oxidative stress in the kidneys and decreased high-salt diet-stimulated xanthine oxidoreductase activity but not nicotinamide adenine dinucleotide phosphate oxidase activity. The high-salt diet did not change urinary excretion of 20-hydroxyeicosatetraenoic acid and decreased cytochrome P450 4A protein expression in the kidneys. Exercise training increased urinary 20-hydoroxyeicosatetraenoic acid excretion and renal cytochrome P450 4A protein expression. Exercise training improved renal disorders without lowering blood pressure in Dahl salt-sensitive rats. Exercise training also decreased oxidative stress and increased 20-hydroxyeicosatetraenoic acid production in the kidneys. These results suggest that improvements in oxidative stress and 20-hydroxyeicosatetraenoic acid production may be potential mechanisms by which exercise training improved renal disorders in Dahl salt-sensitive rats.

The Effect of CB1 Antagonism on Hepatic Oxidative/Nitrosative Stress and Inflammation in Nonalcoholic Fatty Liver Disease.

Dysfunction of the endocannabinoid system (ES) has been identified in nonalcoholic fatty liver disease (NAFLD) and associated metabolic disorders. Cannabinoid receptor type 1 (CB1) expression is largely dependent on nutritional status. Thus, individuals suffering from NAFLD and metabolic syndrome (MS) have a significant increase in ES activity. Furthermore, oxidative/ nitrosative stress and inflammatory process modulation in the liver are highly influenced by the ES. Numerous experimental studies indicate that oxidative and nitrosative stress in the liver is associated with steatosis and portal inflammation during NAFLD. On the other hand, inflammation itself may also contribute to reactive oxygen species (ROS) production due to Kupffer cell activation and increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The pathways by which endocannabinoids and their lipid-related mediators modulate oxidative stress and lipid peroxidation represent a significant area of research that could yield novel pharmaceutical strategies for the treatment of NAFLD. Cumulative evidence suggested that the ES, particularly CB1 receptors, may also play a role in inflammation and disease progression toward steatohepatitis. Pharmacological inactivation of CB1 receptors in NAFLD exerts multiple beneficial effects, particularly due to the attenuation of hepatic oxidative/nitrosative stress parameters and significant reduction of proinflammatory cytokine production. However, further investigations regarding precise mechanisms by which CB1 blockade influences the reduction of hepatic oxidative/nitrosative stress and inflammation are required before moving toward the clinical phase of the investigation.

Association between p22PHOX gene C242T polymorphism and hypertension in end-stage kidney disease patients.

Oxidative stress plays an important role in hypertension associated vascular damage. It is mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. The C242T polymorphism in the p22PHOX gene encoding essential subunit of NADPH oxidase was associated with CVD, hypertension, and endothelial function. The aim of this study was to assess a potential association of C242T polymorphism with hypertension in end-stage kidney disease (ESKD) patients. DNA samples from 495 patients were genotyped by polymerase chain reaction (PCR) with subsequent cleavage with Rsa I restriction endonuclease. There were no significant differences in genotype and allele distribution between ESKD patients and healthy controls. When patients were stratified into male and female subgroups, there were no differences in the frequency of the T allele (0.35 and 0.34, respectively). Genotype and allele frequencies were also comparable between HY+ and HY- subgroups. We analyzed whether there were any differences between genders in the effect of C242T polymorphism on the presence of hypertension by comparing HY+ males with normotensive males and HY+ females with normotensive females. No difference in polymorphism distribution was found in female subgroup. The significant differences were observed in males. In HY+ subgroup, the frequencies of T allele and TT genotype were higher than in HY- males, with OR 1.91 (1.31-2.8), p = 0.0008 and OR 4.2 (1.67-10.6), p = 0.002, respectively. In conclusion, this is the first study to demonstrate significant association of the p22PHOX gene polymorphism with hypertension in male ESKD patients, with T allele as a risk factor for hypertension.

CTRP12 Ameliorated Lipopolysaccharide-Induced Cardiomyocyte Injury.

C1q/tumor necrosis factor (TNF)-related protein 12 (CTRP12) is a secretory protein that participates in the regulation of glucose and lipid metabolism in obesity and diabetes. Its role in cardiovascular disease, particularly sepsis-induced cardiac injury, is unclear. Here, we stimulated cardiomyocytes with lipopolysaccharide (LPS) to establish an in vitro cardiomyocyte injury model and CTRP12 was overexpressed with an adenovirus delivery system. Overexpression of CTRP12 reduced the transcription and release of pro-inflammatory cytokines from LPS-stimulated cardiomyocytes, including TNFα, interleukin-1 (IL-1), and IL-6. Reactive oxygen species (ROS) level increased and the oxidation/redox system was disturbed in LPS-stimulated cardiomyocytes, as evident from the decrease in superoxide dismutase activity and an increase in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and malondialdehyde level. CTRP12 overexpression decreased the increasing level of ROS and ameliorated the unbalance in the oxidation/redox system in LPS-stimulated cardiomyocytes. The viability of cardiomyocytes decreased after LPS stimulation, and the cells underwent apoptosis. CTRP12-overexpressing cardiomyocytes showed a decrease in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells, and the ratio of B cell lymphoma (Bcl)-1/Bax in these cells was recovered. In comparison with the control group, LPS-stimulated cardiomyocytes showed reduced expression of nuclear factor E2-related factor 2 (NRF2), while CTRP12-overexpressing cardiomyocytes showed elevated NRF2 expression. Small-interfering RNA-mediated silencing of NRF2 expression in cardiomyocytes resulted in the inhibition of the protective effects of CTRP12. Thus, CTRP12 ameliorated injury in LPS-stimulated cardiomyocytes in an NRF2-dependent manner.

Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells.

Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host genome. The application of gamma-retroviral long terminal repeat (LTR)-driven vectors, which semirandomly integrate into the genome, has previously caused severe adverse events in some clinical studies due to transactivation of neighboring proto-oncogenes. Consequently, the site-specific integration of a therapeutic transgene into a genomic safe harbor locus would allow stable genetic correction with a reduced risk of insertional mutagenesis. However, recent studies revealed that transgene silencing, especially in case of weaker cell type-specific promoters, can occur in the AAVS1 locus of human pluripotent stem cells (PSCs) and can impede transgene expression during differentiation. In this study, we aimed to correct p47phox deficiency, which is the second most common cause of chronic granulomatous disease, by insertion of a therapeutic p47phox transgene into the AAVS1 locus of human induced PSCs (iPSCs) using CRISPR-Cas9. We analyzed transgene expression and functional correction from three different myeloid-specific promoters (miR223, CatG/cFes, and myeloid-related protein 8 [MRP8]). Upon myeloid differentiation of corrected iPSC clones, we observed that the miR223 and CatG/cFes promoters achieved therapeutically relevant levels of p47phox expression and nicotinamide adenine dinucleotide phosphate oxidase activity, whereas the MRP8 promoter was less efficient. Analysis of the different promoters revealed high CpG methylation of the MRP8 promoter in differentiated cells, which correlated with the transgene expression data. In summary, we identified the miR223 and CatG/cFes promoters as cell type-specific promoters that allow stable transgene expression in the AAVS1 locus of iPSC-derived myeloid cells. Our findings further indicate that promoter silencing can occur in the AAVS1 safe harbor locus in differentiated hematopoietic cells and that a comparison of different promoters is necessary to achieve optimal transgene expression for therapeutic application of iPSC-derived cells.

Effects of Mycobacterium tuberculosis Rv1096 on mycobacterial cell division and modulation on macrophages

Mycobacterium tuberculosis is capable of escaping the clearance of immune system mainly due to its complex constituents of cell wall. Certain studies show that glycoproteins are involved in immune evasion and act as virulence factors. Peptidoglycan deacetylase Rv1096 is a member of mannosylated proteins. Previously, we reported Rv1096 protein contributed to the resistance of Mycobacterium smegmatis (M. smegmatis) to lysozyme, but more characterization of this protein is required where further intracellular function is unknown. Here, Rv1096 was heterologously over-expressed in the fast-growing and nonpathogenic M. smegmatis (named as M. smegmatis/Rv1096). We observed the morphological alterations in M. smegmatis/Rv1096 including an elongated rod-like shape and increased amounts of Z-rings, which implied that Rv1096 facilitated the cell growth and division. Moreover, a series of assays concerning the interaction between M. smegmatis/Rv1096 and host were carried out. The results showed that M. smegmatis/Rv1096 evaded the killing of macrophages due to the inhibition of phagosome-lysosome fusion, nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. The secretion of interleukin-12 and tumor necrosis factor-α was also impaired by Rv1096. In addition, five putative interaction partners of Rv1096 were identified, which possibly cooperated with Rv1096 in cell division and immune regulation. These results suggested that Rv1096 had effects on mycobacterial division and might act as a virulence factor to mediate the immune evasion in macrophage during mycobacterial infection.