Increased NADPH Oxidase Activity Research Articles

Sex differences in subcutaneous adipose tissue redox homeostasis and inflammation markers in control and high-fat diet fed rats.

The development of obesity-related metabolic disorders is more evident in male in comparison with female subjects, but the mechanisms are unknown. Several studies have shown that oxidative stress is involved in the pathophysiology of obesity, but the majority of these studies were performed with male animals. The aim of this study was to evaluate the sex-related differences in subcutaneous adipose tissue redox homeostasis and inflammation of rats chronically fed a high-fat diet. NADPH oxidase (NOX), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities were evaluated in the subcutaneous adipose tissue (SC) of adult male and female rats fed either a standard chow (SCD) or a high-fat diet (HFD) for 11 weeks. NOX2 and NOX4 messenger RNA (mRNA) levels, total reduced thiols, interleukin (IL)-1β, tumor necrosis factor α (TNF-α), and IL-6 were also determined. Higher antioxidant enzyme activities and total reduced thiol levels were detected in SC of control male compared with female rats. Chronic HFD administration increased NOX activity and NOX2 and NOX4 mRNA levels and decreased SOD and GPx activities only in male animals. IL-1β, TNF-α, and IL-6 levels, as well as Adgre1, CD11b, and CD68 mRNA levels, were also higher in SC of males after HFD feeding. In SC of females, catalase activity was higher after HFD feeding. Taken together, our results show that redox homeostasis and inflammation of SC is sexually dimorphic. Furthermore, males show higher oxidative stress in SC after 11 weeks of HFD feeding owing to both increased reactive oxygen species (ROS) production through NOX2 and NOX4 and decreased ROS detoxification.

A15342 Interaction between Ischemic Brain Damage and Amyloid-β Infusion in Cognitive Impairment ∼Possible Amelioration of Cognitive Function by AT2 Receptor Activation∼

Objectives: We examined the possible enhancement of cognitive decline after ischemic brain damage with amyloid-β (Aβ) injection, focusing on the roles of angiotensin II type 2 (AT2) receptor in vascular smooth muscle cells (VSMC). Methods: Adult male wild-type mice (WT) or the mice with VSMC-specific AT2 receptor overexpression (smAT2) were used. Mice were subjected to intracerebroventricular (ICV) injection of Aβ1–40. Ischemic brain injury was induced by bilateral common carotid artery occlusion (BCCAO) after Aβ injection. Cognitive function was evaluated by the Morris water maze test. Results: ICV injection of Aβ in WT showed impaired cognitive function, whereas BCCAO did not decline significantly cognitive function. In contrast, BCCAO following Aβ injection exhibited more marked cognitive impairment compared to Aβ injection alone in concert with the increases in NADPH oxidase activity, expressions of NADPH oxidase subunit p22phox, p40phox and inflammatory cytokine MCP-1, IL1-β in the hippocampus. BCCAO significantly enhanced the expression of Aβ clearance factor, RAGE (receptor for advanced glycation end product). Aβ injection did not increase the neuron pyknosis in the hippocampus, whereas the number of neuron pyknosis was increased significantly with BCCAO. On the other hand, smAT2 did not show cognitive impairment, the changes of the expression for NADPH oxidase subunits, inflammatory cytokines and neuron pyknosis induced by BCCAO with/without Aβ injection in WT. Conclusion: Ischemic brain damage could enhance Aβ-induced cognitive impairment with possible involvements of enhanced oxidative stress, inflammation, neuron degeneration, and breakdown of RAGE-mediated Aβ clearance. AT2 receptor activation in VSMC might play preventive roles in this cognitive decline.

ROS-challenged keratinocytes as a new model for oxidative stress-mediated skin diseases.

In the current study, the effects of the reactive oxygen species (ROS) generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water-soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose-dependent increase in NADPH oxidase activity in intact keratinocytes. The long-term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH-treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O 2- ), while AAPH-treated keratinocytes overproduce hydrogen peroxide (H 2 O 2 ) in extracellular medium. H 2 O 2 is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost-effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes-melanocytes cross-talk studies is also suggested. The model of AAPH-stressed human keratinocytes described here can represent a useful tool for redox cross-talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.

Over-expression of a cardiac-specific human dopamine D5 receptor mutation in mice causes a dilated cardiomyopathy through ROS over-generation by NADPH oxidase activation and Nrf2 degradation

Dilated cardiomyopathy (DCM) is a severe disorder caused by medications or genetic mutations. D5 dopamine receptor (D5R) gene knockout (D5-/-) mice have cardiac hypertrophy and high blood pressure. To investigate the role and mechanism by which the D5R regulates cardiac function, we generated cardiac-specific human D5R F173L(hD5F173L-TG) and cardiac-specific human D5R wild-type (hD5WT-TG) transgenic mice, and H9c2 cells stably expressing hD5F173L and hD5WT. We found that cardiac-specific hD5F173L-TG mice, relative to hD5WT-TG mice, presented with DCM and increased cardiac expression of cardiac injury markers, NADPH oxidase activity, Nrf2 degradation, and activated ERK1/2/JNK pathway. H9c2-hD5F173L cells also had an increase in NADPH oxidase activity, Nrf2 degradation, and phospho-JNK (p-JNK) expression. A Nrf2 inhibitor also increased p-JNK expression in H9c2-hD5F173L cells but not in H9c2-hD5WT cells. We suggest that the D5R may play an important role in the preservation of normal heart function by inhibiting the production of reactive oxygen species, via inhibition of NADPH oxidase, Nrf2 degradation, and ERK1/2/JNK pathways.

MPGES-1 (Microsomal Prostaglandin E Synthase-1) Mediates Vascular Dysfunction in Hypertension Through Oxidative Stress.

mPGES-1 (microsomal prostaglandin E synthase-1), the downstream enzyme responsible for PGE2 (prostaglandin E2) synthesis in inflammatory conditions and oxidative stress are increased in vessels from hypertensive animals. We evaluated the role of mPGES-1-derived PGE2 in the vascular dysfunction and remodeling in hypertension and the possible contribution of oxidative stress. We used human peripheral blood mononuclear cells from asymptomatic patients, arteries from untreated and Ang II (angiotensin II)-infused mPGES-1-/- and mPGES-1+/+ mice, and vascular smooth muscle cells exposed to PGE2 In human cells, we found a positive correlation between mPGES-1 mRNA and carotid intima-media thickness (r=0.637; P<0.001) and with NADPH oxidase-dependent superoxide production (r=0.417; P<0.001). In Ang II-infused mice, mPGES-1 deletion prevented all of the following: (1) the augmented wall:lumen ratio, vascular stiffness, and altered elastin structure; (2) the increased gene expression of profibrotic and proinflammatory markers; (3) the increased vasoconstrictor responses and endothelial dysfunction; (4) the increased NADPH oxidase activity and the diminished mitochondrial membrane potential; and (5) the increased reactive oxygen species generation and reduced NO bioavailability. In vascular smooth muscle cells or aortic segments, PGE2 increased NADPH oxidase expression and activity and reduced mitochondrial membrane potential, effects that were abolished by antagonists of the PGE2 receptors (EP), EP1 and EP3, and by JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular-signal-regulated kinases 1/2) inhibition. Deletion of mPGES-1 augmented vascular production of PGI2 suggesting rediversion of the accumulated PGH2 substrate. In conclusion, mPGES-1-derived PGE2 is involved in vascular remodeling, stiffness, and endothelial dysfunction in hypertension likely through an increase of oxidative stress produced by NADPH oxidase and mitochondria.

KLF2-mediated disruption of PPAR-γ signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Lymphatic abnormalities associated with congenital heart disease are well described, yet the underlying mechanisms remain poorly understood. Using a clinically relevant ovine model of congenital heart disease with increased pulmonary blood flow, we have previously demonstrated that lymphatic endothelial cells (LECs) exposed in vivo to chronically increased pulmonary lymph flow accumulate ROS and have decreased bioavailable nitric oxide (NO). Peroxisome proliferator-activated receptor-γ (PPAR-γ), which abrogates production of cellular ROS by NADPH oxidase, is inhibited by Krüppel-like factor 2 (KLF2), a flow-induced transcription factor. We hypothesized that chronically increased pulmonary lymph flow induces a KLF2-mediated decrease in PPAR-γ and an accumulation of cellular ROS, contributing to decreased bioavailable NO in LECs. To better understand the mechanisms that transduce the abnormal mechanical forces associated with chronically increased pulmonary lymph flow, LECs were isolated from the efferent vessel of the caudal mediastinal lymph node of control ( n = 5) and shunt ( n = 5) lambs. KLF2 mRNA and protein were significantly increased in shunt compared with control LECs, and PPAR-γ mRNA and protein were significantly decreased. In control LECs exposed to shear forces in vitro, we found similar alterations to KLF2 and PPAR-γ expression. In shunt LECs, NADPH oxidase subunit expression was increased, and bioavailable NO was significantly lower. Transfection of shunt LECs with KLF2 siRNA normalized PPAR-γ, ROS, and bioavailable NO. Conversely, pharmacological inhibition of PPAR-γ in control LECs increased ROS equivalent to levels in shunt LECs at baseline. Taken together, these data suggest that one mechanism by which NO-mediated lymphatic function is disrupted after chronic exposure to increased pulmonary lymph flow is through altered KLF2-dependent PPAR-γ signaling, resulting in increased NADPH oxidase activity, accumulation of ROS, and decreased bioavailable NO. NEW & NOTEWORTHY Lymphatic endothelial cells, when exposed in vivo to chronically elevated pulmonary lymph flow in a model of congenital heart disease with increased pulmonary blood flow, demonstrate Krüppel-like factor 2-dependent disrupted peroxisome proliferator-activated receptor-γ signaling that results in the accumulation of reactive oxygen species and decreased bioavailable nitric oxide.

Glypican‐1 Mediates Endothelial Hyper‐permeability in a Model of Acute Heart Failure

Accumulating evidence indicates that the lungs are active participants in the pathogenesis of heart failure (HF). Increases in left ventricular end diastolic pressure (LVEDP) leads to elevated pulmonary capillary pressure (PCP) that causes fluid filtration resulting in dyspnea and, ultimately, frank pulmonary edema (PE). However, the severity of dyspnea does not directly relate to the increase in PCP indicating that the clinical manifestation of HF cannot be solely explained by changes in Starling forces. Acute increases in PCP activate glycocalyx‐dependent signaling (endothelial mechanotransduction) that result in oxidative stress, eNOS activation and endothelial hyper‐permeability. Herein we sought to investigate if glypican‐1, a component of the glycocalyx, contributes to pressure‐dependent hyper‐permeability and PE in a model of acute HF. The isolated perfused lung preparation was used to simulate acute HF in male CD‐1 (wild type, WT) and glypican‐1 knockout (Gpc‐1 KO) mice (6 weeks old). Briefly, mice were anesthetized with isoflurane, tracheostomized and mechanically ventilated. Pulmonary artery and left atria were cannulated and in‐line pressure transducers recorded arterial and left atrial pressure; lungs were perfused at 2 mL/min with a Krebs‐Ringer 3% BSA buffer. Control experiments were done setting left atria pressure (PLA) at 3 cm H2O for 10 minutes; acute heart failure was simulated by setting PLA at 10 cm H2O for 10 minutes. Lung edema was assessed by lung wet‐to‐dry ratio; reactive oxygen species (ROS) production and eNOS activation were assessed as indices of mechanotransduction activation by lucigenin enhanced chemiluminescence and immunoblotting, respectively. NADPH oxidase activity was measured by dihydroethidium fluorescence in isolated membrane‐fracions. WT and Gpc‐1 KO mice had similar increase in pulmonary artery pressure (from 8 to 10 mmHg). Deletion of glypican‐1 protected mice from pressure‐induced PE (wet‐to dry ratio [high and low pressure, respectfully] in WT: 7.3±0.3 vs 4.9±0.3 and in Gpc‐1 KO: 4.8±0.3 vs 4.8±0.2), prevented pressure‐dependen increase in NADPH oxidase activity (15% increase in WT) and in ROS production (80% increase in WT). Moreover, while WT mice showed increased eNOS activation (2‐fold) during acute HF, Gpc‐1 KO mice showed reduced eNOS activation (0.5 fold) indicating that Gpc‐1 is a determinant for mechanotransduction signaling. Taken together, these results suggest that Gpc‐1 activation by pressure leads to endothelial hyper‐permeability due to ROS and NO‐dependent pathways and contributes to PE in an in situ model of acute HF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NADPH oxidase 2 knockout prevents chronic intermittent hypoxia induced sternohyoid muscle weakness in adult male mice

Obstructive sleep apnoea syndrome (OSAS) is the most prevalent form of sleep disordered breathing (SDB) which affects 1 in 5 people worldwide. Chronic intermittent hypoxia (CIH) is a hallmark feature of SDB as a consequence of repetitive upper airway occlusions in patients during sleep. These recurring hypoxia‐reoxygenation cycles result in the overproduction of reactive oxygen species (ROS), ultimately yielding a state of oxidative stress. Excessive levels of ROS have been associated with aberrant plasticity at multiple levels of the respiratory system including impaired upper airway muscle function. However, there is a paucity of information regarding the molecular mechanisms underlying these effects. The NADPH oxidase (NOX) family of enzymes have been linked to a multitude of CIH induced morbidities in a range of organs and systems with NOX subunits also having been identified in skeletal muscle.A mouse model of CIH was generated by the cycling of gas from normoxia (21% O2) for 210 seconds to hypoxia (5% O2 at the nadir) for 90 seconds 8 hr/day for 2 weeks during light hours. 10–11 week old male mice (C57BL/6J) were randomly assigned to one of 3 groups: Normoxic controls (sham; n=24), CIH exposed (n=24), and CIH + Apocynin (2 mM; n=24) treated throughout the gas exposure. 10–11 week old NOX2 null (B6.129S‐Cybbtm1Din/J) male mice were also assigned to a sham (n=12) or CIH (n=12) group. Following this, sternohyoid muscle contractile function was examined ex vivo. Gene expression of the entire family of NOX enzymes was examined by RT‐PCR. Western blot was used to quantify NOX2 and NOX4 protein expression. NOX enzyme activity was determined using a NOX activity assay. Data were expressed as mean±SD and were statistically compared by unpaired Student t‐test or two‐way ANOVA with Bonferroni post‐hoc test.2 weeks of exposure to CIH resulted in a significant decrease of ~45% in the peak specific force (Fmax) of the sternohyoid muscle when compared to normoxic controls (sham). CIH resulted in no significant alteration to the gene expression of any members of the NOX family of enzymes when compared to sham controls. There was no significant difference in NOX2 or NOX4 protein expression between CIH and sham groups. However, CIH exposure resulted in a significant increase in sternohyoid NOX enzyme activity compared to shams. Treatment with Apocynin (2 mM) throughout the 2 week CIH exposure prevented sternohyoid muscle weakness, restoring force to levels equivalent to controls. Furthermore, NOX2 knockout prevented CIH induced sternohyoid muscle weakness.Adult male C57BL/6J mice show signs of profound sternohyoid muscle dysfunction following exposure to 2 weeks of CIH when compared with sham animals. This occurs without alteration to the gene or protein expression of various NOX subunits. Increased NOX activity suggests a CIH induced increase in NOX enzyme assembly rather than abundance. Administration of the putative NOX inhibitor, Apocynin, prevents sternohyoid muscle weakness. NOX2 knockout also entirely prevents sternohyoid muscle weakness confirming its specific role in CIH induced muscle dysfunction. The striking muscle phenotype in the C57 mouse and its prevention in the absence of NOX2 provides a novel and robust platform for the study of mechanisms of hypoxia‐related muscle dysfunction, which may have relevance to respiratory conditions characterised by CIH, such as sleep apnoea.Support or Funding InformationDepartment of Physiology, University College Cork, Cork, IrelandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of NADPH oxidase family members associated with cold stress in strawberry.

NADPH oxidase is encoded by a small gene family (Respiratory burst oxidase homologs, Rbohs) and plays an important role in regulating various biological processes. However, little information about this gene family is currently available for strawberry. In this study, a total of seven Rboh genes were identified from strawberry through genomewide analysis. Gene structure analysis showed the number of exons ranged from 10 to 23, implying that this variation occurred in FvRboh genes by the insertion and distribution of introns; the order and approximate size of exons were relatively conserved. FvRbohC was predicted to localize to the thylakoid membrane of the chloroplast, while other members were computed to localize to the plasma membrane, indicating different functions. Amino acid sequence alignment, conserved domain, and motif analysis showed that all identified FvRbohs had typical features of plant Rbohs. Phylogenetic analysis of Rbohs from strawberry, grape, Arabidopsis, and rice suggested that the FvRbohs could be divided into five subgroups and showed a closer relationship with those from grape and Arabidopsis than those from rice. The expression patterns of FvRboh genes in root, stem, leaf, flower, and fruit revealed robust tissue specificity. The expression levels of FvRbohA and FvRbohD were quickly induced by cold stress, followed by an increase in NADPH oxidase activity, leading to O2− accumulation and triggering the antioxidant reaction by the transient increases in SOD activity. This suggested these two genes may be involved in cold stress and defense responses in strawberry.

Resveratrol attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats: Role of ROS and ROS-mediated cell signaling

Resveratrol, a natural polyphenolic compound has been reported to attenuate angiotensin II -induced vascular smooth muscle cell (VSMC) hypertrophy; however, whether resveratrol could also inhibit hyperproliferation of VSMC from spontaneously hypertensive rats (SHR) is unexplored. The present study investigates the effect of resveratrol on hyperproliferation of VSMC from SHR and the underlying molecular mechanisms responsible for this response. For these studies, aortic VSMC from SHR and Wistar-Kyoto (WKY) rats were used. The proliferation of VSMC was determined by [3H] thymidine incorporation and the levels of proteins were determined by Western blotting. The enhanced proliferation exhibited by VSMC from SHR was attenuated by resveratrol. In addition, resveratrol attenuated the overexpression of cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4), Cdk2, phosphorylated retinoblastoma protein (pRb), Giα proteins and enhanced phosphorylation of ERK1/2 and AKT in VSMC from SHR. Furthermore, the overproduction of superoxide anion, increased NADPH oxidase activity, overexpression of Nox2, Nox4 and p47phox proteins, increased phosphorylation of EGFR, IGF-IR, and c-Src were all abrogated by resveratrol. These results suggest that resveratrol attenuates the hyperproliferation of VSMC from SHR through the inhibition of ROS, c-Src, growth factor receptor activation, MAPK/PI3K, Giα and cell cycle proteins that are implicated in VSMC hyperproliferation.

Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system.

Despite advances in antihypertensive therapeutics, at least 15-20% of hypertensive patients have resistant hypertension through mechanisms that remain poorly understood. In this study, we provide a new mechanism for the regulation of blood pressure (BP) in the central nervous system (CNS) by the (pro)renin receptor (PRR), a recently identified component of the renin-angiotensin system that mediates ANG II formation in the CNS. Although PRR also mediates ANG II-independent signaling, the importance of these pathways in BP regulation is unknown. Here, we developed a unique transgenic mouse model overexpressing human PRR (hPRR) specifically in neurons (Syn-hPRR). Intracerebroventricular infusion of human prorenin caused increased BP in Syn-hPRR mice. This BP response was attenuated by a NADPH oxidase (NOX) inhibitor but not by antihypertensive agents that target the renin-angiotensin system. Using a brain-targeted genetic knockdown approach, we found that NOX4 was the key isoform responsible for the prorenin-induced elevation of BP in Syn-hPRR mice. Moreover, inhibition of ERK significantly attenuated the increase in NOX activity and BP induced by human prorenin. Collectively, our findings indicate that an ANG II-independent, PRR-mediated signaling pathway regulates BP in the CNS by a PRR-ERK-NOX4 mechanism. NEW & NOTEWORTHY This study characterizes a new transgenic mouse model with overexpression of the human (pro)renin receptor in neurons and demonstrated a novel angiotensin II-independent mechanism mediated by human prorenin and the (pro)renin receptor in the central regulation of blood pressure.

Hypoxic challenge of hyperoxic pulmonary artery myocytes increases oxidative stress due to impaired mitochondrial superoxide dismutase activity

Infants with lung disease may be exposed to high O2 concentrations, and may have transient hypoxic episodes due to worsening lung pathophysiology, aggravating pulmonary arterial (PA) oxidative stress. NADPH oxidase (NOX) converts O2 to superoxide. Mitochondrial antioxidants such as superoxide dismutase (SOD) are sensitive to O2 tension. We previously reported decreased SOD2 activity in hypoxic PA myocytes due to nitration. In this study, we examined whether a transient hypoxic episode exposes antioxidant defense defects in hyperoxic PA myocytes.PA myocytes of term newborn piglets were cultured in hyperoxia (60% O2) or normoxia (21% O2) for 72 h; cells from both groups were challenged with transient hypoxia (10% O2) for 2 h. We measured NOX activity, SOD activities (fractionated by centrifugation and concavalin A- Sepharose chromatography), total ROS and superoxide generation, 8-isoprostane, and calcium responses to thromboxane mimetic.NOX activity increased in hyperoxic myocytes. Hyperoxia increased SOD1 activity but decreased SOD2 activity. Total ROS were reduced in hyperoxia, and hyperoxia + hypoxia groups. While hyperoxia alone did not alter superoxide content, superoxide increased after a hypoxic challenge of both normoxic and hyperoxic myocytes. Increased 8-isoprostane was seen only in hyperoxic myocytes challenged by transient hypoxia.We conclude that hyperoxic PA myocytes can limit total ROS despite increased NOX activity, but with inhibited SOD2 activity. Transient hypoxia increases superoxide formation; in the face of impaired SOD2, despite induction of SOD1, this oxidative stress causes increased 8-isoprostane generation. This may contribute to the mechanism of pulmonary arterial reactivity in infants with severe lung disease.

Probucol reverses homocysteine induced inflammatory monocytes differentiation and oxidative stress

Reactive oxygen species have been demonstrated to involve in homocysteine-induced Ly-6Chi monocytes differentiation. Probucol is an anti-oxidant agent that has been used to treat atherosclerosis. We sought to evaluate the effect and potential mechanism of probucol on homocysteine-induced inflammatory monocytes differentiation. The primary mouse splenocytes suspensions were initiated by recombinant interferon-γ and cultured with L-homocysteine in the presence or absence of probucol. The cells were co-incubated with monoclonal antibodies to CD11b–PE and Ly-6C FITC. Flow cytometry analysis was performed on BD FACS caliber. Data were analyzed using the FlowJo software. Mononuclear cells were gated according to the lower granular and larger size, distinguished with granulocytes and lymphocytes. Monocytes were defined as CD11b+ mononuclear cells and further divided into three groups based on their Ly-6C expressions, Ly-6Chi, Ly-6Cmid and Ly-6Clow subsets. The productions of reactive oxygen species in monocytes subsets were detected by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) containing monocytes were marked as DCFH-DA+ cells in both Ly-6C+ and Ly-6C- subsets. The activity of nicotinamide adenine dinucleotide phosphate oxidase in THP-1 cells was measured by assay kit on enzyme-labelling instrument. L-homocysteine promoted inflammatory monocytes differentiation and its reactive oxygen species productions in dose-dependent manner. Probucol dose-dependently suppressed the differentiation and reactive oxygen species productions of inflammatory monocytes induced by L-homocysteine. Furthermore, the increased NADPH oxidase activity induced by L-homocysteine was significantly reversed by probucol in THP-1 cells. Probucol prevented L-homocysteine-induced inflammatory monocytes differentiation and its reactive oxygen species generation probably through inhibiting NADPH oxidase activity.

Exercise intervention attenuates hyperhomocysteinemia-induced aortic endothelial oxidative injury by regulating SIRT1 through mitigating NADPH oxidase/LOX-1 signaling

Coronary artery disease (CAD) is a critical cardiovascular disease and a cause of high morbidity and mortality in this world. Hyperhomocysteinemia (HHcy) has been suggested as a risk factor for CAD. In addition, SIRT1 (sirtuin 1) has been reported to play a protective role in a variety of diseases, especially in the cardiovascular system. The main purpose of this study was to investigate the effects of exercise training on apoptosis and inflammation in HHcy animals. We also tested whether exercise protected against Hhcy-induced dysfunction of endothelium through modulation of SIRT1. C57BL mice (8 in each group) were fed with or without 1% L-methionine (w/w) in water for 4 months to induce HHcy. We found that Hhcy repressed SIRT1 and AMPK expression and increased NADPH oxidase activity. Plasma MDA, endothelium LOX-1 and p-p38 were up-regulated by Hhcy induction. NF-κB and it downstream molecules were activated under Hhcy situation, thereby promoting pro-inflammatory responses. Moreover, we also reported that Hhcy caused endothelium apoptosis involving Akt inhibition and mitochondria-dependent apoptotic pathways. Exercise training significantly protected against endothelium from Hhcy caused oxidative injuries. In addition, EX527 (SIRT1 inhibitor) reduced the therapeutic effects by exercise. Our results had indicated that exercise training prevent the development of atherosclerosis through SIRT1 activation and oxidative stress inhibition under Hhcy situation.

Role of ADP ribosylation factor6− Cytohesin1−PhospholipaseD signaling axis in U46619 induced activation of NADPH oxidase in pulmonary artery smooth muscle cell membrane

Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6−cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.

Abstract 5465: Persistent oxidative stress in mouse intestinal and colonic epithelial cells after exposure to 12C-ion radiation

Abstract Purpose: 12C-ion radiotherapy is demonstrating favorable results compared to photon radiotherapy for select cancers. While normal tissue exposure is lower with 12C-ions relative to γ-rays, it cannot be completely eliminated and therefore, late tissue toxicity, inflammation and secondary carcinogenesis cannot be ruled out. The goal of this study was to assess the extent of persistent oxidative stress (POS) after 12C radiation exposure and compare the results to that after γ radiation exposure. Methods and Materials: Mice (C57BL/6J; 6 to 8 weeks; male) were irradiated with 0.5 or 1.3 Gy of γ or 12C-ion, and intestinal (IEC), colonic (CEC) epithelial cells and tissues were collected 2 months after radiation exposure. In epithelial cells, intracellular ROS, mitochondrial superoxide, mitochondrial membrane potential (MMP), and cardiolipin oxidation were studied by flow-cytometry. Superoxide dismutase (SOD), catalase, and NADPH oxidase activity along with lipid oxidation were also assessed in epithelial cells using biochemical assays. Results: Our results clearly showed radiation quality and dose-dependent induction of POS. Both intestine and colon showed a higher elevation of intracellular ROS, mitochondrial superoxide, NADPH oxidase activity, and mitochondrial cardiolipin oxidation after 12C relative to γ radiation. Moreover, antioxidant enzyme activity in intestine and colon was also significantly reduced in 12C-irradiated mice. Compared to γ radiation, membrane lipid damage was remarkably higher in both intestine and colon of 12C-ion irradiated mice. At 0.5 Gy, persistent oxidative damage indicated by 4-hydroxynonenal was 3-fold in colon and 1.6 fold in intestine after 12C relative γ radiation. Conclusions: Mitochondrial deregulation, increased NADPH oxidase activity and loss of SOD and catalase activities were the major contributory events in 12C-ion-induced POS in mouse GI-tissues. Compared to intestine, colon was more susceptible to POS induction that might be due to progressive cell turnover in intestine resulting in faster elimination of initial damage signal in a given time than colon. Taken together, our data suggest that normal tissue exposure to 12C radiation carries higher long-term risk relative to γ-rays at comparable doses and further detail evaluation is warranted. Citation Format: Shubhankar Suman, Santosh Kumar, Albert J. Fornace, Kamal Datta. Persistent oxidative stress in mouse intestinal and colonic epithelial cells after exposure to 12C-ion radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2017-5465

NADPH oxidase is involved in regulation of gene expression and ROS overproduction in soybean (Glycine max L.) seedlings exposed to cadmium

Cadmium-induced oxidative burst is partially mediated by NADPH oxidase. The aim of the present research was to evaluate the role of NADPH oxidase in soybeans’ response to short-term cadmium stress. The application of an NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), affected expression of two Cd-inducible genes, encoding DOF1 and MYBZ2 transcription factors. This effect was observed after 3 h of treatment. Interestingly, Cd-dependent increases in NADPH oxidase activity occurred only after a period of time ranging from 6 and 24 h of stress. Stimulation of the enzyme correlated in time with a significant accumulation of reactive oxygen species (ROS). Further analysis revealed that pharmacological inhibition of NADPH oxidase activity during 24 h of Cd stress does not affect Cd uptake, seedling growth, or the level of lipid peroxidation. The role of NADPH oxidase in the response of soybean seedlings to short-term Cd exposure is discussed.

Triangular interplay between ROS, ABA and GA in dormancy alleviation of Bunium persicum seeds by cold stratification

Seeds of Bunium persicum (Boiss.) B. Fedtsch. have complex physiological dormancy that can be released by 15 weeks stratification. The present study revealed that cold stratification enhanced content of H2O2, O2 -· and application of GA3 and ROS donors (Fenton reagent, H2O2, methylviologen and menadione) did not affect or only slightly promoted the germination of non-stratified, fully dormant seeds. Dormancy was markedly decreased by ROS-generating reagents, GA3 and fluridone (an inhibitor of ABA biosynthesis) and was enhanced by ROS-decreasing compounds (DMTU, Tiron, SB and DPI), diniconazole (Dinc, an inhibitor of ABA catabolism) and paclobutrazol (PAC, an inhibitor of GA biosynthesis) when dormancy was partially removed by cold stratification. The response to these compounds reduced with increasing time of stratification. ABA inhibited germination by repressing of NADPH oxidase activity and ROS accumulation and conversely, GA triggered germination by promoting an increase of NADPH oxidase activity and ROS levels. Data in this study, for the first time suggest releasing deep complex physiological dormancy by cold stratification is associated with interplay between ROS and ABA/GA.

Advanced glycation end-products decreases expression of endothelial nitric oxide synthase through oxidative stress in human coronary artery endothelial cells

BackgroundAdvanced glycation end-products (AGEs) are elevated under diabetic conditions and associated with insulin resistance, endothelial dysfunction and vascular inflammation in humans. It has been demonstrated that AGEs evoke oxidative and inflammatory reactions in endothelial cells through the interaction with a receptor for AGEs (RAGE). Here, we aimed to identify the cellular mechanisms by which AGEs exacerbate the endothelial dysfunction in human coronary artery endothelial cells (HCAECs).Methods30 type 2 diabetic patients with or without coronary artery atherosclerosis were recruited for this study. Plasma levels of AGE peptides (AGE-p) were analyzed using flow injection assay. Endothelial function was tested by brachial artery flow-mediated vasodilatation (FMD). Further investigations were performed to determine the effects and mechanisms of AGEs on endothelial dysfunction in HCAECs.ResultsAGE-p was inversely associated with FMD in diabetic patients with coronary artery atherosclerosis in our study. After treated with AGEs, HCAECs showed significant reductions of eNOS mRNA and protein levels including eNOS and phospho-eNOS Ser1177, eNOS mRNA stability, eNOS enzyme activity, and cellular nitric oxide (NO) levels, whereas superoxide anion production was significantly increased. In addition, AGEs significantly decreased mitochondrial membrane potential, ATP content and catalase and superoxyde dismutase (SOD) activities, whereas it increased NADPH oxidase activity. Treatment of the cells with antioxidants SeMet, SOD mimetic MnTBAP and mitochondrial inhibitor thenoyltrifluoroacetone (TTFA) effectively blocked these effects induced by AGEs. AGEs also increased phosphorylation of the mitogen-activated protein kinases p38 and ERK1/2, whereas the specific inhibitors of p38, ERK1/2, and TTFA effectively blocked AGEs-induced reactive oxygen species production and eNOS downregulation.ConclusionsAGEs cause endothelial dysfunction by a mechanism associated with decreased eNOS expression and increased oxidative stress in HCAECs through activation of p38 and ERK1/2.

Acute exposure to chlorpyrifos caused NADPH oxidase mediated oxidative stress and neurotoxicity in a striatal cell model of Huntington’s disease

We hypothesized that expression of mutant Huntingtin (HTT) would modulate the neurotoxicity of the commonly used organophosphate insecticide, chlorpyrifos (CPF), revealing cellular mechanisms underlying neurodegeneration. Using a mouse striatal cell model of HD, we report that mutant HD cells are more susceptible to CPF-induced cytotoxicity as compared to wild-type. This CPF-induced cytotoxicity caused increased production of reactive oxygen species, reduced glutathione levels, decreased superoxide dismutase activity, and increased malondialdehyde levels in mutant HD cells relative to wild-type. Furthermore, we show that co-treatment with antioxidant agents attenuated the CPF-induced ROS levels and cytotoxicity. Co-treatment with a NADPH oxidase (NOX) inhibitor, apocynin, also attenuated the CPF-induced ROS production and neurotoxicity. CPF caused increased NOX activity in mutant HD lines that was ameliorated following co-treatment with apocynin. Finally, CPF-induced neurotoxicity significantly increased the protein expression of nuclear factor erythroid 2-related factor (Nrf2) in mutant HD cells as compared to wild-type. This study is the first report of CPF-induced toxicity in HD pathophysiology and suggests that mutant HTT and CPF exhibit a disease-toxicant interaction wherein expression of mutant HTT enhances CPF-induced neurotoxicity via a NOX-mediated oxidative stress mechanism to cause neuronal loss in the full length HTT expressing striatal cells.