Basal Superoxide Production Research Articles

Inspiratory Muscle Strength Training Improves Vascular Endothelial Function in Older Adults by Altering Circulating Factors that Suppress Superoxide and Enhance Nitric Oxide

BackgroundVascular endothelial dysfunction associated with decreased nitric oxide (NO)‐bioavailability secondary to excessive superoxide‐associated oxidative stress is a key antecedent of age‐associated cardiovascular diseases. High‐resistance inspiratory muscle strength training (IMST) is a promising form of time‐efficient physical training for promoting cardiovascular health with aging. However, the efficacy and mechanisms of IMST for improving endothelial function in older adults have not been established. We hypothesized IMST may induce changes in the molecular composition of the circulating blood that improve endothelial function via superoxide suppression‐related increases in NO bioavailability.PurposeTo determine the efficacy of IMST for improving vascular endothelial function in vivo, the influence of IMST‐induced changes in circulating factors on endothelial superoxide and NO production ex vivo, and IMST‐induced changes in the molecular composition of the circulation.MethodsTwenty older adults underwent 6 weeks of IMST (30 breaths/day; 6 days/week; 75% maximum inspiratory pressure [PIMAX]) (n=10, 5M/5F, age: 68±2 years) or low‐resistance sham (15% PIMAX) training (n=10, 6M/4F, age 70±2 years) in a randomized, double‐blind study. Vascular endothelial function was assessed via brachial artery flow‐mediated dilation [FMDBA]. HUVECs were cultured with serum collected at baseline and post‐intervention (n=12; 5 IMST, 7 sham). NO production stimulated with acetylcholine and basal superoxide production from HUVECs were measured using fluorescent microscopy. Plasma metabolite levels were assessed via targeted metabolomics (n=14; 7 IMST, 7 sham) at baseline and post‐intervention with ultra‐high‐performance liquid chromatography‐mass spectrometry.ResultsFMDBA increased 45% after 6 weeks of IMST (pre: 4.7±0.8 Δ%, post: 6.8±0.7 Δ%; p=0.01) but not after sham training (pre: 3.7±0.6 Δ%, post: 3.8±0.6 Δ%; p=0.99), such that FMDBA was 79% higher in the IMST group compared to sham training post‐intervention (p=0.01). HUVEC production of NO was 66% higher (p=0.04) while superoxide production was 38% lower post‐intervention in the IMST group vs. sham, though the latter was not statistically significant (p=0.17). Plasma metabolomics revealed greater levels of arginine (p=0.02) and ornithine (p=0.06) in the IMST group vs. sham post‐intervention, consistent with increased NO production. A greater level of dehydroascorbate (p=0.04) and changes in markers of glutathione synthesis (decreased alanine p=0.02; increased cystine p=0.06) were observed in the IMST group vs. sham post‐intervention, suggesting greater antioxidant defense status with IMST.ConclusionThese findings suggest IMST is a promising intervention for improving vascular endothelial function in older adults, in part by inducing changes in the molecular composition of the blood that increases NO bioavailability by suppressing superoxide.Support or Funding InformationAHA 18POST33990034, R21 AG061677

Alcohol Induced Oxidative Stress in neonatal Cardio myocytes

Alcohol Induced Oxidative Stress in neonatal Cardio myocytes

Substance P receptor blocker, aprepitant, inhibited cutaneous and other neurogenic inflammation side effects of the EGFR1-TKI, erlotinib.

Cutaneous changes like rash and hair loss, as well as other neurogenic inflammation side effects, occur frequently during anticancer treatment with the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), erlotinib. These adverse events may be so severe that they impair the patient's compliance with the treatment or even cause its discontinuation. In the current preclinical study, rats (9.2weeks) were treated with erlotinib (10mg/kg/day) ± aprepitant (2mg/kg/day) for 12weeks. Visual changes in the development of facial skin lesions/hair loss and SP-receptor expression (immunohistochemically) in facial skin tissue were assessed; also changes in plasma magnesium, 8-isoprostane, substance P (SP), neutrophil superoxide production, and cardiac function (echocardiography) were measured. Erlotinib lowered plasma magnesium 14%, elevated SP 65%, caused 3.7-fold higher basal superoxide production, 2.5-fold higher 8-isoprostane levels, 11.6% lower cardiac systolic, and 10.9% lower diastolic function. Facial dermatological changes (alopecia, skin reddening, scabbing, nose crusting) occurred by 4weeks (± + to ++) in erlotinib-treated rats, and progressively worsened (±++ to +++) by week 12. Facial skin SP-receptor upregulation (78% higher) occurred in epidermal and hair follicle cells. All adverse effects were substantially and significantly mitigated by aprepitant, including a 62% lowering of skin SP-receptors (p < 0.05). Elevated SP levels mediated the side effects of erlotinib treatment, but aprepitant's significant prevention of the systemic and cutaneous adverse events indicates a novel potential therapy against the side effects of this anticancer treatment.

Enhanced sensitivity to 4-hydroxynonenal and impaired redox signaling in human endothelial cells in gestational diabetes

Fetal exposure to gestational diabetes (GDM) in utero is strongly associated with a higher risk of cardiovascular disease and insulin resistance in later life, and accumulating evidence suggests this may be a consequence of fetal programming potentially involving epigenetic influences.1 Notably, offspring of mothers with GDM exhibit elevated blood pressure and reduced endothelium-dependent reactivity. We previously reported abnormal nitric oxide production, insulin resistance and reduced cell proliferation in fetal umbilical vein endothelial cells (HUVEC) from GDM pregnancies,2,3 and in parallel studies with endothelial cells from pre-eclamptic pregnancies, associated with in utero oxidative stress, identified abnormalities in NO production and regulation of [Ca2+]i.4,5 These phenotypic changes are maintained during culture, highlighting the involvement of fetal programming and epigenetics. As placental oxidative stress is propagated to the maternal and fetal vasculature via circulating lipid peroxides and H2O2 in GDM, we hypothesised that sustained oxidative stress in the fetal vasculature in utero may impair endogenous antioxidant defences. We established that activation of the redox sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2)6 and its downstream target antioxidant enzymes is markedly inhibited in HUVEC from GDM pregnancies. Fetal endothelial cells exhibited marked deficits in glutathione synthesis, increased basal mitochondrial superoxide production, reduced nuclear translocation of Nrf2, and diminished adaptive increases in the expression of the Nrf2 target genes heme oxygenase-1 (HO-1) and/or NAD(P)H quinone oxidoreductase 1 (NQO1) in response to the lipid peroxidation product 4-hydroxynonenal (4-HNE). A proteomic analysis of normal and GDM HUVEC confirmed the altered GDM phenotype, characterised by markers of increased oxidative stress, reduced antioxidant protection and reduced cell proliferation.7 More recently, we have confirmed increased 4-HNE induced damage and attenuated Nrf2-regulated gene expression in umbilical artery smooth muscle cells from GDM mothers. The altered phenotype of fetal venous endothelial and arterial smooth muscle cells derived from GDM pregnancies may underlie an increased risk of developing type 2 diabetes and cardiovascular disease in childhood and early adulthood.8-12

COX-2 is involved in vascular oxidative stress and endothelial dysfunction of renal interlobar arteries from obese Zucker rats

Obesity is related to vascular dysfunction through inflammation and oxidative stress and it has been identified as a risk factor for chronic renal disease. In the present study, we assessed the specific relationships among reactive oxygen species (ROS), cyclooxygenase 2 (COX-2), and endothelial dysfunction in renal interlobar arteries from a genetic model of obesity/insulin resistance, the obese Zucker rats (OZR). Relaxations to acetylcholine (ACh) were significantly reduced in renal arteries from OZR compared to their counterpart, the lean Zucker rat (LZR), suggesting endothelial dysfunction. Blockade of COX with indomethacin and with the selective blocker of COX-2 restored the relaxations to ACh in obese rats. Selective blockade of the TXA2/PGH2 (TP) receptor enhanced ACh relaxations only in OZR, while inhibition of the prostacyclin (PGI2) receptor (IP) enhanced basal tone and inhibited ACh vasodilator responses only in LZR. Basal production of superoxide was increased in arteries of OZR and involved NADPH and xanthine oxidase activation and NOS uncoupling. Under conditions of NOS blockade, ACh induced vasoconstriction and increased ROS generation that were augmented in arteries from OZR and blunted by COX-2 inhibition and by the ROS scavenger tempol. Hydrogen peroxide (H2O2) evoked both endothelium- and vascular smooth muscle (VSM)-dependent contractions, as well as ROS generation that was reduced by COX-2 inhibition. In addition, COX-2 expression was enhanced in both VSM and endothelium of renal arteries from OZR. These results suggest that increased COX-2-dependent vasoconstriction contributes to renal endothelial dysfunction through enhanced (ROS) generation in obesity. COX-2 activity is in turn upregulated by ROS.

Chronic aldosterone administration causes Nox2-mediated increases in reactive oxygen species production and endothelial dysfunction in the cerebral circulation.

An elevated plasma aldosterone level is an independent cardiovascular risk factor. Although excess aldosterone promotes cardiovascular disease, no studies have examined the effect of increased plasma aldosterone on the cerebral circulation. A major source of vascular reactive oxygen species (ROS) during cardiovascular disease is the NADPH oxidases. Because Nox2-containing NADPH oxidase (Nox2 oxidase) is highly expressed in the cerebral endothelium, we postulated that it might contribute to ROS generation and vascular dysfunction in response to aldosterone. Here, we examined the effect of aldosterone and Nox2 oxidase on ROS production and endothelial dysfunction in the cerebral circulation, and whether the effects of aldosterone are exacerbated in aged mice. In adult (average age ∼24-25 weeks) wild-type and Nox2-deficient (Nox2(/y)) mice, neither vehicle nor aldosterone (0.28 mg/kg per day for 14 days) affected blood pressure (measured using tail-cuff). By contrast, aldosterone treatment reduced dilation of the basilar artery (measured using myography) to the endothelium-dependent agonist acetylcholine in wild-type mice (P < 0.05), but had no such effect in Nox2(/y) mice (P > 0.05). Aldosterone increased basal and phorbol dibutyrate-stimulated superoxide production (measured using L-012-enhanced chemiluminesence) in cerebral arteries from wild-type but not from Nox2(/y) mice. In aged wild-type mice (average age ∼70 weeks), aldosterone treatment increased blood pressure, but had a similar effect on cerebral artery superoxide levels as in adult wild-type mice. These data indicate that Nox2 oxidase mediates aldosterone-induced increases in ROS production and endothelial dysfunction in cerebral arteries from adult mice independently of blood pressure changes. Aldosterone-induced hypertension is augmented during aging.

Influenza A virus and TLR7 activation potentiate NOX2 oxidase-dependent ROS production in macrophages

Influenza A virus infects resident alveolar macrophages in the respiratory tract resulting in Toll like receptor 7 (TLR7) activation that triggers an inflammatory response to resolve the infection. Macrophages are also major sources of reactive oxygen species (ROS) via the NOX2-containing NADPH oxidase. Although ROS are crucial for pathogen clearance, in response to influenza A virus, ROS are touted as being culprit mediators of the lung tissue injury. The aim of the present study was to determine whether influenza A virus infection and TLR7 activation of macrophages, results in alterations in their ROS production. Here we demonstrate using immunofluorescence that influenza A virus (Hong Kong X-31 strain; H3N2) internalizes in RAW264.7 cells and mouse alveolar macrophages within 1 h, resulting in a significant enhancement in the stimulated NOX2 oxidase-dependent oxidative burst, although virus had no effect on basal ROS. The specific TLR7 agonist imiquimod (10 μg/ml) elevated basal superoxide production and, in a similar fashion to influenza A virus, enhanced NOX2 oxidase-dependent oxidative burst. By contrast, the TLR3 agonist, poly I:C (1–100 μg/ml) failed to influence the oxidative burst to NOX2 oxidase. A peptide corresponding to the region 337–348 on p47phox conjugated to a HIV-tat, designed to inhibit the phosphorylation of Ser346 on p47phox suppressed the influenza A virus- and imiquimod-induced enhancement in the oxidative burst. In conclusion, this study demonstrates for the first time that influenza A virus and TLR7 activation enhance the NOX2 oxidase-dependent oxidative burst in macrophages, which might underpin the acute lung injury to influenza A virus infection.

Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase.

Mechanisms of oxidative stress in human aortic aneurysms--association with clinical risk factors for atherosclerosis and disease severity.

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2•−) production in human AAA.Methods and resultsAAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2•− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size.ConclusionsIncreased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.

D-Propranolol protects against oxidative stress and progressive cardiac dysfunction in iron overloaded rats.

d-Propranolol (d-Pro: 2-8 mg·(kg body mass)(-1)·day(-1)) protected against cardiac dysfunction and oxidative stress during 3-5 weeks of iron overload (2 mg Fe-dextran·(g body mass)(-1)·week(-1)) in Sprague-Dawley rats. At 3 weeks, hearts were perfused in working mode to obtain baseline function; red blood cell glutathione, plasma 8-isoprostane, neutrophil basal superoxide production, lysosomal-derived plasma N-acetyl-β-galactosaminidase (NAGA) activity, ventricular iron content, and cardiac iron deposition were assessed. Hearts from the Fe-treated group of rats exhibited lower cardiac work (26%) and output (CO, 24%); end-diastolic pressure rose 1.8-fold. Further, glutathione levels increased 2-fold, isoprostane levels increased 2.5-fold, neutrophil superoxide increased 3-fold, NAGA increased 4-fold, ventricular Fe increased 4.9-fold; and substantial atrial and ventricular Fe-deposition occurred. d-Pro (8 mg) restored heart function to the control levels, protected against oxidative stress, and decreased cardiac Fe levels. After 5 weeks of Fe treatment, echocardiography revealed that the following were depressed: percent fractional shortening (%FS, 31% lower); left ventricular (LV) ejection fraction (LVEF, 17%), CO (25%); and aortic pressure maximum (P(max), 24%). Mitral valve E/A declined by 18%, indicating diastolic dysfunction. Cardiac CD11b+ infiltrates were elevated. Low d-Pro (2 mg) provided modest protection, whereas 4-8 mg greatly improved LVEF (54%-75%), %FS (51%-81%), CO (43%-78%), P(max) (56%-100%), and E/A >100%; 8 mg decreased cardiac inflammation. Since d-Pro is an antioxidant and reduces cardiac Fe uptake as well as inflammation, these properties may preserve cardiac function during Fe overload.

Cyclo-Oxygenase-2 Knockout Genotype in Mice Is Associated With Blunted Angiotensin II-Induced Oxidative Stress and Hypertension

Inflammation and oxidative stress have been identified as integral parts in the pathogenesis of hypertension. Cyclo-oxygenase-2 which could elicit inflammation and free radicals generation appears to be a key enzyme in hypertension. Cyclo-oxygenase-2 expression and oxidative stress in cardiovascular tissues are increased in the angiotensin II model. Cyclo-oxygenase-1 and cyclo-oxygenase-2 deficient mice and their cultured aortic smooth muscle cells were used to investigate the role of these enzymes in angiotensin II induced superoxide production and hypertension. At resting state, the superoxide production in aortic and cardiac tissues was lower in cyclo-oxygenase-2 deficient than in the wild type or in cyclo-oxygenase-1 deficient mice. Chronic angiotensin II infusion increased the superoxide production in these tissues from both cyclo-oxygenase-deficient and wild-type mice whereas the level in cyclo-oxygenase-2 deficient mice was equivalent to the basal level in wild-type mice. The hypertensive effect of angiotensin II was attenuated in cyclo-oxygenase-2 deficient mice. Aspirin treatment reduced the basal superoxide production and blunted the oxidative and hypertensive effect of angiotensin II in wild type and cyclo-oxygenase-1 deficient mice whereas it lost completely its antioxidative property in angiotensin II-treated aortic smooth muscle cells isolated from cyclo-oxygenase-2 deficient mice. Cyclo-oxygenase-2 pathway plays a major role in the superoxide generation as well as in the angiotensin II-induced oxidative stress and blood pressure. Cyclo-oxygenase-1 activity didn't show any influence on these parameters. These results suggest that cyclo-oxygenase-2 is involved in the pathogenesis of hypertension.

142 Atrial sources of reactive oxygen species vary with the substrate and duration of atrial fibrillation: implications for the antiarrhythmic effect of statins

BackgroundAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with altered nitric oxide (NO)-redox balance. The molecular mechanisms and implications of this phenomenon in the management...

Resveratrol Shows Vasoprotective Effect Reducing Oxidative Stress Without Affecting Metabolic Disturbances in Insulin-dependent Diabetes of Rabbits

Resveratrol has been shown to have vasoprotective effects by upregulating oxidative defense mechanisms in a variety of pathophysiological conditions. However, the effect of resveratrol on diabetic oxidative stress and vascular and metabolic abnormalities is not completely understood. Therefore, this study was designed to evaluate whether long-term resveratrol supplementation has a protective effect on vascular function and integrity in association with metabolic parameters and oxidative stress in insulin-dependent diabetes. Diabetes was induced in rabbits with alloxan and maintained for 8 weeks. We used a resveratrol dose of 5 mg/L (10 weeks, starting 14 days before alloxan injection) and 50 mg/L (8 or 10 weeks, starting concomitantly or 14 days before alloxan injection) in the drinking water of rabbits. Relaxation to acetylcholine was impaired (control 75.6 ± 3.59%, versus diabetic 42.23 ± 2.53%) and contractions to phenylephrine increased (control 136.89 ± 2.27%, versus diabetic 159.37 ± 6.27%) in aortas from diabetic animals. These changes were associated with increased basal or NAD(P)H-induced superoxide production, as well as lipid peroxide and superoxide dismutase (SOD) levels in the aortic samples. The maximal relaxation to acetylcholine improved by 75.74 ± 9.04% in diabetic rabbits treated with resveratrol. The increased contractions to phenylephrine were not restored to control values after resveratrol treatments, but sensitivity to the contractions tended to decrease. Resveratrol increased nitrite/nitrate levels and suppressed basal or NAD(P)H-induced superoxide production and lipid peroxide levels in the aortas. Importantly, resveratrol increased serum insulin levels without affecting blood glucose and the lipid profile in diabetic rabbits. Using electron microscopic examinations, resveratrol was found to markedly protect the endothelial integrity from diabetes. Overall, there was no noticeable difference between resveratrol treatment groups on the recovery from diabetes. Our results indicate that resveratrol alleviates type 1 diabetes-induced vasculopathy by decreasing vascular oxidative stress and thereby increasing the bioavailability of nitric oxide without changing metabolic abnormalities.

BAS/BSCR17 Myocardial xanthine oxidase regulates basal inotropy in murine left ventricular myocytes

Xanthine oxidase (XO) is a major source of reactive oxygen species in the cardiovascular system. Enhanced XO activity in the failing myocardium has been associated with a reduction in inotropy;...

Protective role of extracellular superoxide dismutase in renal ischemia/reperfusion injury

Extracellular superoxide dismutase (SOD3) is highly expressed in renal tissues and a critical regulator of vascular function. We hypothesized that deletion of SOD3 would attenuate recovery of renal blood flow (RBF) and increase oxidative stress and injury following renal ischemia/reperfusion (I/R). To test this, we evaluated SOD expression and activity, basal superoxide production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in kidneys from male and female wild-type (WT) and SOD3-knockout mice. RBF, measured using an ultrasonic flow probe, and histological indices of oxidative stress and injury were assessed after 1 h of ischemia. Following ischemia, RBF was attenuated in kidneys from male, but not female, knockout mice compared with their WT counterparts. Total SOD activity was significantly reduced in male knockout compared with WT male mice but was similar in female mice of both genotypes, suggesting upregulated SOD1 activity. Basal superoxide production and NADPH oxidase activity were unrelated to the differences in RBF. After 24 h, kidneys from both genders of knockout mice were found to have more oxidative stress (3-nitrotyrosine immunohistochemistry) and renal cast formation than those from WT mice. Thus, our study found a key role for SOD3 in regulating renal I/R injury.

Endothelin-1 Enhances Superoxide and Prostaglandin E2 Production of Isolated Diabetic Glomeruli

Endothelin-1 (ET-1) plays an important role in the pathogenic mechanism of diabetic nephropathy. However, the regulatory effects of ET-1 on superoxide and prostaglandin E2 (PGE2) in diabetic glomeruli are unclear. The aim of this study was to determine whether ET-1 exerts a differential effect on the production of superoxide and PGE2 in diabetic glomeruli. The regulatory effects of indomethacin, insulin, dexamethasone, and heparin were also investigated. Freshly isolated glomeruli were obtained from normal and streptozotocin-induced diabetic rats for 1 week (DM1W), 1 month (DM1M), and 3 months (DM3M), respectively. Our results showed that the basal superoxide production of isolated glomeruli was significantly higher in DM1M and DM3M than in the normal rats (p < 0.01). ET-1 stimulated superoxide production in normal, DM1W and DM1M glomeruli (p < 0.01) but not in DM3M rats. The basal production of PGE2 in isolated glomeruli did not differ between diabetic and normal rats. ET-1 also stimulated PGE2 production in diabetic rats (p < 0.05). Pretreatment with indomethacin further enhanced ET-1-stimulated superoxide production in all groups of diabetic rats (p < 0.05), while the ET-1-stimulated PGE2 production was attenuated by indomethacin. Insulin, dexamethasone and heparin had no additional effects on ET-1-mediated superoxide and PGE2 production. In conclusion, basal glomerular production of superoxide but not PGE2 was increased in the diabetic glomeruli. ET-1 further stimulated production of both superoxide and PGE2. Indomethacin could enhance ET-1-stimulated superoxide production while attenuating PGE2 production.

Inhibition of Endothelin-1 Receptors Improves Impaired Nitric Oxide Synthase-Dependent Dilation of Cerebral Arterioles in Type 1 Diabetic Rats

Endothelin-1 has been implicated in the pathogenesis of many cardiovascular-related diseases, including diabetes. The goal of this study was to examine the influence of endothelin-1 receptors (ET(A)) in impaired responses of cerebral (pial) arterioles in type-1 diabetic rats. We measured responses of cerebral arterioles in non-diabetic rats to endothelial nitric oxide synthase (eNOS)-dependent (ADP), neuronal nitric oxide synthase (nNOS)-dependent (N-methyl-d-aspartic acid [NMDA]) and NOS-independent (nitroglycerin) agonists before and during application of BQ-123, an ET(A) receptor antagonist. In addition, we harvested brain tissue from non-diabetic and diabetic rats to measure the production of superoxide anion under basal conditions and during inhibition of ET(A) receptors. We found that diabetes specifically impaired eNOS- and nNOS-dependent reactivity of cerebral arterioles, but did not alter NOS-independent vasodilation. In addition, while BQ-123 did not alter responses in non-diabetic rats, BQ-123 restored impaired eNOS- and nNOS-dependent vasodilation in diabetic rats. Further, superoxide production was higher in brain tissue from diabetic rats compared with non-diabetic rats under basal conditions and BQ-123 decreased basal production of superoxide in diabetic rats. We suggest that activation of ET(A) receptors during type-1 diabetes mellitus plays an important role in impaired eNOS- and nNOS-dependent dilation of cerebral arterioles.

The amyloid precursor protein intracellular domain (AICD) disrupts actin dynamics and mitochondrial bioenergetics

The amyloid precursor protein (APP) is critically involved in the pathogenesis of Alzheimer's disease, and is strongly up-regulated in response to traumatic, metabolic, or toxic insults to the nervous system. The processing of APP by gamma/epsilon-secretase activity results in the generation of the APP intracellular domain (AICD). Previously, we have shown that AICD induces the expression of genes (transgelin, alpha2-actin) with functional roles in actin organization and dynamics and demonstrated that the induction of AICD and its co-activator Fe65 (AICD/Fe65) resulted in a loss of organized filamentous actin structures within the cell. As mitochondrial function is thought to be reliant on ordered actin dynamics, we examined mitochondrial function in human SHEP neuroblastoma cells inducibly expressing AICD/Fe65. Confocal analysis of the mitochondrial membrane potential (DeltaPsim) identified a significant decrease in the DeltaPsim in the AICD50/Fe65 over-expressing cells. This was paralleled by significantly reduced ATP levels and decreased basal superoxide production. Overexpression of the proposed AICD target gene transgelin in SHEP-SF parental cells and primary neurons was sufficient to destabilize actin filaments, depolarize DeltaPsim, and significantly alter mitochondrial distribution and morphology. Our data demonstrate that the induction of AICD/Fe65 or transgelin significantly alters actin dynamics and mitochondrial function in neuronal cells.

Augmented Superoxide Production By Nox2-Containing NADPH Oxidase Causes Cerebral Artery Dysfunction During Hypercholesterolemia

We tested the hypothesis that elevated superoxide production by Nox2-NADPH oxidase occurs in cerebral arteries during hypercholesterolemia and causes decreased nitric oxide function. Wild-type (WT), apolipoprotein E-deficient (ApoE(-/-)) and Nox2(-/-)/ApoE(-/-) mice were fed a high-fat diet for 7 to 14 weeks. Basal superoxide production by cerebral arteries was measured using L-012 (100 micromol/L)-enhanced chemiluminescence. Nitric oxide function was assessed in isolated middle cerebral arteries through the constrictor response to N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L). Western blotting was used to measure protein expression of Nox2, p47phox, endothelial nitric oxide synthase, and superoxide dismutases (1-3). Morphology of cerebral arteries was similar in WT and ApoE(-/-) mice. In ApoE(-/-), but not Nox2(-/-)/ApoE(-/-) mice, superoxide production by cerebral arteries was approximately 50% greater than in WT mice (P<0.05). Moreover, the magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE(-/-) mice was <50% of that in WT mice (P<0.05), whereas in Nox2(-/-)/ApoE(-/-) mice, the contractile response was comparable to WT responses. In the presence of the superoxide scavenger, tempol (1 mmol/L), L-NAME-induced contractions of middle cerebral arteries were similar between WT and ApoE(-/-) mice. Expression of p47phox was approximately 2-fold higher in ApoE(-/-) versus WT mice, whereas Nox2, endothelial nitric oxide synthase, and superoxide dismutase isoforms were unchanged. Elevated superoxide production and reduced basal nitric oxide-mediated relaxation occur in cerebral arteries of hypercholesterolemic mice even in the absence of lesions. These changes appear to be exclusively due to increased activity of Nox2-NADPH oxidase, possibly through increased expression of its regulatory subunit p47phox.

Atrial myocardial nox2 containing NADPH oxidase activity contribution to oxidative stress in mitral regurgitation: potential mechanism for atrial remodeling

Oxidative stress is linked with several cardiovascular diseases. However, the NADPH oxidase activity in severe mitral regurgitation patients with and without atrial fibrillation has not yet been explored. This study involved 16 adult patients (eight patients with persistent atrial fibrillation and eight with sinus rhythm) with severe mitral and moderate-to-severe tricuspid regurgitation and five control patients without mitral and tricuspid disease. Atrial tissues of the right and left atrial appendages were obtained during surgery. Superoxide anion production was measured by lucigenin-enhanced chemiluminescence, and the expression of nox2 containing NADPH oxidase mRNA was measured by quantitative real-time RT-PCR. Additionally, immunohistochemical study was performed. NADPH-stimulated superoxide release was significantly higher than basal superoxide production from right [5671.9±3498.7 vs. 232.7±70.0 relative light units per second per milligram of protein (RLU s(-1) mg protein(-1)), P=.008) and left atrial homogenates (6475.1±1890.8 vs. 229.0±79.6 RLU s(-1) mg protein(-1), P=.008) in atrial fibrillation patients. The NADPH-stimulated superoxide release from right atrial homogenates was also significantly higher than basal superoxide production in sinus patients (6809.1±1327.1 vs. 244.2±65.5 RLU s(-1) mg protein(-1), P=.008). Additionally, there was a borderline significant correlation between NADPH-stimulated superoxide production from left atrial homogenates and left atrial sizes (r=0.683, P=.062) in atrial fibrillation patients. Membrane-bound nox2 containing NADPH oxidase mRNA expression was increased and was similar in both the atrial fibrillation patients and sinus patients. The NADPH-stimulated superoxide production in right atrial homogenates in control atrial samples was 1863.7±137.2 RLU s(-1) mg protein(-1). Immunohistochemical study demonstrated increased expression of nox2 in myocytes with moderate-to-severe myolysis and hypertrophy. Results of this study demonstrate that membrane-bound nox2 containing NADPH oxidase activity and expression in the atrial myocardium is increased in patients with severe mitral regurgitation, possibly contributing to atrial remodeling in this clinical setting.