Inhibition of NADPH oxidase alleviates experimental diabetes-induced myocardial contractile dysfunction

Aim: O₂^? production is implicated in cardiac dysfunction for a number of diseases including diabetes. Activation of the O₂^?‐producing enzyme NADPH oxidase is seen in diabetes, although its role in diabetic cardiomyopathy is unclear. This study was designed to evaluate the effect of NADPH oxidase inhibition on cardiac function in diabetes. Methods: Experimental diabetes was induced in adult C57 mice using streptozotocin (STZ, 150 mg/kg, i.p.) prior to the administration of the NADPH oxidase inhibitor apocynin (4 mg/kg/day) for 2 weeks. Left ventricular (LV) and myocyte contractile functions were evaluated using echocardiography and edge‐detection, respectively. Results: STZ elicited hyperglycaemia and reduced body weight gain, which was unaffected by apocynin. STZ significantly reduced fractional shortening, LV wall thickness, peak shortening, maximal velocity and duration of shortening or relengthening, the effects of which – with the exception of wall thickness – were significantly attenuated or ablated by apocynin. Western blot analysis revealed that the effects of comparable Akt phosphorylation, reduced AMPK phosphorylation, downregulation of sarco(endo)plasmic reticulum Ca²⁺‐ATPase and lessened phosphorylation of phospholamban in diabetic myocardium were unaffected by apocynin. Both apocynin and the nitric oxide synthase (NOS) inhibitor l ‐arginine methyl ester (L‐NAME) inhibited elevated O₂^? production in diabetes without any additive effect between the two, indicating the presence of endothelial nitric oxide synthase (eNOS) uncoupling. However, neither diabetes nor apocynin altered the expression of heat shock protein 90 and eNOS phosphorylation (Ser¹¹⁷⁷). In addition, apocynin mitigated elevated levels of nitrotyrosine and nitric oxide in diabetes. Conclusion: Taken together, these data indicate the beneficial role of NADPH oxidase inhibition in diabetes‐induced myocardial contractile dysfunction.     

Fluvastatin-induced reduction of oxidative stress ameliorates diabetic cardiomyopathy in association with improving coronary microvasculature

Diabetic cardiomyopathy is associated with increased oxidative stress and vascular endothelial dysfunction, which lead to coronary microangiopathy. We tested whether statin-induced redox imbalance improvements could ameliorate diabetic cardiomyopathy and improve coronary microvasculature in streptozotocin-induced diabetes mellitus (DM). Fluvastatin (10 mg/kg/day) or vehicle was orally administered for 12 weeks to rats with or without DM. Myocardial oxidative stress was assessed by NADPH (nicotinamide adenine dinucleotide phosphate) oxidase subunit p22^phox and gp91^phox mRNA expression, and myocardial 8-iso-prostaglandin F_2α (PGF_2α) levels. Myocardial vascular densities were assessed using anti-CD31 and anti-α-smooth muscle actin (SMA) antibodies. Fluvastatin did not affect blood pressure or plasma cholesterol, but attenuated increased left ventricular (LV) minimum pressure and ameliorated LV systolic dysfunction in DM rats in comparison with vehicle (LV dP/dt, 8.9 ± 1.8 vs 5.4 ± 1.0 × 10³ mmHg/s, P < 0.05). Myocardial oxidative stress increased in DM, but fluvastatin significantly reduced p22^phox and gp91^phox mRNA expression and myocardial PGF_2α levels. Fluvastatin enhanced myocardial endothelial nitric oxide synthase (eNOS) protein levels and increased eNOS, vascular endothelial growth factor, and hypoxia-inducible factor-1α mRNA expression. CD31-positive cell densities were lower in DM rats than in non-DM rats (28.4 ± 13.2 vs 48.6 ± 4.3/field, P < 0.05) and fluvastatin restored the number (57.8 ± 18.3/field), although there were no significant differences in SMA-positive cell densities between groups. Fluvastatin did not affect cardiac function, oxidative stress, or vessel densities in non-DM rats. These results suggest that beneficial effects of fluvastatin on diabetic cardiomyopathy might result, at least in part, from improving coronary microvasculature through reduction in myocardial oxidative stress and upregulation of angiogenic factor.     

Nebivolol alleviates aortic remodeling through eNOS upregulation and inhibition of oxidative stress in l-NAME-induced hypertensive rats

Purpose: To investigate the effect and mechanism of nebivolol on aortic remodeling in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Methods: Male Sprague–Dawley rats were treated with equal volumes of drinking water or l-NAME (60 mg/kg/day), alone or in combination with nebivolol (8 mg/kg/day) or atenolol (80 mg/kg/day) by gavage for 8 weeks. Systolic blood pressure (SBP), aortic morphometry, plasma nitric oxide (NO) levels, nitric oxide synthase (NOS) activity, and relaxation of aorta to acetylcholine were determined. Protein expression of endothelial NOS (eNOS), Akt, and NADPH oxidase (Nox) was evaluated. Results: l-NAME-treated rats showed an elevated SBP associated with aortic remodeling. l-NAME-treated rats showed reduced plasma NO levels and NOS activity and increased reactive oxygen species (ROS). Protein expression of eNOS, eNOS phosphorylated at Ser¹¹⁷⁷ (p-eNOS), Akt, and Akt phosphorylated at Ser⁴⁷³ (p-Akt) decreased, whereas that of Nox2, Nox4, and p22^phox increased in the aortas from l-NAME-treated rats. Nebivolol treatment reduced SBP and ameliorated aortic remodeling. The effects of nebivolol were accompanied by increasing NO levels, NOS activity, and expression of eNOS, p-eNOS, Akt, and p-Akt, as well as reduction of ROS generation and Nox2, Nox4, and p22^phox expression. These effects of nebivolol were not reproduced by atenolol. Conclusion: Our data indicate a protective role of nebivolol on the high blood pressure and vascular remodeling induced by l-NAME. The beneficial vascular effect of nebivolol is mediated by the upregulation of eNOS and inhibition of oxidative stress.     

Irisin improves endothelial function in type 2 diabetes through reducing oxidative/nitrative stresses

Vascular complications are the major causes of death in patients with diabetes, and endothelial dysfunction is the earliest event in vascular complications of diabetes. It has been reported that plasma irisin level is significantly reduced in patients with type 2 diabetic patients. The present study aimed to investigate whether irisin improved endothelial function in type 2 diabetes as well as the underlying mechanisms. The type 2 diabetes model was established by feeding C57BL/6 mice with high-fat diet. The type 2 diabetic mice exhibited reduced serum irisin level and impaired endothelial function. Irisin treatment (0.5 mg/kg/d) for two weeks improved vascular function based on the evaluation of endothelium-dependent vasorelaxation and p-VASP levels. To investigate the direct endothelial protective effects of irisin, diabetic aortic segments were incubated with irisin (1 μg/ml) ex vivo. Exposure to irisin improved endothelium-dependent vasorelaxation of diabetic aortas. Mechanically, the diabetic aortic segments exhibited increased oxidative/nitrative stresses. Irisin reduced the diabetes-induced oxidative/nitrative stresses evidenced by reducing overproduction of superoxide and peroxynitrite, and down-regulation of iNOS and gp91^phox. To further investigate the protective effects of irisin on endothelial cells and the underlying mechanisms, human umbilical vein endothelial cells (HUVECs) cultured in high-glucose/high-fat (HG/HF) medium were pre-incubated with irisin. Irisin (1 μg/ml) reduced the oxidative/nitrative stresses and apoptosis induced by HG/HF in HUVECs probably via inhibiting activation of PKC-β/NADPH oxidase and NF-κB/iNOS pathways. Taken together, irisin alleviates endothelial dysfunction in type 2 diabetes partially via reducing oxidative/nitrative stresses through inhibiting signaling pathways implicating PKC-β/NADPH oxidase and NF-κB/iNOS, suggesting that irisin may be a promising molecule for the treatment of vascular complications of diabetes.     

NADPH oxidase inhibition prevents beta cell dysfunction induced by prolonged elevation of oleate in rodents

Aims/hypothesis

The activation of NADPH oxidase has been implicated in NEFA-induced beta cell dysfunction. However, the causal role of this activation in vivo remains unclear. Here, using rodents, we investigated whether pharmacological or genetic inhibition of NADPH oxidase could prevent NEFA-induced beta cell dysfunction in vivo.

Methods

Normal rats were infused for 48 h with saline or oleate with or without the NADPH oxidase inhibitor apocynin. In addition, NADPH oxidase subunit p47^phox-null mice and wild-type littermate controls were infused with saline or oleate for 48 h. This was followed by measurement of NADPH oxidase activity, reactive oxygen species (ROS) and superoxide imaging and assessment of beta cell function in isolated islets and hyperglycaemic clamps.

Results

Oleate infusion in rats increased NADPH oxidase activity, consistent with increased total but not mitochondrial superoxide in islets and impaired beta cell function in isolated islets and during hyperglycaemic clamps. Co-infusion of apocynin with oleate normalised NADPH oxidase activity and total superoxide levels and prevented beta cell dysfunction. Similarly, 48 h NEFA elevation in wild-type mice increased total but not mitochondrial superoxide and impaired beta cell function in isolated islets. p47^phox-null mice were protected against these effects when subjected to 48 h oleate infusion. Finally, oleate increased the levels of total ROS, in both models, whereas inhibition of NADPH oxidase prevented this increase, suggesting that NADPH oxidase is the main source of ROS in this model.

Conclusions/interpretation

These data show that NADPH-oxidase-derived cytosolic superoxide is increased in islets upon oleate infusion in vivo; and whole-body NADPH-oxidase inhibition decreases superoxide in concert with restoration of islet function.     

Caloric restriction reverses high-fat diet-induced endothelial dysfunction and vascular superoxide production in C57Bl/6 mice

Obesity is frequently associated with endothelial dysfunction. We hypothesized that high-fat feeding dysregulates the balance between endothelial derived nitric oxide and superoxide formation. Furthermore, we examined whether caloric restriction could reverse the detrimental vascular effects related to obesity. Male C57Bl/6 mice were fed with normal-fat diet (fat 17%) or high-fat diet (fat 60%) for 150 days. After establishment of obesity at day 100, a subgroup of obese mice were put on caloric restriction (CR) (70% of ad libitum energy intake) for an additional 50 days. At day 100, aortic rings from obese mice receiving high-fat diet showed impaired endothelium-dependent vasodilation in response to acetylcholine (ACh). Caloric restriction reversed high-fat diet-induced endothelial dysfunction. At day 150, impaired vasodilatory responses to ACh in obese mice without caloric restriction were markedly improved by preincubation with the tetrahydrobiopterin (BH₄) precursor sepiapterin and l-arginine, a substrate for endothelial nitric oxide synthase (eNOS). Additionally, inhibition of vascular arginase by l-norvaline partially, and superoxide scavenging by Tiron completely, restored endothelial cell function. Obese mice showed increased vascular superoxide production, which was diminished by endothelial denudation, pretreated of the vascular rings with apocynin (an inhibitor of reduced nicotinamide adenine dinucleotide phosphate [NADPH] oxidase), oxypurinol (an inhibitor of xanthine oxidase), N ^G-nitro-l-arginine methyl ester (LNAME; an inhibitor of eNOS), or by adding the BH₄ precursor sepiapterin. Caloric restriction markedly attenuated vascular superoxide production. In obese mice on CR, endothelial denudation increased superoxide formation whereas vascular superoxide production was unaffected by l-NAME. Western blot analysis revealed decreased phosphorylated eNOS (Ser1177)-to-total eNOS expression ratio in obese mice as compared to lean controls, whereas the phospho-eNOS/NOS ratio in obese mice on CR did not differ from the lean controls. In conclusion, the present study suggests that caloric restriction reverses obesityinduced endothelial dysfunction and vascular oxidative stress, and underscores the importance of uncoupled eNOS in the pathogenesis.     

Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet

Hypertension and high serum cholesterol level are important risk factors for atherosclerosis and coronary heart disease. In the present study we tested the hypothesis whether high sodium intake, when given in combination with Western type high-fat diet, induces endothelial dysfunction and promotes atherosclerosis. Furthermore, the role and enzyme sources of increased oxidative stress were examined. Low-density lipoprotein receptor-deficient mice (LDLR^−/−) and control C57Bl/6 mice received either high-fat, normal-sodium diet (fat 18% and cholesterol 0.5%; NaCl 0.7%; w/w) or high-fat, high-sodium diet (7% NaCl w/w) for 12 weeks. Superoxide formation was assessed by lucigenin enhanced chemiluminescence, endothelial functions were examined ex vivo, and atherosclerotic lesions from the aorta were assessed by light microscopy. High-fat, high-sodium diet increased systolic blood pressure in LDLR^−/− mice but not in C57Bl/6 mice, whereas it induced cardiac hypertrophy in both mouse strains. Dietary combination of fat and sodium induced endothelial dysfunction in LDLR^−/− mice. Preincubation with a superoxide scavenger Tiron normalized endothelial dysfunction, whereas the hydrogen peroxide scavenger catalase did not alter endothelial function. High sodium intake induced superoxide formation in LDLR^−/− mice on high-fat diet. Stimulation of muscarinic receptors in the endothelial cells by acetylcholine increased superoxide generation, whereas preincubation with the nitric oxide synthase (NOS) inhibitor L-arginine methyl ester or endothelium removal reduced superoxide production. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by apocynin decreased vascular superoxide formation whereas the xanthine oxidase inhibitor oxypurinol did not significantly affect oxidative stress in LDLR^−/− mice. In conclusion, the detrimental effects of dietary sodium on endothelial function and progression of atherosclerosis in LDLR^−/− mice on high-fat diet are mediated by increased ROS formation mainly through uncoupled NOS and NADPH oxidase. The present study also underscores the importance of superoxide and endothelial NOS uncoupling in the pathogenesis of endothelial dysfunction.     

The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes

Aims/hypothesis  

We have previously shown that NADPH oxidase (NOX) lies upstream of uncoupled endothelial nitric oxide synthase (eNOS), which is known to occur in diabetic endothelium. However, it remains unclear which specific NOX isoform(s) is responsible for eNOS uncoupling and endothelial dysfunction in diabetic mouse models. The aim of the present study was to test the hypothesis that one or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS, which has been shown to occur in patients with diabetes to contribute to endothelial dysfunction.     

Rac1 mediates sex difference in cardiac tumor necrosis factor-α expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia

The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91^phox-NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91^phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts.     

PPARγ ligands regulate NADPH oxidase, eNOS, and barrier function in the lung following chronic alcohol ingestion

Background: Chronic alcohol ingestion increases the incidence and severity of the acute respiratory distress syndrome (ARDS), where reactive species contribute to alveolar‐capillary barrier dysfunction and noncardiogenic pulmonary edema. Previous studies demonstrated that chronic alcohol ingestion increased lung NADPH oxidase and endothelial nitric oxide synthase (eNOS) expression and that ligands for the peroxisome proliferator‐activated receptor gamma (PPARγ) reduced NADPH oxidase expression. Therefore, we hypothesized that the PPARγ ligand, rosiglitazone, would attenuate alcohol‐induced NADPH oxidase expression and pulmonary barrier dysfunction. Methods: C57Bl/6 mice were treated ± alcohol in drinking water (20% w/v) for 12 weeks. During the final week of alcohol treatment, mice were gavaged with rosiglitazone (10 mg/kg/d) or vehicle. Selected animals were treated twice with lipopolysaccharide (LPS, 2 mg/kg IP) prior to sacrifice. Pulmonary barrier dysfunction was estimated from protein content of bronchoalveolar lavage (BAL) fluid. Results: LPS treatment increased BAL protein in alcohol‐fed but not control mice, and rosiglitazone attenuated LPS and alcohol‐induced pulmonary barrier dysfunction. Alcohol‐ and LPS‐induced increases in lung eNOS, Nox1, and Nox4 expression were attenuated by rosiglitazone. In vitro, alcohol (0.10% w/v) increased H₂O₂ production, barrier dysfunction, eNOS, Nox1, and Nox4 expression in human umbilical vein endothelial cell (HUVEC) monolayers, effects also attenuated by rosiglitazone (10 μM). Alcohol‐induced HUVEC barrier dysfunction was attenuated by inhibition of NOS or addition of the eNOS cofactor, tetrahydrobiopterin. Conclusions: These results indicate that PPARγ activation reduced expression of eNOS, Nox1, Nox4, the production of reactive species, and barrier dysfunction caused by chronic alcohol ingestion and suggest that PPARγ represents a novel therapeutic target for strategies designed to reduce the risk of lung injury in patients with a history of chronic alcohol ingestion.     

Caloric restriction improves endothelial dysfunction during vascular aging: Effects on nitric oxide synthase isoforms and oxidative stress in rat aorta

Aging is characterized by activation of inducible over endothelial nitric oxide synthase (iNOS and eNOS), impaired antioxidant activity and increased oxidative stress, which reduces nitric oxide bioavailability and causes endothelial dysfunction. Caloric restriction (CR) blunts oxidative stress. We investigated whether CR impacts endothelial dysfunction in aging and the underlying mechanisms. Aortas from young (YC, 6 months of age) and old (OC, 24 months of age) rats ad-libitum fed and from old rats caloric-restricted for 3-weeks (OR, 26%) were investigated. Endothelium-dependent vasorelaxation was impaired in OC, associated with reduced eNOS and increased iNOS expression (P < 0.05). Aortic nitrite was similar in OC and YC, but the contribution of calcium-independent NOS to total NOS activity was increased whereas that of calcium-dependent NOS was reduced (p ≤ 0.0003). Plasma thiobarbituric acid-reactive substances (TBARS) were elevated in OC as well as aortic nitrotyrosine (P < 0.05). Expression of manganese superoxide dismutase (MnSOD) and total SOD activity were impaired in OC (P < 0.05 vs. YC), whereas copper-zinc (CuZn) SOD expression was similar in OC and YC. CR restored endothelial dysfunction in old rats, reduced iNOS expression, total nitrite and calcium-independent NOS activity in aorta (P < 0.05) without changes in eNOS expression and calcium-dependent NOS activity. Sirtuin-1 expression did not differ among groups. Plasma TBARS and aortic nitrotyrosine were reduced (P < 0.05) in OR compared with OC. In OR CuZnSOD protein and SOD activity increased (P < 0.05) without changes in MnSOD expression. Short-term CR improves age-related endothelial dysfunction. Reversal of altered iNOS/eNOS ratio, reduced oxidative stress and increased SOD enzyme activity rather than enhanced NO production appear to be involved in this effect.     

Endothelial,but not the inducible,nitric oxide synthase is detectable in normal and portal hypertensive rats

Abstract: Background: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. Methods: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1) Partial portal vein ligated rats, (2) Bile duct ligated rats, (3) Carbon tetrachloride treated rats, (4) Sham operated rats, (5) Untreated control rats, and (6) LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. Results: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. Conclusion: In chronic portal hypertension, the main source for NO production depends on eNOS activity.     

Aging-associated insulin resistance predisposes to hypertension and its reversal by exercise: the role of vascular vasorelaxation to insulin

Aging is an independent risk factor for hypertension, and hypertension and insulin resistance commonly coexist in the elderly. This study was designed to examine the effects of aging-related insulin resistance on blood pressure (BP) and its underlying mechanisms, with specific focus on the role of exercise in reversing hypertensive response. Adult (6-month-old) and aging (24-month-old) male Sprague-Dawley rats were subjected to a 10 weeks free-of-loading swim training (60 min/day, 5 days/week). Arterial vasorelaxation, cardiac contraction, eNOS activation, and iNOS and gp91^phox expression were determined. Under aging-related insulin resistance conditions, insulin infusion significantly elevated BP (P < 0.05). Aging caused significant endothelial dysfunction (P < 0.05 − 0.01), which was responsible for decreased arterial vasorelaxation to insulin. Aging attenuated myocardial contractile response to insulin, decreased eNOS expression and its phosphorylation by insulin, and increased iNOS and gp91^phox expression in aging arteries (P < 0.01). Exercise improved insulin sensitivity, potentiated insulin’s positive inotropic effects, facilitated arterial vasorelaxation to insulin, increased arterial eNOS activation in adult and aging rats, and thus attenuated insulin resistance-related hypertensive response to insulin. Moreover, exercise markedly reversed increased iNOS and gp91^phox expression in aging arteries. Inhibition of eNOS with Cavtratin or L-NAME significantly blocked exercise-facilitated arterial vasorelaxation to insulin and exercise-lowered BP response to insulin. In conclusion, these results demonstrate that endothelial dysfunction in response to insulin, but not insulin’s positive inotropic effects, plays an important role in the development of aging-related hypertension. The reversal of hypertensive response to insulin by exercise is most likely associated with improved insulin sensitivity in an eNOS-dependent manner and reduced oxidative and nitrative stresses. Electronic supplementary material:  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Returned for 1. Revision: 18 February 2008 1. Revision received: 11 August 2008 Returned for 2. Revision: 10 September 2008 2. Revision received: 15 September 2008     

Efeitos do Exercício Aeróbico Tardio na Remodelação Cardíaca de Ratos com Infarto do Miocárdio Pequeno

Background:Physical exercise has been considered an important non-pharmacological therapy for the prevention and treatment of cardiovascular diseases. However, its effects on minor cardiac remodeling are not clear.Objective:To evaluate the influence of aerobic exercise on the functional capacity, cardiac structure, left ventricular (LV) function, and gene expression of NADPH oxidase subunits in rats with small-sized myocardial infarction (MI).Methods:Three months after MI induction, Wistar rats were divided into three groups: Sham; sedentary MI (MI-SED); and aerobic exercised MI (MI-AE). The rats exercised on a treadmill three times a week for 12 weeks. An echocardiogram was performed before and after training. The infarction size was evaluated by histology, and gene expression was assessed by RT-PCR. The significance level for statistical analysis was set at 5%.Results:Rats with MI lower than 30% of the LV total area were included in the study. Functional capacity was higher in MI-AE than in Sham and MI-SED rats. The infarction size did not differ between groups. Infarcted rats had increased LV diastolic and systolic diameter, left atrial diameter, and LV mass, with systolic dysfunction. Relative wall thickness was lower in MI-SED than in the MI-AE and Sham groups. Gene expression of the NADPH oxidase subunits NOX2, NOX4, p22^phox, and p47^phox did not differ between groups.Conclusion:Small-sized MI changes cardiac structure and LV systolic function. Late aerobic exercise is able to improve functional capacity and cardiac remodeling by preserving the left ventricular geometry. NADPH oxidase subunits gene expression is not involved in cardiac remodeling or modulated by aerobic exercise in rats with small-sized MI.     

Apocynin Ameliorates Cadmium-Induced Hypertension Through Elevation of Endothelium Nitric Oxide Synthase

Apocynin is reported to have antioxidant and NADPH oxidase inhibitor activities. Cadmium toxicity is reported to causes oxidative damage, resulting in vascular dysfunction, reduced bioavailability of nitric oxide (NO) and hypertension. The study aimed to investigate the protective effects of apocynin in cadmium-induced hypertension. Thirty-six (36) adult male Sprague–Dawley rats were randomly divided into 6 groups. Group 1 served as control, Groups 2 and 3 received 50 and 100 mg/Kg (b.w) apocynin, respectively, Group 4 received 100 ppm CdCl₂ in their drinking water, while Group 5 and 6 received 100 ppm CdCl₂ in their drinking and 50 and 100 mg/Kg (b.w) apocynin, respectively, for 8 weeks. Blood pressure readings were taken weekly using the tail-cuff method. cGMP, endothelial nitric oxide synthase (eNOS), NO and hematological parameters were analyzed at the end of 8 weeks. Apocynin, although a poor antioxidant, caused a significant reduction (p < 0.05) in systolic and mean arterial pressures in the cadmium-induced elevations in blood pressure and amelioration of altered hematological parameters. However, while cadmium exposures did not alter the cGMP, eNOS and nitrate concentrations in serum, apocynin reduced the cGMP and nitrite values while significantly elevating (p < 0.05) the eNOS concentrations and also improved the cadmium-induced anemia. Apocynin was effective in reducing cadmium-induced elevated blood pressures through elevation of eNOS. Inhibition of NADPH oxidase activity may be a useful strategy for prevention and treatment of cadmium-induced hypertension.     

Quercetin inhibits vascular superoxide production induced by endothelin-1: Role of NADPH oxidase, uncoupled eNOS and PKC

Chronic administration of the most abundant dietary flavonoid quercetin exerts antihypertensive effects and improves endothelial function. We have investigated the effects of quercetin and its methylated metabolite isorhamnetin (1-10microM) on endothelial dysfunction and superoxide (O(2*)(-)) production induced by endothelin-1 (ET-1, 10nM). ET-1 increased the contractile response induced by phenylephrine and reduced the relaxant responses to acetylcholine in phenylephrine contracted intact aorta, and these effects were prevented by co-incubation with quercetin, isorhamnetin or chelerythrine (protein kinase C (PKC) inhibitor). This endothelial dysfunction was also improved by superoxide dismutase (SOD), apocynin (NADPH oxidase inhibitor) and sepiapterin (tetrahydrobiopterin synthesis substrate). Furthermore, ET-1 increased intracellular O(2*)(-) production in all layers of the vessel, protein expression of NADPH oxidase subunit p47(phox) without affecting p22(phox) expression and lucigenin-enhanced chemiluminescence signal stimulated by calcium ionophore A23187. All these changes were prevented by both quercetin and isorhamnetin. Moreover, apocynin, endothelium denudation and N(G)-nitro-l-arginine methylester (l-NAME, nitric oxide synthase inhibitor) suppressed the ET-1-induced increase in A23187-stimulated O(2*)(-) generation. Moreover, quercetin but not isorhamnetin, inhibited the increased PKC activity induced by ET-1. Taken together these results indicate that ET-1-induced NADPH oxidase up-regulation and eNOS uncoupling via PKC leading to endothelial dysfunction and these effects were prevented by quercetin and isorhamnetin.     

Polyphenol-containing azuki bean (Vigna angularis) extract attenuates blood pressure elevation and modulates nitric oxide synthase and caveolin-1 expressions in rats with hypertension

Background and aimsAzuki beans (Vigna angularis) contain polyphenols such as proanthocyanidins that exhibit potential radical scavenging activities. We herein investigated the effects of polyphenol-containing azuki bean extract (ABE) on elevated blood pressure, nitric oxide (NO) production, and expressions of endothelial NO synthase (eNOS), inducible NOS (iNOS), and caveolin-1 proteins in the aorta and kidney of chronically hypertensive rats.Methods and resultsSpontaneously hypertensive rats (SHRs/Izm) with approximately 200 mm Hg systolic blood pressure (SBP) were randomly divided into 2 groups fed either 0% or 0.9% ABE-containing diet. Age-matched normotensive Wistar–Kyoto rats were used as the control. The content of 24-h urinary nitrate/nitrite (NOx) excretion was measured to evaluate NO production. After 8 weeks of treatment, the eNOS, iNOS, and caveolin-1 protein expressions in the aorta and kidney were analyzed by western blotting. The SBP of the ABE-treated SHR was significantly lower than that of the untreated SHR. The level of 24-h urinary NOx excretion was significantly higher in the ABE-treated SHR than in the untreated SHR. The eNOS and iNOS expressions in the aorta and kidney were remarkably upregulated in the untreated SHR but suppressed in the ABE-treated SHR. The vascular and renal caveolin-1 expressions were upregulated in the ABE-treated SHR.ConclusionsABE reduced the elevated blood pressure and increased NO production in long-term treatment. It may be associated with the modulation of eNOS and iNOS protein expressions in the aorta and kidney during the development of hypertension.     

Activation of natriuretic peptide receptor-C attenuates the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats: implication of Gialpha protein

We have recently shown that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit enhanced expression of Giα proteins, which was attributed to the enhanced oxidative stress. Since C-ANP_4-23 that specifically interacts with natriuretic peptide C (NPR-C) receptor has been shown to decrease the expression of Giα protein in VSMC, the present study was undertaken to examine if C-ANP_4-23 can also decrease the enhanced expression of Giα protein in VSMC from SHR and whether it is attributed to its ability to attenuate the enhanced oxidative stress. Aortic VSMC from 12-week-old SHR and their age-matched Wistar-Kyoto (WKY) rats were used for the present studies. VSMC from SHR exhibited enhanced expression of Giα-2 and Giα-3 proteins, different subunits of NADPH oxidase such as Nox⁴ and p^47phox proteins but not of p^22phox, enhanced production of superoxide anion as well as NADPH oxidase activity as compared to age-matched WKY rats. Treatment of VSMC from SHR with C-ANP_4-23 decreased towards control levels the enhanced expression of Giα proteins, enhanced superoxide anion production and enhanced NADPH oxidase activity as well as the enhanced expression of Nox⁴ and p^47phox. However, C-ANP_4-23-induced attenuation of the enhanced level of O₂⁻ and NADPH oxidase activity occurs at 4 h before the decrease in the enhanced expression of p^47phox that occurs at 16 h of C-ANP_4-23 treatment. The decreased expression of NADPH oxidase in SHR was also associated with further decrease in O₂⁻ and NADPH oxidase activity. Furthermore, treatment of VSMC from SHR with pertussis toxin (PT) decreased the enhanced levels of superoxide anion as well as NADPH oxidase activity; however, the enhanced levels of different subunits of NADPH oxidase were not attenuated by PT treatment. These results suggest that C-ANP_4-23 decreases the enhanced oxidative stress in SHR by attenuating the enhanced expression of Giα proteins and also the enhanced levels of NADPH oxidase.     

Ethanol Augments PDGF-Induced NADPH Oxidase Activity and Proliferation in Rat Pancreatic Stellate Cells

Background/Aims: Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation. Methods: PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [³H] thymidine incorporation into DNA. Results: mRNA for NADPH oxidase components Nox1, gp91^phox, Nox4, p22P^phoxhox, p47^phox and p67^phox and protein for NADPH oxidase subunits gp91^phox, p22^phox, p47^phox and p67^phox are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented byantioxidant N-ace-tyl-l-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenyleneiodium.Theeffectsof PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47^phox. Conclusions: Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.     

The glucagon-like peptide 1 analog liraglutide reduces TNF-α-induced oxidative stress and inflammation in endothelial cells

ObjectiveGlucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells.Methods and resultsThe effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H₂DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until 30 nM while liraglutide inhibited the induction of gp91^phox and p22^phox, subunit of NADPH oxidase, by TNF-α? In addition, protein levels of SOD-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-3 induced by TNF-α.ConclusionLiraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes.