Vascular Superoxide Generation Research Articles

Syzygium gratum Extract Alleviates Vascular Alterations in Hypertensive Rats.

Background and Objectives: Syzygium gratum (SG) is a local vegetable and widely consumed in Thailand. Previously, a strong antioxidative effect of SG extract has been reported. The effects of SG extract on hypertension have remained unknown. The effect of SG aqueous extract on blood pressure and vascular changes were examined in L-NAME-induced hypertensive rats (LHR), and its potential active constituents were also explored. Materials and Methods: Male Sprague Dawley rats were allocated to control, L-NAME (40 mg/kg/day), L-NAME + SG (100, 300, 500 mg/kg/day), or captopril (5 mg/kg/day) groups. The components of SG extract were analyzed. Results: The analysis of aqueous SG extract was carried out using HPLC-Mass spectroscopy, and phenolic compounds could be identified as predominant components which might be responsible for its antihypertensive effects observed in the LHR model (p < 0.05). Additionally, SG extract also improved vascular responses to acetylcholine and decreased vascular remodeling in LHR (p < 0.05). Enhancements of eNOS expression and plasma nitric oxide metabolite levels, and attenuation of angiotensin converting enzyme (ACE) activity and plasma angiotensin II levels were observed in the LHR group treated with SG. Moreover, SG exhibited strong antioxidant activities by reducing vascular superoxide generation and systemic malondialdehyde in LHRs. Captopril suppressed high blood pressure and alleviated vascular changes and ACE activity in LHRs, similar to those of the SG extract (p < 0.05). Conclusion: Our results suggest that the SG extract exhibited antihypertensive effects, which is relevant to alleviation of vascular dysfunction and vascular remodeling of LHRs. These effects might be mediated by phenolic compounds to inhibit ACE activity and scavenge reactive oxygen species in LHR.

Tangeretin mitigates l-NAME-induced ventricular dysfunction and remodeling through the AT1R/pERK1/2/pJNK signaling pathway in rats.

Tangeretin is a citrus flavonoid that exerts several beneficial effects, including anti-inflammation, anti-oxidation and neuroprotection. In this study, the aim was to test the effect of tangeretin on Nω-Nitro-l-arginine methyl ester (l-NAME)-induced high blood pressure, and left ventricular dysfunction and remodeling in rats. Rats were divided into five groups (n = 8 per each group): a control group, an l-NAME group and three l-NAME groups treated with tangeretin (15 mg kg-1) or tangeretin (30 mg kg-1) or captopril (5 mg kg-1) for the final two weeks. After five weeks of experiment, l-NAME groups had high systolic blood pressures, and ventricular dysfunction and remodeling. Overexpression of angiotensin II type 1 receptor, phosphorylated-extracellular-regulated kinase 1/2 (pERK1/2), and phosphorylated-c-Jun N-terminal kinase (pJNK) protein but downregulation of endothelial nitric oxide synthase (eNOS) protein expression in ventricular tissues were observed in hypertensive rats while the protein expression of phosphorylated-mitogen activated protein kinase p38 did not differ among groups. The decrease in plasma NOx and increase in vascular superoxide generation, plasma malondialdehyde, angiotensin-converting enzyme activity and angiotensin II levels were found in hypertensive rats. These alterations were suppressed in hypertensive rats treated with tangeretin or captopril. In conclusion, tangeretin exhibits antihypertensive effects and alleviates ventricular dysfunction and remodeling in hypertensive rats. These effects are associated with the inhibition of renin angiotensin system activation and restoration of pERK1/2, pJNK, and eNOS protein expressions along with reduced oxidative stress and increased NO bioavailability.

5893Adipose tissue secreted ceramides and related sphingolipids - potential modulators of vascular redox signalling in cardiovascular disease

Abstract Background Adipose tissue (AT) plays a vital role in the modulation of vascular biology via known adipokines and other yet unidentified secreted molecules. Recent technological advances of omics-based approaches have enabled the identification of more metabolites that drive the currently recognised cross talk between the AT and the vasculature. Purpose The main aim of this study is to explore the role of AT secreted ceramides on the regulation of vascular superoxide (O2·−) generation in cardiac patients. Methods Untargeted metabolic profiling for the secretome of paired thoracic and subcutaneous ATs from 48 patients undergoing cardiac surgery (the Oxford cohort for heart, vessels and fat) along with the subsequent sphingolipids-targeted quantification were performed using liquid-chromatography/mass-spectrometry. Immortalized human aortic endothelial cells (teloHAEC) were treated exogenously with C6-ceramide (CerC6) for 20min and O2·− production was measured using lucigenin-enhanced chemiluminescence. Results Metabolomics differential (A) and enrichment analysis (not shown) highlighted the significant differences in sphingolipids secretion levels between the two fat depots. Higher amount of ceramides were produced and secreted from the thoracic depot with C16-ceramide (CerC16) representing the most abundant differentially secreted ceramide (B). Compared to the lowest tertile, the middle and highest tertiles of ceramides in thoracic AT were significantly associated with higher O2·− generation in patients' vessels namely saphenous veins (SV) and internal mammary arteries (IMA) (C). Exogenous treatment of teloHAEC with ceramide increased O2·− generation and eNOS uncoupling evidenced by more negative O2·− after addition of L-NAME, an inhibitor of eNOS (D). Results Overview Conclusions In this study, we demonstrate for the first time that sphingolipids, in particular ceramides, secreted from AT of cardiac patients may modulate their vascular redox state via dysregulating vascular eNOS signalling leading to endothelial dysfunction –a hallmark of cardiovascular disease. Acknowledgement/Funding the NovoNordisk Foundation (NNF15CC0018486)

2 - Oxidative modification of the cardiac sodium potassium pump is worsened in the absence of FXYD1, contributing to cardiac dysfunction and fibrosis

Introduction FXYD1 is a small membrane protein that endogenously protects the cardiac Na+-K+ ATPase from oxidative inhibition. Given the importance of the Na+-K+ ATPase in normal cardiac function, we hypothesized that absence of FXYD1 would worsen reactive oxygen species (ROS)-induced cardiac dysfunction. Methods and Results In studies were performed using wild-type (WT) and FXYD1 knockout (KO) mice. FXYD1 KO mice are prone to exacerbated vascular superoxide generation (P Summary This data defines a new role for FXYD1 in preventing cardiac dysfunction and fibrosis characterized by elevated ROS. Impaired oxidative inhibition of the Na+-K+ ATPase β1 subunit plays a likely role in mediating this protective response.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice.

Hydrogen sulphide (H2S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H2S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO•) bioactivity were significantly reduced ( p < 0.05), and maximal vasorelaxation to the NO• donor sodium nitroprusside was impaired ( p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase ( p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 ( p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO• efficacy and reducing superoxide production in the mouse aorta.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE:We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats.METHODS:Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system.RESULTS:Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group.CONCLUSIONS:In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.

C-Src Inhibition Improves Cardiovascular Function but not Remodeling or Fibrosis in Angiotensin II-Induced Hypertension.

c-Src plays an important role in angiotensin II (Ang II) signaling. Whether this member of the Src family kinases is involved in the development of Ang II-induced hypertension and associated cardiovascular damage in vivo remains unknown. Here, we studied Ang II-infused (400 ng/kg/min) mice in which c-Src was partially deleted (c-Src+/-) and in wild-type (WT, c-Src+/+) mice treated with a c-Src inhibitor (CGP077675; 25 mg/kg/d). Ang II increased blood pressure and induced endothelial dysfunction in WT mice, responses that were ameliorated in c-Src+/- and CGP077675-treated mice. Vascular wall thickness and cross-sectional area were similarly increased by Ang II in WT and c-Src+/- mice. CGP077675 further increased cross-sectional area in hypertensive mice. Cardiac dysfunction (ejection fraction and fractional shortening) in Ang II-infused WT mice was normalized in c-Src+/- mice. Increased oxidative stress (plasma thiobarbituric acid-reactive substances, hydrogen peroxide, and vascular superoxide generation) in Ang II-infused WT mice was attenuated in c-Src-deficient and CGP077675-treated mice. Hyperactivation of vascular c-Src, ERK1/2 (extracellular signal-regulated kinase 1/2), and JNK (c-Jun N-terminal kinase) in hypertensive mice was normalized in CGP077675-treated and c-Src+/- mice. Vascular fibronectin was increased by Ang II in all groups and further augmented by CGP077675. Cardiac fibrosis and inflammation induced by Ang II were amplified in c-Src+/- and CGP-treated mice. Our data indicate that although c-Src downregulation attenuates development of hypertension, improves endothelial and cardiac function, reduces oxidative stress, and normalizes vascular signaling, it has little beneficial effect on fibrosis. These findings suggest a divergent role for c-Src in Ang II-dependent hypertension, where c-Src may be more important in regulating redox-sensitive cardiac and vascular function than fibrosis and remodeling.

Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.

Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice

The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE−/−) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Chronic NaHS Treatment Is Vasoprotective in High-Fat-Fed ApoE−/−Mice

Hydrogen sulfide is emerging as an important mediator of vascular function that has antioxidant and cytoprotective effects. The aim of this study was to investigate the role of endogenous H2S and the effect of chronic exogenous H2S treatment on vascular function during the progression of atherosclerotic disease. ApoE−/− mice were fed a high-fat diet for 16 weeks and treated with the H2S donor NaHS or the cystathionine-γ-lyase (CSE) inhibitor D,L-propargylglycine (PPG), to inhibit endogenous H2S production for the final 4 weeks. Fat-fed ApoE−/− mice displayed significant aortic atherosclerotic lesions and significantly impaired endothelial function compared to wild-type mice. Importantly, 4 weeks of NaHS treatment significantly reduced vascular dysfunction and inhibited vascular superoxide generation. NaHS treatment significantly reduced the area of aortic atherosclerotic lesions and attenuated systolic blood pressure. Interestingly, inhibiting endogenous, CSE-dependent H2S production with PPG did not exacerbate the deleterious vascular changes seen in the untreated fat-fed ApoE−/− mice. The results indicate NaHS can improve vascular function by reducing vascular superoxide generation and impairing atherosclerotic lesion development. Endogenous H2S production via CSE is insufficient to counter the atherogenic effects seen in this model; however exogenous H2S treatment has a significant vasoprotective effect.

Vascular Generation of Tumor Necrosis Factor-α Reduces Nitric Oxide Availability in Small Arteries From Visceral Fat of Obese Patients

The aim of this study was to assess whether small arteries from visceral fat of obese patients show a reduced nitric oxide (NO)-dependent relaxation, as compared with lean control subjects, focusing on the role of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. Visceral obesity is characterized by endothelial dysfunction. Small arteries from 14 obese (body mass index 48.4 ± 11 kg/m(2)) and 14 control subjects (body mass index 24.9 ± 2 kg/m(2)), dissected after a visceral fat biopsy (laparoscopy), were evaluated on a pressurized micromyograph. Endothelium-dependent relaxation was assessed by acetylcholine. The NO availability, superoxide production, and inflammation were assessed by testing acetylcholine under the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methylester, tempol (superoxide scavenger), and infliximab (monoclonal anti-TNF-α antibody), respectively. The roles of nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase (iNOS) were assessed by their selective inhibitors apocynin and S-methylisothiourea (SMT), respectively. Vascular superoxide generation (dihydroethidium staining) protein expression of TNF-α and NOS isoforms (Western Blot) and TNF-α localization (immunohistochemistry) were assessed. Vessels from obese patients displayed a blunted relaxation to acetylcholine and a reduced inhibitory effect of N(ω)-nitro-L-arginine methylester. These alterations were normalized by tempol or infliximab while being partly ameliorated by apocynin and SMT. Vascular superoxide generation was increased (p < 0.01) in obese patients. This condition was abrogated by both tempol and infliximab and partly (p < 0.05 vs. control subjects) reduced by apocynin or SMT. Enhanced TNF-α and iNOS expression together with increased TNF-α localization in the vascular media were detected. Small arteries from visceral fat of obese patients are characterized by an increased TNF-α production, which reduces NO availability by promoting superoxide generation via nicotinamide adenine dinucleotide phosphate oxidase and iNOS activation.

Effects of Kaempferia parviflora Wall. Ex Baker on endothelial dysfunction in streptozotocin-induced diabetic rats

The aim of the present study was to investigate an ethanolic extract of Kaempferia parviflora (KPE) reduces oxidative stress and preserves endothelial function in aortae from diabetic rats. Diabetes was induced in Sprague-Dawley rats by streptozotocin (STZ) treatment (55 mg/kg i.v.). Vascular reactivity and superoxide generation were assessed in aortic rings using standard organ bath techniques and lucigenin-enhanced chemiluminescence, respectively. Eight weeks after STZ treatment blood glucose was elevated compared to citrate treated control rats and there was an increased aortic generation of superoxide anion. In aortic rings acetylcholine-induced relaxation was impaired whereas endothelium-independent relaxation to sodium nitroprusside was unaffected. When aortic rings were acutely exposed to KPE (1, 10 and 100 μg/ml) there was a significant reduction in the detection of superoxide anion and enhanced relaxation to acetylcholine. Two separate groups of rats (control and diabetic) were orally administered daily with KPE (100 mg/kg body weight) for 4 weeks. KPE treatment reduced superoxide generation and increased the nitrite levels in diabetic aortae, and enhanced acetylcholine-induced relaxation. In the presence of N(G)-nitro-L-arginine (L-NNA), the relaxation to acetylcholine in aortic rings of diabetic rats was only partially inhibited, but was totally abolished in aortic rings from the KPE-treated diabetic rats. Indomethacin did not affect relaxation to acetylcholine in aortic rings of any group. These results suggest that KPE, acutely in vitro or after 4 weeks administration in vivo, reduces oxidant stress, increases NO bioavailability and preserves endothelium-dependent relaxation in aortae from diabetic rats.

3′,4′-Dihydroxyflavonol prevents diabetes-induced endothelial dysfunction in rat aorta

Aims Diabetes increases oxidant stress and impairs endothelium-dependent relaxation. We investigated whether the antioxidant 3′,4′-dihydroxyflavonol (DiOHF) reduces the release of superoxide (O 2 −) and preserves endothelial function in aortae from diabetic rats. Main methods Type-1 diabetes was induced in Sprague–Dawley rats by streptozotocin (STZ) treatment (55 mg/kg i.v.) and vascular reactivity and superoxide generation were assessed in aortic rings using standard organ bath techniques and lucigenin-enhanced chemiluminescence respectively. Key findings Eight weeks after STZ treatment blood glucose was elevated (39.4 ± 0.4 mM) compared to citrate treated control rats (5.5 ± 0.1 mM, P < 0.05) and there was an increased aortic generation of O 2 − (control 670 ± 101, diabetic 1535 ± 249 units/mg dry weight, P < 0.05). In aortic rings acetylcholine (ACh)-induced relaxation was impaired ( R max control 78 ± 2, diabetic 66 ± 3%, P < 0.01) whereas endothelium-independent relaxation to sodium nitroprusside (SNP) was unaffected ( R max control 100 ± 1, diabetic 101 ± 2%). When aortic rings were acutely exposed to DiOHF (10 − 5 M) there was a significant reduction in the detection of O 2 − (control 124 ± 15, diabetic 165 ± 21 units/mg, P < 0.01) and enhanced relaxation to ACh ( R max control 84 ± 3, diabetic 87 ± 3%). Two separate groups of rats (control and diabetic) were treated daily with DiOHF (5 mg/kg i.p.) for 7 days. DiOHF treatment reduced superoxide generation in diabetic aortae (untreated diabetic 1471 ± 358, DiOHF-treated diabetic 580 ± 115 units/mg, P < 0.05) and enhanced acetylcholine-induced relaxation ( R max untreated diabetic 58 ± 5, DiOHF-treated diabetic 71 ± 4%, P < 0.05). Significance DiOHF, acutely in vitro or after 1 week treatment in vivo, reduces oxidant stress and preserves endothelium-dependent relaxation in aortae from diabetic rats.

Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.

Abstract 4416: Genetic Variability of Methyl-Tetrahydrofolate-Reductase Modifies eNOS Coupling in Human Arteries and Veins: Effects on Vascular Superoxide Generation and Nitric Oxide Bioavailability

5-methyl-tetrahydrofolate (5MTHF) administration improves endothelial nitric oxide synthase (eNOS) coupling in human vessels. However, it is unclear whether endogenous variations of vascular 5MTHF levels due to the functional 5MTHF-reductase (MTHFR) polymorphism C677T, has an impact on vascular function. We examined the effect of this polymorphism on vascular 5MTHF levels, NO bioavailability and eNOS coupling in human vessels. The C677T polymorphism was determined in 218 patients undergoing coronary bypass surgery. Saphenous veins (SV) and internal mammary arteries (IMA) were obtained to determine vasomotor responses of SV to acetylcholine (ACh) ex-vivo, vascular O2- production (+/− eNOS inhibitor LNAME, by chemilumineschence) and vascular 5MTHF (by HPLC). The genotype distribution was CC:100(46%) CT:94(43%) and TT:24(11%). This polymorphism had a strong effect on vascular (Fig. a ) and plasma 5MTHF (p&lt;0.001). Furthermore, the T allele was associated with lower vasomotor responses of SV to Ach (Fig. b ) as well as higher vascular O2- (Fig. c ) and greater LNAME inhibitable O2- (Fig d ., suggesting eNOS uncoupling) in IMA segments. The C677T polymorphism on MTHFR gene is a strong determinant of vascular 5MTHF, and has a significant effect on NO bioavailability and vascular redox state, by modifying eNOS coupling in human vessels.

Vascular stiffness is related to superoxide generation in the vessel wall

Oxidative stress causes endothelial dysfunction and plays a major role in the pathogenesis of cardiovascular disease. Increased vascular stiffness is an intermediate phenotype in the development of cardiovascular disease. We hypothesized that vascular stiffness is partially determined by oxidative stress. We examined 163 participants out of whom 80 had coronary artery disease. Vascular stiffness was assessed by pulse wave analysis, pulse wave velocity and measurement of aortic compliance by cardiac MRI. Circulating markers of oxidative stress and vascular superoxide generation in saphenous vein were measured. After adjustment for age, sex, BMI, heart rate, blood pressure and lipids only carotid-femoral pulse wave velocity and aortic compliance were different between patients and control group. Aortic compliance was reduced (11.4 +/- 6.3 vs. 13.9 +/- 7.3 ml x 10(-3) per mmHg; P = 0.035) and vascular superoxide generation increased (1.01 +/- 0.45 vs. 0.76 +/- 0.44 nmol/mg per min; P = 0.035) in patients with coronary artery disease compared with those without. In a multiple stepwise regression analysis, aortic compliance was determined by age (P < 0.001) and vascular superoxide production (P = 0.033). CYBA C242T and NOS3 G894T polymorphisms had additive effects on vascular superoxide generation (P = 0.026) and xanthine oxidase activity was increased in patients with CAD (P = 0.043). Genetic factors (P = 0.033) and xanthine oxidase activity (P < 0.001) were also related to aortic compliance. By measuring vascular superoxide generation and aortic compliance using cardiac MRI, we demonstrated a functional relationship between oxidative stress and vascular stiffness. Patients identified with high levels of vascular stiffness are most likely to benefit from strategies to reduce vascular oxidative stress.

Nitric Oxide Generation by NO Donors Is Enhanced Following Balloon Injury in the Porcine Coronary Artery

Vasospasm is a complication of cardiological procedures such as balloon angioplasty and may be related to vascular oxidant stress. Although nitric oxide donor drugs are often administered to prevent vasospasm, the response to these drugs in balloon-injured arteries has not been studied. Pig coronary arteries were balloon-injured in vitro and relaxations to nitric oxide (NO)-donating and NO-independent vasodilators studied. Generation of superoxide in response to injury was assayed using dihydroethidium. NO formation on addition of the NO donor drugs was studied using an amperometric sensor. Expression of nitrotyrosine, a peroxynitrite marker, was probed using immunocytochemistry. In vitro injury enhanced sensitivity to the NO donors SNAP and SpermineNONOate but blunted the response to isoprenaline or chromakalim. With both donors, NO formation was significantly enhanced in the presence of an injured vessel. Vascular superoxide generation was also increased throughout the vessel wall and a small increase in nitrotyrosine was detected in the injured vessel media following addition of SNAP. In conclusion, injured vessels were more sensitive to NO donors and this appears to be due to enhanced NO generation by the donor molecule. Increased formation of peroxynitrite within the injured vessel may contribute to the enhanced relaxation in injured vessels.

Peroxisome Proliferator‐Activated Receptor gamma (PPARgamma) ligand, Rosiglitazone, attenuates vascular oxidative stress in a mouse model of Type 2 Diabetes

Purpose The current study was designed to determine if PPARgamma ligands modulate vascular endothelial superoxide generation in vivo through suppressing expression of selected subunits of NADPH oxidase and/or by increasing the expression and activity of Cu/Zn superoxide dismutase. Methods Lean control (db+/db−) and obese, leptin receptor-deficient (db−/db−) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7 days. Results Compared to db+/db− mice, obese, db−/db− mice had higher serum glucose, insulin, leptin, triglyceride, and fatty acid levels and lower adiponectin levels. Rosiglitazone had no effect on these metabolic derangements. Aortic superoxide generation measured with ESR spectroscopy was significantly increased in db−/db− mice. Aortic tissue from these mice also demonstrated higher relative mRNA levels of the NADPH oxidase subunits, Nox-1 and Nox-4, as measured by real time PCR analysis and lower mRNA levels of PPARgamma. Rosiglitazone treatment decreased superoxide generation and mRNA levels of Nox homologues in db−/db− mice. Conclusions These data indicate that short-term treatment with the rosiglitazone suppressed vascular NADPH oxidase expression and superoxide production in an animal model of vascular oxidative stress. This work is supported by grants from the Veterans Affairs Research Service

Endothelin, Angiotensin, and Oxidative Stress in Hypertension

Endothelin (ET)-1 is a potent vasoconstrictor and mitogen, and because of these properties, it is thought to play a role in the development of hypertension.1,2 The vascular endothelium is a major source of ET-1 production, although a variety of other cell types also have been shown to synthesize and release ET-1. ET-1 is believed to act in a paracrine manner on ETA and ETB receptors on smooth muscle, which mediate contraction, cell proliferation, and hypertrophy. Activation of ETB receptors on endothelial cells stimulates the production of prostacyclin and nitric oxide to induce vasorelaxation and inhibition of sodium transport in renal tubules. Given these properties, considerable attention has been paid to the mechanisms of ET-1 action as it relates to the renal control of blood pressure and the pathogenesis of salt-dependent hypertension. Renal ET synthesis is increased in experimental animals maintained on a high-salt diet and ETA receptor antagonists lower arterial pressure primarily in salt-dependent models of hypertension.1,2 For the past 30 to 40 years, the actions of angiotensin (Ang) II has been arguably the most widely investigated factor in hypertension research. Although physiology textbooks agree on the major actions of Ang II, eg, vasoconstriction and release of aldosterone, recent attention has focused on its ability to stimulate the synthesis of ET-1,3–5 as well as reactive oxygen species.6 There are many reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide that are produced by all cell types and can have profound effects on the vascular system to impact blood pressure regulation. Most recent attention has been paid to the role of superoxide. There are many enzymatic sources of superoxide including NADPH oxidase, xanthine oxidase, nitric oxide synthase, and cytochrome P450. The focus of the current review, however, is be on the …