3-nitrotyrosine and Cardiovascular-Diseases

Several pathologies of the cardiovascular system are associated with an augmented production of nitric oxide and/or superoxide-derived oxidants and/or alteration in the antioxidant detoxification pathways that lead to nitroxidative stress. One important consequence of these reactive intermediates at the biochemical level is the nitration of protein tyrosines, which is performed through either of two of the relevant nitration pathways that operate in vivo, namely peroxynitrite and heme peroxidase-dependent nitration. Proteins nitrated at tyrosine residues have been detected in several compartments of the cardiovascular system. In this review a selection of nitrated proteins in plasma (fibrinogen, plasmin, Apo-1), vessel wall (Apo-B, cyclooxygenase, prostaglandin synthase, Mn-superoxide dismutase) and myocardium (myofibrillar creatine kinase, alpha-actinin, sarcoplasmic reticulum Ca(2+) ATPase) are analyzed in the context of cardiovascular disease. Nitration of tyrosine can affect protein function, which could directly link nitroxidative stress to the molecular alterations found in disease. While some proteins are inactivated by nitration (e.g. Mn-SOD) others undergo a gain-of-function (e.g. fibrinogen) that can have an ample impact on the pathophysiology of the cardiovascular system. Nitrotyrosine is also emerging as a novel independent marker of cardiovascular disease. Pharmacological strategies directed towards inhibiting protein nitration will assist to shed light on the relevance of this post-translational modification to human cardiovascular pathology.

Topics: Animals; Cardiovascular Diseases; Cardiovascular System; Fibrinogen; Humans; Nitric Oxide; Nitrosation; Signal Transduction; Superoxide Dismutase; Tyrosine

Postprandial hyperglycaemia leads to a transient impairment in endothelial function; however, the mechanisms remain largely unknown. Previous work in cell culture models demonstrate that high glucose results in endoplasmic reticulum (ER) stress and, in animal studies, ER stress has been implicated as a cause of endothelial dysfunction. In the present study, we tested the hypothesis that acute oral administration of tauroursodeoxycholic acid (TUDCA, 1500 mg), a chemical chaperone known to alleviate ER stress, would prevent hyperglycaemia-induced endothelial dysfunction. In 12 young healthy subjects (seven men, five women), brachial artery flow-mediated dilation (FMD) was assessed at baseline, and at 60 and 120 min after an oral glucose challenge. Subjects were tested on two separate visits in a single-blind randomized cross-over design: after oral ingestion of TUDCA or placebo capsules. FMD was reduced from baseline during hyperglycaemia under the placebo condition (-32% at 60 min and -28% at 120 min post oral glucose load; P<0.05 from baseline) but not under the TUDCA condition (-4% at 60 min and +0.3% at 120 min post oral glucose load; P>0.05 from baseline). Postprandial plasma glucose and insulin were not altered by TUDCA ingestion. Plasma oxidative stress markers 3-nitrotyrosine and thiobarbituric acid reactive substance (TBARS) remained unaltered throughout the oral glucose challenge in both conditions. These results suggest that hyperglycaemia-induced endothelial dysfunction can be mitigated by oral administration of TUDCA, thus supporting the hypothesis that ER stress may contribute to endothelial dysfunction during postprandial hyperglycaemia.

Topics: Adult; Blood Glucose; Cardiovascular Diseases; Endoplasmic Reticulum Stress; Endothelium, Vascular; Female; Humans; Hyperglycemia; Insulin; Male; Oxidative Stress; Postprandial Period; Taurochenodeoxycholic Acid; Thiobarbituric Acid Reactive Substances; Tyrosine; Young Adult

Intravenous iron is commonly used in conjunction with erythropoietic agents to treat anemia in patients with chronic kidney disease. Iron has been proposed to promote oxidative stress and endothelial dysfunction in vascular tissues. We studied the acute effects of intravenous iron sucrose on homocysteine-induced endothelial dysfunction in the brachial artery of normal human subjects. In all, 40 healthy subjects received intravenous iron sucrose 100 mg or placebo over 30 min immediately before ingestion of 100 mg/kg of oral methionine in a double-blind, randomized study. Flow- and nitroglycerin-mediated dilation in the brachial artery, serum markers of iron stores, and homocysteine and nitrotyrosine levels were measured before and after study drug administration. Intravenous iron significantly increased transferrin saturation and non-transferrin-bound iron (NTBI) when compared with placebo. Flow-mediated dilation significantly decreased from baseline 1 h after administration of iron sucrose when compared with placebo (from 6.66+/-0.47 to 1.93+/-0.35% after iron sucrose vs from 6.00+/-0.40 to 5.61+/-0.46% after placebo, P<0.001), but did not differ between groups at 4 h (1.10+/-0.39 vs 1.33+/-0.51%). Nitroglycerin-mediated vasodilation, and homocysteine and 3-nitrotyrosine levels did not differ after administration of iron sucrose and placebo. Intravenous administration of iron sucrose in the setting of transient hyperhomocysteinemia induced by methionine ingestion significantly increased transferrin saturation and plasma levels of NTBI and significantly attenuated flow-mediated dilation in the brachial artery when compared with placebo. This potential mechanistic link between intravenous iron and endothelial dysfunction warrants further study of cardiovascular effects of intravenous iron in anemic chronic kidney disease populations.

Topics: Adult; Anemia, Iron-Deficiency; Brachial Artery; Cardiovascular Diseases; Chelating Agents; Double-Blind Method; Endothelium, Vascular; Female; Ferric Compounds; Ferric Oxide, Saccharated; Ferritins; Glucaric Acid; Homocysteine; Humans; Iron; Male; Methionine; Nitroglycerin; Oxidative Stress; Razoxane; Regional Blood Flow; Risk Factors; Transferrin; Tyrosine; Vasodilation

Oxidative stress and increased inflammation have been reported to be increased in subjects with diabetes and to be involved in the pathogenesis of cardiovascular complications after myocardial infarction (MI). It is well recognized that red wine has antioxidant and anti-inflammatory activities. We examined the effects of moderate red wine intake on echocardiographic parameters of functional cardiac outcome in addition to inflammatory cytokines and nitrotyrosine (oxidative stress marker), in subjects with diabetes after a first uncomplicated MI.. One hundred and fifteen subjects with diabetes who had sustained a first non-fatal MI were randomized to receive a moderate daily amount of red wine (intervention group) or not (control group). Echocardiographic parameters of ventricular dys-synchrony, circulating levels of nitrotyrosine, tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-18 (IL-18) and C-reactive protein (CRP) were investigated at baseline and 12 months after randomization.. After 1 year of diet intervention, concentrations of nitrotyrosine (P < 0.01), CRP (P < 0.01), TNF-alpha (P < 0.01), IL-6 (P < 0.01) and IL-18 (P < 0.01) were increased in the control group compared with the intervention group. In addition, myocardial performance index (P < 0.02) was higher, and transmitral Doppler flow (P < 0.05), pulmonary venous flow analysis (P < 0.02) and ejection fraction (P < 0.05) were lower in the control group, indicating ventricular dys-synchrony. The concentrations of nitrotyrosine, CRP, TNF-alpha and IL-6 were related to echocardiographic parameters of ventricular dys-synchrony.. In subjects with diabetes, red wine consumption, taken with meals, significantly reduces oxidative stress and pro-inflammatory cytokines as well as improving cardiac function after MI. Moderate red wine intake with meals may have a beneficial effect in the prevention of cardiovascular complications after MI in subjects with diabetes.

Topics: Adult; Aged; C-Reactive Protein; Cardiovascular Diseases; Cytokines; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diet, Mediterranean; Follow-Up Studies; Heart; Humans; Middle Aged; Myocardial Infarction; Oxidative Stress; Prognosis; Treatment Outcome; Tyrosine; Wine

Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction through oxidative stress; however, the distinct role of these two factors is a matter of debate.. Thirty type 2 diabetic patients and 20 normal subjects ate 3 different meals: a high-fat meal; 75 g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia, nitrotyrosine, and endothelial function were assayed during the tests. Subsequently, diabetics took 40 mg/d simvastatin or placebo for 12 weeks. The 3 tests were performed again at baseline, between 3 to 6 days after the start, and at the end of each study. High-fat load and glucose alone produced a decrease of endothelial function and an increase of nitrotyrosine in normal and diabetic subjects. These effects were more pronounced when high fat and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters but reduced the effect on endothelial function and nitrotyrosine observed during each different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations of endothelial function and nitrotyrosine during the tests.. This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug.

Topics: Blood Glucose; Cardiovascular Diseases; Cross-Over Studies; Diabetes Mellitus, Type 2; Double-Blind Method; Drug Administration Schedule; Endothelium, Vascular; Female; Glucose Tolerance Test; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertriglyceridemia; Male; Middle Aged; Oxidative Stress; Postprandial Period; Risk Factors; Simvastatin; Triglycerides; Tyrosine; Vasodilation

Cellular damage by oxidation occurs in numerous chronic diseases, such as obesity, type II diabetes, cardiovascular disease, nonalcoholic fatty liver, etc. The oxidized compound 3-nitrotyrosine is a marker of oxidative stress and protein oxidation damage.. The article aims to assess whether 3-nitrotyrosine levels are higher in young people with obesity than in the same population without obesity.. Anthropometry and blood chemistry analyses were performed on 24 young Mexican participants (18-30 years old), categorized into two groups based on their waist circumference: Withobesity (≥ 80 cm women; ≥ 90 cm men) and without-obesity (<80 cm women; <90 cm men). Additionally, 3-nitrotyrosine blood values were quantified by ELISA.. Except for HDL-cholesterol, the mean values of lipids increased in women and men with obesity (p<0.05), and 3-nitrotyrosine concentration (nM/μg total protein) was higher by 60% in the group with-obesity compared to the group without-obesity, both for women (66.21 ± 23.85 vs. 40.69 ± 16.25, p<0.05) and men (51.72 ± 20.56 vs. 30.52 ± 5.21, p<0.05).. Oxidative damage measured by compound 3-nitrotyrosine was higher in the group with obesity than in the group without obesity, which, if not controlled, could lead to a chronic oxidative condition and thereby to a degree of cellular aging with adverse health effects.

Topics: Adolescent; Adult; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Male; Obesity; Oxidative Stress; Tyrosine; Young Adult

Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD).. Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS.. RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD.. We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.

Topics: Adult; Aged; Apolipoprotein A-I; Arthritis, Rheumatoid; Cardiovascular Diseases; Case-Control Studies; Cholesterol; Female; Halogenation; Humans; Lipoproteins, HDL; Male; Middle Aged; Oxidation-Reduction; Peptide Mapping; Peroxidase; Tandem Mass Spectrometry; Tyrosine

Metabolic syndrome (MetS) confers a greater risk for both cardiovascular disease (CVD) and diabetes. Oxidative stress (OS) could contribute to this excess risk. However, there are few data examining both cellular and circulating biomarkers of OS in MetS uncomplicated by diabetes and CVD.. The aim of the study was to evaluate both cellular and circulating biomarkers of OS in MetS uncomplicated by diabetes or CVD.. At an academic medical center, we compared MetS (n=43) vs. control subjects (n=33). Fasting blood was collected for monocyte isolation and assay of OS biomarkers.. Monocyte nicotinamide adenine dinucleotide phosphate oxidase activity (p22 phox and p47), superoxide anion release, oxidized-low-density lipoprotein (Ox-LDL), nitrotyrosine, and nuclear factor erythroid 2-related factor were measured.. There was significantly increased release of superoxide from the monocytes (basal and after activation) of MetS compared with controls adjusted for body mass index. Body mass index-adjusted plasma levels of Ox-LDL and nitrotyrosine were significantly increased in MetS. There was a linear trend between biomarkers of oxidative stress and increasing number of features of MetS. Also, there was a significant increase in nicotinamide adenine dinucleotide phosphate oxidase membrane expression of p22 phox and p47 phox in MetS. The major cellular antioxidant defense, nuclear factor erythroid 2-related factor was significantly decreased. There were significant correlations between homeostasis model assessment insulin resistance index and both Ox-LDL and nitrotyrosine and superoxide and Ox-LDL.. Collectively, nascent MetS is associated with increased OS as evidenced by both circulating and cellular biomarkers, and this could contribute to the risk for both diabetes and CVD.

Topics: Adult; Biomarkers; Body Mass Index; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Female; Humans; Lipoproteins, LDL; Male; Metabolic Syndrome; Middle Aged; Monocytes; NADPH Oxidases; NF-E2 Transcription Factor; Oxidative Stress; Prevalence; Risk Factors; Superoxides; Tyrosine

Exposure to particulate matter air pollution may be an independent risk factor for cardiovascular morbidity and mortality; however, the biological mechanisms are unclear. We hypothesize that exposure to diesel exhaust (DE), an important source of traffic-related particulate air pollution, promotes changes of atherosclerotic plaque component that may lead to plaque vulnerability.. 30-week old ApoE knockout mice fed with regular chow inhaled DE (at 200 μg/m(3) of particulate) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week) (12 mice/group). Total number of alveolar macrophages (p<0.01) and alveolar macrophages positive for particles (p<0.0001) were more than 8-fold higher after DE inhalation than the control. DE inhalation caused 1.5 to 3-fold increases in plaque lipid content (p<0.02), cellularity (p<0.02), foam cell formation (p<0.04), and smooth muscle cell content (p<0.05). The expression of oxidative stress markers, iNOS, CD36, and nitrotyrosine was significantly increased by 1.5 to 2-fold in plaques, with enhanced systemic lipid and DNA oxidation (p<0.02). Increased foam cells and the expression of iNOS (R(2)=0.72, p=0.0081) and CD36 (R(2)=0.49, p=0.015) in plaques were positively correlated with the magnitude of DE exposure.. Exposure to DE promotes changes in atherosclerotic plaques characteristic of unstable vulnerable plaques. Increased systemic and plaque oxidative stress markers suggest that these changes in plaques could be due to DE-induced oxidative stress.

Topics: Animals; Apolipoproteins E; Cardiovascular Diseases; CD36 Antigens; DNA; Foam Cells; Inhalation Exposure; Macrophages, Alveolar; Male; Mice; Mice, Knockout; Nitric Oxide Synthase Type II; Oxidative Stress; Plaque, Atherosclerotic; Risk Factors; Time Factors; Tyrosine; Vehicle Emissions

We hypothesized that LOX-1 polymorphism may impact on inflammation and cardiovascular risk by modulating systemic resistin expression.. 276 men were randomly selected from a population-based cohort. Metabolic and inflammatory markers were evaluated at baseline and after 6-years follow-up, OLR1 (loxin) IVS4-14 A>G polymorphism was assessed.. Mean plasma resistin and nitrotyrosine values were significantly higher, and TAS was significantly lowered in homozygous for the G allele. The G allele was significantly and directly associated with resistin and nitrotyrosine values.. Enhanced oxidized-LDL uptake by LOX-1 G-allele carriers is associated with increased pro-oxidant status and resistin levels, suggesting a major uptake of ox-LDL by macrophages, smooth muscle cells, and monocytes.

Topics: Antioxidants; C-Reactive Protein; Cardiovascular Diseases; Cholesterol, HDL; Cohort Studies; Cytokines; Gene Frequency; Homozygote; Humans; Inflammation; Lipid Metabolism; Lipoproteins, LDL; Male; Middle Aged; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Resistin; Scavenger Receptors, Class E; Triglycerides; Tyrosine

We tested the hypothesis that older men who perform habitual aerobic exercise do not demonstrate age-associated vascular endothelial oxidative stress compared with their sedentary peers. Older exercising men (n=13, 62±2 years) had higher (P<0.05) physical activity (79±7 vs. 30±6 MET hours per week) and maximal exercise oxygen consumption (42±1 vs. 29±1 mL kg(-1) per minute) vs. sedentary men (n=28, 63±1 years). Brachial artery flow-mediated dilation (FMD), a measure of vascular endothelial function, was greater (P<0.05) in the exercising vs. sedentary older men (6.3±0.5 vs. 4.9±0.4%Δ) and not different than young controls (n=20, 25±1 years, 7.1±0.5%Δ). In vascular endothelial cells sampled from the brachial artery, nitrotyrosine, a marker of oxidative stress, was 51% lower in the exercising vs. sedentary older men (0.38±0.06 vs. 0.77±0.10 AU). This was associated with lower endothelial expression of the oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47(phox) subunit, 0.33±0.05 vs. 0.61±0.09 AU) and the redox-sensitive transcription factor nuclear factor kappa B (NFκB) (p65 subunit, 0.36±0.05 vs. 0.72±0.09 AU). Expression of the antioxidant enzyme manganese superoxide dismutase (SOD) (0.57±0.13 vs. 0.30±0.04 AU) and activity of endothelium-bound extracellular SOD were greater (6.4±0.5 vs. 5.0±0.6 U mL(-1) per minute) in the exercising men (both P<0.05), but differences no longer were significant after correcting for adiposity and circulating metabolic factors. Overall, values for the young controls differed with those for the sedentary, but not the exercising older men. Older men who exercise regularly do not demonstrate vascular endothelial oxidative stress, and this may be a key molecular mechanism underlying their reduced risk of cardiovascular diseases.

Topics: Adult; Aging; Brachial Artery; Cardiovascular Diseases; Endothelial Cells; Endothelium, Vascular; Exercise; Gene Expression; Humans; Male; Middle Aged; NADPH Oxidases; Oxidative Stress; Oxygen Consumption; Risk Factors; Superoxide Dismutase; Transcription Factor RelA; Tyrosine; Vasodilation

Glutaredoxin-1 (Glrx) is a thioltransferase that regulates protein S-glutathiolation. To elucidate the role of endogenous Glrx in cardiovascular disease, Glrx knockout (KO) mice were infused with angiotensin II (Ang II) for 6days. After Ang II infusion, body weight and blood pressure were similar between WT and Glrx KO mice. However, compared to WT mice, Glrx KO mice demonstrated (1) less cardiac and aortic medial hypertrophy, (2) less oxidant generation in aorta as assessed by dihydroethidium staining and nitrotyrosine, (3) decreased phosphorylation of Akt in the heart, and (4) less expression of inducible NOS in aorta and heart. In cultured embryonic fibroblasts from Glrx KO mice, S-glutathiolation of actin was enhanced and actin depolymerization was impaired after hydrogen peroxide stimulation compared with WT cells. Furthermore, oxidant generation in phorbol ester-stimulated fibroblasts and RAW 264.7 macrophage-like cells was lower with Glrx siRNA knockdown. These data indicate that Ang II-induced oxidant production and hypertrophic responses were attenuated in Glrx KO mice, which may result from impaired NADPH oxidase activation.

Topics: Actin Cytoskeleton; Angiotensin II; Animals; Aorta; Cardiovascular Diseases; Cell Line; Glutaredoxins; Hypertrophy; Infusion Pumps; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Nitric Oxide Synthase Type II; Oncogene Protein v-akt; Oxidants; RNA, Small Interfering; Tyrosine

It is important to consider a strategy to halt the development of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). Oral adsorbent AST-120 retards deterioration in renal function, reducing indoxyl sulfate (IS) accumulation. The aim of this study was to determine whether AST-120 improves endothelial dysfunction by reducing oxidative/nitrative stress in a rat-CKD model. Subtotally nephrectomized (Nx) rats aged 17 weeks were divided into two groups: control rats and rats orally treated with AST-120. Two weeks after initiation of AST-120, serum and urinary IS levels, renal histological scores and endothelium-dependent vascular responses (EDVRs) in the aorta were investigated. EDVR in 5-h incubation with 250 microg ml(-1) IS was also examined in normal rat aortas. Nitrotyrosine content, mRNA expression of p47phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component, and expression and phosphorylation (serine-1177) of endothelial nitric oxide synthase (eNOS) in the aorta were examined in untreated and treated Nx rats. At the end of treatment, renal function and histological scores were not different in the two groups. AST-120 prevented the elevation of serum IS level in Nx rats, reducing urinary IS excretion, and ameliorated decreased EDVR in Nx rats. Incubation with IS tended to reduce EDVR in normal aortas, albeit insignificantly. AST-120 also suppressed nitrotyrosine accumulation and inhibited p47phox expression in Nx rats. The eNOS expression and phosphorylation were similar in the two groups. In conclusion, AST-120 ameliorated endothelial dysfunction and alleviated oxidative/nitrative stress in the aorta through reduced accumulation of IS, independent of renal function, in a rat CKD model.

Topics: Acetylcholine; Animals; Aorta; Blotting, Western; Carbon; Cardiovascular Diseases; Endothelium, Vascular; Immunohistochemistry; Kidney; Kidney Function Tests; Male; NADPH Oxidases; Nephrectomy; Nitric Oxide Synthase Type III; Obesity; Oxidative Stress; Oxides; Phosphorylation; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tyrosine

Uncoupling protein 2 (UCP2) is an important regulator of intracellular reactive oxygen species (ROS) production. We determined the effects of calorie restriction (CR) on the dynamic aspects of mitochondrial ROS production, UCP2, and the nitric oxide (NO)-cGMP pathway in the cardiovascular tissues of type II diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Some rats were on restricted diets (30% reduction from free intake) from age 29 to 42 weeks. Blood glucose, hemoglobin A1c, plasma levels of free fatty acid, triacylglycerol, and plasminogen activator inhibitor-1 in OLETF rats were significantly higher than those in nondiabetic control [Long-Evans Tokushima Otsuka (LETO)] rats at 29 weeks. Mitochondrial ROS production and UCP2 expression significantly increased in the heart and aorta of OLETF rats compared with those in LETO rats. A fibrogenic growth factor, transforming growth factor (TGF)-beta1 in the coronary vessels, endothelial nitric-oxide synthase, and aortic nitrotyrosine were increased in OLETF rats at 42 weeks. In contrast, an index of the NO-cGMP pathway, phosphorylated vasodilator-stimulated phosphoprotein, and superoxide dismutase activity in the aorta were significantly diminished. The relationship between UCP2 and ROS production in the cardiovascular function of diabetic rats being fed a calorie-restricted diet is unknown. These abnormalities in OLETF rats were reversed to normal levels by CR. CR significantly improved the NO-cGMP pathway via normalizing ROS generation in OLETF rats. A decrease in UCP2 expression by CR may be a compensatory mechanism to counteract decreased intracellular oxidative stress. The data suggest that CR may prevent cardiovascular tissues from oxidative stress provoked by diabetes mellitus.

Topics: Animals; Body Weight; Caloric Restriction; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Glucose Transporter Type 4; Ion Channels; Lipid Peroxidation; Male; Mitochondria; Mitochondrial Proteins; Nitric Oxide Synthase Type III; Organ Size; Plasminogen Activator Inhibitor 1; Rats; Rats, Long-Evans; Reactive Oxygen Species; Risk Factors; Tyrosine; Uncoupling Protein 2

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.

Topics: Aging; Animals; Calcium-Transporting ATPases; Cardiovascular Diseases; Dithionite; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Kidney; Oxidative Stress; Peroxynitrous Acid; Rabbits; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Superoxide Dismutase; Tyrosine

We recently reported that methionine-loaded human umbilical vein endothelial cells (HUVECs) exported homocysteine (Hcy) and were associated with hydroxyl radical generation and oxidation of lipids in LDL. Herein we have analysed the Hcy-induced posttranslational modifications (PTMs) of LDL protein. PTMs have been characterised using electrophoretic mobility shift, protein carbonyl ELISA, HPLC with electrochemical detection and Western blotting of 3-nitrotyrosine, and LDL uptake by scavenger receptors on monocyte/macrophages. We have also analysed PTMs in LDL isolated from rheumatoid (RA) and osteo-(OA) arthritis patients with cardiovascular disease (CVD). While reagent Hcy (< 50 microM) promoted copper-catalysed LDL protein oxidation, Hcy released from methionine-loaded HUVECs promoted LDL protein nitration. In addition, LDL nitration was associated with enhanced monocyte/macrophage uptake when compared with LDL oxidation. LDL protein nitration and uptake by monocytes, but not carbonyl formation, was elevated in both RA and OA patients with CVD compared with disease-matched patients that had no evidence of CVD. Moreover, a direct correlation between plasma total Hcy (tHcy) and LDL uptake was observed. The present studies suggest that elevated plasma tHcy may promote LDL nitration and increased scavenger receptor uptake, providing a molecular mechanism that may contribute to the clinical link between CVD and elevated plasma tHcy.

Topics: Adult; Aged; Arthritis, Rheumatoid; Blotting, Western; Cardiovascular Diseases; Case-Control Studies; Cells, Cultured; Chromatography, High Pressure Liquid; Copper; Electrophoretic Mobility Shift Assay; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Homocysteine; Humans; Lipoproteins, LDL; Male; Methionine; Middle Aged; Nitrites; Osteoarthritis; Receptors, Scavenger; Tyrosine; Umbilical Veins

Topics: Aerobiosis; Animals; Biomarkers; Calcium-Transporting ATPases; Cardiovascular Diseases; Humans; Oxidative Stress; Peroxynitrous Acid; Protein Processing, Post-Translational; Proteins; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Superoxide Dismutase; Tyrosine

Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-alpha were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during beta-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-alpha and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury.

Topics: Aldehydes; Animals; Blotting, Northern; Blotting, Western; Cardiovascular Diseases; Copper; Diabetes Mellitus, Experimental; Endothelin-1; Glutathione; Immunohistochemistry; Interleukin-6; Iron; Metallothionein; Mice; Mice, Transgenic; Myocardium; Oxidative Stress; RNA, Messenger; Tumor Necrosis Factor-alpha; Tyrosine; Zinc

Topics: Animals; Apolipoprotein A-I; Biomarkers; Cardiovascular Diseases; Cholesterol; Humans; Lipoproteins, HDL; Magnetic Resonance Imaging; Oxidation-Reduction; Peroxidase; Risk Factors; Tyrosine

Formation of nitric oxide-derived oxidants may serve as a mechanism linking inflammation to development of atherosclerosis. Nitrotyrosine, a specific marker for protein modification by nitric oxide-derived oxidants, is enriched in human atherosclerotic lesions and low-density lipoprotein (LDL) recovered from human atheroma.. To determine whether systemic levels of nitrotyrosine are associated with the prevalence of coronary artery disease (CAD) and are modulated by hydroxymethylglutaryl coenzyme-A reductase inhibitor (statin) therapy.. A case-control and interventional study at 2 urban tertiary-care referral centers; recruitment for each was from June 1, 2001, until January 1, 2002. For the case-control study, 100 case-patients with established CAD and 108 patients with no clinically evident CAD were recruited consecutively. In the interventional study, participants aged 21 years or older with hypercholesterolemia (LDL cholesterol > or =130 mg/dL [> or =3.5 mmol/L]) underwent nutrition and exercise counseling. Those whose levels did not decrease with 6 to 8 weeks were enrolled in the study (n = 35). For 12 weeks, they received 10 mg/d of oral atorvastatin therapy.. In the case-control study, the association between systemic levels of protein-bound nitrotyrosine, CAD risk, and presence of CAD. In the interventional study, the change in nitrotyrosine, lipoprotein, and C-reactive protein (CRP) levels.. Nitrotyrosine levels were significantly higher among patients with CAD (median 9.1 micromol/mol [interquartile range, 4.8-13.8 micromol/mol] tyrosine vs 5.2 micromol/mol [interquartile range, 2.2-8.4 micromol/mol]; P<.001). Patients in the upper quartile of nitrotyrosine (29%; P<.001) had a higher odds of CAD compared with those in the lowest quartile (unadjusted odds ratio, 6.1; 95% confidence interval, 2.6-14.0; P<.001). In multivariate models adjusting for Framingham Global Risk Score and CRP, upper quartiles of nitrotyrosine remained associated with CAD (odds ratio, 4.4; 95% confidence interval, 1.8-10.6; P<.001). Statin therapy reduced nitrotyrosine levels significantly (25%; P<.02) with a magnitude similar to reductions in total cholesterol levels (25%; P<.001) and LDL particle number (29%; P<.001) yet were independent of alterations in lipoproteins and inflammatory markers like CRP.. The findings from this preliminary study indicate that nitrotyrosine levels are associated with the presence of CAD and appear to be modulated by statin therapy. These results suggest a potential role for nitric oxide-derived oxidants as inflammatory mediators in CAD and may have implications for atherosclerosis risk assessment and monitoring of anti-inflammatory actions of statins.

Topics: Adult; Aged; Anticholesteremic Agents; Atorvastatin; C-Reactive Protein; Cardiovascular Diseases; Case-Control Studies; Coronary Artery Disease; Female; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Lipoproteins; Male; Middle Aged; Prospective Studies; Pyrroles; Risk Factors; Tyrosine

Adrenomedullin (AM), a potent vasodilator peptide, is produced by posttranslational splicing of pro-adrenomedullin together with proadrenomedullin N-terminal 20 peptide (PAMP), another hypotensive peptide. Although both AM and PAMP have the potential not only to decrease blood pressure but also to protect organs from damage, there is no direct evidence for their individual physiological roles in vivo.. Using knockout mice with the disruption of AM peptide alone, we investigated the organ-protective effect of AM. Although the AM(-/-) mutation in mice was embryonic lethal without any apparent phenotypic changes, AM(+/-) mice were viable and fertile; plasma and organ AM concentrations were almost half of those in AM(+/+) mice. With the administration of angiotensin II (Ang II) on a high-salt diet for 12 days, marked perivascular fibrosis and intimal hyperplasia were found in coronary arteries of Ang II/salt-treated AM(+/-) mice, without the AM upregulation that was observed in Ang II/salt-treated AM(+/+) mice. In AM(+/-) mice, Ang II/salt loading increased both urinary excretion of 8-hydroxydeoxyguanosine and isoprostane, markers of oxidative stress. Consistently, immunostaining of both p67phox and gp91phox, subunits of NAD(P)H oxidase and 3-nitrotyrosine, the metabolites of reactive oxygen species (ROS), and the generation of ROS measured by electron spin resonance spectroscopy apparently increased in the Ang II/salt-treated heart. These data suggested that the overproduction of oxidative stress might be involved in the cardiovascular changes induced by Ang II/salt loading.. The evidence presented supports the hypothesis that endogenous AM possesses a protective action against cardiovascular damage, possibly through the inhibition of oxidative stress production.

Topics: Adrenal Glands; Adrenomedullin; Angiotensin II; Animals; Blood Pressure; Cardiovascular Diseases; Coronary Vessels; Female; Genotype; Lung; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NADH, NADPH Oxidoreductases; NADPH Oxidase 2; NADPH Oxidases; Peptide Fragments; Peptides; Phosphoproteins; Proteins; Reactive Oxygen Species; Sodium, Dietary; Time Factors; Tyrosine

Angiotensin II (ANG II) is a well-established participant in many cardiovascular disorders, but the mechanisms involved are not clear. Vascular cell experiments suggest that ANG II is a potent stimulator of free radicals such as superoxide anion, an agent known to inactivate nitric oxide and promote the formation of peroxynitrite. Here we hypothesized that ANG II reduces the efficacy of NO-mediated vascular relaxation and promotes vascular peroxynitrite formation in vivo. ANG II was infused in rats at sub-pressor doses for 3 days. Systolic blood pressure and heart rate were unchanged on day 3 despite significant reductions in plasma renin activity. Thoracic aorta was isolated for functional and immunohistochemical evaluations. No difference in isolated vascular contractile responses to KCI (125 mM), phenylephrine, or ANG II was observed between groups. In contrast, relaxant response to acetylcholine (ACh) was decreased sixfold without a change in relaxant response to sodium nitroprusside. Extensive prevalence of 3-nitrotyrosine (3-NT, a stable biomarker of tissue peroxynitrite formation) immunoreactivity was observed in ANG II-treated vascular tissues and was specifically confined to the endothelium. Digital image analysis demonstrated a significant inverse correlation between ACh relaxant response and 3-NT immunoreactivity. These data demonstrate that ANG II selectively modifies vascular NO control at sub-pressor exposures in vivo. Thus, endothelial dysfunction apparently precedes other established ANG II-induced vascular pathologies, and this may be mediated by peroxynitrite formation in vivo. Wattanapitayakul, S., Weinstein, D. M., Holycross, B. J., Bauer, J. A. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin-induced cardiovascular disorders.

Topics: Acetylcholine; Angiotensin II; Animals; Aorta, Thoracic; Cardiovascular Diseases; Endothelium, Vascular; Hemodynamics; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Male; Nitrates; Nitroprusside; Phenylephrine; Potassium Chloride; Rats; Rats, Sprague-Dawley; Renin; Tyrosine; Vasoconstriction