3-nitrotyrosine and Diabetic-Angiopathies

Endothelial dysfunction associated with decreased nitric oxide (NO) bioactivity is a major feature of vascular diseases such as atherosclerosis or diabetes. Sodium nitroprusside (SNP)-induced relaxation is entirely dependent on cyclic guanosine monophosphate (cGMP) and preserved in atherosclerosis, suggesting that smooth muscle response to NO donor is intact. However, NO gas activates both cGMP-dependent and -independent signal pathways in vascular smooth muscle cells, and oxidative stress associated with vascular diseases selectively impairs cGMP-independent relaxation to NO. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), which regulates intracellular Ca(2+) levels by pumping Ca(2+) into store, is a major cGMP-independent target for NO. Physiological levels of reactive nitrogen species (RNS) S-glutathiolate SERCA at Cys674 to increase its activity, and the augmentation of RNS in vascular diseases irreversibly oxidizes Cys674 or nitrates tyrosine residues at Tyr296-Tyr297, which are associated with loss of function. S-glutathiolation of various proteins by NO can explain redox-sensitive cGMP-independent actions, and oxidative inactivation of target proteins for NO can be associated with the pathogenesis of cardiovascular diseases. Oxidative inactivation of SERCA is also implicated with dysregulation of smooth muscle migration, promotion of platelet aggregation, and impairment of cardiac function, which can be implicated with restenosis, pathological angiogenesis, thrombosis, as well as heart failure. Analysis of posttranslational oxidative modifications of SERCA and the preservation of SERCA function can be novel strategies against cardiovascular diseases associated with oxidative stress.

Topics: Animals; Antioxidants; Atherosclerosis; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diabetic Angiopathies; Endothelium, Vascular; Glutathione; Heart Failure; Humans; Mice; Models, Animal; Muscle, Smooth, Vascular; Nitric Oxide; Oxidative Stress; Protein Processing, Post-Translational; Rabbits; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tyrosine; Vasodilation

Macro- and microvascular disease are the most common causes of morbidity and mortality in patients with diabetes mellitus. Diabetic cardiovascular dysfunction represents a problem of great clinical importance underlying the development of various severe complications including retinopathy, nephropathy, neuropathy and increase the risk of stroke, hypertension and myocardial infarction. Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, are closely associated with increased oxidative and nitrosative stress, which can trigger the development of diabetic complications. Hyperglycemia stimulates the production of advanced glycosylated end products, activates protein kinase C, and enhances the polyol pathway leading to increased superoxide anion formation. Superoxide anion interacts with nitric oxide, forming the potent cytotoxin peroxynitrite, which attacks various biomolecules in the vascular endothelium, vascular smooth muscle and myocardium, leading to cardiovascular dysfunction. The pathogenetic role of nitrosative stress and peroxynitrite, and downstream mechanisms including poly(ADP-ribose) polymerase (PARP) activation, is not limited to the diabetes-induced cardiovascular dysfunction, but also contributes to the development and progression of diabetic nephropathy, retinopathy and neuropathy. Accordingly, neutralization of peroxynitrite or pharmacological inhibition of PARP is a promising new approach in the therapy and prevention of diabetic complications. This review focuses on the role of nitrosative stress and downstream mechanisms including activation of PARP in diabetic complications and on novel emerging therapeutical strategies offered by neutralization of peroxynitrite and inhibition of PARP.

Topics: Animals; Cardiomyopathies; Diabetes Complications; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Neuropathies; Diabetic Retinopathy; Endothelium, Vascular; Humans; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Superoxides; Tyrosine

Oxidative stress plays an important role in diabetic vascular complications. It has been shown that an imbalance in the ratio of nitric oxide to superoxide anion due to a prevalence of the superoxide anion leads to an alteration in vascular reactivity. Under these conditions an increase in peroxynitrite (ONOO-) production, resulting from the reaction between nitric oxide (NO) and superoxide (O2-), may be hypothesised. ONOO- is responsible for nitration of tyrosine residues in proteins; therefore the presence of nitrotyrosine (NT) in plasma proteins is considered indirect evidence of ONOO- production. NT has been found in the plasma of patients with diabetes, but it is not detectable in the plasma of healthy controls. NT plasma values are correlated with plasma glucose concentrations, and further studies exploring the effects of acute hyperglycaemia on NT formation confirmed that NT is produced both in normal subjects during hyperglycaemic clamp and in working hearts from rats during hyperglycaemic perfusion. Postprandial hypertriglyceridemia and hyperglycaemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridaemia and hyperglycaemia induce an endothelial dysfunction through an oxidative stress; however, the specific roles of these two factors are matters for debate. In a clinical study, high-fat load and glucose alone each produced a decrease in endothelial function and an increase in NT in normal subjects and patients with diabetes. These effects were more pronounced when high-fat load and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters, but reduced the effects of high-fat load, glucose alone, and both high-fat load and glucose on endothelial function and NT Long-term simvastatin treatment was accompanied by a smaller increase in postprandial triglycerides, which was followed by smaller variations in endothelial function and NT. This study showed an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycaemia on endothelial function, suggesting oxidative stress as a common mediator of these effects. 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. These studies indicate that ONOO- is generated in diabetes, suggesting the possible involvement of ONOO- in the development of diabetic complications.

Topics: Animals; Biomarkers; Diabetic Angiopathies; Endothelium, Vascular; Humans; Hyperglycemia; Hypertriglyceridemia; Hypolipidemic Agents; Oxidative Stress; Postprandial Period; Rats; Simvastatin; Tyrosine

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

Endothelial injury plays a vital role in vascular lesions from diabetes mellitus (DM). Therapeutic targets against endothelial damage may provide critical venues for the treatment of diabetic vascular diseases. Peroxisome proliferator-activated receptor β (PPARβ) is a crucial regulator in DM and its complications. However, the molecular signal mediating the roles of PPARβ in DM-induced endothelial dysfunction is not fully understood. The impaired endothelium-dependent relaxation and destruction of the endothelium structures appeared in high glucose incubated rat aortic rings. A high glucose level significantly decreased the expression of PPARβ and endothelial nitric oxide synthase (eNOS) at the mRNA and protein levels, and reduced the concentration of nitric oxide (NO), which occurred in parallel with an increase in the expression of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine. The effect of high glucose was inhibited by GW0742, a PPARβ agonist. Both GSK0660 (PPARβ antagonist) and N

Topics: Animals; Aorta, Thoracic; Diabetic Angiopathies; Down-Regulation; Endothelial Cells; Female; Glucose; Hyperglycemia; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrosative Stress; PPAR-beta; Rats, Sprague-Dawley; Signal Transduction; Tyrosine; Vasodilation

Fibroblast growth factor 21 (FGF21) is an important regulator in glucose and lipid metabolism, and has been considered as a potential therapy for diabetes. The effect of FGF21 on the development and progression of diabetes-induced pathogenic changes in the aorta has not currently been addressed. To characterize these effects, type 1 diabetes was induced in both FGF21 knockout (FGF21KO) and C57BL/6 J wild type (WT) mice via multiple-dose streptozotocin injection. FGF21KO diabetic mice showed both earlier and more severe aortic remodeling indicated by aortic thickening, collagen accumulation and fibrotic mediator connective tissue growth factor expression. This was accompanied by significant aortic cell apoptosis than in WT diabetic mice. Further investigation found that FGF21 deletion exacerbated aortic inflammation and oxidative stress reflected by elevated expression of tumor necrosis factor α and transforming growth factor β, and the accumulation of 3-nitrotyrocine and 4-Hydroxynonenal. FGF21 administration can reverse the pathologic changes in FGF21KO diabetic mice. These findings demonstrate that FGF21 deletion aggravates aortic remodeling and cell death probably via exacerbation of aortic inflammation and oxidative stress. This marks FGF21 as a potential therapy for the treatment of aortic damage due to diabetes.

Topics: Aldehydes; Animals; Aorta; Aortic Diseases; Apoptosis; Collagen; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Fibroblast Growth Factors; Fibrosis; Gene Deletion; Genetic Predisposition to Disease; Male; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type III; Oxidative Stress; Phenotype; Signal Transduction; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tyrosine; Vascular Remodeling

Recently a new rat model for type 2 diabetes the Zucker diabetic Sprague-Dawley (ZDSD/Pco) was created. In this study we sought to characterize the development of diabetic neuropathy in ZDSD rats using age-matched Sprague-Dawley rats as a control. Rats were examined at 34 weeks of age 12 weeks after the onset of hyperglycemia in ZDSD rats. At this time ZDSD rats were severely insulin resistant with slowing of both motor and sensory nerve conduction velocities. ZDSD rats also had fatty livers, elevated serum free fatty acids, triglycerides, and cholesterol, and elevated sciatic nerve nitrotyrosine levels. The corneas of ZDSD rats exhibited a decrease in subbasal epithelial corneal nerves and sensitivity. ZDSD rats were hypoalgesic but intraepidermal nerve fibers in the skin of the hindpaw were normal compared to Sprague-Dawley rats. However, the number of Langerhans cells was decreased. Vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, to acetylcholine and calcitonin gene-related peptide was impaired in ZDSD rats. These data indicate that ZDSD rats develop many of the neural complications associated with type 2 diabetes and are a good animal model for preclinical investigations of drug development for diabetic neuropathy.

Topics: Age Factors; Animals; Arterioles; Blood Glucose; Cholesterol; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Diabetic Neuropathies; Disease Models, Animal; Fatty Acids, Nonesterified; Fatty Liver; Langerhans Cells; Male; Neural Conduction; Nociception; Rats, Sprague-Dawley; Rats, Zucker; Sciatic Nerve; Time Factors; Triglycerides; Tyrosine; Vasodilation

Activation of RhoA/Rho-kinase (ROCK) is increasingly implicated in acute vasospasm and chronic vasoconstriction in major organ systems. Therefore we aimed to ascertain whether an increase in ROCK activity plays a role in the deterioration of coronary vascular function in early stage diabetes.. Synchrotron radiation microangiography was used to determine in vivo coronary responses in diabetic (3 weeks post streptozotocin 65 mg/kg ip) and vehicle treated male Sprague-Dawley rats (n = 8 and 6). Changes in vessel number and calibre during vasodilator stimulation before and after blockade of nitric oxide synthase and cyclooxygenase were compared between rats. Acute responses to ROCK inhibitor, fasudil (10 mg/kg iv) was evaluated. Further, perivascular and myocardial fibrosis, arterial intimal thickening were assessed by histology, and capillary density, nitrotyrosine and ROCK1/2 expressions were evaluated by immunohistochemical staining.. Diabetic rats had significantly elevated plasma glucose (P < 0.001 vs control), but did not differ in fibrotic scores, media to lumen ratio, capillary density or baseline visible vessel number or calibre. Responses to acetylcholine and sodium nitroprusside stimulation were similar between groups. However, in comparison to control rats the diabetic rats showed more segmental constrictions during blockade, which were not completely alleviated by acetylcholine, but were alleviated by fasudil. Further, second order vessel branches in diabetic rats were significantly more dilated relative to baseline (37% vs 12% increase, P < 0.05) after fasudil treatment compared to control rats, while visible vessel number increased in both groups. ROCK2 expression was borderline greater in diabetic rat hearts (P < 0.053).. We found that ahead of the reported decline in coronary endothelial vasodilator function in diabetic rats there was moderate elevation in ROCK expression, more widespread segmental constriction when nitric oxide and prostacyclin production were inhibited and notably, increased calibre in second and third order small arteries-arterioles following ROCK inhibition. Based on nitrotyrosine staining oxidative stress was not significantly elevated in early diabetic rats. We conclude that tonic ROCK mediated vasoconstriction contributes to coronary vasomotor tone in early diabetes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Coronary Angiography; Coronary Circulation; Coronary Vessels; Cyclooxygenase Inhibitors; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Epoprostenol; Fibrosis; Male; Microcirculation; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Tyrosine; Vasoconstriction; Vasodilation; Vasodilator Agents

The functional relevance of NOS3 and ACE genetic variations to endothelial cell function is largely unstudied. Here we tested the functional relevance of the NOS3 (Glu298Asp) polymorphism and ACE (I/D) polymorphism in endothelial cells in vitro. Our hypothesis was that these genetic polymorphisms alter endothelial cell sensitivity to glucose and 3-nitrotyrosine (3NT). Genotyped HUVECs were incubated with glucose, free 3NT or a combination of these two toxicants. Significant differences in glucose-induced cell death and free 3NT-induced cell death were observed among the NOS3 genotypes. Combined glucose/3NT caused increased toxicity among the NOS3 genotypes. No differences were observed among the ACE genotypes in their responses to glucose/3NT. These data demonstrate that the NOS3 genotype may be an important predictor of, or be mechanistically involved in, endothelial vulnerability, whereas the ACE I/D genotype is apparently less important. Thus this NOS3 genetic variation may play a role in vulnerability to endothelium-dependent diabetic vascular complications.

Topics: Caveolin 1; Cell Death; Cells, Cultured; Diabetic Angiopathies; Genotype; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Nitric Oxide Synthase Type III; Nitrites; Peptidyl-Dipeptidase A; Phenotype; Phosphorylation; Polymorphism, Genetic; Proto-Oncogene Proteins c-akt; Tyrosine

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.. The study was performed using diabetic apolipoprotein E/GPx1 (ApoE(-/-)GPx1(-/-))-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.. Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.. Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Topics: Aldehydes; Animals; Antioxidants; Apolipoproteins E; Atherosclerosis; Azoles; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Nephropathies; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Isoindoles; Kidney; Male; Mice; Mice, Knockout; Organoselenium Compounds; Reactive Oxygen Species; Tyrosine; Vascular Cell Adhesion Molecule-1

Recently we showed that lack of the antioxidant enzyme glutathione peroxidase-1 (GPx1) accelerates atherosclerosis and upregulates proatherogenic pathways in diabetic apoE/GPx1-deficient double-knockout mice, thereby establishing GPx1 as an important therapeutic target. In vivo studies now investigate ebselen, a seleno-organic GPx1-mimetic, for its potential to reduce diabetes-associated atherosclerosis.. Lesions were significantly increased in diabetic apoE(-/-) aortas (P<0.001) compared with nondiabetic controls after 20 weeks of diabetes. Ebselen-gavage significantly reduced total aortic lesions (P<0.001), with significant regional reductions in the arch (P<0.001), thoracic (P<0.001), and abdominal regions (P<0.05), but not within the aortic sinus of diabetic apoE(-/-) mice. These reductions were accompanied by significantly lower nitrotyrosine and Nox2 levels, reduced proatherogenic cellularity (macrophages and SMCs), and reduced expression of the proatherogenic mediator RAGE. Within the aortic sinus, ebselen reduced nitrotyrosine, Nox2, and VEGF levels but had no effect on RAGE. Studies in HAECs show that ebselen abrogates H(2)O(2)-induced increases in P-IKK, P-JNK, TNF-alpha, and Nox2.. Ebselen reduces atherosclerotic lesions in most regions of diabetic apoE(-/-) aorta, except within the aortic sinus, suggesting its effectiveness as a potential antiatherogenic therapy in diabetic-macrovascular disease. Ebselen may elicit its effect via modulation of transcription factors such as NF-kappaB and AP-1.

Topics: Administration, Oral; Animals; Antioxidants; Aorta, Abdominal; Aorta, Thoracic; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Azoles; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Endothelial Cells; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; I-kappa B Kinase; Isoindoles; JNK Mitogen-Activated Protein Kinases; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; NADPH Oxidase 2; NADPH Oxidases; Organoselenium Compounds; Phenotype; Phosphorylation; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine; Vascular Endothelial Growth Factor A

Tumor necrosis factor-alpha (TNF-alpha) upregulates the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in type 2 diabetes. We hypothesized that TNF-alpha and MCP-1 may interact to contribute to the evolution of vascular inflammation and endothelial dysfunction in coronary arterioles in type 2 diabetes. To test this hypothesis, we administered anti-MCP-1 to block MCP-1 signaling in genetically modified mice with type 2 diabetes (Lepr(db)) and in heterozygote (m Lepr(db)) lean control. Anti-MCP-1 partially restored vasodilation to the endothelium-dependent vasodilator acetylcholine in isolated, cannulated, and pressurized coronary arterioles in Lepr(db) mice but did not affect vasodilation in m Lepr(db) mice. Anti-MCP-1 attenuated superoxide production and the protein expression of nitrotyrosine, which is an indicator of peroxynitrite production, in isolated coronary arterioles of Lepr(db) mice. Immunostaining results showed that the expression of MCP-1 and vascular cellular adhesion molecule-1 is colocalized with endothelial cells and macrophages. Anti-TNF-alpha or anti-MCP-1 markedly reduced macrophage infiltration and the number of MCP-1-positive endothelium in Lepr(db) mice. The neutralization of TNF-alpha or anti-MCP-1 reduced the expression of adhesion molecules, suggesting that proinflammatory cytokines interact to amplify the signaling process that leads to vascular dysfunction. These findings demonstrate that the endothelial dysfunction occurring in type 2 diabetes is the result of the effects of the inflammatory cytokine TNF-alpha and TNF-alpha-related signaling, including the expression of MCP-1 and adhesion molecules, which further exacerbates vessel inflammation and oxidative stress.

Topics: Animals; Arterioles; Biomarkers; Chemokine CCL2; Coronary Vessels; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; I-kappa B Proteins; Inflammation Mediators; Macrophages; Male; Mice; Mice, Knockout; NF-kappa B; Nitric Oxide; Oxidative Stress; Peroxynitrous Acid; Phosphorylation; Receptors, Leptin; Serine; Signal Transduction; Superoxides; Tumor Necrosis Factor-alpha; Tyrosine; Vascular Cell Adhesion Molecule-1; Vasodilation; Vasodilator Agents

Although mitochondrial reduction-oxidation (redox) stress and increase in membrane permeability play an important role in diabetic-associated renal microvasculopathies, it is unclear whether the intra-renal mitochondrial oxidative stress induces mitochondrial protein modifications, leading to increase mitochondrial membrane permeability. The hypothesis is that mitochondrial oxidative stress induces mitochondrial protein modification and leakage in the mitochondrial membrane in type-2 diabetes. The present study was conducted to determine the involvement of intra-renal mitochondrial oxidative stress in mitochondrial protein modifications and modulation of membrane permeability in the setting of type-2 diabetes. Diabetes was induced by 6-week regimen of a high calorie and fat diet in C57BL/6J mice (Am J Physiol 291:F694-F701, 2006). Subcellular fractionation was carried out in kidney tissue from wild type and diabetic mice. All fractions were highly enriched in their corresponding marker enzyme. Subcellular protein modifications were determined by Western blot and 2-D proteomics. The results suggest that diabetes-induced oxidative stress parallels an increase in NADPH oxidase-4 (NOX-4) and decrease in superoxide dismutase-1, 2 (SOD-1, 2) expression, in mitochondrial compartment. We observed loss of mitochondrial membrane permeability as evidenced by leakage of mitochondrial cytochrome c and prohibitin to the cytosol. However, there was no loss in control tissue. The 2-D Western blots for mitochondrial post-translational modification showed an increase in nitrotyrosine generation in diabetes. We conclude that diabetes-induced intra-renal mitochondrial oxidative stress is reflected by an increase in mitochondrial membrane permeability and protein modifications by nitrotyrosine generation.

Topics: Animals; Cytosol; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Male; Mice; Mice, Inbred C57BL; Microcirculation; Mitochondria; Mitochondrial Membranes; Oxidation-Reduction; Oxidative Stress; Permeability; Reference Values; Tyrosine

Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis.. ApoE-deficient (ApoE-/-) and ApoE/GPx1 double-knockout (ApoE-/- GPx1-/-) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE-/- GPx1-/- aortas compared with diabetic ApoE-/- aortas. This increase was accompanied by increased macrophages, alpha-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE-/- GPx1-/- mouse aortas. These findings were observed despite upregulation of other antioxidants.. Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE-/- mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

Topics: Animals; Aorta; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Connective Tissue Growth Factor; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Fibrosis; Gene Expression Regulation; Glutathione; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Hyperlipoproteinemia Type II; Immediate-Early Proteins; Inflammation; Intercellular Signaling Peptides and Proteins; Isoenzymes; Macrophages; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; NF-kappa B; Oxidation-Reduction; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Sinus of Valsalva; Streptozocin; Superoxide Dismutase; Tyrosine; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Evidence for important roles of the highly reactive oxidant peroxynitrite in diabetic complications is emerging. We evaluated the role of peroxynitrite in early peripheral neuropathy and vascular dysfunction in STZ-diabetic rats. In the first dose-finding study, control and STZ-diabetic rats were maintained with or without the potent peroxynitrite decomposition catalyst Fe(III)tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin (FP15) at 3, 5, or 10 mg.kg(-1).day(-1) in the drinking water for 4 wk after an initial 2 wk without treatment for assessment of early neuropathy. In the second study with similar experimental design, control and STZ-diabetic rats were maintained with or without FP15, 5 mg.kg(-1).day(-1), for vascular studies. Rats with 6-wk duration of diabetes developed motor and sensory nerve conduction velocity deficits, mechanical hyperalgesia, and tactile allodynia in the absence of small sensory nerve fiber degeneration. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve and dorsal root ganglia. All these variables were dose-dependently corrected by FP15, with minimal differences between the 5 and 10 mg.kg(-1).day(-1) doses. FP15, 5 mg.kg(-1).day(-1), also corrected endoneurial nutritive blood flow and nitrotyrosine, but not superoxide, fluorescence in aorta and epineurial arterioles. Diabetes-induced decreases in acetylcholine-mediated relaxation by epineurial arterioles and coronary and mesenteric arteries, as well as bradykinin-induced relaxation by coronary and mesenteric arteries, were alleviated by FP15 treatment. The findings reveal the important role of nitrosative stress in early neuropathy and vasculopathy and provide the rationale for further studies of peroxynitrite decomposition catalysts in long-term diabetic models.

Topics: Animals; Aorta; Arterioles; Blood Glucose; Body Weight; Coronary Vessels; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Neuropathies; Ganglia, Spinal; Hyperalgesia; Hypoglycemic Agents; Male; Mesenteric Arteries; Metalloporphyrins; Neural Conduction; Peroxynitrous Acid; Poly Adenosine Diphosphate Ribose; Rats; Rats, Wistar; Regional Blood Flow; Sciatic Nerve; Superoxides; Tyrosine; Vasodilator Agents

Heme oxygenase (HO) plays a critical role in the regulation of cellular oxidative stress. The effects of the reactive oxygen species scavenger ebselen and the HO inducers cobalt protoporphyrin and stannous chloride (SnCl(2)) on HO protein levels and activity, indices of oxidative stress, and the progression of diabetes were examined in the Zucker rat model of type 2 diabetes. The onset of diabetes coincided with an increase in HO-1 protein levels and a paradoxical decrease in HO activity, which was restored by administration of ebselen. Up-regulation of HO-1 expressed in the early development of diabetes produced a decrease in oxidative/nitrosative stress as manifested by decreased levels of 3-nitrotyrosine, superoxide, and cellular heme content. This was accompanied by a decrease in endothelial cell sloughing and reduced blood pressure. Increased HO activity was also associated with a significant increase in the antiapoptotic signaling molecules Bcl-xl and phosphorylation of p38-mitogen-activated protein kinase but no significant increases in Bcl-2 or BAD proteins. In conclusion, 3-nitrotyrosine, cellular heme, and superoxide, promoters of vascular damage, are reduced by HO-1 induction, thereby preserving vascular integrity and protecting cardiac function involving an increase in antiapoptotic proteins.

Topics: Animals; Antioxidants; Apoptosis; bcl-X Protein; Blood Pressure; Blotting, Western; Diabetic Angiopathies; Endothelial Cells; Heme Oxygenase (Decyclizing); Hypertension; Male; NADPH Oxidases; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Oxygen Consumption; p38 Mitogen-Activated Protein Kinases; Peroxynitrous Acid; Phosphorylation; Rats; Rats, Zucker; Signal Transduction; Tyrosine; Up-Regulation

Stimulation of the local renin-angiotensin system and apoptosis characterize the diabetic heart. Because IGF-1 reduces angiotensin (Ang) II and apoptosis, we tested whether streptozotocin-induced diabetic cardiomyopathy was attenuated in IGF-1 transgenic mice (TGM). Diabetes progressively depressed ventricular performance in wild-type mice (WTM) but had no hemodynamic effect on TGM. Myocyte apoptosis measured at 7 and 30 days after the onset of diabetes was twofold higher in WTM than in TGM. Myocyte necrosis was apparent only at 30 days and was more severe in WTM. Diabetic nontransgenic mice lost 24% of their ventricular myocytes and showed a 28% myocyte hypertrophy; both phenomena were prevented by IGF-1. In diabetic WTM, p53 was increased in myocytes, and this activation of p53 was characterized by upregulation of Bax, angiotensinogen, Ang type 1 (AT(1)) receptors, and Ang II. IGF-1 overexpression decreased these biochemical responses. In vivo accumulation of the reactive O(2) product nitrotyrosine and the in vitro formation of H(2)O(2)-(.)OH in myocytes were higher in diabetic WTM than TGM. Apoptosis in vitro was detected in myocytes exhibiting high H(2)O(2)-(.)OH fluorescence, and apoptosis in vivo was linked to the presence of nitrotyrosine. H(2)O(2)-(.)OH generation and myocyte apoptosis in vitro were inhibited by the AT(1) blocker losartan and the O(2) scavenger TIRON: In conclusion, IGF-1 interferes with the development of diabetic myopathy by attenuating p53 function and Ang II production and thus AT(1) activation. This latter event might be responsible for the decrease in oxidative stress and myocyte death by IGF-1.

Topics: Angiotensin II; Animals; Apoptosis; Cardiomyopathies; Diabetic Angiopathies; DNA; Insulin-Like Growth Factor I; Mice; Mice, Transgenic; Myocardium; Oxidative Stress; Reactive Oxygen Species; Renin-Angiotensin System; Tissue Distribution; Tumor Suppressor Protein p53; Tyrosine; Ventricular Function

Topics: Adult; Biomarkers; Confounding Factors, Epidemiologic; Diabetic Angiopathies; Female; Glucose Clamp Technique; Hemodynamics; Humans; Hydrogen-Ion Concentration; Hyperglycemia; Male; Nitrates; Oxidative Stress; Tyrosine; Vasoconstriction