3-nitrotyrosine and Hyperhomocysteinemia

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among military service members and civilians in the United States. Despite significant advances in the understanding of TBI pathophysiology, several clinical reports indicate that multiple genetic and epigenetic factors can influence outcome. Homocysteine (HCY) is a non-proteinogenic amino acid, the catabolism of which can be dysregulated by stress, lifestyle, aging, or genetic abnormalities leading to hyperhomocysteinemia (HHCY). HHCY is a neurotoxic condition and a risk factor for multiple neurological and cardiovascular disorders that occurs when HCY levels is clinically > 15 µM. Although the deleterious impact of HHCY has been studied in human and animal models of neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease, it has not been addressed in TBI models. This study tested the hypothesis that HHCY has detrimental effects on TBI pathophysiology. Moderate HHCY was induced in adult male Sprague Dawley rats via daily administration of methionine followed by impact-induced traumatic brain injury. In this model, HHCY increased oxidative stress, upregulated expression of proteins that promote blood coagulation, exacerbated TBI-associated blood-brain barrier dysfunction and promoted the infiltration of inflammatory cells into the cortex. We also observed an increase of brain injury-induced lesion size and aggravated anxiety-like behavior. These findings show that moderate HHCY exacerbates TBI outcomes and suggest that HCY catabolic dysregulation may be a significant biological variable that could contribute to TBI pathophysiology heterogeneity.

Topics: Animals; Anxiety; Behavior, Animal; Blood Coagulation; Blood-Brain Barrier; Brain Injuries, Traumatic; Cerebral Cortex; Homocysteine; Hyperhomocysteinemia; Inflammation; Intercellular Adhesion Molecule-1; Male; Methionine; Occludin; Oxidative Stress; Rats, Sprague-Dawley; Tyrosine; Zonula Occludens-1 Protein

In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that nitrative stress plays a role in vascular endothelial injury in hyperhomocysteinemia, as well as induces endothelial progenitor cell injury directly.

Topics: Animals; Endothelial Progenitor Cells; Female; Flow Cytometry; Hyperhomocysteinemia; Immunohistochemistry; In Situ Nick-End Labeling; Random Allocation; Rats, Wistar; Tyrosine

The aim of this study was to determine the levels of regulatory peptides apelin, glucagon-like peptide (GLP-1) and visfatin in hypercholesterolemic and hyperhomocysteinemic state and to examine their relation with nitric oxide (NO) metabolism. 32 Male guinea pigs were divided into four groups and each group was fed as follows: (a) commercial chow, (b) cholesterol (chol)-rich diet, (c) methionine (meth)-rich diet, and (d) chol + meth-rich diet. Blood samples were drawn at the end of 10 weeks, and abdominal aorta was dissected for histopathological examination. Serum insulin, GLP-1, apelin, visfatin, and nitrotyrosine concentrations were measured by the manufacturer's kits based on ELISA; asymmetric dimethylarginine (ADMA) and arginine levels were measured by the high performance liquid chromatography. Homocysteine level was measured by the chemiluminescence immunoassay; glucose, total chol and triglyceride levels were measured by the autoanalyzer. The microscopic examination of aorta indicated varying degrees of vascular disturbance in chol- and chol + meth-fed groups. High levels of chol and homocysteine, accompanied with significantly low levels of apelin and GLP-1 were detected in the plasma. Visfatin, ADMA, and nitrotyrosine levels both in chol- and chol + meth-fed groups were significantly higher than those in control animals, whereas arginine and arginine/ADMA ratio were lower. This study indicated that circulating levels of apelin, GLP-1, and visfatin are markedly altered during the development of atherosclerotic changes in close association with chol, homocysteine, NO, and ADMA levels. The measurements of these peptides in serum may help for the diagnosis and follow-up of vascular dysfunction.

Topics: Animals; Arginine; Cholesterol; Glucagon-Like Peptides; Guinea Pigs; Humans; Hyperhomocysteinemia; Intercellular Signaling Peptides and Proteins; Male; Nicotinamide Phosphoribosyltransferase; Nitric Oxide; Tyrosine

Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.

Topics: Animals; Antioxidants; Blotting, Western; Cystathionine beta-Synthase; Disease Models, Animal; Homocysteine; Hyperhomocysteinemia; Ischemia; Mice, Inbred C57BL; Models, Biological; Muscle, Skeletal; Muscular Diseases; Nitric Oxide Donors; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR gamma; Protein Binding; Transcription Factors; Tyrosine

Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogen α-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine by α-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, after α-zearalanol treatment, the activity of cystathionine β-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionine β-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine β-synthase nitration.

Topics: Animals; Cystathionine beta-Synthase; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Female; Homocysteine; Hyperhomocysteinemia; Liver; Nitrates; Oxidation-Reduction; Phytoestrogens; Rats; Rats, Wistar; Treatment Outcome; Tyrosine; Zeranol

Catecholamine content has been studied in the adrenal gland of rat females whose pregnant mothers were loaded daily with L-methionine administered per os during all the pregnancy period, on the first day of postnatal life, and in one and two months after birth. The animal model of hyperhomocysteinemia used in the experiment has been shown to result in the catecholamine content decreasing in the adrenal gland of both newly born rat offspring with high serum level of homocysteine and one-month old offspring with their homocysteine level decreased to the normal values. It was found that nitrotyrosine level increased significantly in the blood serum of the offspring aged one and two months. The data obtained may testify to oxidative stress development.

Topics: Administration, Oral; Adrenal Glands; Animals; Animals, Newborn; Catecholamines; Disease Models, Animal; Female; Hyperhomocysteinemia; Methionine; Oxidative Stress; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Tyrosine

Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs(+/-) mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs(+/+)). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium.

Topics: Amidines; Animals; Benzylamines; Brain; Endothelium; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Gene Expression Regulation, Enzymologic; Glycine; Homocysteine; Hyperhomocysteinemia; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Phenylacetates; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Stress, Physiological; Tyrosine

A decrease in vascular elasticity and an increase in pulse wave velocity in hyperhomocysteinemic (HHcy) cystathionine-beta-synthase heterozygote knockout (CBS(-/+)) mice has been observed. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-tissue inhibitor of metalloproteinase (TIMP) inhibitory tertiary complex. However, the contribution of the nitric oxide synthase (NOS) isoforms eNOS and iNOS in the activation of latent MMP is unclear. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP, resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the NOS isoforms, endothelial and inducible, in the collagen/elastin switch. Experiments were performed on six groups of animals [wild-type (WT), eNOS(-/-), and iNOS(-/-) with and without homocysteine (Hcy) treatment (0.67 g/l) for 8-12 wk]. In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with trichrome and van Gieson stains, respectively, was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP-2 and -9, and elastin were measured by quantitative PCR and Western blot analyses. The 2% gelatin zymography was used to assess MMP activity. The increase in O(2)(-) and robust activity of MMP-9 in eNOS(-/-), WT+Hcy, and eNOS(-/-)+Hcy was accompanied by the gross disorganization and thickening of the ECM along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that an increase in iNOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance.

Topics: Animals; Aorta; Blood Flow Velocity; Collagen; Compliance; Elastin; Extracellular Matrix; Hyperhomocysteinemia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Tissue Inhibitor of Metalloproteinases; Tyrosine; Ultrasonography

There are few reports about the direct toxic effects of hyperhomocysteinemia on the liver. We investigated oxidative and nitrosative stresses and apoptotic and necrotic changes in the liver of rats fed a high-methionine (HM) diet (2%, w/w) for 6 mo. We also investigated whether taurine, an antioxidant amino acid, is protective against an HM-diet-induced toxicity in the liver.. Lipid peroxide levels, nitrotyrosine formation, and non-enzymatic and enzymatic antioxidants were determined in livers of rats fed an HM diet. In addition, apoptosis-related proteins, proapoptotic Bax and antiapoptotic B-cell lymphoma-2 expressions, apoptotic cell count, histopathologic appearance in the liver, and alanine transaminase and aspartate transaminase activities in the serum were investigated.. Plasma homocysteine levels and serum alanine transaminase and aspartate transaminase activities were increased after the HM diet. This diet resulted in increases in lipid peroxide and nitrotyrosine levels and decreases in non-enzymatic and enzymatic antioxidants in liver homogenates in rats. Bax expression increased, B-cell lymphoma-2 expression decreased, and apoptotic cell number increased in livers of rats fed an HM diet. Inflammatory reactions, microvesicular steatosis, and hepatocyte degeneration were observed in the liver after the HM diet. Taurine (1.5%, w/v, in drinking water) administration and the HM diet for 6 mo was found to decrease serum alanine transaminase and aspartate transaminase activities, hepatic lipid peroxide levels, and nitrotyrosine formation without any change in serum homocysteine levels. Decreases in Bax expression, increases in B-cell lymphoma-2 expression, decreases in apoptotic cell number, and amelioration of histopathologic findings were observed in livers of rats fed with the taurine plus HM diet.. Our results indicate that taurine has protective effects on hyperhomocysteinemia-induced toxicity by decreasing oxidative and nitrosative stresses, apoptosis, and necrosis in the liver.

Topics: Alanine Transaminase; Animals; Antioxidants; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Hyperhomocysteinemia; Inflammation; Lipid Peroxides; Liver; Male; Malondialdehyde; Methionine; Necrosis; Nitrosation; Oxidative Stress; Rats; Rats, Wistar; Taurine; Tyrosine

To investigate the lipotropic action of betaine on plasma lipoproteins and tissue lipids.. Adult mice, wild type (+/+) or heterozygous (+/-) for a disruption of the methylenetetrahydrofolate reductase (Mthfr) gene, were supplemented with betaine for 1 year and compared with mice on control diets. Outcome measures were plasma homocysteine and lipoprotein levels, aortic and liver morphology, and liver staining for 3-nitrotyrosine (oxidative stress marker) and Apolipoprotein A-I (ApoA-I). We also investigated short-term effects of supplemental betaine on plasma lipoproteins in Mthfr +/+ and +/- mice. Both genotypes showed significantly lower plasma homocysteine after long-term betaine supplementation, and lower plasma triglycerides and higher HDL-cholesterol after both short- and long-term betaine. Lipid accumulation in liver and aortic wall tended to be lower in Mthfr+/+ compared to Mthfr+/- mice and in betaine-supplemented compared to unsupplemented mice. Nitrotyrosine staining was higher and ApoA-I staining was lower in livers of Mthfr+/- compared to Mthfr+/+ mice. Betaine did not affect staining of nitrotyrosine but increased ApoA-I staining. A significant negative correlation was observed between plasma homocysteine and liver ApoA-I.. Mild MTHFR deficiency in mice is associated with increased risk for atherosclerotic disease. Betaine has a lipotropic effect, which is associated with a reduction in homocysteine, an increase in ApoA-I and an amelioration of the atherogenic risk profile.

Topics: Animals; Animals, Genetically Modified; Aorta; Apolipoprotein A-I; Betaine; Cholesterol; Disease Models, Animal; Fatty Liver; Homocysteine; Hyperhomocysteinemia; Immunohistochemistry; Lipotropic Agents; Methylenetetrahydrofolate Reductase (NADPH2); Mice; Time; Triglycerides; Tyrosine

High homocysteine levels in vitro promote the expression of inflammatory agents responsible for atherogenesis. We investigated the long-term effects of elevated plasma homocysteine on the expression of inflammatory molecules and attempted to elucidate their mechanisms. Male Sprague-Dawley rats (n = 36) were randomly divided into 3 groups, which received the control AIN-93G diet, the control diet plus 10 g/kg of L-methionine, or that diet without folate (0 m/kg) for 14 wk. Mild hyperhomocysteinemia was then induced in both experimental groups. The mildly hyperhomocysteinemic rats had markedly increased expression of intracellular adhesion molecule-1 (ICAM-1) in the aorta and elevated serum levels of monocyte chemoattractant protein-1 (MCP-1), compared to the control rats. The activation of nuclear factor kappaB and formation of nitrotyrosine in the aorta were greater in rats with mild hyperhomocysteinemia than in control rats. Serum levels of malonyldialdehyde (MDA) were higher in mildly hyperhomocysteinemic rats than in control rats. These results suggest that the oxidative stress resulting from elevated plasma homocysteine stimulates the activation of nuclear factor kappaB, and consequently increases the expression of the inflammatory factors in vivo. Such an effect may contribute to atherogenesis by enhancing the inflammatory response of the vascular endothelium.

Topics: Animals; Aorta; Arteriosclerosis; Chemokine CCL2; Diet; Endothelium, Vascular; Folic Acid; Folic Acid Deficiency; Hyperhomocysteinemia; Inflammation; Intercellular Adhesion Molecule-1; Male; Malondialdehyde; Methionine; NF-kappa B; Nitric Oxide Synthase; Nitrites; Oxidative Stress; Rats; Rats, Sprague-Dawley; Tyrosine

Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Injury of multiple organs, including the kidney, was observed in hyperhomocysteinemic individuals. Activation of a transcription factor, namely, nuclear factor kappa B (NF-kappaB), plays an important role in inflammatory response and can exacerbate organ injury. The objective of the present study was to investigate the effect of hyperhomocysteinemia on renal NF-kappaB activation and the consequence of such activation.. Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. Activation of NF-kappaB was determined by electrophoretic mobility shift assay. Role of inhibitor protein IkappaBalpha was examined by Western immunoblotting analysis.. There was a significant increase in the level of phosphorylated IkappaBalpha protein in kidneys of hyperhomocysteinemic rats. This resulted in a decrease in the IkappaBalpha protein level leading to NF-kappaB activation. As a consequence, the expression of inducible nitric oxide synthase (iNOS) mRNA and protein was significantly elevated in kidneys of hyperhomocysteinemic rats. Increased nitric oxide production (150% of the control) resulted in peroxynitrite formation in these kidneys. Pretreatment of rats with a NF-kappaB inhibitor not only abolished NF-kappaB activation, but also reversed hyperhomocysteinemia-induced iNOS expression in the kidney.. Hyperhomocysteinemia alone can activate NF-kappaB and hence induce iNOS-mediated nitric oxide production in the kidney leading to increased peroxynitrite formation. This may represent one of the mechanisms for renal dysfunction in hyperhomocysteinemia.

Topics: Animals; Diet; Hyperhomocysteinemia; Kidney; Male; Methionine; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thiocarbamates; Tyrosine

Increased homocysteine is associated with the pregnancy complication preeclampsia and with later-life cardiovascular disease. Although elevated homocysteine persists after pregnancy, the vascular changes of preeclampsia abate with delivery, and cardiovascular disease occurs decades later. This suggests the vasculature during pregnancy may manifest increased sensitivity to homocysteine. We used the cystathionine-beta synthase (CBS)-deficient transgenic mouse to investigate whether hyperhomocysteinemia would differentially affect vascular function in nonpregnant and pregnant animals. Mesenteric arteries from nonpregnant and midpregnant (14 to 16 days) wild-type, heterozygous, and homozygous CBS-deficient transgenic mice were investigated for their response to vasoconstriction, endothelial-dependent, and endothelial-independent relaxation using an isometric wire myograph system. Endothelial-dependent vasodilation was similar in arteries from nonpregnant heterozygous and wild-type mice. In contrast, endothelial-dependent relaxation was reduced significantly in arteries from pregnant heterozygous animals compared with wild-type mice. Inhibition of NO synthesis blunted relaxation in arteries from pregnant wild-type but not pregnant heterozygous mice. Endothelial-dependent relaxation was restored by in vitro pretreatment with the tetrahydrobiopterin precursor sepiapterin. These data indicate that in pregnant mice, endothelial-dependent vasodilation is more sensitive to the effect of increased homocysteine than arteries from nonpregnant mice. This effect appears to result from a loss in NO-mediated relaxation that may be mediated by the oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin.

Topics: Animals; Cystathionine beta-Synthase; Endothelium, Vascular; Female; Hyperhomocysteinemia; Mesenteric Arteries; Mice; Mice, Knockout; Mice, Transgenic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Phenylephrine; Pregnancy; Pregnancy Complications; Pterins; Tyrosine; Vasodilation

To test the hypothesis that endothelial dysfunction in hyperhomocysteinemia was due to increased levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in response to antagonism of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), cystathionine beta-synthase (CBS) -/+ mice were bred, tail tissue was analyzed for genotype by PCR, and tail vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry. To induce PPAR-alpha, mice were administered 8 microg/ml of ciprofibrate (CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF (n = 6 in each group). In these four groups of mice, plasma Hcy was 3.0 +/- 0.2, 2.5 +/- 1.2, 15.2 +/- 2.6 (P < 0.05 compared with WT), 11.0 +/- 2.9 micromol/l. Mouse urinary protein was 110 +/- 11, 86 +/- 6, 179 +/- 13, 127 +/- 9 microg.day(-1). kg(-1) by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 +/- 0.012, 0.024 +/- 0.004, 0.132 +/- 0.024 (P < 0.01 compared with WT), 0.05 +/- 0.01 (scan unit) by Western analysis. MMP-2 activity was 0.053 +/- 0.010, 0.024 +/- 0.002, 0.039 +/- 0.009, 0.017 +/- 0.006 (scan unit) by zymography. MMP-9 was specifically induced in CBS -/+ mice and inhibited by CF treatment. Systolic blood pressure (SPB) was 90 +/- 2, 88 +/- 16, 104 +/- 8 (P < 0.05 compared with WT), 96 +/- 3 mmHg. Aortic wall stress [(SPB. radius(2)/wall thickness)/2(radius + wall thickness)] was 10.2 +/- 1.9, 9.7 +/- 0.2, 16.6 +/- 0.8 (P < 0.05 compared with WT), 13.1 +/- 2.1 dyn/cm(2). The results suggest that Hcy increased aortic wall stress by increasing nitrotyrosine and MMP-9 activity.

Topics: Animals; Aorta; Blood Pressure; Clofibric Acid; Cystathionine beta-Synthase; Endothelium, Vascular; Enzyme Activation; Fibric Acids; Genotype; Homocysteine; Hyperhomocysteinemia; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Peroxisome Proliferators; Phenotype; Proteinuria; Tyrosine

Hyperhomocysteinemia is associated with endothelial dysfunction, although the underlying mechanism is unknown. Previous studies have shown that nitric oxide (NO) plays an important role in the regulation of systemic and renal hemodynamics. This study investigated whether hyperhomocysteinemia induces renal oxidative stress and promotes renal dysfunction involving disturbances of the NO-pathway in Wistar rats. During 8 wk, control (C) and hyperhomocysteinemic (HYC) groups had free access to tap water and homocysteine-thiolactone (HTL, 50 mg/kg per d), respectively. At 8 wk, plasma homocysteine concentration, renal superoxide anion (O(2)), nitrotyrosine, and nitrite+nitrate levels, and renal function were measured. To assess NO involvement, the responses to L-Arginine (L-Arg, 300 mg/kg) and N(G)-nitro-L-arginine-methyl-ester (L-NAME, 20 microg/kg per min for 60 min) were analyzed. The HYC group showed higher homocysteine concentration (7.6 +/- 1.7 versus 4.9 +/- 1.0 micromol/L; P < 0.001), (O(2) production (157.92 +/- 74.46 versus 91.17 +/- 29.03 cpm. 10(3)/mg protein), and nitrite+nitrate levels (33.4 +/- 5.1 versus 11.7 +/- 4.3 micro mol/mg protein; P < 0.001) than the control group. Western blot analyses showed a nitrotyrosine mass 46% higher in the HYC group than in the controls. Furthermore, the HYC group showed lower GFR, renal plasma flow (RPF), and higher renal vascular resistance (RVR) than the controls. After L-Arg administration, the responses of GFR, RPF, and RVR were attenuated by 36%, 40%, and 50%, respectively; after L-NAME, the responses of RPF and RVR were exaggerated by 79% and 112%, respectively. This suggests a reduced NO bioavailability to produce vasodilation and an enhanced sensitivity to NO inhibition. In conclusion, hyperhomocysteinemia induces oxidative stress, NO inactivation, and renal dysfunction involving disturbances on the NO-pathway.

Topics: Animals; Cyclic GMP; Homocysteine; Hyperhomocysteinemia; Kidney Diseases; Male; Nitric Oxide; Oxidative Stress; Rats; Rats, Wistar; Renal Circulation; Thiobarbituric Acid Reactive Substances; Tyrosine

The hypothesis is that homocysteine decreases endothelial nitric oxide (NO) availability by generating nitrotyrosine. In the absence of NO, and in an attempt to reduce endocardial load by dilatation, the matrix metalloproteinase (MMP) is activated. To address this hypothesis, homocysteine (0.67 mg/ml) was administered in drinking water of Sprague-Dawley rats for 8 weeks. To elicit the reversible effects of homocysteine, homocysteine was removed from the water after 8 weeks. The plasma levels of homocysteine were 2.79 +/- 0.5 microM in control (n = 6), measured by spectrofluorometry. The levels of homocysteine increased to 22 +/- 1.3 and 17 +/- 2.8 microM following 4 (n = 6) and 8 (n = 6) weeks of homocysteine treatment, respectively. The level of homocysteine decreased to 5.8 +/- 1.0 microM (n = 6) when homocysteine was removed from the drinking water. The mean arterial pressure (MAP) of control rats was 108 +/- 10 mm Hg and increased to 128 +/- 2 and 130 +/- 3 mm Hg following 4 and 8 weeks of homocysteine treatment, respectively. When homocysteine was removed from the drinking water, the MAP was decreased to 118 +/- 3 mm Hg. Left ventricle (LV) parameters were measured by a catheter in the LV through right common carotid artery in anesthetized rats. The LV tissue was analyzed for MMP activity by zymography. Levels of nitrotyrosine and cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) were measured by western blot analysis using the respective antibodies. The specific bands in zymographic gel and western blot were scanned and normalized with beta-actin. The results suggest a continuous increase in nitrotyrosine levels at 4 and 8 weeks after homocysteine administration. The removal of homocysteine did not decrease the levels of nitrotyrosine. The zymographic analysis revealed a temporal increase in MMP-2 activity from 4 to 8 weeks post homocysteine administration. However, removal of homocysteine did not decrease the MMP-2 activity. The cardiac active diastolic function, -dP/dt, was decreased at 4 weeks and stayed depressed up to 12 weeks. The end-diastolic pressure started increasing at 8 weeks; at this point the MMP-2 activity was also increased. The results suggest that in the absence of endothelial NO, and in an attempt to reduce LV load, MMP-2 is activated and CIMP is inactivated, by increasing nitrotyrosine.

Topics: Animals; Blood Pressure; Enzyme Activation; Heart; Heart Rate; Hemodynamics; Hyperhomocysteinemia; Kinetics; Male; Matrix Metalloproteinase 2; Myocardium; Rats; Rats, Sprague-Dawley; Time Factors; Tissue Inhibitor of Metalloproteinase-4; Tissue Inhibitor of Metalloproteinases; Tyrosine; Ventricular Function, Left

Hyperhomocysteinemia (HHcy) is a newly recognized risk factor for myocardial infarction, however, the effect of HHcy on endothelium-dependent flow-induced dilation of coronary arteries is not known. Thus, changes in diameter of small intramural coronary arteries (diameter, approximately 145 microm) isolated from control rats and rats with methionine diet-induced HHcy were investigated by videomicroscopy. Increases in intraluminal flow (from 0 to 40 microl/min) elicited dilations of control vessels (maximum, 25 +/- 2 microm), responses that were absent in HHcy arteries. The nitric oxide (NO) synthase inhibitor L-NAME inhibited flow-induced dilation of control coronaries, whereas it had no effect on responses of HHcy arteries. Dilations of control and HHcy arteries to the NO donor sodium nitroprusside were not different. Responses to flow in HHcy coronary arteries were unaffected by administration of L-arginine or the prostaglandin H(2)/thromboxane A(2) receptor antagonist SQ 29,548. However, in the presence of superoxide dismutase (plus catalase) or the superoxide scavenger Tiron increases in flow elicited L-NAME-sensitive dilations of HHcy coronaries (maximum, 18 +/- 5 microm). Also, superoxide dismutase significantly reduced the enhanced superoxide production of HHcy coronaries (measured by the lucigenin chemiluminescence method). Single vessel Western blotting showed an increased tyrosine nitrosation (a stable biomarker of tissue peroxynitrite formation) in HHcy coronaries. Also, extensive prevalence of 3-nitrotyrosine immunoreactivity was observed in HHcy coronaries that was confined primarily to the subendothelial layers of smooth muscle. We propose that in HHcy an increased level of superoxide scavenges NO forming peroxynitrite, which increases protein nitrosation. The reduced bioavailability of NO impairs flow-induced dilations of coronary arteries, which may contribute to the development of coronary atherosclerosis and ischemic heart disease.

Topics: Animals; Blotting, Western; Coronary Circulation; Coronary Vessels; Enzyme Inhibitors; Hyperhomocysteinemia; Immunohistochemistry; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Rats; Rats, Wistar; Superoxides; Tyrosine; Vasodilation

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Topics: Acetylcholine; Animals; Aorta; Arteriosclerosis; Cystathionine beta-Synthase; Dinoprost; Disease Models, Animal; Endothelium, Vascular; F2-Isoprostanes; Heterozygote; Humans; Hyperhomocysteinemia; In Vitro Techniques; Lipid Peroxides; Mice; Mice, Mutant Strains; Nitroprusside; Reactive Oxygen Species; Risk Factors; Thrombosis; Tyrosine; Vasodilation