3-nitrotyrosine and Thrombosis

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO(-) levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO(-) levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO(-) increase observed in our patients, compared to controls, is most probably due to reaction of NO with O(2)(*-) . These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.

Topics: Aged; Atherosclerosis; Brain Ischemia; Case-Control Studies; Female; Humans; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Reactive Oxygen Species; Thrombosis; Tyrosine

NO in vivo has both beneficial and nonbeneficial effects depending on site and concentration. Peroxynitrite, resulting from the reaction of NO with superoxide radical, causes cellular damage. Nitrotyrosine, end product of NO's toxic effects on cellular proteins, is a stable compound that can be used to detect evidence of harmful quantities of NO. We sought to detect nitrotyrosine in coronary arterioles of DBA/2 mice injected intraperitoneally with Lactobacillus casei cell wall. The inflammatory response induced occurred in perivascular fashion and involved mainly macrophages. It was variable according to time points, being severe on days 10 and 14 and mild to moderate on days 3 and 7. Few basal inflammatory cells appeared in controls injected with phosphate-buffered saline. Western immunoblots of homogenized hearts on days 10 and 14 demonstrated specific nitrated proteins. Immunohistochemistry of frozen sections of diseased hearts showed positive immunoreactivity for nitrotyrosine in coronary arterioles at the same time points. These findings were absent in the controls. We also determined the expression of inducible nitric oxide synthase (iNOS) in controls on days 10 and 14. iNOS colocalized with nitrotyrosine in perivascular macrophages and coronary arterioles of treated mice. Additionally, aneurysms were found on day 10 and intracardiac hemorrhage with consequent death on day 14. These observations supply evidence that NO through its reactive product, peroxynitrite, and its antigen/tissue marker, nitrotyrosine, is directly involved in coronary arteritis and aneurysm development in mice models of Kawasaki disease (KD). This article shows that macrophages are central to this and bolsters the likelihood of L. casei being the cause of KD.

Topics: Aneurysm; Animals; Blotting, Western; Cell Wall; Coronary Vessels; Disease Models, Animal; Hemorrhage; Immunohistochemistry; Inflammation; Lacticaseibacillus casei; Macrophages; Male; Mice; Mucocutaneous Lymph Node Syndrome; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Thrombosis; Time Factors; Tyrosine; Vasculitis

Recently, a novel inhibitor of inducible nitric oxide synthase, ONO-1714, was developed. We evaluated the effect of ONO-1714 on a critical warm I/R model of the pig liver.. Pigs were subjected to 180 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in the serum NO2- + NO3- (NOx), the cellular distribution of endothelial and inducible nitric oxide synthase, thrombocyte-thrombi, and nitrotyrosine by immunohistochemistry. The hepatic tissue blood flow (HTBF) was measured continuously using a laser-Doppler blood flowmeter.. ONO-1714 at 0.05 mg/kg improved the survival rate from 54 (control group) to 100%. The serum NOx levels in the ONO-1714 group were significantly lower than those in the control group at 1, 1.5, 2, 3, and 6 hr after reperfusion. The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels of the ONO-1714 group were significantly lower than the control group, and the HTBF of the ONO-1714 group was significantly higher than the control group. The formation of thrombocyte-thrombi and nitrotyrosine after reperfusion was significantly lower in the ONO-1714 group.. These results indicated that ONO-1714 improved the survival rates and attenuated I/R injury in a critical hepatic warm I/R model of the pig. ONO-1714 will be beneficial for hepatectomy or liver transplantation in the clinical field.

Topics: Amidines; Animals; Aspartate Aminotransferases; Enzyme Inhibitors; Female; Heterocyclic Compounds, 2-Ring; Hot Temperature; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Reperfusion Injury; Survival Analysis; Swine; Thrombosis; Tissue Distribution; Tyrosine

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

Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus. These findings indicate that no perilesional changes in the number of neurons expressing nitric oxide synthase I occur, but that a transient increase in nitric oxide synthase II does take place in the aftermath of small cortical lesions. The results suggest that increased nitric oxide production is limited to certain post-lesional intervals in this experimental model. It is also obvious that the vast majority of nitric oxide synthase-positive cells are nitric oxide synthase II-containing astrocytes three days after lesioning, suggesting that astrocyte-derived nitric oxide plays a significant role in delayed neuronal death. Such a condition points to an important aspect of post-lesional astrocytosis.

Topics: Animals; Cerebral Infarction; Immunohistochemistry; Ischemic Attack, Transient; Isoenzymes; Lectins; Male; NADPH Dehydrogenase; Nitric Oxide Synthase; Rats; Rats, Wistar; Somatosensory Cortex; Thrombosis; Tyrosine