06-284 Sigma-AldrichAnti-Nitrotyrosine Antibody

We investigated the potential of a panel of 22 biomarkers to predict the presence of coronary artery disease (CAD) in type 2 diabetes mellitus (DM2) patients. The study enrolled 96 DM2 patients with (n = 75) and without (n = 21) evidence of CAD. We assessed a biochemical profile that included 22 biomarkers: total cholesterol, LDL, HDL, LDL/HDL, triglycerides, glucose, glycated hemoglobin, fructosamine, homocysteine, cysteine, methionine, reduced glutathione, oxidized glutathione, reduced glutathione/oxidized glutathione, L-arginine, asymmetric dimethyl-L-arginine, symmetric dimethyl-L-arginine, asymmetric dimethyl-L-arginine/L-arginine, nitrate plus nitrite, S-nitrosothiols, nitrotyrosine, and n-acetyl-β-glucosaminidase. Prediction models were built using logistic regression models. We found that eight biomarkers (methionine, nitratate plus nitrite, n-acetyl-β-glucosaminidase, BMI, LDL, HDL, reduced glutathione, and L-arginine/asymmetric dimethyl-L-arginine) along with gender and BMI were significantly associated with the odds of CAD in DM2. These preliminary findings support the notion that emerging biochemical markers might be used for CAD prediction in patients with DM2. Our findings warrant further investigation with large, well-designed studies.

The objective of this study was to assess the possible beneficial skeletal muscle preserving effects of ethanol extract of Schisandrae Fructus (EESF) on sciatic neurectomy- (NTX-) induced hindlimb muscle atrophy in mice. Here, calf muscle atrophy was induced by unilateral right sciatic NTX. In order to investigate whether administration of EESF prevents or improves sciatic NTX-induced muscle atrophy, EESF was administered orally. Our results indicated that EESF dose-dependently diminished the decreases in markers of muscle mass and activity levels, and the increases in markers of muscle damage and fibrosis, inflammatory cell infiltration, cytokines, and apoptotic events in the gastrocnemius muscle bundles are induced by NTX. Additionally, destruction of gastrocnemius antioxidant defense systems after NTX was dose-dependently protected by treatment with EESF. EESF also upregulated muscle-specific mRNAs involved in muscle protein synthesis but downregulated those involved in protein degradation. The overall effects of 500 mg/kg EESF were similar to those of 50 mg/kg oxymetholone, but it showed more favorable antioxidant effects. The present results suggested that EESF exerts a favorable ameliorating effect on muscle atrophy induced by NTX, through anti-inflammatory and antioxidant effects related to muscle fiber protective effects and via an increase in protein synthesis and a decrease in protein degradation.

Damage to the eye from blast exposure can occur as a result of the overpressure air-wave (primary injury), flying debris (secondary injury), blunt force trauma (tertiary injury), and/or chemical/thermal burns (quaternary injury). In this study, we investigated damage in the contralateral eye after a blast directed at the ipsilateral eye in the C57Bl/6J and DBA/2J mouse. Assessments of ocular health (gross pathology, electroretinogram recordings, optokinetic tracking, optical coherence tomography and histology) were performed at 3, 7, 14 and 28 days post-trauma. Olfactory epithelium and optic nerves were also examined. Anterior pathologies were more common in the DBA/2J than in the C57Bl/6 and could be prevented with non-medicated viscous eye drops. Visual acuity decreased over time in both strains, but was more rapid and severe in the DBA/2J. Retinal cell death was present in approximately 10% of the retina at 7 and 28 days post-blast in both strains. Approximately 60% of the cell death occurred in photoreceptors. Increased oxidative stress and microglial reactivity was detected in both strains, beginning at 3 days post-injury. However, there was no sign of injury to the olfactory epithelium or optic nerve in either strain. Although our model directs an overpressure air-wave at the left eye in a restrained and otherwise protected mouse, retinal damage was detected in the contralateral eye. The lack of damage to the olfactory epithelium and optic nerve, as well as the different timing of cell death as compared to the blast-exposed eye, suggests that the injuries were due to physical contact between the contralateral eye and the housing chamber of the blast device and not propagation of the blast wave through the head. Thus we describe a model of mild blunt eye trauma.

Liver X receptor (LXR) plays a critical regulatory role in metabolism and inflammation, and has been demonstrated to be involved in cardiovascular physiology/pathology. In the present study, we investigated the effect of GW3965, a potent LXR agonist, on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice.Non-diabetic db/+ mice and diabetic db/db mice received either vehicle or LXR agonist GW3965 for 12 weeks. Systemic insulin resistance was evaluated by glucose tolerance test and homeostasis model assessment for insulin resistance. Endpoint cardiac function was assessed by echocardiography and catheterization. Ventricular tissue was collected for histology and gene/protein expression analysis. Untreated db/db diabetic mice exhibited diastolic dysfunction with adverse structural remodeling (including myocardial fibrosis and increased apoptosis). Treatment with GW3965 remarkably attenuated myocardial dysfunction and structural remodeling in diabetic db/db mice. Mechanistically, GW3965 restored Akt phosphorylation and inhibited MAP kinases phosphorylation, and reduced oxidative/nitrative stress and inflammation response in the diabetic myocardium.Our data demonstrate that GW3965 exerts a cardioprotective effect against DCM by (at least in part) attenuating insulin resistance, modulating Akt and MAP kinases pathways, and reducing oxidative/nitrative stress and inflammatory response. These findings strongly suggest that LXR agonist may have therapeutic potential in treating DCM.

It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels.We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE-/- mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment.Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE-/- mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels.Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.

This study determined whether acute radiation-induced liver injury seen in Sirtuin3(-/-) mice after exposure to Cs-137 γ-rays was mediated by superoxide anion (O2(•-)).Male wild-type (WT) and SIRT3(-/-) mice were given 2×2 Gy whole-body radiation doses separated by 24 h and livers were harvested 20 h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3(-/-) animals, relative to WT animals, before irradiation, but this increase was not detected 20 h after radiation exposure. In addition, irradiated livers from SIRT3(-/-) animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2 mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30 min before each fraction.Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2(•-) in the liver injury process initiated by whole-body irradiation in SIRT3(-/-) mice.These results support the hypothesis that O2(•-) mediates acute liver injury in SIRT3(-/-) animals exposed to whole-body γ-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo.

Numerous factors contribute to the death of substantia nigra (SN) dopamine (DA) neurons in Parkinson's disease (PD). Compelling evidence implicates mitochondrial deficiency, oxidative stress, and inflammation as important pathogenic factors in PD. Chronic exposure of rats to rotenone causes a PD-like syndrome, in part by causing oxidative damage and inflammation in substantia nigra. Pomegranate juice (PJ) has the greatest composite antioxidant potency index among beverages, and it has been demonstrated to have protective effects in a transgenic model of Alzheimer's disease. The present study was designed to examine the potential neuroprotective effects of PJ in the rotenone model of PD. Oral administration of PJ did not mitigate or prevent experimental PD but instead increased nigrostriatal terminal depletion, DA neuron loss, the inflammatory response, and caspase activation, thereby heightening neurodegeneration. The mechanisms underlying this effect are uncertain, but the finding that PJ per se enhanced nitrotyrosine, inducible nitric oxide synthase, and activated caspase-3 expression in nigral DA neurons is consistent with its potential pro-oxidant activity.

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and β-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

Oxidative stress is becoming increasingly linked to the pathogenesis of abdominal aortic aneurysms (AAAs). The antioxidant activity of flavonoids has attracted attention for their possible role in the prevention of cardiovascular diseases. The purpose of this study was to determine whether an antioxidant mechanism is involved in the aneurysm formation inhibitory effect afforded by quercetin. Male C57/BL6 mice received quercetin continuously from 2 weeks prior to and 6 weeks following the AAA induction with extraluminal CaCl2. Quercetin treatment decreased AAA incidence and inhibited the reactive oxygen species generation, nitrotyrosine formation and lipid peroxidation production in the aortic tissue during AAA development. In addition, quercetin‑treated mice exhibited significantly lower expression of the p47phox subunit of nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, as well as coordinated downregulation of manganese‑superoxide dismutase activities and glutathione peroxidase (GPx)‑1 and GPx‑3 expression. Quercetin also blunted the expression of c‑Jun N‑terminal kinase (JNK) and phospho‑JNK and, in addition, diminished activation of the activator protein (AP)‑1 transcription factor. Gelatin zymography showed that quercetin eliminated matrix metalloproteinase (MMP)‑2 and MMP‑9 activation during AAA formation. In conclusion, the inhibitory effects of quercetin on oxidative stress and MMP activation, through modulation of JNK/AP‑1 signaling, may partly account for its benefit in CaCl2‑induced AAA.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.