Lucigenin-enhanced Chemiluminescence Research Articles

Effect of honey on purified equine myeloperoxidase activity and superoxide radical production in activated Polymorphonuclear neutrophils

Three Algerian honey samples (nectar honey, mixed honey and honeydew honey) were analysed for their total phenolic content (TPC) using the Folin–Ciocalteu reagent and total flavonoid content (TFC) by the aluminium chloride method. The antioxidant activity was tested both against reactive oxygen species (ROS) produced by phorbol 12-myristate 13-acetate (PMA)-stimulated equine polymorphonuclear neutrophils (PMNs) and on purified equine myeloperoxidase (MPO) enzyme activity. The ROS production by stimulated PMNs was measured by lucigenin-enhanced chemiluminescence. Specific immunological extraction followed by enzymatic detection (SIEFED) was used to measure specifically the activity of equine MPO. Nectar honey, mixed honey and honeydew honey had TPCs of 59.52 ± 1.31, 68.36 ± 1.20 and 122.76 ± 4.20 mg gallic acid equivalents (GAE)/100 g honey, respectively (mean ± SD). As for the TPC, the highest TFC was observed for honeydew honey [20.55 ± 0.72 mg catechine equivalents (CE)/100 mg honey], followed by mixed honey (7.47 ± 0.07 CE/100 mg honey) and nectar honey (4.80 ± 0.08 CE/100 mg honey). All the tested honeys displayed a dose-dependent inhibitory effect on the oxidant activities of PMNs. By the SIEFED technique, it was shown that most of the honeys interact directly with MPO: the light honeys (nectar honey and mixed honey) were inhibitors of MPO activity, while the dark honey had less inhibiting effect and even behaved as an enhancer of MPO activity at high concentrations.

L-citrulline for protection of endothelial function from ADMA-induced injury in porcine coronary artery.

Endogenous nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a cardiovascular risk factor. We tested the hypothesis that L-citrulline may ameliorate the endothelial function altered by ADMA in porcine coronary artery (PCA). Myograph study for vasorelaxation, electrochemical measurement for NO, RT-PCR, and Western blot analysis for expression of eNOS, argininosuccinate synthetase (ASS), and p-eNOSser1177 were performed. cGMP was determined by enzyme immunoassay. Superoxide anion (O2.−) production was detected by the lucigenin-enhanced chemiluminescence method. Compare with controls (96.03% ± 6.2%), the maximal relaxation induced by bradykinin was significantly attenuated (61.55% ± 4.8%, p < 0.01), and significantly restored by L-citrulline (82.67 ± 6.4%, p < 0.05) after 24 hours of ADMA exposure. Expression of eNOS, p-eNOSser1177, and ASS in PCA significantly increased after L-citrulline incubation. L-citrulline also markedly restored the NO production, and cGMP level which was reduced by ADMA. The increased O2.− production by ADMA was also inhibited by L-citrulline. L-citrulline restores the endothelial function in preparations treated with ADMA by preservation of NO production and suppression of O2.− generation. Preservation of NO is attributed to the upregulation of eNOS expression along with activation of p-eNOSser1177. L-citrulline improves endothelium-dependent vasodilation through NO/ cGMP pathway.

184 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

<h3>Introduction</h3> Insulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from insulin resistance T2DM. We examined the effects of enhancing EC insulin sensitivity <i>in vivo</i>, by generating a novel transgenic mouse, over-expressing Type A human Insulin Receptor (HIRECO) restricted to EC. <h3>Methods</h3> Western blotting and RT-PCR were carried out on tissues and isolated endothelial cells from lungs to measure protein levels and mRNA expression, respectively. NADPH-dependent lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels. Isolated thoracic aortic rings suspended in an organ bath were used to determine vasomotor functions. eNOS activity was examined by citrulline assay with ¹⁴C-labelled L-arginine. HIRECO were compared to wild type littermates. <h3>Results</h3> Over-expressing human insulin receptors in EC had no significant effect on morphological features, metabolic phenotype or blood pressure of HIRECO. HIRECO demonstrated significant EC dysfunction measured by a blunted endothelium-dependent vasorelaxation to acetylcholine and reduced basal NO release. EC-independent response to sodium nitroprusside remained unchanged. EC dysfunction observed in the organ bath was normalised by a NADPH oxidase-specific inhibitor peptide, gp91ds-tat as well as the superoxide dismutase mimetic, MnTmPyp. HIRECO demonstrated significant increase in superoxide anion release compared to WT littermates. This data was supported by a concomitant increase in NADPH oxidase isoform, NOX2 protein expression. Basal eNOS and Akt phosphorylation levels in isolated EC of HIRECO mice were enhanced compared to WT mice. Interestingly, insulin-stimulated eNOS phosphorylation and activation was decreased, whereas Akt phosphorylation remained unchanged. eNOS tyrosine phosphorylation mediated by proline-rich tyrosine kinase (PYK2) was significantly enhanced in EC from HIRECO mice. In order to investigate, if the perturbations of insulin signalling in the EC have a pathological outcome, HIRECO mice were crossed with ApoE-knockout mice. These mice demonstrated a significant enhancement of plaque formation in aorta, as well as the aortic root. <h3>Conclusions/implications</h3> These data show that increasing EC insulin sensitivity leads to reduced bioavailability of NO. These data demonstrate for the first time that increased insulin signalling in EC increases the generation of superoxide anion via activation of NOX₂ NADPH oxidase and reduced NO production in response to insulin due at least in part to increased EC PYK2 activity leading to a pro-atherosclerotic state.

Therapeutic targeting of oxidative stress with coenzyme Q10 counteracts exaggerated diabetic cardiomyopathy in a mouse model of diabetes with diminished PI3K(p110α) signaling

Diabetes-induced cardiac complications include left ventricular (LV) dysfunction and heart failure. We previously demonstrated that LV phosphoinositide 3-kinase p110α (PI3K) protects the heart against diabetic cardiomyopathy, associated with reduced NADPH oxidase expression and activity. Conversely, in dominant negative PI3K(p110α) transgenic mice (dnPI3K), reduced cardiac PI3K signaling exaggerated diabetes-induced cardiomyopathy, associated with upregulated NADPH oxidase. The goal was to examine whether chronic supplementation with the antioxidant coenzyme Q10 (CoQ10) could attenuate LV superoxide and diabetic cardiomyopathy in a setting of impaired PI3K signaling. Diabetes was induced in 6-week-old nontransgenic and dnPI3K male mice via streptozotocin. After 4 weeks of diabetes, CoQ10 supplementation commenced (10mg/kg ip, 3 times/week, 8 weeks). At study end (12 weeks of diabetes), markers of LV function, cardiomyocyte hypertrophy, collagen deposition, NADPH oxidase, oxidative stress (3-nitrotyrosine), and concentrations of CoQ9 and CoQ10 were determined. LV NADPH oxidase (Nox2 gene expression and activity, and lucigenin-enhanced chemiluminescence), as well as oxidative stress, were increased by diabetes, exaggerated in diabetic dnPI3K mice, and attenuated by CoQ10. Diabetes-induced LV diastolic dysfunction (prolonged deceleration time, elevated end-diastolic pressure, impaired E/A ratio), cardiomyocyte hypertrophy and fibrosis, expression of atrial natriuretic peptide, connective tissue growth factor, and β-myosin heavy chain were all attenuated by CoQ10. Chronic CoQ10 supplementation attenuates aspects of diabetic cardiomyopathy, even in a setting of reduced cardiac PI3K protective signaling. Given that CoQ10 supplementation has been suggested to have positive outcomes in heart failure patients, chronic CoQ10 supplementation may be an attractive adjunct therapy for diabetic heart failure.

Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis

Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14+ Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14+ Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca2+-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca2+ ionophore. Exposure of Mac to increasing concentrations of IFNγ (5–100 ng/ml) or oxidized LDL (5–100 μg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca2+ concentration. Immunohistochemical staining revealed that Nox5 is present in CD68+ Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.

Sex differences in the role of NADPH oxidases in endothelium-dependent vasorelaxation in porcine isolated coronary arteries

The present study examined whether vascular function, expression and activity of NADPH oxidases differ between sexes in porcine isolated coronary arteries (PCAs) using selective Nox inhibitors, ML-171 and VAS2870. Vascular responses of distal PCAs were examined under myographic conditions in the presence of a range of inhibitors. Nox activity in PCA homogenates was assessed using lucigenin-enhanced chemiluminescence. Protein expression of Nox1, Nox2 and Nox4 was compared using Western immunoblotting. The presence of ML-171 or DPI had no effect on the bradykinin-induced vasorelaxation in PCAs from females. In males, DPI shifted the EC50 2.8-fold to the right. In the presence of L-NAME and indomethacin, DPI and ML-171 had no effect in females, but enhanced the bradykinin-induced vasorelaxation in males. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. VAS2870 had no effect on the bradykinin-induced vasorelaxation in both sexes but reduces the EDH-type response in males only. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Protein expression of Nox1 and Nox2 in PCAs was higher in males compared to females whereas Nox4 was higher in females. Inhibition of Nox with ML-171 enhances while VAS2870 reduces the EDH-type response in PCAs from males but not females. This indicates that Nox-generated ROS play a role in the EDH-type response in males with differences attributed to the differential expression of Nox isoforms. This may underlie the greater oxidative stress observed in males.

Des-aspartate angiotensin I (DAA-I) reduces endothelial dysfunction in the aorta of the spontaneously hypertensive rat through inhibition of angiotensin II-induced oxidative stress

Des-aspartate angiotensin I (DAA-I), an endogenous nonapeptide, counteracts several effects of angiotensin II on vascular tone. The aim of this study was to investigate the acute protective effect of DAA-I on endothelial function in the spontaneously hypertensive rat (SHR) as well as its effect on angiotensin II-induced contractions and oxidative stress. Aortic rings were incubated with DAA-I (0.1μM) for 30min prior to the assessment of angiotensin II-induced contractions (0.1nM–10μM) in WKY and SHR aortas. Total nitrate and nitrite levels were assessed using a colorimetric method and reactive oxygen species (ROS) were measured by dihydroethidium (DHE) fluorescence and lucigenin-enhanced chemiluminescence. The effect of DAA-I was also assessed against endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, respectively. Angiotensin II-induced contractions were significantly reduced by DAA-I, losartan and tempol. Incubation with ODQ (soluble guanylyl cyclase inhibitor) and removal of the endothelium prevented the reduction of angiotensin II-induced contractions by DAA-I. Total nitrate and nitrite levels were increased in DAA-I, losartan and tempol treated-SHR tissues while ROS level was reduced by DAA-I and the latter inhibitors. In addition, DAA-I significantly improved the impaired acetylcholine-induced relaxation in SHR aortas whilst sodium nitroprusside-induced endothelium-independent relaxation remained unaffected. The present findings indicate that improvement of endothelial function by DAA-I in the SHR aorta is mediated through endothelium-dependent release of nitric oxide and inhibition of angiotensin II-induced oxidative stress.

Tocotrienol Rich Palm Oil Extract Is More Effective Than Pure Tocotrienols at Improving Endothelium-Dependent Relaxation in the Presence of Oxidative Stress.

Oxidative endothelial dysfunction is a critical initiator of vascular disease. Vitamin E is an effective antioxidant but attempts to use it to treat vascular disorders have been disappointing. This study investigated whether tocotrienols, the less abundant components of vitamin E compared to tocopherols, might be more effective at preserving endothelial function. Superoxide generated by hypoxanthine/xanthine oxidase or rat aorta was measured using lucigenin-enhanced chemiluminescence. The effect of α-tocopherol, α-, δ-, and γ-tocotrienols and a tocotrienol rich palm oil extract (tocomin) on levels of superoxide was assessed. Endothelial function in rat aorta was assessed in the presence of the auto-oxidant pyrogallol. Whilst all of the compounds displayed antioxidant activity, the tocotrienols were more effective when superoxide was produced by hypoxanthine/xanthine oxidase whereas tocomin and α-tocopherol were more effective in the isolated aorta. Tocomin and α-tocopherol restored endothelial function in the presence of oxidant stress but α-, δ-, and γ-tocotrienols were ineffective. The protective effect of tocomin was replicated when the tocotrienols were present with, but not without, α-tocopherol. Tocotrienol rich tocomin is more effective than α-tocopherol at reducing oxidative stress and restoring endothelium-dependent relaxation in rat aortae and although α-, δ-, and γ-tocotrienols effectively scavenged superoxide, they did not improve endothelial function.

Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells.

The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds.

Endothelin-1 contributes to endothelial dysfunction and enhanced vasoconstriction through augmented superoxide production in penile arteries from insulin-resistant obese rats: role of ET(A) and ET(B) receptors.

We assessed whether endothelin-1 (ET-1) inhibits NO and contributes to endothelial dysfunction in penile arteries in a model of insulin resistance-associated erectile dysfunction (ED). Vascular function was assessed in penile arteries, from obese (OZR) and lean (LZR) Zucker rats, mounted in microvascular myographs. Changes in basal and stimulated levels of superoxide (O2 (-) ) were detected by lucigenin-enhanced chemiluminescence and ET receptor expression was determined by immunohistochemistry. ET-1 stimulated acute O2 (-) production that was blunted by tempol and the NADPH oxidase inhibitor, apocynin, but markedly enhanced in obese animals. ET-1 inhibited the vasorelaxant effects of ACh and of the NO donor S-nitroso-N-acetyl-DL-penicillamine in arteries from both LZR and OZR. Selective ETA (BQ123) or ETB receptor (BQ788) antagonists reduced both basal and ET-1-stimulated superoxide generation and reversed ET-1-induced inhibition of NO-mediated relaxations in OZR, while only BQ-123 antagonized ET-1 actions in LZR. ET-1-induced vasoconstriction was markedly enhanced by NO synthase blockade and reduced by endothelium removal and apocynin. In endothelium-denuded penile arteries, apocynin blunted augmented ET-1-induced contractions in OZR. Both ETA and ETB receptors were expressed in smooth muscle and the endothelial layer and up-regulated in arteries from OZR. ET-1 stimulates ETA -mediated NADPH oxidase-dependent ROS generation, which inhibits endothelial NO bioavailability and contributes to ET-1-induced contraction in healthy penile arteries. Enhanced vascular expression of ETB receptors contributes to augmented ROS production, endothelial dysfunction and increased vasoconstriction in erectile tissue from insulin-resistant obese rats. Hence, antagonism of ETB receptors might improve the ED associated with insulin-resistant states.

Abstract 19182: Epicardial Adipose Tissue Volume Selectively Predicts Myocardial Redox State in Patients With Ischemic Heart Disease

Introduction: Myocardial redox state is a strong determinant of heart biology. Epicardial adipose tissue (EpAT) is in close contact with the human heart and it is likely that by secreting a wide range of adipokines it may affect the biology of the underlying myocardium. Hypothesis: We hypothesised that EpAT volume may predict myocardial redox state and consequently myocardial function in ischaemic heart disease (IHD). Methods: We recruited 38 patients undergoing coronary artery bypass grafting surgery. Patients underwent transthoracic echocardiography to assess cardiac function and cardiac computerised tomography to determine the volume of EpAT, pericardial (PerAT) and subcutaneous (ScAT) AT, by using a strictly pre-defined protocol. Samples of right atrium appendage were collected during surgery and myocardial NADPH oxidase activity was assessed by lucigenin-enhanced chemiluminescence (using its substrate -NAPDH 100uM +/- its specific inhibitor VAS2870 40uM). Results: Increased EpAT volume index (EpAT volume/body surface area), but not PerAT or ScAT, was strongly associated with increased myocardial NADPH oxidase activity (Figure). EpAT volume index was not related with left ventricle (LV) mass index or LV systolic/diastolic function (p=NS for all). EpAT volume index was also unrelated to risk factors or body mass index (p=NS for all). Conclusions: These findings introduce the concept of a local cross-talk between EpAT and the underlying myocardium in humans. EpAT volume can be used as a surrogate of myocardial redox state, and this may have direct implications in risk stratification of patients undergoing coronary bypass surgery.

Abstract 15999: The Divergent Effects of Exercise Training After Myocardial Infarction or Pressure Overload Depend Critically on Endothelial Nitric Oxide Synthase

AIMS: Beneficial effects of aerobic exercise training are widely recognized. However, previously we discovered that the positive effects of exercise depend on the underlying cause of cardiac failure. Here we tested the hypothesis that endothelial nitric oxide synthase (eNOS) dependent regulation of the balance between nitric oxide and superoxide (O2•-) is critically involved in determining the effects of exercise. METHODS: Mice were exposed to 8 weeks of voluntary wheel running exercise training (EX) or sedentary housing (SED) immediately following myocardial infarction (MI), pressure overload from a transverse aortic constriction (TAC), or sham (SH) surgery. Subsequently, left ventricular (LV) ejection fraction (EF) was measured by echocardiography and Picrosirius Red staining was performed to measure collagen content. Additionally, total and NOS-dependent LV O2•- were measured using lucigenin-enhanced chemiluminescence without or with NOS inhibitor, L-NAME. eNOS uncoupling was evaluated by determining eNOS monomer dimer protein ratio and peroxynitrite (ONOO-) levels were measured through luminol-enhanced chemiluminescence. RESULTS: Cardiac dysfunction and fibrosis were ameliorated by exercise in MI but not in TAC mice (Table 1). MI and TAC both increased LV O2•- levels. Strikingly, EX diminished O2•- generation in MI, but exacerbated O2•- generation in TAC (Table 1). Furthermore, the EX-induced increase in O2•- levels in TAC were largely NOS-dependent. Accordingly, MI and TAC-induced eNOS uncoupling was normalized by EX in MI but aggravated in TAC mice (Table 1). Similarly, increased ONOO- levels following MI and TAC were diminished by EX in MI, but exacerbated by EX in TAC (Table 1). CONCLUSIONS: EX reduces eNOS-mediated cardiac oxidative stress in MI. In contrast, beneficial effects of EX are lacking in cardiac pressure-overload following TAC, due to EX-induced aggravation of ONOO- formation, eNOS uncoupling and concomitant oxidative stress.

P95 - Differential activation of neutrophil extracellular traps by specific periodontal bacteria

Periodontitis is a chronic inflammatory disease initiated by periodontal pathogens. Neutrophils play a pivotal role within the periodontal lesion, where they have a cytotoxic arsenal at their disposal, including Reactive oxygen species (ROS) and release of neutrophil extracellular trap (NETs). ROS production is essential for NET release and neutrophils in periodontitis patients are hyperactive and hyper-reactive with regard to ROS release (Matthews 2007), which may lead to disease progression. Here we characterise NET and ROS release in response to specific periodontal bacteria.Peripheral blood neutrophils were incubated with a panel of bacteria, including F. nucleatum and C. gingivalis. Neutrophil total ROS and superoxide release were measured (2hours) by luminol and lucigenin-enhanced chemiluminescence respectively. NET release was assayed by incubating the cells with bacteria for 4hours and fluorometrically quantifying NET DNA with sytox green (Palmer 2012). In all assays phorbol myristate acetate [PMA] was used as a positive control.Relative to unstimulated neutrophils S. Gordinii, F. nucleatum, V. parvula and A. viscosus stimulated significantly greater total ROS release (n=5, p=<0.05). S. Gordinii and V. parvula (n=5, p=<0.05) also stimulated significantly higher levels of superoxide.Bacterial stimulation of NETs (n=5) showed marked heterogeneity, with F. nucleatum and S. Gordinii stimulating higher levels of NETs than other species.Collectively, these data demonstrate variability between periodontal bacteria in their ability to stimulate neutrophil total ROS production, superoxide and NET responses. This variability may contribute to altered NET production in-vivo by specific periodontal bacteria and may contribute to the pathogenesis of periodontitis. Mechanisms may include bacterial avoidance of NET stimulation and thus persistence of infection, or excess NET release with associated autoimmunity.

Abstract 614: Anti-inflammatory Effect of Ang-(1-7) non-peptide mimetic (AVE 0991), on T Cell Infiltration in Perivascular Adipose Tissue in ApoE-/- Mice

Angiotensin 1-7 (Ang 1-7) has anti-atherosclerotic effects, possibly through its anti-inflammatory properties. As perivascular inflammation is an important component of early atherosclerosis, we aimed to determine whether angiotensin-(1-7) non peptide mimetic (AVE0991) can modulate these inflammatory mechanisms in the early development of atherosclerosis. Twelve-week old ApoE-/- mice and controls (C57BL/6J) on chow diet were treated with placebo or AVE0991 (0.58μM/g/day, per os). Mice were analysed at 16, 20 and 24 weeks of age. The level of perivascular leukocyte and T cell infiltration was measured by flow cytometry. Atherosclerotic plaque area was assessed by Oil Red O staining of en-face aortic preparations. Endothelial dysfunction was measured by wire myography and ROS production in the aorta by lucigenin-enhanced chemiluminescence (5μM). RESULTS: Levels of endogenous Ang 1-7, measured by mass spectrometry in the perivascular adipose tissue were low in both WT and ApoE-/- (32.9±1.2 vs 31.1±1.1 pg/mg; p=0.1). AVE0991 significantly reduced plaque area in ApoE-/- mice at all time points (14.2±1.9% vs 7.25±1.3% at 24 weeks; p&lt;0.05). During this early stage of atherosclerosis there was no evidence of endothelial dysfunction and no significant increase in ROS generation in ApoE-/- mice. However, there was significant perivascular inflammation in 16 week old ApoE mice: CD45+ leukocytes were 2 fold higher in ApoE-/- mice vs controls (3200±850 vs 1700±267 cells/mg; p&lt;0.05) and the number of CD3+ T in ApoE-/- mice was higher than in C57Bl/6J (638±88 vs 310±64 cells/mg; p&lt;0.05). With aging, the number of leukocytes and T cells further increased in ApoE-/- but not in C57BL/6J mice, an effect abrogated by AVE0991. Moreover in cell culture of SW872 preadipocytes, AVE 0991 preincubation abolished TNF-α induced increase of chemokine RANTES and IL-6 mRNA expression. CONCLUSION: Ang-(1-7) non-peptide mimetic (AVE 0991) effectively inhibits early perivascular inflammation during the development of atherosclerotic plaque. This occurs independently of changes in redox status or endothelial dysfunction.

Hydrogen sulfide treatment reduces blood pressure and oxidative stress in angiotensin II-induced hypertensive mice.

Hydrogen sulfide (H2S) is increasingly recognized as a gasotransmitter with protective effects in the cardiovascular system. The aim of the study was to examine the effects of chronic NaHS treatment on blood pressure, vascular function and oxidative stress in an in vivo model of hypertension and oxidative stress. Male C57Bl6/J mice were rendered hypertensive with 0.7 mg kg(-1) per day angiotensin II (AngII) for 14 days administered via implanted mini-pumps. The mice were treated with NaHS (10 μmol kg(-1) per day) to deliver H2S or an inhibitor of cystathionine-γ-lyase, DL-propargylglycine (PPG 30 mg kg(-1) per day) via intraperitoneal (i.p.) injection. Systolic blood pressure was measured and vascular function examined by myography. Vascular superoxide production was measured by lucigenin-enhanced chemiluminescence. AngII infusion significantly increased systolic blood pressure (P < 0.001). This increase was significantly attenuated by treatment with NaHS (P < 0.001). Both aortic endothelial function and NO bioavailability were significantly attenuated in the AngII group (P < 0.01) but this attenuation was reversed by NaHS treatment. Similarly, aortic superoxide anion production was significantly enhanced by AngII (P < 0.01), and this was reversed by NaHS treatment, and also exacerbated by PPG treatment (P < 0.001). These data show that in a mouse model of hypertension and oxidative stress induced by AngII, exogenous H2S treatment in vivo reduces blood pressure, endothelial dysfunction and vascular oxidative stress, while inhibiting endogenous H2S production in vivo is deleterious. This furthers the evidence that H2S is a vasoprotective molecule that may be a useful treatment target in cardiovascular disease.

Nesfatin-1 alleviates gastric damage via direct antioxidant mechanisms

BackgroundIndomethacin is a nonsteroidal anti-inflammatory drug, which is known to produce serious side effects, causing ulcerative lesions. Nesfatin-1, a newly identified anorexigenic peptide, was recently shown to have neuroprotective effects. The aim of the study was to investigate the anti-inflammatory effects of nesfatin-1 on indomethacin-induced gastric ulcer. Materials and methodsAfter a 24-h starvation period, ulcer was induced in Sprague–Dawley rats by subcutaneous administration of indomethacin (25 mg/kg), whereas control group received vehicle. Fifteen minutes after ulcer induction, rats were treated with either saline or nesfatin-1 (0.1, 0.3, or 1 μg/kg, intraperitoneally). At the fourth hour, all rats were decapitated and their trunk blood was collected for tumor necrosis factor (TNF)-α and interleukin (IL)-6 measurements. Stomach samples were examined microscopically and analyzed for myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), luminol-, and lucigenin-enhanced chemiluminescence (CL) levels. ResultsUlcer induction increased serum TNF-α; and IL-6 levels, gastric CL and MDA levels and MPO activity but decreased gastric GSH content (P < 0.05–0.001). On the other hand, 0.1 μg/kg dose of nesfatin-1 reduced microscopic and macroscopic damage scores, decreased MPO activity and MDA levels, CL and IL-6 levels, whereas gastric GSH was replenished (P < 0.01). However, indomethacin-induced increase in TNF-α level was abolished at only 1 μg/kg dose of nesfatin-1 (P < 0.01). ConclusionsNesfatin-1 alleviated indomethacin-induced gastric injury, suggesting that the anti-inflammatory and gastroprotective effects of nesfatin-1 on oxidative gastric damage could be implemented by supporting the balance in oxidant and antioxidant systems while inhibiting the generation of pro-inflammatory mediators.

Role of AMPK signaling pathway in sevoflurane preconditioning-induced enhancement of antioxidant capacity during hypoxia-reoxygenation in cardiomyocytes of rats

Objective To investigate the role of 5'-AMP-activated protein (AMPK) signaling pathway in sevoflurane preconditioning-induced enhancement of antioxidant capacity during hypoxia-reoxygenation (H/R) in the cardiomyocytes of the rats.Methods The rat embryonic cardiomyocytes (H9C2 cells) were randomly divided into 3 groups (n =12 each) using a random number table:control group (group C),H/R group,and sevoflurane preconditioning group (group SP).AMPK gene knockout (AMPK siRNA) H9C2 cells were randomly divided into 3 groups (n =12 each) using a random number table:control group (group A-C),A-H/R group,and sevoflurane preconditioning group (group A-SP).The cells were exposed to 95％ N2 + 5％ CO2 in an incubator for 14 h followed by reoxygenation with 95％ O2 + 5％ CO2 for 3 h.In SP group,the cells were preconditioned with 15 min sevoflurane (final concentration 2.0 mmol/L) three times at 15 min interval and H/R model was established at 15 min after the end of preconditioning.At 3 h of reoxygenation,the level of superoxide anion was detected using lucigenin-enhanced chemiluminescence and DHE staining,and the release of dehydrogenase (LDH) and caspase3 activity were measured.Results Compared with group C,the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group H/R.Compared with group H/R,the release of LDH and level of superoxide anion were significantly decreased,and caspase-3 activity was weakened in group SP,and the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group A-H/R.Compared with group SP,the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group A-SP.Compared with group A-H/R,the release of LDH and level of superoxide anion were significantly decreased,and caspase-3 activity was weakened in group A-SP.Conclusion AMPK signaling pathway is involved in the mechanism by which sevoflurane preconditioning enhances the antioxidant capacity during H/R in the cardiomyocytes of the rats,but it does not play a major role. Key words: Protein kinases; Anesthetics, inhalation; Myocardial reperfusion injury; Antioxidants; Ischemic preconditioning

173 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

IntroductionInsulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from...

C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells.

In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress.

P51 Effect of type 1 diabetes on the production and vasoactivity of hydrogen sulfide in rat middle cerebral arteries

Hydrogen sulfide (H2S) is produced endogenously in vascular tissue and has both vasoregulation and antioxidant effects. This study examines the effect of diabetes-induced oxidative stress on H2S production and function in rat middle cerebral arteries. Diabetes was induced in rats with streptozotocin (50 mg/kg, i.v.). Middle cerebral artery function was examined using a small vessel myograph and superoxide anion generation measured using nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lucigenin-enhanced chemiluminescence. Cystathionine-c-lyase (CSE) mRNA expression was measured via RT-PCR. Diabetic rats had elevated blood glucose and significantly reduced cerebral artery endothelial function. Maximum vasorelaxation to the H2S donor NaHS was unaffected in diabetic cerebral arteries and was elicited via a combination of K + ,C l, and Ca 2+ channel modulation, although the contribution of Cl channels was significantly less in the diabetic cerebral arteries. Vasorelaxation to the H2S precursor L-cysteine and CSE mRNA were significantly increased in diabetic cerebral arteries. Cerebral artery superoxide production was significantly increased in diabetes, but this increase was attenuated ex vivo by incubation with the H2S donor NaHS. These data confirm that cerebral artery endothelial dysfunction and oxidative stress occurs in diabetes. Endogenous H2S production and activity is upregulated in cerebral arteries in this model of diabetes. Vasorelaxation responses to exogenous H2S are preserved and exogenous H2S attenuates the enhanced cerebral artery generated superoxide observed in the diabetic group. These data suggest that upregulation of endogenous H2S in diabetes may play an antioxidant and vasoprotective role.