Lucigenin-enhanced Chemiluminescence Research Articles

Polyphenol-containing azuki bean ( Vigna angularis) seed coats attenuate vascular oxidative stress and inflammation in spontaneously hypertensive rats

We investigated the effects of azuki bean ( Vigna angularis) seed coats (ABSC), which contain polyphenols, on the vascular oxidative stress and inflammation associated with hypertension. Spontaneously hypertensive rats (SHR) and control normotensive Wistar-Kyoto (WKY) rats were divided into 2 groups each. One group was fed 0% ABSC; the other, a 1.0% ABSC-containing diet. Tail systolic blood pressure (SBP) was examined throughout ABSC treatment. At 8 weeks, vascular superoxide (O 2 −) production was measured by lucigenin-enhanced chemiluminescence. mRNA expressions of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, macrophage chemoattractant protein-1 (MCP-1) and its receptor C-C chemokine receptor 2 (CCR2) in the aorta were analyzed by reverse transcriptase-polymerase chain reaction. Protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by western blotting. Polyphenol-containing ABSC suppressed the elevation of SBP throughout the treatment period. The NADPH-stimulated O 2 − level decreased significantly in the aorta of ABSC-treated SHR compared with the level of untreated SHR. The p47phox and Nox4 mRNA expression increased significantly in untreated SHR compared with that in WKY rats. Conversely, the level of p47phox mRNA was significantly lower in ABSC-treated SHR than in untreated SHR. The protein abundance of both iNOS and COX-2 was significantly decreased in the aorta of the ABSC-treated SHR compared with this abundance in untreated SHR. The MCP-1 and CCR2 mRNA expressions increased in untreated SHR, and these levels were significantly lower in ABSC-treated SHR. In conclusion, our results suggested that polyphenol-containing ABSC could attenuate vascular oxidative stress and inflammation during the progression of hypertension, and this may lead to an improvement in hypertension.

Pentaerythritol Tetranitrate Improves Angiotensin II–Induced Vascular Dysfunction via Induction of Heme Oxygenase-1

The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to the induction of the antioxidant enzyme heme oxygenase (HO)-1. With the present study, we tested whether chronic treatment with pentaerythritol tetranitrate can improve angiotensin II-induced vascular oxidative stress and dysfunction. In contrast to isosorbide-5 mononitrate (75 mg/kg per day for 7 days), treatment with pentaerythritol tetranitrate (15 mg/kg per day for 7 days) improved the impaired endothelial and smooth muscle function and normalized vascular and cardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NO synthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification of dihydroethidine oxidation products in angiotensin II (1 mg/kg per day for 7 days)-treated rats. The antioxidant features of pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-1 protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygen species formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in angiotensin II-treated rats, thereby preventing endothelial NO synthase uncoupling. Haploinsufficiency of HO-1 completely abolished the beneficial effects of pentaerythritol tetranitrate in angiotensin II-treated mice, whereas HO-1 induction by hemin (25 mg/kg) mimicked the effect of pentaerythritol tetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritol tetranitrate largely depends on the induction of the antioxidant enzyme HO-1 and identifies pentaerythritol tetranitrate, in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothelial function.

Neutrophil phenotype and function in partial hepatectomy in man

Hepatic resections are still associated with considerable morbidity mainly because of postoperative infection. Adequate function of neutrophils is a crucial element in host defense. The aim of the study was to characterize neutrophils during partial hepatectomy. Fourteen patients undergoing partial liver resection were enrolled. Twenty-four hours pre-, intra- (after induction of anesthesia, after preparation of the liver, and 15 min after release of the Pringle maneuver), as well as postoperatively (3 h after Pringle; 24, 48, and 120 h after surgery), blood samples were obtained. In addition, healthy volunteers (n = 5) were investigated. Adhesion molecules (CD 62, CD 18), Fcy receptors (CD 16, CD 32), and phagocytosis by neutrophils were characterized by fluorescence-activated cell sorter analysis. Spontaneous and stimulated (formyl-methionyl-leucyl-phenylalanine) oxygen radical generation was measured by lucigenin-enhanced chemiluminescence. Numeric upregulation of CD 62 and CD 18 on neutrophils was seen before the use of Pringle maneuver and persisted thereafter (p < 0.05). Spontaneous numeric expression of Fcy receptors (CD16 and CD 32) was unchanged during liver dissection but downregulated after Pringle maneuver was opened (p < 0.05). Although numeric Fcy receptors were downregulated, phagocytosis of heterologous opsonized Escherichia coli bacteria by neutrophils was unaffected. Spontaneous oxygen radical production peaked sharply 15 min after release of the Pringle maneuver (p < 0.05), contrary to stimulated oxygen radical production, which was depressed 3 h after the release of the Pringle maneuver (ns). Uneventful partial hepatectomy in man resulted already in a significant change in the phenotype but in less significant changes in the functions of neutrophils.

Atrial myocardial nox2 containing NADPH oxidase activity contribution to oxidative stress in mitral regurgitation: potential mechanism for atrial remodeling

Oxidative stress is linked with several cardiovascular diseases. However, the NADPH oxidase activity in severe mitral regurgitation patients with and without atrial fibrillation has not yet been explored. This study involved 16 adult patients (eight patients with persistent atrial fibrillation and eight with sinus rhythm) with severe mitral and moderate-to-severe tricuspid regurgitation and five control patients without mitral and tricuspid disease. Atrial tissues of the right and left atrial appendages were obtained during surgery. Superoxide anion production was measured by lucigenin-enhanced chemiluminescence, and the expression of nox2 containing NADPH oxidase mRNA was measured by quantitative real-time RT-PCR. Additionally, immunohistochemical study was performed. NADPH-stimulated superoxide release was significantly higher than basal superoxide production from right [5671.9±3498.7 vs. 232.7±70.0 relative light units per second per milligram of protein (RLU s(-1) mg protein(-1)), P=.008) and left atrial homogenates (6475.1±1890.8 vs. 229.0±79.6 RLU s(-1) mg protein(-1), P=.008) in atrial fibrillation patients. The NADPH-stimulated superoxide release from right atrial homogenates was also significantly higher than basal superoxide production in sinus patients (6809.1±1327.1 vs. 244.2±65.5 RLU s(-1) mg protein(-1), P=.008). Additionally, there was a borderline significant correlation between NADPH-stimulated superoxide production from left atrial homogenates and left atrial sizes (r=0.683, P=.062) in atrial fibrillation patients. Membrane-bound nox2 containing NADPH oxidase mRNA expression was increased and was similar in both the atrial fibrillation patients and sinus patients. The NADPH-stimulated superoxide production in right atrial homogenates in control atrial samples was 1863.7±137.2 RLU s(-1) mg protein(-1). Immunohistochemical study demonstrated increased expression of nox2 in myocytes with moderate-to-severe myolysis and hypertrophy. Results of this study demonstrate that membrane-bound nox2 containing NADPH oxidase activity and expression in the atrial myocardium is increased in patients with severe mitral regurgitation, possibly contributing to atrial remodeling in this clinical setting.

Effect of Korean Red Ginseng on Testicular Tissue Injury after Torsion and Detorsion

PurposeKorean red ginseng (KRG) is a potent antioxidant and a free radical scavenger. This study was designed to determine whether KRG could protect against dysfunction and oxidative stress induced by torsion-detorsion injury in rat testis.Materials and MethodsSix-week-old male Sprague-Dawley rats were randomly divided into four groups: a sham-operated control group (C), a sham-operated and KRG-treated group (K), a 2 hours torsion and detorsion group (T), and a 2 hours torsion and detorsion and KRG-treated group (T+K). We measured testis weight and hormone levels and reactive oxygen species (ROS) from the left renal vein. Superoxide generation was measured on the basis of lucigenin-enhanced chemiluminescence in testis tissue.ResultsTesticular weight was significantly higher in the T+K group than in the T group; however, there were no significant differences in hormone levels between the 4 groups. The mean level of ROS and superoxide production was significantly higher in the T group than in the C group, whereas administration of KRG attenuated this increase. Upon histologic evaluation, the T group was found to have cellular disarray, a lack of cellular cohesiveness, degenerative changes in the germinal cells, and less distinct changes in the seminiferous tubules, whereas the T+K group had a germinal epithelial layer that appeared nearly normal.ConclusionsThe present study demonstrated that KRG recovered the testis dysfunction in the rat testis by suppressing superoxide production.

Atorvastatin inhibits homocysteine-induced oxidative stress and apoptosis in endothelial progenitor cells involving Nox4 and p38MAPK

Previous studies showed that homocysteine (Hcy) reduces endothelial progenitor cells (EPCs) numbers and impairs functional activity. Atorvastatin, HMG-CoA inhibition has been showed to have protective effects on EPCs. Recent studies have demonstrated that reduced EPCs numbers and activity are associated with EPCs apoptosis. However, the protective mechanisms of atorvastatin on HHcy-induced EPCs apoptosis remain to be determined. This study was designed to examine the effect of atorvastatin on homocysteine-induced reactive oxygen species (ROS) production and apoptosis in EPCs. EPCs were isolated from peripheral blood and characterized, then challenged with Hcy (50–500μmol/L) in the presence or absence of atorvastatin (0.01–1μmol/L) or various stress signaling inhibitors, including mevalonate (100μmol/L), antioxidants N-acetyl cysteine (NAC, 10μmol/L), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI 10μmol/L), the eNOS inhibitor NGmono-methyl-l-arginine LNMA (1mmol/L), and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10μmol/L). Apoptosis was evaluated by FACS analysis and cell viability was determined by MTT assay. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Expression of Nox4 mRNA and p-p38MAPK protein was measured by RT-PCR and Western blot analysis, respectively. Our data revealed that atorvastatin significantly suppressed Hcy-induced ROS accumulation and EPCs apoptosis. Atorvastatin also antagonized homocysteine-induced activation of NADPH oxidase and overexpression of Nox4 mRNA and p-p38MAPK protein. Similar effects occurred with EPCs transfected with Nox4 siRNA. These findings demonstrated that atorvastatin may inhibit Hcy-induced NADPH oxidase activation, ROS accumulation, and EPCs apoptosis through Nox4/p38MAPK dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effects on EPCs function.

Abstract 5574: Cooperation of MT1-MMP and LOX-1 for RhoA- and Rac1-dependent Signaling Pathways in Oxidized LDL-mediated Endothelial Dysfunction and Atherosclerosis

Background: Membrane type 1-matrix metalloproteinase (MT1-MMP) functions not only as a proteolytic enzyme but also as a signaling molecule. Lectin-like oxidized-LDL receptor-1 (LOX-1) is a major receptor for oxidized LDL (ox-LDL). RhoA and Rac1 play crucial roles in endothelial dysfunction including eNOS downregulation and NADPH oxidase-dependent reactive oxygen species (ROS) generation. Hypothesis: MT1-MMP modifies ox-LDL-triggered RhoA- and Rac1-dependent endothelial dysfunction and atherosclerosis. Methods: GTP-loading of RhoA and Rac1 were assessed by pull-down assays. The activity of MT1-MMP was evaluated by fluorescent assay. A neutral antibody to LOX-1 (TS92) was used for inhibition of LOX-1, while MT1-MMP was silenced by siRNA. NADPH oxidase activity was determined by lucigenin-enhanced chemiluminescence. Fluorescent immunohistochemistry and immunoprecipitation were performed using the cultured endothelial cells (ECs) and the aortae of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. Results: Ox-LDL activated RhoA and Rac1 within 5 and 15 minutes, as well as the MT1-MMP activity of membrane fractions after 15 minutes in ECs, which was inhibited by TS92. Ox-LDL-induced activation of RhoA and the downstream events including endothelial cell invasion and eNOS downregulation were blocked by silencing of MT1-MMP. Knockdown of MT1-MMP also markedly attenuated ox-LDL-triggered Rac1 activation, NADPH oxidase activity and ROS generation. Interestingly, LOX-1 and MT1-MMP were partly co-localized in ECs by fluorescent immunohistochemistry, and immunoprecipitation demonstrated that MT1-MMP formed a complex with LOX-1. Furthermore, immunohistochemistry revealed that the two molecules were co-localized in ECs and in the aortic atherosclerotic lesions of WHHLMI rabbits. In addition, immunoprecipitation demonstrated that MT1-MMP and LOX-1 formed a complex in cultured ECs and in the atherosclerotic aortae of WHHLMI rabbits. Conclusions: We show new insights into modulation of endothelial dysfunction and atherosclerosis by an MT1-MMP-LOX-1 axis, suggesting that MT1-MMP is a cell surface modifier of ox-LDL-triggered RhoA and Rac1-activated signaling pathways through LOX-1.

Abstract 5522: Improvement of Angiotensin-II-Induced Vascular Dysfunction and Oxidative Stress by the Organic Nitrate Pentaerithrityl Tetranitrate Depends on Heme Oxygenase-1-Induction

Objective: Pentaerithrityl tetranitrate (PETN)-treatment is devoid of nitrate tolerance which has been attributed to the induction of the antioxidant enzyme heme oxygenase-1 (HO-1). With the present study we tested, whether chronic treatment with PETN can improve angiotensin-II (AT-II)-induced vascular oxidative stress and dysfunction. Methods and Results: In contrast to isosorbide mononitrate (ISMN, 75mg/kg/d/7d), co-treatment with PETN (15mg/kg/d/7d) improved impaired endothelial and smooth muscle function and normalized vascular and cardiac ROS production (mitochondria, NADPH oxidase activity and uncoupled endothelial nitric oxide synthase [eNOS]) as assessed by dihydroethidine (DHE)-staining, lucigenin-enhanced chemiluminescence and quantitative HPLC of DHE-oxidation products in AT-II (1mg/kg/d/7d) treated rats. The antioxidant features of PETN were recapulated in spontaneously hypertensive rats. In addition to increase in HO-1 protein expression, PETN but not ISMN augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase-I and dihydrofolate reductase in AT-II-treated rats, thereby preventing eNOS uncoupling. In AT-II-treated (0.1mg/kg/d/7d) HO-1+/− mice, PETN failed to be antioxidant, whereas HO-1 induction by hemin (25mg/kg) mimicked the PETN effect in AT-II-treated HO-1+/+ mice. Conclusions: PETN improves AT-II-induced vascular oxidative stress and dysfunction via induction of HO-1. Thus, PETN is the first organic nitrate to be beneficial in an experimental model of arterial hypertension.

Abstract 5224: CB1 Receptor Inhibition Leads to Decreased Vascular AT1 Receptor Expression, Inhibition of Oxidative Stress and Improved Endothelial Function

Inhibition of the cannabinoid receptor CB1 (CB1-R) exerts numerous effects such as modulation of blood pressure, insulin sensitivity and lipid concentrations. However, direct vascular effects of CB1-R inhibition remain unclear. Methods and Results: CB1-R expression was validated in vascular smooth muscle cells (VSMC) and aortic tissue of mice (real-time RT-PCR). Treatment of cultured VSMC with the selective CB1-R antagonist rimonabant resulted in reduced angiotensin II (ang II)-mediated reactive oxygen species (ROS) production (DCF fluorescence) and NADPH oxidase activity (lucigenin-enhanced chemiluminescence). Because rimonabant inhibited ang II-induced but not basal ROS production, and ang II activates NADPH oxidase through ang II type-1 receptor (AT1-R) stimulation, we analyzed AT1-R expression in VSMC. CB1-R inhibition with rimonabant or AM251 led to down-regulation of AT1-R protein expression (Western blot), stimulation with the CB1-R agonist CP55,940 resulted in AT1-R up-regulation, and co-incubation with CP55,940 abolished the effect of rimonabant, indicating that AT1-R expression is directly regulated by the CB1-R. The expression of rac1 and MnSOD was not affected. VSMC showed no significant expression of the CB2-R under basal conditions (real-time PCR, Western blot), and CB2-R inhibition had no impact on AT1-R expression in VSMC. To investigate the relevance of CB1-R inhibition in vivo, apolipoprotein E-deficient (ApoE−/−) mice were treated with cholesterol-rich diet and rimonabant or vehicle for 7 weeks. Consistent with the in vitro findings, CB1-R inhibition decreased AT1-R expression (real-time RT-PCR, Western blot), NADPH oxidase activity, and ROS production (L-012 chemiluminescence) in the aortic wall. Importantly, CB1-R inhibition improved endothelium-dependent vasodilation of isolated aortic segments (organ chamber experiments), indicating a functional relevance in vivo. Conclusions: CB1-R inhibition leads to decreased vascular AT1-R expression, NADPH oxidase activity and ROS production in vitro and in vivo. This antioxidative effect is associated with improved endothelial function in ApoE−/− mice, indicating beneficial direct vascular effects of CB1-R inhibition.

Intrarenal artery superoxide is mainly NADPH oxidase-derived and modulates endothelium-dependent dilation in elderly patients

The present study was performed to investigate the contribution of NADPH oxidases (Nox) to superoxide formation in human renal proximal resistance arteries and to test whether superoxide formation contributes to acute vasoconstrictor responses and endothelium-dependent vasodilation in these vessels. Arcuate and proximal interlobular artery segments were from patients who underwent nephrectomy because of a renal tumour. Vessels were dissected from tumour-free parts of the kidneys. Additional intrarenal arteries were obtained from rats. Superoxide formation was measured by lucigenin-enhanced chemiluminescence, expression of Nox isoforms was analysed by RT-PCR, and functional studies were performed by small vessel wire myography. Sixty per cent of superoxide formation in human arcuate and proximal interlobular arteries was due to Nox activity. mRNA expression analyses revealed the presence of Nox2 and Nox4 but not Nox1. Phenylephrine and endothelin-1 induced powerful concentration-dependent vasoconstrictions that were unaffected by superoxide scavengers. Vasopressin elicited small and variable vasoconstrictions with signs of tachyphylaxis. Endothelium-dependent vasodilation was blunted by tiron and Nomega-nitro-L-arginine methyl ester but not by superoxide dismutase or catalase. Exogenous hydrogen peroxide elicited vasoconstriction. Nox activity is the major source of superoxide formation in renal proximal resistance arteries from elderly patients. Acute vasoconstrictor responses to alpha1-adrenoreceptor activation and to endothelin-1 do not depend on superoxide formation, while endothelium-dependent vasodilation in intrarenal arteries is reactive oxygen species-dependent.

JAK/STAT Signaling Pathway Regulates Nox1 and Nox4-Based NADPH Oxidase in Human Aortic Smooth Muscle Cells

Oxidative stress mediated by Nox1- and Nox4-based NADPH oxidase (Nox) plays a key role in vascular diseases. The molecular mechanisms involved in the regulation of Nox are not entirely elucidated. Because JAK/STAT regulates many genes linked to inflammation, cell proliferation, and differentiation, we questioned whether this pathway is involved in the regulation of Nox1 and Nox4 in human aortic smooth muscle cells (SMCs). Cultured SMCs were exposed to interferon gamma (IFNgamma) for 24 hours. Using lucigenin-enhanced chemiluminescence and dihydroethidium assays, real-time polymerase chain reaction, and Western blot analysis, we found that JAK/STAT inhibitors significantly diminished the IFNgamma-dependent upregulation of Nox activity, Nox1 and Nox4 expression. In silico analysis revealed the presence of highly conserved GAS elements within human Nox1, Nox4, p22phox, p47phox, and p67phox promoters. Transient overexpression of STAT1/STAT3 augmented the promoter activities of each subunit. JAK/STAT blockade reduced the Nox subunits transcription. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1/STAT3 proteins with the predicted GAS elements from Nox1 and Nox4 promoters. JAK/STAT is a key regulator of Nox1 and Nox4 in human vascular SMCs. Inhibition of JAK/STAT pathway and the consequent Nox-dependent oxidative stress may be an efficient therapeutic strategy to reduce atherogenesis.

Aggregates of denatured proteins stimulate nitric oxide and superoxide production in macrophages

Denatured proteins are deposited in damaged tissues, around implanted biomaterials, during natural aging as well as in a heterogeneous group of amyloid diseases, such as Alzheimer's disease. There is evidence that tissue damage observed in amyloidosis is mediated mainly by factors released from activated macrophages, such as superoxide and nitric oxide (NO), as opposed to direct interaction between amyloid fibrils and nonimmune cells. Mouse resident peritoneal macrophages were stimulated in serum-free medium with different preparations of nonamyloidogenic proteins: alcohol dehydrogenase (AD), bovine serum albumin (BSA) or fibrinogen (FG). Intra- and extracellular superoxide production was measured by, respectively, nitro blue tetrazolium (NBT) reduction and lucigenin-enhanced chemiluminescence. Levels of nitrite (reflecting NO release) were measured in culture supernatants. Aggregates of denatured, nonamyloidogenic proteins, but not their native or denatured but not aggregated counterparts, stimulated superoxide and/or NO production in macrophages. The NO production was mediated by beta(1) and beta(2) integrins, with a possible contribution of receptor for advanced glycation end products (RAGE). It was catalyzed by inducible NO synthase (iNOS), enhanced synergistically by interferon-gamma (IFN-gamma), and inhibited by covalently modified proteins-components of advanced glycation end products. Although intracellular superoxide production was stimulated significantly by denatured BSA and AD, but not by FG, both denatured BSA and FG strongly enhanced zymosan-stimulated extracellular release of reactive oxygen species. Our results point at similarities in macrophage responses to denatured nonamyloidogenic proteins and to amyloid fibrils. Thus, the tissue injury observed in amyloidosis may result from overstimulation of mechanisms that, under physiological conditions, enable macrophages to recognize and remove denatured proteins.

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling

To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were cultured with Hcy (0.1-5 mmol/L) in the presence or absence of atorvastatin (1-100 micromol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 micromol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 micromol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis. Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 micromol/L. Atorvastatin (10 micromol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17+/-0.33 vs 4.34+/-0.31, P<0.05). Atorvastatin (10 micromol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57+/-0.49 vs 3.33+/-0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7+/-0.1 vs 2.22+/-0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580. Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function.

The novel NO redox sibling, nitroxyl (HNO), prevents cardiomyocyte hypertrophy and superoxide generation via cGMP

We have previously shown that NO•/cGMP signalling is an important antihyper-trophic mechanism in the heart [1-3]. HNO is the one electron reduction of NO•, thought to elicit cardiovascular actions via cGMP and/or calcitonin gene-related peptide (CGRP) [4]; we have recently shown that the HNO donor Angeli's salt inhibits cardiomyocyte hypertrophy and superoxide generation [5]. We now test the hypothesis that isopropylamine/NO (IPA/NO) elicits concentration-dependent anti-hypertrophic and antioxidant actions via HNO/sGC/cGMP-dependent signalling. IPA/NO (0.1–3 μM, replenished 3×/day over 48 h) elicited concentration-dependent inhibition of endothelin-1 (ET1, 60 nM)-stimulated neonatal rat cardiomyocyte (NRCM) hypertrophy (on two dimensional area of live cells). At 3 μM, IPA/NO decreased cell size from 255 ± 28% to 96 ± 27% of paired control (n = 4, p < 0.001). This antihypertrophic action of IPA/NO was significantly attenuated in the presence of the HNO scavenger L-cysteine (3 mM) or the cGMP-dependent protein kinase inhibitor Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by the NO scavenger carboxy-PTIO (200 μM) or the CGRP antagonist, CGRP8–37 (1 μM, both n = 4). For comparison, the NO• donor DEA/NO elicited similar concentration-dependent inhibition of ET1-induced cardiomyocyte hypertrophy; this was inhibited by carboxy-PTIO and Rp-8 PCTP cGMPS (10 μM, both n = 4, p < 0.05), but was unaffected by L-cysteine. Both IPA/NO and DEA/NO also blocked ET1-induced cardiomyocyte superoxide generation (both n = 4, p < 0.001, on NADPH-driven lucigenin-enhanced chemiluminescence), a key trigger of hypertrophy [3]. IPA-NO stimulated purified cell-free sGC activity by 3.2 ± 0.6-fold, and elevated NRCM cGMP content by 3.5 ± 0.4-fold (both n = 5, p < 0.05 via cGMP ELISA, as previously described [2,3]. None of these agents alone, or their respective vehicles, elicited any effect on NRCM. Finally, using an NO•-sensing electrode, we demonstrated that IPA/NO (in contrast to DEA/NO), does not release NO• under these conditions, even at supra-pharmacological concentrations. In conclusion, these results provide convincing evidence that IPA/NO prevents cardiomyocyte hypertrophy via HNO activation of sGC. Although the antihypertrophic and antioxidant efficacy of IPA/NO was comparable to NO•, there is no role for extracellular oxidation of HNO to NO• or CGRP-mediated signalling in these IPA/NO actions. These studies may ultimately facilitate the development of HNO donors such as IPA/NO as novel antihypertrophic therapy for patients at risk of heart failure.

3′,4′-Dihydroxyflavonol prevents diabetes-induced endothelial dysfunction in rat aorta

Aims Diabetes increases oxidant stress and impairs endothelium-dependent relaxation. We investigated whether the antioxidant 3′,4′-dihydroxyflavonol (DiOHF) reduces the release of superoxide (O 2 −) and preserves endothelial function in aortae from diabetic rats. Main methods Type-1 diabetes was induced in Sprague–Dawley rats by streptozotocin (STZ) treatment (55 mg/kg i.v.) and vascular reactivity and superoxide generation were assessed in aortic rings using standard organ bath techniques and lucigenin-enhanced chemiluminescence respectively. Key findings Eight weeks after STZ treatment blood glucose was elevated (39.4 ± 0.4 mM) compared to citrate treated control rats (5.5 ± 0.1 mM, P < 0.05) and there was an increased aortic generation of O 2 − (control 670 ± 101, diabetic 1535 ± 249 units/mg dry weight, P < 0.05). In aortic rings acetylcholine (ACh)-induced relaxation was impaired ( R max control 78 ± 2, diabetic 66 ± 3%, P < 0.01) whereas endothelium-independent relaxation to sodium nitroprusside (SNP) was unaffected ( R max control 100 ± 1, diabetic 101 ± 2%). When aortic rings were acutely exposed to DiOHF (10 − 5 M) there was a significant reduction in the detection of O 2 − (control 124 ± 15, diabetic 165 ± 21 units/mg, P < 0.01) and enhanced relaxation to ACh ( R max control 84 ± 3, diabetic 87 ± 3%). Two separate groups of rats (control and diabetic) were treated daily with DiOHF (5 mg/kg i.p.) for 7 days. DiOHF treatment reduced superoxide generation in diabetic aortae (untreated diabetic 1471 ± 358, DiOHF-treated diabetic 580 ± 115 units/mg, P < 0.05) and enhanced acetylcholine-induced relaxation ( R max untreated diabetic 58 ± 5, DiOHF-treated diabetic 71 ± 4%, P < 0.05). Significance DiOHF, acutely in vitro or after 1 week treatment in vivo, reduces oxidant stress and preserves endothelium-dependent relaxation in aortae from diabetic rats.

Increased cerebral ischemic damage during chronic alcohol consumption: role of NAD(P)H oxidase

We hypothesized that NAD(P)H oxidase may contribute to increased cerebral ischemic damage during chronic alcohol consumption. Sprague‐Dawley rats were randomly divided into four groups: nonalcohol‐fed, alcohol‐fed, nonalcohol‐fed plus apocynin‐treated, and alcohol‐fed plus apocynin‐treated. Alcohol (8 g/kg/day) was mixed into a liquid diet and given to rats for 8 weeks. Apocynin (7.5 mg/kg/day) was orally administrated for 4 weeks prior to the experiment. Rats were subjected to 2 hours of right middle cerebral artery occlusion (MCAO). Infarct volume and neurological score were assessed 24 hours after reperfusion. Superoxide production from ischemic border area was measured by lucigenin‐enhanced chemiluminescence. In addition, expression of NAD(P)H oxidase membrane subunit gp91phox in ischemic border area was analyzed by Western blot. Compared with nonalcohol‐fed rats, alcohol‐fed rats had a larger infarct volume, worse neurological score and higher superoxide production. Apocynin did not alter infarct size, neurological deficit or superoxide production in nonalcohol‐fed rats, but significantly reduced infarct size, improved neurological outcome and attenuated superoxide production in alcohol‐fed rats. Chronic alcohol consumption significantly increased gp91 phox expression compared with nonalcohol‐fed rats, and cerebral ischemia also significantly enhanced gp91 phox expression compared with nonischemic side. Apocynin treatment did not alter gp91 phox expression in either nonalcohol‐fed rats or alcohol‐fed rats. Our findings suggest that NAD(P)H oxidase may play an important role in increased cerebral ischemic damage during chronic alcohol consumption.

Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), is considered to be a risk factor for atherosclerosis. However, the mechanisms relating ADMA with vascular function have been evaluated in vitro and in animal models, but its effect in human vasculature is unclear. We examined the impact of serum ADMA on endothelial nitric oxide (NO) bioavailability and vascular superoxide radical (O2-) production in patients with advanced atherosclerosis. Paired samples of saphenous veins (SVs) and internal mammary arteries (IMAs) were collected from 201 patients undergoing coronary bypass surgery, and serum ADMA was measured pre-operatively. The vasomotor responses of SV segments to acetylcholine (ACh) and bradykinin (Bk) were evaluated ex vivo. Vascular O2- was measured in paired SV and IMA by lucigenin-enhanced chemiluminescence. The l-NAME-inhibitable as well as the NADPH-stimulated vascular O2- generation was also determined by chemiluminescence. High serum ADMA levels were associated with decreased vasorelaxation of SV to ACh (P < 0.05) and Bk (P < 0.05). Similarly, high serum ADMA was associated with higher total O2- production in both SVs and IMAs (P < 0.05) and greater L-NAME-inhibitable vascular O2- (P < 0.05). However, serum ADMA was not associated with NADPH-stimulated vascular O2-. In multivariable linear regression, serum ADMA was independently associated with vascular O2- in both SVs [beta (SE): 0.987 (0.412), P = 0.019] and IMAs [beta (SE): 1.905 (0.541), P = 0.001]. Asymmetrical dimethylarginine was also independently associated with maximum vasorelaxation in response to both ACh [beta (SE): 14.252 (3.976), P = 0.001] and Bk [beta (SE): 9.564 (3.762), P = 0.013]. This is the first study that demonstrates an association between ADMA and important measures of vascular function, such as vascular O2- production and NO bioavailability directly in human vessels. Although serum ADMA has no effect on NADPH-stimulated superoxide in intact vessels, it is associated with greater eNOS uncoupling in the human vascular endothelium of patients with coronary artery disease.

Roles and Mechanisms of Human Immunodeficiency Virus Protease Inhibitor Ritonavir and Other Anti-Human Immunodeficiency Virus Drugs in Endothelial Dysfunction of Porcine Pulmonary Arteries and Human Pulmonary Artery Endothelial Cells

The objective of this study was to determine the effects of highly active antiretroviral therapy (HAART) drugs on pulmonary endothelial function. Porcine pulmonary arteries or human pulmonary arterial endothelial cells (HPAECs) were incubated with eight HAART drugs [ritonavir, indinavir, lopinavir, zidovudine (AZT), abacavir, stavudine, didanosine (ddI), and lamivudine] individually or in combination [three HAART drugs (3-plex; indinavir, stavudine, and ddI)] at their clinical plasma concentrations for 24 hours. Endothelium-dependent vasorelaxation in response to bradykinin was reduced significantly by the ritonavir in a concentration-dependent manner. Five other HAART drugs (indinavir, lamivudine, abacavir, AZT, and ddI) and the 3-plex significantly also impaired endothelium-dependent vasorelaxation in response to bradykinin. Five HAART drugs (ritonavir, indinavir, lamivudine, abacavir, and AZT) significantly decreased endothelial nitric oxide synthase (eNOS) expression and increased superoxide anion levels in both vessels and HPAECs. Furthermore, both ritonavir and AZT substantially activated ERK2 in HPAECs. Additionally, the antioxidants ginsenoside Rb1 and ginkgolide A effectively reversed HAART drug-induced vasomotor dysfunction and eNOS down-regulation. Inhibition of ERK1/2 also partially blocked ritonavir- and AZT-induced down-regulation of eNOS and vasomotor dysfunction. Thus, HAART drugs significantly impair endothelial functions of porcine pulmonary arteries and HPAECs, which may be mediated by eNOS down-regulation, oxidative stress, and ERK1/2 activation. These findings suggest that HAART drugs may contribute to the high incidence of pulmonary artery hypertension in human immunodeficiency virus-infected patients.

Leukotrienes C4 and D4 sensitize human neutrophils for hyperreactivity to chemoattractants

To investigate the sensitizing effects of the cysteinyl leukotrienes (CysLTs) C(4) and D(4) on the proinflammatory responses of chemoattractant-activated human neutrophils in vitro. Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers. Cells were exposed to LTC(4) and LTD(4) (50-300 nM) prior to activation with 1 microM of N-formyl-L-methionyl- L-leucyl-L-phenylalanine (fMLF). A fura-2/AM-based spectrofluorimetric procedure, lucigenin-enhanced chemiluminescence (LECL), a colourimetric method and an ELISA procedure, were used to measure Ca(2+) mobilization, superoxide production, elastase and MMP-8 release respectively following activation of LTC(4)/ D(4)-primed neutrophils with fMLF. Superoxide generation was also measured in the presence and absence of the CysLT receptor 1 antagonist, montelukast (100 nM). Exposure of neutrophils to either LTC(4) or LTD(4) alone had modest effects on Ca(2+) mobilization, while superoxide generation and elastase release were unaffected. However, relative to the responses of neutrophils activated with fMLF in the absence of the CysLTs, pre-treatment of the cells with either LTC(4)or LTD(4) resulted in significant, augmentation of fMLF-activated elastase and MMP-8 release and superoxide generation, which was attenuated by montelukast. These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.

Free radical scavenger specifically prevents ischemic focal ventricular tachycardia

Focal ventricular tachycardia (VT) in acute myocardial ischemia is closely related to triggered activity (TA), which may be blocked by scavenging reactive oxygen species (ROS). This study analyzed effects of acutely administered ROS scavenger-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) on VT in vivo and TA in vitro. Forty-three alpha chloralose anesthetized dogs with coronary artery occlusion were studied. Three-dimensional activation mapping helped to locate the origin of focal or reentrant VT. TEMPO (30 mg/kg intravenously) or vehicle was given. Endocardium excised from the site of origin of VT was studied using standard microelectrode techniques and measures of ROS. Reentry and focal VT induction were both highly reproducible. TEMPO blocked focal VT in 6 of 11 dogs (P <.05), but 9 of 9 dogs with reentrant VT continued to have VT re-induced after TEMPO. TEMPO did not alter effective refractory period (168 +/- 3 to 171 +/- 3 ms), mean blood pressure (88 +/- 3 to 81 +/- 3 mm Hg), and size of ischemia (42% +/- 3% vs 40% +/- 4%). In vitro, TEMPO (10(-3) M, n = 14) produced no change in action potentials. Nevertheless, TA was reversibly attenuated from 5.3 +/- 1.1 to 0.4 +/- 0.4 complexes with TEMPO (n = 15, P <.05). Lucigenin-enhanced chemiluminescence and dihydroethidium staining showed increased ROS in ischemic endocardium; TEMPO dramatically reduced ROS in ischemic sites. TEMPO, a scavenger of ROS, prevented triggered activity associated with focal VT during myocardial ischemia in areas of increased ROS. Antioxidant therapy may play an important role in blockade of focal VT under the conditions of myocardial ischemia.