Stavros Selemidis

Publications of Stavros Selemidis

• Suppressing production of reactive oxygen species (ROS) for influenza A virus therapy.

  Authors: Ross Vlahos, John Stambas, Stavros Selemidis

  Trends in pharmacological sciences. 09/2011; 33(1):3-8.

  Influenza A viral infections claim millions of lives worldwide and continue to impose a major burden on healthcare systems. Current pharmacological strategies to control influenza A virus-inducedInfluenza A viral infections claim millions of lives worldwide and continue to impose a major burden on healthcare systems. Current pharmacological strategies to control influenza A virus-induced lung disease are problematic owing to antiviral resistance and the requirement for strain-specific vaccination. The production of reactive oxygen species (ROS), particularly superoxide, is an important host defence mechanism for killing invading pathogens. However, excessive superoxide may be detrimental following influenza A virus infection. Indeed, suppression of superoxide production by targeting the primary enzymatic source of superoxide in mammalian inflammatory cells, NADPH oxidase 2 (Nox2), markedly alleviates influenza A virus-induced lung injury and virus replication, irrespective of the infecting strain. Therefore, we propose that Nox2 oxidase inhibitors, in combination with current therapeutics (i.e. antivirals and vaccines), could be useful for suppression of influenza A virus-induced lung disease.

• Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets.

  Authors: Grant R Drummond, Stavros Selemidis, Kathy K Griendling, Christopher G Sobey

  Nature reviews. Drug discovery. 06/2011; 10(6):453-71.

  NADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS). The NOX1 (NADPH oxidase 1) and NOX2 oxidases are the major sources of ROS in the artery wall in conditions such asNADPH oxidases are a family of enzymes that generate reactive oxygen species (ROS). The NOX1 (NADPH oxidase 1) and NOX2 oxidases are the major sources of ROS in the artery wall in conditions such as hypertension, hypercholesterolaemia, diabetes and ageing, and so they are important contributors to the oxidative stress, endothelial dysfunction and vascular inflammation that underlies arterial remodelling and atherogenesis. In this Review, we advance the concept that compared to the use of conventional antioxidants, inhibiting NOX1 and NOX2 oxidases is a superior approach for combating oxidative stress. We briefly describe some common and emerging putative NADPH oxidase inhibitors. In addition, we highlight the crucial role of the NADPH oxidase regulatory subunit, p47phox, in the activity of vascular NOX1 and NOX2 oxidases, and suggest how a better understanding of its specific molecular interactions may enable the development of novel isoform-selective drugs to prevent or treat cardiovascular diseases.

• Inhibition of Nox2 oxidase activity ameliorates influenza A virus-induced lung inflammation.

  Authors: Ross Vlahos, John Stambas, Steven Bozinovski, Brad R S Broughton, Grant R Drummond, Stavros Selemidis

  PLoS pathogens. 01/2011; 7(2):e1001271.

  Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. WeInfluenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2(-/y) mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8(+) T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2(-/y) mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2(-/y) mice. The numbers of influenza-specific CD8+D(b)NP(366)+ and D(b)PA(224)+ T cells in the BALF and spleen were comparable in WT and Nox2(-/y) mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner.

• Mechanisms contributing to cerebral infarct size after stroke: gender, reperfusion, T lymphocytes, and Nox2-derived superoxide.

  Authors: Vanessa H Brait, Katherine A Jackman, Anna K Walduck, Stavros Selemidis, Henry Diep, Anja E Mast, Elizabeth Guida, Brad R S Broughton, Grant R Drummond, Christopher G Sobey

  Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 02/2010; 30(7):1306-17.

  Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provideCerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an approximately 15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was approximately sevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.

• Direct evidence of a role for Nox2 in superoxide production, reduced nitric oxide bioavailability and early atherosclerotic plaque formation in ApoE-/- mice.

  Authors: Courtney P Judkins, Henry Diep, Brad Rs Broughton, Anja E Mast, Elizabeth U Hooker, Alyson A Miller, Stavros Selemidis, Gregory J Dusting, Christopher G Sobey, Grant R Drummond

  American journal of physiology. Heart and circulatory physiology. 10/2009;

  The Nox family NADPH oxidases are reactive oxygen species (ROS) generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved.The Nox family NADPH oxidases are reactive oxygen species (ROS) generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here, we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE(-/-)) mice. Wild-type (C57Bl6/J), ApoE(-/-) and Nox2(-/y)/ApoE(-/-) mice were maintained on a high fat (21%) diet from 5 weeks-of-age until they were 12 or 19 weeks-old. Mice were sacrificed and their aortas removed for measurement of Nox2 expression (Western blotting, immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to N(omega)-nitro-L-arginine) and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE(-/-) mice prior to appearance of lesions, and this was associated with elevated ROS levels. In developing plaques, macrophages were also a prominent source of Nox2. Absence of Nox2 in Nox2(-/y)/ApoE(-/-) double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 weeks-of-age. However, en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2(-/y)/ApoE(-/-) versus ApoE(-/-) mice and this was associated with a marked decrease in aortic ROS production and increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability and early lesion development in ApoE(-/-) mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis. Key words: Nox isoforms, reactive oxygen species, endothelial dysfunction, atherogenesis.

• EVIDENCE THAT NITRIC OXIDE INHIBITS VASCULAR INFLAMMATION AND SUPEROXIDE PRODUCTION VIA A P47- DEPENDENT MECHANISM IN MICE.

  Authors: Craig B Harrison, Grant R Drummond, Christopher G Sobey, Stavros Selemidis

  Clinical and experimental pharmacology & physiology. 10/2009;

  Summary 1. Regulation of vascular Nox2-containing NADPH oxidase by p47(phox) plays a pivotal role in atherosclerotic lesion development through superoxide generation. Reduced vascular NOSummary 1. Regulation of vascular Nox2-containing NADPH oxidase by p47(phox) plays a pivotal role in atherosclerotic lesion development through superoxide generation. Reduced vascular NO bioavailability is a major contributing factor in the initiation of atherosclerosis as it leads to an increase in adhesion molecule expression for inflammatory cell recruitment into the vessel wall. 2. The aim of this study was to examine whether the anti-oxidant and anti-inflammatory effects of endogenous NO involve inhibition of NADPH oxidase- dependent superoxide production. 3. To inhibit endogenous NO production, male C57Bl/6 wild-type (WT) mice or age-matched p47(phox-/-) mice were treated with L-NAME (100 mg/kg/day for 4 weeks). Weekly blood pressure measurements were taken via tail cuffing. Basal and phorbol dibutyrate (PDB)-stimulated aortic superoxide production was detected using lucigenin- and L-012-enhanced chemiluminescence, respectively. Aortic Nox2, p47(phox) and VCAM-1 expression were measured via Western blotting. Plasma Ang II levels were determined using radioimmunoassay. 4. L-NAME-treated WT mice had significantly higher systolic blood pressure (n=6, P<0.05) and basal and stimulated aortic extracellular superoxide production (n=6-8, P<0.05), but lower plasma Ang II levels than vehicle-treated WT mice (n=4, P<0.05). Western blotting revealed no change in Nox2 expression following L-NAME treatment (n=6); however, significant increases in both aortic p47(phox) (n=6, P<0.05) and VCAM-1 expression (n=6, P<0.05) were observed. In p47(phox-/-) mice, L-NAME significantly increased systolic blood pressure (n=3-4; P<0.05) but failed to increase aortic superoxide production and VCAM-1 expression. 5. In conclusion, endogenous NO suppresses vascular inflammation, via inhibition of p47(phox) expression leading to attenuation of NADPH oxidase-dependent superoxide production.

• NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival.

  Authors: Hitesh Peshavariya, Gregory Dusting, Fan Jiang, Lesley Halmos, Christopher Sobey, Grant Drummond, Stavros Selemidis

  Naunyn-Schmiedeberg's archives of pharmacology. 05/2009;

  Proliferation and apoptosis of endothelial cells are crucial angiogenic processes that contribute to carcinogenesis and tumor progression. Emerging evidence implicates the regulation of proliferationProliferation and apoptosis of endothelial cells are crucial angiogenic processes that contribute to carcinogenesis and tumor progression. Emerging evidence implicates the regulation of proliferation and apoptosis by reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H(2)O(2)). In the present study, we investigated the roles of the ROS-generating Nox4- and Nox2-containing reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in proliferation of human endothelial cells by examining the impact of these enzyme systems on (1) specific proliferative and tumorigenic kinases, extracellular regulated kinase1/2 (ERK1/2) and Akt, (2) cytoskeletal organization, and (3) the mechanisms that influence cellular apoptosis. ROS production and the expression of NADPH oxidase subunit Nox4, but not Nox2, were markedly higher in proliferating than in quiescent endothelial cells. Addition of the H(2)O(2) scavenger catalase or downregulation of Nox4 protein with specific siRNA reduced ROS levels, cell proliferation, and ERK1/2 phosphorylation but had no effect on either cell morphology or caspase 3/7 activity. Although downregulation of Nox2 protein with siRNA also reduced ROS production and cell proliferation, it caused an increase in caspase 3/7 activity, reduced Akt phosphorylation, and caused cytoskeletal disorganization. Therefore, in endothelial cells, Nox4-derived H(2)O(2) activates ERK1/2 to promote proliferation, whereas Nox2-containing NADPH oxidase maintains the cytoskeleton and prevents apoptosis to support cell survival. Our study provides a new understanding of the molecular mechanisms that underpin endothelial cell survival and a rationale for the combined suppression of Nox4- and Nox2-containing NADPH oxidases for unwanted angiogenesis in cancer.

• Translation-linked mRNA destabilization accompanying serum-induced Nox4 expression in human endothelial cells.

  Authors: Hitesh Peshavariya, Fan Jiang, Caroline J Taylor, Stavros Selemidis, Catherine W T Chang, Greg Dusting

  Antioxidants & redox signaling. 05/2009;

  NADPH oxidase is involved in cell signaling regulating proliferation of vascular cells, especially endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest ofNADPH oxidase is involved in cell signaling regulating proliferation of vascular cells, especially endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest of cultured endothelial cells following serum deprivation paradoxically increases mRNA for Nox4. We have now investigated the relationships between Nox4 mRNA stability and protein expression in human microvascular endothelial cells. Serum starvation increased the steady-state level of Nox4 mRNA but paradoxically diminished Nox4 protein expression. mRNA transcription in the absence of serum is maintained by the p38MAP kinase pathway, for inhibition of p38MAP kinase reduced both Nox4 mRNA and Nox4 promoter activity. In serum-starved cells, reintroduction of serum increased Nox4 protein levels within 30 min and up to 24 hr. In contrast, the mRNA decreased equally rapidly after serum stimulation. Inhibition of Nox4 translation by cycloheximide blocked serum-induced mRNA degradation and Nox4 protein synthesis, and actinomycin-D also delayed Nox4 mRNA decay. Therefore, Nox4 mRNA level falls after serum stimulation because of a translation-initiated mRNA destabilization program. Clearly Nox4 mRNA is regulated at both transcriptional and post-transcriptional levels, and the steady state level of Nox4 mRNA does not accurately reflect Nox4 protein abundance and functions, with implications for regulation of cell proliferation and survival.

• Nox2-Containing NADPH Oxidase and Xanthine Oxidase Are Sources of Superoxide in Mouse Trachea.

  Authors: Alana Westover, Craig B Harrison, Stavros Selemidis

  Clinical and experimental pharmacology & physiology. 12/2008;

  1. Superoxide anion plays an important role in host defense against invading pathogens, and in the inflammation that arises in lungs. The aim of the present study was to elucidate whether the two key1. Superoxide anion plays an important role in host defense against invading pathogens, and in the inflammation that arises in lungs. The aim of the present study was to elucidate whether the two key candidate superoxide producing enzymes in mammalian cells, Nox2- containing NADPH oxidase and xanthine oxidase, are responsible for superoxide production in mouse trachea. 2. Superoxide production by the isolated trachea, as measured by L-012- dependent chemiluminescence, was markedly reduced by superoxide dismutase (300U/ml) and the xanthine oxidase inhibitor, allopurinol (100muM). Tracheas from Nox2(-/-) mice had significantly lower levels of superoxide than control mice. 3. These novel findings suggest that superoxide production by mouse trachea is attributed to both Nox2-containing NADPH oxidase and xanthine oxidase.

• Suppressing NADPH oxidase-dependent oxidative stress in the vasculature with nitric oxide donors.

  Authors: Stavros Selemidis

  Clinical and experimental pharmacology & physiology. 12/2008; 35(11):1395-401.

  1. Reactive oxygen species produced in the vasculature, including superoxide anion, contribute to the pathogenesis of cardiovascular disease states, such as atherosclerosis. A critical source of1. Reactive oxygen species produced in the vasculature, including superoxide anion, contribute to the pathogenesis of cardiovascular disease states, such as atherosclerosis. A critical source of superoxide is vascular NADPH oxidase and upregulation of this enzyme brings about the oxidative stress underlying atherosclerosis. Excessive superoxide in arteries directly inactivates endothelium-derived nitric oxide (NO), compromising its vasoprotective effects. 2. Given that a reduction in NO bioavailability is key in the pathophysiology of atherosclerosis, replacement of NO by exogenously administered NO donors may restore the deficit in NO during disease. Although the organic nitrate family of NO donors is often the first choice for the acute management of symptoms of atherosclerosis and angina pectoris, most of the compounds in this class are unsuitable for long-term therapy because they cause oxidative stress by activation and upregulation of vascular NADPH oxidase and induce tolerance to subsequent nitrate treatment and endogenous NO. These problems of nitrates have not only limited their therapeutic exploitation, but have also stifled interest in newer-generation NO donors. 3. Recent evidence indicates that, in stark contrast with the organic nitrates, the newer-age diazeniumdiolate NONOate class of NO donors suppress vascular NADPH oxidase-dependent superoxide production and are less likely to induce tolerance, making them more suitable for suppression of oxidative stress in atherosclerosis. 4. Here, it is hypothesized that NONOates provide a novel means of suppressing NADPH oxidase-dependent oxidative stress to restore vascular NO levels to prevent, and even reverse, atherosclerosis.

• NADPH oxidases in the vasculature: Molecular features, roles in disease and pharmacological inhibition.

  Authors: Stavros Selemidis, Christopher G Sobey, Harald H H W Schmidt, Kirstin Wingler, Grant R Drummond

  Pharmacology & therapeutics. 09/2008;

  Until the 1970s, reactive oxygen species (ROS) were considered merely harmful by-products of aerobic respiration and the driving force behind the evolution of an array of cellular antioxidant enzymesUntil the 1970s, reactive oxygen species (ROS) were considered merely harmful by-products of aerobic respiration and the driving force behind the evolution of an array of cellular antioxidant enzymes with the purpose of rapidly metabolising ROS to minimise their oxidising effects. However, the perception that ROS are only harmful to cells has since been questioned by a burgeoning body of evidence pointing to the existence of enzymes with the dedicated function of generating ROS. NADPH oxidases represent the only known family of enzymes whose sole purpose is to generate ROS. Members of this enzyme family are expressed across mammalian and non-mammalian cells, and influence a multitude of biological functions including host defence and redox signalling. However, although ROS are deliberately generated by NADPH oxidases during normal cell physiology, the observations that their expression and activity is markedly upregulated in the blood vessel wall in a number of cardiovascular 'high-risk' states (e.g. hypertension, hypercholesterolemia) implicates them in the oxidative stress that gives rise to artery disease and ultimately heart attacks and strokes. These observations highlight the fact that NADPH oxidases are important therapeutic targets in cardiovascular disease and that, hence, there is clearly a need for the development of selective inhibitors of these enzymes. Here we highlight the structural and biochemical characteristics of the NADPH oxidase family and then comprehensively review the literature on the currently available pharmacological inhibitors of these enzymes with a particular emphasis on their mechanisms of action, isoform selectivity and therapeutic potential in cardiovascular disease.

• B(2) kinin receptor activation is the predominant mechanism by which trypsin mediates endothelium-dependent relaxation in bovine coronary arteries.

  Authors: Grant R Drummond, Stavros Selemidis, Thomas M Cocks

  Naunyn-Schmiedeberg's archives of pharmacology. 08/2008; 378(1):33-41.

  The roles of kinin and protease-activated receptors (PAR) in endothelium-dependent relaxations to the serine protease, trypsin, were examined in rings of bovine left anterior descending coronaryThe roles of kinin and protease-activated receptors (PAR) in endothelium-dependent relaxations to the serine protease, trypsin, were examined in rings of bovine left anterior descending coronary artery (LAD). Trypsin (0.01-30 U/ml) caused biphasic, endothelium-dependent relaxations-a high potency (0.01-0.3 U/ml), low efficacy relaxation [maximum relaxation (R (max)), 9.0 +/- 5.1%] followed by a lower potency (1-30 U/ml) but high efficacy (R (max), 90.4 +/- 5.5%) relaxation, which was abolished by aprotinin. Captopril (10 muM) caused an ~10-fold leftward shift of the second phase response such that the first phase was masked. The second phase relaxation to trypsin was inhibited in a concentration-dependent, non-surmountable manner by the B(2) antagonist, HOE-140. At 3 nM HOE-140, the second phase response to trypsin was abolished unmasking the first phase. Kallikrein (0.0003-0.3 U/ml) caused monophasic, endothelium-dependent relaxations (R (max), 33.7 +/- 14.6%), which were potentiated by captopril (R (max), 94.2 +/- 1.0%) and abolished by HOE-140. In the presence of captopril, the second phase relaxation to trypsin was only minimally inhibited by either N(G)-nitro-L: -arginine (100 muM) or 67 mM [K(+)](o) alone but markedly reduced when these two treatments were combined (R (max), 26.1 +/- 11.6% versus 98.6 +/- 2.9% in controls). The PAR1-activating peptide, SFLLRN (0.1-30 muM), but not the PAR2-activating peptide, SLIGRL, caused concentration-dependent relaxations (pEC(50), 5.9 +/- 0.0%; R (max), 43.3 +/- 8.3%). In conclusion, trypsin causes endothelium-dependent relaxations in the bovine LAD predominantly via release of endogenous BK, which in turn activates endothelial B(2) receptors. Only a minor role for PAR1-like receptors was evident in this tissue.

• Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells.

  Authors: Stavros Selemidis, Gregory J Dusting, Hitesh Peshavariya, Barbara K Kemp-Harper, Grant R Drummond

  Cardiovascular research. 08/2007; 75(2):349-58.

  OBJECTIVE: Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here weOBJECTIVE: Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here we investigate the regulation of NADPH oxidase activity by nitric oxide (NO). METHODS: Human cultured microvascular endothelial cells (HMEC-1) were treated with the NO donors, diethylenetriamine (DETA)-NONOate, S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 0.5-24 h. Superoxide production was measured by lucigenin chemiluminescence and dihydroethidium fluorescence, while NADPH oxidase subunit expression was measured via Western blotting. S-nitrosylation was assessed using the 2,3-diaminonapthalene (DAN) assay, and via immunoblotting with an anti-nitrosocysteine antibody. RESULTS: Specific siRNA reduced Nox2 and Nox4 protein expression and markedly decreased superoxide production in HMEC-1. DETA-NONOate (10-300 micromol/L) suppressed superoxide production in HMEC-1 in a concentration- and time-dependent manner, which was not entirely attributable to stoichiometric reaction with NO, for the effect was observed more than 6 h after removing DETA-NONOate from solution. Similarly, sustained attenuation of superoxide production was achieved with SNP (10-100 micromol/L) and SNAP (10-100 micromol/L). The suppressive effect of NO was not dependent on (1) the sGC/cGMP/PKG pathway, (2) peroxynitrite-formation, (3) reduced protein expression of NADPH oxidase subunits or (4) dissociation of NADPH oxidase subunits. Treatment with NO caused S-nitrosylation of the crucial organizer subunit p47phox, and de-nitrosylation with UV light restored superoxide production. CONCLUSIONS: NO causes sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells by S-nitrosylation of p47phox. These findings highlight a novel approach by which vascular oxidative stress might be suppressed by NO donors.

• Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase.

  Authors: Hitesh M Peshavariya, Gregory James Dusting, Stavros Selemidis

  Free radical research. 07/2007; 41(6):699-712.

  All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, weAll methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 microM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 +/- 6% and Nox4 siRNA 83 +/- 7% of control) and lucigenin chemiluminescence (Nox2; 54 +/- 6% and Nox4 74 +/- 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.

• Smooth muscle mediates circumferential conduction of hyperpolarization and relaxation to focal endothelial cell activation in large coronary arteries.

  Authors: Stavros Selemidis, Thomas Cocks

  Naunyn-Schmiedeberg's archives of pharmacology. 05/2007; 375(2):85-94.

  Longitudinal conduction of endothelium-dependent vasodilatation is mediated by intercellular spread of hyperpolarization via gap junctions along the endothelium. If similar electrical signals fromLongitudinal conduction of endothelium-dependent vasodilatation is mediated by intercellular spread of hyperpolarization via gap junctions along the endothelium. If similar electrical signals from the endothelium conduct around the circumference of arteries via smooth muscle cells, then, both longitudinal and circumferential spread of such signals would make it possible for a wide annulus of a large blood vessel like an epicardial coronary artery to dilate to local stimuli. To examine this in vitro, we developed a dual-chambered organ bath in which both membrane potential and force are independently determined in endothelium-intact and -denuded regions of a single annulus of artery. Hyperpolarizations and relaxations to endothelium-dependent vasodilators like bradykinin (BK) and substance P in smooth muscle cells immediately beneath the local endothelium-intact region (local responses) are conducted via smooth muscle cells around the circumference of the artery. The local relaxation was partially inhibited by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG), and subsequently abolished by further treatment with a combination of two characteristic inhibitors of endothelium-dependent hyperpolarization-the Ca2+ -activated potassium channel (KCa) channel inhibitors, apamin and charybdotoxin. The conducted hyperpolarizations and relaxations to BK were unaffected by L-NOARG, but were abolished by apamin and charybdotoxin. In conclusion, these studies demonstrate for the first time that NO acts only as a local vasodilator, whereas endothelium-dependent hyperpolarization (EDH) causes local and remote vasodilatation in large coronary arteries. We propose that such a remote EDH-dependent signalling mechanism compensates for the loss of the local NO-dependent vasodilatation in diseased arteries.

• The 'A's and 'O's of NADPH oxidase regulation: a commentary on "Subcellular localization and function of alternatively spliced Noxo1 isoforms".

  Authors: Nils Opitz, Grant R Drummond, Stavros Selemidis, Sabine Meurer, Harald H H W Schmidt

  Free radical biology & medicine. 02/2007; 42(2):175-9.

• Mechanisms for suppressing NADPH oxidase in the vascular wall.

  Authors: Gregory J Dusting, Stavros Selemidis, Fan Jiang

  Memórias do Instituto Oswaldo Cruz. 04/2005; 100 Suppl 1:97-103.

  Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase.Oxidative stress underlies many forms of vascular disease as well as tissue injury following ischemia and reperfusion. The major source of oxidative stress in the artery wall is an NADPH oxidase. This enzyme complex as expressed in vascular cells differs from that in phagocytic leucocytes both in biochemical structure and functions. The crucial flavin-containing catalytic subunits, Nox1 and Nox4, are not found in leucocytes, but are highly expressed in vascular cells and upregulated with vascular remodeling, such as that found in hypertension and atherosclerosis. The difference in catalytic subunits offers the opportunity to develop "vascular specific" NADPH oxidase inhibitors that do not compromise the essential physiological signaling and phagocytic functions carried out by reactive oxygen and nitrogen species. Nitric oxide and targeted inhibitors of NADPH oxidase that block the source of oxidative stress in the vasculature are more likely to prevent the deterioration of vascular function that leads to stroke and heart attack, than are conventional antioxidants. The roles of Nox isoforms in other inflammatory conditions are yet to be explored.

• Endothelium-dependent hyperpolarization as a remote anti-atherogenic mechanism.

  Authors: Stavros Selemidis, Thomas M Cocks

  Trends in pharmacological sciences. 06/2002; 23(5):213-20.

  Endothelial cell injury and the loss of cytoprotective mechanisms that involve nitric oxide, prostacyclin and endothelium-dependent hyperpolarization (EDH) are thought to underlie atherosclerosis,Endothelial cell injury and the loss of cytoprotective mechanisms that involve nitric oxide, prostacyclin and endothelium-dependent hyperpolarization (EDH) are thought to underlie atherosclerosis, although how these mechanisms are anti-atherogenic is unclear. This is particularly so because thrombus formation, one of the major initiators of the disease, usually occurs at discrete luminal sites; thus, only small numbers of endothelial cells can be recruited to initiate anti-inflammatory responses. However, we, and others, have demonstrated that locally generated EDH spreads to endothelial cells and smooth muscle cells throughout a vessel to cause remote vasodilatation. In this article, we propose that, in addition to a widespread inhibitory signalling mechanism, EDH produced by the endothelium also initiates remote anti-inflammatory actions that prevent large blood vessels developing atherosclerosis.

• NADPH oxidases in the vasculature: Molecular features, roles in disease and pharmacological inhibition

  Authors: Stavros Selemidis, Christopher G. Sobey, Kirstin Wingler, Harald H.H.W. Schmidt, Grant R. Drummond

  Pharmacology & Therapeutics.

  Until the 1970s, reactive oxygen species (ROS) were considered merely harmful by-products of aerobic respiration and the driving force behind the evolution of an array of cellular antioxidant enzymesUntil the 1970s, reactive oxygen species (ROS) were considered merely harmful by-products of aerobic respiration and the driving force behind the evolution of an array of cellular antioxidant enzymes with the purpose of rapidly metabolising ROS to minimise their oxidising effects. However, the perception that ROS are only harmful to cells has since been questioned by a burgeoning body of evidence pointing to the existence of enzymes with the dedicated function of generating ROS. NADPH oxidases represent the only known family of enzymes whose sole purpose is to generate ROS. Members of this enzyme family are expressed across mammalian and non-mammalian cells, and influence a multitude of biological functions including host defence and redox signalling. However, although ROS are deliberately generated by NADPH oxidases during normal cell physiology, the observations that their expression and activity is markedly upregulated in the blood vessel wall in a number of cardiovascular ‘high-risk’ states (e.g. hypertension, hypercholesterolemia) implicates them in the oxidative stress that gives rise to artery disease and ultimately heart attacks and strokes. These observations highlight the fact that NADPH oxidases are important therapeutic targets in cardiovascular disease and that, hence, there is clearly a need for the development of selective inhibitors of these enzymes. Here we highlight the structural and biochemical characteristics of the NADPH oxidase family and then comprehensively review the literature on the currently available pharmacological inhibitors of these enzymes with a particular emphasis on their mechanisms of action, isoform selectivity and therapeutic potential in cardiovascular disease.