Grant R Drummond

Publications of Grant R Drummond

• Importance of T lymphocytes in brain injury, immunodeficiency, and recovery after cerebral ischemia.

  Authors: Vanessa H Brait, Thiruma V Arumugam, 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. 02/2012;

  Following an ischemic stroke, T lymphocytes become activated, infiltrate the brain, and appear to release cytokines and reactive oxygen species to contribute to early inflammation and brain injury.Following an ischemic stroke, T lymphocytes become activated, infiltrate the brain, and appear to release cytokines and reactive oxygen species to contribute to early inflammation and brain injury. However, some subsets of T lymphocytes may be beneficial even in the early stages after a stroke, and recent evidence suggests that T lymphocytes can also contribute to the repair and regeneration of the brain at later stages. In the hours to days after stroke, T-lymphocyte numbers are then reduced in the blood and in secondary lymphoid organs as part of a 'stroke-induced immunodeficiency syndrome,' which is mediated by hyperactivity of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, resulting in increased risk of infectious complications. Whether or not poststroke T-lymphocyte activation occurs via an antigen-independent process, as opposed to a classical antigen-dependent process, is still controversial. Although considerable recent progress has been made, a better understanding of the roles of the different T-lymphocyte subpopulations and their temporal profile of damage versus repair will help to clarify whether T-lymphocyte targeting may be a viable poststroke therapy for clinical use.Journal of Cerebral Blood Flow & Metabolism advance online publication, 1 February 2012; doi:10.1038/jcbfm.2012.6.

• Chlamydia pneumoniae induces a pro-inflammatory phenotype in murine vascular smooth muscle cells independently of elevating reactive oxygen species.

  Authors: Jennifer Rivera, Anna K Walduck, Richard A Strugnell, Christopher G Sobey, Grant R Drummond

  Clinical and experimental pharmacology & physiology. 12/2011; 39(3):218-26.

  NADPH oxidases (Nox) are reactive oxygen species (ROS)-generating enzymes that play important physiological roles in host defence and redox signalling. However, Nox activity is upregulated in theNADPH oxidases (Nox) are reactive oxygen species (ROS)-generating enzymes that play important physiological roles in host defence and redox signalling. However, Nox activity is upregulated in the vascular wall during atherosclerosis and contributes to plaque formation by promoting oxidative stress and inflammation. The bacterium Chlamydia pneumoniae has been detected in vascular smooth muscle cells (VSMC) of human atheroma. We hypothesized that C. pneumoniae infection of VSMC causes Nox activation, which initially limits infection but ultimately causes oxidative stress, activation of pro-inflammatory pathways and an atherogenic phenotype. Chlamydia pneumoniae infection of mouse cultured VSMC significantly increased ROS production by twofold but did not upregulate mRNA expression of Nox1 or Nox4. Chlamydia pneumoniae did increase Nox2 mRNA levels significantly by threefold, but this did not translate to elevated Nox2 protein expression. The Nox inhibitor gp91ds-tat had no effect on C. pneumoniae-induced ROS production. In contrast, apocynin significantly reduced ROS levels by 75% in C. pneumoniae-infected VSMC, an effect most likely attributable to its direct anti-oxidant action. Although apocynin had no effect on C. pneumoniae-induced expression of inflammatory markers, bacteria recovered from apocynin-treated VSMC displayed a higher degree of infectivity in HEp-2 cells. In conclusion, C. pneumoniae infection increases ROS production in VSMC independently of Nox activity. Although elevated ROS production appears to serve a protective role by limiting the spread of infection, we speculate that this response will be detrimental over the long term by causing oxidative stress and a smouldering inflammatory response by maintaining C. pneumoniae persistence within the cell.

• Vasorelaxant and antiaggregatory actions of the nitroxyl donor isopropylamine NONOate are maintained in hypercholesterolemia.

  Authors: Michelle L Bullen, Alyson A Miller, Janahan Dharmarajah, Grant R Drummond, Christopher G Sobey, Barbara K Kemp-Harper

  American journal of physiology. Heart and circulatory physiology. 07/2011; 301(4):H1405-14.

  Nitroxyl (HNO) displays pharmacological and therapeutic actions distinct from those of its redox sibling nitric oxide (NO(•)). It remains unclear, however, whether the vasoprotective actions of HNONitroxyl (HNO) displays pharmacological and therapeutic actions distinct from those of its redox sibling nitric oxide (NO(•)). It remains unclear, however, whether the vasoprotective actions of HNO are preserved in disease. The ability of the HNO donor isopropylamine NONOate (IPA/NO) to induce vasorelaxation, its susceptibility to tolerance development, and antiaggregatory actions were compared with those of a clinically used NO(•) donor, glyceryl trinitrate (GTN), in hypercholesterolemic mice. The vasorelaxant and antiaggregatory properties of IPA/NO and GTN were examined in isolated carotid arteries and washed platelets, respectively, from male C57BL/6J mice [wild-type (WT)] maintained on either a normal diet (WT-ND) or high fat diet (WT-HFD; 7 wk) as well as apolipoprotein E-deficient mice maintained on a HFD (ApoE(-/-)-HFD; 7 wk). In WT-ND mice, IPA/NO (0.1-30 μmol/l) induced concentration-dependent vasorelaxation and inhibition of collagen (30 μg/ml)-stimulated platelet aggregation, which was predominantly soluble guanylyl cyclase/cGMP dependent. Compared with WT-HFD mice, ApoE(-/-)-HFD mice displayed an increase in total plasma cholesterol levels (P < 0.001), vascular (P < 0.05) and platelet (P < 0.05) superoxide (O(2)(·-)) production, and reduced endogenous NO(•) bioavailability (P < 0.001). Vasorelaxant responses to both IPA/NO and GTN were preserved in hypercholesterolemia, whereas vascular tolerance developed to GTN (P < 0.001) but not to IPA/NO. The ability of IPA/NO (3 μmol/l) to inhibit platelet aggregation was preserved in hypercholesterolemia, whereas the actions of GTN (100 μmol/l) were abolished. In conclusion, the vasoprotective effects of IPA/NO were maintained in hypercholesterolemia and, thus, HNO donors may represent future novel treatments for vascular diseases.

• 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.

• Chemokine-related gene expression in the brain following ischemic stroke: no role for CXCR2 in outcome.

  Authors: Vanessa H Brait, Jennifer Rivera, Brad R S Broughton, Seyoung Lee, Grant R Drummond, Christopher G Sobey

  Brain research. 02/2011; 1372:169-79.

  This study sought to identify potential targets for acute stroke therapy that can be exploited pharmacologically beyond the current 4.5h time limit for clinical administration of recombinantThis study sought to identify potential targets for acute stroke therapy that can be exploited pharmacologically beyond the current 4.5h time limit for clinical administration of recombinant tissue-plasminogen activator. We used PCR arrays to initially screen the temporal expression profiles of several chemokine-related genes in the brain at 4, 24 and 72h after stroke. We identified large increases (>10-fold) in mRNA at 24 or 72h for the neutrophil CXCR2 receptor, and for CXCL1 and CXCL2-two chemokine ligands expressed by monocytes and neutrophils with strong neutrophil chemoattractant activity via CXCR2. We then tested the efficacy of a CXCR2 antagonist as a therapeutic. Mice were treated with vehicle (1% DMSO) or SB225002 (2mg/kg per day, ip) commencing at reperfusion, and we evaluated chemokine gene expression, neutrophil infiltration and functional and histological endpoints of stroke outcome. Expression levels of CXCL1, CXCL2 and CXCR2 after 24h were markedly reduced to near normal levels in SB225002-treated mice. Myeloperoxidase-positive cell infiltration was significantly reduced in SB225002-treated mice compared with vehicle-treated mice, and was similar to levels in sham-operated mice. However, although SB225002 evidently antagonised the interaction between CXCR2 and its chemokine ligands in the ischemic brain, mice treated with either SB225002 or vehicle had similar motor impairment and infarct volume at 72h. Thus, the reduced expression of CXC chemokine subfamily genes and neutrophil-related infiltration following SB225002 administration did not improve outcome after cerebral ischemia-reperfusion. CXCR2 antagonists are therefore unlikely to be a potential therapy for ischemic stroke.

• Oxidative stress and endothelial dysfunction in cerebrovascular disease.

  Authors: Sophocles Chrissobolis, Alyson A Miller, Grant R Drummond, Barbara K Kemp-Harper, Christopher G Sobey

  Frontiers in bioscience : a journal and virtual library. 01/2011; 16:1733-45.

  Maintenance of vascular tone by the endothelium involves the production of endothelium-derived nitric oxide (NO). NO, produced from endothelial nitric oxide synthase diffuses to the underlying smoothMaintenance of vascular tone by the endothelium involves the production of endothelium-derived nitric oxide (NO). NO, produced from endothelial nitric oxide synthase diffuses to the underlying smooth muscle to stimulate soluble guanylate cyclase, resulting in increased cyclic GMP levels, and subsequent smooth muscle relaxation and blood vessel dilatation. Endothelial dysfunction, manifested as diminished NO bioavailability, is a common feature of a number of vascular-related diseases.. Oxidative stress can be defined as an imbalance between reactive oxygen species (ROS) production and/or impaired ROS metabolism that favours them being present in excess of physiological levels. Oxidative stress can negatively impact many cell types, including in the vasculature. There is now a wealth of evidence suggesting that oxidative stress is a major cause of endothelial dysfunction in the cerebral circulation. This review will summarize disease models in which both oxidative stress and endothelial dysfunction occur in the cerebral circulation, namely hypertension involving angiotensin II (Ang II), diabetes, subarachnoid hemorrhage, stroke and Alzheimer's disease. Molecular mechanisms by which oxidative stress occurs, (eg increased NADPH-oxidase activity) will also be discussed.

• 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.

• Nox2 oxidase activity accounts for the oxidative stress and vasomotor dysfunction in mouse cerebral arteries following ischemic stroke.

  Authors: T Michael De Silva, Vanessa H Brait, Grant R Drummond, Christopher G Sobey, Alyson A Miller

  PloS one. 01/2011; 6(12):e28393.

  Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms ofPost-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2(-/-)) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting. Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2(-/-) mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2(-/-) mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2(-/-) mice. Vascular CD45 levels were unchanged by ischemia-reperfusion. Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.

• The anti-platelet effects of apocynin in mice are not mediated by inhibition of NADPH oxidase activity.

  Authors: Janahan Dharmarajah, Jane F Arthur, Christopher G Sobey, Grant R Drummond

  Naunyn-Schmiedeberg's archives of pharmacology. 10/2010; 382(4):377-84.

  Apocynin, or a (myelo)peroxidase-derived product thereof, is a powerful inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Apocynin has also been shown to prevent aggregationApocynin, or a (myelo)peroxidase-derived product thereof, is a powerful inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Apocynin has also been shown to prevent aggregation of platelets in response to agonists such as collagen and thrombin. The aims of this study were to establish whether NADPH oxidase activity is required for aggregation of murine platelets to collagen and other agonists and whether the anti-aggregatory effects of apocynin are due to an inhibitory action against this enzyme. Washed platelets were isolated from male C57BL6 (wild-type), Nox2-deficient (Nox2(-/y )), and p47phox-deficient (p47phox(-/-)) mice for assessment of aggregation and NADPH oxidase subunit (Nox2, p47phox) expression. Collagen and U46619 elicited aggregation of murine platelets, and these responses were inhibited by apocynin at concentrations ≥100 μM. By contrast, aggregations to a direct protein kinase C activator, phorbol-12,13-dibutyrate, were insensitive to apocynin. Immunoblotting of platelet protein homogenates from wild-type mice with anti-Nox2 or p47phox antibodies revealed strong bands at 58 and 50 kDa, respectively. While expression of these immunoreactive bands was greatly diminished in platelets from Nox2(-/y ) and p47phox(-/-) mice, collagen still elicited aggregations that were similar to those observed in platelets from wild-types. Moreover, apocynin was an equally effective inhibitor of aggregation in platelets from all three mouse strains. In conclusion, these data suggest that NADPH oxidase-derived reactive oxygen species play no role in the aggregation response of washed murine platelets to collagen. Thus, our observation that apocynin is a powerful inhibitor of platelet aggregation raises further questions about the selectivity of this drug as an NADPH oxidase inhibitor.

• Reduced cerebrovascular remodeling and functional impairment in spontaneously hypertensive rats following combined treatment with suboptimal doses of telmisartan and ramipril: is less really more?

  Authors: Klaudia Budzyn, Christopher G Sobey, Grant R Drummond

  Journal of hypertension. 07/2010; 28(7):1384-9.

• 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.

• Augmented superoxide production by Nox2-containing NADPH oxidase causes cerebral artery dysfunction during hypercholesterolemia.

  Authors: Alyson A Miller, T Michael De Silva, Courtney P Judkins, Henry Diep, Grant R Drummond, Christopher G Sobey

  Stroke; a journal of cerebral circulation. 02/2010; 41(4):784-9.

  We tested the hypothesis that elevated superoxide production by Nox2-NADPH oxidase occurs in cerebral arteries during hypercholesterolemia and causes decreased nitric oxide function. Wild-type (WT),We tested the hypothesis that elevated superoxide production by Nox2-NADPH oxidase occurs in cerebral arteries during hypercholesterolemia and causes decreased nitric oxide function. Wild-type (WT), apolipoprotein E-deficient (ApoE(-/-)) and Nox2(-/-)/ApoE(-/-) mice were fed a high-fat diet for 7 to 14 weeks. Basal superoxide production by cerebral arteries was measured using L-012 (100 micromol/L)-enhanced chemiluminescence. Nitric oxide function was assessed in isolated middle cerebral arteries through the constrictor response to N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L). Western blotting was used to measure protein expression of Nox2, p47phox, endothelial nitric oxide synthase, and superoxide dismutases (1-3). Morphology of cerebral arteries was similar in WT and ApoE(-/-) mice. In ApoE(-/-), but not Nox2(-/-)/ApoE(-/-) mice, superoxide production by cerebral arteries was approximately 50% greater than in WT mice (P<0.05). Moreover, the magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE(-/-) mice was <50% of that in WT mice (P<0.05), whereas in Nox2(-/-)/ApoE(-/-) mice, the contractile response was comparable to WT responses. In the presence of the superoxide scavenger, tempol (1 mmol/L), L-NAME-induced contractions of middle cerebral arteries were similar between WT and ApoE(-/-) mice. Expression of p47phox was approximately 2-fold higher in ApoE(-/-) versus WT mice, whereas Nox2, endothelial nitric oxide synthase, and superoxide dismutase isoforms were unchanged. Elevated superoxide production and reduced basal nitric oxide-mediated relaxation occur in cerebral arteries of hypercholesterolemic mice even in the absence of lesions. These changes appear to be exclusively due to increased activity of Nox2-NADPH oxidase, possibly through increased expression of its regulatory subunit p47phox.

• Nox isoforms in vascular pathophysiology: insights from transgenic and knockout mouse models.

  Authors: Jennifer Rivera, Christopher G Sobey, Anna K Walduck, Grant R Drummond

  Redox report : communications in free radical research. 01/2010; 15(2):50-63.

  Elevated reactive oxygen species (ROS) formation in the vascular wall is a key feature of cardiovascular diseases and a likely contributor to oxidative stress, endothelial dysfunction and vascularElevated reactive oxygen species (ROS) formation in the vascular wall is a key feature of cardiovascular diseases and a likely contributor to oxidative stress, endothelial dysfunction and vascular inflammation. The NADPH oxidases are a family of ROS generating enzymes, of which four members (Nox1, Nox2, Nox4 and Nox5) are expressed in blood vessels. Numerous studies have demonstrated that expression and activity of at least two isoforms of NADPH oxidase - Nox1 and Nox2 - are up-regulated in animal models of hypertension, diabetes and atherosclerosis. However, these observations are merely suggestive of a role for NADPH oxidases in vessel pathology and by no means establish cause and effect. Furthermore, questions surrounding the specificity of current pharmacological inhibitors of NADPH oxidase mean that findings obtained with these compounds must be viewed with caution. Here, we review the literature on studies utilising genetically-modified mouse strains to investigate the roles of NADPH oxidases in experimental models of vascular disease. While several studies on transgenic over-expressing or knockout mice support roles for Nox1- and/or Nox2-containing oxidases as sources of excessive vascular ROS production and causes of endothelial dysfunction in hypertension, atherosclerosis and diabetes, there are still no published reports on the effects of genetic modification of Nox4 or Nox5 in vascular or indeed any other contexts. Further understanding of the roles of specific isoforms of NADPH oxidase in vascular (patho)physiology should provide direction for future programs aimed at developing selective inhibitors of these enzymes as novel therapeutics in cardiovascular disease.

• 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.

• Importance of NOX1 for angiotensin II-induced cerebrovascular superoxide production and cortical infarct volume following ischemic stroke.

  Authors: Katherine A Jackman, Alyson A Miller, Grant R Drummond, Christopher G Sobey

  Brain research. 07/2009;

  Angiotensin II (Ang II) receptor blockade is beneficial in stroke, possibly due to attenuation of vascular oxidative stress. Mice genetically targeted for the superoxide-forming vascular NADPHAngiotensin II (Ang II) receptor blockade is beneficial in stroke, possibly due to attenuation of vascular oxidative stress. Mice genetically targeted for the superoxide-forming vascular NADPH oxidase subunit, NOX1, have a blunted hypertensive response to Ang II. We therefore hypothesised that NOX1 is mechanistically involved in Ang II-induced superoxide production by cerebral arteries, and potentially in stroke outcome. Superoxide production by cerebral arteries and brains from wild-type (WT) and NOX1 deficient (NOX1-KO) mice was measured using L-012-enhanced chemiluminescence. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO; 0.5 h). Cerebral blood flow was measured using transcranial laser-Doppler flowmetry. After 24 h, neurological assessment was performed, mice were euthanased, and infarct and edema volumes calculated. Basal superoxide was similar between WT and NOX1-KO in brain and cerebral artery homogenates, and in intact cerebral arteries. However, Ang II-stimulated increases in superoxide were ~70% smaller in rings from NOX1-KO versus WT. During MCAO, rCBF decreased by ~75% in both WT and NOX1-KO, and increased to similar levels in each strain immediately following reperfusion. No difference in neurological score, total or subcortical cerebral infarct volume or edema volume was observed between WT and NOX1-KO mice. However, cortical infarct volume (which was very modest in WT) was approximately 4-fold greater in brains of NOX1-KO versus WT. Thus, NOX1 is essential for superoxide production in large cerebral arteries in response to Ang II but not under basal conditions. Furthermore, NOX1 does not appear to contribute to stroke size, and it may limit cortical infarct development following cerebral ischemia.

• Gender Influences Cerebral Vascular Responses to Angiotensin II Through Nox2-Derived Reactive Oxygen Species.

  Authors: T Michael De Silva, Brad R S Broughton, Grant R Drummond, Christopher G Sobey, Alyson A Miller

  Stroke; a journal of cerebral circulation. 02/2009;

  BACKGROUND AND PURPOSE: We tested whether gender influences cerebrovascular responses to angiotensin II (AngII) and the role(s) of Nox2. METHODS: AngII-stimulated superoxide (O2(-)) production byBACKGROUND AND PURPOSE: We tested whether gender influences cerebrovascular responses to angiotensin II (AngII) and the role(s) of Nox2. METHODS: AngII-stimulated superoxide (O2(-)) production by cerebral arteries from male and female wild-type (WT) and Nox2(-/-) mice was measured using lucigenin- or L-012-enhanced chemiluminescence. Hydrogen peroxide (H2O2) production was measured using Amplex Red fluorescence. Western Blotting was used to measure expression of Nox2, endothelial nitric oxide synthase (eNOS), angiotensin receptors (AT1 and AT2), and superoxide dismutases (SOD1-3). Immunofluorescence was used to localize Nox2 in middle cerebral arteries (MCA). Vascular responses to AngII were assessed in a perfusion myograph. AngII-stimulated O2(-) and H2O2 production by cerebral arteries from female WT mice was approximately 75% to 85% lower than in males (P<0.05). O2(-) production was approximately 60% lower in Nox2(-/-) versus WT males (P<0.05), whereas Nox2 deletion did not affect O2(-) production in females. Expression of Nox2, eNOS, AT receptors, and SOD isoforms was similar between genders. Nox2 immunofluorescence was similarly localized in adventitial and endothelial cells of MCA from both genders. AngII elicited smaller contractions of MCA from females vs males (P<0.05). Contractions were reduced in male, but not female, Nox2(-/-) mice (P<0.05). The SOD mimetic, tempol, potentiated contractions to AngII in male WT mice (P<0.05), whereas the SOD/catalase mimetic, EUK-134, virtually abolished contractions (P<0.05). CONCLUSIONS: AngII-stimulated O2(-) and H2O2 production are greater in cerebral arteries from male versus female mice, and are associated with greater contractions to AngII mediated by H2O2. These gender differences are dependent on the expression of Nox2.

• NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: Role of Nox2.

  Authors: Alyson A Miller, Grant R Drummond, T Michael De Silva, Anja E Mast, Haruyo Hickey, John P Williams, Brad R S Broughton, Christopher G Sobey

  American journal of physiology. Heart and circulatory physiology. 12/2008;

  We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we firstly tested whether NADPH oxidase activity is also greater inWe previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we firstly tested whether NADPH oxidase activity is also greater in intracranial cerebral arteries than systemic arteries of three other animal species, i.e. mouse, rabbit and pig. Secondly, using Nox2-deficient mice we evaluated the involvement of Nox2-containing NADPH oxidases in any such regional differences. NADPH-stimulated superoxide (O2(-)) production by basilar (BA), middle cerebral (MCA) and common carotid (CA) arteries, and thoracic aorta (AO) from rat, mouse, rabbit and pig was measured using lucigenin-enhanced chemiluminescence. Basal production of O2(-) and hydrogen peroxide (H2O2) by cerebral arteries, AO and CA from wild-type (WT) and Nox2(-/-) mice was measured using L-012-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Western Blotting was used to measure Nox2 and SOD1-3 protein expression, and immunofluorescence was used to localize Nox2, in mouse arteries. In rats, WT mice, rabbits and pigs, NADPH-stimulated O2- production by cerebral arteries was up to 40-fold greater than in AO and CA. In WT mice, basal O2(-) and H2O2 production by cerebral arteries was 9-fold and ~2.5-fold higher, respectively, than in AO and CA, and was associated with ~40% greater expression of Nox2 protein. Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia, in all mouse arteries and appeared to be more intense in endothelium of MCA than AO or CA. In Nox2(-/-) mice, NADPH-stimulated O2(-) production by cerebral arteries was ~35% lower than in WT mice, whereas Nox2 deletion had no significant effect on O2(-) production by AO or CA. Thus, NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species, and is associated with higher cerebrovascular expression and activity of Nox2. Key words: cerebral artery, superoxide, NADPH oxidase, Nox2.

• 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.