Christopher G Sobey

Publications of Christopher G Sobey

• Intravenous immunoglobulin (IVIg) protects neurons against amyloid beta-peptide toxicity and ischemic stroke by attenuating multiple cell death pathways.

  Authors: Alexander Widiapradja, Viktor Vegh, Ker Zhing Lok, Silvia Manzanero, John Thundyil, Mathias Gelderblom, Yi-Lin Cheng, Dale Pavlovski, Sung-Chun Tang, Dong-Gyu Jo, Tim Magnus, Sic L Chan, Christopher G Sobey, David Reutens, Milan Basta, Mark P Mattson, Thiruma V Arumugam

  Journal of neurochemistry. 04/2012;

  Intravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatoryIntravenous immunoglobulin (IVIg) preparations obtained by fractionating blood plasma, are increasingly being used increasingly as an effective therapeutic agent in treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke and Alzheimer's disease (AD) has been proposed, but little is known about the neuroprotective mechanisms of IVIg. In this study we investigated the effect of IVIg on downstream signaling pathways that are involved in neuronal cell death in experimental models of stroke and AD. Treatment of cultured neurons with IVIg reduced simulated ischemia- and amyloid βpeptide (Aβ)-induced caspase-3 cleavage, and phosphorylation of the cell death-associated kinases p38MAPK, JNK and p65, in vitro. Additionally, Aβ-induced accumulation of the lipid peroxidation product 4-HNE was attenuated in neurons treated with IVIg. IVIg treatment also upregulated the anti-apoptotic protein, Bcl2 in cortical neurons under ischemia-like conditions and exposure to Aβ. Treatment of mice with IVIg reduced neuronal cell loss, apoptosis and infarct size, and improved functional outcome in a model of focal ischemic stroke. Together, these results indicate that IVIg acts directly on neurons to protect them against ischemic stroke and Aβ-induced neuronal apoptosis by inhibiting cell death pathways and by elevating levels of the anti-apoptotic protein Bcl2. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

• 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.

• Adiponectin receptor signalling in the brain.

  Authors: John Thundyil, Dale Pavlovski, Christopher G Sobey, Thiruma V Arumugam

  British journal of pharmacology. 06/2011; 165(2):313-27.

  Adiponectin is an important adipocyte-derived hormone that regulates metabolism of lipids and glucose, and its receptors (AdipoR1, AdipoR2, T-cadherin) appear to exert actions in peripheral tissuesAdiponectin is an important adipocyte-derived hormone that regulates metabolism of lipids and glucose, and its receptors (AdipoR1, AdipoR2, T-cadherin) appear to exert actions in peripheral tissues by activating the AMP-activated protein kinase, p38-MAPK, PPARα and NF-kappa B. Adiponectin has been shown to exert a wide range of biological functions that could elicit different effects, depending on the target organ and the biological milieu. There is substantial evidence to suggest that adiponectin receptors are expressed widely in the brain. Their expression has been detected in regions of the mouse hypothalamus, brainstem, cortical neurons and endothelial cells, as well as in whole brain and pituitary extracts. While there is now considerable evidence for the presence of adiponectin and its receptors in the brain, their precise roles in brain diseases still remain unclear. Only a few research studies have looked at this facet of adiponectins in brain disorders. This brief review will describe the evidence for important functions by adiponectin, its structure and known actions, evidence for expression of AdipoRs in the brain, their involvement in brain disorders and the therapeutic potential of agents that could modify AdipoR signalling.

• 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.

• Maternal nicotine exposure and fetal programming of vascular oxidative stress in adult offspring.

  Authors: Rebecca Lim, Christopher G Sobey

  British journal of pharmacology. 05/2011; 164(5):1397-9.

  Despite the well-known harmful effects, many women continue to smoke throughout pregnancy. Consequently, nicotine replacement therapy (NRT) - which has been developed as a pharmacotherapy for smokingDespite the well-known harmful effects, many women continue to smoke throughout pregnancy. Consequently, nicotine replacement therapy (NRT) - which has been developed as a pharmacotherapy for smoking cessation - has been used as an alternative to smoking during pregnancy. However, like cigarette smoking, NRT results in biologically significant levels of nicotine crossing the placenta, leading to both fetal and neonatal exposure to nicotine, and yet, NRT safety during pregnancy has not been extensively evaluated. There is now evidence from studies in rats that maternal nicotine exposure throughout gestation results in fetal programming of vascular oxidative stress in the offspring during adulthood. This phenomenon involves vascular dysfunction mediated by reactive oxygen species in association with decreased superoxide dismutase activity and increased Nox2-NADPH oxidase expression in the vascular wall. If this phenomenon also occurs in humans, either smoking or NRT use during pregnancy may represent a novel risk factor for the unborn that results in accelerated cardiovascular disease in their adulthood.

• Nitroxyl (HNO) as a vasoprotective signaling molecule.

  Authors: Michelle L Bullen, Alyson A Miller, Karen L Andrews, Jennifer C Irvine, Rebecca H Ritchie, Christopher G Sobey, Barbara K Kemp-Harper

  Antioxidants & redox signaling. 05/2011; 14(9):1675-86.

  Nitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO(•)), is rapidly emerging as a novel nitrogen oxide with distinct pharmacology and therapeutic advantages over itsNitroxyl (HNO), the one electron reduced and protonated form of nitric oxide (NO(•)), is rapidly emerging as a novel nitrogen oxide with distinct pharmacology and therapeutic advantages over its redox sibling. Whilst the cardioprotective effects of HNO in heart failure have been established, it is apparent that HNO may also confer a number of vasoprotective properties. Like NO(•), HNO induces vasodilatation, inhibits platelet aggregation, and limits vascular smooth muscle cell proliferation. In addition, HNO can be putatively generated within the vasculature, and recent evidence suggests it also serves as an endothelium-derived relaxing factor (EDRF). Significantly, HNO targets signaling pathways distinct from NO(•) with an ability to activate K(V) and K(ATP) channels in resistance arteries, cause coronary vasodilatation in part via release of calcitonin-gene related peptide (CGRP), and exhibits resistance to scavenging by superoxide and vascular tolerance development. As such, HNO synthesis and bioavailability may be preserved and/or enhanced during disease states, in particular those associated with oxidative stress. Moreover, it may compensate, in part, for a loss of NO(•) signaling. Here we explore the vasoprotective actions of HNO and discuss the therapeutic potential of HNO donors in the treatment of vascular dysfunction.

• Evidence that gamma-secretase-mediated Notch signaling induces neuronal cell death via the nuclear factor-kappaB-Bcl-2-interacting mediator of cell death pathway in ischemic stroke.

  Authors: Thiruma V Arumugam, Yi-Lin Cheng, Yuri Choi, Yun-Hyung Choi, Sunghee Yang, Young-Kwang Yun, Jong-Sung Park, Dong Kwon Yang, John Thundyil, Mathias Gelderblom, Vardan T Karamyan, Sung-Chun Tang, Sic L Chan, Tim Magnus, Christopher G Sobey, Dong-Gyu Jo

  Molecular pharmacology. 03/2011; 80(1):23-31.

  Notch-1 (Notch) is a cell surface receptor that regulates cell-fate decisions in the developing nervous system, and it may also have roles in synaptic plasticity in the adult brain. Binding of itsNotch-1 (Notch) is a cell surface receptor that regulates cell-fate decisions in the developing nervous system, and it may also have roles in synaptic plasticity in the adult brain. Binding of its ligands results in the proteolytic cleavage of Notch by the γ-secretase enzyme complex, thereby causing the release of a Notch intracellular domain (NICD) that translocates to the nucleus, in which it regulates transcription. Here we show that activation of Notch modulates ischemic neuronal cell death in vitro and in vivo. Specifically, our findings from the use of Notch-1 siRNA or the overexpression of NICD indicate that Notch activation contributes to cell death. Using modified NICD, we demonstrate an apoptosis-inducing function of NICD in both the nucleus and the cytosol. NICD transfection-induced cell death was reduced by blockade of calcium signaling, caspase activation, and Janus kinase signaling. Inhibition of the Notch-activating enzyme, γ-secretase, protected against ischemic neuronal cell death by targeting an apoptotic protease, cleaved caspase-3, nuclear factor-κB (NF-κB), and the pro-death BH3-only protein, Bcl-2-interacting mediator of cell death (Bim). Treatment of mice with a γ-secretase inhibitor, compound E, reduced infarct size and improved functional outcome in a model of focal ischemic stroke. Furthermore, γ-secretase inhibition reduced NICD, p-p65, and Bim levels in vivo. These findings suggest that Notch signaling endangers neurons after ischemic stroke by modulating the NF-κB, pro-death protein Bim, and caspase pathways.

• 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.

• Vascular expression, activity and function of indoleamine 2,3-dioxygenase-1 following cerebral ischaemia-reperfusion in mice.

  Authors: Katherine A Jackman, Vanessa H Brait, Yutang Wang, Ghassan J Maghzal, Helen J Ball, Gavin McKenzie, T Michael De Silva, Roland Stocker, Christopher G Sobey

  Naunyn-Schmiedeberg's archives of pharmacology. 02/2011; 383(5):471-81.

  Indoleamine 2,3-dioxygenases-1 (Ido1) and -2 initiate the kynurenine pathway of tryptophan metabolism. In addition to the established immune regulatory effects of Ido1 and the ability of nitric oxideIndoleamine 2,3-dioxygenases-1 (Ido1) and -2 initiate the kynurenine pathway of tryptophan metabolism. In addition to the established immune regulatory effects of Ido1 and the ability of nitric oxide to regulate Ido1 activity, it is now also known that Ido1-mediated metabolism of tryptophan to kynurenine can modulate vascular tone. Ido activity is reportedly elevated in stroke patients and correlates with increased risk of death. Thus, the present goals were to test whether, following cerebral ischaemia, Ido activity and cerebrovascular Ido1 expression are altered and whether expression of Ido1 contributes to stroke outcome. Transient cerebral ischaemia was induced in wild-type and Ido1 gene-deficient (Ido1 (-/-)) mice. Mice were pre-treated with vehicle, the Ido1 inhibitor, 1-methyl-D-tryptophan (1-MT; 50 mg/kg i.p.) or the inducible nitric oxide synthase (Nos2) inhibitor, aminoguanidine (AG, 100 mg/kg i.p.). At 24 h, neurological function, brain infarct size and swelling were assessed. In addition, Ido activity was estimated by plasma kynurenine and tryptophan, and Ido1 expression was examined in cerebral arterioles. Cerebral ischaemia-reperfusion in wild-type mice increased Ido activity and its expression in cerebral arterioles. Ido1 (-/-) and 1-MT-treated wild-type mice had lower Ido activity but similar post-stroke neurological function and similar total brain infarct volume and swelling, relative to control mice. Inhibition of Nos2 with AG also did not affect Ido activity or outcome following stroke. This study provides molecular and pharmacological evidence that the expression and the activity of Ido1 increase following stroke. However, such Ido1 expression does not appear to affect overall outcome following acute ischaemic stroke, and furthermore, a regulatory role of Nos2-derived nitric oxide on Ido activity following cerebral ischaemia-reperfusion appears unlikely.

• 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.

• Pathophysiology, treatment, and animal and cellular models of human ischemic stroke.

  Authors: Trent M Woodruff, John Thundyil, Sung-Chun Tang, Christopher G Sobey, Stephen M Taylor, Thiruma V Arumugam

  Molecular neurodegeneration. 01/2011; 6(1):11.

  Stroke is the world's second leading cause of mortality, with a high incidence of severe morbidity in surviving victims. There are currently relatively few treatment options available to minimizeStroke is the world's second leading cause of mortality, with a high incidence of severe morbidity in surviving victims. There are currently relatively few treatment options available to minimize tissue death following a stroke. As such, there is a pressing need to explore, at a molecular, cellular, tissue, and whole body level, the mechanisms leading to damage and death of CNS tissue following an ischemic brain event. This review explores the etiology and pathogenesis of ischemic stroke, and provides a general model of such. The pathophysiology of cerebral ischemic injury is explained, and experimental animal models of global and focal ischemic stroke, and in vitro cellular stroke models, are described in detail along with experimental strategies to analyze the injuries. In particular, the technical aspects of these stroke models are assessed and critically evaluated, along with detailed descriptions of the current best-practice murine models of ischemic stroke. Finally, we review preclinical studies using different strategies in experimental models, followed by an evaluation of results of recent, and failed attempts of neuroprotection in human clinical trials. We also explore new and emerging approaches for the prevention and treatment of stroke. In this regard, we note that single-target drug therapies for stroke therapy, have thus far universally failed in clinical trials. The need to investigate new targets for stroke treatments, which have pleiotropic therapeutic effects in the brain, is explored as an alternate strategy, and some such possible targets are elaborated. Developing therapeutic treatments for ischemic stroke is an intrinsically difficult endeavour. The heterogeneity of the causes, the anatomical complexity of the brain, and the practicalities of the victim receiving both timely and effective treatment, conspire against developing effective drug therapies. This should in no way be a disincentive to research, but instead, a clarion call to intensify efforts to ameliorate suffering and death from this common health catastrophe. This review aims to summarize both the present experimental and clinical state-of-the art, and to guide future research directions.

• 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.

• Endothelium-dependent relaxation by G protein-coupled receptor 30 agonists in rat carotid arteries.

  Authors: Brad R S Broughton, Alyson A Miller, Christopher G Sobey

  American journal of physiology. Heart and circulatory physiology. 03/2010; 298(3):H1055-61.

  Recent studies have identified that the novel membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), is present in blood vessels. However, the signaling mechanisms associated with GPR30Recent studies have identified that the novel membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), is present in blood vessels. However, the signaling mechanisms associated with GPR30 in the vasculature remain unclear. We examined whether putative agonists of GPR30 exert vasorelaxant and/or antioxidant effects similar to those reported for estrogen. Using wire myography, we assessed the role of the endothelium in relaxation responses to the GPR30 agonists, G-1 and 5408-0877 (1 nM-10 microM), in U-46619-precontracted common carotid arteries from Sprague-Dawley rats. Furthermore, using lucigenin (5 microM)-enhanced chemiluminescence, we tested the effect of G-1 (10 microM) on superoxide levels. Specific immunofluorescence was also used to confirm GPR30 expression in the arterial wall. We found that G-1 and 5408-0877 induced a concentration-dependent relaxation in carotid arteries from both male and female rats. Interestingly, G-1- and 5408-0877-induced relaxation was abolished by endothelium removal and abrogated in the presence of the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester (100 microM). In addition, G-1 significantly decreased NADPH (100 microM)-stimulated superoxide production by carotid and intracranial (pooled basilar and middle cerebral) arteries but also attenuated the superoxide signal detected in a cell-free xanthine/xanthine oxidase assay. Furthermore, GPR30 immunoreactivity was observed in endothelial and vascular smooth muscle cells of carotid arteries from both genders. These findings indicate that GPR30 is expressed throughout the arterial wall and that GPR30 agonists elicit endothelial-derived nitric oxide-dependent relaxation of the carotid artery in male and female rats. Additionally, G-1 appears to directly scavenge superoxide anion.

• 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.

• Vascular dysfunction in cerebrovascular disease: mechanisms and therapeutic intervention.

  Authors: Alyson A Miller, Klaudia Budzyn, Christopher G Sobey

  Clinical science (London, England : 1979). 01/2010; 119(1):1-17.

  The endothelium plays a crucial role in the control of vascular homoeostasis through maintaining the synthesis of the vasoprotective molecule NO* (nitric oxide). Endothelial dysfunction of cerebralThe endothelium plays a crucial role in the control of vascular homoeostasis through maintaining the synthesis of the vasoprotective molecule NO* (nitric oxide). Endothelial dysfunction of cerebral blood vessels, manifested as diminished NO* bioavailability, is a common feature of several vascular-related diseases, including hypertension, hypercholesterolaemia, stroke, subarachnoid haemorrhage and Alzheimer's disease. Over the past several years an enormous amount of research has been devoted to understanding the mechanisms underlying endothelial dysfunction. As such, it has become apparent that, although the diseases associated with impaired NO* function are diverse, the underlying causes are similar. For example, compelling evidence indicates that oxidative stress might be an important mechanism of diminished NO* signalling in diverse models of cardiovascular 'high-risk' states and cerebrovascular disease. Although there are several sources of vascular ROS (reactive oxygen species), the enzyme NADPH oxidase is emerging as a strong candidate for the excessive ROS production that is thought to lead to vascular oxidative stress. The purpose of the present review is to outline some of the mechanisms thought to contribute to endothelial dysfunction in the cerebral vasculature during disease. More specifically, we will highlight current evidence for the involvement of ROS, inflammation, the RhoA/Rho-kinase pathway and amyloid beta-peptides. In addition, we will discuss currently available therapies for improving endothelial function and highlight future therapeutic strategies.