Alyson A Miller

Publications of Alyson A Miller

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• Effect of gender and sex hormones on vascular oxidative stress.

  Authors: Alyson A Miller, T Michael De Silva, Katherine A Jackman, Christopher G Sobey

  Clinical and experimental pharmacology & physiology. 11/2007; 34(10):1037-43.

  1. It is well documented that the incidence and severity of several vascular diseases, such as hypertension, atherosclerosis and stroke, are lower in premenopausal women than men of similar age and1. It is well documented that the incidence and severity of several vascular diseases, such as hypertension, atherosclerosis and stroke, are lower in premenopausal women than men of similar age and post-menopausal women. The mechanisms responsible for gender differences in the incidence and severity of vascular disease are not well understood. However, emerging evidence suggests that sex hormone-dependent differences in vascular oxidative stress may play an important role. The aim of the present brief review is to provide an insight into the effect of gender and sex hormones on vascular oxidative stress. 2. When production of reactive oxygen species (ROS) is enhanced and/or their metabolism by anti-oxidant enzymes is impaired, a condition known as 'oxidative stress' can develop. Oxidative stress is believed to play an important role in both the initiation and progression of a variety of vascular diseases, including hypertension and atherosclerosis. NADPH oxidases are believed to be the major source of vascular ROS. Moreover, excessive production of ROS by NADPH oxidases has been linked to the development of vascular oxidative stress. 3. Increasing evidence suggests that levels of vascular ROS may be lower in women than men during health and disease. Indeed, the activity and expression of vascular NADPH oxidase is lower in female versus male animals under healthy, hypertensive and atherosclerotic conditions. 4. Gonadal sex hormones may play an important role in the regulation of vascular oxidative stress. For example, oestrogens, which are present in highest levels in premenopausal women, have been reported to lower vascular oxidative stress by modulating the expression and function of NADPH oxidases, as well as anti-oxidant enzymes. 5. Further studies are needed to clarify whether lower vascular oxidative stress in women in fact protects against the initiation and development of vascular disease and to further define the roles of gonadal sex hormones in such an effect. Knowledge gained from these studies may potentially lead to advances in the clinical diagnosis and treatment of vascular disease in both genders.

• Effect of gender on NADPH-oxidase activity, expression, and function in the cerebral circulation: role of estrogen.

  Authors: Alyson A Miller, Grant R Drummond, Anja E Mast, Harald H H W Schmidt, Christopher G Sobey

  Stroke; a journal of cerebral circulation. 08/2007; 38(7):2142-9.

  BACKGROUND AND PURPOSE: This study tested whether NADPH-oxidase activity, expression, and functional effects on vascular tone are influenced by gender in the rat cerebral circulation and whether suchBACKGROUND AND PURPOSE: This study tested whether NADPH-oxidase activity, expression, and functional effects on vascular tone are influenced by gender in the rat cerebral circulation and whether such differences are estrogen-dependent. METHODS: NADPH-stimulated superoxide production by cerebral (basilar [BA]; middle cerebral) arteries from male and female Sprague-Dawley rats was measured using lucigenin-enhanced chemiluminescence and dihydroethidium. Protein expression of Nox1, Nox2, Nox4, superoxide dismutase 1 (SOD1), SOD2, and SOD3 was measured using Western blotting. Vascular responses of BA to NADPH were assessed in a myograph. Some female rats were ovariectomized and treated with either vehicle (dimethyl sulfoxide) or 17beta-estradiol. RESULTS: NADPH-stimulated superoxide production by BA and middle cerebral arteries from males was approximately 2-fold greater than vessels from females. Superoxide production was virtually abolished by the NADPH-oxidase inhibitor, diphenyleneiodonium. Protein expression of Nox1 and Nox4 in BA was also higher in males than in females (2.4- and 2.8-fold, respectively), whereas Nox2, SOD1, SOD2, and SOD3 expression did not differ between genders. NADPH induced greater vasorelaxant effects in BA from males versus females (P<0.05). The hydrogen peroxide scavenger, catalase, abolished these NADPH-induced relaxations. NADPH-stimulated superoxide production by BA from ovariectomized rats treated with vehicle was 3-fold greater than levels in intact females. Treatment of ovariectomized rats with 17beta-estradiol decreased superoxide production (P<0.05). NADPH-induced relaxations of BA were smaller in 17beta-estradiol-treated than in vehicle-treated ovariectomized rats (P<0.05). CONCLUSIONS: NADPH-oxidase activity and function are lower in cerebral arteries of female rats. These gender differences are estrogen-dependent and are associated with lower Nox1 and Nox4 expression.

• Novel isoforms of NADPH-oxidase in cerebral vascular control.

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

  Pharmacology & therapeutics. 10/2006; 111(3):928-48.

  Reactive oxygen species (ROS) are thought to play an important role in the initiation and progression of a variety of vascular diseases. Furthermore, accumulating evidence indicates that ROS may alsoReactive oxygen species (ROS) are thought to play an important role in the initiation and progression of a variety of vascular diseases. Furthermore, accumulating evidence indicates that ROS may also serve as important cell signalling molecules for the regulation of normal vascular function. Recently, a novel family of proteins (Nox1, 2 and 4) that act as the catalytic subunit of the superoxide (O2-) producing enzyme NADPH-oxidase has been discovered in vascular cells. There is now preliminary evidence suggesting that NADPH-oxidase-derived ROS may serve as a physiological vasodilator mechanism in the cerebral circulation. Moreover, the activity of NADPH-oxidase is profoundly greater in cerebral versus systemic arteries. Studies have shown that Nox1, Nox2 (also known as gp91phox) and Nox4 are all expressed in cerebral arteries, suggesting that multiple isoforms of NADPH-oxidase may be important for ROS production by cerebral arteries. Enhanced NADPH-oxidase activity is associated with several vascular-related diseases, including hypertension, stroke, subarachnoid haemorrhage and Alzheimer's dementia; however, the consequences of this for cerebral vascular function are controversial. For example, there is some evidence suggesting that NADPH-oxidase-derived O2- may play a role in endothelial dysfunction of cerebral arteries and a subsequent rise in cerebral vascular tone, associated with hypertension. However, activation of NADPH-oxidase elicits cerebral vasodilatation in vivo, and this mechanism is enhanced in chronic hypertension. While further supportive evidence is needed, it is an intriguing possibility that NADPH-oxidase-derived ROS may play a protective role in regulating cerebral vascular tone during disease.

• NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke.

  Authors: Alyson A Miller, Gregory J Dusting, Carli L Roulston, Christopher G Sobey

  Brain research. 10/2006; 1111(1):111-6.

  Reactive oxygen species play a role in neuronal damage following cerebral ischemia-reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebralReactive oxygen species play a role in neuronal damage following cerebral ischemia-reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebral arteries within, adjacent and distant from the ischemic core. The right middle cerebral artery (MCA) of conscious rats was temporarily occluded by perivascular injection of endothelin-1 to induce stroke (ET-1; n=19). Control rats were injected with saline (n=9). At 24 h or 72 h post-administration of ET-1, the MCA and its branches within the ipsilateral penumbra and infarcted core, corresponding arteries in the contralateral hemisphere, and basilar artery were excised. Anatomically similar arteries were excised from saline-injected rats. At 24 h after stroke, NADPH-stimulated superoxide production by arteries from the infarcted core did not differ from levels generated by arteries from control rats, whereas levels were significantly lower 72 h after stroke. However, at both time points after stroke, superoxide production by arteries from the ischemic penumbra was 8-fold greater than levels generated by arteries from control rats. Surprisingly, even in the non-ischemic arteries from the contralateral hemisphere and in the basilar artery, superoxide production was increased approximately 4- to 6-fold at 24 h, but had returned to normal 72 h after stroke. The NADPH-oxidase inhibitor, diphenyleneiodonium, virtually abolished superoxide production by all arteries. Thus, the activity of NADPH-oxidase is enhanced in cerebral arteries from the ischemic penumbra at 24 h and 72 h following cerebral ischemia. Additionally, NADPH-oxidase activity is temporarily enhanced after cerebral ischemia within arteries from non-ischemic parts of the brain.

• Flow-induced cerebral vasodilatation in vivo involves activation of phosphatidylinositol-3 kinase, NADPH-oxidase, and nitric oxide synthase.

  Authors: Tamara M Paravicini, Alyson A Miller, 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. 07/2006; 26(6):836-45.

  Reactive oxygen species (ROS) such as superoxide (O2*-) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamideReactive oxygen species (ROS) such as superoxide (O2*-) and hydrogen peroxide (H2O2) are known cerebral vasodilators. A major source of vascular ROS is the flavin-containing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Activation of NADPH-oxidase leads to dilatation of the basilar artery in vivo via production of H2O2, but the endogenous stimuli for this unique vasodilator mechanism are unknown. Shear stress is known to activate both NADPH-oxidase and phosphatidylinositol-3 kinase (PI3-K) in cultured cells. Hence, this study used a cranial window preparation in anesthetized rats to investigate whether increased intraluminal blood flow could induce cerebral vasodilatation via the activation of NADPH-oxidase and/or PI3-K. Bilateral occlusion of the common carotid arteries to increase basilar artery blood flow caused reproducible, reversible vasodilatation. Topical treatment of the basilar artery with the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) (0.5 and 5 micromol/L) inhibited flow-induced dilatation by up to 50% without affecting dilator responses to acetylcholine. Treatment with the H2O2 scavenger, catalase similarly attenuated flow-induced dilatation, suggesting a role for NADPH-oxidase-derived H2O2 in this response. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) partially reduced flow-induced dilatation, and combined treatment with a ROS inhibitor (DPI or catalase) and L-NAME caused a greater reduction in flow-induced dilatation than that seen with any of these inhibitors alone. Flow-induced dilatation was also markedly inhibited by the PI3-K inhibitor, wortmannin. Increased O2*- production in the endothelium of the basilar artery during acute increases in blood flow was confirmed using dihydroethidium. Thus, flow-induced cerebral vasodilatation in vivo involves production of ROS and nitric oxide, and is dependent on PI3-K activation.

• NADPH-induced contractions of mouse aorta do not involve NADPH oxidase: a role for P2X receptors.

  Authors: Courtney P Judkins, Christopher G Sobey, Thuy T Dang, Alyson A Miller, Gregory J Dusting, Grant R Drummond

  The Journal of pharmacology and experimental therapeutics. 06/2006; 317(2):644-50.

  Reactive oxygen species elicit vascular effects ranging from acute dilatation because of hydrogen peroxide-mediated opening of K(+) channels to contraction arising from superoxide-dependentReactive oxygen species elicit vascular effects ranging from acute dilatation because of hydrogen peroxide-mediated opening of K(+) channels to contraction arising from superoxide-dependent inactivation of endothelium-derived nitric oxide. Given that NADPH oxidases are major sources of superoxide in the vascular wall, this study examined the effects of exogenous NADPH, a substrate of these enzymes, on superoxide generation and isometric tone in mouse isolated aortic rings. NADPH caused concentration-dependent increases in superoxide generation (measured by lucigenin-enhanced chemiluminescence) and vascular tone (isometric tension recordings). However, surprisingly, whereas oxidized NADP(+) was unable to support superoxide production, it was equally as effective as reduced NADPH at stimulating vasocontraction. In addition, an NADPH oxidase inhibitor, diphenyleneiodonium, markedly attenuated NADPH-induced superoxide production, yet had no effect on vasocontractions to NADPH. In contrast, a broad specificity P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, as well as the P2X1 selective antagonist, NF023, markedly attenuated both endothelium-dependent and -independent vasocontractions to NADPH, as did the P2X-desensitizing agent alpha,beta-methylene-ATP. Importantly, alpha,beta-methylene-ATP had no effect on superoxide production induced by NADPH. In conclusion, these findings suggest little role for NADPH oxidase-derived superoxide in the contractile effects of NADPH in the mouse aorta. Rather, NADPH seems to act as an agonist at two distinct P2X receptor populations; one located on the endothelium and the other on smooth muscle layer, both of which ultimately lead to contraction.

• Radicals spark interest in cerebral vasodilator mechanisms. Focus on "TNF-alpha dilates cerebral arteries via NAD(P)H oxidase-dependent Ca2+ spark activation".

  Authors: Christopher G Sobey, Alyson A Miller

  American journal of physiology. Cell physiology. 05/2006; 290(4):C950-1.

• Signalling pathways activated by hydrogen peroxide in vascular smooth muscle.

  Authors: Christopher G Sobey, Alyson A Miller

  Journal of hypertension. 12/2005; 23(11):1961-2.

• NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries.

  Authors: Alyson A Miller, Grant R Drummond, Harald H H W Schmidt, Christopher G Sobey

  Circulation research. 12/2005; 97(10):1055-62.

  Recent studies suggest that the superoxide generating enzyme NADPH oxidase may play a functional role in regulating cerebral vascular tone. We tested whether the activity, function, and expression ofRecent studies suggest that the superoxide generating enzyme NADPH oxidase may play a functional role in regulating cerebral vascular tone. We tested whether the activity, function, and expression of NADPH oxidase differs between rat cerebral and systemic arteries. Superoxide production by basilar (BA), middle cerebral (MCA), carotid (CA), renal (RA), and mesenteric (MA) arteries and aorta (AO) was measured using lucigenin-enhanced chemiluminescence. Superoxide production from NADPH oxidase was localized and semiquantified using dihydroethidium. Vascular functional responses were assessed in a myograph or organ bath. Vascular Nox4 protein expression was measured using Western blotting. Superoxide production (basal or in response to NADPH or angiotensin II) in the intracranial arteries, BA, and MCA was 10- to 100-fold greater than in AO, CA, RA, or MA. Similar results were found using either intact vessels or arterial homogenates, and were associated with 10-fold greater expression of Nox4 in the BA versus AO, CA, and MA. Superoxide production was attenuated by the NADPH oxidase inhibitors, diphenyleneiodonium, apocynin, and gp91ds-tat. NADPH and H2O2 were strong relaxing stimuli in the BA, where the H2O2 scavenger catalase, as well as apocynin, attenuated these relaxations and also augmented contractions to angiotensin II. NADPH oxidase activity is markedly higher in intracranial versus systemic arteries, in association with higher Nox4 expression. In cerebral arteries, endogenous H2O2 derived from NADPH oxidase activation appears to cause relaxation and is able to offset angiotensin II-induced constriction. These data are consistent with the concept that NADPH oxidase-derived reactive oxygen species modulate cerebral vascular tone under physiological conditions.

• Reactive oxygen species in the cerebral circulation: are they all bad?

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

  Antioxidants & redox signaling. 8(7-8):1113-20.

  Reactive oxygen species (ROS) are a diverse family of molecules generated by all cells. ROS may serve as important cell-signalling molecules in the cerebral circulation. Indeed, in contrast toReactive oxygen species (ROS) are a diverse family of molecules generated by all cells. ROS may serve as important cell-signalling molecules in the cerebral circulation. Indeed, in contrast to systemic arteries, major products of superoxide metabolism, including hydrogen peroxide, are powerful cerebral vasodilators, raising the possibility that ROS represent important molecules for increasing local cerebral blood flow. Two major determinants of the overall effects of ROS on cerebrovascular tone are the rate of production of the parent molecule, superoxide, and its rate of metabolism by superoxide dismutases. Although the major enzymatic source of ROS in cerebral arteries has not been clarified, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-oxidases, along with cyclooxygenases and lipoxygenases, are probably the primary sources. In cerebral arteries, activation of NADPH-oxidase elicits both an increase in superoxide production and vasodilatation. The identity of the ROS molecule responsible for the vasodilator effects may be hydrogen peroxide, generated from the dismutation of superoxide. NADPH-oxidase activity and function appears to be profoundly greater in cerebral versus systemic arteries. Furthermore, NADPH-oxidase-derived ROS partly contribute to flow-dependent dilatation and may offset angiotensin II-induced constriction of cerebral arteries, consistent with the hypothesis that NADPH-oxidase-derived ROS may play a physiologic role in the control of cerebrovascular tone.