17649063
NADPH Oxidase p22phox C242T Polymorphism and Ischemic Cerebrovascular Disease: An Updated Meta-Analysis
Background A growing number of studies on the associations between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox C242T polymorphism and risk of ischemic cerebrovascular disease have recently been published, but the results remain inconsistent. Material/Methods We performed an updated meta-analysis to evaluate this association. Eight case-control studies were included, involving 2045 cases and 2102 controls. Heterogeneity was assessed by the Q test and the I2 statistic. Begg and Egger’s tests were conducted to evaluate publication bias. Odds ratio (OR) was tested to identify the associations. Results Significant associations between p22phox gene C242T polymorphism and ischemic cerebrovascular disease (ICVD) risk were observed in the allelic genetic model (OR=1.33, 95% confidence interval [CI] 1.00–1.77, p=0.048). No statistical significant association was found in the dominant model (OR=0.74, 95% CI 0.54–1.02, p=0.064) and recessive model (OR=1.40, 95% CI 0.89–2.19, p=0.146). Subgroup analysis showed an association in European populations for recessive model (OR=2.13, 95% CI 1.06–4.26, p=0.034) and no significant evidence of association in Asian populations was found (dominant model: OR=0.64, 95% CI 0.41–1.00, p=0.05; recessive model: OR=0.98, 95% CI 0.53–1.81, p=0.948; allelic model: OR=1.51, 95% CI 0.98–2.32, p=0.061). Conclusions p22phox gene C242T polymorphism was associated with ICVD risk in the allelic genetic model, as well as in European populations for recessive model. No evidence showed association between p22phox gene C242T polymorphism and ICVD risk in the dominant model and recessive model. Furthermore, no association existed in Asian populations for any of the 3 genetic models and European populations in the dominant model and allelic model.
41169657
Carotid intima media-thickness and genes involved in lipid metabolism in diabetic patients using statins--a pathway toward personalized medicine.
The prognostic importance of large artery structure and function in relation to cardiovascular morbidity and mortality, together with the identification of new genetic risk factors have been two major areas of investigation in recent years. Carotid intima-media thickness (CIMT), as measured by B-mode ultrasound, is a surrogate marker for atherosclerosis and can be used to detect an accelerated disease process as well as subclinical disease. However, the genetic basis for CIMT variation is almost unknown. Cardiovascular genetics has led to numerous clinical studies generally focused on only one candidate gene and were frequently conducted in subjects with cardiovascular diseases and/or taking drugs that could affect CIMT. Pharmacogenetics is the study of the effect of a medication as it relates to single or defined sets of genes. An important goal of pharmacogenetics in cardiovascular disorders is to integrate the two (drugs plus genes) so that true personalized therapy can be delivered. In this paper, we will discuss the interaction between genes involved in lipid metabolism and statin therapy that affects intermediate phenotype (plasma lipid levels) and CIMT in patients with type 2 diabetes.
207554938
Heart Rate Reduction by Ivabradine Reduces Oxidative Stress, Improves Endothelial Function, and Prevents Atherosclerosis in Apolipoprotein E–Deficient Mice
Background— Elevated heart rate is associated with increased cardiovascular morbidity. We hypothesized that selective heart rate reduction may influence endothelial function and atherogenesis and tested the effects of the I(f) current inhibitor ivabradine in apolipoprotein E–deficient mice. Methods and Results— Male apolipoprotein E–deficient mice fed a high-cholesterol diet were treated with ivabradine (10 mg · kg−1 · d−1) or vehicle for 6 weeks (n=10 per group). Ivabradine reduced heart rate by 13.4% (472±9 versus 545±11 bpm; P<0.01) but did not alter blood pressure or lipid levels. Endothelium-dependent relaxation of aortic rings was significantly improved in ivabradine-fed animals (P<0.01). Ivabradine decreased atherosclerotic plaque size in the aortic root by >40% and in the ascending aorta by >70% (P<0.05). Heart rate reduction by ivabradine had no effect on the number of endothelial progenitor cells and did not alter aortic endothelial nitric oxide synthase, phosphorylated Akt, vascular cell adhesion molecule-1, or intercellular adhesion molecule-1 expression but decreased monocyte chemotactic protein-1 mRNA and exerted potent antioxidative effects. Ivabradine reduced vascular NADPH oxidase activity to 48±6% and decreased markers of superoxide production and lipid peroxidation in the aortic wall (P<0.05). The in vivo effects of ivabradine were absent at a dose that did not lower heart rate, in aortic rings treated ex vivo, and in cultured vascular cells. In contrast to ivabradine, treatment with hydralazine (25 mg · kg−1 · d−1 for 6 weeks) reduced blood pressure (−15%) but increased heart rate (37%) and did not improve endothelial function, atherosclerosis, or oxidative stress. Conclusions— Selective heart rate reduction with ivabradine decreases markers of vascular oxidative stress, improves endothelial function, and reduces atherosclerotic plaque formation in apolipoprotein E–deficient mice.
26104841
NADPH oxidase-dependent signaling in endothelial cells: role in physiology and pathophysiology.
Reactive oxygen species (ROS) including superoxide (O(2)(.-)) and hydrogen peroxide (H(2)O(2)) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, "oxidant signaling," has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47(phox), p67(phox) and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91(phox) (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets.
19750358
Polyethylene glycol-conjugated superoxide dismutase in focal cerebral ischemia-reperfusion.
Generation of free radicals during reperfusion after organ ischemia has been implicated in the pathogenesis of ischemic injury. We have previously shown that a combination of intravenous polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) and catalase (PEG-CAT), at a dose of 10,000 U/kg each, is effective in reducing infarct size in a focal cerebral ischemia model in the rat. It is not clear whether PEG-SOD alone is sufficient to reduce ischemic brain injury. In this study we determined the therapeutic efficacy of PEG-SOD and its dose-response curve. In a range of 1,000-30,000 U/kg, PEG-SOD exhibited a U-shaped dose-response curve. Only 10,000 U/kg significantly reduced infarct size [control 121 +/- 12 mm3 (mean +/- SE), n = 35; PEG-SOD 95 +/- 10 mm3, n = 36, P < 0.05]. PEG-SOD at the doses tested did not have significant acute hemodynamic effects but had a tendency to improve postischemic hypotension. This beneficial effect of PEG-SOD on blood pressure did not appear to fully account for the treatment effect of PEG-SOD on infarct size. The narrow therapeutic dose range of PEG-SOD in this study and similar findings of SOD in other investigations may contribute to the inconsistent protective effects of SOD preparations in ischemia-reperfusion injury in the literature.
15650233
Liposome-entrapped superoxide dismutase reduces cerebral infarction in cerebral ischemia in rats.
We studied the role of superoxide radicals in the pathogenesis of ischemic brain injury using a model of focal cerebral ischemia in 102 rats and liposome-entrapped CuZn-superoxide dismutase, which can penetrate the blood-brain barrier and cell membranes efficiently. The bolus intravenous administration of 25,000 units of liposome-entrapped CuZn-superoxide dismutase elevated superoxide dismutase activities in the blood and brain 1, 2, 8, and 24 hours later as well as in the ischemic hemisphere and contralateral cortex. Determined 24 hours after right middle cerebral and bilateral common carotid artery occlusion by the lack of staining for mitochondrial dehydrogenase activity with 2,3,5-triphenyltetrazolium chloride, infarct sizes were reduced by 33%, 25%, and 18% in the anterior, middle, and posterior brain slices, respectively, by treatment with liposome-entrapped CuZn-superoxide dismutase. Our data demonstrate that superoxide radicals are important determinants of infarct size following focal cerebral ischemia and that liposome-entrapped CuZn-superoxide dismutase may have pharmacologic value for the treatment of focal cerebral ischemic injury.
22679758
Polyethylene glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury.
Superoxide dismutase and catalase enzymatically scavenge superoxide and hydrogen peroxide, respectively. Conjugation of polyethylene glycol to superoxide dismutase (PEG-SOD) or catalase (PEG-CAT) prolongs the circulatory half-life of the native enzymes and enhances their intracellular access. We studied the protective effect of these free radical scavengers on ischemic brain injury using a rat model of focal cerebral ischemia, which is suitable for therapeutic trials. Intravenous administration of PEG-SOD (10,000 U/kg) and PEG-CAT (10,000 U/kg) before ischemia reduced the infarct volume (treatment, 139 +/- 9 mm3, means +/- SE, N = 38; placebo, 182 +/- 8 mm3, n = 37, P less than 0.002). This finding supports the concept that superoxide and hydrogen peroxide contribute to brain injury following focal cerebral ischemia.
40340014
Reactive oxygen species in the cerebral circulation: are they all bad?
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 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.
24616658
The role of redox signaling in epigenetics and cardiovascular disease.
SIGNIFICANCE
The term epigenetics refers to the changes in the phenotype and gene expression that occur without alterations in the DNA sequence. There is a rapidly growing body of evidence that epigenetic modifications are involved in the pathological mechanisms of many cardiovascular diseases (CVDs), which intersect with many of the pathways involved in oxidative stress.


RECENT ADVANCES
Most studies relating epigenetics and human pathologies have focused on cancer. There has been a limited study of epigenetic mechanisms in CVDs. Although CVDs have multiple established genetic and environmental risk factors, these explain only a portion of the total CVD risk. The epigenetic perspective is beginning to shed new light on how the environment influences gene expression and disease susceptibility in CVDs. Known epigenetic changes contributing to CVD include hypomethylation in proliferating vascular smooth muscle cells in atherosclerosis, changes in estrogen receptor-α (ER-α) and ER-β methylation in vascular disease, decreased superoxide dismutase 2 expression in pulmonary hypertension (PH), as well as trimethylation of histones H3K4 and H3K9 in congestive heart failure.


CRITICAL ISSUES
In this review, we discuss the epigenetic modifications in CVDs, including atherosclerosis, congestive heart failure, hypertension, and PH, with a focus on altered redox signaling.


FUTURE DIRECTIONS
As advances in both the methodology and technology accelerate the study of epigenetic modifications, the critical role they play in CVD is beginning to emerge. A fundamental question in the field of epigenetics is to understand the biochemical mechanisms underlying reactive oxygen species-dependent regulation of epigenetic modification.
34459452
Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system.
BACKGROUND
Angiotensin II activates NAD(P)H-dependent oxidases via AT1-receptor stimulation, the most important vascular source of superoxide (O2*-). The AT1 receptor is upregulated in vitro by low-density lipoprotein. The present study was designed to test whether hypercholesterolemia is associated with increased NAD(P)H-dependent vascular O2*- production and whether AT1-receptor blockade may inhibit this oxidase and in parallel improve endothelial dysfunction.


METHODS AND RESULTS
Vascular responses were determined by isometric tension studies, and relative rates of vascular O2*- production were determined by use of chemiluminescence with lucigenin, a cypridina luciferin analogue, and electron spin resonance studies. AT1-receptor mRNA was quantified by Northern analysis, and AT1-receptor density was measured by radioligand binding assays. Hypercholesterolemia was associated with impaired endothelium-dependent vasodilation and increased O2*- production in intact vessels. In vessel homogenates, we found a significant activation of NADH-driven O2*- production in both models of hyperlipidemia. Treatment of cholesterol-fed animals with the AT1-receptor antagonist Bay 10-6734 improved endothelial dysfunction, normalized vascular O2*- and NADH-oxidase activity, decreased macrophage infiltration, and reduced early plaque formation. In the setting of hypercholesterolemia, the aortic AT1 receptor mRNA was upregulated to 166+/-11%, accompanied by a comparable increase in AT1-receptor density.


CONCLUSIONS
Hypercholesterolemia is associated with AT1-receptor upregulation, endothelial dysfunction, and increased NADH-dependent vascular O2*- production. The improvement of endothelial dysfunction, inhibition of the oxidase, and reduction of early plaque formation by an AT1-receptor antagonist suggests a crucial role of angiotensin II-mediated O2*- production in the early stage of atherosclerosis.
38697127
Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.
Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.
6055297
Novel NAD(P)H oxidases in the cardiovascular system
In the past 10 years, an enormous amount of research has focused on the role of NAD(P)H oxidases in cardiovascular physiology and pathophysiology. Initially, investigators characterised these enzyme activities biochemically and functionally, but more recently, the molecular identification of a family of Nox (NADPH oxidase) proteins related to the respiratory burst NAD(P)H oxidase of neutrophils has led to an explosion of information about expression patterns, regulation, and functional significance of these enzyme complexes. Nox enzymes have been found to be involved in the regulation of vascular tone, smooth muscle growth, inflammatory responses, and matrix metalloproteinase activity. They have been implicated in hypertension, atherosclerosis, heart failure, diabetic vascular disease, and restenosis. In this article, the current status of the molecular basis for and the functional consequences of activation of these novel enzymes will be discussed.

The first Nox enzyme to be cloned and characterised was termed gp91phox (or Nox2), because it is the catalytic unit of the phagocytic respiratory burst oxidase, which is important for non-specific host defence against invading microbes. This protein exists in a macromolecular complex with p22phox, another membrane subunit that stabilises gp91phox and interacts with the cytosolic regulatory factor p47phox. p47phox has two tandem SH3 groups that interact with auto-inhibitory domains to maintain the protein in an inactive state. Upon phosphorylation of p47phox by protein kinase C, this interaction is lost, and the SH3 groups and proline-rich tail are exposed and bind to the membrane subunits and a second regulatory cytosolic subunit, p67phox. p67phox, in turn, serves as a binding site for the small molecular weight G protein Rac, which modulates enzyme activity.1

Originally, gp91phox was thought to be restricted to phagocytic cells. Recent work, however, indicates that gp91phox is expressed at low, but functionally important levels in many tissues. More importantly, in …
39951100
Oxidative stress and cardiovascular risk: the role of vascular NAD(P)H oxidase and its genetic variants
Several risk factors for coronary artery disease (CAD) induce atherosclerosis through endothelial activation and dysfunction, and ample evidence now suggests that the balance between production and removal of reactive oxygen species (ROS) – a condition termed oxidative stress – is implicated in such processes. A main source of ROS in vascular cells is the reduced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase system. This is a membrane‐associated enzyme, composed of five subunits, catalyzing the one‐electron reduction of oxygen, using NADH or NADPH as the electron donor. One of the system subunits, termed p22‐phox, has a polymorphic site on exon 4, associated with variable enzyme activity. This polymorphism is generated by a point mutation (C242T) producing a substitution of histidine with tyrosine at position 72, which affects one of the heme binding sites essential for the NAD(P)H enzyme activity. The consequent decrease of superoxide production thus characterizes a phenotype candidate for conferring to the carrier a reduced susceptibility to CAD. At present, however, the body of evidence from current literature is not yet sufficient to confirm or exclude the hypothesis that the C242T polymorphism protects from CAD. The functional effects of this polymorphism and the potential and its pathophysiological consequences also need further investigation.
9785654
p22phox Is a Critical Component of the Superoxide-generating NADH/NADPH Oxidase System and Regulates Angiotensin IIinduced Hypertrophy in Vascular Smooth Muscle Cells*
Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 α-subunit, p22phox (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22phox mRNA expression by stable transfection of antisense p22phox cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22phox is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.
27527440
Molecular analysis of 9 new families with chronic granulomatous disease caused by mutations in CYBA, the gene encoding p22(phox).
Chronic granulomatous disease is a rare inherited disorder caused by nonexistent or severely decreased phagocyte superoxide production that results in a severe defect in host defense and consequent predisposition to microbial infection. The enzyme responsible for generating the superoxide, NADPH oxidase, involves at least 5 protein components. The absence of, or a defect in, any 1 of 4 of these proteins (p22(phox), p47(phox), p67(phox), or gp91(phox)) gives rise to the known types of chronic granulomatous disease. One of the rarest forms of the disease is due to defects in the CYBA gene encoding p22(phox), which together with gp91(phox) forms flavocytochrome b(558), the catalytic core of NADPH oxidase. To date, only 9 kindreds with p22(phox) deficiency have been described in the literature comprising 10 mutant alleles. Four polymorphisms in the CYBA gene have also been reported. Here we describe 9 new, unrelated kindreds containing 12 mutations, 9 of which are novel. In addition, we report 3 new polymorphisms. The novel mutations are (a) deletion of exons 2 and 3, (b) a missense mutation in exon 3 (T155-->C), (c) a splice site mutation at the 5' end of intron 3, (d) a missense mutation in exon 2 (G74-->T), (e) a nonsense mutation in exon 1 (G26-->A), (f) a missense mutation in exon 4 (C268-->T), (g) a frameshift in exon 3 due to the insertion of C at C162, (h) a nonsense mutation in exon 2 (G107-->A), and (i) a missense mutation in exon 2 (G70-->A).
461444
Polymorphism of the NADH/NADPH oxidase p22 phox gene in patients with coronary artery disease.
BACKGROUND
Oxidative stress in the vasculature has been implicated in the pathogenesis of coronary artery disease (CAD). NADH/NADPH oxidase is a key enzyme of superoxide production in the vasculature. p22 phox, an essential component of NADH/NADPH oxidase, has four types of polymorphism. The C242T polymorphism changes histidine-72 to tyrosine, located in the potential heme-binding sites, whereas A640G polymorphism is located in the 3' untranslated region.


METHODS AND RESULTS
We investigated whether these polymorphisms were associated with risk of CAD by use of restriction fragment length polymorphism (RFLP). The prevalence of the TC + TT genotype of the C242T polymorphism was significantly more frequent in control subjects (n=201) than in the patients with CAD (n=201). The odds ratio of the TC + TT versus CC genotype of the C242T polymorphism between control subjects and case patients was 0.49 (95% CI, 0.28 to 0.87) (P=.015). The prevalence of the genotypes of the A640G polymorphism was not different between groups. The association of C242T polymorphism of the p22 phox gene with CAD was statistically significant and independent of other risk factors.


CONCLUSIONS
The mutation of the potential heme-binding site of the p22 phox gene may reduce susceptibility to CAD. Our observations suggest that the C242T polymorphism of the p22 phox gene is a novel genetic marker that has a protective effect on coronary risk.
30815413
NADH/NADPH oxidase p22phox C242T polymorphism and coronary artery disease in the Australian population
Oxidative stress induced by the superoxide anion (.O2−) has been implicated in atherogenesis. The NADH/NADPH oxidase system is involved in .O2− production and p22 phox is an essential component of that system.
57382502
C242T Polymorphism of NADPH Oxidase p22 PHOX Gene and Ischemic Cerebrovascular Disease in the Japanese Population
Background and Purpose—Superoxide has been implicated in the pathogenesis of ischemic stroke and atherosclerosis. NADPH oxidase, a major source of superoxide generation in neutrophils and the vascular system, plays a critical role in ischemic injury and atherogenesis. Recently, an association between the C242T polymorphism of p22 PHOX, an essential component of NADPH oxidase, and coronary artery disease (CAD) has been reported in several studies. To investigate the relationship between the C242T polymorphism of p22 PHOX and ischemic cerebrovascular disease (CVD), we conducted a case-control study. Methods—We recruited 226 CVD patients (atherothrombotic infarction, lacunar infarction, and transient ischemic attack) and 301 control subjects and analyzed C242T polymorphism of p22 PHOX by detection of restriction fragment length polymorphism. Results—The TC+TT genotype frequencies in the CVD group and control group were 21.7% and 13.3%, respectively, and the prevalence of the TC+TT genotype was significantly ...
1446412
Statistical heterogeneity in systematic reviews of clinical trials: a critical appraisal of guidelines and practice
Objective: Heterogeneity between study results can be a problem in any systematic review or meta-analysis of clinical trials. Identifying its presence, investigating its cause and correctly accounting for it in analyses all involve difficult decisions for the researcher. Our objectives were: to collate recommendations on the subject of dealing with heterogeneity in systematic reviews of clinical trials; to investigate current practice in addressing heterogeneity in Cochrane reviews; and to compare current practice with recommendations. Methods: We review guidelines for those undertaking systematic reviews and examine how heterogeneity is addressed in practice in a sample of systematic reviews, and their protocols, from the Cochrane Database of Systematic Reviews. Results: Advice to reviewers is on the whole consistent and sensible. However, examination of a sample of Cochrane protocols and reviews demonstrates that the advice is difficult to follow given the small numbers of studies identified in many systematic reviews, the difficulty of pre-specifying important effect modifiers for subgroup analysis or meta-regression and the unresolved debate concerning fixed versus random effects metaanalyses. There was disagreement between protocols and reviews, often either regarding choice of important potential effect modifiers or due to the review identifying too few studies to perform planned analyses. Conclusion: Guidelines that address practical issues are required to reduce the risk of spurious findings from investigations of heterogeneity. This may involve discouraging statistical investigations such as subgroup analyses and meta-regression, rather than simply adopting a cautious approach to their interpretation, unless a large number of studies is available. The notion of a priori specification of potential effect modifiers for a retrospective review of studies is ill-defined, and the appropriateness of using a statistical test for heterogeneity to decide between analysis strategies is suspect.
34104820
NADPH Oxidase Polymorphisms in Cerebral Small Vessel Disease
1 Lovett JK, Dennis MS, Sandercock PA, Bamford J, Warlow CP, Rothwell PM: Very early risk of stroke after a first transient ischemic attack. Stroke 2003; 34:e138–e140. 2 Lovett JK, Coull AJ, Rothwell PM: Early risk of recurrence by subtype of ischemic stroke in population-based incidence studies. Neurology 2004; 62: 569–573. 3 Fairhead JF, Mehta Z, Rothwell PM: Population-based study of delays in carotid imaging and surgery and the risk of recurrent stroke. Neurology 2005; 65: 371–375. 4 Lovblad KO, Laubach HJ, Baird AE, et al: Clinical experience with diffusion-weighted MR in patients with acute stroke. AJNR Am J Neuroradiol 1998; 19: 1061–1066. 5 Kang DW, Latour LL, Chalela JA, Dambrosia J, Warach S: Early ischemic lesion recurrence within a week after acute ischemic stroke. Ann Neurol 2003; 54: 66–74. 6 Wen HM, Lam WW, Rainer T, et al: Multiple acute cerebral infarcts on diffusion-weighted imaging and risk of recurrent stroke. Neurology 2004; 63: 1317–1319. 7 Rothwell PM, Buchan A, Johnston SC: Recent advances in management of transient ischaemic attacks and minor ischaemic strokes. Lancet Neurol 2006; 5: 323–331. 8 Demchuk AM: Acute secondary stroke prevention trials in minor stroke/TIA: prime time for magnetic resonance imaging. Int J Stroke 2006; 1: 91–93. 9 Coutts SB, Hill MD, Simon JE, Sohn CH, Scott JN, Demchuk AM: Silent ischemia in minor stroke and TIA patients identified on MR imaging. Neurology 2005; 65: 513–517. 10 Kang DW, Latour LL, Chalela JA, Dambrosia JA, Warach S: Early and late recurrence of ischemic lesion on MRI: evidence for a prolonged stroke-prone state? Neurology 2004; 63: 2261–2265. 11 World Health Organization: Cerebrovascular Diseases – Prevention, Treatment and Rehabilitation. Geneva, WHO, Tech Rep Ser, 1971. 12 Rothwell PM, Coull AJ, Giles MF, et al: Change in stroke incidence, mortality, case-fatality, severity, and risk factors in Oxfordshire, UK, from 1981 to 2004 (Oxford Vascular Study). Lancet 2004; 363: 1925– 1933. 13 Rothwell PM, Gutnikov SA, Warlow CP: Reanalysis of the final results of the European Carotid Surgery Trial. Stroke 2003; 34: 514–523. 14 Kang DW, Chalela JA, Ezzeddine MA, Warach S: Association of ischemic lesion patterns on early diffusion-weighted imaging with TOAST stroke subtypes. Arch Neurol 2003; 60: 1730–1734. 15 Baird AE, Lovblad KO, Schlaug G, Edelman RR, Warach S: Multiple acute stroke syndrome: marker of embolic disease? Neurology 2000; 54: 674–678. 16 Wong KS, Gao S, Chan YL, et al: Mechanisms of acute cerebral infarctions in patients with middle cerebral artery stenosis: a diffusionweighted imaging and microemboli monitoring study. Ann Neurol 2002; 52: 74–81.
29316645
Functional effects of NAD(P)H oxidase p22(phox) C242T mutation in human leukocytes and association with thrombotic cerebral infarction.
BACKGROUND
Previous study showed that polymorphism of the NAD(P)H oxidase p22(phox) gene is associated with atherosclerosis, although others could not confirm such association. We investigated the association between p22(phox) C242T polymorphism and thrombotic cerebral infarction and the role of this polymorphism on superoxide-production activity in human neutrophils and promyelocytic HL-60 cells as a model system.


METHODS
PCR-RFLP analysis revealed that genotype and allele frequencies of C242T polymorphism in 120 patients with thrombotic cerebral infarction and 177 control subjects. The superoxide-production activity in neutrophils was determined by cytochrome c reduction assay. To clarify the role of p22(phox) C242T polymorphism on NAD(P)H oxidase activity, we used transgenic HL-60 cells transfected with expression plasmids carrying p22(phox) cDNAs with or without C242T polymorphism.


RESULTS
Genotype and allele frequencies of C242T polymorphism in patients and control subjects were not significantly different. The superoxide-production activity in neutrophils with T allele was higher than in neutrophils without T allele. Moreover, expression analysis showed that superoxide-production activity in p22(phox) C242T-expressing HL-60 cells were significantly higher than in p22(phox)-expressing HL-60 cells.


CONCLUSIONS
We conclude that C242T of p22(phox) gene is not involved in thrombotic cerebral infarction but more likely in increased NAD(P)H oxidase activity in phagocytes.
33662345
NAD(P)H oxidase p22phox C242T polymorphism and ischemic stroke in Japan: the Fukuoka Stroke Registry and the Hisayama study
The C242T polymorphism of p22phox, a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi‐squared test revealed that the T‐allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate‐adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72–1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS‐7 cells cotransfected with Nox4 and p22phox of each genotype. The superoxide‐producing activity in those cells expressing p22phox with the T allele was not significantly different from that in cells expressing p22phox with the C allele. The present results suggest that the p22phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.
9530759
The C242T polymorphism of the gene encoding cytochrome b-245 alpha is not associated with paediatric ischaemic stroke: family-based and case-control study.
BACKGROUND AND PURPOSE
Reactive oxygen species play an important role in the physiology and pathology of cerebral arteries, including ischaemic stroke. The cytochrome b-245 alpha gene (CYBA) encodes cytochrome b-245 alpha light chain (p22phox peptide), a critical element of NAD(P)H oxidases, the most important source of superoxide anion in the cerebral arteries. To search for genetic factors associated with paediatric ischaemic stroke, the possible association between CYBA gene C242T polymorphism and the disease was evaluated.


MATERIAL AND METHODS
The study group consisted of 238 individuals: children with ischaemic stroke (n = 70), their biological parents (n = 118) and children without any symptoms of stroke (n = 50). The C242T polymorphism was genotyped using polymerase chain reaction - restriction fragment length methodology. To evaluate the possible association between polymorphism and stroke, the transmission disequilibrium test and the case-control method were applied.


RESULTS
The C242 allele was transmitted more frequently than 242T (62.2% vs. 37.8%) but observed frequencies did not differ significantly from expected (p = 0.10). There were also no significant differences in allele and genotype distribution between patients and control subjects (patients: CC - 50.0%, CT - 38.6%, TT - 11.4% vs. controls: CC - 52.0%, CT - 36.0%, TT - 12.0%).


CONCLUSIONS
The study did not show that the C242T polymorphism of the CYBA gene is a risk factor of ischaemic stroke in children.
36888010
The C242T Polymorphism of the NAD(P)H Oxidase p22phox Subunit Is Associated with an Enhanced Risk for Cerebrovascular Disease at a Young Age
Background and Purpose: Oxidative stress has been proposed as a major contributing factor for vascular disease, that acts independently from its participation in predisposing disorders such as diabetes and arterial hypertension. A functionally relevant C242T polymorphism of the CYBA gene encoding the NAD(P)H oxidase p22phox subunit, is supposed to lead to an abnormal reduction in the generation of reactive oxygen species in vascular smooth-muscle and endothelial cells. Methods: We investigated the p22phox C242T single-nucleotide polymorphism by polymerase chain reaction in consecutive patients with ischemic stroke or transient ischemic attack under the age of 50 (n = 161) and in population-based control subjects (n = 136). Results: Homozygosity for the T variant was associated with an enhanced risk for cerebral ischemia (odds ratio 3.85, confidence interval 1.39–10.64) after adjusting for classical risk factors. Risk for cerebral ischemia was not increased in heterozygous subjects. Conclusion: The p22phox C242T single-nucleotide polymorphism is associated with stroke risk. This finding supports the hypothesis that oxidative stress may contribute to stroke pathogenesis.
57351020
Correlation between NAD(P)H oxidase p22phox C242T polymorphism and cerebral infarction
Objective To investigate the distribution of the gene polymorphism and allele frequency of the NAD(P)H oxidase p22phox C242T,and evaluate the relationship between the polymorphism and cerebral infarction among Han population in Shanghai.Methods The gene C242T was detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique in 176 cerebral infarction patients and 131 healthy elders.The frequency distribution of allele was calculated.Results The serum triglyeride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),blood glucose level and blood pressure in cerebral infarction patients were much higher than those in the healthy elders(P0.05).However,the genotype distributions of the cerebral infarction patients were 92.05%(CC),7.95%(CT) and 0(TT).The genotype distributions of the healthy elders were 93.89%(CC),5.34%(CT) and 0.76%(TT).The allele frequency in the cerebral infarction patients was distributed as 96.02%(C) and 3.98%(T),while that in the healthy elders was distributed as 96.56%(C) and 3.44%(T).There was no significant difference in genotype and allele frequency distribution between the cerebral infarction patients and the healthy elders(P0.05).Conclusions The NAD(P)H oxidase p22phox C242T polymorphism might not associate with cerebral infarction.
18141716
Association of the C242T polymorphism in the NADPH oxidase p22 phox gene with carotid atherosclerosis in Slovenian patients with type 2 diabetes
Oxidative stress plays an important role in the pathogenesis of diabetes and its complications. Genetic variations of enzymes producing reactive oxygen species could change their activity, thus contributing to the susceptibility to oxidative stress. The aim of this study was to examine the role of the NADPH oxidase C242T polymorphism in the development of carotid atherosclerosis in patients with type 2 diabetes. 286 diabetic patients and 150 healthy controls were enrolled in the study. Carotid atherosclerosis was quantified ultrasonographically as carotid intima-media thickness, plaque score (0–6) and plaque type (1–5). Diabetic patients were divided into low and high risk groups based on ultrasound phenotypes of carotid atherosclerosis. Genotypes were determined by real-time PCR. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured by enzyme-linked immunosorbent assay (ELISA). Diabetic patients demonstrated a statistically significant difference compared to healthy controls in the following parameters: age, BMI, waist circumference, smoking prevalence, glucose, triglyceride and 8-OHdG serum levels. Control subjects had significantly higher levels of HDL, LDL and total cholesterol than diabetics (p < 0.001). The NADPH C242T polymorphism was not related with clinical characteristics, lipid parameters and 8-OHdG serum levels. We found no significant difference in the NADPH genotype distribution between diabetics and controls (p = 0.19) nor between low and high risk subgroups of diabetics (mean CIMT: p = 0.67; plaque score: p = 0.49, plaque type: p = 0.56). In the present study the NADPH C242T polymorphism was not associated with the degree of oxidative stress and carotid atherosclerosis. Further studies will show if it can be used as a genetic marker for carotid atherosclerosis in diabetic patients.
18748890
Association between NADPH Oxidase p22phox C242T Polymorphism and Ischemic Cerebrovascular Disease: A Meta-Analysis
Background Epidemiological studies have evaluated the association between nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox C242T polymorphism and risk of ischemic cerebrovascular disease (ICVD), but the results remain inconclusive. This meta-analysis was therefore designed to clarify these controversies. Methodology/Principal Findings Systematic searches of electronic databases Embase, PubMed and Web of Science, as well as hand searching of the references of identified articles and the meeting abstracts were performed. Statistical analyses were performed using software Review Manager (Version 5.1.7) and Stata (Version 11.0). The pooled odds ratios (ORs) with 95% confidence intervals (95%CIs) were performed. Fixed or random effects model was separately used depending on the heterogeneity between studies. Publication bias was tested by Begg's funnel plot and Egger's regression test. A total of 6 studies including 1,948 cases and 2,357 controls were combined showing no statistical evidence of association between NADPH oxidase p22phox C242T polymorphism and overall ICVD (allelic model: OR = 1.08, 95%CI = 0.93–1.26; additive model: OR = 1.33, 95%CI = 0.81–2.17; dominant model: OR = 1.00, 95%CI = 0.86–1.15; recessive model: OR = 1.06, 95%CI = 0.77–1.45). Significant association was found in large-artery atherosclerotic stroke subgroup (allelic model: OR = 1.12, 95%CI = 0.88–1.41; additive model: OR = 1.36, 95%CI = 0.60–3.09; dominant model: OR = 1.25, 95%CI = 0.74–2.11; recessive model: OR = 2.17, 95%CI = 1.11–4.23). No statistical evidence of significant association was observed for small-vessel occlusive stroke, as well as Asian subgroup and Caucasian subgroup. Statistical powers on the combined sample size (total and subgroup) were all lower than 80%. Conclusions/Significance This meta-analysis indicates that NADPH oxidase p22phox C242T polymorphism is more associated with large-artery atherosclerotic stroke than small-vessel occlusive stroke. However, this conclusion should be interpreted with caution due to the small sample size. Larger sample-size studies with homogeneous ICVD patients and well-matched controls are required.
18002811
Meta-analysis of genetic association studies.
Meta-analysis, a statistical tool for combining results across studies, is becoming popular as a method for resolving discrepancies in genetic association studies. Persistent difficulties in obtaining robust, replicable results in genetic association studies are almost certainly because genetic effects are small, requiring studies with many thousands of subjects to be detected. In this article, we describe how meta-analysis works and consider whether it will solve the problem of underpowered studies or whether it is another affliction visited by statisticians on geneticists. We show that meta-analysis has been successful in revealing unexpected sources of heterogeneity, such as publication bias. If heterogeneity is adequately recognized and taken into account, meta-analysis can confirm the involvement of a genetic variant, but it is not a substitute for an adequately powered primary study.
9287220
An analysis of Methylenetetrahydrofolate reductase and Glutathione S-transferase omega-1 genes as modifiers of the cerebral response to ischemia
BackgroundCerebral ischemia involves a series of reactions which ultimately influence the final volume of a brain infarction. We hypothesize that polymorphisms in genes encoding proteins involved in these reactions could act as modifiers of the cerebral response to ischemia and impact the resultant stroke volume. The final volume of a cerebral infarct is important as it correlates with the morbidity and mortality associated with non-lacunar ischemic strokes.MethodsThe proteins encoded by the methylenetetrahydrofolate reductase (MTHFR) and glutathione S-transferase omega-1 (GSTO-1) genes are, through oxidative mechanisms, key participants in the cerebral response to ischemia. On the basis of these biological activities, they were selected as candidate genes for further investigation. We analyzed the C677T polymorphism in the MTHFR gene and the C419A polymorphism in the GSTO-1 gene in 128 patients with non-lacunar ischemic strokes.ResultsWe found no significant association of either the MTHFR (p = 0.72) or GSTO-1 (p = 0.58) polymorphisms with cerebral infarct volume.ConclusionOur study shows no major gene effect of either the MTHFR or GSTO-1 genes as a modifier of ischemic stroke volume. However, given the relatively small sample size, a minor gene effect is not excluded by this investigation.
5625129
Superoxide Production and Expression of Nox Family Proteins in Human Atherosclerosis
Background—NAD(P)H oxidases are important sources of superoxide in the vasculature, the activity of which is associated with risk factors for human atherosclerosis. This study was designed to investigate the localization of superoxide production and the expression of the Nox family of NAD(P)H oxidase proteins (gp91phox, Nox1, and Nox4) in nonatherosclerotic and atherosclerotic human coronary arteries. Methods and Results—In coronary artery segments from explanted human hearts, we examined intracellular superoxide production with dihydroethidium. In nonatherosclerotic coronary arteries, superoxide was present homogenously throughout the intima, media, and adventitia. In atherosclerotic arteries, there was an additional intense area of superoxide in the plaque shoulder, which is rich in macrophages and &agr;-actin–positive cells. p22phox colocalized with gp91phox mainly in macrophages, whereas Nox4 was found only in nonphagocytic vascular cells. Expression of gp91phox and p22phox mRNA was associated with the severity of atherosclerosis. gp91phox correlated with the plaque macrophage content, whereas Nox4 correlated with the content of &agr;-actin–positive cells. Nox1 expression was low both in human coronary arteries and isolated vascular cells. Conclusions—Several Nox proteins, including gp91phox and Nox4, may contribute to increased intracellular oxidative stress in human coronary atherosclerosis in a cell-specific manner and thus may be involved in the genesis and progression of human coronary atherosclerotic disease.
7583204
Functional Effect of the C242T Polymorphism in the NAD(P)H Oxidase p22phox Gene on Vascular Superoxide Production in Atherosclerosis
BackgroundIncreased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels. Methods and ResultsVascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis. ConclusionsAssociation of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.
14507575
Association of NAD(P)H oxidase p22 phox gene variation with advanced carotid atherosclerosis in Japanese type 2 diabetes.
OBJECTIVE
To evaluate the association between the C242T polymorphism of the p22 phox gene, an essential component of NAD(P)H oxidase in the vasculature, with intima-media thickness (IMT) of the carotid artery and risk factors for atherosclerosis in type 2 diabetic subjects.


RESEARCH DESIGN AND METHODS
C242T polymorphism of the p22 phox gene was detected by polymerase chain reaction-restriction fragment-length polymorphism in 200 Japanese type 2 diabetic subjects and 215 nondiabetic subjects. We examined the association with this mutation and carotid atherosclerosis as well as the patients' clinical characteristics and the level of 8-hydroxy-2'deoxyguanosine (8-OHdG) as an index of oxidative DNA damage.


RESULTS
The diabetic subjects with the TC+TT genotypes displayed a significantly lower average IMT (1.13 +/- 0.31 vs. 1.31 +/- 0.34 mm; P = 0.0099) and a not significantly lower serum 8-OHdG level than those with the CC genotype, despite no difference in the risk factors. Stepwise multiple regression analysis showed that the risk factors for increased IMT in the diabetic subjects were systolic blood pressure (P = 0.0042) and p22 phox CC genotype (P = 0.0151). In nondiabetic subjects, the average IMT of the TC+TT group was not different from that of the CC group (0.85 +/- 0.14 vs. 0.94 +/- 0.30 mm, P = 0.417). Fasting plasma insulin concentration (41.4 +/- 15.6 vs. 64.2 +/- 59.4 pmol/l, P = 0.0098) and insulin resistance index of homeostasis model assessment (HOMA-R) (1.58 +/- 0.66 vs. 2.60 +/- 2.56, P = 0.0066) were significantly lower in the TC+TT group than in the CC group.


CONCLUSIONS
These results show that the C242T mutation in the p22 phox gene is associated with progression of asymptomatic atherosclerosis in the subjects with type 2 diabetes and is also associated with insulin resistance in nondiabetic subjects.