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(Circulation Research. 2000;86:e85.)
© 2000 American Heart Association, Inc.

UltraRapid Communications

Vascular Superoxide Production by NAD(P)H Oxidase

Association With Endothelial Dysfunction and Clinical Risk Factors

Tomasz J. Guzik, Nick E. J. West, Edward Black, Denise McDonald, Chandi Ratnatunga, Ravi Pillai, Keith M. Channon

From the Departments of Cardiovascular Medicine (T.J.G., N.E.J.W., D.M., K.M.C.) and Cardiothoracic Surgery (E.B., C.R., R.P.), University of Oxford, John Radcliffe Hospital, Oxford, UK.

Correspondence to Keith M. Channon, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK. E-mail keith.channon{at}cardiov.ox.ac.uk

	Abstract

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Abstract—Superoxide anion plays important roles in vascular disease states. Increased superoxide production contributes to reduced nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of vascular disease. We measured superoxide production by NAD(P)H oxidase in human blood vessels and examined the relationships between NAD(P)H oxidase activity, NO-mediated endothelial function, and clinical risk factors for atherosclerosis. Endothelium-dependent vasorelaxations and direct measurements of vascular superoxide production were determined in human saphenous veins obtained from 133 patients with coronary artery disease and identified risk factors. The predominant source of vascular superoxide production was an NAD(P)H-dependent oxidase. Increased vascular NAD(P)H oxidase activity was associated with reduced NO-mediated vasorelaxation. Furthermore, reduced endothelial vasorelaxations and increased vascular NAD(P)H oxidase activity were both associated with increased clinical risk factors for atherosclerosis. Diabetes and hypercholesterolemia were independently associated with increased NADH-dependent superoxide production. The association of increased vascular NAD(P)H oxidase activity with endothelial dysfunction and with clinical risk factors suggests an important role for NAD(P)H oxidase–mediated superoxide production in human atherosclerosis. The full text of this article is available at http://www.circresaha.org.

Key Words: atherosclerosis • endothelium • superoxide • nitric oxide • diabetes

	Introduction

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Superoxide production by vascular tissues and its interaction with nitric oxide may play important roles in vascular disease pathophysiology.^{1 2} Superoxide reacts rapidly with nitric oxide (NO), reducing NO bioactivity and producing peroxynitrite,^{3 4} a strong oxidant that can nitrosylate cellular proteins and lipoproteins.⁵ Recent evidence suggests that increased superoxide production accounts for a significant proportion of the NO deficit in several animal models of vascular disease, including hypercholesterolemia,^{6 7} hypertension,^{8 9 10} and heart failure.¹¹ In addition to effects mediated by scavenging NO, superoxide directly stimulates mitogenesis in vascular smooth muscle cells^{12 13} and reduces endothelial NO synthase expression and activity in endothelial cells.¹⁴

Potential sources of vascular superoxide production include NAD(P)H-dependent oxidases,^{8 15} xanthine oxidase,^{6 16} lipoxygenase, mitochondrial oxidases, and NO synthases.¹⁷ The NAD(P)H oxidase, originally characterized in neutrophils, is present in vascular smooth muscle cells^{15 18} and endothelial cells^{19 20 21} and is the principal source of superoxide production in some animal models of vascular disease.^{7 8} Furthermore, p22phox, one of the components of the NAD(P)H oxidase, is expressed in atherosclerotic human coronary arteries.²² Genetic polymorphisms in the CYBA gene, encoding p22phox, have recently been associated with coronary artery disease in case-control studies and in a prospective study of coronary artery disease progression or regression.²³ Despite the potential importance of superoxide production by the NAD(P)H oxidase in atherosclerosis, functional studies of human vascular superoxide production have been limited and have not investigated NAD(P)H oxidase activity.^{24 25} Consequently, the relationships between superoxide production by NAD(P)H oxidase, atherosclerotic risk factors, and endothelial dysfunction in human blood vessels remain unclear.

Accordingly, we sought to investigate the importance of NAD(P)H oxidase in human vascular superoxide production, with the use of saphenous veins from patients with systemic risk factors for atherosclerosis. We find that an NAD(P)H oxidase is the principal source of superoxide production in human saphenous veins and that increased vascular NAD(P)H oxidase activity is associated with impaired NO-mediated endothelial function and with increased atherosclerotic risk factor profile.

	Materials and Methods

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Patients
Segments of human saphenous veins were obtained from patients undergoing routine coronary artery bypass surgery at the John Radcliffe Hospital, Oxford, UK. The collection of tissue specimens was approved by the local research ethics committee. Clinical risk factors were categorized as follows: hypercholesterolemia (total plasma cholesterol level >4.8 mmol/L); smoking (current or within last 6 months); diabetes (fasting glucose level >5.5 mmol/L or current treatment with insulin or oral hypoglycemic agents); and hypertension (current treatment with antihypertensive agents).

Vasomotor Studies
Vasomotor responses to phenylephrine, acetylcholine (ACh), calcium ionophore, and sodium nitroprusside (SNP) were determined as previously described.²⁶ All experiments were performed in the presence of indomethacin (10 µmol/L). Responses were expressed as a percentage of the maximal contraction or precontracted tension.

Vascular Superoxide Production
Superoxide production was measured by lucigenin-enhanced chemiluminescence, according to previously described and validated methods.^{6 8 27 28} Vessel segments were equilibrated in oxygenated Krebs-HEPES solution for 30 minutes at 37°C. Lucigenin-enhanced chemiluminescence from intact vessels was measured in buffer (2 mL) containing lucigenin (5 µmol/L),^{27 28} then NADH (100 µmol/L) or other substrates were added. In other experiments, chemiluminescence was measured in vascular homogenates, in buffer containing lucigenin (250 µmol/L), as previously described.^{7 8} Vascular homogenates were separated into soluble (cytosolic) and particulate (membrane-associated) fractions by ultracentrifugation, as previously described.²⁹ Superoxide production was expressed as relative light units (RLU) · s^-1 · mg vessel dry weight^-1 or RLU · s^-1 · µg protein^-1.

We measured NADH-stimulated superoxide production in both vascular homogenates and intact rings prepared from the same patients (n=30) and found a significant correlation (r=0.7, P<0.001), suggesting that either approach provides an equivalent measure of NAD(P)H oxidase activity.

The lucigenin-based assay was validated against an independent measure of superoxide production using superoxide dismutase (SOD)-inhibitable ferricytochrome c reduction, as previously described.³⁰ Equilibrated vessel rings or portions of vascular homogenate were incubated in 1 mL of buffer containing ferricytochrome c (80 µmol/L) at 37°C for 45 minutes, then absorbance was measured at 550 nm. Similar experiments were performed in the presence of NADH (100 µmol/L). All experiments were performed in parallel with and without SOD (400 U/mL). Superoxide-dependent ferricytochrome c reduction was calculated as the portion of ferricytochrome c reduction inhibited by SOD. NADH-stimulated superoxide production measured by ferricytochrome c reduction from both intact vessels and vascular homogenates was closely correlated with measurements determined in parallel by lucigenin-enhanced chemiluminescence (intact vessels: n=9; r=0.91, P<0.0001; homogenates: r=0.94, P=0.001).

Statistical Analysis
Data are expressed as mean±SEM. Vasomotor responses are the average of a mean 3.4 rings for each patient; in all cases n refers to numbers of patients. Statistical significance of differences between superoxide production in response to different substrates or inhibitors was assessed by Student’s t tests. Correlation between vasorelaxations and superoxide production was assessed by simple linear regression. Association between vasorelaxations and number of clinical risk factors was assessed by Spearman’s rank correlation coefficient and between superoxide production and individual risk factors using multiple ANOVA (type III sums of squares). A value of P<0.05 was considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Patient Characteristics
Saphenous vein was obtained from 133 patients (107 men, 26 women) undergoing coronary artery bypass grafting. Demographic and clinical characteristics are shown in Table 1. All patients had at least one clinical risk factor for atherosclerosis, and all patients had been administered multiple drug therapy.

View this table: [in this window] [in a new window]   Table 1. Clinical and Demographic Characteristics of Patients

Superoxide Generation From Human Saphenous Veins
Basal superoxide production was determined by lucigenin-enhanced chemiluminescence from intact vessel rings. Specificity for superoxide was demonstrated by coincubation with either SOD (350 U/mL) or tiron (10 mmol/L). Superoxide production was greatly reduced by diphenyleneiodonium, an inhibitor of flavin-containing oxidases (Table 2). In contrast, neither oxypurinol, rotenone, nor N-methyl-L-arginine significantly inhibited superoxide production. The addition of NADH (100 µmol/L) stimulated superoxide release >10-fold; NADH-stimulated superoxide release was inhibited also by diphenyleneiodonium but not by oxypurinol, rotenone, or N-methyl-L-arginine (Table 2). Because NADH has been reported to stimulate activity of extracellular xanthine oxidase, we measured superoxide release from intact vessel rings in response to hypoxanthine. There was no significant increase in superoxide release in the presence of 1 mmol/L hypoxanthine (n=8; basal 8.2±0.9 versus hypoxanthine 9.3±2.3 RLU · s^-1 · mg^-1; P=NS). We investigated also superoxide release by vascular homogenates in the presence of substrates for specific oxidases (Figure 1A). Again, the greatest stimulation of superoxide production was generated by NADH. NADPH produced 25% of the stimulation seen with NADH. In contrast, neither succinate nor hypoxanthine stimulated superoxide production by vascular homogenates.

View this table: [in this window] [in a new window]   Table 2. Characteristics of Vascular Superoxide Release

View larger version (17K): [in this window] [in a new window]   Figure 1. Superoxide production by vascular homogenates. Segments of saphenous vein were homogenized, and superoxide production was determined by lucigenin-enhanced chemiluminescence. A, Response to potential oxidase substrates: no substrate added (Basal), NADH (100 µmol/L), NADPH (100 µmol/L), succinate (Succ, 5 mmol/L), and hypoxanthine (Xanth, 500 µmol/L). n=9 patients in each group. B, Subcellular location of NAD(P)H oxidase activity was determined by ultracentrifugation of vascular homogenates into soluble (cytosol) and particulate (membrane) fractions. In some vessels, endothelium was removed from vessel segments before homogenization and fractionation (endothelium -). Bars show mean±SEM. Unfractionated and cytosol readings are shown on an enlarged scale for clarity. *P<0.05, **P<0.01 vs Basal or vs endothelium +.

To further characterize this vascular NADH-dependent oxidase activity, we determined the specific activity of the enzyme in subcellular fractions of saphenous vein homogenates generated from intact vessels and from vessels denuded of endothelium (Figure 1B). NADH-dependent superoxide production was reduced by 40% in vascular homogenates prepared after endothelial denudation, suggesting that endothelium contains a substantial proportion of NADH-stimulated oxidase activity. Subcellular fractionation of homogenates by ultracentrifugation into soluble (cytosolic) and particulate (membrane) fractions revealed that >99% of the NADH-stimulated oxidase activity was localized in the particulate fraction.

Taken together, these data suggest that a membrane-associated NAD(P)H-dependent oxidase, rather than xanthine oxidase, mitochondrial oxidases, or NO synthase, is the predominant source of superoxide production in human saphenous veins from patients with atherosclerosis.

Endothelial Vasomotor Function in Human Saphenous Veins
We used isometric vasomotor studies to investigate NO bioactivity in saphenous vein segments. Maximal endothelium-dependent, receptor-mediated relaxation to ACh was 24.7±1.3% (n=118), whereas endothelium-dependent, receptor-independent relaxations to calcium ionophore (A23187) were greater in all patients (39.6±1.2%, P<0.0001 versus ACh). These responses were inhibited by incubation with nitro-L-arginine or by endothelial removal (data not shown). A large variability between patients in relaxations to these endothelium-dependent agonists was demonstrated (ACh: range 1% to 67%; A23187: range 8% to 78%), but maximal relaxations to ACh and calcium ionophore were significantly correlated within patients (r=0.4, P=0.005). In contrast to ACh, SNP produced complete relaxation in all vessels, indicating that reduced ACh relaxations were not due to inability of medial smooth muscle to relax in response to NO.

Relationship Between NADH-Stimulated Superoxide Generation, Endothelial Function, and Clinical Risk Factors
To investigate the potential importance of vascular NAD(P)H oxidase activity in patients with systemic risk factors for atherosclerosis, we compared NADH-dependent superoxide production by vessel rings and NO-mediated, endothelium-dependent vasorelaxations in patients with an increasing number of clinical risk factors. NADH-dependent superoxide production varied by >10-fold between patients, from 79 to 827 RLU · min^-1 · mg^-1 (n=116). However, NADH-dependent superoxide production correlated inversely with maximal relaxation to ACh in individual patients (Figure 2; r=-0.45, P<0.0001) and with maximal relaxation to the receptor-independent agonist A23187 (Figure 2; r=-0.40, P=0.002). In contrast, there was no relationship between superoxide production and maximal relaxation to the direct NO donor SNP (Figure 2; r=0.003, P=0.95).

View larger version (19K): [in this window] [in a new window]   Figure 2. Relationship between NADH-stimulated superoxide production and maximal relaxations to ACh, calcium ionophore (A23187), or SNP in human saphenous veins (n=108 patients). Superoxide production was determined by lucigenin-enhanced chemiluminescence from intact vessels after addition of NADH (100 µmol/L). There was a highly significant correlation between NADH-dependent superoxide generation and relaxations to ACh and A23187.

Furthermore, an increased number of atherosclerotic risk factors was associated with both reduced ACh-dependent vasorelaxations (Figure 3; r=-0.53, P<0.0001) and increased NADH-dependent superoxide production (Figure 3; r=0.49, P<0.0001). Analysis of individual risk factors revealed that diabetes and hypercholesterolemia were independently associated with increased NADH-dependent superoxide production (Table 3). These data indicate that in human saphenous veins, impaired endothelial NO bioactivity is associated with increased NADH-dependent vascular superoxide production and that both of these factors are more marked in patients with increased atherosclerotic risk factors.

View larger version (17K): [in this window] [in a new window]   Figure 3. Box and whisker plots showing relationship between number of clinical risk factors for atherosclerosis and maximal ACh relaxation (top) or vascular NAD(P)H oxidase activity measured by NADH-dependent superoxide generation (bottom) in human saphenous veins. Coronary risk factors are hypertension, diabetes mellitus, smoking, and hypercholesterolemia. Lines within boxes represent median values; upper and lower limits of boxes, 75th and 25th percentiles, respectively; upper and lower bars of whiskers, 90th and 10th percentiles, respectively.

View this table: [in this window] [in a new window]   Table 3. Risk Factors Associated With Vascular NADH-Dependent Superoxide Release

	Discussion

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We have used saphenous veins as a model system to study vascular superoxide production in human blood vessels. Human saphenous veins from patients with atherosclerosis generate superoxide, predominantly by an NAD(P)H-dependent oxidase. Furthermore, we demonstrate associations between NAD(P)H-dependent superoxide release, reduced NO-mediated vasorelaxations, and clinical risk factors for atherosclerosis.

These findings are important because they suggest an association between endothelial dysfunction and increased vascular superoxide production in human atherosclerosis. Our study is in agreement with previous in vivo³¹ and in vitro²⁴ data showing that ACh-mediated vasorelaxations are inversely related to the number of atherosclerotic risk factors present. Huraux et al²⁴ found a large variability in both NO-mediated vascular relaxations and basal superoxide production in internal mammary arteries, as we did in saphenous veins, but did not find consistent associations between these two parameters or clinical risk factors. However, we determined NADH stimulated superoxide release, given that we found this oxidase system to be the predominant source of vascular superoxide production in human saphenous vein and that the activity of this enzyme system is associated with endothelial dysfunction and with clinical risk factors.

Superoxide production is increased in animal models of vascular disease such as hypercholesterolemia,^{6 7} hypertension,^{8 9 10} heart failure,¹¹ and diabetes, and, in most cases, the NAD(P)H oxidase system appears to be the predominant source of superoxide.^{6 7 8} Our identification of the NAD(P)H oxidase system as the principal source of superoxide production in human vessels from patients with atherosclerotic risk factors highlights the potential importance of this oxidase in human atherosclerosis.³² The NAD(P)H oxidase system is present in human vascular smooth muscle cells and endothelial cells in culture^{19 20} and comprises a multisubunit enzyme complex including the p47phox, p67phox, gp91phox, p22phox, and Rac proteins. The p22phox subunit is required for oxidase activity in smooth muscle cells,¹⁵ and activity and expression of the enzyme in animal models and cell culture are stimulated by angiotensin II.^{8 9 18} Of particular relevance to the findings of our study are recent data indicating that the p22phox subunit is expressed in the endothelium, media, and adventitia of human coronary arteries and that p22phox protein is increased in atherosclerotic arteries.²² Our functional data are in agreement with this model, showing that NAD(P)H oxidase activity is present in both the endothelium and media/adventitia. Recent reports suggest a possible association between atherosclerosis and genetic polymorphisms in the CYBA gene encoding p22phox.³³ A larger prospective study, based on the Lipoprotein and Coronary Artery Study (LCAS), identified a strong association between CYBA genotype and the likelihood of coronary artery disease progression or regression over 2.5 years.²³ Our study contributes important functional data to this emerging association between NAD(P)H oxidases and atherosclerosis, by showing that increased atherosclerotic risk factor profile and endothelial dysfunction are associated with increased NAD(P)H oxidase enzyme activity. However, the exact identity of this oxidase, or oxidases, in the cell types present in human saphenous vein remains to be determined. Recent studies revealed NAD(P)H oxidase components other than those found in the neutrophil enzyme, such as the gp91 homolog Mox-1 in vascular smooth muscle cells.¹³ Furthermore, knockout mouse models suggest functional redundancy in the NAD(P)H oxidase components p47phox³⁴ and gp91phox.³⁵ These findings raise the possibility that the vascular NAD(P)H oxidases may have significant differences from the neutrophil enzyme. Future functional studies of NAD(P)H oxidase activity need to identify the NAD(P)H oxidase components present in different cell types in human vessels, address mechanisms of activation and transcriptional regulation of the oxidase components by factors such as angiotensin II and atherosclerotic risk factors, and assess the functional importance of genetic polymorphisms in CYBA and related genes.

We and others have found that vascular NAD(P)H oxidase activity can be stimulated by extracellular NADH applied to intact vessel segments, or intact vascular cells, as well as in homogenates.^{11 32 36 37 38} This could be due to transport of reducing equivalents into cells, thus indirectly increasing intracellular NADH levels. Alternatively, the membrane-associated NAD(P)H oxidase subunits in vascular cells may have different transmembrane orientations from neutrophil NADPH oxidase, as has been observed in fibroblasts.³⁹ Importantly, our finding of a close correlation between NADH-stimulated superoxide release from intact vessels and vascular homogenates from the same patient suggests that either approach to measuring vascular NAD(P)H oxidase activity in blood vessels seems to be valid.

The association between increased vascular NAD(P)H oxidase activity and impaired endothelial vasorelaxations may be due to direct scavenging of NO by superoxide, as has been demonstrated in animal model systems. However, both could result independently from increasing exposure of endothelium, media, and adventitia to factors acting through different signaling pathways. Alternatively, superoxide may directly modulate NO-mediated vascular signaling, for example by peroxynitrite-induced nitration of G proteins or other membrane components,⁴⁰ or reduction of endothelial NO synthase activity,¹⁴ which result in impaired NO production by endothelial NO synthase. Previous data suggest that G protein–coupled receptor function is deficient in atherosclerosis.⁴¹ Our observation that vasorelaxations to ACh were significantly less than maximal relaxations to the calcium ionophore A23187 is consistent with this hypothesis and with observations in human internal mammary arteries.²⁴ However, the significant correlation between ACh- and A23187-induced relaxations and the association of NADH-dependent superoxide production with both ACh- and A231287-stimulated vasorelaxations suggest that a change in G protein–coupled receptor signaling is unlikely to be the sole mechanism underlying reduced NO-mediated vasorelaxations, given that A23187 activates endothelial NO synthase independently of any receptor-mediated pathway. Importantly, increased NADH-dependent superoxide production does appear to be specifically associated with reduced endothelial NO bioactivity rather than the ability of medial smooth muscle to relax to exogenous NO, because maximal endothelium-independent relaxations to SNP were not related to superoxide production or to endothelium-dependent relaxations.

In conclusion, we find that increased vascular NAD(P)H oxidase activity is associated with reduced NO-mediated vasomotor function and with increased atherosclerotic risk factor profile. This suggests a potentially important role for the NAD(P)H oxidase system in the pathophysiology of human atherosclerosis.

	Acknowledgments

 
This work was supported by grants from the Garfield Weston Trust and the British Heart Foundation. T.J.G. is a Crescendum Est Polonia Foundation International Fellow. N.E.J.W is a British Heart Foundation Junior Research Fellow. We are grateful to Dr Martin Farrall for helpful statistical advice.

Received April 7, 2000; accepted April 11, 2000.

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