UltraRapid Communication - Circulation Research

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 and Keith M. Channon 

Circ Res. 2000;86:e85-e90 

doi: 10.1161/01.RES.86.9.e85 

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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 

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. (Circ Res. 2000;86:e85-e90.) 

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

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, 8–10 and heart failure. 11 In addition to 

effects mediated by scavenging NO, superoxide directly 

stimulates mitogenesis in vascular smooth muscle cells12,13 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 cells15,18 and 

endothelial cells19–21 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 

UltraRapid Communication 

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 NOmediated 

endothelial function and with increased atherosclerotic 

risk factor profile. 

Materials and Methods 

Patients 

Segments of human saphenous veins were obtained from patients 

undergoing routine coronary artery bypass surgery at the John 

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

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@cardiov.ox.ac.uk 

© 2000 American Heart Association, Inc. 

Circulation Research is available at http://www.circresaha.org 


http://circres.ahajournals.org/ 1 by guest on January 29, 2013

2 Circulation Research May 12, 2000 

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 �mol/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 �mol/L), 27,28 then NADH (100 �mol/L) 

or other substrates were added. In other experiments, chemiluminescence 

was measured in vascular homogenates, in buffer containing 

lucigenin (250 �mol/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 � �g 

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 �mol/L) at 37°C for 45 minutes, then absorbance was measured 

at 550 nm. Similar experiments were performed in the presence of 

NADH (100 �mol/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 

Patient Characteristics 

Saphenous vein was obtained from 133 patients (107 men, 26 

women) undergoing coronary artery bypass grafting. Demo- 

graphic 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. 

Superoxide Generation From Human 

Saphenous Veins 

Basal superoxide production was determined by lucigeninenhanced 

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 �mol/L) stimulated superoxide release �10fold; 

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. 

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 endo- 

TABLE 1. Clinical and Demographic Characteristics of Patients 

Age (year; mean�SEM) 

Number, % 

64�0.8 

Sex (M:F) 

Risk Factors 

107:26 

Smoking 50 (38) 

Hypertension 97 (73) 

Diabetes mellitus 32 (24) 

Hypercholesterolemia 

Medication 

88 (66) 

�-blockers 80 (60) 

Aspirin 115 (86) 

Nitrates 70 (65) 

Lipid-lowering agents 106 (80) 

Calcium antagonists 69 (52) 

Angiotensin-converting enzyme inhibitors 61 (46) 

Numbers in parentheses are percentages of total number. 



TABLE 2. Characteristics of Vascular Superoxide Release 

Superoxide Production, 

RLU � s �1 � mg �1 

Basal �NADH 

Vessel alone 22.4�3.4 350�31.8 

�Diphenyleneiodonium 6.6�1.7* 94.3�13.4* 

�Oxypurinol 19.1�4.3 371�39.3 

�Rotenone 20.8�2.7 341�35.5 

�SOD 7.2�2.8* 165�24.9* 

�Tiron 5.9�1.0* 80.5�8.9* 

�N-methyl-L-arginine 

Data are mean�SEM. 

26.2�3.9 448�70.6 

Superoxide production was determined in multiple saphenous vein rings 

from 13 patients by lucigenin-enhanced chemiluminescence (5 �mol/L lucigenin) 

in the presence or absence of various inhibitors of oxidase systems 

(diphenyleneiodonium 100 �mol/L, oxypurinol 100 �mol/L, rotenone 100 

�mol/L, or N-methyl-L-arginine 1 mmol/L) or scavengers of superoxide (SOD 

350 U/mL or tiron 10 mmol/L). Vessel rings were incubated with these agents 

for 30 minutes before and during superoxide determination. Other rings were 

stimulated with NADH (100 �mol/L), with or without incubation with inhibitors. 

*P�0.05 vs vessel ring alone. 

thelium contains a substantial proportion of NADHstimulated 

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 membraneassociated 

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 endotheli- 

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 �mol/L), NADPH 

(100 �mol/L), succinate (Succ, 5 mmol/L), and hypoxanthine 

(Xanth, 500 �mol/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 �. 

Guzik et al NAD(P)H Oxidase Activity in Atherosclerosis 3 

um-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, endotheliumdependent 

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, NADHdependent 

superoxide production correlated inversely with 

maximal relaxation to ACh in individual patients (Figure 2; 

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 �mol/L). There was a highly significant correlation between 

NADH-dependent superoxide generation and relaxations to ACh 

and A23187. 




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. 

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). 

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. 

Discussion 

We have used saphenous veins as a model system to study 

vascular superoxide production in human blood vessels. 

TABLE 3. Risk Factors Associated With Vascular NADH-Dependent 

Superoxide Release 

Human saphenous veins from patients with atherosclerosis 

generate superoxide, predominantly by an NAD(P)Hdependent 

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 vivo 31 and in vitro 24 

data showing that ACh-mediated vasorelaxations are inversely 

related to the number of atherosclerotic risk factors 

present. Huraux et al 24 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, 

8–10 heart failure, 11 and diabetes, and, in most cases, the 

NAD(P)H oxidase system appears to be the predominant 

source of superoxide. 6–8 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 

NAD(P)H Oxidase Superoxide Production 

Risk Factor (RF) With RF (n) Without RF (n) With RF Without RF P 

Hypertension 83 33 292�18 275�24 0.4 

Hypercholesterolemia 76 40 324�19 226�17 �0.01 

Smoking 45 71 335�25 258�16 0.08 

Diabetes mellitus 

Data are mean�SEM. 

31 85 360�31 261�14 0.01 

Type III sums-of-squares ANOVA was performed using clinical risk factors as categorical covariates 

and NADH-dependent superoxide production, expressed in RLU � min�1 � mg�1 vessel, as the 

dependent variable. 



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 

p47phox 34 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. 11,32,36–38 This could be due to transport of 

reducing equivalents into cells, thus indirectly increasing 

intracellular NADH levels. Alternatively, the membraneassociated 

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 peroxynitriteinduced 

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 

Guzik et al NAD(P)H Oxidase Activity in Atherosclerosis 5 

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. 

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