In the present study, we sought to evaluate the role of three polymorphisms in the ecNOS (endothelial constitutive nitric oxide synthase) gene in relation to the existence, severity and progression of CAD (coronary artery disease), MI (myocardial infarction) and the occurrence of ischaemia in a predominantly Caucasian population. Patients with CAD (n=760) and age- and sex-matched population-based controls (n=691) were genotyped for the −786T/C, E/D298 and 4a/b polymorphisms. Patients were randomized to pravastatin (40 mg) or placebo. Progression of atherosclerosis was evaluated by sequential angiography. Functionality was assessed by ST segment analysis of ambulant ECGs. The E298 (P=0.003) and 4a (P=0.001) alleles were associated with CAD. Furthermore, E298 (P=0.009) and −786T (P=0.022) alleles were associated with previous MI among patients, predominantly smokers. D/D298 homozygotes, but not −786T/C or 4a/4b mutants, had longer-lasting ischaemia than others (P<0.05). We found no differences in progression of atherosclerosis, irrespective of pravastatin use. We conclude that the E/D298 polymorphism is most consistently associated with CAD, but not with progression of atherosclerosis. The E allele is associated with CAD and MI, whereas the D allele is associated with ischaemia.

INTRODUCTION

NO (nitric oxide) is a key molecule in endothelium-dependent vasodilatation [1,2] and has several potentially anti-atherosclerotic effects, such as inhibition of leucocyte adhesion [35], reduction of platelet-vessel wall interaction [6,7] and endothelial permeability [8,9], and inhibition of vascular smooth muscle cell proliferation and migration [10,11]. The enzyme ecNOS (endothelial constitutive NO synthase) catalyses NO production in the vascular wall [12]. Several modalities, in particular statins, improve disturbed endothelial function possibly by influencing ecNOS expression [13]. In contrast, smoking deteriorates endothelial function [14].

The gene encoding ecNOS is located on chromosome 7q36 and is highly polymorphic [15]. Three polymorphisms, all in strong linkage disequilibrium, have been identified in the promoter region of the ecNOS gene [16]. The −786T/C variant has been described to decrease ecNOS promoter activity, as assessed by luciferase reporter gene assay [16]. In contrast, an exonic polymorphism (G→T) in nucleotide 894 leads to Glu298→Asp substitution of the mature enzyme [17]. This substitution is associated with breakdown of the enzyme [18]. We refer to this polymorphism as E/D298. Furthermore, VNTR (variable number of tandem repeats) in intron 4 (4a/4b) has been associated with lower concentrations of NO metabolites [19,20]. These polymorphisms have all been suggested to be associated with CAD (coronary artery disease) with contradictory results [2123].

We hypothesized that sequence polymorphisms in the gene encoding for ecNOS are associated with coronary atherosclerosis and that (pleiotropic) effects of statin therapy might modify this association. We therefore investigated the relationship of these gene polymorphisms to coronary artery morphology as well as functional parameters, such as the occurrence of ischaemia and MI (myocardial infarction), in the Regression Growth Evaluation Statin Study (REGRESS) and compared the genotype distribution with a sample of healthy male subjects recruited from the general population.

METHODS

Patients with CAD

The patient population consisted of 760 participants recruited from REGRESS, a trial that evaluated the effect of lipid-lowering therapy in non-diabetic male patients who were scheduled to undergo coronary angiography. The original study population consisted of 885 patients. The subgroup for whom DNA was available did not differ significantly in any baseline characteristic from the total group. The details of design and results of REGRESS have been described previously [24]. Briefly, the study subjects were a random sample from the population of patients with CAD, referred for coronary angiography because of anginal complaints. Patients were included if they had a cholesterol level of between 155 and 310 mg/dl (4.0 and 8.0 mmol/l) and triacylglycerols (triglycerides) <354 mg/dl (4.0 mmol/l). Furthermore, the coronary angiogram had to show at least one lesion that, when assessed visually, narrowed the luminal diameter by more than 50%. Patients were randomized to receive pravastatin (40 mg once daily) or a placebo. Patients and their treating physicians were blinded with regard to the randomization throughout the study. Coronary angiography was performed at baseline and after 2 years to assess the progression of the untreated lesions. Clinical follow-up details were available for all patients.

Control group

Age- and sex-matched population-based controls (n=691) were selected from participants of the Cardiovascular Disease Risk Factor Monitoring Project, a large screening project for cardiovascular risk factors that was carried out in three Dutch towns (Amsterdam, Doetinchem and Maastricht) between 1987 and 1991 [25]. Briefly, the study protocol included blood sampling, a physical examination and a self-administered questionnaire. Controls were group matched to cases for comparable selection criteria.

Ethical approval

The study protocol conformed to the Declaration of Helsinki and was approved by the Ethics Committees of each participating institute. Written informed consent was obtained from each participant.

Angiographic methods

Baseline and follow-up coronary angiograms were analysed quantitatively at the Angiographic Core Laboratory using commercially available software (CMS; MEDIS Medical Imaging Systems, Nuenen, The Netherlands). Progression of atherosclerosis was evaluated by the change in average MSD (mean segment diameter) per patient and the change in average MOD (minimum obstruction diameter) per patient. Changes in MSD and MOD reflect diffuse and focal progression–regression of atherosclerosis respectively. If a segment or lesion was adequately visualized in two (preferentially orthogonal) projections and free of significant foreshortening in both views, the average values of the parameters in both projections were calculated. To calculate average MSD and MOD per patient the MSDs and MODs of all qualifying segments or obstructions were added and then divided by the number of contributing segments or obstructions.

Clinical events

The occurrence of major clinical events during the 2 year follow-up of the patients formed a secondary endpoint. Major clinical events were defined as fatal or non-fatal MI, death from CAD other than fatal MI, non-scheduled percutaneous transluminal coronary angioplasty or coronary artery bypass grafting. All cardiovascular clinical events were evaluated and identified according to pre-specified guidelines for inclusion or exclusion in the clinical event analysis by two independent physicians.

Testing of functionality by ST segment analysis on 24 h electrocardiography

In a substudy on 158 patients, the impact of pravastatin on ischaemia was evaluated with ST segment analysis on 24 h AECG (ambulant ECG) registrations. The methods and main results of this study are reported elsewhere [26]. Transient myocardial ischaemia was defined as the presence of episodes showing 0.1 mV horizontal or downsloping ST-segment depression 80 ms after the J-point, lasting for at least 60 s and separated by 60 s from the next ischaemic episode. In this study, only AECG registrations from inclusion up to 3 months after randomization were analysed.

DNA collection and genotyping

Blood samples for genetic studies were obtained at the moment of inclusion. Total genomic DNA was isolated from leucocytes as described previously [27], dissolved in 10 mmol/l Tris/HCl/1 mmol/l EDTA (pH 8.0) and stored at 4 °C. The segments of interest in the ecNOS gene were amplified by PCR. The internal quality control procedures used have been described elsewhere [28]. Genotyping was performed uniformly for all subjects and the analysts were blinded with regard to the phenotype. The E/D298 variant was screened using restriction fragment length polymorphism analysis [17]. The amplified segment was cleaved into fragments by BanII. The −786T/C variant was assessed after cleavage of the PCR product by HaeIII [16]. Finally, PCR and electrophoresis, resulting in 5/5, 4/5 and 4/4 variants, was used to analyse the VNTR variant [22]. This polymorphism is denoted according to Wang et al. [22] as ecNOS4a for four repeats and ecNOS4b for five repeats. Interestingly, we also detected a 6-repeat at 228 bp (n=2) and a 3-repeat at 148 bp (n=3).

Statistical methods

All data are expressed as means±S.D., unless stated otherwise. Differences between genotype groups with respect to baseline parameters were analysed with one-way ANOVA, Kruskal–Wallis test or χ2 test, where appropriate. The changes in angiographic parameters were analysed with two-way covariance analysis with randomized therapy and three possible genotypes as fixed factors and the baseline values as covariates. Only subjects in whom genotyping was unambiguous were included in the analysis. Time to first clinical event was analysed with Cox's model. The AECG data were analysed with the Mann–Whitney U test of the median values of number of ischaemic events, total duration of ischaemia and total sum of ST depression. A P value of 0.05 or less was considered statistically significant.

RESULTS

A total of 760 patients with documented CAD and 691 age- and sex-matched control subjects were investigated. The baseline criteria are described in Table 1. Despite being matched for age, patients were slightly older than controls. As expected, they had higher cholesterol and lower HDL (high-density lipoprotein)-cholesterol concentrations, higher average BP (blood pressure) and higher average BMI (body mass index). The distribution of the three investigated polymorphisms did not deviate from the Hardy–Weinberg equilibrium. There was a significant, albeit weak, linkage disequilibrium between E/D298 and −786T/C (τ=0.390), E/D298 and 4a/4b (τ=0.217) and −786T/C and 4a/4b (τ=−0.421). The population was predominantly of Caucasian origin, with less than 2% individuals of various other races.

Table 1
Baseline characteristics of CAD patients and control subjects

All subjects were non-diabetic males. P values were determined by ANOVA. *P value determined by χ2 analysis. BP, blood pressure.

 CAD patients Control subjects P 
n 760 691  
Age (years) 56.04±8.0 51.28±6.3 <0.0001 
Current smokers (n204 (26.9%) 234 (33.9%) <0.0001* 
Past smokers (n464 (61.1%) 297 (43.0%) <0.0001* 
Total cholesterol (mmol/l) 6.03±0.87 5.85±0.88 <0.0001 
HDL-cholesterol (mmol/l) 0.93±0.22 1.10±0.26 <0.0001 
Systolic BP (mmHg) 135.0±18.4 123.5±13.6 <0.0001 
Diastolic BP (mmHg) 81.5±10.2 78.3±9.5 <0.0001 
BMI (kg/m226.0±2.7 25.5±2.6 <0.0001 
 CAD patients Control subjects P 
n 760 691  
Age (years) 56.04±8.0 51.28±6.3 <0.0001 
Current smokers (n204 (26.9%) 234 (33.9%) <0.0001* 
Past smokers (n464 (61.1%) 297 (43.0%) <0.0001* 
Total cholesterol (mmol/l) 6.03±0.87 5.85±0.88 <0.0001 
HDL-cholesterol (mmol/l) 0.93±0.22 1.10±0.26 <0.0001 
Systolic BP (mmHg) 135.0±18.4 123.5±13.6 <0.0001 
Diastolic BP (mmHg) 81.5±10.2 78.3±9.5 <0.0001 
BMI (kg/m226.0±2.7 25.5±2.6 <0.0001 

CAD

There was an excess of individuals homozygous for E/E298 among the CAD cases (45.4%) when compared with healthy controls (37.6%; P=0.003; Table 2), in contrast with the −786T/C genotype, where no differences were found. The 4a polymorphism was detected more frequently among patients with CAD than healthy controls (Table 2). In comparison with the D/D298 homozygotes, the odds ratio for CAD was 1.77 (95% CI, 1.25–2.51; where CI is confidence interval) for E/E298 homozygotes and 1.37 (95% CI, 0.97–1.94) for heterozygotes. Correction for smoking and age did not change the estimate. The risk of CAD for EE compared with DD homozygotes, stratified according to smoking status, was most evident in ex-smokers (odds ratio, 2.14; 95% CI, 1.32–3.46) and absent in non-smokers (odds ratio, 1.02; 95% CI, 0.45–2.31).

Table 2
Genotype distribution in CAD patients and controls

df, degrees of freedom.

Genotype CAD patients Control subjects  
E/D298 (n755 574 χ2=11.5(2 df), P=0.003 
 EE 343 (45.4%) 216 (37.6%)  
 ED 333 (44.1%) 270 (47.0%)  
 DD 79 (10.5%) 88 (15.3%)  
−786T/C (n709 387 χ2=0.79(2 df), P=0.673 
 TT 281 (39.6%) 164 (42.4%)  
 TC 317 (44.7%) 166 (42.9%)  
 CC 111 (15.7%) 57 (14.7%)  
Intron 4 VNTR* (n752 466 χ2=14.7(2 df), P=0.001 
 4b/4b 545 (72.5%) 381 (81.8%)  
 4b/4a 195 (25.9%) 77 (16.5%)  
 4a/4a 12 (1.6%) 8 (1.7%)  
Genotype CAD patients Control subjects  
E/D298 (n755 574 χ2=11.5(2 df), P=0.003 
 EE 343 (45.4%) 216 (37.6%)  
 ED 333 (44.1%) 270 (47.0%)  
 DD 79 (10.5%) 88 (15.3%)  
−786T/C (n709 387 χ2=0.79(2 df), P=0.673 
 TT 281 (39.6%) 164 (42.4%)  
 TC 317 (44.7%) 166 (42.9%)  
 CC 111 (15.7%) 57 (14.7%)  
Intron 4 VNTR* (n752 466 χ2=14.7(2 df), P=0.001 
 4b/4b 545 (72.5%) 381 (81.8%)  
 4b/4a 195 (25.9%) 77 (16.5%)  
 4a/4a 12 (1.6%) 8 (1.7%)  
*

6-repeat (n=2) and 3-repeat (n=3) genotypes were excluded from this analysis.

Previous MI

Within the CAD group, baseline characteristics did not differ between the genotypes, except a small difference in HDL-cholesterol concentration (results not shown). In contrast, both the E/D298 and the −786T/C polymorphisms differed with regard to the number of patients that previously suffered an MI. There was an excess of patients with a previous MI among individuals homozygous for E/E298 (51%) compared with patients homozygous for D/D298 (32%; P=0.009; Table 3). The prevalence of MI was intermediate (47%) in E/D298 heterozygote patients (Table 3). After stratification according to smoking status, the differences were present among smokers (P=0.006), but not among non-smokers (P=0.07). Similar results were found for the −786T/C polymorphism, but not for 4a/4b (Table 3). Left ventricular ejection fraction at baseline did not differ significantly between the genotypes (E/E298, 0.70±0.12%; E/D298, 0.71±0.13%; D/D298, 0.74±0.10%; P=0.06).

Table 3
Genotype distribution and MI

Values are number of patients with previous MI (% of total).

 Genotype 
 E/D298 −786T/C VNTR 
 EE ED DD P value TT TC CC P value 5/5 5/4 4/4 P value 
All 174 (51%) 157 (47%) 25 (32%) 0.009 143 (51%) 151 (48%) 40 (36%) 0.022 257 (47%) 92 (47%) 5 (42%) 0.93 
Current smokers 36 (45%) 53 (52%) 3 (14%) 0.006 34 (53%) 45 (46%) 8 (26%) 0.042 71 (49%) 21 (37%) 1 (33%) 0.16 
Non-smokers 138 (52%) 104 (45%) 22 (38%) 0.07 109 (50%) 106 (48%) 32 (40%) 0.29 186 (47%) 71 (51%) 4 (44%) 0.33 
 Genotype 
 E/D298 −786T/C VNTR 
 EE ED DD P value TT TC CC P value 5/5 5/4 4/4 P value 
All 174 (51%) 157 (47%) 25 (32%) 0.009 143 (51%) 151 (48%) 40 (36%) 0.022 257 (47%) 92 (47%) 5 (42%) 0.93 
Current smokers 36 (45%) 53 (52%) 3 (14%) 0.006 34 (53%) 45 (46%) 8 (26%) 0.042 71 (49%) 21 (37%) 1 (33%) 0.16 
Non-smokers 138 (52%) 104 (45%) 22 (38%) 0.07 109 (50%) 106 (48%) 32 (40%) 0.29 186 (47%) 71 (51%) 4 (44%) 0.33 

Progression of atherosclerosis

Angiographic follow-up was complete in 573 out of 760 patients. Patients who did and those who did not have follow-up angiography did not differ in any of the baseline characteristics. No differences were present between the different genotypes in multivessel disease, the number of lesions per segment of the coronary artery and MSD and MOD at baseline angiography. There was no association between the genotype and treatment allocation. Genetic variants of ecNOS did not lead to differences in progression of atherosclerosis or the occurrence of events after 2 years of follow-up (Table 4). Finally, an analysis stratified according to placebo or statin treatment did not reveal any differences between the genotypes (results not shown).

Table 4
Progression of atherosclerosis after 2 years

Values are means±S.D. for MSD, medians±interquartile range for MOD, and number (%) for events.

 Genotype 
 E/D298 −786T/C Intron 4 VNTR 
 EE ED DD TT TC CC 4b/4b 4b/4a 4a/4a 
Change in MSD (mm) 0.08±0.22 0.08±0.19 0.11±0.18 0.09±0.21 0.08±0.21 0.10±0.17 0.09±0.21 0.07±0.18 0.06±0.17 
Change in MOD (mm) 0.05±0.21 0.06±0.22 0.09±0.18 0.06±0.22 0.06±0.21 0.10±0.21 0.06±0.21 0.07±0.22 0.07±0.30 
Events in 2 years 54 (16%) 48 (14%) 8 (10%) 41 (15%) 51 (16%) 15 (14%) 77 (14%) 32 (16%) 2 (17%) 
 Genotype 
 E/D298 −786T/C Intron 4 VNTR 
 EE ED DD TT TC CC 4b/4b 4b/4a 4a/4a 
Change in MSD (mm) 0.08±0.22 0.08±0.19 0.11±0.18 0.09±0.21 0.08±0.21 0.10±0.17 0.09±0.21 0.07±0.18 0.06±0.17 
Change in MOD (mm) 0.05±0.21 0.06±0.22 0.09±0.18 0.06±0.22 0.06±0.21 0.10±0.21 0.06±0.21 0.07±0.22 0.07±0.30 
Events in 2 years 54 (16%) 48 (14%) 8 (10%) 41 (15%) 51 (16%) 15 (14%) 77 (14%) 32 (16%) 2 (17%) 

Ischaemia

Baseline 24-h AECG registrations were available from 158 patients. The duration of ST-segment depression was longer in D/D298 homozygotes than in E/E298 homozygotes (43.6 compared with 29.2 min; P<0.05). The mean ST-segment depression tended to be more in D298 carriers (39.5 compared with 16.6 mm; P=0.065). This association was not found for the −786T/C or 4a/4b polymorphisms.

DISCUSSION

The main finding of the present study was that sequence polymorphisms of the ecNOS gene locus were associated with CAD and with previous MI among patients with symptomatic CAD. The results were more consistent for the E/D298 polymorphism than for the −786T/C and 4a/4b polymorphisms. Individuals homozygous for E/E298 had a 1.8-fold increased risk of CAD compared with D/D298 homozygotes. Half of the patients with CAD, who were homozygous for E/E298, had a previous MI compared with one-third of the D/D298 homozygotes. The association with both CAD and MI was present in current and ex-smokers, but not in non-smokers, suggesting a gene–environment interaction. None of the ecNOS polymorphisms investigated was associated with differences in severity or progression of coronary atherosclerosis. However, the occurrence of ischaemia in patients with CAD was associated with genetic variation at the ecNOS gene locus. Patients carrying the D/D298 genotype had a longer duration of ischaemia on AECG than E/E298 genotype carriers. An explanation for this finding could be that individuals with the D/D298 genotype had more ischaemia, resulting in preconditioning of the myocardium and, therefore, less infarctions.

Our present findings contrast with those in two other European studies including predominantly Caucasian individuals [21,29]. Gardemann et al. [29] reported an increased risk of MI in younger individuals carrying the D298 allele, but they did not find an association with CAD. These investigators also studied the intron 4 VNTR, but did not find an association with either MI or CAD. Hingorani et al. [21] reported on CHAOS (Cambridge Heart AntiOxidant Studies), a study comparing CAD and acute MI patients with healthy controls. They found large differences between both the CAD and MI patients and the controls: D298 allele homozygosity was associated with a 4.2-fold increased risk of CAD. In contrast, the results of the French subset of ECTIM (Etude Cas-Temoins de l'Infarctus du Myocarde) [15] were comparable with our present findings in that the E298 allele was associated with MI.

Several studies have investigated the functional significance of ecNOS gene polymorphisms. For example, the 4a allele, which was associated with CAD but not with previous MI in the present study, was previously associated with higher concentrations of metabolites of NO, although the reports disagreed on the allele [19,20]. Other positive associations of 4a compared with 4b were reported in African–Americans with MI [30], Caucasians with MI and CAD [31], Japanese patients with MI [32] and Australians with severely stenosed arteries and a history of MI [22]. No association was found in a German [33] and a Japanese [23] investigation. Since this polymorphism is in a non-coding region, it could merely be a genetic marker that is associated with the functional mutation.

The rare allele of the E/D298 genotype has been associated with higher peroxynitrite concentrations in non-atherosclerotic persons as well [34]. Furthermore, this D allele leads to increased ecNOS cleavage compared with the E allele [18]. In a substudy, we found that carriers of the D allele had longer-lasting ischaemia then their E allele counterparts. This suggests less ecNOS activity in this group of atherosclerotic patients.

The discrepancies between the different reports and our present study might have several explanations. First, the ethnic background of this population is predominantly Caucasian. For instance, in Japanese patients, coronary spasm is a frequent problem [16] and the associations in this ethnic group might differ from our group. Interestingly, in the present study, we found the three different polymorphisms to be in linkage disequilibrium, whereas this is not the case in other ethnic groups [35]. Secondly, these discrepancies might reflect the impact of interactions with environmental factors. In the present study and others [22], smoking seems to be one of those factors. Alternatively, recent reports have suggested that ecNOS is stored in different pools, both subcellular and intracellular [36], and that activity is regulated primarily at a post-transcriptional level [37]. However, the regulation of ecNOS in severely diseased atherosclerotic tissue and the impact of plaque rupture on ecNOS activity leading to MI is not yet known. We did not actually measure ecNOS or NO concentrations to prove the biological effect. The measurement of ischaemia is a functional substitute. Thirdly, considering other studies that reported conflicting findings, the present observations could have been found purely by chance. Since conflicting results have been reported, the functionality of this polymorphism has not yet been proven beyond doubt and post-transcriptional regulation of enzyme activity is also likely, this explanation should be considered.

The data regarding the progression of atherosclerosis rule out the possibility that deleterious ecNOS genotype carriers have a higher incidence of MI because atherosclerosis simply develops at a higher pace. Both the estimates for focal and diffuse progression of atherosclerosis on average changed similarly between the genotypes. The lack of association between ecNOS genotypes and the estimate of severity of coronary atherosclerosis at baseline does support this reasoning. HMG CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitors have been suggested [13] to have both lipid-lowering effects and pleiotropic effects independent of the lipid-lowering potential of these drugs. In REGRESS, we demonstrated previously [24] that pravastatin indeed retarded progression of coronary atherosclerosis and, on an individual basis, was capable of reducing atherosclerosis. In the present study, the ecNOS genotype did not determine the response to statin treatment. Hence there is no role for pharmacogenetics with regard to statins and the ecNOS gene in patients with advanced atherosclerosis.

In conclusion, sequence polymorphisms of the ecNOS gene locus are associated with CAD and MI. The risk of MI is greatest for the E/E298 genotype, especially in smokers. The mechanism by which this variant exerts its effect is not through faster progression of atherosclerosis. The D/D298 ecNOS genotype is associated with longer lasting and more pronounced ischaemia on 24 h AECG registration. This implies that the mechanism behind the observed association might be a difference in endothelial-dependent protection against ischaemia, leading to less collateral vessel protection. Thus the carriers of the E298 allele and the associated −786T allele of the gene encoding ecNOS have a higher risk of MI. Furthermore, the 4a and E298 alleles are associated with CAD. However, it should be noted that conflicting associations have been reported previously in the literature, that functionality of this polymorphism has not been proven beyond doubt and that post-transcriptional regulation of enzyme activity is also likely. Therefore chance might be an alternative explanation of these results.

Since the E/D298 polymorphism is associated with both CAD and MI, this polymorphism seems most useful for future research. The present study is the first report that integrates anatomy, clinical events and functional measurements in relation to the ecNOS polymorphisms and indicates that, for a proper appreciation of associations of genotypes in relation to atherosclerosis, all aspects have to be investigated in an integrated approach.

Abbreviations

     
  • AECG

    ambulant ECG

  •  
  • BMI

    body mass index

  •  
  • BP

    blood pressure

  •  
  • CAD

    coronary artery disease

  •  
  • CI

    confidence interval

  •  
  • HDL

    high-density lipoprotein

  •  
  • MI

    myocardial infarction

  •  
  • MOD

    minimum obstruction diameter

  •  
  • MSD

    mean segment diameter

  •  
  • NO

    nitric oxide

  •  
  • ecNOS

    endothelial constitutive NO synthase

  •  
  • REGRESS

    Regression Growth Evaluation Statin Study

  •  
  • VNTR

    variable number of tandem repeats

W.R.P.A. is supported by grant 1999.210 from the Netherlands Heart Foundation and a grant from the Interuniversity Cardiology Institute of the Netherlands (ICIN). J.M.A.B. is supported by grant 1998.067 from the Netherlands Heart Foundation. J.W.J. is an Established Clinical Investigator of the Netherlands Heart Foundation (2001 D 032).

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