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(Stroke. 2001;32:735.)
© 2001 American Heart Association, Inc.

Original Contributions

A Common Variant of Endothelial Nitric Oxide Synthase (Glu298Asp) Is an Independent Risk Factor for Carotid Atherosclerosis

G. Lembo, MD, PhD; N. De Luca, MD; C. Battagli, DSci; G. Iovino, MD; A. Aretini, DSci; M. Musicco, MD; G. Frati, MD; F. Pompeo, MD; C. Vecchione., MD B. Trimarco, MD

From the Department of Neurocardiology (G.L., C.B., A.A., M.M., G.F., F.P., C.V., B.T.), Neuromed Institute, Pozzilli (Isernia), Italy; Department of Internal Medicine (N. De L., G.I., B.T.), Federico II University, Napoli, Italy; Institute of Advanced Biomedical Technologies CNR (M.M.), Milan, Italy; and Department of Experimental Medicine and Pathology (G.L.), "La Sapienza" University, Rome, Italy.

Correspondence to Giuseppe Lembo, MD, PhD, IRCCS Neuromed, Località Camerelle, 86077 Pozzilli (IS), Italy. E-mail lembo{at}neuromed.it

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose—Endothelium-derived NO is formed from L-arginine by endothelial NO synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because several studies have indicated that NO plays a key role in the development of the atherosclerotic process, we investigated whether common variants in the eNOS gene are associated with an increased risk of plaque on carotid arteries.

Methods—We studied 375 subjects attending the hypertension center of our institution to be screened for arterial hypertension. The examined subjects were classified according to the presence of carotid plaques (intima-media thickness 1.5 mm), and 2 intronic (CA and 27-bp repeats) polymorphisms and 1 exonic (Glu298Asp) polymorphism of the eNOS gene were explored.

Results—Only the Glu298Asp polymorphism of eNOS was associated with the presence of carotid plaques (P<0.05). In particular, there was an excess of homozygotes for the Asp298 variant among subjects with carotid plaques, whereas the number of subjects who had the Glu298 allele in exon 7 of the eNOS gene was equally distributed in both study groups. Interestingly, the risk of having carotid plaques was increased 3 times in subjects who were homozygotic for the Asp298 variant compared with subjects who were homozygotic for the Glu298 variant and was independent of the other common risk factors (age, blood pressure, and smoking).

Conclusions—Homozygosity for Asp298, a common variant of the eNOS gene, is an independent risk factor for carotid atherosclerosis in this study population.

Key Words: atherosclerosis • carotid stenosis • genetics • nitric oxide • polymorphism

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Atherosclerosis is the most common cause of death in industrialized countries.¹ Such a condition is not to be considered a disease in its own right but rather a process that contributes to the pathogenesis of several serious diseases, such as myocardial infarction and stroke. There is no selective agent responsible for the entire atherosclerotic process, which is considered the product of several influences. However, control of previously characterized environmental risk factors has been ineffective in completely predicting development of the atherosclerotic process, suggesting that further advancement could be made by taking into account the specific genetic predisposition.^{2 3} In fact, it has long been recognized that atherosclerosis has a strong genetic component, as evidenced by the greater concordance of the risk of atherosclerosis-induced diseases in monozygotic twins than in dizygotic pairs⁴ and in first-degree relatives of patients than in their healthy controls.⁵ It has also become clear that various genes may contribute singly or in combination to the development of atherosclerosis.⁶ So far, the contribution of the various candidate genes has been mainly in analysis of the major cardiovascular and cerebrovascular events in which vascular atherosclerotic changes are only a component of the entire genetic process. Indeed, the vascular lesion, realized by the atherosclerotic process, is just the plaque that results from an excessive inflammatory fibroproliferative response to various forms of insult to the endothelium and smooth muscle component of the artery wall.¹ In the past years, a noninvasive technique, such as B-mode ultrasound, has allowed great precision and reproducibility in the direct assessment of atherosclerosis through its most intimate trait: presence of plaque on the vascular wall.^{7 8} Therefore, it is now possible to examine the contribution of candidate genes to the development of atherosclerotic lesions particularly at the carotid level, which mainly contribute to the risk of cerebrovascular accident.

It is clear that functional or morphological alterations in endothelial cells appear to be critical to the evolution,⁹ progression, and clinical manifestation of the atherosclerotic process.^{10 11} One of the most important products of endothelial cells is NO, an unusual signaling molecule that is able to exert profound functional and morphological effects on the vascular wall. In endothelial cells, NO is formed from L-arginine by endothelial NO synthase (eNOS). This enzyme is activated by receptor signaling or mechanical forces. There is evidence that NO inhibits several key steps in the atherogenic process,^{12 13 14 15 16 17} suggesting that an impairment of NO production could facilitate the atherogenic process.¹⁸ Therefore, eNOS is a potential candidate gene for atherosclerosis. So far, several intron polymorphic regions of eNOS have been described, but only 1 exonic polymorphism has been identified.¹⁹

The aim of the present study was to investigate whether eNOS polymorphisms are associated with the presence of atherosclerotic plaque in the carotid arteries of subjects who attending our hypertension center.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study Population
The study was approved by an institutional review committee, and the subjects who were recruited gave informed consent. During the study period (March 1997 to June 1998), we enrolled 375 consecutive subjects who were attending for the first time the hypertension center of our institution for screening arterial hypertension. The subjects were divided into 2 study groups according to the presence or absence of carotid plaques. A structured questionnaire was administered to each patient to investigate his or her personal or family history of cardiovascular and cerebrovascular disease, and an accurate medical history was compiled by a physician. Subjects were defined as hypertensive according to World Health Organization criteria²⁰ or if they were receiving any antihypertensive medication. Subjects with a history of diabetes or those receiving any antidiabetic medication were considered to be diabetic. Subjects were deemed dyslipidemic when their had total cholesterol concentration was 220 mg/dL or triglyceride concentration was 200 mg/dL or they were receiving lipid-lowering drugs. Smoking history was coded as never, ex-smoker, <1 pack/d, or >1 pack/d. A complete physical examination was assessed, including the measurement of supine systolic and diastolic blood pressure levels.

After an overnight fast, blood samples were taken for routine laboratory analyses as well as for genomic DNA extraction.

B-Mode Ultrasound
B-mode ultrasonography was performed with subjects in the supine position with the head turned away from the sonographer and the neck extended in mild rotation. The ultrasound protocol included 2 separate components: the scanning and the reading procedures. Comprehensive quality control procedures were integrated within each component and were critical for quality assurance of the database. The aim of the quantitative examination was to video-record continuous longitudinal imaging of a standardized section of the right and left carotid bifurcations. The scanning protocol was performed with an ultrasound device (Hewlett Packard Sonos 2500) equipped with a 7.5-MHz high-resolution transducer with an axial resolution of 0.1 nm.

Carotid ultrasound examinations were recorded on S-VHS videotapes. The images provided a comprehensive view of the carotid arteries and defined the maximum arterial intima-media thickness (IMT) in up to 12 arterial walls, including the right and the left, near and far distal common (1 cm) bifurcation, and proximal internal carotid artery. IMT was estimated offline with an image processing workstation. IMT was estimated by measurement of the linear distance, perpendicular to the luminal axis, between 2 points defined by the ultrasonic interfaces that indicate the boundary between the lumen and the intimal surface and the boundary between the medial and adventitial interfaces. At the end of the reading procedure, the frames showing the maximum IMT were chosen and used to calculate ultrasound outcome measurements. Repeated scans and readings were used to estimate methodological errors and to evaluate intrasonographer and intersonographer reproducibility. Under our experimental conditions, the variability of measurements was 0.01 and 0.03 mm, respectively.

Subjects with a maximum IMT of 1.5 mm were considered to have plaque according to previous studies.^{21 22 23} The sonographers (G.I. and N. De L.) were blinded with respect to subjects’ genetic information.

Analysis of Polymorphisms of eNOS Gene
Genomic DNA was extracted from peripheral blood sample with standardized techniques.²⁴

CA Repeats Polymorphism on Intron 13 of eNOS Gene
The CA repeats was isolated and sequenced with a (GT)10 oligonucleotide as probe and primer. Two PCR primers were designed from the flanking sequences: GT NOS 5'-TGA GGA GAG ACT CAG AAT TGG A-3' and GT NOS BR 5'-GCT TGT GTG GGG TTT CAG GCT-3'. Samples were processed by touchdown PCR, a method used to circumvent spurious priming during gene amplification. PCR products were analyzed on a 6% denaturing polyacrylamide gel.

27-bp Repeat Polymorphism on Intron 4 of eNOS Gene
A PCR analysis was made with oligonucleotide primers flanking the region of 27-bp repeat in intron 4 of the eNOS gene. The sense primer was 5'-GGC CCT ATG GTA GTG CCT TT-3', and the antisense primer was 5'-TCT CTT AGT GCT GTG GTC AC-3'. The PCR products were separated through electrophoresis in a 5% nondenaturing polyacrylamide gel and visualized with silver staining.

Glu298Asp Polymorphism on Exon 7 of eNOS Gene
Glu298Asp eNOS polymorphism was identified with PCR followed by RFLP with the restriction enzymes DpnII and BanII to digest mutant and wild alleles, respectively. PCR primers were generated to amplify the 248-bp fragment encompassing the Glu298Asp variant (sense and antisense primers were 5'-AAG GCA GGA GAC AGT GGA TGGA-3' and 5'-CCC AGT CAA TCC CTT TGG TGC TCA-3', respectively). When a guanine is at nucleotide position 894, resulting in a glutamic acid at amino acid position 298, BanII restriction enzyme produces 2 fragments of 163 and 85 bp. In contrast, when a thymine is at nucleotide position 894, resulting in an aspartic acid in the amino acid sequence, DpnII restriction enzyme produces 2 fragments of 158 and 90 bp. The restriction digest products were analyzed through electrophoresis on 2% agarose gel.

Statistical Analysis
To verify that our population was not selected for particular allele configuration, Hardy-Weinberg equilibrium was assessed with ² analysis. The strength of association with the presence of carotid plaques was quantified as relative risks estimated with odds ratio, and 95% CIs were calculated from the standard errors and coefficients of the logistic regression. Multivariate odds ratios were calculated with all of the considered variables included in the regression to obtain independent risks associated with each variable analyzed. Therefore, the relative risks were estimated as crude risks and as multivariate risks with adjustment for all of the considered variables. The multivariate risks quantify the independent strength of association between each variable and the presence of carotid plaques.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Table 1 shows the characteristics of the examined subjects classified according to the presence of carotid plaques. The subjects with carotid plaques were older but did not differ from subjects without plaques in terms of body mass index or sex. Systolic and diastolic blood pressures were higher and the history of arterial hypertension was more common in subjects presenting with plaques. Also, a metabolic disturbance such as dyslipidemia and cigarette smoking were more frequent in subjects with plaques. The allele frequencies of the 3 polymorphisms in each group satisfied the Hardy-Weinberg equilibrium law. As shown in Tables 2 and 3 the allele frequency of CA repeats polymorphism in intron 13 and the genotype frequency of 27-bp polymorphism in intron 4 were comparable between the 2 study groups. The Glu298Asp polymorphism in exon 7 was significantly associated with the presence of carotid plaques (Table 4). In fact, Asp298 polymorphism of the eNOS gene was about twice as common in subjects who had carotid plaques, whereas the Glu298 allele was equally represented in the 2 study groups.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of the Study Population by Presence of Carotid Plaques

View this table: [in this window] [in a new window]   Table 2. Distribution of CA Repeats Polymorphism on Intron 13 of eNOS Gene in Subjects With or Without Carotid Plaques

View this table: [in this window] [in a new window]   Table 3. Distribution of 27-bp Repeats Polymorphism on Intron 4 of eNOS Gene in Subjects With or Without Carotid Plaques

View this table: [in this window] [in a new window]   Table 4. Distribution of Glu298Asp Polymorphism on Exon 7 of eNOS Gene in Subjects With or Without Carotid Plaques

Table 5 shows analysis of the risk of having carotid plaques associated with each variable. Age was strongly associated with the presence of plaques, and the risk increased for each year of age by 7% to 8%. The risk of having plaques increased significantly with systolic blood pressure as both crude and multivariate estimates, whereas diastolic blood pressure and the history of hypertension were associated with plaques only as crude risks. Likewise, the risk for dyslipidemic subjects was significantly increased only when crude risk was estimated. Smoking was significantly associated with carotid plaques (P=0.034); in particular, the risk for subjects who smoked >1 pack/d was significantly increased with respect to nonsmokers on univariate analysis. This risk was still increased on multivariate analysis but became of borderline statistical significance. With subjects homozygotic for the Glu298 allele considered as the reference category, subjects heterozygotic for the Glu298Asp eNOS polymorphism had no increased risk of plaques. In contrast, subjects homozygotic for the Asp298 variant had a risk increased 3 times with respect to subjects homozygotic for the Glu298 variant. This risk increase was independent of other variables, because it was even higher when the estimates were adjusted for all of the other considered variables.

View this table: [in this window] [in a new window]   Table 5. Relative Risks of Presence of Carotid Plaques by Subjects’ Characteristics and by Glu298Asp eNOS Polymorphism

Finally, the presence of plaques was associated with an increase in wall thickness in each carotid segment, whereas no difference was observed among subjects with different Glu298Asp eNOS polymorphisms (data not shown).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In the present study, we found an excess of homozygotes for the Asp298 variant of eNOS gene among subjects who have atherosclerotic plaques on their carotid arteries compared with subjects without carotid plaques. Multivariate regression analysis showed that the risk of developing carotid plaques was 3-fold higher for the Asp298 homozygotes with respect to subjects who have a Glu298 allele in the eNOS gene, and the risk increase was independent of other factors that commonly influence the occurrence of atherosclerosis.

Atherosclerosis is a multifactorial genetic disease. So far, polymorphic DNA sequence variations within genes involved in lipid metabolism,²⁵ vessel remodeling,²⁶ or platelet aggregation²⁷ have been described as associated with atherosclerotic disease, as assessed through the major cerebrovascular and cardiovascular events that arise from it. Our observation indicates that a polymorphic DNA region in exon 7 of the eNOS gene, resulting from a change in glutamic acid to aspartic acid at amino acid position 298, is associated with the presence of atherosclerotic plaque on carotid artery. So far, Glu298Asp polymorphism has been linked to an increased risk for stroke,²⁸ myocardial infarction,^{29 30} and Alzheimer’s disease³¹ and was not found in another study that focused on patients with ischemic stroke.³² These observations mainly involved major cerebrovascular and cardiovascular events, whereas in the present study, we considered the carotid artery disease, which is a specific component of the cerebrovascular disease.

In the present study, the excess of risk is confined to aspartic acid homozygotes, suggesting that the homozygosity for aspartic acid in position 298 may have a major impact on eNOS. This hypothesis is supported by evidence that other polymorphic regions in the eNOS gene, such as CA repeats in intron 13 and 27-bp repeat in intron 4, are not linked to carotid atheroma, demonstrating that Glu298Asp polymorphism is not in linkage disequilibrium with other genetic variant within the same gene. Actually, despite the apparently conservative nature of this amino acid substitution, the same amino acid substitution in other enzymes alters protein function,³³ and computer analysis has revealed that the Glu298Asp mutation induces a conformational change from helix to tight turn in the eNOS,¹⁸ suggesting that homozygosity for the Asp298 variant may result in a reduction in eNOS activity. Regarding this issue, Philip et al³⁴ recently showed enhanced vasoconstriction in response to phenylephrine in Asp298 homozygotes that may be ascribed to an impaired endothelial NO modulation of adrenergic vasoconstriction. However, the impact of Glu298Asp polymorphism on endothelial NO function remains to be better clarified, and our associative data do not allow any definitive conclusion to be made on the mechanistic contribution of Glu298Asp polymorphism to carotid atherosclerosis.

Several experimental studies have demonstrated that NO inhibits many key steps of the atherosclerotic disease, and a defect of NO production could facilitate the progression of the atherosclerotic process. In fact, NO, besides its potent vasorelaxant effect, plays a major role in the inhibition of platelet adhesion and aggregation,¹² of adhesion molecule and chemokine expression,¹³ of inflammatory cell infiltration,^{14 15} and of smooth muscle cell migration and proliferation.^{16 17} These pathophysiological mechanisms are involved not only in the development of the vascular atherosclerotic lesion but also in the boost of the cardiovascular events that arise from the atherosclerotic disease. Therefore, evidence that the Asp298 variant of eNOS is a major risk for both atherosclerotic plaque and myocardial infarction may suggest that this protein conformation accelerates those mechanisms common to both the early and the late manifestations of the atherosclerotic disease. It is noteworthy that in our study, no association was observed between the Glu298Asp polymorphism and the thickness of the various segments of the carotid arteries, suggesting that the Asp298 variant of the eNOS contributes mainly to focal vascular phenomena such as the atherosclerotic plaque rather than to the generalized architecture of the blood vessels. This conclusion is supported by a recent study that reported the Glu298Asp polymorphism of eNOS is not linked to vascular stiffness, which primarily depends on the generalized structure of the vessels.³⁵ Consequently, it is possible to speculate that in the hierarchy of events that underlie atherosclerotic remodeling of the adult human vessels, alterations in NO production, resulting from the substitution of glutamic acid for aspartic acid at position 298 in eNOS, has a preferential impact on mechanisms involved mainly in the formation of the atherosclerotic lesions, such as platelet aggregation or leukocyte adhesion to the vessel wall, rather than on smooth muscle cell migration and proliferation. This hypothesis is supported by evidence that long-term blockade of whole body NO synthase leads to a proinflammatory vascular phenotype³⁶ and, on the other side of the coin, that eNOS knockout mice show a major increase in carotid wall thickness compared with wild-type mice only when they are properly challenged.³⁷

Our findings may appear to be in contrast with a recent observation showing no linkage between the Glu298Asp polymorphism of eNOS gene and carotid atheroma.³⁸ Actually, careful perusal of the data reveals that the analysis of carotid phenotypic manifestations was mostly targeted to identification of the vascular lesions producing severe carotid stenosis (>50%), a phenotype much different from that explored in our study, which was focused on characterization of the early vascular manifestations of the atherosclerotic disease.

Another distinct feature of the present study is that it involved prominently hypertensive patients, a population study particularly prone to the development of atherosclerotic plaques. Our data confirm that both systolic and diastolic blood pressures are risk factors for carotid atheroma, but in our study population, only systolic blood pressure is an independent predictor of the plaque. It is noteworthy that the increased risk represented by Asp298 homozygosity in eNOS is evidenced independent of blood pressure. Therefore, it seems that our results are not affected by the fact that our population was recruited in a hypertension center. On the other hand, our control population, represented by subjects without carotid atheroma, displays genotype frequencies comparable to those observed in control populations of other studies where an association with this polymorphism was demonstrated in the absence of a hypertensive background.^{29 31}

In conclusion, we identified that Glu298Asp polymorphism of eNOS gene is a risk factor for carotid atheroma, suggesting that such polymorphism represents a genetic marker able to identify subjects prone to the development of atherosclerotic process.

Received October 27, 2000; revision received December 1, 2000; accepted December 8, 2000.

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