Association of a genetic variant of endothelial nitric oxide synthase with the 1 year clinical outcome after coronary stent placement
Olga Gorchakova,
Werner Koch*,
Nicolas von Beckerath,
Julinda Mehilli,
Albert Schömig and
Adnan Kastrati
Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar, Technische Universität München, Lazarettstrasse 36, 80636 München, Germany
* Corresponding author. Tel.: +49-89-12182601; fax: +49-89-12183053
E-mail address: wkoch{at}dhm.mhn.de
Received 8 November 2002;
accepted 20 November 2002
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Abstract
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Aims Endothelial nitric oxide synthase (eNOS) catalyzes the formation of nitric oxide which has vasodilatory, antithrombotic, antiinflammatory and antiproliferative properties. The TT genotype of the single nucleotide polymorphism 894 G/T, located in exon 7 of the eNOS gene, was found to be associated with coronary spasm, coronary artery disease (CAD) and myocardial infarction (MI). We investigated the possibility that the 894 TT genotype has an unfavorable impact on the angiographic and clinical outcome after the placement of stents in coronary arteries.
Methods and results Our study included 1850 patients with CAD who were treated with stent implantation. Major adverse clinical events, including death, MI, and target vessel revascularization, were recorded for 1 year after the intervention. Patients with genotype 894 TT had an increase in the risk of death or MI (hazard ratio 2.14, 95% confidence interval (CI) 1.233.72;
), if compared with G allele carriers. TT patients showed no significant increase in the risk for angiographic restenosis (odds ratio (OR) 1.11, 95% CI 0.781.56;
) and target vessel revascularization (OR 1.21, 95% CI 0.821.78;
).
Conclusions In comparison with eNOS 894 G allele carriers, patients of the TT genotype were at an increased risk of death or MI within 1 year after coronary artery stenting.
Key Words: Endothelial nitric oxide synthase Stents Adverse outcome Myocardial infarction Restenosis Genetics
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1. Introduction
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Endothelial nitric oxide synthase (eNOS) facilitates the production of nitric oxide (NO), a simple free radical gas, which plays an important role as a regulator of vascular tone and a mediator of antiatherogenic functions.13 Acting as an atheroprotective molecule which prevents or limits neointimal response to vascular injury, NOrepresses proliferation of vascular smooth muscle cells, controls formation of extracellular matrix, and regulates thrombotic and inflammatory reactions.2,4 NO is thought to specifically interfere with oxidative stress, that is increasingly recognized as a potentially essential contributor to vessel wall injury.2 In atherosclerotic human arteries, eNOS protein levels and NO release are markedly reduced if compared to normal arteries.5 This finding and other lines of evidence suggested that onset and progression of atherosclerotic lesion formation is, to a major degree, attributable to a graduallyincreasing impairment of NO availability.6,7 Deficiency of NO supply has also been implicated in the occurrence of thrombosis and restenosis at the sites of balloon angioplasty and stent placement in coronary arteries.2,8
The single nucleotide polymorphism (SNP) 894 G/T, located in exon 7 of the eNOS gene, effects an amino acid exchange from glutamic acid (glu) to aspartic acid (asp) at position 298 of the eNOS polypeptide chain.9,10 In clinical studies, this SNP was identified as a genetic marker for cardiovascular risk and the rare genotype 894 TT was implicated with coronary spasm, coronary artery disease (CAD), and myocardial infarction (MI).912
We examined the possibility that the 894 G/T SNP of the eNOS gene is also associated with the occurrence of adverse angiographic and clinical events after the placement of stents in coronary arteries.
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2. Methods
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2.1. Patients
The study included 1850 consecutive Caucasian patients with symptomatic CAD who underwent coronary stent implantation at Deutsches Herzzentrum München and 1. Medizinische Klinik rechts der Isar der Technischen Universität München. All patients participating in this study gave written informed consent for the intervention, follow-up angiography, and genotype determination. The study protocol conformed to the Declaration of Helsinki and was approved by the institutional ethics committee. Stent placement protocols and poststenting therapy have been previously described in detail.13,14 Postprocedural therapy consisted of aspirin (100mg twice daily, indefinitely) and ticlopidine (250mg twice daily for 4 weeks). Patients considered at a higher risk for stent thrombosis received adjunct therapy with glycoprotein IIb/IIIa blocker abciximab that was given as a bolus injection during stent insertion and as a 12-h continuous infusion thereafter. The decision to give abciximab was taken at the operator's discretion.
2.2. Determination of the eNOS gene 894 G/T genotype
Genomic DNA was extracted from 200µl of peripheral blood leukocytes with the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) or the High Pure PCR Template Preparation Kit (Roche Applied Science, Mannheim, Germany). Genotype analysis was performed with allele-specific fluorogenic oligonucleotide probes in an assay combining the polymerase chain reaction (PCR) and the 5'nuclease reaction (TaqMan technique; Applied Biosystems, Weiterstadt, Germany).15 Primers 5'-GTGCTGCCCCTGCTGCT-3' and 5'-TCGGGGGGC AGAAGGA-3' were used to amplify a 62-bp portion of exon 7 containing the polymorphic site. The sequence of the 894 G-specific probe was 5'-CCC CAGATGAGCCCCCAGAACT-3', and the sequence of the 894 T-specific probe was 5'-CCCCAGATGATCC CCCAGAACTCT-3'. FAM, i.e. 6-carboxy-fluorescein, and VIC (proprietary dye of Applied Biosystems) were the fluorogenic dyes used to accomplish allelic discrimination. The two-step thermocycling procedure consisted of 40 cycles of denaturation at 95°C for 15s and primer annealing and extension at 60°C for 1min. As a control, genotyping was repeated for 20% of the samples using DNA prepared separately from the original blood sample. Genotypes were determined without knowledge of patients' clinical and angiographic data.
To identify carriers of homozygous 894 GG and 894 TT DNA, required as standards for allelic discrimination by the TaqMan method, conventional genotype determination was performed using DNA samples of 10 blood donors. This was done by PCR, subsequent digestion of the PCR product with restriction enzyme Bsp143I (MBI Fermentas,St. Leon-Rot, Germany), and separation of the restriction fragments by electrophoresis in a polyacrylamide gel (Invitrogen, Karlsruhe, Germany). The primers 5'-CATGAGGCTCAGCCCCAGAA-3' and 5'-CCAGCAGCATGTTGGACACT-3' were used to amplify a 356-bp sequence containing the variable site. The two-step thermocycling procedure consisted of denaturation at 95°C for 1min and primer annealing and extension at 60°C for 1min, repeated for 35 cycles, followed by a final extension at 72°C for 7min. Bsp143I cleaved the 894 G-specific PCR product in two fragments of 264bp and 92bp and the 894 T-specific PCR product in three fragments of 145, 119, and 92bp.
2.3. Angiographic assessment
Lesion morphology was classified according to the modified American College of Cardiology/American Heart Association grading system in type A, B1, B2, and C,16 and lesions of types B2 and C were considered complex lesions. Digital angiograms were analyzed off-line using the automated edge-detection system CMS (Medis Medical Imaging Systems, Nuenen, The Netherlands). Matched views were selected for angiograms recorded before and immediately after the intervention, and at follow-up. The parameters measured were lesion length, reference diameter, minimal lumen diameter, diameter stenosis, and diameter of the maximally inflated balloon during stent placement. Acute lumen gain was calculated as the differencebetween the final poststenting minimal lumen diameter and the minimal lumen diameter present before stenting. Late lumen loss was calculated as the difference between final poststenting minimal lumen diameter and minimal lumen diameter measured at follow-up angiography. Loss index was calculated as the ratio of late lumen loss and acute lumen gain. Operators who performed the quantitative assessment were unaware of the genotype data.
2.4. Definitions and study endpoints
The diagnosis of unstable angina pectoris at presentation was based on a history of crescendo angina, angina at rest or with minimal exertion, or angina of new onset (within 1 month) in the absence of clear-cut electrocardiographic and cardiac enzyme changes diagnostic of an acute MI.17 Acute MI was diagnosed in the presence of a clinical episode of prolonged chest pain with either the appearance of one or more new pathologic Q waves on the electrocardiogram or an increase in creatine kinase (or its MB isoenzyme) levels to at least twice the upper normal limit. Diabetes mellitus was defined in the presence of an active treatment with insulin or an oral antidiabetic agent; for patients with dietary treatment, documentation of an abnormal fasting blood glucose tolerance test based on the World Health Organization criteria18 was required for establishing this diagnosis. Persons reporting regular smoking in the prior 6 months were considered as current smokers. Systemic arterial hypertension was defined as systolic blood pressure of 90mmHg or greater19 at least on two separate occasions. Hypercholesterolemia was defined as a documented total cholesterol value
6.2mmol/l. The definition of cardiovascular risk factors was based on the data obtained during the actual hospitalization or from the patient's chart.
The primary end point of the study was restenosis defined angiographically as a diameter stenosis of
50% at follow-up angiography performed 6 months after stenting, and clinically as the need for target vessel revascularization (percutaneous transluminal coronary angioplasty or aortic-coronary bypass grafting) due to symptoms or signs of ischemia in the presence of angiographic restenosis at the stented site in the course of 1 year after stent placement. The secondary endpoint of this study was the combined incidence of death from any cause and MI at 1 year after stenting. The follow-up protocol included a phone contact at 30 days, a clinical visit at 6 months, and an additional telephone interview at 1 year after the procedure. For patients reporting cardiac symptoms during the telephone interview, at least one clinical and electrocardiographic follow-up visit was scheduled and performed at the outpatient clinic or by the referring physician. At 1 year, all information derived from hospital readmission records or provided by the referring physician or by theoutpatient clinic was compiled. Persons who performed clinical follow-up were not aware of the patient's genotype.
2.5. Statistical analysis
Discrete variables are expressed as counts or percentages and compared with chi-square or Fisher's exact test, as appropriate. Continuous variables are expressed as mean±SD and compared by means of the unpaired, two-sided t-test or analysis of variance for more than two groups. Risk analysis was performed calculating the odds ratio (OR) and the 95% confidence intervals (CIs). The main analyses consisted of comparisons between homozygous carriers of the 894 T allele and combined heterozygous and homozygous carriers of the 894 G allele. The KaplanMeier method and the logrank test were used to compare the rates of death and MI-free survival within 1 year after stenting. Clinical, lesion-related, and procedural variables with a p value <0.2 in the univariate analysis were included in a multivariate logistic regression model for restenosis and a Cox proportional hazards model for the 1-year outcome. All statistical analyses were performed using S-Plus software (Mathsoft Inc, Seattle, WA). A p value of <0.05 was considered statistically significant.
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3. Results
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3.1. Patient characteristics
Of the patients, 865 (46.8%) were of genotype GG, 792 (42.8%) of genotype GT, and 193 (10.4%) of genotype TT. The genotype distribution was in HardyWeinberg equilibrium
. For comparisons, the patients were distributed into two groups, with group 1 containing the patients of genotype TT, which represented the proposed risk genotype
, and group 2 consisting of the G allele carriers, i.e. patients of genotype GG or GT
. The main baseline characteristics of the patient groups are compared in Table 1 and their angiographic and procedural characteristics at the time of intervention are presented inTable 2. Among the criteria shown in Tables 1 and 2, there were no statistically significant differences between the two genotype groups.
3.2. Acute and subacute thrombotic events
Angiographic stent vessel occlusion occurring within 30 days after stent placement was observed in 2.6% of the TT patients and 1.6% of the G allele carriers
. Within 30 days after the procedure, the mortality rate was 1.0% in the patients carrying genotype TT and 0.9% in the patients carrying the G allele
. The incidence of acute MI was 1.6% in the patients with the TT genotype and 0.9% in the carriers of the G allele
. Death or MI was observed in 2.6% of patients with genotype TT and 1.8% of G allele carriers
. Urgent target vessel revascularization was required in 4.2% of the patients with genotype TT and 2.5% of the G allele carriers
.
3.3. Primary endpoint
Follow-up angiography was performed in 82.9% of the patients with the TT genotype and 84.2% of the G allele carriers
. Continuous measures of restenosis, such as minimal lumen diameter, diameter stenosis, late lumen loss, and loss index, were not significantly different between TT homozygotes and G allele carriers (
; Table 3). The angiographic restenosis rate was 35.0% among TThomozygotes and 32.7% among G allele carriers (OR 1.11, 95% CI 0.781.56;
; Table 3). Patient characteristics that differed between the two groups by a p value <0.2 (arterial hypertension, complex lesions, lesion length, and diameter stenosis immediately after stenting; Tables 1 and 2), were included in a multivariate logistic regression analysis of angiographic restenosis. Upon adjustment, the presence of the TT genotype was associated with an OR of 1.12 (95% CI 0.791.59). Target vessel revascularization, due to restenosis-induced ischemia, was required in 23.8% of the TT genotype carriers and 20.5% of the G allele carriers (OR 1.21, 95% CI 0.821.78;
; Table 3). On the basis of the same multivariate model as that used for assessment of the risk of angiographic restenosis, the TT genotype was not significantly associated with the risk of target vessel revascularization (OR 1.21, 95% CI 0.821.79).
3.4. Secondary endpoint
Within 1 year after stenting, mortality was higher among the carriers of genotype TT (4.7%) than among the GG/GT patients (2.2%;
; Table 4). Fig. 1illustrates the group-specific difference found for 1-year mortality. Patients carrying the TT genotype presented with a higher incidence of MI (3.6%) than G allele carriers (1.6%;
; Table 4). The combined 1-year endpoint of death or MI was observed in 7.8% of the TT patients and 3.7% of the G allele carriers (
; Table 4). On the basis of the Cox proportional hazards model, the TT genotype was associated with a hazard ratio of 2.14 (95% CI 1.233.72) for this endpoint. For death or MI, the divergent 1-year courses of the two patient groups are shown in Fig. 2.

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Fig. 1 One-year mortality curves for patients with genotype TT and patients with genotype GG or GT (G allele carriers). The graph illustrates that patients with genotype TT have a higher risk when compared to G allele carriers.
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Fig. 2 One-year curves illustrating the occurrence of death or MI for patients with genotype TT and patients with genotype GG or GT (G allele carriers). The graph shows that patients with genotype TT have a higher risk when compared to G allele carriers.
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4. Discussion
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The results of this study strongly suggest that the TT genotype of the 894 G/T SNP of the eNOS gene is associated with an increased risk of death and MI within 1 year after coronary artery stenting. In contrast to this finding, the TT genotype is not related to an increase in the risk of clinical and angiographic restenosis. The genotype distribution corresponded well to that of other cohorts recruited in Europe and Australia.10,2022
The pathology of coronary stenting involves early thrombus formation and acute inflammation followed by neointimal growth.23,24 At 6 weeks after stenting, vascular smooth muscle cells form the vessel surface and complete re-endothelilization is first found 12 weeks after stenting.25 Presently, it is not possible to outline a causal relationship between the 894 G/T SNP, compromised eNOS function, subsequent reduced availability of NO, and processes such as atherosclerosis, thrombosis, or restenosis. It is not known whether the substitution of G by T at nucleotide position 894 that results in the presence of asp instead of glu at amino acid position 298 of the polypeptide chain is of relevance for eNOS function. A possible functional effect of the 894 G/T SNP was suggested by the finding that eNOS with asp298, but not with glu298, was susceptible to proteolytic cleavage in cells and hearts of human origin.26 However, it is not known yet whether functional and physiological consequences are associated with asp298-dependent cleavage of eNOS.
Most of the effects of NO on platelets are thought to be mediated by activation of a soluble guanylyl cyclase followed by a rise in intracellular levels of cyclic guanosine 3',5'-monophosphate.27 In stimulated platelets, eNOS produces markedly increased levels of NO that modestly inhibit platelet activation but critically interfere with additional platelet adhesion, suggesting that platelet-derived NO may regulate platelet recruitment to a growing thrombus.28 However, the pathway facilitating NO-dependent inhibition of platelet adhesion and aggregation remains to be established in detail.
Subacute thrombotic occlusion of the stent occurs most frequently during the first 2 weeks after stenting and may result in unfavorable clinical outcome, such as death, acute MI, or need for urgent revascularization.14,29 NO, produced by eNOS in platelets, is thought to inhibit recruitment of platelets to the growing thrombus.28 However, imbalance between the oxidative stress present at the site of stent placement and the antioxidant activity of platelets adherent to the lesion may adversely interfere with platelet function and lead to decreased NO release which, in turn, contributes to enhanced platelet aggregation and subsequent manifestation of acute ischemic coronary syndromes.3032 Despite all these considerations, we did not observe any eNOS genotype-related difference in the 30-day incidence of event rates. Possibly, the genotype-specific association of early adverse events, if present, was outweighed by the use of aspirin, ticlopidin, and the glycoprotein IIb/IIIa inhibitor abciximab for prevention of thrombosis. Antiplatelet therapy was demonstrated to be associated with a significantly reduced rate of adverse events, in particular with a lower incidence of thrombotic occlusion of the stented vessel and significantly reduced rates of MI and repeat interventions.14,3335
Patient-specific variables (e.g. diabetes mellitus), lesion-related factors (e.g. small vessel size), procedural characteristics (e.g. greater extent of the stented segment), stent design (e.g. gold-coated stents), and genetic determinants (e.g. PlA2allele of the gene encoding integrin glycoprotein IIIa) have been identified as predictors of restenosis risk after coronary artery stenting.3645 In-stent neointima formation is predominantly caused by vascular smooth muscle cell migration and proliferation that achieves a peak at 3 months and remains mostly stable after 6 months.46 A protective role of NO against restenosis was suggested by the finding that the NO donor sodium nitroprusside was able to decrease the proliferation of growth factor-stimulated primary cultures of rat vascular smooth muscle cells.47,48 However, our data do not indicate an association of the TT genotype of the eNOS gene with the risk of angiographic restenosis and the need of target vessel revascularization within 1 year after stenting.
The occurrence of death and MI, beyond the acute or subacute phase after stenting, was more pronounced in patients with genotype TT than in G allele carriers. The most likely cause for late MI is the development of an occlusive thrombus either at a so far untreated lesion or on top of neointimal tissue that had formed within the stent. Clinical evidence in support of the second possibility came from a report on the presence of a fresh mural thrombus covering a lesion that developed after angioplasty performed 10 months earlier.49 Data from casecontrol studies showed a significantassociation between the TT genotype and acute MI arising as a spontaneous manifestation of atherosclerosis.1012 Our results provide evidence that the TT genotype is also related to acute MI occurring as a complication of percutaneous coronary interventions. At the time of stenting, about 50% of our patients with genotype TT and 47% of the G allele carriers presented with acute MI or had suffered from MI previously, a difference which is not statistically significant. This observation does not constitute a discrepancy with the results of the earlier casecontrol studies,1012 because they included subjects without apparent CAD as control groups while all participants of our trial presented with significant coronary stenoses.
The role of the TT genotype in cell physiology and its potential contribution to the occurrence of death and MI following stent placement is yet to be defined. It is of particular interest to examine the relationship between asp at position 298 and eNOS function, which, according to our findings and those of other clinical association studies,912 is expected to be different from that involving eNOS with glu at this position. Alternatively, the 894 G/T SNP might represent a genetic marker of no functional importance of its own, which is transmitted together with a functionally critical allele of another variable site present in the eNOS gene locus. It is also possible that only specific combinations of two or more disease-associated alleles or genotypes of different genes, some of which might include the 894 TT genotype of the eNOS gene, have the potential to promote thrombosis, inflammation, and neointima formation associated with coronary interventions. Therefore, it is premature to suggest routine genotype determination of the 894 G/T polymorphism to identify patients at risk of suffering from adverse events after stent placement in coronary arteries. Independent examinations at different institutions are necessary before general conclusions can be drawn.
4.1. Limitations of the study
Our study included a totally Caucasian population and, therefore, it has to be determined separately whether or not the results are valid in populations of different ethnic origin. It is to be noted that previously assessed cohorts including white European and Australian subjects and study groups consisting of Japanese individuals differed greatly in their T allele frequencies, ranging between 0.30 and 0.40 in white populations10,2022 and between 0.02 and 0.09 in Japanese populations.9,11,12 This trial does not provide data on the functional relevance of the 894 G/T polymorphism. Further studies are required to address this importantissue.
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Acknowledgments
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We thank Wolfgang Latz, Marianne Eichinger, Angela Ehrenhaft, and Korinna Griesser for excellent technical assistance.
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