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The Glu298Asp polymorphism in endothelial nitric oxide synthase gene is associated with coronary in-stent restenosis
International Journal of Cardiology 86 (2002) 71–76 www.elsevier.com / locate / ijcard
The Glu298Asp polymorphism in endothelial nitric oxide synthase gene is associated with coronary in-stent restenosis a
a,
b
b
b
Tomomichi Suzuki , Kenji Okumura *, Takahito Sone , Tai Kosokabe , Hideyuki Tsuboi , Junichiro Kondo b , Hiroaki Mukawa b , Hiroki Kamiya a , Takahito Tomida a , Hajime Imai a , Hideo Matsui a , Tetsuo Hayakawa a a
Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466 -8550 Japan b Department of Cardiology, Ogaki Municipal Hospital, 4 -86 Minaminokawa-cho, Ogaki, Japan Received in revised form 11 March 2002; accepted 6 April 2002
Abstract Background: Reduced or impaired synthesis of nitric oxide promotes the proliferation of vascular smooth muscle cells, and thus may induce the neointimal formation leading to coronary in-stent restenosis. Recent reports have suggested that the Glu298Asp polymorphism in exon 7 of the endothelial nitric oxide synthase gene is associated with coronary spasm and acute myocardial infarction. In this study, we have examined the implication of this polymorphism with regard to coronary restenosis after Palmaz–Schatz stent deployment. Methods: Eighty-nine lesions in 85 consecutive patients were treated with Palmaz–Schatz stents, and were prospectively followed up for 6 months. The lesions were classified into a restenosis group (% diameter stenosis550%) and a non-restenosis group. Assessment was made using an automated quantitative angiographic system. We performed polymerase chain reaction-restriction fragment length polymorphism analysis to detect the missense Glu298Asp variant in exon 7 of the endothelial nitric oxide synthase gene. Results: Coronary risk factors and angiographic findings of stenotic lesions did not differ between the groups. Univariate analyses showed that the missense Glu298Asp variant was the only statistically significant predictor of restenosis (odds ratio, 4.27; P50.025). In addition, multiple logistic regression analysis revealed the missense Glu298Asp variant as the only independent predictor for in-stent restenosis (odds ratio, 3.90; P50.036). Conclusions: The missense Glu298Asp variant may be an independent risk factor for in-stent restenosis. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide synthase; Polymorphism; Restenosis; Stents
1. Introduction Coronary stent implantation has brought better procedural results, and lower restenosis rates than balloon angioplasty [1–4]. In addition, their risk of subacute thrombosis has been reduced by antiplatelet *Corresponding author. Tel.: 181-52-744-2168; fax: 181-52-7442177. E-mail address: [email protected] (K. Okumura).
therapy [5,6], high pressure post dilatation, and intracoronary ultrasound. However, the restenosis rate of 20–30% after stent deployment continues to be an important clinical problem [3,4,7]. Many risk factors for restenosis after balloon angioplasty and stent deployment have been identified. Nitric oxide is an important endothelium-derived relaxing factor. Synthesis of nitric oxide from the amino acid L-arginine is catalyzed by nitric oxide synthase families [8]. Three isoforms of nitric oxide
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00192-4
72
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
synthase have been identified so far; endothelial nitric oxide synthase, inducible nitric oxide synthase, and constitutive neuronal nitric oxide synthase [8]. The endothelial nitric oxide synthase is expressed in the endothelium, and synthesizes nitric oxide that penetrates into the vascular smooth muscle cells [8] and serves to maintain vascular relaxation [9–11]. Nitric oxide also inhibits platelet aggregation [12,13], and platelet and leukocyte adhesion to the vascular endothelium [14,15]. Furthermore, nitric oxide protects against mitogenesis and proliferation of vascular smooth muscle cells [16]. In-stent restenosis is secondary to the proliferation of neointima into the stent [17–19]. Therefore, reduced synthesis of nitric oxide may promote the proliferation of vascular smooth muscle cells and induce the in-stent restenosis. The missense Glu298Asp variant in exon 7 of the endothelial nitric oxide synthase gene contributes to endothelial dysfunction [20]. Recent reports have suggested that the Glu298Asp polymorphism is associated with essential hypertension [21], coronary spasm [22] and acute myocardial infarction [23]. In this study, we have examined this polymorphism with regard to coronary restenosis after Palmaz–Schatz stent deployment.
2. Methods
2.1. Subjects The study population included 85 consecutive patients consisting of 60 with stable angina pectoris (50 men and 10 women; mean age, 63.868.9 years; range, 47–84 years), and 25 with unstable angina pectoris (17 men and 8 women; mean age, 66.469.9 years; range, 45–82 years) who were admitted to Ogaki Municipal Hospital. We excluded from this study patients with coronary stent implantation in the setting of acute myocardial infarction. Stable angina pectoris was defined as effort dependent angina, or positive treadmill ECG or stress scintigram. Unstable angina was defined as a recent acceleration of angina, including pain at rest. The 85 patients of the study group had a total of 89 angiographically documented coronary artery stenotic lesions, which were defined as 60% luminal narrowing after intracoronary nitroglycerin administration as determined by quantitative
coronary angiography. All 89 lesions were treated with Palmaz–Schatz stents electively, and evaluated by quantitative coronary angiography at preintervention, postintervention and follow-up (mean, 6.461.7 months). The Glu298Asp genotype in exon 7 of the endothelial nitric oxide synthase gene was determined in all 85 patients by restriction fragment length polymorphism analysis. All subjects enrolled in this study gave their written informed consent.
2.2. Screening of the Glu298 Asp polymorphism by polymerase chain reaction-restriction fragment length polymorphism analysis Genomic DNA was prepared from peripheral leukocytes. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to detect the Glu298Asp polymorphism in exon 7 of the endothelial nitric oxide synthase gene. Primer pairs for polymerase chain reaction were designed to amplify a part of the endothelial nitric oxide synthase gene containing exon 7 as follows: sense 59-TCCC TGAGGAGGGCATGAGGCT-39 and antisense 59-TGAGGGTCACACAGGTTCCT39. Samples were amplified for 35 cycles, consisting of denaturation at 94 8C for 1 min, annealing at 61 8C for 1 min, and extension at 72 8C for 1 min. The 457-bp polymerase chain reaction product was digested with 8U of the restriction enzyme Ban II (New England Biolabs, Beverly, MA) at 37 8C for at least 20 h. Ban II digested the amplified fragments into smaller fragments (137- and 320-bp). A single Ban II site was present in the wild type allele (G at position 1917), and no Ban II site was found in the mutant allele (T at 1917). Therefore, digestion of the wild type (GG) with Ban II yielded 137-bp and 320-bp fragments. In the case of the heterozygous mutant, digestion with Ban II results in three fragments of 457-bp, 320-bp, and 137-bp in size. To separate the restricted fragments, 8% polyacrylamide gel was used with ethidium bromide staining.
2.3. Direct sequencing of amplified DNA The polymerase chain reaction isoform typing results were checked by the direct sequencing of amplified DNA. Primers and d-NTP were removed by columns. Direct sequencing of the polymerase
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
chain reaction products was performed by using a dye terminator cycle-sequencing kit (Perkin-Elmer, Norwalk, CT). All DNA sequences were confirmed by reading both DNA strands using a DNA sequencer (model 377; version 3.2; Perkin-Elmer Applied Biosystems).
2.4. Interventional procedure Quantitative coronary angiography studies were performed for all 89 lesions in 85 patients at preintervention, postintervention (after Palmaz–Schatz stent implantation plus high pressure post dilatation), and follow-up (6.461.7 months later). Forty-six native artery lesions were in the left anterior descending coronary artery, 13 in the left circumflex coronary artery, and 30 in the right coronary artery. Fourteen, 27, 18, and 17 lesions were AHA type A, B1, B2, and C, respectively. All 89 lesions were electively treated with a total of 116 Palmaz–Schatz stents. A single stent was implanted in 62 lesions, and two stents in 27 lesions. Stents were implanted according to standard procedures, delivering by the sheath system. All stents were implanted with high pressure post dilatation (16.062.5 atm) to achieve optimal result. Target lesion revascularization was defined as repeat percutaneous transluminal coronary angioplasty or coronary artery bypass grafting involving the stented lesion, driven by clinical signs of ischemia in the presence of angiographic restenosis.
2.5. Clinical demographics Coronary risk factors were defined as follows. Diabetes mellitus was defined as a prior diagnosis of the disease, a history of antidiabetic medication, or a plasma fasting glucose level of 126 mg / dl on two or more occasions; hypertension as medication dependent or systolic pressure 140 mmHg and / or diastolic pressure 90 mmHg; hypercholesteloremia as medication dependent or serum cholesterol .220 mg / dl; and smoking as continued smoking or cessation of smoking ,6 months before the study. Serum total cholesterol, triglyceride, HDL cholesterol, and glucose levels in blood specimens obtained in the morning after a 12-h fast were measured by an automated enzymatic assay.
73
2.6. Quantitative coronary angiography analysis Qualitative and quantitative coronary angiography were performed before and after the intervention and at follow-up. Two independent investigators, who were unaware of the endothelial nitric oxide synthase genotype of the patients as well as their coronary risk factors, performed quantitative coronary angiography using a computer-assisted Cardiovascular Angiographic Analysis System (CAAS II; Pie Medical, Maastricht, The Netherlands). The external diameter of the contrast-filled catheter was used as the calibration standard. Minimal lumen diameter, reference diameter, and percent diameter stenosis were measured from multiple projections. The sites of the target lesions were classified as ostial, proximal, mid, and distal. AHA type of the lesion was also recorded. Restenosis was defined as 50% stenosis at follow-up in a vessel with ,50% stenosis immediately after coronary angioplasty.
2.7. Statistical analysis Statistical analysis was performed using Stat-View 5.0 (SAS Institute, Cary, NC). Continuous data are presented as the mean6S.D., and categoric data are presented as frequencies. Statistical analysis of frequency counts were performed with the chi-square test or Fisher’s exact test for small samples, and continuous counts with the t-test (two-sided test). The main analysis tested the association of any risk factor with restenosis at follow-up. We made an analysis on a per lesion basis because there is convincing evidence for a lesion dependence of restenosis after stent deployment [24]. However, we checked the validity of this approach by repeating the most important analyses on a per patient basis as well. The odds ratio was used as a measure of the risk of restenosis in the lesions of restenosis with lesions without a risk factor. Multivariate logistic regression analysis was carried out to identify independent correlates of restenosis. The covariates considered were Glu298Asp variant, older age, male sex, diabetes, hypertension, hyperlipidemia, a recent history of smoking, unstable angina as the indication of percutaneous transluminal coronary angioplasty, prior myocardial infarction, prior coronary artery bypass grafting, lesion in the left anterior descending
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
74 Table 1 Clinical characteristics of patients
3.2. Screening of the Glu298 Asp polymorphism of the endothelial nitric oxide synthase gene
Age (years) Male / female Smokers (%) Diabetes mellitus (%) Systemic hypertension (%) Hyperlipidemia (%) Unstable angina (%)
64.669.2 67 / 18 29 (34.1) 23 (27.1) 36 (42.4) 31 (36.5) 25 (31.3)
Data are given as the mean6S.D. or number of patients (%).
coronary artery, complex lesion (AHA type B2 and C), small reference vessel (,3 mm in diameter), multiple stenting, and high pressure balloon dilatation (.16 atm). All covariates were examined as predictors of restenosis in univariate analysis. Univariate predictors of angiographic restenosis with a P value, 0.3 were entered into the multivariate model.
3. Results
In-stent restenosis was present in 16 lesions (18.0%) and absent in 73 lesions (82.0%) (Table 2). The wild type and heterozygote were present in 64 (87.7%) and 9 (12.3%) of 73 lesions without restenosis, respectively. In contrast, the wild type and heterozygote were found in 10 (62.5%) and 6 (37.5%) of 15 lesions with restenosis, respectively. The distribution of the Glu298Asp polymorphism significantly differed between in-stent stenosis and non-restenosis groups. Namely, coronary in-stent restenosis occurred in six of the 15 lesions with the missense Glu298Asp variant, whereas it occurred in only 10 of the 74 lesions without the missense variant. The restenosis rate in the lesions with the missense variant was significantly higher than that in those without the missense variant (40.0 vs. 13.5%; P50.025). Although no homozygote was detected in this study, the genotypic distributions were in keeping with the Hardy–Weinberg equilibrium.
3.1. Clinical characteristics of patients Table 1 shows the clinical characteristics of patients. Most of the patients were men (mean age, 6569 years). Thirty-four percent of the patients were smokers; 27% had diabetes mellitus; 42% had hypertension; 37% had hyperlipidemia; and 31% had unstable angina.
3.3. Influence of endothelial nitric oxide synthase genotype and other risk factors on restenosis The genotype distributions of the Glu298Asp polymorphism were similar to those described previously in Japanese [21]. Univariate analysis revealed that the Glu298Asp variant was the only statistically
Table 2 Univariate analysis for the variable of restenosis Variable
Restenosis group n516
Nonrestenosis group n573
P-value
Odds ratio
95% Confidence limits
Glu298Asp variant (%) Age.65 years (%) Male sex (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) Unstable angina (%) Prior myocardial infarction (%) Prior CABG (%) Lesion in LAD (%) Complex lesion (AHA type B21C) (%) Small vessel (,3 mm in diameter) (%) Multiple stenting (%) Maximal balloon pressure (.16 atm) (%)
6 (38) 8 (50) 13 (81) 3 (19) 5 (31) 4 (25) 4 (25) 4 (25) 5 (31) 1 (6) 8 (50) 9 (56) 9 (56) 7 (44) 7 (44)
9 (12) 32 (44) 58 (79) 20 (27) 32 (44) 28 (38) 28 (38) 22 (30) 18 (25) 2 (3) 38 (52) 35 (48) 41 (56) 20 (27) 20 (27)
0.025 0.65 1.00 0.75 0.35 0.31 0.31 0.77 0.55 0.45 0.88 0.55 0.99 0.23 0.23
4.27 1.28 1.12 0.61 0.58 0.54 0.54 0.77 1.39 2.37 0.92 1.40 1.00 2.06 2.06
1.33–13.78 0.43–3.78 0.28–4.48 0.16–2.35 0.19–1.83 0.16–1.80 0.16–1.80 0.22–2.66 0.48–4.52 0.22–26.01 0.31–2.73 0.47–4.14 0.34–2.99 0.68–6.19 0.68–6.19
Abbreviations: CABG, coronary artery bypass grafting; LAD, left anterior descending coronary artery.
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
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Table 3 Multiple logistic regression analysis for the dependent variable of restenosis Variable
Regression coefficient
Standard error
P-value
Odds ratio
95% Confidence limits
Glu298Asp variant a Multiple stenting b Maximal balloon pressure
1.36 0.61 0.036
0.65 0.60 0.12
0.036 0.31 0.80
3.90 1.84 1.03
1.09–13.95 0.57–5.95 0.81–1.32
a b
Glu298Asp variant51 if yes, 0 if no. Multiple stenting51 if yes, 0 if no.
significant predictor of restenosis (odds ratio 4.27, 95% confidence limits 1.33 to 13.8; P50.025) (Table 2). There were no other statistically significant predictors of restenosis. Table 3 shows the results of multiple logistic regression analysis to determine independent predictors of in-stent restenosis. The analysis revealed that the independent risk factor for restenosis was the missense Glu298Asp variant (odds ratio 3.90, 95% confidence limits 1.09 to 14.0; P5 0.036).
4. Discussion Several common polymorphisms of the endothelial nitric oxide synthase gene have been described. One of the most extensively studied mutations is the Glu298Asp variant and this polymorphism has been associated with coronary artery disease [25]. The present study provides evidence that the missense Glu298Asp variant is an independent risk factor for in-stent restenosis. The odds ratio for in-stent restenosis, which indicates the importance of this risk factor, was four times higher in the lesions with the missense Glu298Asp variant than in those without the missense. The incidence of other risk factors and variables did not significantly differ between the two groups. To our knowledge, this is the first study to implicate the Glu298Asp variant of the endothelial nitric oxide synthase gene as a genetic risk factor for in-stent restenosis. Patient specific factors, lesion related factors, and procedure related factors are considered to influence restenosis after coronary stent deployment [26–28]. We made an analysis on a per lesion basis because there is convincing evidence that restenosis is lesion dependent [24]. However, we checked the validity of this approach by repeating the analyses on a per patient basis, which also showed that the missense Glu298Asp variant is an indepen-
dent risk factor for in-stent restenosis (odds ratio 4.00; P50.031). Endothelium-derived nitric oxide has been shown to inhibit aggregation of platelets [12,13] and adhesion of platelets and leukocytes to the vascular endothelium [14,15]. Nitric oxide also protects against mitogenesis and proliferation of vascular smooth muscle cells [16]. A study performed in the hypercholesterolemic rabbit model showed that animals treated with L-arginine after balloon angioplasty have less marked neo-intimal hyperplasia [29]. We also previously demonstrated that long-term inhibition of nitric oxide synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta [30]. In-stent restenosis is secondary to the proliferation of neointima into the stent [17–19]. The missense Glu298Asp variant contributes to decreased endothelial nitric oxide synthase activity and endothelial dysfunction [20]. Dysregulated nitric oxide production by the mutant endothelial nitric oxide synthase may promote coronary restenosis after stent deployment. These findings support the results in the present study that the missense Glu298Asp variant of the endothelial nitric oxide synthase gene is associated with in-stent restenosis. Recent clinical studies have suggested that the missense Glu298Asp variant is associated with essential hypertension [21], coronary spasm [22], and acute myocardial infarction [23]. In this regard, the data of the present study have added another example of an association between the missense Glu298Asp variant and cardiovascular disorders. In studies investigating the association between a genetic marker and disease, such as the present study, population stratification should be controlled, and genotyping methods should be appropriate. In this study, we recruited 85 consecutive patients prospectively. The prevalence of risk factors for restenosis did not differ between the lesions with and those
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without the missense Glu298Asp variant. In this study no homozygote was detected. However, the genotype frequencies were confirmed by the Hardy– Weinberg equilibrium. A few reports on the functional significance of the missense Glu298Asp variant have been demonstrated [20]. The results of this study, if confirmed by additional larger studies, imply that the missense Glu298Asp variant may be one of the determinants of in-stent restenosis. In conclusion, the present study revealed that the missense Glu298Asp variant is an independent risk factor for in-stent restenosis. Our results may imply a new therapeutic strategy to prevent coronary in-stent restenosis.
References [1] George BS, Voorhees WD, Roubin GS et al. Multicenter investigation of coronary stenting to treat acute or threatened closure after percutaneous transluminal coronary angioplasty: clinical and angiographic outcomes. J Am Coll Cardiol 1993;22:135–43. ¨ [2] Schomig A, Kastrati A, Mudra H et al. Four-year experience with Palmaz–Schatz stenting in coronary angioplasty complicated by dissection with threatened or present vessel closure. Circulation 1994;90:2716–24. [3] Serruys PW, de Jaegere P, Kiemeneij F et al. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. Benestant Study Group. N Engl J Med 1994;331:489–95. [4] Fischman DL, Leon MB, Baim DS et al. A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease: Stent Restenosis Study Investigators. N Engl J Med 1994;331:496–501. ¨ [5] Schomig A, Neumann FJ, Kastrati A et al. A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary-artery stents. N Eng J Med 1996;334:1084–9. ¨ ¨ [6] Schuhlen H, Hadamitzky M, Walter H, Ulm K, Schomig A. Major benefit from antiplatelet therapy for patients at high risk for adverse cardiac events after coronary Palmaz–Schatz stent placement. Analysis of a prospective risk stratification protocol in the intracoronary stenting and antithrombotic regimen (ISAR) trial. Circulation 1997;95:2015–21. ¨ [7] Kastrati A, Schomig A, Dietz R, Neumann FJ, Richardt G. Time course of restenosis during the first year after emergency coronary stenting. Circulation 1993;87:1498–505. [8] Moncada S, Higgs A. The L-arginine–nitric oxide pathway. New Engl J Med 1993;329:2002–12. [9] Chu A, Chambers DE, Lin CC et al. Effects of inhibition of nitric oxide formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs. J Clin Invest 1991;87:1964–8. [10] Lefroy DC, Crake T, Uren NG, Davies GJ, Maseri A. Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation 1993;88:43– 54. [11] Quyyumi AA, Dakak N, Andrews NP, Gilligan DM, Panza JA, Cannon RO III. Contribution of nitric oxide to metabolic coronary vasodilation in the human heart. Circulation 1995;92:320–6.
[12] Furlong B, Henderson AH, Lewis MJ, Smith JA. Endotheliumderived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 1987;90:687–92. [13] Yao S-K, Ober JC, Krishnaswami A et al. Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured srteries. Circulation 1992;86:1302–9. [14] Radomski MW, Palmer RMJ, Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 1987;2:1057–8. [15] Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991;88:4651–5. [16] Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 1989;83:1774–7. [17] Mintz GS, Popma JJ, Hong MK et al. Intravascular ultrasound to discern devise-specific effects and mechanisms of restenosis. Am J Cardiol 1996;78(suppl 3A):18–22. [18] Currier JW, Faxon DP. Restenosis after percutaneous transluminal coronary angioplasty: have we been aiming at the wrong target? J Am Coll Cardiol 1995;25:516–20. [19] Hoffmann R, Mintz GS, Dussaillant GR et al. Patterns and mechanisms of in-stent restenosis: a serial intravascular ultrasound study. Circulation 1996;94:1247–54. [20] Naber CK, Baumgart D, Altmann C, Siffert W, Erbel R, Heusch G. eNOS894T allele and coronary blood flow at rest and during adenosine-induced hyperemia. Am J Physiol 2001;281:H1908–12. [21] Miyamoto Y, Saito Y, Kajiyama N et al. Enodothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension 1998;32:3–8. [22] Yoshimura M, Yasue H, Nakayama M et al. A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese. Hum Genet 1998;103:65–9. [23] Hibi K, Ishigami T, Tamura K et al. Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension 1998;32:521–6. ¨ ¨ [24] Kastrati A, Schomig A, Elezi S, Schuhlen H, Wilhelm M, Dirschinger J. Interlesion dependence of the risk for restenosis in patients with coronary stent placement in multiple lesions. Circulation 1998;97:2396–401. [25] Nassar BA, Bevin LD, Johnstone DE et al. Relationship of the Glu298Asp polymorphism of the endothelial nitric oxide synthase gene and early-onset coronary artery disease. Am Heart J 2001;142(4):586–9. ¨ [26] Kastrati A, Schomig A, Elezi S et al. Predictive factors of restenosis after coronary stent placement. J Am Coll Cardiol 1997;30:1428– 36. [27] Hoffmann R, Mintz GS, Mehran R et al. Intravascular ultrasound predictors of angiographic restenosis in lesions treated with Palmaz– Schatz stents. J Am Coll Cardiol 1998;31:43–9. [28] Bauters C, Hubert E, Prat A et al. Predictors of restenosis after coronary stent implantation. J Am Coll Cardiol 1998;31:1291–8. [29] Le Tourneau TL, Van Belle EV, Corseaux D et al. Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling. J Am Coll Cardiol 1999;33:876–82. [30] Naruse K, Shimizu K, Muramatsu M et al. Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta: PGH 2 does not contribute to impaired endothelium-dependent relaxation. Arterioscler Thromb 1994;14:746–52.
The Glu298Asp polymorphism in endothelial nitric oxide synthase gene is associated with coronary in-stent restenosis a
a,
b
b
b
Tomomichi Suzuki , Kenji Okumura *, Takahito Sone , Tai Kosokabe , Hideyuki Tsuboi , Junichiro Kondo b , Hiroaki Mukawa b , Hiroki Kamiya a , Takahito Tomida a , Hajime Imai a , Hideo Matsui a , Tetsuo Hayakawa a a
Internal Medicine II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466 -8550 Japan b Department of Cardiology, Ogaki Municipal Hospital, 4 -86 Minaminokawa-cho, Ogaki, Japan Received in revised form 11 March 2002; accepted 6 April 2002
Abstract Background: Reduced or impaired synthesis of nitric oxide promotes the proliferation of vascular smooth muscle cells, and thus may induce the neointimal formation leading to coronary in-stent restenosis. Recent reports have suggested that the Glu298Asp polymorphism in exon 7 of the endothelial nitric oxide synthase gene is associated with coronary spasm and acute myocardial infarction. In this study, we have examined the implication of this polymorphism with regard to coronary restenosis after Palmaz–Schatz stent deployment. Methods: Eighty-nine lesions in 85 consecutive patients were treated with Palmaz–Schatz stents, and were prospectively followed up for 6 months. The lesions were classified into a restenosis group (% diameter stenosis550%) and a non-restenosis group. Assessment was made using an automated quantitative angiographic system. We performed polymerase chain reaction-restriction fragment length polymorphism analysis to detect the missense Glu298Asp variant in exon 7 of the endothelial nitric oxide synthase gene. Results: Coronary risk factors and angiographic findings of stenotic lesions did not differ between the groups. Univariate analyses showed that the missense Glu298Asp variant was the only statistically significant predictor of restenosis (odds ratio, 4.27; P50.025). In addition, multiple logistic regression analysis revealed the missense Glu298Asp variant as the only independent predictor for in-stent restenosis (odds ratio, 3.90; P50.036). Conclusions: The missense Glu298Asp variant may be an independent risk factor for in-stent restenosis. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide synthase; Polymorphism; Restenosis; Stents
1. Introduction Coronary stent implantation has brought better procedural results, and lower restenosis rates than balloon angioplasty [1–4]. In addition, their risk of subacute thrombosis has been reduced by antiplatelet *Corresponding author. Tel.: 181-52-744-2168; fax: 181-52-7442177. E-mail address: [email protected] (K. Okumura).
therapy [5,6], high pressure post dilatation, and intracoronary ultrasound. However, the restenosis rate of 20–30% after stent deployment continues to be an important clinical problem [3,4,7]. Many risk factors for restenosis after balloon angioplasty and stent deployment have been identified. Nitric oxide is an important endothelium-derived relaxing factor. Synthesis of nitric oxide from the amino acid L-arginine is catalyzed by nitric oxide synthase families [8]. Three isoforms of nitric oxide
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00192-4
72
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
synthase have been identified so far; endothelial nitric oxide synthase, inducible nitric oxide synthase, and constitutive neuronal nitric oxide synthase [8]. The endothelial nitric oxide synthase is expressed in the endothelium, and synthesizes nitric oxide that penetrates into the vascular smooth muscle cells [8] and serves to maintain vascular relaxation [9–11]. Nitric oxide also inhibits platelet aggregation [12,13], and platelet and leukocyte adhesion to the vascular endothelium [14,15]. Furthermore, nitric oxide protects against mitogenesis and proliferation of vascular smooth muscle cells [16]. In-stent restenosis is secondary to the proliferation of neointima into the stent [17–19]. Therefore, reduced synthesis of nitric oxide may promote the proliferation of vascular smooth muscle cells and induce the in-stent restenosis. The missense Glu298Asp variant in exon 7 of the endothelial nitric oxide synthase gene contributes to endothelial dysfunction [20]. Recent reports have suggested that the Glu298Asp polymorphism is associated with essential hypertension [21], coronary spasm [22] and acute myocardial infarction [23]. In this study, we have examined this polymorphism with regard to coronary restenosis after Palmaz–Schatz stent deployment.
2. Methods
2.1. Subjects The study population included 85 consecutive patients consisting of 60 with stable angina pectoris (50 men and 10 women; mean age, 63.868.9 years; range, 47–84 years), and 25 with unstable angina pectoris (17 men and 8 women; mean age, 66.469.9 years; range, 45–82 years) who were admitted to Ogaki Municipal Hospital. We excluded from this study patients with coronary stent implantation in the setting of acute myocardial infarction. Stable angina pectoris was defined as effort dependent angina, or positive treadmill ECG or stress scintigram. Unstable angina was defined as a recent acceleration of angina, including pain at rest. The 85 patients of the study group had a total of 89 angiographically documented coronary artery stenotic lesions, which were defined as 60% luminal narrowing after intracoronary nitroglycerin administration as determined by quantitative
coronary angiography. All 89 lesions were treated with Palmaz–Schatz stents electively, and evaluated by quantitative coronary angiography at preintervention, postintervention and follow-up (mean, 6.461.7 months). The Glu298Asp genotype in exon 7 of the endothelial nitric oxide synthase gene was determined in all 85 patients by restriction fragment length polymorphism analysis. All subjects enrolled in this study gave their written informed consent.
2.2. Screening of the Glu298 Asp polymorphism by polymerase chain reaction-restriction fragment length polymorphism analysis Genomic DNA was prepared from peripheral leukocytes. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to detect the Glu298Asp polymorphism in exon 7 of the endothelial nitric oxide synthase gene. Primer pairs for polymerase chain reaction were designed to amplify a part of the endothelial nitric oxide synthase gene containing exon 7 as follows: sense 59-TCCC TGAGGAGGGCATGAGGCT-39 and antisense 59-TGAGGGTCACACAGGTTCCT39. Samples were amplified for 35 cycles, consisting of denaturation at 94 8C for 1 min, annealing at 61 8C for 1 min, and extension at 72 8C for 1 min. The 457-bp polymerase chain reaction product was digested with 8U of the restriction enzyme Ban II (New England Biolabs, Beverly, MA) at 37 8C for at least 20 h. Ban II digested the amplified fragments into smaller fragments (137- and 320-bp). A single Ban II site was present in the wild type allele (G at position 1917), and no Ban II site was found in the mutant allele (T at 1917). Therefore, digestion of the wild type (GG) with Ban II yielded 137-bp and 320-bp fragments. In the case of the heterozygous mutant, digestion with Ban II results in three fragments of 457-bp, 320-bp, and 137-bp in size. To separate the restricted fragments, 8% polyacrylamide gel was used with ethidium bromide staining.
2.3. Direct sequencing of amplified DNA The polymerase chain reaction isoform typing results were checked by the direct sequencing of amplified DNA. Primers and d-NTP were removed by columns. Direct sequencing of the polymerase
T. Suzuki et al. / International Journal of Cardiology 86 (2002) 71–76
chain reaction products was performed by using a dye terminator cycle-sequencing kit (Perkin-Elmer, Norwalk, CT). All DNA sequences were confirmed by reading both DNA strands using a DNA sequencer (model 377; version 3.2; Perkin-Elmer Applied Biosystems).
2.4. Interventional procedure Quantitative coronary angiography studies were performed for all 89 lesions in 85 patients at preintervention, postintervention (after Palmaz–Schatz stent implantation plus high pressure post dilatation), and follow-up (6.461.7 months later). Forty-six native artery lesions were in the left anterior descending coronary artery, 13 in the left circumflex coronary artery, and 30 in the right coronary artery. Fourteen, 27, 18, and 17 lesions were AHA type A, B1, B2, and C, respectively. All 89 lesions were electively treated with a total of 116 Palmaz–Schatz stents. A single stent was implanted in 62 lesions, and two stents in 27 lesions. Stents were implanted according to standard procedures, delivering by the sheath system. All stents were implanted with high pressure post dilatation (16.062.5 atm) to achieve optimal result. Target lesion revascularization was defined as repeat percutaneous transluminal coronary angioplasty or coronary artery bypass grafting involving the stented lesion, driven by clinical signs of ischemia in the presence of angiographic restenosis.
2.5. Clinical demographics Coronary risk factors were defined as follows. Diabetes mellitus was defined as a prior diagnosis of the disease, a history of antidiabetic medication, or a plasma fasting glucose level of 126 mg / dl on two or more occasions; hypertension as medication dependent or systolic pressure 140 mmHg and / or diastolic pressure 90 mmHg; hypercholesteloremia as medication dependent or serum cholesterol .220 mg / dl; and smoking as continued smoking or cessation of smoking ,6 months before the study. Serum total cholesterol, triglyceride, HDL cholesterol, and glucose levels in blood specimens obtained in the morning after a 12-h fast were measured by an automated enzymatic assay.
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2.6. Quantitative coronary angiography analysis Qualitative and quantitative coronary angiography were performed before and after the intervention and at follow-up. Two independent investigators, who were unaware of the endothelial nitric oxide synthase genotype of the patients as well as their coronary risk factors, performed quantitative coronary angiography using a computer-assisted Cardiovascular Angiographic Analysis System (CAAS II; Pie Medical, Maastricht, The Netherlands). The external diameter of the contrast-filled catheter was used as the calibration standard. Minimal lumen diameter, reference diameter, and percent diameter stenosis were measured from multiple projections. The sites of the target lesions were classified as ostial, proximal, mid, and distal. AHA type of the lesion was also recorded. Restenosis was defined as 50% stenosis at follow-up in a vessel with ,50% stenosis immediately after coronary angioplasty.
2.7. Statistical analysis Statistical analysis was performed using Stat-View 5.0 (SAS Institute, Cary, NC). Continuous data are presented as the mean6S.D., and categoric data are presented as frequencies. Statistical analysis of frequency counts were performed with the chi-square test or Fisher’s exact test for small samples, and continuous counts with the t-test (two-sided test). The main analysis tested the association of any risk factor with restenosis at follow-up. We made an analysis on a per lesion basis because there is convincing evidence for a lesion dependence of restenosis after stent deployment [24]. However, we checked the validity of this approach by repeating the most important analyses on a per patient basis as well. The odds ratio was used as a measure of the risk of restenosis in the lesions of restenosis with lesions without a risk factor. Multivariate logistic regression analysis was carried out to identify independent correlates of restenosis. The covariates considered were Glu298Asp variant, older age, male sex, diabetes, hypertension, hyperlipidemia, a recent history of smoking, unstable angina as the indication of percutaneous transluminal coronary angioplasty, prior myocardial infarction, prior coronary artery bypass grafting, lesion in the left anterior descending
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74 Table 1 Clinical characteristics of patients
3.2. Screening of the Glu298 Asp polymorphism of the endothelial nitric oxide synthase gene
Age (years) Male / female Smokers (%) Diabetes mellitus (%) Systemic hypertension (%) Hyperlipidemia (%) Unstable angina (%)
64.669.2 67 / 18 29 (34.1) 23 (27.1) 36 (42.4) 31 (36.5) 25 (31.3)
Data are given as the mean6S.D. or number of patients (%).
coronary artery, complex lesion (AHA type B2 and C), small reference vessel (,3 mm in diameter), multiple stenting, and high pressure balloon dilatation (.16 atm). All covariates were examined as predictors of restenosis in univariate analysis. Univariate predictors of angiographic restenosis with a P value, 0.3 were entered into the multivariate model.
3. Results
In-stent restenosis was present in 16 lesions (18.0%) and absent in 73 lesions (82.0%) (Table 2). The wild type and heterozygote were present in 64 (87.7%) and 9 (12.3%) of 73 lesions without restenosis, respectively. In contrast, the wild type and heterozygote were found in 10 (62.5%) and 6 (37.5%) of 15 lesions with restenosis, respectively. The distribution of the Glu298Asp polymorphism significantly differed between in-stent stenosis and non-restenosis groups. Namely, coronary in-stent restenosis occurred in six of the 15 lesions with the missense Glu298Asp variant, whereas it occurred in only 10 of the 74 lesions without the missense variant. The restenosis rate in the lesions with the missense variant was significantly higher than that in those without the missense variant (40.0 vs. 13.5%; P50.025). Although no homozygote was detected in this study, the genotypic distributions were in keeping with the Hardy–Weinberg equilibrium.
3.1. Clinical characteristics of patients Table 1 shows the clinical characteristics of patients. Most of the patients were men (mean age, 6569 years). Thirty-four percent of the patients were smokers; 27% had diabetes mellitus; 42% had hypertension; 37% had hyperlipidemia; and 31% had unstable angina.
3.3. Influence of endothelial nitric oxide synthase genotype and other risk factors on restenosis The genotype distributions of the Glu298Asp polymorphism were similar to those described previously in Japanese [21]. Univariate analysis revealed that the Glu298Asp variant was the only statistically
Table 2 Univariate analysis for the variable of restenosis Variable
Restenosis group n516
Nonrestenosis group n573
P-value
Odds ratio
95% Confidence limits
Glu298Asp variant (%) Age.65 years (%) Male sex (%) Diabetes (%) Hypertension (%) Hyperlipidemia (%) Smoking (%) Unstable angina (%) Prior myocardial infarction (%) Prior CABG (%) Lesion in LAD (%) Complex lesion (AHA type B21C) (%) Small vessel (,3 mm in diameter) (%) Multiple stenting (%) Maximal balloon pressure (.16 atm) (%)
6 (38) 8 (50) 13 (81) 3 (19) 5 (31) 4 (25) 4 (25) 4 (25) 5 (31) 1 (6) 8 (50) 9 (56) 9 (56) 7 (44) 7 (44)
9 (12) 32 (44) 58 (79) 20 (27) 32 (44) 28 (38) 28 (38) 22 (30) 18 (25) 2 (3) 38 (52) 35 (48) 41 (56) 20 (27) 20 (27)
0.025 0.65 1.00 0.75 0.35 0.31 0.31 0.77 0.55 0.45 0.88 0.55 0.99 0.23 0.23
4.27 1.28 1.12 0.61 0.58 0.54 0.54 0.77 1.39 2.37 0.92 1.40 1.00 2.06 2.06
1.33–13.78 0.43–3.78 0.28–4.48 0.16–2.35 0.19–1.83 0.16–1.80 0.16–1.80 0.22–2.66 0.48–4.52 0.22–26.01 0.31–2.73 0.47–4.14 0.34–2.99 0.68–6.19 0.68–6.19
Abbreviations: CABG, coronary artery bypass grafting; LAD, left anterior descending coronary artery.
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Table 3 Multiple logistic regression analysis for the dependent variable of restenosis Variable
Regression coefficient
Standard error
P-value
Odds ratio
95% Confidence limits
Glu298Asp variant a Multiple stenting b Maximal balloon pressure
1.36 0.61 0.036
0.65 0.60 0.12
0.036 0.31 0.80
3.90 1.84 1.03
1.09–13.95 0.57–5.95 0.81–1.32
a b
Glu298Asp variant51 if yes, 0 if no. Multiple stenting51 if yes, 0 if no.
significant predictor of restenosis (odds ratio 4.27, 95% confidence limits 1.33 to 13.8; P50.025) (Table 2). There were no other statistically significant predictors of restenosis. Table 3 shows the results of multiple logistic regression analysis to determine independent predictors of in-stent restenosis. The analysis revealed that the independent risk factor for restenosis was the missense Glu298Asp variant (odds ratio 3.90, 95% confidence limits 1.09 to 14.0; P5 0.036).
4. Discussion Several common polymorphisms of the endothelial nitric oxide synthase gene have been described. One of the most extensively studied mutations is the Glu298Asp variant and this polymorphism has been associated with coronary artery disease [25]. The present study provides evidence that the missense Glu298Asp variant is an independent risk factor for in-stent restenosis. The odds ratio for in-stent restenosis, which indicates the importance of this risk factor, was four times higher in the lesions with the missense Glu298Asp variant than in those without the missense. The incidence of other risk factors and variables did not significantly differ between the two groups. To our knowledge, this is the first study to implicate the Glu298Asp variant of the endothelial nitric oxide synthase gene as a genetic risk factor for in-stent restenosis. Patient specific factors, lesion related factors, and procedure related factors are considered to influence restenosis after coronary stent deployment [26–28]. We made an analysis on a per lesion basis because there is convincing evidence that restenosis is lesion dependent [24]. However, we checked the validity of this approach by repeating the analyses on a per patient basis, which also showed that the missense Glu298Asp variant is an indepen-
dent risk factor for in-stent restenosis (odds ratio 4.00; P50.031). Endothelium-derived nitric oxide has been shown to inhibit aggregation of platelets [12,13] and adhesion of platelets and leukocytes to the vascular endothelium [14,15]. Nitric oxide also protects against mitogenesis and proliferation of vascular smooth muscle cells [16]. A study performed in the hypercholesterolemic rabbit model showed that animals treated with L-arginine after balloon angioplasty have less marked neo-intimal hyperplasia [29]. We also previously demonstrated that long-term inhibition of nitric oxide synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta [30]. In-stent restenosis is secondary to the proliferation of neointima into the stent [17–19]. The missense Glu298Asp variant contributes to decreased endothelial nitric oxide synthase activity and endothelial dysfunction [20]. Dysregulated nitric oxide production by the mutant endothelial nitric oxide synthase may promote coronary restenosis after stent deployment. These findings support the results in the present study that the missense Glu298Asp variant of the endothelial nitric oxide synthase gene is associated with in-stent restenosis. Recent clinical studies have suggested that the missense Glu298Asp variant is associated with essential hypertension [21], coronary spasm [22], and acute myocardial infarction [23]. In this regard, the data of the present study have added another example of an association between the missense Glu298Asp variant and cardiovascular disorders. In studies investigating the association between a genetic marker and disease, such as the present study, population stratification should be controlled, and genotyping methods should be appropriate. In this study, we recruited 85 consecutive patients prospectively. The prevalence of risk factors for restenosis did not differ between the lesions with and those
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without the missense Glu298Asp variant. In this study no homozygote was detected. However, the genotype frequencies were confirmed by the Hardy– Weinberg equilibrium. A few reports on the functional significance of the missense Glu298Asp variant have been demonstrated [20]. The results of this study, if confirmed by additional larger studies, imply that the missense Glu298Asp variant may be one of the determinants of in-stent restenosis. In conclusion, the present study revealed that the missense Glu298Asp variant is an independent risk factor for in-stent restenosis. Our results may imply a new therapeutic strategy to prevent coronary in-stent restenosis.
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