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Endothelin-converting enzyme-1b C-338A polymorphism is associated with the increased risk of coronary artery disease in Chinese population
Clinica Chimica Acta 384 (2007) 75 – 79 www.elsevier.com/locate/clinchim
Endothelin-converting enzyme-1b C-338A polymorphism is associated with the increased risk of coronary artery disease in Chinese population Lian-Sheng Wang a,b,1 , Na-Ping Tang a,1 , Huai-Jun Zhu a , Bo Zhou a , Li Yang c , Bin Wang a,⁎ a
Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu Province 210029, China Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China b
c
Received 5 April 2007; received in revised form 6 June 2007; accepted 6 June 2007 Available online 14 June 2007
Abstract Background: Endothelin-converting enzyme-1 (ECE-1), the key enzyme responsible for endothelin-1 generation, has been linked to coronary artery disease (CAD). Recently, a genetic polymorphism (ECE-1b C-338A) located in ECE-1 gene promoter was identified. However, it is unclear whether this polymorphism is associated with the risk of CAD. Methods: We conducted a study with CAD patients and controls matched by age and sex to examine the prevalence of ECE-1b C-338A polymorphism in CAD. Results: The frequencies of ECE-1b-338CC, CA, and AA genotypes in cases (40.1%, 42.2%, and 17.7%) were significantly different from those of controls (50.6%, 40.5%, and 8.9%, χ2 = 9.989, P = 0.007). Subjects with the variant genotypes (CA+ AA) had a 58% increased risk of CAD relative to CC carriers (adjusted OR = 1.58, 95% CI = 1.07–2.32). Furthermore, the adjusted OR of AA genotype for CAD was 2.33 (95% CI = 1.25–4.35). In stratified analyses, the A allele was significantly associated with increased risk of CAD in female (adjusted OR = 2.86, 95% CI = 1.40–5.84) and subjects with age ≥ 64 y (adjusted OR = 2.96, 95% CI = 1.73–5.08). Moreover, the frequency of patients with variant genotypes increased gradually from single- to triple-vessel disease although without statistical significance (P = 0.069 for trend). Conclusion: Our results suggested that ECE-1b-338C to A variant might be associated with increased risk of CAD in Chinese population. © 2007 Elsevier B.V. All rights reserved. Keywords: Coronary artery disease; Endothelin-1; Endothelin-converting enzyme-1; Polymorphism
1. Introduction Endothelin-1 (ET-1), a 21-amino-acid residue peptide, is predominantly produced by endothelial cell from which it is released toward smooth muscle cell [1,2]. ET-1 has the most potent vasoconstrictive effect on vascular smooth muscle [1]. In addition, findings by Morbidelli et al. showed that ET-1 could enhance the mitogenesis and proliferation of smooth muscle cell indicating that ET-1 might play an important role in the atherogenic process [3]. Endothelin-converting enzyme-1 (ECE-1) is the main protease responsible for ET-1 generation by cleavage of its functionally inactive precursor, big-ET-1 [4]. Several lines of ⁎ Correspondence author. Tel./fax: +86 25 86862884. E-mail address: [email protected] (B. Wang). 1 Contributed equally to this work. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.06.003
evidence supported the involvement of ECE-1 in the development of coronary artery disease (CAD) [5–10]. The expression of ECE-1 mRNA was found to be greatly increased in rat balloon-injured arteries and human coronary atherosclerosis [5– 7]. In addition, the activity of ECE-1 was inversely correlated with plasma low density lipoprotein levels and positively associated with fibrinogen in human vascular tissue [8]. Furthermore, the ECE-1 enzyme activity was significantly elevated in atherosclerotic human coronary arteries [5,9,10]. ECE-1 gene is located on chromosome 1 (1p36) [11]. The ECE protein exits in four isoforms (ECE-1a, ECE-1b, ECE-1c and ECE-1d), which only differ by their N-terminal amino-acid tails and result from the existence of 4 isoform-specific alternative promoters in the gene [12]. Recently, a genetic polymorphism (ECE-1b C-338A) located in the ECE-1 gene promoter (338 bp upstream from the translation start site) has been identified [13]. It has been reported that the A allele was
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associated with significantly increased transcriptional activity than C allele, because of the creation of a binding site for transcription factor E2F-2 [13]. To date, there have been relatively few studies assessing the association between ECE-1b C-338A polymorphism and human diseases [13–16]. Funke-Kaiser et al. and Funalot et al. showed that ECE-1b-338AA genotype was associated with increased risk of hypertension [13,14]. However, a protective effect of AA genotype was showed on late-onset Alzheimer's disease in the study by Funalot et al. [15]. In addition, Reiterova et al. reported that no association was found between the ECE-1b C-338A polymorphism and the progression of autosomal dominant polycystic kidney disease [16]. To our knowledge, the association between the ECE-1b C338A polymorphism and the risk of CAD has so far not been investigated. Therefore, the purpose of the current study was to evaluate the relationship of the ECE-1b C-338A polymorphism with the risk of CAD in Chinese population. 2. Material and methods 2.1. Subjects Consecutive 237 CAD patients were recruited from the inpatients who were admitted to Nanjing Medical University Affiliated Hospital because of suspected coronary artery disease. The diagnosis of CAD was confirmed by coronary angiography performed with the Judkins technique using a quantitative coronary angiographic system [17]. CAD was defined as angiographic evidence of at least 1 segment of a major coronary artery including the left anterior descending, left circumflex or right coronary artery with N50% organic stenosis. The number of diseased vessels was determined according to the Coronary Artery Surgery Study classification. Two cardiologists who were unaware that the patients were to be included in this study assessed the angiograms. The severity of CAD was expressed as single-, double- and triple-vessel disease depending on the number of the main vessels with stenosis. One control per case was randomly selected from persons who had slight disease during the same period in the same hospital. Considering that it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, the following inclusion criteria were used: no history of angina, without any symptoms or signs of other atherosclerotic vascular diseases. They were matched with CAD patients for sex and 5-y age groups. Patients and control subjects with familial history of CAD, determined by interviewing, were also excluded from the study. All subjects enrolled in this study were Han Chinese and residing in or nearby Jiangsu province. They had no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. Hypertension, diabetes mellitus, and dyslipidemia were defined as those reported in our previous study [18]. Briefly, hypertension was defined as resting systolic blood pressure N140 mm Hg and/or diastolic blood pressure N90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose N7.8 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol level of N6.2 mmol/l or on drugs. Smoking was defined as ≥ 10 cigarettes per day. This study was approved by the Nanjing Medical University Affiliated Hospital Ethics Committee and informed consent was obtained from each participant.
2.2. Laboratory measurements Approximately 5 ml venous blood sample was drawn from each subject into tubes containing EDTA after an overnight fast. The blood sample was centrifuged at 2000 × g for 15 min immediately after collection and stored at − 80 °C until analysis. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured enzymatically (First Chemical Co. Japan) on a chemistry analyzer (Olympus Au2700).
2.3. Genotyping The protocols used for genomic DNA extracting and genotyping were described in our previous study [19]. The ECE-1b C-338A polymorphism was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism. A 446 bp DNA fragment (nucleotide positions-759 to-314 of the promoter) containing the polymorphic site was amplified by PCR in the T1 Thermocycler (Biometra, Goettingen, Germany) using the forward primer 5′TAG GGT TAT AGG AGA GGG CTC AGG-3′ and the reverse primer 5′-AAG TAT CAG GAA GGT GCC CTC AAT-3′. Reverse primer was modified at position 22 (nucleotide-335), thus creating a recognition site for the restriction endonuclease Tsp509I (TTAA) in the presence of A allele at position-338. The PCR reaction was performed in a total volume of 20 μl containing 2 μl 10 × PCR buffer, 1.125 mmol/l MgCl2, 0.1 mmol/l dNTPs, 0.25 μmol/l each primer, 200 ng of genomic DNA and 1 U of Taq DNA polymerase (MBI Fermentas). The PCR conditions were 94 °C for 9 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 56 °C and 30 s at 72 °C, with a final elongation at 72 °C for 8 min. The PCR product was digested with Tsp509I (New England BioLabs, Waltham, MA) at 65 °C for 3 h. After electrophoresis through 3% agarose gel, the digestion products were visualized by staining with 0.5 μg/ml of ethidium bromide. Two researchers, blinded to the clinical data, scored the genotypes independently. In the case of discordance between them, a third reviewer would assess the genotype and determine whether it was necessary to repeat the assay. At last, about 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant.
2.4. Statistical analysis Statistical analyses were conducted with Stata ver. 8.0 (STATA Corp., College Station, TX) and SPSS 13.0 (SPSS Inc., Chicago, IL). Normality was tested using the Shapiro–Wilk statistics. Student's t-test was used to identify differences between the means of quantitative variables without skewness. Mann–Whitney rank sum test was used to test differences between the medians of quantitative variables departing from the normal distribution. Qualitative variables were represented as frequencies and were compared by the Pearson χ2test. Pearson χ2-test was also used to test for differences in allele distribution between the groups. Hardy–Weinberg equilibrium was assessed by a χ2 goodness-of-fit test. Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the association between ECE-1b C-338A polymorphism and the risk of CAD. The Woolf approximation method was used to calculate the crude OR. Unconditional logistic regression analysis was used to assess the OR
Table 1 Baseline characteristics of cases and controls Characteristics
Age a(y) Sex (male), n (%) BMI (kg/m2) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%) Smoking, n (%) TC a(mmol/l) TG a(mmol/l) HDL-C a(mmol/l) LDL-C a(mmol/l)
Cases
Controls
(n = 237)
(n = 237)
64 (56–72) 164 (69.2) 24.9±3.3 176 (74.3) 51 (21.5) 16 (6.8) 108 (45.6) 4.13 (3.49–4.69) 1.49 (1.08–2.10) 1.01 (0.86–1.16) 2.32 (1.79–2.80)
64 (56–71) 164 (69.2) 24.3±3.8 126 (53.2) 38 (16.0) 7 (3.0) 76 (32.1) 3.90 (3.34–4.44) 1.15 (0.83–1.62) 1.07 (0.90–1.28) 2.10 (1.61–2.47)
NS NS NS b0.001 NS 0.054 0.003 0.030 b0.001 0.007 0.003
– – –
– – –
Number of diseased vessels Single vessel, n (%) 85 (35.9) Double vessels, n (%) 68 (28.7) Triple vessels, n (%) 84 (35.4)
P
BMI, body mass index; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. a Median (25th–75th percentiles).
L.-S. Wang et al. / Clinica Chimica Acta 384 (2007) 75–79 adjusted for the risk factors including age sex, body mass index, smoking status, hypertension, diabetes and dyslipidemia. The ECE polymorphism might affect risk primarily in different age and sex groups [14,20]. Thus, to test whether the relationship between the ECE-1b C-338A polymorphism and the risk of CAD was modified by the effects of age and sex, genotype ⁎ age and genotype ⁎ sex, representing the interaction between genotype and age and sex respectively, were included in the analysis models, and stratified analyses were conducted by the median age of controls and sex although the small sample size after stratifying by age and sex might limit the statistical power. The linear trend in the association of ECE-1b C-338A polymorphism with CAD severity was evaluated by χ2-test for trend. All statistical tests were performed using two-tailed tests at 5% level of significance.
3. Results Overall, 237 CAD cases and 237 age- and sex-matched controls were enrolled in the study. The baseline characteristics are given in Table 1. As expected, there was no significant difference in age and sex distribution between the case and control groups. CAD cases smoked more cigarettes, had a higher TC, TG, LDL-C, numbers of hypertension and lower HDL-C. However, no significant difference was found in the proportion of diabetic and dyslipidemia between the two groups and the levels of BMI were also similar. By coronary angiography, 85 (35.9%) CAD cases had single-vessel disease, 68 (28.7%) had double-vessel disease and 84 (35.4%) had triple-vessel disease. Within cases and controls, the genotype distributions were consistent with those predicted by the Hardy–Weinberg equilibrium (P = 0.086 and P = 0.774, respectively). The distribution of ECE-1b C-338A genotypes (CC, CA and AA) was significantly different between cases (40.1%, 42.2%, and 17.7%) and controls (50.6%, 40.5%, and 8.9%, χ2 = 9.989, P = 0.007). In addition, the allele frequency of ECE-1b-338A was higher in CAD patients than those observed in the control subjects (χ2 = 9.952, P = 0.002) (Table 2). Table 2 also shows Table 2 Distribution of the ECE-1b C-338A genotype and the risk estimates for the variant ECE-1b genotypes Cases (n = 237)
Controls (n = 237)
Crude OR (95% CI)
P
Genotype b, n (%) CC 95 (40.1) 120 (50.6) 1.00 CA 100 (42.2) 96 (40.5) 1.32 NS (0.89–1.94) AA 42 (17.7) 21 (8.9) 2.53 0.002 (1.40–4.55) 0.021 CA+ AA 142 (59.9) 117 (49.4) 1.53 (1.07–2.21)
Adjusted a OR (95% CI)
P
1.00 1.40 NS (0.93–2.12) 2.33 0.008 (1.25–4.35) 1.58 0.020 (1.07–2.32)
c
Allele , n (%) C-allele 290 (61.2) 336 (70.9) – A-allele 184 (38.8) 138 (29.1) –
– –
– –
– –
Distributions of the ECE-1b C-338A genotype in cases and controls were in Hardy–Weinberg equilibrium (P = 0.086 and P = 0.774, respectively). CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia. b Pearson χ2 = 9.989, P = 0.007 for genotype. c Pearson χ2 = 9.952, P = 0.002 for allele.
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Table 3 Stratified analyses by age and sex for the variant ECE-1b genotypes in cases and controls Variable
AA+ CA/CC cases controls
Age (median) b64 y 64/47 70/43 ≥64 y
78/48 47/77
Sex Males
95/69 90/74
Females
47/26 27/46
Crude OR (95% CI)
Adjusted a OR (95% CI)
P
0.84 (0.49–1.43) 2.66 (1.60–4.44)
NS
1.13 (0.73–1.75) 3.08 (1.57–6.05)
NS
b0.001
0.001
P
0.73 (0.40–1.31) 2.96 (1.73–5.08)
NS
1.17 (0.74–1.85) 2.86 (1.40–5.84)
NS
b0.001
0.004
CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia.
the risk estimates for the variant ECE-1b genotypes among cases compared with controls. After being adjusted for the risk factors including age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the OR for subjects with the variant genotypes (CA+ AA) was 1.58 (95% CI = 1.07–2.32, P = 0.020) relative to CC carriers. And we also found that AA carriers had more than 2 fold risk of CAD compared with the CC carriers (adjusted OR = 2.33, 95% CI = 1.25–4.35, P = 0.008). In the logistic regression analyses, there was a significant interaction between genotype categories and sex or age (P = 0.036 and 0.001 respectively, data not shown), indicating evidence for sex- and age-specific effect of variant in the ECE-1 gene on the CAD risk. Therefore, stratified analyses were performed by the median age of controls (64 y) and sex (Table 3). We noted that A allele was significantly associated with the increased risk of CAD in female (adjusted OR = 2.86, 95% CI = 1.40–5.84, P = 0.004) and subjects with age ≥ 64 y (adjusted OR = 2.96, 95% CI = 1.73–5.08, P b 0.001), while not in male (adjusted OR = 1.17, 95% CI = 0.74–1.85, P = NS) and subjects with ageb64 y (adjusted OR = 0.73, 95% CI = 0.40– 1.31, P = NS). Patients with CAD were subclassified into 3 subgroups (single-, double- and triple-vessel disease) according to the number of affected coronary arteries. And graded increased proportions of patients with the variant genotypes presenting with single- to triple-vessel disease were noted in our analysis although without statistical significance (P = 0.069 for trend) (Table 4). Table 4 ECE-1b C-338A genotypes and CAD severity CAD severity
SVD (n = 85) DVD (n = 68) TVD (n = 84)
Genotype, n (%) CC
CA+ AA
P for trend a
39 (45.9) 29 (42.6) 27 (32.1)
46 (54.1) 39 (57.4) 57 (67.9)
0.069
CAD, coronary artery disease; DVD, double-vessel disease; SVD, single-vessel disease; TVD, triple-vessel disease. a χ2 = 3.315 for trend.
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4. Discussion In this hospital-based case-control study, we for the first time evaluated the frequency of ECE-1b C-338A polymorphism and its potential relevance for angiographic CAD in Chinese population. In our control subjects, the A allele frequency was observed to be 29.1%. It was slightly higher than those observed in healthy Czech controls (26.3%, n = 160) [16] and in French Caucasian (28.0%, n = 461) [15], indicating that the distribution of the ECE-1b genotype might vary among populations. However, the genotype distribution of controls in our study did not deviate from Hardy–Weinberg proportions. In the analyses of the relationship between ECE–1b C-338A polymorphism and CAD, we noted that the ECE-1b-338C to A variant was significantly associated with the increased risk of CAD. ECE-1b C-338A polymorphism is located in the 5′-regulatory region of the ECE-1 gene [13]. To date, very few studies have associated the ECE-1b C-338A polymorphism with cardiovascular disease. Funke-Kaiser et al. showed that ECE-1b-338A allele was associated with higher daytime and nighttime 24 h systolic blood pressure (SBP) as well as higher daytime and nighttime 24 h diastolic blood pressure (DBP) (P = 0.049, P = 0.028, P = 0.017, P = 0.035, respectively) in untreated German women [13]. Funalot et al. reported the statistically significant association between the ECE-1b AA homozygote and higher SBP, DBP and mean blood pressure (P = 0.01, P = 0.02, P = 0.006, respectively) in French women [14]. In the present study, a significant difference of ECE-1b C338A genotype distribution was found between CAD cases and controls. A 58% increased risk of CAD was observed in subjects with ECE-1b-338CA/AA compared with the CC carriers (adjusted OR = 1.58, 95% CI = 1.07–2.32, P = 0.020). And a higher risk of CAD was also observed in individuals with AA genotype (adjusted OR = 2.33, 95% CI = 1.25–4.35, P = 0.008). ECE-1b C-338A polymorphism is a functional variant. It has been reported that the E2F-2 transcription factor displayed an increased affinity to the ECE-1b-338A allele carrying promoter [13]. And the A allele was expected to have higher levels of ECE-1 gene transcription and enzymatic activity than C allele [13]. Recently, multiple lines of evidence supported the involvement of ECE-1 in the pathogenesis of atherosclerotic cardiovascular disease. Investigators have shown that the expression of ECE-1 mRNA was significantly increased in rat balloon-injured arteries [5]. Findings also confirmed that the ECE-1 was upregulated in patients with coronary atherosclerosis and diabetes [5–7,21]. And the ECE-1 enzyme activity was greatly enhanced in atherosclerotic human coronary arteries [5,9,10]. Since ECE-1 has been considered to be a rate-limiting enzyme of the biosynthesis of ET-1, the enhanced ECE-1 activity could increase the production of ET-1 [2,22]. Importantly, ET-1 could stimulate the mitogenesis and proliferation of intimal smooth muscle cell, an essential process of coronary atherosclerosis [3]. It could also promote the formation of reactive oxygen species and pro-inflammatory molecules, which played key roles in the pathogenesis of CAD [23,24]. Therefore, it was reasonable to suppose that our
observation of an association between the ECE-1b C-338A polymorphism and the increased risk of CAD might be due to the elevated ECE-1 activity and higher ET-1 concentration. However, more studies are needed to confirm this hypothesis. In this study we also observed an interaction between ECE1b polymorphism and sex. In female subjects, those who carried the variant genotypes had significantly higher risk of CAD than CC carriers. However, it was without significance among male subjects. Several investigators have reported the sex-specific effects of genotypes [13,15]. Funalot et al. speculated that the sex-specific association might be due to a past effect of female hormones on ECE-1 gene regulation or alternatively to the effects of androgens in men, which would abolish the influence of the genetic variants [14]. This might be also a possible mechanism for our results, because most of the women included in our study were postmenopausal (90%). 17-beta-oestradiol has been shown to inhibit the endothelin-1 gene expression in rat cardiac fibroblasts and aortic smooth muscle cells [25,26]. Importantly, estradiol also could decrease ET-1 levels in the coronary circulation of postmenopausal women with CAD [27]. However, the androgen had an inverse effect that could increase the ET levels [28]. Thus, the elevated levels or activity of ET-1 caused by androgens might mask the effect of the ECE-1b338C to A variant in male subjects. We also investigated the interaction between ECE-1b C338A polymorphism and age. The results showed an increased risk of CAD associated with the mutant genotypes (CA+AA) of ECE-1b in subjects with age ≥ 64 y, but not in subjects with age b64 y. The mechanisms by which the ECE-1b C-338A polymorphism affected the risk of CAD in subjects with age ≥ 64 y remain unclear. However, our data suggested that other genetic factors might contribute to the disease among subjects with age b64 y. Additionally, the antecedents and promoters of CAD are complex and multifactorial, and the impact of genetic variation on CAD likely differs in the presence of other risk factors. Hence, we could not exclude the possibility that other factors, whose prevalence differed between the age groups but was not accounted for in our analyses, might impact the association and, thus explained the age-differences in the association of this polymorphism with CAD. We also conducted a stratified analysis by the severity of CAD. There was a trend toward greater number of diseased coronary artery in variant genotypes (CA+AA), although the differences were not statistically significant. Studies showed that the A allele with higher levels of ECE-1 gene transcription and enzymatic activity contributed to enhanced levels of ET-1 [13]. This could also provide a mechanism for its association with the severity of the CAD. Findings by Lanza et al. demonstrated that ET-1 levels were higher in patients who had 3-vessel disease on angiography [29]. And Salomone et al. also reported that the concentration of ET-1 was associated with severity of coronary artery disease [30]. However, our results should be confirmed by other large studies. Our study does have certain limitations. Firstly, the relatively small sample size of our study, especially after stratifying by age and sex, may limit the statistical power. However, this internally consistent pilot study certainly provides valuable insights and
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interesting information and may serve to guide future studies in this area. Secondly, although we selected controls from individuals with no history of angina and no symptoms or signs of other atherosclerotic vascular diseases, without performing coronary angiography, we could not rule out CAD completely. Nevertheless, the prevalence of CAD in an asymptomatic population appeared to be low [31]. Thirdly, the plasma levels of ECE-1 or ET-1 in cases and controls were not measured and no functional analysis of the C-338A polymorphism was conducted. It was therefore not possible to link plasma ECE-1 or ET-1 levels with CAD. However, the association between ECE-1 or ET-1 and CAD has been confirmed [5–10] and the mechanism linking the polymorphism with the increased gene transcription and enzymatic activity also has been explored [13]. Fourthly, this is a single SNP approach rather than a haplotype approach. At last, our study was only conducted in Chinese population. Data should be extrapolated to other ethnic groups cautiously. In conclusion, our results showed that the ECE-1b-338 C to A variant might be associated with the increased risk of CAD in Chinese population. The interactions between the ECE-1b338C/A genotype and age or sex were also demonstrated. In order to reach a more definitive conclusion, further large sample studies are needed to investigate the genotypic effects of ECE1b polymorphism on the CAD severity. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (no. 30672486), the Natural Science Foundation of Jiangsu Province (no. BK2006525) and the development program of Nanjing Science and Technology Bureau (no. 200401068-2). References [1] Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5. [2] Wagner OF, Christ G, Wojta J, et al. Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem 1992;267:16066–8. [3] Morbidelli L, Orlando C, Maggi CA, Ledda F, Ziche M. Proliferation and migration of endothelial cells is promoted by endothelins via activation of ETB receptors. Am J Physiol 1995;269:H686–95. [4] Xu D, Emoto N, Giaid A, et al. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994;78:473–85. [5] Minamino T, Kurihara H, Takahashi M, et al. Endothelin-converting enzyme expression in the rat vascular injury model and human coronary atherosclerosis. Circulation 1997;95:221–30. [6] Ihling C, Szombathy T, Bohrmann B, Brockhaus M, Schaefer HE, Loeffler BM. Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis. Circulation 2001;104:864–9. [7] Hai E, Ikura Y, Naruko T, et al. Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis. Int J Mol Med 2004;13:649–54. [8] Ruschitzka F, Moehrlen U, Quaschning T, et al. Tissue endothelinconverting enzyme activity correlates with cardiovascular risk factors in coronary artery disease. Circulation 2000;102:1086–92. [9] Maguire JJ, Davenport AP. Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for upregulation of endothelin-converting enzyme activity in disease. Br J Pharmacol 1998;125:238–40.
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[10] Shirai N, Naruko T, Ohsawa M, et al. Expression of endothelin-converting enzyme, endothelin-1 and endothelin receptors at the site of percutaneous coronary intervention in humans. J Hypertens 2006;24:711–21. [11] Valdenaire O, Rohrbacher E, Mattei MG. Organization of the gene encoding the human endothelin-converting enzyme (ECE-1). J Biol Chem 1995;270:29794–8. [12] Valdenaire O, Lepailleur-Enouf D, Egidy G, et al. A fourth isoform of endothelin-converting enzyme (ECE-1) is generated from an additional promoter molecular cloning and characterization. Eur J Biochem 1999;264: 341–9. [13] Funke-Kaiser H, Reichenberger F, Kopke K, et al. Differential binding of transcription factor E2F-2 to the endothelin-converting enzyme-1b promoter affects blood pressure regulation. Hum Mol Genet 2003;12: 423–33. [14] Funalot B, Courbon D, Brousseau T, et al. Genes encoding endothelinconverting enzyme-1 and endothelin-1 interact to influence blood pressure in women: the EVA study. J Hypertens 2004;22:739–43. [15] Funalot B, Ouimet T, Claperon A, et al. Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer's disease. Mol Psychiatry 2004;9:1122–8. [16] Reiterova J, Merta M, Stekrova J, et al. The influence of the endothelinconverting enzyme-1 gene polymorphism on the progression of autosomal dominant polycystic kidney disease. Ren Fail 2006;28:21–4. [17] Santamore WP, Kahl FR, Kutcher MA, et al. A microcomputer based automated, quantitative coronary angiographic analysis system. Ann Biomed Eng 1988;16:367–77. [18] Wang B, Pan J, Wang L, Zhu H, Yu R, Zou Y. Associations of plasma 8isoprostane levels with the presence and extent of coronary stenosis in patients with coronary artery disease. Atherosclerosis 2006;184:425–30. [19] Zhu H, Yang L, Zhou B, Yu R, Tang N, Wang B. Myeloperoxidase G463A polymorphism and the risk of gastric cancer: a case-control study. Carcinogenesis 2006;27:2491–6. [20] Iemitsu M, Maeda S, Otsuki T, et al. Polymorphism in endothelin-related genes limits exercise-induced decreases in arterial stiffness in older subjects. Hypertension 2006;47:928–36. [21] Anstadt MP, Hutchinson J, Portik-Dobos V, et al. Vascular endothelin converting enzyme-1 expression and activity is upregulated in clinical diabetes. Ethn Dis 2002;12:S3–S5–S9. [22] Bur D, Dale GE, Oefner C. A three-dimensional model of endothelinconverting enzyme (ECE) based on the X-ray structure of neutral endopeptidase 24.11 (NEP). Protein Eng 2001;14:337–41. [23] Lund AK, Peterson SL, Timmins GS, Walker MK. Endothelin-1-mediated increase in reactive oxygen species and NADPH Oxidase activity in hearts of aryl hydrocarbon receptor (AhR) null mice. Toxicol Sci 2005;88:265–73. [24] Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension 2003;42:1075–81. [25] Chao HH, Chen JJ, Chen CH, et al. Inhibition of angiotensin II induced endothelin-1 gene expression by 17-beta-oestradiol in rat cardiac fibroblasts. Heart 2005;91:664–9. [26] Hong HJ, Liu JC, Chan P, et al. 17beta-estradiol downregulates angiotensin-II-induced endothelin-1 gene expression in rat aortic smooth muscle cells. J Biomed Sci 2004;11:27–36. [27] Webb CM, Ghatei MA, McNeill JG, Collins P. 17beta-estradiol decreases endothelin-1 levels in the coronary circulation of postmenopausal women with coronary artery disease. Circulation 2000;102:1617–22. [28] Polderman KH, Stehouwer CD, van Kamp GJ, Dekker GA, Verheugt FW, Gooren LJ. Influence of sex hormones on plasma endothelin levels. Ann Intern Med 1993;118:429–32. [29] Lanza GA, Bencardino G, Sestito A, et al. Association of endothelin-1 with transient myocardial ischemia in patients with unstable angina pectoris. Am J Cardiol 2005;95:492–4. [30] Salomone OA, Elliott PM, Calvino R, Holt D, Kaski JC. Plasma immunoreactive endothelin concentration correlates with severity of coronary artery disease in patients with stable angina pectoris and normal ventricular function. J Am Coll Cardiol 1996;28:14–9. [31] Parmley WW. Prevalence and clinical significance of silent myocardial ischemia. Circulation 1989;80:IV68–73.
Endothelin-converting enzyme-1b C-338A polymorphism is associated with the increased risk of coronary artery disease in Chinese population Lian-Sheng Wang a,b,1 , Na-Ping Tang a,1 , Huai-Jun Zhu a , Bo Zhou a , Li Yang c , Bin Wang a,⁎ a
Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu Province 210029, China Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of General Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China b
c
Received 5 April 2007; received in revised form 6 June 2007; accepted 6 June 2007 Available online 14 June 2007
Abstract Background: Endothelin-converting enzyme-1 (ECE-1), the key enzyme responsible for endothelin-1 generation, has been linked to coronary artery disease (CAD). Recently, a genetic polymorphism (ECE-1b C-338A) located in ECE-1 gene promoter was identified. However, it is unclear whether this polymorphism is associated with the risk of CAD. Methods: We conducted a study with CAD patients and controls matched by age and sex to examine the prevalence of ECE-1b C-338A polymorphism in CAD. Results: The frequencies of ECE-1b-338CC, CA, and AA genotypes in cases (40.1%, 42.2%, and 17.7%) were significantly different from those of controls (50.6%, 40.5%, and 8.9%, χ2 = 9.989, P = 0.007). Subjects with the variant genotypes (CA+ AA) had a 58% increased risk of CAD relative to CC carriers (adjusted OR = 1.58, 95% CI = 1.07–2.32). Furthermore, the adjusted OR of AA genotype for CAD was 2.33 (95% CI = 1.25–4.35). In stratified analyses, the A allele was significantly associated with increased risk of CAD in female (adjusted OR = 2.86, 95% CI = 1.40–5.84) and subjects with age ≥ 64 y (adjusted OR = 2.96, 95% CI = 1.73–5.08). Moreover, the frequency of patients with variant genotypes increased gradually from single- to triple-vessel disease although without statistical significance (P = 0.069 for trend). Conclusion: Our results suggested that ECE-1b-338C to A variant might be associated with increased risk of CAD in Chinese population. © 2007 Elsevier B.V. All rights reserved. Keywords: Coronary artery disease; Endothelin-1; Endothelin-converting enzyme-1; Polymorphism
1. Introduction Endothelin-1 (ET-1), a 21-amino-acid residue peptide, is predominantly produced by endothelial cell from which it is released toward smooth muscle cell [1,2]. ET-1 has the most potent vasoconstrictive effect on vascular smooth muscle [1]. In addition, findings by Morbidelli et al. showed that ET-1 could enhance the mitogenesis and proliferation of smooth muscle cell indicating that ET-1 might play an important role in the atherogenic process [3]. Endothelin-converting enzyme-1 (ECE-1) is the main protease responsible for ET-1 generation by cleavage of its functionally inactive precursor, big-ET-1 [4]. Several lines of ⁎ Correspondence author. Tel./fax: +86 25 86862884. E-mail address: [email protected] (B. Wang). 1 Contributed equally to this work. 0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2007.06.003
evidence supported the involvement of ECE-1 in the development of coronary artery disease (CAD) [5–10]. The expression of ECE-1 mRNA was found to be greatly increased in rat balloon-injured arteries and human coronary atherosclerosis [5– 7]. In addition, the activity of ECE-1 was inversely correlated with plasma low density lipoprotein levels and positively associated with fibrinogen in human vascular tissue [8]. Furthermore, the ECE-1 enzyme activity was significantly elevated in atherosclerotic human coronary arteries [5,9,10]. ECE-1 gene is located on chromosome 1 (1p36) [11]. The ECE protein exits in four isoforms (ECE-1a, ECE-1b, ECE-1c and ECE-1d), which only differ by their N-terminal amino-acid tails and result from the existence of 4 isoform-specific alternative promoters in the gene [12]. Recently, a genetic polymorphism (ECE-1b C-338A) located in the ECE-1 gene promoter (338 bp upstream from the translation start site) has been identified [13]. It has been reported that the A allele was
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associated with significantly increased transcriptional activity than C allele, because of the creation of a binding site for transcription factor E2F-2 [13]. To date, there have been relatively few studies assessing the association between ECE-1b C-338A polymorphism and human diseases [13–16]. Funke-Kaiser et al. and Funalot et al. showed that ECE-1b-338AA genotype was associated with increased risk of hypertension [13,14]. However, a protective effect of AA genotype was showed on late-onset Alzheimer's disease in the study by Funalot et al. [15]. In addition, Reiterova et al. reported that no association was found between the ECE-1b C-338A polymorphism and the progression of autosomal dominant polycystic kidney disease [16]. To our knowledge, the association between the ECE-1b C338A polymorphism and the risk of CAD has so far not been investigated. Therefore, the purpose of the current study was to evaluate the relationship of the ECE-1b C-338A polymorphism with the risk of CAD in Chinese population. 2. Material and methods 2.1. Subjects Consecutive 237 CAD patients were recruited from the inpatients who were admitted to Nanjing Medical University Affiliated Hospital because of suspected coronary artery disease. The diagnosis of CAD was confirmed by coronary angiography performed with the Judkins technique using a quantitative coronary angiographic system [17]. CAD was defined as angiographic evidence of at least 1 segment of a major coronary artery including the left anterior descending, left circumflex or right coronary artery with N50% organic stenosis. The number of diseased vessels was determined according to the Coronary Artery Surgery Study classification. Two cardiologists who were unaware that the patients were to be included in this study assessed the angiograms. The severity of CAD was expressed as single-, double- and triple-vessel disease depending on the number of the main vessels with stenosis. One control per case was randomly selected from persons who had slight disease during the same period in the same hospital. Considering that it was unethical to perform coronary angiography to rule out the presence of asymptomatic CAD, the following inclusion criteria were used: no history of angina, without any symptoms or signs of other atherosclerotic vascular diseases. They were matched with CAD patients for sex and 5-y age groups. Patients and control subjects with familial history of CAD, determined by interviewing, were also excluded from the study. All subjects enrolled in this study were Han Chinese and residing in or nearby Jiangsu province. They had no history of significant concomitant diseases including cardiomyopathy, bleeding disorders, renal failure, previous thoracic irradiation therapy and malignant diseases. Hypertension, diabetes mellitus, and dyslipidemia were defined as those reported in our previous study [18]. Briefly, hypertension was defined as resting systolic blood pressure N140 mm Hg and/or diastolic blood pressure N90 mm Hg or in the presence of active treatment with antihypertensive agents. Diabetes was defined as fasting blood glucose N7.8 mmol/l or a diagnosis of diabetes needing diet or antidiabetic drug therapy. Dyslipidemia was defined as total cholesterol level of N6.2 mmol/l or on drugs. Smoking was defined as ≥ 10 cigarettes per day. This study was approved by the Nanjing Medical University Affiliated Hospital Ethics Committee and informed consent was obtained from each participant.
2.2. Laboratory measurements Approximately 5 ml venous blood sample was drawn from each subject into tubes containing EDTA after an overnight fast. The blood sample was centrifuged at 2000 × g for 15 min immediately after collection and stored at − 80 °C until analysis. The levels of plasma total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured enzymatically (First Chemical Co. Japan) on a chemistry analyzer (Olympus Au2700).
2.3. Genotyping The protocols used for genomic DNA extracting and genotyping were described in our previous study [19]. The ECE-1b C-338A polymorphism was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism. A 446 bp DNA fragment (nucleotide positions-759 to-314 of the promoter) containing the polymorphic site was amplified by PCR in the T1 Thermocycler (Biometra, Goettingen, Germany) using the forward primer 5′TAG GGT TAT AGG AGA GGG CTC AGG-3′ and the reverse primer 5′-AAG TAT CAG GAA GGT GCC CTC AAT-3′. Reverse primer was modified at position 22 (nucleotide-335), thus creating a recognition site for the restriction endonuclease Tsp509I (TTAA) in the presence of A allele at position-338. The PCR reaction was performed in a total volume of 20 μl containing 2 μl 10 × PCR buffer, 1.125 mmol/l MgCl2, 0.1 mmol/l dNTPs, 0.25 μmol/l each primer, 200 ng of genomic DNA and 1 U of Taq DNA polymerase (MBI Fermentas). The PCR conditions were 94 °C for 9 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 56 °C and 30 s at 72 °C, with a final elongation at 72 °C for 8 min. The PCR product was digested with Tsp509I (New England BioLabs, Waltham, MA) at 65 °C for 3 h. After electrophoresis through 3% agarose gel, the digestion products were visualized by staining with 0.5 μg/ml of ethidium bromide. Two researchers, blinded to the clinical data, scored the genotypes independently. In the case of discordance between them, a third reviewer would assess the genotype and determine whether it was necessary to repeat the assay. At last, about 10% of the samples were randomly selected to perform the repeated assays, and the results were 100% concordant.
2.4. Statistical analysis Statistical analyses were conducted with Stata ver. 8.0 (STATA Corp., College Station, TX) and SPSS 13.0 (SPSS Inc., Chicago, IL). Normality was tested using the Shapiro–Wilk statistics. Student's t-test was used to identify differences between the means of quantitative variables without skewness. Mann–Whitney rank sum test was used to test differences between the medians of quantitative variables departing from the normal distribution. Qualitative variables were represented as frequencies and were compared by the Pearson χ2test. Pearson χ2-test was also used to test for differences in allele distribution between the groups. Hardy–Weinberg equilibrium was assessed by a χ2 goodness-of-fit test. Odds ratio (OR) and 95% confidence interval (CI) were calculated to estimate the association between ECE-1b C-338A polymorphism and the risk of CAD. The Woolf approximation method was used to calculate the crude OR. Unconditional logistic regression analysis was used to assess the OR
Table 1 Baseline characteristics of cases and controls Characteristics
Age a(y) Sex (male), n (%) BMI (kg/m2) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%) Smoking, n (%) TC a(mmol/l) TG a(mmol/l) HDL-C a(mmol/l) LDL-C a(mmol/l)
Cases
Controls
(n = 237)
(n = 237)
64 (56–72) 164 (69.2) 24.9±3.3 176 (74.3) 51 (21.5) 16 (6.8) 108 (45.6) 4.13 (3.49–4.69) 1.49 (1.08–2.10) 1.01 (0.86–1.16) 2.32 (1.79–2.80)
64 (56–71) 164 (69.2) 24.3±3.8 126 (53.2) 38 (16.0) 7 (3.0) 76 (32.1) 3.90 (3.34–4.44) 1.15 (0.83–1.62) 1.07 (0.90–1.28) 2.10 (1.61–2.47)
NS NS NS b0.001 NS 0.054 0.003 0.030 b0.001 0.007 0.003
– – –
– – –
Number of diseased vessels Single vessel, n (%) 85 (35.9) Double vessels, n (%) 68 (28.7) Triple vessels, n (%) 84 (35.4)
P
BMI, body mass index; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. a Median (25th–75th percentiles).
L.-S. Wang et al. / Clinica Chimica Acta 384 (2007) 75–79 adjusted for the risk factors including age sex, body mass index, smoking status, hypertension, diabetes and dyslipidemia. The ECE polymorphism might affect risk primarily in different age and sex groups [14,20]. Thus, to test whether the relationship between the ECE-1b C-338A polymorphism and the risk of CAD was modified by the effects of age and sex, genotype ⁎ age and genotype ⁎ sex, representing the interaction between genotype and age and sex respectively, were included in the analysis models, and stratified analyses were conducted by the median age of controls and sex although the small sample size after stratifying by age and sex might limit the statistical power. The linear trend in the association of ECE-1b C-338A polymorphism with CAD severity was evaluated by χ2-test for trend. All statistical tests were performed using two-tailed tests at 5% level of significance.
3. Results Overall, 237 CAD cases and 237 age- and sex-matched controls were enrolled in the study. The baseline characteristics are given in Table 1. As expected, there was no significant difference in age and sex distribution between the case and control groups. CAD cases smoked more cigarettes, had a higher TC, TG, LDL-C, numbers of hypertension and lower HDL-C. However, no significant difference was found in the proportion of diabetic and dyslipidemia between the two groups and the levels of BMI were also similar. By coronary angiography, 85 (35.9%) CAD cases had single-vessel disease, 68 (28.7%) had double-vessel disease and 84 (35.4%) had triple-vessel disease. Within cases and controls, the genotype distributions were consistent with those predicted by the Hardy–Weinberg equilibrium (P = 0.086 and P = 0.774, respectively). The distribution of ECE-1b C-338A genotypes (CC, CA and AA) was significantly different between cases (40.1%, 42.2%, and 17.7%) and controls (50.6%, 40.5%, and 8.9%, χ2 = 9.989, P = 0.007). In addition, the allele frequency of ECE-1b-338A was higher in CAD patients than those observed in the control subjects (χ2 = 9.952, P = 0.002) (Table 2). Table 2 also shows Table 2 Distribution of the ECE-1b C-338A genotype and the risk estimates for the variant ECE-1b genotypes Cases (n = 237)
Controls (n = 237)
Crude OR (95% CI)
P
Genotype b, n (%) CC 95 (40.1) 120 (50.6) 1.00 CA 100 (42.2) 96 (40.5) 1.32 NS (0.89–1.94) AA 42 (17.7) 21 (8.9) 2.53 0.002 (1.40–4.55) 0.021 CA+ AA 142 (59.9) 117 (49.4) 1.53 (1.07–2.21)
Adjusted a OR (95% CI)
P
1.00 1.40 NS (0.93–2.12) 2.33 0.008 (1.25–4.35) 1.58 0.020 (1.07–2.32)
c
Allele , n (%) C-allele 290 (61.2) 336 (70.9) – A-allele 184 (38.8) 138 (29.1) –
– –
– –
– –
Distributions of the ECE-1b C-338A genotype in cases and controls were in Hardy–Weinberg equilibrium (P = 0.086 and P = 0.774, respectively). CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia. b Pearson χ2 = 9.989, P = 0.007 for genotype. c Pearson χ2 = 9.952, P = 0.002 for allele.
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Table 3 Stratified analyses by age and sex for the variant ECE-1b genotypes in cases and controls Variable
AA+ CA/CC cases controls
Age (median) b64 y 64/47 70/43 ≥64 y
78/48 47/77
Sex Males
95/69 90/74
Females
47/26 27/46
Crude OR (95% CI)
Adjusted a OR (95% CI)
P
0.84 (0.49–1.43) 2.66 (1.60–4.44)
NS
1.13 (0.73–1.75) 3.08 (1.57–6.05)
NS
b0.001
0.001
P
0.73 (0.40–1.31) 2.96 (1.73–5.08)
NS
1.17 (0.74–1.85) 2.86 (1.40–5.84)
NS
b0.001
0.004
CI, confidence interval; OR, odds ratio. a Adjusted for age, sex, body mass index, smoking status, hypertension, diabetes, dyslipidemia.
the risk estimates for the variant ECE-1b genotypes among cases compared with controls. After being adjusted for the risk factors including age, sex, BMI, smoking status, hypertension, diabetes and dyslipidemia, the OR for subjects with the variant genotypes (CA+ AA) was 1.58 (95% CI = 1.07–2.32, P = 0.020) relative to CC carriers. And we also found that AA carriers had more than 2 fold risk of CAD compared with the CC carriers (adjusted OR = 2.33, 95% CI = 1.25–4.35, P = 0.008). In the logistic regression analyses, there was a significant interaction between genotype categories and sex or age (P = 0.036 and 0.001 respectively, data not shown), indicating evidence for sex- and age-specific effect of variant in the ECE-1 gene on the CAD risk. Therefore, stratified analyses were performed by the median age of controls (64 y) and sex (Table 3). We noted that A allele was significantly associated with the increased risk of CAD in female (adjusted OR = 2.86, 95% CI = 1.40–5.84, P = 0.004) and subjects with age ≥ 64 y (adjusted OR = 2.96, 95% CI = 1.73–5.08, P b 0.001), while not in male (adjusted OR = 1.17, 95% CI = 0.74–1.85, P = NS) and subjects with ageb64 y (adjusted OR = 0.73, 95% CI = 0.40– 1.31, P = NS). Patients with CAD were subclassified into 3 subgroups (single-, double- and triple-vessel disease) according to the number of affected coronary arteries. And graded increased proportions of patients with the variant genotypes presenting with single- to triple-vessel disease were noted in our analysis although without statistical significance (P = 0.069 for trend) (Table 4). Table 4 ECE-1b C-338A genotypes and CAD severity CAD severity
SVD (n = 85) DVD (n = 68) TVD (n = 84)
Genotype, n (%) CC
CA+ AA
P for trend a
39 (45.9) 29 (42.6) 27 (32.1)
46 (54.1) 39 (57.4) 57 (67.9)
0.069
CAD, coronary artery disease; DVD, double-vessel disease; SVD, single-vessel disease; TVD, triple-vessel disease. a χ2 = 3.315 for trend.
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4. Discussion In this hospital-based case-control study, we for the first time evaluated the frequency of ECE-1b C-338A polymorphism and its potential relevance for angiographic CAD in Chinese population. In our control subjects, the A allele frequency was observed to be 29.1%. It was slightly higher than those observed in healthy Czech controls (26.3%, n = 160) [16] and in French Caucasian (28.0%, n = 461) [15], indicating that the distribution of the ECE-1b genotype might vary among populations. However, the genotype distribution of controls in our study did not deviate from Hardy–Weinberg proportions. In the analyses of the relationship between ECE–1b C-338A polymorphism and CAD, we noted that the ECE-1b-338C to A variant was significantly associated with the increased risk of CAD. ECE-1b C-338A polymorphism is located in the 5′-regulatory region of the ECE-1 gene [13]. To date, very few studies have associated the ECE-1b C-338A polymorphism with cardiovascular disease. Funke-Kaiser et al. showed that ECE-1b-338A allele was associated with higher daytime and nighttime 24 h systolic blood pressure (SBP) as well as higher daytime and nighttime 24 h diastolic blood pressure (DBP) (P = 0.049, P = 0.028, P = 0.017, P = 0.035, respectively) in untreated German women [13]. Funalot et al. reported the statistically significant association between the ECE-1b AA homozygote and higher SBP, DBP and mean blood pressure (P = 0.01, P = 0.02, P = 0.006, respectively) in French women [14]. In the present study, a significant difference of ECE-1b C338A genotype distribution was found between CAD cases and controls. A 58% increased risk of CAD was observed in subjects with ECE-1b-338CA/AA compared with the CC carriers (adjusted OR = 1.58, 95% CI = 1.07–2.32, P = 0.020). And a higher risk of CAD was also observed in individuals with AA genotype (adjusted OR = 2.33, 95% CI = 1.25–4.35, P = 0.008). ECE-1b C-338A polymorphism is a functional variant. It has been reported that the E2F-2 transcription factor displayed an increased affinity to the ECE-1b-338A allele carrying promoter [13]. And the A allele was expected to have higher levels of ECE-1 gene transcription and enzymatic activity than C allele [13]. Recently, multiple lines of evidence supported the involvement of ECE-1 in the pathogenesis of atherosclerotic cardiovascular disease. Investigators have shown that the expression of ECE-1 mRNA was significantly increased in rat balloon-injured arteries [5]. Findings also confirmed that the ECE-1 was upregulated in patients with coronary atherosclerosis and diabetes [5–7,21]. And the ECE-1 enzyme activity was greatly enhanced in atherosclerotic human coronary arteries [5,9,10]. Since ECE-1 has been considered to be a rate-limiting enzyme of the biosynthesis of ET-1, the enhanced ECE-1 activity could increase the production of ET-1 [2,22]. Importantly, ET-1 could stimulate the mitogenesis and proliferation of intimal smooth muscle cell, an essential process of coronary atherosclerosis [3]. It could also promote the formation of reactive oxygen species and pro-inflammatory molecules, which played key roles in the pathogenesis of CAD [23,24]. Therefore, it was reasonable to suppose that our
observation of an association between the ECE-1b C-338A polymorphism and the increased risk of CAD might be due to the elevated ECE-1 activity and higher ET-1 concentration. However, more studies are needed to confirm this hypothesis. In this study we also observed an interaction between ECE1b polymorphism and sex. In female subjects, those who carried the variant genotypes had significantly higher risk of CAD than CC carriers. However, it was without significance among male subjects. Several investigators have reported the sex-specific effects of genotypes [13,15]. Funalot et al. speculated that the sex-specific association might be due to a past effect of female hormones on ECE-1 gene regulation or alternatively to the effects of androgens in men, which would abolish the influence of the genetic variants [14]. This might be also a possible mechanism for our results, because most of the women included in our study were postmenopausal (90%). 17-beta-oestradiol has been shown to inhibit the endothelin-1 gene expression in rat cardiac fibroblasts and aortic smooth muscle cells [25,26]. Importantly, estradiol also could decrease ET-1 levels in the coronary circulation of postmenopausal women with CAD [27]. However, the androgen had an inverse effect that could increase the ET levels [28]. Thus, the elevated levels or activity of ET-1 caused by androgens might mask the effect of the ECE-1b338C to A variant in male subjects. We also investigated the interaction between ECE-1b C338A polymorphism and age. The results showed an increased risk of CAD associated with the mutant genotypes (CA+AA) of ECE-1b in subjects with age ≥ 64 y, but not in subjects with age b64 y. The mechanisms by which the ECE-1b C-338A polymorphism affected the risk of CAD in subjects with age ≥ 64 y remain unclear. However, our data suggested that other genetic factors might contribute to the disease among subjects with age b64 y. Additionally, the antecedents and promoters of CAD are complex and multifactorial, and the impact of genetic variation on CAD likely differs in the presence of other risk factors. Hence, we could not exclude the possibility that other factors, whose prevalence differed between the age groups but was not accounted for in our analyses, might impact the association and, thus explained the age-differences in the association of this polymorphism with CAD. We also conducted a stratified analysis by the severity of CAD. There was a trend toward greater number of diseased coronary artery in variant genotypes (CA+AA), although the differences were not statistically significant. Studies showed that the A allele with higher levels of ECE-1 gene transcription and enzymatic activity contributed to enhanced levels of ET-1 [13]. This could also provide a mechanism for its association with the severity of the CAD. Findings by Lanza et al. demonstrated that ET-1 levels were higher in patients who had 3-vessel disease on angiography [29]. And Salomone et al. also reported that the concentration of ET-1 was associated with severity of coronary artery disease [30]. However, our results should be confirmed by other large studies. Our study does have certain limitations. Firstly, the relatively small sample size of our study, especially after stratifying by age and sex, may limit the statistical power. However, this internally consistent pilot study certainly provides valuable insights and
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interesting information and may serve to guide future studies in this area. Secondly, although we selected controls from individuals with no history of angina and no symptoms or signs of other atherosclerotic vascular diseases, without performing coronary angiography, we could not rule out CAD completely. Nevertheless, the prevalence of CAD in an asymptomatic population appeared to be low [31]. Thirdly, the plasma levels of ECE-1 or ET-1 in cases and controls were not measured and no functional analysis of the C-338A polymorphism was conducted. It was therefore not possible to link plasma ECE-1 or ET-1 levels with CAD. However, the association between ECE-1 or ET-1 and CAD has been confirmed [5–10] and the mechanism linking the polymorphism with the increased gene transcription and enzymatic activity also has been explored [13]. Fourthly, this is a single SNP approach rather than a haplotype approach. At last, our study was only conducted in Chinese population. Data should be extrapolated to other ethnic groups cautiously. In conclusion, our results showed that the ECE-1b-338 C to A variant might be associated with the increased risk of CAD in Chinese population. The interactions between the ECE-1b338C/A genotype and age or sex were also demonstrated. In order to reach a more definitive conclusion, further large sample studies are needed to investigate the genotypic effects of ECE1b polymorphism on the CAD severity. Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (no. 30672486), the Natural Science Foundation of Jiangsu Province (no. BK2006525) and the development program of Nanjing Science and Technology Bureau (no. 200401068-2). References [1] Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5. [2] Wagner OF, Christ G, Wojta J, et al. Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem 1992;267:16066–8. [3] Morbidelli L, Orlando C, Maggi CA, Ledda F, Ziche M. Proliferation and migration of endothelial cells is promoted by endothelins via activation of ETB receptors. Am J Physiol 1995;269:H686–95. [4] Xu D, Emoto N, Giaid A, et al. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994;78:473–85. [5] Minamino T, Kurihara H, Takahashi M, et al. Endothelin-converting enzyme expression in the rat vascular injury model and human coronary atherosclerosis. Circulation 1997;95:221–30. [6] Ihling C, Szombathy T, Bohrmann B, Brockhaus M, Schaefer HE, Loeffler BM. Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis. Circulation 2001;104:864–9. [7] Hai E, Ikura Y, Naruko T, et al. Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis. Int J Mol Med 2004;13:649–54. [8] Ruschitzka F, Moehrlen U, Quaschning T, et al. Tissue endothelinconverting enzyme activity correlates with cardiovascular risk factors in coronary artery disease. Circulation 2000;102:1086–92. [9] Maguire JJ, Davenport AP. Increased response to big endothelin-1 in atherosclerotic human coronary artery: functional evidence for upregulation of endothelin-converting enzyme activity in disease. Br J Pharmacol 1998;125:238–40.
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