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(Hypertension. 1997;30:569.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin-Converting Enzyme Gene I/D Polymorphism and Carotid Plaques in Japanese

Yasujirou Watanabe; Tomoaki Ishigami; Yoshiyuki Kawano; Takahiko Umahara; Akitoshi Nakamori; Shunsaku Mizushima; Kiyoshi Hibi; Izumi Kobayashi; Kouichi Tamura; Hisao Ochiai; Satoshi Umemura; Masao Ishii

From the Department of Internal Medicine, Nanasawa Rehabilitation Hospital (Y.W., Y.K., T.U., A.N.); the Second Department of Internal Medicine (T.I., K.H., I.K., K.T., H.O., S.U., M.I.); and the Department of Public Health (S.M.), Yokohama City University School of Medicine, Kanagawa, Japan.

Correspondence to Satoshi Umemura, MD, Second Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.

	Abstract

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Abstract To clarify the role of genetic factors in atherosclerotic plaque formation in the carotid artery and magnetic resonance imaging abnormalities in the brain, we investigated the association of these abnormalities with the angiotensin-converting enzyme (ACE) genotype. One hundred sixty-nine subjects (age, 59.2±0.8 years, mean±SE) admitted to our hospital for health checkups underwent brain magnetic resonance imaging to evaluate lacunar infarction. B-mode ultrasound examinations of the carotid arteries were performed to detect atherosclerotic plaque. The I/D polymorphism of the ACE gene was determined by the polymerase chain reaction method. Multivariate regression analysis was performed to assess the effects of the following variables on the presence of plaque, mean plaque thickness, and number of plaques: fibrinogen, sex, age, body mass index, mean blood pressure, glycosylated hemoglobin, LDL cholesterol, HDL cholesterol, hematocrit, and the D allele of the ACE gene. The frequency of carotid atherosclerotic plaque was significantly (P=.034) higher in subjects with the D allele than in those without this allele. However, the frequency of lacunar stroke was similar in these groups. A multivariate regression analysis showed that the presence of plaque was independently associated with the D allele (odds ratio=3.27, P=.016). However, mean plaque thickness and the number of plaques were not associated with the D allele. The D allele of the ACE gene may be involved in the presence of carotid plaque but not in the extent of this plaque or asymptomatic lacunar stroke in Japanese subjects.

Key Words: angiotensin-converting enzyme • genes • atherosclerosis, carotid arteries • ultrasonography • asymptomatic lacunar stroke

	Introduction

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The renin-angiotensin system (RAS) plays an important role in the pathogenesis of cardiovascular diseases, and recent studies, including ours,^{1 2} have shown the genetic contributions of the RAS to these diseases.^{3 4 5 6} The ACE catalyzes the conversion of angiotensin I to angiotensin II and the breakdown of bradykinin to kinin degradation products. ACE activity is associated with carotid wall thickness,⁷ and the ACE gene I/D polymorphism in intron 16 has been found to be associated with plasma ACE levels^{8 9} as well as atherosclerotic diseases, such as myocardial infarction,^{3 4} atheromatous renal artery stenosis,⁵ and restenosis after coronary angioplasty.⁶ Furthermore, this polymorphism has been reported to be associated with cerebrovascular diseases^{10 11} and carotid arterial wall thickness.^{12 13 14} Therefore, to further clarify the genetic factors in cerebrovascular diseases, we investigated the association between these polymorphisms and the ultrasonographic findings of carotid arteries and MRI in asymptomatic subjects who visited our hospital for health checkups.

	Methods

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Subjects
In Japan, health checkups for the prevention of cardiovascular diseases are very common. We studied 220 subjects who were voluntarily admitted to Nanasawa Rehabilitation Hospital for health checkups. Medical histories were compiled for all subjects, with an emphasis on cerebrovascular, cardiovascular, and metabolic diseases; smoking habits; and long-term drug treatment. In some of the 220 subjects, health checkups had been performed as part of a follow-up of their disease, such as cerebral infarction, cerebral hemorrhage, and coronary artery disease. Thus, these subjects were in fact patients, and their number increased as study enrollment continued. To reduce the bias due to the subjects, we excluded them from the study; there were 18 subjects with cerebrovascular disease and 3 subjects with coronary artery disease. Thirty subjects who were receiving ACE inhibitors or antithrombotic drugs were also excluded, because these medications may confound the biochemical data, such as plasma ACE activity and fibrinogen level. On the morning of the second hospital day, blood pressure was measured with a sphygmomanometer, with the patient in the supine position. Body mass index was calculated as body weight (in kilograms) divided by the square of height (in meters). After an overnight fast, blood samples were taken for measurement of plasma ACE activity, glycosylated hemoglobin, plasma fibrinogen concentration, hematocrit, and HDL and LDL cholesterol levels. LDL cholesterol was calculated with the following formula: total cholesterol-.2xtriglycerides-HDL cholesterol. Plasma ACE activity was determined colorimetrically by quantifying the quinonimine dye produced from the substrate p-hydroxyhippuryl-L-histidyl-L-leucine according to a modification of the method of Kasahara and Ashihara.¹⁵ All subjects underwent carotid ultrasonography and brain MRI.

B-Mode Ultrasonography
The right and left carotid arteries were investigated in the longitudinal and transverse projections by high-resolution, real-time ultrasonography with a 7.5-MHz in-line Sectascanner (Toshiba Medical System Co Ltd) operated by the same technician. The common carotid arteries, the carotid bifurcation, and the origin (first 2 cm) of the internal carotid arteries were scanned. We also measured the IMT at the site of greatest thickness in the longitudinal projection. Both near and far walls of all arterial segments were scanned longitudinally and transversely to assess the occurrence of plaques, defined as a localized echo structure encroaching into the vessel lumen when the distance between the medial-adventitial interface and the internal side of the lesion was 1.0 mm or more.¹⁶ When several plaques were present on the same arterial segment (ie, common carotid artery, bifurcation, or origin of the internal carotid artery), the examination was focused on that segment showing the greatest encroachment into the lumen. When a plaque was mineralized, the sonographer had to obtain the best incidence so that the plaque could be visualized on the far wall, and an estimate of IMT was made by extrapolating the adjacent medial-adventitial interface. To assess the extent of plaque, we considered the plaque score and the number of plaques. A plaque score was computed by averaging the plaque thicknesses as measured on longitudinal views and grading them as follows: 0, no plaque; 1, mean thickness 1 mm and 2 mm; 2, mean thickness >2 mm and 3 mm; and 3, mean thickness >3 mm.¹⁶

Brain MRI
MRI was performed with a General Electric imaging unit equipped with a superconducting magnet that generated a field strength of 1.5 T. Image acquisition was performed by the spin-echo technique, the pulse sequences having TR of 600 to 800 ms and a TE of 20 to 25 ms for T1-weighted images and TR of 2500 to 3000 ms and TE of 20 to 80 ms for T2-weighted images. Sagittal, axial, and coronal scans were available for every subject and were interpreted by a neuroradiologist and a neurologist blinded to the clinical diagnoses. A lacune was strictly defined as a low-signal-intensity area (<1 cm diameter) on T1-weighted images that was also visible as a hyperintense lesion on T2-weighted images.¹⁷ We classified the subjects into a lacune-absent group and a lacune-present group.

Detection of the ACE I/D Polymorphism
Detection of the ACE I/D polymorphism was performed as reported previously.² Genomic DNA was extracted from peripheral leukocytes. The ACE genotype was determined by the PCR method. To reduce the incidence of mistyping the I/D genotype, 5% dimethylsulfoxide was added to the reaction mixture.^{18 19} The oligonucleotide sequences of the PCR primers were 5'-CTGCAGACCACTCCCATCCTTTCT-3' (sense primers) and 5'-GATGTGGCCATCACATTCGTCAGAT-3' (antisense primers). DNA was amplified for 30 cycles. Each cycle consisted of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 1 minute. The PCR product was electrophoresed in 1.6% agarose gel and visualized by ethidium bromide staining. The I/D polymorphism of the ACE gene was detected; homozygotes for this polymorphism had either a 490-bp band, named II, or a 190-bp band, named DD, whereas heterozygotes had both bands, named ID.

Statistical Analysis
Data are expressed as mean±SE. We used the ² test for analysis of the categorical variables and Student’s t test or the Mann-Whitney U test for analysis of the continuously distributed variables. Hardy-Weinberg equilibrium was tested by the ² test. Multivariate logistic regression analysis was performed to assess the combined influence of variables on the presence of atherosclerotic plaque. Multivariate linear regression analysis was performed to assess the combined influence of variables on plaque score and the number of plaques. For the presence of plaque, plaque score, and the number of plaques, age, sex, smoking, body mass index, mean blood pressure, glycosylated hemoglobin, LDL cholesterol, HDL cholesterol, hematocrit, fibrinogen, and ACE genotype effect were considered independent variables. IMT was excluded because it was not considered an independent variable. The ACE genotype effect was determined to be a dominant model (with scores of 0 for II and 1 for ID and DD combined), a recessive model (with scores of 0 for II and ID combined and 1 for DD), an additive model (with scores of 0 for II, 1 for ID, and 2 for DD), or a codominant model (II versus ID versus DD) of inheritance. These statistical analyses were carried out with SPSS version 6.1. Values of P<.05 were considered significant.

	Results

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The clinical and laboratory characteristics of the subjects are shown in Table 1. Subjects with plaque differed significantly from those without plaque with respect to age (P=.013), IMT (P<.0001), plasma fibrinogen concentration (P=.042), and lacunar lesions (P=.0007). The other factors were similar between the two groups.

View this table: [in this window] [in a new window]   Table 1. Comparison of Clinical and Metabolic Characteristics Between Plaque-Absent and Plaque-Present Subjects

Table 2 shows the genotype and derived allele frequency with respect to plaque (absent or present) and lacunes (absent or present). The genotype distribution did not deviate significantly from Hardy-Weinberg equilibrium in any subgroup. There were also no significant differences between groups in genotype or allele frequency. Because the frequency of the presence of plaque was higher in the ID (25/[51+25], or .33) and DD (8/[23+8], or .26) groups than the II group (10/[52+10], or .16), the dominant model was used in later analysis.

View this table: [in this window] [in a new window]   Table 2. Distribution of I/D Polymorphism of the ACE Gene and Frequencies of I and D Alleles in Relation to the Presence or Absence of Plaque and Cerebral Lacunar Lesions

We grouped the subjects on the basis of the D allele of the ACE gene: those carrying the ID or DD genotype and those carrying the II genotype (dominant models of inheritance). The groups differed significantly with regard to plasma ACE activity (P<.0001) and the presence of carotid atherosclerotic plaque (P=.034) but not with regard to IMT, plaque score, or the number of plaques (Table 3). The mean plasma ACE activity was 10.21±0.46, 13.08±0.55, and 15.02±0.61 IU/L at 37°C for II, ID, and DD, respectively (II vs ID, P=.0001; II vs DD, P<.0001; ID vs DD, P=.045). However, in the other models of inheritance, ie, recessive, additive, and codominant models, we could not find any difference in the frequencies of the presence of plaque, plaque score, or the number of plaques between the two groups.

View this table: [in this window] [in a new window]   Table 3. Results of Characteristics According to the D Allele of the ACE Gene

Multivariate logistic regression analysis showed that the presence of carotid atherosclerotic plaque was independently associated with the D allele of the ACE gene (OR, 3.27; P=.016) and inversely with HDL cholesterol (OR, 0.95; P=.015; Table 4).

View this table: [in this window] [in a new window]   Table 4. Multivariate Logistic Regression Analysis With Presence of Carotid Atherosclerotic Plaque as the Dependent Variable

Multivariate linear regression analysis showed that plaque score was independently associated with mean blood pressure (P=.001) and LDL cholesterol level (P=.023) and that the number of plaques was associated with age (P=.013) and inversely with HDL cholesterol (P=.009; Table 5). However, no association was found between the ACE D allele and plaque score or number. Additionally, IMT was significantly greater in subjects with silent lacunar lesions than in those without such lesions (1.20±0.06 vs 1.03±0.03 mm, P=.0057).

View this table: [in this window] [in a new window]   Table 5. Multivariate Linear Regression Analysis of Independent Variables to Plaque Score and the Number of Plaques

	Discussion

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In the present study, we found that the frequency of the presence of plaque in the carotid artery was higher in the ID+DD group than in the II group. Furthermore, multivariate regression analysis showed that plaque was correlated with the D allele, suggesting that the ACE D allele is involved in the presence of carotid plaque. Although we could not find any correlation between the D allele and the extent of plaque (Table 5; ie, plaque score or the number of plaques), the extent of plaque was correlated with age, HDL cholesterol, LDL cholesterol, and mean blood pressure, which accords with previous findings.^{20 21 22 23} The present study also showed that the IMT of the common carotid artery was not different between the ID+DD group and the II group. Cigarette smoking has been found to be a significant risk factor for carotid atherosclerosis in whites^{20 21} but not in Japanese.²² We also did not find any association between carotid atherosclerotic plaque and cigarette smoking.

Increased IMT of the carotid artery has been reported in patients with the ACE D allele or the DD genotype.^{12 13 14} However, another study demonstrated a negative correlation between IMT and the ACE D allele.¹⁰ In the former studies, the subjects were either non–insulin-dependent diabetics or drawn from the general population, but in the latter study, the subjects were stroke patients. Therefore, the discrepancy may be due to population bias. The reasons why we could not find any associations between IMT and ACE genotypes are unclear. One possibility is the differences among the subjects in our study. Atherosclerotic lesions are usually divided into three basic types according to severity: the fatty streak, appearing as increased IMT; the fibrous plaque; and the complicated lesion.²⁴ In addition, early changes in vascular thickness may result in atheroma.²⁵ Therefore, in our study, only the relatively severe atherosclerotic lesion, ie, plaque, but not increased IMT, might correlate with the ACE D allele. Furthermore, we measured the IMT at the site of greatest thickness of common carotid artery but did not measure or average other arteries, such as the bifurcation and internal carotid artery. This methodological difference may be another reason for the discrepancy.

Increased IMT of the carotid artery was reported to be associated with increased plasma ACE activity.⁷ However, we could not find any association between the presence of plaque and plasma ACE activity. The actual reasons why we found no association between these variables are unclear. Because a recent report²⁶ showed that local ACE activity in samples of the left ventricle from organ donors without cardiac disease was higher in persons with the ACE D allele, local ACE activity might be associated with ACE gene polymorphism, although this speculation is too premature without direct evidence of such a correlation in the carotid artery.

Markus et al¹⁰ demonstrated a positive association between the D allele or DD genotype and the risk of lacunar infarction in patients with stroke.¹⁰ However, we did not find any association between the D allele and lacunar infarction. The reasons for this discrepancy are unclear, but Markus et al¹⁰ studied white patients with stroke, whereas we studied asymptomatic Japanese patients who were hospitalized for health checkups. These differences may partially explain the difference between our results and theirs. However, in subjects with plaque, the frequency of lacunar infarction was higher than in the subjects without plaque. Kobayashi et al²⁸ reported that cerebral blood flow was decreased in subjects with silent lacunes, and thus, the extent of plaque may be more important for decreased cerebral blood flow than the mere presence of plaque. Because our study demonstrated that the ACE D allele was associated with the presence of plaque but not with the extent of plaque, there might be no association between the ACE D allele and lacunar infarction. Confirmation of this hypothesis requires demonstration of an association between cerebral blood flow and the extent of plaque or the presence of plaque.

ACE gene polymorphism has been associated with several cardiovascular diseases,^{3 4 5 6} although prospective studies have failed to find the expected association of the ACE I/D alleles with myocardial infarction²⁸ or left ventricular mass.²⁹ These differences in findings might be due to mistyping the ACE gene polymorphism, population bias, and ethnic differences.³⁰ In our study, 5% dimethylsulfoxide was added to the reaction mixture to reduce the incidence of mistyping the I/D genotype.^{18 19} All subjects in the present study were Japanese and had no symptoms of cerebrovascular disease, but the number of subjects enrolled was relatively small compared with study groups of recent studies. Therefore, a large population study is required to confirm the present findings.

In conclusion, the D allele of the ACE gene may be involved in the presence of carotid atherosclerotic plaque, but not the extent of plaque and asymptomatic lacunar stroke. For the extent of plaque and lacunar infarction, conventional risk factors like age, HDL cholesterol, LDL cholesterol, and blood pressure may be more important than the ACE D allele. However, to confirm this assumption, a large, prospective study is necessary.

	Acknowledgments

 
This work was supported in part by grants from the Ministry of Education, Science, and Culture of Japan (No. 08258222, 08407020, 02670404, and 05670956) and by a grant from the Uehara Memorial Foundation. We thank Yasunobu Hirabayashi for ultrasonic examinations.

Received March 17, 1997; first decision April 17, 1997; accepted April 30, 1997.

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