Angiotensin-Converting Enzyme Gene Polymorphism Is Associated With Carotid Arterial Wall Thickness in Non–Insulin-Dependent Diabetic Patients
Background The insertion/deletion (I/D) polymorphism of the ACE gene has been shown to be associated with cardiovascular disease in healthy subjects as well as in patients with non–insulin-dependent diabetes mellitus (NIDDM). We investigated the relationship between the ACE gene polymorphism and the wall thickness of both carotid and femoral arteries in NIDDM patients.
Methods and Results We measured the intimal plus medial thickness (IMT) of both carotid and femoral arteries using high-resolution B-mode ultrasonography in 288 Japanese NIDDM patients (160 men, 128 women). No significant differences among the three genotypes were found with respect to age, sex, duration of diabetes, body mass index, blood pressure, plasma glucose, hemoglobin A1C, total cholesterol, triglycerides, HDL cholesterol, or cigarette-years. Plasma ACE levels were strongly associated with I/D polymorphism, with an additive effect of the D alleles. The carotid IMT of the patients carrying the D allele (DD+ID genotype) was significantly higher than that of the patients not carrying the D allele (II genotype) (P=.037), whereas the femoral IMT was not affected by the I/D polymorphism. Multiple regression analysis demonstrated that the risk factors for carotid IMT of patients with NIDDM were age, non-HDL cholesterol, and D allele of the ACE gene (R2=.155, P<.0001).
Conclusions The D allele of the ACE gene may be a risk factor for the development of wall thickening of the carotid but not the femoral artery in NIDDM patients.
It is well known that diabetic angiopathy, including atherosclerosis and coronary heart disease, is a pivotal complication of NIDDM. However, the mechanism underlying diabetic angiopathy is still little known. It is not fully explained by metabolic factors, such as hyperglycemia, hypertension, or hyperlipidemia. Some genetic factors are considered to be involved in the development of vascular complications of NIDDM.
ACE has been considered a candidate gene for the development of atherosclerosis, because ACE inhibitor has been shown to reduce the degree of atherosclerosis in some animal models.1 The ACE gene has an I/D polymorphism in intron 16, which determines the plasma level in humans.2 This I/D polymorphism of the ACE gene has been shown to be associated with myocardial infarction in the nondiabetic population3 as well as in NIDDM patients,4 with restenosis after percutaneous transluminal coronary angioplasty,5 and with diabetic nephropathy.6
Recently, we reported that hemodialysis patients have advanced atherosclerosis in the carotid arteries as measured by high-resolution B-mode ultrasonography, which allows the noninvasive imaging and quantitative assessment of atherosclerotic lesions.7 In this study, to clarify the genetic factors that play some role in the development of diabetic macroangiopathy, we investigated the association between the ACE genotype and both carotid and femoral IMT measured with high-resolution B-mode imaging in NIDDM patients.
Subjects and Clinical Characteristics
We evaluated 288 Japanese inpatients and outpatients, 18 to 79 years old, with NIDDM (160 men and 128 women). The inclusion criterion was the presence of ascertained NIDDM, defined as previously reported.4 Patients treated with ACE inhibitor were excluded. Each subject gave informed consent to participate in this study.
Blood pressure was measured as previously described.7 Fasting blood was withdrawn for the analysis of serum concentrations of glucose, total cholesterol, triglyceride, HDL cholesterol, and Hb A1C by standard laboratory methods. Plasma ACE activity was measured according to a method described by Kasahara and Ashihara.8
Lifelong exposure to smoking was estimated as previously described.7 Body mass index was calculated as body weight divided by the square of height (kg/m2).
Ultrasonographic scanning of the carotid and femoral arteries was performed as previously reported.7 Briefly, the bilateral carotid and femoral arteries were investigated in the longitudinal and the transverse projections with high-resolution real-time ultrasonography with a 10-MHz in-line Sectascanner (SSD650CL, Aloka Co Ltd). The carotid artery was scanned at the level of the bifurcation and the common carotid artery. The femoral arteries were scanned where the artery divides into the superficial and profound femoral arteries. At each longitudinal projection, the IMT, as defined by Wendelhag et al,9 was measured from the site of the greatest thickness. The three determinations were averaged. To assess the intraobserver variability of the recording and measurement of IMT in the carotid and femoral arteries, a total of 20 subjects were examined on two different occasions separated by 7 to 14 days. The coefficient of variation for IMT in the carotid artery was 3.2%, and that in the femoral artery was 3.0%.
Data are expressed as mean±SD. Clinical characteristic values among the three genotypes were compared with a one-way ANOVA with Scheffe´'s F test. The relationship between IMT values and age was examined by linear regression analysis. Multiple regression analysis was performed to assess the combined influence of variables on IMT values. For IMT values, age, non-HDL cholesterol, systolic blood pressure, Hb A1C, history of smoking (cigarette-years), and the ACE genotype (II, 0; ID, 1; DD, 2) were considered independent variables. These statistical analyses were carried out with StatView IV on a personal computer (Macintosh Centris 650). Statistical significance was set at P<.05.
The ACE genotype was determined in all 288 NIDDM patients. In the total population, the frequency of the I allele was 62.8% and that of the D allele, 37.2%. The genotype distribution (II, 43.1%; ID, 39.6%; DD, 17.4%) was consistent with other published reports in Japanese NIDDM patients without myocardial infarction.12 No significant differences were found among the three genotypes with respect to sex, age, body mass index, duration of diabetes, cigarette-years, blood pressure, fasting plasma glucose, Hb A1C, total cholesterol, triglycerides, or HDL cholesterol (Table 1⇓).
Plasma ACE Activity
Plasma ACE level was strongly associated with the ACE I/D polymorphism, with an additive effect of the D alleles. The plasma ACE levels in DD, ID, and II were 23.2±1.02, 19.4±0.70, and 16.6±0.59 IU/L, respectively (DD versus II, ID versus II, and ID versus DD, P<.02) (Table 1⇑). This result was consistent with previous results in healthy subjects.2
Carotid and Femoral IMTs
There were no significant differences in the IMT values of the carotid or femoral artery among the three genotypes (1.00±0.04, 1.11±0.06, and 1.20±0.09 mm in patients with the II, ID, and DD genotypes, respectively). Those of the femoral artery in the three genotypes were 1.45±0.07, 1.55±0.09, and 1.65±0.13 mm in the II, ID, and DD genotypes, respectively (not significant). With respect to the D allele of the ACE gene, the carotid IMT of the patients carrying the D allele (DD plus ID type) was significantly higher than that of the patients not carrying the D allele (II type) (1.14±0.05 versus 1.00±0.04 mm, P=.037). The femoral IMT was not different between these two groups (1.58±0.07 mm in the DD+ID group and 1.45±0.07 mm in the II group, P=.209). The IMTs of carotid and femoral arteries were both significantly correlated with age (r=.319, P<.0001 and r=.366, P<.0001, respectively).
Analysis of Risk Factors in NIDDM
Multiple regression analysis demonstrated that the risk factors for carotid IMT of patients with NIDDM were age, non-HDL cholesterol, and the D allele of the ACE gene (II, 0; ID, 1; and DD, 2) (R2=.155, P<.0001), as shown in Table 2⇓. The risk factors for femoral IMT were shown to be age, systolic blood pressure, and smoking (R2=.175, P<.0001; Table 2⇓).
In this study, we first demonstrated the significant association of the ACE I/D polymorphism and the wall thickness of the carotid artery in NIDDM patients by direct imaging with ultrasonography. The extent of the carotid artery atherosclerosis as measured by ultrasound B-mode imaging has been shown to be strongly correlated with the presence of coronary atherosclerotic disease and to be a marker for an early phase of the atherosclerotic process.13 The D allele of the ACE gene has been shown to be an independent risk factor for coronary heart disease in nondiabetic subjects3 as well as NIDDM patients.4 We were able to demonstrate that the D allele of the ACE gene is associated with carotid IMT in NIDDM patients. Just recently, in a general population in Italy, the ACE DD genotype was shown to be significantly associated with common carotid IMT.14
The risk factors for the carotid and femoral arterial wall thicknesses were not identical. Multiple regression analysis reveals that age contributed independently to the extent of both the carotid and femoral IMTs, whereas the D allele of the ACE gene contributed significantly only to the carotid IMT. The extent and severity of the carotid atherosclerosis and coronary atherosclerosis were strongly correlated, whereas the relationship between the coronary and femoral atherosclerosis was weak in autopsy studies.15 Thus, it may be agreed that the D allele is the risk factor for both carotid and coronary atherosclerosis in NIDDM patients4 but not for the femoral artery.
Angiotensin II is demonstrated to modulate the growth of vascular smooth muscle cells. ACE inhibitor is established to have vascular protection benefits via direct antiatherogenic, antihypertensive, and antiproliferative effects on smooth muscle cells.1 In diabetic rats, mesenteric ACE levels were increased concomitantly with increased vessel weight, and ACE inhibitor attenuated this vascular hypertrophy as well as ACE levels.16 Using an in vivo gene transfer technique, Morishita et al17 demonstrated a vascular hypertrophy in the ACE-transfected blood vessel. Thus, accumulating evidence strongly suggests the role of ACE in vascular hypertrophy.18 Recently, cardiac tissue ACE activity was demonstrated to be increased in subjects with the DD genotype,19 allowing us to speculate on the possible involvement of the ACE genotype in the regulation of tissue ACE activity as well as plasma ACE activity.
The effect of the D allele on plasma ACE was additive, but on the carotid IMT, it was not. The absence of a gene dosage effect in IMT may be because (1) tissue ACE activity may be more important and may be influenced by gene polymorphism differently from serum ACE activity and (2) there may be no mechanistic relationship between the ACE polymorphism and IMT. We cannot exclude the possibility that other DNA sequence differences in the ACE gene may contribute to genetic susceptibility to diabetic macroangiopathy in NIDDM.
Thus, the ACE I/D polymorphism may be a genetic factor that predicts the progress of carotid atherosclerosis in patients with NIDDM.
Selected Abbreviations and Acronyms
|IMT||=||intimal plus medial thickness|
|NIDDM||=||non–insulin-dependent diabetes mellitus|
This work was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture. The authors are grateful for the expert technical assistance of Keiko Kawanishi and Mayuko Murano.
Reprint requests to Masayuki Hosoi, MD, PhD, Second Department of Internal Medicine, Osaka City University Medical School, 1-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan.
- Received November 28, 1995.
- Revision received February 1, 1996.
- Accepted February 7, 1996.
- Copyright © 1996 by American Heart Association
Lonn E, Yusuf S, Jha PMT, Teo K, Benedict C, Pitt B. Emerging role of angiotensin-converting enzyme inhibitors in cardiac and vascular protection. Circulation. 1994;90:2056-2069.
Rigat B, Hubert C, Alhenc GF, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990;86:1343-1346.
Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D, Luc G, Bard JM, Bara L, Ricard S, Tiret L, Amouyel P, Alhenc-Gelas F, Soubrier F. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature. 1992;359:641-644.
Ruiz J, Blanche H, Cohen N, Velho G, Cambien F, Cohen D, Passa P, Froguel P. Insertion/deletion polymorphism of the angiotensin-converting enzyme gene is strongly associated with coronary heart disease in non-insulin-dependent diabetes mellitus. Proc Natl Acad Sci U S A. 1994;91:3662-3665.
Marre M, Bernadet P, Gallolis Y, Savagner F, Guyene T-T, Hallab M, Cambien F, Passa P, Alhene-Gelas F. Relationships between angiotensin I converting enzyme gene polymorphism, plasma levels, and diabetic retinal and renal complications. Diabetes. 1994;43:384-388.
Kasahara Y, Ashihara Y. Colorimetry of angiotensin-I converting enzyme activity in serum. Clin Chem. 1981;27:1922-1925.
Wendelhag I, Wiklund O, Wikstrand J. Atherosclerotic changes in the femoral and carotid arteries in familial hypercholesterolemia: ultrasonographic assessment of intima-media thickness and plaque occurrence. Arterioscler Thromb. 1993;13:1404-1411.
Kawasaki ES. Sample preparation from blood cells, and other fluids. In: PCR Protocols. San Diego, Calif: Academic Press, Inc; 1990:146-152.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res. 1992;20:1433.
Fujisawa T, Ikegami H, Shen G-Q, Yamato E, Takekawa E, Nakagawa Y, Hamada Y, Ueda H, Rakugi H, Higaki J, Ohishi M, Fujii K, Fukuda M, Ogihara T. Angiotensin I-converting enzyme gene polymorphism is associated with myocardial infarction, but not with retinopathy or nephropathy, in NIDDM. Diabetes Care. 1995;18:983-985.
Wofford J, Kahl F, Howard G, McKinney W, Toole J, Crouse JI. Relation of extent of extracranial carotid artery atherosclerosis as measured by B-mode ultrasound to the extent of coronary atherosclerosis. Arterioscler Thromb. 1991;11:1786-1794.
Castellano M, Muiesan M, Rizzoni D, Beschi M, Pasini G, Cinelli A, Salvetti M, Porteri E, Betoni G, Kreutz R, Lindpaintner K, Rosei E. Angiotensin-converting enzyme I/D polymorphism and arterial wall thickness in a general population: the Vobarno study. Circulation. 1995;91:2721-2724.
Sternby N. Atherosclerosis in a defined population: an autopsy survey in Malmo¨, Sweden. Acta Pathol Microbiol Scand. 1968;194(suppl I):111-118.
Cooper M, Rumble J, Komers R, He-Cheng D, Jandeleit K, Sheung-To C. Diabetes-associated mesenteric vascular hypertrophy is attenuated by angiotensin-converting enzyme inhibition. Diabetes. 1994;43:1221-1228.
Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H, Kaneda Y, Ogihara T, Dzau VJ. Evidence for direct local effect of angiotensin in vascular hypertrophy: in vivo gene transfer of angiotensin converting enzyme. J Clin Invest. 1994;94:978-984.
Johnston C. Tissue angiotensin converting enzyme in cardiac and vascular hypertrophy, repair, and remodeling. Hypertension. 1994;23:258-268.
Danser JAH, Schalekamp MADH, Bax WA, van der Brink AM, Saxena PR, Riegger GAJ, Schunkert H. Angiotensin-converting enzyme in the human heart: effect of the deletion/insertion polymorphism. Circulation. 1995;92:1387-1388.