Introduction

The renin–angiotensin (RA) system is one of the major determinants of the vascular system. Dysfunction of the RA system promotes the development of cardiovascular disease.^1,2 Renin secretion, physio-logically triggered by a reduction in blood pressure, intravasal volume or renal perfusion, initiates the RA system cascade by converting angiotensinogen to angiotensin I. Angiotensin I-converting enzyme (ACE) converts angiotensin I into angiotensin II (AT-II), which physiologically induces vasoconstriction and aldosterone secretion.¹ However, AT-II may also exert a strong atherogenic effect by promoting vascular cell proliferation and neointima formation.^1,2 The release of other vasoactive substances with a distinct atherogenic potential is induced by the RA system, such as endothelins (ETs) and vasopressin (VP).^2,3,4 The ET system consists of three vasoactive peptides, ET-1, -2 and -3, that are synthesized by endothelial cells, smooth muscle cells and macrophages.^5,6 ET-1 is a potent vasoconstrictor, promotes smooth muscle cell proliferation and the synthesis of extracellular matrix substances. ET-1 therefore promotes its own production by smooth muscle cells. The strong atherogenic impact of ET-1 is increased by AT-II.^4,5,7 ET-1 production may in turn be induced by AT-II and VP, emphasizing the cooperation of the RA system with ET-1 and VP in the stimulation of neointimal growth.^2,3,8 Increased local expression of ET-1 was demonstrated in atherosclerosis in different vascular regions.^7,9,10 VP secretion is stimulated by AT-II.² In addition to its antidiuretic function, VP exerts vasoconstrictive and growth-promoting effects on the vasculature.¹¹

ACE has been shown to influence VP and ET-1 through the activation of AT-II. Pharmacological inhibition of ACE leads to a decrease in AT-II, ET-1 and VP plasma levels.^1,11,12 The insertion/deletion (I/D) polymorphism in intron 16 of the human ACE gene leads to changes in plasma ACE concentration and activity. Homozygotes for the shorter allele (DD) show increased ACE plasma concentrations and activity.^13,14 The D allele is also associated with coronary artery disease (CAD) in younger Caucasian males.¹⁵ The association of the ACE I/D gene polymorphism with the risk of ischaemic heart disease in the Caucasian population has been intensively investigated and discussed.^{14,15,16,17,18,19} The ACE I/D gene polymor-phism is not associated with changes in plasma levels of renin, AT-II or aldosterone in spite of an increased ACE activity.^20,21,22,23 This may be explained by compensation within the RA system. However, the ACE I/D gene polymorphism accounts for 47% of phenotypic variance in ACE plasma levels.¹³ Homozygotes for the D allele show an 85% increase in ACE activity compared to I allele homozygotes.¹⁴ Since AT-II stimulates ET-1 production directly⁴ or via an increased VP production,^2,8 ET-1 and VP secretion could be stimulated through increased ACE activity. A sustained upregulation especially of ETs, leading to increased plasma levels, could partly result from a self-stimulatory production by vessel wall cells in atherosclerosis.^3,5,6 We therefore hypothesized that changes of ACE activity through the ACE I/D polymorphism may not lead to pronounced changes in plasma levels of RA system components, but could result in an increase in vasoactive agents associated with the RA system such as ETs or VP. Changes in ET-1 or VP levels could possibly lie within the range of ACE activity differences between the ACE I/D genotypes. So far the association of ACE activity and genetic polymorphisms in the ACE gene with changes in ET-1 and VP plasma levels has not been examined. This study investigated the association of the ACE I/D gene polymorphism with plasma levels of ET-1 and VP, as well as with levels of renin, AT-II and ACE activity in patients with advanced coronary atherosclerosis.

Materials and methods

Patient profiles

ACE I/D polymorphism was analysed in 98 male and female Caucasian individuals admitted to the Clinic for Cardiovascular Surgery for coronary artery bypass surgery. The patients were evaluated according to a detailed study protocol containing anamnestic and clinical information about CAD risk factors. All subjects gave informed consent. With the help of a preceding coronary angiography, the study population was divided into subgroups according to severity of CAD (individuals with single-vessel disease and coronary artery stenoses >50%, double-vessel disease and triple-vessel disease) and Gensini score.²⁴ All patients were admitted to the Clinic for Cardiovascular Surgery in a stable condition, an acute myocardial infarction or instable angina pectoris was excluded according to established criteria.²⁵ Patients with ischaemic or idiopathic dilated cardiomyopathy were excluded from the study population because of a possible association with the DD genotype.²⁶ Patients with chronic ACE inhibitor medication (>1 month) or with signs of renal insufficiency (serum creatinine >180 mol/l) were also excluded. Before blood samples were drawn, patients were allowed to rest in a horizontal position for 30 min.

Measurements of enzymes, substrates and vasoactive peptides

Serum ACE activity was measured using an ACE colorimetric assay kit (Bühlmann Lab., Switzerland) on a Cobas Mira Plus system (Roche Diagnostics, Germany). The analytical intra-assay precision of the test was 5.2–7.3% determined by using samples with varying ACE activities, and the accuracy was below 1.0%. Plasma levels of VP were measured using a Vasopressin RIA kit (Bühlmann Lab., Switzerland); intra-assay precision was 2.1–15.3% and accuracy was 3.0–9.0%. Active renin was measured using a Renin III generation IRMA kit (Bio-Rad, CA, USA), with an intra-assay precision of 3.7–7.2% and an accuracy of 1.0–7.0%. AT-II was analysed using an AT-II human EIA kit (Phoenix Pharmaceuticals, CA, USA); intra-assay precision was 3.0–4.8% and accuracy was 0.8–11.0%. ET-1 was determined using a human ET-1 EIA kit (Assay Designs, MI, USA); intra-assay precision was 2.1–2.9% and accuracy was 3.3–11.8%. All patient samples were measured in one series for each of these tests. Total cholesterol was measured by the CHOD-PAP (cholesterol oxidase) photometric–enzymatic method on a Hitachi 917R system (Roche Diagnostics, Germany). All other measurements were done according to established clinical–chemical methods in routine laboratory testing.

PCR detection of ACE I/D polymorphism

The ACE gene insertion defining the ACE I/D polymorphism consists of a 287 bp sequence.²⁷ The presence (insertion) or absence (deletion) of this sequence was analysed as described previously,^14,15 based on established methods.²⁷ Amplification products were electrophoresed in 2% NA agarose gels (Pharmacia) and visualized by ethidium bromide staining, whereby the I allele (insertion polymorphism) was demonstrated by a 490 bp fragment and the D allele (deletion polymorphism) by a 190 bp fragment. Mistyping of I/D heterozygotes was excluded as previously described ¹⁵ according to Lindpaintner and et al.¹⁸

Statistical analysis

Statistical analysis was performed using SPSS for Windows software, version 9.0. A probability value of P<0.05 indicated statistical significance, and P>0.05 indicated the disapproval of the tested hypothesis. By univariate analysis, genotype was related to different types of variables: Continuous variables were sex, age, severity of CAD (extension of CAD, ie single-, double- and triple-vessel disease; Gensini score), body mass index (BMI), cholesterol and pack years of nicotine abuse (one pack year: 20 cigarettes per day for 1 year). To evaluate the distribution of continuous variables, the Shapiro–Wilk test was employed. Since variables did not all follow normal distribution, genotype was related to continuous variables by nonparametric one-way ANOVA with the help of the Kruskal–Wallis test. Genotype was also related to binary variables by means of ² anaylsis, that is, arterial hypertension, diabetes mellitus, family history of CAD, and additional atherosclerotic disease in one or more other arterial regions (carotid, femoral or iliac, brachial or subclavian, mesenteric region). Genotype was then related to the plasma levels of ACE, VP, AT-II, ET-1 and renin by Kruskal–Wallis one-way ANOVA. By multiple regression analysis, the relation of the ACE I/D polymorphism to plasma levels of ACE, VP, AT-II, ET-1 and renin was tested individually considering different quantitative and categorical variables, such as severity of CAD, BMI, cholesterol, arterial hypertension or diabetes mellitus. The number of subjects included in the study was based on power calculation using previously described allele frequencies¹⁵ and probability values =5% and =10%.

Results

Patient characteristics and ACE I/D gene polymorphism

The study population consisted of 98 individuals, 70 males and 28 females, with patient mean age 66.3 years (range 41–83 years). The genotype subgroups contained comparable patient samples according to patient age and sex (Table 1). The relative allele frequencies were 0.47 for the I and 0.53 for the D allele in the total study population. Genotypes were found to be distributed according to the Hardy–Weinberg equilibrium, as previously shown.^14,15,27

Table 1 - Patient characteristics of the total study population and of ACE I/D genotype groups II, ID and DD, indicated as absolute or percental number of patients or mean values.

Full table

The variables BMI, cholesterol plasma level, nicotine consumption, arterial hypertension, diabetes mellitus, family history of CAD and atherosclerosis in different arterial regions showed a corresponding distribution between the ACE I/D genotype subgroups ( Table 1). Univariate analysis revealed no association of ACE I/D genotype to patient variables (sex, age, severity of CAD, BMI, cholesterol, pack years of nicotine abuse, arterial hypertension, diabetes mellitus, family history of CAD or atherosclerosis in other arterial regions). Multivariate analysis revealed no association of ACE I/D genotype and severity of CAD considering the other patient variables (data not shown).

ACE I/D genetic polymorphism, the RA system and related vasoactive peptides

Univariate analysis of plasma level of ACE, VP, AT-II, ET-1 or renin by ACE I/D polymorphism revealed a positive relation between the D allele and the enzymatic activity of plasma ACE, also indicated by increasing ACE activity from II (median activity: 15.0 U/L) over ID (31.0 U/L) to DD (54.0 U/L) genotype subgroup. In contrast, the plasma levels of VP, ET-1, AT-II or renin were not statistically related to the ACE I/D polymorphism in univariate analysis. Table 2 shows the median values and ranges of ACE, VP, ET-1, AT-II and renin plasma concentrations, or activities in the total study population and in genotype subgroups, with the corresponding reference range limits.

Table 2 - Vasoactive peptides and enzymes analysed in the total study population and by genotype groups.

Full table

Through multivariate analysis, we investigated individually the association of ACE, VP, AT-II, ET-1 and renin plasma levels to the ACE I/D polymorphism, considering different patient variables. The positive association between the ACE I/D genotype and ACE activity was not additionally influenced by patient variables. As in univariate analysis, plasma levels of VP, AT-II, ET-1 and renin were not associated with ACE I/D genotype in multiple regression analysis, considering possible effects of patient variables (P values >0.05). An influence of the ACE I/D gene polymorphism on VP and ET-1 plasma levels or on RA system components in patients with CAD was, therefore, not demonstrated in this study.

Discussion

Dysfunction of the RA system is considered an important determinant for the development of atherosclerosis.^2,33 Especially, AT-II has a strong atherogenic effect.^1,2 AT-II is released by the catalytic activity of ACE that plays a key role in the RA system. Additional vasoactive substances with an atherogenic potential are released by the RA system, namely ET-1 and VP,^2,3,4 which are investigated here. The RA system cooperates with ET-1 and VP in promoting atherosclerosis.^2,3,8 ET-1 promotes vasoconstriction and neointima development,^4,5,7 thereby locally producing a strong proatherogenic effect.^7,9,10 ET-1 production is induced by AT-II, VP and by a paracrine pathway; its atherogenic effect is increased by AT-II.^3,5,6 Also, VP acts proatherogenically¹¹ and is induced by AT-II.²

A central role in the RA system has been attributed to ACE mainly because of the activation of AT-II. The catalytic activity of ACE may influence the balance within the RA system, and indirectly of the RA system with ET-1 and VP.^1,11,12 The enzymatic activity and plasma concentration of ACE is genetically determined³⁴ by ACE I/D polymorphism.^13,27 An association of ACE I/D gene polymorphism with an increased risk of CAD in the Caucasian population has been controversial.¹⁵ Several investigators found no association,^18,19 while others demonstrated a positive association between the D allele and ischaemic heart disease generally¹⁶ or for certain subgroups of patients.^14,15 A meta-analysis including most investigations on ACE gene polymorphism and the risk of ischaemic heart disease found a trend for the association of the D allele with myocardial infarction, but no clear association.¹⁷ An association between CAD according to the Gensini score severity and younger age (<61.7 years) in DD homozygotes was observed.¹⁵ The study population in the present investigation consisted of individuals with CAD and showed allele frequencies comparable to a Caucasian male study population consisting of individuals with and without CAD as published previously.¹⁵ A genetically determined increased ACE activity related to the D allele was demonstrated as described previously.^13,14 The composition of the patient group was based on the study hypothesis that the influence of increased ACE activity could show measurable effects on ET-1 and VP plasma levels in atherosclerosis. ET-1 and VP production is stimulated by RA system components. Neointimal growth could lead to a sustained ET-1 production by a paracrine pathway.^2,3,4,5,6,8

In the present study, plasma concentrations of VP, AT-II, ET-1 or renin were not associated with ACE I/D polymorphism in CAD patients. These parameters were also not associated taking into consideration different patient variables. The study hypothesis was therefore disapproved according to the data presented. Other authors^20,21,22,23 were also unable to relate plasma levels of renin, AT-II and aldosterone with ACE I/D gene polymorphism. The association of the ACE I/D polymorphism with ET-1 and VP, as RA system-related factors, has not been investigated previously. However, this study found no association of the ACE I/D polymorphism with plasma levels of ET-1 and VP in patients with CAD. Plasma ACE enzymatic activity and the D allele of the ACE I/D genotype showed a strong association not related with increasing age, severity of CAD or other patient variables, but was evenly distributed throughout the study population. An increased activity of ACE, genetically determined by the D allele of ACE I/D polymorphism, may be compensated directly within the RA system and/or through counter-regulation of non-ACE pathways of AT-II generation.^35,36 This could explain that plasma levels of RA system components as well as levels of ET-1 and VP show no differences between genotype subgroups.

The present study did not find an association of ACE I/D genotype with severity of CAD (extension of CAD, Gensini score) or an age dependency of this phenomenon. However, this study was designed to relate ACE genotype to RA system-related components and probably lacked sufficient statistical power because of the size of the study group to prove such effects within subgroups. The study population, sample size and the distribution of patient characteristics in the study sample were appropriate to test for a possible association of peptide plasma levels and genotype within the total population. Patient characteristics, that is, sex, BMI, cholesterol, pack years of nicotine abuse, arterial hypertension, diabetes mellitus, family history of CAD or atherosclerosis in other arterial regions, were evenly distributed between the population subgroups. The association of ACE I/D polymorphism and severity of CAD with established risk factors of CAD, such as hypertension, smoking, diabetes mellitus or lipoprotein-a plasma level, has already been investigated in previous studies.^14,15

Consequently, this study could not demonstrate an association of the ACE I/D genotype with plasma levels of RA system peptides, that is, renin and AT-II, and of the RA system-related substances ET-1 and VP in patients with CAD. An increased ACE activity related to the D allele may be compensated within the RA system and/or within non-ACE pathways of AT-II generation, adjusting the balance of vasoactive substances to a physiological level. The RA system-related substances ET-1 and VP may therefore remain unchanged, leading to plasma concentrations independent of the ACE I/D genotype.

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Acknowledgements

We thank W Pabst, Institute of Medical Informatics, Justus Liebig University Giessen, for assistance with the statistical analysis employed in this study. The valuable assistance of A Hirst Ourmazd, PhD, Berlin, in the preparation of the text is gratefully acknowledged.