Author + information
- Received July 8, 1996
- Revision received October 9, 1996
- Accepted November 12, 1996
- Published online March 1, 1997.
- Carole Amant, BSA,
- Martial Hamon, MDB,
- Christophe Bauters, MDB,
- Florence Richard, MDA,
- Nicole Helbecque, PhDA,
- Eugène P McFadden, MRCPIB,
- Xavier Escudero, MDB,
- Jean-Marc Lablanche, MDB,
- Philippe Amouyel, MD, PhDA,* and
- Michel E Bertrand, MDB
- ↵*Dr. Philippe Amouyel, Service d’Epidémiologie et de Santé Publique/INSERM CJF 95-05, Institut Pasteur de Lille, 1 rue Calmette, 59019 Lille Cedex, France.
Objectives. This study sought to assess the potential association of the angiotensin-converting enzyme (ACE) and angiotensin II type 1 (AT1) receptor gene polymorphisms on coronary vasomotion in humans.
Background. Abnormal coronary vasomotion plays a role in the clinical expression of coronary atherosclerosis. The components of the renin–angiotensin system are important determinants of vasomotor tone. Furthermore, epidemiologic evidence suggests that these components are involved in the pathogenesis of coronary artery disease. Indeed, two genetic polymorphisms of the ACEand AT1receptor genes were synergistically associated with the occurrence of myocardial infarction. The influence of these genetic polymorphisms on the risk of myocardial infarction may be related, at least in part, to a deleterious effect on coronary vasomotion.
Methods. We studied the response of angiographically normal human coronary arteries after intravenous injection of methylergonovine maleate, a potent vasoconstrictor whose effects have been previously explored in various aspects of coronary artery disease. We characterized the ACEand AT1receptor genotypes in a consecutive series of 140 patients with normal coronary arteries. Coronary vasomotion was assessed with quantitative coronary angiography.
Results. No effect of the ACEgene polymorphism was detected. Conversely, the patients carrying the AT1receptor CCgenotype (n = 13) had significantly greater vasoconstriction in distal coronary vessels (p < 0.009).
Conclusions. The AT1receptor gene polymorphism is associated with coronary vasomotion in humans.
(J Am Coll Cardiol 1997;29:486–90)
The clinical manifestations of coronary artery disease are not linearly related to the extent of coronary atherosclerosis. Indeed, acute myocardial infarction generally occurs at atherosclerotic plaques that do not significantly narrow the coronary artery lumen (). The ultimate step in the pathogenesis of acute myocardial infarction is the formation of occlusive thrombus in a coronary artery at the site of a ruptured atherosclerotic plaque (); however, evidence of plaque rupture is common at autopsy in patients dying of noncardiac causes, suggesting that plaque rupture is a necessary but not the sole factor involved in coronary artery occlusion (). Intense vasoconstriction at the site of plaque rupture may also be an important contributory factor; direct evidence that this is so has been provided by angiographic observations during acute infarction ().
The components of the renin–angiotensin system, involved in blood pressure regulation and vascular smooth muscle cell proliferation, are important determinants of vasomotor tone (). Angiotensin-converting enzyme (ACE), a key component of the renin–angiotensin system, is involved in the generation of angiotensin II, a potent vasoconstrictor peptide, and in the degradation of bradykinin, a potent vasodilator (). The circulating and cellular levels of ACE are partly genetically determined through the insertion/deletion polymorphism of the gene coding for ACE (ACE) ([7, 8]). Subjects bearing the ACEdeletion (D) allele have a higher level of circulating enzymes than subjects bearing the insertion (I) allele. Recent studies ([9–11]) have suggested that ACE Dallele bearers may have a higher risk of myocardial infarction and sudden death.
The effects of angiotensin II are exerted through the activation of specific high affinity receptors. Most of the cardiovascular effects of angiotensin II are mediated by the angiotensin II type 1 (AT1) receptor, expressed by vascular smooth muscle cells (). Recently, a polymorphism located in the 3′ untranslated region of the AT1 receptor gene (AT1) (corresponding to an adenine/cytosine (A/C) base substitution at the 1166 position) has been shown () to modify the association of the I/D ACEpolymorphism with the occurrence of myocardial infarction. The AT1receptor Callele increased synergistically the risk of myocardial infarction in subjects carrying the ACE Dallele. The interaction of these two genetic susceptibility risk factors on the occurrence of myocardial infarction could be related to a deleterious effect on coronary vasomotion. To explore this hypothesis, we analyzed the vasomotor responses of angiographically normal coronary arteries to methylergonovine maleate, the most commonly used vasoconstrictor stimulus in clinical practice (), and to isosorbide dinitrate (ISDN), an endothelium-independent smooth muscle dilator.
We studied patients admitted for diagnostic coronary angiography who were found to have an angiographically normal coronary tree. The study included 140 such patients recruited consecutively. Age, gender, body mass index, family and personal history of coronary artery disease, and drug and tobacco consumption were recorded. Total cholesterol and triglyceride plasma levels were measured by enzymatic methods (Boehringer Mannheim, Germany) adapted to a Hitachi 717 analyzer. Patients with dilated cardiomyopathy or a history of myocardial infarction or who had valvular heart disease were excluded.
1.2 Angiography and provocative testing.
All drugs were discontinued 48 h before angiography. After diagnostic coronary angiography, an appropriate view of a coronary artery was selected, and a baseline arteriogram was obtained. Methylergonovine maleate (100 μg) was injected intravenously, and an angiogram was obtained 3 min later. An additional dose of methylergonovine maleate (300 μg) was then injected, and another angiogram was recorded 3 min later. After that, 200 μg of ISDN was injected into the coronary artery, and another angiogram was recorded 2 min later. Finally, 2 mg of ISDN was injected into the coronary artery, and a final angiogram was recorded 2 min later.
1.3 Quantitative coronary angiographic analysis.
The coronary angiograms were analyzed with the CAESAR System (Computer Assisted Evaluation of Stenosis and Restenosis), a computerized automatic analysis system (). The angiographic catheter was used for calibration. The mean diameters of proximal and distal segments, identified by their distance from side branches or from the origin of the vessel, were determined. Maximal response to methylergonovine maleate and ISDN was expressed relative to baseline diameter after 300 μg of methylergonovine maleate and 2 mg of ISDN, according to the formula: Maximal constriction by 300 μg of methylergonovine maleate = [(Diameter after ergonovine − Baseline diameter)/Baseline diameter] × 100 (%), and Maximal dilation by 2 mg of ISDN = [(Diameter after ISDN − Baseline diameter)/Baseline diameter] × 100 (%).
1.4 Genetic analyses.
Genomic DNA was extracted from white blood cells. The ACEfragment () containing the I/Dsequence and AT1receptor fragment () containing the A/C1166 substitution were amplified with a Perkin Elmer DNA thermal cycler and Thermus aquaticus DNA polymerase (Amersham). The ACEpolymorphism was detected as previously described, except for addition of dimethylsulfoxide to enhance amplification of the ACE Iallele (). The AT1receptor A/Cpolymorphism was analyzed by allele-specific oligonucleotide hybridizations after amplification ().
1.5 Statistical analyses.
Statistical analyses were performed with the SAS Software release 6.10 (SAS Institute Inc.). Mean value ± SD were calculated; error of the mean (SEM) was used to plot quantitative data. Quantitative variables were compared according to the ACEgenotypes (i.e., II, ID, DD) and the AT1receptor genotypes (i.e., AA, AC, CC). Given the unequal number of observations for each group of genotype, we performed an analysis of variance within the framework of general linear models (GLM procedure). This GLM procedure was also used to conduct a multivariate analysis of covariance to take into account the potential effect of quantitative and qualitative confounding factors, and to test for interaction (crossed effects) between the two polymorphisms. A synergistic effect between the two genetic variables was tested by including both variables simultaneously in the model together with the interaction between the ACEand AT1receptor genotypes. In these multivariate models, the normal distribution of the residues was verified; the estimate of the coefficients and standard errors from the multivariate analysis were computed for each variable. The genetic variables were tested with 2 degrees of freedom. Significance levels were set at p < 0.01.
Sixty-eight percent of our patients were male and had a mean age of 54 ± 10 years; 27% were smokers; 12% had diabetes mellitus; 48% had systemic hypertension; and 41% had a family history of coronary artery disease. The frequencies of the ACE Dand Ialleles were 0.57 and 0.43, respectively; the frequencies of the AT1receptor Aand Calleles were 0.70 and 0.30, respectively. These frequencies were close to values reported in other white patient groups ([9, 13]). Genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium. Proximal and distal baseline segment diameters were similar among the different groups of genotypes (Table 1). In the whole cohort, proximal and distal segments constricted in response to methylergonovine maleate and relaxed after ISDN. No patient developed occlusive epicardial spasm. No differences in vasoconstrictor or vasodilator responses were observed in subgroups with different ACEgenotypes (Fig. 1). However, patients who had the AT1receptor CCgenotype had significantly greater maximal vasoconstriction (p < 0.009) in distal coronary segments than patients with the AAor ACgenotypes (22.9%, 11.3% and 11.5%, respectively) (Fig. 2). This enhanced constriction was independent (adjusted test, p = 0.008) of age, gender, family history of coronary heart disease, plasma lipid levels, diabetes, systemic hypertension and cigarette consumption (Table 2). Maximal vasodilation in response to ISDN did not differ among the different groups of genotypes. No significant association between the AT1receptor genotypes and systemic hypertension existed in this sample. No significant interaction on coronary vasomotion could be detected between ACEand AT1receptor genotypes.
Our results suggest that subjects with the AT1receptor CCgenotype had an exaggerated constrictor response to methylergonovine maleate, a vasoconstrictor agent used to explore coronary vasomotion in clinical practice. By contrast, no effect of the ACEgenotype on coronary vasomotion could be detected.
These results are at variance with those of a Japanese study () reporting that the ACEbut not the AT1receptor polymorphism was associated with coronary artery spasm. Although Japanese study and ours relate to coronary vasomotion, they addressed different issues in a different ethnic background. Indeed, the case-control study developed by Oike et al. () recruited specific contrasted groups of patients: The cases presented a total occlusive spasm during ergonovine provocation and significant ST segment alterations, whereas the controls had no reaction after the provocative test. Conversely, none of our patients developed occlusive spasm. Moreover, the normal distributions of the vasomotor tone indexes allowed us to perform a quantitative analysis. The differences in ethnic background may also account for the different results: In the Japanese study, the ACE Dallele frequency was 0.36, consistent with other Japanese reports () but significantly lower than those in white patients (0.56) (). The frequency reported in the Japanese cohort for the Callele of the AT1receptor was 0.09, significantly lower than that observed in white patients (0.30) (). Finally, the AT1receptor CCgenotype was totally absent in the Japanese study, and consequently, its effect on vasomotion could not be tested.
3.1 Potential mechanisms.
We previously reported () the effects of methylergonovine maleate on the coronary circulation in patients with diverse clinical manifestations of coronary artery disease. Localized segmental hypereactivity in the infarct-related vessel was observed in 20% of patients with recent myocardial infarction. The influence of the AT1receptor CCgenotype on coronary artery vasomotion in response to methylergonovine maleate may be related to an effect on smooth muscle cells. Methylergonovine maleate has a direct constrictor effect on vascular smooth muscle cells due to a stimulant effect on alpha-adrenergic and serotoninergic receptors (). These arterial smooth muscle cells express AT1 receptors that mediate most of the known vascular effects of angiotensin II, a potent vasoconstrictor peptide ().
In our patients, the influence of the AT1receptor polymorphism on coronary vasomotion was observed predominantly in distal coronary segments. This observation is consistent with previous studies () that have documented a heterogeneous response in the coronary tree to vasoactive stimuli: Distal coronary segments have greater vasoreactivity in response to ergonovine and nitrates than proximal segments.
This influence on coronary vasoconstriction was observed in subjects homozygous for the Callele of the AT1receptor polymorphism, suggesting a recessive effect. Conversely, no effect of ACEgene polymorphism on coronary vasomotion was detected. However, because methylergonovine maleate predominantly acts through endothelium-independent direct smooth muscle cell constriction (), we cannot exclude an effect of the ACEgenotype on endothelium-dependent responses.
A recent study () described an increased frequency of the AT1receptor Callele in subjects with essential hypertension. We were not able to detect this association in our sample. Given the small effect reported for this polymorphism on hypertension, our study was lacking statistical power to draw any conclusion. However, the enhanced vasoconstriction associated with the AT1receptor polymorphism may partly account for high blood pressure levels in Callele bearers. A recent report () has associated the AT1receptor CCgenotype with aortic stiffness in hypertensive patients. Our observation of increased vasoconstriction in AT1receptor CCgenotype bearers could support the hypothesis that this increased aortic stiffness may reflect an excessive constrictor response of systemic resistance vessels in such patients.
The present study demonstrates that the AT1receptor genotype is associated with exaggerated coronary vasoconstriction. Given the low frequency of the Callele and CCgenotypes in human populations, further investigations are needed to confirm these results and to clarify the possible mechanisms involved in this phenomenon. In particular, it is important to differentiate between a direct but less probable role of the AT1receptor C1166 mutation and a strong linkage disequilibrium of this polymorphism with a causal variant ([13, 23]). However, this observation might explain, at least in part, the synergistic effect of this genetic polymorphism together with the ACE I/Dpolymorphism on the occurrence of myocardial infarction.
We thank Claudine Mercier and Valérie Codron for excellent scientific and technical assistance.
☆ Dr. Hamon was supported by a grant from the Société Française de Cardiologie, Paris, and Ms. Amant by the Conseil Régional du Nord-Pas de Calais, Lille, France. This work was supported by grants from the Direction de la Recherche et des Etudes Doctorales, Lille; the Centre Hospitalier Universitaire de Lille, Lille; the Institut National de la Santé et de la Recherche Médicale, Paris; and the Institut Pasteur de Lille, Lille, France.
- angiotensin-converting enzyme
- angiotensin II type 1 receptor
- general linear models
- isosorbide dinitrate
- Received July 8, 1996.
- Revision received October 9, 1996.
- Accepted November 12, 1996.
- The American College of Cardiology
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