Estrogen Receptor-α Polymorphisms and Angiographic Outcome After Coronary Artery Stenting
Objective— Because of the receptor-mediated antiproliferative effects of estradiol on vascular smooth muscle cells, our study aimed at identifying a role of PvuII and XbaI polymorphisms of the α-estrogen receptor (αER) gene in the occurrence of restenosis after coronary stent implantation (in-stent restenosis [ISR]).
Methods and Results— In 858 patients (148 women), 955 lesions were treated with stent implantation, and the PvuII C/T and XbaI G/A polymorphisms of the αER gene were determined. Quantitative angiography was performed before and after stenting and at 6-month follow-up. The allelic frequencies were similar between sexes (C/T allele, 0.43/0.57 and 0.44/0.56; P=0.9; G/A allele, 0.35/0.65 and 0.38/0.62; P=0.8; in women and men, respectively). A significantly higher ISR rate in women than in men homozygous for the T-allele of the PvuII polymorphism (42.6% versus 26.9%, P=0.03) or the G-allele of the XbaI polymorphism (41.2% versus 19.4%, P=0.04) was observed. At multivariate analysis, T/T genotype was the only independent predictor of ISR in women but not in men (odds ratio, 1.5; 95% CI, 1.0 to 2.1; P=0.03). XbaI polymorphism was no longer associated with ISR in both sexes.
Conclusions— Women homozygous for the T-allele of the PvuII polymorphism of the αER gene treated with coronary stent implantation have a higher risk of ISR than men.
The low incidence of coronary events in premenopausal women is related to the protective effects that female hormones exert on the cardiovascular system.1,2 The atheroprotective effects of estrogens are in part attributable to the receptor-mediated genomic, long-term influence on metabolisms of lipids, glucose, and hemocoagulative system and to the interaction with the autonomous nervous system.2
Functional estrogen receptors are present in endothelial3 and vascular smooth muscle cells,4 where they mediate acute and short-term effects on vasoreactivity. In this setting, different sex-related functional responses to the administration of estradiol have been reported.5
The PvuII and XbaI polymorphisms of the α-estrogen receptor (αER) gene are associated with receptor expression and function in nonatherosclerotic diseases like breast cancer and osteoporosis.6–8 Recent studies have evaluated the role of these 2 polymorphisms in coronary artery disease (CAD), but results about the possible functional role of these polymorphisms in disease/phenotype expression seem discrepant.9–13 To the best of our knowledge, there are no data available exploring the association between αER polymorphisms and restenosis after coronary stent implantation.
The αER gene is located on the chromosome 6. The PvuII polymorphism is caused by a T/C transition in intron 1. The XbaI polymorphism is a G/A transition located 50 bp apart from the PvuII polymorphism site, with a high degree of linkage disequilibrium with the former one.14 Because of the receptor-mediated antiproliferative effects of estradiol on vascular smooth muscle cells,15 the trophic effects on endothelial cells,16 and the influence of αER expression on the progression of CAD,17 we hypothesized that the response to vascular injury after percutaneous coronary intervention could be modulated by the αER polymorphisms. Therefore, the role of the PvuII and XbaI αER polymorphisms in the 6-month angiographic outcome after coronary stent implantation in coronary lesions of female and male patients was evaluated.
The occurrence of angiographic in-stent restenosis (ISR) was analyzed in 995 lesions of 858 patients genotyped for the αER PvuII and XbaI polymorphisms, with appropriate quantitative coronary analysis (QCA) before and after intervention and at 6-month follow-up. These patients underwent coronary angioplasty with stent implantation and DNA extraction between 1993 and 2000 and were scheduled for elective repeat angiographic control with QCA. Patients gave their informed consent to the diagnostic and interventional procedures. The female population included mostly postmenopausal women (92%), none of whom were undergoing hormone replacement therapy during the follow-up period. A total of 32% of patients were treated with ACE inhibitors, and 30% were treated with statins. Insulin-dependent diabetic patients were excluded from the analysis because of their known excessive propensity to develop restenosis after angioplasty. Another 15 patients were excluded because polymerase chain reaction could not reliably identify the αER polymorphisms. Nearly 10% of patients did not repeat angiography systematically because of advanced age, impaired general conditions, or patient’s preference. The angiographic parameters of ISR are analyzed in the whole population and also separately in women and men to look for αER polymorphism sex-related differences.
Balloon Angioplasty and Stent Implantation
Balloon angioplasty and stent implantation were performed according to standard clinical practice. The use of IIb-IIIa glycoprotein inhibitors was left to operator’s preference. After the procedure, all patients received 250 mg of ticlopidine twice daily for the first month and 165 mg of aspirin daily indefinitely. QCA was performed at baseline, immediately after stent implantation, and at 6 months. Measurements were obtained in at least 2 orthogonal projections after intracoronary nitrate administration.
Determination of αER Genotypes
Blood was obtained from peripheral veins of all recruited patients. Genomic DNA was extracted from 200 μL of whole blood with a QIAamp Blood Kit (QIAGEN). The PvuII polymorphism in the estrogen receptor gene was determined according to the method of Yaich et al.18 A 1.6-kb DNA fragment of αER gene containing the 2 polymorphic sites in intron 1 was amplified by polymerase chain reaction and digested with PvuII enzyme (Roche) at 37°C for 3 hours; the T-allele was digested, whereas the C-allele was not. These products were electrophoresed in 2.5% agarose gel and visualized directly with ethidium bromide staining. The same conditions were used for genotyping the XbaI polymorphism. The polymerase chain reaction product was digested with the XbaI enzyme (Roche) at 37°C for 3 hours; the A-allele was digested, whereas the G-allele was not cut.
Comparison with the allele nomenclature, already described in literature, is as follows: PvuII C (=P1=P), T (=P2=p), XbaI G (=1=X1=X), A (=2=X2=x). We adopted the nucleotide nomenclature to avoid any ambiguity.
ISR was defined as in-stent percent diameter stenosis ≥50% observed at QCA follow-up. Minimal lumen diameter (MLD) was obtained from the QCA analysis of the luminogram. Acute gain in MLD was calculated as the difference between postprocedural and preprocedural data. Late loss in MLD was calculated as the difference between postprocedural and follow-up values. Late loss index was calculated as (late loss/acute gain in MLD)×100. Net gain was obtained as difference between acute gain and late loss.
Continuous data are expressed as means and SDs; discrete variables are given as absolute values and percentages. Two-tailed Student’s t test was used for comparison of parametric variables, and the χ2 or exact test was used for discrete variables. For multivariate analysis, a model including the following variables was elaborated: sex, age, hypertension, dyslipidemia, non–insulin-dependent diabetes mellitus (NIDDM), smoking habit, presence of acute coronary syndrome on admission, complex morphology of the target lesion (B2 and C, according to the American Heart Association/American College of Cardiology classification), use of multiple stents, QCA parameters, and PvuII and XbaI genotypes. Statistical analyses were made using the SPSS software (7.5 release for Windows).
Clinical and angiographic baseline characteristics of the 858 patients analyzed by sex are described in Table 1, and procedural and angiographic results of the 955 lesions are described in Table 2. As expected, women presented a higher-risk clinical profile and significantly smaller reference vessel diameters before and after stenting. The incidence of restenosis was 26.8% in the entire population, with a slight, nonsignificant difference between the 2 sexes (32.1% in women, 25.7% in men; P=0.1). Despite similar MLD and acute gain at the end of the procedure, 6-month MLD and net gain were significantly smaller in women (Table 2).
The allelic frequencies of the whole population were similar to those expected in European subjects,19 as follows: PvuII C-allele, 0.44; T-allele, 0.56; XbaI G-allele, 0.37; A-allele, 0.63; with a similar distribution between sexes (PvuII C/T, 0.43/0.57 versus 0.44/0.56; P=0.9; XbaI G/A allele, 0.35/0.65 versus 0.38/0.62; P=0.8; in women and men, respectively).
The analysis of the whole population showed similar 6-month restenosis rates between polymorphisms. Patients carrying the T-allele of the PvuII polymorphism had a slightly higher, although not significant, restenosis rate (PvuII C/C, 19.4%; C/T, 23.9%; T/T, 28.5%; P=0.1). A genetic effect of the XbaI polymorphism was less apparent (XbaI G/G, 20%; G/A, 23.4%; A/A, 27.2%; P=0.3).
The occurrence of ISR at follow-up in the whole population, analyzed as categorical variable, was higher after multiple stent implantation (OR, 1.8; 95% CI, 1.4 to 2.3; P=0.00001), in NIDDM patients (OR, 1.7; 95% CI, 1.4 to 2.2; P=0.00003), and in hypertensive patients (OR, 1.3; 95% CI, 1.1 to 1.6; P=0.008). Considering the continuous variables, an increased risk of ISR per unit of measure was observed for the vessel diameter before (OR, 0.4; 95% CI, 0.33 to 0.6; P=0.00001) and after the procedure (OR, 0.4; 95% CI, 0.30 to 0.59; P=0.00001), the MLD before (OR, 0.7; 95% CI, 0.49 to 0.94; P=0.02) and after the procedure (OR, 0.5; 95% CI, 0.38 to 0.70; P=0.00001), and the length of the stented segment (OR, 1.03; 95% CI, 1.03 to 1.05; P=0.0001).
In consideration of the different relevance that estrogen and ER have in women compared with men, a subgroup analysis according to sex was performed. The T/T genotype of the PvuII polymorphism was the only variable associated with ISR in women (OR, 1.4; 95% CI, 1.02 to 2.1; P=0.04). In men, the following variables were associated with ISR: the use of multiple stents (OR, 1.9; 95% CI, 1.5 to 2.6; P=0.00001), NIDDM (OR, 1.9; 95% CI, 1.5 to 2.5; P=0.00001), hypertension (OR, 1.4; 95% CI, 1.1 to 1.8; P=0.005), complex morphology of the target lesion (OR, 1.3; 95% CI, 1.1 to 1.7; P=0.01), vessel diameter before (OR, 0.4; 95% CI, 0.3 to 0.57; P=0.00001) and after the procedure (OR, 0.3; 95% CI, 0.23 to 0.49; P=0.00001), MLD before (OR, 0.6; 95% CI, 0.39 to 0.8; P=0.001) and after the procedure (OR, 0.4; 95% CI, 0.29 to 0.60; P=0.0001), and length of the stented segment (OR, 1.04; 95% CI, 1.02 to 1.06; P=0.0001).
Occurrence of ISR, analyzed for polymorphisms in female and male subjects separately, showed a significantly higher rate for the T/T genotype in women (P=0.04) but not in men (P=0.6), whereas the 3 genotypes of the XbaI polymorphism showed similar ISR rate in both sexes (Table 3). The incidence of restenosis for the T/T genotype of the PvuII and the G/G genotype of the XbaI was higher in women than in men (P=0.01 and P=0.04, respectively) (Figure 1).
The analysis of restenosis as a continuous variable shows that women carriers of the T/T genotype have the worst long-term angiographic result when the late loss in MLD is compared according to polymorphisms and sex (Table 4). The influence of the genotypes on the angiographic outcome according to sex is shown in Figure 2.
In the whole population, the use of multiple stents (OR, 1.6; 95% CI, 1.3 to 1.9; P=0.00001), NIDDM (OR, 1.5; 95% CI, 1.2 to 1.8; P=0.0002), hypertension (OR, 1.2; 95% CI, 1.0 to 1.4; P=0.01), and reference vessel diameter after treatment (OR, 0.6; 95% CI, 0.4 to 1.0; P=0.05) were independent predictors of ISR. NIDDM (OR, 1.6; 95% CI, 1.2 to 2.0; P=0.0001), hypertension (OR, 1.3; 95% CI, 1.1 to 1.5; P=0.002), the length of the stented segment (OR, 1.05; 95% CI, 1.0 to 1.1; P=0.00001), and the reference vessel diameter after angioplasty (OR, 0.3; 95% CI, 0.2 to 0.4; P=0.00001) emerged as predictors of ISR in men. In the smaller group of women only, homozygosity for the T-allele of the PvuII polymorphism was the only independent predictor of ISR (OR, 1.5; 95% CI, 1.0 to 2.1; P=0.03).
To our knowledge, this is the first study that analyzes the association between the PvuII and XbaI polymorphisms of the αER gene and ISR in humans. The polymorphisms of the αER gene did not correlate with the occurrence of ISR in the whole population and in male patients, whereas a significant association was observed in women for the PvuII polymorphism. Compared with men, women homozygous for the T-allele of the PvuII or for the G-allele of the XbaI polymorphisms revealed a higher ISR rate. Of particular importance is the observation that the T/T genotype emerged as the only independent predictor of ISR in women. The observation that the association between genetic polymorphisms and restenosis may differ between sexes has been reported previously by other authors.20
Indirect Evidence of the Role of the αER Polymorphisms in CAD
It is known that premenopausal women have fewer estrogen receptors in atherosclerotic than in normal coronary arteries17 and that the absence of functional estrogen receptors in men with a disruptive mutation in the estrogen receptor gene may be a novel risk factor for CAD.9 Therefore, estrogen receptors seem to play a role in the prevention or in the occurrence of cardiovascular disease. However, it is not known how and to what extent the polymorphisms of the αER gene studied in our report may function as genetic markers of vascular pathology.
αER polymorphisms have raised great interest in the last few years, and the PvuII is one of the most extensively investigated. In 1997, Matsubara et al10 showed the lack of association of the PvuII and XbaI polymorphisms with the severity of coronary stenosis measured by angiography in a series of 87 cases (65 male and 22 female) compared with 94 healthy control subjects. However, the small number of patients included in that case-control analysis does not strongly support the lack of association. Recently, the length of the nucleotide repeat in the regulatory region of the αER gene has been associated with severity of CAD in men. The authors suggest that carriers of the long-repeat variant have a lower expression of the ER gene and that this may reduce the benefits derived from the cardiovascular protective effects of the ER.11
The role of αER polymorphisms on lipid metabolism has been recently assessed by Lu et al.13 In patients with familial hypercholesterolemia, the G/G genotype of the XbaI polymorphism was an independent predictor of CAD. In women under treatment with hormone replacement therapy, a significantly higher increment of HDL cholesterol level according to baseline was observed in patients homozygous for the C-allele of the IVS1-401 polymorphism compared with treated women carrying other genotypes. Similar patterns of response were observed for other closely related genotypes in intron 1.12
αER as Predictor of ISR
The assessment of the incidence of ISR according to the genotypes of the PvuII and XbaI polymorphisms of the αER gene suggests sex-related differences of angiographic outcome after stent implantation. The biological mechanism through which a polymorphic expression of the αER gene can influence the reaction of the vessel wall to stent implantation is unknown. Based on available evidence, we speculate that the healing response to vascular injury may differ according to the lower expression of αER that derives from its polymorphisms, in particular when the circulating level of estrogens is low. This hypothesis seems to apply to women only, suggesting that a possible deficient expression and function of the αER may be more relevant as a cause of restenosis in women, in whom premenopause estrogen levels play a key role in keeping a normal vascular biology. In men, the low estrogen level throughout lifetime suggests either a less-determinant role of the hormone in normal vascular biology or a less important role of ER and its polymorphic expression. Nevertheless, our interpretation remains speculative, because the functional relevance of the αER polymorphisms cannot be addressed in this type of association studies but rather needs a specific determination of the quantitative trait of heritable phenotype, with particular interest in female subjects.
Genetic Polymorphisms and Targeted Medical Treatment
The finding of a causal association between a genetic marker and restenosis could support a strategy of individual treatment scheme. The enthusiasm derived from the recent achievements of drug-eluting stent technology may be also seen in this context. Indeed, whether local administration of estradiol (or other antiproliferative drugs) will be effective in all patients or in responders only will keep alive the interest for identifying patients and drugs for the best-suited therapy.
Because the role of estrogens seems more important in women than in men, the determination of αER polymorphisms may help for a tailored treatment focused on responders rather than on unselected patients. However, such a kind of association did not hold in a study designed to test this concept. In fact, contrary to expectation, the use of ACE inhibitor quinapril in patients with high ACE enzyme activity after stent implantation was not beneficial in the randomized controlled PARIS study.21 Therefore, at the present stage, either the concept of genetically targeted medical treatment of vascular diseases is too much simplified or the genetic markers tested so far are not specific enough.
Conclusion and Limitations
The main finding of our study is a sex-specific role of the PvuII and XbaI polymorphisms of the αER gene in the occurrence of ISR in humans and a detrimental effect of the T/T genotype of the PvuII polymorphism in women only.
It is already well established that the statistical power (an inadequate sample size) is a major source of biased genetic associations.22 Although our study has been performed on one of the largest published series of patients treated with stent implantation, DNA extraction, and elective QCA follow-up, the possibility of a spurious association cannot be fully ruled out. Furthermore, the genetic heterogeneity of complex processes such as ISR is not likely to depend on a single but rather on several polymorphisms that may cluster in the same gene locus or, more probably, in different loci. The functional relevance of the polymorphisms remains to be assessed by the specific determination of the quantitative trait of heritable phenotype that in our hypothesis would be related to the density of expression and degree of function of receptors or to the linkage disequilibrium with other regulatory or functional polymorphisms.
As with any novel genetic-association study, the results of our investigation should be considered exploratory and await confrontation with others experiences, in particular with larger comparison between premenopausal and postmenopausal women.
V.F. was supported by an educational grant of the European Society of Cardiology as a Research Fellow at the OLV Cardiovascular Center, Aalst, Belgium.
- Received July 23, 2003.
- Accepted September 18, 2003.
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