Recurrent In-Stent Restenosis is Not Associated with the Angiotensin-Converting Enzyme D/I, Angiotensinogen Thr174Met and Met235
Despite great progress in reducing death from cardiovascular disease over the past few decades, coronary artery disease (CAD) remains the most common cause of morbidity and mortality in Western countries. Coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are alternative methods of revascularization in patients with CAD.1 The mechanism leading to restenosis after balloon angioplasty includes elastic recoil and vascular remodeling. However, excessive neointimal formation, a process determined by smooth-muscle cell proliferation and extracellular matrix synthesis, is the primary contributor to lumen renarrowing after deployment of stents.2,3 Although intracoronary stent implantation has been shown to significantly reduce angiographic restenosis in humans, the issue remains, even after drug-eluting stents (DES) have emerged as one of the most promising technologies in the field of interventional cardiology.4–8 Several components of the RAS play a pivotal role in various cellular processes, e.g., cell migration, proliferation of smooth-muscle cells, extracellular matrix deposition, thrombosis, and generation of reactive oxygen species. Therefore, they are reasonable candidate genes for ISR.9 We selected 3 genes that encode for proteins with an important pathophysiological role: AGT, ACE, and the AGTR1. Previous studies have shown a positive correlation between the ACE D/I polymorphism and ISR, indicating that the RAS is involved in neointimal proliferation after stenting.10,11
The aim of the present study was to test the hypothesis that genetic polymorphisms of the RAS are associated with recurrent ISR. Four specific functional polymorphisms, i.e., ACE-D/I, AGT T174M and M235T, and A1166C of the AGTR1, have been characterized in patients with ISR and in patients with RISR, respectively (Figure 1).
Materials and Methods
Patients. Coronary stent implantation was performed in 272 Caucasian subjects with clinical symptoms or objective signs of ischemia. All patients received bare-metal stents (BMS). Following successful target lesion revascularization, all patients were again directed to heart catheterization 6 months after. Eighty-one patients (29.8%) displayed ISR; these patients underwent balloon angioplasty and were scheduled for a further 6 months of clinical and angiographic follow up. A total of 42 patients with repeated ISR (RISR group, 89.7% males; aged 33–79 years; mean age 59.7 ± 1.5 years), and 39 patients who showed only 1 ISR event (ISR group, 83% males; aged 34–72 years; mean age 58.7 ± 1.4 years) were examined for the 4 genetic RAS variants. All procedures were performed according to established institutional guidelines and with the patients’ written informed consent.
Angiographic analysis. A single operator who was unaware of the patients’ genotype analyzed all angiograms by using the automated edge detection algorithm (CAAS II, PIE Medical System, Maastricht, The Netherlands). Quantitative analysis was performed for each lesion at baseline and at follow up. After selection of two orthogonal projections, minimal lumen diameter, lesion length and diameter stenosis (%) were measured. Acute gain, net gain and late loss were calculated for each follow-up interval. ISR was defined as ≥ 50% diameter reduction. To determine in-stent diameter restenosis rate, reoccluded vessels were not excluded by statistical analysis. All patients were scheduled for clinical examination, physical examination, electrocardiography, echocardiography, stress testing and angiographic follow up. Periprocedural complications and follow up, including cardiac events, were recorded in a predetermined manner. The angina pectoris status according to the Canadian Cardiovascular Society classification of stable angina pectoris was classified at baseline and at each visit.
Genotyping. Genomic DNA was extracted from peripheral blood leukocytes by Puregene kits using an Autopure LS instrument (Qiagen, Hilden, Germany). The ACE D/I genotypes were determined by polymerase-chain-reaction (PCR) and agarose gel electrophoresis as suggested by O’Dell et al.12 Genotyping for AGT Thr174Met (C>T) and Met235Thr (T>C), and for the AGTR1 1166 A>C polymorphisms was performed using PCR and restriction-fragment length polymorphism (RFLP) analysis as previously described.13–16 For validation, genotyping was repeated in some randomly chosen samples. The results of genotyping were independently checked by two researchers. Statistical analysis. Discrete variables are expressed as counts or percentages and compared using the chi-square test. Continuous variables are expressed as mean value ± standard error. Comparison between groups for continuous data was performed by using the unpaired two-sided t-test.
Table 1 summarizes the demographic, clinical, biochemical and angiographic characteristics of patients with and without repeated restenosis. ACE inhibitors, beta-blockers, statins and antiplatelets were distributed equally across the two groups. A total of 76.3% of the patients in the ISR group were treated with ACE inhibitors versus 75.6% in the RISR group. No significant differences were found between the two groups for nominal stent diameter and stent length (Table 1). There was a tendency towards a higher incidence of diabetes mellitus in the RISR group. The invasively measured aortic blood pressure did not differ between groups (Table 1). In the ISR group, the culprit lesions were located in the left anterior descending artery (LAD) in 56.4% of patients, the right coronary artery (RCA) in 28.2%, and the left circumflex artery (LCX) in 15.4% of all patients. Target vessels in the RISR group included 54.8% in the LAD, 26.2% in the RCA and 19.0% in the LCX. The initial minimal lumen diameter (MLD) in the ISR group was 0.56 ± 0.07 mm vs. 0.65 ± 0.06 mm in the RISR (p = 0.16). The in-segment stenosis in the ISR group was 76.5 ± 2.7% versus 70.9 ± 2.6% in the RISR group (p = 0.07). After stenting, in-segment stenosis declined to 18.3 ± 0.9% (MLD: 2.6 ± 0.07 mm) in the ISR group, and to 18.5 ± 1.3% (MLD: 2.6 ± 0.08 mm; p = 0.48) in the RISR group, respectively. At the first follow-up angiography, in-stent restenosis in the ISR group was 64.7 ± 10.4% vs. 75.2 ± 11.9% in the RISR group (p < 0.01). The corresponding MLD were 0.81 ± 0.13 mm in the ISR group and 0.52 ± 0.08 in the RISR (p < 0.01), respectively. After balloon angioplasty, further follow up was performed angiographically in all patients after 6.0 ± 0.9 months in the ISR group and 5.0 ± 0.9 months in the RISR patients. Twenty patients in the RISR and 10 patients in the ISR group were admitted to the hospital with suspected restenosis prior to the 6-month interval. Coronary angiography revealed RISR in 42 patients (51.9%), whereas 39 patients (48.1%) displayed no significant restenosis.
Angiographic restenosis at the stented site was 84.1 ± 13.5% in the RISR group and 19.2 ± 3.1% in the ISR group, respectively. There was no significant difference in Mehran classification between the ISR and RISR groups.17 The restenosis pattern in the ISR group was focal in 21 patients and diffuse in 15 patients; 3 stents were occluded. In the RISR group, a focal restenosis pattern was found in 24 patients, 15 displayed a diffuse restenosis pattern, and 1 patient showed a multifocal restenosis pattern. In two cases, the stent was completely occluded (Table 1).
Genotype analysis revealed that the incidence of RISR was not associated with any of the polymorphisms examined in this study. The distribution of ACE D/I genotypes was D/D 8 (20.5%), D/I 29 (74.4%), and I/I 2 (5.1%) in the ISR group versus D/D 11 (26.2%), D/I 27 (64.3%) and I/I 4 (9.5%) in the RISR group (chi-square 1.1, n.s., Table 2). Furthermore, there were no significant differences in allele findings in the AGT Thr174Met (C/C 79.5%, T/C 18%, T/T 2.5%, vs. 69%, 26.2%, 4.8%, chi-square 1.18, n.s.), and AGT Met235Thr (T/T 38.4%, T/C 46.2%, C/C 15.4% vs. 35.7%, 47.6%, 16.7%, chi-square 0.07, n.s.) genotype. The distribution of AGTR1 alleles was A/A 23 (59%), A/C 14 (35.9%) and C/C 2 (5.1%) in the ISR group, and A/A 19 (45.3%), A/C 20 (47.6%) and C/C 3 (7.1%) in the RISR group, respectively.
Neointimal hyperplasia, rather than arterial remodeling, plays a pivotal role in the pathophysiology of restenosis after deployment of stents in coronary arteries.18 It is a healing process that results from deep focal injury caused by the penetration of stent struts and the presence of foreign body material.9 The vascular response comprises smooth-muscle cell proliferation and migration and extracellular matrix deposition.19–21 Furthermore, the denudation of endothelium and substantial damage to deeper layers of the arterial wall leads to the exposure of thrombogenic subendothelium to blood, inducing adhesion, aggregation and activation of platelets. Activated platelets release several adhesion molecules, growth factors, cytokines and chemokines such as platelet-derived growth factor, transforming growth factor-b, thromboxan A2 and serotonin.22,23
RAS has been implicated in the development of neointimal hyperplasia, and a major role for angiotensin II (ANG II) in the migration and proliferation of smooth-muscle cells in restenotic lesions has been proposed.9 ANG is hydrolyzed into angiotensin I (ANG I) by renin. Subsequently, ANG I is converted to ANG II by ACE. ANG II exerts its deleterious effects on coronary arteries after stenting through the stimulation of AGTR1 (Figure 1).
The gene encoding ACE is located on chromosome 17 in humans. The best studied genetic variant is the insertion or deletion of a 287 base Alu element within intron 16 of the ACE gene. The ACE D/D polymorphism is associated with higher plasma and tissue ANG II levels.11,15 Although the pathophysiological significance of high ANG II levels is controversially discussed in literature, two published studies have shown a positive correlation between the ACE D/I polymorphism and in-stent restenosis.10,24 To our knowledge, the known polymorphisms of RAS have not yet been studied in high-risk patients with RISR. We therefore examined a possible link between the ACE D/I, ANG Thr174Met, ANG Met235Thr and the AGTR1 A1166C polymorphisms and RISR. We failed, however, to demonstrate a significant association between the polymorphisms with repeated restenosis, indicating that the underlying mechanism leading to repeat restenosis after balloon angioplasty for ISR may be a different pathophysiological process, e.g., a combination of neointimal proliferation and elastic recoil. Since vessel diameter, MLD, stent diameter and length were similar between the ISR and RISR groups, we think the impact of these parameters on restenosis were comparable. A further explanation for the lack of correlation may be the slight differences in ACE inhibition by concomitant medication with ACE-inhibitors. Koch et al. demonstrated that carriers of the ACE D/D polymorphism are neither at higher risk of ISR nor of recurrent myocardial infarction when treated with ACE-inhibitors, suggesting a beneficial role of ACE-inhibitors in patients who are predisposed to cell proliferation after stenting of coronary arteries.24 In the present study, 76.3% of the patients in the ISR group, and 75.6% in the RISR group were treated with ACE-inhibitors. Although there was a tendency towards a higher incidence of diabetes mellitus in the RISR group only, we cannot exclude a correlation of diabetes with recurrent restenosis that is independent of the underlying genotype.
Study limitations. The size of the study population is a limitation in our study. We think, however, that our finding is not spurious due to the following considerations: Both ISR and RISR patients were ethnically homogenous, thus excluding population-based differences in allele frequencies. Phenotyping was performed without knowledge of the patient’s genotype to avoid observer bias. Strict diagnostic criteria and good measurable parameters ensured phenotypic homogeneity of the patient group. Finally, we used a sensitive method for genotyping and tried to reduce mistyping by scoring the gels by two independent researchers.
In conclusion, the ACE D/I, ANG Thr174Met, ANG Met235Thr and the AGTR1 A1166C polymorphisms were not associated with RISR in this study. However, we think that further efforts are needed to conclusively eliminate the possibility of a positive association between ISR and RAS gene polymorphisms.
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- Lasala JM, Stone GW, Dawkins KD, et al. An Overview of the TAXUS Express, Paclitaxel-Eluting Stent Clinical Trial Program. J Interv Cardiol 2006;19:422–431.
- Moses JW, Leon MB, Popma JJ, et al. Sirolimus-eluting stents versus standard stents in patients with stenosis in a native coronary artery. N Engl J Med 2003;349:1315–1323.
- Moses JW, Stone GW, Nikolsky E, et al. Drug-eluting stents in the treatment of intermediate lesions: Pooled analysis from four randomized trials. J Am Coll Cardiol 2006;47:2164–2171.
- Schampaert E, Cohen EA, Schluter M, et al. The Canadian study of the sirolimus-eluting stent in the treatment of patients with long de novo lesions in small native coronary arteries (C-SIRIUS). J Am Coll Cardiol 2004;43:1110–1115.
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- Mehran R, Dangas G, Abizaid AS, et al. Angiographic patterns of in-stent restenosis: Classification and implications for long-term outcome. Circulation 1999;100:1872–1878.
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- Koch W, Mehilli J, von Beckerath N, et al. Angiotensin I-converting enzyme (ACE) inhibitors and restenosis after coronary artery stenting in patients with the DD genotype of the ACE gene. J Am Coll Cardiol 2003;41:1957–1961.