Heme oxygenase-1 gene promotor microsatellite polymorphism is associated with angiographic restenosis after coronary stenting

Ying-Hwa Chena,b, Lee-Young Chauc, Ming-Wei Linb,d, Lung-Ching Chena, Ming-Hui Yoc, Jaw-Wen Chena,e and Shing-Jong Lina,b,e,*

a Division of Cardiology, Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
b Institute of Clinical Medicine, National Yang-Ming University, Taipei Taiwan
c Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
d Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
e Cardiovascular Research Center, National Yang-Ming University, Taipei, Taiwan

* Correspondence: Shing-Jong Lin, MD, PhD, Division of Cardiology, Department of Internal Medicine, Taipei Veterans General Hospital; 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan. Tel: +886-2-28757507; Fax: +886-2-28748374
E-mail address: sjlin{at}vghtpe.gov.tw

Received 13 May 2003; revised 23 September 2003; accepted 16 October 2003

Abstract

Aims Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in heme degradation, leading to the generation of free iron, biliverdin, and carbon monoxide (CO). CO exerts potent antiproliferative and anti-inflammatory effects in the vascular walls, thereby influencing neointimal formation after vascular injury. A dinucleotide GT repeat in the promotor region of human HO-1 gene shows a length polymorphism that modulates the level of gene transcription. This study aimed to assess the association of the length of (GT)nrepeats in HO-1 gene promotor with restenosis and adverse cardiac events after coronary stenting.

Methods and results Quantitative coronary angiography was evaluated before, immediately after and 6 months after stent implantation in 323 consecutive patients with successful coronary stenting. In each patient, the allele frequency of (GT)nrepeats in HO-1 gene promotor was examined. Compared with those with shorter (S, <26) GT repeats, patients with longer (L, ≥26) GT repeats on either allele had more frequent angiographic restenosis with an adjusted odds ratio (OR) of 3.74 (95% confidence interval, 1.61 to 8.70, P=0.002). Such association was even more prominant in patients with small coronary arteries or complex lesions before stenting. Besides, carriers of L/L genotype had an increased risk (adjusted OR, 3.26; 95% confidence interval, 1.58 to 6.72, P=0.001) for adverse cardiac events during follow-up.

Conclusions The length polymorphism of GT repeat in HO-1 gene promoter is an independent risk factor for angiographic restenosis as well as adverse cardiac events after coronary stenting. These findings suggest the genetic contribution to stent restenosis and support the notion that the long dinucleotide GT repeat in promotor region may interfere with HO-1 gene transcription, leading to decreased vascular protection upon injury.

Key Words: Genes • Stents • Restenosis

1. Introduction

Percutaneous coronary intervention with stent implantation has proved efficient to reduce complications and restenosis compared with balloon angioplasty alone. The major drawback of this technique is intimal hyperplasia developed by migration and proliferation of smooth muscle cells, a cell growth process that causes significant stent restenosis in around 30% of cases.1–3Several factors, such as pre-stenting vessel size, lesion type, and the presence of diabetes mellitus have been identified to be associated with restenosis or clinical events in patients receiving coronary stenting. However, little information is available for the role of genetic background in the development of stent restenosis.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in heme degradation, leading to the generation of free iron, biliverdin, and carbon monoxide (CO).4It is a stress-responsive protein, which could be induced by various oxidative agents.4,5Recently, there is increasing evidence showing that HO-1 exerts potent antiproliferative6–8and anti-inflammatory effects9–11via its byproducts derived from heme degradation in the vascular walls, thereby influencing vascular smooth muscle cell activation and neointimal formation after injury.

Human HO-1 gene was mapped to chromosome 22q12,12and a (GT)ndinucleotide repeat was identified in the proximal promoter region.13We and others have recently showed that the (GT)nrepeat is highly polymorphic, and longer (GT)nrepeat exhibits lower transcriptional activity and is associated with susceptibility to coronary artery disease.14,15It is interesting to see whether the genetic variation influencing HO-1 expression might interact with traditional risk factors and contribute to the development of restenosis after placement of coronary stents.

The present study was conducted to determine whether the length polymorphism of the (GT)nrepeats in the HO-1 gene promoter is an independent risk factor for angiographic restenosis as well as adverse cardiac events after coronary stenting (CS) in patients with coronary artery disease (CAD).

2. Methods

2.1. Subjects
The study population consisted of 389 consecutive patients underwent single coronary stent implantation for symptomatic CAD at the catheterization laboratory in a single medical centre between January 1999 and June 2000. Coronary stenting was performed using standard techniques. Before stenting, no additional alternative technique (i.e., rotational ablation, laser, or other debulking devices) was used. All patients received antiplatelet therapy. Aspirin (100 to 325mg/day) was started at least 24h before CS and continued indefinitely. Ticlopidine (500mg/day) was started immediately after CS and continued for 6 to 8 weeks. Only patients with successful single coronary stenting for a critical lesion (>60% luminal diameter stenosis of coronary artery) were included into the study. In each patient, angiographic follow-up was scheduled 6 months after the coronary stenting. However, coronary angiography might be repeated earlier if there was any angina or adverse event occurred. While entering the study, every patient gave a written informed consent for the intervention, angiographic follow-up and genotype determination. The study protocol conformed to the Declaration of Helsinki and was approved by the institutional ethics committee.

2.2. Determination of length polymorphism of (GT)nrepeats in HO-1 gene promoter
Genomic DNAs were extracted from leukocytes by conventional procedures. The 5'-flanking region containing (GT)nrepeats of the HO-1 gene was amplified by PCR with a FAM-labelled sense primer, 5'-AGAGCCTGCAGCTTCTCAGA-3', and an antisense primer, 5'-ACAAAGTCTGGCCATAGGAC-3', according to the published procedure.16The PCR products were mixed together with GenoTypeTM TAMRA DNA ladder (size range 50–500bp) (GibcoBRL) and analysed with automated DNA sequencer (ABI PrismTM 377). Each size of the (GT)nrepeat was calculated using the GeneScan Analysis software (PE Applied Biosystems). To further confirm the sizes of the (GT)nrepeats, some of the PCR products were subcloned into pCRTMII vector (Invitrogen) and purified plasmid DNAs were subjected to sequence analysis.

2.3. Angiographic assessment
Lesions were classified according to the modified American College of Cardiology/American Heart Association grading system.17A quantitative computer-assisted angiographic analysis was performed off-line on angiograms obtained just before stenting, immediately after stenting, and at follow-up using the automated edge-detection system CMS (Medis Medical Imaging Systems). Operators were unaware of the patients’ genotype. Identical projections of the target lesions were used for all assessed angiograms. Minimal lumen diameter (MLD), interpolated reference diameter, percent diameter stenosis, and lesion length were the angiographic parameters obtained with this analysis system. Late loss was calculated as the difference between MLD at the end of intervention and MLD at the time of follow-up angiography. Loss index was calculated as the ratio between late lumen loss and acute lumen gain.

2.4. Definitions and study end-points
The primary end-point of the study was angiographically restenosis at 6-month follow-up. It was defined as a newly developed =50% of coronary luminal diameter stenosis in the index lesion originally treated with a stent. Major adverse cardiac events included death, non-fatal myocardial infarction (MI) and unstable angina (Canadian Heart Class III–IV) necessitating target lesion revascularization (balloon angioplasty or aortocoronary bypass surgery). The diagnosis of acute MI was based on the presence of new pathological Q waves or a value of creatine kinase or its MB isoenzyme at least three times the upper limit.18

2.5. Statistical analysis
All statistical analyses were conducted using the SPSS statistical package, version 11.0. Distributions of continuous variables in groups were expressed as mean±standard deviation (SD) and compared by Student's t tests. Categorical variables were analysed by chi-square test or Fisher exact test if the expected value of any one of cells is less than five. Association of restenosis or adverse cardiac events with specific classes of alleles was analysed, and odds ratios and 95% confidence intervals (CIs) were calculated to assess the relative risk conferred by a particular allele or genotype. Multiple logistic regression models were developed for the development of restenosis or adverse cardiac events. Multiple logistic regression models for subgroup analysis for the development of restenosis in patients with small coronary arteries and with complex lesions were also built up, respectively. Factors including complex lesions, reference diameter before stenting, minimal lumen diameter immediately after stenting, and HO-1 promoter genotypes revealing significant association with restenosis in the univariate analysis (P<0.05) were included in a multiple logistic regression model with forward conditional stepwise selection procedure allowing for the adjustment of age and sex. The multivariate model was then obtained with entry significance of P<0.05 and removal significance P>0.10. Continuous variable such as reference diameter before stenting, minimal lumen diameter immediately after stenting was entered in the logistic regression model using their original data. Significance was accepted at P<0.05.

3. Results

3.1. Characteristics of the study population
Of the 389 patients initially evaluated, 323 (83%) had a follow-up coronary angiography performed at a median of 6.2 months (range: 2.0–12.1 months) after stent implantation. Angiographic restenosis was present in 111 patients (34.4%) and absent in the other 212. Baseline characteristics of the study population are shown in Table 1. There were no significant differences between patients with and without restenosis with regard to age, sex, percentage of risk factors, serum cholesterol, triglycerides and fasting blood glucose levels. The angiographic and procedural characteristics at the time of intervention and at follow-up were listed in Table 2and Table 3. Patients with restenosis had more complex lesions (B2 or C) before CS. The reference diameter (RD) immediately before and minimal lumen diameter immediately after CS were larger in patients without restenosis.


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Table 1 Baseline characteristics of study patientsa

 

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Table 2 Coronary interventional detailsa

 

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Table 3 Quantitative coronary angiography data

 
3.2. Allele and genotypic frequencies of HO-1 microsatellite polymorphism and other risk factors for angiographic restenosis after coronary stenting
The allele frequencies of (GT)nmicrosatellite polymorphism in HO-1 promoter region found in the studied individuals were shown in Fig. 1. The GT repeat numbers ranged from 16 to 38, and (GT)23and (GT)30are the two most common alleles in our study population. Since the proportion of allele frequencies below or above 26GT repeats was around 50%, so we classified the alleles into two subgroups: the lower component, with repeat number <26, was designated as ‘class S’; and the upper component, with ≥26GT repeats, was designated as ‘class L’ Table 4listed the proportions of allele and genotypic frequencies of the study population. The proportion of L allele frequency was significantly higher in patients with restenosis (63.1%) than in those without restenosis (49.8%) (P=0.002). The distribution of allele and genotypic frequencies of patients without restenosis was similar to that of control subjects as shown in our previous study.14



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Fig. 1 Frequency distribution of the (GT)nrepeats in study population: patients without restenosis (n=212), and those with restenosis (n=111).

 

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Table 4 Distribution of HO-1 promoter genotypes and allele frequencies of all study population with or without restenosis

 
With regard to the genotypic distribution, the odds ratio (OR) of angiographic restenosis was 3.17 for patients carrying L alleles (L/L and L/S) (P=0.001), 0.29 in each 1mm increment of pre-stenting RD (P<0.001) and 2.19 for patients with complex lesions (P=0.005) by univariate analysis. Multivariate analysis of correlates of restenosis was performed with the variables of age, gender, complex lesions and vessel size, the presence of L allele (OR 3.74, 95% CI 1.61–8.70, P=0.002), pre-stenting vessel size (OR 0.24 per increase of 1mm, 95% CI 0.12–0.47, P<0.001) and complex lesions were still independently (OR 2.71, 95% CI 1.44–5.10, P=0.002) associated with an increased risk for angiographicrestenosis.

3.3. Allele and genotypic frequencies of HO-1 microsatellite polymorphism and their association with stent restenosis in patients with small coronary arteries and in patients with complex lesions
Since pre-stenting vessel size is different between patients with and without restenosis, further analysis was done in patients with large coronary arteries (reference diameter ≥3.0mm, n=236) or with small ones (reference diameter <3.0mm, n=87) according to the pre-stenting RD (Table 3). For complex lesions also had more stent restenosis, further analysis was done to evaluate the association of the length polymorphism of GT repeat in HO-1 gene promotor with stent restenosis in patients with complex lesions (AHA type ≥B2 or C, n=173) or those with simple ones (AHA type A or B1, n=150). The results showed that HO-1 microsatellite polymorphism was significantly associated with restenosis only in patients with small coronary arteries or with complex lesions but not those with large vessels or with simple lesions.

In patients with small coronary arteries or with complex lesions, there were no significant differences between those with and those without restenosis regarding to age, sex, risk factors, serum cholesterol, triglycerides, and fasting blood glucose levels. The angiographic and procedural characteristics at the time of intervention and at follow-up were also similar between the two groups. In patients with small coronary arteries, the reference diameter before stenting was comparable between these two groups of patients (2.81±0.18mm vs 2.77±0.18mm; P=0.293). However, the proportion of L allele frequency was significantly higher in patients with restenosis (65.9%) than in those without restenosis (39.5%) (P=0.019) (Table 5). Further, the odds ratio of angiographic restenosis was 5.84 among subjects carrying L alleles (L/L and L/S) (95% CI, 1.50 to 22.75; P=0.011). Similarly, in patients with complex lesions, the proportion of L allele frequency was also significantly higher in patients with restenosis (64.4%) than in those without restenosis (47.2%) (P=0.002) (Table 5). Further, the odds ratio of angiographic restenosis was 4.27 among subjects carrying L alleles (L/L and L/S) (95% CI, 1.67 to 10.87, P=0.002). After adjustment for variables including age, gender and complex lesions, the presence of L allele was still an independent risk factor for stent restenosis in patients with small coronary arteries (adjusted OR, 6.96; 95% CI, 1.67 to 29.11; P=0.008) (Table 6). Similarly, after adjustment for variables including age, gender and vessel size, the presence of L allele was also an independent risk factor for stent restenosis in patients with complex lesions (adjusted OR, 5.08; 95% CI, 1.73 to 14.96; P=0.003) (Table 7).


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Table 5 Distribution of HO-1 promoter genotypes and allele frequencies of in patients with small coronary arteries and complex lesions

 

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Table 6 Logistic regression analysis of determinants of restenosis after stenting adjusted for age and sex in patients with small coronary arteries

 

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Table 7 Logistic regression analysis of determinants of restenosis after stenting adjusted for age and sex in patients with complex lesions

 
3.4. Allele and genotypic frequencies of HO-1 microsatellite polymorphism and their association with adverse cardiac events after coronary stenting
Among the study population, 59 of them (18%) had adverse cardiac events during the 20.3±5.1 months of clinical follow-up period. The most common adverse event was unstable angina necessitating target lesion revascularization (n=51). Seven patients suffered from non-fatal myocardial infarction and one patient died suddenly 2 years after coronary stenting. There were no significant differences between patients with and without cardiac events with regard to age, sex, percentage of risk factors, serum cholesterol, triglycerides and fasting blood glucose levels. The angiographic and procedural characteristics at the time of intervention and at follow-up were also similar except that reference diameter before stenting (3.07±0.42mm vs 3.29±0.47mm; P=0.005) and MLD immediately after stenting (2.92±0.50mm vs 3.10±0.52mm; P=0.041) were smaller in patients with adverse events. Table 8displayed the proportions of allelic and genotypic frequencies of the study population. The proportion of L allele frequency was significantly higher in patients with adverse events (P=0.032). Besides, patients with L/L genotype had more adverse outcome (odds ratio, 3.17; 95% CI, 2.09 to 3.75, P=0.014). Even after adjusting for other factors, the presence of L/L genotype was still associated with an increased risk for adverse outcome. (Adjusted odds ratio, 3.26; 95% CI, 1.58 to 6.72; P=0.001) (Table 9).


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Table 8 Distribution of HO-1 promoter genotypes and allele frequencies of study population with or without adverse cardiac events

 

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Table 9 Logistic regression analysis of determinants of adverse cardiac events after stenting adjusted for age and sex

 
4. Discussion

Restenosis after percutaneous coronary interventions has represented a large clinical problem since the introduction of balloon angioplasty, and it remains the major drawback of this technique even after the introduction of stent implantation.1–3The formation of restenosis after balloon angioplasty is a complex process involving recoil of the vessel and cell migration/proliferation, whereas stent restenosis is almost solely a result of neointimal growth.19–21Recently, there is increasing evidence that genetic background may explain at least part of the excessive risk for restenosis observed in certain patients. The most frequently studied polymorphisms in this regard are those of the genes encoding for angiotensin-converting enzyme. However, the published studies were heterogeneous, especially those addressing in-stent restenosis.22In this study, for the first time, we demonstrated that a polymorphism in the gene encoding HO-1 could be associated with angiographic restenosis as well as adverse cardiac events after CS in a cohort of CAD patients. Our findings may not only indicate the contribution of genetic background to stent restenosis but also support the notion that the long dinucleotide GT repeat in promotor region may interfere with HO-1 gene transcription, and consequently reduce vascular protection upon injury.

HO-1 is a stress-responsive protein that can be generated by HO-1 gene under a variety of stimulations associated with oxidative stress.4,5Upon stimulation, HO-1 can produce CO to inhibit vascular smooth muscle cell growth23and protect endothelial cells from tumour necrosis factor-alpha-mediated apoptosis.24It may also inhibit the monocyte transmigration through the production of antioxidants biliverdin and bilirubin.25Though the importance of HO-1 in human atherosclerosis has not been fully defined, it was demonstrated to have in vivo anti-atherogenic properties possibly through the inhibition of lipid peroxidation.26,27Further, treatment with hemin, a HO-1 inducer, markedly inhibited balloon injury-induced neointimal formation in animals.28,29Recent studies also indicated that adenovirus-mediated HO-1 gene delivery could inhibit atherogenesis in apo-E deficiency mice30and attenuates neointimal formation after experimental vascular injury.31

(GT)nrepeat is the most frequent simple repeat in human genome and often exhibit length polymorphism, which may affect transcriptional activity.32Human HO-1 gene was mapped to chromosome 22q12,12and a (GT)ndinucleotide repeat was identified in the proximal promoter region.13It is suggested that the (GT)nrepeat in HO-1 gene is highly polymorphic that could modulate the gene transcription. By using HO-1 promoter/luciferase reporter genes carrying different lengths of (GT)nrepeats, we previously demonstrated that the more (GT)nrepeats in promoter region, the less transcription of HO-1 gene in rat aortic smooth muscle cells.14The similar result was also shown earlier in Hep3B cells.33Recently, the role of microsatellite polymorphism of HO-1 gene promoter has been reported in some human diseases. It was shown that longer (GT)nrepeat was associated with emphysema in smokers.33Our recent study further demonstrated that type 2 diabetics carrying longer (GT)nrepeats may have higher oxidative stress and increased susceptibility to the development of CAD.14More recently, in concordant with the findings of present study, short GT repeats of HO-1 gene promoter was found to be associated with reduced restenosis at 6 months after percutaneous transluminal angioplasty in the femoro-popliteal arteries.34

It has been demonstrated that patients with small arteries and complex lesions presented a higher risk for an adverse outcome after CS because of a higher incidence of restenosis.35–38Restenosis after CS ranges from 35% to 67% in small coronary arteries,39,40and around 33.2% in complex lesions and 24.9% in simple lesions (P<0.001).38In the present study, the length polymorphism of GT repeat in HO-1 gene promoter is an independent risk factor for angiographic restenosis as well as adverse cardiac events after CS. However, such association was mainly in patients with small coronary arteries or those with complex lesions before stenting. In fact, small artery size is also known as one of the independent risk factors for angiographic stenosis as well as adverse cardiac events after CS. One of the possible mechanisms is that during coronary intervention, the process of lumen gain may require a higher degree of vessel stretch and injury in small rather than in large coronary artery.41Moreover, arterial medial disruption by stent struts could be associated with increased neointimal growth and inflammatory cell infiltration in vascular wall.42Accordingly, compared to the large ones, small coronary arteries may suffer more severe vascular injury during coronary intervention, which could then lead to more frequent restenosis after CS. It has been shown that upon vascular injury, the inducible CO production by HO-1 is important in vascular protection.6,23,43Since the long GT repeats in the promotor region could impair HO-1 gene transcription and consequently reduce vascular HO-1 and CO production, one may speculate that this polymorphism could be particularly important to stent restenosis while there is more severe vascular injury in small coronary arteries. On the other hand, it has also been shown that complex lesions were more prone to restenosis after CS as compared with the simple ones. The similar speculation about the importance of HO-1 gene in severe vascular injury could also be applied to the finding that microsatellite polymorphism in the promoter of HO-1 gene was associated with late restenosis mainly in patients with complex lesions rather than in those with simple ones. Further clinical studies are needed to validate the above issues.

In conclusion, this is the first study demonstrating that the microsatellite polymorphism in the promoter of HO-1 gene is related to angiographic restenosis as well as adverse cardiac events after CS, especially in patients with small coronary arteries or complex target lesions. These findings support the notion that genetic variation such as that in HO-1 gene may modify the long-term prognosis after CS, which provide important clinical implications to identify a subgroup of patients who with a special genetic background that might not benefit from stent implantation for their CAD. In these patients, alternative therapies including medical treatment and bypass surgery should be first considered.

Acknowledgments

This work was supported by grants from the Clinical Research Center of Institute of Biomedical Sciences, Academia Sinica; the National Science Council NSC 91-2314-B-010-059; the Taipei Veterans General Hospital VGH 91-190, VGH 91-256, and VTY 90-9p03, and the Yen Tjing-Ling Medical Foundation (CI-90-6-1), Taiwan.

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