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Human Molecular Genetics Advance Access originally published online on January 9, 2007
Human Molecular Genetics 2007 16(6):592-599; doi:10.1093/hmg/ddl483
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Polymorphisms in pro- and anti-inflammatory cytokine genes and susceptibility to atherosclerosis: a pathological study of 1503 consecutive autopsy cases

Kanae Oda1, Noriko Tanaka2, Tomio Arai3, Jungo Araki1, Yixuan Song1, Ling Zhang1, Aya Kuchiba2, Takayuki Hosoi4, Takuji Shirasawa5, Masaaki Muramatsu1,* and Motoji Sawabe3

1 Department of Molecular Epidemiology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan, 2 Department of Clinical Bioinformatics, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan, 3 Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo 173-0015, Japan, 4 Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Aichi 474-8511, Japan and 5 Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan

* To whom correspondence should be addressed. Tel: +81 03 5280 8060; Fax: +81 03 5280 8061; Email: muramatsu.epi{at}mri.tmd.ac.jp

Received October 28, 2006; Revised December 22, 2006; Accepted December 28, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 SUBJECTS AND METHODS
 REFERENCES
 
Atherosclerosis is a chronic inflammatory disease in the intima of the arterial wall, where cytokines play a crucial role in the pathogenesis of this disease. However, the question of whether or not genetic variations in the cytokine genes could influence the development of atherosclerosis has been poorly investigated. We investigated the relationship of nine common single-nucleotide polymorphisms (SNPs) in tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-1ß, IL-10, IL-4 and transforming growth factor (TGF)-ß1 with the atherosclerotic severity in 10 different arteries based on 1503 consecutive autopsies of elderly Japanese subjects registered in the Japanese SNPs for geriatric research (JG-SNP) study. The –1031C allele of TNF-{alpha} was a significant protective factor for atherogenesis in the carotid, femoral and intracranial arteries [odds ratio (OR): 0.72, 0.73 and 0.70, respectively]. The –511T of IL-1ß and the +29T of TGF-ß1 were significant risk factors for atherogenesis in the subclavian and intracranial arteries (OR: 1.35 and 1.48, respectively). In contrast, conventional risk factors for atherogenesis, such as hypertension and diabetes mellitus, conferred independent risks for almost all arteries. Functional SNPs in TNF-{alpha}, IL-1ß and TGF-ß1 genes play a role in atherogenesis, although their influences are less pronounced than those of conventional risk factors and appear to be limited to specific arteries in the Japanese elderly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 SUBJECTS AND METHODS
 REFERENCES
 
Atherosclerosis, which is one of the leading causes of ischemic heart disease and cerebrovascular disease, is now widely accepted to be a chronic inflammatory disease that is characterized by lipid accumulation in the arterial wall (1–5). In the early stage of the disease, immunocompetent cells, such as monocyte-derived macrophages and T-lymphocytes, infiltrate the arterial intima, and the immune responses evoked in the lesion may play a pivotal role in the progression of atherogenesis. The expression of pro-inflammatory cytokines is observed predominantly in atherosclerotic lesions, whereas the expression of anti-inflammatory cytokines in these lesions is rare (6). Thus, it is likely that both pro- and anti-inflammatory mediators produced by activated immunocompetent cells, endothelia and vascular smooth-muscle cells in the lesion are important for the development of atherosclerosis (7). However, it remains to be seen whether or not the genetic factors for these inflammation-related mediators underlie atherogenesis.

The heritability of atherosclerosis is around 40%, according to studies measuring the intima-media thickness, which is a clinical measure of atherosclerosis (8,9). Pathological investigations have also been carried out, such as the Helsinki Sudden Death Study (HSDS) and the Pathobiological Determinants of Atherosclerosis in Youth (PDAY). These studies investigated the autopsies of sudden death cases and put forward several candidate polymorphisms for atherosclerosis (10–16). However, there are very few reports about cytokine genetic polymorphisms.

To this end, we determined the relationship between single-nucleotide polymorphisms (SNPs) of pro- and anti-inflammatory cytokines and atherosclerotic severity in 10 different arteries based on 1503 consecutive autopsies of elderly Japanese subjects. We examined nine common functional SNPs in the cytokine genes that are known to be related to atherosclerosis: –1031T/C, –863C/A and –857C/T in the promoter of the tumor necrosis factor-{alpha} (TNF-{alpha}) gene, –511C/T in the promoter of the interleukin (IL)-1ß gene, –1082A/G, –819T/C and –592A/C in the promoter of the IL-10 gene, –589T/C in the promoter of the IL-4 gene, and C29T (with a proline to leucine amino-acid change in codon 10) in the transforming growth factor (TGF)-ß1 gene (7). These SNPs have been previously associated with many inflammatory diseases such as Crohn's disease (17), and with some atherosclerosis-related diseases such as myocardial infarction (18) and diabetes mellitus (19). However, the question of whether or not they are related to atherosclerosis itself has been poorly investigated. Only SNPs in the IL-10 genes, to our knowledge, have been shown to be associated with atherosclerotic renal artery stenosis in a small population (20) and with the carotid artery intima-media thickness in hemodialysis patients (21).

In the present study, we showed for the first time that functional SNPs in TNF-{alpha} (-1031T/C), IL-1ß (-511C/T) and TGF-ß1 (C29T) play a role in atherogenesis at specific arteries in the Japanese elderly. We also demonstrated that conventional risk factors, such as hypertension and diabetes mellitus, are associated with atherogenesis at almost all arteries, suggesting that the genetic variants would affect atherogenesis less than the conventional risk factors, at least in the elderly.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 SUBJECTS AND METHODS
 REFERENCES
 
Allele frequency and linkage disequilibrium
Tables 1 and 2 show the distributions of the allele and haplotype frequencies observed for the 1503 autopsy cases, respectively. The allele frequencies of the SNPs were similar to those previously reported in Japanese subjects or to the information in the database for Japanese SNPs (JSNP database) (22–26). All of the SNPs were in Hardy–Weinberg equilibrium. Strong linkage disequilibrium was observed between the –1031T/C and –863C/A SNPs of the TNF-{alpha} gene (r2 = 0.71, |D'| = 1) and between the –819T/C and –592A/C SNPs of the IL-10 gene (r2 = 1, |D'| = 1). These results are consistent with the results of previous studies on Japanese subjects (27,28).


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  		Table 1. Allele frequencies and primer pairs for the genotyping of each SNP

 


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  		Table 2. Haplotype frequencies of TNF-{alpha} and IL-10

 
Association of individual minor alleles of TNF-{alpha}, IL-1ß, IL-10 and TGF-ß1 with atherosclerotic severity
Table 3 shows the results of multiple logistic regression analysis for the individual minor alleles of TNF-{alpha}, IL-1ß, IL-4, IL-10 and TGF-ß1 and the atherosclerotic severity.


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  		Table 3. The results of multiple logistic regression analysis for the third quartile point of the degree of atherosclerosis and each SNP

 
The –1031C allele of TNF-{alpha}, the –511T allele of IL-1ß and the +29T allele of TGF-ß1 were found to be significantly associated with atherosclerotic severity in the intracranial, carotid and femoral arteries [P < 0.05; odds ratio (OR): 0.70, 0.72 and 0.73, respectively], the subclavian artery (P < 0.05; OR: 1.35), and the intracranial arteries (P < 0.05; OR: 1.48) (Table 3).

No significant association was observed between the –863A and –857T alleles of TNF-{alpha}, the –1082G, –819C and –592C alleles of IL-10, and the –589C allele of IL-4, and atherosclerotic severity. No SNPs that affect atherogenesis in the aorta, coronary, splenic, superior mesenteric and common iliac arteries were identified.

These results imply that the –1031C (TNF-{alpha}) SNP has an inhibitory effect on atherogenesis in each artery, while the –511T (IL-1ß) and +29T (TGF-ß1) SNPs promote atherogenesis.

Lack of association of TNF-{alpha} and IL-10 haplotypes with atherosclerotic severity
To analyze the association of the haplotypes of TNF-{alpha} and IL-10 with atherosclerotic severity in each artery, multiple logistic regression analyses were performed. The conventional risk factors of atherogenesis, i.e. gender, age (at death), drinking status, smoking status, and histories of diabetes mellitus, hypertension and hyperlipidemia, were included as explanatory variables. No significant association was observed between the haplotypes of TNF-{alpha} and IL-10 and atherosclerotic severity in any artery (data not shown).

Conventional risk factors and atherosclerotic severity
The results obtained using the conventional non-genetic risk factors of atherogenesis are summarized in Table 4. Overall, the conventional risk factors were confirmed to be independent risk factors for the atherosclerosis of systemic arteries, but our results also revealed that their effects differed somewhat from artery to artery. Aging is an independent high-risk factor for all arteries except the coronary, and smoking is an independent high-risk factor for all arteries except the coronary and the intracranial. Females are less susceptible to atherogenesis of the carotid and coronary arteries, while they are more susceptible to atherogenesis of the splenic artery. No significant association was observed between drinking status and atherosclerotic severity in any of the arteries.


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  		Table 4. Results of multiple logistic regression analysis for the third quartile point of the degree of atherosclerosis and the conventional risk factors

 
Hypertension was found to be an independent risk factor for the atherogenesis of all arteries (OR: 1.72–2.60), while diabetes mellitus and hyperlipidemia affected atherosclerotic severity in seven (OR: 1.42–1.97) and six (OR: 1.45–2.22) specific arteries, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
SUBJECTS AND METHODS
 REFERENCES
 
The view that atherosclerosis is a chronic inflammatory disease initiated by the adhesion of immunocompetent cells to the endothelium is verified by many experiments using cholesterol-fed animals, genetic engineering and histological methods. It is now well accepted that pro-inflammatory cytokines promote atherogenesis, and that anti-inflammatory cytokines or agents have the potential to prevent the complications of atherosclerosis (29–31).

In the present study, we investigated the genetic background of atherogenesis, focusing on its inflammatory aspects. We selected nine SNPs of five cytokine genes, all of which have been reported to be associated with altered gene function.

The –1031C allele of TNF-{alpha} was found to have significant protective effects on atherogenesis of the intracranial, carotid and femoral arteries. An in vitro study has shown that the –863A allele of TNF-{alpha}, which is in strong linkage disequilibrium with the variant at position –1031 of TNF-{alpha}, reduced the interaction with nuclear protein complexes in electromobility shift assays, and was associated with a 31% decrease in transcriptional activity in chloramphenicol acetyltransferase assays (32). The TNF-{alpha} gene is located in the human leukocyte antigen (HLA) class III region in the 6p21.3 band of the short arm of chromosome 6, and the –1031C/–863A allele has been reported to be in significant linkage disequilibrium with HLA-B61, -B39 and -DRB1*0901 (33). Although it is plausible that TNF-{alpha} gene variants are associated with inflammatory disease, we cannot exclude the possibility that these associations might be due to the polymorphisms in the adjacent HLA genes. Since our results show that a functional genetic variation that lowers the transcriptional activity of TNF-{alpha} has a significantly protective effect, we prefer to think that this might be the causative polymorphism.

The –511T allele of IL-1ß is associated with increased IL-1ß protein production, according to the results of an ex vivo blood stimulation assay (34). This supports our finding that the –511T allele is a risk factor for atherogenesis in the subclavian artery.

It has been reported that the homozygote for C in the +29 allele of TGF-ß1 is associated with higher mRNA expression, quantified by means of TGF-ß1 gene-specific competitor constructs in competitive PCR (35), and also by means of enzyme-linked immunosorbent assay (ELISA) with higher serum concentrations of TGF-ß1 (18). Although TGF-ß1 has also been reported to have pro-atherogenic properties through its role in vascular remodeling (36,37), our results reveal that the +29T allele is a risk factor for atherogenesis of the intracranial arteries. This might be due to the fact that the anti-inflammatory activity of TGF-ß1 is more preponderant than the pro-atherogenic property of TGF-ß1 in the formation of atherosclerosis in intracranial arteries.

Therefore, overall, we detected some SNPs that are related to atherogenesis. In general, pro-inflammatory alleles confer risk and anti-inflammatory alleles are protective, although their influence is selective and limited to specific arteries. The reason why inflammation-related SNPs confer site-specific atherogenic risks is difficult to explain in a straightforward manner. However, our pathological observation revealed that individual differences in atherosclerotic severity at each artery have a wide range of variety. For example, one subject suffered from atherosclerosis only at the aortic and femoral arteries, while another's was confined to the coronary arteries. The anatomy of the vessels, blood flow and shear stress regulate the expression of specific genes in the endothelium, especially those encoding cell-adhesion and inflammation-related molecules (38,39), and they are thought to contribute to such differences. Along the same lines, a previous report from the Honolulu heart study has shown that risk factors for the coronary arteries and aorta have different components (40). Recently, Vanderlaan et al. (41) have proposed that regional differences in the hemodynamic profile prime the endothelial phenotype to respond distinctly to systemic risk factors, such as hypercholesterolemia, genetics, immune status, gender and oxidative stress. We speculate that cytokine polymorphisms interact with these artery-specific atherogenic factors and enhance atherosclerosis. If this hypothesis does not hold, then an unknown mechanism may underlie the involvement of cytokines in the inflammation of the arteries in a site-specific manner. It seems worthwhile to investigate how local differences influence site-specific atherogenesis. In particular, our results raise the possibility that the development of atherosclerosis in the coronary arteries is more affected by aging, smoking at the early stage of the disease, and being female than the development of atherosclerosis in other arteries. In fact, the coronary arteries have been shown to be the first sites of atherosclerotic involvement among the systemic arteries (42). Therefore, further investigations are needed to evaluate the factors that affect the progression of coronary atherosclerosis in younger populations.

We also evaluated the association between the conventional risk factors of atherosclerosis and atherosclerotic severity in each artery. Advanced age and smoking were associated with atherosclerotic severity in all arteries, except the coronary and the coronary and intracranial, respectively. Hypertension was associated with atherosclerotic severity in all arteries, while diabetes mellitus and hyperlipidemia affected it in most, if not all, of the arteries, which is consistent with the fact that these are bona fide risk factors. Generally, the conventional risk factors showed higher ORs than the genetic variants, and their influence tended to be systemic (Table 4).

In the present study, we demonstrated that functional SNPs in TNF-{alpha} (–1031T/C), IL-1ß (–511C/T) and TGF-ß1 (C29T) play a role in atherogenesis, although their influence was less than that of systemic diseases such as hypertension and diabetes, as well as smoking and aging, and limited to specific arteries. Clearly, the subjects of our study are elderly persons who escaped from fatal cardiovascular diseases, and environmental factors would be more influential. Because the observed ORs of the genetic variants were close to 1, it is possible that they were mere chance observations. Nevertheless, even after excluding the risk-allele-bearing subjects, we could detect associations between genetic variants and atherosclerosis. We believe that our results point out the importance of inflammation-related genetic diversity in atherosclerosis. However, factors other than those found in this study must also be involved. Since progress in understanding the genetic factors of atherogenesis will enable us to develop preventive and therapeutic strategies for atherosclerosis, we intend to expand our investigation of the relationship between genes and atherosclerosis, especially with respect to the inflammatory aspects, by increasing the number of subjects and polymorphisms in future studies.

Limitation of the study
The subjects of this study were autopsy cases of patients in a community-based geriatric hospital. Therefore, our approach had two limitations. The first is the selection bias from chance of admission, cause of death and autopsy practice. The rate of autopsy in this hospital was ~40%. Those who died suddenly outside the hospital and were medicolegal cases were not autopsied for this study. According to the 2001 population survey report of Japan (http://www.dbtk.mhlw.go.jp/toukei/data/010/2001/toukeihyou/0003845/t0066001/c080_001.html), more than 81% of elderly persons (70–89 years) die in a hospital, whereas 12% die at home. Although the rate of in-hospital death for the elderly is high, this might be a minor confounding factor for the data. We therefore compared the direct causes of death between our subjects and the census data in ‘Abridged Life Tables For Japan 2003’ by the Ministry of Health, Labour and Welfare of Japan (http://www.mhlw.go.jp/english/database/db-hw/lifetb03/index.html). Although almost all death rates for major diseases per 100 000 between 80 and 84 years of age in Tokyo, Japan were consistent with our autopsy data, those for malignancy and cerebrovascular disease were ~15% lower and 10% higher, respectively, than our autopsy data. Hasuo et al. compared the death certificates issued without knowledge of the autopsy findings with pathological reports of autopsy cases originating from the Hisayama study (43). They indicated that cerebral stroke and cardiac disease tended to be overdiagnosed, whereas malignant neoplasms were underdiagnosed. Thus, the subjects in our study did not differ greatly from the standard elderly residents of Tokyo, Japan. Second, since post-mortem blood samples were not collected, we cannot check the inflammatory markers at death, but had to rely on patients' clinical charts retrospectively to obtain available clinical information.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 SUBJECTS AND METHODS
REFERENCES
 
Subjects
The subjects were 1503 consecutive autopsy cases of elderly patients who had suffered in-hospital death. The autopsies were performed at the Tokyo Metropolitan Geriatric Hospital in Tokyo, Japan from 1995 to 2004 (men: 696; women: 807; mean age at death: 80.3 ± 8.9 years) (Table 5). The subjects were registered in the Internet-based database of Japanese SNPs for geriatric research (the JG-SNP) (http://www.tmgh.metro.tokyo.jp/jg-snp/english/E_top.html) (44). The major clinical diagnoses and direct cause of death in this population are summarized in Table 5 (see the JG-SNP for detailed information). The direct cause of death was determined based on the descriptions given in the death certificate issued after the autopsy. The details of the underlying disease, history of smoking and drinking, serum lipid data and presence of atherosclerotic complications were obtained from the clinical records of the patients.


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  		Table 5. The characteristics of the subjects (N = 1503)

 
Written informed consent was obtained from the bereaved families of the patients at the time of autopsy. This study was approved by the ethical committees of the Tokyo Metropolitan Geriatric Hospital and the Tokyo Medical and Dental University.

Pathological assessment of atherosclerosis
Macroscopic evaluation of the degree of atherosclerosis was performed for the following large- and medium-sized arteries: the carotid, subclavian, splenic, superior mesenteric, common iliac, external iliac, femoral, coronary and intracranial, as well as the aorta.

We adopted the method of pathological assessment of atherosclerosis reported previously by us (45–47). For eight arteries, i.e. the carotid, subclavian, splenic, superior mesenteric, common iliac, external iliac, femoral and the aorta, the atherosclerotic severity is scored according to the ratio of the occupying atheroma to the entire intimal area, while for the coronary and intracranial arteries, it is the sum of stenotic scores of their respective branches. At a medium-size artery, such as the coronary or intracranial, the stenotic degree is clinically valuable, and basically parallels the atherosclerotic severity as the ratio of the occupying atheroma. On the other hand, at a large-size artery such as the aorta, the stenotic degree is not useful, and is evaluated conventionally based on the ratio of the occupying lesion. Although we evaluated atherosclerosis by means of two pathological methods, together they both indicate the degree of atherosclerosis.

Selected SNPs and genotyping using melting curve analysis
We selected nine SNPs in five genes that are considered to be relevant to atherosclerosis, especially with respect to the inflammatory aspect, i.e. –1031T/C, –863C/A and –857C/T in the promoter region of the TNF-{alpha} gene, –511C/T in the promoter of the IL-1ß gene, –1082A/G, –819T/C and –592A/C in the promoter of the IL-10 gene, –589T/C in the promoter of the IL-4 gene, and C29T (with a proline to leucine amino-acid change in codon 10) in the TGF-ß1 gene.

DNA samples were extracted from the renal cortex by the phenol/chloroform method, and 10-ng DNA aliquots were used for the genotyping reaction. The PCR primer pairs used in the study are summarized in Table 1. The PCR reactions and melting curve analyses carried out for the purpose of detecting polymorphisms in TNF-{alpha} (–1031T/C, –863C/A and –857C/T), IL-1ß (–511C/T), IL-10 (–1082A/G, –819T/C and –592A/C), IL-4 (–589T/C) and TGF-ß1 (C29T) were performed using the LightTyper (Roche Diagnostics) according to the protocols previously reported by us (48). The rates of genotyping error were 0.3–3.5%. The pathological assessment and genotyping were done at different facilities in double-blind fashion.

Statistical analysis
The Hardy–Weinberg equilibrium of the alleles at individual loci was assessed using Fisher's exact test. Lewontin's D prime (D') and correlation coefficient (r2) were calculated as two measures of linkage disequilibrium (LD) between SNPs.

As shown in Figure 1, the distributions of the atherosclerotic severity differed greatly from artery to artery: some were continuous, while others were discrete. For the sake of unity of the analysis methods, we considered all arteriosclerosis degrees as discrete variables by dividing them into upper and lower than each 75% point (shown by dashed lines at each artery). For the arteries whose distributions seemed to be continuous, we also analyzed them as continuous variables by means of multiple regression analysis, but the results were the same as those of the analysis using discrete variables (data not shown). To identify the SNPs that affect atherogenesis, we determined their relationships according to the genotypes in a dominant model for each minor allele. All of the variables of atherosclerotic severity, minor allele and haplotype distributions were categorized on a scale of 0 or 1, depending on whether they were lower or higher than each 75% point, and whether or not each minor allele or haplotype was absent or present, respectively. The conventional risk factors of age (at death), gender, drinking status and smoking status, as well as histories of hypertension, diabetes mellitus and hyperlipidemia, were also categorized as follows: gender (male versus female), years of age at death (60 versus 90, 70 versus 90, 80 versus 90), and histories of smoking, drinking, hypertension, diabetes mellitus and hyperlipidemia (absent versus present) as 0 versus 1, respectively.


Figure 1
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  		Figure 1. Distributions of atherosclerotic severity in individual arteries. These graphs show the distributions of atherosclerotic severity in the individual arteries. The Y-axis is the atherosclerotic severity and the X-axis is the number of subjects. The dashed line indicates the 75% point of each distribution. These graphs show that the atherosclerotic severity differs greatly from artery to artery.

 
Multiple logistic regression analyses were performed to assess the associations of individual minor SNPs and haplotypes with the risk of atherosclerosis, with adjustment for the conventional risk factors. Using the EM algorithm, we estimated the frequencies in this population of the four and three most common (>1% frequency) haplotypes of TNF-{alpha} and IL-10, respectively, and imputed the subject-specific expected haplotypes.

The statistical analyses were performed using the SAS 9.1.3 statistical software. The subject-specific haplotypes were estimated using the existing procedure in SAS/GENETICS version 9.1.3. In general, association studies involving many genes have the problem of multiple statistical comparisons. This study, however, was a hypothesis-generative search, and the groups with severe atherosclerosis differed greatly from artery to artery (Fig. 1), so we considered two-tailed P-values <0.05 to be statistically significant with no correction.


   ACKNOWLEDGEMENTS
 
This work was partly supported by a Grant-in-Aid from the Comprehensive Research on Aging and Health (No. 16090101) project, under the aegis of the Ministry of Health, Labor and Welfare, Japan, and by a Japan Heart Foundation Grant for Research on Arteriosclerosis Update.

Conflict of Interest statement. The authors hereby declare that there is no financial conflict of interest.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 SUBJECTS AND METHODS
 REFERENCES
 

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