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C Polymorphism of the Interleukin-6 Gene Promoter is Associated with Peripheral Artery Occlusive Disease
Eur J Vasc Endovasc Surg 24, 264±268 (2002) doi:10.1053/ejvs.2002.1711, available online at http://www.idealibrary.com on
The ÿ174 G/C Polymorphism of the Interleukin-6 Gene Promoter is Associated with Peripheral Artery Occlusive Disease A. Flex1,2 , E. Gaetani1,2 , R. Pola1,3 , A. Santoliquido4 , F. Aloi1, P. Papaleo2 , A. Dal Lago2, E. Pola1, M. Serricchio2, P. Tondi4 and P. Pola1,2,4 1
Laboratory of Vascular Biology & Genetics, 2Department of Internal Medicine & Angiology, 3Department of Geriatric Medicine and 4Division of Instrumental Vascular Medicine, A. Gemelli University Hospital, UniversitaÁ Cattolica del Sacro Cuore School of Medicine, Rome, Italy
Objective and design: high plasma levels of Interleukin-6 (IL-6) are found in patients with atherosclerotic disorders. Recently, a common polymorphism of the IL-6 gene promoter, influencing the transcription rate of the gene, has been described and associated with atherosclerosis of carotid and coronary arteries. The objective of this study was to test whether IL-6 gene promoter polymorphism is associated with peripheral artery occlusive disease (PAOD) in a case-control study. Methods: IL-6 gene promoter polymorphism was evaluated by polymerase chain reaction followed by restriction enzyme analysis in 84 patients affected by PAOD and 183 controls. Results: the distribution of IL-6 genotypes was: patients with PAOD: 44 GG, 30 GC, 10 CC; control subjects: 53 GG, 80 GC, 50 CC. The GG genotype was significantly more common in the PAOD group (p 5 0.0001), while the CC genotype was significantly more common in control patients (p 0.005). Conclusions: this study indicates a strong association between IL-6 gene polymorphism and PAOD and support the hypothesis that IL-6 and IL-6 gene polymorphism are important in the pathophysiology and evolution of ischaemic diseases of the lower limbs. Key Words: Interleukin-6; Gene polymorphism; Peripheral artery occlusive disease.
Introduction Several observations hint at the role of inflammatory mechanisms in the development, maturation and destabilisation of atherosclerotic plaque.1±4 Interleukin-6 (IL-6) is a multifunctional cytokine produced by several cell types, including fibroblasts, monocytes, adipocytes, and endothelial cells, which plays a key role in driving the acute inflammatory response, and orchestrates the production of acute phase proteins.5 IL-6 may induce endothelial activation6 or stimulate fibrinogen synthesis,7 which may itself be directly pathogenic to the endothelium. Elevated levels of IL-6 are found in patients with carotid atherosclerosis1 and coronary syndromes8 and have been associated with increased risk of cerebral ischaemia9 and future myocardial infarction in an apparently healthy population.10 Recently, a common genetic polymorphism has been reported in the ÿ174 promoter region of the IL-6 Please address all correspondence to: A. Flex, Istituto di Patologia Speciale Medica, A. Gemelli University Hospital, UniversitaÁ Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy.
gene.11 Two different alleles can be identified (G and C allele), resulting in three possible genotypes, GG, GC, and CC. This polymorphism is functionally important, since it influences the transcription rate of the gene and the plasma concentration of IL-6 protein appears to be influenced by the G/C genotype.11±13 Interestingly, both carotid atherosclerosis and coronary artery disease (CAD) have been associated with the ÿ174 G/C polymorphism of the IL-6 gene promoter.14 The aim of this study was to test the hypothesis that the IL-6 gene polymorphism is associated with peripheral artery occlusive disease (PAOD). We analysed the distribution of IL-6 genotypes and alleles in a case-control study including 84 PAOD patients and 183 control subjects. Methods Subjects Both patients and controls were recruited among subjects consecutively admitted to the Department of
1078±5884/02/030264 05 $35.00/0 # 2002 Elsevier Science Ltd. All rights reserved.
IL-6 Gene Polymorphism and PAOD
Internal Medicine and Angiology of the A. Gemelli University Hospital of Rome, from 1 December 2000 to 30 November 2001. Inclusion criteria for the PAOD group were Caucasian race and presence of PAOD. Diagnosis of PAOD was performed in accordance with the criteria established by the Ad Hoc Committee on Reporting Standards of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery.15 All patients had an ankle/arm pressure index lower than 0.8 and were at Fontaine's stage II, with intermittent claudication and no rest pain or trophic lesions. Inclusion criteria for the control group were Caucasian race, absence of PAOD, and no relationship with cases. All control subjects presented an ankle/arm pressure index 1 and normal findings on bilateral high-resolution Bmode ultrasonography evaluation (Echocolor Doppler Acuson 128XP/10, Acuson, Mountain View, CA, U.S.A., with an 4 MHz transducer). Exclusion criteria from the study were tumours, chronic inflammatory diseases, and autoimmune diseases. At the end of the recruitment period, a total of 84 PAOD patients and 183 controls were enrolled. All subjects belonged to independent pedigrees. Informed consent was obtained from all patients. The study protocol was accepted by the Ethics Committee of our University Hospital. Sample preparation and PCR detection of IL-6 DNA polymorphism Nucleic acid isolation from 200 ml of peripheral blood was carried out by using a DNA extraction kit (NucleoSpin Blood QuickPure, Macherey-Nagel, Duren, Germany), as recommended by the supplier. Genomic DNAs were assayed with polymerase chain reaction (PCR) for the detection of IL-6 gene, using the published primer set: 50 -TGACTTCAGCTTTACTCTTTGT-30 (sense primers) and 50 -CTGATTGGAAACCTTATTAGG-30 (antisense primers). Briefly, PCR reaction, containing 0.2 mmol/L of each primer, 0.2 mmol/L of dNTPs (Boehringer GmbH, Mannheim, Germany), 1.5 mmol/L MgCl2, and 2.5 U of AmpliTaq polymerase (Perkin-Elmer, Cetus, Norwalk, CT, U.S.A.) in a final volume of 20 ml, was performed in a GeneAmp PCR System 9700 (Perkin±Elmer) with an initial denaturation step of 10 min at 94 C and a final extension step of 10 min at 72 C. The following thermal profile was repeated for 35 cycles: denaturation for 1 min at 94 C, annealing for 1 min and 35 s at 55 C and extension for 1 min at 72 C. The amplified sequence was digested by SfaNI restriction enzyme (New England BioLabs, Beverly, MA,
265
U.S.A.) at 37 C overnight. The digested products were electrophoresed in 2% agarose gel and visualised by ethidium bromide staining. The GG genotype corresponded to the contemporary presence of 140 and 58 bp fragments. The GC genotype corresponded to the contemporary presence of 198, 140, and 58 bp fragments. The CC genotype corresponded to a 198 bp fragment. Statistical analysis Demographic and clinical data between groups were compared by t-test and Chi-squared test. Genotype and allele ratio distribution between groups was analysed by Chi-squared test. A logistic regression analysis was performed to estimate the association between genotype and PAOD. Odds ratios were calculated with 95% confidence interval (CI). Analyses were done with Intercooled STATA 6.0 for Windows (Statistics/Data Analysis, Stata Corporation, College Station, TX, U.S.A.). Statistical significance was established at p 5 0.05. Results Demographic and clinical characteristics of patients and controls are reported in Table 1. Diabetes and abuse of tobacco were significantly more frequent in patients than controls (p 0.001 and p 5 0.0001, respectively). Hypertension and gender ratio were also close to being significantly different. Other risk factors for PAOD, such as hypercholesterolaemia, history of coronary artery disease (CAD), stroke or transient ischaemic attacks (TIA) of the brain, were not differently distributed between patients and controls. The distribution of IL-6 genotypes and the allele ratio in cases and controls is shown in Table 2. Genotypes were in Hardy-Weinberg equilibrium. The genotype distribution was 44 GG, 30 GC, 10 CC in the 84 patients with PAOD and 53 GG, 80 GC, 50 CC in Table 1. Demographic and clinical data in PAOD patients and controls.
Age (years SD) Male gender Hypertension Hypercholesterolaemia Diabetes History of CAD Stroke/TIA Smoking
Patients (n 84)
Controls (n 183)
75 7 51 (61%) 59 (70%) 25 (30%) 39 (46%) 33 (39%) 22 (26%) 55 (65%)
76 7 89 (49%) 108 (59%) 59 (32%) 40 (22%) 55 (30%) 44 (24%) 60 (33%)
p 0.41 0.06 0.06 0.68 0.001 0.13 0.70 50.0001
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Table 2. IL-6 genotype distribution and allele ratio between groups. Patients
Controls
All GG GC CC G/C ratio
n 84 44 (52%) 30 (36%) 10 (12%) 2.3
n 183 53 (29%) 80 (56%) 50 (27%) 1.0
50.0001 0.23 0.005 50.0001
Males GG GC CC G/C ratio
n 51 28 (55%) 17 (33%) 6 (12%) 2.5
n 89 32 (36%) 34 (38%) 23 (26%) 1.2
0.029 0.56 0.004 50.0001
Females GG GC CC G/C ratio
n 33 16 (49%) 13 (39%) 4 (12%) 2.1
n 94 21 (22%) 46 (49%) 27 (29%) 0.9
0.004 0.34 0.05 0.003
p
Table 3. Risk factors for PAOD based on logistic regression analysis.
G/G genotype G/C genotype Diabetes Sex M Age Hypertension Hypercholesterolaemia History of CAD Stroke/TIA Smoking
Odds ratio
95% CI
4.6 1.7 3.1 0.8 0.9 1.7 1.0 1.7 0.6 5.7
1.9±11.04 0.74±4.22 1.7±9.34 0.44±1.59 0.95±1.02 0.94±3.36 0.55±2.03 0.94±3.40 0.34±1.40 2.47±13.1
p 0.001 0.197 0.007 0.607 0.598 0.074 0.855 0.073 0.311 50.0001
the 183 control subjects. The frequency of the GG genotype in patients with PAOD (52%) was significantly higher than in controls (29%) (p 5 0.0001). In contrast, the frequency of the CC genotype was more than two times higher in controls than in subjects with PAOD (27 vs 12%, p 0.005). Likewise, allele ratio was significantly different between the two groups: the ratio between the G and C allele frequency was 2.3 in patients with PAOD and 1.0 in controls (p 5 0.0001). In order to evaluate whether the distribution of IL-6 genotypes was influenced by sex, male and female subjects were separately analysed (Table 2). The same trend was observed in both men and women: the incidence of GG genotype was significantly higher in men, as well as in women affected by PAOD (p 0.029 and p 0.004, respectively). The role of the GG genotype in the development of PAOD was then investigated by logistic regression analysis (Table 3). We found that the GG genotype is an independent risk for PAOD in our population and that subjects carrying the GG genotype have a risk more than four times higher than CC homozygous Eur J Vasc Endovasc Surg Vol 24, September 2002
individuals to be affected by the disease [Odds ratio 4.6 (95% CI 1.9±11.04), p 0.001]. Discussion The initiation and progression of atherosclerosis involve vessel conformation, haemodynamic forces, exposure to multiple risk factors, cellular composition, and inflammation.4,16 The endothelium expresses procoagulant, instead of anticoagulant, properties and produces vasoactive molecules, inflammatory cytokines, and growth factors. This leads to the accumulation and activation of macrophages and lymphocytes within the plaque. Activation of these inflammatory cells leads to the further release of hydrolytic enzymes, cytokines, chemokines, and growth factors,17 inducing damage and eventually leading to focal necrosis.18 IL-6 is a prototypical inflammatory cytokine able to damage the endothelium and initiate atherosclerosis.19 Immunohistochemical studies of the human arterial atherosclerotic wall have demonstrated that IL-6 is expressed in the cellular and extracellular deposits in the connective tissue matrix and that atherosclerotic plaques have a significantly high level of IL-6.20 Studies in ApoE-knockout mice, that spontaneously develop systemic atherosclerosis, also show elevated levels of IL-6 mRNA in the plaque area compared with normal mice.21 These data are consistent with the observation of elevated levels of IL-6 in patients with coronary syndromes and atherosclerosis-related diseases.8,22±24 Interestingly, plasma levels of IL-6 are influenced by the ÿ174 G/C polymorphism of the IL-6 gene promoter.11 Recent studies have investigated the possible clinical significance of this polymorphism and analysed its association with atherosclerotic and ischaemic diseases, including carotid atherosclerosis,19 CAD,14 and abdominal aortic aneurysm.13 However, no studies have been performed in order to test whether the G/C IL-6 gene polymorphism is associated with atherosclerosis of the peripheral circulation of lower limbs. Our study shows for the first time that the distribution of IL-6 genotypes and alleles is different between patients affected by PAOD and controls. Indeed, the frequency of the GG genotype and G allele is significantly higher in PAOD patients. In contrast, control subjects exhibit a significantly higher frequency of the CC genotype and C allele. When male and female patients were separately analysed, a similar distribution of IL-6 genotypes and alleles was found, demonstrating that the G/C polymorphism of the IL-6 gene is involved in the pathophysiology of PAOD in both men and
IL-6 Gene Polymorphism and PAOD
women. We also show, by using logistic regression analysis, that subjects carrying the G allele have increased risk of PAOD. In particular, subjects GG homozygous have a risk 4.6 times higher to develop the disease, when compared to CC homozygous patients. Taken together, our data are consistent with the hypothesis that the ÿ174 G/C polymorphism of the IL-6 gene promoter is relevant for the pathogenesis and/or evolution of systemic atherosclerosis. Several studies have focused on the measurement and detection of the IL-6 protein in serum or tissue. However, IL-6 protein levels are influenced by many pathologic and physiologic conditions, such as infections, autoimmune disorders, malignancies, trauma, ischaemia, and drug treatment.25±28 Therefore, a single measurement might be not predictive of disease in a given individual. For this reason, our study focuses on the evaluation of a polymorphism that genetically determines the plasmatic levels of IL-6.11±13 This study has some potential limitations. In particular, it is a case-control study and a possible survival bias cannot be excluded for the patients with PAOD. In addition, the size of the studied population is relatively small and our findings need to be confirmed in larger samples. Likewise, the association between IL-6 gene polymorphism and PAOD should be tested in groups of different ethnic origin. Finally, we cannot exclude a role played by a gene or by several genes in linkage disequilibrium with the IL-6 gene, nor that a variant of an other gene, closely linked to the IL-6 gene, causes the observed association. In particular, there is strong linkage disequilibrium between the IL-6 gene promoter polymorphism and the variable number of tandem repeat polymorphism of the 30 flanking region of the IL-6 gene.29 In conclusion, this study evaluates for the first time the association between PAOD and IL-6 gene promoter polymorphism and shows that the GG genotype and the G allele are significantly more frequent in patients affected by PAOD. Although further investigations are needed, our data support the hypothesis that the IL-6 G/C polymorphism is important in the pathophysiology of clinically relevant atherosclerotic disorders.
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References 1 Milei J, Parodi JC, Fernandez Alonso G et al. Carotid Atherosclerosis. Immunocytochemical analysis of the vascular and cellular composition in endarterectomies. Cardiologia 1996; 41: 535±542. 2 van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thromboses coronary atherosclerotic plaques is characterized by an inflammatory process
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irrespective of the dominant plaque morphology. Circulation 1994; 89: 36±44. Buja LM, Willerson JT. Role of inflammation in coronary plaque distruption. Circulation 1994; 89: 503±505. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340: 115±126. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990; 265: 621±636. Romano M, Sironi M, Toniatti C et al. A. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997; 6: 315±325. Dalmon J, Laurent M, Courtois G. The human beta fibrinogen promoter contains a hepatocyte nuclear factor 1-dependent interleukin-6-responsive element. Mol Cell Biol 1993; 13: 1183±1193. Biasucci LM, Vitelli A, Liuzzo G et al. Elevated levels of interleukin-6 in unstable angina. Circulation 1996; 94: 874±847. Grau AJ, Aulmann M, Lichy C et al. Increased cytokine release by leucocytes in survivors of stroke at young age. Eur J Clin Invest 2001; 31: 999±1006 Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 2000; 101: 1767±1772. Fishman D, Faulds G, Jeffery R. The effect of novel polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic onset juvenile chronic arthritis. J Clin Invest 1998; 102: 1369±1376. Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin-6 transcriptional regulation. J Biol Chem 2000; 275: 18138±18144. Jones KG, Brull DJ, Brown LC, Sian M et al. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation 2001; 103: 2260±2265. Humphries SE, Luong LA, Ogg MS, Hawe E, Miller GJ. The interleukin-6 -174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J 2001; 22: 2243±2252 Rutherford RB, Flanigan DP, Gupta SK, and the Ad Hoc Committee on Reporting Standards SVS/ISCVS. Suggested standards for reports dealing with lower extremity ischaemia. J Vasc Surg 1986; 4: 80±94. Ross R. The pathogenesis of atherosclerosis: a prospective for the 1990s. Nature 1993; 362: 801±809. Libby P, Ross R. Cytokines and growth regulatory molecules. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Vol. 1. Philadelphia: Lippincott-Raven, 1996: 585±594. Falk E, Shan PK, Fuster V. Pathogenesis of plaque distruption. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Vol. 2. Philadelphia:lippincott.Raven 1996: 492±510. Rauramaa R, Vaisanen SB, Luong LA et al. Stromelysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol 2000; 20: 2657±2662. Rus HG, Vlaicu R, Niculescu F. Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis 1996; 127: 263±271. Sukovich DA, Kauser K, Shirley FD et al. Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17û-estradiol. Arterioscler Thromb Vasc Biol 1998; 18: 1498±1505. Miyao Y, Yasue H, Ogawa H et al. Elevated plasma interleukin6 levels in patients with acute myocardial infarction. Am Heart J 1993; 126: 1299±1304. Guillen I, Blanes M, Gomez-Lechon MJ, Castell JV. Cytokine signalling during myocardial infarction: sequential appearance of IL-1 beta and IL-6. Am J Physiol 1995; 269: R229±R235. Biasucci LM, Liuzzo G, Fantuzzi G et al. Increasing levels of interleukin (IL)-1Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased Eur J Vasc Endovasc Surg Vol 24, September 2002
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risk of in-hospital coronary events. Circulation 1999; 99: 2079±2084. 25 Van Snick J. Interleukin-6: an overview. Ann Rev Immunol 1990; 8: 253±278. 26 Papanicolau DA, Wilder RL, Manolagas SC, Chrousos GP. The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 1998; 128: 127±137. 27 Marian AJ. On genetics, inflammation, and abdominal aortic aneurysm. Circulation 2001; 103: 2222±2224.
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28 Ikonomidis I, Andreotti F, Economou E et al. Increased proinflammatory cytokines in patients with chronic stable angina and their reduction by aspirin. Circulation 1999; 100: 793±798. 29 Bagli M, Papassotiropoulos A, Knapp M et al. Association between an interleukin-6 promoter and 30 flanking region haplotype and reduced Alzheimer's disease risk in a German population. Neurosci Lett 2000; 283: 109±112. Accepted 17 May 2002
The ÿ174 G/C Polymorphism of the Interleukin-6 Gene Promoter is Associated with Peripheral Artery Occlusive Disease A. Flex1,2 , E. Gaetani1,2 , R. Pola1,3 , A. Santoliquido4 , F. Aloi1, P. Papaleo2 , A. Dal Lago2, E. Pola1, M. Serricchio2, P. Tondi4 and P. Pola1,2,4 1
Laboratory of Vascular Biology & Genetics, 2Department of Internal Medicine & Angiology, 3Department of Geriatric Medicine and 4Division of Instrumental Vascular Medicine, A. Gemelli University Hospital, UniversitaÁ Cattolica del Sacro Cuore School of Medicine, Rome, Italy
Objective and design: high plasma levels of Interleukin-6 (IL-6) are found in patients with atherosclerotic disorders. Recently, a common polymorphism of the IL-6 gene promoter, influencing the transcription rate of the gene, has been described and associated with atherosclerosis of carotid and coronary arteries. The objective of this study was to test whether IL-6 gene promoter polymorphism is associated with peripheral artery occlusive disease (PAOD) in a case-control study. Methods: IL-6 gene promoter polymorphism was evaluated by polymerase chain reaction followed by restriction enzyme analysis in 84 patients affected by PAOD and 183 controls. Results: the distribution of IL-6 genotypes was: patients with PAOD: 44 GG, 30 GC, 10 CC; control subjects: 53 GG, 80 GC, 50 CC. The GG genotype was significantly more common in the PAOD group (p 5 0.0001), while the CC genotype was significantly more common in control patients (p 0.005). Conclusions: this study indicates a strong association between IL-6 gene polymorphism and PAOD and support the hypothesis that IL-6 and IL-6 gene polymorphism are important in the pathophysiology and evolution of ischaemic diseases of the lower limbs. Key Words: Interleukin-6; Gene polymorphism; Peripheral artery occlusive disease.
Introduction Several observations hint at the role of inflammatory mechanisms in the development, maturation and destabilisation of atherosclerotic plaque.1±4 Interleukin-6 (IL-6) is a multifunctional cytokine produced by several cell types, including fibroblasts, monocytes, adipocytes, and endothelial cells, which plays a key role in driving the acute inflammatory response, and orchestrates the production of acute phase proteins.5 IL-6 may induce endothelial activation6 or stimulate fibrinogen synthesis,7 which may itself be directly pathogenic to the endothelium. Elevated levels of IL-6 are found in patients with carotid atherosclerosis1 and coronary syndromes8 and have been associated with increased risk of cerebral ischaemia9 and future myocardial infarction in an apparently healthy population.10 Recently, a common genetic polymorphism has been reported in the ÿ174 promoter region of the IL-6 Please address all correspondence to: A. Flex, Istituto di Patologia Speciale Medica, A. Gemelli University Hospital, UniversitaÁ Cattolica del Sacro Cuore, L.go A. Gemelli 8, 00168 Rome, Italy.
gene.11 Two different alleles can be identified (G and C allele), resulting in three possible genotypes, GG, GC, and CC. This polymorphism is functionally important, since it influences the transcription rate of the gene and the plasma concentration of IL-6 protein appears to be influenced by the G/C genotype.11±13 Interestingly, both carotid atherosclerosis and coronary artery disease (CAD) have been associated with the ÿ174 G/C polymorphism of the IL-6 gene promoter.14 The aim of this study was to test the hypothesis that the IL-6 gene polymorphism is associated with peripheral artery occlusive disease (PAOD). We analysed the distribution of IL-6 genotypes and alleles in a case-control study including 84 PAOD patients and 183 control subjects. Methods Subjects Both patients and controls were recruited among subjects consecutively admitted to the Department of
1078±5884/02/030264 05 $35.00/0 # 2002 Elsevier Science Ltd. All rights reserved.
IL-6 Gene Polymorphism and PAOD
Internal Medicine and Angiology of the A. Gemelli University Hospital of Rome, from 1 December 2000 to 30 November 2001. Inclusion criteria for the PAOD group were Caucasian race and presence of PAOD. Diagnosis of PAOD was performed in accordance with the criteria established by the Ad Hoc Committee on Reporting Standards of the Society for Vascular Surgery and the International Society for Cardiovascular Surgery.15 All patients had an ankle/arm pressure index lower than 0.8 and were at Fontaine's stage II, with intermittent claudication and no rest pain or trophic lesions. Inclusion criteria for the control group were Caucasian race, absence of PAOD, and no relationship with cases. All control subjects presented an ankle/arm pressure index 1 and normal findings on bilateral high-resolution Bmode ultrasonography evaluation (Echocolor Doppler Acuson 128XP/10, Acuson, Mountain View, CA, U.S.A., with an 4 MHz transducer). Exclusion criteria from the study were tumours, chronic inflammatory diseases, and autoimmune diseases. At the end of the recruitment period, a total of 84 PAOD patients and 183 controls were enrolled. All subjects belonged to independent pedigrees. Informed consent was obtained from all patients. The study protocol was accepted by the Ethics Committee of our University Hospital. Sample preparation and PCR detection of IL-6 DNA polymorphism Nucleic acid isolation from 200 ml of peripheral blood was carried out by using a DNA extraction kit (NucleoSpin Blood QuickPure, Macherey-Nagel, Duren, Germany), as recommended by the supplier. Genomic DNAs were assayed with polymerase chain reaction (PCR) for the detection of IL-6 gene, using the published primer set: 50 -TGACTTCAGCTTTACTCTTTGT-30 (sense primers) and 50 -CTGATTGGAAACCTTATTAGG-30 (antisense primers). Briefly, PCR reaction, containing 0.2 mmol/L of each primer, 0.2 mmol/L of dNTPs (Boehringer GmbH, Mannheim, Germany), 1.5 mmol/L MgCl2, and 2.5 U of AmpliTaq polymerase (Perkin-Elmer, Cetus, Norwalk, CT, U.S.A.) in a final volume of 20 ml, was performed in a GeneAmp PCR System 9700 (Perkin±Elmer) with an initial denaturation step of 10 min at 94 C and a final extension step of 10 min at 72 C. The following thermal profile was repeated for 35 cycles: denaturation for 1 min at 94 C, annealing for 1 min and 35 s at 55 C and extension for 1 min at 72 C. The amplified sequence was digested by SfaNI restriction enzyme (New England BioLabs, Beverly, MA,
265
U.S.A.) at 37 C overnight. The digested products were electrophoresed in 2% agarose gel and visualised by ethidium bromide staining. The GG genotype corresponded to the contemporary presence of 140 and 58 bp fragments. The GC genotype corresponded to the contemporary presence of 198, 140, and 58 bp fragments. The CC genotype corresponded to a 198 bp fragment. Statistical analysis Demographic and clinical data between groups were compared by t-test and Chi-squared test. Genotype and allele ratio distribution between groups was analysed by Chi-squared test. A logistic regression analysis was performed to estimate the association between genotype and PAOD. Odds ratios were calculated with 95% confidence interval (CI). Analyses were done with Intercooled STATA 6.0 for Windows (Statistics/Data Analysis, Stata Corporation, College Station, TX, U.S.A.). Statistical significance was established at p 5 0.05. Results Demographic and clinical characteristics of patients and controls are reported in Table 1. Diabetes and abuse of tobacco were significantly more frequent in patients than controls (p 0.001 and p 5 0.0001, respectively). Hypertension and gender ratio were also close to being significantly different. Other risk factors for PAOD, such as hypercholesterolaemia, history of coronary artery disease (CAD), stroke or transient ischaemic attacks (TIA) of the brain, were not differently distributed between patients and controls. The distribution of IL-6 genotypes and the allele ratio in cases and controls is shown in Table 2. Genotypes were in Hardy-Weinberg equilibrium. The genotype distribution was 44 GG, 30 GC, 10 CC in the 84 patients with PAOD and 53 GG, 80 GC, 50 CC in Table 1. Demographic and clinical data in PAOD patients and controls.
Age (years SD) Male gender Hypertension Hypercholesterolaemia Diabetes History of CAD Stroke/TIA Smoking
Patients (n 84)
Controls (n 183)
75 7 51 (61%) 59 (70%) 25 (30%) 39 (46%) 33 (39%) 22 (26%) 55 (65%)
76 7 89 (49%) 108 (59%) 59 (32%) 40 (22%) 55 (30%) 44 (24%) 60 (33%)
p 0.41 0.06 0.06 0.68 0.001 0.13 0.70 50.0001
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Table 2. IL-6 genotype distribution and allele ratio between groups. Patients
Controls
All GG GC CC G/C ratio
n 84 44 (52%) 30 (36%) 10 (12%) 2.3
n 183 53 (29%) 80 (56%) 50 (27%) 1.0
50.0001 0.23 0.005 50.0001
Males GG GC CC G/C ratio
n 51 28 (55%) 17 (33%) 6 (12%) 2.5
n 89 32 (36%) 34 (38%) 23 (26%) 1.2
0.029 0.56 0.004 50.0001
Females GG GC CC G/C ratio
n 33 16 (49%) 13 (39%) 4 (12%) 2.1
n 94 21 (22%) 46 (49%) 27 (29%) 0.9
0.004 0.34 0.05 0.003
p
Table 3. Risk factors for PAOD based on logistic regression analysis.
G/G genotype G/C genotype Diabetes Sex M Age Hypertension Hypercholesterolaemia History of CAD Stroke/TIA Smoking
Odds ratio
95% CI
4.6 1.7 3.1 0.8 0.9 1.7 1.0 1.7 0.6 5.7
1.9±11.04 0.74±4.22 1.7±9.34 0.44±1.59 0.95±1.02 0.94±3.36 0.55±2.03 0.94±3.40 0.34±1.40 2.47±13.1
p 0.001 0.197 0.007 0.607 0.598 0.074 0.855 0.073 0.311 50.0001
the 183 control subjects. The frequency of the GG genotype in patients with PAOD (52%) was significantly higher than in controls (29%) (p 5 0.0001). In contrast, the frequency of the CC genotype was more than two times higher in controls than in subjects with PAOD (27 vs 12%, p 0.005). Likewise, allele ratio was significantly different between the two groups: the ratio between the G and C allele frequency was 2.3 in patients with PAOD and 1.0 in controls (p 5 0.0001). In order to evaluate whether the distribution of IL-6 genotypes was influenced by sex, male and female subjects were separately analysed (Table 2). The same trend was observed in both men and women: the incidence of GG genotype was significantly higher in men, as well as in women affected by PAOD (p 0.029 and p 0.004, respectively). The role of the GG genotype in the development of PAOD was then investigated by logistic regression analysis (Table 3). We found that the GG genotype is an independent risk for PAOD in our population and that subjects carrying the GG genotype have a risk more than four times higher than CC homozygous Eur J Vasc Endovasc Surg Vol 24, September 2002
individuals to be affected by the disease [Odds ratio 4.6 (95% CI 1.9±11.04), p 0.001]. Discussion The initiation and progression of atherosclerosis involve vessel conformation, haemodynamic forces, exposure to multiple risk factors, cellular composition, and inflammation.4,16 The endothelium expresses procoagulant, instead of anticoagulant, properties and produces vasoactive molecules, inflammatory cytokines, and growth factors. This leads to the accumulation and activation of macrophages and lymphocytes within the plaque. Activation of these inflammatory cells leads to the further release of hydrolytic enzymes, cytokines, chemokines, and growth factors,17 inducing damage and eventually leading to focal necrosis.18 IL-6 is a prototypical inflammatory cytokine able to damage the endothelium and initiate atherosclerosis.19 Immunohistochemical studies of the human arterial atherosclerotic wall have demonstrated that IL-6 is expressed in the cellular and extracellular deposits in the connective tissue matrix and that atherosclerotic plaques have a significantly high level of IL-6.20 Studies in ApoE-knockout mice, that spontaneously develop systemic atherosclerosis, also show elevated levels of IL-6 mRNA in the plaque area compared with normal mice.21 These data are consistent with the observation of elevated levels of IL-6 in patients with coronary syndromes and atherosclerosis-related diseases.8,22±24 Interestingly, plasma levels of IL-6 are influenced by the ÿ174 G/C polymorphism of the IL-6 gene promoter.11 Recent studies have investigated the possible clinical significance of this polymorphism and analysed its association with atherosclerotic and ischaemic diseases, including carotid atherosclerosis,19 CAD,14 and abdominal aortic aneurysm.13 However, no studies have been performed in order to test whether the G/C IL-6 gene polymorphism is associated with atherosclerosis of the peripheral circulation of lower limbs. Our study shows for the first time that the distribution of IL-6 genotypes and alleles is different between patients affected by PAOD and controls. Indeed, the frequency of the GG genotype and G allele is significantly higher in PAOD patients. In contrast, control subjects exhibit a significantly higher frequency of the CC genotype and C allele. When male and female patients were separately analysed, a similar distribution of IL-6 genotypes and alleles was found, demonstrating that the G/C polymorphism of the IL-6 gene is involved in the pathophysiology of PAOD in both men and
IL-6 Gene Polymorphism and PAOD
women. We also show, by using logistic regression analysis, that subjects carrying the G allele have increased risk of PAOD. In particular, subjects GG homozygous have a risk 4.6 times higher to develop the disease, when compared to CC homozygous patients. Taken together, our data are consistent with the hypothesis that the ÿ174 G/C polymorphism of the IL-6 gene promoter is relevant for the pathogenesis and/or evolution of systemic atherosclerosis. Several studies have focused on the measurement and detection of the IL-6 protein in serum or tissue. However, IL-6 protein levels are influenced by many pathologic and physiologic conditions, such as infections, autoimmune disorders, malignancies, trauma, ischaemia, and drug treatment.25±28 Therefore, a single measurement might be not predictive of disease in a given individual. For this reason, our study focuses on the evaluation of a polymorphism that genetically determines the plasmatic levels of IL-6.11±13 This study has some potential limitations. In particular, it is a case-control study and a possible survival bias cannot be excluded for the patients with PAOD. In addition, the size of the studied population is relatively small and our findings need to be confirmed in larger samples. Likewise, the association between IL-6 gene polymorphism and PAOD should be tested in groups of different ethnic origin. Finally, we cannot exclude a role played by a gene or by several genes in linkage disequilibrium with the IL-6 gene, nor that a variant of an other gene, closely linked to the IL-6 gene, causes the observed association. In particular, there is strong linkage disequilibrium between the IL-6 gene promoter polymorphism and the variable number of tandem repeat polymorphism of the 30 flanking region of the IL-6 gene.29 In conclusion, this study evaluates for the first time the association between PAOD and IL-6 gene promoter polymorphism and shows that the GG genotype and the G allele are significantly more frequent in patients affected by PAOD. Although further investigations are needed, our data support the hypothesis that the IL-6 G/C polymorphism is important in the pathophysiology of clinically relevant atherosclerotic disorders.
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