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Two polymorphisms in the fracalkine receptor CX3CR1 are not associated with peripheral arterial disease
Atherosclerosis 166 (2003) 339 /343 www.elsevier.com/locate/atherosclerosis
Two polymorphisms in the fracalkine receptor CX3CR1 are not associated with peripheral arterial disease Alexander Gugl a,*, Wilfried Renner a,b, Gerald Seinost a, Marianne Brodmann a, Edmund Pabst a, Thomas C. Wascher b, Bernhard Paulweber c, Bernhard Iglseder d, Ernst Pilger a a
b
Division of Angiology, Department of Medicine, Karl Franzens University, Graz, Austria Diabetic Angiopathy Research Group, Department of Medicine, Karl Franzens University, Graz, Austria c Department of Medicine, Landeskliniken Salzburg, Salzburg, Austria d Department of Neurology, Landeskliniken Salzburg, Salzburg, Austria Received 13 May 2002; received in revised form 28 August 2002; accepted 18 September 2002
Abstract Objective: CX3CR1 is a novel chemokine receptor located on monocytes. Recently, two polymorphisms were linked to coronary artery disease (CAD), V249I and T280M. Carriers of at least one I-allele or one M-allele were found less frequently among patients with CAD compared to controls. The aim of the present study was to investigate the influence of these polymorphisms on the development of peripheral arterial disease (PAD). Methods: 522 human subjects with documented PAD and 522 age and sex matched controls were genotyped by polymerase chain reaction followed by restriction digestion. Results: Adjusted odds ratio (OR) of carriers of the I-allele for PAD was 1.34 (95% confidential interval (CI) from 0.86 to 2.09; P/0.19). The OR associated with the M-allele for PAD was 0.65 (95% CI from 0.41 to 1.04; P/0.07), when tested in the same regression analysis with the V249I genotypes. The genotypes were not linked to age at onset or severity of the disease. A subgroup of 137 CAD patients of whom 131 could be genotyped and who did not differ in baseline parameters from the remaining PAD patients, showed VV-genotype in 52.0%, VI in 42.7% and II in 5.3% CAD (OR associated with the I-allele for CAD: 1.29; 95% CI: 0.66 /2.51; P/0.46). The distribution of the T280M genotypes was 67.1, 29.8, 3.1% (TT, TM, MM) also showing no association with CAD (OR /0.77; 95% CI 0.36 /1.46; P/0.37). Conclusion: In this study we could not detect a difference in genotype frequencies of the V249I and T280M polymorphisms in CX3CR1 between PAD patients and controls. CAD concomitant with PAD was also not affected by the I- or the M-allele. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CX3CR1; Fractalkine; Peripheral arterial disease; Polymorphism; Epidemiology
1. Introduction Peripheral arterial disease (PAD) is caused by atherosclerosis, a multifactorial process. Cell adhesion molecules and chemokines are now considered to play a major role in plaque formation by enhancement of leucocyte trafficking and sticking to the vessel-walls representing early steps towards vascular damage [1 /4].
* Corresponding author. Klinische Abteilung fu¨r Angiologie, Medizinische Universita¨tsklinik, Auenbruggerplatz 15, A 8036 Graz, Austria. Tel.: /43-316-385-2911; fax: /43-316-385-3788 E-mail address: [email protected] (A. Gugl).
Fractalkine is a molecule of interest, since it is the only member of a recently described chemokine subfamily presenting with a CX3C phenotype with the chemokine domain on top of a mucin like stalk [5]. It exists in a soluble as well as a membrane bound form, attached to TNF-a and IL-1 activated endothelial cells [5,6]. It mediates monocyte capture and firm adhesion to the endothelium in vitro [7]. Shed forms of fractalkine operate in an integrin-dependent pathway by G-protein activation. Furthermore in vitro studies confirmed that fractalkine-receptor expressing cells can adhere to purified fractalkine in an integrin-independent manner [7,8]. Recent published work provides evidence for
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 3 6 2 - 3
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A. Gugl et al. / Atherosclerosis 166 (2003) 339 /343
higher levels of chromosome 16q13 chemokines like fractalkine in atherosclerotic lesions, particularly on macrophages located in plaques characterized by neovascularisation [9]. The fractalkine receptor CX3CR1 has a seven-transmembrane-domain G-protein coupled structure [8], a feature typical for all chemokine receptors [10]. It is expressed on CD 14 monocytes, interleukin-2 activated CD 4 and CD 8 T-cells [8], CD 16 natural killer cells [11] and microglia [12], being involved in migration and adherence of these cells. At a molecular level CX3CR1 and other chemokine receptors act as activators for tyrosine kinases [13]. CX3CR1-fractalkine interactions enable leucocyte capture and firm adhesion under physiologic conditions in vitro without requirement of other cell-adhesion molecules like selectins, integrins and vascular cell adhesion molecules [14]. Two polymorphisms in the sixth and seventh membranepassing domain of the fractalkine receptor CX3CR1 have recently been identified in Caucasian subjects, V249I and T280M [15]. Heterozygosity at position 249 results in a decrease in the number of receptor binding sites for fractalkine on monocytes [16]. V249I and T280M heterozygosity and homozygosity for the I- or M-allele have been found to be associated with a reduced risk of acute coronary events. This protective effect could be explained by the decreased ability of monocytes to adhere to vascular endothelium. The I249M280 haplotype has been associated with a rapid progression to AIDS [15]. Both genotypes are in linkage disequilibrium forming 6 of 9 possible genotypes [15]. Other investigators found an association between the I249 allele and improved vascular endothelial function [17]. In the present study we asked whether allele frequencies of the V249I and T280M polymorphisms of CX3CR1 differ between patients with manifestation of PAD and controls.
Department of Internal Medicine, LKH Universita¨tsklinikum Graz. The study was performed according to the Austrian Gene Technology Act and to the guidelines of the local Ethical Committee of the LKH Universita¨tsklinikum Graz, written informed consent was obtained from all participating subjects. The investigation conforms with the principles outlined in the Declaration of Helsinki. Cardiovascular risk profile and previous or current cardiovascular disease were identified using the following sources: detailed self-reported medical and medication history, medical records provided by general practitioners, and medical records from the Division of Angiology. Measurement of ankle/brachial index (ABI) was performed according to Sanchez and Veith [18]. PAD was diagnosed by the following criteria: clinical symptoms of PAD (intermittent claudication, rest pain, or gangrene) accompanied by an ankle-brachial-index B/0.9 and significant stenoses of leg arteries confirmed by FCDS and/or angiography. Subjects were also considered as PAD patients if they had previous aortofemoral or femoropopliteal bypass surgery. Coronary artery disease (CAD) was characterized by angina pectoris, atypical chest pain with pathological resting or stress echocardiography or coronary angiography confirming at least 50% stenosis in any epicardial coronary artery. Control subjects were characterized as follows: (I) no clinical symptoms of PAD, CAD, or cerebrovascular disease (CVD), and (II) no medical history of PAD, CAD, or CVD. Subjects were considered to be hypertensive if their blood pressure was /140 mmHg systolic, or /90 mmHg diastolic, or if they were taking antihypertensive medication. Diabetes was diagnosed according to the criteria of the World Health Organisation. Patients were considered as hypercholesterolemic, when their fasting total cholesterol exceeded 200 mg/dl or when they received cholesterol lowering medication. Former and actual smokers were combined in one group for statistical analysis.
2. Methods 2.2. Genetic analysis 2.1. Study population Five hundred and twenty two white patients with documented PAD at Fontaine stage IIa/IIb (intermittent claudication), III (rest pain) or IV (gangrene) were enrolled in our study. Patients were recruited between December 1997 and July 1999 from those admitted to the Division of Angiology, LKH Universita¨tsklinikum Graz, Austria. A control group consisted of 522 white age- and sex-matched subjects. One hundred and ninety nine of them participated in the SAPHIR study, Landeskliniken Salzburg, Austria, 115 were recruited in the community of Graz, from a diabetes screening program, 208 were subjects without any known cardiovascular disease recruited among patients and staff from the
Venous blood was collected in 5 ml EDTA tubes, genomic DNA was isolated using a Nucleospin Blood kit (Macherey-Nagel) and stored at 4 8C. Genetic analysis was performed by polymerase chain reaction (PCR) amplifying of a CX3CR1 fragment containing 588 base-pairs. The amplified DNA fragment containing the polymorphic site was flanked by the primers 5?CCGAGGTCCTTCAGGAAATCT-3? (forward) and 5?-TCAGCATCAGGTTCAGGAACTC-3? (reverse). PCR was performed according to previously published protocols [16]. Restriction fragment length polymorphism analysis was conducted incubating PCR products with AclI at 378 and Bsma1 at 558 (both New England Biolabs) for at least 4 h. Fragments were separated on
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2.5% agarose gel and visualized by ethidium bromide staining.
Table 2 Genotype frequencies of the V249I and T280M polymorphism of CX3CR1
2.3. Statistics
Genotype
PAD (n 492)
Controls (n 503)
V249I VV VI II
261 (53) 198 (40.3) 33 (6.7)
268 (53.2) 210 (41.7) 25 (5)
T280M TT TM MM
333 (67.7) 143 (29) 16 (3.3)
326 (64.8) 165 (32.8) 12 (2.4)
Values are expressed as mean9/S.D. for continuous variables. x2-test was applied to analyze dichotomous variables, Mann /Whitney U -test for abnormally distributed variables. Adjusted odds ratios (OR) were computed performing logistic regression analysis. Statistical significance was considered as P B/0.05. All statistical analyses were performed with statistical software package SPSS version 10.0.
Table 3 Combined genoptypes
3. Results 3.1. Clinical and biochemical investigations Groups were well matched with respect to age and gender. Clinical characteristics and biochemical markers of patients and controls are presented in Table 1. Smoking, hypercholesterolemia, hypertension and diabetes were significantly more frequent among patients. 3.2. Genotype distribution Genotyping was successful in 492 patients and 503 controls. In the remaining subjects PCR was considered as uninterpretable after three repeats. Data are displayed in Table 2. The genotype distribution among controls and patients did not deviate from the Hardy / Weinberg equilibrium. The unadjusted OR associated of the I-allele carriers versus non carriers for PAD was 0.99 (95% CI 0.77 /1.27; P /0.96) and did not substantially change after adjustment for smoking status, diabetes, hypertension and hypercholesteremia (OR: 0.99; 95% CI 0.74 /1.32; P/0.92). Since the second polymorphism T280M is in complete linkage disequilibrium with the V249I polymorphism, forming 6 of 9 possible genotypes and only three haplotypes (Table 3) as described by Table 1 Distribution of risk factors among patients and controls
Mean age, years Range Male gender Current or recent smokers Hypercholesterolemia Hypertension Diabetes Symptomatic CAD
Cases (n 522)
Controls (n 522)
64.9911.9 31 /96 305 (58.4) 225 (43.1)* 379 (72.6)** 316 (60.5)* 240 (46)* 137 (26.2)*
64.9911.9 30 /96 305 (58.4) 85 (16.3) 342 (65.5) 205 (39.3) 67 (12.8) 0
Values represent mean9S.D. or number of subjects (%), *P B 0.01, **P B 0.05 cases versus controls.
Combined genotype
V249I T280M Cases (n 492)
Controls (n 503)
1 2 3 4 5 6
VV II II II VI VI
268 (53.3%) 2 (0.4%) 12 (2.4%) 11 (2.2%) 56 (11.1%) 154 (30.6%)
TT TT MM TM TT TM
261 (53%) 3 (0.6) 16 (3.3%) 14 (2.9%) 69 (14%) 129 (26.2%)
Faure et al. [15], we tested also the effect of the M-allele alone and in the same logistic regression analysis with the V249I polymorphism (MM/TM versus TT). The unadjusted OR of the M allele carriers for PAD was 0.88 (95% CI 0.68 /1.14; P /0.34) and after the correction for smoking status, diabetes, hypertension and hypercholesteremia 0.82 (CI 0.60 /1.22; P /0.21). Even with both variables in the same regression analysis no significant level was reached: I-allele (II/VI versus VV): OR /1.34 (95% CI 0.86 /2.09; P /0.19); M-allele (MM/TM versus TT): OR /0.65 (95% CI 0.41 /1.04; P /0.07).
3.3. Fontaine stages of peripheral arterial disease Based upon Fontaine stages the study cohort was subdivided into two groups (stage IIa/IIb and stage III/ IV). The influence of general risk-factors and genotypes on the severity of peripheral artery disease was estimated after adjusting for age and gender within the patient group. Diabetes had a major influence (OR: 2.99; 95% CI 1.89 /4.71; P B/0.001). Age was weakly linked to PAD-stage (OR: 1.04; 95% CI 1.02 /1.06; P B/ 0.001). Hypertension, hypercholesterolemia and smoking were not related to PAD stage. Presence of the I249 allele or M-allele of CX3CR1 were not associated with disease severity of PAD (I-allele: OR 1.27; 95% CI 0.67 / 2.42; P /0.46; M-allele: OR /0.93; 95% CI: 0.47 /1.82; P /0.82).
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3.4. Genotypes in patients and comorbidity of PAD and CAD One hundred and thirty-seven (26%) of 492 PAD patients suffered also from symptomatic CAD. Genotyping was possible in 131 subjects. This subgroup did not differ in age, gender, hypertension, diabetic and smoking status compared to PAD patients without CAD, but had a lower frequency of hypercholesterolemia, (P B/0.01). V249I genotype distribution in PAD patients with CAD was 68 (52.0%), 56 (42.7%), 7 (5.3%) (VV, VI, II) and did not differ from patients without CAD (OR associated with the I-allele for CAD: 1.29; 95% CI: 0.66 /2.51; P /0.46). T280M genotype distribution was 88 (67.1%), 39 (29.8%), 4 (3.1%) (TT, TM, MM) also showing no association with CAD (OR associated with the M-allele: 0.77; 95% CI 0.36 /1.46 P /0.37).
4. Discussion The present study investigates the role of the V249I and T280M polymorphism in the CX3CR1 gene for the presence and severity of PAD. Our results do not reveal any effect of the I249- and the M280-allele on the presence of PAD. No evidence for any influence of the CX3CR1 polymorphisms on the Fontaine stages of the disease could be detected. Previously published data describe correlations between V249I polymorphism and reduced risk of acute coronary syndromes [16] and CAD [17]. After logistic regression analysis only the I-allele remained significant in predicting acute coronary syndromes in the study by Moatti et al. Quantitative analysis of fractalkine binding sites on the surface of monocytes revealed significantly lower CX3CR1 levels in patients carrying the VI genotype. Another study conducted by Faure et al. showed that the I249M280 haplotype influences the progression of AIDS [15]. These findings provide evidence for the functional importance of the V249I and the T280M polymorphism on CX3CR1-expression. Monocyte adhesion and migration precedes the transformation into foamcells, an important step towards plaque formation. Upcoming discussions on involvement of CX3CR1-polymorphisms and fractalkine in atherosclerosis [19,20] prompted us to undertake this investigation of PAD patients. Since PAD reflects a general atherosclerotic process, which is frequently accompanied by CAD [21], a possible link between genetic risk factors for CAD and PAD was examined. In our study we found no effect of either the I- or the M-allele on the presence of symptomatic CAD, which is in contrast to data published by McDermott et al. [17]. This fact could be explained (I) by ethnic diversity of study populations and (II) by the investigation of CAD
concomitant with PAD and (III) by the small number of CAD patients in our study. It has to be emphasized that we cannot conclude that the I- or the M-allele does not influence the presence of CAD in patients free of PAD. Although CAD and PAD seem to be driven by the same process, both manifestations of atherosclerosis seem to have different epidemiologic backgrounds in that some classical risk factors for atherosclerosis have a different influence on PAD and CAD. Smoking, e.g. has a stronger impact on PAD than on CAD suggesting PAD to be more life style influenced [22]. Some genetic variations which are discussed in involvement with CAD showed no influence on PAD [23,24]. The current investigation shows once more that genetic factors in association with atherosclerosis in peripheral arteries are not the same as in the coronary vascular bed. Recent studies showed fractalkine expression in interleukin-4 and interleukin-13 stimulated macrophages in atherosclerotic lesions [9] and in TNF-a activated human umbilical vein endothelial cells [7], which underlines the possible role of this molecule in vascular disorders. Along with these findings the localization of fractalkine on the surface of dermal dendrocytes in psoriasis [25], its involvement in pathogenesis of glomerulonephritis [26] and Crohn’s disease [27] confirm the importance of fractalkine in the inflammation process. In vitro models identified fractalkine/CX3CR1 and other chemokines as potential therapeutic targets in inflammatory diseases [28]. Atherosclerosis exhibits features of inflammation like involvement of cell adhesion molecules, chemokines, cytokines and their receptors [29]. In addition CX3CR1 polymorphisms V249I and T280M have been investigated in AIDS patients. The I249M280 haplotype showed reduced fractalkine binding affinity and a rapid progression of AIDS [15]. McDermott et al. found a correlation between the I249 allele and improved endothelial function in CAD patients diagnosed with angiography [17]. In summary this is the first investigation of the fractalkine-receptor CX3CR1 polymorphisms V249I and T280M in PAD patients. No effect of allelic variation at position 249 and 280 in the fractalkine receptor CX3CR1 on disease progression and severity could be detected. No association of the V249I and T280M polymorphism with the presence of CAD among PAD patients was found. Further investigations are required to clarify the role of the CX3CR1 gene locus in the pathogenesis of atherosclerotic vascular disease.
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[16] Moatti D, Faure S, Fumeron F, Amara Mel-W, Seknadji P, McDermott DH, et al. Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery disease. Blood 2001;97:1925 /8. [17] McDermott DH, Halcox JP, Schenke WH, Waclawiw MA, Merrell MN, Epstein N, et al. Association between polymorphism in the chemokine receptor CX3CR1 and coronary vascular endothelial dysfunction and atherosclerosis. Circ Res 2001;89:401 /7. [18] Sanchez LA, Veith FJ. Diagnosis and treatment of chronic lower extremity ischemia. Vasc Med 1998;3:291 /309. [19] Alexander RW. Cytokine receptor CX3CR-1 and fractalkine: new factors in the atherosclerosis drama? Circ Res 2001;89:376 /7. [20] Umehara H, Bloom E, Okazaki T, Domae N, Imai T. Fractalkine and vascular injury. Trends Immunol 2001;22:602 /7. [21] McDermott MM, Fried L, Simonsick, Ling S, Guralnik. Asymptomatic peripheral arterial disease is independently associated with impaired lower extremity functioning: the women’s health and aging study. Circulation 2000;101:1007 /12. [22] Management of peripheral arterial disease (PAD). TransAtlantic Inter-Society Consensus (TASC). Int Angiol. 2000;19(I / XXIV):1-304. [23] Renner W, Brodmann M, Winkler M, Washer TC, Pilger E. The PLA1/A2 polymorphism of platelet glycoprotein IIIa is not associated with peripheral arterial disease. Thromb Haemost 2001;85:745 /6. [24] Renner W, Schallmoser K, Gallippi P, Krauss C, Toplak H, Wascher TC, et al. C242T polymorphism of the p22 phox gene is not associated with peripheral arterial occlusive disease. Atherosclerosis 2000;152(1):175 /9. [25] Raychaudhuri SP, Jiang WY, Farber EM. Cellular localization of fractalkine at sites of inflammation: antigen-presenting cells in psoriasis express high levels of fractalkine. Br J Dermatol 2001;144:1105 /13. [26] Furuichi K, Wada T, Iwata Y, Sakai N, Yoshimoto K, Shimizu M, et al. Upregulation of fractalkine in human crescentic glomerulonephritis. Nephron 2001;87:314 /20. [27] Muehlhoefer A, Saubermann LJ, Gu X, Luedtke-Heckenkamp K, Xavier R, Blumberg RS, et al. Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J Immunol 2000;164:3368 /76. [28] Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P, et al. Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication. Kidney Int 1999;56:612 /20. [29] Epstein SE, Zhou YF, Zhu J. Infection and atherosclerosis: emerging mechanistic paradigms. Circulation 1999;100:e20 /8.
Two polymorphisms in the fracalkine receptor CX3CR1 are not associated with peripheral arterial disease Alexander Gugl a,*, Wilfried Renner a,b, Gerald Seinost a, Marianne Brodmann a, Edmund Pabst a, Thomas C. Wascher b, Bernhard Paulweber c, Bernhard Iglseder d, Ernst Pilger a a
b
Division of Angiology, Department of Medicine, Karl Franzens University, Graz, Austria Diabetic Angiopathy Research Group, Department of Medicine, Karl Franzens University, Graz, Austria c Department of Medicine, Landeskliniken Salzburg, Salzburg, Austria d Department of Neurology, Landeskliniken Salzburg, Salzburg, Austria Received 13 May 2002; received in revised form 28 August 2002; accepted 18 September 2002
Abstract Objective: CX3CR1 is a novel chemokine receptor located on monocytes. Recently, two polymorphisms were linked to coronary artery disease (CAD), V249I and T280M. Carriers of at least one I-allele or one M-allele were found less frequently among patients with CAD compared to controls. The aim of the present study was to investigate the influence of these polymorphisms on the development of peripheral arterial disease (PAD). Methods: 522 human subjects with documented PAD and 522 age and sex matched controls were genotyped by polymerase chain reaction followed by restriction digestion. Results: Adjusted odds ratio (OR) of carriers of the I-allele for PAD was 1.34 (95% confidential interval (CI) from 0.86 to 2.09; P/0.19). The OR associated with the M-allele for PAD was 0.65 (95% CI from 0.41 to 1.04; P/0.07), when tested in the same regression analysis with the V249I genotypes. The genotypes were not linked to age at onset or severity of the disease. A subgroup of 137 CAD patients of whom 131 could be genotyped and who did not differ in baseline parameters from the remaining PAD patients, showed VV-genotype in 52.0%, VI in 42.7% and II in 5.3% CAD (OR associated with the I-allele for CAD: 1.29; 95% CI: 0.66 /2.51; P/0.46). The distribution of the T280M genotypes was 67.1, 29.8, 3.1% (TT, TM, MM) also showing no association with CAD (OR /0.77; 95% CI 0.36 /1.46; P/0.37). Conclusion: In this study we could not detect a difference in genotype frequencies of the V249I and T280M polymorphisms in CX3CR1 between PAD patients and controls. CAD concomitant with PAD was also not affected by the I- or the M-allele. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CX3CR1; Fractalkine; Peripheral arterial disease; Polymorphism; Epidemiology
1. Introduction Peripheral arterial disease (PAD) is caused by atherosclerosis, a multifactorial process. Cell adhesion molecules and chemokines are now considered to play a major role in plaque formation by enhancement of leucocyte trafficking and sticking to the vessel-walls representing early steps towards vascular damage [1 /4].
* Corresponding author. Klinische Abteilung fu¨r Angiologie, Medizinische Universita¨tsklinik, Auenbruggerplatz 15, A 8036 Graz, Austria. Tel.: /43-316-385-2911; fax: /43-316-385-3788 E-mail address: [email protected] (A. Gugl).
Fractalkine is a molecule of interest, since it is the only member of a recently described chemokine subfamily presenting with a CX3C phenotype with the chemokine domain on top of a mucin like stalk [5]. It exists in a soluble as well as a membrane bound form, attached to TNF-a and IL-1 activated endothelial cells [5,6]. It mediates monocyte capture and firm adhesion to the endothelium in vitro [7]. Shed forms of fractalkine operate in an integrin-dependent pathway by G-protein activation. Furthermore in vitro studies confirmed that fractalkine-receptor expressing cells can adhere to purified fractalkine in an integrin-independent manner [7,8]. Recent published work provides evidence for
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 3 6 2 - 3
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A. Gugl et al. / Atherosclerosis 166 (2003) 339 /343
higher levels of chromosome 16q13 chemokines like fractalkine in atherosclerotic lesions, particularly on macrophages located in plaques characterized by neovascularisation [9]. The fractalkine receptor CX3CR1 has a seven-transmembrane-domain G-protein coupled structure [8], a feature typical for all chemokine receptors [10]. It is expressed on CD 14 monocytes, interleukin-2 activated CD 4 and CD 8 T-cells [8], CD 16 natural killer cells [11] and microglia [12], being involved in migration and adherence of these cells. At a molecular level CX3CR1 and other chemokine receptors act as activators for tyrosine kinases [13]. CX3CR1-fractalkine interactions enable leucocyte capture and firm adhesion under physiologic conditions in vitro without requirement of other cell-adhesion molecules like selectins, integrins and vascular cell adhesion molecules [14]. Two polymorphisms in the sixth and seventh membranepassing domain of the fractalkine receptor CX3CR1 have recently been identified in Caucasian subjects, V249I and T280M [15]. Heterozygosity at position 249 results in a decrease in the number of receptor binding sites for fractalkine on monocytes [16]. V249I and T280M heterozygosity and homozygosity for the I- or M-allele have been found to be associated with a reduced risk of acute coronary events. This protective effect could be explained by the decreased ability of monocytes to adhere to vascular endothelium. The I249M280 haplotype has been associated with a rapid progression to AIDS [15]. Both genotypes are in linkage disequilibrium forming 6 of 9 possible genotypes [15]. Other investigators found an association between the I249 allele and improved vascular endothelial function [17]. In the present study we asked whether allele frequencies of the V249I and T280M polymorphisms of CX3CR1 differ between patients with manifestation of PAD and controls.
Department of Internal Medicine, LKH Universita¨tsklinikum Graz. The study was performed according to the Austrian Gene Technology Act and to the guidelines of the local Ethical Committee of the LKH Universita¨tsklinikum Graz, written informed consent was obtained from all participating subjects. The investigation conforms with the principles outlined in the Declaration of Helsinki. Cardiovascular risk profile and previous or current cardiovascular disease were identified using the following sources: detailed self-reported medical and medication history, medical records provided by general practitioners, and medical records from the Division of Angiology. Measurement of ankle/brachial index (ABI) was performed according to Sanchez and Veith [18]. PAD was diagnosed by the following criteria: clinical symptoms of PAD (intermittent claudication, rest pain, or gangrene) accompanied by an ankle-brachial-index B/0.9 and significant stenoses of leg arteries confirmed by FCDS and/or angiography. Subjects were also considered as PAD patients if they had previous aortofemoral or femoropopliteal bypass surgery. Coronary artery disease (CAD) was characterized by angina pectoris, atypical chest pain with pathological resting or stress echocardiography or coronary angiography confirming at least 50% stenosis in any epicardial coronary artery. Control subjects were characterized as follows: (I) no clinical symptoms of PAD, CAD, or cerebrovascular disease (CVD), and (II) no medical history of PAD, CAD, or CVD. Subjects were considered to be hypertensive if their blood pressure was /140 mmHg systolic, or /90 mmHg diastolic, or if they were taking antihypertensive medication. Diabetes was diagnosed according to the criteria of the World Health Organisation. Patients were considered as hypercholesterolemic, when their fasting total cholesterol exceeded 200 mg/dl or when they received cholesterol lowering medication. Former and actual smokers were combined in one group for statistical analysis.
2. Methods 2.2. Genetic analysis 2.1. Study population Five hundred and twenty two white patients with documented PAD at Fontaine stage IIa/IIb (intermittent claudication), III (rest pain) or IV (gangrene) were enrolled in our study. Patients were recruited between December 1997 and July 1999 from those admitted to the Division of Angiology, LKH Universita¨tsklinikum Graz, Austria. A control group consisted of 522 white age- and sex-matched subjects. One hundred and ninety nine of them participated in the SAPHIR study, Landeskliniken Salzburg, Austria, 115 were recruited in the community of Graz, from a diabetes screening program, 208 were subjects without any known cardiovascular disease recruited among patients and staff from the
Venous blood was collected in 5 ml EDTA tubes, genomic DNA was isolated using a Nucleospin Blood kit (Macherey-Nagel) and stored at 4 8C. Genetic analysis was performed by polymerase chain reaction (PCR) amplifying of a CX3CR1 fragment containing 588 base-pairs. The amplified DNA fragment containing the polymorphic site was flanked by the primers 5?CCGAGGTCCTTCAGGAAATCT-3? (forward) and 5?-TCAGCATCAGGTTCAGGAACTC-3? (reverse). PCR was performed according to previously published protocols [16]. Restriction fragment length polymorphism analysis was conducted incubating PCR products with AclI at 378 and Bsma1 at 558 (both New England Biolabs) for at least 4 h. Fragments were separated on
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2.5% agarose gel and visualized by ethidium bromide staining.
Table 2 Genotype frequencies of the V249I and T280M polymorphism of CX3CR1
2.3. Statistics
Genotype
PAD (n 492)
Controls (n 503)
V249I VV VI II
261 (53) 198 (40.3) 33 (6.7)
268 (53.2) 210 (41.7) 25 (5)
T280M TT TM MM
333 (67.7) 143 (29) 16 (3.3)
326 (64.8) 165 (32.8) 12 (2.4)
Values are expressed as mean9/S.D. for continuous variables. x2-test was applied to analyze dichotomous variables, Mann /Whitney U -test for abnormally distributed variables. Adjusted odds ratios (OR) were computed performing logistic regression analysis. Statistical significance was considered as P B/0.05. All statistical analyses were performed with statistical software package SPSS version 10.0.
Table 3 Combined genoptypes
3. Results 3.1. Clinical and biochemical investigations Groups were well matched with respect to age and gender. Clinical characteristics and biochemical markers of patients and controls are presented in Table 1. Smoking, hypercholesterolemia, hypertension and diabetes were significantly more frequent among patients. 3.2. Genotype distribution Genotyping was successful in 492 patients and 503 controls. In the remaining subjects PCR was considered as uninterpretable after three repeats. Data are displayed in Table 2. The genotype distribution among controls and patients did not deviate from the Hardy / Weinberg equilibrium. The unadjusted OR associated of the I-allele carriers versus non carriers for PAD was 0.99 (95% CI 0.77 /1.27; P /0.96) and did not substantially change after adjustment for smoking status, diabetes, hypertension and hypercholesteremia (OR: 0.99; 95% CI 0.74 /1.32; P/0.92). Since the second polymorphism T280M is in complete linkage disequilibrium with the V249I polymorphism, forming 6 of 9 possible genotypes and only three haplotypes (Table 3) as described by Table 1 Distribution of risk factors among patients and controls
Mean age, years Range Male gender Current or recent smokers Hypercholesterolemia Hypertension Diabetes Symptomatic CAD
Cases (n 522)
Controls (n 522)
64.9911.9 31 /96 305 (58.4) 225 (43.1)* 379 (72.6)** 316 (60.5)* 240 (46)* 137 (26.2)*
64.9911.9 30 /96 305 (58.4) 85 (16.3) 342 (65.5) 205 (39.3) 67 (12.8) 0
Values represent mean9S.D. or number of subjects (%), *P B 0.01, **P B 0.05 cases versus controls.
Combined genotype
V249I T280M Cases (n 492)
Controls (n 503)
1 2 3 4 5 6
VV II II II VI VI
268 (53.3%) 2 (0.4%) 12 (2.4%) 11 (2.2%) 56 (11.1%) 154 (30.6%)
TT TT MM TM TT TM
261 (53%) 3 (0.6) 16 (3.3%) 14 (2.9%) 69 (14%) 129 (26.2%)
Faure et al. [15], we tested also the effect of the M-allele alone and in the same logistic regression analysis with the V249I polymorphism (MM/TM versus TT). The unadjusted OR of the M allele carriers for PAD was 0.88 (95% CI 0.68 /1.14; P /0.34) and after the correction for smoking status, diabetes, hypertension and hypercholesteremia 0.82 (CI 0.60 /1.22; P /0.21). Even with both variables in the same regression analysis no significant level was reached: I-allele (II/VI versus VV): OR /1.34 (95% CI 0.86 /2.09; P /0.19); M-allele (MM/TM versus TT): OR /0.65 (95% CI 0.41 /1.04; P /0.07).
3.3. Fontaine stages of peripheral arterial disease Based upon Fontaine stages the study cohort was subdivided into two groups (stage IIa/IIb and stage III/ IV). The influence of general risk-factors and genotypes on the severity of peripheral artery disease was estimated after adjusting for age and gender within the patient group. Diabetes had a major influence (OR: 2.99; 95% CI 1.89 /4.71; P B/0.001). Age was weakly linked to PAD-stage (OR: 1.04; 95% CI 1.02 /1.06; P B/ 0.001). Hypertension, hypercholesterolemia and smoking were not related to PAD stage. Presence of the I249 allele or M-allele of CX3CR1 were not associated with disease severity of PAD (I-allele: OR 1.27; 95% CI 0.67 / 2.42; P /0.46; M-allele: OR /0.93; 95% CI: 0.47 /1.82; P /0.82).
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3.4. Genotypes in patients and comorbidity of PAD and CAD One hundred and thirty-seven (26%) of 492 PAD patients suffered also from symptomatic CAD. Genotyping was possible in 131 subjects. This subgroup did not differ in age, gender, hypertension, diabetic and smoking status compared to PAD patients without CAD, but had a lower frequency of hypercholesterolemia, (P B/0.01). V249I genotype distribution in PAD patients with CAD was 68 (52.0%), 56 (42.7%), 7 (5.3%) (VV, VI, II) and did not differ from patients without CAD (OR associated with the I-allele for CAD: 1.29; 95% CI: 0.66 /2.51; P /0.46). T280M genotype distribution was 88 (67.1%), 39 (29.8%), 4 (3.1%) (TT, TM, MM) also showing no association with CAD (OR associated with the M-allele: 0.77; 95% CI 0.36 /1.46 P /0.37).
4. Discussion The present study investigates the role of the V249I and T280M polymorphism in the CX3CR1 gene for the presence and severity of PAD. Our results do not reveal any effect of the I249- and the M280-allele on the presence of PAD. No evidence for any influence of the CX3CR1 polymorphisms on the Fontaine stages of the disease could be detected. Previously published data describe correlations between V249I polymorphism and reduced risk of acute coronary syndromes [16] and CAD [17]. After logistic regression analysis only the I-allele remained significant in predicting acute coronary syndromes in the study by Moatti et al. Quantitative analysis of fractalkine binding sites on the surface of monocytes revealed significantly lower CX3CR1 levels in patients carrying the VI genotype. Another study conducted by Faure et al. showed that the I249M280 haplotype influences the progression of AIDS [15]. These findings provide evidence for the functional importance of the V249I and the T280M polymorphism on CX3CR1-expression. Monocyte adhesion and migration precedes the transformation into foamcells, an important step towards plaque formation. Upcoming discussions on involvement of CX3CR1-polymorphisms and fractalkine in atherosclerosis [19,20] prompted us to undertake this investigation of PAD patients. Since PAD reflects a general atherosclerotic process, which is frequently accompanied by CAD [21], a possible link between genetic risk factors for CAD and PAD was examined. In our study we found no effect of either the I- or the M-allele on the presence of symptomatic CAD, which is in contrast to data published by McDermott et al. [17]. This fact could be explained (I) by ethnic diversity of study populations and (II) by the investigation of CAD
concomitant with PAD and (III) by the small number of CAD patients in our study. It has to be emphasized that we cannot conclude that the I- or the M-allele does not influence the presence of CAD in patients free of PAD. Although CAD and PAD seem to be driven by the same process, both manifestations of atherosclerosis seem to have different epidemiologic backgrounds in that some classical risk factors for atherosclerosis have a different influence on PAD and CAD. Smoking, e.g. has a stronger impact on PAD than on CAD suggesting PAD to be more life style influenced [22]. Some genetic variations which are discussed in involvement with CAD showed no influence on PAD [23,24]. The current investigation shows once more that genetic factors in association with atherosclerosis in peripheral arteries are not the same as in the coronary vascular bed. Recent studies showed fractalkine expression in interleukin-4 and interleukin-13 stimulated macrophages in atherosclerotic lesions [9] and in TNF-a activated human umbilical vein endothelial cells [7], which underlines the possible role of this molecule in vascular disorders. Along with these findings the localization of fractalkine on the surface of dermal dendrocytes in psoriasis [25], its involvement in pathogenesis of glomerulonephritis [26] and Crohn’s disease [27] confirm the importance of fractalkine in the inflammation process. In vitro models identified fractalkine/CX3CR1 and other chemokines as potential therapeutic targets in inflammatory diseases [28]. Atherosclerosis exhibits features of inflammation like involvement of cell adhesion molecules, chemokines, cytokines and their receptors [29]. In addition CX3CR1 polymorphisms V249I and T280M have been investigated in AIDS patients. The I249M280 haplotype showed reduced fractalkine binding affinity and a rapid progression of AIDS [15]. McDermott et al. found a correlation between the I249 allele and improved endothelial function in CAD patients diagnosed with angiography [17]. In summary this is the first investigation of the fractalkine-receptor CX3CR1 polymorphisms V249I and T280M in PAD patients. No effect of allelic variation at position 249 and 280 in the fractalkine receptor CX3CR1 on disease progression and severity could be detected. No association of the V249I and T280M polymorphism with the presence of CAD among PAD patients was found. Further investigations are required to clarify the role of the CX3CR1 gene locus in the pathogenesis of atherosclerotic vascular disease.
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