Genetic Variation at the Chemokine Receptors CCR5/CCR2 in Myocardial Infarction

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Genes and Immunity (2001) 2, 191–195  2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene Genetic variation at the chemokine receptors CCR5/CCR2 in myocardial infarction

P Gonza´lez1, R Alvarez1, A Batalla2, JR Reguero2, V Alvarez1, A Astudillo3, GI Cubero2, A Cortina2 and E Coto1 1Gene´tica Molecular-Instituto Reina Sofıá de Investigacioń Nefrolo´gica, Spain; 2Cardiologıá and 3Anatomıá Patolo´gica, Hospital Central de Asturias, 33006, Oviedo, Spain

Our objective was to examine the association between myocardial infarction (MI) and two DNA-polymorphisms at the proinflammatory chemokine receptors CCR2 (I64V) and CCR5 (32 bp deletion, ⌬ccr5), defining if these polymorphisms influence the age for the onset of MI. A total of 214 patients with an age at the first MI episode Ͻ55 years, 96 patients that suffered the first MI episode when older than 60 years, and 360 population controls were polymerase chain reaction genotyped for the CCR2-V64I and CCR5-⌬32/wt polymorphisms. Patients and controls were male from the same Caucasian population (Asturias, northern Spain). The frequency of the ⌬ccr5 allele was significantly higher in controls compared to patients Ͻ55 years (P = 0.004), or in patients Ͼ60 years compared to patients Ͻ55 years (P = 0.002). Taking the patients Ͼ60 years as the reference group, non-carriers of the ⌬ccr5-allele would have a three-fold higher risk of suffering an episode of MI at Ͻ55 years of age (OR = 3.06; 95% CI = 1.46–6.42). Gene and genotype frequencies for the CCR2 polymorphism did not differ between patients Ͻ55 years and controls or patients Ͼ60 years. Our data suggest that the variation at the CCR5 gene could modulate the age at the onset of MI. Patients carrying the ⌬ccr5-allele would be protected against an early episode of MI. CCR5 and the CCR5-ligands are expressed by cells in the arteriosclerotic plaque. Thus, the protective role of ⌬ccr5 could be a consequence of an attenuated inflammatory response, that would determine a slower progression of the arteriosclerotic lesion among ⌬ccr5-carriers. Our work suggests that the pharmacological blockade of CCR5 could be a valuable therapy in the treatment of MI. Genes and Immunity (2001) 2, 191–195.

Keywords: myocardial infarction; inﬂammation; chemokine receptors; DNA-polymorphisms

Introduction receptors. Two of these, CCR2 and CCR5, bind MCP-1 (CCR2) and RANTES, MIP-1alpha or MIP-1beta The immune inflammatory response is involved in the (CCR5).4,13 As part of the proinflammatory response, 1,2 development of the arteriosclerotic lesion. Chemokines these receptors could be involved in the initiation and are proinflammatory cytokines that recruit leukocytes progression of arteriosclerosis. CCR2 and CCR5 have from the blood into tissues, thus playing a pivotal role in recently been identified on vascular smooth muscle cells, 3,4 human inflammatory diseases. Among these chemo- where they may influence proliferation and kines, MCP-1 (monocyte chemoattractant protein-1), migration.14,15 Apolipoprotein E (ApoE) null mice RANTES (regulated on activation, normal T cell develop severe atherosclerosis, and these ApoE-null mice expressed and secreted), and the macrophage inflamma- also lacking CCR2 showed a decreased arteriosclerotic tory proteins-1 (MIP-1) alpha and beta act on lympho- lesion, an effect mediated through a reduced recruitment cytes, eosinophils, monocyte/macrophages, and baso- of monocytes/macrophages into the vessel wall.16 phils, and have been detected in arteriosclerotic In addition to their function in the inflammatory 5–8 plaques. MCP-1 is expressed by stimulated cultured response, CCR2 and CCR5 act as coreceptors for the HIV- 9,10 vascular smooth muscle cells. In addition, MCP-1 is 1 virus.17 Several mutations (polymorphisms) at the upregulated in arteries of primates on a hypercholestero- CCR2 and CCR5 genes have been described, being laemic diet, and there is a growing evidence that the involved in the resistence to HIV-1 infection and the rate lipid-ladden macrophages (foam cells) that characterise of AIDS progression.18–23 Thus, individuals who are the fatty streak arteriosclerotic lesions could be recruited homozygous for a 32-bp deletion at the CCR-5 gene (the 11,12 to the vessel wall by these chemokines. ⌬ccr5 mutation) are almost completely resistant to HIV- Chemokines exert their biological functions through 1 infection, and ⌬ccr5 heterozygotes showed a slow pro- binding to seven-transmembrane G-protein coupled gression towards AIDS compared to wild-type homozygotes. The ⌬ccr5 allele encodes a truncated protein that is not expressed on membrane cells, with ⌬ccr5/⌬ccr5 Correspondence: Dr Eliecer Coto Garcıá, Laboratorio de Gene´tica Mole- individuals having a complete lack of the receptor while cular, Hospital Central de Asturias, 33006 – Oviedo – Spain. E-mail: ⌬ ecotoȰhcas.insalud.es ccr5 heterozygotes express lower amounts of the recep- 24 This work was supported by a grant from the Spanish Health Sys- tor compared to wild-type homozygotes. A polymor- tem (Fondo de Investigaciones Sanitarias 99/0924). phism at the CCR2 gene (V64I) has been described, and Genetic variation of chemokine receptors P Gonza´lez et al 192 Table 1 Anthropometric characteristics and average values of total Table 3 Gene and genotype frequenciess for the CCR5 polymor- cholesterol (TC), triglycerides (TG), and HDL-cholesterol in patients phism in myocardial infarction patients and controls and controls. Numbers in parenthesis represent percentages CCR5 (⌬32bp) Patients Ͻ55 Patients Ͼ60 Controls = = = n 214 n 96 n 360 Genotypes* Alleles** ± ± ± Average age 45 5657426 wt/wt wt/⌬32 ⌬32/⌬32 wt ⌬32 Hypertensives 75 (35) 38 (40) 54 (15) Smokers 203 (95) 81 (84) 160 (44) ± ± ± Patients 199 15 0 413 15 TC (mg/dl) 222 56 197 42 203 40 Ͻ HDL-C (mg/dl) 33 ± 933± 851± 12 55 years (93%) (7%) (0%) (0.96) (0.04) ± ± ± TG 180 130 140 72 127 82 Controls 307 49 4 663 57 (95%) (14%) (1%) (0.92) (0.08) Patients 78 17 1 173 19 Ͼ carriers of Isoleucine at codon 64 (the 64 I-allele) would 60 years (81%) (18%) (1%) (0.92) (0.08) 22,23 be protected against HIV disease progression. = = = ⌬ + ⌬ ⌬ The ⌬ccr5 and 64I alleles exist at a high frequency in *P 0.009; OR 0.44; 95% CI 0.23, 0.82. ( 32/wt 32/ 32) vs wt/wt, patients Ͻ55 vs controls. **P = 0.004; OR = 0.42; 95% CI = most Caucasian populations. In this study we genotyped 0.23, 0.77. ⌬32 vs wt, patients Ͻ55 vs controls. a number of patients with early (Ͻ55 years) and late (Ͼ60 years) myocardial infarction (MI), as well as healthy controls from the same Caucasian population. Our purpose Gene and genotype frequencies for the CCR5 polymor- was to determine whether the CCR5 and CCR2 polymorphism are summarised in Table 3. We found a signifi- phisms contribute to the susceptibility to develop myo- cantly lower frequency of the ⌬ccr5 allele among the cardial infarction or influences the age at the onset of youngest patients compared to controls (P = 0.004; OR = the disease. 0.42, 95% CI = 0.23–0.77) or to oldest patients (P = 0.002; OR = 0.42, 95% CI = 0.16, 0.68). No patient Ͻ55 years was ⌬ Results ccr5-homozygote, compared to 1% among the controls. While 7% of the patients Ͻ55 years were ⌬ccr5-carriers Table 1 summarises the characteristics of patients and (⌬/wt + ⌬/⌬), this frequency was 15% among the con- controls. All the individuals in both groups of patients trols and 19% among the patients Ͼ60 years. These data were either smokers and/or hypertensives and/or had suggest that non-carriers of the ⌬ccr5 allele have an early hypercholesterolaemia, three well recognised risk factors onset of the disease. Taking the patients Ͼ60 years as the for coronary artery disease. Compared to controls, reference group, wt/wt individuals would have a three- patients Ͻ55 years had a significantly higher frequency fold higher risk of suffering an early episode (Ͻ55 years) of smokers (P Ͻ 0.0001; OR = 23, 95% CI = 12.44, 44.70) of MI (OR = 3.06; 95% CI = 1.46–6.42). and hypertensives (P Ͻ 0.0001; OR = 3.06, 95% CI = 2.01, The CCR2 and CCR5 genes are closely linked on chro- 4.66). Average cholesterol and triglycerides values were mosome 3. We analysed if any genotype combination higher in patients Ͻ55 years compared to controls, while was more frequent in patients compared to controls. As the HDL-cholesterol value was lower in patients. How- summarised in Table 4, CCR2/CCR5 genotype fre- ever, these values were not significantly different quencies did not differ between the three groups. between patients and controls. Coronary angiograms were performed on 104 of the Table 2 summarises the CCR2 frequencies (V64I polymorphism) in patients and controls. The CCR2 allele and genotype frequencies did not differ between the Table 4 Distribution of CCR2-genotypes according to the CCR5- genotypes in patients and controls. None of the ⌬ccr5 carriers was three groups, suggesting that this polymorphism neither CCR2-II, indicating a linkage disequilibrium between the two poly- contributes to the risk of developing MI nor delays the morphisms outcome of coronary heart disease. CCR5 CCR2 Table 2 Gene and genotype frequencies for the CCR2 polymorphism in patients (Ͻ55 years and Ͼ60 years) and controls VV IV II Total (100%) CCR2 (V64I) wt/wt Ͻ Genotypes Alleles Patients 55 149 (75%) 48 (24%) 2 (1%) 199 Controls 240 (78%) 65 (21%) 2 (1%) 307 Patients Ͼ60 62 (79%) 14 (18%) 2 (3%) 78 VV IV II V I ⌬/wt Ͻ Patients 164 48 2 376 52 Patients 55 15 (100%) 0 0 15 Ͻ55 years (77%) (22%) (1%) (0.88) (0.12) Controls 46 (94%) 3 (6%) 0 49 Patients Ͼ60 17 (100%) 0 0 17 Controls 290 68 2 648 72 ⌬/⌬ (80%) (19%) (1%) (0.90) (0.10) Patients Ͻ55 0 0 0 0 Patients 80 14 2 174 18 Controls 4 (100%) 0 0 4 Ͼ60 years (83%) (15%) (2%) (0.91) (0.09) Patients Ͼ60 1 0 0 1

Genes and Immunity Genetic variation of chemokine receptors P Gonza´lez et al 193 Table 5 CCR5 genotype frequencies in patients and controls according to risk factors, and average lipid values for each genotype (⌬+= ⌬/wt + ⌬/⌬; parenthesis indicate percentage)

Patients Ͻ55 Patients Ͼ60 Controls

wt/wt ⌬+ wt/wt ⌬+ wt/wt ⌬+

Smokers* 189 (93) 14 (7) 66 (79) 15 (21) 134 (84) 26 (16) Non-smokers 10 (90) 1 (10) 12 (80) 3 (20) 173 (86) 27 (14) Hypertensives** 72 (96) 3 (4) 32 (84) 6 (16) 47 (87) 7 (13) Normotensives 127 (91) 12 (9) 46 (79) 12 (21) 260 (85) 46 (15) TC (mg/dl) 224 ± 45 220 ± 62 203 ± 45 189 ± 32 208 ± 42 193 ± 30 HDL-C (mg/dl) 33 ± 10 36 ± 13 34 ± 10 32 ± 953± 12 48 ± 13 TG 183 ± 135 187 ± 177 135 ± 74 140 ± 69 124 ± 79 140 ± 104

*Smokers, Ͻ55 years vs controls, P = 0.008; Ͻ55 vs Ͼ60, P = 0.007. **Hypertensives, Ͻ55 vs controls, P = 0.12; Ͻ55 vs Ͼ60, P = 0.03. patients Ͻ55 years, and only three showed non-diseased artery smooth muscle cells, and the MIP-1beta (a CCR5 vessels. Among the patients Ͼ60 years, a total of 36 were ligand) was detected in smooth muscle cells and macro- angiographically evaluated, and only two had non- phages of the atherosclerotic plaque.15 diseased vessels. Gene and genotype frequencies for the CCR5 also acts as an HIV-1 coreceptor, and the ⌬ccr5 CCR5 polymorphism did not differ between total mutation has recently acquired particular relevance by patients and patients with angiographically confirmed confering resistance to HIV infection.17–23,28 The ⌬ccr5 diseased vessels. A similar result was obtained for the allele encodes a truncated protein that does not reach the CCR2 polymorphism. cell surface, with ⌬ccr5/⌬ccr5 homozygotes having a We also analysed the CCR5 genotypes distribution complete lack of the CCR5 on cells membrane, and ⌬ccr5 according to cardiovascular risk factors. As summarised carriers expressing a reduced amount of the receptor in Table 5, ⌬ccr5 carriers were at a similar frequency compared to wild-type homozygotes.24 Although they do among hypertensives/normotensives, or smokers/non- not express CCR5, individuals homozygous for the ⌬ccr5 smokers. In addition, average lipid values did not differ allele are healthy, probably due to the fact that the CCR5 between the different genotypes. When only smokers ligands can exert their function by binding to other were compared, ⌬ccr5 carriers were at a higher frequency chemokine receptors. However, it is possible that the among patients Ͻ55 years compared to controls (P = 0.08) total or partial absence of CCR5 determines differencies or to patients Ͼ60 years (P = 0.007). in the rates of progression for diseases in which the inflammatory response is involved, particularly those in ⌬ Discussion which RANTES and MIP-1 play a role. In this way, ccr5 carriers could have a reduced inflammatory response in Our work suggests that carriers of the ⌬ccr5 mutation vascular endothelium. As a consequence, the atheroscler- are at a lower frequency among patients with early MI, otic lesion could develop at a slower rate in ⌬ccr5 carriers compared to controls and patients with a late-onset of the compared to wild-type homozygotes, who would suffer disease. These data support a role for this polymorphism the MI at an earlier age. modulating the age at the onset of the disease. A similar result has been found in asthma and in rheu- CCR5 and CCR2 are proinflammatory receptors that matoid arthritis. These are diseases with an inflammatory bind RANTES, MIP-1, and MCP-1. These chemokines are component, and ⌬ccr5 carriers would be protected produced by the smooth muscle cells from the vascular against the disease, or would develop a less severe form endothelium, thus recruiting monocyte/macrophages to of the disease, compared to CCR5 wild-type homozy- the vessel wall. This leukocyte contributes to the develop- gotes.29–32 ment of the arteriosclerotic lesion, a process that involves The V64I polymorphism at the CCR2 gene has also interactions between endothelial cells, vascular smooth been associated with differencies in HIV disease pro- muscle cells, macrophages and lymphocytes.1,2,25 gression, with the 64I-allele having a protective role.22 It Monocytes/macrophages in the vessel wall accumulate is unlikely that the presence of valine or isolecine deter- lipids and become the foam cells (lipid-laden mines major functional changes in CCR2, and the differ- macrophages) that characterise the arterial fatty streak, ent biological properties of this polymorphism could be a which is the precursor of a more complex and danger- consequence of a linkage disequilibrium with a different ous lesion. polymorphism at the CCR2 and/or CCR5 genes, closely The importance of CCR2 in the initiation of atheroscler- linked on chromosome 3. Because the CCR2-gene and osis has been recently demonstrated by Boring et al.16 genotype frequencies were similar in MI patients and These authors reported a decreased atherosclerotic lesion healthy controls, this polymorphism would not contrib- formation in mice (knock-out) lacking both apoE and ute to the susceptibility to develop the disease. In CCR2, identifying CCR2 as a genetic determinant of addition, our data do not support a role for the CCR2- murine arteriosclerosis. This effect could be mediated polymorphism in modulating the onset of MI. through a reduced leukocyte adhesion and monocyte Some previous reports have analysed the genetic vari- extravasation in CCR2-null mice.26,27 In addition to CCR2, ation at several inflammatory molecules in coronary CCR5 has also been found in human aortic and coronary artery disease.33 To our knowledge, our work represents

Genes and Immunity Genetic variation of chemokine receptors P Gonza´lez et al 194 the first attempt to define a role for polymorphisms at hypertensives. A smoking history from all subjects was the proinflammatory receptors in MI. Apart from CCR2 obtained, and a lipid profile in blood that included total and CCR5, other chemokine receptors are actively cholesterol, triglycerides, and HDL-cholesterol was also expressed on monocytes, and polymorphisms at these determined for each patient and control. Hypercholester- genes could contribute to the susceptibility to develop oloemia was defined as a total cholesterol Ͼ250 mg/dl. MI, or determine differencies in the rate of disease progression. Genotyping of the CCR5 and CCR2 polymorphisms Our work has several limitations. First of all, data from Genomic DNA was obtained from patients and controls. case-control studies are subjected to a number of possible For the analysis of the ⌬ccr5-allele, genomic DNA was confounding factors, the total number of patients and PCR-amplyfied in a final volume of 30 ␮l containing 30 controls and the homogeneity of the population (in terms pmol of each primer (forward -CTGTGTTTGCG of sex, geographical origin, and age) among others. TCTCTCCCA and reverse-CCTCTTCTTCTCATTTCGA- × Although based on a reduced number of patients, our CAC CG), 2 mm of each dNTP, 2 mm MgCl2,1 Taq study included only males from the same region. In polymerase buffer, and 0.5 U of Taq DNA polymerase. addition, the CCR5 and CCR2-polymorphisms were in After 32 cycles of 95°C for 20 s, 62°C for 1 min and 72°C the Hardy–Weinberg equilibrium in our population, sug- for 30 s, 15 ␮l of the reaction was electrophoresed on a 3% gesting the absence of population biases. The role of the agarose gel and alleles visualised, after ethidium bromide CCR5 polymorphism in modulating the origin of the staining, as fragments of 222 bp (wild-type allele) and 190 atherosclerotic plaque cannot be established from our bp (⌬ccr5-allele). work. This would require us to genotype a group of old- For the analysis of the G/A polymorphism at codon healthy controls with MI risk factors and without dis- 64 (Val/Ile) of the CCR2 gene a 215 bp sequence was eased vessels. However, coronariographic analysis is amplified with primers fwd.-ATGCTGTCCACATC expensive and potentially dangerous, and was only per- TCGTTCT and rvs.-TTTTTGCAGTTTATTAAGATGA formed in the patients as a diagnostic tool. GT. PCR conditions were as described for the CCR5 Finally, the role of CCR5 (and other proinflammatory sequence, with an annealing temperature of 62°C. Ten ␮l receptors) in HIV infection and AIDS development has of each reaction were digested with 10 U of the restriction made the development of drugs that target these recep- enzyme RsaI, and electrophoresed on a 4% agarose gel. tors a major goal of research. Homozygotes for the ⌬ccr5 Alleles were visualised as fragments of 191 bp (64V) and mutation are a natural knock-out for the CCR5 gene. 215 bp (64I). These individuals are healthy, and the specific pharmacological targeting of CCR5 in order to abolish its function Statistical analysis would not have undesirable symptoms. If works on other The chi-square test was used to compare gene and geno- populations confirm the role of the genetic variation at type frequencies between the groups. Odds Ratios (OR’s) and their 95% confidence interval (CI) were also calcu- the CCR5 in modulating the onset of MI, anti-CCR5 Ͻ drugs could be a valuable therapy in the treatment of lated. 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