There is increasing evidence from numerous studies to indicate that the over-recruitment of leukocytes, especially monocytes, is central to the pathology of atherosclerosis [1–4]. Consequently, atherosclerosis is considered now to be an inflammatory disease. Chemokines are inflammatory chemotactic cytokines that play a role in leucocyte recruitment and the chemokine CCL2 (monocyte chemoattractant protein-1 or MCP-1) has been found to be highly expressed in human atherosclerotic plaques [5]. CCL2 attracts monocytes as well as T lymphocytes and mediates its effects by binding to G protein coupled receptors expressed on the surface of target cells [6]. The most prominent receptor for CCL2 is CCR2 [7] and its binding to this receptor has been shown to result in recruitment of monocytes into the subendothelial space [8]. Further evidence for the role of CCL2 and its receptor CCR2 in the pathology of atherosclerosis has come from gene knock-out experiments in mice. CCR2 (-/-) mice show defects in leukocyte adhesion and monocyte/macrophage recruitment [9]. The absence of CCR2 on macrophages protects those mice from developing atherosclerosis [10–12]. Moreover, mice lacking the ligand CCL2 (-/-) also showed decreased lesion size and a significant reduction of macrophages in lesions [13]. In addition, overexpression of CCL2 results in an increase in macrophage number within the lesions [14]. Taken together, these studies strongly suggest a direct role for CCL2 and CCR2 in monocyte recruitment and in the pathology of atherosclerosis. The CCR2 gene comprises two exons and is subject to alternative splicing [6]. A G to A coding polymorphism in exon 2 of the gene has been reported which results in a Val to Ile substitution at position 64. Evidence for this polymorphism having an effect on the function of CCR2 comes from studies of HIV infected individuals. CCR2 and CCR5 are required for the entry of the virus into lymphocytes. Those individuals carrying the CCR2 Ile allele were found to progress to AIDS 2-4 years later than individuals homozygous for the common Val allele [15]. We and others have reported on the role of this polymorphism in the development of coronary artery disease, but the findings do not all appear to be in agreement. In our previous study [16] we looked at the effect of the Val64Ile polymorphism in a cohort of first degree relatives of patients with premature coronary artery disease. The extent of coronary artery calcification was found to be significantly lower in subjects with the CCR2 Ile64 variant than in subjects carrying two Val64 alleles which we interpreted as being beneficial. A similar conclusion was drawn by Szalai et. al. [17] based on the absence of any CCR2 Ile64 homozygotes in the cases group of a CAD case-control study. In contrast, Ortlepp et. al. [18] found the Ile64 allele to be associated with an increased risk for MI (myocardial infarction) and heart failure in subjects of age <65 y in a large cohort of patients who underwent left ventricular catheterization and also Gonzalez et. al. [19] were unable to find a protective effect of the Ile64 allele MI in a case control study. To investigate further our previous suggestion for a protective effect of Ile64 in coronary atherosclerosis, we examined the role of the Val64Ile polymorphism in a nested case-control sample from the West of Scotland Coronary Prevention (WOSCOPS) study.

For a complex pathology such as coronary artery disease, there are likely to be many susceptibility genes which play a role in determining the phenotype [22]. Based upon previous work implicating the CCR2 gene in the progression of atherosclerosis, we sought to further assess the role of the Val64Ile polymorphism in the clinical endpoint of coronary artery disease. In this study we examined the genotypes of 443 cases and 1003 control individuals from the WOSCOPS study. The results obtained for both the combined and single endpoint analyses did not indicate a significant difference in genotype frequencies between cases and controls and so do not suggest a significant role for this polymorphism in the development of coronary artery disease. A number of recent studies have looked at the association of the CCR2 Val64Ile polymorphism and clinical endpoints associated with atherosclerosis. In our previous study the extent of coronary artery calcification was significantly lower in subjects with the CCR2 Ile64 variant than in subjects carrying two Val64 alleles [16] suggesting a beneficial effect for the Ile64 allele. Coronary artery calcification has previously been shown to be correlated with the extent of coronary atherosclerotic lesions [23]. The results of our present study are not in agreement with our previous study and there are several potential explanations for this. Firstly the definition of cases was different. Valdes et al. defined cases as male or female first degree relatives of subjects with premature coronary artery disease and there were various exclusion criteria including smoking status. Subjects were non-Hispanic whites from the Pennsylvania area. In the Woscops study, all subjects were men from the west of Scotland with pre-determined cut off values for cholesterol and LDL/cholesterol and smokers were included. The phenotypes are also different between the studies. Valdes et al. measured coronary artery calcification (CAC) using tomography. In Woscops the endpoints were death from coronary artery disease (CAD), non-fatal acute myocardial infarction, or re-vascularisation. In addition the sample sets were different with 400 cases and 1003 controls for this study and 662 subjects in the CAC study. In Valdes et al, the genotype frequencies were compared between individuals in a high or low CAC value group whereas the cases in Woscops were those with the aforementioned endpoints. Also in Woscops, each case was matched to 2 controls. It is possible that these differences in study design or geographical location of subjects could account for the different conclusions from the two studies examining the same Val64 Ile polymorphism. Other evidence attributing a role for CCR2 in the development of atherosclerosis has been shown [18]. In contrast, here the Ile allele was found to be positively associated with myocardial infarction and heart failure in patients less than 65 years of age. There was no association of the CCR2 genotype with atherosclerosis, but the CCR2 Ile allele appeared to predispose patients to myocardial infarction before 65 years of age. In other work, the role of CCR2 Val64Ile was not supported in studies of both myocardial infarction and coronary artery disease as diagnosed by angiography in patients versus controls [19, 24]. Also recently Bjarnadottir et al. [25] were unable to find any effect of the CCR2 Val64Ile mutation in a large Icelandic retrospective nested case-control study of MI survivors. Again it cannot be excluded that the different conclusions of these studies could be related to differences in study design, clinical endpoints of the coronary artery disease phenotype or that subjects were derived from different geographical populations where there are differences in genetic or environmental factors [26]. The WOSCOPS sample set is a large, well characterised cohort comprising individuals from the same geographical location. In summary, the results of this present study do not support a role for the CCR2Val64Ile polymorphism in the endpoints of death from coronary artery disease (CAD), non-fatal acute myocardial infarction, or re-vascularisation.

This CCR2 association study does not confirm a role for the CCR2 Val64Ile polymorphism in the development of coronary artery disease.