点击显示 收起
From Laboratoire d’Immunologie Cellulaire, INSERM U543 (E.L., M.D., P. Debré, P. Deterre, C.C.) and the INSERM U525 (F.C.), H?pital Pitié-Salpêtrière, Paris, France; and the Service de Neurologie (J.L., P.A.), CHU Bichat, Paris, France.
Correspondence to Christophe Combadière, INSERM U543, Laboratoire d’Immunologie Cellulaire, H?pital Pitié-Salpêtrière, 91 Boulevard de l’H?pital, 75654 Paris, cedex 13, France. E-mail combad@ccr.jussieu.fr
Abstract
Objective— We investigated the role of monocyte-recruiting chemokines in cerebrovascular diseases among the subjects of the GENIC case-control study of brain infarction (BI).
Methods and Results— Of the genotypes tested, only homozygosity for the rare CX3CR1 alleles was more frequent in cases than in controls: the I249 and M280 alleles were associated with an increased risk of BI (OR, 1.66 and OR, 2.62 with P<0.05, respectively). This effect was independent of other established risk factors and uncorrelated with disease severity. The study confirmed previous reports of a dominant protective association between CX3CR1-I249 allele and the risk of cardiovascular history. The risk of BI associated with homozygosity for the rare CX3CR1 alleles was enhanced in patients with no previous cardiovascular events. Ex vivo studies showed that the number of monocytes adhering to immobilized CX3CL1, the CX3CR1 ligand, increased proportionally to the number of CX3CR1 mutated alleles carried by the individual.
Conclusions— The rare CX3CR1 alleles were associated with an increased risk of BI and with reduced frequency of cardiovascular history. We propose that the extra adhesion of monocytes observed in individuals carrying rare alleles of CX3CR1 may favor mechanisms leading to stroke.
We analyzed the role of monocyte-recruiting chemokines in cerebrovascular diseases among the subjects of a case-control study of brain infarction (BI). Of the alleles tested, only homozygosity for the CX3CR1 rare alleles was associated with increased risk of BI. The associations may be related to CX3CL1-mediated monocyte adhesion.
Key Words: cerebral infarction ? genetics ? leukocytes ? chemokines ? inflammation ? risk factors
Introduction
Identifying factors that predispose to atherosclerosis is a major public health challenge for this disease that can have profound clinical effects, including myocardial infarction (MI) and brain infarction (BI), both leading causes of death in developed countries.1 Environmental factors, disease conditions, and known and yet-to-be-discovered genetic determinants may control the pathogenesis of this disease,2–4 which can be considered to be an unusual inflammatory process occurring within the artery wall and leading to leukocyte infiltration into the lesion.5 Monocyte infiltration and macrophage accumulation beneath the endothelial cell layer appear to be critical events in the genesis of early lesions, in disease progression (chronic lesions), and during acute complications.
Recent studies show that chemokines and their receptors play a key role in atherogenesis.6,7 These molecules orchestrate leukocyte migration and thus may directly control the infiltration of monocytes, T cells, and smooth muscle cells into atherosclerotic lesions. Their role in atherogenesis has been inferred from murine models, as well as from the expression of chemokines in human atherosclerotic lesions and of their receptors by inflammatory cells within lesions. Deletion of genes coding for CCR2,8,9 its ligand CCL2,10 CX3CR1,11,12 or CXCR213 decreases lesion formation in murine models of atherogenesis. As potent mediators of monocyte migration and macrophage differentiation, chemokines, in particular CCL2,14 CCL3,15 CCL13, CCL22, and CX3CL1,16,17 have been detected in primary human atherosclerotic lesions. In humans, these chemokines are assumed to enable the selective recruitment of monocytes that express their cognate receptors (such as CCR2 and CX3CR1).
Epidemiological studies have also helped to uncover the critical effect of chemokines and their receptors in cardiovascular disease susceptibility. We identified 2 common single-nucleotide polymorphisms in the open reading frame of the CX3CR1 gene, called V249I and T280M;18 both are associated with a reduced risk of MI.19 Two studies by McDermott et al report that less common alleles of CX3CR1 are associated with a reduced risk of coronary artery diseases (CAD) in a case-control study20 and with protection from cardiovascular disease in a population-based study.21 All of the published studies consistently report these associations between the CX3CR1 polymorphism and the risk of CAD. More recent findings associate rare CX3CR1 alleles with a reduced risk of internal carotid artery occlusive disease and carotid plaque stability.22 More conflicting or unconfirmed reports connect other chemokine and receptor polymorphisms with CAD as well.23
Although many studies have focused on CAD, very few reports examine the potential role of chemokines and their receptors in cerebrovascular pathologies. We decided to extend our investigation of the role of chemokines in these syndromes and therefore evaluated a number of chemokines and their receptors in the GENIC case-control study of BI.
Methods
Study Population
The GENIC ("profil GENétique de l’Infarctus Cérébral") case-control study recruited 510 consecutive patients from all patients admitted to 12 French neurological departments. Controls (n=510) with no history of stroke were recruited from individuals hospitalized at the same institutions for any reason other than neurological disease. Stratification by age (±5 years) and sex was used to match case and control patients. Controls with a cardiovascular history other than stroke were eligible if they had 2 white parents. Two neurologists classified patients into etiologic BI subtypes, as previously described.24
Data Collection and Risk Factor Definition
A structured questionnaire was used to collect information about all subjects. We defined hypertension as a history of treated hypertension and a positive cardiovascular history as a history of MI, angioplasty, coronary artery bypass surgery, or lower-limb arterial disease. We classified subjects as having diabetes if they reported being treated for insulin-dependent or noninsulin-dependent diabetes. Use of lipid-lowering drugs was assessed.
CX3CR1, CCR2, CCR5, and CCL2 polymorphisms were identified with Minor Groove Binder polymerase chain reaction-based amplification technology from Perkin Elmer (Applied Biosystems Division, Cheshire, UK). The primer express program (Perkin Elmer) was used to design the probes and primers (Table I, available online at http://atvb.ahajournals.org). Each polymerase chain reaction contained 10 to 200 ng of genomic DNA, 900 nM primers, 200 nM probes, and 6 μL of TaqMan Universal polymerase chain reaction master mix (Perkin Elmer). Amplification took place under the following conditions: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 62°C for 1 minute. Real-time detection and analysis were performed on an ABI 7700 thermocycler (Perkin Elmer).
Cells
Peripheral blood mononuclear cells were isolated from heparinized venous blood from healthy volunteers by 1-step centrifugation on a Ficoll separating solution (Biochrom KG, Berlin, Germany). Monocytes isolated by a negative procedure (Dynal Biotech ASA, Oslo, Norway) according to the manufacturer’s instructions were 80% to 95% pure (confirmed by flow cytometric analysis). CX3CL1-expressing human embryonic kidney (HEK) cells were obtained as previously described.25
Chemotaxis was performed in a 96-well chemotaxis chamber with a filter porosity of 10 μm (NeuroProbe, Cabin John, Md) as previously described.25 Adhesion experiments used the conventional parallel-plate flow technique.25 Briefly, CMFDA-labeled monocytes were allowed to interact with HEK cells in the flow chamber at controlled shear stress and the adherent cells were counted.
Statistical Analysis
Allelic frequencies were calculated by gene counting. Hardy-Weinberg equilibrium was tested with the 2 statistic. To compare the overall distribution of the genotypes of each polymorphism among cases and controls, we used the –2 log-likelihood statistic of a logistic regression model adjusted for matching variables (ie, age, sex, and center). To ensure all genotyped controls and cases were entered in the analysis, we did not use a conditional logistic regression analysis for matched sets. For each CX3CR1 polymorphism, genotypic odds ratios (ORs) for BI were calculated by a logistic regression adjusted for matching variables (ie, assuming a codominant genetic model). No excess risk of BI was associated with heterozygosity for either CX3CR1 polymorphism. ORs were therefore computed on the assumption of a recessive genetic model. The log-likelihood of this model was not significantly different from that of a codominant genetic model. Our analyses concerned the entire study group and were subsequently stratified according to the main BI subtypes (ie, atherothrombotic strokes, lacunar strokes, cardioembolic strokes, and strokes of unknown cause); in each stratum, cases were compared with their matched controls. Analyses concerning strokes of undetermined cause are not reported, because this is, by definition, a highly heterogeneous group. The homogeneity across the main subtypes of the association between each CX3CR1 polymorphism and the disease was assessed with the Breslow-Day heterogeneity test. Haplotype analysis was performed to better-characterize the contribution of CX3CR1 polymorphisms to the risk of BI. Pair-wise haplotype disequilibrium was estimated with a log-linear model analysis.26 As previously reported,18–21 the CX3CR1-V249I and T280M polymorphisms were in complete linkage disequilibrium; they generated 6 combined genotypes and 3 haplotypes (VT, IT, and IM). ORs for BI associated with IT and IM by reference to VT were calculated by logistic regression adjusted for matching variables, assuming dominant and recessive models. Polymorphisms and haplotype analysis were adjusted for cerebrovascular risk factors (ie, history of hypertension and diabetes, current smoking, total and low-density lipoprotein cholesterol, and cardiovascular history). The homogeneity of the ORs across cerebrovascular risk factors was tested by introducing the corresponding interaction term into models. Statistical testing was 2-tailed, with set at 0.05. Data were analyzed with the SAS package (SAS Institute, Cary, NC).
Results
Association Between BI and Established Risk Factors
To evaluate the impact of chemokines and their receptors in stroke, we selected a panel of compelling candidate molecules (those previously reported to have genetic variations involved in atherosclerosis, such as CCR532, CCR2-I64 and its ligand CCL2-G2578, CX3CR1-I249, and CX3CR1-M280) and studied their allelic distribution in the GENIC cohort. Table 1 summarizes the general characteristics of the study subjects. Case subjects had a higher frequency of cerebrovascular risk factors (smoking, hypertension, hypercholesterolemia, and diabetes) and reported a cardiovascular history more frequently than controls.
TABLE 1. General Characteristics and Conventional Risk Factors for BI in Patients and Controls in the GENIC Study
Distribution of Chemokine and Chemokine Receptor Polymorphisms in the GENIC Study Population
Table 2 shows the distribution of the various genotypes among the case and control subjects. The allelic frequency of each rare allele fell within the range reported in previous studies. The frequencies of the polymorphisms of chemokine receptors and chemokines did not deviate from Hardy-Weinberg equilibrium among cases or controls (data not shown). The similarity of the genotype distribution of the CCR532 and CCL2-G2578 alleles among cases and controls (P=0.98 and P=0.45, respectively) indicated that these polymorphisms had no significant effect on the BI risk. The proportion of individuals carrying the less common CCR2 allele was slightly higher for cases than controls, but not significantly (P=0.13). The difference in the proportions of those carrying the rare CCR2-I64 allele (heterozygous or homozygous) between the case and control groups was just on the borderline of significance (P=0.05). Cases were homozygous for the CX3CR1-I249 or the CX3CR1-M280 alleles more often than controls were (Table 2; P=0.07 and P=0.04, respectively). When we consider the reference group of subjects homozygous for the frequent allele (VV or TT, respectively), we see that heterozygosity for CX3CR1 polymorphisms did not increase BI risk (Table 3). We therefore computed ORs associated with homozygosity for the I249 allele (OR, 1.68; 95% confidence interval [CI],1.04 to 2.74) and for the M280 allele (OR, 2.89; 95% CI,1.23 to 6.47). After adjustments for other cerebrovascular risk factors such as history of hypertension and diabetes, total and low-density lipoprotein cholesterol, current smoking, and cardiovascular history, the associations between BI and the CX3CR1 polymorphisms were slightly attenuated but remained significant (Table 3). No heterogeneity in the association between CX3CR1 polymorphisms and BI was observed according to BI subtype (Table 4), but we note that the power of the analysis was limited by the relatively small number of patients in each category. In addition, the CX3CR1 polymorphisms were not statistically associated with disease severity, as assessed by intima-media thickness, handicap scores, or mortality (data not shown).
TABLE 2. Distribution of Genotypes for Polymorphisms in Chemokines and Chemokine Receptors in Cases and Controls
TABLE 3. Odd Ratios for BI Associated With CX3CR1 Polymorphisms
TABLE 4. Association Between CX3CR1 Polymorphisms and Main BI Subtypes
Enhanced Risk of BI Associated With Rare CX3CR1 Alleles in Patients With No Cardiovascular History
The association of CX3CR1-I249 and CX3CR1-M280 alleles with an increased risk of BI was intriguing because previous work, including ours, demonstrates that CX3CR1 alleles are associated with a reduced risk of cardiovascular events.19–21 Consistently with these previous findings, among cases, cardiovascular history was less frequent in those with at least 1 CX3CR1-I249 allele (26.7% VV, 18.7% VI, 10.9% II; P=0.01). A similar association was observed with the CX3CR1-M280 allele (24.8% TT, 18.6% TM, 4.8% MM; P=0.04). Haplotype analysis sought to identify alleles associated with risk of BI (Table 5). Only the IM haplotype was positively associated with BI under the recessive model (OR, 2.68; 95% CI, 1.16 to 6.19). Similar results were found after adjustment for cerebrovascular risk factors. Because we had found an interaction between CX3CR1 polymorphisms and cardiovascular history (P<0.05), we performed a subgroup analysis according to cardiovascular history. IM homozygosity was associated with an increased risk of BI only among subjects with no cardiovascular history, with an adjusted OR of 4.18 (95% CI, 1.49 to 11.74). In subjects with cardiovascular history, the IM haplotype was associated with a tendency toward reduced risk of BI (OR, 0.09; 95% CI, 0.01 to 1.96). These results were limited, however, by the relatively small number of homozygotes for the rare allele with cardiovascular history.
TABLE 5. Odd Ratios for BI Associated With IT and IM Haplotypes
CX3CR1 Genotypes and Monocyte Cell Functions
To investigate how CX3CR1 polymorphisms might affect predisposition to BI or CAD, we evaluated the functional impact of these CX3CR1 allelic variants on monocytes, the cells considered to be key players in atherogenesis. An in vitro chemotactic assay tested cells’ response to soluble CX3CL1 and a cell adherence assay tested their response to membrane-anchored CX3CL1. First, we compared the transmigration of monocytes from subjects homozygous for the CX3CR1-V249 allele with that of monocytes from those heterozygous and homozygous for the CX3CR1-I249 allele (Figure A). We detected no difference in the migratory capacity of monocytes from individuals carrying these various genotypes. Comparing the chemotactic response of monocytes from individuals with the various CX3CR1 T280M genotypes yielded similar results (data not shown). We then compared the adhesion of monocytes from individuals with different CX3CR1 genotypes to a monolayer of CX3CL1-expressing HEK (parallel-plate adhesion method). At a low perfusion rate (1.5 dynes.cm–2), the monocytes carrying 2 CX3CR1-I249 alleles were captured in significantly larger numbers than the cells with 1 I249 allele (Figure B, left). The latter, in turn, were more adherent than monocytes with 2 V249 alleles. Similar effects were observed when monocyte adherence was analyzed as a function of the M280 allele (Figure B, right). These effects also remained after increasing the shear stress to 15 dynes.cm–2 (data not shown). The differences we observed in the adhesion behavior of cells with the CX3CR1 variants are not because of differences in receptor expression; flow cytometric analysis indicated the similarity of the frequency and mean fluorescence intensity of CX3CR1-positive monocytes in peripheral blood mononuclear cells from individuals carrying the different CX3CR1 genotypes.
Migration and adhesive capacities of monocytes from individuals with various CX3CR1 genotypes. A, Monocytes from individuals homozygous for the CX3CR1-V249 allele (VV; black square, n=5) and heterozygous or homozygous for the CX3CR1-I249 allele (VI; white square, n=3; and II; white circle, n=3) were assayed in a transmigration assay. Data are summarized as the mean±SEM of triplicates from 4 independent experiments and presented as chemotactic index. B, Monocytes from the same individuals were also tested for adhesion in a parallel-plate laminar flow chamber, with coverslips coated with adherent CX3CL1-expressing HEK cells. Adherent monocytes were counted after each flow change. Monocytes from each individual were tested in duplicate in 2 independent experiments. Numbers of individuals per group are indicated above each bar. *P<0.05 between 2 genotype groups.
Discussion
This study shows that only the rarer CX3CR1 alleles, of all the chemokine and receptor alleles tested, were associated with an increased BI risk and they had no effect on disease severity. We also characterized the impact of mutated CX3CR1 alleles on monocyte functional response and found that the number of monocytes adhering to CX3CL1 increases with the number of I249 alleles that an individual carries. These results support the hypothesis that CX3CR1 plays a pathogenic role in cerebrovascular disease.
A few studies report that some chemokine receptor gene variants are associated with susceptibility to CAD.19–21,27–29 Our study is the first to our knowledge to indicate that the CX3CR1-I249 allele and its genetically linked partner, the CX3CR1-M280 allele, are associated with an increased risk of BI. Because of the linkage disequilibrium between M280 and I249, it is difficult to distinguish the independent contribution of each of these polymorphisms to BI risk. These polymorphisms are not, however, associated with disease severity, as measured by intima-media thickness, handicap scoring, or mortality (data not shown). These results are especially intriguing because previous work, including ours, demonstrated that the rare CX3CR1 allele is associated with a reduced risk of cardiovascular diseases.19–21 The effect in each of these studies was dominant, whereas the association with increased BI risk we describe here appeared to be recessive. This difference suggests that different cellular pathways may underlie the mechanisms of these 2 conditions.
Of the other alleles tested, the 32-bp deletion in the CCR5 open reading frame, which causes the loss of CCR5 receptors on the lymphoid cell surface,30 was not significantly associated with either BI risk or cardiovascular history. This finding is concordant with previous epidemiological data20 as well as with a model of atherogenesis in mice deficient for CCR5 and apolipoprotein E genes.31 Other reports indicate that the CCR532 allele reduces risk for early MI32 and severe CAD27 by attenuating inflammatory responses. The role of the CCR2/CCL2 axis in atherogenesis is well-established in murine models: both types of deficient mice have fewer and less severe atherosclerotic lesions.8–10 CCL2 antagonist has proven potent in controlling plaque formation in mice.33 In humans, a substitution in the distal regulatory region of the CCL2 gene (CCL2-G2578) has been shown to modulate CCL2 production34 and to be associated with a reduced risk of severe CAD.27 Here, we found no association between the less common CCL2 allele and the risk of BI or cardiovascular history. The situation is even more complex for the CCR2-I64 allele, which has a dominant borderline association with increased BI risk (P=0.05). These results are ambiguous, probably because of this allele’s low frequency, and they do not clarify the initial observation that CCR2-I64 is associated with a reduced risk of MI27 or the subsequent reports that it predisposes its carriers to MI.28,29 Further work is needed to clarify the pathogenic impact of these alleles.
In view of its prognostic power for AIDS18,35 and cardiovascular and cerebrovascular diseases,19–21 understanding the molecular defect caused by CX3CR1 mutations is an important challenge. A recent study reports a global impairment of the CX3CR1-M280 cell response to CX3CL1, ie, ligand binding, calcium response, and adhesive and chemotactic functions.21 We could not confirm these results and obtained opposite results. Monocytes from individuals carrying the CX3CR1-I249-M280 allele were captured in larger numbers than monocytes from those carrying CX3CR1-V249-T280 allele. This set of results was similar to those we obtained with human peripheral blood mononuclear cells of various genotypes and HEK cells transfected with CX3CR1 variants,25 and with 2 different techniques of testing adherence. We do not yet have a reasonable explanation for the discrepancies with the previous report from McDermott, but it may be interesting to consider sets of data as reflecting different CX3CR1 functional states. The apparent discrepancies may be artifactual and stem from subtle differences in the techniques or cell environment used. Further work to identify the various transduction steps underlying the CX3CR1-I249-M280 effect might help to clarify these conflicting data.
Monocytes recruited in the intima layer to form atherosclerotic plaque first adhere to and cross the endothelial barrier.7,36 Umehara et al suggested, based on an in vitro chemotaxis assay, that membrane-bound CX3CL1 expressed on endothelial cells strengthens monocyte adhesion and thus decreases transmigration of monocytes across endothelial cell monolayers.37 This phenomenon may reduce atherosclerosis. The decreased lesion infiltration may mimic the pathological phenotype observed in mice deficient for apolipoprotein E and CX3CR1 genes; the first step of monocyte recruitment is unfavorable in these mice because there are fewer monocyte anchors to the endothelial wall.11,12 Platelets that express CX3CR138 play a key role in the pathogenesis of atherothrombotic conditions, eg, acute coronary syndromes and cerebrovascular events. In these disease conditions, overexpression of CX3CL1 by vascular endothelial cells may promote platelet adhesion and activation. It is thus likely that CX3CR1 rare allele may favor proatherogenic conditions. Interestingly, platelet activity as measured by mean platelet volume was positively associated with increased risk of BI but not with major coronary events.39 We propose that the level of chemokines produced by different vascular beds may determine the proatherogenic effect of platelets. In addition, Yoneda et al report a positive correlation between NK cell adhesion to endothelial cells and susceptibility to NK cell-mediated cytolysis.40 They also found that soluble CX3CL1 markedly suppressed both of these enhanced functions. Thus, CX3CL1, in addition to its monocyte and platelet tethering roles, may favor NK cell-mediated endothelium damage, which can ultimately promote atherothrombotic conditions, as McDermott et al have proposed.20 More globally, it is conceivable that the risk of cardiovascular or cerebrovascular disease depends on the relation between the systemic release of CX3CL1 that promotes CX3CR1-expressing cell activation and the competing locally expressed membrane-bound CX3CL1, which promotes CX3CR1-expressing cell adherence. To clarify these issues, the CX3CR1-mediated cellular mechanisms predisposing to BI must be characterized.
Consideration of potential mechanisms whereby the CX3CR1-I249M280 haplotype might predispose carriers to BI is also important. We observed the relation between the CX3CR1 polymorphism and BI only with the homozygous genotype, even though the in vitro data from the adhesion assay were significant with the heterozygous genotype. One way to reconcile these 2 sets of data is to hypothesize that the extra monocyte adhesion is not biologically relevant until it exceeds a threshold reached only by individuals homozygous for these rare alleles. The in vitro assay that revealed the specific functional characteristic of the rare allele may not reflect physiological flow conditions; therefore, the discrimination between heterozygous and homozygous cells may be artifactual.
This work showed that naturally occurring mutations in CX3CR1 have a severe impact on susceptibility to BI and CAD. Because of the complexity of pathologies underlying ischemic stroke, which develop over many years and are likely affected by many different factors, further studies are needed to confirm these findings. This work further revealed that CX3CR1 might be considered a powerful prognostic tool and an excellent therapeutic target for cardiovascular and cerebrovascular diseases.
Acknowledgments
This study was supported by a grant from the Fondation de France.
References
Lopez AD, Murray CC. The global burden of disease, 1990–2020. Nat Med. 1998; 4: 1241–1243.
Marenberg ME, Risch N, Berkman LF, Floderus B, de Faire U. Genetic susceptibility to death from coronary heart disease in a study of twins. N Engl J Med. 1994; 330: 1041–1046.
Nora JJ, Lortscher RH, Spangler RD, Nora AH, Kimberling WJ. Genetic-epidemiologic study of early-onset ischemic heart disease. Circulation. 1980; 61: 503–508.
Izawa H, Yamada Y, Okada T, Tanaka M, Hirayama H, Yokota M. Prediction of genetic risk for hypertension. Hypertension. 2003; 41: 1035–1040.
Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.
Lucas AD, Greaves DR. Atherosclerosis: role of chemokines and macrophages. Expert Rev Mol Med. 2001; 3: 1–18.
Reape TJ, Groot PH. Chemokines and atherosclerosis. Atherosclerosis. 1999; 147: 213–225.
Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998; 394: 894–897.
Dawson TC, Kuziel WA, Osahar TA, Maeda N. Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice. Atherosclerosis. 1999; 143: 205–211.
Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998; 2: 275–281.
Lesnik P, Haskell CA, Charo IF. Decreased atherosclerosis in CX3CR1–/– mice reveals a role for fractalkine in atherogenesis. J Clin Invest. 2003; 111: 333–340.
Combadiere C, Potteaux S, Gao JL, Esposito B, Casanova S, Lee EJ, Debre P, Tedgui A, Murphy PM, Mallat Z. Decreased Atherosclerotic Lesion Formation in CX3CR1/Apolipoprotein E Double Knockout Mice. Circulation. 2003; 107: 1009–1016.
Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice. J Clin Invest. 1998; 101: 353–363.
Nelken NA, Coughlin SR, Gordon D, Wilcox JN. Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 1991; 88: 1121–1127.
Wilcox JN, Nelken NA, Coughlin SR, Gordon D, Schall TJ. Local expression of inflammatory cytokines in human atherosclerotic plaques. J Atheroscler Thromb. 1994; 1 (Suppl 1): S10–S13.
Greaves DR, Hakkinen T, Lucas AD, Liddiard K, Jones E, Quinn CM, Senaratne J, Green FR, Tyson K, Boyle J, Shanahan C, Weissberg PL, Gordon S, Yla-Hertualla S. Linked chromosome 16q13 chemokines, macrophage-derived chemokine, fractalkine, and thymus- and activation-regulated chemokine, are expressed in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2001; 21: 923–929.
Lucas AD, Bursill C, Guzik TJ, Sadowski J, Channon KM, Greaves DR. Smooth muscle cells in human atherosclerotic plaques express the fractalkine receptor CX3CR1 and undergo chemotaxis to the CX3C chemokine fractalkine (CX3CL1). Circulation. 2003; 108: 2498–2504.
Faure S, Meyer L, Costagliola D, Vaneensberghe C, Genin E, Autran B, Delfraissy JF, McDermott DH, Murphy PM, Debre P, Theodorou I, Combadiere C. Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1. Science. 2000; 287: 2274–2277.
Moatti D, Faure S, Fumeron F, Amara M, Seknadji P, McDermott DH, Debre P, Aumont MC, Murphy PM, de Prost D, Combadiere C. Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery disease. Blood. 2001; 97: 1925–1928.
McDermott DH, Halcox JP, Schenke WH, Waclawiw MA, Merrell MN, Epstein N, Quyyumi AA, Murphy PM. Association between polymorphism in the chemokine receptor CX3CR1 and coronary vascular endothelial dysfunction and atherosclerosis. Circ Res. 2001; 89: 401–407.
McDermott DH, Fong AM, Yang Q, Sechler JM, Cupples LA, Merrell MN, Wilson PW, D’Agostino RB, O’Donnell CJ, Patel DD, Murphy PM. Chemokine receptor mutant CX3CR1–M280 has impaired adhesive function and correlates with protection from cardiovascular disease in humans. J Clin Invest. 2003; 111: 1241–1250.
Ghilardi G, Biondi ML, Turri O, Guagnellini E, Scorza R. Internal carotid artery occlusive disease and polymorphisms of fractalkine receptor CX3CR1: a genetic risk factor. Stroke. 2004; 35: 1276–1279.
Bursill CA, Channon KM, Greaves DR. The role of chemokines in atherosclerosis: recent evidence from experimental models and population genetics. Curr Opin Lipidol. 2004; 15: 145–149.
Elbaz A, Poirier O, Canaple S, Chedru F, Cambien F, Amarenco P. The association between the Val34Leu polymorphism in the factor XIII gene and brain infarction. Blood. 2000; 95: 586–591.
Daoudi M, Lavergne E, Garin A, Tarantino N, Debre P, Pincet F, Combadiere C, Deterre P. Enhanced adhesive capacities of the naturally occurring Ile249-Met280 variant of the chemokine receptor CX3CR1. J Biol Chem. 2004; 279: 19649–19657.
Tiret L, Amouyel P, Rakotovao R, Cambien F, Ducimetiere P. Testing for association between disease and linked marker loci: a log-linear-model analysis. Am J Hum Genet. 1991; 48: 926–934.
Szalai C, Duba J, Prohaszka Z, Kalina A, Szabo T, Nagy B, Horvath L, Csaszar A. Involvement of polymorphisms in the chemokine system in the susceptibility for coronary artery disease (CAD). Coincidence of elevated Lp(a) and MCP-1–2518 G/G genotype in CAD patients. Atherosclerosis. 2001; 158: 233–239.
Ortlepp JR, Vesper K, Mevissen V, Schmitz F, Janssens U, Franke A, Hanrath P, Weber C, Zerres K, Hoffmann R. Chemokine receptor (CCR2) genotype is associated with myocardial infarction and heart failure in patients under 65 years of age. J Mol Med. 2003; 81: 363–367.
Petrkova J, Cermakova Z, Drabek J, Lukl J, Petrek M. CC chemokine receptor (CCR)2 polymorphism in Czech patients with myocardial infarction. Immunol Lett. 2003; 88: 53–55.
Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, Goedert JJ, Buchbinder SP, Vittinghoff E, Gomperts E, Donfield S, Vlahov D, Kaslow R, Saah A, Rinaldo C, Detels R, O’Brien SJ. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study . Science. 1996; 273: 1856–1862.
Kuziel WA, Dawson TC, Quinones M, Garavito E, Chenaux G, Ahuja SS, Reddick RL, Maeda N. CCR5 deficiency is not protective in the early stages of atherogenesis in apoE knockout mice. Atherosclerosis. 2003; 167: 25–32.
Gonzalez P, Alvarez R, Batalla A, Reguero JR, Alvarez V, Astudillo A, Cubero GI, Cortina A, Coto E. Genetic variation at the chemokine receptors CCR5/CCR2 in myocardial infarction. Genes Immun. 2001; 2: 191–195.
Ni W, Egashira K, Kitamoto S, Kataoka C, Koyanagi M, Inoue S, Imaizumi K, Akiyama C, Nishida KI, Takeshita A. New anti-monocyte chemoattractant protein-1 gene therapy attenuates atherosclerosis in apolipoprotein E-knockout mice. Circulation. 2001; 103: 2096–2101.
Rovin BH, Lu L, Saxena R. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun. 1999; 259: 344–348.
Faure S, Meyer L, Genin E, Pellet P, Debre P, Theodorou I, Combadiere C. Deleterious Genetic Influence of CX3CR1 Genotypes on HIV-1 Disease Progression. J Acquir Immune Defic Syndr. 2003; 32: 335–337.
Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell. 2001; 104: 503–516.
Umehara H, Goda S, Imai T, Nagano Y, Minami Y, Tanaka Y, Okazaki T, Bloom ET, Domae N. Fractalkine, a CX3C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1. Immunol Cell Biol. 2001; 79: 298–302.
Schafer A, Schulz C, Eigenthaler M, Fraccarollo D, Kobsar A, Gawaz M, Ertl G, Walter U, Bauersachs J. Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion. Blood. 2004; 103: 407–412.
Bath P, Algert C, Chapman N, Neal B. Association of mean platelet volume with risk of stroke among 3134 individuals with history of cerebrovascular disease. Stroke. 2004; 35: 622–626.
Yoneda O, Imai T, Goda S, Inoue H, Yamauchi A, Okazaki T, Imai H, Yoshie O, Bloom ET, Domae N, Umehara H. Fractalkine-mediated endothelial cell injury by NK cells. J Immunol. 2000; 164: 4055–4062.