Published online before print October 1, 2007, doi: 10.1161/CIRCULATIONAHA.107.706986
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(Circulation. 2007;116:1931-1941.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
CC Chemokine Ligand-5 (CCL5/RANTES) and CC Chemokine Ligand-18 (CCL18/PARC) Are Specific Markers of Refractory Unstable Angina Pectoris and Are Transiently Raised During Severe Ischemic Symptoms
From the Department of Cardiology and Einthoven Laboratory of Experimental Vascular Medicine (A.O.K., J.W.J.) and Department of Medical Statistics and Bioinformatics (H.P.), Leiden University Medical Center, Leiden, The Netherlands; Department of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research (A.O.K., S.C.A.d.J., T.J.C.v.B., E.A.L.B.), Leiden University, Leiden, The Netherlands; Department of Paediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s Hospital (W.J.d.J., B.J.P.), Utrecht, The Netherlands; School of Molecular and Biomedical Science (S.R.M.), The University of Adelaide, Adelaide, Australia; Division of Immunological and Infectious Diseases (I.H., L.N.), TNO Prevention and Health, Leiden, The Netherlands; and Department of Pathology (E.A.L.B.), Academic University Hospital Maastricht, Maastricht, The Netherlands.
Correspondence to J.W. Jukema, MD, PhD, Department of Cardiology and Einthoven Laboratory of Experimental Vascular Medicine C5-P, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail j.w.jukema{at}lumc.nl
Received April 24, 2007; accepted August 8, 2007.
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Abstract |
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Background— Chemokines play an important role in atherogenesis and in ischemic injury and repair; however, prospective data on individual chemokines in unstable angina pectoris (UAP) are scarce. Therefore, we assessed chemokine patterns in a prospective cohort of patients with UAP.
Methods and Results— Plasma samples of 54 patients with Braunwald class IIIB UAP were examined at baseline for 11 chemokines and 5 inflammatory mediators via multiplex analysis. Levels of CC chemokine ligand (CCL)-5 (also known as RANTES [regulated on activation, normally T-cell expressed, and secreted]; 32.7 versus 23.1 ng/mL, P=0.018) and CCL18 (also known as PARC [pulmonary and activation-regulated chemokine]; 104.4 versus 53.7 ng/mL, P=0.011) were significantly elevated in patients with refractory ischemic symptoms versus stabilized patients. Temporal monitoring by ELISA of CCL5, CCL18, and soluble CD40 ligand (sCD40) levels revealed a drop in CCL5 and sCD40L levels in all UAP patients from day 2 onward (CCL5 12.1 ng/mL, P<0.001; sCD40L 1.35 ng/mL, P<0.05), whereas elevated CCL18 levels were sustained for at least 2 days, then were decreased at 180 days after inclusion (34.5 ng/mL, P<0.001). Peripheral blood mononuclear cells showed increased protein expression of chemokine receptors CCR3 and CCR5 in CD3+ and CD14+ cells at baseline compared with 180 days after inclusion, whereas mRNA levels were downregulated, which was attributable in part to a postischemic release of human neutrophil peptide-3–positive neutrophils and in part to negative feedback. Finally, elevated CCL5 and CCL18 levels predicted future cardiovascular adverse events, whereas C-reactive protein and sCD40L levels did not.
Conclusions— We are the first to report that CCL18 and CCL5 are transiently raised during episodes of UAP, and peak levels of both chemokines are indicative of refractory symptoms. Because levels of both chemokines, as well as of cognate receptor expression by circulating peripheral blood mononuclear cells, are increased during cardiac ischemia, this may point to an involvement of CCL5/CCL18 in the pathophysiology of UAP and/or post-UAP responses.
Key Words: atherosclerosis • chemokines • ligands • ischemia • leukocytes
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Introduction |
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Acute coronary syndromes, including unstable angina pectoris (UAP), are associated with a high morbidity and mortality. In general, UAP results from erosion or rupture of a vulnerable atherosclerotic plaque superimposed by occlusive thrombus formation and distal ischemia.1 Atherosclerosis is increasingly regarded as a dyslipidemic disorder with a strong inflammatory character.2 These inflammatory processes are orchestrated in part by chemokines, which participate in the inflammatory process by mediating monocyte recruitment to sites of injury, vascular smooth muscle cell proliferation, neovascularization, and platelet activation.3–5 Furthermore, chemokines appear to play a role in cardiac ischemia as well. Indeed, ischemia was reported to lead to induced expression of chemokines in the myocardium or in the circulation, which translates into the recruitment of leukocyte subsets and progenitor cells to the injury zone to contribute to injury repair.6 Given their diverse and deep impact on cardiovascular diseases, chemokines not only might serve as biomarkers of atherosclerosis, plaque disruption, or ischemia but might also represent attractive therapeutic targets.7
Clinical Perspective p 1941
Approximately 50 chemokines have been characterized thus far, and some of these are implicated in atherosclerosis and atherothrombosis.5 In fact, plasma levels of RANTES (regulated on activation, normally T-cell expressed and secreted [also known as CC chemokine ligand-5, or CCL5]), fractalkine (CX3CL1), and monocyte chemotactic protein-1 (MCP-1, or CCL2) have already been shown in various studies to be altered in UAP or myocardial infarction.8–11 Still, prospective data on chemokine plasma levels and chemokine receptor expression by circulating leukocyte subsets in acute coronary syndromes are lacking.
Therefore, the aim of the present study was to asses the levels of 11 chemokines in refractory UAP. We have examined baseline chemokine plasma patterns of a prospective cohort of patients with UAP by a novel, custom-made, high-throughput multiplex assay that allows simultaneous quantification of multiple chemokines in a single plasma sample.12 For prospective analysis, differentially expressed chemokines at baseline were analyzed in follow-up samples by ELISA. Furthermore, peripheral blood mononuclear cells (PBMCs) were examined for chemokine receptor expression. Not only do we show that the rise in CCL5 and CCL18 levels is more pronounced in patients with refractory UAP than in stabilized patients, we also demonstrate that the increase of these 2 chemokines in UAP is transient and accompanied by changes in chemokine receptor expression by circulating leukocytes.
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Methods |
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Study Population
All chemokines and inflammatory parameters were determined in plasma samples of a patient cohort derived from the well-defined APRAIS (Acute Phase ReAction and Ischemic Syndromes) study.13 In brief, 54 patients who were admitted to the emergency department of the Leiden University Medical Center between March and September 1995 with Braunwald class IIIB UAP were included and followed up for as long as 18 months. Venous blood samples were obtained at the time of admission (time [t]=0) and 2 (t=2) and 180 days (t=180) after admission and centrifuged, and plasma aliquots were stored at –80°C until further analysis. All patients had received standard medical therapy (ie, aspirin 300 mg orally, nitroglycerine intravenously, and heparin infusion titrated on the basis of the activated partial thromboplastin time). A clinical end point of the APRAIS study was the occurrence of refractory UAP during hospitalization. UAP was considered refractory if angina at rest, despite medical treatment, remained or reoccurred, prompting invasive coronary assessment and subsequent revascularization therapy. Although the study cohort was relatively small, it constituted a clearly defined, well-documented population with a similar starting point. All subjects gave written informed consent, and the study protocol was approved by the Ethics Committee of the Leiden University Medical Center.
Isolation of Cells
PBMCs from patients (t=0 and t=180) and from 6 healthy age-matched volunteers were isolated as described in the online-only Data Supplement.
Multiplex Chemokine Assay
Circulating levels of the chemokines CCL2, CCL3, CCL5, CCL11, CCL17, CCL18, CCL22, CXCL8, CXCL9, CXCL10, and chemokine-like factor MIF (macrophage migration inhibitory factor), the cytokines OSM (oncostatin M), interferon-
, and osteoprotegerin (OPG), and adhesion molecules sRANKL (soluble receptor activator of nuclear factor-
B ligand), sVCAM (soluble vascular cell adhesion molecule), and sICAM (soluble intercellular adhesion molecule) were determined in t=0 samples with a custom-made multiplex bioassay using the Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, Calif) as described in detail previously14 and in the online-only Data Supplement.
ELISA and Other Assays
For temporal analysis of human CCL5 and CCL18 plasma levels during follow-up, the t=0, t=2, and t=180 samples were assayed with a CCL5 instant ELISA kit (Bender MedSystems, Vienna, Austria) and a CCL18 ELISA (RayBiotech, Norcross, Ga), respectively, according to the manufacturer’s protocol. Baseline inflammatory parameters such as C-reactive protein (CRP), fibrinogen, erythrocyte sedimentation rate, and plasminogen activator inhibitor-1 were determined as described in detail previously.13 Soluble CD40 ligand (sCD40L) and interleukin-6 were determined via a highly sensitive immunoassay (Quantakine HS, R&D Systems, Minneapolis, Minn) and t=180 CRP samples via a turbidimetric assay on a fully automated Modular P800 U (Roche, Almere, The Netherlands).
Assessment of Heterophilic CCL5 and CCL18 Interaction
SDS-PAGE and matrix-assisted laser desorption/ionization–time of flight mass spectrometry was used to assess whether recombinant CCL5 and synthetic CCL18 engage in heterophilic interactions (see online-only Data Supplement).
Reverse-Transcription Polymerase Chain Reaction Analyses
To assess expression of CCL5, CCL18, CCR1, CCR2, CCR3, CCR4, CCR5, CX3CR1, and human neutrophil peptide-3 (HNP-3) in PBMCs, mRNA was isolated and analyzed as described in the online-only Data Supplement.
Flow Cytometry
CCR3 and CCR5 surface expression on CD3+ and CD14+ PBMCs was assessed by flow cytometry (see online-only Data Supplement for further details).
PBMC Stimulation Assay
PBMCs from 6 healthy volunteers were stimulated with CCL5 and CCL18 and assessed for chemokine receptor expression as described in the online-only Data Supplement.
Statistical Analysis
Differences between the present study populations and the original cohort were examined by the Fisher exact test and the Student unpaired t test. Plasma levels of chemokines and inflammatory markers were tested for normal gaussian distribution, and values were log-transformed in the case of a skewed distribution when appropriate. Regarding the latter, geometric instead of arithmetic means are given. Means were compared by unpaired 2-tailed Student t test or Mann-Whitney U test when appropriate. To assess the predictive value of CCL5 and CCL18 for the occurrence of refractory symptoms, independent of potentially confounding factors, a multivariate analysis was performed, with correction for age, HDL cholesterol, and erythrocyte sedimentation rate levels, as well as for other established cardiovascular risk factors (eg, hypertension, hypercholesterolemia, use of lipid- and blood pressure–lowering medication, diabetes mellitus, smoking behavior, body mass index, and history of cardiovascular disease) and biomarkers sCD40L and CRP. Quartile distribution was assessed and used for Spearman’s correlation coefficient and Pearson’s 
2 testing to determine the association of chemokine plasma levels and levels of sCD40L and CRP with the occurrence of refractory UAP. Receiver operating characteristic curves were generated to assess the predictive value of chemokines for refractory ischemic symptoms. Correlation analysis between multiplex and ELISA values and between chemokines and inflammatory parameters were performed by Spearman’s rank correlation test. Fluorescence-activated cell sorter results were analyzed via paired t test; the stimulation assay was analyzed via ANOVA. A 2-sided probability value <0.05 was considered significant. All analyses were performed with SPSS version 14.0 software (SPSS, Chicago, Ill).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Study Population
Plasma analyses on chemokines were performed in a subcohort of previously unfrozen plasma samples of 54 consecutive patients, which precludes selection bias. This subcohort, which consisted of 31 patients with stabilized and 23 with refractory ischemic symptoms, was matched with the original cohort on the basis of cardiovascular risk factors, history of myocardial infarction or percutaneous transluminal coronary angioplasty/coronary artery bypass grafting, and laboratory parameters (Tables 1 and 2
). Because not all 54 patients returned to donate blood after 180 days, ELISA analysis at that point was performed for 47 patients (29 stabilized versus 18 refractory), but the baseline characteristics of this subcohort matched those of the original cohort (data not shown). Comparison for baseline demographics in the chemokine cohort showed no striking differences between patients with refractory versus stabilized symptoms, except for a small but significant difference in gender composition (87% versus 67% males; P=0.05); the mean age of all patients was 65 years (41 to 85 years). With regard to clinical and plasma lipid parameters at baseline, total cholesterol levels in stabilized and refractory patients did not differ (5.92 versus 6.16 mmol/L; P=0.56), whereas HDL cholesterol levels were lower (1.23 versus 0.99 mmol/L; P=0.02) in the refractory group. This group also displayed an increased tendency toward a higher inflammatory status, as illustrated by elevated levels of the erythrocyte sedimentation rate (14.15 versus 20.7 mm/h, P=0.03), although fibrinogen and CRP levels were essentially similar. No differences were observed in baseline sCD40L levels between groups.
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Multiplex Analysis: Upregulation of CCL5 and CCL18
All of the chemokine and cytokine data as determined by multiplex analysis (t=0 samples) were log-transformed before further statistical analysis because of their skewed distribution profiles, except for OPG. Plasma levels of the majority of chemokines and cytokines did not differ between stabilized and refractory patients. CCL5 (23.1 versus 32.7 ng/mL, P=0.018) and CCL18 (53.7 versus 104.4 ng/mL, P=0.011) levels, however, appeared to be significantly increased in refractory patients, whereas a borderline significant increase in CCL3 levels occurred (53.6 versus 73.7 pg/mL, P=0.09; Table 3; Figure 1A). Moreover, the observed differences in CCL5 levels remained significant after multivariate analysis that adjusted for cardiovascular risk factors and sCD40L and CRP levels (P=0.023), whereas differences in CCL18 levels were of borderline significance (P=0.06). However, differences in CCL18 levels reached significance after multivariate analysis for all confounding factors except HDL cholesterol (P=0.021). Therefore, CCL5 and CCL18 appear to be independent predictors of the occurrence of refractory ischemic symptoms, even when adjustment was made for sCD40L and CRP levels. Furthermore, CCL5 and CCL18 levels showed no mutual correlation (R=0.05; P=0.7), which indicates that these chemokines are regulated or operate in an independent manner. Still, although no significant heterophilic interactions between CCL5 and CCL18 were observed, it is conceivable that both chemokines, which share CCR3 as a common target receptor, will interact functionally (online-only Data Supplement, Figures I and II). CXCL10 had a tendency to rise in stabilized patients, although this increase was not quite significant (221.6 versus 157.5 pg/mL, P=0.12), which could point toward a protective effect of this specific chemokine. Levels of interferon- were nearly undetectable and therefore are not shown.
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Next, we sought to assess whether CCL5 and CCL18 levels have diagnostic potential. Given the cohort size, levels of CCL5 and CCL18 were categorized into quartiles and analyzed for correlation with the occurrence of future refractory ischemic symptoms (for quartile distribution, see online-only Data Supplement, Table I). The risk of refractory ischemic symptoms was seen to be increased in the upper quartiles of CCL5 (R=0.32, P=0.017; linear-by-linear association 2 5.53, P=0.019), whereas this trend was even more pronounced for CCL18 (R=0.392, P=0.003; linear-by-linear association 2 8.105, P=0.004; Figure 2A). Elevated CCL18 levels were slightly more predictive than those of CCL5, as indicated by the receiver operating characteristic curve (area under the curve 0.71 versus 0.69). Cutoff values of >40 ng/mL for CCL5 and >130 ng/mL for CCL18 yielded a sensitivity of 73.9% and 65.2%, respectively, and a specificity of 67.7% and 61.3%. Combined analysis of the upper 2 quartiles of CCL5 and CCL18 for the occurrence of refractory ischemic symptoms revealed a very significant relationship (2 with continuity correction 8.12, P<0.01). Although the sensitivity reached 47.8%, the specificity of the combined analysis was a remarkably high 90.3%. The positive predictive value of combined analysis for CCL5 and CCL18 levels was 78.5%, with a concomitant negative predictive value of 70.0%. The addition of sCD40L or CRP levels to the analysis did not yield any further increases in sensitivity, specificity, or predictive value (data not shown).
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CCL5 and CCL18 ELISA Verification and Follow-Up Analysis
Mean and individual ELISA and multiplex CCL5 levels showed excellent correspondence (P<0.001). Moreover, CCL5 plasma levels were also seen to be increased in refractory compared with stabilized patients at day 0 when assessed by ELISA (36.4 versus 26.5 ng/mL). Interestingly, even after just 2 days, a marked decrease in plasma CCL5 levels was observed in the entire cohort (12.1 versus 30.3 ng/mL, P<0.001), and reduced CCL5 levels were also observed at t=180, which demonstrates that CCL5 is transiently raised during an episode of UAP (Figure 1B). We did not observe any differences between the stabilized and refractory groups at 2 and 180 days after inclusion. Plasma levels of CCL18 showed a different temporal pattern after ischemic symptoms. ELISA analysis confirmed the differential expression of CCL18 at day 0 between refractory and stabilized patients (56.2 versus 41.1 ng/mL, P=0.02). Although absolute values were slightly lower with the ELISA than with the multiplex assay, statistical analysis revealed an excellent correlation between the 2 assays (Spearman test P<0.001). Interestingly, CCL18 levels of the total cohort on day 2 did not differ from baseline levels (day 0), which suggests that CCL18 and CCL5 levels might be regulated via separate mechanisms. At 180 days, CCL18 levels were significantly downregulated compared with the day 2 values (48.4 versus 34.5 ng/mL, P<0.001), which suggests a role of CCL18 in cardiac ischemia-reperfusion–related processes (Figure 1C).
sCD40L and CRP
Levels of both sCD40L and CRP were significantly elevated at t=0 compared with t=180 (sCD40L 2.04 versus 0.69 ng/mL, P<0.001; CRP 2.36 versus 0.96 mg/L, P<0.001; Figure 1D and 1E). However, sCD40L levels began to decline at t=2 (1.35 ng/mL, P<0.05), which indicates that elevated levels at baseline reflect a platelet activation–related acute-phase response. Because sCD40L t=0 and t=2 levels correlated significantly with CCL5 t=0 and t=2 levels (t=0 R=0.40, P<0.01; t=2 R=0.35, P=0.01), elevated CCL5 levels may be caused primarily by platelet activation as well. sCD40L, however, showed a significant negative correlation with CCL18 levels at t=0 (R=–0.36, P=0.01), which suggests that the latter represents a feedback response to platelet activation. CRP levels were increased even further at t=2 (6.43 mg/L, P<0.001), which is in keeping with previous reports15,16 and presumably indicative of an enhanced postischemic systemic inflammatory status in these patients 2 days after ischemia and/or coronary intervention. CRP levels showed no correlations with CCL5 or CCL18 levels. Quartile levels of sCD40L and CRP did not have any potential to predict refractory ischemic symptoms (R=0.043 and R=–0.034, P=NS; Figure 2A; for quartile distribution, see online-only Data Supplement, Table II).
Inflammation and Clinical Follow-Up
Correlation analysis for all chemokines with the systemic inflammatory parameters fibrinogen, interleukin-6, plasminogen activator inhibitor-1, and erythrocyte sedimentation rate revealed no association, except for a weak correlation between CXCL10 and interleukin-6 levels (R=0.29; P=0.02, other data not shown). Importantly, the baseline upper-quartile levels of CCL5 as determined by multiplex assay were seen to correlate with the need for revascularization procedures within the next 18 months (R=0.35, P=0.01). Furthermore, baseline upper-quartile levels of CCL18 correlated with the reoccurrence of UAP during hospitalization (R=0.36, P=0.007) and with the occurrence of an acute coronary syndrome during the 18-month period of follow-up (R=0.31, P=0.02; Figure 2B through 2D). Baseline levels of sCD40L and CRP did not correlate with any of the follow-up parameters (data not shown).
PBMC Chemokine and Chemokine Receptor Expression Analysis
Although the interaction of CCL5 with CCR1, CCR3, CCR4, and CCR5 is well described, the actual receptor for CCL18 is unknown, which makes CCL18 currently an orphan ligand.17 However, CCL18 has been reported to be a competitive inhibitor of CCL11 (eotaxin) binding to CCR3.18 Therefore, we examined mRNA expression of the chemokine receptors CCR1, CCR3, CCR4, and CCR5 and that of CCL5 and CCL18 in PBMCs. We observed a remarkable, highly significant downregulation of all 4 involved chemokine receptors at baseline (t=0) compared with PBMCs at t=180 (Figure 3). A similar temporal pattern was seen for CCL5 and CCL18, with CCL5 being abundantly expressed in PBMCs and CCL18 being expressed at only minor levels. To our surprise, subsequent fluorescence-activated cell sorter analysis to detect CCR3 and CCR5 expression on CD3+ T cells and CD14+ monocytes revealed significantly elevated protein expression of CCR3 and CCR5 in both CD3+ and CD14+ cells at t=0 compared with t=180 (Figure 4A through 4D). Triple staining for CD3 or CD14 with CCR3 and CCR5 showed increased chemokine receptor expression in the CD3+ population (3.1% triple-positive cells at t=0 versus 2.3% at t=180, P=0.007), even more prominently so in CD14+ cells (32.1% versus 5.1% triple-positive cells at t=0 and t=180, respectively; P<0.001). An identical pattern was seen for the percentage of CCR3+ and CCR5+ cells and the percentage of combined CCR3+/CCR5+ cells in the total PBMC population (Figure 4G through 4I).
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2-fold downregulated at baseline (B). Values represent mean±SEM. *P<0.05 and **P<0.001.
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To assess whether the reduced gene expression pattern at baseline was caused by transient shifts in the leukocyte distribution profile, we monitored the total percentage of CD14+ (monocytes) and CD3+ cells (T lymphocytes) in the PBMCs. Monocyte counts were not different between the 2 time points, whereas CD3+ cells were slightly decreased at t=0 (54.2% versus 66.6%, P=0.01; online-only Data Supplement, Figure IIA). A further study revealed no differences in the expression ratio of CCR2:CX3CR1, a measure of monocyte subset distribution,19 in PBMCs. We did, however, observe significantly elevated expression levels of HNP-3, a selective neutrophil marker, at t=0, which points to an enhanced release of neutrophils during UAP (online-only Data Supplement, Figure IIB). Conceivably, the observed changes in chemokine receptor expression at t=0 may be attributed at least in part to the increased neutrophil counts. In contrast to chemokine plasma levels, no differences in expression level were seen for chemokine receptors between patients with stabilized and refractory symptoms at t=0 (data not shown).
PBMC Stimulation Assay
In part, however, the chemokine receptor downregulation may reflect a feedback response on the immunomodulator burst after UAP. To verify whether the observed expressional regulation of CCR1, CCR3, CCR4, and CCR5 in PBMCs was related to the elevated CCL5 and CCL18 levels during ischemic events, we stimulated PBMCs with recombinant CCL5 and/or synthetic CCL18. After 6 hours of stimulation, we observed no differential effect on CCR1, CCR4, and CCR5 mRNA expression. In sharp contrast to this, however, synthetic CCL18 caused a dramatic downregulation in CCR3 expression, and this effect was further amplified by coincubation with recombinant CCL5 (P<0.01; Figure 5A through 5D). Therefore, the downregulation of CCR3 mRNA in PBMCs observed in vivo could be caused by the increased levels of CCL18. The downregulation of CCR1, CCR4, and CCR5 in vivo might well be regulated by ligands other than CCL5 and CCL18.
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Discussion |
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To the best of our knowledge, the present study is the first to describe the profiling of an extensive panel of chemokines by multiplex assay in plasma of UAP patients in a prospective manner. Of all chemokines tested, only CCL5 and CCL18 levels, independent of other inflammatory markers and sCD40L, were seen to be transiently elevated in refractory versus stabilized patients at baseline and to decline within 6 months after onset of the UAP symptoms. These phenomena were accompanied by a sharp, probably CCL18-induced decrease in mRNA expression of the cognate chemokine receptors CCR3 and CCR5 in PBMCs at day 0 versus day 180. Concomitantly, CCR3 and CCR5 surface expression was found to be increased at baseline, possibly reflecting a rapid receptor exposure by PBMCs during ischemic symptoms. Both CCL5 and CCL18 also showed predictive features with regard to clinical outcome.
The multiplex panel contained various chemokines that previously have been linked with atherosclerosis or cardiovascular disease, such as CCL2, CCL5, CCL11, CXCL8, and CXCL10.5 CCL5 and CCL18 were the only 2 chemokines that were differentially regulated at baseline between refractory and stabilized patients. Refractory patients had severe sustained ischemic complaints despite antianginal medication that warranted coronary angiography with or without percutaneous coronary intervention. Therefore, although the levels of other chemokines that have been implicated in cardiovascular disease were relatively unaltered, and although refractory patients do not generally differ from stabilized patients in the extent of general systemic inflammation, CCL5 and CCL18 might be exclusive chemokine markers of ischemia severity in patients with UAP.
CCL5 and CCL18 were selected for further temporal analysis for a 180-day follow-up. As mentioned previously, the role of CCL5 as an inflammatory mediator in cardiovascular disease is widely recognized, and CCL5 levels were indeed seen to be raised in patients with acute coronary syndromes.9,20 However, these previous studies examined CCL5 levels at hospitalization and, with 1 exception, did not include a prospective study design. Only the study by Nomura et al9 showed a drop in CCL5 levels in UAP patients 30 days after percutaneous coronary intervention to levels comparable to the 180-day levels seen in the present study. The present data extend this observation, because they demonstrate that the decline in CCL5 levels is not a consequence of percutaneous coronary intervention but an intrinsic feature of stabilized UAP patients. Although data on CCL5 reference levels are still lacking, CCL5 at 2 and 180 days after inclusion was very comparable to values reported in healthy control subjects by Parissis et al,21 which suggests that CCL5 levels had returned to baseline within 2 days after onset of the ischemic symptoms.
To gain further insight into the contribution of activated platelets to CCL5 peak levels, we performed a temporal assessment of sCD40L.22 We observed significantly elevated levels of sCD40L at baseline, which is in concordance with earlier studies and reflective of the enhanced platelet activation status in UAP.23,24 However, the observed progressive decline in sCD40L levels at t=2 and t=180 after UAP has never been documented in patients with UAP and may illustrate the rapid restoration of sCD40L homeostasis after UAP. Furthermore, t=0 and t=2 levels correlated with CCL5 levels, which suggests that activated platelets may, directly or indirectly, be a major source of CCL5. Apart from its massive secretion by activated platelets, elevated CCL5 levels during UAP could also arise from activated T lymphocytes and as a result of altered homeostasis in the ischemic tissue distal to the occlusion.25,26 Because Rothenbacher et al27 observed reduced CCL5 levels in patients with stable coronary heart disease compared with control subjects, acute inflammation per se is unlikely to be responsible for the transient increase in CCL5 during UAP. This is underscored by the present findings, because we observed a downregulation of CCL5 mRNA expression in PBMCs at baseline compared with 180 days after onset of the ischemia. Whether the increased response in refractory patients reflects a more extensive platelet (or T cell) activation or a higher capacity of platelets and T cells to elaborate CCL5 remains to be determined.
Interestingly, CCL18 has not yet been associated with cardiovascular disorders in patient cohorts. CCL18 is present at high levels in blood, and it is produced by antigen-presenting cells and by eosinophils. It is thought to act in the primary immune response functioning as an attractant for T cells, B lymphocytes, and monocytes.17 As mentioned previously, however, its receptor has not been identified, although CCL18 was reported to function as a neutral CCR3 antagonist.18 Evidence of a direct role of CCL18 in cardiovascular disease is not conclusive and is limited to 2 descriptive studies documenting CCL18 expression in atherosclerotic plaques, particularly at sites of reduced stability.28,29 We now show that CCL18 plasma levels are increased in UAP patients and even more so in patients with refractory symptoms. CCL18 elevation is sustained transiently, but levels are lowered after 180 days. The actual source of the persistent CCL18 increase after UAP is less clear. CCL18 expression was downregulated in PBMCs at baseline, which disqualifies abundant production by these cells as major source of plasma CCL18. Conceivably, plasma levels may reflect a release from CCL18-containing vulnerable plaques.28 CCL18 levels were negatively correlated with sCD40L levels, which possibly points to a negative feedback response on platelet activation. Further research will be needed to clarify its role in acute coronary syndromes.
It has been suggested that several chemokines can act in the pathogenesis of noninfarcted ischemic cardiomyopathy, because the prevailing reactive oxygen generation and hypoxia in the ischemic tissue will induce a chemokine response.30 Illustratively, MCP-1 was seen to be upregulated in the myocardium at least 7 days after ischemia in mice and was associated with interstitial fibrosis and left ventricular dysfunction in the absence of myocardial infarction.6 CCL18 levels persisted at a high level for at least 2 days as well, and given its capacity to activate fibroblasts and increase collagen production, it is tempting to propose a similar role of CCL18 in injury healing.31 CCL18 may not only modulate the attraction of leukocyte subsets, but, as shown by Wimmer et al,32 it may also play a facilitative role in bone marrow hematopoietic stem cell function. Therefore, elevated CCL18 levels could contribute to the inflammatory response but also to progenitor cell mobilization toward areas of myocardial ischemia in anticipation of the myocardial repair process.
To further stress the role of CCL5 and CCL18 in the pathophysiology of myocardial ischemia, we observed a significant increase in surface exposure of CCR3 and CCR5 by CD3+ T cells and CD14+ monocytes and a paradoxical mRNA downregulation of CCR1, CCR3, CCR4, and CCR5 at baseline. This is an intriguing and counterintuitive observation, although we are not the first to observe such a discrepancy between protein and mRNA chemokine receptor expression in PBMCs from UAP patients. In fact, Damas et al33 have reported a similar but opposite effect for CXCR4 (ie, downregulation at the protein level but upregulation at the mRNA level in UAP patients compared with healthy control subjects), whereas levels of its ligand CXCL12 were lowered in patients with UAP compared with control subjects. The rapid increase in surface protein exposure may result from acute mobilization of intracellular receptors in response to enhanced plasma levels of the cognate ligands or of other actors that are released in unstable angina. The relative mRNA downregulation of chemokine receptors in PBMCs may reflect in part a shifted leukocyte profile in UAP with a rapid mobilization of HNP-3+ neutrophils, as judged from the enhanced HNP-3 expression in PBMC mRNA at t=0,34 and a minor decrease in CD3+ cells, whereas total CD14+ levels remained unaffected. However, it may also be attributable in part to a negative feedback response to normalize exposed receptor levels, as appears from the present in vitro CCL18 regulation studies (Figure 5). The transcriptional feedback may be effected in direct response to exposure of the surface receptors to CCL18, because CCR3 mRNA levels were decreased dramatically after exposure to sCCL18, thus identifying a new modulatory role of CCL18 in cardiac ischemia.
Examination of CCL5 and CCL18 quartile distribution shows a clear-cut relation with the occurrence of refractory symptoms. Furthermore, upper-quartile levels also correlated with future cardiovascular events and revascularization procedures, whereas sCD40L and CRP, which have been shown to have strong prognostic power in other studies,35–37 did not in the present cohort size. Given the major cellular sources of CCL5 and CCL18 (activated platelets and ischemic tissue), the increased levels in refractory UAP may reflect a more pronounced thrombosis- and ischemia-related induction in these patients. Whether it is causal in the refractory disease progression remains to be clarified. With regard to the prognostic capacities of CCL5 and CCL18, the sensitivity and specificity of the upper-quartile levels of the chemokines separately did not exceed 80%. The combination of the upper 2 quartiles of both chemokines yielded a viable specificity of 90.3%, which thereby quite effectively rules out refractory symptoms for low CCL5 and CCL18 levels. However, although CCL5 and CCL18 may have potential as independent prospective biomarkers for disease, the correlations we observed between these chemokines and clinical severity of the symptoms, as well as various follow-up parameters, although very significant, are currently not strong enough on their own. Therefore, the determination of plasma CCL5 and CCL18 levels in combination with other clinical diagnostic parameters could add prognostic features to the evaluation of patients with UAP. This issue needs to be addressed in future, larger-scale studies.
A few issues and limitations of the present study should be noted. First, our setup principally precluded the study of control levels of these chemokines before UAP. Nevertheless, we believe that because prospective analyses were performed in the same patients, conclusions about the temporal profile of CCL5 and CCL18 are justified. Because all patients were largely symptom-free at 180 days after UAP, we may safely assume that the latter values will approach the pre-UAP levels of patients with coronary artery disease. Second, it has recently been shown that statins can influence chemokine serum levels and chemokine receptor expression on PBMCs.8,38 Because we were in the fortunate circumstance that cohort sampling had taken place when statin therapy had just begun to emerge, only 8.2% of the patients in the present cohort were taking statin therapy. Because the present data were corrected for this minor statin use, we believe that our results are not biased by statin therapy. Finally, the multiplex panel also comprised chemokines that have previously been linked to atherosclerosis or myocardial ischemia, including CCL2, CCL3, CXCL8, and CXCL10.21,39,40 In the present study, refractory UAP patients did not show significant differences with regard to these chemokines or for the other immunomodulators that had been assayed. These cytokines were, therefore, not selected for further temporal analysis, but we cannot a priori rule out that these cytokines may affect UAP and myocardial ischemia.
To conclude, we identified CCL5 and particularly CCL18 as relevant chemokines in UAP. Regardless of whether they play a causative role in the pathogenesis of this disease or are more indirectly involved via other mechanisms, the question of whether these markers harbor any further diagnostic potential and are suitable therapeutic targets needs to be addressed in future studies.
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Acknowledgments |
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Sources of Funding
This work was supported by the Netherlands Heart Foundation (grant M93.001). Drs Jukema and Biessen are (Clinical) Established Investigators of the Netherlands Heart Foundation (2001-D032 and D2003T201). Drs van Berkel and Biessen belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Sciences, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
Disclosures
None.
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Footnotes |
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The online-only Data Supplement, which consists of an expanded Methods section, as well as tables and figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.706986/DC1.
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