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Keywords:

  • CCL5;
  • CCR5;
  • CD16;
  • coronary artery disease;
  • monocyte–platelet aggregates;
  • monocytes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

Background

Monocytes and platelets are important cellular mediators of atherosclerosis. Human monocytes can be divided into CD14++CD16, CD14++CD16+ and CD14+CD16++ cells, which differ in their functional properties. The aim of this study was to examine monocyte subset distribution, monocyte–platelet aggregate (MPA) formation and expression of CCR5, the receptor of the platelet-derived chemokine CCL5, and to determine whether these parameters are altered in individuals with coronary atherosclerosis.

Methods

Peripheral blood cells from 64 healthy blood donors (HBDs) and 60 patients with stable coronary artery disease (CAD) were stained with antibodies against CD14, CD16, CD42b and CCR5 and analysed by flow cytometry. Circulating CCL5 levels were determined using an enzyme-linked immunosorbent assay.

Results

In patients with CAD, the relative proportion of the CD14++CD16 monocyte subset was elevated (< 0.05) and of the CD14+CD16++ subset was reduced (< 0.001) compared with the HBD group. Furthermore, MPA formation significantly increased in patients with CAD in all three monocyte subsets. In both study groups, the majority of CCR5+ cells was detected in CD14++CD16+ monocytes (< 0.001 versus CD14++CD16 and CD14+CD16++), although the CCR5+ monocyte number was reduced in patients with CAD (CD14++CD16/CD14+CD16++, < 0.001; CD14++CD16+, < 0.05) compared with the HBD group, particularly in those who were not taking statins. Ex vivo incubation of monocytes from HBDs with plasma from patients with CAD also decreased CCR5+ expression (< 0.05 versus plasma from HBDs). Serum CCL5 levels were similar in both groups.

Conclusions

The increased monocyte–platelet cross-talk in patients with CAD might have contributed to atherosclerosis progression. The decreased CCR5+ monocyte numbers in patients with CAD could have resulted from CCR5+ cell recruitment into atherosclerotic lesions or CCR5 downregulation in response to circulating factors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

Monocytes are crucial cellular mediators of atherosclerotic lesion formation [1]. Atherosclerosis is a chronic inflammatory disease [2]; circulating monocytes are attracted by pro-inflammatory molecules, such as the chemokine CCL5, into the developing lesion, where they differentiate into macrophages. These cells take up modified lipoproteins and secrete a variety of cytokines leading to attraction of smooth muscle cells, T lymphocytes and monocytes.

Based on the surface expression of the lipopolysaccharide receptor CD14 and the Fc receptor γIII (CD16), human monocytes can be divided into classical CD14++CD16, intermediate CD14++CD16+ and nonclassical CD14+CD16++ monocytes [3]. Monocyte subsets differ in their functional properties [4-6]. Previous studies revealed increased numbers of CD16+ monocytes in the blood of patients with atherosclerosis [7] as well as elevated numbers of CD14++CD16+ and CD14+CD16++ monocytes in individuals with the cardiovascular disease risk factors obesity and hyperglycaemia [8] or peripheral artery disease [9]. The potential significance of CD16+ monocytes in atherosclerosis is underscored by their pro-inflammatory/proatherogenic features [10] as well as their binding affinity to the endothelium [11]. On the other hand, CD14++CD16 monocytes were also reported to be increased in subjects with cardiovascular disease risk factors [12].

Platelets are critically involved in arterial thrombosis, but also play a role during all stages of atherosclerotic lesion formation [13]. Activated platelets promote monocyte arrest on the endothelium, thus enhancing monocyte recruitment into developing atherosclerotic lesions [14]. In this regard, it was demonstrated that CCL5 deposition by platelets triggers monocyte arrest on atherosclerotic endothelium [15]. CCL5 is known to interact with, amongst others, the chemokine receptor CCR5 [16], suggesting that the interaction between CCL5 and CCR5 may be important for monocyte recruitment into developing atherosclerotic lesions.

The formation of monocyte–platelet aggregates (MPAs) has been shown to be a sensitive and early indicator of in vivo platelet activation [17]. Moreover, MPAs may directly modulate vascular inflammation, atherosclerosis and thrombosis [13], as they modify monocyte function [14]. However, with which of the three monocyte subsets platelets primarily interact to form MPAs in patients with coronary artery disease (CAD) remains largely unclear.

In this study, we examined the relative proportion of CD14++CD16, CD14++CD16+ and CD14+CD16++ monocyte subsets as well as MPA formation in healthy blood donors (HBDs) and patients with stable CAD. In addition, surface expression of CCR5 on monocyte subsets and circulating levels of CCL5 and other platelet activation markers were determined.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

Study population

In total, 64 HBDs and 60 patients with angiographically confirmed CAD were prospectively recruited between April 2011 and April 2012 at the Department of Transfusion Medicine and the Department of Cardiology and Pulmonary Medicine, University Medical Center Göttingen, respectively. In general, individuals were excluded if they suffered from an acute or chronic inflammatory condition, active malignant disorder, nephropathy or a recent (<8 weeks) acute coronary syndrome (ACS). In control subjects, diabetes mellitus, arterial hypertension, hypercholesterolaemia or current smoking as well as use of medication for cardiovascular disease were additional exclusion criteria. CAD was defined as stenosis ≥50% in a major coronary artery and/or a history of coronary revascularization. Cardiovascular disease risk factors were defined as follows: arterial hypertension – blood pressure ≥140/90 mmHg or antihypertensive treatment; diabetes mellitus – elevated glycosylated fraction of the major component of adult haemoglobin (HbA1c) or antidiabetic treatment; hypercholesterolaemia – low-density lipoprotein cholesterol (LDL-C) level >3.9 mmol L−1 or previously diagnosed hypercholesterolaemia; current smoking – active smoking or smoking cessation <6 months before study entry. In HBDs, we used a standardized questionnaire to record a potential history of cardiovascular disease, including risk factors. Therefore, we cannot completely rule out the possibility of unknown cardiovascular disease in this control group. Clinical information regarding patients with CAD was documented from hospital medical records. Written informed consent was obtained from each study participant. The study protocol confirmed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the human research committee (application no. 11/8/10) at the University Medical Center Göttingen.

Biochemical analyses

In order to characterize the study population and to control for study exclusion criteria, routine laboratory analyses were performed at the Department of Clinical Chemistry, University Medical Center Göttingen. Measurements included determination of blood levels of creatinine, aspartate aminotransferase, total cholesterol, LDL-C, high-density lipoprotein (HDL) cholesterol, HbA1c and high-sensitivity C-reactive protein (hs-CRP). Differential blood counts were obtained using an automated cell counter (CELL-DYN Sapphire; Abbott Diagnostics, Abbott Park, IL, USA).

Measurement of serum markers

Serum samples were stored in small aliquots at −80 °C until further analysis. Serum concentrations of CCL5 (PeproTech, Rocky Hill, NJ, USA), soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1; eBioscience, San Diego, CA, USA) and soluble platelet (sP)-selectin (eBioscience) were measured by commercially available enzyme-linked immunosorbent assays, according to the manufacturer's instructions. All measurements were performed in duplicate.

Flow cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood by density gradient centrifugation using Histopaque (Sigma-Aldrich, St. Louis, MO, USA). After fixation with 1% paraformaldehyde, PBMCs were stained with fluorescein isothiocyanate-conjugated anti-human CD14 (clone MφP9; BD, Franklin Lakes, NJ, USA), phycoerythrin (PE)-conjugated anti-human CD16 (clone 3G8; BD) and allophycocyanin (APC)-conjugated anti-human CD195 (i.e. CCR5; clone 2D7; BD) antibodies, or the respective isotype controls. For the detection of MPAs, 100 μL citrated blood was treated with BD fluorescence-activated cell sorting (FACS) lysing solution, and cells were stained with anti-human CD14, anti-human CD16 and APC-conjugated anti-human CD42b (clone HIP1; BD), or isotype-matched antibodies. Samples were kept at 4 °C in the dark until further analysis within a maximum of 2 h from sample preparation. Analyses were performed on a BD FACS Canto II flow cytometer and evaluated using BD FACS Diva software. Monocytes were first gated according to their forward (FSC) and sideward (SSC) scatter profiles in a PBMC suspension or in citrated blood, respectively. Next, monocytes were subclassified according to CD14 and CD16 expression into CD14++CD16, CD14++CD16+ and CD14+CD16++ monocytes. Subsets were then further analysed with regard to CD42b and CCR5 expression. At least 10 000 events in the monocyte gate were measured per experiment. Results are presented as percentage of positive cells. Cell surface CD42b and CCR5 expression on individual cells were quantified by determination of the mean fluorescence intensity minus the respective isotype control (MFI-FMO). In some experiments, PBMCs from healthy individuals were incubated in a humidified incubator (37 °C, 5% CO2) for 4 h in Opti-MEM medium (Life Technologies, Paisley, UK) containing 10% heparinized plasma of representative HBDs or CAD patients, before being analysed for CD14 and CCR5.

Statistical analysis

Results are expressed as median and quartiles (25th percentile; 75th percentile) for continuous variables and as number (frequency) for categorical variables. Data are presented as histograms showing the median as well as the upper and lower quartiles. Comparisons were performed using the nonparametric Mann–Whitney U-test (for continuous values) or chi-squared test (for categorical values). For correlation analyses, Spearman's rank correlation coefficient was calculated. All statistical tests were two-sided. A probability (P) value of <0.05 was considered significant. Calculations were performed with spss (version 19.0; SPSS Inc., Chicago, IL, USA) or GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) software.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

Characterization of study participants

The clinical characteristics and laboratory findings of the study participants are shown in Table 1. In contrast to patients with CAD , subjects in the HBD group had no history of cardiovascular disease or either risk factors or use of medication for cardiovascular disease. As it was difficult to recruit older subjects without any of the predefined exclusion criteria, the HBD and CAD groups differed with regard to their median age (< 0.001). Patients with CAD had higher leucocyte counts (< 0.01) and blood levels of creatinine (< 0.01), hs-CRP (< 0.001) and HbA1c (< 0.001) compared with HBDs, whereas the number of thrombocytes was reduced (< 0.01). Total cholesterol (< 0.001) and LDL-C (< 0.01) levels were found to be decreased in CAD subjects, probably due to effective cholesterol-lowering therapy.

Table 1. Characteristics of HBDs and patients with CAD
 HBD (= 64)CAD (= 60)P-value
  1. ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BMI, body mass index; CAD, coronary artery disease; HbA1c, glycosylated haemoglobin; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; HBDs, healthy blood donors; LDL-C, low-density lipoprotein cholesterol; s/p, status post.

  2. Values are presented as median (25th; 75th percentile) as number of individuals (%). aThree values are missing in the HBD group. b10 values are missing in the HBD group. Statistically significant differences are highlighted in bold.

Male, n (%)46 (71.9)46 (76.7)0.544
Age, years53.5 (47.0; 57.8)70.0 (63.3; 73.0) <0.001
BMI, kg m−225.4 (23.6; 27.4)26.4 (23.9; 30.1)0.061
Diabetes mellitus, n (%)0 (0)12 (20.0) <0.001
Arterial hypertension, n (%)0 (0)49 (81.7) <0.001
Hypercholesterolaemia, n (%)0 (0)37 (61.7) <0.001
Current smoking, n (%)0 (0)8 (13.3) <0.01
CAD, n (%)0 (0)60 (100.0) <0.001
One-vessel disease0 (0)17 (28.3) <0.001
Two-vessel disease0 (0)14 (23.3) <0.001
Three-vessel disease0 (0)29 (48.3) <0.001
s/p bypass surgery, n (%)0 (0)24 (40.0) <0.001
s/p stroke, n (%)0 (0)4 (6.7) <0.05
Peripheral artery disease, n (%)0 (0)2 (3.3)0.142
ACE inhibitor/ARB, n (%)0 (0)54 (90.0) <0.001
Acetylsalicylic acid, n (%)0 (0)49 (81.7) <0.001
Beta-blocker, n (%)0 (0)49 (81.7) <0.001
Oral antidiabetics, n (%)0 (0)11 (18.3) <0.001
Insulin, n (%)0 (0)3 (5.0)0.071
Phenprocoumon, n (%)0 (0)18 (30.0) <0.001
Thienopyridine derivates, n (%)0 (0)11 (18.3) <0.001
Statins, n (%)0 (0)48 (80.0) <0.001
Ezetimibe, n (%)0 (0)11 (18.3) <0.001
Leucocytes, ×103 μL−15.9 (4.7; 6.8)6.6 (5.5; 8.0) <0.01
Monocytes, ×103 μL−1a0.51 (0.41; 0.63)0.51 (0.41; 0.60)0.750
Haemoglobin, g dL−114.1 (13.5; 14.9)14.1 (13.1; 15.1)0.717
Thrombocytes, ×103 μL−1225 (195; 275)200 (166; 248) <0.01
hs-CRP, mg L−10.74 (0.43; 2.00)2.55 (1.23; 4.70) <0.001
Creatinine, mg dL−10.88 (0.78; 0.97)0.95 (0.83; 1.09) <0.01
Aspartate transaminase, U L−126.5 (23.0; 33.8)30.0 (24.3; 35.5)0.251
Total cholesterol, mg dL−1206 (183; 222)172 (149; 211) <0.001
LDL-C, mg dL−1120 (104; 138)97 (78; 130) <0.01
HDL-C, mg dL−158.5 (46.5; 76.5)51.0 (41.0; 61.8) <0.05
HbA1c, mmol/molb37.7 (35.5; 39.9)41.0 (37.7; 44.3) <0.001

Comparison of monocyte subset distribution

The distribution of CD14++CD16, CD14++CD16+ and CD14+CD16++ monocytes was determined by flow cytometry. First, monocytes were gated according to their characteristic FSC/SSC profiles in a PBMC suspension (Fig. 1a) and then further subclassified according to their CD14 and CD16 surface expression (Fig. 1b). In HBDs, 84.9% of total monocytes were CD14++CD16, compared with only 7.1% and 7.8% of CD14++CD16+ and CD14+CD16++ cells, respectively (Fig. 1c). In patients with CAD, the relative percentage of CD14++CD16 monocytes was increased (86.6%; < 0.05 versus the HBD group), whereas CD14++CD16+ monocyte levels were similar (7.7%; = 0.134) and there were fewer CD14+CD16++ cells (5.9%; < 0.001). In both HBD and CAD groups, the number of CD14++CD16 monocytes was higher compared with CD14++CD16+ (< 0.001) and CD14+CD16++ monocytes (< 0.001), whereas CD14+CD16++ monocytes were less abundant than CD14++CD16+ cells in patients with CAD (< 0.001) and more common in HBDs (< 0.05). Comparison of the absolute numbers of the circulating monocyte subsets (determined by multiplying the relative monocyte subset percentage with its respective absolute monocyte number obtained from automated blood counts) showed that CD14+CD16++ monocytes remained less common in patients with CAD (< 0.001 versus the HBD group; Fig. 1d).

image

Figure 1. Flow cytometric analysis of monocyte subset distribution. Monocytes were gated according to their forward (FSC) and sideward (SSC) scatter profiles in a peripheral blood mononuclear cell (PBMC) suspension (a) and classified according to CD14 and CD16 expression (b). Representative dot blots are shown in (a) and (b). The relative number of the three monocyte subsets (as a percentage of the total monocytes) in the healthy blood donor (HBD;= 64) group and in patients with coronary artery disease (CAD;= 60) (c). Absolute monocyte numbers in each subset (d). Data are presented as histograms showing the median as well as upper and lower quartiles. The level of significance is indicated for intergroup comparisons (i.e. between HBD and CAD groups). For intragroup comparisons (i.e. within each monocyte subset): +++< 0.001 versus CD14++CD16; ###< 0.001, ##< 0.01 and #< 0.05 versus CD14++CD16+.

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Quantification of MPA formation in monocyte subsets

Next, we determined differences between monocyte subsets in the formation of MPAs, identified by immunofluorescence positivity for both monocyte (CD14) and platelet (CD42b) antigens. As shown in Fig. 2(a), MPA formation in HBDs was highest in CD14++CD16+ (18.7%) and CD14+CD16++ (17.8%) compared with CD14++CD16 monocytes (15.0%; < 0.01 for both). In patients with CAD, MPA formation was significantly elevated in CD14++CD16 (20.1%; < 0.001 versus the HBD group), CD14++CD16+ (21.7%; < 0.05 versus the HBD group) and CD14+CD16++ (22.5%; < 0.01 versus the HBD group) monocytes. In order to exclude the possibility that differences in the median age between the CAD and HBD groups may have contributed to the observed increase in MPA formation in patients with CAD, we also examined a subgroup of age-matched individuals in whom CAD was excluded by coronary angiography [= 9; median age 72.0 (70.5; 73.5) years; = not significant versus CAD]. Importantly, these analyses confirmed significantly higher MPA numbers in the CAD group for all three monocyte subsets (Fig. S1). By contrast, the median CD42b expression per monocyte, indicating the number of bound platelets per monocyte, on all three monocyte subsets did not differ significantly between the CAD and HBD groups (Fig. 2b). In CAD but not in HBD subjects, thrombocyte counts were found to correlate with the percentage of MPA formation in CD14++CD16 (= 0.576; < 0.001), CD14++CD16+ (= 0.434; = 0.001) and CD14+CD16++ (= 0.539; < 0.001) monocytes.

image

Figure 2. Monocyte–platelet aggregate (MPA) formation and CCR5 expression in monocyte subsets. The percentage of CD42b+ MPAs (a) and CCR5+ monocytes (c) in each monocyte subset was analysed in the healthy blood donor (HBD;= 64) group and in patients with coronary artery disease (CAD;= 60). The mean fluorescence intensity minus the respective isotype control (MFI-FMO) for CD42b (b) and CCR5 (d) expression on monocyte subsets is also shown. Study participants with CAD were stratified according to those taking (= 48) and not taking (= 12) statin medication (e), and the percentage of CCR5+ monocytes in each subset was determined. Data are presented as histograms showing the median as well as upper and lower quartiles. The level of significance is indicated for intergroup comparisons (i.e. between HBD and CAD groups). For intragroup comparisons (i.e. within each monocyte subset): +++< 0.001, ++< 0.01 and +< 0.05 versus CD14++CD16; ###< 0.001 versus CD14++CD16+.

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Expression of CCR5 on monocyte subsets

To determine potential causes underlying the observed differences in MPA formation between monocyte subgroups from patients with CAD and HBD subjects, the number of monocytes expressing CCR5, the receptor of the platelet chemokine CCL5, was determined. In HBDs, the number of CCR5+ monocytes was highest in CD14++CD16+ (38.0%) compared with CD14++CD16 (8.1%) and CD14+CD16++ (10.9%) monocytes (< 0.001 for both; Fig. 2c). Although a similar distribution was seen in patients with CAD, that is, the number of CCR5+ monocytes was highest in CD14++CD16+ (35.5%) compared with CD14++CD16 (5.5%; < 0.001) and CD14+CD16++ (8.4%; < 0.01) monocytes, the percentage of CCR5+ monocytes was found to be reduced compared with HBDs in each of the three subsets (CD14++CD16/CD14+CD16++, < 0.001; CD14++CD16+, < 0.05). On the other hand, median CCR5 expression per monocyte did not differ between the study groups (Fig. 2d), suggesting that the number of CCR5+ monocytes was reduced and not the expression of the receptor on individual cells. It is interesting that CCR5+ monocyte numbers were significantly decreased in all three monocyte subsets in individuals with CAD taking statin medication (= 48) compared with those without medication (= 12; CD14++CD16/CD14++CD16+, < 0.01; CD14+CD16++, < 0.05; Fig. 2e).

Paracrine plasma effects on monocyte CCR5 expression

To explore possible mechanisms underlying the reduced number of CCR5+ monocytes observed in patients with CAD and the causal role of circulating factors, PBMCs isolated from healthy subjects were incubated with medium containing 10% heparinized plasma from either HBDs or patients with CAD, followed by the analysis of monocyte CCR5 expression. Notably, a decreased percentage of CCR5+ monocytes was observed after incubation of PBMCs from HBDs with plasma from patients with CAD (< 0.05 versus plasma from HBDs; Fig. 3a), similar to the observed reduction of circulating CCR5+ monocytes in patients with CAD. Again, CCR5 MFI-FMO was not significantly altered in monocytes from HBDs treated with plasma from patients with CAD (Fig. 3b). Of note, serum CCL5 levels did not differ between the two study groups (0.520 vs. 0.520 ng mL−1; = 0.776; Fig. 3c).

image

Figure 3. CCR5 expression on monocytes after exposure to plasma from healthy blood donors (HBDs) or patients with coronary artery disease (CAD), and serum CCL5 levels. Human peripheral blood mononuclear cells from the control group (HBD;= 4) were incubated with medium supplemented with 10% heparinized plasma from either (HBDs) subjects or patients with CAD. Monocytes were analysed with respect to the percentage of CCR5+ cells (a) and the CCR5 mean fluorescence intensity minus the respective isotype control (MFI-FMO; b). Serum CCL5 levels were measured in 64 HBDs and 60 patients with CAD (c); three CCL5 values were above the upper detection limit and were excluded. Data are presented as histograms showing the median as well as upper and lower quartiles. The level of between-group significance is indicated.

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Circulating markers of platelet activation

Finally, we examined serum levels of additional, noncellular markers of platelet activation in both study groups. In contrast to our findings of increased monocyte–platelet interaction and MPA formation in patients with CAD, serum levels of sPECAM-1 (60.1 vs. 71.8 ng mL−1; < 0.001; Fig. 4a) and sP-selectin (151 vs. 182 ng mL−1; < 0.001; Fig. 4b) were found to be significantly lower in patients with CAD compared with HBDs. There was a positive association between sPECAM-1 and sP-selectin levels (= 0.369; < 0.001), which persisted if patients with CAD alone were analysed (= 0.276; < 0.05).

image

Figure 4. Serum levels of platelet activation markers. Levels of soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1; a) and soluble platelet (sP)-selectin (b) were measured in serum from the healthy blood donor (HBD;= 64) group and from patients with coronary artery disease (CAD;= 60). Data are presented as histograms showing the median as well as upper and lower quartiles. The level of between-group significance is indicated.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

The analysis of murine monocyte subpopulations has recently revealed important new insights into their subset-specific contribution to atherosclerotic lesion formation. However, human monocyte heterogeneity and its relation to CAD remain poorly understood. In the present study, we observed an increased relative percentage of CD14++CD16 and decreased relative percentage of CD14+CD16++ monocytes in stable patients with CAD compared with healthy control subjects. Furthermore, an increase in monocyte–platelet interaction was detected in patients with CAD, as indicated by increased aggregate formation in all three monocyte subsets. Although blood levels of the platelet-borne chemokine CCL5, an important mediator of monocyte–platelet cross-talk, did not differ between subjects in the HBD and CAD groups, circulating CCR5+ monocyte numbers were significantly decreased in patients with CAD. This decrease could be reproduced ex vivo by incubating monocytes from HBDs with plasma obtained from patients with CAD, and to a significantly lesser degree in monocytes obtained from patients with CAD on statin medication.

Leucocytes, and especially monocytes, are important contributors to atherosclerosis [18]. Whereas differential functional roles of monocyte subsets have been implicated in atherosclerotic lesion formation in mice [19], the relative subset contribution is less clear in humans. Although previous studies found that a shift towards CD16+ monocytes was associated with the presence of CAD [7], vulnerable atherosclerotic plaques [20] or fibrous cap thickness [21], CD16+ monocytes had not been further subdivided according to the strength of the CD14 expression signal. Interestingly, in a recent cohort analysis of more than 900 subjects with cardiovascular disease or a high burden of risk factors, it was found that CD14++CD16+ monocytes predicted future cardiovascular events [22]. By contrast, it has also been reported that the percentage of classical CD14++CD16 monocytes is increased in individuals with an ischaemic cardiovascular event compared with event-free subjects [23] and in subjects with cardiovascular disease risk factors [12]. In the present study, we also observed an increased relative percentage of CD14++CD16 and a decrease in CD14+CD16++ monocytes in CAD, whereas the absolute CD14++CD16 monocyte number was similar in the two study groups.

The cross-talk between monocytes and platelets is regarded as a crucial pathophysiological mechanism linking inflammation and thrombosis [24]. In addition, the monocyte–platelet interaction may favour the development of a proatherogenic monocyte phenotype [11], as platelet adherence enhances monocyte cytokine production [25]. The interaction of platelets and monocytes in the circulation can be monitored by the detection of MPA formation, and probably more sensitively mirrors vascular inflammation than cell counts or serum markers [26]. To date, it has been demonstrated that patients with stable CAD [27] or ischaemic heart disease [28] as well as individuals with cardiovascular disease risk factors, such as arterial hypertension [29], smoking [30] or diabetes [31], have increased circulating MPA levels. However, little is known about which of the three monocyte subsets primarily interact with platelets and how this cross-talk is altered in patients with stable CAD. In the present study, we have demonstrated that in particular the so-called inflammatory (and presumably proatherogenic) CD16+ monocytes (CD14++CD16+ and CD14+CD16++) possess a higher tendency to form MPAs, although an increased MPA level was also observed in CD14++CD16 monocytes in patients with CAD.

In contrast to our findings regarding MPA formation, serum levels of known circulating markers of platelet activation were found to be decreased in the stable CAD group. In a previous study, no difference in sPECAM-1 levels was observed between patients with atherosclerosis and healthy control subjects [32], in contrast to the report of elevated sPECAM-1 and sP-selectin levels in individuals with stable or unstable CAD [33]. Our own data might be explained by the fact that the study population was highly selected for individuals with stable CAD but without other concomitant diseases. Additionally, all patients were optimally treated with cardiovascular therapeutic agents, some of which are known to have beneficial pleiotropic effects on the cardiovascular system.

The recruitment of monocytes and their subsets into atherosclerotic lesions is largely determined by their response to chemokines and regulated by the expression of specific chemokine receptors. Activated platelets adhering to the endothelium help monocytes to invade atherosclerotic lesions [14, 34]. The platelet chemokine CCL5, also termed regulated on activation normal T-cell expressed and secreted (RANTES), is known to be involved in monocyte recruitment [35]. Moreover, the causal role of CCL5 in vascular lesion growth is underlined by the finding that CCR5 blockade results in experimental atherosclerosis reduction [36-38]. Previous studies have shown that CCR5 is highly expressed on CD16+ monocytes, especially CD14++CD16+ cells [39-41]. Given the prominent role of CCR5 in atherosclerosis, these findings might favour CD14++CD16+ monocytes as drivers of human atherosclerosis. In this regard, our data revealed that the percentage of CCR5+ cells is highest in the CD14++CD16+ monocyte fraction. In addition, we found that the CCR5+ cell percentage in all three monocyte subsets was significantly decreased in patients with CAD compared with healthy HBDs. Although the design of our study does not allow any conclusion as to causal relationships, increased CCR5+ cell recruitment into atherosclerotic lesions may have contributed to the reduced presence of CCR5+ monocyte subsets in the blood of patients with CAD.

Serum levels of CCL5 were determined in order to investigate whether the reduced number of CCR5+ monocyte subsets in patients with CAD may be the result of changes in the number of these cells in response to chronically elevated CCL5 levels. However, in contrast to a previous report of increased CCL5 levels in middle-aged subjects with coronary atherosclerosis [42], we did not detect a significant difference between the CAD and HBD groups. In line with the hypothesis that factors other than CCL5 may have contributed to the observed decrease in CCR5+ cells in patients with CAD, reduced CCR5+ cell numbers were also observed in monocytes from HBDs after exposure to plasma from patients with CAD. However, precisely which factors are responsible for regulating the CCR5 positivity on monocytes remains to be determined. Interestingly, in vitro studies have shown that inhibitors of the enzyme HMG-CoA reductase involved in cholesterol synthesis (i.e. statins) downregulate CCR5 in primary macrophages, probably due to their anti-inflammatory properties [43]. We also observed a reduction in the number of CCR5+ monocytes in patients with CAD being treated with statins.

In conclusion, we have shown that the relative percentage of CD14++CD16 monocytes is increased, consistent with previous findings in mice [19], and that of CD14+CD16++ monocytes is decreased in patients with CAD compared with healthy control subjects. The formation of MPAs is significantly increased in monocytes from patients with stable CAD, and the proatherogenic monocyte–platelet interaction occurs in CD14++CD16+, CD14+CD16++ and, particularly, CD14++CD16 monocytes. Although our findings cannot prove (i) direct causality between surface factor expression on monocyte subsets and atherosclerotic lesion formation or (ii) that platelet passage through atherosclerotic lesions might have induced platelet activation without pathogenic contribution to atherosclerosis, we hypothesize that the enhanced monocyte–platelet cross-talk might have contributed to monocyte activation, recruitment into vascular lesions (as possibly reflected by decreased numbers of CCR5+ monocyte subsets in patients with CAD) and thus atherosclerotic plaque progression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

This research project was supported by a ‘Heidenreich von Siebold-Programm’ grant from the University Medical Center Göttingen (to FSC).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conflict of interest statement
  8. Acknowledgements
  9. References
  10. Supporting Information
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joim12145-sup-0001-FigS1.docWord document217KFigure S1 Monocyte–platelet aggregate (MPA) formation in monocyte subsets.

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