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Monocytes are crucial cellular mediators of atherosclerotic lesion formation . Atherosclerosis is a chronic inflammatory disease ; 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 . Monocyte subsets differ in their functional properties [4-6]. Previous studies revealed increased numbers of CD16+ monocytes in the blood of patients with atherosclerosis  as well as elevated numbers of CD14++CD16+ and CD14+CD16++ monocytes in individuals with the cardiovascular disease risk factors obesity and hyperglycaemia  or peripheral artery disease . The potential significance of CD16+ monocytes in atherosclerosis is underscored by their pro-inflammatory/proatherogenic features  as well as their binding affinity to the endothelium . On the other hand, CD14++CD16− monocytes were also reported to be increased in subjects with cardiovascular disease risk factors .
Platelets are critically involved in arterial thrombosis, but also play a role during all stages of atherosclerotic lesion formation . Activated platelets promote monocyte arrest on the endothelium, thus enhancing monocyte recruitment into developing atherosclerotic lesions . In this regard, it was demonstrated that CCL5 deposition by platelets triggers monocyte arrest on atherosclerotic endothelium . CCL5 is known to interact with, amongst others, the chemokine receptor CCR5 , 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 . Moreover, MPAs may directly modulate vascular inflammation, atherosclerosis and thrombosis , as they modify monocyte function . 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.
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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 . Whereas differential functional roles of monocyte subsets have been implicated in atherosclerotic lesion formation in mice , 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 , vulnerable atherosclerotic plaques  or fibrous cap thickness , 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 . 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  and in subjects with cardiovascular disease risk factors . 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 . In addition, the monocyte–platelet interaction may favour the development of a proatherogenic monocyte phenotype , as platelet adherence enhances monocyte cytokine production . 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 . To date, it has been demonstrated that patients with stable CAD  or ischaemic heart disease  as well as individuals with cardiovascular disease risk factors, such as arterial hypertension , smoking  or diabetes , 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 , in contrast to the report of elevated sPECAM-1 and sP-selectin levels in individuals with stable or unstable CAD . 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 . 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 , 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 . 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 , 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.