International Journal of Cardiology 186 (2015) 219–225

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Prevalence of M4 macrophages within human coronary atherosclerotic plaques is associated with features of plaque instability☆ Christian Erbel a,b,1, Antonia Wolf a,1, Felix Lasitschka c, Fabian Linden a, Gabriele Domschke a, Mohammadreza Akhavanpoor a, Andreas O. Doesch a,b, Hugo A. Katus a,b, Christian A. Gleissner a,b,⁎ a b c

Department of Cardiology, University of Heidelberg, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg, Germany Institute of Pathology, University of Heidelberg, Heidelberg, Germany

a r t i c l e

i n f o

Article history: Received 24 November 2014 Received in revised form 5 February 2015 Accepted 15 March 2015 Available online 17 March 2015 Keywords: Atherosclerosis Coronary artery disease Macrophage Unstable plaque

a b s t r a c t Background: The platelet chemokine CXCL4 induces monocyte differentiation resulting in a macrophage phenotype called “M4”, which co-expresses CD68, MMP7, and S100A8. We hypothesized that M4 macrophages are associated with plaque destabilization. Methods: Atherosclerotic arteries were obtained from explanted hearts of patients with severe coronary artery disease (CAD, n = 32) and of patients with dilated cardiomyopathy and no or mild CAD (controls, n = 19). Coronary arteries were stained with H&E, and immuno-fluorescence was performed against CD68, MMP7, and S100A8. Results: Both CD68+ macrophages representing the entire macrophage population and MMP7+S100A8+CD68+ M4 macrophages could be reproducibly identified within all arterial layers. The average proportion of the M4 macrophage phenotype amongst all CD68+ macrophages was 31.7 ± 16.2%. The highest number of M4 macrophages was found in the adventitia, followed by the intima. CD68+ and M4 macrophage numbers were significantly higher in patients with severe CAD. The presence of M4 macrophages within the intima and the media was significantly associated with plaque instability as determined by Stary class. Multivariate analysis showed a highly significant contribution of cardiovascular risk factors (P = 0.008) to plaque instability, while only trends were observed for age (P = 0.060) and intimal prevalence of M4 macrophages (P = 0.098). Conclusions: We demonstrate for the first time that M4 macrophages can be reproducibly found in coronary artery plaques. The prevalence of M4 macrophages is associated with indexes of plaque instability, most likely representing a surrogate marker of inflammatory activity. These findings suggest a pathogenetic role of M4 macrophages in vulnerable atherosclerotic plaques. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Atherosclerosis and its consequences such as myocardial infarction and stroke represent the major cause of mortality and morbidity worldwide [1]. During atherogenesis, blood monocytes enter the vascular intima and differentiate towards macrophages and foam cells [2]. This process is orchestrated by a plethora of growth factors, cytokines and chemokines [3]. Depending on the vascular micromilieu, macrophages may assume different phenotypes [4]. Certain phenotypes may promote inflammation thereby favoring plaque development and destabilization. Abbreviations: CAD, coronary artery disease; DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy; MMP, matrix metalloprotease. ☆ All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. ⁎ Corresponding author at: Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany. E-mail address: [email protected] (C.A. Gleissner). 1 Equal contribution.

http://dx.doi.org/10.1016/j.ijcard.2015.03.151 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.

A prototype of this pro-inflammatory macrophage type is the M1 macrophage induced by tumor necrosis factor-α and lipopolysaccharide [5]. On the other hand, anti-inflammatory macrophages may dampen the inflammatory process thereby leading to plaque stabilization. Platelets and platelet-derived chemokines play an important role during atherogenesis [6,7]. CXCL4 (also known as platelet factor-4) is one of the most abundant platelet chemokines released upon platelet activation to the plasma in micromolar concentrations [7]. CXCL4 has been shown to be pro-atherogenic in the Apoe−/− mouse model, where knock out of the Pf4 gene coding for murine CXCL4 resulted in decreased size of atherosclerotic lesions [8,9]. Furthermore, we and others have previously demonstrated that CXCL4 promotes macrophage differentiation towards a specific, previously unknown phenotype [10–12]. Based on its cytokine profile and surface receptor repertoire, we suggested to call these macrophages “M4” [13]. Recently, we have demonstrated that M4 macrophages can be distinguished from other macrophage polarization types applying an immunohistochemistry approach staining for CD68, MMP7, and S100A8

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Table 1 Patient characteristics (mean ± SD or number (percentage)). All patients (n = 51)

ICM (n = 32)

Non-ICM (n = 19)

P

Demographic data Male sex Age (years)

41 (80.4%) 51.6 ± 11.7

25 (78.1%) 55.8 ± 5.2

16 (84.2%) 44.4 ± 15.7

Risk profile Arterial hypertension Diabetes mellitus Hypercholesterolemia Obesity Family history of CAD Active Smoker

25 (49.0%) 18 (35.3%) 26 (51.0%) 11 (21.6%) 16 (31.4%) 21 (41.2%)

19 (59.4%) 14 (43.8%) 24 (75.0%) 8 (25.0%) 11 (34.4%) 15 (46.9%)

6 (31.6%) 4 (21.1%) 2 (10.5%) 3 (15.8%) 5 (26.5%) 6 (31.6%)

0.079 0.076 b0.001*** 0.498 0.455 0.192

0.443 0.022*

Cardiovascular medical history Previous myocardial 23 (45.1%) infarction Previous PCI 19 (37.3%) Previous CABG 10 (19.6%) Previous Stroke 7 (13.7%)

24 (75.0%)

0 (0.0%)

b0.001***

19 (59.4%) 10 (31.3%) 4 (12.5%)

0 (0.0%) 0 (0.0%) 3 (15.8%)

b0.001*** 0.004** 0.543

Clinical chemistry (biochemistry) Creatinine (mg/dL) 1.29 ± 0.46 White blood count (/nL) 9.3 ± 3.8 Hemoglobin (g/dL) 11.8 ± 2.5 LDL (mg/dL) 75 ± 32 HDL (mg/dL) 38 ± 16 Triglycerides (mg/dL) 147 ± 108 C-reactive protein (mg/L) 18.3 ± 25.6 Troponin T (μg/L) 1.1 ± 2.2 HbA1c (%) 6.24 ± 0.93

1.31 ± 0.44 9.1 ± 3.2 11.8 ± 1.8 71 ± 25 37 ± 15 160 ± 121 19.9 ± 32.3 1.1 ± 2.5 6.38 ± 1.06

1.26 ± 0.39 9.7 ± 3.9 12.3 ± 1.7 85 ± 36 40 ± 18 132 ± 91 15.4 ± 13.8 1.0 ± 1.8 6.04 ± 0.70

0.654 0.444 0.484 0.437 0.533 0.566 0.305 0.297 0.253

Medication ASS Beta blocker ACE inhibitor AT1 antagonist Aldosterone antagonist Diuretic Nitrate Statin Antidiabetic Coumarin Heparin

7 (21.9%) 24 (75.0%) 19 (59.4%) 11 (34.4%) 19 (59.4%) 23 (71.9%) 5 (15.6%) 22 (68.8%) 11 (34.4%) 17 (53.1%) 4 (12.5%)

1 (5.3%) 14 (73.7%) 13 (68.4%) 6 (31.6%) 15 (78.9%) 17 (89.5%) 0 (0.0%) 9 (47.4%) 1 (5.3%) 8 (42.1%) 6 (31.6%)

0.162 0.550 0.640 0.645 0.820 0.334 0.074 0.088 0.026* 0.510 0.107

8 (15.7%) 38 (74.5%) 32 (62.7%) 17 (33.3%) 38 (74.5%) 41 (80.4%) 5 (9.8%) 31 (60.8%) 12 (17.6%) 25 (49.0%) 10 (19.6%)

[14]. Using this combination allows unequivocal identification of M4 macrophages in vitro and in vivo without an overlap with M1, M2, or other macrophage types. We have also demonstrated that these cells can be identified in human coronary atherosclerotic plaques post mortem. However, thus far it was unclear whether the presence of M4 macrophages was associated with clinical parameters, plaque morphology and most importantly plaque stability. To answer these questions, we investigated the prevalence of M4 macrophage within human coronary atherosclerotic arteries. Coronary arteries were obtained from hearts explanted during allograft transplantation due to terminal heart failure. Coronary arteries from patients with ischemic cardiomyopathy caused by severe coronary artery disease (CAD) were compared to those derived from patients with dilated cardiomyopathy displaying no or only mild CAD. Our hypothesis was that the presence of M4 macrophages within atherosclerotic plaques may be associated with plaque destabilization. We therefore specifically sought to analyze whether the prevalence of M4 macrophages was associated with advanced plaque morphology. 2. Methods 2.1. Patient samples Tissue sections of coronary arteries were provided by the tissue bank for inflammatory diseases Heidelberg (GEZEH) and were taken from explanted hearts from patients having undergone heart transplantation

between 2005 and 2009 at the University of Heidelberg. We included patients with ischemic cardiomyopathy (ICM) caused by severe coronary artery disease (CAD) and patients with dilated cardiomyopathy displaying no or mild CAD served as controls (non-ICM). In both groups, diagnosis was based on coronary angiography; DCM was furthermore confirmed by cardiac magnetic resonance tomography. For all patients, a complete set of demographic, clinical and laboratory parameters was available. Information on traditional risk factors for CAD, including arterial hypertension (blood pressure ≥ 140/90 mm Hg or antihypertensive therapy) [15], hyperlipidemia (low-density lipoprotein cholesterol (LDL-C) ≥130 mg/dL or statin therapy) [16], current or prior smoking, diabetes mellitus, and a family history of CAD was available for all patients. In accordance with the ethical guidelines of the 1975 Declaration of Helsinki, ethics approval was obtained a priori from the local ethics committee (No. 206/2005). 2.2. Immunohistochemistry Slides of formalin-fixed, paraffin-embedded human coronary arteries were deparaffinized and dehydrated, and subsequently heatinduced antigen retrieval was performed using commercially available antigen retrieval solution (DAKO). Five micrometer tissue sections from each patient were co-stained with primary antibodies against CD68 (DAKO, prediluted), MMP7 (Sigma-Aldrich, 1:100) and S100A8 (BMA Biomedicals, 1:100) overnight at 4 °C. To detect nonspecific binding, one additional section per specimen was stained with isotype controls as follows: mouse-IgG1 (1:100, DAKO), mouse-IgG3 (1:800, R&D Systems) and rabbit-IgG (1:2250, R&D Systems). After washing three times in TBS, the slides were incubated with secondary antibodies for 60 min at room temperature applying donkey-anti-rabbit Cy2 (1:200) to detect MMP7, Streptavidin-Cy3 (1:1000) to detect S100A8 and donkey-anti-mouse-Cy5 (1:200, all Jackson ImmunoResearch) to identify CD68. After washing, the slides were mounted with DAPIcontaining mounting medium. From each patient, three adjacent sections were stained for CD68+ macrophages and MMP7+S100A8+CD68+ M4 macrophages. For intima, media and adventitia of each section, five independent representative fields of view each were analyzed using an automatic wide field fluorescence microscope (Nikon Ni-E) and NIS-Elements AR Software (Nikon, version 4.20) at a magnification of 20 ×. For grading of the plaques according to the Stary classification [17], adjacent sections were stained with hematoxylin/eosin (Sigma Aldrich). 2.3. Statistical analysis All results are indicated as means ± standard deviation or absolute numbers and percentages. D'Agostino Pearson omnibus normality testing was used to test for normal distribution. Continuous variables were compared by non-parametric Mann–Whitney testing and categorical variables using Chi square testing. Correlations were analyzed by nonparametric Pearson's testing. The interaction between cardiovascular risk factors, prevalence of M4 macrophages within atherosclerotic Table 2 Plaque characteristics according to the classification suggested by Stary [29]. All patients (n = 51) Lesion type Stary I 1 (2.0%) Stary IIa 4 (7.8%) Stary IIb 5 (9.8%) Stary III 3 (5.9%) Stary IV 6 (1.8%) Stary Va 7 (13.7%) Stary Vb 6 (11.8%) Stary Vc 8 (15.7%) Stary VI 11 (21.6%) ** P b 0.01

ICMP (n = 32)

Non-ICM (n = 19)

P

0 (0.0%) 0 (0.0%) 1 (3.1%) 3 (9.4%) 1 (3.1%) 5 (15.6%) 5 (15.6%) 7 (21.9%) 10 (31.3%)

1 (5.3%) 4 (21.1%) 4 (21.1%) 0 (0.0%) 5 (26.3%) 2 (10.5%) 1 (5.3%) 1 (5.3%) 1 (5.3%)

0.001**

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Fig. 1. M4 macrophages within the coronary artery wall. (A) Representative immunohistochemical staining of an explanted coronary artery against MMP7 (green), S100A8 (red), and CD68 (cyan). DAPI as nuclear stain. Yellow arrow indicates a MMP7+S100A8+CD68+ M4 macrophage, and white arrow indicates a MMP7−S100A8−CD68+ macrophage. White bar = 10 μm. (B) Overlay of the stainings shown in (A). (C) Absolute numbers of all CD68+ macrophages and MMP7+S100A8+CD68+ M4 macrophages within the entire vessel wall. (D) Average proportion of MMP7+S100A8+CD68+ M4 macrophages indicated as percentage of all CD68+ macrophages. (E) Absolute numbers of all CD68+ macrophages and MMP7+S100A8+CD68+ M4 macrophages divided by the different compartments of the vascular wall; *P b 0.05, **P b 0.01, ***P b 0.001.

Table 3 Distribution of CD68+ and M4 macrophages within the different compartments of the arterial wall in all patients and divided in the patients with ICM and non-ICM (means ± SD).

Plaque macrophages Entire vessel wall CD68+ macrophages (n) M4 macrophages (n) M4 macrophages (%) Intima CD68+ macrophages (n) M4 macrophages (n) M4 macrophages (%) Media CD68+ macrophages (n) M4 macrophages (n) M4 macrophages (%) Adventitia CD68+ macrophages (n) M4 macrophages (n) M4 macrophages (%) * P b 0.05 ** P b 0.01 *** P b 0.001

All patients (n = 51)

ICMP (n = 32)

Non-ICM (n = 19)

P

178.1 ± 135.5 56.8 ± 47.3 31.7

191.4 ± 159.8 69.5 ± 48.8 37.9

155.7 ± 78.7 35.53 ± 36.7 21.4

0.397 0.001** b0.001***

59.0 ± 48.8 17.3 ± 18.8 27.8

62.0 ± 49.3 21.3 ± 15.9 36.3

54.0 ± 48.7 10.5 ± 21.6 14.1

0.539 0.002** b0.001***

2.4 ± 4.1 0.5 ± 1.0 23.3

2.9 ± 5.0 0.6 ± 1.1 28.2

1.42 ± 1.7 0.21 ± 0.7 12.5

0.319 0.046* 0.111

116.8 ± 108.1 39.1 ± 33.7 34.7

126.5 ± 130.4 47.5 ± 36.1 40.7

100.3 ± 52.0 24.8 ± 23.7 24.6

0.763 0.006** 0.001**

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plaques and plaque instability were assessed by analysis of variance. P b 0.05 was considered statistically significant. All analyses were performed using SPSS (IBM) or PRISM (GraphPad). 3. Results 3.1. Patient characteristics In total, coronary arteries from 51 hearts explanted during heart allograft transplantation were stained for CD68+ macrophages and MMP7+S100A8+CD68+ M4 macrophages. The majority of coronary arteries were obtained from 32 patients with ischemic cardiomyopathy (ICM) caused by severe coronary artery disease (CAD); nineteen coronary arteries with mild CAD were derived from patients with dilated

cardiomyopathy (DCM) and served as controls. In all patients, the diagnostic workup included coronary angiography and in suspected DCM cardiac magnetic resonance tomography. The demographic and clinical patient characteristics are given in Table 1. The mean age was 51.6 ± 11.7 years. At the time of heart transplantation, ICM patients were significantly older than subjects with nonischemic DCM (55.8 ± 5.2 versus 44.4 ± 15.7 years, P = 0.022). About 80% of patients were male. Cholesterol levels were higher in ICM patients (P b 0.001). There was a trend for a higher prevalence of arterial hypertension (P = 0.079) and diabetes mellitus (P = 0.076) in the ICM group. None of the non-ICM patients had a history of previous myocardial infarction (P b 0.001 versus ICM), percutaneous coronary intervention (P b 0.001), or coronary aortic bypass grafting (P = 0.004). There were no significant differences regarding blood work. ICM patients were

Fig. 2. Representative immunohistochemical stainings of (A) a coronary artery with severe ICM and (B) a non-ICM control coronary artery against MMP7 (green), S100A8 (red), and CD68 (cyan). DAPI as nuclear stain. Shown are representative sections of intima, media, and adventitia. Lower panels are magnifications of the white boxes shown in the upper row. White arrows indicate MMP7+S100A8+CD68+ M4 macrophages. * denotes lumen. White bar = 100 μm.

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Fig. 3. Number of M4 macrophages (A) within the entire vessel wall and (B, C, D) within the different compartments of the vessel wall in ICM and non-IMC patients. *P b 0.05, ***P b 0.001.

more often on antidiabetic drugs (P = 0.026). There were trends for a higher usage of nitrates (P = 0.074) and statins (P = 0.088). As expected, coronary arteries from ICM patients displayed significantly more atherosclerotic lesions with more complex plaque composition (Table 2): In ICM patients, 28 of 32 (87.5%) plaques showed characteristics of advanced lesions (Stary class IV and higher), while 9 of 19 (47.3%) control patients displayed characteristics of early lesions (Stary class I–III).

(Fig. 2A/B, Table 3). Here, both the number and the percentage of M4 macrophages were significantly higher in coronary arteries from ICM patients than in controls (Table 3). This was true for the entire vessel wall, but also for the three compartments intima, media, and adventitia (Fig. 3).

3.2. Distribution of CD68+ macrophages and MMP7+S100A8+CD68+ M4 macrophages within the arterial wall

To assess whether the prevalence of M4 macrophages was associated with advanced lesion morphology, all plaques were graded according to the classification suggested by Stary et al. [17]. In fact, there was a significant positive correlation between the Stary class and the number of M4 macrophages within the intima and the media, but not within the adventitia (Table 4, Figs. 4, 5). Notably, no significant correlation was seen between Stary class and the overall number of CD68+ macrophages in any compartment of the vascular wall (data not shown). Other factors significantly associated with the quantity of M4 macrophages within the arterial wall were the diagnosis of ICM and age (Table 4). To test whether the prevalence of M4 macrophages in the intima represents an independent risk factor for plaque destabilization as determined by Stary class or rather a consequence of an overall proatherosclerotic environment, we performed analysis of variance to identify the contribution of sex, age, cardiovascular risk factors, and the presence of CD68+ macrophages or MMP7+S100A8+CD68+ M4 macrophages in the intima. This analysis showed that plaque instability was significantly associated with the number of cardiovascular risk factors (P = 0.008); there were trends for age (P = 0.060) and prevalence of M4 macrophages (P = 0.098). Sex and overall numbers of CD68+

Both CD68+ macrophages representing the entire macrophage population and MMP7+S100A8+CD68+ M4 macrophages were reproducibly identified within all layers of coronary arteries (Fig. 1A–C and Table 3). The average proportion of M4 macrophages of all CD68+ macrophages was 31.7 ± 16.2% (Fig. 1D). The highest number of CD68+ macrophages was found in the adventitia, followed by the intima. As expected, there were only few CD68+ macrophages in the media. Similar results were found for M4 macrophages (Fig. 1E). 3.3. M4 macrophages in ICM and non-ICM controls While the overall number of CD68+ macrophages within the arterial wall was higher in ICM than in non-ICM control patients, the difference was not statistically significant (P = 0.397, Table 3). Similar results were found when analyzing intima, media, and adventitia separately (P = 0.593, P = 0.319, and P = 0.763, respectively). M4 macrophages could be identified in the arterial wall of both ICM patients and controls

3.4. Prevalence of M4 macrophages in the intima and features of plaque instability

Table 4 Correlation of the number of M4 macrophages in the vessel wall with clinical parameters (correlation coefficient, P value). Entire vessel wall Diagnosis of ICM Male sex Age Arterial hypertension Diabetes mellitus Hypercholesterolemia Obesity Family history Smoker Stary classification * P b 0.05 ** P b 0.01 *** P b 0.001

0.452 −0.079 0.283 −0.065 −0.061 0.154 −0.067 −0.030 0.070 0.182

Intima 0.001** 0.582 0.045* 0.635 0.676 0.286 0.644 0.843 0.628 0.200

0.447 0.050 0.210 −0.075 −0.003 0.172 0.047 −0.100 0.198 0.361

Media 0.001** 0.725 0.139 0.605 0.984 0.232 0.746 0.513 0.168 0.009**

0.282 0.300 0.063 0.244 −0.112 0.220 0.187 0.234 0.347 0.434

Adventitia 0.045* 0.032* 0.660 0.087 0.440 0.125 0.194 0.122 0.013* 0.001**

0.391 −0.133 0.255 −0.050 −0.079 0.143 −0.087 0.034 −0.021 0.090

0.004** 0.354 0.071 0.731 0.583 0.322 0.548 0.825 0.885 0.053

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Fig. 4. Representative H&E staining of (A) a coronary artery with an early lesion (Stary IIb) and (C) a coronary artery with a complicated lesion (Stary VI). (B) and (D) show representative immunohistochemical staining of sections adjacent to (A) and (C) stained for MMP7 (green), S100A8 (red), and CD68 (cyan). DAPI as nuclear stain. * denotes lumen. White bar = 500 μm. Panels on the right are magnifications of the white boxes shown in the mid column.

macrophage were not associated with plaque stability (P = 0.239 and P = 0.337, respectively). 4. Discussion Atherosclerosis remains the major cause of death worldwide [1]. Macrophages are essential players in atherogenesis [2]. Macrophage phenotypic differentiation largely depends on the local micromilieu [3, 4]. We have recently shown that the platelet chemokine CXCL4 promotes macrophage differentiation towards a specific phenotype, which we suggested to call “M4” [11,12]. We here demonstrate that (1) M4 macrophages can be reproducibly found in human atherosclerotic coronary arteries, (2) the prevalence of M4 macrophages is greater in patients with severe CAD, and (3) that there is a significant correlation between M4 macrophage accumulation within the intima and plaque destabilization, which is independent of the overall number of CD68+ macrophages in the vascular wall. There is good evidence for a pro-atherogenic role of CXCL4: Activated platelets release CXCL4 at high concentrations and promote atherogenesis in mice [18], the underlying mechanisms most likely involve deposition of platelet chemokines on the endothelial surface. It is

known that the platelet chemokines CXCL4 and CCL5 form heterodimers that promote monocyte adhesion to the endothelial surface [19]. Interruption of CXCL4/CCL5 heterodimer formation using a specifically designed small molecule was demonstrated to inhibit atherogenesis [20]. Knock out of the Pf4 gene coding for murine CXCL4 in atherosclerosis-prone Apoe−/− mice resulted in decreased lesion development confirming a pro-atherogenic role for CXCL4 [8]. Recent data comparing the basic mechanisms of immunity and inflammation in mice and humans have revealed substantial interspecies differences [21,22]. Therefore, it seems important to confirm findings from murine models in the human system before aiming at translating these findings into the clinical setting. In fact, the presence of CXCL4 within carotid atherosclerotic plaques was found to correlate with both lesion severity and symptomatic state of the patients [23]. Over the past few years, we have studied he effects of CXCL4 on human monocyte macrophage differentiation. As described by Scheuerer et al., CXCL4 prevents monocyte apoptosis and promotes macrophage differentiation [10]. We were able to show that these CXCL4-induced macrophages display features different from previously described macrophage polarizations such as M1 or M2 [5]. Thus, while M4 macrophages lose the hemoglobin–haptoglobin receptor CD163 (which is typically

Fig. 5. Correlation between the prevalence of M4 macrophages within (A) the entire vessel wall and the different compartments of the arterial wall (B, C, D) and plaque morphology as represented by the Stary classification.

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expressed by M2 macrophages), they display a cytokine profile similar to that of M1 macrophages [11,12]. Expanding this work, we were able to identify a marker combination that would allow identification of M4 macrophages both in vitro and in vivo. Based on transcriptomic data from M4, and also M1 and M2 macrophages, we found that staining against CD68, MMP7 and S100A8 unequivocally identifies M4 macrophages in atherosclerotic coronary arteries [14]. We have now for the first time studied the prevalence of M4 macrophages MMP7+S100A8+CD68+ within human coronary atherosclerotic lesions and its association with plaque instability. Similar to CD68+ macrophages, M4 macrophages are mostly located within the intima and the adventitia; cell numbers in the media were low for both. Most strikingly, the intima advanced unstable lesions contain significantly more M4 cells, while the number of CD68+ was not significantly increased as compared to controls. The significant association between M4 macrophages within the intima underlines the important role of this compartment for plaque destabilization. Current understanding is that plaque destabilization is mainly the result of matrix degradation caused by matrix metalloproteases predominantly secreted by plaque macrophages [24,25]. Accordingly, the phenotype of plaque macrophages may have a great influence on the fate of an atherosclerotic lesion. Our data suggest that not the overall number of CD68+ macrophages is important, but rather the number of plaque-destabilizing cells; M4 macrophages seem to represent such a plaque-destabilizing phenotype. At this point in time, it remains unclear whether M4 macrophages directly contribute to plaque destabilization or are rather a bystander. There are data suggesting a potential pathogenic role since M4 macrophages show increased MMP7 expression [14]. There are data suggesting a role for MMP7 in atherogenesis [26] and plaque destabilization [27,28]. Whether M4 macrophages destabilize plaques via increased MMP7 activity needs to be tested in future studies. Other (indirect) mechanisms of MMP7 effects on atherosclerosis are conceivable, thus it has been demonstrated that MMP7 may modulate apoptosis of vascular smooth muscle cells thereby contributing to plaque rupture [28]. Furthermore, increased levels of pro-inflammatory cytokines such as interleukin-6 or TNF-α secreted by M4 macrophages may also play a role [11]. In conclusion, our data suggest that the presence of M4 macrophages in the intima of coronary arteries is associated with advanced atherosclerosis and plaque instability. Accordingly, M4 macrophages may represent an attractive target to image and modulate unstable lesions in coronary atherosclerosis. Further studies are needed to elucidate the molecular pathways involved in M4-dependent pathomechanisms and to test whether inhibition of M4 macrophages is an option to achieve plaque stabilization. Conflict of interest The authors report no relationships that could be construed as a conflict of interest. Grant support This work was supported by the Otto Hess Stipend (German Cardiac Society) to A.W., by grants from the German Heart Foundation to C.E. and C.A.G., by the German Research Foundation (DFG) to C.E. (ER 682/ 2-1) and to F.L. (SFB938/TP Z2), by the DZHK (German Centre for Cardiovascular Research), and by the BMBF (German Ministry of Education and Research) to H.A.K.

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Prevalence of M4 macrophages within human coronary atherosclerotic plaques is associated with features of plaque instability.

The platelet chemokine CXCL4 induces monocyte differentiation resulting in a macrophage phenotype called "M4", which co-expresses CD68, MMP7, and S100...
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