High Levels of Myeloid-Related Protein 14 in Human Atherosclerotic Plaques Correlate With the Characteristics of Rupture-Prone Lesions
Objective— Atherosclerotic plaque rupture can lead to severe complications such as myocardial infarction and stroke. Myeloid related protein (Mrp)-14, Mrp-8, and Mrp-8/14 complex are inflammatory markers associated with myocardial infarction. It is, however, unknown whether Mrps are associated with a rupture-prone plaque phenotype. In this study, we determined the association between Mrp-14, -8, -8/14 plaque levels and plaque characteristics.
Methods and Results— In 186 human carotid plaques, levels of Mrp-14, -8, and -8/14 were quantified using ELISA. High levels of Mrp-14 were found in lesions with a large lipid core, high macrophage staining, and low smooth muscle cell and collagen amount. Plaques with high levels of Mrp-14 contained high interleukin (IL)-6, IL-8, matrix metalloprotease (MMP)-8, MMP-9, and low MMP-2 concentrations. Mrp-8 and Mrp-8/14 showed a similar trend. Within plaques, a subset of nonfoam macrophages expressed Mrp-8 and Mrp-14 and the percentage of Mrp-positive macrophages was higher in rupture-prone lesions compared to stable ones. In vitro, this subset of macrophages does not acquire a foamy phenotype when fed oxLDL.
Conclusion— Mrp-14 is strongly associated with the histopathologic features and the inflammatory status of rupture-prone atherosclerotic lesions, identifying Mrp-14 as a local marker for these plaques.
Rupture of an atherosclerotic plaque and subsequent thrombosis is the underlying cause of the majority of acute coronary syndromes (ACS) and strokes.1 The major determinants of a rupture-prone plaque are the size of the lipid core, the thickness of the fibrous cap covering the core, and ongoing inflammation and repair within the cap.2 Macrophages can weaken the fibrous cap by secreting matrix degrading proteases and inflammatory proteins, leading to plaque disruption and subsequent thrombosis. Although macrophages are considered a hallmark of the rupture-prone plaque, it is unknown which proteins expressed by these macrophages can be used to identify rupture-prone plaques.
Myeloid related proteins (Mrp)-14 and -8 (also named S100A9 and S100A8 or calgranulin B and A) are 2 calcium-binding proteins mainly expressed in cells of myeloid origin, particularly in monocytes and neutrophils.3 Both proteins are secreted by activated monocytes and neutrophils and have proinflammatory effects.4,5 Mrp-14 and Mrp-8 are expressed by subsets of macrophages during inflammation in different tissues,6 as well as in atherosclerotic lesions.7 On cell activation, the 2 proteins can form a complex, Mrp-8/14, that translocates to the cytoskeleton and plasma membrane where it is secreted.8 Intracellularly, Mrp-8 and Mrp-14 essentially regulate phagocyte (monocytes and neutrophils) migration by integrating the calcium and mitogen-activated protein kinase (MAPK) transduction pathways, thereby controlling reorganization of the phagocyte microtubular system.9 The secreted Mrp-8/14 complex exerts antimicrobial activity,10 stimulates IL-8 production by airway epithelial cells,11 and transports arachidonic acid to endothelial cell (EC) targets affecting pathological responses in inflammation and atherosclerosis.10 The receptor for advanced glycation endproducts (RAGE)12 and toll like receptor (TLR)-413 are 2 putative receptors for Mrp-8, Mrp-14, and Mrp-8/14 complex on phagocytes. The Mrp-8/14 complex is emerging as a new blood biomarker that can discriminate between patients with ACS and those with stable coronary heart disease.14 Systemically as well as at the site of coronary occlusion, the Mrp-8/14 complex is a novel, early, and sensitive marker of ACS and is elevated before necrotic factors such as myoglobin, CK-MB, and troponin.14 A recent study of the platelet transcriptome led to the identification of Mrp-14 as a biomarker that can predict future cardio-vascular events in healthy individuals.15
Taken together, these results suggest that Mrp-8, Mrp-14, and Mrp-8/14 reflect biological events in plaque progression toward rupture leading to the hypothesis that Mrp plaque levels correlate with the characteristics of high-risk rupture-prone atherosclerotic lesions. Until now, it is unknown whether these proteins are associated with the rupture-prone plaque phenotype. To address this issue, we determined Mrp-8, Mrp-14, and Mrp-8/14 levels in a large cohort of human atherosclerotic specimens and assessed the association with plaque characteristics and the presence of clinically manifest atherosclerotic disease. We found high levels of Mrp-14 in the ruptured-prone lesions which make this protein a suitable candidate for the imaging of high-risk rupture-prone plaques in humans. A subset of nonfoam macrophages expressing Mrp-8 and -14 was predominant in the rupture-prone lesions; in vitro, the Mrp-macrophage subset did not acquire a foamy phenotype when fed with human oxidized low density lipoprotein (oxLDL).
Athero-Express is an ongoing longitudinal cohort study, initiated in 2002 by 2 Dutch hospitals: the University Medical Center Utrecht and the St Antonius Hospital in Nieuwegein.16 The study has been approved by the institutional boards of both hospitals, and written informed consent was obtained from all participants. The study is designed to investigate the expression of atherosclerotic tissue–derived biological markers in relation to plaque phenotype of patients undergoing carotid endarterectomy (CEA) and adverse cardiovascular events during follow-up. Patients who undergo carotid endarterectomy (CEA) fill in an extensive questionnaire, and diagnostic examinations are performed.
In this study a random set of 186 plaques from symptomatic (n=154) and asymptomatic (n=32) patients undergoing carotid endarterectomy (CEA) were included. The indication for CEA for asymptomatic patients was based on the recommendations published by the Asymptomatic Carotid Surgery Trial (ACST) and for symptomatic patients was based on recommendations based on the European Carotid Surgery Trial (ECST) and the North American Symptomatic Carotid Endarterectomy Trial (NASCET).17–20 All patients were reviewed by the vascular surgeon or neurologist before CEA to assess the nature and timing of clinical symptoms.
All carotid plaques were carefully dissected from the carotid arteries and immediately transferred to the laboratory for further processing as described previously16 and in detail in the supplemental materials (available online at http://atvb.ahajournals.org). In short, in the laboratory the atherosclerotic fragments were dissected by a dedicated technician into 0.5-cm-thick cross-sectional segments along the longitudinal axis of the vessel. The plaque segment showing the largest plaque burden was called the culprit lesion and was used for histological analysis to determine plaque morphology. The definitions of each staining category (H&E, Elastin von Gieson, picrosirius red, α-actin, and CD68) have been described previously.16
Levels of interleukin (IL)-6 and IL-8 were measured by a multiplex suspension array system according to the manufacturer’s protocol (Bender Med Systems). Matrix metalloproteinase (MMP)-2, MMP-8, and MMP-9 activities were measured using the Biotrak activity assays RPN 2631, RPN 2635, and RPN 2634 (Amersham Biosciences), respectively.
Immunoassays for Mrp-8, Mrp-14, and Mrp-8/14
Concentrations of Mrp-8, Mrp-14, and Mrp-8/14 were measured with a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available kits (BMA Biomedicals AG) according to the manufacturer’s protocols. The detection limits for Mrp-8 homodimers, Mrp-14 homodimers, and Mrp-8/14 heterodimers were 0.69, 0.31, and 4.69 ng/mL, respectively. Each ELISA kit was specific for the target Mrp-protein, and the cross-reactivity was minimal (according to the manufacturer). All concentrations were corrected (normalized) for the amount of protein in each sample.
Immunohistochemistry for Mrp-8 and Mrp-14
To determine the cellular source of Mrp-8 and Mrp-14, a random set of 80 plaques, from the total 186 plaques included in the present study, was selected for immunohistological analysis. Sections were pretreated with EDTA and stained with mouse antihuman Mrp-8 (mouse IgG2b, dilution 1:750; Santa-Cruz Biotechnologies) monoclonal antibody. Consecutive sections were boiled in citrate buffer (M=294.1 g/mol, pH 6.0, 20 minutes) and stained with a monoclonal antihuman Mrp-14 antibody (mouse IgG1, dilution 1:200; Santa-Cruz Biotechnologies). Powervision poly HRP-antimouse IgG (Immunologic) was used as secondary antibody. Mouse IgG of the same isotype and same subclass as the primary antibody was used as negative control. The signal was visualized using diaminobenzidine. Sections were counterstained with hematoxylin.
The CD68 staining and the Mrp-8 staining were quantified using image-analyzing software (Soft Imaging Systems). Expression of Mrp-8 and Mrp-14 was detected in nonfoam CD68-positive macrophages. We therefore decided to select only the nonfoam CD68-positive macrophage areas for quantitative analysis. Areas rich in macrophage foam cells were excluded from the analysis. CD68 positive macrophage foam cells were identified by their classical morphology (increased cell size, lipid droplets in the cytoplasm, and nucleus pushed to the membrane side of the cytoplasm).
In Vitro Generation of Human oxLDL-Laden Macrophages
Human monocyte-derived macrophages were generated as previously described21; monocytes were isolated from anonymous healthy blood donors’ buffy coats using Ficoll and Percoll density gradients (density: 1.077 g/mL and 1.063 g/mL, respectively). Next, the monocytes were differentiated into macrophages by culturing the monocytes under nonadherent conditions in RPMI 1640 medium (BioWhittaker) supplemented with 25 nM Hepes, Ultraglutamin1, and 5% human AB serum and without further cytokine stimulation. After 7 days, macrophages were sorted based on CD14 and Mrp-8/14 membrane expression as described below. The 2 sorted macrophage populations, namely Mrp-8/14 negative and Mrp-8/14 positive, were plated into 12-well (flow cytometric analysis) or 96-well (oil red O staining) flat-bottom culture plates (Nunc). The macrophages were incubated during 24 hours with 10 μg/mL human oxLDL, isolated and oxidized as described previously22,23 or with culture medium as control. OxLDL uptake was detected with oil red O, which stains neutral lipids, as previously described.24
Flow Cytometry and Fluorescence-Activated Cell Sorting
Fluorescence-activated cell sorting on a FACS Aria (BD Biosciences) was performed to sort the CD14-positive Mrp-8/14–negative and CD14-positive Mrp-8/14–positive cells. CD14-positive cell sorting was based on forward light scattering (FSC) and sideward light scattering (SSC), and subsequent gating of cells negative for CD3-PerCP (BD Biosciences), CD19-APC (BD Biosciences), and CD56-RPE (Bio-Connect BV). Mrp-8/14 FITC (27E10) (mouse IgG1, Santa Cruz) antibody, recognizing only the Mrp-8/14 heterodimers, was used to identify the Mrp-8/14–negative and Mrp-8/14–positive cells within the CD14 positive population.
For flow cytometry, the following antibodies were used: CD14-PerCP (BD Biosciences); CD68-APC (R&D Systems); Mrp-8/14-FITC (27E10), Mrp-8-FITC, and Mrp-14-FITC (all from Santa Cruz). The samples were measured on a LSR II (BD Biosciences) and analyzed using FACS Diva version 6.1.1. (BD biosciences) and Flow Jo Version 7.2.5 (Tree Star Inc) software.
Data are expressed as means±SEM. Correlations between different parameters were assessed using Spearman correlation test; the statistical significance of the difference between two groups was determined using Mann–Whitney test; probability values <0.05 were considered significant.
The authors had full access to the data and take responsibility for its integrity. All authors have read and agreed to the manuscript as written.
Mrp Plaque Levels and Atherosclerotic Plaque Histology
The clinical characteristics of the patients and the histological features of the atherosclerotic specimens are shown in the supplemental materials.
Protein levels of Mrp-8, Mrp-14, and Mrp-8/14 were quantified in 186 atherosclerotic specimens and compared with different characteristics of the atherosclerotic plaque: size of the lipid core, the amount of collagen, the frequency of macrophages, and SMCs (Figure 1). Mrp-8, -14, and -8/14 levels were associated with the size of the lipid core (Figure 1); this positive association was statistically significant (P=0.001, P=0.001, P=0.004 for Mrp-8, -14, and -8/14, respectively). Higher Mrp-8, -14, and -8/14 levels were observed in plaques with a lipid core greater than 40% of the plaque area compared to plaques with a lipid core smaller than 10%.
An inverse association was observed between plaque Mrp-14 levels and the amount of collagen and SMC. High Mrp-14 levels correlated with low collagen levels in the plaque (P=0.01; Figure 1B) and with low SMC content in the plaque (P=0.001; Figure 1B). Mrp-8 and -8/14 showed a similar trend, although without reaching statistical significance (Figure 1A and 1C).
A positive correlation was observed between the macrophage content of the plaque and high levels of Mrp-14 (Figure 1B); this correlation was statistically significant (P=0.008). In plaques with heavy macrophage staining, the concentrations of Mrp-14 were higher compared to plaques with minor macrophage staining. High Mrp-8 levels were significantly associated with heavy macrophage content (P=0.001; Figure 1A), whereas for Mrp-8/14 a similar but nonsignificant trend was observed (Figure 1C). The absolute levels of Mrp-8/14 were higher compared to Mrp-8 and Mrp-14 levels, suggesting that Mrp-8/14 is more abundant in plaques.
In the lesions of patients with clinical symptoms (n=154) we detected higher levels of Mrp-14 and Mrp-8/14 compared to the asymptomatic lesions (n=32; supplemental Figure I).
Mrp Plaque Levels in Relation to MMPs and Cytokines
To investigate the relationship between plaque Mrp levels and matrix degradation and inflammation as features of the rupture-prone plaque, we determined the levels of the proteases MMP-2, MMP-8, MMP-9, and the proinflammatory molecules IL-6 and IL-8 in the lesion (Table). Mrp-14 showed significant positive correlations with IL-6, IL-8, MMP-8, and MMP-9 and a significant negative correlation with MMP-2. Mrp-8 was associated with IL-6, IL-8, and MMP-8 and was not associated with MMP-2 and MMP-9. Mrp-8/14 was associated with IL-8, MMP-8, and MMP-9 plaque levels.
Mrp-8 and Mrp-14 Expression in a Subset of Plaque Macrophages
Mrp-8 and Mrp-14 were expressed by the same cells within atherosclerotic plaques (Figure 2a and 2b). Mrp-8 and Mrp-14 expression was observed in a subset of CD68 positive macrophages (Figure 2c through 2f). The Mrp-positive macrophages did not exhibit a foamy phenotype as CD68-foam macrophages stain negative for Mrp (Figure 2e and 2f). Within nonfoam CD68-positive macrophage areas, the percentage of Mrp-positive macrophages is significantly higher in rupture-prone (n=55, mean 33.57±3.74%) than in stable lesions (n=25, mean 9.56±3.5%; P=0.003; Figure 3).
In addition to macrophages, Mrp expression was observed in a small number of neutrophilic granulocytes (supplemental Figure II).
Mrp and In Vitro Development of Monocytes Into Foamy Macrophages
Because Mrp-8 and Mrp-14 were expressed in nonfoam CD68 plaque macrophages, we asked whether human oxLDL will induce foam morphology in Mrp-expressing macrophages. We monitored Mrp-expression during the development of healthy human monocytes via monocyte-derived macrophages into oxLDL-loaded foamy macrophages using an in vitro system21 (Figure 4a). Monocytes were identified based on their CD14 positivity. These monocytes lacked Mrp-8/14 membrane expression and intracellular CD68 expression but all showed intracellular Mrp-8, -14, and -8/14 positivity (Figure 4b). After 7 days of culture under nonadherent conditions and without further cytokine stimulation as expected, the CD14 monocyte population developed into monocyte-derived macrophages, without losing CD14 membrane expression (Figure 4c). These primary macrophages were now also CD68-positive. All macrophages showed intracellular Mrp-8, -14, and -8/14 expression, but only half of these cells expressed Mrp-8/14 on the membrane (Figure 4c). Subsequent sorting based on FSC, SSC, and Mrp-8/14 surface expression resulted in 2 human primary macrophage populations, namely Mrp-8/14-positive and -negative. Both populations were fed for 24 hours with 10 μg/mL oxLDL, and changes in the morphology were determined by light microscopy and by oil red O staining to detect intracellular neutral lipids. Macrophages expressing membrane-bound Mrp-8/14 did not acquire a foamy phenotype, whereas the vast majority of the macrophages lacking Mrp-8/14 membrane expression acquired a foamy phenotype and contained lipid droplets in the cytoplasm (Figure 5). The Mrp-8/14 membrane expressing macrophages maintained intracellular Mrp-8, Mrp-14, and Mrp-8/14 expression (Figure 4d). In contrast, almost all macrophages that developed a foamy morphology lacked intracellular and membrane Mrp-8/14 as well as intracellular Mrp-14, and approximately half of these cells lacked intracellular Mrp-8 expression (Figure 4d).
Rupture of an atherosclerotic plaque and subsequent thrombosis is the most common cause of acute coronary syndromes and stroke, and markers are needed to identify these dangerous lesions.
The present study identifies Mrp-14 as a marker of the high-risk prone-to-rupture plaque. We report that high levels of Mrp-14 are associated with atherosclerotic lesions displaying features of a rupture-prone plaque. Levels of Mrp-8 and Mrp-8/14 complex showed similar trends but did not always reach statistical significance.
High Mrp-14 plaque levels are significantly associated with high levels of IL-6, IL-8, MMP-8, and MMP-9 and low levels of MMP-2. Mrp-8 showed no significant correlation with MMP-2 and MMP-9, whereas Mrp-8/14 was not correlated with IL-6 and MMP-2. Within the atherosclerotic plaque, MMP-2 is a molecule associated with a stable plaque phenotype.25 Proinflammatory cytokines IL-6 and IL-8 are associated with active plaque inflammation. The levels of proteases, such as MMP-8 and MMP-9, are elevated in the rupture-prone plaques and these MMPs are very active in the most vulnerable regions of the plaque: cap and shoulder.26 The cellular source of these cytokines (IL-6, IL-8) and proteases (MMP-2, -8, -9) in atherosclerotic lesions is heterogeneous; IL-6 is secreted by active plaque macrophages, smooth muscle cells, endothelial cells, and T-cells whereas IL-8 is secreted by active macrophages, endothelial cells, and T-cells.27 MMP-2 and MMP-9 colocalize with activated smooth muscle cells and macrophages within atherosclerotic plaques.26 MMP-8 is an extremely efficient type I collagenolytic enzyme in humans28 and is traditionally considered a neutrophil product, a cell type not commonly observed in atheroma29; however, within human atherosclerotic lesions also vascular endothelial cells, smooth muscle cells, and macrophages express MMP-8.30
Mrp-8 and Mrp-14 are expressed in healthy human blood monocytes and neutrophils and in subpopulation of macrophages in inflammatory tissues.31 In our study, Mrp-8 and Mrp-14 were detected in a subset of macrophages in atherosclerotic plaques. This observation is in accordance with a previous report of subsets of Mrp-positive macrophages,7 suggesting differential activation of plaque macrophages and underlining existence of subsets of macrophages in atherosclerotic lesions. Expression of plaque Mrp-8 and -14 was only observed in nonfoamy macrophages with a higher percentage of Mrp-8– and 14–positive area of the nonfoam CD68 area in the rupture-prone lesions compared to stable ones. This points to Mrps as markers for macrophages that do not develop into foam cells. In vitro data confirmed this observation; showing that Mrp-8/14 membrane bound expressing macrophages did not acquire a foamy phenotype when oxLDL was added to the culture. In contrast, the majority of macrophages lacking Mrp-8/14 membrane expression accumulated lipid droplets in the cytoplasm when fed with oxLDL. A variety of intracellular functions have been implied for Mrp-8, -14, and -8/14 in phagocyte physiology,32 however nothing is documented regarding involvement of these proteins in phagocytosis. A mouse study suggested that secreted CP-10 (58% amino acid identity with human Mrp-8) has chemotactic properties for monocytes and enhances scavenger receptor expression and uptake of modified LDL by these attracted macrophages.33 The Mrp-positive macrophage subset is associated with a high plaque inflammatory status as reflected by the levels of proinflammatory interleukins and proteases, suggesting that this subset might be involved in plaque destabilization (see supplemental Table V). Involvement of Mrp in determining foam cell development or inflammatory active macrophages, however, remains to be determined.
In addition, Mrp-8 and -14 staining was observed in a small number of neutrophilic granulocytes within the plaque area (supplemental Figure II). This observation coupled with the positive correlation between IL-8 and Mrp-8, -14, -8/14 plaque levels might suggest a possible role for neutrophils in plaque associated inflammation. A recent study34 demonstrated that the infiltrated neutrophils within atherectomy specimens of patients with unstable angina were Mrp-8/14–positive. Although the focus of the present study was on the Mrp-subset of nonfoam plaque macrophages, it does not exclude the importance of other possible Mrp-cellular sources within plaque (eg, the neutrophils).
We found high levels of Mrp-14 and Mrp-8/14 in clinically symptomatic atherosclerotic plaques. This observation is in accordance with previous studies showing that symptomatic plaques often exhibit a rupture-prone phenotype.35 Altwegg et al14 recently identified Mrp-8/14 as a new biomarker that can discriminate between patients with ACS and patients with stable coronary heart disease. They report that Mrp-8/14 is markedly elevated in ACS culprit lesions (thrombus and plaque material) when compared with systemic levels, suggesting that Mrp-8/14 is locally expressed at the site of coronary occlusion because of plaque rupture or erosion. Our study clearly demonstrates that high levels of Mrp-14 but probably also Mrp-8 and Mrp-8/14 plaque are associated with rupture-prone atherosclerotic plaques. This has important implications for possible noninvasive imaging techniques to detect in vivo, high-risk, hidden plaque destabilization and rupture before this leads to dangerous cardiovascular complications and therefore adds to patient stratification for therapy.
In summary, we show that high levels of Mrp-14 and to a lesser extent also Mrp-8 and Mrp-8/14, expressed by a subset of nonfoam macrophages in human plaques, are strongly associated with both histopathologic features and the inflammatory status of rupture prone lesions. These results identify Mrps as possible imaging markers to detect the hidden rupture-prone plaque.
The nonsignificant results obtained for Mrp-8 and -8/14 might be attributable to limitations of the commercial ELISA kits used for the detection of those proteins. The Mrp-8 levels might be underestimated because of lower sensitivity of the Mrp-8 ELISA kit compared to the Mrp-14 ELISA kit. The detection of the Mrp-8/14 heterodimer could be influenced by different conditions used when performing the assays; we performed all assays under the same conditions, and little variation between tests was observed. It is important to note that the present study is purely observational and no implications regarding causality can be drawn. However, considering the facts that Mrp-positive cells are absent in the normal vessel wall,7 are differentially expressed in stable versus rupture-prone plaques, with high levels in the rupture-prone plaque, it is more than reasonable to speculate that these proteins play a role in plaque destabilization. The exact function of Mrps in atherosclerotic lesions, however, remains unclear at this point.
The authors acknowledge Evelyn Velema, Santusha S. Karia, and E.F.E. de Haas for technical assistance. We thank Prof Johan Kuiper (Leiden University) for providing human oxLDL.
Sources of Funding
This work was supported by the grant from the European Community’s Sixth Framework Program contract LSHMCT-2006-037400 (IMMUNATH). Work in the laboratory of J.D.L. is supported by the Dutch MS Research Foundation.
The authors report that Dominique P.V. de Kleijn, Frans Moll, and Gerard Pasterkamp are cofounders of Cavadis, a biomarker company. The other authors report no significant conflicts of interest.
Received November 7, 2008; revision accepted May 27, 2009.
Falk E, Fuster V. Angina pectoris and disease progression. Circulation. 1995; 92: 2033–2035.
Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000; 20: 1262–1275.
Hessian PA, Edgeworth J, Hogg N. MRP-8 and MRP-14, two abundant Ca2+)-binding proteins of neutrophils and monocytes. J Leukoc Biol. 1993; 53: 197–204.
Frosch M, Strey A, Vogl T, Wulffraat NM, Kuis W, Sunderkotter C, Harms E, Sorg C, Roth J. Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis. Arthritis Rheum. 2000; 43: 628–637.
Edgeworth J, Gorman M, Bennett R, Freemont P, Hogg N. Identification of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J Biol Chem. 1991; 266: 7706–7713.
McCormick MM, Rahimi F, Bobryshev YV, Gaus K, Zreiqat H, Cai H, Lord RSA, Geczy CL. S100A8 and S100A9 in human arterial wall: implications for atherogenesis. J Biol Chem. 2005; 280: 41521–41529.
Teigelkamp S, Bhardwaj RS, Roth J, Meinardus-Hager G, Karas M, Sorg C. Calcium-dependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14. J Biol Chem. 1991; 266: 13462–13467.
Vogl T, Ludwig S, Goebeler M, Strey A, Thorey IS, Reichelt R, Foell D, Gerke V, Manitz MP, Nacken W, Werner S, Sorg C, Roth J. MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. Blood. 2004; 104: 4260–4268.
Sunahori K, Yamamura M, Yamana J, Takasugi K, Kawashima M, Yamamoto H, Chazin W, Nakatani Y, Yui S, Makino H. The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis. Arthritis Res Ther. 2006; 8: R69.
Altwegg LA, Neidhart M, Hersberger M, Muller S, Eberli FR, Corti R, Roffi M, Sutsch G, Gay S, von Eckardstein A, Wischnewsky MB, Luscher TF, Maier W. Myeloid-related protein 8/14 complex is released by monocytes and granulocytes at the site of coronary occlusion: a novel, early, and sensitive marker of acute coronary syndromes. Eur Heart J. 2007; 28: 941–948.
Healy AM, Pickard MD, Pradhan AD, Wang Y, Chen Z, Croce K, Sakuma M, Shi C, Zago AC, Garasic J, Damokosh AI, Dowie TL, Poisson L, Lillie J, Libby P, Ridker PM, Simon DI. Platelet expression profiling and clinical validation of myeloid-related protein-14 as a novel determinant of cardiovascular events. Circulation. 2006; 113: 2278–2284.
Verhoeven B, Velema E, Schoneveld A, Vries J, de Bruin P, Seldenrijk C, Kleijn D, Busser E, der Graaf Y, Mol F, Pasterkamp G. Athero-express: Differential atherosclerotic plaque expression of mRNA and protein in relation to cardiovascular events and patient characteristics. Rationale and design. Eur J Epidemiol. 2004; 19: 1127–1133.
Barnett HJM, Taylor DW, Eliasziw M, Fox AJ, Ferguson GG, Haynes RB, Rankin RN, Clagett GP, Hachinski VC, Sackett DL, Thorpe KE, Meldrum HE, Spence JD, The North American Symptomatic Carotid Endarterectomy Trial Collaborators. Benefit of carotid endarterectomy in patients with symptomatic moderate or severe stenosis. N Engl J Med. 1998; 339: 1415–1425.
Boven LA, Van Meurs M, Van Zwam M, Wierenga-Wolf A, Hintzen RQ, Boot RG, Aerts JM, Amor S, Nieuwenhuis EE, Laman JD. Myelin-laden macrophages are anti-inflammatory, consistent with foam cells in multiple sclerosis. Brain. 2006; 129: 517–526.
Van Berkel TJ, De Rijke YB, Kruijt JK. Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells. J Biol Chem. 1991; 266: 2282–2289.
Chayen J, Bitensky L. Analysis of chemical components of cells and tissues: reactions for lipids. Practical Histochemistry. New York: Chichester; 1991: 45.
Sluijter JPG, Pulskens WPC, Schoneveld AH, Velema E, Strijder CF, Moll F, De Vries JP, Verheijen J, Hanemaaijer R, De Kleijn DPV, Pasterkamp G. Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms. Stroke. 2006; 37: 235–239.
Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatory pathways. Physiol Rev. 2006; 86: 515–581.
Hasty KA, Jeffrey JJ, Hibbs MS, Welgus HG. The collagen substrate specificity of human neutrophil collagenase. J Biol Chem. 1987; 262: 10048–10052.
Herman MP, Sukhova GK, Libby P, Gerdes N, Tang N, Horton DB, Kilbride M, Breitbart RE, Chun M, Schonbeck U. Expression of neutrophil collagenase matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation. 2001; 104: 1899–1904.
Zwadlo G, Schlegel R, Sorg C. A monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues. J Immunol. 1986; 137: 512–518.
Miyamoto S, Ueda M, Ikemoto M, Naruko T, Itoh A, Tamaki Si, Nohara R, Terasaki F, Sasayama S, Fujita M. Increased serum levels and expression of S100A8/A9 complex in infiltrated neutrophils in atherosclerotic plaque of unstable angina. Heart. 2008;hrt.
Verhoeven B, Hellings WE, Moll FL, De Vries JP, De Kleijn DPV, de Bruin P, Busser E, Schoneveld AH, Pasterkamp G. Carotid atherosclerotic plaques in patients with transient ischemic attacks and stroke have unstable characteristics compared with plaques in asymptomatic and amaurosis fugax patients. J Vasc Surg. 2005; 42: 1075–1081.