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Chemokine (C-C motif) ligand 2 and coronary artery disease: Tissue expression of functional and atypical receptors
Cytokine 126 (2020) 154923
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Chemokine (C-C motif) ligand 2 and coronary artery disease: Tissue expression of functional and atypical receptors
T
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Anna Hernández-Aguileraa,1, , Montserrat Fiblab,1, Noemí Cabréc, Fedra Luciano-Mateoc, Jordi Campsa, Salvador Fernández-Arroyoc,d, Vicente Martín-Parederoe, Javier A. Menendezf,g, ⁎ Juan J. Sirventb, Jorge Jovena,c,d, a
Unitat de Recerca Biomèdica, Hospital Universitari Sant Joan, Institut d’Investigació Sanitària Pere Virgili, Reus 43201, Tarragona, Spain Department of Pathology, Hospital Universitari Joan XXIII, Tarragona 43005, Spain c Universitat Rovira i Virgili, Departament de Medicina i Cirurgia, Unitat de Recerca Biomèdica, Tarragona 43003, Spain d The Campus of International Excellence Southern Catalonia, Tarragona 43003, Spain e Department of Vascular Surgery, Hospital Universitari Joan XXIII, Tarragona 43005, Spain f ProCURE (Program Against Cancer Therapeutic Resistance), Metabolism and Cancer Group, Catalan Institute of Oncology, Salt 17190, Girona, Spain g Girona Biomedical Research Institute (IDIBGI), Salt 17190, Girona, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis CCL2 CCR2 ACKR Inflammation
Chemokines, particularly chemokine (C-C- motif) ligand 2 (CCL2), control leukocyte migration into the wall of the artery and regulate the traffic of inflammatory cells. CCL2 is bound to functional receptors (CCR2), but also to atypical chemokine receptors (ACKRs), which do not induce cell migration but can modify chemokine gradients. Whether atherosclerosis alters CCL2 function by influencing the expression of these receptors remains unknown. In a necropsy study, we used immunohistochemistry to explore where and to what extent CCL2 and related receptors are present in diseased arteries that caused the death of men with coronary artery disease compared with unaffected arteries. CCL2 was marginally detected in normal arteries but was more frequently found in the intima. The expression of CCL2 and related receptors was significantly increased in diseased arteries with relative differences among the artery layers. The highest relative increases were those of CCL2 and ACKR1. CCL2 expression was associated with a significant predictive value of atherosclerosis. Findings suggest the need for further insight into receptor specificity or activity and the interplay among chemokines. CCL2-associated conventional and atypical receptors are overexpressed in atherosclerotic arteries, and these may suggest new potential therapeutic targets to locally modify the overall anti-inflammatory response.
1. Introduction Reversing atherosclerosis is a desirable and formidable challenge because acute coronary syndrome (ACS) and strokes remain the leading causes of death and affliction worldwide [1,2]. Preventive measures based on treating systemic risk factors are necessary and may slow the progression, but this strategy is insufficient because atherosclerosis development is likely regulated by the local microenvironment in
diseased arteries [3]. The inside-out theory of vascular inflammation indicates that lipid deposition in the subendothelial layer of arteries and retention in smooth muscle cells entail structural changes that stimulate inflammation and the transformation of macrophages into foam cells in a self-maintaining progressive cycle [4–7]. By contrast, an outside-in theory assumes that the inflammatory cells gain entry to the vessel wall via the adventitial vasa vasorum [8]. Both theories may be integrated into a model to understand atherogenesis, but the task involves
Abbreviations: ACKRs, atypical chemokine receptors; CCBP2, chemokine binding protein 2; CCL2, chemokine (C-C motif) ligand 2; CCR2, C-C chemokine receptor type 2; CD68, cluster of differentiation 68; DARC, Duffy antigen receptor for chemokines; GPCRs, G protein-coupled receptors; H&E, hematoxylin and eosin ⁎ Corresponding authors at: Unitat de Recerca Biomèdica - Hospital Universitari Sant Joan, Institut d’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201 Reus, Spain. E-mail addresses: [email protected] (A. Hernández-Aguilera), montsefi[email protected] (M. Fibla), [email protected] (N. Cabré), [email protected] (F. Luciano-Mateo), [email protected] (J. Camps), [email protected] (S. Fernández-Arroyo), [email protected] (V. Martín-Paredero), [email protected] (J.A. Menendez), [email protected] (J.J. Sirvent), [email protected] (J. Joven). 1 These authors contributed equally. https://doi.org/10.1016/j.cyto.2019.154923 Received 24 April 2019; Received in revised form 6 November 2019; Accepted 8 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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manipulating inflammation under strategies focused on interfering with this vicious cycle. Many anti-inflammatory treatments for atherosclerosis have been tested but most of them needs to be further studied in clinical trials [9,10]. The lack of diagnostic, prognostic and treatment response markers sets hurdles in the improvement of drugs targeting relevant processes [10]. Recent interventions that facilitate macrophage egress or that attempt to solubilize cholesterol regress the pre-existing atherosclerotic plaques and reveal that cholesterol efflux alters the local production and deposition of cytokines [3,11,12]. Among cytokines, chemokine (CC motif) ligand 2 (CCL2), through conventional receptors, mainly CCR2, belonging to the 7-transmembrane-spanning family of G proteincoupled receptors (GPCRs), regulates pleiotropic signaling pathways that influence numerous cellular activities involved in atherosclerosis development [13–18]. The role of circulating CCL2 as a potential biomarker has been examined [19–21] with controversial results [22,23] derived from ill-defined effects of atypical chemokine receptors (ACKRs), which are expressed in non-leukocyte cell types but do not induce cell migration. ACKRs are those kinds of receptors that do not transduce signal through G-protein signaling but may modify chemokine availability. The best-known ACKRs that bind CCL2 and other chemokines are ACKR1 (Duffy antigen receptor for chemokines; DARC) and ACKR2 (chemokine binding protein 2; CCBP2 or D6) [24–27]. We envisioned that atherosclerosis could promote the expression and interplay of conventional and atypical CCL2 receptors in cells that affect the development of advanced coronary artery disease (CAD). What we know in humans is modest, and there have been no clinical studies designed to examine this relationship, probably because doing so would require specific biological assays or excessively invasive methods [28,29]. We reasoned that a necropsy study could provide additional knowledge by exploring, for the first time, where and to what extent CCL2 and the expression of related receptors may increase in diseased arteries that have been responsible for the death of patients with CAD compared with those in healthy arteries. Our data reinforce CCL2 as a major determinant of atherosclerosis progression and might support the quest to new druggable targets.
Fig. 1. Histologic changes in coronary arteries reveal atherosclerosis. (A) Lesions, especially the thickness of the media and intima, were evident with hematoxylin and eosin staining. (B) Alizarin Red Staining was used to assess calcium deposits, and (C) Masson’s trichrome staining was mostly used to assess variations in the overall structure and remodeling. (L is Lumen, I is Intima, M is Media and A is Adventitia). All sections were examined at different magnifications, but automated analysis was performed at 20 × . Representative images include the scale bar.
fixation and was deemed irrelevant for our purposes.
2. Materials and methods
2.2. Histological examination
2.1. Research design
The specimens were fixed in 10% neutral-buffered formalin and were embedded in paraffin. Serial sections of 4 µm were placed in a 45 °C water bath before the staining procedures with hematoxylin and eosin (H&E), Alizarin Red (Sigma-Aldrich, Steinheim, Germany) and Masson’s Trichrome reagents (Dako, Glostrup, Denmark) to assess the structure and extent of fibrosis in the arteries and to visualize the localization of calcium phosphate. The intima and media thickness (IMT) and ratio (IMR) were measured and calculated in H&E-stained sections (Fig. 1). Slides were loaded in an automated slide stainer, Autostainer Universal™ (Dako), including sections (in triplicate) for immunohistochemical analyses after conducting the standard procedures for manual de-waxing and heat-induced epitope retrieval (citrate solution pH = 6). Because the tissues contained endogenous peroxidase, we added a blocking step (0.3% hydrogen peroxide in PBS) for 25 min at room temperature after antigen retrieval. Before primary antibody incubation, we also used BSA 1% to block non-specific interactions. Macrophages were detected by the presence of the CD68 antigen (clone: KP1) with reagents provided by Dako (M0876). Primary antibodies against CCL2 (ab9669), CCR2 (ab21667) and ACKR2 (ab1658) were obtained from Abcam (Cambridge, UK) and were used at dilutions of 1:200, 1:100, and 1:500, respectively. ACKR1 (PAB13254) antibody was from Abnova (Taipei, Taiwan) and was used at a dilution of 1:200. Biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used at a dilution of 1:200. Detection was performed using the Vectastain ABC HRP kit (PK-4000, Vector Laboratories) and 3, 3-diaminobenzidine as a substrate (Dako). The slides were
We designed a pre-set duration, observational, cross-sectional study in consecutive male decedents who underwent autopsy between December 2012 and November 2014 at our settings. Based on the autopsy, CAD was firmly established as the cause of death. The local Ethics Committee, which acts as the Institutional Review Board, approved the study protocol and procedures (EPINOLS/12-03-29/3proj6 approved on 29th March 2012). Written informed consent was obtained from next of kin of the deceased for autopsy and access to tissues. The described association between CCL2 and other complex diseases [21] provided pre-established criteria for excluding decedents with evidence of recent infection (n = 2), diabetes (n = 10), liver disturbances (n = 2), neoplasms (n = 3) or with sudden, out of hospital, death (n = 4). As the disease is more prevalent in men and in order to avoid possible sex differences, we finally included 68 men donors in whom the heart was dissected following standard autopsy protocols. In each corpse, we obtained at least one major coronary artery that was narrowed by > 80% of the cross-sectional area by atherosclerotic plaque or associated thrombus. For comparisons, we obtained coronary arteries without visible signs of atherosclerosis from 19 men who had died as a result of road traffic accidents during the same period of time. For other associations, we determined arterial stenosis and arbitrarily classified a total of 123 specimens into mild (less than 20% obstruction), moderate (between 21% and 50%) or severe (more than 50%) atherosclerosis. The expected decrease in the dimensional changes associated with fixation and processing (3–7%) correlated with the values before 2
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counterstained with Mayer’s hematoxylin. The results with these antibodies were compared with sections of previously obtained peripheral arteries [30] and selected tumor sections from primary breast cancer tissues [31] that were used as positive or negative controls (Fig. 3). We also used negative controls from study samples by omitting the primary antibodies from the incubations. Comparisons with other ACKR1 and ACKR2 antibodies (HPA013819 and HPA016421, from Sigma; PA121614, from Affinity Bioreagents; and NB100-2421 from Novus Biologicals) did not yield divergent results. To ensure the localization of CCL2 and their atypical receptors (ACKR1 and ACKR2), we also performed additional double-color immunohistochemistry combining these markers with those related with arterial layers: CD34 for endothelial cells (intima) and the adventitia and α-smooth muscle actin (αSMA) for smooth muscle cells (media). Both primary antibodies were also obtained from Abcam (CD34, ab8536; αSMA, ab21027) and used at dilutions 1:1000 and 1:250 respectively. In this case, detection of these markers was performed with EnVision FLEX HRP Magenta Substrate Chromogen System (Dako). Microphotographs were acquired using an optical microscope (Nikon, Eclipse E600, Madrid, Spain) and an image analysis system (AnalySIS™, Soft Imaging System, Münster, Germany), as described previously [30]. When the arteries were greater than the histological field used for analyses (20 × magnification), several pictures were combined to calculate the percentage of stained area with respect to the total area of the artery. Once images were obtained, colors of the molecules of interest were defined and automatically detected in all samples. The stained area was related to the total area of the image by using phase analysis. More details about the quantification method have been already described and used in different studies [32–34].
Table 1 Main clinical variables. Clinical characteristics
Control group (N = 19)
CAD group (N = 68)
p-value
Age, years Body mass index, kg/m2 No smokers, n (%) Alcohol consumption, n (%) Heart weight (g) Ventricular thickness (mm) Fibrosis, n (%) Affected valves, n (%) Hypertension, n (%)
45 (33–69) 25.7 (4.1) 15 (78.4) 2 (10.5) 380 (300–450) 16 (14–18) 0 2 (10.5) 2 (10.5)
71 (61–77) 24.8 (4.0) 42 (61.8) 9 (14.3) 480 (400–600) 19 (15–20) 10 (15.9) 31 (45.6) 35 (51.5)
0.001 0.217 0.164 0.126 0.002 0.005 0.110 0.005 0.001
*Results are expressed as n (%), median and interquartile range (IQR, 25–75%) or mean (standard deviation).
IMT (intima-media thickness) values of control and diseased arteries, which were significantly different (p < 0.001): in medians (IQR), 586 μm (452.7–865.1) for diseased arteries and 379 μm (265.8–467.8) for control. Moreover, modified tissue architecture of disease arteries was evident when assessed by the different stainings (Fig. 1). Macrophages infiltrated the subendothelial space with a considerable extracellular network of collagen and lipid deposition. CD68stained cells with foamy cytoplasm were scarcely observed in minimally affected arteries but were apparent in the neointima, tunica media and adventitia of affected arteries (Fig. 2). In severe lesions, fibrofatty plaques with cholesterol clefts and mineralized material were frequent. In some cases, lesions were characterized by fibrosis and necrotic debris. Irregular calcium deposits were abundant in severe lesions, which were mostly limited to the intima but also occurred deep in the plaque. Of note, adventitial inflammation and vascularity were significantly increased in affected arteries. When the interactions between each receptor and CCL2 were examined, the functional networks appeared to be similarly complex (S1 figure A-B). Moreover, we found no evidence of any predicted proteinprotein interactions or associations among ACKRs (S1 figure C). Exploring this issue further would require functional assays and additional in vitro designs. Quantitative measurements of the immunohistochemical variables are summarized in Table 2 and are illustrated in Fig. 2. Details segregated among the layers are illustrated in S2 and S3 Figures for the control and affected arteries, respectively. The immediate finding is that the selected markers were significantly more prominent in diseased arteries. The highest relative increase in detection was that of CCL2 (> 9-fold), which closely resembled the higher abundance in macrophages. CCL2 expression was only evident in 26% of normal arteries and was mostly restricted to smooth muscle cells with faint staining in the intima and adventitia. In affected arteries, CCL2 was detected in all specimens and remained more evident in the tunica media but was significantly increased in all layers, particularly in the adventitial tissue. CCR2 expression was observed in more cells and with higher intensity in affected arteries than in controls, but the differences were only statistically significant in the adventitia. The distribution of both CCL2 and CCR2 was qualitatively similar in the smooth muscle cells of the tunica media, probably indicating a functional relationship. The structural role of CCR2 is also likely because CCR2 was expressed in all specimens and because the CCR2 relative abundance in the intima and media was similar in both controls and diseased arteries. In the affected arteries, however, the relative expression was 3-fold higher in the adventitial tissue. The expression of atypical receptors was significantly higher in diseased arteries, and the distribution was qualitatively different. This differential scattering was already observed in those tumor sections used as controls, indicating that not all cells have the ability to respond. ACKR2 did not co-express with macrophages but CCL2, CCR2 and ACKR1 were closely related (Fig. 3). For instance, ACKR1 was not detected in all specimens (63% in controls and 81% in affected arteries);
2.3. Statistical analyses Inter-observer agreement was assessed, and discrepancies were resolved by consensus. Student’s t-test or Mann-Whitney’s test was used to assess the difference between groups according to the distribution of variables. The chi-squared test was used to compare categorical variables. Spearman correlation coefficients were used to evaluate the degree of association between variables and linear regression was performed to check dependences between quantitative variables. To correct for multiple testing, we used the False Discovery Rate estimated using the Q-value. The true- and false-positive rates at various threshold settings were used to plot receiver operating characteristic (ROC) curves. The Statistical Package for the Social Sciences, version 19.0 (SPSS Inc, IBM Corp, Chicago, IL, USA) and MetaboAnalyst 3.0® (www. metaboanalyst.ca) were used for analyses and for obtaining random forest plots and ROC curves. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, http://string-db.org) and HumanBase (Flatiron Institute, http://hb.flatironinstitute.org/) were used as a biological databases and web resources of known and predicted proteinprotein interactions [35–37]. 3. Results The clinical variables of donors confirmed the influence of some expected systemic risk factors (Table 1). In decedents from CAD, compared with controls, hypertension was more prevalent, and age, heart weight and ventricular thickness were significantly increased. However, the proportion of daily smokers and alcohol consumption (10–20 g/day) was similar between the groups, and there were no differences in the body mass index relating the weight to the height. Atherosclerosis is an aging-related disease. To ascertain that the expression of these markers was associated with atherosclerosis and not with age itself, we assessed this point through linear regression models. Results did not support dependence on age (Supplementary Table S1 and S2). Standard histopathology revealed atherosclerosis. We assessed the 3
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Table 2 Main immunohistochemical variables. Immunohistochemical variables* Positive staining expressed as a percentage (%) of the arterial area
CD68 CCL2 CCR2 ACKR1 ACKR2
Control group (N = 19)
CAD group (N = 68)
p-value
0.75 (0–1.8) 2.4 (1.3–5.8) 24.4 (14.4–49.6) 6.8 (2.4–12.6) 21.9 (8.1–35.5)
3.8 (1.6–6.2) 17.2 (9.1–27.3) 40.1 (24.3–54.1) 13.3 (6–43.1) 34.8 (25.2–53.7)
0.002 < 0.001 0.037 0.001 0.020
Percentage (%) of stained area in positive specimens CCL2 (n = 5; 26%) (n = 68; 100%) Intima 4.1 (2.4–5.3) 8.5 (5.9–11.2) Media 10.5 (3.5–19.1) 35.5 (22.4–59.6) Adventitia 3.6 (2.7–5.5) 21.5 (18.4–26.2) CCR2 (n = 19; 100%) (n = 68; 100%) Intima 24.5 (19.2–29.3) 29.1 (24.4–33.3) Media 70.1 (64.3–74.2) 75.6 (70.7–84.6.6) Adventitia 10.4 (7.5–14.1) 31.8 (27.2–34.7) ACKR1 (n = 12; 63%) (n = 55; 81%) Intima 5.4 (3.1–7.3) 74.1 (64.3–78.2) Media 15.8 (11.2–20.5) 82.7 (73.2–84.9) Adventitia 3.9 (2.1–4.9) 65.8 (61.6–68.9) ACKR2 (n = 16; 84%) (n = 68; 100%) Intima 19.5 (11.9–26.3) 21.2 (17–24.4) 24.9 (17.7–26.1) 30.7 (22.9–40.3) Media Adventitia 50.7 (44.2–67.3) 74.2 (61.2–85.1)
< 0.001 < 0.001 < 0.001 0.120 0.087 0.001 0.001 0.001 0.001 0.102 0.087 0.001
Statistically significant differences are marked up in bold. * Staining was measured using automated image analysis and expressed as median and interquartile range (IQR, 25–75%).
tissue. In the affected arteries, ACKR2 was present in all specimens, but the expression was not quantitatively different in the intima and media with respect to the controls. The increased expression in adventitia was statistically significant. To confirm the expression of CCL2 and their atypical receptors in endothelial cells and cells in the adventitia (CD34) and also in smooth muscle cells (αSMA), we performed double-color immunohistochemistry. In control arteries, although there were no clear differences between tunica media and tunica intima, CCL2 was found in smooth muscle cells. ACKR1 was also mainly found in smooth muscle cells. On the contrary, ACKR2 seemed to be more located in the adventitia, although some staining was also observed in tunica media (Fig. 4). Regarding CAD group, CCL2 was mainly expressed in the adventitia although some specimens showed intense expression of this marker in the media. ACKR1 was observed in all arterial layers and ACKR2 was mainly restricted to the adventitia (Fig. 5). We also explored the correlation between these inflammatory markers and the potential value of CCL2 and related receptors as a marker of both the presence and severity of atherosclerosis in terms of stenosis degree. We found no dependence between the measured variables using a Spearman-based correlation matrix, where strong associations can be found in red color and weak association are displayed in blue (Fig. 6 A). Although we found that CCL2 and CCR2 were expressed in the same layers, there was no quantitative correlation among them.
Fig. 2. Expression of CCL2 and related receptors in coronary arteries. The immunohistochemical expression in non-affected arteries and atherosclerotic arteries of CCL2, CD68-stained cells (macrophages), and functional and putatively silent receptors for CCL2 displayed significant differences (see Table 2 for quantitative analysis). CCL2 and CCR2 expression levels were apparently in the same layers in the artery wall. Conversely, the expression of both ACKR1 and ACKR2 differed in intensity and arterial distribution (L is Lumen, I is Intima, M is Media, and A is Adventitia). All sections were examined at different magnifications, but automated analysis was performed at 20 × . Representative images include the scale bar. Representative images at higher magnifications were included in supplementary Figures S2 (control arteries) and S3 (affected arteries).
however, if present, the differences between the controls and affected arteries were significant. Moreover, healthy arteries were stained faintly with some preponderance in smooth muscle cells, and the affected arteries were more strongly stained in all layers. Conversely, the expression of ACKR2 in healthy arteries was more intense and widely distributed than that of ACKR1 (Table 2) but was undetectable in 16% of control arteries. Of note, ACKR2 was detected in endothelial cells, smooth muscle cells and, more importantly, in the perivascular adipose
Fig. 3. The expression of CCL2 and related receptors is tissue-specific. Sections of human tissue of breast carcinoma (Mag. 20 × ) expressing all of the antigens were used as positive controls and were treated exactly as study samples. Selected specimens of breast tissue without ACKR1 or ACKR2 expression were used as a source of negative tissue controls to confirm the absence of background staining and illustrate tissue specificity in ACKR expression (images not shown). 4
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classification constructed using Random forest plots, which is a nonparametric and bias-free method that classifies variables according their discriminant capacity between two groups, CCL2 expression was the variable with the best discriminant ability (Fig. 6C). CCL2 detection, but not related receptors, discriminated controls from CAD samples in a mild, moderate or severe state (Fig. 7) with a global cut-off of 6.335 (89.3% sensitivity and 85% specificity). Collectively, the data indicate that CCL2 and related receptors are expressed in different cell types and might suggest that the presence of CCL2 is associated with the occlusion of the arteries. Co-expression among the examined proteins, although suggestive, was not verified or discarded. 4. Discussion Mammals have over 45 inflammatory or homeostatic chemokines divided into subfamilies according to the differences in their function and mature protein sequences. The biological complexity and variety of contexts in which they are found complicate the knowledge on their precise relationships [38]. Indeed, recent literature have linked diet composition and liver energy sensors with the circulating inflammatory monocyte pool [39]. Although CCL2 action is mediated by binding to numerous (over 15), promiscuous and probably redundant canonical receptors, the receptor/ligand interactions are further obscured by an undetermined number of ACKRs that can modulate the expression, or signaling, of that conventional chemokine receptors [29,40]. In the chemokine system, the role of CCL2 and the CCL2/CCR2 axis appears to be critical in the molecular control of inflammation by promoting the response and migration of immune cells, transforming monocytes into macrophages, stimulating smooth muscle cells proliferation and maintaining the continuous and local release of proinflammatory molecules [5,41,42]. This axis is also involved in atherosclerosis development. As seen in double-deficient CCR2-/- and apolipoprotein E-/- mice, the absence of this receptor reduces the atherosclerosis lesion area and slows down lesion formation [43,44]. The potential role of ACKR1 and ACKR2 in atherogenesis is plausible [45,46]. As atherosclerosis is an age-related disease, obtaining samples from age-matched control population without any sign of atherosclerosis is almost impossible. Despite these limitations and those derived from autopsy studies, namely that patients could be not representative of the general population and that women were not included, our findings indicate that the presence of widely distributed CCL2 and conventional and atypical receptors in coronary arteries and perivascular tissue of decedents from CAD may have implications in further research and drug development. The high levels of plasma CCL2 in patients with atherosclerosis and other chronic inflammatory conditions most likely reflect its accumulation in inflamed tissue and the subsequent drainage through the lymphatic system, which is influenced by the active roles of CCR2 and chemokine-scavenging ACKRs [21,28,47,48]. The complex relationships among receptors may explain that CCL2 remained as the only variable with predictive value. Interestingly, CCL2 and ACKR1 expression levels were increased in all layers of the affected arteries, but the higher expression of ACKR1 and ACKR2 was particularly prominent in the adventitia and perivascular fat. Accordingly, the adventitia becomes populated with exogenous cell types that cause an increase in the local expression of CCL2 and related receptors. In humans, both structure and function of adventitial vasa vasorum may associate microvascular dysfunction with early coronary atherosclerosis [49], and CCL2 appears to activate adventitia during angioplasty, inducing sequential patterns in receptor expression [50]. Conversely, it is unclear whether receptors may influence CCL2 expression. Our measurements do not provide insight into receptor specificity or activity but highlight the importance of understanding the influence of CCL2 and the expression of related receptors in the compensatory role of adventitia [51]. Human studies on inflammation in CAD have been focused on single nucleotide polymorphisms, microRNA and lipid determinations in plasma samples [52–57]. Obtaining high quality mRNA
Fig. 4. Double-color immunohistochemical expression of CCL2, ACKR1 and ACKR2 together with CD34 or αSMA in control coronary arteries. Images were segregated by markers (rows) to illustrate layer specific staining (columns) and were selected as representative.
Fig. 5. Double-color immunohistochemical expression of CCL2, ACKR1 and ACKR2 together with CD34 or αSMA in atherosclerotic coronary arteries. Images were segregated by markers (rows) to illustrate layer specific staining (columns) and were selected as representative.
CCL2 expression displayed the most important variable indicating atherosclerosis compared to CCR2 expression when using Receiver Operating Characteristics (ROC) Curve (Fig. 6B). In a model of 5
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Fig. 6. Histologic variables do not correlate, but CCL2 retains predictive value. (A) The correlation matrix was used to investigate the dependence among multiple variables at the same time (based on % of stained area), and no significant association was found. (B) We performed a ROC curve with CCL2 and CCR2 but only CCL2 expression had an acceptable predictive value (great Area Under the Curve). (C) We used Random forests to rank the importance of variables in a likely classification of predictors, and CCL2 was significantly different.
expression in cell types, the ability of ligands to bind several receptors and the formation of dimers and oligomers are major hurdles [59]. For instance, a CCR5 antagonist that was recently introduced for HIV therapy and also another dual CCR5 and CCR2 inhibitor may show promise in the management of atherosclerosis [60,61]. The higher adventitial inflammation, neovascularization and ACKR expression in affected arteries add force to the notion that ACKRs participate in the process and might be druggable targets. In this case, ACKRs have the potential to neutralize chemokine activity, and the pharmacological approach could be the development of their inducers rather than
from these samples was unsuccessful but ongoing studies should clarify this point. Our study provides a first approach about chemokine availability in tissue, which will serve for future mechanistic insights or even for novel therapeutic strategies. To assess potentially effective anti-inflammatory therapies, future research will require the development of novel assays to assess the formation of CCL2 gradients and an accurate characterization of conventional and atypical receptor-expressing cells [58]. The current pharmacological approach is to develop antagonists of chemokine receptors that are involved in leukocyte migration. However, promiscuous ligand binding, the differential
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Fig. 7. CCL2 expression is associated with the percentage of lumen occlusion. Receiving operating characteristic curves were plotted using true- and false-positive results. Most values were associated with coronary artery disease (CCL2, CD68 and atypical receptors), but CCL2 was the most significant (A). Only the predictive value of CCL2 remained significant to distinguish among mild, moderate and severe occlusion (B).
5. Conclusions
antagonists. Because ACKRs may be key components of the regulatory network of inflammation, patient-relevant outcomes are likely. The concept is currently being examined in the field of tumorigenesis with the rationale that chemokine scavenger activity may prevent the recruitment of cells that sustain tumor growth [31,62].
Although this is an observational study and it does not prove any causality, we conclude that the continuous production of CCL2 in chronic inflammation may activate mechanisms with biological functions in atherosclerosis. The increase in ACKRs expression could be a 7
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compensatory mechanism to regulate the increased CCL2 expression. The assessment of how and to what extent the binding of CCL2 to associated receptors transduce signals and/or mediate scavenging may result in potential therapeutic targets.
[7] F. Abdolmaleki, S.M. Gheibi Hayat, V. Bianconi, T.P. Johnston, A. Sahebkar, Atherosclerosis and immunity: A perspective. Vol. 29, Trends Cardiovascular Med. (2019) 363–371. [8] M.J. Mulligan-Kehoe, M. Simons, Vasa vasorum in normal and diseased arteries, Circulation 129 (24) (2014) 2557–2566. [9] Y. Zhu, X. Xian, Z. Wang, Y. Bi, Q. Chen, X. Han, et al., Research progress on the relationship between atherosclerosis and inflammation, Biomolecules 8 (2018) 80. [10] P. Raggi, J. Genest, J.T. Giles, K.J. Rayner, G. Dwivedi, R.S. Beanlands, et al., Role of inflammation in the pathogenesis of atherosclerosis and therapeutic interventions, Atherosclerosis 276 (2018 Sep) 98–108. [11] R.E. Gerszten, A.M. Tager, The monocyte in atherosclerosis–should I stay or should I go now? N. Engl. J. Med. 366 (18) (2012 May) 1734–1736. [12] S. Zimmer, A. Grebe, S.S. Bakke, N. Bode, B. Halvorsen, T. Ulas, et al., Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming, Sci. Transl. Med. 8 (333) (2016) 333ra50. [13] D. Manka, T.K. Chatterjee, L.L. Stoll, J.E. Basford, E.S. Konaniah, R. Srinivasan, et al., Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1, Arterioscler Thromb Vasc Biol. 34 (8) (2014) 1723–1730. [14] B. Yu, M.M. Wong, C.M.F. Potter, R.M.L. Simpson, E. Karamariti, Z. Zhang, et al., Vascular stem/progenitor cell migration induced by smooth muscle cell-derived chemokine (C-C Motif) ligand 2 and chemokine (C-X-C motif) ligand 1 contributes to neointima formation, Stem Cells 34 (9) (2016 Sep) 2368–2380. [15] V. Frodermann, J. van Duijn, M. van Pel, P.J. van Santbrink, I. Bot, J. Kuiper, et al., Mesenchymal stem cells reduce murine atherosclerosis development, Sci Rep. 5 (1) (2015) 15559. [16] E. Sierra-Filardi, C. Nieto, A. Dominguez-Soto, R. Barroso, P. Sanchez-Mateos, A. Puig-Kroger, et al., CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile, J. Immunol. 192 (8) (2014 Apr) 3858–3867. [17] J.L. Schultze, S.V. Schmidt, Molecular features of macrophage activation, Semin Immunol. 27 (6) (2015) 416–423. [18] Y.S. Roh, E. Seki, Chemokines and Chemokine Receptors in the Development of NAFLD, Advances in experimental medicine and biology, 2018, pp. 45–53. [19] D. Ding, D. Su, X. Li, Z. Li, Y. Wang, J. Qiu, et al., Serum levels of monocyte chemoattractant protein-1 and all-cause and cardiovascular mortality among patients with coronary artery disease, PLoS One 10 (3) (2015) e0120633. [20] A.L.M. Eikendal, A.M.V. Evelein, C.S.P.M. Uiterwaal, C.K. van der Ent, F.L.J. Visseren, M.L. Bots, et al., Relation between circulating inflammatory chemokines and vascular characteristics in healthy, Young Children. J Am Heart Assoc. 4 (12) (2015). [21] B. Coll, C. Alonso-Villaverde, J. Joven, Monocyte chemoattractant protein-1 and atherosclerosis: Is there room for an additional biomarker? Clin Chim Acta. 383 (1–2) (2007 Aug) 21–29. [22] A. Rull, A. Hernandez-Aguilera, M. Fibla, J. Sepulveda, E. Rodríguez-Gallego, M. Riera-Borrull, et al., Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities, Vasc. Med. 19 (6) (2014) 442–451. [23] G. Aragones, A. Ercilla, M.M. Barreda, A. Rull, R. Beltran-Debon, E. RodriguezGallego, et al., Human Duffy blood group alloantigen system influences the measurement of monocyte chemoattractant protein-1 (MCP-1) in serum but not in plasma, Clin. Lab. 58 (1–2) (2012) 185–188. [24] T. Tabata, S. Mine, C. Kawahara, Y. Okada, Y. Tanaka, Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells, Biochem. Biophys. Res. Commun. 305 (2) (2003) 380–385. [25] L.B. Ford, V. Cerovic, S.W.F. Milling, G.J. Graham, C.A.H. Hansell, R.J.B. Nibbs, Characterization of conventional and atypical receptors for the chemokine CCL2 on mouse leukocytes, J. Immunol. 193 (1) (2014) 400–411. [26] V. Tomasdottir, A. Vikingsson, I. Hardardottir, J. Freysdottir, Murine antigen-induced inflammation–a model for studying induction, resolution and the adaptive phase of inflammation, J. Immunol. Meth. 15 (415) (2014) 36–45. [27] C.A.H. Hansell, C.E. Hurson, R.J.B. Nibbs, DARC and D6: silent partners in chemokine regulation? Immunol. Cell Biol. 89 (2) (2011 Feb) 197–206. [28] R.B. Schnabel, J. Baumert, M. Barbalic, J. Dupuis, P.T. Ellinor, P. Durda, et al., Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators, Blood 115 (26) (2010) 5289–5299. [29] R. Bonecchi, G.J. Graham, Atypical chemokine receptors and their roles in the resolution of the inflammatory response, Front Immunol. 10 (7) (2016) 224. [30] A. Hernández-Aguilera, J. Sepúlveda, E. Rodríguez-Gallego, M. Guirro, A. GarcíaHeredia, N. Cabré, et al., Immunohistochemical analysis of paraoxonases and chemokines in arteries of patients with peripheral artery disease, Int J Mol Sci. 16 (5) (2015) 11323–11338. [31] K.D. Yu, X. Wang, C. Yang, X.H. Zeng, Z.M. Shao, Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse, Oncotarget. 6 (28) (2015) 26519–26527. [32] A. Hernández-Aguilera, J. Sepúlveda, E. Rodríguez-Gallego, M. Guirro, A. GarcíaHeredia, N. Cabré, et al., Immunohistochemical analysis of paraoxonases and chemokines in arteries of patients with peripheral artery disease, Int. J. Mol. Sci. 16 (5) (2015) 11323–11338. [33] F. Rodriguez-Sanabria, A. Rull, R. Beltran-Debon, G. Aragones, J. Camps, B. Mackness, et al., Tissue distribution and expression of paraoxonases and chemokines in mouse: the ubiquitous and joint localisation suggest a systemic and coordinated role, J. Mol. Histol. 41 (6) (2010 Dec) 379–386. [34] J. Marsillach, J. Camps, R. Beltran-Debón, A. Rull, G. Aragones, C. MaestreMartínez, et al., Immunohistochemical analysis of paraoxonases-1 and 3 in human
6. Funding Current work in our laboratories is supported by grants from the Instituto de Salud Carlos III (Grants PI15/00285 and PI18/00921 cofounded by the European Regional Development Fund [FEDER]) to JJ. We also acknowledge the support by the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (Grants 2017SGR436), and the Fundació La Marató de TV3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 7. Data availability All relevant data are within the paper and supporting information files and details available upon request. Author Contributions Conceptualization: VMP, JJS, JJ. Funding acquisition: JAM, JJ. Data curation: MF, AHA, SFA. Formal analysis: AHA, MF, NC, FLM, JJS. Investigation: AHA, MF, NC, FLM, JC, SFA, JJS, JJ. Methodology: MF, SFA, AHA, NC, FLM. Project administration: AHA, JJ. Resources: VMP, JAM, JJS, JJ. Supervision: JC, SFA. Validation: MF, AHA, JC, JJS. Visualization: AHA, MF. Writing – original draft: AHA, JJ. Writing – review & editing: AHA, MF, NC, FLM, JC, SFA, VMP, JAM, JJS, JJ. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cyto.2019.154923. References [1] A. Arbab-Zadeh, V. Fuster, The risk continuum of atherosclerosis and its implications for defining CHD by coronary angiography, J. Am. Coll. Cardiol. 68 (22) (2016) 2467–2478. [2] M.V. Madhavan, B.J. Gersh, K.P. Alexander, C.B. Granger, G.W. Stone, Coronary artery disease in patients ≥80 years of age, J. Am. Coll. Cardiol. 71 (18) (2018) 2015–2040. [3] A.J. Yurdagul, A.C. Finney, M.D. Woolard, A.W. Orr, The arterial microenvironment: the where and why of atherosclerosis, Biochem. J. 473 (10) (2016 May) 1281–1295. [4] J. Viola, O. Soehnlein, Atherosclerosis - A matter of unresolved inflammation, Semin Immunol. 27 (3) (2015) 184–193. [5] R.L. Sun, C.X. Huang, J.L. Bao, J.Y. Jiang, B. Zhang, S.X. Zhou, et al., CC-Chemokine ligand 2 (CCL2) suppresses high density lipoprotein (HDL) Internalization and cholesterol efflux via CC-chemokine receptor 2 (CCR2) induction and p42/44 mitogen-activated protein kinase (MAPK) activation in human endothelial cells, J. Biol. Chem. 291 (37) (2016) 19532–19544. [6] J.A. Dubland, G.A. Francis, So Much Cholesterol: the unrecognized importance of smooth muscle cells in atherosclerotic foam cell formation, Curr Opin Lipidol. 27 (2) (2016) 155–161.
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A. Hernández-Aguilera, et al.
[49] K.H. Park, T. Sun, F. Diez-Delhoyo, Z. Liu, S.W. Yang, R.J. Lennon, et al., Association between coronary microvascular function and the vasa vasorum in patients with early coronary artery disease, Atherosclerosis 253 (2016 Oct) 144–149. [50] A. Jabs, E. Okamoto, J. Vinten-Johansen, G. Bauriedel, J.N. Wilcox, Sequential patterns of chemokine- and chemokine receptor-synthesis following vessel wall injury in porcine coronary arteries, Atherosclerosis 192 (1) (2007) 75–84. [51] K. Werth, R. Forster, Active shaping of chemokine gradients by atypical chemokine receptors: A 4D live-cell imaging migration assay, Meth. Enzymol. 570 (2016) 293–308. [52] J.S. Kim, K. Pak, T.S. Goh, D.C. Jeong, M.E. Han, J. Kim, et al., Prognostic value of MicroRNAs in coronary artery diseases: a meta-analysis, Yonsei. Med. J. 59 (4) (2018) 495. [53] R. Kumari, S. Kumar, M.K. Ahmad, R. Singh, S. Kant Kumar, A. Pradhan, et al., Promoter variants of TNF-α rs1800629 and IL-10 rs1800871 are independently associated with the susceptibility of coronary artery disease in north Indian, Cytokine 4 (110) (2018) 131–136. [54] N. Moradi, R. Fadaei, R. Ahmadi, E. Kazemian, S. Fallah, Lower expression of miR10a in coronary artery disease and its association with pro/anti-inflammatory cytokines, Clin Lab. 64 (5) (2018) 847–854. [55] R. Ahmadi, E. Heidarian, R. Fadaei, N. Moradi, M. Malek, S. Fallah, miR-342-5p expression levels in coronary artery disease patients and its association with inflammatory cytokines, Clin Lab. 64 (4) (2018) 603–609. [56] A.E. Bildirici, S. Arslan, N. Özbilüm Şahin, Ö. Berkan, O. Beton, M.B. Yılmaz, MicroRNA-221/222 expression in atherosclerotic coronary artery plaque and peripheral blood, Biomarkers 8 (2018) 1–19. [57] X. Zhao, D. Sun, R.X. Xu, Y.L. Guo, C.G. Zhu, N.Q. Wu, et al., Low-density lipoprotein-associated variables and the severity of coronary artery disease: an untreated Chinese cohort study, Biomarkers 7 (2018) 1–23. [58] R. Khan, V. Spagnoli, J.C. Tardif, P.L. L’Allier, Novel anti-inflammatory therapies for the treatment of atherosclerosis, Atherosclerosis 240 (2) (2015) 497–509. [59] T.J. Schall, A.E.I. Proudfoot, Overcoming hurdles in developing successful drugs targeting chemokine receptors, Nat. Rev. Immunol. 11 (5) (2011 May 15) 355–363. [60] S. Piconi, D. Pocaterra, V. Rainone, M. Cossu, M. Masetti, G. Rizzardini, et al., Maraviroc reduces arterial stiffness in PI-Treated HIV-infected Patients, Sci Rep. 6 (1) (2016) 28853. [61] V.G. Kramer, S. Hassounah, S.P. Colby-Germinario, M. Oliveira, E. Lefebvre, T. Mesplede, et al., The dual CCR5 and CCR2 inhibitor cenicriviroc does not redistribute HIV into extracellular space: implications for plasma viral load and intracellular DNA decline, J. Antimicrob. Chemother. 70 (3) (2015) 750–756. [62] M. Massara, O. Bonavita, A. Mantovani, M. Locati, R. Bonecchi, Atypical chemokine receptors in cancer: friends or foes? J. Leukoc. Biol. 99 (6) (2016 Jun) 927–933.
atheromatous plaques, Eur. J. Clin. Invest. 41 (3) (2011) 308–314. [35] D. Szklarczyk, A. Franceschini, S. Wyder, K. Forslund, D. Heller, J. Huerta-Cepas, et al., STRING v10: Protein-protein interaction networks, integrated over the tree of life, Nucleic Acids Res. 43 (D1) (2015 Jan) D447–D452. [36] C.S. Greene, A. Krishnan, A.K. Wong, E. Ricciotti, R.A. Zelaya, D.S. Himmelstein, et al., Understanding multicellular function and disease with human tissue-specific networks, Nat. Genet. 47 (6) (2015) 569–576. [37] A. Krishnan, R. Zhang, V. Yao, C.L. Theesfeld, A.K. Wong, A. Tadych, et al., Genome-wide prediction and functional characterization of the genetic basis of autism spectrum disorder, Nat. Neurosci. 19 (11) (2016) 1454–1462. [38] A. Zlotnik, O. Yoshie, H. Nomiyama, The chemokine and chemokine receptor superfamilies and their molecular evolution, Genome Biol. 7 (12) (2006) 243. [39] S. Jordan, N. Tung, M. Casanova-Acebes, C. Chang, C. Cantoni, D. Zhang, et al., Dietary intake regulates the circulating inflammatory monocyte pool, Cell. 178 (5) (2019) 1102–1114.e17. [40] F. Bachelerie, G.J. Graham, M. Locati, A. Mantovani, P.M. Murphy, R. Nibbs, et al., New nomenclature for atypical chemokine receptors, Nat. immunol. United States 15 (2014) 207–208. [41] D.P. Ramji, T.S. Davies, Cytokines in atherosclerosis: key players in all stages of disease and promising therapeutic targets, Cytokine Growth Factor Rev. 26 (6) (2015) 673–685. [42] A. Ridiandries, J.T.M. Tan, C.A. Bursill, The role of CC-chemokines in the regulation of angiogenesis, Int. J. Mol. Sci. 17 (11) (2016) 1856. [43] L. Boring, J. Gosling, M. Cleary, I.F. Charo, Decreased lesion formation in CCR2-/mice reveals a role for chemokines in the initiation of atherosclerosis, Nature. 394 (6696) (1998) 894–897. [44] T.C. Dawson, W.A. Kuziel, T.A. Osahar, N. Maeda, Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice, Atherosclerosis 143 (1) (1999) 205–211. [45] W. Wan, Q. Liu, M.S. Lionakis, A.P.M.P.P. Marino, S.A. Anderson, M. Swamydas, et al., Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoEknockout mice, Cardiovasc. Res. 106 (3) (2015) 478–487 Jun 1. [46] C. Cochain, C. Auvynet, L. Poupel, J. Vilar, E. Dumeau, A. Richart, et al., The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction, Arterioscler. Thromb. Vasc. Biol. 32 (9) (2012) 2206–2213. [47] L. Gao, Z. Xu, Z. Yin, K. Chen, C. Wang, H. Zhang, Association of hydrogen sulfide with alterations of monocyte chemokine receptors, CCR2 and CX3CR1 in patients with coronary artery disease, Inflamm Res. 64 (8) (2015) 627–635. [48] K.M. Lee, R. Danuser, J.V. Stein, D. Graham, R.J.B. Nibbs, G.J. Graham, The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density, EMBO J. 33 (21) (2014) 2564–2580.
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Chemokine (C-C motif) ligand 2 and coronary artery disease: Tissue expression of functional and atypical receptors
T
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Anna Hernández-Aguileraa,1, , Montserrat Fiblab,1, Noemí Cabréc, Fedra Luciano-Mateoc, Jordi Campsa, Salvador Fernández-Arroyoc,d, Vicente Martín-Parederoe, Javier A. Menendezf,g, ⁎ Juan J. Sirventb, Jorge Jovena,c,d, a
Unitat de Recerca Biomèdica, Hospital Universitari Sant Joan, Institut d’Investigació Sanitària Pere Virgili, Reus 43201, Tarragona, Spain Department of Pathology, Hospital Universitari Joan XXIII, Tarragona 43005, Spain c Universitat Rovira i Virgili, Departament de Medicina i Cirurgia, Unitat de Recerca Biomèdica, Tarragona 43003, Spain d The Campus of International Excellence Southern Catalonia, Tarragona 43003, Spain e Department of Vascular Surgery, Hospital Universitari Joan XXIII, Tarragona 43005, Spain f ProCURE (Program Against Cancer Therapeutic Resistance), Metabolism and Cancer Group, Catalan Institute of Oncology, Salt 17190, Girona, Spain g Girona Biomedical Research Institute (IDIBGI), Salt 17190, Girona, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis CCL2 CCR2 ACKR Inflammation
Chemokines, particularly chemokine (C-C- motif) ligand 2 (CCL2), control leukocyte migration into the wall of the artery and regulate the traffic of inflammatory cells. CCL2 is bound to functional receptors (CCR2), but also to atypical chemokine receptors (ACKRs), which do not induce cell migration but can modify chemokine gradients. Whether atherosclerosis alters CCL2 function by influencing the expression of these receptors remains unknown. In a necropsy study, we used immunohistochemistry to explore where and to what extent CCL2 and related receptors are present in diseased arteries that caused the death of men with coronary artery disease compared with unaffected arteries. CCL2 was marginally detected in normal arteries but was more frequently found in the intima. The expression of CCL2 and related receptors was significantly increased in diseased arteries with relative differences among the artery layers. The highest relative increases were those of CCL2 and ACKR1. CCL2 expression was associated with a significant predictive value of atherosclerosis. Findings suggest the need for further insight into receptor specificity or activity and the interplay among chemokines. CCL2-associated conventional and atypical receptors are overexpressed in atherosclerotic arteries, and these may suggest new potential therapeutic targets to locally modify the overall anti-inflammatory response.
1. Introduction Reversing atherosclerosis is a desirable and formidable challenge because acute coronary syndrome (ACS) and strokes remain the leading causes of death and affliction worldwide [1,2]. Preventive measures based on treating systemic risk factors are necessary and may slow the progression, but this strategy is insufficient because atherosclerosis development is likely regulated by the local microenvironment in
diseased arteries [3]. The inside-out theory of vascular inflammation indicates that lipid deposition in the subendothelial layer of arteries and retention in smooth muscle cells entail structural changes that stimulate inflammation and the transformation of macrophages into foam cells in a self-maintaining progressive cycle [4–7]. By contrast, an outside-in theory assumes that the inflammatory cells gain entry to the vessel wall via the adventitial vasa vasorum [8]. Both theories may be integrated into a model to understand atherogenesis, but the task involves
Abbreviations: ACKRs, atypical chemokine receptors; CCBP2, chemokine binding protein 2; CCL2, chemokine (C-C motif) ligand 2; CCR2, C-C chemokine receptor type 2; CD68, cluster of differentiation 68; DARC, Duffy antigen receptor for chemokines; GPCRs, G protein-coupled receptors; H&E, hematoxylin and eosin ⁎ Corresponding authors at: Unitat de Recerca Biomèdica - Hospital Universitari Sant Joan, Institut d’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201 Reus, Spain. E-mail addresses: [email protected] (A. Hernández-Aguilera), montsefi[email protected] (M. Fibla), [email protected] (N. Cabré), [email protected] (F. Luciano-Mateo), [email protected] (J. Camps), [email protected] (S. Fernández-Arroyo), [email protected] (V. Martín-Paredero), [email protected] (J.A. Menendez), [email protected] (J.J. Sirvent), [email protected] (J. Joven). 1 These authors contributed equally. https://doi.org/10.1016/j.cyto.2019.154923 Received 24 April 2019; Received in revised form 6 November 2019; Accepted 8 November 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.
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manipulating inflammation under strategies focused on interfering with this vicious cycle. Many anti-inflammatory treatments for atherosclerosis have been tested but most of them needs to be further studied in clinical trials [9,10]. The lack of diagnostic, prognostic and treatment response markers sets hurdles in the improvement of drugs targeting relevant processes [10]. Recent interventions that facilitate macrophage egress or that attempt to solubilize cholesterol regress the pre-existing atherosclerotic plaques and reveal that cholesterol efflux alters the local production and deposition of cytokines [3,11,12]. Among cytokines, chemokine (CC motif) ligand 2 (CCL2), through conventional receptors, mainly CCR2, belonging to the 7-transmembrane-spanning family of G proteincoupled receptors (GPCRs), regulates pleiotropic signaling pathways that influence numerous cellular activities involved in atherosclerosis development [13–18]. The role of circulating CCL2 as a potential biomarker has been examined [19–21] with controversial results [22,23] derived from ill-defined effects of atypical chemokine receptors (ACKRs), which are expressed in non-leukocyte cell types but do not induce cell migration. ACKRs are those kinds of receptors that do not transduce signal through G-protein signaling but may modify chemokine availability. The best-known ACKRs that bind CCL2 and other chemokines are ACKR1 (Duffy antigen receptor for chemokines; DARC) and ACKR2 (chemokine binding protein 2; CCBP2 or D6) [24–27]. We envisioned that atherosclerosis could promote the expression and interplay of conventional and atypical CCL2 receptors in cells that affect the development of advanced coronary artery disease (CAD). What we know in humans is modest, and there have been no clinical studies designed to examine this relationship, probably because doing so would require specific biological assays or excessively invasive methods [28,29]. We reasoned that a necropsy study could provide additional knowledge by exploring, for the first time, where and to what extent CCL2 and the expression of related receptors may increase in diseased arteries that have been responsible for the death of patients with CAD compared with those in healthy arteries. Our data reinforce CCL2 as a major determinant of atherosclerosis progression and might support the quest to new druggable targets.
Fig. 1. Histologic changes in coronary arteries reveal atherosclerosis. (A) Lesions, especially the thickness of the media and intima, were evident with hematoxylin and eosin staining. (B) Alizarin Red Staining was used to assess calcium deposits, and (C) Masson’s trichrome staining was mostly used to assess variations in the overall structure and remodeling. (L is Lumen, I is Intima, M is Media and A is Adventitia). All sections were examined at different magnifications, but automated analysis was performed at 20 × . Representative images include the scale bar.
fixation and was deemed irrelevant for our purposes.
2. Materials and methods
2.2. Histological examination
2.1. Research design
The specimens were fixed in 10% neutral-buffered formalin and were embedded in paraffin. Serial sections of 4 µm were placed in a 45 °C water bath before the staining procedures with hematoxylin and eosin (H&E), Alizarin Red (Sigma-Aldrich, Steinheim, Germany) and Masson’s Trichrome reagents (Dako, Glostrup, Denmark) to assess the structure and extent of fibrosis in the arteries and to visualize the localization of calcium phosphate. The intima and media thickness (IMT) and ratio (IMR) were measured and calculated in H&E-stained sections (Fig. 1). Slides were loaded in an automated slide stainer, Autostainer Universal™ (Dako), including sections (in triplicate) for immunohistochemical analyses after conducting the standard procedures for manual de-waxing and heat-induced epitope retrieval (citrate solution pH = 6). Because the tissues contained endogenous peroxidase, we added a blocking step (0.3% hydrogen peroxide in PBS) for 25 min at room temperature after antigen retrieval. Before primary antibody incubation, we also used BSA 1% to block non-specific interactions. Macrophages were detected by the presence of the CD68 antigen (clone: KP1) with reagents provided by Dako (M0876). Primary antibodies against CCL2 (ab9669), CCR2 (ab21667) and ACKR2 (ab1658) were obtained from Abcam (Cambridge, UK) and were used at dilutions of 1:200, 1:100, and 1:500, respectively. ACKR1 (PAB13254) antibody was from Abnova (Taipei, Taiwan) and was used at a dilution of 1:200. Biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) were used at a dilution of 1:200. Detection was performed using the Vectastain ABC HRP kit (PK-4000, Vector Laboratories) and 3, 3-diaminobenzidine as a substrate (Dako). The slides were
We designed a pre-set duration, observational, cross-sectional study in consecutive male decedents who underwent autopsy between December 2012 and November 2014 at our settings. Based on the autopsy, CAD was firmly established as the cause of death. The local Ethics Committee, which acts as the Institutional Review Board, approved the study protocol and procedures (EPINOLS/12-03-29/3proj6 approved on 29th March 2012). Written informed consent was obtained from next of kin of the deceased for autopsy and access to tissues. The described association between CCL2 and other complex diseases [21] provided pre-established criteria for excluding decedents with evidence of recent infection (n = 2), diabetes (n = 10), liver disturbances (n = 2), neoplasms (n = 3) or with sudden, out of hospital, death (n = 4). As the disease is more prevalent in men and in order to avoid possible sex differences, we finally included 68 men donors in whom the heart was dissected following standard autopsy protocols. In each corpse, we obtained at least one major coronary artery that was narrowed by > 80% of the cross-sectional area by atherosclerotic plaque or associated thrombus. For comparisons, we obtained coronary arteries without visible signs of atherosclerosis from 19 men who had died as a result of road traffic accidents during the same period of time. For other associations, we determined arterial stenosis and arbitrarily classified a total of 123 specimens into mild (less than 20% obstruction), moderate (between 21% and 50%) or severe (more than 50%) atherosclerosis. The expected decrease in the dimensional changes associated with fixation and processing (3–7%) correlated with the values before 2
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counterstained with Mayer’s hematoxylin. The results with these antibodies were compared with sections of previously obtained peripheral arteries [30] and selected tumor sections from primary breast cancer tissues [31] that were used as positive or negative controls (Fig. 3). We also used negative controls from study samples by omitting the primary antibodies from the incubations. Comparisons with other ACKR1 and ACKR2 antibodies (HPA013819 and HPA016421, from Sigma; PA121614, from Affinity Bioreagents; and NB100-2421 from Novus Biologicals) did not yield divergent results. To ensure the localization of CCL2 and their atypical receptors (ACKR1 and ACKR2), we also performed additional double-color immunohistochemistry combining these markers with those related with arterial layers: CD34 for endothelial cells (intima) and the adventitia and α-smooth muscle actin (αSMA) for smooth muscle cells (media). Both primary antibodies were also obtained from Abcam (CD34, ab8536; αSMA, ab21027) and used at dilutions 1:1000 and 1:250 respectively. In this case, detection of these markers was performed with EnVision FLEX HRP Magenta Substrate Chromogen System (Dako). Microphotographs were acquired using an optical microscope (Nikon, Eclipse E600, Madrid, Spain) and an image analysis system (AnalySIS™, Soft Imaging System, Münster, Germany), as described previously [30]. When the arteries were greater than the histological field used for analyses (20 × magnification), several pictures were combined to calculate the percentage of stained area with respect to the total area of the artery. Once images were obtained, colors of the molecules of interest were defined and automatically detected in all samples. The stained area was related to the total area of the image by using phase analysis. More details about the quantification method have been already described and used in different studies [32–34].
Table 1 Main clinical variables. Clinical characteristics
Control group (N = 19)
CAD group (N = 68)
p-value
Age, years Body mass index, kg/m2 No smokers, n (%) Alcohol consumption, n (%) Heart weight (g) Ventricular thickness (mm) Fibrosis, n (%) Affected valves, n (%) Hypertension, n (%)
45 (33–69) 25.7 (4.1) 15 (78.4) 2 (10.5) 380 (300–450) 16 (14–18) 0 2 (10.5) 2 (10.5)
71 (61–77) 24.8 (4.0) 42 (61.8) 9 (14.3) 480 (400–600) 19 (15–20) 10 (15.9) 31 (45.6) 35 (51.5)
0.001 0.217 0.164 0.126 0.002 0.005 0.110 0.005 0.001
*Results are expressed as n (%), median and interquartile range (IQR, 25–75%) or mean (standard deviation).
IMT (intima-media thickness) values of control and diseased arteries, which were significantly different (p < 0.001): in medians (IQR), 586 μm (452.7–865.1) for diseased arteries and 379 μm (265.8–467.8) for control. Moreover, modified tissue architecture of disease arteries was evident when assessed by the different stainings (Fig. 1). Macrophages infiltrated the subendothelial space with a considerable extracellular network of collagen and lipid deposition. CD68stained cells with foamy cytoplasm were scarcely observed in minimally affected arteries but were apparent in the neointima, tunica media and adventitia of affected arteries (Fig. 2). In severe lesions, fibrofatty plaques with cholesterol clefts and mineralized material were frequent. In some cases, lesions were characterized by fibrosis and necrotic debris. Irregular calcium deposits were abundant in severe lesions, which were mostly limited to the intima but also occurred deep in the plaque. Of note, adventitial inflammation and vascularity were significantly increased in affected arteries. When the interactions between each receptor and CCL2 were examined, the functional networks appeared to be similarly complex (S1 figure A-B). Moreover, we found no evidence of any predicted proteinprotein interactions or associations among ACKRs (S1 figure C). Exploring this issue further would require functional assays and additional in vitro designs. Quantitative measurements of the immunohistochemical variables are summarized in Table 2 and are illustrated in Fig. 2. Details segregated among the layers are illustrated in S2 and S3 Figures for the control and affected arteries, respectively. The immediate finding is that the selected markers were significantly more prominent in diseased arteries. The highest relative increase in detection was that of CCL2 (> 9-fold), which closely resembled the higher abundance in macrophages. CCL2 expression was only evident in 26% of normal arteries and was mostly restricted to smooth muscle cells with faint staining in the intima and adventitia. In affected arteries, CCL2 was detected in all specimens and remained more evident in the tunica media but was significantly increased in all layers, particularly in the adventitial tissue. CCR2 expression was observed in more cells and with higher intensity in affected arteries than in controls, but the differences were only statistically significant in the adventitia. The distribution of both CCL2 and CCR2 was qualitatively similar in the smooth muscle cells of the tunica media, probably indicating a functional relationship. The structural role of CCR2 is also likely because CCR2 was expressed in all specimens and because the CCR2 relative abundance in the intima and media was similar in both controls and diseased arteries. In the affected arteries, however, the relative expression was 3-fold higher in the adventitial tissue. The expression of atypical receptors was significantly higher in diseased arteries, and the distribution was qualitatively different. This differential scattering was already observed in those tumor sections used as controls, indicating that not all cells have the ability to respond. ACKR2 did not co-express with macrophages but CCL2, CCR2 and ACKR1 were closely related (Fig. 3). For instance, ACKR1 was not detected in all specimens (63% in controls and 81% in affected arteries);
2.3. Statistical analyses Inter-observer agreement was assessed, and discrepancies were resolved by consensus. Student’s t-test or Mann-Whitney’s test was used to assess the difference between groups according to the distribution of variables. The chi-squared test was used to compare categorical variables. Spearman correlation coefficients were used to evaluate the degree of association between variables and linear regression was performed to check dependences between quantitative variables. To correct for multiple testing, we used the False Discovery Rate estimated using the Q-value. The true- and false-positive rates at various threshold settings were used to plot receiver operating characteristic (ROC) curves. The Statistical Package for the Social Sciences, version 19.0 (SPSS Inc, IBM Corp, Chicago, IL, USA) and MetaboAnalyst 3.0® (www. metaboanalyst.ca) were used for analyses and for obtaining random forest plots and ROC curves. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, http://string-db.org) and HumanBase (Flatiron Institute, http://hb.flatironinstitute.org/) were used as a biological databases and web resources of known and predicted proteinprotein interactions [35–37]. 3. Results The clinical variables of donors confirmed the influence of some expected systemic risk factors (Table 1). In decedents from CAD, compared with controls, hypertension was more prevalent, and age, heart weight and ventricular thickness were significantly increased. However, the proportion of daily smokers and alcohol consumption (10–20 g/day) was similar between the groups, and there were no differences in the body mass index relating the weight to the height. Atherosclerosis is an aging-related disease. To ascertain that the expression of these markers was associated with atherosclerosis and not with age itself, we assessed this point through linear regression models. Results did not support dependence on age (Supplementary Table S1 and S2). Standard histopathology revealed atherosclerosis. We assessed the 3
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Table 2 Main immunohistochemical variables. Immunohistochemical variables* Positive staining expressed as a percentage (%) of the arterial area
CD68 CCL2 CCR2 ACKR1 ACKR2
Control group (N = 19)
CAD group (N = 68)
p-value
0.75 (0–1.8) 2.4 (1.3–5.8) 24.4 (14.4–49.6) 6.8 (2.4–12.6) 21.9 (8.1–35.5)
3.8 (1.6–6.2) 17.2 (9.1–27.3) 40.1 (24.3–54.1) 13.3 (6–43.1) 34.8 (25.2–53.7)
0.002 < 0.001 0.037 0.001 0.020
Percentage (%) of stained area in positive specimens CCL2 (n = 5; 26%) (n = 68; 100%) Intima 4.1 (2.4–5.3) 8.5 (5.9–11.2) Media 10.5 (3.5–19.1) 35.5 (22.4–59.6) Adventitia 3.6 (2.7–5.5) 21.5 (18.4–26.2) CCR2 (n = 19; 100%) (n = 68; 100%) Intima 24.5 (19.2–29.3) 29.1 (24.4–33.3) Media 70.1 (64.3–74.2) 75.6 (70.7–84.6.6) Adventitia 10.4 (7.5–14.1) 31.8 (27.2–34.7) ACKR1 (n = 12; 63%) (n = 55; 81%) Intima 5.4 (3.1–7.3) 74.1 (64.3–78.2) Media 15.8 (11.2–20.5) 82.7 (73.2–84.9) Adventitia 3.9 (2.1–4.9) 65.8 (61.6–68.9) ACKR2 (n = 16; 84%) (n = 68; 100%) Intima 19.5 (11.9–26.3) 21.2 (17–24.4) 24.9 (17.7–26.1) 30.7 (22.9–40.3) Media Adventitia 50.7 (44.2–67.3) 74.2 (61.2–85.1)
< 0.001 < 0.001 < 0.001 0.120 0.087 0.001 0.001 0.001 0.001 0.102 0.087 0.001
Statistically significant differences are marked up in bold. * Staining was measured using automated image analysis and expressed as median and interquartile range (IQR, 25–75%).
tissue. In the affected arteries, ACKR2 was present in all specimens, but the expression was not quantitatively different in the intima and media with respect to the controls. The increased expression in adventitia was statistically significant. To confirm the expression of CCL2 and their atypical receptors in endothelial cells and cells in the adventitia (CD34) and also in smooth muscle cells (αSMA), we performed double-color immunohistochemistry. In control arteries, although there were no clear differences between tunica media and tunica intima, CCL2 was found in smooth muscle cells. ACKR1 was also mainly found in smooth muscle cells. On the contrary, ACKR2 seemed to be more located in the adventitia, although some staining was also observed in tunica media (Fig. 4). Regarding CAD group, CCL2 was mainly expressed in the adventitia although some specimens showed intense expression of this marker in the media. ACKR1 was observed in all arterial layers and ACKR2 was mainly restricted to the adventitia (Fig. 5). We also explored the correlation between these inflammatory markers and the potential value of CCL2 and related receptors as a marker of both the presence and severity of atherosclerosis in terms of stenosis degree. We found no dependence between the measured variables using a Spearman-based correlation matrix, where strong associations can be found in red color and weak association are displayed in blue (Fig. 6 A). Although we found that CCL2 and CCR2 were expressed in the same layers, there was no quantitative correlation among them.
Fig. 2. Expression of CCL2 and related receptors in coronary arteries. The immunohistochemical expression in non-affected arteries and atherosclerotic arteries of CCL2, CD68-stained cells (macrophages), and functional and putatively silent receptors for CCL2 displayed significant differences (see Table 2 for quantitative analysis). CCL2 and CCR2 expression levels were apparently in the same layers in the artery wall. Conversely, the expression of both ACKR1 and ACKR2 differed in intensity and arterial distribution (L is Lumen, I is Intima, M is Media, and A is Adventitia). All sections were examined at different magnifications, but automated analysis was performed at 20 × . Representative images include the scale bar. Representative images at higher magnifications were included in supplementary Figures S2 (control arteries) and S3 (affected arteries).
however, if present, the differences between the controls and affected arteries were significant. Moreover, healthy arteries were stained faintly with some preponderance in smooth muscle cells, and the affected arteries were more strongly stained in all layers. Conversely, the expression of ACKR2 in healthy arteries was more intense and widely distributed than that of ACKR1 (Table 2) but was undetectable in 16% of control arteries. Of note, ACKR2 was detected in endothelial cells, smooth muscle cells and, more importantly, in the perivascular adipose
Fig. 3. The expression of CCL2 and related receptors is tissue-specific. Sections of human tissue of breast carcinoma (Mag. 20 × ) expressing all of the antigens were used as positive controls and were treated exactly as study samples. Selected specimens of breast tissue without ACKR1 or ACKR2 expression were used as a source of negative tissue controls to confirm the absence of background staining and illustrate tissue specificity in ACKR expression (images not shown). 4
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classification constructed using Random forest plots, which is a nonparametric and bias-free method that classifies variables according their discriminant capacity between two groups, CCL2 expression was the variable with the best discriminant ability (Fig. 6C). CCL2 detection, but not related receptors, discriminated controls from CAD samples in a mild, moderate or severe state (Fig. 7) with a global cut-off of 6.335 (89.3% sensitivity and 85% specificity). Collectively, the data indicate that CCL2 and related receptors are expressed in different cell types and might suggest that the presence of CCL2 is associated with the occlusion of the arteries. Co-expression among the examined proteins, although suggestive, was not verified or discarded. 4. Discussion Mammals have over 45 inflammatory or homeostatic chemokines divided into subfamilies according to the differences in their function and mature protein sequences. The biological complexity and variety of contexts in which they are found complicate the knowledge on their precise relationships [38]. Indeed, recent literature have linked diet composition and liver energy sensors with the circulating inflammatory monocyte pool [39]. Although CCL2 action is mediated by binding to numerous (over 15), promiscuous and probably redundant canonical receptors, the receptor/ligand interactions are further obscured by an undetermined number of ACKRs that can modulate the expression, or signaling, of that conventional chemokine receptors [29,40]. In the chemokine system, the role of CCL2 and the CCL2/CCR2 axis appears to be critical in the molecular control of inflammation by promoting the response and migration of immune cells, transforming monocytes into macrophages, stimulating smooth muscle cells proliferation and maintaining the continuous and local release of proinflammatory molecules [5,41,42]. This axis is also involved in atherosclerosis development. As seen in double-deficient CCR2-/- and apolipoprotein E-/- mice, the absence of this receptor reduces the atherosclerosis lesion area and slows down lesion formation [43,44]. The potential role of ACKR1 and ACKR2 in atherogenesis is plausible [45,46]. As atherosclerosis is an age-related disease, obtaining samples from age-matched control population without any sign of atherosclerosis is almost impossible. Despite these limitations and those derived from autopsy studies, namely that patients could be not representative of the general population and that women were not included, our findings indicate that the presence of widely distributed CCL2 and conventional and atypical receptors in coronary arteries and perivascular tissue of decedents from CAD may have implications in further research and drug development. The high levels of plasma CCL2 in patients with atherosclerosis and other chronic inflammatory conditions most likely reflect its accumulation in inflamed tissue and the subsequent drainage through the lymphatic system, which is influenced by the active roles of CCR2 and chemokine-scavenging ACKRs [21,28,47,48]. The complex relationships among receptors may explain that CCL2 remained as the only variable with predictive value. Interestingly, CCL2 and ACKR1 expression levels were increased in all layers of the affected arteries, but the higher expression of ACKR1 and ACKR2 was particularly prominent in the adventitia and perivascular fat. Accordingly, the adventitia becomes populated with exogenous cell types that cause an increase in the local expression of CCL2 and related receptors. In humans, both structure and function of adventitial vasa vasorum may associate microvascular dysfunction with early coronary atherosclerosis [49], and CCL2 appears to activate adventitia during angioplasty, inducing sequential patterns in receptor expression [50]. Conversely, it is unclear whether receptors may influence CCL2 expression. Our measurements do not provide insight into receptor specificity or activity but highlight the importance of understanding the influence of CCL2 and the expression of related receptors in the compensatory role of adventitia [51]. Human studies on inflammation in CAD have been focused on single nucleotide polymorphisms, microRNA and lipid determinations in plasma samples [52–57]. Obtaining high quality mRNA
Fig. 4. Double-color immunohistochemical expression of CCL2, ACKR1 and ACKR2 together with CD34 or αSMA in control coronary arteries. Images were segregated by markers (rows) to illustrate layer specific staining (columns) and were selected as representative.
Fig. 5. Double-color immunohistochemical expression of CCL2, ACKR1 and ACKR2 together with CD34 or αSMA in atherosclerotic coronary arteries. Images were segregated by markers (rows) to illustrate layer specific staining (columns) and were selected as representative.
CCL2 expression displayed the most important variable indicating atherosclerosis compared to CCR2 expression when using Receiver Operating Characteristics (ROC) Curve (Fig. 6B). In a model of 5
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Fig. 6. Histologic variables do not correlate, but CCL2 retains predictive value. (A) The correlation matrix was used to investigate the dependence among multiple variables at the same time (based on % of stained area), and no significant association was found. (B) We performed a ROC curve with CCL2 and CCR2 but only CCL2 expression had an acceptable predictive value (great Area Under the Curve). (C) We used Random forests to rank the importance of variables in a likely classification of predictors, and CCL2 was significantly different.
expression in cell types, the ability of ligands to bind several receptors and the formation of dimers and oligomers are major hurdles [59]. For instance, a CCR5 antagonist that was recently introduced for HIV therapy and also another dual CCR5 and CCR2 inhibitor may show promise in the management of atherosclerosis [60,61]. The higher adventitial inflammation, neovascularization and ACKR expression in affected arteries add force to the notion that ACKRs participate in the process and might be druggable targets. In this case, ACKRs have the potential to neutralize chemokine activity, and the pharmacological approach could be the development of their inducers rather than
from these samples was unsuccessful but ongoing studies should clarify this point. Our study provides a first approach about chemokine availability in tissue, which will serve for future mechanistic insights or even for novel therapeutic strategies. To assess potentially effective anti-inflammatory therapies, future research will require the development of novel assays to assess the formation of CCL2 gradients and an accurate characterization of conventional and atypical receptor-expressing cells [58]. The current pharmacological approach is to develop antagonists of chemokine receptors that are involved in leukocyte migration. However, promiscuous ligand binding, the differential
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Fig. 7. CCL2 expression is associated with the percentage of lumen occlusion. Receiving operating characteristic curves were plotted using true- and false-positive results. Most values were associated with coronary artery disease (CCL2, CD68 and atypical receptors), but CCL2 was the most significant (A). Only the predictive value of CCL2 remained significant to distinguish among mild, moderate and severe occlusion (B).
5. Conclusions
antagonists. Because ACKRs may be key components of the regulatory network of inflammation, patient-relevant outcomes are likely. The concept is currently being examined in the field of tumorigenesis with the rationale that chemokine scavenger activity may prevent the recruitment of cells that sustain tumor growth [31,62].
Although this is an observational study and it does not prove any causality, we conclude that the continuous production of CCL2 in chronic inflammation may activate mechanisms with biological functions in atherosclerosis. The increase in ACKRs expression could be a 7
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compensatory mechanism to regulate the increased CCL2 expression. The assessment of how and to what extent the binding of CCL2 to associated receptors transduce signals and/or mediate scavenging may result in potential therapeutic targets.
[7] F. Abdolmaleki, S.M. Gheibi Hayat, V. Bianconi, T.P. Johnston, A. Sahebkar, Atherosclerosis and immunity: A perspective. Vol. 29, Trends Cardiovascular Med. (2019) 363–371. [8] M.J. Mulligan-Kehoe, M. Simons, Vasa vasorum in normal and diseased arteries, Circulation 129 (24) (2014) 2557–2566. [9] Y. Zhu, X. Xian, Z. Wang, Y. Bi, Q. Chen, X. Han, et al., Research progress on the relationship between atherosclerosis and inflammation, Biomolecules 8 (2018) 80. [10] P. Raggi, J. Genest, J.T. Giles, K.J. Rayner, G. Dwivedi, R.S. Beanlands, et al., Role of inflammation in the pathogenesis of atherosclerosis and therapeutic interventions, Atherosclerosis 276 (2018 Sep) 98–108. [11] R.E. Gerszten, A.M. Tager, The monocyte in atherosclerosis–should I stay or should I go now? N. Engl. J. Med. 366 (18) (2012 May) 1734–1736. [12] S. Zimmer, A. Grebe, S.S. Bakke, N. Bode, B. Halvorsen, T. Ulas, et al., Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming, Sci. Transl. Med. 8 (333) (2016) 333ra50. [13] D. Manka, T.K. Chatterjee, L.L. Stoll, J.E. Basford, E.S. Konaniah, R. Srinivasan, et al., Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1, Arterioscler Thromb Vasc Biol. 34 (8) (2014) 1723–1730. [14] B. Yu, M.M. Wong, C.M.F. Potter, R.M.L. Simpson, E. Karamariti, Z. Zhang, et al., Vascular stem/progenitor cell migration induced by smooth muscle cell-derived chemokine (C-C Motif) ligand 2 and chemokine (C-X-C motif) ligand 1 contributes to neointima formation, Stem Cells 34 (9) (2016 Sep) 2368–2380. [15] V. Frodermann, J. van Duijn, M. van Pel, P.J. van Santbrink, I. Bot, J. Kuiper, et al., Mesenchymal stem cells reduce murine atherosclerosis development, Sci Rep. 5 (1) (2015) 15559. [16] E. Sierra-Filardi, C. Nieto, A. Dominguez-Soto, R. Barroso, P. Sanchez-Mateos, A. Puig-Kroger, et al., CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile, J. Immunol. 192 (8) (2014 Apr) 3858–3867. [17] J.L. Schultze, S.V. Schmidt, Molecular features of macrophage activation, Semin Immunol. 27 (6) (2015) 416–423. [18] Y.S. Roh, E. Seki, Chemokines and Chemokine Receptors in the Development of NAFLD, Advances in experimental medicine and biology, 2018, pp. 45–53. [19] D. Ding, D. Su, X. Li, Z. Li, Y. Wang, J. Qiu, et al., Serum levels of monocyte chemoattractant protein-1 and all-cause and cardiovascular mortality among patients with coronary artery disease, PLoS One 10 (3) (2015) e0120633. [20] A.L.M. Eikendal, A.M.V. Evelein, C.S.P.M. Uiterwaal, C.K. van der Ent, F.L.J. Visseren, M.L. Bots, et al., Relation between circulating inflammatory chemokines and vascular characteristics in healthy, Young Children. J Am Heart Assoc. 4 (12) (2015). [21] B. Coll, C. Alonso-Villaverde, J. Joven, Monocyte chemoattractant protein-1 and atherosclerosis: Is there room for an additional biomarker? Clin Chim Acta. 383 (1–2) (2007 Aug) 21–29. [22] A. Rull, A. Hernandez-Aguilera, M. Fibla, J. Sepulveda, E. Rodríguez-Gallego, M. Riera-Borrull, et al., Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities, Vasc. Med. 19 (6) (2014) 442–451. [23] G. Aragones, A. Ercilla, M.M. Barreda, A. Rull, R. Beltran-Debon, E. RodriguezGallego, et al., Human Duffy blood group alloantigen system influences the measurement of monocyte chemoattractant protein-1 (MCP-1) in serum but not in plasma, Clin. Lab. 58 (1–2) (2012) 185–188. [24] T. Tabata, S. Mine, C. Kawahara, Y. Okada, Y. Tanaka, Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells, Biochem. Biophys. Res. Commun. 305 (2) (2003) 380–385. [25] L.B. Ford, V. Cerovic, S.W.F. Milling, G.J. Graham, C.A.H. Hansell, R.J.B. Nibbs, Characterization of conventional and atypical receptors for the chemokine CCL2 on mouse leukocytes, J. Immunol. 193 (1) (2014) 400–411. [26] V. Tomasdottir, A. Vikingsson, I. Hardardottir, J. Freysdottir, Murine antigen-induced inflammation–a model for studying induction, resolution and the adaptive phase of inflammation, J. Immunol. Meth. 15 (415) (2014) 36–45. [27] C.A.H. Hansell, C.E. Hurson, R.J.B. Nibbs, DARC and D6: silent partners in chemokine regulation? Immunol. Cell Biol. 89 (2) (2011 Feb) 197–206. [28] R.B. Schnabel, J. Baumert, M. Barbalic, J. Dupuis, P.T. Ellinor, P. Durda, et al., Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators, Blood 115 (26) (2010) 5289–5299. [29] R. Bonecchi, G.J. Graham, Atypical chemokine receptors and their roles in the resolution of the inflammatory response, Front Immunol. 10 (7) (2016) 224. [30] A. Hernández-Aguilera, J. Sepúlveda, E. Rodríguez-Gallego, M. Guirro, A. GarcíaHeredia, N. Cabré, et al., Immunohistochemical analysis of paraoxonases and chemokines in arteries of patients with peripheral artery disease, Int J Mol Sci. 16 (5) (2015) 11323–11338. [31] K.D. Yu, X. Wang, C. Yang, X.H. Zeng, Z.M. Shao, Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse, Oncotarget. 6 (28) (2015) 26519–26527. [32] A. Hernández-Aguilera, J. Sepúlveda, E. Rodríguez-Gallego, M. Guirro, A. GarcíaHeredia, N. Cabré, et al., Immunohistochemical analysis of paraoxonases and chemokines in arteries of patients with peripheral artery disease, Int. J. Mol. Sci. 16 (5) (2015) 11323–11338. [33] F. Rodriguez-Sanabria, A. Rull, R. Beltran-Debon, G. Aragones, J. Camps, B. Mackness, et al., Tissue distribution and expression of paraoxonases and chemokines in mouse: the ubiquitous and joint localisation suggest a systemic and coordinated role, J. Mol. Histol. 41 (6) (2010 Dec) 379–386. [34] J. Marsillach, J. Camps, R. Beltran-Debón, A. Rull, G. Aragones, C. MaestreMartínez, et al., Immunohistochemical analysis of paraoxonases-1 and 3 in human
6. Funding Current work in our laboratories is supported by grants from the Instituto de Salud Carlos III (Grants PI15/00285 and PI18/00921 cofounded by the European Regional Development Fund [FEDER]) to JJ. We also acknowledge the support by the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (Grants 2017SGR436), and the Fundació La Marató de TV3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 7. Data availability All relevant data are within the paper and supporting information files and details available upon request. Author Contributions Conceptualization: VMP, JJS, JJ. Funding acquisition: JAM, JJ. Data curation: MF, AHA, SFA. Formal analysis: AHA, MF, NC, FLM, JJS. Investigation: AHA, MF, NC, FLM, JC, SFA, JJS, JJ. Methodology: MF, SFA, AHA, NC, FLM. Project administration: AHA, JJ. Resources: VMP, JAM, JJS, JJ. Supervision: JC, SFA. Validation: MF, AHA, JC, JJS. Visualization: AHA, MF. Writing – original draft: AHA, JJ. Writing – review & editing: AHA, MF, NC, FLM, JC, SFA, VMP, JAM, JJS, JJ. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cyto.2019.154923. References [1] A. Arbab-Zadeh, V. Fuster, The risk continuum of atherosclerosis and its implications for defining CHD by coronary angiography, J. Am. Coll. Cardiol. 68 (22) (2016) 2467–2478. [2] M.V. Madhavan, B.J. Gersh, K.P. Alexander, C.B. Granger, G.W. Stone, Coronary artery disease in patients ≥80 years of age, J. Am. Coll. Cardiol. 71 (18) (2018) 2015–2040. [3] A.J. Yurdagul, A.C. Finney, M.D. Woolard, A.W. Orr, The arterial microenvironment: the where and why of atherosclerosis, Biochem. J. 473 (10) (2016 May) 1281–1295. [4] J. Viola, O. Soehnlein, Atherosclerosis - A matter of unresolved inflammation, Semin Immunol. 27 (3) (2015) 184–193. [5] R.L. Sun, C.X. Huang, J.L. Bao, J.Y. Jiang, B. Zhang, S.X. Zhou, et al., CC-Chemokine ligand 2 (CCL2) suppresses high density lipoprotein (HDL) Internalization and cholesterol efflux via CC-chemokine receptor 2 (CCR2) induction and p42/44 mitogen-activated protein kinase (MAPK) activation in human endothelial cells, J. Biol. Chem. 291 (37) (2016) 19532–19544. [6] J.A. Dubland, G.A. Francis, So Much Cholesterol: the unrecognized importance of smooth muscle cells in atherosclerotic foam cell formation, Curr Opin Lipidol. 27 (2) (2016) 155–161.
8
Cytokine 126 (2020) 154923
A. Hernández-Aguilera, et al.
[49] K.H. Park, T. Sun, F. Diez-Delhoyo, Z. Liu, S.W. Yang, R.J. Lennon, et al., Association between coronary microvascular function and the vasa vasorum in patients with early coronary artery disease, Atherosclerosis 253 (2016 Oct) 144–149. [50] A. Jabs, E. Okamoto, J. Vinten-Johansen, G. Bauriedel, J.N. Wilcox, Sequential patterns of chemokine- and chemokine receptor-synthesis following vessel wall injury in porcine coronary arteries, Atherosclerosis 192 (1) (2007) 75–84. [51] K. Werth, R. Forster, Active shaping of chemokine gradients by atypical chemokine receptors: A 4D live-cell imaging migration assay, Meth. Enzymol. 570 (2016) 293–308. [52] J.S. Kim, K. Pak, T.S. Goh, D.C. Jeong, M.E. Han, J. Kim, et al., Prognostic value of MicroRNAs in coronary artery diseases: a meta-analysis, Yonsei. Med. J. 59 (4) (2018) 495. [53] R. Kumari, S. Kumar, M.K. Ahmad, R. Singh, S. Kant Kumar, A. Pradhan, et al., Promoter variants of TNF-α rs1800629 and IL-10 rs1800871 are independently associated with the susceptibility of coronary artery disease in north Indian, Cytokine 4 (110) (2018) 131–136. [54] N. Moradi, R. Fadaei, R. Ahmadi, E. Kazemian, S. Fallah, Lower expression of miR10a in coronary artery disease and its association with pro/anti-inflammatory cytokines, Clin Lab. 64 (5) (2018) 847–854. [55] R. Ahmadi, E. Heidarian, R. Fadaei, N. Moradi, M. Malek, S. Fallah, miR-342-5p expression levels in coronary artery disease patients and its association with inflammatory cytokines, Clin Lab. 64 (4) (2018) 603–609. [56] A.E. Bildirici, S. Arslan, N. Özbilüm Şahin, Ö. Berkan, O. Beton, M.B. Yılmaz, MicroRNA-221/222 expression in atherosclerotic coronary artery plaque and peripheral blood, Biomarkers 8 (2018) 1–19. [57] X. Zhao, D. Sun, R.X. Xu, Y.L. Guo, C.G. Zhu, N.Q. Wu, et al., Low-density lipoprotein-associated variables and the severity of coronary artery disease: an untreated Chinese cohort study, Biomarkers 7 (2018) 1–23. [58] R. Khan, V. Spagnoli, J.C. Tardif, P.L. L’Allier, Novel anti-inflammatory therapies for the treatment of atherosclerosis, Atherosclerosis 240 (2) (2015) 497–509. [59] T.J. Schall, A.E.I. Proudfoot, Overcoming hurdles in developing successful drugs targeting chemokine receptors, Nat. Rev. Immunol. 11 (5) (2011 May 15) 355–363. [60] S. Piconi, D. Pocaterra, V. Rainone, M. Cossu, M. Masetti, G. Rizzardini, et al., Maraviroc reduces arterial stiffness in PI-Treated HIV-infected Patients, Sci Rep. 6 (1) (2016) 28853. [61] V.G. Kramer, S. Hassounah, S.P. Colby-Germinario, M. Oliveira, E. Lefebvre, T. Mesplede, et al., The dual CCR5 and CCR2 inhibitor cenicriviroc does not redistribute HIV into extracellular space: implications for plasma viral load and intracellular DNA decline, J. Antimicrob. Chemother. 70 (3) (2015) 750–756. [62] M. Massara, O. Bonavita, A. Mantovani, M. Locati, R. Bonecchi, Atypical chemokine receptors in cancer: friends or foes? J. Leukoc. Biol. 99 (6) (2016 Jun) 927–933.
atheromatous plaques, Eur. J. Clin. Invest. 41 (3) (2011) 308–314. [35] D. Szklarczyk, A. Franceschini, S. Wyder, K. Forslund, D. Heller, J. Huerta-Cepas, et al., STRING v10: Protein-protein interaction networks, integrated over the tree of life, Nucleic Acids Res. 43 (D1) (2015 Jan) D447–D452. [36] C.S. Greene, A. Krishnan, A.K. Wong, E. Ricciotti, R.A. Zelaya, D.S. Himmelstein, et al., Understanding multicellular function and disease with human tissue-specific networks, Nat. Genet. 47 (6) (2015) 569–576. [37] A. Krishnan, R. Zhang, V. Yao, C.L. Theesfeld, A.K. Wong, A. Tadych, et al., Genome-wide prediction and functional characterization of the genetic basis of autism spectrum disorder, Nat. Neurosci. 19 (11) (2016) 1454–1462. [38] A. Zlotnik, O. Yoshie, H. Nomiyama, The chemokine and chemokine receptor superfamilies and their molecular evolution, Genome Biol. 7 (12) (2006) 243. [39] S. Jordan, N. Tung, M. Casanova-Acebes, C. Chang, C. Cantoni, D. Zhang, et al., Dietary intake regulates the circulating inflammatory monocyte pool, Cell. 178 (5) (2019) 1102–1114.e17. [40] F. Bachelerie, G.J. Graham, M. Locati, A. Mantovani, P.M. Murphy, R. Nibbs, et al., New nomenclature for atypical chemokine receptors, Nat. immunol. United States 15 (2014) 207–208. [41] D.P. Ramji, T.S. Davies, Cytokines in atherosclerosis: key players in all stages of disease and promising therapeutic targets, Cytokine Growth Factor Rev. 26 (6) (2015) 673–685. [42] A. Ridiandries, J.T.M. Tan, C.A. Bursill, The role of CC-chemokines in the regulation of angiogenesis, Int. J. Mol. Sci. 17 (11) (2016) 1856. [43] L. Boring, J. Gosling, M. Cleary, I.F. Charo, Decreased lesion formation in CCR2-/mice reveals a role for chemokines in the initiation of atherosclerosis, Nature. 394 (6696) (1998) 894–897. [44] T.C. Dawson, W.A. Kuziel, T.A. Osahar, N. Maeda, Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice, Atherosclerosis 143 (1) (1999) 205–211. [45] W. Wan, Q. Liu, M.S. Lionakis, A.P.M.P.P. Marino, S.A. Anderson, M. Swamydas, et al., Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoEknockout mice, Cardiovasc. Res. 106 (3) (2015) 478–487 Jun 1. [46] C. Cochain, C. Auvynet, L. Poupel, J. Vilar, E. Dumeau, A. Richart, et al., The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction, Arterioscler. Thromb. Vasc. Biol. 32 (9) (2012) 2206–2213. [47] L. Gao, Z. Xu, Z. Yin, K. Chen, C. Wang, H. Zhang, Association of hydrogen sulfide with alterations of monocyte chemokine receptors, CCR2 and CX3CR1 in patients with coronary artery disease, Inflamm Res. 64 (8) (2015) 627–635. [48] K.M. Lee, R. Danuser, J.V. Stein, D. Graham, R.J.B. Nibbs, G.J. Graham, The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density, EMBO J. 33 (21) (2014) 2564–2580.
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