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Cartilage Oligomeric Matrix Protein Associates With a Vulnerable Plaque Phenotype in Human Atherosclerotic Plaques

Originally publishedhttps://doi.org/10.1161/STROKEAHA.119.026457Stroke. 2019;50:3289–3292

Abstract

Background and Purpose—

Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE−/− mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow–derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease.

Methods—

In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry.

Results—

Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7–14.3] versus 5.6% [2.8–9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=−0.16; P=0.024), elastin (r=−0.14; P=0.041), and smooth muscle cells (r=−0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006).

Conclusions—

The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.

In atherosclerotic disease plaques are formed in the vessel wall. Atherosclerotic plaques that rupture, leading to thrombus formation, may cause arterial occlusion and result in myocardial infarction or stroke. Rupture-prone, so-called vulnerable plaques, are characterized by a large necrotic core with high content of lipids, inflammatory cells, and intraplaque hemorrhage covered by a thin fibrous cap, whereas stable plaques are characterized by thick fibrous caps with smooth muscle cells (SMCs) producing collagen.1

Extracellular matrix proteins are important in the atherosclerotic disease not only by influencing plaque stability and cellular behavior but also by regulating inflammation.2 COMP (cartilage oligomeric matrix protein) interacts with several proteins, which could influence plaque stability, including collagen type I and growth factors.3 Furthermore, COMP is a substrate for several proteases.3 COMP is present in the medial layer of nonatherosclerotic arteries as well as in intimal SMCs of human primary atherosclerotic and restenotic lesions4 and regulates SMC differentiation and functions.5,6 In murine atherosclerosis, COMP is present in both inflammatory and fibrous plaques.7 Recent studies in mice revealed a protective role of COMP in atherosclerosis, where deficiency of COMP resulted in increased plaque size7,8 accompanied by increased plaque calcification.8 Interestingly, bone marrow transplantation experiments revealed a significant role also for COMP in bone marrow–derived cells in plaque development.8

Despite the evidence of COMP playing a role in murine atherosclerosis, knowledge is lacking on COMP in human atherosclerotic plaques. Here, we analyzed COMP expression in 211 human carotid plaques, and its association to plaque vulnerability.

Methods

Because of the sensitive nature of the data collected for this study, requests to access the dataset from qualified researchers trained in human subject confidentiality protocols may be sent to Isabel Goncalves, Lund University.

Detailed Materials and Methods are deposited in the online-only Data Supplement.

Informed consent was given by each patient. The study was approved by the local ethical committee.

Immunohistochemical Analysis

Transversal tissue sections from plaques were stained for COMP. The specificity of the COMP antibody was confirmed by staining of transfected cells overexpressing COMP9 (Figure I in the online-only Data Supplement). Stained areas were quantified blindly, normalized to total plaque area, and expressed as % of plaque area stained.

Results

COMP Is Associated With a Vulnerable Plaque Phenotype

COMP levels were determined in tissue sections from a plaque biobank consisting of 110 plaques from patients with cerebrovascular symptoms and 101 plaques from asymptomatic patients. Baseline characteristics of the patients are shown in Table I in the online-only Data Supplement. COMP staining was found in every plaque and varied between 0.03% and 38.6% staining of total plaque area. COMP was significantly increased in atherosclerotic plaques from symptomatic patients compared with lesions from asymptomatic patients, and in plaques with a high vulnerability index (Figure 1). Furthermore, COMP was positively associated with plaque lipid area and with macrophages determined by CD68 staining but was negatively associated with collagen and elastin levels as well as with SMCs areas (Table).

Table. Correlation of COMP to Plaque Components

COMP*P Value
R
Collagen−0.160.024
Elastin−0.140.041
Lipids (Oil Red O)*0.320.000002
SMCs α-actin*−0.250.0002
Macrophages CD68*0.150.036
CD163*0.370.00000006
Glycophorin A*0.280.00006

COMP indicates cartilage oligomeric matrix protein, and SMCs, smooth muscle cells.

*Percentage of total plaque area.

†Milligram per gram plaque tissue.

Figure 1.

Figure 1. COMP (cartilage oligomeric matrix protein) is increased in symptomatic and vulnerable human plaques. A, COMP is increased in carotid plaques associated with symptoms. B, COMP is increased in plaques with a high vulnerability index (above median). Vulnerability index was calculated based on the ratio between lipid-, CD68-, hemorrhage- and smooth muscle cell, and collagen-stainings.

COMP was localized to areas rich in lipids and CD68-positive cells, and to some extent to areas with SMCs α-actin expression (Figure II in the online-only Data Supplement). Co-staining of COMP together with either CD68 or SMC-α-actin revealed that COMP staining was present in both CD68 cells and SMCs (Figure IIIA and IIIB in the online-only Data Supplement). In the majority of the plaques, COMP was present in the shoulder regions, in the core, in the interface between the core and media, and between the core and cap, but was only detected in the cap region in 2% of the plaques (Figure IV in the online-only Data Supplement).

COMP Is Associated With CD163-Positive Cells

Recent data show that COMP-deficient macrophages correlated inversely with the gene profile of M2c macrophages.8 CD163, a receptor for hemoglobin-haptoglobin, is a marker of M2c and Mhem macrophages. In accordance, COMP was positively correlated with CD163 in the lesions (Table). COMP and CD163 were localized to the same plaque areas, but COMP often showed a more widespread localization (Figure 2). Furthermore, COMP was present in CD163-positive cells of human carotid plaques (Figure V in the online-only Data Supplement). To investigate if COMP colocalization with CD163 reflects an upregulated COMP expression, we stimulated monocyte-derived macrophages with heme and measured CD163, COMP, and the Mhem-associated cytokine IL-10 by real-time PCR. IL-10 expression peaked after 48 hours (P<0.05, ANOVA, Dunnett multiple comparison test) and was preceded by CD163 expression. COMP expression displayed a similar pattern as IL-10 with a transient but nonsignificant peak after 48 hours of stimulation (Figure VIA through VIC in the online-only Data Supplement). CD163 macrophages are present at sites of intraplaque hemorrhage.10,11 In agreement, COMP correlated with the erythrocyte marker glycophorin A (Table). Intraplaque hemorrhage was increased in symptomatic plaques compared with asymptomatic plaques (score 2.1±0.9 versus 1.7±0.8; P=0.012).

Figure 2.

Figure 2. Human carotid plaques (AC) stained for COMP (cartilage oligomeric matrix protein) and CD163. Boxed regions are magnified. c indicates core; fc, fibrous caps; l, lumen; and sh, shoulder regions.

Discussion

Previous studies suggest that COMP has a protective role in atherosclerosis. COMP is present in human vascular SMCs, maintains the contractile phenotype,5 and promotes collagen fibrillogenesis. COMP deficiency in mice results in larger plaques,7,8 accompanied by increased collagen and thicker collagen fibrils.7 In the present study, we investigated the association of COMP with human plaque vulnerability. Unexpectedly, we found that COMP was increased in human plaques associated with symptoms and was positively correlated with lipid- and CD68-positive areas, but negatively with collagen, elastin, and SMCs. Altogether, these data indicate that COMP is associated with a vulnerable carotid plaque phenotype in humans. Interestingly, COMP was associated and colocalized with CD163 cells, which could indicate that COMP modulates macrophage phenotype. This is supported by a previous study, where COMP-deficient macrophages correlated negatively with the gene profile of M2c macrophages and shifted macrophages to a more atherogenic and osteogenic phenotype.8 Although, lack of COMP in bone marrow–derived cells resulted in increased plaque area indicating a protective role of COMP in plaque progression,8 this does not exclude that COMP could have an opposite effect in advanced plaques, increasing plaque vulnerability. CD163 cells, previously considered as anti-inflammatory macrophages, were recently shown to be increased in ruptured/rupture-healed human plaques and associated with intraplaque angiogenesis, increased vascular permeability, and inflammatory cell recruitment in mice.12 Thus, it is possible that COMP affects the polarization or function of CD163 macrophages, which in turn increases plaque vulnerability.

Limitations

The current study has some limitations, which should be noted. First, this is not a mechanistic study, and thus conclusions of the functional role of COMP in plaque vulnerability are not possible. Second, we are only assessing COMP in the most stenotic part of the plaque and cannot rule out that the amount of COMP may be different in other parts of the lesions. Third, in the present study, only advanced plaques were used, and it is possible that the associations of COMP to plaque components may be different in early lesions. However, since the purpose of the study was to assess COMP in relation to plaque vulnerability, it makes sense to analyze advanced lesions.

Future Directions

Increased knowledge about molecules involved in plaque vulnerability is important for the discovery of new targets aiming to stabilize the plaques. Future directions should include mechanistic studies to determine the functional role of COMP in plaque vulnerability, aiming for therapeutic interventions to stabilize the plaques possibly via macrophage phenotype modulation.

Conclusions

In conclusion, the current study shows that COMP is increased in carotid atherosclerotic plaques from symptomatic patients and associates with plaque vulnerability and CD163-positive macrophages.

Acknowledgments

We are grateful to Professor Anna Blom, Department of Translational Medicine, Lund University for kindly providing transfected cells overexpressing COMP9 and to PhD Birgitta Gullstrand, Department of Clinical Science, Lund University for valuable input to the article.

Footnotes

*Drs Bengtsson and Gonçalves contributed equally to this work.

Current address for Dr Hultman: Novo Nordisk Foundation, Innovation Department, Hellerup, Denmark.

The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.119.026457.

Correspondence to Eva Bengtsson, PhD, Clinical Research Center, Lund University, Malmö, Sweden. Email

References

  • 1. Bentzon JF, Otsuka F, Virmani R, Falk E. Mechanisms of plaque formation and rupture.Circ Res. 2014; 114:1852–1866. doi: 10.1161/CIRCRESAHA.114.302721LinkGoogle Scholar
  • 2. Sorokin L. The impact of the extracellular matrix on inflammation.Nat Rev Immunol. 2010; 10:712–723. doi: 10.1038/nri2852CrossrefMedlineGoogle Scholar
  • 3. Acharya C, Yik JH, Kishore A, Van Dinh V, Di Cesare PE, Haudenschild DR. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.Matrix Biol. 2014; 37:102–111. doi: 10.1016/j.matbio.2014.06.001CrossrefGoogle Scholar
  • 4. Riessen R, Fenchel M, Chen H, Axel DI, Karsch KR, Lawler J. Cartilage oligomeric matrix protein (thrombospondin-5) is expressed by human vascular smooth muscle cells.Arterioscler Thromb Vasc Biol. 2001; 21:47–54.LinkGoogle Scholar
  • 5. Wang L, Zheng J, Du Y, Huang Y, Li J, Liu B, et al. Cartilage oligomeric matrix protein maintains the contractile phenotype of vascular smooth muscle cells by interacting with alpha(7)beta(1) integrin.Circ Res. 2010; 106:514–525. doi: 10.1161/CIRCRESAHA.109.202762LinkGoogle Scholar
  • 6. Yu H, Jia Q, Feng X, Chen H, Wang L, Ni X, et al. Hypoxia decrease expression of cartilage oligomeric matrix protein to promote phenotype switching of pulmonary arterial smooth muscle cells.Int J Biochem Cell Biol. 2017; 91(pt A):37–44. doi: 10.1016/j.biocel.2017.08.007CrossrefGoogle Scholar
  • 7. Bond AR, Hultgårdh-Nilsson A, Knutsson A, Jackson CL, Rauch U. Cartilage oligomeric matrix protein (COMP) in murine brachiocephalic and carotid atherosclerotic lesions.Atherosclerosis. 2014; 236:366–372. doi: 10.1016/j.atherosclerosis.2014.07.029CrossrefMedlineGoogle Scholar
  • 8. Fu Y, Gao C, Liang Y, Wang M, Huang Y, Ma W, et al. Shift of macrophage phenotype due to cartilage oligomeric matrix protein deficiency drives atherosclerotic calcification.Circ Res. 2016; 119:261–276. doi: 10.1161/CIRCRESAHA.115.308021LinkGoogle Scholar
  • 9. Englund E, Bartoschek M, Reitsma B, Jacobsson L, Escudero-Esparza A, Orimo A, et al. Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer.Oncogene. 2016; 35:5585–5596. doi: 10.1038/onc.2016.98CrossrefGoogle Scholar
  • 10. Boyle JJ, Harrington HA, Piper E, Elderfield K, Stark J, Landis RC, et al. Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.Am J Pathol. 2009; 174:1097–1108. doi: 10.2353/ajpath.2009.080431CrossrefMedlineGoogle Scholar
  • 11. Finn AV, Nakano M, Polavarapu R, Karmali V, Saeed O, Zhao X, et al. Hemoglobin directs macrophage differentiation and prevents foam cell formation in human atherosclerotic plaques.J Am Coll Cardiol. 2012; 59:166–177. doi: 10.1016/j.jacc.2011.10.852CrossrefMedlineGoogle Scholar
  • 12. Guo L, Akahori H, Harari E, Smith SL, Polavarapu R, Karmali V, et al. CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.J Clin Invest. 2018; 128:1106–1124. doi: 10.1172/JCI93025CrossrefMedlineGoogle Scholar

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