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Increased proteolytic cleavage of osteoglycin is associated with a stable plaque phenotype and lower risk of cardiovascular events

      Highlights

      • Plaque levels of cleaved osteoglycin are higher in plaques from asymptomatic patients and patients without diabetes.
      • High plaque levels of cleaved osteoglycin are associated with a stable plaque phenotype and a lower risk for cardiovascular events.
      • The cleavage process of osteoglycin may be a potential therapeutic target for the stabilization of vulnerable plaques.

      Abstract

      Background and aims

      Extracellular matrix (ECM) remodeling is one of the key components in the formation of vulnerable atherosclerotic plaques and cardiovascular events. We recently showed that the full-length ECM-proteoglycan osteoglycin was associated with plaque vulnerability and future cardiovascular events. In the present study, we aimed to investigate the association of cleaved osteoglycin with plaque phenotype.

      Methods

      Two-hundred human carotid plaques were analyzed by immunohistochemistry. Cleaved osteoglycin and active caspase-3 were assessed by ELISA. ECM components (collagen, elastin and glycosaminoglycans) were assessed by colorimetric assays in plaque tissue homogenates. Matrix metalloproteinases (MMPs) were assessed using Milliplex. MMP-cleavage of osteoglycin and its effect on apoptosis were studied in vitro. Cardiovascular events were recorded during follow-up using national registries.

      Results

      Plaque levels of cleaved osteoglycin were significantly higher in asymptomatic plaques and correlated to α-actin plaque area, collagen, elastin and inversely to lipids, active.
      caspase-3 and a histological vulnerability index. Cleaved osteoglycin correlated to several MMPs, especially MMP-12, which was also shown to cleave osteoglycin in vitro. In vitro cleavage of osteoglycin was also associated with less smooth muscle cell apoptosis. Patients with high plaque levels of cleaved osteoglycin had a significantly lower risk to suffer from future cardiovascular events.

      Conclusions

      The current study shows that cleaved osteoglycin is associated with a stable plaque phenotype and lower risk for future cardiovascular events. Potentially due to reduced cell apoptosis and ability to retain LDL. These results indicate that targeting the cleavage of osteoglycin may be a potential therapeutic strategy to stabilize plaques.

      Graphical abstract

      Keywords

      1. Introduction

      Atherosclerotic plaques are formed when lipids and immune cells accumulate in the arterial wall. They are commonly divided into vulnerable and stable plaques based on plaque composition. Vulnerable plaques are at high risk to rupture and cause clinical complications [
      • Naghavi M.
      • Libby P.
      • Falk E.
      • Casscells S.W.
      • Litovsky S.
      • Rumberger J.
      • et al.
      From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I.
      ]. The extracellular matrix (ECM) is a complex acellular network surrounding cells in all tissues of the human body. Apart from providing mechanical support to the surrounding cells, the ECM plays a key role in different biological processes such as cell migration and lipoprotein binding. The ECM contains different proteins including collagens, elastin, glycoproteins, glycosaminoglycans and proteoglycans. Depending on the protein composition, the ECM could either be associated to a stable or vulnerable plaque phenotype [
      • Katsuda S.
      • Kaji T.
      Atherosclerosis and extracellular matrix.
      ].
      Osteoglycin, also known as mimecan, is a proteoglycan belonging to the small leucin-rich proteoglycans (SLRPs) family involved in angiogenesis [
      • Wu Q.H.
      • Ma Y.
      • Ruan C.C.
      • Yang Y.
      • Liu X.H.
      • Ge Q.
      • et al.
      Loss of osteoglycin promotes angiogenesis in limb ischaemia mouse models via modulation of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 signalling pathway.
      ] and collagen fibrillogenesis [
      • Tasheva E.S.
      • Koester A.
      • Paulsen A.Q.
      • Garrett A.S.
      • Boyle D.L.
      • Davidson H.J.
      • et al.
      Mimecan/osteoglycin-deficient mice have collagen fibril abnormalities.
      ]. During ECM remodeling, osteoglycin can be cleaved by matrix metalloproteinases (MMPs) [
      • Barascuk N.
      • Vassiliadis E.
      • Zheng Q.
      • Wang Y.
      • Wang W.
      • Larsen L.
      • et al.
      Levels of circulating MMCN-151, a degradation product of mimecan, reflect pathological extracellular matrix remodeling in apolipoprotein E knockout mice.
      ]. Recently, we showed high levels of full-length osteoglycin to be associated with plaque vulnerability and increased risk for future cardiovascular events [
      • Tengryd C.
      • Nielsen S.H.
      • Cavalera M.
      • Bengtsson E.
      • Genovese F.
      • Karsdal M.
      • et al.
      The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.
      ]. In the current study, we aimed to investigate whether the cleavage of osteoglycin is associated to a stable plaque phenotype.

      2. Materials and methods

      A detailed description of methods is provided in the Supplementary Methods. The dataset is not publicly available due to the sensitive nature of the data regulated by GDPR regarding living subjects. Sensible requests to access the dataset from qualified researchers trained in human subject confidentiality protocols may be sent to Prof. Isabel Goncalves at Lund University.

      2.1 Human carotid plaques

      Human carotid plaques (n = 200) were obtained from the Carotid Plaque Imaging Project (CPIP) Biobank (Lund University, Malmö, Sweden). All patients had undergone carotid endarterectomy (CEA) at the Vascular Department at Skåne University Hospital in Malmö and had given both oral and written informed consent for their inclusion. The study was approved by the Regional Ethical Committee (472/2005) and the study followed the declaration of Helsinki. Clinical data regarding cardiovascular risk factors (age, sex, body mass index, hypertension, smoking, diabetes) as well as medications (statins, anti-hypertensive drugs) of the study cohort were recorded during patient enrollment. Blood samples were collected 24 h prior to surgery.
      Of the 200 patients included, 112 patients had suffered from symptoms (amaurosis fugax, transient ischemic attack (TIA) or stroke; 97 patients <1month prior to surgery and 15 patients >1month prior to surgery) and had carotid plaque with a degree of stenosis >70%. Eighty-eight patients were asymptomatic but had carotid plaques with a degree of stenosis >80%. The degree of stenosis was evaluated preoperatively with doppler ultrasound. All patients were assessed by a neurologist.

      2.2 Sample preparation and histology

      Plaques were snap-frozen in liquid nitrogen immediately after being collected from the operation theater during a CEA surgery. From the most stenotic region of the plaque, a 1 mm thick fragment was obtained and cryo-sectioned into 8 μm sections in order to be used for histological analyses. The remaining part of the plaque was homogenized in a buffer using a previously described methodology [
      • Goncalves I.
      • Moses J.
      • Dias N.
      • Pedro L.M.
      • Fernandes e Fernandes J.
      • Nilsson J.
      • et al.
      Changes related to age and cerebrovascular symptoms in the extracellular matrix of human carotid plaques.
      ].
      The 8 μm sections were stained for α-actin (vascular smooth muscle cells), Oil Red O (neutral lipids), CD68 (macrophages), glycophorin A (intraplaque hemorrhage) and Movat pentachrome (from which collagens were measured) according to previously described protocols [
      • Gonçalves I.
      • Edsfeldt A.
      • Ko N.Y.
      • Grufman H.
      • Berg K.
      • Björkbacka H.
      • et al.
      Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation.
      ,
      • Tomas L.
      • Edsfeldt A.
      • Mollet I.G.
      • Perisic Matic L.
      • Prehn C.
      • Adamski J.
      • et al.
      Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques.
      ]. Also, 14 plaque sections were stained for MMP-12 using a rabbit polyclonal primary antibody (Abcam, ab137443, Cambridge, England) and the secondary reagent MACH3 (Biocare, Medical, M3R531HM Lot:041621, CA, USA).
      Osteoglycin was stained using a primary rabbit polyclonal antibody (PA5-48255, Invitrogen, Waltham, MA) and MACH3 rabbit probe and horseradish peroxidase-polymer (RP531H, Biocare Medical, Pacheco, CA) as secondary antibody. A rabbit IgG polyclonal isotype antibody was used as a control (ab27478, Abcam, Cambridge, UK). A histological vulnerability index (VI) was calculated for each plaque (a ratio between the destabilizing components CD68, Oil Red O, glycophorin A and the stabilizing components α-actin and Movat collagen) as previously published [
      • Goncalves I.
      • Sun J.
      • Tengryd C.
      • Nitulescu M.
      • Persson A.F.
      • Nilsson J.
      • et al.
      Plaque vulnerability index predicts cardiovascular events: a histological study of an endarterectomy cohort.
      ]. Subsequently, the stained plaques were scanned using ScanScope Console Version 8.2 (LRI imaging AB, CA, USA) and photographed with Aperio ImageScope v.8.0 (Aperio Technologies Inc, Vista, CA, USA). The positively stained areas in % of the total plaque area as well as the macrocalcified regions of the plaque were quantified blindly using Biopix iQ 2.1.8 (Gothenburg, Sweden). QuPath 0.2.3 was used to quantify MMP-12 stained areas [
      • Bankhead P.
      • Loughrey M.B.
      • Fernández J.A.
      • Dombrowski Y.
      • McArt D.G.
      • Dunne P.D.
      • et al.
      QuPath: open source software for digital pathology image analysis.
      ].

      2.3 Analysis of cleaved osteoglycin

      Cleaved osteoglycin was measured in the carotid plaque homogenates using a competitive ELISA assay, as described by Barascuk et al. [
      • Barascuk N.
      • Vassiliadis E.
      • Zheng Q.
      • Wang Y.
      • Wang W.
      • Larsen L.
      • et al.
      Levels of circulating MMCN-151, a degradation product of mimecan, reflect pathological extracellular matrix remodeling in apolipoprotein E knockout mice.
      ].

      2.4 Detection of the ECM components

      The detection of the ECM components (collagen, elastin and glycosaminoglycans) in the plaque homogenate was performed using colorimetric assays as described previously [
      • Goncalves I.
      • Moses J.
      • Dias N.
      • Pedro L.M.
      • Fernandes e Fernandes J.
      • Nilsson J.
      • et al.
      Changes related to age and cerebrovascular symptoms in the extracellular matrix of human carotid plaques.
      ]. All ECM components were normalized to plaque wet weight.

      2.5 Analyses of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and active caspase-3 in plaque tissue homogenates

      Plaque levels of six different MMPs; MMP-1, -2, -3, -9, -10 and −12 were measured using Mesoscale Human MMP Ultra-Sensitive kit (Mesoscale, Gaithersburg, MD, USA). The levels of TIMPs (TIMP-1 and TIMP-2) were evaluated using MILLIPLEX MAP Human TIMP Magnetic Bead Panel (Milliplex, MA, USA). The method used was performed as previously described by Edsfeldt et al. [
      • Edsfeldt A.
      • Goncalves I.
      • Grufman H.
      • Nitulescu M.
      • Duner P.
      • Bengtsson E.
      • et al.
      Impaired fibrous repair: a possible contributor to atherosclerotic plaque vulnerability in patients with type II diabetes.
      ]. Active caspase-3 was measured using the active caspase-3 ELISA kit (Invitrogen Caspase-3 [active] Human ELISA, Invitrogen Corporation, Camarillo, CA) and Luminex 100 IS 2.3 (Austin, TX). The values were normalized to plaque wet weight.

      2.6 In vitro cleavage of osteoglycin

      Human recombinant osteoglycin (cat. No. 13478-H08H, Sinobiological, USA) was cleaved with activated MMPs (−1/-2/-3/-9/-10 and-12).

      2.7 In vitro studies of smooth muscle cell apoptosis

      Human coronary artery smooth muscle cells (Thermo Scientific, Göteborg, Sweden) were cultured in Medium 231 supplemented with smooth muscle growth supplement (Thermo Scientific) on 12-well glass slides (ibidi, Gräfelfing, Germany). Apoptosis was induced by 40 μM tert-Butyl hydroperoxide (Sigma-Aldrich/Merck, Darmstadt, Germany) incubation for 18 h in the presence or absence of full-length or cleaved osteoglycin. Apoptotic cells were labelled by TUNEL staining using the Roche in situ cell death detection kit (Sigma-Aldrich/Merck) and mounted in Vectashield Antifade medium with DAPI (Vector Laboratories, Burlingame, USA). To quantify the percentage of apoptotic cells, 5 images per condition were taken at 100× magnification. DAPI-stained nuclei were automatically counted using ImageJ v1.53, and TUNEL-positive nuclei in the respective images were counted manually.

      2.8 RNA sequencing of osteoglycin (OGN) in carotid plaque tissue

      The OGN mRNA levels were compared between symptomatic and asymptomatic plaques using RNA sequencing. A detailed description is provided as supplementary material.

      2.9 Clinical follow up

      To explore if cleaved osteoglycin could predict future cardiovascular events, the study cohort was divided into two groups, high and low plaque levels of cleaved osteoglycin (based on the median). By using the Swedish National in-patient Health Register and the ICD-10 codes G45.3, G45.9, G46, I12-22, 124.8-9, I25.1–2, I25.5–6, I25.8, I63.1–2, I63.3–5, I63.8–9 and I64.5, the CV events were identified. Moreover, verification of the events was done through standardized telephone interviews with the patients and by medical chart reviews. If the patients experienced several CV events, only the first event was included in the analysis. CEA-related events, defined as cardiovascular events occurring within 72 h after the planned CEA, were excluded. Clinical characteristics of the study cohort are provided in Supplementary Table S1.

      2.10 Statistics

      Student's T test and Mann-Whitney U test were used for comparisons of continuous variables and presented as mean and standard deviation (SD) or median and interquartile range (IQR), depending on the distribution of the variables. Pearson's or Spearman's correlation tests were used for correlations analyses, when appropriate. Categorical data were analyzed using Chi2 test. Kaplan-Meier survival analyses followed by log-rank test were used for the follow-up analyses together with uni- and multivariate Cox regression analyses. To compare osteoglycin gene expression between symptomatic and asymptomatic patients, a linear regression was conducted. p-values were adjusted using Benjamin & Hochberg method. Statistical significance was defined as a p-value <0.05. For statistical analysis, SPSS 24.0 (IBM Corp., Amonk, NY, USA) and GraphPad Prism v7.03 (GraphPad Software, San Diego, California, USA) were used.

      3. Results

      3.1 Plaque levels of cleaved osteoglycin and mRNA levels of osteoglycin gene (OGN) are lower in symptomatic plaques

      Plaques from patients that experienced symptoms (amaurosis fugax, TIA or stroke) within one month prior to surgery had lower levels of cleaved osteoglycin compared to plaques from asymptomatic patients (19.5 (IQR 12.6–29.2) vs 16.6 (IQR 11.9–23.0) pg/gram wet weight plaque, p = 0.045; Fig. 1A). Cleaved osteoglycin was lower in plaques from patients with diabetes (14.2 (IQR 11.7–23.7) vs 19.4 (IQR 13.6–27.7) pg/gram wet weight plaque, p = 0.03, Fig. 1B) and in plaques from males compared to plaques from females (15.9 (IQR 11.8–22.0) vs 22.8 (IQR 14.7–31.4) pg/gram wet weight plaque, p < 0.001).
      Fig. 1
      Fig. 1Higher levels of cleaved osteoglycin are associated with asymptomatic carotid plaques.
      (A) Plaques not associated with symptoms <1 month prior to surgery had significantly higher levels of cleaved osteoglycin compared to symptomatic plaques (p = 0.045). (B) Plaque levels of cleaved osteoglycin were significantly higher in plaques from patients without diabetes compared to plaques from diabetic patients (p = 0.026). Lines and bars represent median and IQR. Mann-Whitney U test was used to determine the level of significance. (C) Plaque OGN gene expression levels were significantly higher in asymptomatic plaques compared to symptomatic plaques. Students T-test was used for the comparison. Lines indicate- median and boxes 25% and 75%. OGN, osteoglycin.
      Interestingly, the osteoglycin mRNA levels were significantly higher in plaques not associated with symptoms (mean 7.99 log2 CPM (95% Cl 7.42–8.56) compared to symptomatic plaques (mean 7.01 log2 CPM (95% Cl 6.70–7.32); log2 fold change 0.98, p = 0.0034; Fig. 1C).

      3.2 High plaque levels of cleaved osteoglycin are associated with a lower risk for future cardiovascular events

      In line with the higher levels of cleaved osteoglycin in plaques from asymptomatic patients, patients with levels of cleaved osteoglycin above median had a significantly lower risk to suffer from future cardiovascular events (p = 0.007, Fig. 2). Cleaved osteoglycin remained a significant predictor of future cardiovascular events after adjusting for age, gender, diabetes and preoperative cerebrovascular symptoms in a multivariate Cox regression analysis (HR 0.55, 95% CI 0.31–0.98; p = 0.043).
      Fig. 2
      Fig. 2Higher levels of cleaved osteoglycin are associated with a lower risk to suffer from future cardiovascular events.
      Kaplan-Meier curve for cardiovascular events comparing patients with high (green) or low (blue; divided by the median) plaque levels of cleaved osteoglycin. In patients with high levels of cleaved osteoglycin there was a lower risk for future cardiovascular events (p = 0.007). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

      3.3 Cleaved osteoglycin is associated with smooth muscle cells and stabilizing ECM proteins

      Plaque levels of cleaved osteoglycin showed a weak correlation to α-actin plaque area (r = 0.17, p = 0.02) and an inverse weak correlation to plaque area of neutral lipids (Oil Red O; r = −0.18, p = 0.01) and VI (r = −0.24, p = 0.001). However, plaque levels of cleaved osteoglycin did not significantly correlate to neither the plaque area of CD68 (r = −0.08, p = 0.25), glycophorin A (intraplaque hemorrhage; r = −0.14, p = 0.06) nor to calcium (r = −0.07, p = 0.33). Plaque levels of the ECM-proteins collagen and elastin, but not glycosaminoglycans, showed good correlations to cleaved osteoglycin (r = 0.51, p < 0.0001; r = 0.59, p < 0.0001; respectively).

      3.4 Markers of ECM remodeling are associated to cleaved osteoglycin

      Plaque levels of cleaved osteoglycin correlated to MMP-2, -3, -10 and −12 (r = 0.39, p < 0.00001; r = 0.28, p < 0.00001 r = 0.17, p = 0.02; r = 0.62, p < 0.00001; respectively; Fig. 3A). Also, MMP-12 levels were identified to be significantly higher in asymptomatic compared to symptomatic plaques (2.9 × 107 (IQR 1.7 × 107-4.9 × 107) vs 1.6 × 107 (IQR 1 × 107-2.9 × 107) arbitrary units/gram wet weight plaque, p < 0.001; Fig. 3B). TIMP-1 correlated inversely to cleaved osteoglycin (r = −0.16, p = 0.025) whereas TIMP-2 correlated positively (r = 0.40, p < 0.0001). Additional in vitro experiments confirmed that the cleavage of osteoglycin is mainly driven by MMP-12, but also MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 are able to generate the fragment (Fig. 3C). There was no reactivity towards the MMPs without osteoglycin present, and a slight reactivity towards full-length osteoglycin was observed.
      Fig. 3
      Fig. 3Matrix metalloproteinase-12 (MMP-12) is a strong inducer of osteoglycin cleavage.
      (A) Plaque levels of cleaved osteoglycin correlated to plaque levels of MMP-12. Spearman rank correlation test was used. (B) Plaques associated with symptoms had lower levels of MMP-12 then asymptomatic plaques (p < 0.001). Lines and bars represents median and IQR. Mann-Whitney U test was used. (C) MMP-12 was a strong inducer of osteoglycin cleavage in vitro, n = 1. Levels are corrected for background (MMP-buffer). Bars represent mean levels of cleaved osteoglycin. MMP, matrix metalloproteinase. (D) Plaque areas stained positive for MMP-12 were more commonly identified in close proximity to plaque areas stained positive for osteoglycin among asymptomatic plaques compared to symptomatic. MMP-12+ and osteoglycin+ plaque areas in brown. Scale bars 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
      Next, MMP-12 was identified by immunohistochemistry in 14 plaques together with full-length osteoglycin to test if MMP-12 co-localized with osteoglycin to a greater extent in asymptomatic plaques compared to symptomatic plaques, as an indirect indication of increased MMP-12 induced osteoglycin cleavage in asymptomatic plaques. Interestingly, MMP-12+ plaque areas were identified in close proximity with osteoglycin+ plaque areas in 60% of the asymptomatic plaques but only in 17% of the symptomatic plaques (Fig. 3D). Finally, no correlation was identified between full-length osteoglycin and cleaved osteoglycin (r = −0.096, p = 0.18; n = 194).

      3.5 A ratio between cleaved/full-length osteoglycin is associated with a stable plaque phenotype

      As we have previously showed that full-length osteoglycin is associated with a vulnerable plaque phenotype, here a ratio between cleaved and full-length osteoglycin was calculated in order to asses if the balance between the two would show any association with plaque phenotype. Importantly, the calculated ratio showed a similar pattern of associations to plaque phenotype markers as the plaque levels of cleaved osteoglycin. The ratio correlated positively to plaque homogenate levels of MMP-12 and plaque area stained positive for α-actin and inversely to plaque area of intraplaque hemorrhage, CD68 and the calculated VI (Fig. 4).
      Fig. 4
      Fig. 4A ratio of cleaved and full-length osteoglycin is associated to markers of the stable plaque phenotype.
      Heatmap visualizing correlations between plaque phenotype markers and a calculated ratio between plaque levels of cleaved osteoglycin (pg/gram wet weight plaque) and plaque area stained positive for osteoglycin (% of total plaque area). Spearman's correlation tests were used. All analyses with a correlation coefficient r > 0.16 or r < 0.16 were significant. CD68, cluster of differentiation 68. MMP-12, matrix metalloproteinase-12. VI, vulnerability index.

      3.6 Cleaved osteoglycin levels are associated with less plaque cell apoptosis

      Finally, as full-length osteoglycin has been suggested to be associated with smooth muscle cell apoptosis we explored if the cleavage of osteoglycin could potentially protect cells from undergoing apoptosis. Human coronary artery smooth muscle cells were stimulated with either full-length or cleaved osteoglycin and then exposed to tert-Butyl hydroperoxide to induce cell apoptosis. Apoptotic cells were identified by TUNEL fluorescence, representative images are presented in Fig. 5A. Interestingly, full-length osteoglycin was associated with a greater percentage of apoptotic cells compared to cleaved osteoglycin (2.9 SEM 0.2 cells vs 2.0 SEM 0.2 log % apoptotic cells, p = 0.04; Fig. 5B). Next, to explore if plaque cell apoptosis was associated with cleaved osteoglycin, active caspase-3 was assessed in plaque tissue homogenates. Importantly, plaque levels of cleaved osteoglycin were negatively correlated to plaque levels of active caspase-3 (r = −0.32, p < 0.0001; Fig. 5C).
      Fig. 5
      Fig. 5Cleaved osteoglycin is associated to lower levels of plaque cell apoptosis.
      (A) Vascular smooth muscle cells were treated in vitro with osteoglycin or cleaved osteoglycin (both 20 μg/mL) and exposed to tert-Butyl hydroperoxide (40 μM for 18 h) to explore if the cleavage of full-length osteoglycin would affect cell apoptosis. Representative images are presented in A. (B) Cleaved osteoglycin induced less apoptosis (Log2% TUNEL+cells) compared to full-length osteoglycin. Lines and bars represents mean and standard error of the mean. One-way ANOVA was used. N = 10. (C) Scatter plots showing an inversely correlation between plaque homogenate levels of active-caspase-3 and cleaved osteoglycin (r = 32, p < 0.0001). Spearman's correlations coefficient was used.

      4. Discussion

      Synthesis and degradation of ECM proteins is central in plaque formation and stability. In this study, we showed that cleaved osteoglycin is associated with a stable plaque phenotype and a lower risk of suffering from future cardiovascular complications.
      Plaque levels of cleaved osteoglycin correlated to the VSMC marker α-actin as well as collagen and elastin, two ECM proteins synthesized by VSMCs. Zhang et al. [
      • Zhang H.J.
      • Wang J.
      • Liu H.F.
      • Zhang X.N.
      • Zhan M.
      • Chen F.L.
      Overexpression of mimecan in human aortic smooth muscle cells inhibits cell proliferation and enhances apoptosis and migration.
      ] have shown that OGN gene overexpression may enhance VSMC apoptosis and inhibit proliferation. As VSMCs are critical for the formation of the collagen and elastin-rich fibrous cap, an increased VSMCs apoptosis would reduce the number of VSMCs and thereby collagen, leading to a rupture prone thin fibrous cap. In the present study, we showed that cleaved osteoglycin was inversely correlated to plaque levels of the apoptosis marker active caspase-3 and that cleaved osteoglycin was associated to lower levels of VSMC apoptosis in vitro. These findings imply that cleavage of osteoglycin reduces VSMC apoptosis, which would result in more VSMCs present in the plaque and higher levels of collagen and elastin. Moreover, cleaved osteoglycin correlated positively to both MMP-2 and collagen. MMP-2 is known to promote VSMCs migration and proliferation [
      • Katsuda S.
      • Kaji T.
      Atherosclerosis and extracellular matrix.
      ]. This finding further elucidates the fact that cleavage of osteoglycin enhances the presence of the VSMCs in the plaque and a fibrous plaque phenotype potentially through limiting VSMCs apoptosis. However, in opposite to previous studies suggesting a negative effect of OGN gene overexpression on VSMC apoptosis and migration, we showed that OGN mRNA levels were significantly lower in plaques from patients with symptoms compared to asymptomatic plaques. Together with our previous and the present study, this indicates that the balance between synthesis and degradation of osteoglycin is of greater importance than the gene expression itself.
      Another characteristic of vulnerable plaques is increased lipid content [
      • Naghavi M.
      • Libby P.
      • Falk E.
      • Casscells S.W.
      • Litovsky S.
      • Rumberger J.
      • et al.
      From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I.
      ]. It is well established that proteoglycans play a role in lipoprotein retention [
      • O'Brien K.D.
      • Lewis K.
      • Fischer J.W.
      • Johnson P.
      • Hwang J.Y.
      • Knopp E.A.
      • et al.
      Smooth muscle cell biglycan overexpression results in increased lipoprotein retention on extracellular matrix: implications for the retention of lipoproteins in atherosclerosis.
      ,
      • Klezovitch O.
      • Edelstein C.
      • Zhu L.
      • Scanu A.M.
      Apolipoprotein(a) binds via its C-terminal domain to the protein core of the proteoglycan decorin. Implications for the retention of lipoprotein(a) in atherosclerotic lesions.
      ]. Tengryd et al. [
      • Tengryd C.
      • Nielsen S.H.
      • Cavalera M.
      • Bengtsson E.
      • Genovese F.
      • Karsdal M.
      • et al.
      The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.
      ] showed that full-length osteoglycin correlated to plaque lipid area. On the contrary, we found that cleaved osteoglycin correlated inversely to lipid area. In line with this, the calculated ratio of cleaved and full-length osteoglycin were even more negatively associated to plaque area of lipids, suggesting that plaques with a greater osteoglycin cleavage in relation to the full-length osteoglycin retain less lipids. The ratio was also negatively associated to plaque area of other known plaque vulnerability markers, including CD68 (myeloid cell marker), glycophorin A (intraplaque hemorrhage) and the histological VI. The negative correlations between cleaved osteoglycin, osteoglycin ratio and VI (reflecting the balance between vulnerable and stable plaque components) is of particular interest as the VI has previously been shown to be an independent predictor of future cardiovascular events [
      • Goncalves I.
      • Sun J.
      • Tengryd C.
      • Nitulescu M.
      • Persson A.F.
      • Nilsson J.
      • et al.
      Plaque vulnerability index predicts cardiovascular events: a histological study of an endarterectomy cohort.
      ]. Collectively, these findings indicate that osteoglycin may be another proteoglycan that affects plaque phenotype and that its cleavage may affect LDL-retention, inflammatory cell accumulation and plaque cell apoptosis. However, further studies are needed to confirm these findings.
      MMPs are enzymes that degrade the ECM [
      • Katsuda S.
      • Kaji T.
      Atherosclerosis and extracellular matrix.
      ] and in atherosclerosis different types of MMPs have been shown to play different roles. Generally, MMPs have been associated with plaque vulnerability, however some studies have challenged that insight and presented conflicting results that highlight the divergent effect of these enzymes. Besides MMP-2, cleaved osteoglycin correlated positively to MMP-3, MMP-10 and MMP-12. MMP-3 is considered to be atheroprotective as it has previously been shown that MMP-3−/−ApoE−/− mice form larger plaques with decreased VSMCs content compared to wild-type [
      • Johnson J.L.
      • George S.J.
      • Newby A.C.
      • Jackson C.L.
      Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries.
      ]. However, a recent mendelian randomization study showed that MMP-3 was associated with an increased risk to suffer from ischemic stroke [
      • Folkersen L.
      • Gustafsson S.
      • Wang Q.
      • Hansen D.H.
      • Hedman Å K.
      • Schork A.
      • et al.
      Genomic and drug target evaluation of 90 cardiovascular proteins in 30,931 individuals.
      ]. The aforementioned study also showed that MMP-12 was associated with a lower risk to suffer from ischemic stroke, also conflicting previous mouse studies showing smaller lesions, more VSMCs and less macrophages in MMP-12−/−ApoE−/− plaques [
      • Johnson J.L.
      • George S.J.
      • Newby A.C.
      • Jackson C.L.
      Divergent effects of matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability in mouse brachiocephalic arteries.
      ]. These conflicting results highlight the complexity of the functionality of MMPs. Barascuk et al. [
      • Barascuk N.
      • Vassiliadis E.
      • Zheng Q.
      • Wang Y.
      • Wang W.
      • Larsen L.
      • et al.
      Levels of circulating MMCN-151, a degradation product of mimecan, reflect pathological extracellular matrix remodeling in apolipoprotein E knockout mice.
      ] have shown that MMP-12 cleaves osteoglycin and in our study, we confirmed an increased cleavage of osteoglycin particularly by MMP-12 but also by MMP-3 and MMP-10. In support for the previous human study indicating a protective role of MMP-12 against ischemic stroke, we showed that MMP-12 levels were significantly higher among asymptomatic plaques compared to symptomatic. Based on histology, MMP-12 was also more commonly identified in plaque regions with osteoglycin in asymptomatic plaques, supporting a greater MMP-12 induced cleavage in stable plaques. Our data also indicate that an increased osteoglycin cleavage may reduce VSMCs apoptosis and thereby contribute to a greater collagen synthesis. A plaque with more cleaved osteoglycin is more likely to have an efficient remodeling with more stabilizing ECM proteins and a subsequent lower risk to rupture.
      The imbalance between the expression of MMPs and TIMPs is suggested to enhance plaque progression [
      • Raffetto J.D.
      • Khalil R.A.
      Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease.
      ]. In our study, cleaved osteoglycin was negatively correlated to TIMP-1 while TIMP-2 correlated positively. Several studies have demonstrated that TIMP-2 has a more prominent anti-atherogenic role than TIMP-1 and that loss of TIMP-2 expression is associated to plaque vulnerability [
      • Di Gregoli K.
      • George S.J.
      • Jackson C.L.
      • Newby A.C.
      • Johnson J.L.
      Differential effects of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 on atherosclerosis and monocyte/macrophage invasion.
      ,
      • Johnson J.L.
      • Baker A.H.
      • Oka K.
      • Chan L.
      • Newby A.C.
      • Jackson C.L.
      • et al.
      Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis.
      ].
      We recently showed that full-length osteoglycin is associated to future cardiovascular events [
      • Tengryd C.
      • Nielsen S.H.
      • Cavalera M.
      • Bengtsson E.
      • Genovese F.
      • Karsdal M.
      • et al.
      The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.
      ]. Interestingly, opposite to full-length osteoglycin, we show that high levels of cleaved osteoglycin are associated to asymptomatic plaques and a lower risk to suffer from future cardiovascular events.
      There are some limitations in our study that should be taken into consideration. The plaque levels of cleaved osteoglycin were measured in plaque tissue homogenate, while the plaque area of the different plaque components stained with immunohistochemistry were measured in 8 μm thick sections and may not represent the entire lesion. The cleaved isoform of osteoglycin was not possible to locate histologically as there are no antibodies available to target these isoforms specifically. It should be considered that the osteoglycin antibody used in Tengryd et al. [
      • Tengryd C.
      • Nielsen S.H.
      • Cavalera M.
      • Bengtsson E.
      • Genovese F.
      • Karsdal M.
      • et al.
      The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.
      ] also targets the cleaved form of osteoglycin, which could potentially lead to an overestimation of the full-length osteoglycin positive plaque area. However, plaque levels of cleaved osteoglycin did not correlate to plaque areas stained positive for full-length osteoglycin (r = −0.096, p = 0.18; n = 194), suggesting that the overlap between full-length and cleaved osteoglycin has not had a major impact on the results. Also, the assay used for measuring MMPs and TIMPs did not measure activity but protein concentrations of these proteins.
      Moreover, in the present study, symptomatic plaques were surgically removed within one month post event (median 16 days, IQR 9–25 days). Since the healing process of the plaque may have been initiated, the morphology of the plaque obtained from surgery may not fully represent the plaque morphology that contributed to the event. However, it has been shown that the remodeling of symptomatic plaques and the alteration in plaque morphology is most prominent >30 days after rupture with a successive increase of stabilizing components and a decrease of inflammation [
      • Peeters W.
      • Hellings W.E.
      • de Kleijn D.P.
      • de Vries J.P.
      • Moll F.L.
      • Vink A.
      • et al.
      Carotid atherosclerotic plaques stabilize after stroke: insights into the natural process of atherosclerotic plaque stabilization.
      ].
      In conclusion, this study shows that increased cleavage of osteoglycin is associated to a stable plaque phenotype and lower risk of cardiovascular events. Thereby, targeting the cleavage of osteoglycin may be a therapeutic strategy for the stabilization of vulnerable plaques. However further research is needed to identify target proteins responsible for the osteoglycin cleavage.

      Financial support

      The work was supported by the Swedish Society for Medical Research , Emil and Wera Cornell foundation , the Swedish Research Council , Crafoord foundation , The Swedish Society of medicine , Swedish Heart and Lung Foundation , Åke Wiberg foundation , SUS foundations and funds and Lund University Diabetes Center ( Swedish Research Council - Strategic Research Area Exodiab Dnr 2009-1039 , Swedish Foundation for Strategic Research Dnr IRC15-0067 ). The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Skåne are acknowledged for generous financial support. In addition, we would like to thank the Danish Research foundation and Innovation Fund Denmark. No funding agents had any role in the study design, collection of data, analysis, interpretation of data, writing of the report or in the decision to submit the paper for publication.

      CRediT authorship contribution statement

      Dania Al-Sharify: Investigation, Formal analysis, Visualization, Writing – original draft. Signe Holm Nielsen: Investigation, Formal analysis, Conceptualization, Visualization, Writing – review & editing. Frank Matthes: Investigation, Formal analysis, Visualization, Writing – review & editing. Christoffer Tengryd: Investigation, Formal analysis, Writing – review & editing. Jiangming Sun: Investigation, Formal analysis, Visualization, Writing – review & editing. Federica Genovese: Investigation, Visualization, Writing – review & editing. Morten A. Karsdal: Resources, Conceptualization, Writing – review & editing. Jan Nilsson: Resources, Conceptualization, Writing – review & editing. Isabel Goncalves: Investigation, Resources, Formal analysis, Conceptualization, Writing – original draft, Supervision, Funding acquisition. Andreas Edsfeldt: Investigation, Resources, Formal analysis, Conceptualization, Writing – original draft, Supervision, Funding acquisition.

      Declaration of interests

      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
      S.H.N and F.G.E are full-time employees at Nordic Bioscience a biotechnology company located in Copenhagen. They both own stocks at Nordic Bioscience. The other authors have nothing to disclose.

      Acknowledgments

      The authors would like to thank Bettina Jung, Ana Persson, Mihaela Nitulescu and Lena.
      Sundius for their technical support. The graphical abstract was created using Biorender.com.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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