The rupture of an atherosclerotic plaque is one of the main causes of coronary artery thrombotic occlusion, leading to myocardial infarction. However, the exact mechanism and causal risk factors for plaque rupture remain unclear. To identify a potential molecule that can influence atherosclerotic plaque rupture, we investigated protein expression in serum from patients with acute myocardial infarction (AMI) and stable angina (SA), using proteomic analysis. The expression of six proteins, including fibrinogen, fetuin-B, keratin 9, proapolipoprotein and fibrinogen, were altered in serum from patients with AMI compared with serum from those with SA. Of these, fetuin-B, proapolipoprotein, fibrinogen γ-B-chain precursors and fibrinogen expression were greater in serum from patients with AMI than from patients with SA. Increased fetuin-B expression in serum from AMI patients was also confirmed by Western blot analysis. Treatment with recombinant human fetuin-B increased the migration in monocytes and macrophages in a concentration-dependent manner. Fetuin-B also affected vascular plaque-stabilizing factors, including lipid deposition and cytokine production in macrophages, the activation of matrix metalloproteinase (MMP)-2 in monocytes, and the activation of apoptosis and MMP-2 in vascular smooth muscle cells. Moreover, in vivo administration of fetuin-B decreased the collagen accumulation and smooth muscle cell content and showed an increased number of macrophages in the vascular plaque. From these results, we suggest that fetuin-B may act as a modulator in the development of AMI. This study may provide a therapeutic advantage for patients at high risk of AMI.

CLINICAL PERSPECTIVES

  • Atherosclerotic plaque rupture, a main cause of coronary artery thrombotic occlusion, can lead to myocardial infarction. However, the exact mechanism and causal risk factors for plaque rupture are unclear. To identify a potential molecule that can influence atherosclerotic plaque rupture, the present study investigated protein expression in sera from AMI and SA patients, using proteomic analysis.

  • Fetuin-B was one of the highly expressed serum proteins in AMI compared with the SA patient group. This protein increased the migration in monocytes and macrophages, and also affected vascular plaque-stabilizing factors, including lipid deposition and cytokine production in macrophages, MMP-2 activation in monocytes, and the activation of apoptosis and MMP-2 in VSMCs. In vivo administration of fetuin-B decreased the cuff placement-induced vascular plaque stability.

  • In conclusion, fetuin-B may play an important role in modulating AMI development, which may provide therapeutically beneficial information for patients at high risk of AMI.

INTRODUCTION

Atherosclerosis is a chronic inflammatory disease characterized by the formation of a plaque in the intimal layer of the vessel walls [1]. Coronary artery disease (CAD) frequently progresses in an abrupt fashion, and the occlusion of vessels develops rapidly, which is largely related to thrombosis resulting from the disruption of atherosclerotic plaque [2]. It is known that both innate and acquired immune responses are implicated in the development of atherosclerotic plaque [3]. Immune cells are commonly found in the plaque during all stages of CAD [4]. They are particularly abundant at the leading edge of the injured part of vessels and the shoulder region of the lipid core in the plaques. It is known that atherosclerotic plaques with enriched immune cells are more vulnerable and tend to rupture [5]. Inflammation, haemorrhage and abnormal apoptosis in the plaque region are well-known features of vulnerable plaques that are prone to rupture [1,6]. The accumulated data support macrophages and T-cells playing a critical role in the development of plaque rupture through the production of matrix metalloproteinases (MMPs) [7]. The activation of MMPs consequently results in destruction of the thin cap and potentiation of the rupture [7,8]. Moreover, an intimate interaction between T-cells and macrophages stimulates the production of MMPs that were derived from macrophages. The CD40 ligand on T-cells binds to its receptor on macrophages, which leads to MMP synthesis [9]. It is known that tumour necrosis factor α (TNF-α) is derived from various cells of the plaque region and stimulates monocyte recruitment to the atherosclerotic plaque [10]. Moreover, monocytes, as well as T-cells, infiltrated into endothelial walls during atherosclerosis can produce proinflammatory cytokines, chemokines and metalloproteinases. The molecules produced not only are involved in plaque growth but also contribute to plaque remodelling and stabilization [1,11]. However, the clear mechanisms of plaque formation and rupture need to be established.

Proteomics, the global method for the study of proteins, is a powerful tool for identifying proteins involved in the physiology and pathogenesis of specific diseases. It also provides a rich view of cellular processes and unique insights into biological complexity in a variety of cells [1214]. Moreover, the serum proteomics from disease models or patients can provide us with clues for identifying disease development mechanisms. This approach may guide the development of new diagnostics and therapies for human diseases [15]. There are several reports that the expressions of serum proteins change during acute coronary syndrome (ACS), including acute myocardial infarction (AMI) [1619]. However, these results focused mainly on alterations in protein expression.

In the present study, we tested whether protein expression is changed in serum from AMI patients with unstable plaques, and we identified six proteins that were altered in the AMI group using proteomic analysis. Furthermore, we explored a possible involvement of fetuin-B, which was up-regulated in the AMI group, in the activation of cells including monocytes, macrophages and vascular smooth muscle cells (VSMCs) which potently influence plaque stability related to plaque rupture.

MATERIALS AND METHODS

Materials

TNF-α, Oil Red O and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Recombinant human fetuin-B was obtained from BD Biosciences. Anti-fetuin-B, anti-fetuin-A, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-MMP-2, anti-cleaved caspase 3 (Santa Cruz Biotechnology), anti-stromal cell-derived factor-1 (SDF-1), anti-α-smooth muscle actin (SMA), anti-collagen and anti-CD68 (Abcam), anti-TNF-α, anti-CD14 and anti-interleukin (IL)-6 (eBioscience) antibodies were used. All chemicals used for 2D electrophoresis (2DE) and protein identification were purchased from either Bio-Rad Laboratories or Applied Biosystems.

Patients and specimens

Patients

A total of 47 patients who had the complaint of chest pain and a significant coronary stenosis on coronary angiography were investigated in this study. No patient had any type of previous cardiovascular disease, such as congestive heart failure, MI, hypertrophic cardiomyopathy or peripheral artery disease. Patients who had anaemia, chronic kidney disease, malignancy or any evidence of infection were also excluded. Smokers were defined as participants who had smoked regularly for >1 year. Hypertension was defined as systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg or current use of antihypertensive agents. Diabetes was defined as the use of insulin, oral hypoglycaemic agents or a fasting plasma glucose level ≥126 mg/dl. Hyperlipidaemia was defined as total cholesterol ≥200 mg/dl or current use of anti-cholesterol agents. After coronary angiography and the laboratory test, the patients were subjected to confirmation of clinical diagnosis and divided into two groups: AMI and stable angina (SA). The definition of AMI was a typical rise and/or gradual fall (troponin) or a more rapid rise and fall (CKMB or creatine kinase-MB fraction) of biochemical markers of myocardial necrosis, with at least one ischaemic symptom (chest pain) within 12 h or electrocardiograph (ECG) changes indicative of ischaemia. SA was considered as the case with typical exertional chest pain over the previous 3 months with no elevation in biochemical markers. The clinical baseline characteristics of the studied patient groups are summarized in Table 1. There was no significant difference in age distribution, sex composition (male) and risk factors (diabetes mellitus, hypertension, hypercholesterolaemia and stroke) between AMI and SA patient groups.

Table 1
Clinical baseline characteristics of the study groups
Stable angina (n=15)AMI (n=32)P value
Age (years) 60.2±12.4 60.7±12.9 >0.05 
Male, n (%) 11 (73.3) 28 (87.5) >0.05 
Risk factors, n (%)    
Diabetes mellitus 6 (40.0) 10 (31.2) >0.05 
Hypertension 12 (80.0) 17 (53.1) >0.05 
Hypercholesterolaemia 5 (33.3) 9 (28.1) >0.05 
Stroke 2 (13.3) 1 (3.1) >0.05 
Medication on admission, n (%)    
Anti-platelet agents 10 (66.7) 6 (18.8) <0.05 
β Blockers 8 (53.3) 4 (12.5) <0.05 
ACEIs or ARBs 10 (66.7) 7 (21.9) <0.05 
Calcium channel blockers 6 (40.0) 8 (25.0) >0.05 
Statins 5 (33.3) 2 (6.2) <0.05 
Diuretics 7 (46.7) 2 (6.2) <0.05 
Number of diseased vessels, n (%)   >0.05 
4 (26.7) 19 (59.4)  
5 (33.3) 7 (21.9)  
6 (40.0) 6 (18.8)  
Stable angina (n=15)AMI (n=32)P value
Age (years) 60.2±12.4 60.7±12.9 >0.05 
Male, n (%) 11 (73.3) 28 (87.5) >0.05 
Risk factors, n (%)    
Diabetes mellitus 6 (40.0) 10 (31.2) >0.05 
Hypertension 12 (80.0) 17 (53.1) >0.05 
Hypercholesterolaemia 5 (33.3) 9 (28.1) >0.05 
Stroke 2 (13.3) 1 (3.1) >0.05 
Medication on admission, n (%)    
Anti-platelet agents 10 (66.7) 6 (18.8) <0.05 
β Blockers 8 (53.3) 4 (12.5) <0.05 
ACEIs or ARBs 10 (66.7) 7 (21.9) <0.05 
Calcium channel blockers 6 (40.0) 8 (25.0) >0.05 
Statins 5 (33.3) 2 (6.2) <0.05 
Diuretics 7 (46.7) 2 (6.2) <0.05 
Number of diseased vessels, n (%)   >0.05 
4 (26.7) 19 (59.4)  
5 (33.3) 7 (21.9)  
6 (40.0) 6 (18.8)  

ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.

Specimens

Patients were subjected to coronary angiography via the radial or femoral artery approach using a 5 Fr-guided catheter system and iso-osmolar contrast medium (Visipaque, GE Healthcare) at Konkuk University Hospital (Seoul, Korea). Angiography was performed using a manual injection of contrast dye and images were acquired digitally (Allura, Philips). At the start of the procedure, 10 ml of whole blood was acquired in a heparin tube and transferred to a core laboratory. Blood samples were obtained from patients who were enrolled according to the same inclusion criteria, with each experimental group as described above, namely 32 patients (age 60.7±12.9 years) diagnosed with AMI accompanied by unstable plaque and 15 SA patients (age 60.2±12.4 years). Serum samples were separated from the whole blood samples, put into aliquots and stored at −80°C. This study was carried out in accordance with the World Medical Association's Declaration of Helsinki (2013) and approved by the Institutional Review Board of Konkuk University Hospital. All patients gave their informed consent to participate in the study.

Protein isolation and identification

The sera from patients with AMI or SA were treated with a Multi Affinity Removal Column (Agilent Technologies), specifically to remove six high-abundance plasma proteins that can interfere with exact proteomic analysis and make identification of the low-abundant proteins difficult [20]. The supernatant was diluted with a rehydration buffer including 8 M urea, 0.28% (w/v) DTT, 0.5% (w/v) CHAPS, 10% (v/v) glycerol, 0.5% (v/v) appropriate ampholyte and 0.002% (w/v) Bromophenol Blue. After isoelectric focusing, protein separation was carried out using SDS/10% PAGE. The gels were silver stained and visualized with a densitometer (VersaDoc Imaging System 1000, Bio-Rad Laboratories). Images of silver-stained gels from six different sets of experiments were normalized and counted by automated and manual spot-detection methods, and analysed using PDQuest statistical software (version 7.1.1, Bio-Rad Laboratories). The mean matching rates for gels were about 72–79% for the same set and 62–67% between gels for a different set. The spots that differed significantly by1.5-fold between two groups were selected and identified.

The trypsin-digested samples for LC/tandem mass spectrometry (MS/MS) analysis were vacuum dried and resuspended in 5% acetonitrile containing 0.1% formic acid. Peptides were analysed using a Synapt High Definition Mass Spectrometer (Waters) equipped with a nanoACQUITY Ultra Performance LC system. In brief, 2 μl of the peptide solution was injected on to a 75×100 mm Atlantis dC18 column. Peptides were initially separated using 100-min gradients and electrosprayed at a flow rate of 300 nl/min into the MS machine fitted with a nanoLock-Spray source (Waters). Mass spectra were acquired from m/z 300 to m/z 1600 for 1 s, followed by four data-dependent MS/MS scans from m/z 50 to m/z 1900 for 1 s each. The collision energy used to perform MS/MS was varied according to the mass and charge state of the eluting peptide. (Glu1)-fibrinopeptide B was infused at a rate of 350 nl/min, and an MS scan was acquired for 1 s every 30 s throughout the run. A database search was performed using MASCOT (Matrix Science) with the following parameters: NCBInr.08.03.26 database, Homo sapiens species and maximum number of missed cleavages by trypsin set to one. Mass tolerance ranged from 750 parts per million (ppm) to 7100 ppm. The peptide modification allowed during the search was carbamidomethylation in the fixed-modification mode.

Cell migration measurement

Cell lines of human monocyte, U937, cells and murine macrophage, RAW264.7, cells were purchased from ATCC and maintained in RPMI-1640 medium

Western blotting

Cell lysates were centrifuged at 16 000 g for 15 min at 4°C. Protein concentrations of the supernatants were determined using Bio-Rad DC protein assay reagents (Bio-Rad Laboratories), diluted with SDS sample buffer containing 40 mM Tris/HCl, pH 6.8, 8 mM EGTA, 4% (v/v) 2-mercaptoethanol, 40% (v/v) glycerol, 0.01% (w/v) Bromophenol Blue and 4% (w/v) SDS, and then boiled for 5 min. An equivalent number of samples were mounted on SDS/12% PAGE and then transferred electrophoretically to polyvinylidene difluoride (PVDF). The membrane was blocked with PBS containing 5% (w/v) fat-free dried milk and incubated overnight at 4°C, with each antibody diluted 1:1000–5000. Immune complexes were incubated for 1 h with a peroxidase-conjugated antibody diluted 1:5000. After application of the secondary antibody, the blots were incubated in enhanced chemiluminescence kits (Amersham Pharmacia). Band intensity was quantified using a Luminescent Image Analyzer LAS-3000 (Fujifilm).

Apoptosis assay

VSMCs were enzymatically isolated from the aortas of male SD rats at 10 weeks (n=5) purchased from Orient Bio. The isolated cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 units/ml of penicillin, 100 μg/ml of streptomycin and 200 mM glutamine. For all experiments, the cells were used at passages 5–8, grown to 70–80% confluence and starved in DMEM without FBS for 24 h. Apoptosis was analysed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL, Roche, Basel) method, in accordance with the manufacturer's protocol. Its detailed protocol is described in the Supplementary Expanded Methods.

Oil Red O staining

To analyse the population of lipid-laden macrophages, RAW264.7 cells were stained routinely with Oil Red O. Briefly, RAW264.7 cells were incubated with Oil Red O (0.5% in propan-2-ol) for 30 min at room temperature. The counting of the staining against the nucleus was performed using haematoxylin (Sigma-Aldrich). After washing for 10 min in PBS, the stained cells were mounted on glass slides and observed using a BX51 light microscope.

Flow cytometry analysis

To determine the effects of fetuin-B on cytokine production of RAW264.7 cells, flow cytometry analysis was performed using a FACSCaliberTM flow cytometer (BD Biosciences). A detailed protocol is described in the Supplementary Expanded Methods.

Animal surgery and immunostaining

Mice (C57BL/6J, n=18) aged 9 weeks were purchased from Orient Bio. All experiments using animals were performed in conformity with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23) and approved by the Animal Subjects Committee and the Institutional Guidelines of Konkuk University, Korea. Animals were divided into three groups and provided with a freely available high-cholesterol and fat-containing diet (D12336, Research Diets, Inc.) throughout the experimental period, starting 1 week before vascular ligation. The first group (n=6) was subjected only to carotid artery ligation; the second group (n=6) was subjected to both carotid artery vascular ligation and cuff placement; the third group (n=6) was subjected to vascular ligation, cuff placement and fetuin-B treatment–injected intraperitoneally at a concentration of 4 μg/kg every 2 days, starting 1 week before cuff placement, and at a concentration of 4 μg/kg per day for 4 days after cuff placement. Detailed protocols are described in the Supplementary Expanded Methods.

Statistical analysis

The experimental results are expressed as means±S.E.M.s. ANOVA was used for multiple comparisons (GraphPad Prism, version 5.0). If there was a significant variation between treated groups, Dunnett's test was applied. The data were considered significant when P<0.05.

RESULTS

Identification of proteins altered in sera from AMI or SA patients

The sera were collected from 32 patients with AMI accompanied by unstable plaque (mean age 60.7±12.9 years) and 15 SA patients (mean age 60.2±12.4 years), who were enrolled according to each inclusion criterion of AMI and SA. To increase the sensitivity for detection of low-abundant proteins in serum samples, the six most abundant plasma proteins (albumin, IgG, IgA, haptoglobin, transferrin and antitrypsin) were specifically removed from each sample by affinity chromatography using the Multiple Affinity Removal Column (see Supplementary Figure S1). Silver-stained 2DE gels showed >500 proteins spots (see Supplementary Figure S1C). The statistical differences in protein expression levels between sera from AMI and SA patients were analysed using PDQuest (n=6). Moreover, we identified the six protein spots that revealed significant 1.5-fold differences between two groups in silver-stained 2DE gels (Figure 1). Among these proteins, expression of fibrinogen (γ-A precursor), proapolipoprotein, fetuin-B (fetuin-like protein IRL685) and fibrinogen (γ-B chain precursor) was significantly increased in sera from AMI patients compared with SA patients (Figure 1). In contrast, keratin 9 expression declined in sera from AMI patients (Figure 1). Table 2 shows the properties of the altered proteins identified with an LC-MS/MS analyser, highlighting representative peptide sequences and their sequence coverage, noting both the theoretical isoelectric point (pI) and Mr values, and providing accession numbers from the National Center for Biotechnology Information (NCBI) databases.

Enlargement of representative silver-stained 2DE images of proteins altered in sera from SA and AMI patients

Figure 1
Enlargement of representative silver-stained 2DE images of proteins altered in sera from SA and AMI patients

Proteins extracted from serum were loaded on to non-linear (NL), immobilized pH gradient (IPG) strips (pH 4–7, 17 cm), subjected to isoelectric focusing, and transferred to SDS/10% PAGE. The protein spots were visualized with silver staining and analysed using PDQuest statistical software. Protein spots altered by at least 1.5-fold were identified using LC-MS/MS, as shown in Table 2. The images shown are representative of six independent experiments. Right-hand side: statistical results from the left-hand side. *P<0.05 vs SA control. A, absorbance; Mr, molecular mass.

Figure 1
Enlargement of representative silver-stained 2DE images of proteins altered in sera from SA and AMI patients

Proteins extracted from serum were loaded on to non-linear (NL), immobilized pH gradient (IPG) strips (pH 4–7, 17 cm), subjected to isoelectric focusing, and transferred to SDS/10% PAGE. The protein spots were visualized with silver staining and analysed using PDQuest statistical software. Protein spots altered by at least 1.5-fold were identified using LC-MS/MS, as shown in Table 2. The images shown are representative of six independent experiments. Right-hand side: statistical results from the left-hand side. *P<0.05 vs SA control. A, absorbance; Mr, molecular mass.

Table 2
Identification of differentially expressed serum proteins between AMI and SA patients
No.ChangesProtein namePeptide sequencesScore/Sequence coverage (%)pI/Mr (kDa)Accession no./Database
2202 +3.3 Fibrinogen γ-A precursor K.IIPFNR.L 592/39 5.07/50.0 gi|70906437/NC 
   K.NWIQYK.E    
   R.LDGSVDFK.K    
   K.MLEEIMK.Y    
   K.MLEEIMK.Y + oxidation (M)    
   K.MLEEIMK.Y + oxidation (M)    
   K.MLEEIMK.Y + 2 oxidation (M)    
   R.VELEDWNGR.T    
   R.TSTADYAMFK.V    
   R.TSTADYAMFK.V + oxidation (M)    
   K.QSGLYFIKPLK.A    
   R.TSTADYAMFK.V    
   R.TSTADYAMFK.V + oxidation (M)    
   K.QSGLYFIKPLK.A    
   K.QSGLYFIKPLK.A    
   K.YEASILTHDSSIR.Y    
   R.YLQEIYNSNNQK.I    
   R.LTIGEGQQHHLGGAK.Q    
   K.IHLISTQSAIPYALR.V    
   K.IHLISTQSAIPYALR.V    
   K.ASTPNGYDNGIIWATWK.T    
   K.ASTPNGYDNGIIWATWK.T    
   K.ASTPNGYDNGIIWATWK.T    
   K.EGFGHLSPTGTTEFWLGNEK.I    
   K.EGFGHLSPTGTTEFWLGNEK.I    
   K.AIQLTYNPDESSKPNMIDAATLK.S    
   K.AIQLTYNPDESSKPNMIDAATLK.S + oxidation (M)    
3002 +1.5 Proapolipoprotein K.VSFLSALEEYTK.K 67/4 5.45/28.9 gi|178775/NC 
4202 +1.8 Fetuin-B, fetuin-like protein IRL685 K.DGYVLR.L 250/17 6.87/42.9 gi|6562434/NC 
   K.AIFYMNNPSR.V    
   K.AIFYMNNPSR.V    
   K.QYSLFK.V    
   R.ASSQWVVGPSYFVEYLIK.E    
   R.ASSQWVVGPSYFVEYLIK.E    
   R.GSVQYLPDLDDKNSQEK.G    
   K.LVVLPFPK.E    
4205 +1.8 Fibrinogen γ-B-chain precursor DNCCILDER 2 propionamide (C) 68/3 5.37/71.8 gi|71828/NC 
   VELEDWNGR    
5202 −1.9 Keratin 9 K.VQALEEANNDLENK.I 291/22 5.14/62.1 gi|453155/NC 
   K.NYSPYYNTIDDLKDQIVDLTVGNNK.T    
   K.TLLDIDNTR.M    
   R.MTLDDFR.I    
   R.IKFEMEQNLR.Q    
   R.QGVDADINGLR.Q    
   R.QVLDNLTMEK.S    
   R.QEYEQLIAK.N    
   K.DIENQYETQITQIEHEVSSSGQEVQSSAK.E    
   R.HGVQELEIELQSQLSK.K    
5206 +3.2 Fibrinogen γ-B-chain precursor DNCCILDER 2 propionamide (C) 333/13 5.37/51.4 gi|71828/NC 
   EGFGHLSPTGTTEFWLGNEK    
   IHLISTQSAIPYALR    
   VELEDWNGR    
   IIPFNR    
No.ChangesProtein namePeptide sequencesScore/Sequence coverage (%)pI/Mr (kDa)Accession no./Database
2202 +3.3 Fibrinogen γ-A precursor K.IIPFNR.L 592/39 5.07/50.0 gi|70906437/NC 
   K.NWIQYK.E    
   R.LDGSVDFK.K    
   K.MLEEIMK.Y    
   K.MLEEIMK.Y + oxidation (M)    
   K.MLEEIMK.Y + oxidation (M)    
   K.MLEEIMK.Y + 2 oxidation (M)    
   R.VELEDWNGR.T    
   R.TSTADYAMFK.V    
   R.TSTADYAMFK.V + oxidation (M)    
   K.QSGLYFIKPLK.A    
   R.TSTADYAMFK.V    
   R.TSTADYAMFK.V + oxidation (M)    
   K.QSGLYFIKPLK.A    
   K.QSGLYFIKPLK.A    
   K.YEASILTHDSSIR.Y    
   R.YLQEIYNSNNQK.I    
   R.LTIGEGQQHHLGGAK.Q    
   K.IHLISTQSAIPYALR.V    
   K.IHLISTQSAIPYALR.V    
   K.ASTPNGYDNGIIWATWK.T    
   K.ASTPNGYDNGIIWATWK.T    
   K.ASTPNGYDNGIIWATWK.T    
   K.EGFGHLSPTGTTEFWLGNEK.I    
   K.EGFGHLSPTGTTEFWLGNEK.I    
   K.AIQLTYNPDESSKPNMIDAATLK.S    
   K.AIQLTYNPDESSKPNMIDAATLK.S + oxidation (M)    
3002 +1.5 Proapolipoprotein K.VSFLSALEEYTK.K 67/4 5.45/28.9 gi|178775/NC 
4202 +1.8 Fetuin-B, fetuin-like protein IRL685 K.DGYVLR.L 250/17 6.87/42.9 gi|6562434/NC 
   K.AIFYMNNPSR.V    
   K.AIFYMNNPSR.V    
   K.QYSLFK.V    
   R.ASSQWVVGPSYFVEYLIK.E    
   R.ASSQWVVGPSYFVEYLIK.E    
   R.GSVQYLPDLDDKNSQEK.G    
   K.LVVLPFPK.E    
4205 +1.8 Fibrinogen γ-B-chain precursor DNCCILDER 2 propionamide (C) 68/3 5.37/71.8 gi|71828/NC 
   VELEDWNGR    
5202 −1.9 Keratin 9 K.VQALEEANNDLENK.I 291/22 5.14/62.1 gi|453155/NC 
   K.NYSPYYNTIDDLKDQIVDLTVGNNK.T    
   K.TLLDIDNTR.M    
   R.MTLDDFR.I    
   R.IKFEMEQNLR.Q    
   R.QGVDADINGLR.Q    
   R.QVLDNLTMEK.S    
   R.QEYEQLIAK.N    
   K.DIENQYETQITQIEHEVSSSGQEVQSSAK.E    
   R.HGVQELEIELQSQLSK.K    
5206 +3.2 Fibrinogen γ-B-chain precursor DNCCILDER 2 propionamide (C) 333/13 5.37/51.4 gi|71828/NC 
   EGFGHLSPTGTTEFWLGNEK    
   IHLISTQSAIPYALR    
   VELEDWNGR    
   IIPFNR    

Validation of fetuin expression in sera from AMI or SA patients

Among the altered proteins, fetuin-B is known to have a structural similarity to fetuin-A, which plays a potent role as a cardiovascular risk factor [21,22]. We thus selected fetuin-B as a target protein and determined the expression using Western blot analysis to validate the distribution of fetuin-B in sera from patients with AMI. As shown in Figure 2(A), the expression level of fetuin-B was significantly increased in patients with AMI compared with those with SA. In contrast, fetuin-A did not show a difference in expression between AMI and SA patients (Figure 2B).

Validation of the expression of fetuin proteins in sera from patients with AMI or SA

Figure 2
Validation of the expression of fetuin proteins in sera from patients with AMI or SA

Western blot analysis was performed with sera from AMI and SA patients. Upper panels: representative images of the expression of (A) fetuin-B and (B) fetuin-A. GAPDH served as a loading control. Lower panels: the statistical data from the upper panels were quantified with densitometry, expressed as means±S.E.M.s (n=7). **P<0.01 vs SA.

Figure 2
Validation of the expression of fetuin proteins in sera from patients with AMI or SA

Western blot analysis was performed with sera from AMI and SA patients. Upper panels: representative images of the expression of (A) fetuin-B and (B) fetuin-A. GAPDH served as a loading control. Lower panels: the statistical data from the upper panels were quantified with densitometry, expressed as means±S.E.M.s (n=7). **P<0.01 vs SA.

Effects of recombinant human fetuin protein on functions in immune cells

Migratory responses in immune cells play a crucial role in the development of atherosclerotic plaque, thereby making the plaque more vulnerable and susceptible to rupture [23,24]. Therefore, to determine the possible involvement of fetuin-B in the development of atherosclerosis, particularly in plaque formation, we first carried out monocyte migration analysis using U937 cells. Treatment with recombinant human fetuin-B significantly increased U937 cell migration in a concentration-dependent manner (1–50 ng/ml); these effects started at 1 ng/ml and reached a maximum at 50 ng/ml (Figure 3A). We also investigated whether fetuin-B enhances the activation of MMP-2 in monocytes. As shown in Figure 3B, fetuin-B (50 ng/ml) significantly elevated the activation of MMP-2 in U937 monocytes. It is widely known that the stimulation of TNF-α or LPS leads to increased activation of MMP-2, and this up-regulation of MMP-2 is linked to increased cell migration [25,26]. Therefore, we also determined the effects of fetuin-B on TNF-α- and LPS-induced MMP-2 activation in U937 cells. Treatment with TNF-α (20 ng/ml) and LPS (100 ng/ml) increased the activation of MMP-2, but these enhancements were not affected by treatment with fetuin-B (Figure 3B).

Effects of fetuin-B on migration and MMP-2 activation in U937 monocytes

Figure 3
Effects of fetuin-B on migration and MMP-2 activation in U937 monocytes

(A) The effect of fetuin-B on U937 monocyte migration. U937 cell migrations were examined using the Neuroprobe ChemoTx system, as described under Materials and Methods. The cells (1×106 cells/ml) were loaded in the upper migration chamber with a conditioned medium, and fetuin-B (1–50 ng/ml) was added to the bottom components of the migration chamber. Migrated cells were determined using CellTiter-Glo Luminescent Cell Viability Assay. The statistical results are expressed as means±S.E.M.s of seven independent experiments. *P<0.05 and **P<0.01 vs non-treated control. RLU, relative light unit. (B) The effect of fetuin-B on the activation of MMP-2 in U937 monocytes. Confluent cells were pretreated in the presence or absence of 50 ng/ml of fetuin-B in serum-free medium for 1 h and stimulated with 20 ng/ml of TNF-α or 100 ng/ml of LPS for an additional 24 h. Band intensity was quantified with densitometry. Active MMP-2 expression of the non-treated control is represented as 100%. The statistical results are expressed as means±S.E.M.s (n=5). **P<0.01 vs non-treated control in the absence of fetuin-B.

Figure 3
Effects of fetuin-B on migration and MMP-2 activation in U937 monocytes

(A) The effect of fetuin-B on U937 monocyte migration. U937 cell migrations were examined using the Neuroprobe ChemoTx system, as described under Materials and Methods. The cells (1×106 cells/ml) were loaded in the upper migration chamber with a conditioned medium, and fetuin-B (1–50 ng/ml) was added to the bottom components of the migration chamber. Migrated cells were determined using CellTiter-Glo Luminescent Cell Viability Assay. The statistical results are expressed as means±S.E.M.s of seven independent experiments. *P<0.05 and **P<0.01 vs non-treated control. RLU, relative light unit. (B) The effect of fetuin-B on the activation of MMP-2 in U937 monocytes. Confluent cells were pretreated in the presence or absence of 50 ng/ml of fetuin-B in serum-free medium for 1 h and stimulated with 20 ng/ml of TNF-α or 100 ng/ml of LPS for an additional 24 h. Band intensity was quantified with densitometry. Active MMP-2 expression of the non-treated control is represented as 100%. The statistical results are expressed as means±S.E.M.s (n=5). **P<0.01 vs non-treated control in the absence of fetuin-B.

In addition to monocytes, we tested whether fetuin-B affects functions of macrophages. Migration in RAW264.7 macrophages was increased by treatment with fetuin-B (1–50 ng/ml) and the migration level showed a dose dependency (Figure 4A). Treatment with 50 ng/ml of fetuin-B results in significant lipid accumulation in RAW264.7 cells (Figure 4B). Moreover, proinflammatory cytokine production was determined in macrophages using flow cytometry. As shown in Figure 4(C), the proportion of TNF-α-secreting RAW264.7 cells was significantly increased by treatment with 50 ng/ml of fetuin-B compared with the control group. An IL-6-secreting cell population was also higher in the group treated with 50 ng/ml of fetuin-B than in the untreated control group (Figure 4C).

Effects of fetuin-B on RAW264.7 cell responses

Figure 4
Effects of fetuin-B on RAW264.7 cell responses

(A) The effect of fetuin-B on RAW264.7 cell migration. Migration of RAW264.7 cells was tested using the Neuroprobe ChemoTx system. The cells (1×106 cells/ml) were loaded in the upper migration chamber and 1–50 ng/ml fetuin-B was added to the bottom chamber. Migrated cells were determined using CellTiter-Glo Luminescent Cell Viability Assay. *P<0.05 vs non-treated control (n=7). RLU, relative light unit. (B) Representative photomicrographs of lipid-laden RAW264.7 cells. RAW264.7 macrophages were exposed to 50 ng/ml of fetuin-B and incubated for 72 h at 37°C. Lipids in the cells were stained with Oil Red O. The counting of the staining against the nucleus was performed using haematoxylin. Arrows indicate Oil Red O-positive responses in the cells. Scale bar: 100 μm. (C) Production of TNF-α and IL-6 in RAW264.7 cells. The RAW264.7 cells were exposed to 50 ng/ml of fetuin-B for 72 h at 37°C, and then stained using anti-TNF-α and anti-IL-6 antibodies. The expression of TNF-α and IL-6 in the cells was observed using a FACSCaliber flow cytometer. Flow cytometry was performed using FlowJO software. The graphs on the right represent the statistical data obtained from the left panels (n=6). **P<0.01 vs fetuin B-non-treated cells.

Figure 4
Effects of fetuin-B on RAW264.7 cell responses

(A) The effect of fetuin-B on RAW264.7 cell migration. Migration of RAW264.7 cells was tested using the Neuroprobe ChemoTx system. The cells (1×106 cells/ml) were loaded in the upper migration chamber and 1–50 ng/ml fetuin-B was added to the bottom chamber. Migrated cells were determined using CellTiter-Glo Luminescent Cell Viability Assay. *P<0.05 vs non-treated control (n=7). RLU, relative light unit. (B) Representative photomicrographs of lipid-laden RAW264.7 cells. RAW264.7 macrophages were exposed to 50 ng/ml of fetuin-B and incubated for 72 h at 37°C. Lipids in the cells were stained with Oil Red O. The counting of the staining against the nucleus was performed using haematoxylin. Arrows indicate Oil Red O-positive responses in the cells. Scale bar: 100 μm. (C) Production of TNF-α and IL-6 in RAW264.7 cells. The RAW264.7 cells were exposed to 50 ng/ml of fetuin-B for 72 h at 37°C, and then stained using anti-TNF-α and anti-IL-6 antibodies. The expression of TNF-α and IL-6 in the cells was observed using a FACSCaliber flow cytometer. Flow cytometry was performed using FlowJO software. The graphs on the right represent the statistical data obtained from the left panels (n=6). **P<0.01 vs fetuin B-non-treated cells.

Effects of fetuin-B treatment on apoptosis in VSCMs

It is reported that death or diminishment of VSMCs may be involved in weakening the fibrous cap in atherosclerotic plaque, leading to plaque rupture [27]. To determine whether fetuin-B promotes VSMC death, we examined its effects on apoptosis induction and expression of its signalling proteins in VSMCs using TUNEL assay and immunoblot analysis, respectively. As shown in Figure 5(A), treatment with 50 ng/ml of fetuin-B induced a TUNEL-positive response in VSMCs. Moreover, fetuin-B increased the expression of cleaved caspase 3 and active MMP-2 in VSMCs (Figure 5B). Fetuin-B (50 ng/ml) also stimulated SDF-1 expression in VSMCs (Figure 5B).

Effects of fetuin-B on VSMC responses

Figure 5
Effects of fetuin-B on VSMC responses

(A) Apoptosis induction of VSMCs in response to fetuin-B. VSMCs isolated from rat aortas were treated with 50 ng/ml of fetuin-B for 24 h, and then stained for 1 h using the TUNEL method. Green colours in the photomicrograph represent the presence of TUNEL-positive nuclei in the cells. The cells were counterstained with DAPI (blue). Scale bar: 50 μm. Lower panel shows the statistical data from the upper panel (n=6). The percentage of apoptotic cells was determined by comparing the number of TUNEL-positive cells with the total number of DAPI-stained cells. Results are expressed as means±S.E.M.s. (B) Expressions of cleaved caspase 3, MMP-2 and SDF-1 in fetuin B-treated VSMCs. Cells were cultured without or with 50 ng/ml of fetuin-B for 24 h or 48 h, and the lysates were immunoblotted with the indicated antibodies. Each graph was obtained from the results in the upper panels (n=6). The response in the cells under the quiescent state was considered to be 100%. *P<0.05 and **P<0.01 vs untreated cells.

Figure 5
Effects of fetuin-B on VSMC responses

(A) Apoptosis induction of VSMCs in response to fetuin-B. VSMCs isolated from rat aortas were treated with 50 ng/ml of fetuin-B for 24 h, and then stained for 1 h using the TUNEL method. Green colours in the photomicrograph represent the presence of TUNEL-positive nuclei in the cells. The cells were counterstained with DAPI (blue). Scale bar: 50 μm. Lower panel shows the statistical data from the upper panel (n=6). The percentage of apoptotic cells was determined by comparing the number of TUNEL-positive cells with the total number of DAPI-stained cells. Results are expressed as means±S.E.M.s. (B) Expressions of cleaved caspase 3, MMP-2 and SDF-1 in fetuin B-treated VSMCs. Cells were cultured without or with 50 ng/ml of fetuin-B for 24 h or 48 h, and the lysates were immunoblotted with the indicated antibodies. Each graph was obtained from the results in the upper panels (n=6). The response in the cells under the quiescent state was considered to be 100%. *P<0.05 and **P<0.01 vs untreated cells.

Effects of in vivo administration of fetuin-B on plaque stability in mice

In addition, we confirmed fetuin-B expression in vascular neointima formed by carotid artery ligation. As shown in Supplementary Figure S2, the expression of fetuin-B was enhanced in the neointima layer compared with the media layer after carotid artery ligation. On the basis of findings in immune cells and VSMCs, we further tested the effect of fetuin-B administration on plaque stability using an animal model for atherosclerotic plaque rupture induced by mixed modification of previous methods [2830]. The cuff placement in ligated arteries caused atherosclerotic plaque rupture and this rupture was increased by treatment with 4 μg/kg of fetuin-B (Figure 6). In addition, we also observed potential components in the plaques that can affect atherosclerotic plaque stability. As shown in Figure 6, the expression of α-SMA and collagen were attenuated in the plaques of ligated arteries of the cuff-placed group compared with the ligation-alone group. Such expression in the plaques of ligated arteries of the cuff-placed group was observed more weakly by administration of 4 μg/kg of fetuin-B. On the other hand, CD68-positive macrophages were increased by cuff placement in the plaque of ligated arteries, and this response was elevated more strongly by administration of fetuin-B (Figure 6).

Representative images showing atherosclerotic plaques after fetuin-B administration

Figure 6
Representative images showing atherosclerotic plaques after fetuin-B administration

Mice were subjected to ligation of the bilateral carotid arteries. After 21 days, a polyethylene cuff was placed on the common carotid artery for 4 days, as described under Materials and Methods. Fetuin-B treatment was performed by intraperitoneal injection at a concentration of 4 μg/kg every 2 days, starting 1 week before cuff placement, and at a concentration of 4 μg/kg per day for 4 days after cuff placement. For each staining test, the histological cross-section of left ligated carotid arteries was performed at 4 days after cuff placement. The sections were treated with antibodies overnight and then stained with Alexa 488-conjugated antibody (n=6). Each antibody-positive response is shown in green. Some sections were stained with haematoxylin and eosin for analysis of the plaque features (n=6). Scale bar: 100 μm.

Figure 6
Representative images showing atherosclerotic plaques after fetuin-B administration

Mice were subjected to ligation of the bilateral carotid arteries. After 21 days, a polyethylene cuff was placed on the common carotid artery for 4 days, as described under Materials and Methods. Fetuin-B treatment was performed by intraperitoneal injection at a concentration of 4 μg/kg every 2 days, starting 1 week before cuff placement, and at a concentration of 4 μg/kg per day for 4 days after cuff placement. For each staining test, the histological cross-section of left ligated carotid arteries was performed at 4 days after cuff placement. The sections were treated with antibodies overnight and then stained with Alexa 488-conjugated antibody (n=6). Each antibody-positive response is shown in green. Some sections were stained with haematoxylin and eosin for analysis of the plaque features (n=6). Scale bar: 100 μm.

DISCUSSION

In the present study, we found that six proteins showed altered expression in sera from patients with AMI compared with patients with SA. Among these proteins, fetuin-B is reported to be similar in structure to fetuin-A, a known cardiovascular risk factor [21]. In the present study, there was a significantly higher level of fetuin-B in patients with AMI than in those with SA, implying that this protein may be associated with atherosclerotic plaque vulnerability. Atherosclerotic plaque rupture is considered to be a key event in the pathogenesis of ACS [31]. The severity of the culprit lesion or thrombus formation after plaque rupture may be responsible for the development of ACS [32]. Results obtained using ECG and serum biomarkers often failed to identify the disease in outpatients and high-risk patients. Thus, it is important to find new target molecules that can be used to identify patients with AMI [33]. Moreover, finding a regulator capable of controlling the vulnerable plaque would be of great clinical benefit, because it would enable patients at high risk of AMI to be diagnosed early or to be treated. Therefore, the regulation of this molecule may contribute to the pathophysiology of AMI and overcoming it clinically.

Fetuins, also known as α2-Heremans–Schmid glycoproteins, are serum proteins with diverse functions including the regulation of osteogenesis, systemic inflammation and mineralization [22,34,35]. Recent investigations have focused on the potential role of fetuin, especially fetuin-A, as a non-traditional cardiovascular risk factor [21]. Serum fetuin-A is known as a circulating inhibitor of calcification which is inversely related to valvular calcification [36,37]; however, no significant association was identified between serum fetuin-A and aortic stiffness in dialysis patients [38]. Calcification occurs at sites of atherosclerotic plaques, where there is a combination of cellular necrosis, inflammation and cholesterol deposition [39]. Plaque calcification initially destabilizes a plaque that is prone to rupture [40,41]. However, evidence indicates that calcified plaque may be more stable and, thus, less prone to rupture than non-calcified lesions [40]. Huang et al. [42] demonstrated that calcium provides the stability of the fibrous cap. In addition, Beckman et al. [43] suggested that calcification of the responsible culprit lesions is reduced for AMI compared with lesions associated with SA. Thus, plaque that is less calcified may be more unstable and more prone to rupture than calcified plaque [41].

Although possible roles of fetuin-A in plaque calcification have been suggested, the physiological and pathophysiological roles of fetuin-B in the stability of plaques have not yet been reported. In the present study, fetuin-B expression was enhanced in the vascular neointima of mice subjected to carotid artery ligation, implying a potential role in artery plaque formation. Moreover, in vivo administration of fetuin-B caused a stronger artery plaque rupture induced by cuff placement on the ligated carotid artery of mice, and the plaque with rupture showed a diminishment of VSMC contents and collagen formation. Moreover, fetuin-B induced apoptosis and increased cleaved caspase 3 in VSMCs. These findings are similar to events known to weaken the stability of the plaque [27]. As it is known that VSMCs are probably involved in the maintenance of plaque stability [5], we suggest a possibility that fetuin-B may contribute to atherosclerotic plaque stability, probably by inhibition of VSMC populations and collagen formation. To the best of our knowledge, our data may be the first indication that fetuin-B could be a molecule involved in plaque formation and rupture.

The infiltration of immune cells, including monocytes, macrophages, activated T-cells and mast cells, is a process essential to the development of atherosclerosis and its stability. Monocytes contribute directly to atherosclerosis as effectors of innate immunity. MMPs are involved in the monocyte functions [44] and participate in the destabilization of atherosclerotic plaques [45,46]. Moreover, MMPs can degrade extracellular matrix (ECM) proteins and are implicated in the dysfunction of connective tissue and remodelling of atherosclerotic lesions [47,48]. MMP-2, a member of the MMP family, plays an important role in the regulation of angiogenesis and vascular permeability, because it degrades and breaches ECM proteins surrounding each cell and the internal elastic lamina [49,50]. MMP-2 has also been associated with cap thickness in atherosclerotic plaque and consequently promotes the rupture [51,52]. Several studies have demonstrated the expression of MMP-2 in atherosclerotic plaques [53,54]. Diabetes and hypertension, the main risk factors of atherosclerosis, have been associated with MMP-2 [54,55]. In the present study, we found that fetuin-B, TNF-α or LPS elevated the activation of MMP-2 in monocytes, but a co-operative effect of fetuin-B and TNF-α or LPS was not observed. We also demonstrated that treatment with fetuin-B increased SDF-1 expression in VSMCs and migration of monocytes and macrophages, implying activation of monocytes and macrophages by fetuin-B. Previously, we showed that SDF-1 stimulated the action of T-cells via the epigenetic regulation of C-X-C chemokine receptor type 4 (CXCR4) receptor expression [56]. Moreover, flow cytometry analysis revealed that fetuin-B increased the deposition of lipid and production of the proinflammatory cytokines, TNF-α and IL-6, in macrophages which may be involved in plaque stability [57,58]. Actually, the present study showed that fetuin-B increased MMP-2 activation in monocytes and VSMCs, so it is possible that fetuin-B could stimulate the infiltration of immune cells in damaged intima and consequently stimulate vascular cells that potently influence plaque destabilization.

In summary, we identified six proteins that were altered in sera from patients with AMI compared with SA, by using proteomic analysis. Among these proteins, fetuin-B was one of the up-regulated proteins in the AMI group, as confirmed by Western blot analysis. Moreover, treatment with this protein increased migration in monocytes and macrophages. Fetuin-B also affected vascular plaque-stabilizing factors, including the deposition of lipid and production of cytokines in macrophages, the activation of MMP-2 in monocytes, and the activation of apoptosis and MMP-2 in VSMCs. Moreover, in vivo administration of fetuin-B decreased cuff placement-induced vascular plaque stability. The results from the present study demonstrate that fetuin-B may be a possible contributor to AMI. Therefore, we suggest that fetuin-B may be useful as a candidate target molecule for the treatment of unstable plaque or a potential diagnostic marker of plaque instability.

AUTHOR CONTRIBUTION

Seung Hyo Jung, KyungJong Won and Bokyung Kim conceived and designed this research; Seung Hyo Jung, HyunJoong Kim, Kang Pa Lee, Hwan Myung Lee, EunHye Seo and Eun Seok Park were involved with data acquisition; KyungJong Won, HyunJoong Kim, EunHye Seo, Seung Hyun Lee and Bokyung Kim analysed the data; KyungJong Won, Seung Hyun Lee and Bokyung Kim interpreted the data; KyungJong Won, Seung Hyun Lee and Bokyung Kim drafted the manuscript; Seung Hyun Lee, HyunJoong Kim, Kang Pa Lee and Bokyung Kim prepared the figures; and Seung Hyun Lee, KyungJong Won, Kang Pa Lee, HyunJoong Kim, EunHye Seo, Hwan Myung Lee, Eun Seok Park, Seung Hyun Lee and Bokyung Kim approved the final version of manuscript.

FUNDING

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea, NRF, funded by the Ministry of Education, Science and Technology (2011-0029583) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (A090812).

Abbreviations

     
  • 2DE

    2D electrophoresis

  •  
  • ACS

    acute coronary syndrome

  •  
  • AMI

    acute myocardial infarction

  •  
  • CAD

    coronary artery disease

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • ECG

    electrocardiograph

  •  
  • ECM

    extracellular matrix

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • IL

    interleukin

  •  
  • LPS

    lipopolysaccharide

  •  
  • MMP

    matrix metalloproteinase

  •  
  • ppm

    parts per million

  •  
  • SA

    stable angina

  •  
  • SDF-1

    stromal cell-derived factor-1

  •  
  • SMA

    α-smooth muscle actin

  •  
  • TNF-α

    tumour necrosis factor α

  •  
  • VSMC

    vascular smooth muscle cell

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Author notes

1

These authors contributed equally to this work.

Supplementary data