Introduction

Inflammation plays a central role throughout the entire atherosclerotic process and monocytes are the primary inflammatory cell type that infiltrates early atherosclerotic plaques [1, 2]. Their recruitment into plaques drives disease progression. It has been recently shown that a population of monocytes/macrophages emigrates from atherosclerotic lesions and re-enters into the blood [1, 2]. Thus, monocytes may serve as reporters and play a pivotal role in providing information which can directly state the condition of disease. Since monocytes are essential cells that participate throughout the atherosclerotic process, analytical approaches such as proteomics can be a useful strategy for investigating their role in atherosclerosis.

Proteomics is a powerful and expanding field of investigation and is being explored for identification of proteins that actively participate in pathophysiological processes and/or novel protein candidates that can potentially serve as useful clinical biomarkers of disease [3–5]. A few recent studies have reported the proteome of monocytes in basal and under pro-inflammatory conditions [6–10]. However, studies on differential expression of monocyte proteome in coronary artery disease (CAD) are lacking. Thus, a comparative protein profiling of monocytes derived from CAD patients with and healthy controls can provide information reflecting the role of monocytes in the atherosclerotic process. In the present study, we have performed proteomic analysis of circulating monocytes from CAD patients to identify proteins potentially involved in atherosclerotic processes and which could serve as individual markers or define a characteristic profile of CAD.

Materials and methods

Subject selection

Twenty angiographically confirmed male, CAD patients (≥50% stenosis) attending cardiology clinic of Nehru Hospital, PGIMER, Chandigarh, and who were not receiving any lipid lowering therapy were enrolled in the study. The exclusion criteria were previous history of stroke, renal diseases, diabetes mellitus, inflammatory diseases or cancer. Twenty unrelated, age and sex matched healthy individuals were enrolled as controls. The inclusion criteria consisted of normal clinical examination, no family history of ischemic heart disease, normal ECG and negative stress test. The subjects with abnormal ECG or coronary angiogram or positive stress test, hypertension, diabetes mellitus, dyslipidemia, renal diseases, chronic liver diseases, inflammatory or neoplasic diseases were excluded from control group. The study was approved by Institute ethics committee’s and informed consent was obtained from all participating individuals.

Monocyte isolation

20 ml of blood (overnight fasting) was collected in EDTA coated vials from each subject and monocytes were isolated by density gradient centrifugation in Ficoll–Histopaque. A band corresponding to peripheral blood mononuclear cells (PBMCs) was recovered at the interface over Ficoll, and washed three times with PBS (pH 7.2). Monocytes were isolated by passing the PBMCs through a magnetic cell separation system (AutoMACS; Miltenyi Biotec). The purity of the monocytes was evaluated by flow cytometry. The purified monocytes were washed with equal volume of sucrose (350 mM) and centrifuged at 2500 rpm for 20 min at 4°C and the pellet was suspended in lysis buffer containing protease inhibitors cocktail. The cells were lysed by sonication at 20% power, five cycles for 30 s each. Finally, the sample was treated with 2D clean-up kit (Bio-Rad, USA) to remove salts and contaminants. Protein concentration of the samples was estimated by Bradford protein estimation method [11].

Two-dimensional electrophoresis (2-DE)

An individual gel was run for each subject in this study. Monocyte lysate (150 μg protein) was diluted in rehydration solution and used for the first dimension of 2-DE (isoelectric focusing) on IEF IPG gel strips (equilibrated strips (IPGs), pH 3–10 and pH 4–7 range; Bio-Rad). The IEF was developed horizontally in a protean IEF cell system (Bio-Rad). After completing IEF, the strips were equilibrated in equilibration buffer I (6 M urea, 0.375 mM Tris–HCl, pH 8.8, 2% SDS, 20% glycerol, and 2% DTT w/v) and equilibration buffer II (6 M urea, 0.375 mM Tris–HCl, pH 8.8, 2% SDS, 20% glycerol, and 2.5% iodoacetamide). Second dimension of 2-DE was performed by applying the IPGs onto 12% polyacrylamide gels in the presence of SDS. The gels were fixed in 5% v/v acetic acid, 30% v/v ethanol, and silver stained. IPG strip (pH 4–7) showed optimal resolution and was used for all subsequent experiments.

Image analysis of 2D gels

The gel images were analyzed using PDQuest 2DE gel analysis software (Bio-Rad version 7.1) to identify the protein spots. The position of the spots was detected with the same software, followed by characterization with respect to their pI and apparent molecular weight (MW). The intensity level of each spot (protein) in the gel was standardized reporting it as the ratio to the total intensity of all the spots present in the gel. Further, this software normalizes each gel to “housekeeping proteins”, filters out background noise, and creates virtual gels using ideal Gaussian representations of experimental gels. At each stage, PDQuest shows the spot of interest on all of the gels. During the analysis, the software identifies the protein spots and subsequently the two gel images of our interest are compared by superimposing each other. Later the software provides the number of matching protein spots and unmatched protein spots. Each gel was thoroughly matched and verified, identified by two or three observers blinded for the study. Every protein spot matched, software provides the expression of proteins spots keeping control as a reference gel. Based on the proteins spots which were fivefold significantly changed in their expression levels were further selected for identification. The coordinates were expressed in relation to a reference point in each 2-DE gel. The mean values and coefficients of variation of the differentiated spots (as expression or presence/absence) were calculated using the same software. Spots between patient and control groups were compared using Student’s t test (statistical software packages SPSS 10.0.). Statistical significance was accepted when P < 0.05 (two-tailed).

In-gel digestion of proteins and sample preparation for mass spectrometric analysis

Protein spots were excised manually, destained, and digested. Briefly, gel plugs were rinsed with 200 mM ammonium bicarbonate dehydrated with 50 μl acetonitrile, and vacuum dried for 30 min. Modified porcine trypsin (sequencing grade; Promega, Madison WI, USA) at a final concentration of 16 ng/ml in 50 mM ammonium bicarbonate was added to the dry gel pieces and the digestion proceeded at 37°C for 6 h. The reaction was stopped by adding 0.5% TFA for peptide extraction. Trypsin digest solution was vacuum dried, saturated with equal volume of α-cyano-4-hydroxycinnamic acid (HCCA) in solution of 100% acetonitrile and 0.1% TFA (1:2, v/v) and subjected directly on the matrix plate and air dried for 20 min. This was followed by peptide analysis of these spots by MALDI-MS.

MALDI-MS measurements were performed in the reflector mode for positive ion detection at an acceleration voltage of 25 kV and delay of 50 ns. Mass spectra were calibrated using peptide calibration standard mixture (Bruker, Germany) and peptide masses were measured as monoisotropic masses. The resulting spectra were processed using Flex analysis software (Bruker, Germany) and the peaks for autolytic fragments of trypsin, keratin, and sodium and potassium adduct peaks were removed from the spectra. Spectra were processed and analyzed in Bio-Tools software (Bruker, Germany). Peak lists were entered into MASCOT search engine database and searched for peptide mass fingerprints in a protein database of Humans generated by the annotation of Homo sapiens sequence information available at the Swiss Prot and Expasy databases. The search was allowed for further modifications such as alkylation of cysteine during the tryptic digest procedure and the possible formation of methionine sulfoxide. No restrictions were placed on the species of origin of the protein, and no variable modifications were allowed. A MOWSE score of greater than 51 was considered significant (P < 0.05).

Statistical analysis

The results are expressed as mean ± standard deviation. For the comparison of 2-DE gels from each group, spots were tested automatically with Student’s t test by PDQuest 2-DE gel statistical analysis software (Bio-Rad, version 6.2). The software compared the CAD group with control group. The values of protein spots whose expression was found to be significantly different between two groups were subsequently analyzed using appropriate test with statistical package SPSS 14.0.

Results

Baseline characteristics of the subjects are shown in Table 1.

Table 1 Demographic profile of CAD patients and controls
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Protein profile of circulating monocytes

Approximately 350 protein spots were observed per gel. Reproducibility was tested by comparison of the variation within the different gels in the same group. A greater than 50% of all protein spots on each gel were successfully matched to a respective protein spot on the reference gel. Figure 1 shows representative 2D gel profile of a CAD patient and a control subject. We observed altered expression of 63 proteins spots in the circulating monocytes from CAD patients. 13 spots showed fivefold and 31 spots exhibited threefold increased expression in CAD group as compared to control group, respectively. Two protein spots showed fivefold and 17 protein spots showed threefold reduced expression in CAD patients as compared to controls (Table 2). Protein spots with fivefold changed expression between CAD patients and controls were selected for protein identification analysis by MALDI-TOF. The protein spots with their SSP number selected for identification by MALDI-TOF are given in Supplementary material.

Fig. 1
figure 1

Representative 2DGE gel images of a control and b patient samples. Monocyte lysate (200 μg of protein) was applied to immobilized pH gradient strips (pH 4–7 and 7 cm long), subjected to IEF and separated on 12% SDS-PAGE followed by silver staining. The selected proteins for further investigation are represented with their SSP number provided by the software

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Table 2 Comparative protein profile of monocytes in CAD patients and controls
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Protein identification by MALDI-TOF

Through MALDI-MS, we could successfully identify five proteins, as only these five proteins showed appropriate MASCOT score and sequence coverage. Table 3 shows the theoretical Mr and pI, the access key, the sequence coverage according to PMF, of the identified proteins. The differentially expressed proteins were identified as follows: glutathione S-transferase (GST), mannose binding receptor protein, vimentin, heat Shock protein-70 (HSP-70), and S100 calcium-binding protein A8. Expression of protein spots corresponding to vimentin, mannose binding receptor protein, and S100 calcium-binding protein A8 was significantly increased and that of GST proteins and HSP-70 was significantly decreased in CAD patients as compared to controls. The proteins identified are represented in Fig. 1. The experiments were performed in triplicates for identification of protein spots.

Table 3 Differentially expressed proteins in CAD patients versus controls
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Discussion

Monocytes play a crucial role in the development of atherosclerosis and have been suggested to be involved in all stages of atherosclerotic plaque development. These cells have been proposed as a potential biomarker of disease progression and to be candidates for therapeutic intervention and response to therapy. In this study, we have compared the proteomic profile of circulating monocytes derived from CAD patients with proteomic profile of monocytes from healthy controls. We observed the altered expression of 63 spots in circulating monocytes; out of these 63 spots, 13 protein spots showed fivefold increased expression in monocytes of CAD patients as compared to control group by 2-DGE. We identified five differentially expressed proteins by MALDI. The expression of some of these proteins, such as HSP-70 was recently reported to be normalized in the monocytes of patients with acute coronary syndrome by atorvastatin therapy [10]. These differentially expressed proteins are involved in several pathways which are involved in regulation of lipids, inflammation, and contractile, structural representing multi-factorial nature of monocytes. The putative role of these proteins in the CAD is discussed below.

Glutathione transferase (GST)

We observed significantly decreased GST expression in the monocytes from CAD patients as compared to controls. Barderas et al. [9] have reported the absence of expression of this enzyme in the monocytes of ACS patients. GST is an important antioxidant enzyme and catalyzes the conjugation of glutathione with a variety of reactive electrophilic compounds, thereby neutralizing their active electrophilic sites and subsequently making the parent compound more water soluble. In addition to catalytic functions, the GSTs can also bind covalently/non-covalently to a wide number of hydrophobic compounds, such as heme, drugs, and carcinogens. GST enzymes have been shown to be expressed in human artherosclerotic plaques [12]. Glutathione transferases provide protection against oxidative stress and accompanying cellular processes as evidenced by several experimental studies: for example, transfection of endothelial cells with cDNA of GST A4-4 protects these cells from H2O2-induced apoptosis [13] and Cao and his colleagues have shown that the induction of GST and other antioxidant enzymes in rat smooth muscle cells increased their resistance to oxidative vascular injury [14]. In addition, absence of GST gene has been shown to be associated with increased incidence of stroke and MI in diabetic smokers [15]. Barderas et al. [9] have suggested that absence of GST can be a triggering factor to the progression of the ACS. The observed decreased expression of GST in CAD monocytes as compared to monocytes from healthy subjects, in our study, indicates that deficient GST expression might be associated with increased oxidative stress in the monocytes. Torzewski et al. [16] recently showed that activity of red blood cell GST was inversely associated with the risk of cardiovascular events in patients with CAD.

Vimentin

The cytoskeleton is a structure composed of microtubules, microfilaments, and intermediate filaments. Proteins involved in cellular organization are represented by cytoskeletal proteins; in addition to its known structural role, cytoskeletal proteins regulate important aspects of leukocyte functions such as cell mobility and migration, immunological synapse formation and apoptosis [17]. Vimentin is a cytoskeletal protein and has been shown to be present in cells of the blood vessel walls. It is known to have important role in vessel wall structural stability. Previous studies have demonstrated that vimentin is up-regulated in proliferating fibroblasts and vascular smooth muscle cells [18, 19]. Vimentin provides support for the contractile apparatus [20] and was overexpressed in VSMC’s that comprise human restenotic lesions. Vimentin has been reported to be involved in vascular smooth muscle cell differentiation [21]. This protein is a ubiquitous component of the intermediate filaments that build up the cytoskeleton of almost all eukaryotic cells and expressed during cell differentiation. In this study, we observed increased expression of vimentin protein in monocytes from CAD patients as compared to controls. Barderas et al. [9] ACS patients suggesting that it aggravates triggering of ACS. The expression of vimentin is increased in vessel walls following angioplasty and it has been suggested to play important role in arterial remodeling. Vimentin has been shown to be one of the major endothelial antigens recognized in the sera of patients and antivimentin antibodies were shown to be an independent predictor of transplant associated CAD [22]. Recently, vimentin expression has been shown to be up-regulated in THP-1 human monocytes stimulated by ox-LDL [23]. It was reported that vimentin binds to ox-LDL in macrophages and may play a role in the intracellular processing of this lipoprotein in CVD [24]. Increased expression of vimentin seen by us in CAD further suggests role of this protein in CAD and may reflect an adaptive response of monocytes to increased ox-LDL which is seen in monocytes in CAD. However, the mechanisms involved in CAD etiology need to be further elucidated.

Mannose binding lectin receptor protein (MBL)

We observed increased expression of MBL in monocytes from CAD patients as compared to controls. Keller et al. [25] have also reported that mannose binding protein was associated with increased risk of CAD. This increase in MBL may be due to enhanced inflammation seen in CAD. Inflammation plays a major role in all phases of atherogenesis from plaque initiation to plaque rupture. Several inflammatory markers have been associated with an increased risk of atherosclerotic vascular disease. Recently, evidence has emerged concerning the role of MBL in the development of atherosclerosis. MBL activates the complement system which has been implicated in atherogenesis and was recently shown to be associated with increased cardiovascular risk in patients with advanced atherosclerosis MBL has been shown to be a part of the complement cascade and plays an important role in the first line of defense of the innate immune system against pathogenic microorganisms [25–27]. MBL recognizes sugar patterns on the surface of many pathogens, [28] phospholipids, immune complexes [29], and apoptotic cells [30]. In circulation, MBL forms a complex with MBL-associated serine proteases (MASPs). This complex becomes enzymatically active and activates the classical complement route. MBL binds directly to granulocytes, monocytes, and macrophages, which may stimulate the production of pro-inflammatory cytokines [31]. Since innate immunity has been implicated in atherogenesis, MBL has been suggested to play a role in the formation of atherosclerotic plaque [32]. Indeed, increased deposition of complement iC3b in ruptured and vulnerable plaques suggests a role for complement activation in acute coronary syndromes [33]. Furthermore, endothelial oxidative stress, which plays a major role in atherogenesis, activates complement via the lectin complement pathway in human cell cultures. Increased MBL expression in CAD monocytes thus indicates interplay of inflammation and oxidative stress in the development of atherosclerosis and its prominent clinical manifestation in CAD.

Heat shock protein (HSP-70)

We observed decreased expression of HSP-70 protein within monocytes of CAD patients as compared to controls. Barderas et al. [10] have recently reported downregulation or absence of HSP-70 and its constitutive form, HSC-70 in monocytes of ACS patients on conventional therapy patients. Decreased serum HSP-70 levels have been earlier shown to be associated with increased risk of CAD [34, 35]. Shamaei-Tousi et al. [36] have also shown absence of detectable HSP-60 in mononuclear cells of all patients with CAD and suggested that it was due to the leakage of this protein from monocytes into the plasma, as its pro-inflammatory activity remained unexpressed while it is sequestered inside the cells. Thus lower HSP-70 levels seen in monocytes may also be due to leakage of this protein from monocytes. Expression of HSP70 protein is increased in atherosclerotic lesions, which correspond to sites of LDL oxidation and/or of oxidized LDL (ox-LDL) accumulation [37, 38]. In addition, plasma concentrations of HSP-70 were shown to increase in patients with CVD, including atherosclerosis. Treatment of macrophages with ox-LDL significantly increased HSP-70 concentrations in culture supernatants, which could induce pro-inflammatory cytokines such as IL-1 and IL-12 secretion in naive macrophages [39]. HSP-70 protects endothelial function and is involved in anti-apoptotic mechanisms preserving the myocardium from hypoxia [34, 35]. Thus, the decreased expression of HSP-70 in CAD patients in the present study could enhance apoptotic death of macrophages, favoring the atherogenic process. In addition, future studies are required to assess of HSP-70 proteins expression level in CVD patients.

S100A8 calcium-binding protein

We observed increased expression of S100A8 calcium-binding protein in monocytes of CAD patients as compared to controls. S100 are a family of calcium-binding proteins that have been shown to be biomarkers for diseases like stroke and other brain injuries [40, 41]. They are receptor ligands for advanced glycation end products, which are important mediators of vascular injury, and shown to accelerate atherogenesis in animal models [42]. S100A8 is expressed constitutively in high concentrations by granulocytes and during early differentiation stages of monocytes [42]. It is involved in regulating the phosphorylation, NADPH-oxidase activity, and fatty acid transport in monocytes and neutrophils [43]. This protein also has chemo attractant properties and has been related to inflammatory conditions, including atherosclerosis [42]. It was shown to be present in the macrophages of the human atheroma [43]. These observations suggest this protein to be very pro-atherogenic; thus increased expression of this protein could lead to increased generation of free radicals, inflammation, and aberrant lipid transport, thereby contributing to atherogenic processes in CAD. Barderas et al. [9] have reported absence of S100A8 calcium-binding protein in monocytes from ACS patients, whereas it was expressed normally in chronic patients. They suggested that absence of S100 A8 calcium-binding protein expression could be related to ACS triggering. Further studies are needed to elucidate the mechanism of it action in the etiology of CAD.

In summary, our results show altered expression of several proteins in circulating monocytes in patients with stable CAD. The molecular mechanisms which modulate the expression of these proteins and their role in atherosclerosis are not well defined and needs to be investigated further. However, many of these proteins have been shown to be differentially expressed in human monocytes which confirm their functional role in CAD patients [9]. These identified proteins are also attractive candidates as biomarkers involved in the pathogenesis of atherosclerosis and prospective studies examining their plasma levels in CAD patients are needed. Future studies will be carried out to validate these proteins in a larger cohort of CAD patients and to determine the functional role of these proteins in CAD.