Inflammation and ECM (extracellular matrix) remodelling play important roles in LV (left ventricular) remodelling following acute MI (myocardial infarction). Previous studies show elevated plasma MMP (matrix metalloproteinase) levels in patients with acute MI, but their sources are not clear. The recruitment of mononuclear cells into the infarcted myocardium is critical for inflammatory responses, but their exact roles in LV remodelling have not been fully investigated, as it is difficult to isolate and study the function of regional inflammatory cells. To address these questions, we isolated PBMCs (peripheral blood mononuclear cells) from blood samples of patients with acute MI or stable angina, or healthy controls (n=14, 8 and 12 respectively). PBMCs were cultured for 24 h and the MMP9 level in the culture medium was measured by gelatin zymography, and MMP9 gene expression was measured by real-time PCR. Two superarrays (ECM and adhesion molecules, and common cytokines; 84 genes included in each array) were employed to screen gene expression profiles by PBMCs in five patients with acute MI and five controls. We found that MMP9 expression by PBMCs at both the mRNA and protein levels was increased 2-fold (both P<0.05) in patients with acute MI compared with the two control groups. Notably, MMP2 was not expressed by PBMCs. Superarray screening revealed that PBMCs not only expressed MMPs, TIMPs (tissue inhibitors of metalloproteinases) and matrix proteins, but also served as an important source of cell adhesion molecules, inflammatory cytokines and growth factors. A total of 42 genes were differentially expressed in patients with acute MI compared with controls. Expression of selected genes was confirmed by real-time PCR. In conclusion, PBMCs constitute a key cellular source for elevated plasma MMP9, but not for MMP2. PBMCs also contribute to systemic and regional inflammation and matrix remodelling in acute MI.

INTRODUCTION

Acute MI (myocardial infarction) triggers inflammatory responses, including the release of a variety of inflammatory mediators and infiltration of leucocytes into the infarcted myocardium, which is a prerequisite for healing and scar formation [1]. However, enhanced inflammatory response is associated with further myocardial injury and LV (left ventricular) remodelling. Inflammatory cells recruited into the infarcted myocardium have been suggested to be an important source of regional MMPs (matrix metalloproteinases), especially MMP9 [2], which are elevated in infarcted hearts [35]. Studies using genetic manipulation of MMPs/TIMP1 (tissue inhibitor of metalloproteinases 1) or synthetic MMP inhibitors have indicated a causal role for activation of MMPs (which mediates degradation of collagen network) in post-MI remodelling [69]. Therefore inflammation and ECM (extracellular matrix) degradation contribute significantly to post-MI LV remodelling. A number of animal studies have shown beneficial effects of anti-inflammatory strategies in reducing infarct size or attenuating LV remodelling [10,11]. However, in clinical practice, anti-inflammatory therapies are generally unsuccessful [12]. Therefore a more complete understanding of the inflammatory response in acute MI is required before the successful application of inflammation-related interventions.

The recruitment of mononuclear cells into the infarcted myocardium, which is mediated by cell adhesion molecules, is a critical step in the inflammatory response following MI, but the exact roles of mononuclear cells in myocardial injury, infarct healing and ventricular remodelling have not been fully investigated, as it is difficult to isolate and study the function of regional inflammatory cells. Thus it is interesting to study PBMCs (peripheral blood mononuclear cells) infiltrating into the infarcted heart thereby representing local inflammatory cells.

In addition to the up-regulation of regional MMPs in the infarcted heart, plasma levels of several MMPs are elevated in patients with acute MI [1318], but the sources of MMP species in plasma are not very clear. Moreover, two important MMP species (MMP2 and MMP9) display diverse changes in their plasma levels after MI. Although plasma MMP9 is consistently elevated [13,14,17], the changes in plasma MMP2 remain inconsistent [13,14,17,18]. In addition, plasma MMP9 increases early after MI [13,14,17]; however, a delayed increase in plasma MMP2 is observed [13,18]. The reasons for such diversities are not fully understood.

Our previous study has reported that MMP9 gene expression was increased in PBMCs from mice with acute MI compared with those from sham-operated mice, indicating that PBMCs are activated and are an important source of MMP9 in acute MI in mice [4]. In the present study, we aimed to determine whether PBMCs from patients with acute MI also produce higher amount of MMP9 compared with cells from patients with stable angina or healthy controls. We further examined changes in gene expression profiles of pathways on ECM, cell adhesion molecules and common cytokines expressed by PBMCs from patients with acute MI.

MATERIALS AND METHODS

Study subjects

We studied 14 patients with acute MI based on the symptom of persistent chest pain and a plasma level of troponin 2.5 times greater than the laboratory reference value within 48 h after the onset of pain. Aspirin, heparin and nitrates were given to all patients. The use of β-blockers, calcium channel blockers, ACE (angiotensin-converting enzyme)-inhibitors and nitrates was decided by attending physicians. Another eight patients with chronic stable angina and 12 healthy volunteers served as controls. Patients with stable angina complained of angina on effort without evidence of recent deterioration or rest pain in the previous 6 months. The healthy volunteers had no evidence of cardiovascular disease. The present study did not include patients or control subjects with significant renal or hepatic dysfunction, apparent infectious disease or any surgical procedure in the preceding 6 months.

The study complied with the Declaration of Helsinki and was approved by the Ethics Committee of Alfred Hospital. Informed consent was obtained from all subjects.

Blood collection and isolation and culture of PBMCs

Blood samples were obtained from patients with acute MI on day 3 and patients with stable angina and healthy controls. Among the 14 acute MI patients, ten underwent PCI (percutanenous coronary intervention), of whom nine received stents. Blood was taken from nine patients after PCI, and one patient before PCI. PBMCs were isolated with the use of a Ficoll-Paque plus (Amersham Biosciences), according to the manufacturer's instructions. Blood was diluted 1:2 with PBS and was layered on to the top of Ficoll-Paque and centrifuged at 400 g for 30 min at room temperature (21°C). The mononuclear cell layer was carefully collected and rinsed twice with PBS. PBMCs were suspended in RPMI 1640 medium supplemented with L-glutamine and antibiotics/antimyocyocotic, and plated at a density of 2.5×106 cells/ml in 24-well plates and incubated at 37°C in a 5% CO2 atmosphere. After 24 h of incubation, the supernatants were collected and stored at −80°C until assay.

Gelatin zymography

Gelatin zymography was performed using an equal amount of cell supernatants for measurement of gelatinases, MMP2 and MMP9, as described previously [4]. Aliquots (50 μl) of the medium were loaded on to 7.5% acrylamide gels containing 0.5 mg/ml gelatin (Sigma–Aldrich) and were electrophoresed. Following electrophoresis, the gel was washed twice with 0.25% Triton X-100, and then incubated with the incubation buffer containing 50 mM Tris/HCl (pH 7.5), 10 mM CaCl2, 1 mM ZnCl2, 1% Triton X-100 and 0.2 mM NaN3 at 37°C overnight (>16 h). The gel was stained with 0.1% Coomassie Brilliant Blue solution containing 40% isopropanol for 1 h at room temperature and then de-stained with 20% methanol containing 7% acetic acid with shaking until bands were visualized. The gel was scanned, images were inverted and densitometry levels were determined by using the software Quantity One (Bio-Rad Laboratories).

Gene expression of MMP9

RNA was isolated from fresh PBMCs and MMP9 gene expression was measured by real-time PCR. Total RNA was isolated from cells with TRIzol® reagent (Invitrogen) following the manufacturer's instructions. After DNase (Promega) treatment, RNA was reverse-transcribed into first-strand cDNA with the use of random primers and MMLV (Moloney-murine-leukaemia virus) reverse transcriptase (Invitrogen), following the manufacturer's instructions. Real-time quantitative PCR was performed using an SYBR Green kit (Invitrogen). Detection and analysis were performed on an ABI Prism 7500 system (Applied Biosystems). The transcript abundance was expressed as the fold increase over the value of healthy control group calculated using the 2−ΔΔCt method. The expression of targeted genes was normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Superarray

A portion (2 μg) of total RNA isolated from PBMCs was treated with Turbo DNase (Turbo DNA-free kit; Ambion) and reverse-transcribed into cDNA with the use of the RT2 first strand kit (Superarray Bioscience) following the manufacturer's instructions. Two superarrays [human ECM and adhesion molecules (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-013A.html), and human common cytokine (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-021A.html) RT2 Profiler™ PCR Arrays) were selected for the present study. Each array profiles the expression of 84 genes (see Supplementary Tables 1 and 2 at www/clinsci.org/cs/119/cs1190175add.htm). The RT2 Profiler™ PCR Array was set up following the manufacturer's instructions and was performed according to the manufacturer's protocol. An experimental cocktail was prepared for each plate made up of the processed cDNA and 2×instrument-specific and ready-to-use Superarray RT2 qPCR master mix, containing SYBR Green and a reference dye. A portion (25 μl) of the experimental cocktail was placed into each well of the PCR Array plate containing the pre-dispensed gene-specific primer sets, and PCR was performed on the ABI Prism 7500 Sequence Detection System. A two-step cycling program was used (10 min at 95°C to activate the HotStart DNA polymerase, followed by 40 cycles of denaturing for 15 s at 95°C and annealing for 1 min at 60°C). Data collected were entered into online software PCR Array Data Analysis Web Portal provided by the manufacturer for data analysis. Gene expression levels were normalized against the housekeeping genes including GUSB (glucuronidase β), HPRT1 (hypoxanthine guanine phosphoribosyl transferase 1), HSP90AB1 (heat-shock protein α class B member 1), GAPDH and ACTB (β-actin). Fold changes in gene expression were calculated using the 2−ΔΔCt method by the software. On the basis of previous studies [19], a 1.5-fold or greater change was determined to be the threshold cut-off point for what is considered a change in gene expression.

Statistical analysis

Values are expressed as means±S.E.M. One-way ANOVA was used to detect the difference among groups and was followed by the Newman–Keuls multiple comparison post-hoc tests. A difference at P<0.05 was considered as statistically significant.

RESULTS

Baseline characteristics of study subjects

The characteristics of the MI distribution, ST-segment elevation and enzyme profiles in acute MI patients are shown in Table 1. There were no significant differences in age, gender, BMI (body mass index), frequencies of smoking, hypertension, diabetes mellitus, hyperlipidaemia, family history of heart disease, previous history of MI and the number of angiographically significant stenosis between groups of stable angina and acute MI (Table 1).

Table 1
Baseline characteristics of study subjects

Values are means±S.E.M. *P<0.05 compared with control. CK, creatine kinase; ND, not done; NA, not applicable; STEMI/NSTEMI, ST-elevation MI/non-ST-elevation MI.

CharacteristicControl (n=12)Stable angina (n=8)Acute MI (n=14)P value (ANOVA)
Age (years) 56.6±3.3 63.9±3.8 58.4±2.8 0.364 
Gender (n) (male/female) 7/5 7/1 9/5 0.370 
BMI (kg/m224.6±1 30.4±1.3* 29.8±1.1* 0.002 
Smoker (%) 16.7 25 28.6 0.771 
Hypertension (%) 8.3 37.5 42.9 0.133 
Diabetes (%) 8.3 12.5 14.3 0.893 
Hypercholesterolaemia (%) 16.7 75* 71.4* 0.007 
Previous MI (%) NA 25 42.9 0.402 
Family history of heart disease (%) 8.3 50 42.9 0.082 
Stenosed vessels (nNA 1.6±0.4 1.6±0.2  
STEMI/NSTMI (nNA NA 7/7  
Location of MI (nNA NA Anterior, 6; interior, 6; posterior, 2  
Peak troponin (μg/l) ND ND 49.5±19.6  
Peak CK-MB (units/l) ND ND 1665±532.9  
CharacteristicControl (n=12)Stable angina (n=8)Acute MI (n=14)P value (ANOVA)
Age (years) 56.6±3.3 63.9±3.8 58.4±2.8 0.364 
Gender (n) (male/female) 7/5 7/1 9/5 0.370 
BMI (kg/m224.6±1 30.4±1.3* 29.8±1.1* 0.002 
Smoker (%) 16.7 25 28.6 0.771 
Hypertension (%) 8.3 37.5 42.9 0.133 
Diabetes (%) 8.3 12.5 14.3 0.893 
Hypercholesterolaemia (%) 16.7 75* 71.4* 0.007 
Previous MI (%) NA 25 42.9 0.402 
Family history of heart disease (%) 8.3 50 42.9 0.082 
Stenosed vessels (nNA 1.6±0.4 1.6±0.2  
STEMI/NSTMI (nNA NA 7/7  
Location of MI (nNA NA Anterior, 6; interior, 6; posterior, 2  
Peak troponin (μg/l) ND ND 49.5±19.6  
Peak CK-MB (units/l) ND ND 1665±532.9  

Increased MMP9 production by PBMCs at the protein and mRNA levels

As MMP9 plays an important role in post-MI LV remodelling [2,47], we first examined whether PBMCs from patients with acute MI produced a higher amount of MMP9 compared with PBMCs from patients with stable angina or healthy controls. MMP9 activity detected in culture medium of PBMCs was increased by 2-fold in acute MI compared with the control groups (Figure 1B). MMP9 mRNA levels in PBMCs were also increased 2-fold in patients with acute MI compared with the two control groups (Figure 1C). These results indicate that PBMCs are activated in the setting of acute MI to transcribe and secrete more MMP9, forming an important cellular source of elevated level of plasma MMP9 [13,14,17]. In the present study, we did not find any significant difference in MMP9 levels between patients receiving (n=9) or not receiving (n=5) PCI. Interestingly, MMP2 was undetectable in the culture medium of PBMCs from either patients or the control subjects (Figure 1A), and MMP2 mRNA was also not detected by real-time PCR either (results not shown). Thus PBMCs are not a cellular source for MMP2.

MMP protein and gene expression in patients with MI, patients with stable angina and healthy controls

Figure 1
MMP protein and gene expression in patients with MI, patients with stable angina and healthy controls

(A) Representative gelatin zymographic gel performed on the culture medium of PBMCs from patients with acute MI or stable angina (SA), and healthy controls (Ctrl), and (B) quantification of the MMP9 band in these three groups. Bands from the infarct zone (IZ) of a mouse heart served as positive control. (C) Gene expression of MMP9 in PBMCs from the three groups. *P<0.05 compared with healthy controls; #P<0.05 compared with patients with stable angina.

Figure 1
MMP protein and gene expression in patients with MI, patients with stable angina and healthy controls

(A) Representative gelatin zymographic gel performed on the culture medium of PBMCs from patients with acute MI or stable angina (SA), and healthy controls (Ctrl), and (B) quantification of the MMP9 band in these three groups. Bands from the infarct zone (IZ) of a mouse heart served as positive control. (C) Gene expression of MMP9 in PBMCs from the three groups. *P<0.05 compared with healthy controls; #P<0.05 compared with patients with stable angina.

Superarray analysis of ECM and adhesion molecules

Next, we screened gene expression profiles of ECM and adhesion molecules in PBMCs from five patients with acute MI and five healthy controls by superarray analysis (the superarray was not done on patients with stable angina as there were no significant differences between controls and patients with stable angina regarding MMP9 levels by gelatine zymography and real-time PCR). Among 84 genes in the ECM and adhesion molecule array, 18 genes were not detected in PBMCs (Supplementary Table 1). On the basis of the cut-off value of ±1.5-fold relative to control value [19], 13 genes were up-regulated whereas 14 genes were down-regulated (Table 2). These 27 genes were distributed among nine functional gene groups included in this array (Figure 2A). Among the five MMPs expressed by PBMCs, four MMPs (MMP1, MMP8, MMP9 and MMP14) were up-regulated in acute MI (Table 2). MMP9 was expressed most abundantly, indicating further that PBMCs are an important cellular source of MMP9. Three TIMPs (TIMP1, TIMP2 and TIMP3), especially TIMP1 and TIMP2, were expressed by PBMCs, but only TIMP2 showed a small increase in acute MI (Table 2). Thus an imbalance exists between the expression of MMPs and TIMPs by PBMCs in acute MI. PBMCs also express a number of genes encoding matrix proteins. Expression of several genes, such as COL7A1 (collagen 7α1), COL6A1 (collagen 6α1) and COL6A2 (collagen 6α2), LAMA2 (lamina α2) and LAMA3 (lamina α3), FN1 (fibronectin 1), ON (osteonectin) and OPN (osteopontin), were changed in PBMCs from patients with acute MI compared with controls (Table 2). Notably, FN1 was weakly expressed by PBMCs, but markedly increased in acute MI (by ~9-fold). All of these matrix proteins serve as adhesive proteins. PMBCs are also a cellular source of other cell adhesion molecules. PBMCs expressed most integrins which display differential expression patterns. Although the gene expression of ITGAM (integrin αM), ITGB3 (integrin β3) and ITGB5 (integrin β5) was up-regulated, the gene expression of ITGA3 (integrin α3), ITGA6 (integrin α6) and ITGB4 (integrin β4) was down-regulated. In addition, the gene expression of VCAM1 (vascular cell adhesion molecule 1), PECAM1 (platelet/endothelial cell adhesion molecule 1), CDH1 (cadherin 1), VCAN (versican) and CLEC3B (C-type lectin family 3, member B) was also changed in acute MI (Table 2).

Table 2
Changes in ECM and adhesion molecules in PBMCs

ADAMST, ADAM (a disintegrin and metalloproteinase) metalloproteinase with thrombospondin.

(a) Up-regulated genes
Array locationGene nameFold change
B05 COL7A1 1.87 
B07 VCAN 1.74 
C02 FN1 9.07 
D02 ITGAM 1.50 
D06 ITHB3 5.43 
D08 ITGB5 1.77 
E04 MMP1 2.85 
E09 MMP14 1.70 
F03 MMP8 3.14 
F04 MMP9 2.44 
F11 ON 2.95 
G07 TIMP2 1.50 
G09 CLEC3B 1.54 
(b) Down-regulated genes
Array locationGene nameFold change
A01 ADAMTS1 −2.16 
A02 ADAMTS13 −1.94 
A03 ADAMTS8 −2.33 
A05 CDH1 −1.55 
B03 COL6A1 −2.46 
B04 COL6A2 −1.98 
C07 ITGA3 −1.74 
C10 ITGA6 −1.50 
D07 ITGB4 −1.97 
D11 LAMA2 −1.67 
D12 LAMA3 −3.49 
F06 PECAM1 −3.50 
G01 OPN −2.50 
G11 VCAM1 −2.47 
(a) Up-regulated genes
Array locationGene nameFold change
B05 COL7A1 1.87 
B07 VCAN 1.74 
C02 FN1 9.07 
D02 ITGAM 1.50 
D06 ITHB3 5.43 
D08 ITGB5 1.77 
E04 MMP1 2.85 
E09 MMP14 1.70 
F03 MMP8 3.14 
F04 MMP9 2.44 
F11 ON 2.95 
G07 TIMP2 1.50 
G09 CLEC3B 1.54 
(b) Down-regulated genes
Array locationGene nameFold change
A01 ADAMTS1 −2.16 
A02 ADAMTS13 −1.94 
A03 ADAMTS8 −2.33 
A05 CDH1 −1.55 
B03 COL6A1 −2.46 
B04 COL6A2 −1.98 
C07 ITGA3 −1.74 
C10 ITGA6 −1.50 
D07 ITGB4 −1.97 
D11 LAMA2 −1.67 
D12 LAMA3 −3.49 
F06 PECAM1 −3.50 
G01 OPN −2.50 
G11 VCAM1 −2.47 

Superarray analysis of common cytokines

We employed another superarray to measure the gene expression of a wide range of common cytokines in PBMCs from five patients with acute MI and five healthy controls. Out of the 84 genes in this array, 16 genes were not expressed by PBMCs (Supplementary Table 2). On the basis of the cut-off value of ±1.5-fold relative to the control value [19], although seven genes were up-regulated, eight genes were down-regulated in the acute MI group (Table 3). This array was divided into six functional gene groups and the percentage of genes changed in each group is listed in Figure 2(B). Among the 15 genes altered in acute MI, six genes were from the IL (interleukin) family [IL1A (IL-1α), IL-12A, IL-17B, IL-17C, IL-4 and IL-5], two genes were from the TNFSF [TNF (tumour necrosis factor) ligand superfamily] [TNFSF13 (TNFSF member 13) and TNFSF7 (TNFSF member 7)] and one gene from IFN (interferon) family [IFNB1 (IFNβ1)]. Five genes were from the TGF (transforming growth factor) and BMP (bone morphogenetic proteins) family, such as TGFB2 (TGFβ2; ~2.4-fold), BMP6, GDF8 (growth differentiation factor 8) and GDF10 (growth differentiation factor 10), and INHBA (inhibin βA) were altered (Table 3). In addition, the expression of PDGFA (platelet-derived growth factor α) was up-regulated in PBMCs from patients with acute MI (~2-fold) (Table 3).

Table 3
Changes in common cytokines in PBMCs
(a) Up-regulated genes
Array locationGene nameFold change
A06 BMP6 1.54 
D07 IL1A 1.99 
F04 INHBA 2.55 
F09 PDGFA 2.32 
F12 TGFB2 2.49 
G7 TNFSF13 1.50 
G11 TNFSF7 1.54 
(b) Down-regulated genes
Array locationGene nameFold change
B02 GDF10 −2.51 
B07 GDF8 −1.59 
C02 IFNB1 −3.00 
C07 IL12A −1.51 
D02 IL17B −2.42 
D03 IL17C −1.66 
E09 IL4 −1.87 
E10 IL5 −1.73 
(a) Up-regulated genes
Array locationGene nameFold change
A06 BMP6 1.54 
D07 IL1A 1.99 
F04 INHBA 2.55 
F09 PDGFA 2.32 
F12 TGFB2 2.49 
G7 TNFSF13 1.50 
G11 TNFSF7 1.54 
(b) Down-regulated genes
Array locationGene nameFold change
B02 GDF10 −2.51 
B07 GDF8 −1.59 
C02 IFNB1 −3.00 
C07 IL12A −1.51 
D02 IL17B −2.42 
D03 IL17C −1.66 
E09 IL4 −1.87 
E10 IL5 −1.73 

Percentage of genes altered in different functional gene groups in the ECM and adhesion molecule superarray (A) and the common cytokine superarray (B) in acute MI

Figure 2
Percentage of genes altered in different functional gene groups in the ECM and adhesion molecule superarray (A) and the common cytokine superarray (B) in acute MI

The horizontal bars represent the percentage number of altered genes in the total number of genes in a particular functional gene group, as indicated by the superarray [ECM and adhesion molecules (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-013A.html), and common cytokines (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-021A.html]. The numbers in parentheses indicate the total number of genes in a functional gene group. N.B. some genes belong to more than one functional gene groups in the ECM and adhesion molecule superarray.

Figure 2
Percentage of genes altered in different functional gene groups in the ECM and adhesion molecule superarray (A) and the common cytokine superarray (B) in acute MI

The horizontal bars represent the percentage number of altered genes in the total number of genes in a particular functional gene group, as indicated by the superarray [ECM and adhesion molecules (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-013A.html), and common cytokines (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-021A.html]. The numbers in parentheses indicate the total number of genes in a functional gene group. N.B. some genes belong to more than one functional gene groups in the ECM and adhesion molecule superarray.

Validation of superarray results by real-time PCR

In order to validate the array data, in addition to MMP9 measured by real-time PCR above, we selected four genes from two superarrays, and performed real-time PCR in all 34 subjects from the three subject groups in the present study. The selection was based on their functional significance and was representative of the gene groups included in the superarrays. Gene expression of MMP8, ITGAM and TNFSF7 was increased, but ITGA6 was decreased in acute MI compared with the two other control groups, confirming findings from the two superarrays. Table 4 shows the fold changes detected by superarray and real-time PCR, which are comparable with each other.

Table 4
Validation of the superarray data by real-time PCR
GeneArray (MI compared with control)Real-time PCR (MI compared with control)
MMP9 2.44 2.00 
MMP8 3.14 2.68 
ITGAM 1.50 1.91 
ITGA6 −1.50 −2.22 
TNFSF7 1.54 2.27 
GeneArray (MI compared with control)Real-time PCR (MI compared with control)
MMP9 2.44 2.00 
MMP8 3.14 2.68 
ITGAM 1.50 1.91 
ITGA6 −1.50 −2.22 
TNFSF7 1.54 2.27 

DISCUSSION

In the present study, we examined PBMCs from patients with acute MI and have made several major findings. First, PBMCs from patients with acute MI produced a higher amount of MMP9 compared with those from patients with stable angina or healthy controls, indicating that PBMCs are activated and become an important cellular source of plasma MMP9, which is known to be elevated in MI and contribute to post-MI LV remodelling [47]. Secondly, gene screening of two important pathways (ECM and adhesion molecules, and common cytokines) from PBMCs revealed different expression profiles in acute MI relative to healthy controls. These findings imply significant roles for PBMCs in systemic and regional inflammatory responses and ECM remodelling in acute MI. Thus activation of PBMC, which reflects the degree of overall and regional inflammation, could be related to LV remodelling and even prognosis of MI patients. Assays for PBMC activation might serve as biochemical markers to evaluate patients with acute MI in regard to prognosis and LV remodelling, which merits further investigation.

Activation of MMPs, particularly MMP2 and MMP9, is suggested to play an important role in the pathogenesis of LV remodelling following MI [49]. Diverse changes in plasma MMP9 and MMP2 in acute MI have been reported [1318], but the mechanism is not clear yet. In the present study, PBMCs from patients with acute MI produced a greater amount of MMP9 detected by both real-time PCR and gelatin zymography, and superarray data also demonstrated that MMP9 was the most abundantly expressed MMP by PBMCs among all of MMP species, confirming that circulating inflammatory cells are an important source for plasma MMP9. PBMCs mainly comprise T-lymphocytes, B-lymphocytes and monocytes. Previous studies have shown that MMP9 is expressed in all main cell types of PBMCs [2023]. In contrast, MMP2 was not detected at either the mRNA or protein level in PBMCs, suggesting that PBMCs are not a cellular source of plasma MMP2. These results may explain diverse changes in plasma MMP9 and MMP2 in patients with acute MI as well as their clinical implication. Our results indicate that MMP9 and MMP2 are derived from distinct cellular sources. Inflammatory cells are an important source of MMP9, but MMP2 is mainly expressed by cardiac cells in the heart [24]. Indeed, our previous work in the mouse infarct model also showed that a great number of inflammatory cells accumulated in infarcted hearts during the acute phase of MI, accompanied by an early and marked increase in cardiac MMP9, but a modest increase in cardiac MMP2 [4,5]. Thus MMP9, which comes from inflammatory cells, plays a critical role in acute LV remodelling. Not surprisingly, the plasma MMP9 concentration was positively correlated with LV remodelling [15,17,25] and has also been reported to predict fatal events in patients with known coronary artery disease [26], MMP2 concentrations, however, were unrelated [25] or inversely [27] related to LV volume after MI. In addition to MMP2 and MMP9, two collagenases (MMP8 and MMP1) and MMP14 (membrane type 1 MMP) were also up-regulated in PBMCs in patients with MI, accompanied by a moderate increase in TIMP2. Thus an imbalance between MMPs/TIMPs towards proteolysis exists in PBMCs in the setting of acute MI, which is in keeping with the altered ratio of MMP/TIMP seen in the infarcted mouse heart [4].

The recruitment of leucocytes into the infarcted myocardium is mediated by cell adhesion molecules. PBMCs serve as an important source of cell adhesion molecules, such as integrins and other cell adhesion molecules, including a number of matrix proteins that also act as adhesive proteins. PBMCs from the acute MI group also showed a differential expression pattern of integrins known to participate in cell adhesion and cell-surface-mediated signalling. Expression of ITGAM, ITGB3 and ITGB5 was up-regulated, but expression of ITGA3, ITGA6, and ITGB4 was down-regulated. Notably, integrin αM combines with the β2 chain to form Mac-1, which plays an essential role in mediating leucocyte adhesion through binding to ICAM1 (intercellular cell adhesion molecule 1) expressed on activated vascular endothelium. In addition, integrin β3 and β5 also mediate cell adhesion and migration. [28,29]. Expression of several other adhesion molecules also changed in PBMCs from MI patients. These results suggest an important role of PBMCs in mediating cell adhesion and migration, which is an essential step in inflammatory responses.

PBMCs also serve as a cellular source for matrix proteins. Thus inflammatory cells recruited into infarcted hearts are involved in the healing process by secreting matrix proteins. Matrix proteins also serve as adhesive proteins, and regulate cellular function and signalling through interactions with specific receptors. In acute MI, gene expression of a number of matrix proteins by PBMCs changed significantly. Notably, a marked increase in the FN1 gene was observed. Fibronectin is a major component of the cellular basement membrane and also acts as an adhesive protein and a major player in cell–matrix interaction through its binding to integrins. An increase in plasma fibronectin has also been reported in patients with acute MI [30]. In addition to collagen, lamina and fibronectin, gene expression of another class of matrix proteins, matricellular proteins such as OPN and ON by PBMCs, was also changed in acute MI. The function of matricellular proteins in acute MI is not clear yet, but studies have suggested that matricellular proteins regulate the phenotype and function of many cell types involved in LV remodelling and wound healing [31].

PBMCs are capable of producing a wide range of inflammatory cytokines, such as ILs, TNFs and IFNs, and growth factors, including TGFβ, PDGF, VEGF (vascular endothelial growth factor) and CSF (colony stimulating factor). Following MI, the release of inflammatory cytokines promotes the recruitment of inflammatory cells into the infarcted site. The present study supports further the concept that inflammatory cells provide an additional source of local inflammatory cytokine production and amplification of the inflammatory response. Expression of several genes from the IL family was altered in PBMCs after MI. Notably, IL1α (IL1A), which induces apoptosis and is involved in various inflammatory processes, was up-regulated in acute MI. In contrast, both IL4 and IL5 were down-regulated. IL4 and IL5 are Th (helper T-cell) 2 cytokines, and IL4 suppresses acute inflammation and macrophage activation [1]. Thus an imbalance between Th1 and Th2 cytokines may exist in PBMCs after acute MI. Such change has also been implicated in atherosclerotic plaque instability [32]. In addition, gene expression of TNFSF13 and TNFSF7 was also increased in PBMCs from patients with acute MI. TNFSF7 contributes to T- and B-lymphocyte activation [33], and up-regulation of these two cytokines may also be important in the inflammatory response after MI. On the other hand, mononuclear cells are also necessary in post-infarct healing by secreting TGF, PDGF and other growth factors. Gene expression of TGFB2 and PDGFA, molecules that promote fibroblast activation and fibrotic healing [34,35], was up-regulated in PBMCs from MI patients.

Our results suggest that PBMCs in patients with acute MI are activated, but the cause of such systemic activation of PBMCs remains undefined, which forms the basis for future work. Several mechanisms may be suggested: (i) inflammatory cytokines released by infarcted myocardium may enter into the circulation and then stimulate PBMCs; (ii) sustained neurohormonal activation, such as of the renin–angiotensin–aldosterone system and the adrenergic system could also contribute to the activation of PBMCs [3638]; and (iii) other factors, such as hypoxia and oxidative stress, may activate PBMCs [39].

In conclusion, PBMCs are activated in patients with acute MI. PBMCs constitute a key cellular source for plasma MMP9 in acute MI, but not for plasma MMP2. PBMCs also contribute to ECM remodelling, and amplification of the inflammatory response as well as infarct healing by producing MMPs/TIMPs, matrix proteins, inflammatory cytokines and growth factors. Activity of PBMCs may serve as a biochemical marker to evaluate patients with acute MI in regard to regional inflammation, ventricular remodelling and the efficacy of anti-inflammatory interventions.

FUNDING

This study was supported by the National Health and Medical Research Council (NHMRC) of Australia [grant number 472600]. X.J.D. and A.M.D. are NHMRC Research Fellows.

Abbreviations

     
  • BMI

    body mass index

  •  
  • BMP

    bone morphogenetic protein

  •  
  • CDH

    cadherin

  •  
  • CLEC3B

    C-type lectin family 3, member B

  •  
  • COL7A1 etc.

    collagen 7α1 etc.

  •  
  • ECM

    extracellular matrix

  •  
  • FN1

    fibronectin 1

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • GDF

    growth differentiation factor

  •  
  • ICAM1

    intercellular adhesion molecule 1

  •  
  • IFN

    interferon

  •  
  • IL

    interleukin

  •  
  • INHBA

    inhibin βA

  •  
  • ITGAM etc.

    integrin αM etc.

  •  
  • LAMA2 etc.

    lamina α2 etc.

  •  
  • LV

    left ventricular

  •  
  • MI

    myocardial infarction

  •  
  • MMP

    matrix metalloproteinase

  •  
  • ON

    osteonectin

  •  
  • OPN

    osteopontin

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PCI

    percutanenous coronary intervention

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • PECAM1

    platelet/endothelial cell adhesion molecule 1

  •  
  • TGF

    transforming growth factor

  •  
  • Th

    helper T-cell

  •  
  • TIMP

    tissue inhibitor of metalloproteinases

  •  
  • TNF

    tumour necrosis factor

  •  
  • TNFSF

    TNF ligand superfamily

  •  
  • VCAM1

    vascular cell adhesion molecule 1

  •  
  • VCAN

    versican

  •  
  • VEGF

    vascular endothelial growth factor

References

References
1
Frangogiannis
 
N. G.
Smith
 
C. W.
Entman
 
M. L.
 
The inflammatory response in myocardial infarction
Cardiovasc. Res.
2002
, vol. 
53
 (pg. 
31
-
47
)
2
Lindsey
 
M.
Wedin
 
K.
Brown
 
M. D.
Keller
 
C.
Evans
 
A. J.
Smolen
 
J.
Burns
 
A. R.
Rossen
 
R. D.
Michael
 
L.
Entman
 
M.
 
Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion
Circulation
2001
, vol. 
103
 (pg. 
2181
-
2187
)
3
Cavasin
 
M. A.
Tao
 
Z.
Menon
 
S.
Yang
 
X. P.
 
Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice
Life Sci.
2004
, vol. 
75
 (pg. 
2181
-
2192
)
4
Fang
 
L.
Gao
 
X. M.
Moore
 
X. L.
Kiriazis
 
H.
Su
 
Y.
Ming
 
Z.
Lim
 
Y. L.
Dart
 
A. M.
Du
 
X. J.
 
Differences in inflammation, MMP activation and collagen damage account for gender difference in murine cardiac rupture following myocardial infarction
J. Mol. Cell. Cardiol.
2007
, vol. 
43
 (pg. 
535
-
544
)
5
Fang
 
L.
Gao
 
X. M.
Samuel
 
C. S.
Su
 
Y.
Lim
 
Y. L.
Dart
 
A. M.
Du
 
X. J.
 
Higher levels of collagen and facilitated healing protect against ventricular rupture following myocardial infarction
Clin. Sci.
2008
, vol. 
115
 (pg. 
99
-
106
)
6
Ducharme
 
A.
Frantz
 
S.
Aikawa
 
M.
Rabkin
 
E.
Lindsey
 
M.
Rohde
 
L. E.
Schoen
 
F. J.
Kelly
 
R. A.
Werb
 
Z.
Libby
 
P.
, et al 
Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction
J. Clin. Invest.
2000
, vol. 
106
 (pg. 
55
-
62
)
7
Heymans
 
S.
Luttun
 
A.
Nuyens
 
D.
Theilmeier
 
G.
Creemers
 
E.
Moons
 
L.
Dyspersin
 
G. D.
Cleutjens
 
J. P.
Shipley
 
M.
Angellilo
 
A.
, et al 
Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure
Nat. Med.
1999
, vol. 
5
 (pg. 
1135
-
1142
)
8
Hayashidani
 
S.
Tsutsui
 
H.
Ikeuchi
 
M.
Shiomi
 
T.
Matsusaka
 
H.
Kubota
 
T.
Imanaka-Yoshida
 
K.
Itoh
 
T.
Takeshita
 
A.
 
Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction
Am. J. Physiol. Heart Circ. Physiol.
2003
, vol. 
285
 (pg. 
H1229
-
H1235
)
9
Matsumura
 
S.
Iwanaga
 
S.
Mochizuki
 
S.
Okamoto
 
H.
Ogawa
 
S.
Okada
 
Y.
 
Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice
J. Clin. Invest.
2005
, vol. 
115
 (pg. 
599
-
609
)
10
Vinten-Johansen
 
J.
 
Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury
Cardiovasc. Res.
2004
, vol. 
61
 (pg. 
481
-
497
)
11
Weisman
 
H. F.
Bartow
 
T.
Leppo
 
M. K.
Marsh
 
Jr, H. C.
Carson
 
G. R.
Concino
 
M. F.
Boyle
 
M. P.
Roux
 
K. H.
Weisfeldt
 
M. L.
Fearon
 
D. T.
 
Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis
Science
1990
, vol. 
249
 (pg. 
146
-
151
)
12
Faxon
 
D. P.
Gibbons
 
R. J.
Chronos
 
N. A.
Gurbel
 
P. A.
Sheehan
 
F.
 
The effect of blockade of the CD11/CD18 integrin receptor on infarct size in patients with acute myocardial infarction treated with direct angioplasty: the results of the HALT-MI study
J. Am. Coll. Cardiol.
2002
, vol. 
40
 (pg. 
1199
-
1204
)
13
Kai
 
H.
Ikeda
 
H.
Yasukawa
 
H.
Kai
 
M.
Seki
 
Y.
Kuwahara
 
F.
Ueno
 
T.
Sugi
 
K.
Imaizumi
 
T.
 
Peripheral blood levels of matrix metalloproteases-2 and -9 are elevated in patients with acute coronary syndromes
J. Am. Coll. Cardiol.
1998
, vol. 
32
 (pg. 
368
-
372
)
14
Webb
 
C. S.
Bonnema
 
D. D.
Ahmed
 
S. H.
Leonardi
 
A. H.
McClure
 
C. D.
Clark
 
L. L.
Stroud
 
R. E.
Corn
 
W. C.
Finklea
 
L.
Zile
 
M. R.
, et al 
Specific temporal profile of matrix metalloproteinase release occurs in patients after myocardial infarction: relation to left ventricular remodeling
Circulation
2006
, vol. 
114
 (pg. 
1020
-
1027
)
15
Sundstrom
 
J.
Evans
 
J. C.
Benjamin
 
E. J.
Levy
 
D.
Larson
 
M. G.
Sawyer
 
D. B.
Siwik
 
D. A.
Colucci
 
W. S.
Sutherland
 
P.
Wilson
 
P. W.
, et al 
Relations of plasma matrix metalloproteinase-9 to clinical cardiovascular risk factors and echocardiographic left ventricular measures: the Framingham Heart Study
Circulation
2004
, vol. 
109
 (pg. 
2850
-
2856
)
16
Soejima
 
H.
Ogawa
 
H.
Sakamoto
 
T.
Miyamoto
 
S.
Kajiwara
 
I.
Kojima
 
S.
Hokamaki
 
J.
Sugiyama
 
S.
Yoshimura
 
M.
Suefuji
 
H.
, et al 
Increased serum matrix metalloproteinase-1 concentration predicts advanced left ventricular remodeling in patients with acute myocardial infarction
Circ. J.
2003
, vol. 
67
 (pg. 
301
-
304
)
17
Kaden
 
J. J.
Dempfle
 
C. E.
Sueselbeck
 
T.
Brueckmann
 
M.
Poerner
 
T. C.
Haghi
 
D.
Haase
 
K. K.
Borggrefe
 
M.
 
Time-dependent changes in the plasma concentration of matrix metalloproteinase 9 after acute myocardial infarction
Cardiology
2003
, vol. 
99
 (pg. 
140
-
144
)
18
Hojo
 
Y.
Ikeda
 
U.
Ueno
 
S.
Arakawa
 
H.
Shimada
 
K.
 
Expression of matrix metalloproteinases in patients with acute myocardial infarction
Jpn Circ. J.
2001
, vol. 
65
 (pg. 
71
-
75
)
19
Veeriah
 
S.
Miene
 
C.
Habermann
 
N.
Hofmann
 
T.
Klenow
 
S.
Sauer
 
J.
Bohmer
 
F.
Wolfl
 
S.
Pool-Zobel
 
B. L.
 
Apple polyphenols modulate expression of selected genes related to toxicological defence and stress response in human colon adenoma cells
Int. J. Cancer
2008
, vol. 
122
 (pg. 
2647
-
2655
)
20
Bar-Or
 
A.
Nuttall
 
R. K.
Duddy
 
M.
Alter
 
A.
Kim
 
H. J.
Ifergan
 
I.
Pennington
 
C. J.
Bourgoin
 
P.
Edwards
 
D. R.
Yong
 
V. W.
 
Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis
Brain
2003
, vol. 
126
 (pg. 
2738
-
2749
)
21
Brunner
 
S.
Kim
 
J. O.
Methe
 
H.
 
Relation of matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio in peripheral circulating CD14+ monocytes to progression of coronary artery disease
Am. J. Cardiol.
2010
, vol. 
105
 (pg. 
429
-
434
)
22
Oviedo-Orta
 
E.
Bermudez-Fajardo
 
A.
Karanam
 
S.
Benbow
 
U.
Newby
 
A. C.
 
Comparison of MMP-2 and MMP-9 secretion from T helper 0, 1 and 2 lymphocytes alone and in coculture with macrophages
Immunology
2008
, vol. 
124
 (pg. 
42
-
50
)
23
Trocme
 
C.
Gaudin
 
P.
Berthier
 
S.
Barro
 
C.
Zaoui
 
P.
Morel
 
F.
 
Human B lymphocytes synthesize the 92-kDa gelatinase, matrix metalloproteinase-9
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
20677
-
20684
)
24
Cheung
 
P. Y.
Sawicki
 
G.
Wozniak
 
M.
Wang
 
W.
Radomski
 
M. W.
Schulz
 
R.
 
Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart
Circulation
2000
, vol. 
101
 (pg. 
1833
-
1839
)
25
Kelly
 
D.
Cockerill
 
G.
Ng
 
L. L.
Thompson
 
M.
Khan
 
S.
Samani
 
N. J.
Squire
 
I. B.
 
Plasma matrix metalloproteinase-9 and left ventricular remodelling after acute myocardial infarction in man: a prospective cohort study
Eur. Heart J.
2007
, vol. 
28
 (pg. 
711
-
718
)
26
Blankenberg
 
S.
Rupprecht
 
H. J.
Poirier
 
O.
Bickel
 
C.
Smieja
 
M.
Hafner
 
G.
Meyer
 
J.
Cambien
 
F.
Tiret
 
L.
 
Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease
Circulation
2003
, vol. 
107
 (pg. 
1579
-
1585
)
27
Squire
 
I. B.
Evans
 
J.
Ng
 
L. L.
Loftus
 
I. M.
Thompson
 
M. M.
 
Plasma MMP-9 and MMP-2 following acute myocardial infarction in man: correlation with echocardiographic and neurohumoral parameters of left ventricular dysfunction
J. Card. Failure
2004
, vol. 
10
 (pg. 
328
-
333
)
28
Hein
 
S.
Schaper
 
J.
 
The extracellular matrix in normal and diseased myocardium
J. Nucl. Cardiol.
2001
, vol. 
8
 (pg. 
188
-
196
)
29
Lee
 
B. H.
Bae
 
J. S.
Park
 
R. W.
Kim
 
J. E.
Park
 
J. Y.
Kim
 
I. S.
 
βig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through αvβ5 integrin
Exp. Mol. Med.
2006
, vol. 
38
 (pg. 
153
-
161
)
30
Orem
 
C.
Celik
 
S.
Orem
 
A.
Calapoglu
 
M.
Erdol
 
C.
 
Increased plasma fibronectin levels in patients with acute myocardial infarction complicated with left ventricular thrombus
Thromb. Res.
2002
, vol. 
105
 (pg. 
37
-
41
)
31
Schellings
 
M. W.
Pinto
 
Y. M.
Heymans
 
S.
 
Matricellular proteins in the heart: possible role during stress and remodeling
Cardiovasc. Res.
2004
, vol. 
64
 (pg. 
24
-
31
)
32
Methe
 
H.
Brunner
 
S.
Wiegand
 
D.
Nabauer
 
M.
Koglin
 
J.
Edelman
 
E. R.
 
Enhanced T-helper-1 lymphocyte activation patterns in acute coronary syndromes
J. Am. Coll. Cardiol.
2005
, vol. 
45
 (pg. 
1939
-
1945
)
33
Huang
 
J.
Kerstann
 
K. W.
Ahmadzadeh
 
M.
Li
 
Y. F.
El-Gamil
 
M.
Rosenberg
 
S. A.
Robbins
 
P. F.
 
Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer immunotherapy
J. Immunol.
2006
, vol. 
176
 (pg. 
7726
-
7735
)
34
Bassols
 
A.
Massague
 
J.
 
Transforming growth factor β regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans
J. Biol. Chem.
1988
, vol. 
263
 (pg. 
3039
-
3045
)
35
Zymek
 
P.
Bujak
 
M.
Chatila
 
K.
Cieslak
 
A.
Thakker
 
G.
Entman
 
M. L.
Frangogiannis
 
N. G.
 
The role of platelet-derived growth factor signaling in healing myocardial infarcts
J. Am. Coll. Cardiol.
2006
, vol. 
48
 (pg. 
2315
-
2323
)
36
Brasier
 
A. R.
Recinos
 
A.
Eledrisi
 
M. S.
 
Vascular inflammation and the renin–angiotensin system
Arterioscler. Thromb. Vasc. Biol.
2002
, vol. 
22
 (pg. 
1257
-
1266
)
37
Ahokas
 
R. A.
Warrington
 
K. J.
Gerling
 
I. C.
Sun
 
Y.
Wodi
 
L. A.
Herring
 
P. A.
Lu
 
L.
Bhattacharya
 
S. K.
Postlethwaite
 
A. E.
Weber
 
K. T.
 
Aldosteronism and peripheral blood mononuclear cell activation: a neuroendocrine-immune interface
Circ. Res.
2003
, vol. 
93
 (pg. 
e124
-
e135
)
38
Werner
 
C.
Werdan
 
K.
Ponicke
 
K.
Brodde
 
O. E.
 
Impaired β-adrenergic control of immune function in patients with chronic heart failure: reversal by β1-blocker treatment
Basic Res. Cardiol.
2001
, vol. 
96
 (pg. 
290
-
298
)
39
Janabi
 
M.
Yamashita
 
S.
Hirano
 
K.
Sakai
 
N.
Hiraoka
 
H.
Matsumoto
 
K.
Zhang
 
Z.
Nozaki
 
S.
Matsuzawa
 
Y.
 
Oxidized LDL-induced NF-κB activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients
Arterioscler. Thromb. Vasc. Biol.
2000
, vol. 
20
 (pg. 
1953
-
1960
)

Supplementary data