Atherosclerosis is an inflammatory disease in which systemic inflammation correlates with disease activity. Matrix metalloproteinases (MMPs) contribute to collagen breakdown in atherosclerotic plaques. In the present study, we investigated whether the ratio of MMP-9 and its endogenous inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1, in circulating monocytes correlates with the clinical stages of coronary artery disease. We studied 18 patients with stable angina pectoris (SAP), 14 patients with unstable angina pectoris and non–ST-segment elevation myocardial infarction (UAP/NSTEMI), 14 patients with ST-elevation myocardial infarction (STEMI), and 16 healthy controls. The protein and mRNA levels of MMP-9 and TIMP-1 in CD14+ monocytes were analyzed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The activity of serum MMP-9 was assessed using zymography. Compared to the controls (0.07 ± 0.01 relative units) and patients with SAP (0.25 ± 0.1 relative units, p = NS), the monocytic MMP-9 mRNA levels were increased in those with UAP/NSTEMI (0.9 ± 0.3 relative units, p <0.05 vs SAP) or STEMI (1.6 ± 0.4 relative units, p <0.05 vs UAP/NSTEMI). In contrast, the protein and mRNA expression of monocytic TIMP-1 levels was 4.5- to 4.7-fold lower in patients with STEMI than in the controls or those with SAP or UAP/NSTEMI (p <0.05). Changes in monocytic expression of MMP-9 and TIMP-1 tracked with the serum levels of MMP-9 and TIMP-1. The activity of serum MMP-9 correlated with the individual MMP-9/TIMP-1 ratio in the peripheral circulating monocytes (r 2 = 0.82, p <0.02). In conclusion, the progression of coronary artery disease was mirrored by an increasing MMP-9/TIMP-1 ratio in the peripheral circulating CD14+ monocytes and serum, respectively. Circulating monocytes displayed the same pattern of imbalance in the expression of MMP-9 and TIMP-1 as previously reported for monocyte-derived macrophages within atherosclerotic plaques, supporting the notion of atherosclerosis as a systemic inflammatory disease.
Atherosclerosis is an inflammatory disease in which the balance between synthesis and degradation of extracellular matrix components is crucial for plaque stability. Vulnerable lesions with a tendency to rupture are rich in activated macrophages, suggesting the macrophage as a key regulator of atherosclerotic plaque stability. Monocytes, on activation, and tissue-invading macrophages are able to secrete several classes of neutral extracellular proteases, including matrix metalloproteinases (MMPs). Experimental evidence from several animal models and histologic sections of human specimens has suggested that degradation of extracellular matrix by MMPs plays an important role in plaque destabilization. In particular, MMP-1, MMP-3, and MMP-9 have been localized in the rupture-prone shoulder regions of atherosclerotic plaques. Inactivation by binding to 4 tissue inhibitors of MMPs (TIMPs) normally prevents MMP activity from becoming excessive. Under normal circumstances, TIMPs are in balance with MMPs; however, elevated MMP levels and dysregulation within the MMP/TIMP system can be detected in the serum of patients with advanced stages of coronary artery disease. In our study, we investigated the levels of MMP-9 and its inhibitor TIMP-1 in peripheral circulating CD14+ monocytes in patients with different stages of coronary artery disease to link this with the serum concentration and activity of MMPs.
Methods
The ethics committee of the Ludwig-Maximilians-University, Munich, Germany approved the present study. All subjects provided informed consent. A total of 46 patients and 16 healthy controls were studied. Blood samples were obtained from all patients as soon as possible after admission. The concentrations of C-reactive protein, white blood cell count, cholesterol, glucose, troponin I, and creatine kinase were measured according to routine protocols. According to the clinical characteristics at admission, the patients were assigned to 1 of 3 groups: group I included 18 patients with stable angina pectoris (SAP) who had undergone diagnostic coronary angiography; and group II included 9 patients with a diagnosis of unstable angina pectoris (UAP) according to Braunwald’s class II and III and 5 patients with non–ST-elevation myocardial infarction (NSTEMI). All patients with UAP had experienced chest pain at rest within the preceding 48 hours but had no evidence of myocardial necrosis by an increase of creatine kinase or ST-segment elevation. Patients with NSTEMI had elevated troponin I levels. Transient ST-segment depression and/or T wave inversion were present in all cases. Group III included 14 patients with ST elevation myocardial infarction (STEMI) and a characteristic pattern of necrotic myocardial serum enzymes. The exclusion criteria for the present study included myocardial infarction within the previous 6 months, admission more than 6 hours from the onset of symptoms, inflammatory conditions likely to be associated with an acute phase response, and autoimmune and neoplastic disease. None of the included patients had advanced liver disease, renal failure, or valvular heart disease. Group IV included 16 healthy volunteers with no clinical signs of coronary artery disease, who were without coronary risk factors. All 16 control patients had normal electrocardiographic and echocardiographic findings and no evidence of atherosclerosis by carotid artery sonography.
Mononuclear cells were isolated from 16 ml heparinized blood (Vacutainer CPT, Becton Dickinson, Heidelberg, Germany) using Ficoll density gradient centrifugation (20 minutes, 1,500 g , 4°C). The cells were pooled, washed with phosphate-buffered saline (containing 2 mmol/L ethylenediaminetetraacetic acid, and 0.5% bovine serum albumin), and the monocytes were separated by magnetic cell sorting on a MACS LS+ separation column after incubation with CD14− MicroBeads, according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity was consistently >95% as revealed by flow cytometry (data not shown). Monocytic RNA was isolated using the monophasic phenol-guanidine isothiocyanate TRIzol reagent (Invitrogen, Karlsruhe, Germany). Reverse transcription was performed using the ImProm-IITM Reverse Transcription System (Promega, Madison, Wisconsin) according to the manufacturer’s protocol. cDNA samples were analyzed by quantitative reverse transcriptase-polymerase chain reaction using the following human primers (MWG-Biotech AG, Ebersberg, Germany): glyceraldehyde phosphate dehydrogenase (sense, 5′-TGA CAA CAG CCT CAA GAT CA-3′; antisense, 5′-CTG TGG TCA TGA GTC CTT CC-3′); MMP-9 (sense, 5′-CTT GCA TAA GGA CGA CGT G-3′; antisense, 5′-ACA GTA GTG GCC GTA GAA GG-3′); and TIMP-1 (sense, 5′-GTT GTT GCT GTG GCT GAT AG-3′; antisense, 5′-GCT GGT ATA AGG TGG TCT GG-3′). Quantitative reverse transcriptase-polymerase chain reaction was performed using the SYBR Green Reaction Mix (Eurogentec, Cologne, Germany) on an ABI PRISM 7900HT Detection System (Applied Biosystems, Foster City, California). Each sample was run in duplicate. The expression of each gene was quantified relative to the H4 mRNA expression levels according to the Sequence Detector User Bulletin (Applied Biosystems).
The serum levels of MMP-9 (Oncogene, Cambridge, Massachusetts) and TIMP-1 (R&D Systems, Wiesbaden, Germany) were determined by enzyme-linked immunosorbent assay. The serum was obtained by centrifugation at 3,000 rpm. for 10 minutes within 15 minutes of collection. The serum was frozen and stored at −70°C for subsequent analysis after a single thaw. The extinction of samples was measured by a spectrophotometer at 450 and 540 nm (Titerek Multiskan MCC/340, Flow Laboratories, Huntsville, Alabama).
For gelatin zymography, the total serum protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, Illinois), and aliquots of 150-μg serum samples were analyzed on 10% (wt/vol) polyacrylamide gels containing 0.1% gelatin. A precision protein molecular weight standard (Bio-Rad Laboratories, Hercules, California) was included with the sample in the gel. After electrophoresis, the gels were washed in Triton X-100 (25 ml/L) and incubated for 24 hours at 37°C in enzyme incubation buffer (containing 50 mM Tris-HCl, pH 7.5, 5 mM CaCl 2 , 100 mM NaCl, 1 mM ZnCl 2 , 0.3 mM NaN 3 , 0.2 g/L Brij-35, and 2.5 ml/L Triton X-100). For activation of the zymogens, 2 mM p -amino phenyl-mercuric acetate was added. After staining with Coomassie blue, enzyme activity appeared as clear bands against the blue-stained background. The gelatinolytic bands disappeared in parallel zymograms in which the development buffer contained 5 mM ethylenediaminetetraacetic acid and 1,10-phenanthroline, confirming that the gelatinolytic activity was caused by the MMPs. MMP-9 activity was identified by molecular weight. The gels were scanned into Adobe Photoshop, version 4.0 (Adobe Systems, New York, New York) as black and white images and inverted; the area of lysis for each band detected was analyzed using densitometric software (Phoretix International, Newcastle Upon Tyne, United Kingdom). The results are expressed as densitometric units. To assess the reproducibility of this assay, selected samples were run in duplicate, and the coefficient of variation was <10%.
Statistical analysis was performed with JMP statistical software (SAS Institute, Cary, North Carolina). The Shapiro-Wilk test was used to determine whether the values followed a normal distribution. Because the C-reactive protein, troponin I, and creatine kinase values did not follow a normal distribution, the comparisons between groups were conducted using the Wilcoxon test for 2 groups and the Kruskal-Wallis test for more than 2 groups. The remaining continuous variables were compared using the Student t tests for 2- and 1-way analysis of variance for more than 2 groups. Categorical variables were compared using the chi-square test. Significant values were not adjusted for multiple comparisons. C-reactive protein, troponin I, and creatine kinase values are presented as the median and range, the remaining variables are expressed as the mean ± SD or number (percentage). Spearman’s correlation was used to determine the relation between serum MMP-9 gelatinolytic activity and the ratio of MMP-9/TIMP-1 mRNA expression in circulating CD14+ monocytes. p Values <0.05 were considered statistically significant.
Results
No significant differences were found in age, gender, lipid or blood glucose level, frequency of smoking, or use of medications in patients with SAP, UAP/NSTEMI, or STEMI ( Table 1 ). Patients with UAP/NSTEMI or STEMI were treated with standard antianginal medications, intravenous heparin, aspirin, clopidogrel, and nitroglycerin.
| Variable | Control (n = 16) | SAP (n = 18) | UAP/NSTEMI (n = 14) | STEMI (n = 14) |
|---|---|---|---|---|
| Age (years) | 56 ± 11 | 66 ± 13 | 72 ± 10 | 65 ± 14 |
| Men | 8 | 9 | 8 | 7 |
| Laboratory parameters | ||||
| C-reactive protein (mg/dl) | 0.1 (0–0.3) | 0.2 (0–0.5) | 1.0 (0.3–3.6) | 0.9 (0.1–4.5) |
| White blood cells (U/L) | 7.1 ± 0.3 | 7.2 ± 0.5 | 7.4 ± 1.0 | 7.3 ± 2.2 |
| Cholesterol (mg/dl) | 181 ± 27 | 179 ± 22 | 183 ± 27 | 192 ± 23 |
| Glucose (mg/dl) | 109 ± 9 | 114 ± 8 | 113 ± 12 | 118 ± 19 |
| Troponin I (ng/ml) | ND | ND | 6.9 (0.9–10.5) | 87 (34–123) ⁎ |
| Creatine kinase (U/L) | 35 (14–51) | 33 (20–43) | 34 (16–53) | 394 (297–521) † |
| Medication at admission (%) | ||||
| β Blockers | — | 12 (67) | 10 (71) | 10 (71) |
| Aspirin | — | 16 (89) | 11 (79) | 11 (79) |
| Angiotensin-converting enzyme inhibitors | — | 8 (44) | 7 (50) | 7 (50) |
| Statins | — | 14 (78) | 11 (79) | 10 (71) |
Circulating CD14+ monocytes were isolated and analyzed for mRNA expression of MMP-9 and its endogenous inhibitor TIMP-1 in the different patient groups using quantitative real-time polymerase chain reaction. mRNA expression of MMP-9 in patients with SAP was not significantly greater than that in controls (0.25 ± 0.1 vs 0.07 ± 0.01 relative units, p = 0.61). Circulating monocytes from patients with UAP/NSTEMI revealed a 3.7-fold increased mRNA MMP-9 expression compared with patients with SAP (0.9 ± 0.3 relative units, p <0.05 vs SAP). Patients with STEMI displayed the greatest mRNA expression of MMP-9 in circulating monocytes (1.6 ± 0.4 relative units, p <0.05 vs UAP/NSTEMI; Figure 1 ). In contrast, mRNA expression of TIMP-1 in the circulating CD14+ monocytes was 4.5- to 4.7-fold lower in patients with STEMI than in patients with UAP/NSTEMI or SAP or controls (p <0.05). TIMP-1 expression in the latter 3 groups did not differ significantly from each other ( Figure 1 ). Thus, the ratio of MMP-9 to TIMP-1 mRNA expression in the circulating CD14+ monocytes was increased from that in controls to patients with SAP to patients with UAP/NSTEMI, reaching a maximum in patients with STEMI ( Figure 1 ).
