Matrix metalloproteinase-3 (MMP-3), or stromelysin-1, is a matrix metalloproteinase which is expressed in atherosclerotic plaques and which has been implicated in the pathogenesis of acute coronary syndrome (ACS). Functional polymorphisms in the promoter region of the human MMP-3 gene resulting in an increased expression of MMP-3 have been shown to predict the risk of incident myocardial infarction (MI). However, there have been no studies that have specifically examined the utility of baseline plasma MMP-3 levels for the prediction of long-term MI. In this study, baseline plasma MMP-3 levels were measured in 355 male patients who were referred for coronary angiography and followed prospectively for the development of enzymatically confirmed MI out to 5 years. After adjustment for a variety of baseline clinical, angiographic, and laboratory parameters, plasma MMP-3 levels were an independent predictor of MI at 5 years (hazards ratio 1.42, 95% CI 1.13 to 1.79; p = 0.0023). Furthermore, in 5 additional multivariate models that included a variety of contemporary biomarkers associated with adverse outcomes and MI, MMP-3 remained an independent predictor of MI at 5 years. Similar results were obtained when the analyses were restricted to the subpopulation of patients presenting with ACS. In conclusion, elevated levels of MMP-3 are associated with an increased risk of long-term MI in patients with and without ACS referred for coronary angiography. Furthermore, this association is independent of a variety of clinical, angiographic, laboratory variables, including biomarkers with established prognostic efficacy for the prediction of MI.
Plaque rupture with superimposed thrombosis is now considered to be the main cause of acute coronary syndrome (ACS), including unstable angina pectoris and acute myocardial infarction (MI). Matrix metalloproteinases, or MMPs, are a family of extracellular matrix degrading enzymes implicated in coronary atherosclerotic plaque destabilization and, by extension, ACS. They are expressed by macrophages, vascular smooth muscle cells, and endothelial cells in response to inflammatory stimuli. One matrix metalloproteinase in particular, MMP-3 or stromelysin-1, has garnered interest as a potential mediator of atherothrombotic disease. MMP-3 is a broad spectrum protease which is expressed in atherosclerotic plaques and is capable of cleaving several nonfibrillar extracellular matrix proteins and proteolytically activating other MMPs. Expression of MMP-3 is regulated primarily at the level of transcription, where the promoter of the gene responds to various stimuli. Functional polymorphisms in the promoter region of the human MMP-3 gene associated with higher promoter activity have been reported. More importantly, and consistent with the biologic actions of MMP-3, polymorphisms associated with higher promoter activity have been shown to predict the risk of incident MI. Such studies suggest that genetic variation in MMP-3 expression may contribute to vascular risk. However, to date, there have been no studies that have specifically examined the utility of baseline plasma MMP-3 protein levels for predicting the development of future MI. Accordingly, the objective of the present study was to investigate the prognostic utility of baseline plasma MMP-3 levels for the prediction of MI in patients with known or suspected coronary artery disease, including those with ACS, and do so in the context of well-established covariates and biomarkers known to be associated with poor outcomes and MI.
Methods
The study population and design have been described in detail elsewhere. The study database was generated at an urban Veterans Administration Medical Center and was approved by the local institutional review board. Written informed consent was obtained from all patients. All patients who were referred to the cardiac catheterization laboratory for coronary angiography at the Bronx Veterans Affairs Medical Center between January 13, 1999 and October 17, 2002 were eligible for inclusion in the database. Patients with active gastrointestinal bleeding or a hemoglobin concentration <8 gm/dl were excluded. During the period of study enrollment, a total of 523 unique and consecutive male patients underwent diagnostic coronary angiography. Of these 523 patients, 50 could not be enrolled because of an unexpected loss of key study personnel between January 29, 2001 and July 2, 2001. Of the remaining 473 patients, 84 patients were either unwilling or unable to provide informed consent. Thus, a total of 389 patients provided informed consent and constituted the final study population. Of these 389 patients, MMP-3 values were not available for 34 patients. The present study represents an analysis of both the entire cohort of 355 patients and the subpopulation of 175 patients with ACS (i.e., ST-segment elevation MI or STEMI, non–ST-segment elevation MI or NSTEMI, and unstable angina pectoris).
Fasting whole blood was obtained from all patients at the time of angiography before injection of contrast. The whole blood was centrifuged (4 g ) for 10 minutes at 4°C, the plasma aliquoted into 1.5 ml vials and then stored at −70°C for subsequent analysis. The samples were thawed only once when ready for analysis. Commercially available kits were used to measure the plasma levels of high-sensitivity C-reactive protein (hs-CRP; Life Diagnostics, West Chester, Pennsylvania), N-terminal-pro–B-type natriuretic peptide (NT-proBNP; Diagnostic Automation, Calabasas, California), myeloperoxidase (MPO; Assay Designs, Ann Arbor, Michigan), and MMP-3 (R & D Systems, Minneapolis, Minnesota). For the MMP-3 assay, the sensitivity was 0.04 ng/ml, the range was 0.1 to 10 ng/ml and the intraassay and interassay coefficients of variation were 6.3% and 5.2%, respectively.
All patients were followed prospectively for the development of MI. MI during follow-up (i.e., as a clinical outcome) was defined by a history of chest pain with an associated increase in troponin I >1.0 ng/ml or troponin T >0.1 ng/ml.
Patients were divided into 2 groups using the upper tertile of MMP-3 values for the entire cohort (i.e., ≥20.56 ng/ml and <20.56 ng/ml). Summary statistics for continuous variables were presented both as means (with SDs) and medians (with interquartile ranges), and comparisons between the 2 groups were performed with the nonparametric Kruskall–Wallis test. All biomarkers were log transformed and normalized to reduce skewness and kurtosis of data. Categorical data were summarized as frequencies and percentages, and comparisons between the 2 groups were performed with Pearson chi-square test or Fisher’s exact test.
The predictors of MI at 5 years were identified by univariate Cox regression. All the variables (baseline characteristics) listed in Table 1 were studied by univariate analysis for their association with the subsequent development of MI. All biomarkers were analyzed as continuous variables, with the hazard ratios (HRs) representing an increase of one SD in the respective log-transformed biomarker. Only those univariate predictors with p <0.05 were subsequently entered into multivariate models. Multivariate Cox proportional hazard analyses were then performed as stepwise regressions with backward elimination to identify the independent predictors of MI. The variable “heart failure on presentation” was not included as a covariate in the multivariate models because of collinearity with the variable left ventricular (LV) function, which was used instead. Similarly, the estimated glomerular filtration rate using the Modification of Diet in Renal Disease formula (or eGFR-MDRD) was used instead of serum creatinine in the multivariate models. Six multivariate models were created based on pathophysiological rationale that included various combinations of clinical variables and biomarkers. The proportional hazards model assumption was tested by interacting covariates with time within the statistical model; lack of significant effects for these covariates thus indicated constant hazard over time. Time-to-event at 5 years was presented with Kaplan–Meier curves for the outcome of MI. For patients who experienced more than one MI during the follow-up period, only the first MI was used in the generation of the Kaplan–Meier curves. Comparison between the 2 groups defined by the upper tertile value was performed with the log-rank test. All analyses used 2-sided tests with an overall significance level of alpha = 0.05. All statistical analyses were performed using SAS (version 8) statistical software package (Cary, North Carolina).
| Characteristics | MMP-3 (ng/ml) | p Value | |
|---|---|---|---|
| <20.56 (N = 237) | >=20.56 (N = 118) | ||
| Age (yrs) | 0.0058 | ||
| Mean | 64.4 (10.1) | 67.5 (9.4) | |
| Median (25 th , 75 th ) | 65.1 (55.3, 73.1) | 68.6 (61.5, 74.7) | |
| 0.9462 | |||
| Black | 76 (32.1%) | 38 (32.2%) | |
| Hispanic | 68 (28.7%) | 32 (27.1%) | |
| White | 93 (39.2%) | 48 (40.7%) | |
| Family history of premature coronary artery disease | 63 (26.6%) | 24 (20.3%) | 0.1976 |
| Diabetes mellitus ∗ | 95 (40.1%) | 59 (50.0%) | 0.0758 |
| Hypertension † | 194 (81.9%) | 102 (86.4%) | 0.2744 |
| Hyperlipidemia ‡ | 122 (51.5%) | 68 (57.6%) | 0.2737 |
| Active tobacco use | 81 (34.2%) | 34 (28.8%) | 0.3090 |
| BMI (kg/m 2 ) | 0.8751 | ||
| Mean (SD) | 28.6(5.8) | 28.5(5.5) | |
| Median (25 th , 75 th ) | 28.2 (24.2, 31.7) | 27.3 (25.4, 30.8) | |
| Atrial fibrillation | 16 (6.8%) | 12 (10.2%) | 0.2603 |
| Aspirin use | 200 (84.4%) | 102 (86.4%) | 0.6092 |
| Beta blocker use | 163 (68.8%) | 78 (66.1%) | 0.6111 |
| ACE inhibitor use | 130 (54.9%) | 86 (72.9%) | 0.0010 |
| ARB use | 14 (5.9%) | 7 (5.9%) | 0.9925 |
| Statin use | 118 (49.8%) | 64 (54.2%) | 0.4236 |
| Fibrate use | 12 (5.1%) | 3 (2.5%) | 0.2630 |
| LV systolic function | 0.0828 | ||
| EF >=55% | 85 (39.0%) | 34 (29.6%) | |
| EF 45-54% | 53 (24.3%) | 29 (25.2%) | |
| EF 31-44% | 54 (24.8%) | 27 (23.5%) | |
| EF <=30% | 26 (11.9%) | 25 (21.7%) | |
| Number of narrowed coronary arteries § | 0.7680 | ||
| 0 | 47 (19.8%) | 22 (18.6%) | |
| 1 | 35 (14.8%) | 20 (17.0%) | |
| 2 | 64 (27.0%) | 29 (24.6%) | |
| 3 | 77 (32.5%) | 43 (36.4%) | |
| 4 | 14 (5.9%) | 4 (3.4%) | |
| Prior CABG | 22 (9.3%) | 9 (7.6%) | 0.6027 |
| Heart failure on presentation | 56 (23.6%) | 40 (33.9%) | 0.0402 |
| MI on presentation | 72 (30.4%) | 31 (26.3%) | 0.4217 |
| STEMI | 15 (6.3%) | 7 (5.9%) | 0.8838 |
| NSTEMI | 57 (24.1%) | 24 (20.3%) | 0.4324 |
| Unstable angina | 47 (19.8%) | 25 (21.4%) | 0.7355 |
| ACS | 119 (50.2%) | 56 (47.5%) | 0.6250 |
| Troponin I (ng/ml) | 0.5397 | ||
| Mean (SD) | 10.5(51.7) | 10.7(42.1) | |
| Median (25 th , 75 th ) | 0.3(0.2,2.3) | 0.3(0.2, 1.0) | |
| Serum creatinine (mg/dl) | <0.0001 | ||
| Mean (SD) | 1.1 (0.3) | 1.7 (2.1) | |
| Median (25 th , 75 th ) | 1.0 (0.9, 1.2) | 1.2 (1.0, 1.4) | |
| eGFR by MDRD (ml/min/1.73 m 2 ) | <0.0001 | ||
| Mean (SD) | 86.2 (23.2) | 67.7 (24.4) | |
| Median (25 th , 75 th ) | 83 (71, 94) | 69 (58,80) | |
| hs-CRP (mg/l) | 0.3925 | ||
| Mean (SD) | 21.5 (34.9) | 30.3 (51.1) | |
| Median (25 th , 75 th ) | 9.2 (3.6,18.2) | 9.7 (4.3, 23.8) | |
| NT-pro-BNP (ng/l) | 0.0217 | ||
| Mean (SD) | 1244 (978) | 1593 (1229) | |
| Median (25 th , 75 th ) | 881 (499, 1697) | 1197 (647, 2218) | |
| Myeloperoxidase (ng/ml) | 0.0994 | ||
| Mean (SD) | 24.9 (20.2) | 26.5 (16.8) | |
| Median (25 th , 75 th ) | 19.0 (13.2, 29.1) | 20.4 (15.4, 35.0) | |
| Hemoglobin (g/dl) | 0.0022 | ||
| Mean (SD) | 13.6 (1.6) | 12.9 (1.9) | |
| Median (25 th , 75 th ) | 13.8 (12.6, 14.8) | 13.2 (12.0, 14.3) | |
∗ Patients were diagnosed with diabetes mellitus if they had ever been treated with an insulin regimen or an oral hypoglycemic agent, or if they were documented to have a random glucose value of ≥ 200 mg/dl before the current presentation.
† Patients were diagnosed with hypertension if they were documented to have a blood pressure of ≥ 140/90 mm Hg on ≥ 2 occasions or were on antihypertensive therapy.
‡ Patients were diagnosed with hyperlipidemia if they had been given lipid-lowering medications or had a history of total cholesterol levels > 240 mg/dl.
§ Takes into account the left main, left anterior descending, left circumflex, and right coronary arteries.
Results
A total of 389 patients were enrolled in the study. However, MMP-3 values were available for only 355 of these patients and formed the basis for the current analysis. As stated in the Methods section, patients were categorized into 2 groups using the upper tertile of MMP-3 values as a cutoff. The baseline characteristics of the study population using this stratification are listed in Table 1 . Elevated MMP-3 levels were associated with older age, the presence of heart failure, greater use of angiotensin-converting enzyme (ACE) inhibitor, worse renal function, and anemia. In addition, the levels of MMP-3 were also positively correlated with those of NT-proBNP.
There were a total of 104 MIs in the entire cohort during the 5 years of follow-up period. The following baseline variables were significant for their association with the development of subsequent MI at 5 years with p <0.05 on univariate analysis: diabetes mellitus, heart failure on presentation, LV function, the extent of angiographically defined coronary artery disease, MI on presentation, a history of previous coronary artery bypass graft (CABG) surgery, serum creatinine, eGFR-MDRD, as well as the biomarkers MMP-3, NT-proBNP, MPO, hs-CRP, and hemoglobin. Together with MMP-3 (analyzed as a continuous variable), all baseline clinical, laboratory, and angiographic variables that were significant for their association with the subsequent development of MI with p <0.05 on univariate analysis were entered into 6 different multivariate models ( Table 2 ). All 6 models included MMP-3 in the selection process, as it was significant on univariate analysis and the focus of the investigation. In each and every one of these models, MMP-3 emerged as an independent predictor of the subsequent development of MI out to 5 years, with a HR ranging between 1.25 and 1.42.
| Multivariate Model | Variable | p-value | HR (95% CI) |
|---|---|---|---|
| Model 1 | MMP-3 | 0.0023 | 1.42 (1.13, 1.79) |
| Diabetes mellitus | 0.0088 | 1.74 (1.15, 2.63) | |
| LV function | 0.0047 | 1.32 (1.09, 1.59) | |
| CAD extent | 0.0055 | 1.29 (1.08, 1.54) | |
| Model 2 | MMP-3 | 0.0023 | 1.42 (1.13, 1.79) |
| Diabetes mellitus | 0.0088 | 1.74 (1.15, 2.63) | |
| LV function | 0.0047 | 1.32 (1.09, 1.59) | |
| CAD extent | 0.0055 | 1.29 (1.08, 1.54) | |
| Model 3 | MMP-3 | 0.0110 | 1.32 (1.07, 1.63) |
| Diabetes mellitus | 0.0007 | 1.99 (1.34, 2.96) | |
| CAD extent | 0.0015 | 1.32 (1.12, 1.56) | |
| NT-proBNP | 0.0002 | 1.49 (1.21, 1.83) | |
| Model 4 | MMP-3 | 0.0054 | 1.39 (1.10, 1.75) |
| Diabetes mellitus | 0.0014 | 2.05 (1.32, 3.17) | |
| LV function | 0.0056 | 1.31 (1.08, 1.59) | |
| CAD extent | 0.0211 | 1.24 (1.03, 1.50) | |
| Myeloperoxidase | 0.0022 | 1.37 (1.12, 1.69) | |
| Model 5 | MMP-3 | 0.0217 | 1.32 (1.04, 1.65) |
| Diabetes mellitus | 0.0306 | 1.59 (1.04, 2.41) | |
| LV function | 0.0045 | 1.32 (1.09, 1.60) | |
| CAD extent | 0.0248 | 1.23 (1.03, 1.48) | |
| Hemoglobin | 0.0022 | 0.77 (0.66, 0.91) | |
| Model 6 | MMP-3 | 0.0459 | 1.25 (1.00, 1.56) |
| NT-proBNP | 0.0026 | 1.39 (1.12, 1.73) | |
| Myeloperoxidase | 0.0262 | 1.23 (1.03, 1.49) | |
| Hemoglobin | 0.0037 | 0.78 (0.65, 0.92) |
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