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Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are thought to be associated with coronary artery disease. The association of these markers with incident coronary artery disease has not been well described.
Using a case–cohort design, we selected 216 individuals who had incident coronary artery disease (case group) and 225 individuals from a cohort random sample (comparison group) from participants enrolled in the Atherosclerosis Risk in Communities study. We measured plasma levels of MMP-1 and TIMP-1, traditional risk factors, and other markers of inflammation.
We found no significant difference in TIMP-1 levels between the case group (827.8 ± 23.8 ng/mL) and the comparison group (819.31 ± 16.1 ng/mL) (P=0.77), and no significant difference in the frequency of MMP-1 levels that were dichotomized at the minimum detectable value of 1.7 ng/mL (P=0.49). In models adjusted for age, sex, race, body mass index, hypertension, diabetes, total cholesterol, high-density lipoprotein cholesterol, triglycerides, fibrinogen, von Willebrand factor, and white blood cell count, the hazard-rate ratio for incident coronary artery disease was 1.14 (95% confidence interval, 0.63–2.04; P=0.67) for individuals whose TIMP-1 levels were above, versus at or below the mean, and 1.17 (95% confidence interval, 0.63–2.19; P=0.62) for individuals whose MMP-1 levels were above 1.7 ng/mL.
We conclude that TIMP-1 and MMP-1 levels in plasma were not predictive of incident coronary artery disease in a case–cohort random sample of the Atherosclerosis Risk in Communities study, a population study of asymptomatic middle-aged adults who had no prevalent atherosclerosis upon enrollment.
Components of the inflammatory cascade that is thought to be associated with atherogenesis and plaque rupture have been identified during the last 2 decades and have been examined as risk factors for atherosclerotic vascular disease. Among these are matrix metalloproteinases (MMPs), a family of enzymes that play an important role in the degradation of collagen and other extracellular matrix macromolecules.1,2 By degrading the insoluble fibrillar components of the fibrous cap,3 MMPs are also thought to have a strong influence on plaque rupture. The activity of MMP is tightly regulated by various endogenous inhibitors, including another family of proteins—tissue inhibitors of matrix metalloproteinases (TIMPs).2,4,5
Inflammatory cytokines induce MMP expression in endothelial cells, smooth muscle cells, and macrophages. It has been proposed that an imbalance between MMPs and their respective tissue inhibitors constitutes a causal factor for cardiovascular disease.
Although results of research studies have suggested a role for MMP-1 and TIMP-1 in the pathophysiology of atherosclerosis, relatively few prospective epidemiologic studies have investigated the relationship between circulating plasma MMP-1 and TIMP-1 levels and the occurrence of coronary artery disease (CAD). We hypothesized that elevated levels of MMP-1 and TIMP-1 are associated with incident CAD, and we sought to discover a possible relationship between levels of these substances and incident CAD in the Atherosclerosis Risk in Communities (ARIC) study, a prospective study of atherosclerosis and cardiovascular disease incidence.
We selected all of our group from 15,792 participants in the ARIC study. The individuals were initially 45 to 64 years of age and had been sampled from 4 communities in the United States: Forsyth County, North Carolina; Jackson, Mississippi (black persons only); the northwestern suburbs of Minneapolis, Minnesota; and Washington County, Maryland. A complete description of the design, objectives, sampling approaches, and examination techniques of the ARIC study has been published previously.6 For our analysis, we used a case–cohort design: we compared 216 patients who had incident CAD (case group) with 225 individuals (comparison group) whom we selected from a cohort random sample of ARIC participants. Both groups were stratified on the basis of age, sex, ultrasonographic examination of the carotid arteries, and community of enrollment. Only persons who had no prevalent CAD at visit 2 of the ARIC study (from 1990 through 1992—the baseline for this analysis) were eligible for the analysis. We excluded individuals whose CAD prevalence data were missing and those who were neither black nor white. The patients chosen for our case group had developed incident CAD events between visit 2 of the ARIC study and 31 December 2001. Such events were defined as coronary revascularization, a definite or probable myocardial infarction, a between-examinations silent myocardial infarction that was later detected on electrocardiography, or death that was definitely due to CAD. Incident CAD events were identified by means of an annual questionnaire, clinic visits at approximately 3-year intervals, and hospital and death-certificate surveillance.7 The diagnoses were made by a panel of physicians who had used all available information and standard criteria.
All of our laboratory measurements were performed on frozen samples that had been obtained at visit 2 and had been stored at −70 °C. We ascertained baseline measurements (as of visit 2 of the ARIC study, for this analysis) for the variables, including cigarette smoking, hypertension, diabetes mellitus, body mass index (BMI), and waist and hip circumferences, in accordance with previously described definitions and methods.8,9 Plasma lipid levels and hemostatic factors were measured at centralized laboratories by standardized and validated methods.10–12 We measured the levels of MMP-1 and TIMP-1 in the plasma samples by using commercially available assays (R&D Systems Inc.; Minneapolis, Minn). The minimum detectable value of MMP-1 was 1.7 ng/mL, and the minimum detectable value for TIMP-1 was 1.25 ng/mL. The reliability coefficients for 11 blinded split-pair samples of MMP-1 and for 20 such samples of TIMP-1 were 0.74 and 0.62, respectively.
Because of different sampling fractions across the strata for the cohort random sample (comparison group), all analyses were weighted. The proportions, means, and standard errors of established cardiovascular risk factors were reported as results of weighted analyses for the 2 groups (baseline characteristics, Table I) and were calculated by use of STATA Release 9 (StataCorp; College Station, Tex).
We used Cox proportional hazards models to evaluate TIMP-1 that was dichotomized at the population mean (>856.9 ng/mL vs ≤856.9 ng/mL) and to evaluate MMP-1 that was dichotomized at the minimum detectable level (>1.7 ng/mL vs ≤1.7 ng/mL) (Table II). The method of Barlow13 was used to adjust for the sampling approach in the Cox proportional hazards modeling. For the case group, the follow-up interval was defined as the time between visit 2 of the ARIC study and the date of the 1st CAD event, the date of death, the date of last contact, or 31 December 2001, whichever occurred first. For the comparison group from the cohort random sample, follow-up was from visit 2 of the ARIC study until the date of death, the date of last contact, or 31 December 2001, whichever occurred first. The established cardiovascular risk factors that we evaluated as potential confounders in the 3 Cox models included age, sex, race, BMI, hypertension, diabetes mellitus, von Willebrand factor, and white blood cell count, and levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, and fibrinogen. Covariates were evaluated for statistical significance by use of the Wald χ2 statistic; P <0.05 was considered statistically significant.
Using weighted t tests, we evaluated possible associations by considering the comparison group's mean levels of various variables. These variables included age, BMI, waist-to-hip ratio, white blood cell count, fibrinogen, von Willebrand factor, and levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and HDL-C. We associated these with TIMP-1 that was dichotomized at its mean (856.9 ng/mL) and for MMP-1 that was dichotomized at 1.7 ng/mL, the minimum level of detection (Table III).
Individuals with incident CAD (case group) were more likely than those in the comparison group from the cohort random sample to have diabetes mellitus, higher waist-to-hip ratios, higher white blood cell counts, higher levels of total cholesterol, LDL-C, and triglycerides, and lower levels of HDL-C. However, no significant differences were noted between the groups with respect to measurements of hemostasis, such as fibrinogen and von Willebrand factor. There were no significant differences between the groups in MMP-1 levels >1.7 ng/mL (P=0.49) or in mean TIMP-1 levels (case group, 827.8 ± 23.8 ng/mL vs comparison group, 819.31 ± 16.1 ng/mL; P=0.77).
When dichotomized at the mean (>856.9 vs ≤856.9 ng/mL), TIMP-1 levels were not associated with incidence of CAD in the various analytical models (Table II). For TIMP-1 dichotomized at the mean, the hazard-rate ratios were 1.25 (95% confidence interval [CI], 0.80–1.95; P=0.33) in a model adjusted for age, sex, and race; 1.03 (95% CI, 0.6–1.77; P=0.91) in a model adjusted for age, sex, race, BMI, hypertension, diabetes mellitus, total cholesterol, and HDL-C; and 1.14 (95% CI, 0.63–2.04; P=0.67) in a model adjusted for age, sex, race, BMI, hypertension, diabetes mellitus, total cholesterol, HDL-C, triglycerides, fibrinogen, von Willebrand factor, and white blood cell count. When evaluated as a dichotomous variable (levels >1.7 ng/mL vs levels ≤1.7 ng/mL), MMP-1 level was also not significantly associated with incident CAD in the respective models, with hazard-rate ratios of 1.20 (95% CI, 0.77–1.88; P=0.43), 0.92 (95% CI, 0.52–1.65; P= 0.78), and 1.17 (95% CI, 0.63–2.19; P= 0.67) (Table II).
When potential associations of TIMP-1 with various variables were examined in the comparison group from the cohort random sample, participants with a TIMP-1 level above the mean were older (P <0.01) and had a higher BMI (P=0.01), a higher waist-to-hip ratio (P <0.01), significantly higher levels of fibrinogen (P=0.04) and von Willebrand factor (P=0.03), and significantly lower HDL-C levels (P <0.01) (Table III). There were no differences in the prevalence of diabetes mellitus or hypertension, smoking, or BMI between individuals in the comparison group who had TIMP-1 levels above the mean, versus below. On the other hand, when MMP-1 levels were dichotomized at 1.7 ng/mL, there were no significant differences between comparison-group individuals who had MMP-1 levels >1.7 ng/mL versus ≤1.7 ng/mL (Table III).
In the ARIC study, TIMP-1 and MMP-1 levels were tested, and no association with incident CAD was found. The results of animal and other human studies into the possible role of different MMPs and TIMPs in CAD have been inconsistent. Although many have shown an association between MMPs or TIMPs and CAD,14–18 another study19 found no connection. Similarly, there has been inconsistent association between the various members of the MMP or TIMP families and CAD—for example, a 2002 study20 showed an atheroprotective counter-regulatory function of TIMP-1, whereas a more recent study4 raised the question of whether TIMP-1 is atherogenic. When plasma levels of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were measured in 53 men who had premature stable CAD,16 MMP-9 and TIMP-1 were significantly higher and MMP-3 and TIMP-2 were significantly lower in the CAD patients than in the control group of 133 age-matched men. Similarly, MMP-9 and TIMP-1 were increased and MMP-2 and TIMP-2 were decreased in 200 men who had premature CAD, in comparison with a control group of 201 age-matched men.17
In other studies,4,19,21 TIMP-1 has been associated with all-cause mortality, myocardial infarction, and acute coronary syndromes. An elevated TIMP-1 level was associated with the presence of carotid plaque in 238 men who were known to be free of CAD, in a multivariate model that was adjusted for age, BMI, smoking, total cholesterol and triglycerides, C-reactive protein (CRP) level, diabetes mellitus, systolic blood pressure, and heart rate (odds ratio; 2.89; 95% CI, 1.12–7.47; P <0.01). Although TIMP-1 was also associated with common carotid artery intima–media thickness in a univariate analysis,22 the association was not present in a multivariate analysis.22 On the other hand, in yet another report,23 MMP-9 and TIMP-2, but not TIMP-1, were elevated in 204 patients who had stable CAD, in comparison with a control group.
Investigations into the role of MMPs and TIMPs in the pathobiology of atherosclerosis have also produced varying results. Increased expression of interstitial collagenase (MMP-1) has been described more often in vulnerable atherosclerotic plaques than in lesion-free areas of the vessels.24 Another investigation25 showed that CRP (an inflammatory marker that is associated with CAD) augmented MMP-1 and MMP-10 mRNA expression in human umbilical-vein endothelial cells, and that MMP-1 and MMP-10 were significantly elevated in persons whose CRP levels were greater than 3 mg/L.
Overexpression of TIMP-1 by adenovirus-mediated gene transfer has been shown to inhibit smooth-muscle-cell migration and neointimal formation in human saphenous veins,26 and the adenovirus-mediated overexpression of TIMP-1 in atherosclerosis-susceptible apolipoprotein E-deficient mice significantly reduced atherosclerotic lesions.27 However, the investigators of that murine model also reported28 that in apolipoprotein E-deficient mice that were overexpressing TIMP-1, the formation of aneurysms was decreased, but the development of atherosclerosis was not. Similarly, MMP-1 and MMP-3 levels were reported to be higher in aneurysmal human atherosclerotic plaques than in occlusive lesions, whereas TIMP-1 was associated with calcification, a marker of plaque stability.29 In another apolipoprotein E-deficient murine model, in which the mice were maintained on a high-fat diet, transfection with TIMP-2 but not with TIMP-1 resulted in the inhibition of atherosclerotic plaque development and of plaque destabilization in the brachial artery. The authors concluded that TIMP-2 (and not TIMP-1) was an effective inhibitor of plaque growth, and that this inhibition may be secondary to the effect of TIMP-2 on the behaviors of smooth muscle cells and macrophages.30
Hence, the available human and animal data on the potential associations of MMPs and TIMPs with CAD have not been conclusive. Our study has the advantages of 1) being a population-based study that provides information on a middle-aged U.S. population rather than on a high-risk patient cohort that has established atherosclerosis, and 2) having a longer follow-up than that in other investigations. The lack of association of TIMP-1 and MMP-1 with incident CAD in the current study provides important additional information on the unclear relationship between MMPs or TIMPs and CAD. Perhaps the balance or the ratio between MMPs and TIMPs is more important in determining the contribution of these markers to atherosclerosis. Matrix metalloproteinases are known to have a role in degrading collagen,1 and TIMPs, as described, act as counter-regulatory enzymes. Therefore, the TIMP level may increase only when the MMP level also increases. In our study, there was no difference between the case and comparison groups with respect to detectable MMP levels, and perhaps this also accounted for there being no difference in the TIMP-1 levels.
Matrix metalloproteinases and TIMPs have been associated with established risk factors for atherosclerosis.31–34 The Framingham Heart Study investigators31 reported that TIMP-1 was associated with all major cardiovascular risk factors and hypothesized that the risk factors influence vascular and cardiac remodeling via extracellular matrix degradation. A report from another study32 described significantly higher MMP-9 and TIMP-1 levels in patients who had hypertension than in control-group patients, and that MMP-9 levels decreased and TIMP-1 levels increased after antihypertensive treatment. In our investigation, the prevalence of hypertension was not significantly different between our 2 groups; however, there were significant differences in serum cholesterol levels and diabetes mellitus (adjusted for in the various models). In our comparison group, TIMP-1 level was associated with some markers, such as fibrinogen and von Willebrand factor. Further evaluation will be required to understand better whether MMPs and TIMPs are primarily involved in atherogenesis and plaque rupture, whether they are primarily responsive to other traditional risk factors, and what their effects are on the vasculature and on the inflammatory process that promotes atherosclerosis development.
Several limitations of our study should be noted. The assay with which we analyzed MMP-1 detected only levels greater than 1.7 ng/mL, and most of our patients had levels of less than 1.7 ng/mL; therefore, ratios of MMP to TIMP could not be determined. Only MMP-1 and TIMP-1 were measured, and not the other metalloproteinases (including MMP-9) and their inhibitors. The tested samples were stored at −70 °C until they were analyzed, and the protracted storage may have led to the degradation or modification of MMP-1 or TIMP-1; however, any effect would have been similar for both the case and comparison groups. Finally, we examined only plasma levels of metalloproteinases, which may not correlate with tissue levels in arterial plaques.
Despite these limitations, the data from our analyses suggest that plasma levels of MMP-1 and TIMP-1 were not predictive of incident CAD events in the ARIC population of asymptomatic middle-aged adults who had no atherosclerosis upon enrollment. The exact role of MMP-1 and TIMP-1 in CAD will need better characterization.
The authors thank the staff and participants of the ARIC study for their important contributions, and Joanna Brooks, BA, for her editorial assistance.
Address for reprints: Vijay Nambi, MD, STE-B-160/MS-A601;6565 Fannin St., Houston, TX 77030. E-mail: ude.cmt.mcb@ibmanv
Grant Support: The Atherosclerosis Risk in Communities study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts N01-HC-55015 and N01-HC-55016, and N01-HC-55018 through N01-HC-55022.