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High plasma levels of matrix metalloproteinase-8 in patients with unstable angina
Atherosclerosis 209 (2010) 206–210
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
High plasma levels of matrix metalloproteinase-8 in patients with unstable angina Yukihiko Momiyama a,∗ , Reiko Ohmori c , Nobukiyo Tanaka b , Ryuichi Kato b , Hiroaki Taniguchi b , Takeshi Adachi b , Haruo Nakamura b , Fumitaka Ohsuzu b a b c
Division of Cardiology, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan First Department of Internal Medicine, National Defense Medical College, Saitama, Japan Faculty of Education, Utsunomiya University, Utsunomiya, Japan
a r t i c l e
i n f o
Article history: Received 5 June 2009 Received in revised form 7 July 2009 Accepted 8 July 2009 Available online 25 July 2009 Keywords: Metalloproteinase Unstable angina
a b s t r a c t Matrix metalloproteinases (MMPs) play a role in collagen breakdown, leading to plaque instability. High levels of MMPs mRNA and proteins, especially MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, were shown in human atherosclerotic plaques. However, among various MMPs, only MMP-1, MMP-8 and MMP-13, socalled interstitial collagenases, can initiate collagen breakdown. To elucidate whether MMP-1, MMP-8 and MMP-13 levels in blood were high in patients with unstable angina (UAP), we measured serum MMP-1 and plasma MMP-8 and MMP-13 levels in 45 patients with UAP, 175 with stable coronary artery disease (CAD), and 45 controls. Plasma C-reactive protein levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l). Regarding blood levels of MMPs, MMP-13 levels were above the lower detection limit in only one patient with UAP (2%), one with stable CAD (1%), and none in controls. MMP-1 levels did not differ among patients with UAP, stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml). Notably, MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005), however, MMP-8 levels in patients with UAP were much higher than those in stable CAD (3.9 ng/ml vs. 3.5 ng/ml, P < 0.05). In multivariate analysis, MMP-8 level was an independent factor for UAP. Thus, plasma MMP-8 levels were found to be high in patients with UAP, suggesting that MMP-8 levels in UAP may reflect coronary plaque instability and that MMP-8 is a promising biomarker for UAP. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interstitial collagen, especially type I, is a major component of atherosclerotic plaques [1–3]. Matrix metalloproteinases (MMPs) play an important role in the collagen breakdown that can cause plaque instability and rupture, thus leading to acute myocardial infarction (AMI) and unstable angina pectoris (UAP). High levels of mRNA and proteins of MMPs, especially MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, have been demonstrated in human atherosclerotic plaques, most profoundly in unstable plaques prone to rupture [4–9]. With respect to blood levels of MMPs, Kai et al. [10] reported serum MMP-2 and plasma MMP-9 levels in 11 patients with UAP to be higher than those in 17 with stable angina and 17 controls. Tziakas et al. [11] also showed serum MMP-2 and MMP-9 levels to be higher in 20 patients with UAP than in 16 controls. These findings suggest that MMP-2 and MMP-9 levels in blood would be biomarkers for UAP. However, among the various known MMPs,
∗ Corresponding author. Tel.: +81 03 3411 0111; fax: +81 03 3412 9811. E-mail address: [email protected] (Y. Momiyama). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.07.037
only MMP-1, MMP-8 and MMP-13, so-called interstitial collagenases, can initiate collagen breakdown, thereby making collagen fragments susceptible to further degradation by other MMPs, such as MMP-2 and MMP-9 [12]. Although three small studies reported serum MMP-1 levels to not be high in patients with UAP [11,13,14], there has so far been no report showing blood levels of either MMP8 or MMP-13 in patients with UAP. Therefore, our study was carried out to elucidate if MMP-1, MMP-8 and MMP-13 levels in blood were high in patients with UAP and if these levels could be biomarkers for UAP. 2. Methods 2.1. Study patients We measured serum MMP-1 and plasma MMP-8 and MMP-13 levels in 45 consecutive patients who had UAP at rest during the preceding 48 h, so-called class III UAP according to Braunwald’s classification [15], and 175 patients with stable coronary artery disease (CAD). They underwent coronary angiography for suspected CAD at the National Defense Medical College Hospital and were
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found to have CAD which was defined as at least one coronary artery having >50% luminal diameter stenosis on angiograms. Coronary angiograms were recorded by a femoral approach using the Judkins technique and cineangiogram system (Toshiba, Tokyo, Japan). All coronary angiograms were evaluated by Y.M., who was blinded to MMP data. The results were then compared with those of 45 age- and gender-matched controls who had coronary angiography for suspected CAD but were found to have angiographically normal coronary arteries. Because MMPs have been shown to be released from the myocardium with infarction [16–18], any patients with AMI who showed a significant increase (more than the upper normal range) in serum creatine kinase levels were excluded. Patients with a history of MI within 6 months, those with a history of percutaneous coronary intervention or coronary artery bypass surgery, or those with heart failure, cardiomyopathies or valvular heart disease were also excluded. Our study was approved by the ethics committee of the hospital. After written informed consent was obtained, blood samples in patients with UAP were taken within 24 h after their admission, and blood samples in patients with stable CAD and controls were taken on the day of angiography. 2.2. Measurements of serum MMP-1 and plasma MMP-8 and MMP-13 levels After blood samples were centrifuged at 2000 × g for 15 min at 4 ◦ C, the serum and plasma were frozen and stored at −80 ◦ C until analyzed. Serum MMP-1 levels were measured by a one-step sandwich enzyme immunoassay using a commercially available kit (Daiichi Pharmaceutical, Toyoma, Japan). This kit measures the total concentration of the precursor form, the active form, and the TIMP-1 or -2 complex forms of MMP-1 in the serum, but it is highly specific and does not cross-react with MMP-8 and MMP13 [19]. The lower detection limit of this assay was 0.1 ng/ml. Plasma MMP-8 levels were measured by a two-site sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (MMP-8 Human Biotrak ELISA System, Amersham Biosciences, Buckinghamshire, UK). This kit measures the total concentration of both the precursor and active forms of MMP-8 in the plasma, but it is highly specific and does not cross-react with either MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 or MT1MMP [20]. The lower detection limit was 2.5 ng/ml. Plasma MMP-13 levels were also measured by a two-site sandwich ELISA with a commercially available kit (MMP-13 Human Biotrak ELISA System, Amersham Biosciences). This kit measures the total concentration of the precursor and active forms of MMP-13 in the plasma, but
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it is highly specific and does not cross-react with either MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 or MT1-MMP. The lower detection limit was 0.09 ng/ml. MMP-1, MMP-8 and MMP-13 levels of all samples were measured in duplicate, and the results were then averaged. Plasma high sensitivity C-reactive protein (hsCRP) levels were also measured using a BNII nephelometer (Dade Behring, Tokyo, Japan). In patients with UAP, a rapid qualitative test for troponin T (TnT) was performed using a commercially available kit (TROP T sensitive, Roche Diagnostics, Tokyo, Japan). The lower detection limit of this test was 0.1 ng/ml, and positive TnT was defined as >0.1 ng/ml. Serum lipid levels were measured by standard laboratory methods. 2.3. Statistics Any differences between the two groups were evaluated by the unpaired t-test for parametric variables, by the Mann–Whitney Utest for nonparametric variables, and by the chi-square test for categorical variables. Any differences among the three groups were evaluated by ANOVA with the Scheffe’s test for parametric variables, by the Kruskal–Wallis test for nonparametric variables, and by the chi-square test for categorical variables. The correlation between MMP-8 and hsCRP levels was evaluated by the Spearman’s rank correlation test. A forward stepwise multiple logistic regression analysis was used to elucidate the independent association between MMP-8 levels and UAP. A P-value of <0.05 was considered to be statistically significant. The results are presented as the mean value ±SD. Since the distributions of the measured MMP-1, MMP-8 and hsCRP levels were highly skewed, these results are presented as the median value. 3. Results As shown in Table 1, there was no difference in age or gender among the three groups. Total cholesterol levels were lower in patients with UAP than in those with stable CAD and controls. The percentage of patients taking statin was 29% in patients with UAP, 29% in those with stable CAD, and 18% in controls, respectively (P = NS). The number of >50% stenotic coronary vessels was similar in patients with UAP and those with stable CAD (2.0 ± 0.8 and 1.9 ± 0.8), and 1-vessel, 2-vessel, and 3-vessel disease was present in 31%, 36%, and 33% of patients with UAP vs. 38%, 37%, and 25% of those with stable CAD (P = NS). Of the 45 patients with UAP, 10 (22%) showed positive TnT without a significant increase in creatine kinase levels.
Table 1 Clinical characteristics in three groups.
Age (years) Gender (male) Hypertension Systolic BP (mmHg) Hyperlipidemia Total cholesterol (mg/dl) HDL-cholesterol (mg/dl) Statin Diabetes mellitus Current smoker Number of >50% stenotic coronary vessels 1-Vessel disease 2-Vessel disease 3-Vessel disease
UAP (n = 45)
UAP vs. CAD
CAD (n = 175)
CAD vs. control
Controls (n = 45)
UAP vs. control
67 ± 9 34 (76%) 33 (73%) 138 ± 25 24 (53%) 190 ± 34 51 ± 12 13 (29%) 14 (31%) 18 (40%) 2.0 ± 0.8 14 (31%) 16 (36%) 15 (33%)
NS NS NS NS NS < 0.02 NS NS NS NS NS NS NS NS
66 ± 8 141 (81%) 119 (68%) 137 ± 18 76 (43%) 203 ± 32 49 ± 14 51 (29%) 62 (35%) 56 (32%) 1.9 ± 0.8 67 (38%) 65 (37%) 43 (25%)
NS NS NS NS NS NS <0.001 NS <0.01 NS
66 ± 7 34 (76%) 26 (58%) 132 ± 14 15 (33%) 206 ± 30 60 ± 15 8 (18%) 6 (13%) 12 (27%)
NS NS NS NS NS <0.05 <0.005 NS NS NS
Data are presented as the mean value ± SD or the number (%) of patients. UAP, unstable angina pectoris; CAD, coronary artery disease; BP, blood pressure. Hypertension was defined as blood pressures ≥140/90 mmHg or on medication. Hyperlipidemia was defined as total cholesterol levels >240 mg/dl or on medication. Diabetes mellitus was defined as fasting glucose levels ≥126 mg/dl or on insulin or hypoglycemic drugs.
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Fig. 1. Plasma hsCRP levels in the three groups. Plasma hsCRP levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l), but these differences did not reach statistical significance. The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
Plasma hsCRP levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l), but these differences did not reach statistical significance (Fig. 1). With respect to blood levels of MMPs, plasma MMP-13 levels were above the lower detection limit (>0.09 ng/ml) in only one (2%) patient with UAP, one (1%) patient with stable CAD, and none (0%) in controls, thus suggesting very low levels of MMP13 in the plasma. Serum MMP-1 levels were not different among patients with UAP, those with stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml, P = NS) (Fig. 2). Notably, plasma MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005) (Fig. 3). In patients with stable CAD, a stepwise increase in MMP8 levels was found depending on the number of >50% stenotic coronary vessels: 3.2 in 1-vessel, 3.5 in 2-vessel, and 4.2 ng/ml in 3vessel disease (P < 0.001). However, MMP-8 levels in patients with UAP were much higher than those in stable CAD (median 3.9 ng/ml vs. 3.5 ng/ml, P < 0.05) (Fig. 3). In patients with UAP, MMP-8 levels tended to be higher in patients with positive TnT than in those
Fig. 3. Plasma MMP-8 levels in the three groups. Plasma MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005), but MMP-8 levels in patients with UAP were much higher than those in stable CAD (3.9 ng/ml vs. 3.5 ng/ml, P < 0.05). The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
without positive TnT (4.2 ng/ml vs. 3.8 ng/ml, P = NS), but this difference did not reach statistical significance. In patients with UAP, no difference was observed in MMP-8 levels among patients with 1vessel, 2-vessel and 3-vessel disease (3.9, 4.2 and 3.7 ng/ml, P = NS). As a result, patients with UAP more often had a MMP-8 level of >3.5 ng/ml than those with stable CAD and controls (67% vs. 48% and 29%, P < 0.05). However, a significant correlation was observed between MMP-8 and hsCRP levels (r = 0.29, P < 0.001). To elucidate the independent association between MMP-8 levels and UAP, atherosclerotic risk factors (age, gender, hypertension, diabetes, smoking, hyperlipidemia, and HDL-cholesterol levels) and hsCRP and MMP-8 levels were entered into a multiple logistic regression model. Among the 265 study patients, a multivariate analysis revealed that high MMP-8 level (>3.5 ng/ml) was the only independent factor associated with UAP (odds ratio = 2.28, 95%CI = 1.14 to 4.58, P < 0.025). Even among the 220 patients with CAD (45 with UAP and 175 with stable CAD), high MMP-8 level was found to be the only independent factor associated with UAP (odds ratio = 2.10, 95%CI = 1.05 to 4.19, P < 0.05). However, hsCRP levels did not show such associations. 4. Discussion
Fig. 2. Serum MMP-1 levels in the three groups. Serum MMP-1 levels were not different among patients with UAP, those with stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml, P = NS). The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
The present study investigated serum MMP-1 and plasma MMP8 and MMP-13 levels in 45 patients with UAP compared with 175 with stable CAD and 45 controls. Although MMP-1 and MMP-13 levels were not high in patients with UAP, MMP-8 levels were found to be significantly higher in patients with UAP than in those with stable CAD and controls. In the multivariate analysis, MMP-8 levels were an independent factor associated with UAP. Among the various known MMPs, only MMP-1, MMP-8, and MMP-13, the so-called interstitial collagenases, can initiate the collagen breakdown of atherosclerotic plaques, thereby making collagen fragments susceptible to further degradation by other MMPs [12]. Although increased expressions of MMP-1 and MMP-13 were shown in atherosclerotic plaques [4,6,7], the role of MMP-8 in atherogenesis had been neglected until recently, because MMP8 had been thought to be produced only by neutrophils that are uncommonly present in plaques [23]. However, in 2001, Herman et al. [8] first demonstrated that endothelial cells, smooth muscle cells, and macrophages in atherosclerotic plaques expressed MMP-
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8 mRNA and protein, especially in lipid-rich plaques with a thin fibrous cap. They suggested that MMP-8 may play a role in the collagen breakdown of atherosclerotic plaques. Moreover, MMP-8 was reported to degrade type I collagen, which is a major component of atherosclerotic plaques, three times more potently than MMP-1 and MMP-13 [21,22]. In 2004, Molloy et al. [24] investigated MMP-1, MMP-8 and MMP-13 concentrations within carotid plaques from 159 patients undergoing carotid endarterectomy. They reported total and active MMP-8 concentrations to be higher in the plaques of symptomatic patients, those with embolism, or those with histological evidence of rupture than in those of asymptomatic patients or those with no evidence of rupture. However, no differences were observed in MMP-1 or MMP-13 concentrations. These findings suggested that, among the interstitial collagenases, MMP-8 plays the most important role in the collagen breakdown of atherosclerotic plaques leading to plaque instability. With respect to MMP-8 levels in blood, Krupinski et al. [25] measured plasma MMP-8 levels in 87 diabetic patients undergoing carotid endarterectomy. They reported patients with ulcerated carotid plaques to have higher MMP-8 levels than those with stable, fibrous plaques. Recently, Tuomainen et al. [26] investigated MMP-8 levels in 905 men with no history of CAD with a 10-year follow-up. They showed MMP-8 levels to be associated with cardiovascular events, such as AMI and death from CAD. Although we previously reported plasma MMP-8 levels in patients with stable CAD to be higher than those without CAD and to correlated with the severity of coronary stenosis [27], our present study, for the first time, showed plasma MMP-8 levels to be much higher in patients with UAP than in those with stable CAD and to be associated with UAP independent of traditional risk factors and hsCRP levels. Our findings suggest that MMP-8 levels could be a biomarker for UAP and that plasma MMP-8 levels in patients with UAP may reflect coronary plaque instability. Regarding MMP-1 levels, Fukuda et al. [14] reported serum MMP-1 levels to not be higher in 23 patients with UAP than in 19 with stable angina. The other small studies also showed serum MMP-1 levels to not be high in patients with UAP [11,13]. Based on the same line of evidence, our study confirmed no significant difference to exist in serum MMP-1 levels among patients with UAP, those with stable CAD, or controls. Although there is no previous report showing MMP-13 levels in patients with UAP, our study, for the first time, showed MMP-13 to be detected within plasma in only 2% of patients with UAP and 1% of patients with stable CAD, thus suggesting a relatively low abundance of MMP-13 in the plasma of patients with UAP. Thus, among the blood levels of interstitial collagenases, plasma MMP-8 levels are considered to be the most promising biomarker for UAP. Our study has several limitations. First, in our study, 29% of patients with UAP, 29% of those with stable CAD and 18% of controls were taking statin. Because statins have been reported to reduce MMP-1 and MMP-9 expressions in rabbit atheroma [28] and to inhibit MMP-1 and MMP-9 secretion from rabbits smooth muscle cells and macrophages [29], the inclusion of patients who were taking statins may therefore have confounded our results of blood levels of MMPs. However, now that statin use in patients with CAD is very common, the exclusion of patients taking statins would render our results inapplicable to the general patients with CAD. Second, since we did not measure MMP-8 levels in the coronary sinus, our study could not determine the main sources of plasma MMP-8 in patients with UAP. Peripheral leukocytes/macrophages or atherosclerotic plaques in other vascular beds than the coronary arteries may be a source of high MMP-8 levels. Moreover, as shown in Fig. 3, there was some overlap in MMP-8 levels between patients with UAP and those with stable CAD. Plasma MMP-8 levels in patients with stable CAD correlated with the number of stenotic coronary vessels, thus suggesting that MMP-8 levels in patients with stable CAD may reflect the degree of coronary atherosclerosis.
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Therefore, plasma MMP-8 levels in patients with UAP may reflect the degree of coronary atherosclerosis as well as coronary plaque instability. Third, UAP with positive TnT has become recently considered as non-ST elevation MI [30]. However, in our study, UAP was defined as Braunwald class III UAP without a significant increase in creatine kinase levels, like the previous studies [10,13,14] showing MMP levels in UAP. As a result, of the 45 patients with UAP, 10 (22%) showed positive TnT, suggestive of some myocardial injury and nonST elevation MI. MMP-8 levels tended to be higher in UA patients with positive TnT than in those without positive TnT, but this difference did not reach statistical significance. To elucidate whether or not MMP-8 levels were much higher in UAP patients with positive TnT than without positive TnT, a further study in a larger number of UAP patients with and without positive TnT is needed. Finally, the number of our patients with UAP was relatively small (45 patients), and our study had no follow-up data. To confirm the diagnostic value of plasma MMP-8 levels for UAP and to elucidate the prognostic value of MMP-8 levels in patients with UAP, a further study with follow-up in a larger number of patients with UAP is needed. In conclusion, among the blood levels of MMP-1, MMP-8, and MMP-13, the so-called interstitial collagenases, only plasma MMP-8 levels were found to be high in patients with UAP and to be associated with UAP independent of hsCRP levels. Our results suggest that plasma MMP-8 levels in patients with UAP may therefore reflect coronary plaque instability and that MMP-8 levels would be the most promising biomarker for UAP among interstitial collagenases. References [1] Shekhonin BV, Domogatsky SP, Muzykantov VR, et al. Distribution of type I, III, IV and V collagen in normal and atherosclerotic human arterial wall: immunomorphological characteristics. Collagen Related Res 1985;5:335–68. [2] Mayne R. Collagenous proteins of blood vessels. Arteriosclerosis 1986;6:585–93. [3] Rekhter MD, Zhang K, Narayanan AS, et al. Type I collagen gene expression in human atherosclerosis: localization to specific plaque regions. Am J Pathol 1993;143:1634–48. [4] Galis ZS, Sukhova GK, Lark MW, et al. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493–503. [5] Brown DL, Hibbs MS, Kearney M, et al. Identification of 92-kDa gelatinase in human coronary atherosclerotic lesions: association of active enzyme synthesis with unstable angina. Circulation 1995;91:2125–31. [6] Nikkari ST, O’Brien KD, Ferguson M, et al. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation 1995;92:1393–8. [7] Sukhova GK, Schonbeck U, Rabkin E, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 1999;99:2503–9. [8] Herman MP, Sukhova GK, Libby P, et al. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation 2001;104:1899–904. [9] Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863–8. [10] Kai H, Ikeda H, Yasukawa H, et al. Peripheral blood levels of matrix metalloproteases-2 and -9 are elevated in patients with acute coronary syndromes. J Am Coll Cardiol 1998;32:368–72. [11] Tziakas DN, Chalikias GK, Parissis JT, et al. Serum profiles of matrix metalloproteinases and their tissue inhibitor in patients with acute coronary syndromes: the effects of short-term atorvastatin administration. Int J Cardiol 2004;94:269–77. [12] Owen CA, Campbell EJ. The cell biology of leukocyte-mediated proteolysis. J Leukoc Biol 1999;65:137–50. [13] Inoue T, Kato T, Takayanagi K, et al. Circulating matrix metalloproteinase-1 and -3 in patients with an acute coronary syndrome. Am J Cardiol 2003;92:1461–4. [14] Fukuda D, Shimada K, Tanaka A, et al. Comparison of levels of serum matrix metalloproteinase-9 in patients with acute myocardial infarction versus unstable angina pectoris versus stable angina pectoris. Am J Cardiol 2006;97:175–80. [15] Braunwald E. Unstable angina: a classification. Circulation 1989;80:410–4. [16] Etoh T, Joffs C, Deschamps AM, et al. Myocardial and interstitial matrix metalloproteinase activity after acute myocardial infarction in pigs. Am J Physiol Heart Circ Physiol 2001;281:H987–94. [17] Bradham WS, Gunasinghe H, Holder JR, et al. Release of matrix metalloproteinases following alcohol septal ablation in hypertrophic obstructive cardiomyopathy. J Am Coll Cardiol 2002;40:2165–73. [18] Webb CS, Bonnema DD, Ahmed SH, et al. Specific temporal profile of matrix metalloproteinase release occurs in patients with myocardial infarction: relation to left ventricular remodeling. Circulation 2006;114:1020–7.
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Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
High plasma levels of matrix metalloproteinase-8 in patients with unstable angina Yukihiko Momiyama a,∗ , Reiko Ohmori c , Nobukiyo Tanaka b , Ryuichi Kato b , Hiroaki Taniguchi b , Takeshi Adachi b , Haruo Nakamura b , Fumitaka Ohsuzu b a b c
Division of Cardiology, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan First Department of Internal Medicine, National Defense Medical College, Saitama, Japan Faculty of Education, Utsunomiya University, Utsunomiya, Japan
a r t i c l e
i n f o
Article history: Received 5 June 2009 Received in revised form 7 July 2009 Accepted 8 July 2009 Available online 25 July 2009 Keywords: Metalloproteinase Unstable angina
a b s t r a c t Matrix metalloproteinases (MMPs) play a role in collagen breakdown, leading to plaque instability. High levels of MMPs mRNA and proteins, especially MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, were shown in human atherosclerotic plaques. However, among various MMPs, only MMP-1, MMP-8 and MMP-13, socalled interstitial collagenases, can initiate collagen breakdown. To elucidate whether MMP-1, MMP-8 and MMP-13 levels in blood were high in patients with unstable angina (UAP), we measured serum MMP-1 and plasma MMP-8 and MMP-13 levels in 45 patients with UAP, 175 with stable coronary artery disease (CAD), and 45 controls. Plasma C-reactive protein levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l). Regarding blood levels of MMPs, MMP-13 levels were above the lower detection limit in only one patient with UAP (2%), one with stable CAD (1%), and none in controls. MMP-1 levels did not differ among patients with UAP, stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml). Notably, MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005), however, MMP-8 levels in patients with UAP were much higher than those in stable CAD (3.9 ng/ml vs. 3.5 ng/ml, P < 0.05). In multivariate analysis, MMP-8 level was an independent factor for UAP. Thus, plasma MMP-8 levels were found to be high in patients with UAP, suggesting that MMP-8 levels in UAP may reflect coronary plaque instability and that MMP-8 is a promising biomarker for UAP. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interstitial collagen, especially type I, is a major component of atherosclerotic plaques [1–3]. Matrix metalloproteinases (MMPs) play an important role in the collagen breakdown that can cause plaque instability and rupture, thus leading to acute myocardial infarction (AMI) and unstable angina pectoris (UAP). High levels of mRNA and proteins of MMPs, especially MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, have been demonstrated in human atherosclerotic plaques, most profoundly in unstable plaques prone to rupture [4–9]. With respect to blood levels of MMPs, Kai et al. [10] reported serum MMP-2 and plasma MMP-9 levels in 11 patients with UAP to be higher than those in 17 with stable angina and 17 controls. Tziakas et al. [11] also showed serum MMP-2 and MMP-9 levels to be higher in 20 patients with UAP than in 16 controls. These findings suggest that MMP-2 and MMP-9 levels in blood would be biomarkers for UAP. However, among the various known MMPs,
∗ Corresponding author. Tel.: +81 03 3411 0111; fax: +81 03 3412 9811. E-mail address: [email protected] (Y. Momiyama). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.07.037
only MMP-1, MMP-8 and MMP-13, so-called interstitial collagenases, can initiate collagen breakdown, thereby making collagen fragments susceptible to further degradation by other MMPs, such as MMP-2 and MMP-9 [12]. Although three small studies reported serum MMP-1 levels to not be high in patients with UAP [11,13,14], there has so far been no report showing blood levels of either MMP8 or MMP-13 in patients with UAP. Therefore, our study was carried out to elucidate if MMP-1, MMP-8 and MMP-13 levels in blood were high in patients with UAP and if these levels could be biomarkers for UAP. 2. Methods 2.1. Study patients We measured serum MMP-1 and plasma MMP-8 and MMP-13 levels in 45 consecutive patients who had UAP at rest during the preceding 48 h, so-called class III UAP according to Braunwald’s classification [15], and 175 patients with stable coronary artery disease (CAD). They underwent coronary angiography for suspected CAD at the National Defense Medical College Hospital and were
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found to have CAD which was defined as at least one coronary artery having >50% luminal diameter stenosis on angiograms. Coronary angiograms were recorded by a femoral approach using the Judkins technique and cineangiogram system (Toshiba, Tokyo, Japan). All coronary angiograms were evaluated by Y.M., who was blinded to MMP data. The results were then compared with those of 45 age- and gender-matched controls who had coronary angiography for suspected CAD but were found to have angiographically normal coronary arteries. Because MMPs have been shown to be released from the myocardium with infarction [16–18], any patients with AMI who showed a significant increase (more than the upper normal range) in serum creatine kinase levels were excluded. Patients with a history of MI within 6 months, those with a history of percutaneous coronary intervention or coronary artery bypass surgery, or those with heart failure, cardiomyopathies or valvular heart disease were also excluded. Our study was approved by the ethics committee of the hospital. After written informed consent was obtained, blood samples in patients with UAP were taken within 24 h after their admission, and blood samples in patients with stable CAD and controls were taken on the day of angiography. 2.2. Measurements of serum MMP-1 and plasma MMP-8 and MMP-13 levels After blood samples were centrifuged at 2000 × g for 15 min at 4 ◦ C, the serum and plasma were frozen and stored at −80 ◦ C until analyzed. Serum MMP-1 levels were measured by a one-step sandwich enzyme immunoassay using a commercially available kit (Daiichi Pharmaceutical, Toyoma, Japan). This kit measures the total concentration of the precursor form, the active form, and the TIMP-1 or -2 complex forms of MMP-1 in the serum, but it is highly specific and does not cross-react with MMP-8 and MMP13 [19]. The lower detection limit of this assay was 0.1 ng/ml. Plasma MMP-8 levels were measured by a two-site sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (MMP-8 Human Biotrak ELISA System, Amersham Biosciences, Buckinghamshire, UK). This kit measures the total concentration of both the precursor and active forms of MMP-8 in the plasma, but it is highly specific and does not cross-react with either MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 or MT1MMP [20]. The lower detection limit was 2.5 ng/ml. Plasma MMP-13 levels were also measured by a two-site sandwich ELISA with a commercially available kit (MMP-13 Human Biotrak ELISA System, Amersham Biosciences). This kit measures the total concentration of the precursor and active forms of MMP-13 in the plasma, but
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it is highly specific and does not cross-react with either MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 or MT1-MMP. The lower detection limit was 0.09 ng/ml. MMP-1, MMP-8 and MMP-13 levels of all samples were measured in duplicate, and the results were then averaged. Plasma high sensitivity C-reactive protein (hsCRP) levels were also measured using a BNII nephelometer (Dade Behring, Tokyo, Japan). In patients with UAP, a rapid qualitative test for troponin T (TnT) was performed using a commercially available kit (TROP T sensitive, Roche Diagnostics, Tokyo, Japan). The lower detection limit of this test was 0.1 ng/ml, and positive TnT was defined as >0.1 ng/ml. Serum lipid levels were measured by standard laboratory methods. 2.3. Statistics Any differences between the two groups were evaluated by the unpaired t-test for parametric variables, by the Mann–Whitney Utest for nonparametric variables, and by the chi-square test for categorical variables. Any differences among the three groups were evaluated by ANOVA with the Scheffe’s test for parametric variables, by the Kruskal–Wallis test for nonparametric variables, and by the chi-square test for categorical variables. The correlation between MMP-8 and hsCRP levels was evaluated by the Spearman’s rank correlation test. A forward stepwise multiple logistic regression analysis was used to elucidate the independent association between MMP-8 levels and UAP. A P-value of <0.05 was considered to be statistically significant. The results are presented as the mean value ±SD. Since the distributions of the measured MMP-1, MMP-8 and hsCRP levels were highly skewed, these results are presented as the median value. 3. Results As shown in Table 1, there was no difference in age or gender among the three groups. Total cholesterol levels were lower in patients with UAP than in those with stable CAD and controls. The percentage of patients taking statin was 29% in patients with UAP, 29% in those with stable CAD, and 18% in controls, respectively (P = NS). The number of >50% stenotic coronary vessels was similar in patients with UAP and those with stable CAD (2.0 ± 0.8 and 1.9 ± 0.8), and 1-vessel, 2-vessel, and 3-vessel disease was present in 31%, 36%, and 33% of patients with UAP vs. 38%, 37%, and 25% of those with stable CAD (P = NS). Of the 45 patients with UAP, 10 (22%) showed positive TnT without a significant increase in creatine kinase levels.
Table 1 Clinical characteristics in three groups.
Age (years) Gender (male) Hypertension Systolic BP (mmHg) Hyperlipidemia Total cholesterol (mg/dl) HDL-cholesterol (mg/dl) Statin Diabetes mellitus Current smoker Number of >50% stenotic coronary vessels 1-Vessel disease 2-Vessel disease 3-Vessel disease
UAP (n = 45)
UAP vs. CAD
CAD (n = 175)
CAD vs. control
Controls (n = 45)
UAP vs. control
67 ± 9 34 (76%) 33 (73%) 138 ± 25 24 (53%) 190 ± 34 51 ± 12 13 (29%) 14 (31%) 18 (40%) 2.0 ± 0.8 14 (31%) 16 (36%) 15 (33%)
NS NS NS NS NS < 0.02 NS NS NS NS NS NS NS NS
66 ± 8 141 (81%) 119 (68%) 137 ± 18 76 (43%) 203 ± 32 49 ± 14 51 (29%) 62 (35%) 56 (32%) 1.9 ± 0.8 67 (38%) 65 (37%) 43 (25%)
NS NS NS NS NS NS <0.001 NS <0.01 NS
66 ± 7 34 (76%) 26 (58%) 132 ± 14 15 (33%) 206 ± 30 60 ± 15 8 (18%) 6 (13%) 12 (27%)
NS NS NS NS NS <0.05 <0.005 NS NS NS
Data are presented as the mean value ± SD or the number (%) of patients. UAP, unstable angina pectoris; CAD, coronary artery disease; BP, blood pressure. Hypertension was defined as blood pressures ≥140/90 mmHg or on medication. Hyperlipidemia was defined as total cholesterol levels >240 mg/dl or on medication. Diabetes mellitus was defined as fasting glucose levels ≥126 mg/dl or on insulin or hypoglycemic drugs.
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Fig. 1. Plasma hsCRP levels in the three groups. Plasma hsCRP levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l), but these differences did not reach statistical significance. The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
Plasma hsCRP levels tended to be higher in patients with UAP than in those with stable CAD and controls (median 0.94 vs. 0.69 and 0.51 mg/l), but these differences did not reach statistical significance (Fig. 1). With respect to blood levels of MMPs, plasma MMP-13 levels were above the lower detection limit (>0.09 ng/ml) in only one (2%) patient with UAP, one (1%) patient with stable CAD, and none (0%) in controls, thus suggesting very low levels of MMP13 in the plasma. Serum MMP-1 levels were not different among patients with UAP, those with stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml, P = NS) (Fig. 2). Notably, plasma MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005) (Fig. 3). In patients with stable CAD, a stepwise increase in MMP8 levels was found depending on the number of >50% stenotic coronary vessels: 3.2 in 1-vessel, 3.5 in 2-vessel, and 4.2 ng/ml in 3vessel disease (P < 0.001). However, MMP-8 levels in patients with UAP were much higher than those in stable CAD (median 3.9 ng/ml vs. 3.5 ng/ml, P < 0.05) (Fig. 3). In patients with UAP, MMP-8 levels tended to be higher in patients with positive TnT than in those
Fig. 3. Plasma MMP-8 levels in the three groups. Plasma MMP-8 levels were higher in patients with stable CAD than in controls (median 3.5 ng/ml vs. 2.8 ng/ml, P < 0.005), but MMP-8 levels in patients with UAP were much higher than those in stable CAD (3.9 ng/ml vs. 3.5 ng/ml, P < 0.05). The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
without positive TnT (4.2 ng/ml vs. 3.8 ng/ml, P = NS), but this difference did not reach statistical significance. In patients with UAP, no difference was observed in MMP-8 levels among patients with 1vessel, 2-vessel and 3-vessel disease (3.9, 4.2 and 3.7 ng/ml, P = NS). As a result, patients with UAP more often had a MMP-8 level of >3.5 ng/ml than those with stable CAD and controls (67% vs. 48% and 29%, P < 0.05). However, a significant correlation was observed between MMP-8 and hsCRP levels (r = 0.29, P < 0.001). To elucidate the independent association between MMP-8 levels and UAP, atherosclerotic risk factors (age, gender, hypertension, diabetes, smoking, hyperlipidemia, and HDL-cholesterol levels) and hsCRP and MMP-8 levels were entered into a multiple logistic regression model. Among the 265 study patients, a multivariate analysis revealed that high MMP-8 level (>3.5 ng/ml) was the only independent factor associated with UAP (odds ratio = 2.28, 95%CI = 1.14 to 4.58, P < 0.025). Even among the 220 patients with CAD (45 with UAP and 175 with stable CAD), high MMP-8 level was found to be the only independent factor associated with UAP (odds ratio = 2.10, 95%CI = 1.05 to 4.19, P < 0.05). However, hsCRP levels did not show such associations. 4. Discussion
Fig. 2. Serum MMP-1 levels in the three groups. Serum MMP-1 levels were not different among patients with UAP, those with stable CAD, and controls (median 4.8, 5.3, and 5.4 ng/ml, P = NS). The central line represents the median value, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.
The present study investigated serum MMP-1 and plasma MMP8 and MMP-13 levels in 45 patients with UAP compared with 175 with stable CAD and 45 controls. Although MMP-1 and MMP-13 levels were not high in patients with UAP, MMP-8 levels were found to be significantly higher in patients with UAP than in those with stable CAD and controls. In the multivariate analysis, MMP-8 levels were an independent factor associated with UAP. Among the various known MMPs, only MMP-1, MMP-8, and MMP-13, the so-called interstitial collagenases, can initiate the collagen breakdown of atherosclerotic plaques, thereby making collagen fragments susceptible to further degradation by other MMPs [12]. Although increased expressions of MMP-1 and MMP-13 were shown in atherosclerotic plaques [4,6,7], the role of MMP-8 in atherogenesis had been neglected until recently, because MMP8 had been thought to be produced only by neutrophils that are uncommonly present in plaques [23]. However, in 2001, Herman et al. [8] first demonstrated that endothelial cells, smooth muscle cells, and macrophages in atherosclerotic plaques expressed MMP-
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8 mRNA and protein, especially in lipid-rich plaques with a thin fibrous cap. They suggested that MMP-8 may play a role in the collagen breakdown of atherosclerotic plaques. Moreover, MMP-8 was reported to degrade type I collagen, which is a major component of atherosclerotic plaques, three times more potently than MMP-1 and MMP-13 [21,22]. In 2004, Molloy et al. [24] investigated MMP-1, MMP-8 and MMP-13 concentrations within carotid plaques from 159 patients undergoing carotid endarterectomy. They reported total and active MMP-8 concentrations to be higher in the plaques of symptomatic patients, those with embolism, or those with histological evidence of rupture than in those of asymptomatic patients or those with no evidence of rupture. However, no differences were observed in MMP-1 or MMP-13 concentrations. These findings suggested that, among the interstitial collagenases, MMP-8 plays the most important role in the collagen breakdown of atherosclerotic plaques leading to plaque instability. With respect to MMP-8 levels in blood, Krupinski et al. [25] measured plasma MMP-8 levels in 87 diabetic patients undergoing carotid endarterectomy. They reported patients with ulcerated carotid plaques to have higher MMP-8 levels than those with stable, fibrous plaques. Recently, Tuomainen et al. [26] investigated MMP-8 levels in 905 men with no history of CAD with a 10-year follow-up. They showed MMP-8 levels to be associated with cardiovascular events, such as AMI and death from CAD. Although we previously reported plasma MMP-8 levels in patients with stable CAD to be higher than those without CAD and to correlated with the severity of coronary stenosis [27], our present study, for the first time, showed plasma MMP-8 levels to be much higher in patients with UAP than in those with stable CAD and to be associated with UAP independent of traditional risk factors and hsCRP levels. Our findings suggest that MMP-8 levels could be a biomarker for UAP and that plasma MMP-8 levels in patients with UAP may reflect coronary plaque instability. Regarding MMP-1 levels, Fukuda et al. [14] reported serum MMP-1 levels to not be higher in 23 patients with UAP than in 19 with stable angina. The other small studies also showed serum MMP-1 levels to not be high in patients with UAP [11,13]. Based on the same line of evidence, our study confirmed no significant difference to exist in serum MMP-1 levels among patients with UAP, those with stable CAD, or controls. Although there is no previous report showing MMP-13 levels in patients with UAP, our study, for the first time, showed MMP-13 to be detected within plasma in only 2% of patients with UAP and 1% of patients with stable CAD, thus suggesting a relatively low abundance of MMP-13 in the plasma of patients with UAP. Thus, among the blood levels of interstitial collagenases, plasma MMP-8 levels are considered to be the most promising biomarker for UAP. Our study has several limitations. First, in our study, 29% of patients with UAP, 29% of those with stable CAD and 18% of controls were taking statin. Because statins have been reported to reduce MMP-1 and MMP-9 expressions in rabbit atheroma [28] and to inhibit MMP-1 and MMP-9 secretion from rabbits smooth muscle cells and macrophages [29], the inclusion of patients who were taking statins may therefore have confounded our results of blood levels of MMPs. However, now that statin use in patients with CAD is very common, the exclusion of patients taking statins would render our results inapplicable to the general patients with CAD. Second, since we did not measure MMP-8 levels in the coronary sinus, our study could not determine the main sources of plasma MMP-8 in patients with UAP. Peripheral leukocytes/macrophages or atherosclerotic plaques in other vascular beds than the coronary arteries may be a source of high MMP-8 levels. Moreover, as shown in Fig. 3, there was some overlap in MMP-8 levels between patients with UAP and those with stable CAD. Plasma MMP-8 levels in patients with stable CAD correlated with the number of stenotic coronary vessels, thus suggesting that MMP-8 levels in patients with stable CAD may reflect the degree of coronary atherosclerosis.
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Therefore, plasma MMP-8 levels in patients with UAP may reflect the degree of coronary atherosclerosis as well as coronary plaque instability. Third, UAP with positive TnT has become recently considered as non-ST elevation MI [30]. However, in our study, UAP was defined as Braunwald class III UAP without a significant increase in creatine kinase levels, like the previous studies [10,13,14] showing MMP levels in UAP. As a result, of the 45 patients with UAP, 10 (22%) showed positive TnT, suggestive of some myocardial injury and nonST elevation MI. MMP-8 levels tended to be higher in UA patients with positive TnT than in those without positive TnT, but this difference did not reach statistical significance. To elucidate whether or not MMP-8 levels were much higher in UAP patients with positive TnT than without positive TnT, a further study in a larger number of UAP patients with and without positive TnT is needed. Finally, the number of our patients with UAP was relatively small (45 patients), and our study had no follow-up data. To confirm the diagnostic value of plasma MMP-8 levels for UAP and to elucidate the prognostic value of MMP-8 levels in patients with UAP, a further study with follow-up in a larger number of patients with UAP is needed. In conclusion, among the blood levels of MMP-1, MMP-8, and MMP-13, the so-called interstitial collagenases, only plasma MMP-8 levels were found to be high in patients with UAP and to be associated with UAP independent of hsCRP levels. Our results suggest that plasma MMP-8 levels in patients with UAP may therefore reflect coronary plaque instability and that MMP-8 levels would be the most promising biomarker for UAP among interstitial collagenases. References [1] Shekhonin BV, Domogatsky SP, Muzykantov VR, et al. Distribution of type I, III, IV and V collagen in normal and atherosclerotic human arterial wall: immunomorphological characteristics. Collagen Related Res 1985;5:335–68. [2] Mayne R. Collagenous proteins of blood vessels. Arteriosclerosis 1986;6:585–93. [3] Rekhter MD, Zhang K, Narayanan AS, et al. Type I collagen gene expression in human atherosclerosis: localization to specific plaque regions. Am J Pathol 1993;143:1634–48. [4] Galis ZS, Sukhova GK, Lark MW, et al. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493–503. [5] Brown DL, Hibbs MS, Kearney M, et al. Identification of 92-kDa gelatinase in human coronary atherosclerotic lesions: association of active enzyme synthesis with unstable angina. Circulation 1995;91:2125–31. [6] Nikkari ST, O’Brien KD, Ferguson M, et al. Interstitial collagenase (MMP-1) expression in human carotid atherosclerosis. Circulation 1995;92:1393–8. [7] Sukhova GK, Schonbeck U, Rabkin E, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 1999;99:2503–9. [8] Herman MP, Sukhova GK, Libby P, et al. Expression of neutrophil collagenase (matrix metalloproteinase-8) in human atheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation 2001;104:1899–904. [9] Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863–8. [10] Kai H, Ikeda H, Yasukawa H, et al. Peripheral blood levels of matrix metalloproteases-2 and -9 are elevated in patients with acute coronary syndromes. J Am Coll Cardiol 1998;32:368–72. [11] Tziakas DN, Chalikias GK, Parissis JT, et al. Serum profiles of matrix metalloproteinases and their tissue inhibitor in patients with acute coronary syndromes: the effects of short-term atorvastatin administration. Int J Cardiol 2004;94:269–77. [12] Owen CA, Campbell EJ. The cell biology of leukocyte-mediated proteolysis. J Leukoc Biol 1999;65:137–50. [13] Inoue T, Kato T, Takayanagi K, et al. Circulating matrix metalloproteinase-1 and -3 in patients with an acute coronary syndrome. Am J Cardiol 2003;92:1461–4. [14] Fukuda D, Shimada K, Tanaka A, et al. Comparison of levels of serum matrix metalloproteinase-9 in patients with acute myocardial infarction versus unstable angina pectoris versus stable angina pectoris. Am J Cardiol 2006;97:175–80. [15] Braunwald E. Unstable angina: a classification. Circulation 1989;80:410–4. [16] Etoh T, Joffs C, Deschamps AM, et al. Myocardial and interstitial matrix metalloproteinase activity after acute myocardial infarction in pigs. Am J Physiol Heart Circ Physiol 2001;281:H987–94. [17] Bradham WS, Gunasinghe H, Holder JR, et al. Release of matrix metalloproteinases following alcohol septal ablation in hypertrophic obstructive cardiomyopathy. J Am Coll Cardiol 2002;40:2165–73. [18] Webb CS, Bonnema DD, Ahmed SH, et al. Specific temporal profile of matrix metalloproteinase release occurs in patients with myocardial infarction: relation to left ventricular remodeling. Circulation 2006;114:1020–7.
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