- Email: [email protected]
Plasma levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 are increased in the coronary circulation in patients with acute coronary syndrome
Plasma levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 are increased in the coronary circulation in patients with acute coronary syndrome Yoichi Inokubo, MD, Hiroyuki Hanada, MD, Hiroshi Ishizaka, MD, Tomohisa Fukushi, MD, Takaatsu Kamada, MD, and Ken Okumura, MD Hirosaki, Japan
Background Previous studies on atherectomy specimens from patients with acute coronary syndrome (ACS) implicated the role of proteolytic enzymes. We examined whether the plasma levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were increased in the coronary circulation in ACS.
Methods and Results The plasma levels (nanograms per milliliter) of MMP-9 and TIMP-1 in the aorta (Ao) and great cardiac vein (GCV) were measured in 29 patients with ACS (20 with acute myocardial infarction [group 1] and 9 with unstable angina [group 2]), 17 with stable effort angina (group 3), and 20 control subjects (group 4). Group 1 patients had occlusion in the left anterior descending artery (LAD), and groups 2 and 3 patients had culprit lesion in the LAD. In group 1 blood samples were obtained at the time of direct coronary angioplasty done within 12 hours after the onset. The Ao level of either MMP-9 or TIMP-1 did not differ among the 4 groups. The GCV-Ao differences in MMP-9 and TIMP-1 were both significantly increased in groups 1 and 2 compared with those in group 4. Neither of them was different between groups 3 and 4. Neither the GCV-Ao difference in MMP-9 or TIMP-1 level was correlated with the maximal creatine kinase level in group 1. Conclusions Increased plasma levels of MMP-9 and TIMP-1 were detected in the coronary circulation in ACS patients, suggesting a process of active plaque rupture in ACS. (Am Heart J 2001;141:211-7.)
Rupture of the coronary artery atherosclerotic plaque and subsequent thrombus formation in the artery has been indicated to be responsible for the pathogenesis of acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina pectoris (UAP).1-4 Two types of atherosclerotic plaques consisting of a soft, lipid-rich plaque and a hard, fibrous plaque have been reported for the coronary artery stenotic lesions.3,5 In the former type of the plaque especially obtained from the patients who died of AMI, inflammatory cells including activated macrophages and mast cells are shown to accumulate in high concentration.6 Also, histologic examination of specimens obtained by directional atherectomy demonstrated a high content of macrophages in patients with AMI or UAP compared with those with stable angina pectoris (SEA).7 There is an increased rate of formation of metalloproteinase From the Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan. Submitted July 2, 1999; accepted October 1, 2000. Reprint requests: Ken Okumura, MD, Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan. E-mail: [email protected] Copyright © 2001 by Mosby, Inc. 0002-8703/2001/$35.00 + 0 4/1/112238 doi:10.1067/mhj.2001.112238
enzymes such as collagenase, gelatinase, and stromelysin in the ruptured atherosclerotic plaque.4,8-10 These proteinases are indicated to be elaborated by activated macrophages and mast cells and to degrade the interstitial matrix isolating the lipid core and activated macrophages from the bloodstream.11,12 Thus active rupture of the vulnerable plaque by these proteinases is one of the triggers causing subsequent thrombus formation and ACS. Despite the histologic evidence of the participation of inflammatory proteinases in the pathogenesis of ACS, information about the dynamic change of these proteinases during the acute phase of the syndrome has been limited. Recently, Kai et al13 demonstrated serial changes in the peripheral blood levels of matrix metalloproteinase (MMP)-2 and MMP-9 in patients with ACS, which implicated the role of these enzymes in the molecular mechanism of plaque destabilization in the syndrome. In this recent study, however, the origin of the enzymes was not clarified. In the current study we collected blood samples simultaneously from the aorta and great cardiac vein in the patients with AMI, UAP, and SEA, all of whom had a culprit lesion in the left anterior descending artery (LAD), and in control subjects with angiographically normal coronary arteries. The plasma levels of MMP-9 and tissue inhibitor of metallopro-
American Heart Journal February 2001
212 Inokubo et al
teinase-1 (TIMP-1) were measured and the differences in the plasma levels between the great cardiac vein and aorta were estimated in each group of patients. By comparing the differences among the groups, we investigated the role of inflammatory proteinases in the pathogenesis of ACS.
Methods Study patients Four groups of patients were included in this study. Group 1 consisted of 20 patients with anterior AMI (15 men and 5 women with a mean age of 65 years, ranging from 40 to 77 years). All these patients were admitted to our institution within 12 hours after the onset of AMI, and coronary angiography performed immediately after admission (average 6.6 ± 3.0 hours after onset, mean ± SD) revealed total occlusion in the LAD (12 patients) or subtotal occlusion in the LAD with a flow grade of 1 or 2 defined by Thrombolysis in Myocardial Infarction (TIMI) study group (8 patients). No patient was in cardiogenic shock. Primary angioplasty of the culprit lesion in the LAD was done in 16 of the 20 patients. Group 2 consisted of 9 patients with UAP (7 men and 2 women with a mean age of 66 years, ranging from 57 to 73 years), all of whom had anginal episodes at rest or angina during a mild degree of exertion within 48 hours of the study. All these patients had a highly stenotic lesion in the LAD that was considered to be the culprit lesion on the basis of the electrocardiographic finding during the anginal attack. An abnormal increase in the creatine phosphokinase level was not detected during hospitalization in any of the patients. Group 3 consisted of 17 patients with SEA (13 men and 4 women with a mean age of 64 years, ranging from 42 to 77 years), all of whom had exertional angina but not rest angina. All patients had a highly stenotic lesion in the LAD that was considered to be the culprit lesion on the basis of electrocardiographic findings during treadmill exercise testing. Group 4 consisted of 20 patients with angiographically normal coronary arteries (14 men and 6 women with a mean age of 62 years, ranging from 39 to 81 years). All these patients underwent coronary angiographic examination for atypical chest pain and showed negative treadmill exercise testing. Intracoronary acetylcholine study, which was done after the blood sampling study, failed to induce coronary spasm in any of the patients. Written informed consent was obtained from all patients before the study. The protocol of the study was in agreement with the guidelines approved by the ethics committee at our institution.
Cardiac catheterization The study was done in the fasting state in all but group 1 patients. All group 1 patients received aspirin (162 mg) at the time of admission and some nitroglycerin or isosorbide dinitrate. On the day of cardiac catheterization, all group 2 patients were administered aspirin (81 mg) and isosorbide dinitrate and most of them also metoprolol, a β-adrenergic blocking agent; all group 3 patients were administered aspirin (81 mg), but other antianginal drugs except for sublingual nitroglycerin were withdrawn 24 hours before the study in this group. None of group 4 patients received any drugs affecting the cardiovascular system within 3 days before the study.
Under fluoroscopic control, a 5F multipurpose catheter (Cordis, Miami, Fla) for venous blood sampling was percutaneously inserted in the great cardiac vein through the internal jugular vein. Then a 6F pig tail catheter (Cordis) for arterial blood sampling was percutaneously inserted in the aortic root through the femoral artery after administration of 5000 units of heparin. Blood samples (7 mL each) for MMP-9 and TIMP-1 were collected simultaneously from the great cardiac vein and the aortic root at the same speed and put into the tubes containing 10.5 mg of ethylenediamine tetra-acetic acid (EDTA). The plasma was immediately separated with a low-temperature centrifugation at 3000 revolutions/min and stored at –80°C until assayed. In 16 group 1 patients undergoing primary angioplasty, blood samples were obtained before the angioplasty to avoid the influences of the procedure and reperfusion of ischemic or infarcted myocardium to the measurements. Coronary angiographic examination for the evaluation of the ischemia- or infarction-related culprit lesion was performed from the multiple projections after intracoronary administration of isosorbide dinitrate (1 mg) in group 1, 2, and 3 patients. In group 4 patients a provocative test for coronary spasm was performed with intracoronary acetylcholine and then coronary angiograms were filmed after intracoronary isosorbide dinitrate to evaluate any fixed stenotic lesion.
Serial blood sampling in patients with AMI In 9 of the 20 group 1 patients, peripheral venous blood was sampled before cardiac catheterization (day 1) and in the morning (in the fasting state) of the days 3, 5, 7, 14, and 21 after admission for assays of the plasma levels of MMP-9 (n = 9) and TIMP-1 (n = 8). Each blood sample was put into tubes containing 10.5 mg of EDTA, and the plasma was immediately separated with low-temperature centrifugation at 3000 revolutions/min and stored at –80°C until assayed.
Assays of MMP-9 and TIMP-1 The plasma MMP-9 and TIMP-1 levels were measured with the use of commercially available kits with monoclonal antibodies against MMP-9 and TIMP-1 (Fuji Chemical, Takaoka, Japan). All the measurements were carried out in the ranges at which the standard curves were linear (r2 = 0.9847 for MMP-9 and r2 = 0.9935 for TIMP-1) and were done in duplicate. The intrasample variation was within 10%. MMP-9. A specimen (50 µL) was mixed with 300 µL of antiMMP-9 monoclonal antibody–peroxidase conjugate (0.42 µg/mL) in 30 mmol/L sodium phosphate buffer (pH 7.0) containing 10 g/L bovine serum albumin, 0.1 mol/L sodium chloride (NaCl), and 10 mmol/L EDTA. A bead previously coated with the monoclonal antibody was added into each tube, and the tube was allowed to stand for 60 minutes at room temperature without shaking. The tube was then washed three times with 10 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1 mol/L NaCl, and the bead was transferred to another tube. The assay for peroxidase activity bound on the bead was started by adding 300 µL of hydrogen peroxide (0.02%) and ophenylenediamine (0.2%) for 30 minutes at room temperature. The reaction was stopped by the addition of 1500 µL of sulfuric acid (1.33N), and the absorbance at 492 nm was measured by a microplate reader.
American Heart Journal Volume 141, Number 2
Inokubo et al 213
Table I. Clinical profiles of study patients
No. Age (y) Sex (male/female) Smoking Diabetes mellitus Hypertension (>140/90 mm Hg) Total cholesterol (mg/dL) Triglycerides (mg/dL)
Group 1
Group 2
Group 3
Group 4
Statistical significance
20 65 ± 2 15:5 11/20 (55%) 11/20 (55%) 8/20 (40%) 193 ± 8 103 ± 8
9 66 ± 2 7:2 7/9 (78%) 4/9 (44%) 5/9 (56%) 186 ± 16 113 ± 21
17 64 ± 2 13:4 9/17 (53%) 10/17 (59%) 9/17 (53%) 199 ± 10 170 ± 41
20 62 ± 3 14:6 6/20 (30%) 2/20 (10%) 7/20 (35%) 201 ± 8 126 ± 9
NS NS NS P = .0079 NS NS NS
Values are means ± SEM.
TIMP-1. A specimen (10 µL) diluted at 1:40 with normal saline solution was mixed with 150 µL of anti-human TIMP-1 monoclonal antibody–peroxidase conjugate (50 µg/mL) in 30 mmol/L sodium phosphate buffer (pH 7.0) containing 10 g/L bovine serum albumin, 0.1 mol/L NaCl, and 10 mmol/L EDTA. A 100-µL aliquot of the solution was transferred to the microplate well previously coated with the monoclonal antibody, and the plate was allowed to stand for 30 minutes at room temperature without shaking. The plate was then washed 4 times with 0.4 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1% Tween 20. The assay for peroxidase activity bound on the plate was started by adding 100 µL of hydrogen peroxide (0.02%) and o-phenylenediamine (0.05%) for 15 minutes at room temperature. The reaction was stopped by the addition of 100 µL of sulfuric acid (2N) and the absorbance at 492 nm was measured by a microplate reader. In the preliminary study, the effects of heparin on the plasma levels of MMP-9 and TIMP-1 were examined in 6 patients without angina and with normal coronary arteriograms. A blood sample was obtained from the aortic root before and 5 minutes after heparin administration (5000 units). The plasma MMP-9 levels before and after heparin were 21 ± 6 ng/mL and 19 ± 4 ng/mL, respectively (mean ± SEM, P not significant [NS] by a 2-tailed paired t test). The plasma TIMP-1 levels before and after heparin were 77 ± 6 ng/mL and 80 ± 8 ng/mL, respectively (P NS).
Data analysis All data are shown as the mean ± SEM. Comparison of the plasma levels of MMP-9 and TIMP-1, the great cardiac vein–aortic root differences in the plasma levels of MMP-9 and TIMP-1, age, and serum cholesterol and triglyceride levels among groups was done with 1-way analysis of variance (ANOVA) followed by the Dann test. Serial changes in the plasma MMP-9 and TIMP-1 levels in group 1 patients were analyzed with ANOVA for repeated measures followed by the Dunnett test. Comparison of the other clinical characteristics was done by a chi-square test. Correlation between each of the plasma levels of MMP-9 and TIMP-1 and each of the maximal values of creatine phosphokinase and C-reactive protein (CRP) level after admission in group 1 was analyzed with a linear regression analysis. Probability levels less than .05 were considered to be statistically significant.
Results Clinical characteristics of the study groups Table I shows the clinical characteristics of the 4 study group patients. There was no statistical difference among 4 groups in the following variables: age, sex, hypertension, smoking, serum cholesterol, and serum triglycerides. The incidence of diabetes mellitus was different among the 4 groups and it was high in groups 1, 2, and 3 compared with group 4.
Plasma MMP-9 levels The plasma level of MMP-9 (nanograms per milliliter) in the aortic root was 25 ± 3 in group 1, 20 ± 5 in group 2, 16 ± 3 in group 3, and 17 ± 2 in group 4 (P NS among groups) (Figure 1, left). That in the great cardiac vein was 37 ± 5 in group 1, 25 ± 6 in group 2, 15 ± 3 in group 3, and 15 ± 2 in group 4 (P < .05, group 1 vs groups 3 and 4) (Figure 1, center). The great cardiac vein–aortic root difference in the plasma MMP-9 level was significantly higher in groups 1 and 2 than in group 4: the difference was 12 ± 4 in group 1 (P < .05 vs groups 3 and 4), 5 ± 2 in group 2 (P < .05 vs group 4), –2 ± 2 in group 3 (P NS vs group 4), and –2 ± 1 in group 4 (Figure 1, right). Of the 20 group 1 patients, 12 had totally occluded vessels and the other 8 a subtotally occluded one with a TIMI flow grade 1 or 2. The great cardiac vein–aortic root difference in the plasma MMP-9 level in the former patients with total occlusion was 16 ± 4, whereas that in the latter patients with subtotal occlusion was 6 ± 8 (P = .064). There was no significant correlation between the plasma MMP-9 levels and the maximal creatine phosphokinase levels (r = 0.372, P NS) or the CRP level (r = 0.101, P NS) in group 1. Also, there was no significant correlation between the great cardiac vein–aortic root difference in the MMP-9 level and the maximal creatine phosphokinase level (r = 0.00028, P NS) or the CRP level (r = 0.103, P NS) in group 1. In 9 of the 20 group 1 patients, the serial change in the plasma MMP-9 level after admission was examined.
American Heart Journal February 2001
214 Inokubo et al
Figure 1
Comparisons of plasma levels of MMP-9 in aortic root (Ao) (left) and great cardiac vein (GCV) (center) and the great cardiac vein–aortic root difference (GCV-Ao) in the plasma MMP-9 level (right) among 4 groups. Open circles and vertical bars indicate mean values and SEMs, respectively.
Figure 2
Figure 2 (upper) shows the changes in the plasma MMP-9 level for 21 days after the onset of AMI for each patient. No significant change in the plasma level was noted for the period (P = .238), but in 7 of the 9 patients the level was decreased on day 3 compared with day 1.
Plasma TIMP-1 levels The plasma level of TIMP-1 (nanograms per milliliter) in the aortic root was 80 ± 8 in group 1, 106 ± 16 in group 2, 79 ± 8 in group 3, and 78 ± 7 in group 4 (P NS among groups) (Figure 3, left). That in the great cardiac vein was 94 ± 8 in group 1, 121 ± 14 in group 2, 82 ± 9 in group 3, and 73 ± 7 in group 4 (P < .05, group 2 vs group 4) (Figure 3, center). The great cardiac vein–aortic root difference in the plasma level of TIMP-1 was significantly higher in groups 1 and 2 than in group 4: the difference was 14 ± 4 in group 1 (P < .05 vs group 4), 15 ± 5 in group 2 (P < .05 vs group 4), 3 ± 3 in group 3 (P NS vs group 4) and –5 ± 3 in group 4 (Figure 3, right). The great cardiac vein–aortic root difference in the plasma TIMP-1 level in the group 1 patients with total occlusion was 15 ± 6, whereas that in the
Serial changes in plasma MMP-9 (upper) and TIMP-1 (lower) levels in the peripheral vein for 21 days after admission in patients with AMI. Although no significant change in the plasma MMP-9 level was noted, the mean plasma TIMP-1 level was significantly increased on the day 2 and showed sustained high levels during the period.
American Heart Journal Volume 141, Number 2
Inokubo et al 215
Figure 3
Comparisons of plasma levels of TIMP-1 in the aortic root (Ao) (left) and great cardiac vein (GCV) (center) and the great cardiac vein–aortic root difference (GCV-Ao) in the plasma TIMP-1 level (right) among 4 groups. Open circles and vertical bars indicate mean values and SEMs, respectively.
other group 1 patients with subtotal occlusion 13 ± 7 (P NS). There was no significant correlation between the plasma TIMP-1 level and the maximal creatine phosphokinase level (r = 0.174, P NS) or CRP level (r = 0.00128, P NS) in group 1. Also, there was no significant correlation between the great cardiac vein–aortic root difference in the TIMP-1 level and the maximal creatine phosphokinase level (r = 0.142, P NS) or CRP level (r = 0.0426, P NS) in group 1. The correlations between the plasma levels of MMP-9 and TIMP-1 and between the great cardiac vein–aortic root differences in the MMP-9 and TIMP-1 levels were analyzed for all study patients, but no significant correlation was found (r = 0.199, P NS for the former; r = 0.207, P NS for the latter). In 8 of the 20 group 1 patients, the serial change in the plasma TIMP-1 level after admission was examined. Figure 2 (lower) shows the change in the plasma TIMP1 level for 21 days after the onset of AMI for each patient. The mean plasma TIMP-1 level was significantly increased on the day 3 compared with that on day 1 (62 ± 8 on day 1 and 91 ± 8 on day 3, P < .05) and showed sustained high levels during the period of 21 days after the onset (104 ± 14 on day 7, 105 ± 12 on day 14, and 102 ± 16 on day 21, all P < .05 vs the value on day 1).
Discussion By sampling blood simultaneously from the aortic root and great cardiac vein, the current study showed that the great cardiac vein–aortic root differences in
plasma MMP-9 and TIMP-1 levels in patients with ACS were both significantly higher than those in patients with SEA and control subjects with normal coronary arteries. Neither of the great cardiac vein–aortic root differences in the MMP-9 level nor that in the TIMP-1 level was correlated with any of the maximal creatine phosphokinase and CRP levels in patients with AMI. All the patients with AMI, UAP, and SEA had culprit lesions in the LAD. Furthermore, blood sampling was done early after the onset of AMI or within 48 hours from the last angina episode in UAP. The current findings therefore indicate that MMP-9 and TIMP-1 are formed in the coronary circulation in the patients with AMI and UAP and strongly suggest the role of the proteolytic enzyme in the development of ACS. Rupture of the lipid-rich atherosclerotic plaque and subsequent thrombus formation in the coronary artery have been shown to be involved in the pathogenesis of ACS.1-4 In the atherosclerotic plaque obtained from the patients who died of AMI, activated macrophages and mast cells accumulate in high concentrations.6 Histologic examination of specimens obtained by directional atherectomy also demonstrated a high content of macrophages in patients with AMI or UAP compared with those with SEA.7 Kaartinen et al14 also showed that mast cells accumulate in the shoulder region of stable atherosclerotic plaque as well as at sites of plaque rupture. These authors recently reported that in the atherectomy specimens obtained from unstable angina patients the numbers of mast cells and T lymphocytes increased compared with those in the specimens
American Heart Journal February 2001
216 Inokubo et al
obtained from patients with stable angina and, furthermore, large numbers of tumor necrosis factor-α (TNFα)–positive mast cells and of MMP-9–positive macrophages were noted.12 It has been shown that TNF-α, a proinflammatory cytokine, can stimulate the production of MMP-9 by macrophages.16 Macrophages and smooth muscle cells of human atherosclerotic plaques have been shown to synthesize MMP-1, MMP-3, and MMP-9.8,16,17 After activated by neutral proteases such as tryptase18 and chymase19 secreted by stimulated mast cells, these MMPs degrade extracellular matrix and seem to make the plaque prone to disruption. The MMP activity has been measured in coronary atherectomy specimens by the use of zymography and the greatest activity resided in the 92-kd band, indicating the presence of active MMP-9 in the specimens.20 In addition to the role of MMP in the unstable atherosclerotic plaque, it is suggested that the synthesis of collagen by the vascular smooth muscle cells is inhibited in the vulnerable regions of the plaque’s fibrous cap through the elaboration of interferon gamma from the activated T lymphocytes, which also renders the plaque weak and prone to disruption.5 The degrading effects of activated MMP on extracellular matrix are inhibited by TIMP.11 Human vascular smooth muscle cells express major isoforms of TIMPs (ie, TIMP-1 and TIMP-2) and a 72-kd gelatinase (ie, MMP2).21 This constitutively expressed MMP-2 seems to exist as a complex with TIMP-2, which is also constitutively expressed.22 In the atherosclerotic plaque the synthesis of proteolytic enzymes is up-regulated through the inflammatory process and thus it is speculated that there is a net excess of degrading activity in the plaque.5 For MMP-9, TIMP-1 shows a potent inhibitory effect,22 but it is still unknown whether the plasma levels of TIMP-1 are increased in patients with ACS. In the current study we examined the plasma levels of MMP-9 and TIMP-1 in the patients with and without ACS. No significant difference was detected in the plasma MMP-9 and TIMP-1 levels in the aortic root among the patient groups with AMI, UAP, or SEA and healthy controls. Recently Kai et al13 demonstrated serial changes in the peripheral blood levels of MMP-2 and MMP-9 in patients with ACS. They showed that both serum MMP-2 and plasma MMP-9 levels in patients with UAP and AMI are increased on the day of admission compared with those in patients with SEA and control subjects. In this recent study, however, the origin of these enzymes was not clarified. In the current study we collected blood samples simultaneously from the aorta and great cardiac vein and showed significant elevation in the plasma MMP-9 and TIMP-1 levels in the coronary circulation in patients with AMI and UAP, each of whom had culprit lesion in the LAD, indicating the formation of MMP-9 and TIMP-1 in the coronary artery with an unstable atherosclerotic plaque. The increase in the
TIMP-1 level in ACS patients seems to be a response to the increase in MMP-9 in the vulnerable plaque. Alternatively, the increased levels of MMP-9 and TIMP-1 in the coronary circulation may reflect the increased production of these enzymes in the ischemic or infarcted myocardium. The increased levels were also found in the current patients with UAP in whom the serum creatine phosphokinase level was not elevated. The absence of the differences in the plasma MMP-9 and TIMP-1 levels in the aortic root among the groups may be explained by the small differences of the levels in systemic circulation, especially in the early phase after the onset of AMI. We further showed that the peripheral plasma level of TIMP-1 was increased significantly on the third day of AMI and showed sustained high levels at least for 3 weeks. We failed to demonstrate the increase in the peripheral plasma MMP-9 level during an acute phase of AMI, which seems to be inconsistent with the findings by Kai et al.13 Because TIMP-1 is a potent inhibitor of MMP-9, its increase during the acute phase of AMI may indicate the induced production of MMP-9 in the infarcted myocardium.23 The measured plasma level of MMP-9 does not necessarily indicate the level of activated MMP-9 because anti-MMP-9 monoclonal antibody used for the plasma level assay cannot distinguish the active enzyme form from proenzyme forms. We thank Drs Y. Fujino, T. Miura, and T. Osanai for their help and comments on the current study.
References 1. Davies MJ, Thomas A. Thrombosis and acute coronary artery lesions in sudden cardiac ischemic death. N Engl J Med 1984; 310:1137-40. 2. Fuster V, Badimon L, Badimon JJ, et al. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242-50. 3. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation 1995;92:657-71. 4. Shah PK. New insights into the pathogenesis and prevention of acute coronary syndromes. Am J Cardiol 1997;79:17-23. 5. Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995;91:2844-50. 6. Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarction. Circulation 1995;92:1084-8. 7. Moreno PR, Falk E, Palacios IF, et al. Macrophage infiltration in acute coronary syndromes. Circulation 1994;90:775-8. 8. Brown DL, Hibbs MS, Kearney M, et al. Identification of 92-kD gelatinase in human coronary atherosclerotic lesions. Circulation 1995;91:2125-31. 9. Libby P, Geng YJ, Aikawa M, et al. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996;7:330-5. 10. Theroux P, Fuster V. Acute coronary syndromes. Circulation 1998; 97:1195-206. 11. Shah PK, Falk E, Badimon JJ, et al. Human monocyte-derived
American Heart Journal Volume 141, Number 2
12.
13.
14.
15.
16.
17.
Inokubo et al 217
macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Circulation 1995;92:1565-9. Kaartinen M, van der Wal AC, van der Loos CM, et al. Mast cell infiltration in acute coronary syndromes: implications for plaque rupture. J Am Coll Cardiol 1998;32:606-12. Kai H, Ikeda H, Yasukawa H, et al. Peripheral blood levels of matrix metalloproteinases-2 and -9 are elevated in patients with acute coronary syndromes. J Am Coll Cardiol 1998;32:368-72. Kaartinen M, Penttila A, Kovanen PT. Accumulation of activated mast cells in the shoulder region of human coronary atheroma, the predilection site of atheromatous rupture. Circulation 1994;90:1669-78. Saren P, Welgus HG, Kovanen PT. TNF-a and IL-1b selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996;157:4159-65. Henney AM, Wakeley PR, Davies MJ, et al. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A 1991;88:8154-8. 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.
18. Gruber BL, Marchese MJ, Suzuki K, et al. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J Clin Invest 1989;84: 1657-62. 19. Saarinen J, Kalkkinen N, Welgus HG, et al. Activation of human interstitial procollagenase through direct cleavage of the Leu83Thr84 bond by mast cell chymase. J Biol Chem 1994;27:1813440. 20. Tyagi SC, Meyer L, Schmaltz RA, et al. Proteinases and restenosis in the human coronary artery: extracellular matrix production exceeds the expression of proteolytic activity. Atherosclerosis 1995;116:43-57. 21. Galis ZS, Muszynski M, Sukhova GK, et al. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann N Y Acad Sci 1995;748:501-7. 22. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863-8. 23. Cleutjens JPM, Kandala JC, Guarda E, et al. Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 1995;27:1281-92.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of the American Heart Journal for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one third off on quantities of 24 or more. Please write to Mosby Subscription Customer Service, 6277 Sea Harbor Dr, Orlando, FL 32887, or call 800-654-2452 or 407-345-4000 for information on availability and prices of particular issues. If unavailable from the publisher, photocopies of complete issues may be purchased from Bell & Howell Information and Learning, 300 N. Zeeb Rd., Ann Arbor, MI 48106, (734)761-4100 or (800) 521-0600.
Background Previous studies on atherectomy specimens from patients with acute coronary syndrome (ACS) implicated the role of proteolytic enzymes. We examined whether the plasma levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were increased in the coronary circulation in ACS.
Methods and Results The plasma levels (nanograms per milliliter) of MMP-9 and TIMP-1 in the aorta (Ao) and great cardiac vein (GCV) were measured in 29 patients with ACS (20 with acute myocardial infarction [group 1] and 9 with unstable angina [group 2]), 17 with stable effort angina (group 3), and 20 control subjects (group 4). Group 1 patients had occlusion in the left anterior descending artery (LAD), and groups 2 and 3 patients had culprit lesion in the LAD. In group 1 blood samples were obtained at the time of direct coronary angioplasty done within 12 hours after the onset. The Ao level of either MMP-9 or TIMP-1 did not differ among the 4 groups. The GCV-Ao differences in MMP-9 and TIMP-1 were both significantly increased in groups 1 and 2 compared with those in group 4. Neither of them was different between groups 3 and 4. Neither the GCV-Ao difference in MMP-9 or TIMP-1 level was correlated with the maximal creatine kinase level in group 1. Conclusions Increased plasma levels of MMP-9 and TIMP-1 were detected in the coronary circulation in ACS patients, suggesting a process of active plaque rupture in ACS. (Am Heart J 2001;141:211-7.)
Rupture of the coronary artery atherosclerotic plaque and subsequent thrombus formation in the artery has been indicated to be responsible for the pathogenesis of acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina pectoris (UAP).1-4 Two types of atherosclerotic plaques consisting of a soft, lipid-rich plaque and a hard, fibrous plaque have been reported for the coronary artery stenotic lesions.3,5 In the former type of the plaque especially obtained from the patients who died of AMI, inflammatory cells including activated macrophages and mast cells are shown to accumulate in high concentration.6 Also, histologic examination of specimens obtained by directional atherectomy demonstrated a high content of macrophages in patients with AMI or UAP compared with those with stable angina pectoris (SEA).7 There is an increased rate of formation of metalloproteinase From the Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan. Submitted July 2, 1999; accepted October 1, 2000. Reprint requests: Ken Okumura, MD, Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan. E-mail: [email protected] Copyright © 2001 by Mosby, Inc. 0002-8703/2001/$35.00 + 0 4/1/112238 doi:10.1067/mhj.2001.112238
enzymes such as collagenase, gelatinase, and stromelysin in the ruptured atherosclerotic plaque.4,8-10 These proteinases are indicated to be elaborated by activated macrophages and mast cells and to degrade the interstitial matrix isolating the lipid core and activated macrophages from the bloodstream.11,12 Thus active rupture of the vulnerable plaque by these proteinases is one of the triggers causing subsequent thrombus formation and ACS. Despite the histologic evidence of the participation of inflammatory proteinases in the pathogenesis of ACS, information about the dynamic change of these proteinases during the acute phase of the syndrome has been limited. Recently, Kai et al13 demonstrated serial changes in the peripheral blood levels of matrix metalloproteinase (MMP)-2 and MMP-9 in patients with ACS, which implicated the role of these enzymes in the molecular mechanism of plaque destabilization in the syndrome. In this recent study, however, the origin of the enzymes was not clarified. In the current study we collected blood samples simultaneously from the aorta and great cardiac vein in the patients with AMI, UAP, and SEA, all of whom had a culprit lesion in the left anterior descending artery (LAD), and in control subjects with angiographically normal coronary arteries. The plasma levels of MMP-9 and tissue inhibitor of metallopro-
American Heart Journal February 2001
212 Inokubo et al
teinase-1 (TIMP-1) were measured and the differences in the plasma levels between the great cardiac vein and aorta were estimated in each group of patients. By comparing the differences among the groups, we investigated the role of inflammatory proteinases in the pathogenesis of ACS.
Methods Study patients Four groups of patients were included in this study. Group 1 consisted of 20 patients with anterior AMI (15 men and 5 women with a mean age of 65 years, ranging from 40 to 77 years). All these patients were admitted to our institution within 12 hours after the onset of AMI, and coronary angiography performed immediately after admission (average 6.6 ± 3.0 hours after onset, mean ± SD) revealed total occlusion in the LAD (12 patients) or subtotal occlusion in the LAD with a flow grade of 1 or 2 defined by Thrombolysis in Myocardial Infarction (TIMI) study group (8 patients). No patient was in cardiogenic shock. Primary angioplasty of the culprit lesion in the LAD was done in 16 of the 20 patients. Group 2 consisted of 9 patients with UAP (7 men and 2 women with a mean age of 66 years, ranging from 57 to 73 years), all of whom had anginal episodes at rest or angina during a mild degree of exertion within 48 hours of the study. All these patients had a highly stenotic lesion in the LAD that was considered to be the culprit lesion on the basis of the electrocardiographic finding during the anginal attack. An abnormal increase in the creatine phosphokinase level was not detected during hospitalization in any of the patients. Group 3 consisted of 17 patients with SEA (13 men and 4 women with a mean age of 64 years, ranging from 42 to 77 years), all of whom had exertional angina but not rest angina. All patients had a highly stenotic lesion in the LAD that was considered to be the culprit lesion on the basis of electrocardiographic findings during treadmill exercise testing. Group 4 consisted of 20 patients with angiographically normal coronary arteries (14 men and 6 women with a mean age of 62 years, ranging from 39 to 81 years). All these patients underwent coronary angiographic examination for atypical chest pain and showed negative treadmill exercise testing. Intracoronary acetylcholine study, which was done after the blood sampling study, failed to induce coronary spasm in any of the patients. Written informed consent was obtained from all patients before the study. The protocol of the study was in agreement with the guidelines approved by the ethics committee at our institution.
Cardiac catheterization The study was done in the fasting state in all but group 1 patients. All group 1 patients received aspirin (162 mg) at the time of admission and some nitroglycerin or isosorbide dinitrate. On the day of cardiac catheterization, all group 2 patients were administered aspirin (81 mg) and isosorbide dinitrate and most of them also metoprolol, a β-adrenergic blocking agent; all group 3 patients were administered aspirin (81 mg), but other antianginal drugs except for sublingual nitroglycerin were withdrawn 24 hours before the study in this group. None of group 4 patients received any drugs affecting the cardiovascular system within 3 days before the study.
Under fluoroscopic control, a 5F multipurpose catheter (Cordis, Miami, Fla) for venous blood sampling was percutaneously inserted in the great cardiac vein through the internal jugular vein. Then a 6F pig tail catheter (Cordis) for arterial blood sampling was percutaneously inserted in the aortic root through the femoral artery after administration of 5000 units of heparin. Blood samples (7 mL each) for MMP-9 and TIMP-1 were collected simultaneously from the great cardiac vein and the aortic root at the same speed and put into the tubes containing 10.5 mg of ethylenediamine tetra-acetic acid (EDTA). The plasma was immediately separated with a low-temperature centrifugation at 3000 revolutions/min and stored at –80°C until assayed. In 16 group 1 patients undergoing primary angioplasty, blood samples were obtained before the angioplasty to avoid the influences of the procedure and reperfusion of ischemic or infarcted myocardium to the measurements. Coronary angiographic examination for the evaluation of the ischemia- or infarction-related culprit lesion was performed from the multiple projections after intracoronary administration of isosorbide dinitrate (1 mg) in group 1, 2, and 3 patients. In group 4 patients a provocative test for coronary spasm was performed with intracoronary acetylcholine and then coronary angiograms were filmed after intracoronary isosorbide dinitrate to evaluate any fixed stenotic lesion.
Serial blood sampling in patients with AMI In 9 of the 20 group 1 patients, peripheral venous blood was sampled before cardiac catheterization (day 1) and in the morning (in the fasting state) of the days 3, 5, 7, 14, and 21 after admission for assays of the plasma levels of MMP-9 (n = 9) and TIMP-1 (n = 8). Each blood sample was put into tubes containing 10.5 mg of EDTA, and the plasma was immediately separated with low-temperature centrifugation at 3000 revolutions/min and stored at –80°C until assayed.
Assays of MMP-9 and TIMP-1 The plasma MMP-9 and TIMP-1 levels were measured with the use of commercially available kits with monoclonal antibodies against MMP-9 and TIMP-1 (Fuji Chemical, Takaoka, Japan). All the measurements were carried out in the ranges at which the standard curves were linear (r2 = 0.9847 for MMP-9 and r2 = 0.9935 for TIMP-1) and were done in duplicate. The intrasample variation was within 10%. MMP-9. A specimen (50 µL) was mixed with 300 µL of antiMMP-9 monoclonal antibody–peroxidase conjugate (0.42 µg/mL) in 30 mmol/L sodium phosphate buffer (pH 7.0) containing 10 g/L bovine serum albumin, 0.1 mol/L sodium chloride (NaCl), and 10 mmol/L EDTA. A bead previously coated with the monoclonal antibody was added into each tube, and the tube was allowed to stand for 60 minutes at room temperature without shaking. The tube was then washed three times with 10 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1 mol/L NaCl, and the bead was transferred to another tube. The assay for peroxidase activity bound on the bead was started by adding 300 µL of hydrogen peroxide (0.02%) and ophenylenediamine (0.2%) for 30 minutes at room temperature. The reaction was stopped by the addition of 1500 µL of sulfuric acid (1.33N), and the absorbance at 492 nm was measured by a microplate reader.
American Heart Journal Volume 141, Number 2
Inokubo et al 213
Table I. Clinical profiles of study patients
No. Age (y) Sex (male/female) Smoking Diabetes mellitus Hypertension (>140/90 mm Hg) Total cholesterol (mg/dL) Triglycerides (mg/dL)
Group 1
Group 2
Group 3
Group 4
Statistical significance
20 65 ± 2 15:5 11/20 (55%) 11/20 (55%) 8/20 (40%) 193 ± 8 103 ± 8
9 66 ± 2 7:2 7/9 (78%) 4/9 (44%) 5/9 (56%) 186 ± 16 113 ± 21
17 64 ± 2 13:4 9/17 (53%) 10/17 (59%) 9/17 (53%) 199 ± 10 170 ± 41
20 62 ± 3 14:6 6/20 (30%) 2/20 (10%) 7/20 (35%) 201 ± 8 126 ± 9
NS NS NS P = .0079 NS NS NS
Values are means ± SEM.
TIMP-1. A specimen (10 µL) diluted at 1:40 with normal saline solution was mixed with 150 µL of anti-human TIMP-1 monoclonal antibody–peroxidase conjugate (50 µg/mL) in 30 mmol/L sodium phosphate buffer (pH 7.0) containing 10 g/L bovine serum albumin, 0.1 mol/L NaCl, and 10 mmol/L EDTA. A 100-µL aliquot of the solution was transferred to the microplate well previously coated with the monoclonal antibody, and the plate was allowed to stand for 30 minutes at room temperature without shaking. The plate was then washed 4 times with 0.4 mmol/L sodium phosphate buffer (pH 7.0) containing 0.1% Tween 20. The assay for peroxidase activity bound on the plate was started by adding 100 µL of hydrogen peroxide (0.02%) and o-phenylenediamine (0.05%) for 15 minutes at room temperature. The reaction was stopped by the addition of 100 µL of sulfuric acid (2N) and the absorbance at 492 nm was measured by a microplate reader. In the preliminary study, the effects of heparin on the plasma levels of MMP-9 and TIMP-1 were examined in 6 patients without angina and with normal coronary arteriograms. A blood sample was obtained from the aortic root before and 5 minutes after heparin administration (5000 units). The plasma MMP-9 levels before and after heparin were 21 ± 6 ng/mL and 19 ± 4 ng/mL, respectively (mean ± SEM, P not significant [NS] by a 2-tailed paired t test). The plasma TIMP-1 levels before and after heparin were 77 ± 6 ng/mL and 80 ± 8 ng/mL, respectively (P NS).
Data analysis All data are shown as the mean ± SEM. Comparison of the plasma levels of MMP-9 and TIMP-1, the great cardiac vein–aortic root differences in the plasma levels of MMP-9 and TIMP-1, age, and serum cholesterol and triglyceride levels among groups was done with 1-way analysis of variance (ANOVA) followed by the Dann test. Serial changes in the plasma MMP-9 and TIMP-1 levels in group 1 patients were analyzed with ANOVA for repeated measures followed by the Dunnett test. Comparison of the other clinical characteristics was done by a chi-square test. Correlation between each of the plasma levels of MMP-9 and TIMP-1 and each of the maximal values of creatine phosphokinase and C-reactive protein (CRP) level after admission in group 1 was analyzed with a linear regression analysis. Probability levels less than .05 were considered to be statistically significant.
Results Clinical characteristics of the study groups Table I shows the clinical characteristics of the 4 study group patients. There was no statistical difference among 4 groups in the following variables: age, sex, hypertension, smoking, serum cholesterol, and serum triglycerides. The incidence of diabetes mellitus was different among the 4 groups and it was high in groups 1, 2, and 3 compared with group 4.
Plasma MMP-9 levels The plasma level of MMP-9 (nanograms per milliliter) in the aortic root was 25 ± 3 in group 1, 20 ± 5 in group 2, 16 ± 3 in group 3, and 17 ± 2 in group 4 (P NS among groups) (Figure 1, left). That in the great cardiac vein was 37 ± 5 in group 1, 25 ± 6 in group 2, 15 ± 3 in group 3, and 15 ± 2 in group 4 (P < .05, group 1 vs groups 3 and 4) (Figure 1, center). The great cardiac vein–aortic root difference in the plasma MMP-9 level was significantly higher in groups 1 and 2 than in group 4: the difference was 12 ± 4 in group 1 (P < .05 vs groups 3 and 4), 5 ± 2 in group 2 (P < .05 vs group 4), –2 ± 2 in group 3 (P NS vs group 4), and –2 ± 1 in group 4 (Figure 1, right). Of the 20 group 1 patients, 12 had totally occluded vessels and the other 8 a subtotally occluded one with a TIMI flow grade 1 or 2. The great cardiac vein–aortic root difference in the plasma MMP-9 level in the former patients with total occlusion was 16 ± 4, whereas that in the latter patients with subtotal occlusion was 6 ± 8 (P = .064). There was no significant correlation between the plasma MMP-9 levels and the maximal creatine phosphokinase levels (r = 0.372, P NS) or the CRP level (r = 0.101, P NS) in group 1. Also, there was no significant correlation between the great cardiac vein–aortic root difference in the MMP-9 level and the maximal creatine phosphokinase level (r = 0.00028, P NS) or the CRP level (r = 0.103, P NS) in group 1. In 9 of the 20 group 1 patients, the serial change in the plasma MMP-9 level after admission was examined.
American Heart Journal February 2001
214 Inokubo et al
Figure 1
Comparisons of plasma levels of MMP-9 in aortic root (Ao) (left) and great cardiac vein (GCV) (center) and the great cardiac vein–aortic root difference (GCV-Ao) in the plasma MMP-9 level (right) among 4 groups. Open circles and vertical bars indicate mean values and SEMs, respectively.
Figure 2
Figure 2 (upper) shows the changes in the plasma MMP-9 level for 21 days after the onset of AMI for each patient. No significant change in the plasma level was noted for the period (P = .238), but in 7 of the 9 patients the level was decreased on day 3 compared with day 1.
Plasma TIMP-1 levels The plasma level of TIMP-1 (nanograms per milliliter) in the aortic root was 80 ± 8 in group 1, 106 ± 16 in group 2, 79 ± 8 in group 3, and 78 ± 7 in group 4 (P NS among groups) (Figure 3, left). That in the great cardiac vein was 94 ± 8 in group 1, 121 ± 14 in group 2, 82 ± 9 in group 3, and 73 ± 7 in group 4 (P < .05, group 2 vs group 4) (Figure 3, center). The great cardiac vein–aortic root difference in the plasma level of TIMP-1 was significantly higher in groups 1 and 2 than in group 4: the difference was 14 ± 4 in group 1 (P < .05 vs group 4), 15 ± 5 in group 2 (P < .05 vs group 4), 3 ± 3 in group 3 (P NS vs group 4) and –5 ± 3 in group 4 (Figure 3, right). The great cardiac vein–aortic root difference in the plasma TIMP-1 level in the group 1 patients with total occlusion was 15 ± 6, whereas that in the
Serial changes in plasma MMP-9 (upper) and TIMP-1 (lower) levels in the peripheral vein for 21 days after admission in patients with AMI. Although no significant change in the plasma MMP-9 level was noted, the mean plasma TIMP-1 level was significantly increased on the day 2 and showed sustained high levels during the period.
American Heart Journal Volume 141, Number 2
Inokubo et al 215
Figure 3
Comparisons of plasma levels of TIMP-1 in the aortic root (Ao) (left) and great cardiac vein (GCV) (center) and the great cardiac vein–aortic root difference (GCV-Ao) in the plasma TIMP-1 level (right) among 4 groups. Open circles and vertical bars indicate mean values and SEMs, respectively.
other group 1 patients with subtotal occlusion 13 ± 7 (P NS). There was no significant correlation between the plasma TIMP-1 level and the maximal creatine phosphokinase level (r = 0.174, P NS) or CRP level (r = 0.00128, P NS) in group 1. Also, there was no significant correlation between the great cardiac vein–aortic root difference in the TIMP-1 level and the maximal creatine phosphokinase level (r = 0.142, P NS) or CRP level (r = 0.0426, P NS) in group 1. The correlations between the plasma levels of MMP-9 and TIMP-1 and between the great cardiac vein–aortic root differences in the MMP-9 and TIMP-1 levels were analyzed for all study patients, but no significant correlation was found (r = 0.199, P NS for the former; r = 0.207, P NS for the latter). In 8 of the 20 group 1 patients, the serial change in the plasma TIMP-1 level after admission was examined. Figure 2 (lower) shows the change in the plasma TIMP1 level for 21 days after the onset of AMI for each patient. The mean plasma TIMP-1 level was significantly increased on the day 3 compared with that on day 1 (62 ± 8 on day 1 and 91 ± 8 on day 3, P < .05) and showed sustained high levels during the period of 21 days after the onset (104 ± 14 on day 7, 105 ± 12 on day 14, and 102 ± 16 on day 21, all P < .05 vs the value on day 1).
Discussion By sampling blood simultaneously from the aortic root and great cardiac vein, the current study showed that the great cardiac vein–aortic root differences in
plasma MMP-9 and TIMP-1 levels in patients with ACS were both significantly higher than those in patients with SEA and control subjects with normal coronary arteries. Neither of the great cardiac vein–aortic root differences in the MMP-9 level nor that in the TIMP-1 level was correlated with any of the maximal creatine phosphokinase and CRP levels in patients with AMI. All the patients with AMI, UAP, and SEA had culprit lesions in the LAD. Furthermore, blood sampling was done early after the onset of AMI or within 48 hours from the last angina episode in UAP. The current findings therefore indicate that MMP-9 and TIMP-1 are formed in the coronary circulation in the patients with AMI and UAP and strongly suggest the role of the proteolytic enzyme in the development of ACS. Rupture of the lipid-rich atherosclerotic plaque and subsequent thrombus formation in the coronary artery have been shown to be involved in the pathogenesis of ACS.1-4 In the atherosclerotic plaque obtained from the patients who died of AMI, activated macrophages and mast cells accumulate in high concentrations.6 Histologic examination of specimens obtained by directional atherectomy also demonstrated a high content of macrophages in patients with AMI or UAP compared with those with SEA.7 Kaartinen et al14 also showed that mast cells accumulate in the shoulder region of stable atherosclerotic plaque as well as at sites of plaque rupture. These authors recently reported that in the atherectomy specimens obtained from unstable angina patients the numbers of mast cells and T lymphocytes increased compared with those in the specimens
American Heart Journal February 2001
216 Inokubo et al
obtained from patients with stable angina and, furthermore, large numbers of tumor necrosis factor-α (TNFα)–positive mast cells and of MMP-9–positive macrophages were noted.12 It has been shown that TNF-α, a proinflammatory cytokine, can stimulate the production of MMP-9 by macrophages.16 Macrophages and smooth muscle cells of human atherosclerotic plaques have been shown to synthesize MMP-1, MMP-3, and MMP-9.8,16,17 After activated by neutral proteases such as tryptase18 and chymase19 secreted by stimulated mast cells, these MMPs degrade extracellular matrix and seem to make the plaque prone to disruption. The MMP activity has been measured in coronary atherectomy specimens by the use of zymography and the greatest activity resided in the 92-kd band, indicating the presence of active MMP-9 in the specimens.20 In addition to the role of MMP in the unstable atherosclerotic plaque, it is suggested that the synthesis of collagen by the vascular smooth muscle cells is inhibited in the vulnerable regions of the plaque’s fibrous cap through the elaboration of interferon gamma from the activated T lymphocytes, which also renders the plaque weak and prone to disruption.5 The degrading effects of activated MMP on extracellular matrix are inhibited by TIMP.11 Human vascular smooth muscle cells express major isoforms of TIMPs (ie, TIMP-1 and TIMP-2) and a 72-kd gelatinase (ie, MMP2).21 This constitutively expressed MMP-2 seems to exist as a complex with TIMP-2, which is also constitutively expressed.22 In the atherosclerotic plaque the synthesis of proteolytic enzymes is up-regulated through the inflammatory process and thus it is speculated that there is a net excess of degrading activity in the plaque.5 For MMP-9, TIMP-1 shows a potent inhibitory effect,22 but it is still unknown whether the plasma levels of TIMP-1 are increased in patients with ACS. In the current study we examined the plasma levels of MMP-9 and TIMP-1 in the patients with and without ACS. No significant difference was detected in the plasma MMP-9 and TIMP-1 levels in the aortic root among the patient groups with AMI, UAP, or SEA and healthy controls. Recently Kai et al13 demonstrated serial changes in the peripheral blood levels of MMP-2 and MMP-9 in patients with ACS. They showed that both serum MMP-2 and plasma MMP-9 levels in patients with UAP and AMI are increased on the day of admission compared with those in patients with SEA and control subjects. In this recent study, however, the origin of these enzymes was not clarified. In the current study we collected blood samples simultaneously from the aorta and great cardiac vein and showed significant elevation in the plasma MMP-9 and TIMP-1 levels in the coronary circulation in patients with AMI and UAP, each of whom had culprit lesion in the LAD, indicating the formation of MMP-9 and TIMP-1 in the coronary artery with an unstable atherosclerotic plaque. The increase in the
TIMP-1 level in ACS patients seems to be a response to the increase in MMP-9 in the vulnerable plaque. Alternatively, the increased levels of MMP-9 and TIMP-1 in the coronary circulation may reflect the increased production of these enzymes in the ischemic or infarcted myocardium. The increased levels were also found in the current patients with UAP in whom the serum creatine phosphokinase level was not elevated. The absence of the differences in the plasma MMP-9 and TIMP-1 levels in the aortic root among the groups may be explained by the small differences of the levels in systemic circulation, especially in the early phase after the onset of AMI. We further showed that the peripheral plasma level of TIMP-1 was increased significantly on the third day of AMI and showed sustained high levels at least for 3 weeks. We failed to demonstrate the increase in the peripheral plasma MMP-9 level during an acute phase of AMI, which seems to be inconsistent with the findings by Kai et al.13 Because TIMP-1 is a potent inhibitor of MMP-9, its increase during the acute phase of AMI may indicate the induced production of MMP-9 in the infarcted myocardium.23 The measured plasma level of MMP-9 does not necessarily indicate the level of activated MMP-9 because anti-MMP-9 monoclonal antibody used for the plasma level assay cannot distinguish the active enzyme form from proenzyme forms. We thank Drs Y. Fujino, T. Miura, and T. Osanai for their help and comments on the current study.
References 1. Davies MJ, Thomas A. Thrombosis and acute coronary artery lesions in sudden cardiac ischemic death. N Engl J Med 1984; 310:1137-40. 2. Fuster V, Badimon L, Badimon JJ, et al. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med 1992;326:242-50. 3. Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation 1995;92:657-71. 4. Shah PK. New insights into the pathogenesis and prevention of acute coronary syndromes. Am J Cardiol 1997;79:17-23. 5. Libby P. Molecular bases of the acute coronary syndromes. Circulation 1995;91:2844-50. 6. Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarction. Circulation 1995;92:1084-8. 7. Moreno PR, Falk E, Palacios IF, et al. Macrophage infiltration in acute coronary syndromes. Circulation 1994;90:775-8. 8. Brown DL, Hibbs MS, Kearney M, et al. Identification of 92-kD gelatinase in human coronary atherosclerotic lesions. Circulation 1995;91:2125-31. 9. Libby P, Geng YJ, Aikawa M, et al. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol 1996;7:330-5. 10. Theroux P, Fuster V. Acute coronary syndromes. Circulation 1998; 97:1195-206. 11. Shah PK, Falk E, Badimon JJ, et al. Human monocyte-derived
American Heart Journal Volume 141, Number 2
12.
13.
14.
15.
16.
17.
Inokubo et al 217
macrophages induce collagen breakdown in fibrous caps of atherosclerotic plaques. Circulation 1995;92:1565-9. Kaartinen M, van der Wal AC, van der Loos CM, et al. Mast cell infiltration in acute coronary syndromes: implications for plaque rupture. J Am Coll Cardiol 1998;32:606-12. Kai H, Ikeda H, Yasukawa H, et al. Peripheral blood levels of matrix metalloproteinases-2 and -9 are elevated in patients with acute coronary syndromes. J Am Coll Cardiol 1998;32:368-72. Kaartinen M, Penttila A, Kovanen PT. Accumulation of activated mast cells in the shoulder region of human coronary atheroma, the predilection site of atheromatous rupture. Circulation 1994;90:1669-78. Saren P, Welgus HG, Kovanen PT. TNF-a and IL-1b selectively induce expression of 92-kDa gelatinase by human macrophages. J Immunol 1996;157:4159-65. Henney AM, Wakeley PR, Davies MJ, et al. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A 1991;88:8154-8. 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.
18. Gruber BL, Marchese MJ, Suzuki K, et al. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J Clin Invest 1989;84: 1657-62. 19. Saarinen J, Kalkkinen N, Welgus HG, et al. Activation of human interstitial procollagenase through direct cleavage of the Leu83Thr84 bond by mast cell chymase. J Biol Chem 1994;27:1813440. 20. Tyagi SC, Meyer L, Schmaltz RA, et al. Proteinases and restenosis in the human coronary artery: extracellular matrix production exceeds the expression of proteolytic activity. Atherosclerosis 1995;116:43-57. 21. Galis ZS, Muszynski M, Sukhova GK, et al. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann N Y Acad Sci 1995;748:501-7. 22. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995;77:863-8. 23. Cleutjens JPM, Kandala JC, Guarda E, et al. Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 1995;27:1281-92.
AVAILABILITY OF JOURNAL BACK ISSUES As a service to our subscribers, copies of back issues of the American Heart Journal for the preceding 5 years are maintained and are available for purchase from Mosby until inventory is depleted. The following quantity discounts are available: 25% off on quantities of 12 to 23, and one third off on quantities of 24 or more. Please write to Mosby Subscription Customer Service, 6277 Sea Harbor Dr, Orlando, FL 32887, or call 800-654-2452 or 407-345-4000 for information on availability and prices of particular issues. If unavailable from the publisher, photocopies of complete issues may be purchased from Bell & Howell Information and Learning, 300 N. Zeeb Rd., Ann Arbor, MI 48106, (734)761-4100 or (800) 521-0600.