Pravastatin suppresses the increase in matrix metalloproteinase-2 levels after acute myocardial infarction

International Journal of Cardiology 105 (2005) 67 – 73 www.elsevier.com/locate/ijcard

Pravastatin suppresses the increase in matrix metalloproteinase-2 levels after acute myocardial infarction Reiko Nakaya, Hiroyasu Uzui, Hiromasa Shimizu, Akira Nakano, Yasuhiko Mitsuke, Taketoshi Yamazaki, Takanori Ueda, Jong-Dae LeeT First Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, 23 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui, 910-1193, Japan Received 6 July 2004; received in revised form 29 December 2004; accepted 30 December 2004 Available online 5 March 2005

Abstract Background: Matrix metalloproteinase (MMP) may contribute to myocardial remodeling after myocardial infarction. The goal of this study was to characterize the effects of pravastatin on circulating levels of MMP and on left ventricular dilatation after acute myocardial infarction (AMI). Methods: Thirty-four consecutive patients with successful reperfusion following AMI were assigned to either pravastatin group (group P, n=12) or non-pravastatin group (group NP, n=22). Serum MMP-2 and tissue inhibitor of MMP (TIMP)-2 were measured immediately after reperfusion, on days 2, 3, 7, 30, and at 6 months after MI. Left ventriculography was performed after reperfusion and at 4 weeks and 6 months. Results: MMP-2 levels were higher in patients with MI than control on days 1, 30, and at 6 months. Left ventricular end-diastolic volume index (LVEDVI) at 6 months correlated with MMP-2 levels on day 30 (r=0.47, pb0.01) and at 6 months (r=0.56, pb0.001). MMP-2 levels at 6 months were significantly lower in group P than group NP. Further, LVEDVI at 6 months tended to be smaller and DLVEDVI was significantly smaller in group P when compared with group NP. Conclusion: Serum MMP-2 varied in a time-dependent manner following AMI and correlated with late changes in LVEDVI. Serum MMP-2 levels were significantly lower in treatment group than in non-treatment group and DLVEDVI was significantly smaller in treatment group after long-term pravastatin administration. Use of statins in AMI patients may provide beneficial effects in terms of preventing heart failure over and above its lipid-lowering effects. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Pravastatin; Matrix metalloproteinase; Remodeling; Myocardial infarction

1. Introduction Left ventricular (LV) remodeling after myocardial infarction (MI) is an important modulator of long-term ventricular function and affects morbidity and mortality [1]. The matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that regulate collagen degradation and remodeling and thereby play a role in maintenance of LV geometry and function [2]. Indeed, studies have demonstrated specific

* Corresponding author. Tel.: +81 776 61 3111; fax: +81 776 61 8109. E-mail address: [email protected] (J.-D. Lee). 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2004.12.024

increases in MMP levels (e.g., MMP-1, MMP-2, MMP-3 and MMP-9) in human and experimental animal models during the remodeling process following MI [3–6]. MMP-2 is a 72-kDa type IV collagenase/gelatinase with activity towards basement membranes and partially degraded collagen. A recent study demonstrated that targeted deletion of MMP-2 attenuated early left ventricular rupture and late remodeling after MI in mice [7]. Thus, MMP-2 is a potential mediator of ventricular remodeling after MI. The major action of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or bstatinsQ, is the inhibition of hepatic cholesterol synthesis. Statins reduce morbidity and mortality in patients with coronary artery

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disease [8,9] by lowering serum LDL levels and by stabilizing atherosclerotic plaques. However, these agents may also exert their beneficial effects via other mechanisms. For example, investigators have reported that fluvastatin may improve LV remodeling and function after myocardial infarction via attenuation of increased MMP-2 and MMP-13 activity in mice [10]. However, the clinical effect of longterm administration of statins on MMPs and LV remodeling after acute myocardial infarction remain unclear. Therefore, the purpose of this study was to evaluate the effects of long-term administration of pravastatin on MMP2 levels and LV dilatation following acute myocardial infarction (AMI) and subsequent reperfusion.

2. Materials and methods 2.1. Patients and study design Thirty-nine consecutive patients (65F8 years old, 23 men) with AMI and 11 normal volunteers (control subjects; 61F10 years old, 6 men) were enrolled in this study. Control subjects had no history of cardiovascular disease. Patients or control subjects with a history of neoplastic, hepatic, infectious or autoimmune disease were excluded from the study. A diagnosis of AMI was based on the presence of typical prolonged chest pain accompanied by serial changes on the standard 12-lead electrocardiogram (ECG) and significant (2-fold more than the upper normal range) increases in creatine kinase (CK). All patients underwent emergency coronary angiography (CAG) and primary percutaneous transluminal coronary angioplasty (PTCA). Consequently, all experimental group patients in the present study underwent reperfusion. The time interval from onset of symptoms to admission ranged from 1.0 to 11.0 h (mean: 4.6F2.5 h). Heparin and nitroglycerin were administered intravenously during PTCA and heparin administration was continued for 1–2 days after admission. Aspirin was given to all patients. Administration of h-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and nitrate preparations was determined on a case-by-case basis by the attending physician as dictated by the clinical situation. Informed consent was obtained from all patients prior to enrollment in the study. Patients with hypercholesterolemia (total cholesterol level R200 mg/dL) received pravastatin (10 mg/day) beginning after 2 weeks following MI (group P, n=12). Patients without hypercholesterolemia (total cholesterol level b200 mg/dL) did not receive lipid-lowering therapy (group NP, n=22). 2.2. Blood sampling and enzyme immunoassay Blood samples were drawn from peripheral veins. Peripheral blood samples for CK assay were obtained

immediately after admission and every 4 h thereafter for 48 h. Fasting serum total cholesterol, triglyceride, HDL cholesterol and LDL cholesterol levels were measured on day 7, 30, and at 6 months after symptom onset using commercially available assays. Serial blood samples were also collected from the patients immediately after admission and on days 2, 3, 7, and 30 and at 6 months after symptom onset for assays of serum MMP-2 and tissue inhibitor of MMP (TIMP)-2. Serum was immediately obtained by centrifugation at 2000 g for 15 min at 4 8C and stored at 80 8C until use. Sandwich enzyme immunoassay was performed to measure concentrations of serum MMP-2 and TIMP-2. These immunoassays employed commercially available kits with monoclonal antibodies against each substance according to the manufacturer’s instructions (Fuji Chemical Industries, Takaoka, Japan) [6,11,12] to detect proMMP-2 and proMMP-2 complexed with TIMP-2. Active MMP-2 levels were measured using MMP-2 activity assay kits (Amersham Pharmacia Biotech, Buckinghamshire, UK). 2.3. Left ventriculography Left ventriculography (LVG) was performed immediately after reperfusion and approximately four weeks and six months after symptom onset. The left ventriculogram was analyzed using a digitizer and a computer (Kontron Elektronik Cardio 98, Munich, Germany). The end-diastolic frame was determined as the frame nearest to the peak of the R wave. The frame with the smallest ventricular volume was taken to show the end-systolic volume and ventricular volume was calculated by a modification of Dodge’s formula [13]. LVEDVI over time (DLVEDVI) referred to immediately after reperfusion and at 6 months. 2.4. Statistical analysis Results are expressed as mean valueFSD, except in figures, where SE values are shown. Differences between the two groups were analyzed using Student’s unpaired t test. Differences between the parameters during the course of AMI were determined by analysis of variance (ANOVA). Person’s correlation analysis was used to characterize the relationships between MMP-2, TIMP-2, and left ventricular volume indices. A p value b0.05 was considered statistically significant.

3. Results 3.1. Baseline characteristics Table 1 summarizes the clinical characteristics of experimental and control subjects. There were no significant differences in terms of average age, gender distribution, or prevalence of diabetes mellitus, smoking or hypertension

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Table 1 Clinical characteristics

Age (years) Men/women Hypertension Diabetes mellitus Smoker

Controls (n=11)

Group P (n=12)

Group NP (n=22)

p value

61F10 6/5 5 2 4

62F10 8/4 6 3 8

66F7 15/7 11 7 11

NS NS NS NS NS

6 1 5

12 2 8

NS NS NS

11/1/0

18/4/0

NS

0 12 9 2

2 22 18 3

NS NS NS NS

3 3154F1650 4 (33.3%)

10 2637F1404 5 (22.7%)

NS NS NS

Infarct-related artery Left anterior descending Left circumflex Right Extent of coronary artery disease 1-vessel/2-vessels/ 3-vessels Medication h-blockers Aspirin ACE inhibitors Angiotensin receptor blockers Calcium antagonists Peak CK (IU/L) Restenosis

Fig. 1. Time-dependent alteration of serum MMP-2 and TIMP-2 levels after AMI. (A) Serum MMP-2 levels in AMI patients were significantly higher at admission, on day 30 and at 6 months after symptom onset (o) than those in control subjects ( ). (B) Serum TIMP-2 levels were not different when comparing patients with AMI and control subjects. Values are expressed as meansFSEM. *pb0.05 compared with control subjects.

.

when comparing the three groups. Further, medications profiles (except for lipid-lowering drugs), the number of angiographically significant stenoses and peak CK were not significantly different when comparing groups P and NP. During the 6 months of follow-up, 4 of 12 (33.3%) patients in group P and 5 of 22 (22.7%) patients in group NP experienced restenosis with a culprit lesion. However, there was no significant difference in prevalence of restenosis when comparing the two study groups. Despite these restenoses, none of the patients with AMI experienced reocclusion. 3.2. Time course of serum MMP-2 and TIMP-2 levels Fig. 1 shows the time-dependent alterations in serum MMP-2 (Fig. 1A) and TIMP-2 (Fig. 1B) levels following AMI. Serum MMP-2 levels were higher in patients with MI than control on days 1, 30, and at 6 months. In contrast, serum TIMP-2 levels did not change with time following AMI. Neither serum MMP-2 nor TIMP-2 levels significantly correlated with CK max or CK release at any time point. 3.3. Relationship to left ventricular indices Table 2 summarizes the relationship between serum MMP-2 levels and left ventricular indices. Left ventricular end-diastolic volume index (LVEDVI) immediately after reperfusion and at 4 weeks after symptom onset did not significantly correlated with serum MMP-2 concentrations

at any time point. However, serum MMP-2 levels on day 30 significantly correlated with LVEDVI at 6 months after symptom onset (r=0.47, pb0.01) and serum MMP-2 levels at 6 months after symptom onset significantly correlated with LVEDVI at the same time point (r=0.56, pb0.001) (Fig. 2A) and with change in LVEDVI over time (r=0.50, pb0.01) (Fig. 2B). In contrast, left ventricular ejection fraction (LVEF) did not significantly correlate with serum MMP-2 concentrations at any time point. Active MMP-2 levels were also measured and analyzed in AMI patients with and without concomitant cardiac diseases, including dilated cardiomyopathy, valvular disease, and old myocardial infarction. Active MMP-2 levels significantly correlated with serum MMP-2 levels measured by immunoassay (n=214, r=0.52, pb0.0001). Further, serum TIMP-2 levels did not correlate with any left ventricular indices.

Table 2 Relationship of MMP-2 levels to left ventricular indices LVEDVI (4 weeks) Serum MMP-2 levels Day 30 p=NS 6 months p=NS

LVEDVI (6 months)

DLVEDVI

pb0.01, r=0.47 pb0.001, r=0.56

p=NS pb0.01, r=0.50

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significantly to 194F37 mg/dL ( pb0.05 vs. on day 7) in pravastatin group, but there were no significant differences in total cholesterol levels when comparing groups P and NP on day 30 and at 6 months. Further, serum triglycerides and HDL cholesterol levels did not differ when comparing the two study groups at any time point. Serum LDL cholesterol levels on day 7 were significantly higher in group P when compared with group NP (149F43 vs. 93F30 mg/dL, pb0.001). Serum LDL cholesterol levels decreased significantly in group P on day 30 ( pb0.05 vs. on day 7), but there were no significant differences in LDL cholesterol levels when comparing groups P and NP on day 30 and at the 6month follow-up time point. 3.5. Comparison of serum MMP-2 levels in groups P and NP Prior to administration of pravastatin, no significant difference was observed in serum MMP-2 levels when comparing groups P and NP. However, at the 6-month follow-up time point, serum MMP-2 concentrations were significantly lower in group P than in group NP (653F202 vs. 836F228 ng/mL pb0.03) (Fig. 3A). In contrast, serum

Fig. 2. Correlations between serum MMP-2 levels and left ventricular indices. (A) LVEDVI at 6 months significantly correlated with serum MMP-2 levels at the same time point (r=0.56, pb0.001). (B) DLVEDVI significantly correlated with serum MMP-2 levels at 6 months after symptom onset (r=0.50, pb0.01).

3.4. Serum lipid data Table 3 summarizes changes in serum lipid levels before and after pravastatin treatment. Serum total cholesterol levels on day 7 were significantly higher in group P when compared with group NP (221F52 vs. 165F30 mg/dL, pb0.001). On day 30, total cholesterol levels decreased

Table 3 Serum lipid levels Total cholesterol (mg/dL)

Triglycerides (mg/dL)

HDL cholesterol (mg/dL)

LDL cholesterol (mg/dL)

Day 7 Group P Group NP

221F52a 165F30

142F60 111F60

43F16 45F11

149F43a 93F30

Day 30 Group P Group NP

194F37b 185F34

122F37 133F56

44F14 47F15

124F38b 112F30

6 months Group P Group NP

187F23b 182F22

138F68 130F74

45F10 47F13

115F21b 109F25

a b

pb0.001 vs. group NP (day 7). pb0.05 vs. group P (day 7).

Fig. 3. Comparison of serum MMP-2 levels in groups P and NP. (A) Before pravastatin treatment, no significant difference was observed in serum MMP-2 levels when comparing groups P (hatched bars) and NP (open bars). After 6 months of pravastatin treatment, serum MMP-2 levels were significantly lower in group P with compared with group NP. (B) Serum TIMP-2 levels were not different when comparing the two groups. Values are expressed as meansFSEM.

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Fig. 4. Comparison of left ventricular indices in groups P and NP. (A) LVEDVI at 6 months after onset tended to be smaller in group P (hatched bars) than in group NP (open bars). (B) DLVEDVI was significantly smaller in group P than in group NP. Values are expressed as meansFSEM.

TIMP-2 levels were not different when comparing the two groups (Fig. 3B). 3.6. Comparison of left ventricular indices in groups P and NP LVEDVI after reperfusion and at 4 weeks after symptom onset were not different when comparing groups P and NP. At the 6-month follow-up time point, LVEDVI tended to be smaller in group P when compared with group NP (67F8 vs. 80F25 ml/m2, p=0.08) (Fig. 4A) and DLVEDVI was significantly smaller in group P when compared with group NP ( 4F15 vs. 15F24 ml/m2, pb0.03) (Fig. 4B). In contrast, LVEF was not different when comparing groups P and NP at any time point.

4. Discussion The present study demonstrated that serum MMP-2 levels were significantly lower in pravastatin group than in non-pravastatin group and DLVEDVI was significantly smaller in prvastatin group after long-term administration of pravastatin. Further, MMP-2 levels of the subacute phase positively correlated with LVEDVI of the chronic phase. The restenosis rate in the pravastatin group was higher than in non-pravastatin group, though non-significantly in this study. Serum total cholesterol and LDL cholesterol

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levels prior to administration of pravastatin were significantly higher in the pravastatin group. Thus, patients in the pravastatin group might have more complicated culprit lesions. Furthermore, a few patients received stent implantation in our study. These factors might affect the restenosis rate. In the present patient population, MMP-2 levels were higher in patients with MI than control on days 1, 30, and at 6 months. Further, serum TIMP-2 levels did not change following AMI. MMP-2 levels did not correlate with CK levels or with LV indices immediately after reperfusion or at 4 weeks after the infarct. This suggests that the extent of myocardial damage with ischemia/reperfusion injury did not affect circulating MMP-2 levels. A previous report demonstrated that the increase of MMP-2 in plaques was related to atherosclerotic arterial remodeling in the human coronary artery [14]. In our patient population, serum MMP-2 levels were higher in subjects with unstable angina than in control subjects. Increased MMP-2 on admission may be associated with the progression and destabilization of coronary atherosclerosis. However, it is unlikely that elevated MMP originated from atherosclerotic plaques, as elevated MMP-2 was observed in patients with restenosis as well as patients without restenosis. Further studies to clarify sources of MMP-2 production during the chronic phase of AMI would be of benefit. Serum MMP-2 levels on day 30 and at 6 months after the infarct positively correlated with LVEDVI at the 6-month time point. This suggests that the increase in MMP-2 is associated with LV dilatation after MI. Indeed, MMP-2 expression has previously been observed to be associated with the progression of heart failure [15,16,17] and targeted deletion of MMP-2 attenuated late remodeling after MI in mice [7]. Substrates of MMP-2 include the basement membrane components, collagen IV and laminin [2]. Thus, increased MMP-2 activity could contribute to a discontinuity in the basement membrane, thereby disrupting the normal myocyte–matrix interface and contributing to LV remodeling. Clinical trials have indicated that treatment with statins reduce death from cardiovascular causes in patients with coronary artery disease [8,9]. While this has largely been attributed to the lipid-lowering effect of statins, the effect of statins in preventing heart failure cannot be excluded. Indeed, long-term treatment with simvastatin reduced the occurrence of heart failure in patients with coronary heart disease without previous evidence of congestive heart failure [18]. Further, statins improved LV remodeling and function after MI in mice and rats [10,19]. In this study, LVEDVI tended to be smaller in pravastatin group than in non-pravastatin group after 6-month treatment and DLVEDVI was significantly smaller in pravastatin group than in non-pravastatin group. In this study, 94% of patients with AMI received angiotensin-converting enzyme (ACE) inhibitors or angio-

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tensin receptor blockers. Despite this, medication profiles (excepting lipid-lowering drugs) were not significantly different when comparing the two groups. ACE inhibitors are associated with attenuated LV remodeling, an improvement in survival, and reduced morbidity and mortality due to major cardiovascular events [20,21]. However, heart failure can occur despite the use of ACE inhibitors. Administration of statins may represent a novel adjunctive strategy to prevent progressive ventricular dilatation in patients already receiving conventional treatment for MI. Recent studies demonstrated that statin administration resulted in decreases in MMP levels in human carotid plaques [22], vascular endothelial cells [23] and serum [24]. Further, fluvastatin improved LV structural remodeling and contraction in a murine model of post-infarct heart failure and this effect was associated with the attenuation of MMP-2 and MMP-13 [10]. Statins may also attenuate LV remodeling through inhibition of MMP-2 production. Several other mechanisms may account for the attenuation of LV remodeling with pravastatin treatment. First, simvastatin significantly reduced LV hypertrophy and cardiac tissue ACE activity in rats [25] and ACE inhibition attenuated death and improved LV remodeling after MI [20,21]. However, it seems to be unlikely that ACE inhibition by statins contributed to the prevention of heart failure after MI, as LV dilatation after MI was attenuated by pravastatin treatment in patients who received ACE-inhibitors. Second, pro-inflammatory cytokines such as IL-6 and TNF-a play an important role in LV remodeling [26] and previous studies indicated that statins inhibit pro-inflammatory cytokine induction [27]. Further, nitric oxide synthase inhibition abolished the positive effects of cerivastatin on LV remodeling in rats after MI [19]. In our study, serum MMP-2 levels were significantly lower in pravastatin group than in nonpravastatin group and DLVEDVI was significantly smaller in prvastatin group after long-term pravastatin treatment. However, the conclusion that pravastatin-mediated attenuation of LV remodeling following AMI may occur via inhibition of MMP-2 production is premature due to lack of randomisation and small study group. Further studies are required. The present study possesses several limitations. First, this study was a nonrandomized study and had small number subjects, but administration of pravastatin was divided into two groups after measuring the serum lipid profile, eventually pravastatin was given only to AMI patients with hypercholesterolemia. Previous study reported that lipid lowering by diet reduced MMP activity [28]. In our study, total cholesterol levels decreased significantly in pravastatin group and increased in non-pravastatin group on day 30. However, there were no significant differences in MMP-2 levels when comparing pravastatin group and non-treatment group in day 30. This factor might suggest that it is unlikely that differences in serum lipid levels between the two study groups could affect the results. Furthermore, the immuno-

assay employed in this study detects MMP-2 as well as MMP-2 complexed with TIMP-2. Since we measured TIMP-2 together with TIMP-2 complexed with MMPs, we cannot comment on the balance between MMP and TIMP.

5. Conclusions Our study demonstrated that serum MMP-2 levels were significantly lower in treatment group than in non-treatment group and DLVEDVI was significantly smaller in treatment group after long-term pravastatin administration in patients with MI. Use of statins in AMI patients with hypercholesterolemia may provide beneficial effects in terms of preventing heart failure over and above its lipid-lowering effects.

Acknowledgments This study was supported in part by a Grant-in-Aid for General Scientific Research (No. 12770339) from the Ministry of Education, Science, and Culture of Japan. We are also grateful to Miss Hiromi Nishimura, Miss Makoto Matsumoto and Miss Yumie Tanaka for their technical assistance.

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