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Decreased plasma and cardiac matrix metalloproteinase activities in patients with coronary artery disease and treated with pravastatin
European Journal of Pharmacology 594 (2008) 146–151
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Decreased plasma and cardiac matrix metalloproteinase activities in patients with coronary artery disease and treated with pravastatin Takayuki Fujiwara a, Shin Saito a, Tomohiro Osanai a, Kunihiko Kameda a, Naoki Abe a, Takumi Higuma a, Jin Yokoyama a, Hiroyuki Hanada a, Kozo Fukui b, Ikuo Fukuda b, Ken Okumura a,⁎ a b
Division of Cardiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan Division of Cardiovascular Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
a r t i c l e
i n f o
Article history: Received 2 January 2008 Received in revised form 16 July 2008 Accepted 23 July 2008 Available online 31 July 2008 Keywords: Pravastatin Matrix metalloproteinase Oxidative stress Coronary artery disease
a b s t r a c t Matrix metalloproteinase (MMP), which is activated by oxidative stress, plays an important role in the development of ventricular remodeling in coronary artery disease. Pravastatin is shown to reduce oxidative stress. We tested the hypothesis that cardiac oxidative stress and MMP activity are reduced in patients with coronary artery disease and treated with pravastatin. Forty-eight patients who underwent coronary artery bypass graft surgery (CABG) were studied. Twenty-four patients had the serum low-density lipoprotein (LDL) cholesterol level N2.59 mM, and were treated with pravastatin (10 mg/day) for 2 months before CABG (pravastatin group). The other 24 had LDL cholesterol ≤ 2.59 mM, and were untreated (control group). The plasma and pericardial MMP-2 and MMP-9 activities were measured by gelatin zymography, and MMP-2 and MMP-9 levels, and pericardial 8-iso-prostagrandin F2α (8-iso-PGF2α) level, a maker of oxidative stress, by enzyme-linked immunosorbent assay. The plasma and pericardial MMP-2 and MMP-9 activities and levels were all lower by 20–30% in pravastatin than in control group (all P b 0.05). The pericardial 8-iso-PGF2α level was lower in pravastatin than in control group (38 ± 4 vs 64 ± 7 pg/ml, P b 0.05). The pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level (r = 0.57 and 0.47, respectively, both P b 0.01). Thus, cardiac oxidative stress and MMP activities are reduced in patients with coronary artery disease and treated with pravastatin, which may be beneficial in preventing and reducing ventricular remodeling. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Left ventricular remodeling after myocardial infarction is an important modulator of long-term ventricular function and both morbidity and mortality (Preffer et al., 1990). Matrix metalloproteinases (MMPs) were shown to play an important role in the development of left ventricular remodeling in the experimental myocardial infarction model and are involved deeply in the pathogenesis of cardiovascular diseases (Peterson et al., 2001; Rohde et al., 1999; Nian et al., 2004). We recently reported that the circulating level of gelatinolysis activity predicts the development of ventricular remodeling in patients with acute myocardial infarction (AMI) (Matsunaga et al., 2005). Oxidative stress has been implicated in the pathogenesis of congestive heart failure (McMurray et al., 1993; Cappola et al., 2001). Mallat et al. reported that the pericardial level of 8-isoprostaglandin F2α (8-iso-PGF2α), a specific and quantitative biochem-
⁎ Corresponding author. The Division of Cardiology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan. Tel.: +81 172 39 5057; fax: +81 172 35 9190. E-mail address: [email protected] (K. Okumura). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.07.039
ical marker for oxidative stress in vivo, is increased as the functional severity of congestive heart failure is increased (Mallat et al., 1998). In addition, anti-oxidant agents such as allopurinol and carvedilol were shown to improve cardiac function in human failing heart (Cappola et al., 2001; Nakamura et al., 2002). Recently, we showed that the pericardial level of 8-iso-PGF2α is positively correlated with those of MMP-2 and MMP-9 activities in patients with coronary artery disease (Kameda et al., 2003). 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) improve endothelial dysfunction, increase nitric oxide bioavailability, show anti-oxidant properties, inhibit inflammatory response, and stabilize atherosclerotic plaques; thereby improving the prognosis for patients with coronary artery disease independently of their cholesterol-lowing effects (Davignon et al., 2004). In fact, statin reduced morbidity and mortality in patients with coronary artery disease by lowering serum low-density lipoprotein (LDL) cholesterol levels and by stabilizing atherosclerotic plaques (Scandinavian Simvastatin Survival Study Group, 1994; Sacks et al., 1996). Simvastatin reduced the occurrence of heart failure in patients with coronary artery disease and without previous evidence of congestive heart failure (Kjekshus et al., 1997). In an experimental mice model, fluvastatin improved left ventricular remodeling and
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function after myocardial infarction via attenuation of increased MMP activity (Hayashidani et al., 2002). However, it is unknown how in patients with coronary artery disease, treatment with statin affects cardiac MMP levels and activities and oxidative stress. To clarify it, we measured both the plasma and pericardial MMP activities and their levels and the pericardial 8-iso-PGF2α level in patients with coronary artery disease treated with and without pravastatin prior to coronary artery bypass graft surgery. 2. Methods 2.1. Study patients The study protocol was approved by the ethical committee on human research at our institution. All patients gave informed consent before the study. Forty-eight patients with coronary artery disease who underwent coronary artery bypass graft surgery at our institution were studied. None of the patients had been administered with statin before the study. Of these 48 patients, 24 had the serum LDL cholesterol level N2.59 mM (mean ± S.E.M., 3.13 ± 0.10 mM) at the time when coronary artery bypass graft surgery was indicated, and were treated with pravastatin (10 mg/day) (Mevalotin, Daiichi-Sankyo Pharmaceutical Inc., Japan) for 2 months before coronary artery bypass graft surgery (pravastatin group). The other 24 patients had LDL cholesterol level ≤2.59 mM (2.44 ± 0.78 mM, P b 0.01 vs pravastatin group), and were not treated (control group). The patients with AMI, severe liver, renal, or brain dysfunction, malignant tumor, autoimmune disease, and other serious complications were excluded from the present Table 1 Comparison of clinical and angiographic characteristics between 2 groups
Age Gender (male/female) Blood chemistry Total cholesterol (mM) LDL cholesterol (mM) Triglyceride (mM) Creatinine (mg/dl) Blood sugar (mg/dl) HbA1C (%) Drug Beta-blocker Spironolactone ACE inhibitor ARB Nicorandil (oral administration) No. of diseased vessel 1VD 2VD 3VD LMT Use of IABP after CABG LVG parameters before CABG LVEDVI LVESVI LVEF LVG parameters after CABG LVEDVI LVESVI LVEF Inhospital cardiac events after CABG Heart failure Af VT, VF
Pravastatin (n = 24)
Control (n = 24)
P value
68 ± 2 20/4
67 ± 2 19/5
NS NS
4.53 ± 0.21 2.69 ± 0.18 1.25 ± 0.08 1.0 ± 0.1 126 ± 11 6.1 ± 0.3
4.66 ± 0.18 2.59 ± 0.18 1.30 ± 0.09 1.0 ± 0.1 130 ± 8 6.1 ± 0.2
NS NS NS NS NS NS
17/24 8/24 10/24 12/24 9/24
18/24 7/24 12/24 9/24 12/24
NS NS NS NS NS
4/24 9/24 11/24 11/24 2/24
4/24 11/24 9/24 12/24 1/24
NS NS NS NS NS
81 ± 4 41 ± 3 51 ± 2
79 ± 5 41 ± 4 51 ± 3
NS NS NS
77 ± 5 37 ± 3 53 ± 2
74 ± 5 35 ± 3 53 ± 2
NS NS NS
1/24 4/24 1/24
2/24 11/24 0/24
NS 0.0599 NS
Variables are shown as mean ± one standard error (S.E.M.). LDL, low-density lipoprotein; angiotensin receptor blocker; VD, vessel disease; LMT, left main trunk; CABG,ACE, angiotensin converting enzyme; ARB, coronary artery bypass graft; IABP, intra-aortic balloon pumping; LVEDVI, left ventricular end-diastolic volume index; LVESVI, left ventricular end-systolic volume index; LVEF, left ventricular ejection fraction; Af, atrial fibrillation; VT, ventricular tachycardia; VF, ventricular fibrillation.
Fig. 1. (A) Representative gelatin zymographies of the pericardial fluid (PF) in pravastatin group and control group shown with MMP-2 and MMP-9 standard (STD). (B) The change in the pericardial MMP-2 and MMP-9 activities in control group (open bar) and in pravastatin group (closed bar). ⁎P b 0.05.
study. All patients were followed up at the outpatient clinic for a 2month period before the admission to the hospital for coronary artery bypass graft surgery. Some of the study patients (25 male patients) also were included in our previous study in which the effects of pravastatin on the gene expressions of adipokines and protein carbonyl group, an indicator of oxidative stress, in the visceral and subcutaneous adipose tissues were examined with the use of the same study design (Saito et al., 2008). In this previous study, we showed that adiponectin expression and generation in the visceral adipose tissue was increased in men with coronary artery disease treated with pravastatin, and as its mechanism, we suggested pravastatin-induced attenuation of oxidative stress in the adipose tissue. All patients underwent cardiac catheterization including coronary angiography and biplane left ventriculography (LVG) before the study, i.e.,N2 months before coronary artery bypass graft surgery, and 2 weeks after surgery. Two cardiologists who were unaware of the treatment assignment (with or without pravastatin pretreatment) analyzed the LVG using a LVG analysis system (Cardio 500, Kontron Instruments, Eching, Germany), and determined left ventricular enddiastolic (LVEDVI) and end-systolic volume indexes (LVESVI) and left ventricular ejection fraction (LVEF). 2.2. Sampling of venous blood and pericardial fluid during surgery We measured MMP activities in the plasma and the pericardial fluid. This was because several studies including our previous ones have shown that the pericardial fluid, which strongly reflects the circumstances in the myocardial tissue, is useful in investigating the pathophysiology of ischemic heart disease (Mallat et al., 1998; Kameda et al., 2003; Abe et al., 2006). Immediately after a small incision of the pericardium, undiluted samples of pericardial fluid were obtained. The samples were collected in sterile tubes, placed immediately on ice, clarified by centrifugation at 3000 ×g for 10 min at 4 °C, and rapidly frozen at −80 °C until analyses. The venous blood was drawn before coronary artery bypass graft surgery. The samples were collected in the tubes containing 10.5 mg of EDTA and immediately placed on ice. After clarification by removing the cellular components by centrifugation at 3000 ×g for 15 min at 4 °C, the plasma samples were rapidly stored at −80 °C until analyses.
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2.4. SDS-PAGE zymography To determine MMP-2 and MMP-9 activities, the quantitative gelatin zymography was performed (Fujita et al., 2001; Kai et al., 1998). Briefly, the plasma and pericardial samples (3 μl) were separated by dilution into zymography sample buffer. The samples and MMP-2 standard (1.25 × 10− 3 unit/lane, Wako chemical, Japan) were electrophoresed in a 10% gelatin gel, and incubated in renaturing buffer (2.5% Triton X-100). The gel was incubated with development buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 1 μM ZnCl2, 0.02% Briji-35) at 37 °C for 18 h, and stained with 0.5% Coomassie blue G-250 for 3 h. The gels were digitized using a scanning digitizing system and analyzed using NIH image software. The MMP-2 (72–62 kDa) and MMP-9 (92–85 kDa) activities were expressed as the ratio to MMP-2 standard to avoid the differences among gels. 2.5. Statistical analysis
Fig. 2. (A) Representative gelatin zymographies of the plasma in pravastatin group and control group shown with MMP-2 and MMP-9 standard (STD). (B) The change in the plasma MMP-2 and MMP-9 activities in control group (open bar) and in pravastatin group (closed bar). ⁎P b 0.05.
2.3. Measurements of MMP-2, MMP-9 and 8-iso-PGF2α levels The plasma and pericardial fluid levels of MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacture's instructions (MMP-2, MMP-9 and TIMP-2: Biotrak, Amersham Pharmacia Biotech, UK; TIMP-1: Daiichi Fine Chemical Industries, Japan). The pericardial fluid level of 8-iso-PGF2α was measured by a specific enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI).
Fig. 3. Relationships between the pericardial MMP-2 (A) and MMP-9 (B) activities and the plasma MMP-2 and MMP-9 activities, respectively. PF = pericardial fluid.
All results are expressed as mean ± S.E.M. Clinical characteristics, LVG parameters and inhospital cardiac events after coronary artery bypass graft surgery were compared between the 2 groups using unpaired t-test and the chi-square test. A linear regression analysis was performed to examine the correlation between MMP activities in the pericardial fluid and each of 8-iso-PGF2α and MMP activities in the plasma. A P value b0.05 was considered as a significant. 3. Results 3.1. Comparison of clinical and angiographic profiles There was no significant difference in the age, gender, the number of diseased vessel and the use of intra-aortic balloon pumping after coronary artery bypass graft surgery between the 2 groups. There was no significant difference in the levels of blood chemistries including LDL cholesterol measured just before surgery and in the drugs administered prior to surgery. In pravastatin group, LDL cholesterol level was decreased from 3.13 ± 0.10 to 2.69 ± 0.18 mM after the treatment (P b 0.05). There was no difference in in-hospital cardiac
Fig. 4. (A) The changes in the pericardial MMP-2 and MMP-9 levels determined by ELISA in control group (open bar) and in pravastatin group (closed bar). (B) The changes in the plasma MMP-2 and MMP-9 levels in control group (open bar) and in pravastatin group (closed bar). PF = pericardial fluid, ⁎P b 0.05.
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the plasma MMP-9 activity and the pericardial MMP-9 activity (r = 0.46, P b 0.01) (Fig. 3B). By ELISA, both the pericardial fluid levels of MMP-2 and MMP-9 in pravastatin group were significantly lower than those in control group (both P b 0.05, Fig. 4A). Also, both the plasma levels of MMP-2 and MMP-9 in pravastatin group were significantly lower than those in control group (both P b 0.05, Fig. 4B). There were significant positive correlations between the plasma MMP-2 level and the pericardial MMP-2 level (r = 0.48, P b 0.05) and between the plasma MMP-9 level and the pericardial MMP-9 level (r = 0.65, P b 0.05). There were no differences in the pericardial TIMP-1 and TIMP-2 levels between the 2 groups (TIMP-1, 1449 ± 68 in pravastatin group vs 1391 ± 82 ng/ml in control group; TIMP-2, 222 ± 25 vs 223 ± 22 ng/ml). Also there were no differences in the plasma TIMP-1 and TIMP-2 levels between the 2 groups (TIMP-1, 89 ± 7 in pravastatin group vs 98 ± 9 ng/ml in control group; TIMP-2, 42 ± 8 vs 42 ± 5 ng/ml). 3.3. Comparison of the pericardial level of 8-iso-PGF2α In pravastatin group, the pericardial level of 8-iso-PGF2α was significantly lower than that in control group (P b 0.05, Fig. 5A). Both the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial level of 8-iso-PGF2α (Fig. 5B and C, respectively). The pericardial MMP levels were also positively correlated with the pericardial level of 8-iso-PGF2α (MMP-2, r = 0.46; MMP-9, r = 0.45, both P b 0.05). 4. Discussion 4.1. Major findings The present study showed that, in patients with coronary artery disease and treated with pravastatin for 2 months, both of the pericardial and plasma MMP-2 and MMP-9 activities and levels were decreased, and also the pericardial 8-iso-PGF2α level was decreased compared with those without pravastatin treatment. Both the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level. Thus, the pericardial oxidative stress and MMP activities are reduced in patients with coronary artery disease and treated with pravastatin, which may be beneficial in preventing and reducing ventricular remodeling. 4.2. Effect of pravastatin on MMP activity and oxidative stress in patients with coronary artery disease Fig. 5. (A) The change in the pericardial 8-iso-PGF2α level in control group (open bar) and in pravastatin group (closed bar). (B, C) Relationship between the pericardial MMP-2 and MMP-9 activities and the pericardial 8-iso-PGF2α level. PF = pericardial fluid, ⁎P b 0.05.
events between the 2 groups. There were no differences in the LVG parameters before and after surgery between the 2 groups. Atrial fibrillation occurred after surgery less frequently in pravastatin group (4/24) than in control group (11/24), but the difference did not reach a statistical significance (P = 0.0599) (Table 1). 3.2. Comparison of the pericardial and plasma MMP activities and levels Representative examples of gelatin zymography in pravastatin and control groups are shown in the pericardial fluid (Fig. 1A) and the plasma (Fig. 2A). In pravastatin group, both MMP-2 and MMP-9 activities in the pericardial fluid were significantly lower than those in control group (both P b 0.05, Fig. 1B). Both MMP-2 and MMP-9 activities in the plasma were significantly lower in pravastatin group than those in control group (both P b 0.05, Fig. 2B). There were significant positive correlations between the plasma MMP-2 activity and the pericardial MMP-2 activity (r = 0.51, P b 0.01) (Fig. 3A) and between
In addition to the inhibition of hepatic cholesterol synthesis, statins have pleiotropic, cardioprotective effects (Davignon et al., 2004). In the previous study, statin reduced cardiovascular events in patients with coronary artery disease (Scandinavian Simvastatin Survival Study Group, 1994; Sacks et al., 1996). A previous experimental study showed that fluvastatin improves left ventricular remodeling and function after myocardial infarction via attenuation of increased MMP activity in mice (Hayashidani et al., 2002). Oxidative stress and activation of MMP seem to be involved deeply in the pathogenesis of ventricular remodeling and heart failure (Peterson et al., 2001; Rohde et al., 1999; Nian et al., 2004; McMurray et al., 1993; Cappola et al., 2001). It was shown that oxidative stress activates MMP-2 and MMP-9 in cultured smooth muscle cells (Rajagopalan et al., 1996). Nakamura et al. recently reported that carvedilol, a beta blocker having an anti-oxidative effect, decreased the elevated level of oxidative stress and improved function of failing myocardium in patients with dilated cardiomyopathy (Nakamura et al., 2002). This, in part, may be explained by the suppression of MMP activity by carvedilol via its anti-oxidant effect. The pericardial fluid is derived through the lymphatic channels and therefore seems to reflect the circumstances of the myocardial
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interstitial tissue (Fujita et al., 2001). In several studies including our previous ones, the pericardial fluid is useful in investigating the pathophysiology of ischemic heart disease (Mallat et al., 1998; Kameda et al., 2003; Abe et al., 2006). The present study showed that MMP-2 and MMP-9 activities and levels in the pericardial fluid were lower in pravastatin than in control group. The pericardial 8-iso-PGF2α level also was significantly decreased in pravastatin group compared with control group. The baseline characteristics were similar between the patient groups with and without pravastatin treatment except for LDL cholesterol level before the treatment. Thus, the reduction of MMP activities and 8-iso-PGF2α level in the pericardial fluid appears to be caused by pravastatin. The present study further showed that the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level, suggesting their mechanistic link. It is therefore suggested that pravastatin decreased MMP activities by reducing oxidative stress in the myocardial tissue. It was recently reported that cardiac production of MMP-2 and -9 determined by the difference in their serum levels between the coronary sinus and arterial blood samples is enhanced in patients with AMI, being associated with the enlargement of LVEDVI, and pravastatin treatment for 3–4 weeks after the onset of AMI is associated with the reduction of cardiac MMPs production (Yasuda et al., 2007). MMPs have been shown to play an important role in the progression of cardiovascular diseases, including atherosclerosis, plaque vulnerability (Kai et al., 1998; Brown et al., 1995; Inokubo et al., 2001), restenosis after coronary angioplasty and ventricular remodeling after myocardial infarction and pacing-induced heart failure (Peterson et al., 2001; Rohde et al., 1999; Miyamoto et al., 2002; Spinale et al., 1998; Mukherjee et al., 2003). MMP-2 and MMP-9 are released from several inflammatory cells activated by oxidative stress (Galis et al., 1994; Lindsey et al., 2001; Schwartz et al., 1998). Oxidative stress is related to the progression of heart failure and atherosclerotic plaque instability, and is increased in the ischemic myocardium and failing heart (Delanty et al., 1997; Ohara et al., 1993). We reported that the pericardial fluid levels of MMP-2 and MMP-9 activities, which were positively correlated with a marker of oxidative stress, 8-iso-PGF2α, were related to the development of left ventricular remodeling in patients with coronary artery disease (Kameda et al., 2003). We also reported that the circulating MMP activity at day 14 after the onset of AMI was correlated with the changes in LVEDVI and LVESVI at 6 months after the onset of AMI (Matsunaga et al., 2005). In the present study, although pravastatin treatment for 2 months before surgery was associated with reduced MMP-2 and MMP-9 activities and levels, it was not with the decreases in LVEDVI and LVESVI. This may be explained by a relatively short period of pravastatin treatment or by a type 2 statistical error due to a small number of study patients. A previous study showed that in patients with idiopathic dilated cardiomyopathy, simvastatin treatment for 14 weeks was associated with a lower New York Heart Association functional class and improved left ventricular ejection fraction compared with placebo (Node et al., 2003). Further studies on the effect of statins on the development of ventricular remodeling would be required. 4.3. Study limitations We administered pravastatin to the patients with a serum LDL cholesterol level N2.59 mM for a 2-month period before coronary artery bypass graft surgery in accord with the guidelines (National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult treatment panel III), 2002). We then compared MMP activities and levels and 8-iso-PGF2α level between the patient groups, those with or without pravastatin treatment. Therefore, it may be pointed out that the present observation was not necessarily related to the pravastatin's effects, but rather to pre-existing hypercholesterolemia. To exclude this possibility, a prospective, randomized study should
be required at least in the patient group with LDL cholesterol level b2.59 mM in which either pravastatin or placebo can be randomly administered. The present study included only the patients undergoing surgical therapy for coronary artery disease, and control subjects without coronary artery disease, i.e., normal subjects, were not included. Thus, it is unclear whether the levels and activities measured in the present patients are abnormally high or not. A study on the effect of pravastatin on MMP activities in patients without overt coronary artery disease is required. 5. Conclusion Oxidative stress seems to play an important role in the regulation of MMP activity, and the augmented MMP activity may be involved in the development of ventricular remodeling in patients with coronary artery disease. 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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Decreased plasma and cardiac matrix metalloproteinase activities in patients with coronary artery disease and treated with pravastatin Takayuki Fujiwara a, Shin Saito a, Tomohiro Osanai a, Kunihiko Kameda a, Naoki Abe a, Takumi Higuma a, Jin Yokoyama a, Hiroyuki Hanada a, Kozo Fukui b, Ikuo Fukuda b, Ken Okumura a,⁎ a b
Division of Cardiology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan Division of Cardiovascular Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
a r t i c l e
i n f o
Article history: Received 2 January 2008 Received in revised form 16 July 2008 Accepted 23 July 2008 Available online 31 July 2008 Keywords: Pravastatin Matrix metalloproteinase Oxidative stress Coronary artery disease
a b s t r a c t Matrix metalloproteinase (MMP), which is activated by oxidative stress, plays an important role in the development of ventricular remodeling in coronary artery disease. Pravastatin is shown to reduce oxidative stress. We tested the hypothesis that cardiac oxidative stress and MMP activity are reduced in patients with coronary artery disease and treated with pravastatin. Forty-eight patients who underwent coronary artery bypass graft surgery (CABG) were studied. Twenty-four patients had the serum low-density lipoprotein (LDL) cholesterol level N2.59 mM, and were treated with pravastatin (10 mg/day) for 2 months before CABG (pravastatin group). The other 24 had LDL cholesterol ≤ 2.59 mM, and were untreated (control group). The plasma and pericardial MMP-2 and MMP-9 activities were measured by gelatin zymography, and MMP-2 and MMP-9 levels, and pericardial 8-iso-prostagrandin F2α (8-iso-PGF2α) level, a maker of oxidative stress, by enzyme-linked immunosorbent assay. The plasma and pericardial MMP-2 and MMP-9 activities and levels were all lower by 20–30% in pravastatin than in control group (all P b 0.05). The pericardial 8-iso-PGF2α level was lower in pravastatin than in control group (38 ± 4 vs 64 ± 7 pg/ml, P b 0.05). The pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level (r = 0.57 and 0.47, respectively, both P b 0.01). Thus, cardiac oxidative stress and MMP activities are reduced in patients with coronary artery disease and treated with pravastatin, which may be beneficial in preventing and reducing ventricular remodeling. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Left ventricular remodeling after myocardial infarction is an important modulator of long-term ventricular function and both morbidity and mortality (Preffer et al., 1990). Matrix metalloproteinases (MMPs) were shown to play an important role in the development of left ventricular remodeling in the experimental myocardial infarction model and are involved deeply in the pathogenesis of cardiovascular diseases (Peterson et al., 2001; Rohde et al., 1999; Nian et al., 2004). We recently reported that the circulating level of gelatinolysis activity predicts the development of ventricular remodeling in patients with acute myocardial infarction (AMI) (Matsunaga et al., 2005). Oxidative stress has been implicated in the pathogenesis of congestive heart failure (McMurray et al., 1993; Cappola et al., 2001). Mallat et al. reported that the pericardial level of 8-isoprostaglandin F2α (8-iso-PGF2α), a specific and quantitative biochem-
⁎ Corresponding author. The Division of Cardiology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan. Tel.: +81 172 39 5057; fax: +81 172 35 9190. E-mail address: [email protected] (K. Okumura). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.07.039
ical marker for oxidative stress in vivo, is increased as the functional severity of congestive heart failure is increased (Mallat et al., 1998). In addition, anti-oxidant agents such as allopurinol and carvedilol were shown to improve cardiac function in human failing heart (Cappola et al., 2001; Nakamura et al., 2002). Recently, we showed that the pericardial level of 8-iso-PGF2α is positively correlated with those of MMP-2 and MMP-9 activities in patients with coronary artery disease (Kameda et al., 2003). 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) improve endothelial dysfunction, increase nitric oxide bioavailability, show anti-oxidant properties, inhibit inflammatory response, and stabilize atherosclerotic plaques; thereby improving the prognosis for patients with coronary artery disease independently of their cholesterol-lowing effects (Davignon et al., 2004). In fact, statin reduced morbidity and mortality in patients with coronary artery disease by lowering serum low-density lipoprotein (LDL) cholesterol levels and by stabilizing atherosclerotic plaques (Scandinavian Simvastatin Survival Study Group, 1994; Sacks et al., 1996). Simvastatin reduced the occurrence of heart failure in patients with coronary artery disease and without previous evidence of congestive heart failure (Kjekshus et al., 1997). In an experimental mice model, fluvastatin improved left ventricular remodeling and
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function after myocardial infarction via attenuation of increased MMP activity (Hayashidani et al., 2002). However, it is unknown how in patients with coronary artery disease, treatment with statin affects cardiac MMP levels and activities and oxidative stress. To clarify it, we measured both the plasma and pericardial MMP activities and their levels and the pericardial 8-iso-PGF2α level in patients with coronary artery disease treated with and without pravastatin prior to coronary artery bypass graft surgery. 2. Methods 2.1. Study patients The study protocol was approved by the ethical committee on human research at our institution. All patients gave informed consent before the study. Forty-eight patients with coronary artery disease who underwent coronary artery bypass graft surgery at our institution were studied. None of the patients had been administered with statin before the study. Of these 48 patients, 24 had the serum LDL cholesterol level N2.59 mM (mean ± S.E.M., 3.13 ± 0.10 mM) at the time when coronary artery bypass graft surgery was indicated, and were treated with pravastatin (10 mg/day) (Mevalotin, Daiichi-Sankyo Pharmaceutical Inc., Japan) for 2 months before coronary artery bypass graft surgery (pravastatin group). The other 24 patients had LDL cholesterol level ≤2.59 mM (2.44 ± 0.78 mM, P b 0.01 vs pravastatin group), and were not treated (control group). The patients with AMI, severe liver, renal, or brain dysfunction, malignant tumor, autoimmune disease, and other serious complications were excluded from the present Table 1 Comparison of clinical and angiographic characteristics between 2 groups
Age Gender (male/female) Blood chemistry Total cholesterol (mM) LDL cholesterol (mM) Triglyceride (mM) Creatinine (mg/dl) Blood sugar (mg/dl) HbA1C (%) Drug Beta-blocker Spironolactone ACE inhibitor ARB Nicorandil (oral administration) No. of diseased vessel 1VD 2VD 3VD LMT Use of IABP after CABG LVG parameters before CABG LVEDVI LVESVI LVEF LVG parameters after CABG LVEDVI LVESVI LVEF Inhospital cardiac events after CABG Heart failure Af VT, VF
Pravastatin (n = 24)
Control (n = 24)
P value
68 ± 2 20/4
67 ± 2 19/5
NS NS
4.53 ± 0.21 2.69 ± 0.18 1.25 ± 0.08 1.0 ± 0.1 126 ± 11 6.1 ± 0.3
4.66 ± 0.18 2.59 ± 0.18 1.30 ± 0.09 1.0 ± 0.1 130 ± 8 6.1 ± 0.2
NS NS NS NS NS NS
17/24 8/24 10/24 12/24 9/24
18/24 7/24 12/24 9/24 12/24
NS NS NS NS NS
4/24 9/24 11/24 11/24 2/24
4/24 11/24 9/24 12/24 1/24
NS NS NS NS NS
81 ± 4 41 ± 3 51 ± 2
79 ± 5 41 ± 4 51 ± 3
NS NS NS
77 ± 5 37 ± 3 53 ± 2
74 ± 5 35 ± 3 53 ± 2
NS NS NS
1/24 4/24 1/24
2/24 11/24 0/24
NS 0.0599 NS
Variables are shown as mean ± one standard error (S.E.M.). LDL, low-density lipoprotein; angiotensin receptor blocker; VD, vessel disease; LMT, left main trunk; CABG,ACE, angiotensin converting enzyme; ARB, coronary artery bypass graft; IABP, intra-aortic balloon pumping; LVEDVI, left ventricular end-diastolic volume index; LVESVI, left ventricular end-systolic volume index; LVEF, left ventricular ejection fraction; Af, atrial fibrillation; VT, ventricular tachycardia; VF, ventricular fibrillation.
Fig. 1. (A) Representative gelatin zymographies of the pericardial fluid (PF) in pravastatin group and control group shown with MMP-2 and MMP-9 standard (STD). (B) The change in the pericardial MMP-2 and MMP-9 activities in control group (open bar) and in pravastatin group (closed bar). ⁎P b 0.05.
study. All patients were followed up at the outpatient clinic for a 2month period before the admission to the hospital for coronary artery bypass graft surgery. Some of the study patients (25 male patients) also were included in our previous study in which the effects of pravastatin on the gene expressions of adipokines and protein carbonyl group, an indicator of oxidative stress, in the visceral and subcutaneous adipose tissues were examined with the use of the same study design (Saito et al., 2008). In this previous study, we showed that adiponectin expression and generation in the visceral adipose tissue was increased in men with coronary artery disease treated with pravastatin, and as its mechanism, we suggested pravastatin-induced attenuation of oxidative stress in the adipose tissue. All patients underwent cardiac catheterization including coronary angiography and biplane left ventriculography (LVG) before the study, i.e.,N2 months before coronary artery bypass graft surgery, and 2 weeks after surgery. Two cardiologists who were unaware of the treatment assignment (with or without pravastatin pretreatment) analyzed the LVG using a LVG analysis system (Cardio 500, Kontron Instruments, Eching, Germany), and determined left ventricular enddiastolic (LVEDVI) and end-systolic volume indexes (LVESVI) and left ventricular ejection fraction (LVEF). 2.2. Sampling of venous blood and pericardial fluid during surgery We measured MMP activities in the plasma and the pericardial fluid. This was because several studies including our previous ones have shown that the pericardial fluid, which strongly reflects the circumstances in the myocardial tissue, is useful in investigating the pathophysiology of ischemic heart disease (Mallat et al., 1998; Kameda et al., 2003; Abe et al., 2006). Immediately after a small incision of the pericardium, undiluted samples of pericardial fluid were obtained. The samples were collected in sterile tubes, placed immediately on ice, clarified by centrifugation at 3000 ×g for 10 min at 4 °C, and rapidly frozen at −80 °C until analyses. The venous blood was drawn before coronary artery bypass graft surgery. The samples were collected in the tubes containing 10.5 mg of EDTA and immediately placed on ice. After clarification by removing the cellular components by centrifugation at 3000 ×g for 15 min at 4 °C, the plasma samples were rapidly stored at −80 °C until analyses.
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2.4. SDS-PAGE zymography To determine MMP-2 and MMP-9 activities, the quantitative gelatin zymography was performed (Fujita et al., 2001; Kai et al., 1998). Briefly, the plasma and pericardial samples (3 μl) were separated by dilution into zymography sample buffer. The samples and MMP-2 standard (1.25 × 10− 3 unit/lane, Wako chemical, Japan) were electrophoresed in a 10% gelatin gel, and incubated in renaturing buffer (2.5% Triton X-100). The gel was incubated with development buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 1 μM ZnCl2, 0.02% Briji-35) at 37 °C for 18 h, and stained with 0.5% Coomassie blue G-250 for 3 h. The gels were digitized using a scanning digitizing system and analyzed using NIH image software. The MMP-2 (72–62 kDa) and MMP-9 (92–85 kDa) activities were expressed as the ratio to MMP-2 standard to avoid the differences among gels. 2.5. Statistical analysis
Fig. 2. (A) Representative gelatin zymographies of the plasma in pravastatin group and control group shown with MMP-2 and MMP-9 standard (STD). (B) The change in the plasma MMP-2 and MMP-9 activities in control group (open bar) and in pravastatin group (closed bar). ⁎P b 0.05.
2.3. Measurements of MMP-2, MMP-9 and 8-iso-PGF2α levels The plasma and pericardial fluid levels of MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were determined by a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacture's instructions (MMP-2, MMP-9 and TIMP-2: Biotrak, Amersham Pharmacia Biotech, UK; TIMP-1: Daiichi Fine Chemical Industries, Japan). The pericardial fluid level of 8-iso-PGF2α was measured by a specific enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI).
Fig. 3. Relationships between the pericardial MMP-2 (A) and MMP-9 (B) activities and the plasma MMP-2 and MMP-9 activities, respectively. PF = pericardial fluid.
All results are expressed as mean ± S.E.M. Clinical characteristics, LVG parameters and inhospital cardiac events after coronary artery bypass graft surgery were compared between the 2 groups using unpaired t-test and the chi-square test. A linear regression analysis was performed to examine the correlation between MMP activities in the pericardial fluid and each of 8-iso-PGF2α and MMP activities in the plasma. A P value b0.05 was considered as a significant. 3. Results 3.1. Comparison of clinical and angiographic profiles There was no significant difference in the age, gender, the number of diseased vessel and the use of intra-aortic balloon pumping after coronary artery bypass graft surgery between the 2 groups. There was no significant difference in the levels of blood chemistries including LDL cholesterol measured just before surgery and in the drugs administered prior to surgery. In pravastatin group, LDL cholesterol level was decreased from 3.13 ± 0.10 to 2.69 ± 0.18 mM after the treatment (P b 0.05). There was no difference in in-hospital cardiac
Fig. 4. (A) The changes in the pericardial MMP-2 and MMP-9 levels determined by ELISA in control group (open bar) and in pravastatin group (closed bar). (B) The changes in the plasma MMP-2 and MMP-9 levels in control group (open bar) and in pravastatin group (closed bar). PF = pericardial fluid, ⁎P b 0.05.
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the plasma MMP-9 activity and the pericardial MMP-9 activity (r = 0.46, P b 0.01) (Fig. 3B). By ELISA, both the pericardial fluid levels of MMP-2 and MMP-9 in pravastatin group were significantly lower than those in control group (both P b 0.05, Fig. 4A). Also, both the plasma levels of MMP-2 and MMP-9 in pravastatin group were significantly lower than those in control group (both P b 0.05, Fig. 4B). There were significant positive correlations between the plasma MMP-2 level and the pericardial MMP-2 level (r = 0.48, P b 0.05) and between the plasma MMP-9 level and the pericardial MMP-9 level (r = 0.65, P b 0.05). There were no differences in the pericardial TIMP-1 and TIMP-2 levels between the 2 groups (TIMP-1, 1449 ± 68 in pravastatin group vs 1391 ± 82 ng/ml in control group; TIMP-2, 222 ± 25 vs 223 ± 22 ng/ml). Also there were no differences in the plasma TIMP-1 and TIMP-2 levels between the 2 groups (TIMP-1, 89 ± 7 in pravastatin group vs 98 ± 9 ng/ml in control group; TIMP-2, 42 ± 8 vs 42 ± 5 ng/ml). 3.3. Comparison of the pericardial level of 8-iso-PGF2α In pravastatin group, the pericardial level of 8-iso-PGF2α was significantly lower than that in control group (P b 0.05, Fig. 5A). Both the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial level of 8-iso-PGF2α (Fig. 5B and C, respectively). The pericardial MMP levels were also positively correlated with the pericardial level of 8-iso-PGF2α (MMP-2, r = 0.46; MMP-9, r = 0.45, both P b 0.05). 4. Discussion 4.1. Major findings The present study showed that, in patients with coronary artery disease and treated with pravastatin for 2 months, both of the pericardial and plasma MMP-2 and MMP-9 activities and levels were decreased, and also the pericardial 8-iso-PGF2α level was decreased compared with those without pravastatin treatment. Both the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level. Thus, the pericardial oxidative stress and MMP activities are reduced in patients with coronary artery disease and treated with pravastatin, which may be beneficial in preventing and reducing ventricular remodeling. 4.2. Effect of pravastatin on MMP activity and oxidative stress in patients with coronary artery disease Fig. 5. (A) The change in the pericardial 8-iso-PGF2α level in control group (open bar) and in pravastatin group (closed bar). (B, C) Relationship between the pericardial MMP-2 and MMP-9 activities and the pericardial 8-iso-PGF2α level. PF = pericardial fluid, ⁎P b 0.05.
events between the 2 groups. There were no differences in the LVG parameters before and after surgery between the 2 groups. Atrial fibrillation occurred after surgery less frequently in pravastatin group (4/24) than in control group (11/24), but the difference did not reach a statistical significance (P = 0.0599) (Table 1). 3.2. Comparison of the pericardial and plasma MMP activities and levels Representative examples of gelatin zymography in pravastatin and control groups are shown in the pericardial fluid (Fig. 1A) and the plasma (Fig. 2A). In pravastatin group, both MMP-2 and MMP-9 activities in the pericardial fluid were significantly lower than those in control group (both P b 0.05, Fig. 1B). Both MMP-2 and MMP-9 activities in the plasma were significantly lower in pravastatin group than those in control group (both P b 0.05, Fig. 2B). There were significant positive correlations between the plasma MMP-2 activity and the pericardial MMP-2 activity (r = 0.51, P b 0.01) (Fig. 3A) and between
In addition to the inhibition of hepatic cholesterol synthesis, statins have pleiotropic, cardioprotective effects (Davignon et al., 2004). In the previous study, statin reduced cardiovascular events in patients with coronary artery disease (Scandinavian Simvastatin Survival Study Group, 1994; Sacks et al., 1996). A previous experimental study showed that fluvastatin improves left ventricular remodeling and function after myocardial infarction via attenuation of increased MMP activity in mice (Hayashidani et al., 2002). Oxidative stress and activation of MMP seem to be involved deeply in the pathogenesis of ventricular remodeling and heart failure (Peterson et al., 2001; Rohde et al., 1999; Nian et al., 2004; McMurray et al., 1993; Cappola et al., 2001). It was shown that oxidative stress activates MMP-2 and MMP-9 in cultured smooth muscle cells (Rajagopalan et al., 1996). Nakamura et al. recently reported that carvedilol, a beta blocker having an anti-oxidative effect, decreased the elevated level of oxidative stress and improved function of failing myocardium in patients with dilated cardiomyopathy (Nakamura et al., 2002). This, in part, may be explained by the suppression of MMP activity by carvedilol via its anti-oxidant effect. The pericardial fluid is derived through the lymphatic channels and therefore seems to reflect the circumstances of the myocardial
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interstitial tissue (Fujita et al., 2001). In several studies including our previous ones, the pericardial fluid is useful in investigating the pathophysiology of ischemic heart disease (Mallat et al., 1998; Kameda et al., 2003; Abe et al., 2006). The present study showed that MMP-2 and MMP-9 activities and levels in the pericardial fluid were lower in pravastatin than in control group. The pericardial 8-iso-PGF2α level also was significantly decreased in pravastatin group compared with control group. The baseline characteristics were similar between the patient groups with and without pravastatin treatment except for LDL cholesterol level before the treatment. Thus, the reduction of MMP activities and 8-iso-PGF2α level in the pericardial fluid appears to be caused by pravastatin. The present study further showed that the pericardial MMP-2 and MMP-9 activities were positively correlated with the pericardial 8-iso-PGF2α level, suggesting their mechanistic link. It is therefore suggested that pravastatin decreased MMP activities by reducing oxidative stress in the myocardial tissue. It was recently reported that cardiac production of MMP-2 and -9 determined by the difference in their serum levels between the coronary sinus and arterial blood samples is enhanced in patients with AMI, being associated with the enlargement of LVEDVI, and pravastatin treatment for 3–4 weeks after the onset of AMI is associated with the reduction of cardiac MMPs production (Yasuda et al., 2007). MMPs have been shown to play an important role in the progression of cardiovascular diseases, including atherosclerosis, plaque vulnerability (Kai et al., 1998; Brown et al., 1995; Inokubo et al., 2001), restenosis after coronary angioplasty and ventricular remodeling after myocardial infarction and pacing-induced heart failure (Peterson et al., 2001; Rohde et al., 1999; Miyamoto et al., 2002; Spinale et al., 1998; Mukherjee et al., 2003). MMP-2 and MMP-9 are released from several inflammatory cells activated by oxidative stress (Galis et al., 1994; Lindsey et al., 2001; Schwartz et al., 1998). Oxidative stress is related to the progression of heart failure and atherosclerotic plaque instability, and is increased in the ischemic myocardium and failing heart (Delanty et al., 1997; Ohara et al., 1993). We reported that the pericardial fluid levels of MMP-2 and MMP-9 activities, which were positively correlated with a marker of oxidative stress, 8-iso-PGF2α, were related to the development of left ventricular remodeling in patients with coronary artery disease (Kameda et al., 2003). We also reported that the circulating MMP activity at day 14 after the onset of AMI was correlated with the changes in LVEDVI and LVESVI at 6 months after the onset of AMI (Matsunaga et al., 2005). In the present study, although pravastatin treatment for 2 months before surgery was associated with reduced MMP-2 and MMP-9 activities and levels, it was not with the decreases in LVEDVI and LVESVI. This may be explained by a relatively short period of pravastatin treatment or by a type 2 statistical error due to a small number of study patients. A previous study showed that in patients with idiopathic dilated cardiomyopathy, simvastatin treatment for 14 weeks was associated with a lower New York Heart Association functional class and improved left ventricular ejection fraction compared with placebo (Node et al., 2003). Further studies on the effect of statins on the development of ventricular remodeling would be required. 4.3. Study limitations We administered pravastatin to the patients with a serum LDL cholesterol level N2.59 mM for a 2-month period before coronary artery bypass graft surgery in accord with the guidelines (National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult treatment panel III), 2002). We then compared MMP activities and levels and 8-iso-PGF2α level between the patient groups, those with or without pravastatin treatment. Therefore, it may be pointed out that the present observation was not necessarily related to the pravastatin's effects, but rather to pre-existing hypercholesterolemia. To exclude this possibility, a prospective, randomized study should
be required at least in the patient group with LDL cholesterol level b2.59 mM in which either pravastatin or placebo can be randomly administered. The present study included only the patients undergoing surgical therapy for coronary artery disease, and control subjects without coronary artery disease, i.e., normal subjects, were not included. Thus, it is unclear whether the levels and activities measured in the present patients are abnormally high or not. A study on the effect of pravastatin on MMP activities in patients without overt coronary artery disease is required. 5. Conclusion Oxidative stress seems to play an important role in the regulation of MMP activity, and the augmented MMP activity may be involved in the development of ventricular remodeling in patients with coronary artery disease. 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