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Atorvastatin lowers plasma matrix metalloproteinase-9 in patients with acute coronary syndrome.

Atheromatous plaque rupture and subsequent thrombosis are the main causes of acute coronary syndrome (ACS) including acute myocardial infarction (AMI), unstable angina pectoris (UAP), and sudden cardiac death (1, 2). Plaque instability is associated with a high macrophage content and a thin fibrous cap. Matrix metalloproteinases (MMPs) have the capability to degrade the extracellular matrix of the fibrous cap, predisposing to plaque rupture (3). Macrophages are the major source of the MMPs, of which MMP-9 is the most prevalent form. Several lines of evidence suggest that MMP-9 could play a potential role in atheromatous plaque disruption and in the molecular mechanism of ACS (4, 5). Clinical trials have demonstrated that statin therapy reduces cardiovascular events and mortality (6, 7). The MIRACL Study demonstrated that atorvastatin treatment during the acute phase of ACS reduced recurrent ischemic events (8). In addition to the effects on lipid concentrations, stabilization of atherosclerotic plaques and attenuation of the inflammatory response may account for the clinical benefits of statins in ACS. Animal experiments and clinical studies have shown that statins can stabilize plaque by increasing the collagen content and inhibiting metalloproteinases (9-11). Plaque stabilization could be achieved by direct inhibition of MMPs by statins.

For this study, we enrolled 40 patients with ACS (including 17 with AMI and 23 with UAP), who were in their first episode. All of the UAP patients fulfilled the criteria for class IIIB of the Ham and Braunwald (12) classification of unstable angina. The diagnosis of AMI was based on a history of ischemic chest pain, characteristic electrocardiographic changes, and increased creatine kinase-MB activity at least twice the upper reference limit (>48 U/L) within 24 h after the onset of pain. Exclusion criteria included body temperature >38.0[degrees]C, severe heart failure (NYHA class 3 or 4, Killip class III or IV for AMI patients), inflammatory diseases, impaired liver function, renal failure (plasma creatinine <178 [micro]mol/L), previous lipid-lowering therapy, and recent major surgery. The patients were randomly separated into two groups to receive conventional therapy (group A; n = 20) or a combination of conventional therapy plus 10 mg/day atorvastatin (group B; n = 20) for 4 weeks. Conventional therapy included aspirin, beta-blocker, angiotensin-converting enzyme inhibitor, low-molecular-weight heparin, and urokinase (for AMI patients). All patients gave written informed consent before study entry. The study protocol was approved by the Ethical Committee of Central South University. Twenty healthy individuals who were age- and sex-matched with patients served as controls.

Peripheral venous blood was drawn in heparin-containing tubes after overnight fasting at baseline and after 4 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Shanghai Biotech) gradient centrifugation. The PBMCs were aspirated and washed with Hanks Balanced Salt Solution (Sigma) twice and with RPMI 1640 (Gibco Life Technologies) once. The final pellet was suspended in serum-free RPMI 1640, and the cells were plated at a density 1 x [10.sup.7] cells/flask. Cell viability was routinely >95% as demonstrated by trypan blue exclusion. Nonadhering cells were moved after 6 h of incubation. Adhering monocytes were cultured for 24 h with RPMI 1640 supplemented with 2 mmol/L N-acetyl-L-alanyl-L-glutamine, 100 000 units/L penicillin, 100 mg/L streptomycin, 20 g/L sodium pyruvate, 20 mmol/L HEPES (Gibco Life Technologies), 100 mL/L heat-inactivated fetal calf serum (Gibco Life Technologies) and 1 mg/L lipopolysaccharide at 37[degrees]C in 5% C[O.sub.2]. For the in vitro study, monocytes were exposed to various concentrations of atorvastatin (0, 0.1, 1, and 10 [micro]mol/L) dissolved in dimethyl sulfoxide (Sigma) for 24 h. Cell culture supernatants were collected and stored at -70[degrees]C for MMP-9 analysis.

All cell supernatants and plasma were analyzed for MMP-9 by ELISA (Quantikine[TM]; R&D Systems) according to the manufacturer's instructions. The lowest detection limit for MMP-9 was 0.156 mg/L. The imprecision (CV) was 2.9% at the upper reference limit and 2.3% in ACS patients. Total cholesterol and triglyceride concentrations were measured by enzymatic methods. HDL- and LDL-cholesterol were measured by direct methods.

Statistical analysis was performed with the SPSS 10.0 statistical software package (SPSS Inc.). Data with a gaussian distribution are reported as the mean (SD), and skewed data are presented as the median (25th-75th percentiles) if not otherwise mentioned. Comparisons between groups were analyzed by t-test (two-sided) or one-way ANOVA followed by the Bonferroni test for experiments with more than two subgroups when appropriate. Comparison of categorical variables was by [chi square] test. Because some data were skewed, nonparametric tests (Wilcoxon signed-ranks test for paired data and Mann-Whitney U-test for unpaired data) were used. A P value <0.05 (two-tailed) was considered statistically significant.

There were no significant differences in baseline characteristics between the two treatment groups (Table 1). Patients with ACS had higher plasma MMP-9 concentrations than the controls [median (25th-75th percentile), 27.9 (7.4-48.7) vs 11.3 (4.8-29.0) mg/L; P <0.05]. MMP-9 concentrations in monocyte cultures from patients with ACS were significantly higher than concentrations in cultures from the healthy controls [165 (87.0-215.0) vs 84.3 (53.9-139.3) mg/L; P <0.05]. MMP-9 concentrations in plasma and monocyte supernatants from patients with AMI were significantly higher than in patients with UAP [34.9 (22.3-75.8) vs 15.7 (5.4-46.8) mg/L and 188.0 (147.7-234.3) vs 116.6 (82.2-181.0) mg/L, respectively; P <0.05 for both]. There was no significant change in lipid profiles at the end of 4 weeks in the group receiving conventional therapy, whereas total cholesterol, triglycerides, and LDL-cholesterol concentrations were significantly lower (data not shown) in the group receiving conventional therapy plus atorvastatin. After 4 weeks, plasma MMP-9 had decreased significantly in both groups: from 26.6 (13.8-45.9) to 17.7 (13.0-29.8) mg/L the group receiving conventional therapy; and from 28.3 (10.8-50.1) to 12.1 (6.5-22.1) mg/L in the group receiving conventional therapy plus atorvastatin (P <0.05 for both). The decrease in plasma MMP-9 in the group receiving conventional therapy plus atorvastatin was significantly greater than in the group receiving conventional alone (P <0.05).

MMP-9 in PBMC supernatants was significantly lower in two the ACS patients groups after therapy (P <0.05 for both), but the differences in MMP-9 between baseline and the values after 4 weeks in the two groups were not significant (Fig. 1A).

Atorvastatin significantly decreased the production of MMP-9 in PBMCs in vitro up to 62% (P <0.05), in a dose-dependent manner. The differences in MMP-9 concentrations produced in the presence of various concentrations of atorvastatin were also significant (P <0.05; Fig. 1B).

The present investigation shows that plasma concentrations of MMP-9 in patients with ACS are significantly higher than those in controls, which is consistent with other studies (5). Furthermore, our study showed that the amount of MMP-9 released by PBMCs from patients with ACS is significantly higher than the amount release by PBMCs from healthy individuals, which indicates that circulating monocytes may be one of the sources of circulating MMP-9 and could thus contribute to plaque degradation by secreting MMP-9.

Recently, MMP-9 was identified as a risk factor for unfavorable outcome in patients with coronary heart disease (13), which suggests that MMP-9 could serve as an indicator for estimating the effectiveness of treatment in patients with ACS. In the present investigation, the MMP-9 concentrations in blood samples from the group receiving conventional therapy plus atorvastatin showed a larger decrease than those in the group receiving conventional alone. From the above findings, it is tempting to speculate that the atorvastatin-induced reduction in plasma MMP-9 may, in part, provide clinical benefits in patients with ACS, although this hypothesis was not formally confirmed in the present investigation. Interestingly, plasma MMP-9 in the group receiving conventional therapy alone also decreased significantly, which could be attributable to aspirin and angiotensin-converting enzyme inhibitor, which have been shown to decrease MMP-9 production (14,15). We found that atorvastatin lowered the production of MMP-9 released by monocytes from health donors in vitro in a concentration-dependent manner, which is in accordance with previous studies (16,17). Porter and Turner (18) observed that simvastatin inhibited MMP-9 secretion after much longer incubation. Further study is needed to determine whether the effects of atorvastatin on MMP-9 secretion by monocytes are also concentration dependent in vivo. Although productions of MMP-9 in PBMCs from patients with ACS was significantly lower after 4 weeks of therapy in both the conventional and conventional plus atorvastatin groups, the differences between baseline and the values after 4 weeks in the two groups were not significant, which may be attributed to the low atorvastatin dose used in the present study.

[FIGURE 1 OMITTED]

We are grateful to the nursing staff of the Department of Cardiology of the Second XiangYa Hospital of Central South University for providing expert clinical assistance. We thank Donghai Huangfu for assistance in the preparation of this manuscript.

References

(1.) Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndrome. N Engl J Med 1992;326:242-50.

(2.) Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation 1995;92: 657-71.

(3.) Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135-43.

(4.) Galis ZS, Sukhova GK, Lark MW. Increased expression of matrix metalloproteinase and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493-503.

(5.) Kai H, Ikeda H, Yasukawa H, Kai M, Seki Y, Kuwahara F, et al. Peripheral blood levels of matrix metalloproteinase-2 and -9 are elevated in patients with acute coronary syndromes. J Am Coll Cardiol 1998;32:368-72.

(6.) The Scandinavian Simvastatin Survival Study Group. Randomized trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383-9.

(7.) Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med 1995;333:1301-7.

(8.) Schwartz GG, Olsson AG, Ezekowitz MD, Ganz P, Oliver MF, Waters D, et al.; Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) Study investigators. Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. JAMA 2001;285:1711-8.

(9.) Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, et al. An HMG-COA reductase inhibitors, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation 2001;103:276-83.

(10.) Sukhova G, Williams JK, Libby P. Statins reduce inflammation in atheroma of nonhuman primates independent of effects on serum cholesterol. Arterioscler Thromb Vasc Biol 2002;22:1452-8.

(11.) Crisby M, Nordin-Fredriksson G, Shah PK, Yano J, Zhu J, Nilsson J. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaque: implications for plaque stabilization. Circulation 2001;103:92633.

(12.) Hamm CW, Braunwald E. A classification of unstable angina revisited. Circulation 2000;102:118-22.

(13.) Blankenberg S, Rupprecht HJ, Poirier 0, Bickel C, Smieja M, Hafner G, et al.; AtheroGene Investigators. Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease. Circulation 2003;107:1579-85.

(14.) Murono S, Yoshizaki T, Sato H, Takeshita H, Furukawa M, Pagano JS. Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression. Cancer Res 2000;60:2555-61.

(15.) Li H, Simon H, Bocan TM, Peterson JT. MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE- and MMP-inhibition. Cardiovasc Res 2000;46:298-306.

(16.) Grip O, Janciauskiene S, Lindgren S. Atorvastatin activates PPAR-y and attenuates the inflammatory response in human monocytes. Inflamm Res 2002;51:58-62.

(17.) Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Paoletti R, et al. HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 1998;18:1671-8.

(18.) Porter KE, Turner NA. Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9. Biochem Soc Trans 2002;30: 120-6.

DOI: 10.1373/clinchem.2003.026070

Zhumei Xu, Shuiping Zhao, * Hongnian Zhou, Huijun Ye, and Jiang Li (Department of Cardiology, the Second XiangYa Hospital, Central South University, Changsha, Hunan, China; * address correspondence to this author at: Department of Cardiology, The Second XiangYa Hospital, Central South University, Middle Renmin Road, No. 86, Changsha, Hunan 410011, China; fax 86-731-4895989, e-mail ZhaoSP@public.cs.hn.cn or zhumeixu2001@yahoo.com.cn)
Table 1. Baseline characteristics of ACS patients treated (group B)
and or not treated (group A) with atorvastatin.

 Group A Group B
 (n = 20) (n = 20) P

Mean (SD) age, years 63.3 (9.1) 61.7 (7.8) 0.828
M/F, n 17/3 16/4 0.5
AMI, n (%) 8 (40%) 9 (45%) 0.5
Mean (SD) body mass index, 23.4 (3.8) 24.5 (4.5) 0.721
 kg/[m.sup.2]
Smoking, n (%) 9 (45%) 8 (40%) 0.5
Hypertension, n (%) 7 (35%) 8 (40%) 0.5
Diabetes mellitus, n (%) 3 (15%) 3 (15%) 0.5
Mean (SD) blood pressure,
mmHg
 Systolic 129.2 (26.6) 125.5 (23.9) 0.612
 Diastolic 76.9 (16.8) 77.5 (15.1) 0.541
Mean (SD) TC, (a) mmol/L 5.44 (1.26) 5.57 (1.39) 0.793
Mean (SD) TG, mmol/L 2.30 (1.57) 2.34 (1.69) 0.588
Mean (SD) HDL-C, mmol/L 1.12 (0.26) 1.15 (0.27) 0.312
Mean (SD) fasting glucose, 5.7 (2.3) 5.8 (2.8) 0.176
 mmol/L
Mean (SD) creatinine, 120.5 (23.9) 120.6 (21.9) 0.754
 [micro]mol/L
Mean (SD) creatine 108.1 (135.8) 106.8 (132.8) 0.223
 kinase-MB, U/L
Mean (SD) WBC, 7.02 (1.98) 7.13 (2.01) 0.28
 [10.sup.9]/L
Medications, n (%)
 Beta-blockers 18 (90%) 18 (90%) 0.5
 ACE inhibitors 19 (95%) 18 (90%) 0.5
 Nitrates 12 (60%) 11 (55%) 0.5
 Aspirin 18 (90%) 19 (95%) 0.5
 LMWH 17 (85%) 18 (90%) 0.5

(a) TC, total cholesterol; TG, triglycerides; HDL-C, HDL-cholesterol;
WBC, white blood cell count; ACE, angiotensin-converting enzyme;
LMWH; low-molecular-weight heparin.
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Title Annotation:Technical Briefs
Author:Zhumei, Xu; Shuiping, Zhao; Hongnian, Zhou; Huijun, Ye; Jiang, Li
Publication:Clinical Chemistry
Date:Apr 1, 2004
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