C-reactive protein-induced upregulation of extracellular matrix metalloproteinase inducer in macrophages: Inhibitory effect of fluvastatin

Abstract

Objective

Extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMP)-9 were reported to be expressed at the macrophage-rich area in human coronary atherosclerotic plaque. We examined whether C-reactive protein (CRP) activates macrophages to express EMMPRIN and MMP-9 in vitro and whether statins inhibit it.

Methods and results

Rat peritoneal macrophages were collected by peritoneal lavage, and were incubated in the presence or absence of CRP. CRP at 5 μg/ml increased the gene expression of EMMPRIN relative to GAPDH, measured by RT-PCR, by 1.67 ± 0.07 fold at 24 h and by 1.85 ± 0.49 fold at 48 h (both p < 0.05). The gene expression of MMP-9 in the presence of CRP at 5 μg/ml was followed by 1.36 ± 0.11 fold increase at 24 h and by 3.95 ± 0.81 fold at 48 h (both p < 0.05). CRP at 5 μg/ml for 48 h increased by 6 fold MMP-9 activity, measured by zymography, without affecting tissue inhibitor of metalloproteinases-1. Boiled CRP at 5 μg/ml for 48 h unaffected MMP-9 activity. Fluvastatin blocked the CRP-induced increases in EMMPRIN and MMP-9 expression and activity. Diphenylene iodonium, an inhibitor of NADPH oxidase, had a similar effect on MMP-9 activity. Fluvastatin suppressed the CRP-induced increases in 8-epi-prostaglandin F_2α levels in the condition media.

Conclusions

CRP is an activator for macrophages to enhance EMMPRIN and MMP-9 expression. Fluvastatin inhibits them presumably through its antioxidant effect.

Introduction

Atherosclerotic plaques are characterized by a lipid core covered by a fibrous cap composed of vascular smooth muscle cells (VSMCs) and extracellular matrix (Ross, 1993). These plaques have chronic inflammatory infiltrates of macrophages and lymphocytes, and plaque disruption is the main cause for acute coronary syndrome including unstable angina, acute myocardial infarction, and sudden death. Plaque instability including ulceration of the fibrous cap, hemorrhage of the intraplaque, or plaque rupture is characterized by the presence of activated macrophages and high lipid content (Falk et al., 1995). Extracellular matrix metalloproteinase inducer (EMMPRIN) induces matrix metalloproteinases (MMPs) (van et al., 1997, Yang et al., 2003), and MMPs play an important role in plaque disruption (Bellosta et al., 1998). In human coronary atherosclerotic samples (atherectomy materials from unstable angina), EMMPRIN was shown to be present in CD68-positive macrophage-rich areas as well as in the areas with MMP-9 expressions (Major et al., 2002). However, little is known about the relationship between macrophage activation and EMMPRIN or MMP-9 expression and what is involved in the activation of macrophages at plaque sites.

C-reactive protein (CRP) is not only a biomarker for atherosclerotic events (Ridker et al., 2000) but a potent vasoactive mediator to promote atherogenesis. In monocytes, CRP induces the production of inflammatory cytokines and promotes monocyte chemotaxis and tissue factor expression (Cermak et al., 1993). In endothelial cells, CRP increases the expression of cell adhesion molecules, chemokines, and endothelin-1 (ET-1), decreases endothelial nitric oxide synthase (eNOS) expression and activity, and augments monocyte-endothelial cell adhesion (Pasceri et al., 2001, Venugopal et al., 2002, Verma et al., 2002). In VSMCs, CRP promotes its migration and proliferation and increases the production of reactive oxygen species (ROS) (Wang et al., 2003). In human monocyte-derived macrophages, CRP was shown to increase MMP-1 production (Williams et al., 2004). The 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors or statins have an antioxidant effect (Wassmann et al., 2002) and were shown to prevent events of coronary artery disease (Heart Protection Study Collaborative Group, 2002) and stabilize atherosclerotic plaques (Fukumoto et al., 2001). In the present study, we tested the hypothesis that CRP activates macrophages to express EMMPRIN and MMP-9 and that statins suppress it through the antioxidant effect.