Elsevier

Atherosclerosis

Volume 188, Issue 1, September 2006, Pages 51-58
Atherosclerosis

Simvastatin modulates chemokine and chemokine receptor expression by geranylgeranyl isoprenoid pathway in human endothelial cells and macrophages

https://doi.org/10.1016/j.atherosclerosis.2005.10.015Get rights and content

Abstract

Objective

Atherosclerosis is a chronic immuno-inflammatory disease involving the recruitment of monocytes and T lymphocytes to the vascular wall of arteries. Chemokines and their receptors, known to induce leukocyte migration, have recently been implicated in atherogenesis. Recent in vitro and in vivo studies have suggested that statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors) have anti-inflammatory properties beyond their lipid-lowering effects. Thus, the aim of the present study was to investigate whether simvastatin reduces the expression of chemokines and chemokine receptors in two major cell types implicated in atherogenesis and to test isoprenoid intermediates involved in their regulation.

Methods and results

We performed in vitro experiments on human vascular endothelial cells and human primary macrophages. First, we have shown by ELISA that 1 μM simvastatin significantly reduced MCP-1 in endothelial cells (ECs) and macrophages stimulated with TNF-α or IFN-γ, respectively. Messenger RNA analysis revealed that expression of the chemokines MCP-1, MIP-1α and MIP-1β, as well as the chemokine receptors CCR1, CCR2, CCR4 and CCR5, was decreased by simvastatin, both in ECs and macrophages. Furthermore, the statin effects were reversed by mevalonate and mimicked by the geranylgeranyl transferase inhibitor (GGTI), whereas the farnesyl transeferase inhibitor (FTI) had no effect. These results suggests that statins act via inhibition of the geranylgeranylation of proteins.

Conclusions

Our results demonstrate that statins reduce chemokine and chemokine receptor expressions in human ECs and macrophages via inhibition of the geranylgeranylpyrophosphate pathway. Thus, our data provide further evidence that statins have anti-inflammatory properties beyond their lipid-lowering effects. These findings highlight specific novel therapeutic targets for cardiovascular diseases to reduce inflammation mediated by chemokines and their receptors.

Introduction

Over the past decades, atherosclerosis has been recognized as an inflammatory disease and established as the most common cause of death in western countries, responsible for half of the morbidity and mortality. Atherosclerotic lesions are characterized by accumulation of lipids, fibrous elements and immune infiltrates. Endothelial cells (ECs) and monocyte/macrophages are major cell types implicated during atherogenesis. During the whole process of atherogenesis, monocytes migrate through the endothelium and differentiate into foam cells within the neo-intima of the vessel wall [1]. Chemotactic cytokines or chemokines, are known to induce leukocyte migration, growth and activation through G protein-coupled cell-surface receptors on target cells and regulate leukocyte trafficking during inflammation [2]. Several studies have demonstrated a crucial role of chemokines and their receptors during the development of atherosclerosis [3]. Inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase or statins, are potent lipid-lowering drugs, which have been shown to reduce cardiovascular events and mortality in patients with or without coronary artery disease [4]. Besides their lipid-lowering effects, increasing evidence suggest that statins possess also anti-inflammatory as well as immunomodulatory properties [5], [6]. Indeed, by inhibiting l-mevalonic acid synthesis, statins also prevent the catabolism of several isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) [7], which are known to induce prenylation of numerous cellular proteins. Prenylation of proteins is a pre-requisite for cell membrane association to both plasma and internal membrane and is essential for their functions [8]. Prenylated proteins include the γ-subunit of heterotrimeric G protein, Heme-a, nuclear lamins and the GTP-binding proteins Ras and Ras-like proteins, such as Rho, Rab, Rac, Ral and Rap [9]. Numerous pleiotropic effects of statins seem to be mediated by regulation of these isoprenoid intermediates [7], [10], [11].

Previous studies have shown that statins inhibit the expression of chemokines, such as IL-8 and MCP-1 [12], [13]. However, the mechanism implicated in their regulation is still unknown. In this study, we chose to investigate the effect of statins on the expression of MCP-1, RANTES, MIP-1α and MIP-1β and their receptors CCR1, CCR2, CCR4 and CCR5. Indeed, these chemokines belong to the largest chemokine family known, which is the CC chemokines [2]. These chemokines tend to attract mononuclear cells and are found at sites of chronic inflammation [14]. Furthermore, MCP-1 [15], [16], [17], as well as MIP-1α, MIP-1β and RANTES [18], [19] have been detected in atherosclerotic lesions and are also expressed by atheroma-associated cells, such as endothelial cells and macrophages. Thus, the aim of this study was to determine the effect of statins on the expression of the principal chemokines and their receptors implicated in atherosclerosis and also to analyze the possible signaling pathway of this regulation.

Section snippets

Reagents

Human recombinant TNF-α, IFN-γ, MCP-1, as well as RANTES were obtained from R&D Systems (Abingdon, UK). The statin simvastatin was obtained from commercial sources and diluted in 10% ethanol. Mevalonate was obtained from Sigma–Aldrich (Steinheim, Germany). Farnesyl transferase inhibitor (FTI-277) and geranylgeranyl transferase inhibitor type I (GGTI-286) were purchased from Calbiochem (Darmstadt, Germany). Antibodies for human RANTES and MCP-1 were obtained from R&D systems. All experiments

Simvastatin blocks inflammatory mediators at low concentrations

In order to investigate the effect of statins on chemokine and chemokine receptor expressions in ECs and macrophages, we first performed simvastatin dose-response experiments. Cells were stimulated with TNF-α or IFN-γ, with or without increasing concentrations of simvastatin. We used TNF-α and IFN-γ to stimulate ECs and macrophages, respectively, to obtain the maximum chemokines and chemokine receptors expression in each group. Expression of the chemokine receptor CCR4 in ECs (Fig. 1A) and the

Discussion

Increasing evidence suggest that chemokines and their receptors play crucial roles in the pathogenesis of atherosclerosis, in particular for leukocyte recruitment. Statins are known to reduce diapedesis, notably by chemokine and chemokine receptor inhibition. However, this mechanism is less understood. However, chemokines are not only involved in atherosclerosis, recent studies also report that they may be implicated in vascular remodeling and restenosis following percutaneous transluminal

Acknowledgements

This work was supported by grants from the Swiss National Science Foundation #3200-065121.01/1 to François Mach, a grant from Fondation pour la Recherche Médicale (France) to Claire Arnaud, by grant from the Foundation for Medical Research (Geneva) to Niels Veillard and the European Community, 6 and Frame Program (EVGN #LSHM-CT-2003-503254) to F.M.

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    These authors equally contributed to this work.

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