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Atherosclerosis 188 (2006) 51–58
Simvastatin modulates chemokine and chemokine receptor expressionby geranylgeranyl isoprenoid pathway in human endothelial
cells and macrophages
Niels R. Veillard 1, Vincent Braunersreuther 1, Claire Arnaud, Fabienne Burger,Graziano Pelli, Sabine Steffens, Francois Mach ∗
Cardiology Division, Department of Medicine, Geneva University Hospital, Foundation for Medical Research,64 Avenue Roseraie, 1211 Geneva, Switzerland
Received 19 May 2005; received in revised form 23 September 2005; accepted 11 October 2005Available online 29 November 2005
bstract
bjective: Atherosclerosis is a chronic immuno-inflammatory disease involving the recruitment of monocytes and T lymphocytes to theascular wall of arteries. Chemokines and their receptors, known to induce leukocyte migration, have recently been implicated in atherogenesis.ecent in vitro and in vivo studies have suggested that statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors)ave anti-inflammatory properties beyond their lipid-lowering effects. Thus, the aim of the present study was to investigate whether simvastatineduces the expression of chemokines and chemokine receptors in two major cell types implicated in atherogenesis and to test isoprenoidntermediates involved in their regulation.
ethods and results: We performed in vitro experiments on human vascular endothelial cells and human primary macrophages. First, weave shown by ELISA that 1 �M simvastatin significantly reduced MCP-1 in endothelial cells (ECs) and macrophages stimulated with TNF-�r IFN-�, respectively. Messenger RNA analysis revealed that expression of the chemokines MCP-1, MIP-1� and MIP-1�, as well as thehemokine receptors CCR1, CCR2, CCR4 and CCR5, was decreased by simvastatin, both in ECs and macrophages. Furthermore, the statinffects were reversed by mevalonate and mimicked by the geranylgeranyl transferase inhibitor (GGTI), whereas the farnesyl transeferasenhibitor (FTI) had no effect. These results suggests that statins act via inhibition of the geranylgeranylation of proteins.onclusions: Our results demonstrate that statins reduce chemokine and chemokine receptor expressions in human ECs and macrophagesia inhibition of the geranylgeranylpyrophosphate pathway. Thus, our data provide further evidence that statins have anti-inflammatory
roperties beyond their lipid-lowering effects. These findings highlight specific novel therapeutic targets for cardiovascular diseases to reducenflammation mediated by chemokines and their receptors.2005 Elsevier Ireland Ltd. All rights reserved.
eywords: Atherosclerosis; Inflammation; Statin; Chemokines
fl
. Introduction
Over the past decades, atherosclerosis has been recog-ized as an inflammatory disease and established as the mostommon cause of death in western countries, responsible
∗ Corresponding author. Tel.: +41 22 382 7234; fax: +41 22 382 7245.E-mail address: [email protected] (F. Mach).
1 These authors equally contributed to this work.
eacaata
021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2005.10.015
or half of the morbidity and mortality. Atheroscleroticesions are characterized by accumulation of lipids, fibrouslements and immune infiltrates. Endothelial cells (ECs)nd monocyte/macrophages are major cell types impli-ated during atherogenesis. During the whole process of
therogenesis, monocytes migrate through the endotheliumnd differentiate into foam cells within the neo-intima ofhe vessel wall [1]. Chemotactic cytokines or chemokines,re known to induce leukocyte migration, growth and
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ctivation through G protein-coupled cell-surface receptorsn target cells and regulate leukocyte trafficking duringnflammation [2]. Several studies have demonstrated arucial role of chemokines and their receptors duringhe development of atherosclerosis [3]. Inhibitors of the-hydroxy-3-methylglutaryl coenzyme A (HMG CoA)eductase or statins, are potent lipid-lowering drugs, whichave been shown to reduce cardiovascular events andortality in patients with or without coronary artery disease
4]. Besides their lipid-lowering effects, increasing evidenceuggest that statins possess also anti-inflammatory asell as immunomodulatory properties [5,6]. Indeed, by
nhibiting l-mevalonic acid synthesis, statins also preventhe catabolism of several isoprenoid intermediates of theholesterol biosynthetic pathway, such as farnesylpyrophos-hate (FPP) and geranylgeranylpyrophosphate (GGPP) [7],hich are known to induce prenylation of numerous cellularroteins. Prenylation of proteins is a pre-requisite for cellembrane association to both plasma and internal membrane
nd is essential for their functions [8]. Prenylated proteinsnclude the �-subunit of heterotrimeric G protein, Heme-a,uclear lamins and the GTP-binding proteins Ras andas-like proteins, such as Rho, Rab, Rac, Ral and Rap [9].umerous pleiotropic effects of statins seem to be mediatedy regulation of these isoprenoid intermediates [7,10,11].
Previous studies have shown that statins inhibit the expres-ion of chemokines, such as IL-8 and MCP-1 [12,13]. How-ver, the mechanism implicated in their regulation is stillnknown. In this study, we chose to investigate the effectf statins on the expression of MCP-1, RANTES, MIP-� and MIP-1� and their receptors CCR1, CCR2, CCR4nd CCR5. Indeed, these chemokines belong to the largesthemokine family known, which is the CC chemokines [2].hese chemokines tend to attract mononuclear cells and are
ound at sites of chronic inflammation [14]. Furthermore,CP-1 [15–17], as well as MIP-1�, MIP-1� and RANTES
18,19] have been detected in atherosclerotic lesions and arelso expressed by atheroma-associated cells, such as endothe-ial cells and macrophages. Thus, the aim of this study was toetermine the effect of statins on the expression of the princi-al chemokines and their receptors implicated in atheroscle-osis and also to analyze the possible signaling pathway ofhis regulation.
. Material and method
.1. Reagents
Human recombinant TNF-�, IFN-�, MCP-1, as well asANTES were obtained from R&D Systems (Abingdon,K). The statin simvastatin was obtained from commer-
ial sources and diluted in 10% ethanol. Mevalonate wasbtained from Sigma–Aldrich (Steinheim, Germany). Farne-yl transferase inhibitor (FTI-277) and geranylgeranyl trans-erase inhibitor type I (GGTI-286) were purchased from
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rosis 188 (2006) 51–58
albiochem (Darmstadt, Germany). Antibodies for humanANTES and MCP-1 were obtained from R&D systems.ll experiments were performed by adding correspondingilutions of the simvastatin vehicle (10% ethanol) to the non-tatin treated conditions.
.2. Cell isolation and culture
Human vascular endothelial cells (ECs) were isolatedrom saphenous veins and cultured as previously described20]. ECs were grown in M199 (BioWhittaker), supple-ented with 10% fetal bovine serum (FBS) and 50 �g/ml
ndothelial cell growth factor (ECGF) and used at pas-ages 2–4 for all experiments. Monocyte/macrophages weresolated from human peripheral blood mononuclear cellsPBMCs) obtained by Ficoll gradient centrifugation (Amer-ham Biosciences) and grown for 10 days in RPMI 1640ontaining 5% human AB+ serum (Sigma–Aldrich). Twenty-our hours prior to cell stimulation as well as during stimula-ion, macrophages were incubated in serum-free conditions,hereas stimulation of ECs was performed in the presencef 0.1% human serum albumin, without pre-incubating theells in serum-free condition. Phenotype of endothelial cellsnd macrophages was tested by FACS. Cells were stimulateduring 4 h with TNF-� (10 ng/ml) or IFN-� (5000 U/ml) inhe presence or absence of simvastatin (1 �M), mevalonate500 �M), geranylgeranyl transferase inhibitor (4 �M) or far-esyl transeferase inhibitor (200 nM).
.3. ELISA
After 4 h of stimulation, cell culture supernatants werearvested for the detection of the chemokines MCP-1 andANTES, using paired antibodies (R&D Systems).
.4. mRNA isolation and analysis
Total mRNA from ECs and macrophages was extractedith Tri reagent (MRC Inc., Cincinnati, OH) according to
he manufacturer’s instructions. Real-time quantitative RT-CR (ABI Prism 7000 Sequence Detection System, Appliediosystems, Foster City, CA) was used to determine theRNA levels of RANTES, MCP-1, MIP-1� and MIP-1�
ABI Prism, Pre-Developed TaqMan Assay Reagents, 6-arboxyfluorescent (6-FAM)-labeled). Each sample was ana-yzed in triplicate and normalized in multiplex reactions usingaqMan eukaryotic 18S control (TaqMan Reagent, Appliediosystems, Foster City, CA, VIC-labeled). The fold induc-
ions in mRNA expression of inflammatory mediators at eachime point relative to the reference unstimulated cells condi-ion were analyzed by the comparative computed tomography
ethod. Expression of chemokine receptors was detected
y ribonuclease protection assay (RPA) using the commer-ial RPA Multi-Probe Template Set (Pharmingen, probe setCR-5) as recommended by the supplier. RPAs were car-ied out with 10 �g of mRNA per reaction. Signals were
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N.R. Veillard et al. / Ath
uantified using a Phosphor Imager (Molecular Dynamics,unnyvale, CA) and normalized to GAPDH and L32 house-eeping genes.
.5. Statistical analysis
All results are expressed as mean ± S.E.M. Differencesetween the groups were considered significant at P < 0.05sing the Student’s T-test. If data distribution failed to meetequirements for use of the parametric T-test, the non-arametric Mann–Whitney U-Wilcoxon sum test was used.
. Results
.1. Simvastatin blocks inflammatory mediators at lowoncentrations
In order to investigate the effect of statins onhemokine and chemokine receptor expressions in ECs andacrophages, we first performed simvastatin dose-response
xperiments. Cells were stimulated with TNF-� or IFN-�,
ig. 1. Dose-dependent inhibition of CCR4 and MCP-1 by simvastatin.Nase protection assays performed on ECs for CCR4 (A). Real-time semi-uantitative PCR performed on macrophages for MCP-1 (B). ECs andacrophages under unstimulated conditions (control), treated with TNF-�
10 ng/ml, 4 h) or IFN-� (5000 U/ml, 4 h) alone or in the presence of simvas-atin (10 �M–20 nM). Results (n = 3 from different donors) are expressed asercentage compared to TNF-� or IFN-� stimulated cells (4 h). Data wereormalized to GAPDH or 18S. *P < 0.05 vs. TNF-� or IFN-�.
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rosis 188 (2006) 51–58 53
ith or without increasing concentrations of simvastatin. Wesed TNF-� and IFN-� to stimulate ECs and macrophages,espectively, to obtain the maximum chemokines andhemokine receptors expression in each group. Expressionf the chemokine receptor CCR4 in ECs (Fig. 1A) andhe chemokine MCP-1 in macrophages (Fig. 1B) was sig-ificantly increased by TNF-� (10 ng/ml, 4 h) or IFN-�5000 U/ml, 4 h). In both cell types, treatment with simvas-atin significantly decreased CCR4 and MCP-1 expression indose-dependent manner. Although maximal inhibition wasbserved at 10 �M of simvastatin, efficient inhibition was stillbserved at a lower concentration (100 nM) (Fig. 1A and B).e also measured LDH activity in culture supernatants and
ound no cytotoxic effect neither on ECs nor macrophages, atny statin concentration used (data not shown). Since 1 �Mimvastatin represents a concentration close to those mea-ured in the serum of patients during statin therapy, we chosehis concentration for our following experiments.
.2. Simvastatin inhibits MCP-1 secretion from
ndothelial cells and monocyte/macrophagesWe analyzed by ELISA whether simvastatin could reg-late the secretion of RANTES and MCP-1. As shown in
ig. 2. Simvastatin reduces MCP-1 secretion. ELISA for RANTES andCP-1 within supernatants of the different cell stimulations performed with
Cs (A) and macrophages (B). Stimulation abbreviations—T: TNF-�, I:FN-�, Simva: simvastatin, Meva: mevalonate. Results (n = 5 from differentonors) are expressed as percentage of mean OD value of TNF-� or IFN-�timulated cells (4 h). *P < 0.05 vs. TNF-� or IFN-�.
54 N.R. Veillard et al. / Atherosclerosis 188 (2006) 51–58
Fig. 3. Simvastatin inhibits chemokine mRNA expression. Real-timequantitative PCR performed on ECs (A) and macrophages (B) forthe chemokines RANTES, MCP-1, MIP-1� and MIP-1�. Stimulationabbreviations—Simva: simvastatin, Meva: mevalonate. Results (n = 5 fromdifferent donors) are expressed as percentage compared to TNF-� or IFN-�sI
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Fig. 4. Simvastatin inhibits chemokine receptor mRNA expression. RNaseprotection assays performed on ECs for CCR4 (A) and macrophagesfor the chemokine receptors CCR1, CCR2 and CCR5 (B). Stimulationabbreviations—S or Simva: simvastatin, M or Meva: mevalonate, T: TNF-�
dc
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timulated cells (4 h). Data were normalized to 18S. *P < 0.05 vs. TNF-� orFN-�.
ig. 2, simvastatin significantly reduced the expression ofCP-1 in both ECs and macrophages. On the other hand, we
id not observe significant effect on RANTES protein secre-ion. Addition of mevalonate in the culture medium of thetatin-treated cells reversed the inhibitory effect induced byimvastatin. In contrast, secretion of RANTES from ECs oracrophages was not altered by simvastatin.
.3. Simvastatin inhibits chemokine and chemokineeceptor mRNA expression in ECs and macrophages
To further investigate the regulation of these chemokinesy statins, we analyzed expression levels of RANTES, MCP-, MIP-1� and MIP-1� by semi-quantitative real-time RT-CR. RANTES, MCP-1, MIP-1� and MIP-1� play a centralole in leukocytes recruitment into the vessel’s wall dur-ng atherogenesis. We observed that TNF-�-induced mRNAxpression of RANTES, MCP-1 and MIP-1� in ECs, whereasnalysis of MIP-1� expression revealed no detectable mRNAevels (data not shown). Although no effect was detected onANTES mRNA levels, simvastatin significantly reduced
RNA expression of MCP-1 and MIP-1�, within stimu-ated ECs (Fig. 3A). Macrophages showed similar response totatins, as simvastatin reduced IFN-�-induced expression of
CP-1, MIP-1� and MIP-1�. Addition of mevalonate couldsp
. Data were normalized to GAPDH and L32. Results (n = 5 from differentonors) are expressed as percentage compared to TNF-� or IFN-� stimulatedells (4 h). *P < 0.05 vs. TNF-� or IFN-�.
everse inhibitory effects of simvastatin. However, as shownor ECs, no significant effect was observed on RANTESRNA expression (Fig. 3B). Based on our previous results
n chemokines, we hypothesized that simvastatin might alsoodulate the expression of corresponding chemokine recep-
ors. When stimulated with TNF-�, ECs expressed a highmount of the chemokine receptor CCR4, which was reducedy simvastatin (Fig. 4A). Similarly, macrophages were stim-lated with IFN-� in the presence or absence of 1 �M simvas-atin. Expression of the chemokine receptors CCR1, CCR2nd CCR5 was induced on stimulated macrophages, an effecthat could be blocked by the addition of simvastatin. Foroth ECs and macrophages, the effects of simvastatin onhemokine receptors were reversed by the addition of meval-nate (Fig. 4A and B).
.4. The effect of statins on chemokines and chemokineeceptors is mimicked by the inhibition of proteineranylgeranylation
By inhibiting the HMG-CoA reductase, statins reduce theynthesis of isoprenoid intermediates, leading to a decrease inrotein prenylation. Thus, in order to determine whether sim-
N.R. Veillard et al. / Atherosclerosis 188 (2006) 51–58 55
Fig. 5. Effect of simvastatin on chemokine mRNA expression is GGPP-dependent. Real-time quantitative PCR performed on ECs (A) andmacrophages (B) for the chemokines RANTES, MCP-1, MIP-1� and MIP-1�. Stimulation abbreviations, GGTI: geranylgeranyl transferase inhibitor,FTI: farnesyl transferase inhibitor. Results (n = 5 from different donors) areeD
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Fig. 6. Effect of simvastatin on chemokine receptor mRNA expression isGGPP-dependent. RNase protection assays performed on ECs for CCR4(A) and macrophages for the chemokine receptors CCR1, CCR2 and CCR5(B). Stimulation abbreviations, T: TNF-�, GGTI: geranylgeranyl transferaseinhibitor, FTI: farnesyl transferase inhibitor. Results (n = 5 from differentdonors) are expressed as percentage compared to TNF-� or IFN-� stimulatedco
rp
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xpressed as percentage compared to TNF-� or IFN-� stimulated cells (4 h).ata were normalized to 18S. *P < 0.05 vs. TNF-� or IFN-�.
astatin inhibits the expression of chemokines and chemokineeceptors via prenylated proteins, we used GGTI and FTIo inhibit the geranylgeranyl and farnesyl pathways, respec-ively. While neither GGTI nor FTI could significantly inhibitANTES expression (Fig. 5), we showed that GGTI, butot FTI, mimicked the effect of statin by reducing bothhemokines (Fig. 5A) and chemokine receptors (Fig. 6A)n ECs. Similar results were obtained in macrophagesFigs. 5B and 6B).
. Discussion
Increasing evidence suggest that chemokines and theireceptors play crucial roles in the pathogenesis of atheroscle-osis, in particular for leukocyte recruitment. Statins arenown to reduce diapedesis, notably by chemokine andhemokine receptor inhibition. However, this mechanism isess understood. However, chemokines are not only involvedn atherosclerosis, recent studies also report that they may be
mplicated in vascular remodeling and restenosis followingercutaneous transluminal coronary angioplasty, as well asn bypass vein graft occlusion after coronary bypass surgery.herefore, more specific inhibition of chemokine/chemokinesnei
ells (4 h). Data were normalized to GAPDH or 18S. *P < 0.05 vs. TNF-�r IFN-�.
eceptor interactions could provide novel and efficient thera-eutic strategies in cardiovascular diseases.
In the present study, we proposed an explanation ofow statins reduce the chemokine and chemokine recep-or expression. We first have demonstrated that simvastatineduces secretion of MCP-1 from ECs and macrophageshereas the decrease in RANTES, which looks like consis-
ent, was not significant. In a previous study, it has been shownhat cerivastatin and fluvastatin reduced RANTES expres-ion from murine ECs that were stimulated with IFN-� andNF-�, whereas pravastatin had no effect [21]. Furthermore,iomede et al. observed in vivo that the effect of lovastatinn the synthesis of RANTES was linked to the inflammatory
timulus applied. When carrageenan was used, lovastatin sig-ificantly lowered RANTES production, whereas it had noffect in mice treated with LPS [22]. Thus, the lack of signif-cant decrease of RANTES expression in macrophages and
56 N.R. Veillard et al. / Atherosclerosis 188 (2006) 51–58
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ig. 7. Hypothesized regulation pathway of chemokines and chemokinexpression could be mediated via two mechanisms. (1) Statins inhibit geranL-8 and IP-10. (2) By increasing the activity of the transcriptional represso
Cs under simvastatin treatment could be due, first to thetatin used and second, to the inflammatory stimulus. Then,n agreement with several recent studies [12,13,23,24], weave shown that simvastatin inhibits the expression of thehemokines MCP-1, MIP-1� and MIP-1� and the chemokineeceptors CCR1, CCR2, CCR4 and CCR5 at the mRNA level.hese effects of statin were HMG-CoA reductase-dependantince they were reversed by addition of mevalonate. We fur-her observed that the effect of simvastatin on chemokinend chemokine receptor expression was dose-dependent,oth in endothelial cells and macrophages. Interestingly,imvastatin-induced inhibition of chemokine and chemokineeceptor expression was still observed at low doses of statin,losed to those measured in human plasma during statin ther-py. Then, we have investigated by which pathway, statinseduce the expression of the chemokines ant their recep-ors. Besides their role on lipid-lowering, statins also possessnti-inflammatory properties, which seem to be mediatedy non-steroidal products of the mevalonate pathway [4].y blocking l-mevalonic acid synthesis, statins also inhibit
ts conversion into farnesylpyrophosphate and geranylger-nylpyrophosphate. Thus, to investigate which prenylationathway might be involved in statins inhibitory expressionf chemokines and chemokine receptors, we used GGTI andTI, two specific inhibitors of the geranylgeranyl transferasend the farnesyl transferase, respectively. We demonstrated
hat GGTI, but not FTI, mimicked the effect of simvastatin,n both endothelial cells and macrophages, indicating thathemokines and chemokine receptors are regulated by GGPPsoprenoids.RrflO
rs by statins. The effect of statin on chemokine and chemokine receptorylation of RhoA and thus, NF-�B, leading to a downregulation of MCP-1,, statins regulate CCR2 and CCR5 expression via geranylgeranyl pathway.
Previous studies have described various transcription fac-ors involved in chemokine/chemokine receptor expression.vidence indicates a role for AP-1 involving cooperative
nteractions with NF-�B, in the regulation of chemokine generanscription, such as activation of IL-8 and MCP-1 genexpression [25–28]. Concerning the regulation of MIP-1�nd MIP-1� expression, activities of their proximal promot-rs in transfected cells are very similar [29]. Regulation ofIP-1� is mediated by NF-�B, whereas MIP-1� has been
emonstrated to contain nuclear-binding sites in its proximalromoter, which bind AP-1 proteins [29,30]. Previous studiesave reported that statins inhibit the expression of MCP-, IP-10 and IL-8 via inhibition of NF-�B [12,13,23,31].oreover, it is well known that isoprenylation of protein is
ecessary for the activation of small G proteins, such as Rhond Rac implicated in the NF-�B signal transduction [32];nd either farnesylated or geranylgeranylated members ofhe Rho family GTPases have been involved in the regulationf several cellular functions and implicated in the regula-ion of some cytokines and chemokines [33,34]. Our resultsemonstrate that regulation of chemokines by simvastatins geranylgeranylpyrophosphate-dependent. Since RhoA, aeranylgeranylated protein, has been implicated in the acti-ation of NF-�B [35,36], we can hypothesize that regulationf MCP-1, MIP-1� and MIP-1� expression could be relatedo an inhibitory effect of statin on geranylgeranylation of
hoA and thus, NF-�B activity. Similarly, the chemokineeceptors CCR2 and CCR5 contain within their proximal 5′anking region cis-regulatory elements, such as GATA- andct-1-binding domains [37,38]. Ortego et al. have previously
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eported that statins increase the activity of the transcrip-ional repressor Oct-1 [39]. As Oct-1 regulates CCR2 andCR5 expression, the reduction of these chemokine recep-
ors by simvastatin might be correlated to Oct-1 nuclear factorctivation; and since expression of the chemokine receptorsCR1, CCR2, CCR4 and CCR5 are dependent on geranylger-nylated isoprenoids regulation, statins could possibly exertts effect via GGPP isoprenoids (Fig. 7).
In conclusion, our data suggest that statins reduce expres-ion of crucial chemokines and chemokine receptors impli-ated during the inflammatory process of atherosclerosis.urthermore, we also show that this effect of statins seems toe mediated through the inhibition of protein geranylgerany-ation. These findings might open new therapeutic strategieso reduce inflammation in several disorders.
cknowledgements
This work was supported by grants from the Swissational Science Foundation #3200-065121.01/1 to Francoisach, a grant from Fondation pour la Recherche Medicale
France) to Claire Arnaud, by grant from the Foundation foredical Research (Geneva) to Niels Veillard and the Euro-
ean Community, 6 and Frame Program (EVGN #LSHM-T-2003-503254) to F.M.
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