Microvascular Research 77 (2009) 364–369

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Microvascular Research

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Regular Article

Clopidogrel attenuates atheroma formation and induces a stable plaque phenotype inapolipoprotein E knockout mice

Arnon Afek, Evgeny Kogan, Sofia Maysel-Auslender, Adi Mor, Ehud Regev, Ardon Rubinstein,Gad Keren, Jacob George ⁎The Department of Cardiology, Tel Aviv Sourasky Medical Center, Tel Aviv University, Sackler School of Medicine, Tel Aviv, Israel

⁎ Corresponding author. Fax: +972 3 5469832.E-mail address: jacobg@post.tau.ac.il (J. George).

0026-2862/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.mvr.2009.01.009

a b s t r a c t

a r t i c l e i n f o

Article history:

Aim: Clopidogrel is a widely Received 23 May 2008Revised 29 December 2008Accepted 8 January 2009Available online 31 January 2009

Keywords:AtherosclerosisT cellsImmune system clopidogrelEndothelial progenitors

used anti-thrombotic for the prevention of stent thrombosis and cardiovascularevents in patients with coronary atherosclerosis. Clopidogrel has been shown to exhibit anti-inflammatoryeffects that are related to the attenuated activation of platelets. Atherosclerosis is a complex process in whichthe immune system and the endothelium appear to play a prominent role. Herein, we tested the hypothesisthat clopidogrel will influence plaque size and composition in the atherosclerosis prone apolipoprotein Eknockout (apoE KO) mouse model.Methods and results: Eight week old mice were fed daily with either PBS, 1 mg or 2 mg of clopidogrel for10 weeks. Plaque size was evaluated in the aortic sinus and cellular and humoral responses were studied aswell as splenic and bone marrow endothelial progenitors by FACS.Treatment with either 1 mg and 2 mg of clopidogrel significantly reduced plaque size and augmented itsstability by increasing atheromatous fibrous area. Whereas antigen specific oxLDL immune response was notinfluenced by clopidogrel feeding, the number of atheroprotective regulatory CD4+CD25+ T cells wassignificantly increased. Moreover, clopidogrel treatment resulted in a prominent rise in splenic but not bonemarrow derived Sca-1+/flk-1+ endothelial progenitors.Conclusion: Clopidogrel significantly reduces atheroma burden and stabilizes aortic sinus plaques in apoE KOmice. These effects may partially be mediated by upregulation of the regulatory T cell pool and splenicendothelial progenitor cells. These findings may expand the potential applications of clopidogrel in humansubjects.

© 2009 Elsevier Inc. All rights reserved.

Introduction

The role of the immune system in the pathogenesis of theatheroma is well established (Hansson, 2005; Libby, 2002; Binderet al., 2002). Among the studies lending support to this contention, arereports showing that immunemodulating strategies influence the sizeand the composition of the plaque. T cells are present in human(Hansson et al., 1989) and murine (Roselaar et al., 1996) athero-sclerotic plaques and adoptive transfer studies confirm the role ofcellular immunity in the pathogenesis of atherosclerosis (Zhou et al.,2000; George et al., 2000, 2001).

Clopidogrel is a platelet P2Y12 receptor inhibitor that was approvedfor the prevention of ischemic stroke, myocardial infarction, andvascular death in patients with symptomatic atherosclerosis (CAPRIESteering Committee, 1996). Following publication of the CURE trial(Yusuf et al., 2001), the use of clopidogrel added to standard therapywas approved for the reduction of atherothrombotic events in patientswith acute coronary syndromes. Recently, an important beneficial role

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has also been established for clopidogrel in patients with ST elevationacute myocardial infarction (Sabitine et al., 2005).

Platelet P2Y12 receptor is of critical importance in attenuation ofplatelet activation and thus represents an effective pharmacologicaltarget for the inhibition of platelet aggregation and prevention ofatherothrombotic events (Herbert, 2004). However, immunemediated responses that are implicated in the pathogenesis ofatherosclerosis and atherothrombosis and depend on platelets, arealso reduced by ADP-receptor antagonism (Hermann et al., 2001).Thus, the release of CD40 ligand (CD154) from platelets is inhibited bytreatment with clopidogrel (Klinkhardt et al., 2003). Additionally, theexpression of P-selectin (an adhesion protein involved in platelet–leukocyte interactions) on stimulated platelets is also reduced byclopidogrel treatment (Klinkhardt et al., 2003, 2002). In human trials,there is also evidence that supports the role of clopidogrel as an antiinflammatory agent other than its well known anti-thromboticproperties (Solheim et al.; 2006, Azar et al., 2006; Graff et al., 2005).

In view of the potential influence of clopidogrel on the immunesystem, we tested the hypothesis that clopidogrel could attenuateplaque formation and stability in the atherosclerosis prone apolipo-protein E knockout mouse.

Fig. 1. The effect of clopidogrel on lymphocyte proliferation. Splenocytes were obtainedfrom clopidogrel (2 mg) or PBS treated animals upon sacrifice. Proliferative capacityfrom 4 animals in each group to MDA-LDL and copper oxLDL was assessed as describedin Materials and methods (A). Splenocytes from PBS treated mice were incubated in thepresence of different concentrations of clopidogrel for 72 h and proliferation wasdetermined by XTT method as described in Materials and methods (B). Both the 5 and10 mcg/ml concentrations yielded significant (Pb0.05) reduction as compared withcontrols. Results are presented as means±SD.

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Materials and methods

Animals

ApoE-KO mice on a C57BL/6 background (Plump et al.; 1992) andtheir wild-type litters were purchased from Jackson Laboratories andgrown at the local animal house. Mice were fed a normal chow dietcontaining 4.5% fat by weight (0.02% cholesterol).

Experimental groups

Eight week old female, apoE KO mice (10/group) were fed oncedaily with clopidogrel 1 mg, 2 mg (both, dissolved in PBS) or PBS ascontrol. Animals were fed for 10 weeks until sacrifice, when theirhearts and aortas were obtained for measurement of atheroscleroticplaque size.

Lipid profile

Total plasma cholesterol and triglyceride levels were determinedusing an automated enzymatic technique (Boehringer Mannheim,Germany).

Splenocyte proliferation assays

Splenocytes from clopidogrel 1 mg, 2 mg and PBS treated mice(1×106 cells/ml) were incubated in triplicates in 0.2ml of cultureme-dium in microtiter wells in the presence or in the absence of 1 μg/mloxLDL or MDA-LDL for 72 h. In brief, after 72 h of proliferation, XTTreagent was added to wells according to manufacturer's instructions.After 2 h of incubation orange color that developed is measured onELISA reader and it is proportional to amount of cells in the well.

For in vitro proliferation splenocytes from apoE KO mice(1×106 cells/ml) were incubated for 72 h in presence of clopidogrel(0.5–10 μg/ml) in 0.2 ml of culture medium. Detection of proliferationwas done as described above.

Detection of anti-oxidized-LDL and anti-MDA LDL antibodies by ELISA

Ninety-six-well polystyrene plates (Nunc) were coated with eithercopper-oxidized LDL, native LDL (at a concentration of 5 μg/ml inPBS), or PBS alone overnight at 4 °C. After 4 washes with PBScontaining 0.05% Tween and 0.001% aprotinin (Sigma), the plateswere blocked with 2% BSA for 2 h at room temperature. Serumfractions were diluted to 1:50 in PBS–0.05% Tween–0.2% BSA andadded to the wells. After additional overnight incubation the plateswere washed, and alkaline phosphatase-conjugated goat anti-mouseIgG (Jackson ImmunoResearch laboratories Inc), diluted 1:10 000 inPBS–0.05% Tween–0.2% BSA, was added for 1 h at room temperature.After extensivewashing,1mg/ml p-nitrophenyl phosphate (Sigma) in50 mmol/l carbonate buffer containing 1 mmol/l MgCl2, pH 9.8, wasadded as a substrate. The reactionwas stopped after 30 min by adding1mol/l NaOH. The optical density was read at a 405-nmwavelength ina Titertek ELISA reader (SLT Laboratory Instruments). Levels of anti-oxLDL antibodies were calculated as the level of binding to native LDLsubtracted from that for the binding to oxLDL (George et al., 1998).

Analysis Tregs by FACS

Splenocytes from sacrificed animals were co-stained with thefollowing monoclonal antibodies: FITC-labeled anti-CD4 (7D4, Mil-tenyi Biotec), phycoerythrin (PE)-labeled anti-CD25 (GK1.5, MiltenyiBiotec), FITC-labeled mouse IgG2bκ isotypic control (KLH/G2b-1-2from SouthernBiotech) and phycoerythrin (PE)-labeled mouse IgMκisotypic control (RTK2118 from BioLegend).

In addition, staining was performed on splenocytes incubated for72 h with Clopidogrel (0.5–10 μg/ml).

Assessment of bone marrow and spleen cell derivedendothelial progenitors

Spleen and bone marrow cells were stained with the followingantibodies: PE-anti mouse Flk-1 (Avas12a1; e-Bioscience), FITC-antimouse-Sca-1 (D7 e-Bioscience) antibodies and corresponding isotypecontrols.

Assessment of aortic sinus atherosclerosis

Atherosclerotic fatty-streak lesions were quantified by calculatingthe lesion size in the aortic sinus as previously described (Paigen et al.,1987) with a fewmodifications. Briefly, the heart and upper section ofthe aorta were removed from the animals, and the peripheral fat wascarefully cleaned. The upper section was embedded in OCT mediumand frozen. Every other section (10 μm thick) throughout the aorticsinus (400 μm) was taken for analysis. The distal portion of the aorticsinus is recognized by the 3 valve cusps, which are the junctions of theaorta to the heart.

The extent of atherosclerosis was evaluated blindly by two expertpathologists. Processing and staining of the tissue with oil red O were

Fig. 2. Antibodies to modified LDL in clopidogrel treated mice. Upon sacrifice, sera fromclopidogrel and PBS treated mice were tested for the presence of antibodies to copperoxidized LDL (A) and MDA-LDL (B) by ELISA as described in Materials and methods.Sera from all mice were taken for the analysis. Results are means±SD.

Fig. 3. Clopidogrel augments the Treg numbers in apoE KOmice. Upon sacrifice, spleenswere obtained frommice treatedwith clopidogrel or PBS and tested for Treg numbers asdescribed in Materials and methods (A). Representative FACS sheets are presented in Band C. Pb0.05.

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carried out according to Paigen et al. (1987). All animals in the study(n=9) were used for analysis.

Staining with Masson's trichrome was employed to determinefibrous area. The proportional area of the plaque stained positive forcollagenous fibrosis was determined by quantitative morphometry.

Statistical analysis

All parameters were evaluated by the one way ANOVA test.Pb0.05 was considered statistically significant. Results are expressedas mean±SEM unless otherwise specified in the text.

Results

The effect of clopidogrel on lipid profile

We have found that treatment with clopidogrel at either the 1 mgor 2 mg daily doses, did not influence total cholesterol levels.Moreover, triglyceride values also did not change following the tworegimens of clopidogrel treatment (data not shown).

The effect of clopidogrel on cellular immune responses to oxLD

Next, we tested the effects of oral treatment with clopidogrel, onproliferative capacity of splenocytes to copper oxidized LDL. We havefound that treatment with clopidogrel (either 1 mg or 2 mg) did notinfluence the proliferation of splenocytes to MDA-LDL and copper-oxLDL (Fig. 1A). However, when splenocytes were incubated in vitrowith clopidogrel, the 5 and 10 mcg/ml concentrations yielded asignificant decrease in spontaneous proliferation (Fig. 1B).

The effect of treatment with clopidogrel on humoral immune markers

In the apoE KO mouse model, antibodies to oxidized LDL developas cholesterol levels rise and atherosclerotic lesions progress. We

tested 2 types of IgG antibodies to modified LDL. Treatment withclopidogrel with either 1 mg or 2 mg daily did not alter the levels ofIgG antibodies to either copper oxidized LDL or MDA modified LDLmeasured at sacrifice (Fig. 2).

The effect of clopidogrel on Treg numbers

We evaluated the effects of clopidogrel on Tregs by assaying CD4+CD25high in the spleens from both clopidogrel treatment groups incomparison with controls. We have found that both doses ofclopidogrel significantly increased the number of peripheral naturallyoccurring Tregs as compared with the PBS treated group (Fig. 3).

Fig. 4. Clopidgrel treatment augments peripheral endothelial progenitor cell numbers. Bone marrow (A) and spleen cells (B) were obtained from 4 animals from each group andstained with labeled antibodies to Sca-1 and flk-1 as well as their respective isotype controls. Representative FACS sheets are provided from splenocytes from PBS (C) andclopoidogrel (D) treatment. ⁎Pb0.01.

Fig. 5. Clopidogrel reduces atherosclerosis and stabilizes plaques in apoE KO mice.Clopidogel and PBS treated animals were sacrificed and their aortic sinus obtained foranalysis of plaque size and composition. The extent of atherosclerosis was determinedafter oil Red O staining (A), and fibrous area signifying plaque stability was determinedemploying Masson's trichrome staining (B). ⁎Pb0.05; ⁎⁎Pb0.01.

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The effect of clopidogrel on the number of endothelial progenitorcells (EPC)

One of the methods by which EPC in the mouse is by co-expressionof Sca-1 and VEGF-receptor 2 (FLK-1) (Werner et al., 2002). Weevaluated the numbers of bone marrow and peripheral (splenocyte)EPC by FACS analysis for the respective markers. Treatment withclopidogrel with either 1 mg or 2 mg significantly increased thenumbers of EPC in the spleen as compared with PBS (Fig. 4).Clopidogrel treatment did not influence the numbers of bone marrowderived EPC as compared with PBS.

Both clopidogrel regimens resulted in a significant attenuation ofaortic sinus atherosclerotic lesion (Fig. 5). Whereas 1 mg ofclopidogrel given for 10 week was associated with a reduction of45% in lesions size, the 2 mg dose resulted in a 48% decrease inatheroma as compared with PBS (Pb0.01 for both comparisons).

Aiming to study the effects of clopidogrel on plaque stability weperformed Masson's trichrome staining to detect relative plaquefibrous content. Treatment with a daily dose of 1 mg clopidogrel wasassociated with a 31% increase in fibrous area, whereas administrationof 2 mg daily, increased relative plaque fibrous area by 46% in compa-rison with PBS (Pb0.05 for both comparisons).

Discussion

The pathogenesis of atherosclerosis is complex and, other thanderanged lipid metabolism, immune responses and endothelialmalfunction have been shown to play a prominent role.

Herein, we tested the effects of the widely employed ADP inhibitorclopidogrel on the size and of stability of the atherosclerotic plaque inthe apoE KOmousemodel. Despite thewell-known role of clopidogrel

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in the prevention of stent thrombosis and adverse myocardial events,there is currently no evidence supporting its effects on plaque size andcomposition. Such studies are inherently easier to perform inexperimental murine atherosclerosis models. Indeed, we have foundthat treatment with clopidogrel given at 1 mg and 2 mg daily dosesequivalent to those given to humans (75 mg and 150 mg, respec-tively), for a period of 10 weeks significantly reduced the size ofatherosclerotic plaques (Fig. 6).

We have studied several mechanisms that could mediate thebeneficial effects of clopidogrel. Cellular immune responses areknown to influence the size and composition of murine plaques.Indeed, oxLDL reactive lymphocytes have been demonstrated withinhuman plaques (Stemme et al., 1995), and active immunizationstudies in with different antigens have been shown influence plaqueprogression (George et al., 1998; Xu et al., 1992; Palinski et al., 1995).

Although no effect of clopidgorel was evident on antibodies tooxLDL, it is nevertheless possible that different timing of clopidgorelwith respect to the development of the plaqueswould have influencedhumoral responses.

Additionally, adoptive transfer of antigen-specific and non specificlymphocytes was demonstrated to promote atherosclerosis (Zhouet al., 2000; George et al., 2000, 2001). In this study we found thattreatment with clopidogrel did not influence cellular responsesevident by splenocyte proliferation to either oxLDL or the non specificmitogen Concavalin-A, suggesting this mechanisms may not havebeen involved in the atheroprotective effect of clopidogrel. Interest-ingly, in vitro proliferation to clopidogrel did produce a suppressiveeffect onmurine splenocytes that was not evident when the treatment

Fig. 6. Representative photomicrographs of aortic sinus from clopoidgrel and PBS treated mMasson's trichrome (middle panel magnification ×100) and Elastica Von Giesen (lower panClopidogrel 2 mg (right panel); 1 mg (middle panel) or PBS (left panel).

was given in vivo. This finding may be explained by the attenuation ofclopidogrel's effect in vivo due to various possible systemic factorssuch as lipids and cytokines that are not present in the in vitro isolatedsystem.

A relatively new role has recently been ascribed to naturallyoccurring CD4+CD25+ Tregs in atherosclerosis (reviewed in George,2008). Consistent with the well documented role of Tregs in sup-pressing autoimmune responses (Sakaguchi, 2004), others (Ait-Oufella et al., 2006) and we (Mor et al., 2007) have shown that thispopulation of cells exhibit anti-atherosclerotic properties in mice. Wehave also shown that peripheral population and functional suppres-sive properties of Tregs are compromised in humans and suggested itmay have a role in destabilization of human plaques with consequentdevelopment of acute coronary syndromes (Mor et al., 2006). In thisstudy we have found that treatment with clopidogrel resulted in asignificant increase in the number of spleen cell derived Treg, a findingthat has not yet been associated with the agent. This effect ofclopidogrel may be related to a direct effect of clopidogrel on Tregs ortheir transcriptional activator, or alternatively to a reduction plateletderived cyotokine secretion that may influence indirectly, Treg pool.Regardless of the potential mechanism of Treg upregulation, this effectof clopidogrel may be responsible for its anti-atherosclerotic effects.

EPC are reduced in patients with risk factors for atherosclerosis(Hill et al., 2003; Vasa et al., 2001), and mobilized to the periphery inacute coronary syndrome patients (George et al., 2004). Contrastingresults have been provided with regard to the role of EPC transfer onatherosclerosis, some of which showed anti-atherogenic (Rauscheret al., 2003) and others showing enhanced atherosclerosis (George

ice. Aortic sinus sections were stained with Oil red O (upper panel magnification ×40)el magnification×100). Representative atherosclerotic plaques in apoE KO treated with

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et al., 2005). In any case, upregulation of the endogeneous EPC pool isof possible benefit in atherosclerosis (Vasa et al., 2001) whereas thepotential effects of cell transfer remain to be resolved. In the currentstudy, we have found that clopidogrel significantly increased thenumber of EPC as compared to PBS, thus potentially contributing tothe anti-atherogenic properties of the compound. The mechanismresponsible for the upregulated EPC pool has not been explored. Itcould occur due to mobilization from the bone marrow, oralternatively a decrease in the peripheral apoptotic destruction EPC.Indeed, we have recently shown that in humans, a circulating pool ofapoptotic progenitors is detectable and measurable, and is increasedin acute coronary syndrome patients (Shwartzenberg et al., 2007).

In conclusion, we have shown here that clopidogrel has, other thanits well known anti-thrombotic properties, a role as an anti-atherogenic and plaque stabilizing compound in apoE KO mice.These findings may be explained by two potentially novel mecha-nisms of clopidogrel that include upregulation of the peripheral poolof naturally occurring Tregs and EPC, both of which have been shownto possess anti-atherogenic properties. If these findings are to bevalidated in additional experimental models, the use of clopidogrelcould be further expanded to humans with proven atherosclerosis.

Acknowledgments

Supported in part by a grant from the Israeli Science foundation(JG Grant No. 832/06) and Chief Scientist of Israel (JG).

References

Ait-Oufella, H., Salomon, B.L., Potteaux, S., Robertson, A.K., Gourdy, P., Zoll, J., Merval, R.,Esposito, B., Cohen, J.L., Fisson, S., Flavell, R.A., Hansson, G.K., Klatzmann, D., Tedgui,A., Mallat, Z., 2006. Natural regulatory T cells control the development ofatherosclerosis in mice. Nat. Med. 12, 178–180.

Azar, R.R., Kassab, R., Zoghbi, A., Aboujaoude, S., El-Osta, H., Ghorra, P., Germanos, M.,Salame, E., 2006. Effects of clopidogrel on soluble CD40 ligand and on high-sensitivity C-reactive protein in patients with stable coronary artery disease. Am.Heart J. 521, e1–521 e4.

Binder, C.J., Chang, M.K., Shaw, P.X., Miller, Y.I., Hartvigsen, K., Dewan, A., Witztum, J.L.,2002. Innate and acquired immunity in atherogenesis. Nat. Med. 8, 1218–1226.

CAPRIE Steering Committee, 1996. A randomised, blinded, trial of clopidogrel versusaspirin in patients at risk of ischaemic events (CAPRIE). Lancet 348, 1329–1339.

George, J., 2008. Mechanisms of disease: the evolving role of regulatory T cells inatherosclerosis. Nat. Clin. Pract. Cardiovasc. Med. 5, 531–540.

George, J., Afek, A., Gilburd, B., Blank, M., Levy, Y., Aron-Maor, A., Levkovitz, H., Shaish, A.,Goldberg, I., Kopolovic, J., Harats, D., Shoenfeld, Y., 1998. Induction of earlyatherosclerosis in LDL receptor deficient mice immunized with beta 2 glycoproteinI. Circulation 15, 1108–1115.

George, J., Harats, D., Gilburd, B., Afek, A., Shaish, A., Kopolovic, J., Shoenfeld, Y., 2000.Adoptive transfer of beta(2)-glycoprotein I-reactive lymphocytes enhances earlyatherosclerosis in LDL receptor-deficient mice. Circulation 10, 1822–1827.

George, J., Afek, A., Gilburd, B., Shoenfeld, Y., Harats, D., 2001. Cellular and humoralimmune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice. J. Am. Coll. Cardiol. 38, 900–905.

George, J., Goldstein, E., Abashidze, S., Deutsch, V., Shmilovich, H., Finkelstein, A., Herz, I.,Miller, H., Keren, G., 2004. Circulating endothelial progenitor cells in patientswith unstable angina: association with systemic inflammation. Eur. Heart J. 25,1003–1008.

George, J., Afek, A., Abashidze, A., Shmilovich, H., Deutsch, V., Kopopolivch, Y., Miller, H.,Keren, G., 2005. Transfer of endothelial progenitor and bone marrow cellsinfluences atherosclerotic plaque size and composition in apolipoprotein Eknockout mice. Arterioscler. Thromb. Vasc. Biol. 25, 2636–2641.

Graff, J., Harder, S., Wahl, O., Scheuermann, E.H., Gossmann, J., Anti-inflammatory effectsof clopidogrel intake in renal transplant patients: effects on platelet–leukocyteinteractions, platelet CD40 ligand expression, and proinflammatory biomarkers.2005; 78: 468–76.

Hansson, G.K., 2005. Inflammation, atherosclerosis, and coronary artery disease.N. Engl. J. Med. 352, 1685–1695.

Hansson, G.K., Holm, J., Jonasson, L., 1989. Detection of activated T lymphocytes in thehuman atherosclerotic plaque. Am. J. Pathol. 135, 169–175.

Herbert, J.M., 2004. Effects of ADP-receptor antagonism beyond traditional inhibition ofplatelet aggregation. Expert Opin. Investig. Drugs 13, 457–460.

Hermann, A., Rauch, B.H., Braun, M., Schrör, K., Weber, A.A., 2001. Platelet CD40 ligand(CD40L)-subcellular localization, regulation of expression, and inhibition byclopidogrel. Platelets 12, 74–82.

Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., Finkel, T.,2003. Circulating endothelial progenitor cells, vascular function, and cardiovascularrisk. N. Engl. J. Med. 348, 593–600.

Klinkhardt, U., Graff, J., Harder, S., 2002. Clopidogrel, but not abciximab, reduces plateletleukocyte conjugates and P-selectin expression in a human ex vivo in vitro model.Clin. Pharmacol. Ther. 71, 176–185.

Klinkhardt, U., Bauersachs, R., Adams, J., 2003. Clopidogrel but not aspirin reduces P-selectin expression and formation of platelet–leukocyte aggregates in patients withatherosclerotic vascular disease. Clin. Pharmacol. Ther. 73, 232–241.

Libby, P., 2002. Inflammation in atherosclerosis. Nature 420, 868–874.Mor, A., Luboshits, G., Planer, D., Keren, G., George, J., 2006. Altered status of CD4(+)

CD25(+) regulatory T cells in patients with acute coronary syndromes. Eur. Heart J.27, 2530–2537.

Mor, A., Planer, D., Luboshits, G., Afek, A., Metzger, S., Chajek-Shaul, T., Keren, G., George,J., 2007. Role of naturally occurring CD4+ CD25+ regulatory T cells in experimentalatherosclerosis. Arterioscler. Thromb. Vasc. Biol. 27, 893–900.

Paigen, B., Morrow, A., Holmes, P.A., Mitchell, D., Williams, R.A., 1987. Quantitativeassessment of atherosclerotic lesions in mice. Atherosclerosis 68, 231–240.

Palinski, W., Miller, E., Witztum, J.L., 1995. Immunization of low density lipoprotein(LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDLreduces atherogenesis. Proc. Natl. Acad. Sci. U. S. A. 92, 821–825.

Plump, A.S., Smith, J.D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J.G., Rubin, E.M.,Breslow, J.L., 1992. Severe hypercholesterolemia and atherosclerosis in apolipopro-tein-E deficient mice created by homologous recombination in ES cells. Cell 71,343–353.

Rauscher, F.M., Goldschmidt-Clermont, P.J., Davis, B.H., Wang, T., Gregg, D., Ramaswami,P., Pippen, A.M., Annex, B.H., Dong, C., Taylor, D.A., 2003. Aging, progenitor cellexhaustion, and atherosclerosis. Circulation 108, 457–463.

Roselaar, S.E., Kakkanthu, P.X., Daugherty, A., 1996. Lymphocyte population inatherosclerosis lesions of apoE −/− and LDL receptor −/− mice. Decreasingdensity with disease progression. Arterioscler. Thromb. Vasc. Biol. 16, 1013–1018.

Sabitine, M.S., Cannon, C.P., Gibson, C.M., López-Sendón, J.L., Montalescot, G., Theroux,P., Claeys, M.J., Cools, F., Hill, K.A., Skene, A.M., McCabe, C.H., Braunwald, E., CLARITY-TIMI 28 Investigators, 2005. Addition of clopidogrel to aspirin and fibrinolytictherapy for myocardial infarction with ST-segment elevation. N. Engl. J. Med. 352,1179–1189.

Sakaguchi, S., 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22,531–562.

Shwartzenberg, S., Deutsch, V., Maysel-Auslander, S., Kisil, S., Keren, G., George, J., 2007.Circulating apoptotic progenitor cells. A novel biomarker in patients with acutecoronary syndromes. Arterioscler. Thromb. Vasc. Biol. 27, e27–e31.

Solheim, S., Pettersen, A.A., Arnesen, H., Seljeflot, I., 2006. No difference in the effects ofclopidogrel and aspirin on inflammatory markers in patients with coronary heartdisease. Thromb. Haemost. 96, 660–664.

Stemme, S., Faber, B., Holm, J., 1995. T lymphocytes from human atherosclerotic plaquesrecognize oxidized low density lipoprotein. Proc. Natl. Acad. Sci. U. S. A. 92,3893–3897.

Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., Zeiher, A.M.,Dimmeler, S., 2001. Number and migratory activity of circulating endothelialprogenitor cells inversely correlate with risk factors for coronary artery disease.Circ. Res. 89, E1–7.

Werner, N., Priller, J., Laufs, U., et al., 2002. Bone marrow-derived progenitor cellsmodulate vascular reendothelialization and neointimal formation: effect of3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition. Arterioscler.Thromb. Vasc. Biol. 22, 1567–1572.

Xu, Q., Dietrich, H., Steiner, H.J., Gown, A.M., Schoel, B., Mikuz, G., Kaufman, S.H., Wick,G., 1992. Induction of arteriosclerosis in normocholesterolemic mice rabbits byimmunization with heat shock protein 65. Arterioscler. Thromb. 12, 789–799.

Yusuf, S., Zhao, F., Mehta, S.R., 2001. Effects of clopidogrel in addition to aspirin inpatients with acute coronary syndromes without ST-segment elevation. N. Engl. J.Med. 345, 494–502.

Zhou, X., Nicoletti, A., Elhage, R., Hansson, G.K., 2000. Transfer of CD4 (+) T cellsaggravates atherosclerosis in immunodeficient apolipoprotein E knockout mice.Circulation 12, 2919–2922.