The RXR Agonist Bexarotene Improves Cholesterol Homeostasis and Inhibits Atherosclerosis Progression in a Mouse Model of Mixed Dyslipidemia

The RXR Agonist Bexarotene Improves CholesterolHomeostasis and Inhibits Atherosclerosis Progression in a

Mouse Model of Mixed DyslipidemiaFanny Lalloyer, Catherine Fievet, Sophie Lestavel, Gerard Torpier, Jelske van der Veen,

Veronique Touche, Stephanie Bultel, Saıd Yous, Folkert Kuipers, Rejane Paumelle,Jean-Charles Fruchart, Bart Staels, Anne Tailleux

Objective—The activity of the antitumoral agent bexarotene (Targretin, Bexarotene) depends on its binding to the nuclearretinoid-X receptor (RXR) and subsequent transcriptional regulation of target genes. Through RXR activation,bexarotene may modulate numerous metabolic pathways involved in atherosclerosis. Here, we investigated the effect ofbexarotene on atherosclerosis progression in a dyslipidemic murine model, the human apolipoprotein E2 knockinmouse, that develops essentially macrophage-laden lesions.

Methods and Results—Atherosclerotic lesions together with different metabolic pathways involved in atherosclerosis wereinvestigated in mice treated or not with bexarotene. Bexarotene protects from atherosclerosis development in mice, atleast in part by improving the circulating cholesterol distribution profile likely via a marked decrease of dietarycholesterol absorption caused by modulation of intestinal expression of genes recently identified as major players in thisprocess, Niemann-Pick-C1-Like1 (NPC1L1) and CD13. This atheroprotection appears despite a strong hypertriglycer-idemia. Moreover, bexarotene treatment only modestly modulates inflammatory gene expression in the vascular wall,but markedly enhanced the capacity of macrophages to efflux cellular lipids.

Conclusion—These data provide evidence of a favorable pharmacological effect of bexarotene on atherosclerosis despitethe induction of hypertriglyceridemia, likely via a beneficial action on intestinal absorption and macrophage efflux.(Arterioscler Thromb Vasc Biol. 2006;26:2731-2737.)

Key Words: atherosclerosis � cholesterol homeostasis � intestinal cholesterol absorption � rexinoid � triglycerides

Bexarotene [Targretin, 4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphtalenyl) ethenyl] benzoic acid] is an

antitumoral agent used as chemotherapy in the treatment ofcutaneous T-cell lymphoma.1 Bexarotene is currently beingevaluated for the treatment of other cancers1 and psoriasis.2

Thus, bexarotene is both an element of the current antitu-moral therapeutic arsenal and a molecule with emerging andpromising effects in various pathologies.

Atherosclerosis is a complex inflammatory pathology ofthe vascular wall, precipitated by systemic factors, such asqualitative or quantitative abnormalities of circulating lipidsand lipoproteins. Blood lipid concentrations reflect an equi-librium between absorption of dietary lipids in the smallintestine, production after endogenous synthesis in the liver,and removal by different peripheral tissues and the liver. Inpathological conditions, circulating atherogenic lipoproteinscan be taken up by macrophages in the vascular wall, thusinitiating an inflammatory process leading to a progressive

evolution of atherosclerosis. Through the action of locallyproduced cytokines and other inflammatory proteins leadingto cell migration and proliferation, the vascular wall iscontinuously remodeled. Atherosclerosis progressivelyevolves from the simple fatty streak to advanced atheroscle-rotic plaques, which may ultimately lead to plaque ruptureand thrombus formation.

The pharmacological responses to bexarotene originatefrom the transcriptional control of gene programs via activa-tion of a member of the nuclear receptor superfamily, theretinoid-X receptor (RXR). As other nuclear receptors, RXRacts as a transcription factor that, on activation by binding ofa specific ligand, binds to gene regulatory DNA sequencesand subsequently modulates the transcription of target genes.A growing number of studies have reported effects of RXRligands on plasma lipid and apolipoprotein concentrations,cell migration, proliferation, apoptosis, matrix remodeling,and inflammation, all of which impinge on atherogenesis.3–5

Original received March 23, 2006; final version accepted September 14, 2006.From Institut Pasteur de Lille (F.L., C.F., S.L., G.T., V.T., S.B., R.P., J.-C.F., B.S., A.T.), Departement d’Atherosclerose, Lille, France; Inserm (F.L.,

C.F., S.L., G.T., V.T., S.B., R.P., J.-C.F., B.S., A.T.), U545, Lille, France; Universite de Lille 2 (F.L., C.F., S.L., G.T., V.T., S.B., R.P., J.-C.F., B.S.,A.T.), Lille, France; Center for Liver, Digestive, and Metabolic Disease (J.v.d.V., F.K.), University Medical Center Groningen, Groningen, TheNetherlands; Institut de Chimie Pharmaceutique Albert Lespagnol (S.Y.), Universite de Lille 2, Lille, France.

Correspondence to Anne Tailleux, Inserm U545, Institut Pasteur de Lille, 1 rue du Professeur Calmette, F-59019 Lille, France. [email protected]

© 2006 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000248101.93488.84

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A beneficial effect on atherogenesis in vivo has been reportedonly once, with the rexinoid LG100364, a molecule that wasnever further developed, and thus has no clinical applications, inthe apolipoprotein E (apoE) knockout (apoE-KO) mouse.6 In thepresent report, we studied the apoE2-KI mouse model, whichdiffers from the apoE-KO mouse model, because it ratherdevelops a mixed dyslipidemia combining hypertriglyceridemiaand hypercholesterolemia as frequently found in humans,whereas apoE-KO mice are characterized by an isolated hyper-cholesterolemia. Furthermore, apoE2-KI mice appear a betterpharmacological model to test agonists of nuclear receptors thanapoE2-KO mice.7 Because the concept of selective nuclearreceptor modulator (SNRM) agonists, which postulates thatdifferent agonists of a nuclear receptor lead to overlapping butalso distinct biological effects, also pertains to RXR modulatorsand because apoE2-KI mice display hypertriglyceridemia that isexacerbated by rexinoid treatment as observed in humans treatedwith rexinoids, we decided to study the effects of bexarotene, arexinoid used in humans, on atherosclerosis and related meta-bolic pathways in the apoE2-KI model.

We show here that bexarotene protects apoE2-KI micefrom atherosclerotic lesion development, despite a markedincrease in triglycerides, at least in part, by decreasing theatherogenic cholesterol-containing lipoprotein fraction likelydue to a marked decrease of dietary cholesterol absorption inrelation to decreased intestinal expression of Niemann-PickC1-Like1 (NPC1L1)8 and CD13.9 Bexarotene treatment onlymodestly modulates the expression of inflammatory genes inthe vascular wall, but enhanced the capacity of macrophagesto efflux cellular lipids. Thus pharmacological atheroprotec-tion can be obtained despite triglyceride elevation whenatherogenic lipoprotein concentrations decrease.

Materials and MethodsAnimals and TreatmentHomozygous female human apoE2-KI mice on a C57BL6 geneticbackground were used.10 The animal experiments were performedaccording to the institutional guidelines. Mice were euthanized bycervical dislocation.

Bexarotene was synthesized in the Laboratoire de Chimie Phar-maceutique (Faculte des Sciences Pharmaceutiques, Universite deLille 2, France).

In three different experiments, 2 separate groups of apoE2-KImice were matched for age. Doses of bexarotene, within the range ofdoses previously used in rodents,11 efficient on lipid parameters andnontoxic during the treatment duration were used.

Twenty-four mice (n�12/group) aged 7 to 10 weeks were fed aWestern-style diet containing 0.2% cholesterol and 21% fat (SAFE,Augy, France) supplemented or not with bexarotene (0.018% wt/wt)for 11 weeks. Based on food consumption, this dose corresponds to�35 mg per kg. At the end of the treatment, blood was collected byretro-orbital venipuncture under isoflurane anesthesia after 4 hoursof fasting (9:00 AM to 1:00 PM) and plasma was separated. Livers andintestines were removed, duodenum and jejunum separated, longi-tudinally opened, and enterocytes were scrapped. Hearts wereremoved and treated as further described.

Sixteen chow fed mice (n�8/group) aged 6 months were dosed for14 days with bexarotene (300 mpk) or with vehicle alone (carboxym-ethylcellulose 1%/polyethylene glycol [PEG] 400/Tween, 90/9.95/0.05, by volume). Hearts were removed and the upper half contain-ing the aortic sinus was sectioned at a plane parallel to a line drawnbetween the tips of the atria. The intestine was removed andenterocytes obtained as noted.

For these experiments, tissues and cells were frozen in liquidnitrogen (LN2) and stored at �80°C until mRNA analysis.

Sixteen chow diet fed mice (n�8/group) aged 6 months weredosed once daily first with bexarotene (300 mpk) or vehicle(carboxymethylcellulose 1%/PEG 400/Tween, 90/9.95/0.05, by vol-ume) for 5 days, and then with a single dose of [14C]cholesterol (1�Ci) and [3H]sitostanol (2 �Ci) in 150 �L of olive oil. The micewere subsequently dosed once daily with bexarotene or vehicle for 4additional days, totaling 9 consecutive treatment days. Feces werecollected during the 4 last days to calculate intestinal cholesterolabsorption (see http://atvb.ahajournals.org).

For expanded Materials and Methods used in this article, pleasesee http://atvb.ahajournals.org.

ResultsBexarotene Decreases Atherosclerosis Developmentin ApoE2-KI MiceThe apoE2-KI mice were fed a Western diet with or withoutbexarotene for 11 weeks. The treated group gained weight toa comparable extent as the control group, indicating absenceof toxicity (data not shown).

Bexarotene-treated mice displayed a marked decrease inatherosclerotic lesion areas as compared with control mice(median 0.028 versus 0.129 mm2, P�0.001) (Figure 1A).Microphotographs representative of treated and control mice(Figure 1B) show the decrease of lipid-stained surfacesinduced by treatment. Moreover, as revealed by MOMA-2-

Figure 1. Bexarotene decreases atherosclerosis development inapoE2-KI mice. Female apoE2-KI mice were fed a Western-stylediet supplemented (BEXA) or not (CONT) with 0.018% bexarotene(n�12/group) for 11 weeks. A, Oil-Red-O staining of atheroscle-rotic lesions in aortas of control (E) and treated (● ) mice. Graphsrepresent mean lesion area of the analyzed portion. Each pointcorresponds to one mouse. Horizontal bars indicate the median ofthe mean lesion area. ***P�0.01 vs controls. B, Representativemicrographs showing atherosclerotic lesions in the aortic sinus ofcontrol (CONT) or treated (BEXA) mice. Bar�0.5 mm. Arrows indi-cate traces of remaining Oil-Red-O staining parts.

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specific staining of the lesions, macrophage content wasreduced on bexarotene treatment and colocalized with Oil-Red-O–stained areas (data not shown).

Bexarotene Increases Plasma TriglycerideConcentrations in ApoE2-KI MiceCompared with controls, bexarotene-treated mice showed amarked increase in plasma triglyceride concentrations(�50%, P�0.001) (Figure 2A). The triglyceride distributionprofile showed that triglycerides were associated with very-low-density lipoprotein (VLDL) (data not shown). To analyzethe mechanisms involved in this increase, the hepatic expres-sion of proteins controlling triglyceride metabolism weremeasured. Stearyl-coenzyme A (CoA) desaturase 1 (SCD1)and fatty acid synthase (FAS) are lipogenic enzymes, whereasangiopoietin-like 3 (Angptl3) is implicated in triglyceridecatabolism. The expression of genes encoding SCD1 andFAS was strongly increased in livers of treated mice ascompared with controls (P�0.001), whereas Angptl3 mRNAwas only slightly increased (P�0.05) (Figure 2B).

Because the activity of SCD1 can be evaluated by deter-mining the ratio of oleic acid to stearic acid (desaturationindex; C18:1/C18:0),12 the fatty acid composition of cho-lesteryl esters in plasma was measured (see http://atvb.ahajournals.org, Table II). Expressed as percentage of totalfatty acid mass in cholesteryl esters, saturated fatty acids with16 and 18 carbon atoms were significantly decreased(P�0.001) in treated mice, whereas monounsaturated fattyacids with the same number of carbons were unchanged,leading to a significant increase of the ratios C18:1/C18:0 andC16:1/C16:0, indicative of an enhanced SCD1 activity.

Bexarotene Decreases AtherogenicCholesterol-Containing Lipoproteins inApoE2-KI MiceBexarotene-treated mice exhibited a reduction in total cho-lesterol levels (P�0.05), which was entirely caused byreduced non–high-density lipoprotein (HDL) cholesterolconcentrations (P�0.01), whereas HDL cholesterol levelswere comparable in treated and control mice (Figure 3A).Cholesterol distribution profile analysis in control mice (Fig-ure 3B) clearly showed that in the apoE2-KI mouse model,cholesterol is mainly transported by VLDL, intermediary-density lipoprotein (IDL), and low-density lipoprotein (LDL),whereas the HDL fraction represents only �20% of totalcholesterol as previously observed.13 Treatment with bexaro-tene led to a decrease in non-HDL cholesterol exclusively bydecreasing the IDL–LDL fraction without any change in theHDL fraction (Figure 3B). The reduction of the atherogeniccholesterol-rich remnant particles in treated mice was asso-ciated with reduced plasma concentrations of its main proteincomponent, apoB (P�0.01) (Figure 3C), but hepatic apoBmRNA levels were unchanged (Figure 3D). Because markedchanges were observed in the cholesterol distribution profilein treated mice, the mechanisms that could explain thesemodifications were further analyzed by measuring the hepaticexpression of genes controlling cholesterol homeostasis.While LDL receptor mRNA levels were markedly enhanced(P�0.001) in treated mice as compared with controls (Figure

Figure 2. Bexarotene increases plasma triglycerides in apoE2-KImice. Female apoE2-KI mice were fed a Western-style diet sup-plemented (BEXA) or not (CONT) with 0.018% bexarotene(n�12/group) for 11 weeks. Mean�SD. *P�0.05, ***P�0.001 vscontrols. A, Plasma triglyceride concentrations in control (whitebars) and treated (black bars) mice. B, Hepatic SCD1, FAS andAngptl3 mRNA levels. Data are expressed as percentages, arbi-trary values of 100% being attributed to the control group.

Lalloyer et al Atheroprotective Effect of Bexarotene in Mice 2733



3E), 3-hydroxy-3-methylglutaryl (HMG)-CoA-synthase(HMG-CoA-S) gene expression was not altered (Figure 3F),suggesting that hepatic removal of LDL from circulationcould be enhanced by treatment without concomitant changesin cholesterol synthesis.

Bexarotene Decreases Intestinal CholesterolAbsorption and Modulates Expression of IntestinalGenes in ApoE2-KI MiceTo gain insight in the mechanisms underlying the reducedLDL/IDL levels in treated mice, intestinal cholesterol absorptionwas determined. Bexarotene treatment led to a 64% reduction ofcholesterol absorption efficiency (P�0.001) (Figure 4). SinceNPC1L1 and CD13 have recently been identified as criticalcomponents of the intestinal cholesterol absorption machinery,intestinal mRNA levels of these genes were measured. NPC1L1and CD13 mRNA levels were significantly decreased in bothduodenum and jejunum of bexarotene-treated mice as comparedwith controls (Figure 5). In addition, as ABCA1 has been shownto be implicated in intestinal cholesterol metabolism, its expres-sion was also measured. ABCA1 mRNA levels were signifi-cantly lower in treated mice than in controls (Figure 5). Com-

parable decreases both in cholesterol absorption and expressionof these genes were observed in mice fed a Western diet for 11weeks (data not shown).

Bexarotene Improves Macrophage LipidHomeostasis In Vivo and In Vitro, and InducesABCA1 and ABCG1 ExpressionThe efficiency of plasma from treated and control mice topromote cholesterol efflux ex vivo in Fu5AH hepatoma cellswas comparable (30.5�4.7% versus 27.1�6.2%, treatedversus controls, P�0.118).

Figure 3. Bexarotene decreases concentrations of non-HDL cho-lesterol-containing lipoproteins in apoE2-KI mice. Female apoE2-KImice were fed a Western-style diet supplemented (BEXA) or not(CONT) with 0.018% bexarotene (n�12/group) for 11 weeks.*P�0.05, **P�0.01, ***P�0.001 vs controls. A, Plasma cholesterolconcentrations in control (white bars) and treated (black bars) mice.Mean�SD. B, Representative cholesterol profile obtained after sizeexclusion chromatography of lipoproteins from control (E) andtreated (● ) mice. C, Plasma apoB concentrations. D, Hepatic apoBmRNA levels. Data are expressed as percentages, arbitrary valuesof 100% being attributed to the control group. Mean�SD. E and F,Hepatic LDL-R and HMG-CoA synthase mRNA levels. Data areexpressed as percentages, arbitrary values of 100% being attrib-uted to the control group. Mean�SD.

Figure 4. Bexarotene decreases intestinal cholesterol absorption.Cholesterol absorption in control and treated mice was measuredusing the fecal dual-isotope method (n�8/group). After a 5-daytreatment, mice received an intragastric single dose of [14C]choles-terol and [3H]sitostanol and were subsequently treated for 4 addi-tional days. Feces from the 4 last days of treatment were pooled,lipids were extracted, and radiolabeled isotopes were measured.Mean�SD, ***P�0.001 vs controls.

Figure 5. Bexarotene decreases expression of cholesterol-handling genes in the intestine. Female apoE2-KI mice fed achow diet were treated with bexarotene (300 mpk) (BEXA) orvehicle (CONT) for 14 days (n�8/group). Data are expressed aspercentages, arbitrary values of 100% being attributed to thecontrol group. Mean�SD. *P�0.05, **P�0.01 vs controls.




Interestingly, mice fed a Western diet displayed an increasednumber of Oil-Red-O–stained peritoneal macrophages (73% oftotal cells) compared with mice fed a chow diet (14% of totalcells), and bexarotene-treatment strongly reduced (33% of totalcells) the Western diet-induced lipid-loading of these peritonealmacrophages (Figure 6A). To further analyze the mechanism,cholesterol-loaded peritoneal macrophages isolated fromapoE2-KI mice and in vitro treated with bexarotene wereincubated with either apoA-I or HDL as cholesterol acceptors,and cellular cholesterol efflux was measured. The cholesterolefflux was significantly higher in bexarotene-treated macro-phages than in controls whatever the acceptor (P�0.05) (Figure6B). Because ABCA1 and ABCG1 are the main cholesteroltransporters mediating apoA-I–dependent and HDL-dependentcholesterol efflux pathways, respectively, the expression of thesetransporters was compared in the aortic sinus of bexarotene-treated and control mice. The mRNA levels of both proteinswere significantly induced by bexarotene treatment (P�0.01)(Figure 6C).

Bexarotene Only Modestly Modulates GeneExpression of Cytokines in the Aortic Sinus ofApoE2-KI MiceThe expression of genes encoding several molecules im-plicated in the inflammatory process was measured in the

aortic sinus of 6-month-old chow fed female mice after a14-day treatment with bexarotene. At this age, femaleapoE2-KI mice display large macrophage-laden lesions inthe aortic sinus (personal unpublished results), whichallows to study gene expression in the context of inflam-matory protein producing cells. Monocyte chemoattractantprotein-1 (MCP-1) and macrophage colony-stimulatingfactor (M-CSF) mRNA levels were minimally altered inbexarotene-treated mice (MCP-1 161�75 versus 100�28,treated versus controls, P�0.05; macrophage colony-stimulating factor 72�18 versus 100�17, treated versuscontrols, P�0.05). Expression of other genes studied,IL-6, IL-12, tumor necrosis factor-�, cyclo-oxygenase 2,tissue factor, and vascular cellular adhesion molecule-1,was not significantly changed by treatment (supplementalFigure I, available online at http://atvb.ahajournals.org).

DiscussionIn the present report, we show that bexarotene, a rexinoid usedin humans, inhibits atherogenesis in the apoE2-KI mouse.Bexarotene treatment induces effects both on systemic plasmalipid parameters and on macrophage lipid homeostasis. Bexaro-tene likely exerts atheroprotection in apoE2-KI mice by lower-ing circulating atherogenic cholesterol-containing lipoproteins.Interestingly, the pronounced effect of bexarotene treatment on

Figure 6. Bexarotene increases macro-phage lipid efflux. A, Representativemicrographs of in vivo lipid-loaded peri-toneal macrophages. Female apoE2-KImice were fed a chow diet (left), a West-ern diet (middle), or a Western diet sup-plemented with bexarotene for 12 days(right). Peritoneal macrophages wererecruited using thioglycollate and stainedex vivo by Oil-Red-O. Bar�50 �m. B,Apo A-I and HDL mediated-cholesterolefflux in peritoneal macrophagesobtained from apoE2-KI mice. Macro-phages were in vitro treated with bexaro-tene (1 �mol/L) for 24 hours and choles-terol efflux measured. Results areexpressed as fold induction as com-pared with controls. Mean�SD. *P�0.05vs controls. C, ABCA1 and ABCG1 geneexpression in the aortic sinus. FemaleApoE2-KI mice fed a chow diet weretreated with bexarotene (300 mpk) orvehicle for 12 days (n�8/group). Aorticsinuses were removed from individualmice, RNA extracted, and gene expres-sion analyzed. Data are expressed aspercentages, arbitrary values of 100%being attributed to the control group.Mean�SD. **P�0.01 vs controls.




plasma cholesterol concentrations coincides with a decrease inintestinal cholesterol absorption, which is associated with areduced intestinal gene expression of NPC1L1 and CD13.Indeed, NPCL1 has been recently identified as a target of thehypocholesterolemic drug ezetimibe through regulating intesti-nal cholesterol absorption.14–18 Thus, inhibiting NPC1L1 activ-ity by ezetimibe in apoE-KO mice14 or gene expression bybexarotene in apoE2-KI mice leads to a decreased susceptibilityto atherosclerosis. CD13 has been identified as a moleculepotentially implicated in cholesterol absorption19 and could alsobe a molecular target of ezetimibe.9 Interestingly, bexarotenedecreased the expression of ABCA1 in the intestine. This resultdiffers from observation with an other rexinoid, LG268, show-ing an increased ABCA1 expression in the intestine associatedwith a decreased intestinal cholesterol absorption.20 However,the potential effect of ABCA1 in cholesterol absorption has beenhighly debated21–23 and a recent study24 showed its implicationin intestinal HDL biogenesis.

One level of complexity of RXR biology relates to its abilityto activate transcription as an obligate partner of heterodimer-ization with many nuclear receptors. Thus, bexarotene activationof RXR could elicit responses of the permissive heterodimersand thus modulate cholesterol absorption. The question address-ing which partner(s) of heterodimerization modulate the bexaro-tene responses in intestinal cholesterol absorption is still open.Despite being expressed in the intestine, Peroxisomeproliferator-activated receptor (PPAR)� is unlikely to be in-volved, because a previous study showed no effect of itsactivation on NPC1L1 gene expression.25 Conversely, PPAR�could be implicated because NPC1L1 is known to be reduced inthe intestine on PPAR� activation.16 However, PPAR� mRNAlevels are decreased in bexarotene-treated mice as comparedwith controls (data not shown), suggesting the existence of analternative partner. Finally, the most attractive partner of RXRheterodimerization in the intestine is the liver-X receptor (LXR).Indeed, NPC1L1 is reduced after LXR activation.25 Surpris-ingly, expression of the ABCA1 gene, a positive LXR targetgene, is decreased in the intestine of bexarotene-treated mice. Inaddition, expression of ABCG5 and ABCG8, other well-characterized LXR target genes,26 are also decreased in theintestine of bexarotene-treated mice (data not shown). Interest-ingly, treatment with bexarotene did increase aortic ABCA1expression, but only modestly altered expression of genesimplicated in the parietal inflammatory process, whereas LXRactivation has been shown to exert anti-inflammatory effects.27

Thus it is unclear to what extent the LXR/RXR pathwaymediates the effects of bexarotene and bexarotene certainly actsas a selective, tissue-specific, and gene-specific modulator of theLXR/RXR pathway.

Cholesterol efflux, the obligatory first step of reverse choles-terol transport, is a “bi-partner process.” On the one side are theacceptors of cholesterol, namely lipoproteins present in plasmaand intercellular fluids, and on the other side the cells and theircholesterol transporters. Among them, ABCA1 and ABCG1have been shown to specifically mediate cholesterol efflux fromcells to lipid-poor apoA-I and to HDL, respectively.28,29 Treatingmice with bexarotene did not significantly modify the capacityof plasma to accept cholesterol from cells ex vivo. By contrast,in vitro incubation of murine cholesterol-laden peritoneal mac-

rophages with bexarotene resulted in a significant increase oflipid-free apoA-I-mediated cholesterol efflux. Moreover, bex-arotene enhanced cholesterol efflux mediated by HDL, suggest-ing that both ABCA1 and ABCG1-mediated cholesterol effluxare facilitated on treatment of macrophages in vitro. Finally, theexpression of these cholesterol transporters was enhanced in theaortic sinus, ie, the area enriched in macrophages, on in vivotreatment. Moreover, in vivo treatment decreased the number oflipid-loaded peritoneal macrophages, which correlates with lesslipid-loading of the cell population present in the atheroscleroticlesions. Taken together, our present results suggest that macro-phages in the aortic sinus of treated mice display enhancedcapacity to efflux cholesterol, thus preventing foam cell forma-tion and subsequent lesion development.

As the role of inflammation in atherosclerosis has beenincreasingly recognized at all stages of its pathogenesis, wemeasured the expression of genes encoding different cytokinesand proteins implicated in the inflammatory process and previ-ously shown to be regulated in vitro by retinoids (natural RXRligands) or rexinoids (synthetic RXR ligands).30–34 Bexarotenetreatment induced only modest variations in the genes encodingMCP-1 and macrophage colony-stimulating factor, two strongmodulators of foam cell formation. In addition, in the subcuta-neous dorsal pouch acute inflammatory model,35 bexarotenetreatment did not exert any anti-inflammatory activity (data notshown) indicating that bexarotene has no marked effect in theinflammatory process in the vascular wall in vivo.

In our model, bexarotene-treatment induced a marked in-crease in triglyceridemia as it does in humans.1 Our resultsdemonstrate that the biosynthesis of triglycerides could beaffected by bexarotene treatment, because hepatic expression ofSCD1 and FAS was increased. Interestingly, these genes aretargets of the LXR/RXR heterodimer and LXR agonists alsoincrease hepatic lipogenesis and plasma triglycerides.36 SCD1protein activity appeared also increased by bexarotene treatment,as assessed by the higher desaturation index (C18:1/C18:0) ofplasma cholesteryl esters in treated mice as compared withcontrols. In addition, the slight increase of hepatic expression ofAngptl3, a protein identified as an inhibitor of lipoprotein–lipase,37 the enzyme responsible for catabolism of triglyceridesin the vascular compartment, could also enhance the triglycer-idemia. Although epidemiological and clinical studies demon-strated the association of elevated plasma triglyceride levels withincreased risk of cardiovascular disease,38 it is interesting to notethat increasing plasma triglyceride levels, as observed not onlyafter bexarotene but also LXR agonist treatment, is not sufficientto aggravate atherosclerosis progression when associated with adecrease in non-HDL cholesterol and an improvement of lipidhomeostasis in macrophages.

AcknowledgmentsWe thank Dr Hafid Mezdour for providing the mouse strain, PrJean-Paul Bonte and Brigitte Lacroix for fatty acid compositionanalyses, and Emmanuelle Vallez, Bruno Derudas, and JonathanVanhoutte for excellent technical assistance.

Sources of FundingThis work was supported by grants of the Leducq Foundation, ACI02 20475 (French Research Ministry and Servier laboratory) and theEuropean project X-TRA-NET (018882).




DisclosuresNone.

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Jean-Charles Fruchart, Bart Staels and Anne TailleuxVéronique Touche, Stéphanie Bultel, Saïd Yous, Folkert Kuipers, Réjane Paumelle,

Fanny Lalloyer, Catherine Fiévet, Sophie Lestavel, Gérard Torpier, Jelske van der Veen,Atherosclerosis Progression in a Mouse Model of Mixed Dyslipidemia

The RXR Agonist Bexarotene Improves Cholesterol Homeostasis and Inhibits

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 2006 American Heart Association, Inc. All rights reserved.

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The RXR agonist bexarotene improves cholesterol homeostasis and inhibits

atherosclerosis progression in a mouse model of mixed dyslipidemia.

by Fanny Lalloyer et al.

Supplementary information published online

1) Materials and Methods

Lipid and lipoprotein analyses

Plasma concentrations of total cholesterol (TC) and triglycerides (TG) were

measured using commercially available kits (Biomerieux, France). HDL cholesterol

(HDL-C) concentrations were determined in plasma after precipitation of

apolipoprotein (apo) B-containing lipoproteins by phosphotungstic acid/Mg

(Boehringer Mannheim, Germany). Non-HDL cholesterol (N-HDL-C) was obtained by

subtraction of HDL-C values from TC.

Lipoproteins were separated from 200 µl of pooled plasma by gel filtration

chromatography using a Superose 6 HR 10/30 column (Pharmacia, Sweden). The

gel was allowed to equilibrate with 10 mM phosphate–buffered saline (PBS)

containing 0.01% (wt/vol) EDTA and 0.01% (wt/vol) sodium azide. Plasma was eluted

with the buffer at room temperature at a flow rate of 0.2 ml.min-1. The effluents were

collected in 0.22-ml fractions. Cholesterol and triglyceride concentrations were

determined in the eluted fractions as described above.

Plasma levels of mouse B were measured by immunonephelometric assays using

specific polyclonal antibodies.

Fatty acid composition of plasma cholesteryl esters.

Lipids were extracted from plasma according to the Folch method (Folch et al.). The

lipid extract was dried under nitrogen, dissolved in chloroform/methanol (2:1, v:v),

and separated by thin layer chromatography using petroleum ether/ether/acetic acid

(90:10:5, v:v:v). Cholesteryl esters were identified by simultaneous migration of

standards. Cholesteryl esters were scrapped, hydrolysed and the resulting fatty acids

were methylated by treatment with methalonic H2SO4 for 2 h at 70°C. The fatty acid

methyl esters were extracted with heptane and analysed by gas chromatography on

a Varian 3400 chromatograph (Palo Alto, CA) equipped with a flame ionisation

detector and a capillary column. A gradient temperature from 160°C to 210°C at

1.5°C min-1 and a N2 pressure of 85 kPA were used. Identification of fatty acids was

achieved using commercially available fatty acid methyl esters.

Reference :

Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and

purification of total lipids from animal tissues. J Biol Chem 1957;226:497-509.

Quantification of atherosclerotic lesions

After euthanasia, the heart was perfused from its apex with cold Krebs-Ringer buffer

and fixed in a solution containing 4% paraformaldehyde in PBS, pH 7.4. The heart,

containing the aortic origin, was carefully removed and fixed in paraformaldehyde

overnight. The upper half of the heart was equilibrated in 30% sucrose, embedded in

Tissue-Tek OCT compound and frozen in liquid nitrogen in combination with

isopentane. Serial 10-µm-thick cryosections were obtained from the aortic arch to the

ventricles and were used for detection of lipid deposition using Oil Red O and

hematoxylin. Quantitative analysis of the atherosclerotic lesion area including the

positive Oil Red O staining area was performed every 5 sections, each 50 µm apart.

Images were captured by use of a JVC 3-CCD video camera and analysed blindly by

one observer using computer assisted-Quips Image analysis system from Leica

(Leica Mikroskopic und System GmbH, Wetzlar, Germany). To avoid morphological

heterogeneity between mice, the expression of average atherosclerotic lesion areas

of the aortic root, from the appearance to the disappearance of the aortic valves, was

normalized at ten equal sections.

Cholesterol efflux experiments

Cellular cholesterol efflux ability of plasma was determined using Fu5AH rat

hepatoma cells following the procedure described by de la Llera Moya et al. Briefly,

the cells were maintained in minimal essential medium containing 5% fetal calf

serum. Fu5AH were plated on 24 multiwell plates (BD, Le Pont de Claix, France)

using 1 mL per well. Two days after plating, cellular cholesterol was labelled during a

72 hour-incubation with 3H-cholesterol (Amersham, Orsay, France) (0.5 µCi/well). To

allow equilibration of the label, the cells were washed and incubated for 24 hours in

minimal essential medium with 0.5% wt/vol bovine serum albumin (BSA). Then the

cells were washed with phosphate-buffered saline (PBS) and incubated at 37°C for 4

hours with 2.5% (v:v) diluted plasma. At the end of the incubation, the medium was

removed and centrifuged. The monolayer cells were washed 3 times with PBS and

harvested with 0.5 mL of 1 mol/L NaOH. Radioactivity was then measured in both

medium and cells, and percentage of cholesterol efflux calculated. Measurements

were performed in triplicate.

Thioglycollate-elicited mouse peritoneal macrophages plated at 105 cells/ml were

loaded with 50 µg/ml [3H] acetylated low-density lipoproteins for 24 h in serum-free

DMEM containing 0.2 % (v:v) BSA. Loaded cells were then washed, activated for 24

hours with bexarotene (1µM) and efflux was initiated in medium containing 10 µg/ml

of human apo A-I or 30 µg/ml of human HDL3 isolated from plasma by

ultracentrifugation at density 1.063<d<1.21. Twenty-four hours later, the medium was

removed and centrifuged to remove cell debris. The cells were dissolved in 1 M

NaOH. Radioactivity was then measured in both medium and cells, and percentage

of cholesterol efflux calculated. Measurements were performed in triplicate.

Reference :

de la Llera MM, Atger V, Paul JL, Fournier N, Moatti N, Giral P, Friday KE, Rothblat

G. A cell culture system for screening human serum for ability to promote cellular

cholesterol efflux. Relations between serum components and efflux, esterification,

and transfer. Arterioscler Thromb 1994;14:1056-1065.

Analysis of lipid-loading of peritoneal macrophages

Mouse peritoneal macrophages (MPMs) were collected by peritoneal lavage with

PBS from mice given a 3-mL intraperitoneal injection of 4% thioglycollate in water.

The MPMs were pelleted, washed twice with serum-free RPMI (GIBCO), plated at a

density of 1.5x106 cells/35-mm dish, and allowed to adhere to dishes for 2 hours in

RPMI. Then, plates were washed 3 times with PBS to remove nonadherent cells.

Lipids were stained by Oil-Red-O according to the method of Lillie and Ashburn

(1943). Cells were counterstained with hematoxylin.

Intestinal cholesterol absorption

Intestinal cholesterol absorption was measured by the dual fecal isotope ratio

method, as described by Wang and Carey. Fecal lipids were extracted and

[14C]cholesterol and [3H]sitostanol levels determined. The ratio of the two

radiolabeled isotopes in the fecal extracts and the dosing mixture was used for

calculation of percent cholesterol absorption :

% absorption = [14C] / [3H] dosing mixture - [14C] / [3H] feces x 100

[14C] / [3H] dosing mixture

Reference :

Wang DQ, Carey MC. Measurement of intestinal cholesterol absorption by plasma

and fecal dual-isotope ratio, mass balance, and lymph fistula methods in the mouse:

an analysis of direct versus indirect methodologies. J Lipid Res 2003;44:1042-1059.

Real-time PCR-based quantitative gene expression analysis

Total RNA was isolated from cells or tissues by the acid guanidium

thiocyanate/phenol/chloroform method as previously described (Chomczynski 1987)

and reverse transcribed using random hexamer primers and Superscript reverse

transcriptase (Life Technologies, France). For quantitative PCR, reverse transcribed

cDNA were quantified by real-time PCR on a MX 4000 apparatus (Stratagene), using

specific oligonucleotide primers listed in Table 1. PCR amplification was performed in

a volume of 25 µl containing 100 nM of each primer, 4 mM MgCl2, the Brilliant

Quantitative PCR Core Reagent mix as recommended by the manufacturer

(Stratagene), and SYBR Green 0.33 X (Sigma-Aldrich, France). The conditions were

95°C for 10 min, followed by 40 cycles of 30 sec at 95°C, 30 sec at 55°C, and 30 sec

at 72°C. All samples are run in duplicate. Results are expressed normalized to 28S

RNA.

Reference :

Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium

thiocyanate- phenol-chloroform extraction. Anal Biochem 1987;162:156-159.)

Table I : Primers used for real-time PCR

Genes Forward primers Reverse primers

28S 5’-AAA CTC TGG TGG AGG TCC GT-3’ 5’-CTT ACC AAA AGT GGC CCA CTA-3’

MCP-1 5’-GCC AAC TCT CAC TGA AGC C-3’ 5’-GCT GGT GAA TGA GTA GCA GC-3’

M-CSF 5’-AGC ATG GAC AGG CAG GGA C-3’ 5’-CTG CGT GCC TTT ATG CCT TT-3’

IL-6 5’-CCA GTT GCC TTC TTG GGA CTG-3’ 5’-CAG GTC TGT TGG GAG TGG TAT CC-3’

IL12b 5’-GAC ATC ACC TGG ACC TCA GAC-3’ 5’-AGT CTC GCC TCC TTT GTG GC-3’

TNFα 5’-AGC A CA GAA AGC ATG ATC CG-3’ 5’-CCC GAA GTT CAG TAG ACA GAA GAG-3’

TF 5’-CAA GTG CTT CTC GAC CAC AGA CAC C-3’ 5’-TCC CCA TGA ACT GAG TTC CTC CG-3’

COX-2 5’-GGC CCT TCC TCC CGT AGC AG-3’ 5’-AGA CCA GGC ACC AGA CCA AAG ACT-3’

VCAM-1 5’-TCT CCA GCT TCT CTC AGG AAA TGC C-3’ 5’-AAC CGA ATC CCC AAC TTG TGC AG-3’

ApoB 5’-GTG GCT TCT GTC ACT GCT AAA G-3’ 5’-GTC TGA TTT CCC CTC AAT GG-3’

LDL-R 5’-CAG GCA GCA GGA ACG AGT TC-3’ 5’-GGA GTC AGG AAT GCA TCG GC-3’

HMGCoA-S 5’-TGG TGG ATG GGA AGC TGT CTA-3’ 5’TTC TTG CGG TAG GCT GCA TAG-3’

SCD1 5’-TTC CCT CCT GCA AGC TCT ACA CCT G-3’ 5’-AGC CGT GCC TTG TAA GTT CTG TGG-3’

FAS 5’-GGT ATG TCC GGG AAA TTG CC-3’ 5’-ATT GTG TGT GCC TGC TTG GG-3’

Angptl3 5’-AGG GCT TTG GAG GAG CAG CTA ACC-3’ 5’-GCA GTC GGC AGG AAG GTC ATC TTG-3’

NPC1L1 5’-ATC CTC ATC CTG GGC TTT GC-3’ 5’-GCA AGG TGA TCA GGA GGT TGA-3’

CD13 5’-GCA ACA AGG AGC GGG TGG TC-3’ 5’-AAG CCC TCG TTC AGC CAC AG-3’

ABCA1 5’-ACA CCT GAC ACA CCA GCT AGA AGG C-3’ 5’-TGA GTT CTT CCC ACA TGC CTT CC-3’

Statistical analysis

Values are reported as mean±SD. Statistical differences were determined by the

non-parametric Mann-Whitney U-test. A value of p<0.05 was accepted as statistically

significant.

2) Results

Figure 7 : Influence of bexarotene on the expression of genes encoding

cytokines and inflammatory proteins in the aortic sinus of apoE2-KI mice.

Female apoE2-KI mice were treated with bexarotene (BEXA) (n=6) for 12 days and

compared to control mice (CONT) (n=6). Data are expressed as percentages,

TNFα

50

100

150

CONT BEXA

M-CSF

50

100

150

CONT BEXA

TF

50

100

150

CONT BEXA

IL6

50

100

150

200

CONT BEXA

IL12

50

100

150

CONT BEXA

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

COX2

50

100

150

200

CONT BEXA

mR

NA

(%)

*

MCP-1

50

100

150

200

250

CONT BEXA

mR

NA

(%)

*

VCAM-1

50

100

150

CONT BEXA

mR

NA

(%)

TNFα

50

100

150

CONT BEXA

TNFα

50

100

150

CONT BEXA

M-CSF

50

100

150

CONT BEXA

M-CSF

50

100

150

CONT BEXA

TF

50

100

150

CONT BEXA

TF

50

100

150

CONT BEXA

IL6

50

100

150

200

CONT BEXA

IL6

50

100

150

200

CONT BEXA

IL12

50

100

150

CONT BEXA

IL12

50

100

150

CONT BEXA

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

mR

NA

(%)

COX2

50

100

150

200

CONT BEXA

mR

NA

(%)

COX2

50

100

150

200

CONT BEXA

COX2

50

100

150

200

CONT BEXA

mR

NA

(%)

*

MCP-1

50

100

150

200

250

CONT BEXA

mR

NA

(%)

*MCP-1

50

100

150

200

250

CONT BEXA

mR

NA

(%)

*

VCAM-1

50

100

150

50

100

150

CONT BEXA

mR

NA

(%)

arbitrary values of 100% being attributed to the control group. Mean +/- SD. *p<0.05

versus controls.

Documents

The RXR Agonist Bexarotene Improves Cholesterol Homeostasis and Inhibits Atherosclerosis Progression in a Mouse Model of Mixed Dyslipidemia