The Effect of T0901317 on ATP-binding Cassette Transporter A1 and Niemann-Pick Type C1 in ApoE−/− Mice

Abstract

Although a range of studies indicated Liver X receptor (LXR) activation inhibited the development of atherosclerosis in animal models, the mechanism of this effect for LXR agonists has not been fully understood. A recent study has suggested LXR activators increased the amount of free cholesterol in the plasma membrane of human macrophages by inducing Niemann-Pick type C1 (NPC1) gene expression. Therefore, we hypothesize that LXRs may also promote NPC1 expression in vivo. Here we investigated the effect of a synthetic LXR agonist T0901317 on ATP-binding cassette transporter A1 (ABCA1) and NPC1 in apolipoprotein E knockout (apoE−/−) mice. Male apoE−/− mice were randomized into four groups: baseline group (n = 10), vehicle group (n = 14), prevention group (n = 14), and treatment group (n = 14). En face analysis and Oil red O staining were used to examine the aortic atherosclerotic lesions. Macrophage content of aortic root atherosclerotic lesions and cholesterol efflux form peritoneal macrophages were measured. Gene and protein expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting, respectively. T0901317 treatment reduced aortic atherosclerotic lesion area by 64.2% in prevention group (P < 0.001) and 58.3% in treatment group (P < 0.001) and resulted in a reduction in macrophage content. Plasma triglyceride, total cholesterol, high-density lipoprotein cholesterol, and apoA-I concentrations were markedly increased in T0901317-treated groups. T0901317 also promoted ABCA1 and NPC1 gene and protein levels in the aorta, liver, and small intestine of apoE−/− mice and significantly increased cholesterol efflux from peritoneal macrophages. T0901317 upregulates ABCA1 and NPC1. This study gives us a new insight into the mechanism for antiatherogenic effect of LXR synthetic agonists.

Liver X receptors (LXRs), LXRα and LXRβ, are members of the nuclear hormone receptor superfamily and activated by oxysterol ligands.¹ LXRs bind to DNA as obligate heterodimers with retinoid X receptors (RXRs). LXRα is mainly expressed in the liver, intestine, and macrophages, whereas LXRβ is ubiquitously expressed in many cell types. These sterol-responsive transcription factors control the expression of a set of genes involved in cholesterol absorption, transport, efflux, and excretion, as well as fatty acid and glucose metabolism.^2-4

LXRs inhibit atherosclerosis initiation and development through regulation of genes involved in both cholesterol elimination and inflammation pathways. LXRs directly induce the expression of ATP-binding cassette transporters ABCA1 and ABCG1^5,6 and apolipoprotein (apo) E, which mediate cellular cholesterol export in the presence of acceptors, such as high-density lipoprotein (HDL) and apoA-I. Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism and caused by mutations in ABCA1 gene. It is characterized by absence of HDL and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes.^7,8 disease is also a severe autosomal recessive lipidosis characterized by the accumulation of unesterified cholesterol in the endosomal/lysosomal system.⁹ It is caused by mutations in two genes: Niemann-Pick type C (NPC) 1, which accounts for 95% of NPC patients, and NPC2, which accounts for the remaining 5% of patients. NPC proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane.^10,11 On the other hand, LXR agonists negatively regulate macrophage inflammatory gene expression.¹² Recent data indicate that pathogens which contribute to the initiation and progression of atherosclerosis interfere with macrophage cholesterol metabolism by inhibition of the LXR signaling pathway.¹³

In recent years, many in vitro studies about ABCA1 have been conducted. Our observations have demonstrated that oxidized low-density lipoprotein (Ox-LDL) and apoA-I elevate ABCA1 mRNA and protein expression in THP-1 macrophage-derived foam cells and promote cholesterol efflux from these cells,^14,15 whereas oleate reduces cholesterol efflux and the level of ABCA1 protein in THP-1 macrophage-derived foam cells.¹⁶ We have also previously demonstrated that NO-1886 suppressing atherosclerosis in high-fat/high-sucrose/high-cholesterol-fed Bama minipigs is related to upregulating ABCA1.¹⁷ Furthermore, Rigamonti et al have shown that LXR activation controls intracellular cholesterol trafficking and esterification in human macrophages in vitro by inducing NPC1 gene expression, which indicates that LXR activation enhances cholesterol trafficking to the plasma membrane. Here, it becomes available for efflux at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.¹⁸ Therefore, we hypothesize that LXR agonist may also promote NPC1 expression in vivo, resulting in decreased cholesterol accumulation in foam cells and reduced atherosclerotic lesions in the artery wall. Herein we investigate the effect of LXR agonist T0901317 on ABCA1 and NPC1 in apoE−/− mice.

MATERIALS AND METHODS

Materials

T0901317 (Catalog No. 71810, CAS Registry No. 293754-55-9, purity: ≥98%, supplied as a crystalline solid) was obtained from Cayman Chemical (Ann Arbor, MI) and dissolved in vehicle (PEG400: Tween 80, 4:1). DyNAmoTM SYBR Green qPCR kit was obtained from Finnzymes Oy (Espoo, Finland). RevertAidTM First Strand cDNA Synthesis Kit (#k1622) was purchased from Fermentas (St. Leon-Roth, Germany).

Animals and Diets

Male 8-week-old apoE−/− mice on a C57BL/6 background (purchased from Laboratory Animal Center of Peking University, China) were randomized into four groups-baseline group (n = 10), vehicle group (n = 14), prevention group (n = 14) and treatment group (n = 14)-and housed 4 or 5 per cage at 25°C on a 12-hour light/dark cycle. All of the mice were fed a high-fat, high-cholesterol diet containing 15% fat and 0.25% cholesterol (obtained from Laboratory Animal Center of Peking University, China). The baseline group treated with vehicle was sacrificed after 8 weeks of the diet to evaluate whether the atherosclerotic lesions have formed. The vehicle group and the prevention group were treated with either vehicle (PEG400: Tween 80, 4:1) or LXR agonist (T0901317, 10 mg/kg body weight)¹⁹ daily by oral gavage (0.2 mL per mouse) for 14 weeks. The treatment group was treated with vehicle for 8 weeks, and then was treated with the agonist T0901317 for additional 6 weeks (as described above). Body weight was monitored at regular intervals. At week 14, the mice were sacrificed, blood was obtained, and tissues were collected for further analysis. All animal experiments were done in accordance with the Institutional Animal Ethics Committee and the University of South China Animal Care guidelines for use of experimental animals.

Analysis of Atherosclerotic Lesion

For en face analysis, aortas from different groups were opened longitudinally from the heart to the iliac arteries, and lesions were stained with Sudan IV. En face aortic lesion areas were digitized by a Nikon S6 digital camera, analyzed using Image-Pro Plus image analysis software (Media Cybernetics), and expressed as the percentage of the total aortic surface area covered by lesions.^19,20

Oil Red O Staining

The upper portion of the heart and proximal aorta were obtained, embedded in Optimal Cutting Temperature (OCT) compound (Fisher, Tustin, CA), and stored at −70°C. Serial 10-μm thick cryosections of aorta, beginning at the aortic root, were collected for a distance of 400 μm. Sections were stained with Oil red O and hematoxylin. The Oil red O-positive areas in digitized color images of stained aortic root sections (three equally spaced sections per mouse; n = 5 per group) were quantified using Image-Pro Plus image analysis software (Media Cybernetics), and the data are expressed as percent of total section area.

Immunohistochemistry

For detection of macrophages, aortic sections were incubated with mouse macrophage-specific antibody (MOMA-2, Accurate Chemical & Scientific Corp, San Diego, CA) dissolved in 1% bovine serum albumin/phosphate-buffered saline (PBS) at a final concentration of 1:200 for 2 hours at 25°C. After several washes with PBS, the sections were stained with biotinconjugated goat anti-rat immunoglobulin G (Boster Bioengineering Inc., Wuhan, China) for 1 hour. To prevent endogenous peroxidase reactions, the samples were pretreated with 0.3% H₂O₂ in cold methanol for 30 minutes. Finally, 0.1 mg/mL of 3,3′-diaminobenzidine tetrahydrochloride was applied to sections for 10 minutes. The sections were counterstained with hematoxylin. The macrophage-positive areas in digitized color images of stained aortic root sections (three equally spaced sections per mouse; n = 5 per group) were quantified using Image-Pro Plus image analysis software (Media Cybernetics) and the data are expressed as percent of total lesion area.

Lipid and Lipoprotein Analyses

Mice were fasted overnight and euthanized, and blood samples were obtained from the retro-orbital plexus. Triglyceride (TG), total cholesterol (TC), and HDL-C were determined by commercially enzymatic methods (test kits, Shanghai Rongsheng Biotech Inc. Shanghai, China). ApoA-I and apoB were measured by immunoturbidimetry (Shanghai Rongsheng Biotech, Shanghai, China).

Assay for Plasma Transaminase Activities

Blood was obtained from the retro-orbital plexus for analysis of serum alanine aminotransferase (ALT) as an index of hepatocellular injury. Measurements of serum ALT were made using a diagnostic kit (Wiener Laboratories, Rosario, Argentina).

RNA Isolation and Real-time Quantitative Polymerase Chain Reaction Analysis

Total RNA from aortas, livers and intestines of apoE−/− was extracted by using TRIzol reagent (BBI, Canada) in accordance with the manufacturer's instructions. Real-time quantitative polymerase chain reaction (PCR), using SYBR Green detection chemistry, was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System. The following primers were used for quantification of mouse ABCA1 and NPC1 mRNA levels: mouse ABCA1 mRNA, forward primer: 5′-GCC GTC TTT CCA GGA CAG TAT G-3′ and reverse primer 5′-CAG GGT GGC TCT TCT CAT CAA T-3′, and mouse NPC1 mRNA, forward primer: 5′-TGA ATG CGG TCT CCT TGG TC-3′ and reverse primer 5′-CTC ACT CGG CTT CCT TTG GTA-3′. Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. Quantitative measurements were determined using the ΔΔCt method and expression of β-actin was used as the internal control.

Western Blot Analysis

All murine tissues were harvested immediately after sacrifice and snap frozen in liquid nitrogen until use. Total aorta, liver, and small intestinal cells lysates were prepared using tissue lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% Sodium Dodecyl Sulfate (SDS) supplemented with protease inhibitors. The protein concentration in cellular supernatant was determined by the Bicinchoninic Acid (BCA) assay. Equal amounts of proteins (typically 50 μg) were separated on 6-10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose (NC) membrane (Millipore Corporation). The transferred NC membranes were incubated overnight with a mouse monoclonal antibody to mouse ABCA1 (ab18180, Abcam) or NPC1 (SC-18201, Santa Cruz) at a dilution of 1:1000 on a rotating platform at 4°C. Subsequently, membranes were rinsed in Tris-Buffered Saline Tween-20 (TBST) (pH 7.6) and incubated with horseradish peroxidase-conjugated antimouse or antigoat immunoglobulin G antibodies (Boster Bioengineering Inc., Wuhan, China) diluted in TBST (1:2000) for 2 hours on a rotating platform at 4°C. Bands were visualized using a HRP developer, and background-subtracted signals were quantified on a laser densitometer (Bio-Rad). Blots were probed with mouse anti-β-actin monoclonal antibody (Boster Bioengineering Inc., Wuhan, China) to ensure equal protein loading. All protein levels were assessed by densitometry with β-actin used as a control.

Assessment of Cholesterol Efflux

Peritoneal macrophages were isolated from the apoE−/− mice and seeded at a density of 400,000 cells per well in a 24-well plate, and maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing 10% Lipoprotein Deficient Serum (LPDS) and antibiotics (Pen/Strep/L-glutamine, Sigma). Macrophages were loaded with medium containing 1uCi/mL ³H-cholesterol for 4 hours. After being labeled with ³H-cholesterol, cells were washed and incubated for an additional 24 hours in serum-free media containing bovine serum albumin 2 g/L to allow for equilibration of ³H-cholesterol with intracellular cholesterol. Cholesterol efflux was initiated by adding the indicated amount of apoA-I (Fluka Biochemika), usually 10 mg/L, in serum-free medium. After 12 hours, media were harvested and cells were dissolved in HEPES 1 mmol/L, pH 7.5 containing 0.5% Triton X-100. Media were briefly centrifuged to remove nonadherent cells, and then aliquots of both supernatants and dissolved cells were subjected to FJ-2107P type liquid scintillator to determine radioactivity. Cholesterol efflux is expressed as a percentage, calculated as ³H-cholesterol l in medium/(³H-cholesterol in medium + ³H-cholesterol in cells) × 100%.

Statistical Analyses

Data are expressed as means ± SEM. Results were analyzed by Newman-Keuls Multiple Comparisons (for one-way analysis of variance) or Student unpaired t test, using Graph-Pad Prism (GraphPad Software Inc). Statistical significance was obtained when P values were less than 0.05.

RESULTS

T0901317 Inhibits Atherosclerosis Initiation and Progression in ApoE−/− Mice

To investigate whether T0901317 exerts preventive and reversal effect on atherosclerosis, we determined the atherosclerotic lesion area by en face analysis and Oil red O staining. We found that apoE−/− mice in the baseline group, which were fed a high-fat, high-cholesterol diet without T0901317, developed advanced lesions throughout the aortic arch and distal portions of thoracic and abdominal aortic regions, which continued to progress in the vehicle treated group during the 6-week treatment period (Fig. 1B). LXR agonist treatment resulted in a 64.2% and 58.3% reduction of aortic atherosclerotic lesion area in prevention group and treatment group, respectively, compared with vehicle-treated controls (P < 0.001, Fig. 1C), demonstrating a preventive and reversal effect of the LXR agonist on lesion development. Furthermore, LXR agonist in treatment group resulted in a significant 41.7% reduction in lesion areas in comparison to mice assessed at baseline group (P < 0.001, Fig. 1C), demonstrating that the LXR agonist induced regression of established atherosclerotic lesions. Quantification of lesions after Oil red O staining again revealed a statistically significant 43.7% and 36.1% reduction in aortic root atherosclerotic lesion area in prevention group and treatment group, respectively, compared with vehicle-treated controls (P < 0.001, Fig. 2).

FIGURE 1: T0901317 inhibited atherosclerosis initiation and development in apoE−/− mice fed a high-fat, high-cholesterol diet. A, Experimental timeline. B, Representative Sudan IV-stained aortas from experimental apoE−/− mice. En face aortic preparations show distribution of sudanophilic (red) atherosclerotic lesion areas in aortas. C, Quantification of atherosclerosis in apoE−/− mice (n = 5 mice per group). Aortic surface covered by Sudan IV-stained lesions was quantified and expressed as a percent of total aortic area. Data are mean ± SEM. *P < 0.005 versus baseline; **P < 0.001 versus vehicle and baseline.

FIGURE 2: Oil red O staining. A, Representative Oil red O-stained aortic root sections from experimental apoE−/− mice (three equally spaced sections per mouse; n = 5 mice per group). Original magnification: ×100. B, Quantification of atherosclerotic lesion area in aortic sections (n = 5 mice per group). The Oil red O-positive areas in digitized color images of stained aortic root sections were quantified and are expressed as percent of total aortic section area. Data are mean ± SEM. *P < 0.005 vs baseline; **P < 0.001 versus vehicle and baseline.

LXR Activation Attenuates Infiltration of Inflammatory Cells in ApoE−/− mice

At end of experiment, aortic walls in the apoE−/− mice were severely infiltrated by various inflammatory cells (Fig. 3A). The major cell type among those inflammatory cells was macrophages, as depicted by the positive staining with MOMA-2 antibody (Fig. 3A). Inflammation in the aortic walls of T0901317-treated apoE−/− mice was less severe compared with vehicle-treated apoE−/− mice (Fig. 3A), as reflected by reduced areas of MOMA-2-positive staining (P < 0.001, Fig. 3B).

FIGURE 3: Macrophage content detected by immunohistochemistry. A, Representative photomicrographs of sections of advanced atherosclerotic plaques (immunohistochemical staining for MOMA-2) from the aortic sinus of experimental apoE−/− mice (three equally spaced sections per mouse; n = 5 mice per group). Arrows indicate macrophage-positive areas within total aortic root lesions. Original magnification: ×100. B, Quantitative analysis of MOMA-2 staining in sections from apoE−/− mice (n = 5 mice per group). Data are mean ± SEM. *P < 0.005 versus baseline; **P < 0.001 versus vehicle.

Examination of Plasma Lipid Concentrations in ApoE−/− Mice

Because LXRs are involved in regulation of cholesterol and lipid metabolism, we examined the terminal plasma lipid levels from experimental mice. As shown in Table 1, treatment of apoE−/− mice fed a high-fat/high-cholesterol diet with T0901317 led to an 80% to 95% increase in plasma TG levels. A modest 28.9% to 34.7% increase in total cholesterol is accompanied by a 72.5% to 76.7% increase in HDL-C; however, no significant alternation occurred in LDL-C. Also, no difference occurred in body weight between groups at end of experiments.

TABLE 1: Terminal Plasma Lipid Levels in Apoe−/− Mice Fed a High-Fat, High-Cholesterol Diet With or Without T0901317

Effect of T0901317 on Hepatic Injury of ApoE−/− Mice

Because the previous study have shown that administration of the synthetic LXR ligands to mice triggers induction of the lipogenic pathway and elevates plasma and hepatic triglyceride levels,^21,22 we next assessed whether T0901317 had any effect on liver injury of apoE−/− mice. Serum levels of alanine aminotransferase (ALT) were measured; the data showed T0901317 administration contributed to significant increased serum levels of ALT compared with vehicle-treated apoE−/− mice (P < 0.005, Fig. 4).

FIGURE 4: Effect of T0901317 on hepatic injury of apoE−/− mice. Serum levels of ALT were measured by using standard spectrophotometric assays as described in Materials and Methods. Data are expressed as mean ± SEM from three independent experiments, each performed in triplicate. *P < 0.005 versus vehicle.

Effect of T0901317 on ABCA1 and NPC1 Gene Expression in the Aorta, Liver, and Small Intestine of ApoE−/− Mice

To explore the effect of T0901317 on ABCA1 and NPC1 gene expression, we collected the aorta, liver, and small intestine of apoE−/− mice to perform real-time quantitative PCR. Our observations suggested the gene expression of ABCA1 and NPC1 had been markedly upregulated, as shown in Figure 5. In the small intestine, strong induction of ABCA1 was observed in response to T0901317 (P < 0.001). However, the largest induction of NPC1 was found in the aorta (P < 0.001).

FIGURE 5: Regulation of ABCA1and NPC1 mRNA expression by T0901317 in the aorta, liver, and small intestine in apoE−/− mice fed a high-fat/high-cholesterol diet. Gene expression was measured by real-time quantitative PCR (Taqman) assays. Data are presented as mRNA expression relative to vehicle control. The results are expressed as mean ± SEM from three independent experiments, each performed in triplicate. *P < 0.005 versus vehicle; **P < 0.001 versus vehicle.

Effect of T0901317 on ABCA1 and NPC1 Protein Levels in the Aorta, Liver, and Small Intestine of ApoE−/− Mice

To examine the protein expression of ABCA1 and NPC1, we further performed Western blot analysis. ABCA1 and NPC1 were significantly increased in the aorta, liver, and small intestine of apoE−/− mice treated with T0901317, as shown in Figure 6. The levels of ABCA1 were markedly upregulated by T0901317 in small intestine, with increases of 2.54- and 2.05-fold (P < 0.001) in prevention group and treatment group, respectively. But, NPC1 protein levels were most significantly promoted in the aorta in response to T0901317, as shown by increases of 1.37 and 1.12 fold (P < 0.001) in the prevention and treatment groups, respectively. These results are consistent with the data from real-time quantitative PCR analysis. All protein levels were assessed by densitometry with β-actin as a control.

FIGURE 6: Western blot analysis of ABCA1 protein levels in the aorta, liver, and small intestine of the atherogenic apoE−/− mice treated with or without T0901317. The details of experiments are described in Materials and Methods. All protein levels were assessed by densitometry with β-actin as a control. A, ABCA1 and NPC1 proteins were analyzed from various tissues. B, Statistical graphs of ABCA1 and NPC11protein levels. Data are expressed as mean ± SEM from three independent experiments, each performed in triplicate. *P < 0.005 versus vehicle; **P < 0.001 versus vehicle.

Increased Macrophage Cholesterol Efflux to ApoA-I

To assess the effect of the increased ABCA1 expression, cholesterol efflux to apoA-I from peritoneal macrophages isolated from apoE−/− mice was measured (Fig. 7). Macrophages from T0901317-treated mice showed increased cholesterol efflux compared to the vehicle-treated mice (24.8 ± 1.2 and 21.6 ± 0.9 vs 14.3 ± 0.6%, P < 0.005).

FIGURE 7: T0901317 increased cholesterol efflux from peritoneal macrophages of apoE−/− mice in the presence of apoA-I 10 mg/L. Data are expressed as mean ± SEM from three independent experiments, each performed in triplicate. *P < 0.005 versus vehicle.

DISCUSSION

LXRα and LXRβ are nuclear hormone receptors whose native ligands are oxysterols, such as 22(R)-hydroxycholesterol. T0901317 is a potent and selective agonist for both LXRα and LXRβ, with an EC₅₀ of about 50 nM.⁴ In the present study, we observed for the first time the effect of a synthetic LXR agonist T0901317 on NPC1 in apoE−/− mice fed a high-fat, high-cholesterol diet. The most significant finding is that T0901317 induced both ABCA1 and NPC1 gene and protein expression in atherogenic apoE−/− mice. At the same time, our results demonstrated that the LXR agonist T0901317 not only inhibited the initiation of atherogenesis but induced regression of preexisting atherosclerotic lesions (Figs. 1 and 2). The following observations suggested that the ability of the LXR agonist to inhibit initiation of atherosclerosis and induce regression of established atherosclerotic lesions is linked to increased cholesterol efflux (Fig. 7) through induction of expression of the ABCA1 and NPC1 (Figs. 5 and 6).

The reduction in atherosclerosis observed in apoE−/− mice was accompanied by significant increase in plasma TG, TC, HDL-C, and apoA-I levels, but no significant change in LDL-C and apoB levels. Although previous work has shown that in apoE−/− mice, total cholesterol levels and HDL cholesterol were unchanged but triglycerides were mildly increased in response to LXR agonist GW3965,²³ we indeed found LXR agonist T0901317 markedly elevated TC and HDL-C and led to an 80% to 95% increase in plasma TG levels. We consider this difference was caused by the different agonist we used.

Consistent with previous studies,^19,24 treatment with T0901317 markedly promoted hepatic lipogenesis and increased plasma triglyceride levels (Table 1). LXRs seem to regulate both cholesterol and fatty acid metabolism in the liver. LXRs regulate genes participating in the control of cholesterol balance, including Cyp7α, the rate-limiting enzyme that promotes conversion of cholesterol to bile acids for excretion into bile, and ABCG5 and ABCG8, which regulate cholesterol trafficking.^24-27 Recently, one study has suggested that transgenic expression of Cyp7α in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia.²⁸ LXRs also regulate fatty acid synthesis in the liver through control of sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1.^21,29,30 Synthetic LXR agonists increase plasma triglyceride levels²¹ and reduce dietary cholesterol absorption. Thus, hepatic LXR activity could influence atherosclerosis by modifying processes that directly impact plasma lipid levels. However, we also found that LXR activation had effect on liver injury.

Previous work has confirmed that bile acids are avidly reabsorbed in the terminal ileum via the intestinal bile acid transporter. Similarly, biliary cholesterol can be reabsorbed from the intestinal lumen and between 50% and 80% of luminal cholesterol is reabsorbed.³¹ The mechanisms of cholesterol absorption are still being worked out, aided in part by the discovery of ezetimibe, a small molecule that specifically inhibits intestinal cholesterol absorption. Recent data have suggested that a key molecule in intestinal cholesterol absorption is NPC1-like 1^32,33 and this molecule is the direct target of ezetimibe. However, other molecules may also be involved in cholesterol absorption and the detailed molecular mechanisms have yet to be fully clarified. Once imported from the intestinal lumen into the enterocyte, cholesterol may be packaged into chylomicrons or, as noted earlier, effluxed via ABCA1 to lipid-free apoA-I. Although the role of ABCA1 in intestinal cholesterol absorption is controversial, one recent study has shown tissue-specific induction of intestinal ABCA1 expression with a LXR agonist raises plasma HDL-C levels.³⁴ We found that the intestinal ABCA1 mRNA and protein expression was greatly induced by T0901317 (Figs. 5 and 6). Also, the small intestinal NPC1 gene and protein expression was significantly upregulated by the synthetic LXR agonist T0901317 (Figs. 5 and 6). The net effect of LXR activation on intestine may cause the reduced cholesterol absorption and then decrease plasma cholesterol levels.

Considerable evidence has emerged to indicate that, in addition to inducing genes involved in reverse cholesterol transport, LXRs reciprocally negatively regulate an array of inflammatory genes after bacterial, lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), or interleukin (IL)-1β stimulation.¹² Inhibition of inflammatory signaling by LXR activation is not limited to isolated macrophages³⁵ but also manifests itself in vivo. Experiments in several different models have confirmed the anti-inflammatory effects of LXRs. When challenged intraperitoneally with LPS, LXRαβ-/- mice exhibit an exacerbated systemic inflammatory response and increased hepatic expression of inducible nitric oxide synthase (iNOS), TNF-α, and IL-1β.¹² Synthetic LXR agonists also reduce inflammation in a model of irritant contact dermatitis. A similar result was reported by Fowler et al, who found that LXR ligands showed activity comparable to that of a steroid-based drug in an oxazolone-induced allergic dermatitis model.³⁶ Furthermore, administration of LXR ligands to mice inhibits tissue factor expression in the kidney and lung after an LPS challenge.³⁷ However, Dai et al have indicated that T0901317 has paradoxical roles in hepatic gene expression of proinflammatory cytokines in apoE−/− mice.³⁸ Our results indicated that inflammation in the aortic walls of T0901317-treated apoE−/− mice was less severe compared with vehicle-treated apoE−/− mice. This may explain, at least in part, why despite the above less-favorable lipid profile (elevated plasma triglyceride concentrations), apoE−/− mice also showed a substantial reduction in atherosclerosis in response to T0901317.

In conclusion, the synthetic LXR agonist T0901317 increases the expression of ABCA1 and NPC1 in apoE−/− mice, resulting in decreased cholesterol accumulation in foam cells and reduced atherosclerotic lesions in the artery wall. This study gives us a new insight into the mechanism for antiatherogenic effects of LXR synthetic agonists.