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Deficiency of PXR decreases atherosclerosis in apoE-deficient mice

2011; Elsevier BV; Volume: 52; Issue: 9 Linguagem: Inglês

10.1194/jlr.m017376

ISSN

1539-7262

Autores

Yipeng Sui, Jinxian Xu, Jennifer Rios-Pilier, Changcheng Zhou,

Tópico(s)

Retinoids in leukemia and cellular processes

Resumo

The pregnane X receptor (PXR, also known as SXR) is a nuclear hormone receptor activated by xenobiotics as well as diverse sterols and their metabolites. PXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Recent evidence indicates that PXR may also play an important role in lipid homeostasis and atherosclerosis. To define the role of PXR in atherosclerosis, we generated PXR and apoE double knockout (PXR−/−apoE−/−) mice. Here we show that deficiency of PXR did not alter plasma triglyceride and cholesterol levels in apoE−/− mice. However, PXR−/−apoE−/− mice had significantly decreased atherosclerotic cross-sectional lesion area in both the aortic root and brachiocephalic artery by 40% (P < 0.01) and 60% (P < 0.001), respectively. Interestingly, deficiency of PXR reduced the expression levels of CD36, lipid accumulation, and CD36-mediated oxidized LDL uptake in peritoneal macrophages of PXR−/−apoE−/− mice. Furthermore, immunofluorescence staining showed that PXR and CD36 were expressed in the atherosclerotic lesions of apoE−/− mice, and the expression levels of PXR and CD36 were diminished in the lesions of PXR−/−apoE−/− mice. Our findings indicate that deficiency of PXR attenuates atherosclerosis development, which may result from decreased CD36 expression and reduced lipid uptake in macrophages. The pregnane X receptor (PXR, also known as SXR) is a nuclear hormone receptor activated by xenobiotics as well as diverse sterols and their metabolites. PXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Recent evidence indicates that PXR may also play an important role in lipid homeostasis and atherosclerosis. To define the role of PXR in atherosclerosis, we generated PXR and apoE double knockout (PXR−/−apoE−/−) mice. Here we show that deficiency of PXR did not alter plasma triglyceride and cholesterol levels in apoE−/− mice. However, PXR−/−apoE−/− mice had significantly decreased atherosclerotic cross-sectional lesion area in both the aortic root and brachiocephalic artery by 40% (P < 0.01) and 60% (P < 0.001), respectively. Interestingly, deficiency of PXR reduced the expression levels of CD36, lipid accumulation, and CD36-mediated oxidized LDL uptake in peritoneal macrophages of PXR−/−apoE−/− mice. Furthermore, immunofluorescence staining showed that PXR and CD36 were expressed in the atherosclerotic lesions of apoE−/− mice, and the expression levels of PXR and CD36 were diminished in the lesions of PXR−/−apoE−/− mice. Our findings indicate that deficiency of PXR attenuates atherosclerosis development, which may result from decreased CD36 expression and reduced lipid uptake in macrophages. Despite enormous research efforts and advances in treatments over the past few decades, atherosclerotic cardiovascular disease remains the leading cause of death in the developed world (1Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4667) Google Scholar, 2Sanz J. Fayad Z.A. Imaging of atherosclerotic cardiovascular disease.Nature. 2008; 451: 953-957Crossref PubMed Scopus (420) Google Scholar). Nuclear receptors have become attractive targets for the development of therapeutic agents for treatment of atherosclerosis (3Barish G.D. Evans R.M. PPARs and LXRs: atherosclerosis goes nuclear.Trends Endocrinol. Metab. 2004; 15: 158-165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Glass C.K. Going nuclear in metabolic and cardiovascular disease.J. Clin. Invest. 2006; 116: 556-560Crossref PubMed Scopus (82) Google Scholar). A number of nuclear receptors, such as liver X receptor (LXR) and peroxisome proliferator-activated receptors (PPARs), are key regulators of lipid homeostasis and inflammation and play important roles in atherosclerosis development (3Barish G.D. Evans R.M. PPARs and LXRs: atherosclerosis goes nuclear.Trends Endocrinol. Metab. 2004; 15: 158-165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Glass C.K. Going nuclear in metabolic and cardiovascular disease.J. Clin. Invest. 2006; 116: 556-560Crossref PubMed Scopus (82) Google Scholar, 5Hageman J. Herrema H. Groen A.K. Kuipers F. A role of the bile salt receptor FXR in atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1519-1528Crossref PubMed Scopus (80) Google Scholar, 6Huang W. Glass C.K. Nuclear receptors and inflammation control: molecular mechanisms and pathophysiological relevance.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1542-1549Crossref PubMed Scopus (103) Google Scholar). The pregnane X receptor (PXR; also known as steroid and xenobiotic receptor, or SXR) is a nuclear receptor activated by a diverse array of endogenous hormones, dietary steroids, pharmaceutical agents, and xenobiotic compounds (7Blumberg B. Sabbagh Jr, W. Juguilon H. Bolado Jr, J. van Meter C.M. Ong E.S. Evans R.M. SXR, a novel steroid and xenobiotic-sensing nuclear receptor.Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (814) Google Scholar, 8Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. et al.An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway.Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 9Zhou C. Verma S. Blumberg B. The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism.Nucl. Recept. Signal. 2009; 7: e001Crossref PubMed Scopus (136) Google Scholar). PXR functions as a xenobiotic sensor that regulates clearance via induction of genes involved in drug and xenobiotic metabolism, including oxidation (e.g., cytochrome P450), conjugation (e.g., glutathione transferase), and transport (e.g., multidrug resistance 1) (9Zhou C. Verma S. Blumberg B. The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism.Nucl. Recept. Signal. 2009; 7: e001Crossref PubMed Scopus (136) Google Scholar, 10Kliewer S.A. Goodwin B. Willson T.M. The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism.Endocr. Rev. 2002; 23: 687-702Crossref PubMed Scopus (724) Google Scholar). The broad response profile of PXR has led to the development of "the steroid and xenobiotic sensor hypothesis." In the past decade, PXR's function in drug and xenobiotic metabolism has been extensively studied by many laboratories (9Zhou C. Verma S. Blumberg B. The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism.Nucl. Recept. Signal. 2009; 7: e001Crossref PubMed Scopus (136) Google Scholar). Recent evidence indicates that PXR may also play an important role in lipid homeostasis and atherosclerosis. Many clinically relevant PXR ligands (e.g., rifampicin, ritonavir) have been shown to affect lipid levels in patients and may have pro-atherogenic effects (11Khogali A.M. Chazan B.I. Metcalf V.J. Ramsay J.H. Hyperlipidaemia as a complication of rifampicin treatment.Tubercle. 1974; 55: 231-233Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 12Carr A. Samaras K. Burton S. Law M. Freund J. Chisholm D.J. Cooper D.A. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors.A IDS. 1998; 12: F51-F58Google Scholar, 13Shafran S.D. Mashinter L.D. Roberts S.E. The effect of low-dose ritonavir monotherapy on fasting serum lipid concentrations.HIV Med. 2005; 6: 421-425Crossref PubMed Scopus (128) Google Scholar, 14Barbaro G. Metabolic and cardiovascular complications of highly active antiretroviral therapy for HIV infection.Curr. HIV Res. 2006; 4: 79-85Crossref PubMed Scopus (71) Google Scholar, 15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In addition to playing an important role in xenobiotic metabolism, PXR can mediate sterol-regulatory element binding protein (SREBP)-independent lipogenic pathways by activating the FFA uptake transporter CD36 and several accessory lipogenic enzymes, such as stearoyl CoA desaturase-1 and long-chain FFA elongase (16Zhou J. Zhai Y. Mu Y. Gong H. Uppal H. Toma D. Ren S. Evans R.M. Xie W. A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway.J. Biol. Chem. 2006; 281: 15013-15020Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 17Zhou J. Febbraio M. Wada T. Zhai Y. Kuruba R. He J. Lee J.H. Khadem S. Ren S. Li S. et al.Hepatic fatty acid transporter Cd36 is a common target of LXR, PXR, and PPARgamma in promoting steatosis.Gastroenterology. 2008; 134: 556-567Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). We also found that chronic activation of PXR increases CD36 expression and lipid accumulation in peritoneal macrophages of wild-type (WT) and apoE knockout (apoE−/−) mice (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Moreover, acute PXR activation can regulate SREBP-dependent lipogenic pathways by inducing Insig-1 expression, resulting in decreased levels of active SREBP-1 and reduced triglyceride synthesis (18Roth A. Looser R. Kaufmann M. Blaettler S. Rencurel F. Huang W. Moore D.D. Meyer U.A. Regulatory cross-talk between drug metabolism and lipid homeostasis: constitutive androstane receptor and pregnane X receptor increase Insig-1 expression.Mol Pharmacol. 2008; 73: 1282-1289Crossref PubMed Scopus (119) Google Scholar). We and other groups have shown that activation of PXR also affects the expression of other hepatic genes involved in lipid homeostasis, including apoA-IV, oxysterol 7α-hydroxylase (CYP39A1), and 7-dehydrocholesterol reductase (DHCR7) in several animal models including WT, apoE−/−, or LDL receptor knockout (LDLR−/−) mice (9Zhou C. Verma S. Blumberg B. The steroid and xenobiotic receptor (SXR), beyond xenobiotic metabolism.Nucl. Recept. Signal. 2009; 7: e001Crossref PubMed Scopus (136) Google Scholar, 19de Haan W. de Vries-van der Weij J. Mol I.M. Hoekstra M. Romijn J.A. Jukema J.W. Havekes L.M. Princen H.M. Rensen P.C. PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice.Biochim. Biophys. Acta. 2009; 1791: 191-197Crossref PubMed Scopus (31) Google Scholar, 20Hoekstra M. Lammers B. Out R. Li Z. Van Eck M. Van Berkel T.J. Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice.Mol. Pharm. 2009; 6: 182-189Crossref PubMed Scopus (32) Google Scholar). These studies indicate that PXR can mediate cholesterol and lipid homeostasis at multiple levels. Modulation of PXR activity has been found to affect plasma lipid levels in several animal models. Activation of PXR by pregnenolone 16α-carbonitrile (PCN) treatment can increase plasma total cholesterol and VLDL levels in apoE*3-Leiden (E3L) mice and decrease HDL levels in E3L mice with human cholesteryl ester transfer protein (E3L.CETP)-transgenic mice, a well-established model for human-like lipoprotein metabolism (19de Haan W. de Vries-van der Weij J. Mol I.M. Hoekstra M. Romijn J.A. Jukema J.W. Havekes L.M. Princen H.M. Rensen P.C. PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice.Biochim. Biophys. Acta. 2009; 1791: 191-197Crossref PubMed Scopus (31) Google Scholar). We found that chronic activation of PXR by feeding apoE−/− mice the PXR agonist PCN can decrease HDL levels without affecting total cholesterol levels (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Another report showed that short-term activation of PXR increases plasma triglyceride levels but decreases LDL cholesterol levels in LDLR−/− mice (20Hoekstra M. Lammers B. Out R. Li Z. Van Eck M. Van Berkel T.J. Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice.Mol. Pharm. 2009; 6: 182-189Crossref PubMed Scopus (32) Google Scholar). Similar treatment also caused increased plasma triglyceride levels in apoE−/− mice (20Hoekstra M. Lammers B. Out R. Li Z. Van Eck M. Van Berkel T.J. Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice.Mol. Pharm. 2009; 6: 182-189Crossref PubMed Scopus (32) Google Scholar). Interestingly, a recent meta-analysis of seven genome-wide association studies indicates that common genetic variants in PXR can strongly affect plasma lipid levels in humans, and 19 PXR single nucleotide polymorphisms were determined to significantly affect plasma LDL cholesterol levels (21Lu Y. Feskens E.J. Boer J.M. Muller M. The potential influence of genetic variants in genes along bile acid and bile metabolic pathway on blood cholesterol levels in the population.Atherosclerosis. 2010; 210: 14-27Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). All of the evidence suggests that modulation of PXR activity may regulate lipid homeostasis and affect atherosclerosis development. The identification of PXR as a xenobiotic sensor has provided an important tool for studying new mechanisms through which diet, drugs, and chemical exposures might impact human health. Several nuclear receptors (LXR, PPAR) that regulate lipid metabolism and modulate inflammation have been extensively studied for their roles in atherosclerosis (3Barish G.D. Evans R.M. PPARs and LXRs: atherosclerosis goes nuclear.Trends Endocrinol. Metab. 2004; 15: 158-165Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Glass C.K. Going nuclear in metabolic and cardiovascular disease.J. Clin. Invest. 2006; 116: 556-560Crossref PubMed Scopus (82) Google Scholar). However, the role of PXR in atherosclerosis remains to be determined. In our previous study, we found that chronic activation of PXR by feeding apoE−/− mice the PXR agonist PCN significantly increases atherosclerosis (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To further define the role of PXR in atherosclerosis, we generated PXR and apoE double knockout (PXR−/−apoE−/−) mice. Here we show that PXR−/−apoE−/− mice had significantly decreased atherosclerotic cross-sectional lesion area in both the aortic root and brachiocephalic artery, without altering plasma lipid levels. Fresh isolated peritoneal macrophages from PXR−/−apoE−/− mice showed reduced expression of CD36 mRNA and lipid accumulation compared with apoE−/− mice. Deficiency of PXR also decreased CD36-mediated oxidized LDL (oxLDL) uptake in peritoneal macrophages. Furthermore, immunofluorescence staining showed that PXR was present in the atherosclerotic lesions of apoE−/− mice, and the expression levels of CD36 were diminished in the lesions of PXR−/−apoE−/− mice. Our findings indicate that deficiency of PXR attenuates atherosclerosis development, which may result from decreased expression of CD36 and reduced lipid uptake in macrophages. ApoE−/− mice on the C57BL/6 background (stock no. 002052) were purchased from The Jackson Laboratory. PXR−/− mice were originally generated in the laboratory of Dr. Ronald Evans at Salk Institute (22Xie W. Barwick J.L. Downes M. Blumberg B. Simon C.M. Nelson M.C. Neuschwander-Tetri B.A. Brunt E.M. Guzelian P.S. Evans R.M. Humanized xenobiotic response in mice expressing nuclear receptor SXR.Nature. 2000; 406: 435-439Crossref PubMed Scopus (602) Google Scholar) and were backcrossed with C57BL/6 for several generations before we obtained them. These animals were backcrossed five additional generations onto the C57BL/6 background (>99% C57BL/6) using the marker-assisted Microsatellite Genotyping technique (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). PXR−/−apoE−/− mice were generated by crossing PXR−/− with apoE−/− mice and maintained on standard chow diet. All animals were housed by the Division of Laboratory Animal Resources, University of Kentucky, in a specific pathogen-free environment with a 12 h light-dark cycle, and approved by the Institutional Animal Care and Use Committee. Four week-old experimental male apoE−/− or PXR−/−apoE−/− littermates were weaned and fed a modified semisynthetic low-fat AIN76 diet containing 0.02% cholesterol (Research Diet; New Brunswick, NJ) (23Teupser D. Persky A.D. Breslow J.L. Induction of atherosclerosis by low-fat, semisynthetic diets in LDL receptor-deficient C57BL/6J and FVB/NJ mice: comparison of lesions of the aortic root, brachiocephalic artery, and whole aorta (en face measurement).Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1907-1913Crossref PubMed Scopus (132) Google Scholar, 24Wolfrum S. Teupser D. Tan M. Chen K.Y. Breslow J.L. The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes.Proc. Natl. Acad. Sci. U SA. 2007; 104: 18601-18606Crossref PubMed Scopus (78) Google Scholar) for 12 weeks until euthanasia at 16 weeks of age. On the day of euthanasia, mice were fasted for 6 h following the dark (feeding) cycle. Immediately prior to euthanasia,the fasting plasma glucose was measured, and mice were then anesthetized by intraperitoneal injection with sodium pentobarbital (Henry Schein; Melville, NY). Mice were exsanguinated by left ventricular puncture, and blood was collected into EDTA-containing syringes. Plasma was prepared by spinning at 16,000 g for 10 min. The circulation was flushed with PBS, and the heart was removed and stored frozen in Tissue-Tek OCT compound as described previously (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Liver and other tissues were collected and stored in RNAlater solution (Life Technologies; Carlsbad, CA). Plasma total cholesterol and triglyceride concentrations were determined enzymatically by colorimetric methods as described previously (Roche; Indianapolis, IN) (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Plasma from multiple mice (n = 6) was pooled, and plasma lipoprotein cholesterol distributions were determined by fast-performance liquid chromatography (FPLC). Lipoprotein fractions were also isolated by centrifuging at 70,000 rpm for 3 h in a Beckman Optima TL-100 tabletop ultracentrifuge at its own density (1.006 g/ml). Then the infranatant was adjusted to a density of 1.063 g/ml with solid KBr in order to harvest the HDL fraction by spinning at 70,000 rpm for 18 h. The cholesterol content of HDL infranatant was measured enzymatically. To quantify the lesion areas at the aortic root, OCT compound-embedded hearts were sectioned at a 12 µm thickness, keeping all three valves of the aortic root in the same plane, and stained with Oil Red O as described previously (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Teupser D. Persky A.D. Breslow J.L. Induction of atherosclerosis by low-fat, semisynthetic diets in LDL receptor-deficient C57BL/6J and FVB/NJ mice: comparison of lesions of the aortic root, brachiocephalic artery, and whole aorta (en face measurement).Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1907-1913Crossref PubMed Scopus (132) Google Scholar). To quantify atherosclerosis at the brachiocephalic artery, the OCT-embedded brachiocephalic artery was sectioned distal to proximal at a thickness of 10 µm. Atherosclerotic lesions lumenal to the internal elastic lamina were quantified in three equidistant Oil Red O-stained sections 200, 400, and 600 µm proximal from the branching point of the brachiocephalic artery into the carotid and subclavian arteries (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Total RNA was isolated from mouse tissues using TRIzol Reagent (Life Technologies) as per the manufacturer-supplied protocol. Quantitative real-time PCR (QPCR) was performed using gene-specific primers and the SYBR green PCR kit (Life Technologies) in an ABI 7900 system (Life Technologies) as described previously (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). All samples were quantified using the comparative Ct method for relative quantification of gene expression, and normalized to GAPDH (25Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123298) Google Scholar). The primer sets used in this study are shown in supplementary Table I. Mice were injected intraperitoneally with 1 ml of 3% thioglycollate, and peritoneal macrophages were collected 4 days later as described previously (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). After centrifugation at 500 g for 5 min, the cells were resuspended in DMEM. Three to five million peritoneal macrophages from each group were allowed to attach to either coverslips or one 10 cm culture dish for 4 h. Then the culture dishes and coverslips were washed three times with PBS to remove nonadherent cells. RNA was isolated from the macrophages on culture dishes, and Oil Red O/hematoxylin staining was performed on the macrophages adhering to one coverslip. Cells containing lipid droplets (>10) were counted as foam cells and at least 10 fields per condition were counted (26Rahaman S.O. Lennon D.J. Febbraio M. Podrez E.A. Hazen S.L. Silverstein R.L. A CD36-dependent signaling cascade is necessary for macrophage foam cell formation.Cell Metab. 2006; 4: 211-221Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 27Wang Z. Klipfell E. Bennett B.J. Koeth R. Levison B.S. Dugar B. Feldstein A.E. Britt E.B. Fu X. Chung Y.M. et al.Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.Nature. 2011; 472: 57-63Crossref PubMed Scopus (3459) Google Scholar). The macrophages on the other coverslip were incubated with DMEM containing 100 µg/ml of oxLDL (Biomedical Technologies, Inc.; Stoughton, MA) for 24 h, followed by washing with PBS and staining with Oil Red O/hematoxylin. Macrophages were also stained with FITC-conjugated monocyte/macrophage antibody MOMA-2 (AbD Serotec; Raleigh, NC) or CD36 antibody (AbD Serotec), followed by Alexa 488-conjugated secondary antibody (Life Technologies). Immunohistochemistry was performed on 12 μm sections of aortic roots freshly embedded in OCT. Sections were first fixed in 100% ice-cold acetone for 15 min and then washed with PBS for 20 min. Sections were permeabilized with PBS + 0.1% Triton X-100 (PBST) for 10 min. Nonspecific binding was reduced by incubating slides in 10% rabbit sera diluted in PBST for 20 min at room temperature. Sections were then incubated with rat anti-mouse monocyte/macrophage marker MOMA-2 antibody conjugated with FITC (1:100; AbD Serotec), rabbit anti-mouse PXR antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or rat anti-mouse CD36 antibody (1:100; AbD Serotec) at 4°C for 12–15 h. Sections were rinsed with PBS and incubated with Alexa 594-labeled goat anti-rabbit (for PXR) or Alexa 488-labeled donkey anti-rat (for CD36) secondary antibodies (1:500; Life Technologies). The nuclei were stained by mounting the slides with 4',6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories; Burlingame, CA). Images were acquired with a Nikon fluorescence microscope (Nikon; Melville, NY). All data are expressed as mean ± SD unless indicated otherwise. Statistically significant differences between two groups were analyzed by t-test for data normally distributed and by the Mann-Whitney test for data not normally distributed, using Prism software, version 5.0. A P value of less than 0.05 was considered to be significant. To determine the role of PXR in the development of atherosclerosis, we generated PXR−/−apoE−/− double knockout mice by crossing PXR−/− mice with apoE−/− mice. PXR−/−apoE−/− mice are variable and appear indistinguishable from apoE−/− mice. To investigate whether deficiency of PXR affects atherosclerosis, male apoE−/− and PXR−/−apoE−/− mice were fed a low-fat semi-synthetic AIN76a diet containing 0.02% cholesterol for 12 weeks. The low-fat and low-cholesterol AIN76a diet has been successfully used to induce atherosclerosis development in apoE−/− or LDLR−/− mice without increasing obesity and insulin resistance (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 23Teupser D. Persky A.D. Breslow J.L. Induction of atherosclerosis by low-fat, semisynthetic diets in LDL receptor-deficient C57BL/6J and FVB/NJ mice: comparison of lesions of the aortic root, brachiocephalic artery, and whole aorta (en face measurement).Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1907-1913Crossref PubMed Scopus (132) Google Scholar, 24Wolfrum S. Teupser D. Tan M. Chen K.Y. Breslow J.L. The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes.Proc. Natl. Acad. Sci. U SA. 2007; 104: 18601-18606Crossref PubMed Scopus (78) Google Scholar). At 16 weeks of age, apoE−/− and PXR−/−apoE−/− mice had similar body weights and fasting blood glucose levels (see supplementary Fig. I). The effect of deficiency of PXR on plasma lipid and lipoprotein levels was next examined. As shown in Fig. 1, deficiency of PXR did not affect plasma triglyceride and total and HDL cholesterol levels in PXR−/−apoE−/− mice. In addition, FPLC analysis showed that apoE−/− and PXR−/−apoE−/− mice had similar plasma cholesterol distribution patterns (Fig. 1D). The apoE−/− and PXR−/−apoE−/− mice were euthanized at 16 weeks of age, and atherosclerotic lesion areas were determined in the aortic root and brachiocephalic artery as shown in Fig. 2. ApoE−/− mice have a mean atherosclerotic lesion area of 202,405 ± 49,489 μM2 in the aortic root and 44,945 ± 18,344 μM2 in the brachiocephalic artery. Compared with apoE−/− mice, PXR−/−apoE−/− mice had significantly decreased cross-sectional lesion areas of 40% at the aortic root (121,713 ± 69,511 μM2; P < 0.01) and 60% at the brachiocephalic artery (17,899 ± 11,918 μM2; P < 0.001) (Fig. 2A, B, respectively). Thus, deficiency of PXR decreases atherosclerosis in apoE−/− mice. To elucidate possible molecular mechanisms through which PXR deficiency decreases atherosclerosis, a panel of hepatic atherosclerosis modifier genes were measured by QPCR. As shown in supplementary Fig. II, the expression levels of the selected genes, including SREBP1a, SREBP1c, SREBP2, HMGCR, LDLR, and SR-BI, did not change in the liver of PXR−/−apoE−/− mice. In addition to liver, PXR is also expressed in immune cells such as T cells, B cells, monocytes, and peritoneal macrophages (15Zhou C. King N. Chen K.Y. Breslow J.L. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.J. Lipid Res. 2009; 50: 2004-2013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 28Dubrac S. Elentner A. Ebner S. Horejs-Hoeck J. Schmuth M. Modulation of T lymphocyte function by the pregnane X receptor.J. Immunol. 2010; 184: 2949-2957Crossref PubMed Scopus (49) Google Scholar, 29Albermann N. Schmitz-Winnenthal F.H. Z'Graggen K. Volk C. Hoffmann M.M. Haefeli W.E. Weiss J. Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver.Biochem. Pharmacol. 2005; 70: 949-958Crossref PubMed Scopus (236) Google Scholar, 30Owen A. Chandler B. Back D.J. Khoo S.H. Expression of pregnane-X-receptor transcript in peripheral blood mononuclear cells and correlation with MDR1 mRNA.Antivir. Ther. 2004; 9: 819-821PubMed Google Scholar, 31Siest G. Jeannesson E. Mart

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