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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2606.)
© 2007 American Heart Association, Inc.

Vascular Biology

Farnesoid X Receptor Ligands Inhibit Vascular Smooth Muscle Cell Inflammation and Migration

Yoyo T.Y. Li; Karen E. Swales; Gareth J. Thomas; Timothy D. Warner; David Bishop-Bailey

From the Centre of Translational Medicine & Therapeutics (Y.T.Y.L., K.E.S., T.D.W., D.B.B.), William Harvey Research Institute, and the Tumour Biology Laboratory (G.J.T.), Cancer Research UK Clinical Centre, Barts & The London, Queen Mary University of London, UK.

Correspondence to Dr. David Bishop-Bailey, Centre of Translational Medicine & Therapeutics, William Harvey Research Institute, Barts & The London, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, United Kingdom, E-mail d.bishop-bailey{at}qmul.ac.uk

	Abstract

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Objective— The farnesoid X receptor/bile acid receptor (FXR; NR1H4) is a ligand-activated transcription factor that regulates bile acid and lipid homeostasis, and is highly expressed in enterohepatic tissue. FXR is also expressed in vascular tissue. We have investigated whether FXR regulates inflammation and migration in vascular smooth muscle cells.

Methods and Results— The FXR target gene, small heterodimer partner (SHP), was induced in vascular smooth muscle cells after treatment with synthetic FXR ligands, GW4064, or 6-ethyl-chenodeoxycholic acid. FXR ligands induced smooth muscle cell death and downregulated interleukin (IL)-1β–induced inducible nitric oxide synthase and cyclooxygenase-2 expression. In addition, FXR ligands suppressed smooth muscle cell migration stimulated by platelet-derived growth factor-BB. Reporter gene assays showed that FXR ligands activated an FXR reporter gene and suppressed IL-1β–induced nuclear factor (NF)-B activation and iNOS in a manner that required functional FXR and SHP.

Conclusion— Our observations suggest that a FXR-SHP pathway may be a novel therapeutic target for vascular inflammation, remodeling, and atherosclerotic plaque stability.

FXR is expressed in vascular smooth muscle cells. Here we show that in addition to antiproliferative properties, activation of FXR inhibits inflammation and migration of vascular smooth muscle cells. FXR may therefore be a novel direct target for vascular disease.

Key Words: FXR • vascular smooth muscle • iNOS • COX-2 • cell migration

	Introduction

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Atherosclerosis is considered as a progressive inflammatory disease characterized by the accumulation of lipids and fibrous elements in the large arteries. This view is supported by the massive expression of activated inflammatory cells such as macrophages and T lymphocytes in atherosclerotic plaques.¹ The levels of synthetic enzymes for inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are also upregulated in both infiltrating inflammatory cells and local vascular smooth muscle cells (VSMCs), as well as endothelial cells inside the atherosclerotic lesions.^2,3 The levels of these and other proinflammatory mediators and enzymes are controlled by the activation of proinflammatory transcription factor signaling pathways, such as nuclear factor (NF)B and activator protein (AP)-1.^4,5 In addition to inflammation, inappropriate vascular remodeling has been reported to underlie the pathogenesis of atherosclerosis.⁶ VSMC proliferation and migration as well as extracellular matrix remodeling are important in atherosclerosis.^7,8 These events are mediated by various cytokines and growth factors, and also depend on the degradation of extracellular matrix by proteinases such as matrix metalloproteinases.

The farnesoid X receptor/bile acid receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that binds and acts as a heterodimer with retinoid X receptors, and is highly expressed in liver, kidney, adrenals, and intestine.⁹ FXR expression was recently found in vascular tissue especially in the VSMCs, where its activation led to apoptosis.¹⁰ FXR can be activated by high levels of farnesol, but is now recognized as a bile acid receptor with ligands including chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA).¹¹ Synthetic FXR ligands have also been identified, such as GW4064 and 6-ethyl-chenodeoxycholic acid (6ECDCA).^12,13 FXR activation leads to induction of an orphan nuclear receptor, small heterodimer partner (SHP), that mediates some of the inhibitory effects of FXR ligands on bile acid and lipid metabolism.^14,15

Here we report that FXR activation leads to a downregulation of the proinflammatory enzymes iNOS and COX-2, as well as cell migration in VSMCs. FXR may therefore be a novel therapeutic target through which to reduce vascular inflammation, remodeling, and atherosclerotic plaque stability.

	Methods

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Materials and Reagents
Polyclonal antibodies to iNOS (INOS22-S) and FXR (sc-13063) were purchased from Autogen Bioclear. Anti–COX-2 polyclonal antibody (Cay-160106) and 1400W were purchased from Axxora. IL-1β was from R&D Systems. pNF-B-Luc was from BD Biosciences Clontech. FXR-responsive IR-1 reporter gene, pcDNA-rFXR, and pcDNA dominant-negative-(DN)-rFXR were generous gifts from Dr Tom Kocarek (Wayne State University). All RT-PCR reagents were obtained from Promega. NovaFECTOR was from VennNova. Platelet-derived growth factor (PDGF)-BB was from Calbiochem. Rat specific siRNA to SHP and control siRNA were from Ambion Applied Biosystems. 6ECDCA and GW4064 were kindly donated by Dr Roberto Pellicciari (U. Perugia) and Dr Eric Niesor (ILEX Corp), respectively. All other reagents were from Sigma.

Cell Culture
Rat aortic smooth muscle cells (RASMCs; WKY3m-22), HepG2, and HEK293 cells were grown and maintained as previously described.¹⁶ Human aortic vascular smooth muscle cells were cultured according to the supplier’s instructions (Promocell).

iNOS Activity and Measurement of RASMC Death
FXR ligands, 6ECDCA (0.1 to 30 µmol/L), GW4064 (0.1 to 30 µmol/L), or vehicle control(s) were added 1 hour prior to incubation with IL-1β (0.01 to 10 ng/mL, 24 hour). In some experiments, the selective iNOS inhibitor, 1400W (10 µmol/L) was included 1 hour before IL-1β addition, or cells were preincubated with control or SHP specific siRNA using NovaFECTOR 24 hours before drug treatment. The cellular supernatants were then removed from each well for nitrite analysis.¹⁷ Total accumulated nitrite was determined spectrophotometrically (OD₅₅₀) using the Griess assay.¹⁸ Cell viability was measured by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 0.2 mg/mL) assay,¹⁹ as previously described.²⁰

Semi-Quantitative Reverse Transcription–Polymerase Chain Reaction
RT-PCR was performed as using standard techniques. Primers for rat FXR and SHP¹⁰ were as previously described. Rat β-actin (452 bp) was chosen as a housekeeping gene internal control.²¹ In parallel reactions where reverse transcriptase or cDNA was omitted, no bands were visible (data not shown). Quantification of the relative intensity of each band was determined and analyzed using ImageJ (http://rsb.info.nih.gov/ij/).

Western Blot Analysis
Protein was extracted from RASMCs and Western Blot analysis was essentially as previously described²² using rabbit anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-FXR (1:500) polyclonal antibodies, or a mouse monoclonal anti–β-actin (1:1000) antibody as a loading control. HepG2 protein was used as a positive control for FXR protein expression.

Transient Transfection and Reporter Gene Assays
Transfection and luciferase reporter gene assays were performed as previously described.¹⁶ In HEK293 cells the IR-1 luciferase reporter gene²³ or pNF-B-Luc was cotransfected using NovaFECTOR, with pcDNA-rFXR in the presence or absence of pcDNA dominant-negative-(DN)-rFXR, or SHP-PCMV-SPORT6 (Geneservice), with pcDNA3.1 as control. In RASMCs, the miNOSpG2Basic (a kind gift from Dr Mark A. Perrella, Brigham and Women’s Hospital, Harvard, Boston) was cotransfected using Lipofectamine2000 (Invitrogen) with SHP-PCMV-SPORT6 or pcDNA3.1 control plasmid. 16 hours posttransfection, cells were treated with 6ECDCA or IL-1β (10 ng/mL). In some experiments 6ECDCA was added 1 hour before the addition of IL-1β. After 18 hours, cells were lysed for measurement of luciferase activity and protein concentration.

Cell Migration Assays
Smooth muscle cell migration across growth factor reduced Matrigel coated Transwell filters (8µ pore size, Transwell Corning), was essentially as previously described,²⁴ using either IL-1β (10 ng/mL) or PDGF-BB (25 ng/mL) as the stimulant for migration.

	Results

Top Abstract Introduction Methods Results Discussion References

 
FXR Is Functionally Expressed in RASMCs
FXR protein (Figure 1a) and mRNA (Figure 1b) were detected in RASMCs. Consistent with alternative FXR activators,¹⁰ activation of RASMCs by either of the selective FXR ligands, GW4064 or 6ECDCA, induced expression of the FXR target gene, SHP (Figure 1c), and RASMC death (Figure 1d).

View larger version (34K): [in this window] [in a new window]   Figure 1. FXR is functionally expressed in VSMCs. a, Western Blot for FXR in cultures of RASMCs (HepG2 cells, positive control). b, RT-PCR for FXR in n=3 (1 to 3) separate cultures of RASMCs. c, FXR ligands, 6ECDCA (6E; 30 µmol/L) and GW4064 (GW; 3 µmol/L) induce the FXR target gene, SHP, in RASMCs as detected by RT-PCR. β-actin levels remained unchanged. Data are representative of n=5 experiments. d, FXR ligands induce RASMC death. Cell viability was measured by the MTT assay with 100% defined as vehicle-treated cells. Data are mean±SEM of 9 determinations from 3 experiments.

FXR Ligands Inhibit IL-1β–Induced iNOS Activity in RASMCs
Incubation of cultured RASMC with IL-1β (0.01 to 10 ng/mL) for 24 hours resulted in a marked increase in NO synthesis determined by nitrite accumulation using the Griess assay (Figure 2). Both basal and IL-1β–induced nitrite release were strongly inhibited by the selective iNOS inhibitor, 1400W (Figure 2a). Incubation of RASMCs with GW4064 (Figure 2b), 6ECDCA (Figure 2c), or CDCA (supplemental Figure Ia, available online at http://atvb.ahajournals.org) inhibited both basal and IL-1β–induced nitrite release in a concentration-dependant manner. The synthetic ligand GW4064 was 3-fold more potent than the semisynthetic ligand 6ECDCA, consistent with their relative potencies on FXR. Western blot analysis showed that both basal and IL-1β–induced iNOS protein was inhibited by coincubation with either GW4064 (3 µmol/L, a concentration that did not significantly affect RASMC viability), 6ECDCA (30 µmol/L; Figure 2d), or CDCA (supplemental Figure I). Similarly, GW4064 and 6ECDCA also inhibited the induction of COX-2 protein by IL-1β in RASMCs (Figure 2d).

View larger version (34K): [in this window] [in a new window]   Figure 2. Synthetic FXR ligands inhibit IL-1β–induced inflammatory responses in RASMCs. a, Nitrite release from RASMCs induced by IL-1β (0.1 to 10 ng/mL; 24 hours) in the absence (filled squares) or presence (unfilled squares) of the selective iNOS inhibitor, 1400W (10 µmol/L). Data represents mean±SEM of 9 determinations from 3 experiments. Selective FXR ligands, GW4064 (b) and 6ECDCA (c) inhibit basal and IL-1β (10 ng/mL; 24 hours)–induced nitrite release from RASMCs. Data represents mean±SEM of 9 determinations from 3 experiments. d, FXR ligands inhibit IL-1β–induced iNOS and COX-2 protein in RASMCs. GW4064 (3 µmol/L) or 6ECDCA (30 µmol/L) inhibit basal and IL-1β–induced (10 ng/mL; 24 hours) iNOS (130kDa) and COX-2 (72kDa) protein levels in RASMCs. Data are representative of n=3 separate experiments. *P<0.05.

FXR Ligands Downregulate RASMC Migration
PDGF-BB (25 ng/mL) but not IL-1β (10 ng/mL) stimulated RASMC migration (Figure 3). Cell migration, basally and in the presence of IL-1β or stimulated by PDGF-BB was abolished in the presence of 6ECDCA (30 µmol/L; Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. FXR ligands reduce PDGF-BB–induced RASMC migration. RASMC migration induced by either IL-1β (10 ng/mL) or PDGF-BB (25 ng/mL) compared with basal migration in the presence or absence of the FXR ligand 6ECDCA (6E; 30 µmol/L) at 72 hours. Data are mean±SEM of results from 4 experiments. *P<0.05 between FXR ligand and respective control. P<0.05 between control and PDGF-BB.

FXR Ligands Inhibit Human Aortic Vascular Smooth Muscle Cell Responses
FXR mRNA was present in human aortic vascular smooth muscle cells (HASMCs; Figure 4a). 6ECDCA induced SHP expression (Figure 4b), inhibited IL-1β–induced COX-2 expression (Figure 4c and 4d), and abolished PDGF-BB induced cell migration (Figure 4d).

View larger version (28K): [in this window] [in a new window]   Figure 4. FXR expression and activation in primary human aortic vascular smooth muscle cells (HASMCs). a, RT-PCR for FXR in 2 separate HASMC cultures (HepG2 as positive control). b, 6ECDCA (6E; 30 µmol/L) induces SHP, in HASMCs as detected by RT-PCR. Data are representative of n=4 experiments. Representative blot (c) and densitometry (d) analysis showing 6ECDCA (30 µmol/L) inhibits IL-1β–induced (10 ng/mL; 24 hours) COX-2 in HASMCs. e, HASMC migration induced by PDGF-BB (25 ng/mL) in the presence or absence of the FXR ligand 6ECDCA (6E; 30 µmol/L) at 72 hours. Data are mean±SEM of results from 4 separate cultures. *P<0.05 between FXR ligand treatment and respective control, P<0.05 between control and PDGF-BB.

NF-B Activation by IL-1β Is Reduced by FXR and SHP
In HEK293 cells, 6ECDCA activated the IR-1 FXR reporter gene in the presence, but not absence of FXR. DN-FXR strongly inhibited 6ECDCA-induced FXR activation (Figure 5a). Incubation of HEK293 cells with IL-1β (10 ng/mL) induced NFB luciferase activity in transfected cells (Figure 5b). IL-1β–induced NFB reporter gene activation was reduced to baseline by 6ECDCA only when cells were transfected with FXR. 6ECDCA had no inhibitory effects on IL-1β–induced NFB activation in cells without FXR or in the presence of FXR cotransfected with DN-FXR. The inhibitory effect of FXR activation on IL-1β–induced NFB activity in HEK293 cells was mimicked by cotransfection of SHP but not control plasmid pcDNA (Figure 6a). Overexpression of SHP in RASMCs also suppressed IL-1β–induced iNOS reporter gene activation compared with control plasmid pcDNA (Figure 6b). Moreover, siRNA knockdown of SHP in RASMCs (Figure 6c), abolished the ability of 6ECDCA to inhibit IL-1β- induced iNOS activity (nitrite formation; Figure 6d).

View larger version (17K): [in this window] [in a new window]   Figure 5. 6ECDCA inhibits IL-1β–induced NFB activity in HEK293 cells. a, Activation of an FXR reporter gene (fold luciferase activity normalized to total protein) by 6ECDCA (30 µmol/L) in cells transfected with or without wild-type FXR or DN-FXR. b, Activation of an NFB reporter gene by IL-1β (10 ng/mL) in the presence or absence of 6ECDCA (30 µmol/L), in cells transfected with or without wild-type FXR or DN-FXR. *P<0.05 as shown. P<0.05 between FXR and DN-FXR–transfected cells, P<0.05 between control and IL-1β.

View larger version (29K): [in this window] [in a new window]   Figure 6. 6ECDCA inhibits IL-1β–induced NFB and iNOS through SHP. a, Activation of an NFB reporter gene in HEK293 cells by IL-1β (10 ng/mL) in the presence or absence of control plasmid (pcDNA) or SHP. b, Activation of an iNOS promoter reporter gene in RASMCs by IL-1β (10 ng/mL) in the presence or absence of control plasmid (pcDNA) or SHP. c, SHP siRNA but not control siRNA inhibits 6ECDCA (6E; 30 µmol/L)-induced SHP expression in RASMCs. Top panel shows representative blot, whereas figure shows densitometry analysis of SHP expression normalized to β-actin (arbitrary units). d, SHP siRNA but not control siRNA abolishes 6ECDCA (6E; 30 µmol/L) inhibition of iNOS induction (nitrite induction as % of IL-1β induced). Data represents mean±SEM of 12 determinations from 4 experiments. *P<0.05 as shown; P<0.05 between control and IL-1β.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Here we show FXR activation inhibits vascular smooth muscle inflammatory responses and migration. We previously reported that FXR is expressed and induced apoptosis in VSMCs.¹⁰ These newer selective FXR ligands, 6ECDCA and GW4064, similarly induced the FXR target gene SHP and cause VSMC death at higher concentrations, consistent with their relative potencies on FXR.^11,25,26Although we find in other systems that high concentrations of GW4064 induce caspase-3–dependent apoptosis via FXR²⁷ to look at the effects of FXR on VSMC activation, "nonapoptotic" concentrations of these FXR ligands were used.

IL-1β and PDGF-BB are well established activators of VSMCs. The induction of iNOS and COX-2 by IL-1β were both inhibited by FXR ligands. Consistent with known VSMC responses, PDGF-BB but not IL-1β induced VSMC migration.²⁸ RASMC migration under basal, IL-1β, or PDGF-BB–stimulated conditions was abolished by 6ECDCA. In slight contrast, PDGF-BB–stimulated but not basal HASMC migration was inhibited by 6ECDCA. This difference most likely reflects that the RASMC cell line is already partially active in culture (as seen by low levels of basally expressed iNOS and COX-2). By contrast, in hepatic stellate cells²⁹ FXR ligands induce matrix metalloproteinase (MMP)-2 activity by suppressing (TIMP)-1 and -2, whereas they induce vascular endothelial cell migration by inducing MMP-9 expression.³⁰ The effects of FXR on cell movement or MMPs are therefore highly tissue-specific.

As FXR ligands inhibit distinct proinflammatory pathways, it is likely that a common mechanism(s) exists for such inhibition, similar to those found with related nuclear receptors. The peroxisome proliferator-activated receptors, for example, inhibit vascular inflammation by a variety of mechanisms including induction of the NFB inhibitor (IB), inhibition of c-Jun or c-Fos binding to AP-1,^31,32and by direct interactions with AP-1, NFB, and STAT signaling pathways via competition for essential cofactors.³³ NFB can regulate the expression of iNOS³⁴ and COX-2.³⁵ Moreover, FXR has previously been shown to inhibit both AP-1 and NFB activation in endothelial cells.³⁶ In HEK293 cells the FXR ligand 6ECDCA only induced an FXR-responsive reporter gene activity, or inhibited IL-1β–induced NFB activity in the presence of active FXR. SHP overexpression in HEK293 cells or RASMCs suppresses IL-1β–induced NFB or iNOS reporter gene activation, respectively. In contrast, specific siRNA knockdown of SHP in RASMC removed the ability of FXR activation to inhibit iNOS activity.

FXR knockout mice have severe dyslipidaemia³⁷ and when crossed with apolipoprotein E (apoE) knockouts produce male offspring with enhanced atherosclerotic lesion formation as well as increased mortality when fed with a high-fat Western style diet, as might be expected.³⁸ In contrast, male FXR^–/–/ low-density lipoprotein receptor (Ldlr)^–/– double knockout mice³⁹ and female FXR^–/–/apoE^–/– double-null mice⁴⁰ fed with similar diets have reduced atherosclerotic lesion formation, despite a proatherosclerotic lipid profile. Although these reports have different and yet to be fully explained results, our current findings of FXR having an antiinflammatory influence on RASMCs are consistent with both of the male double-null mice where hepatic inflammatory genes such as tumor necrosis factor- were upregulated thereby promoting hepatic inflammation.^38,39

FXR is expressed in human and rat VSMCs and endothelial cells.^10,36 The majority of published studies so far have been unable to establish expression or a direct role of FXR in inflammatory cells such as monocytes.^38–40An antiinflammatory profile would in theory be of benefit in atherosclerosis. However, a high smooth muscle cell/inflammatory cell ratio correlates with stable atherosclerotic lesions. If the antiproliferative or antiinflammatory effects of FXR were solely limited to smooth muscle cells and not inflammatory cells, atherosclerotic plaques might then become unbalanced and actually more prone to rupture. The local vascular roles of FXR in the atherosclerotic lesions of these complex models have yet to be ascertained. FXR ligands attributable to their metabolic effects have also been suggested as novel therapeutics for dyslipidemia and diabetes,^41,42 established cardiovascular risk factors. The effect of FXR activation on atherosclerosis and plaque stability is clearly in need of further investigation.

In conclusion, FXR is expressed and induces its target gene SHP in VSMCs, and activation of FXR and SHP lead to downregulation of important contributors to vascular inflammation and migration, notably COX-2 and iNOS. FXR may therefore be a novel therapeutic target for the treatment of vascular inflammation, remodeling, and atherosclerotic plaque stability.

	Acknowledgments

 
Sources of Funding

Y.T.Y.L. holds a British Heart Foundation PhD studentship (FS/04/049/17115). D.B.B is the recipient of a British Heart Foundation Basic Science Lectureship (BS/02/002). This work was also funded by grants from the Wellcome Trust (074361/Z/04/Z; to K.E.S.), and from European Community FP6 funding (LSHM-CT-2004-0050333).

Disclosures

None.

	Footnotes

 
Original received November 1, 2006; final version accepted September 3, 2007.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986; 6: 131–138.[Medline] [Order article via Infotrieve]

2. Baker CS, Hall RJ, Evans TJ, Pomerance A, Maclouf J, Creminon C, Yacoub MH, Polak JM. Cyclooxygenase-2 is widely expressed in atherosclerotic lesions affecting native and transplanted human coronary arteries and colocalizes with inducible nitric oxide synthase and nitrotyrosine particularly in macrophages. Arterioscler Thromb Vasc Biol. 1999; 19: 646–655.[Abstract/Free Full Text]

3. Behr D, Rupin A, Fabiani JN, Verbeuren TJ. Distribution and prevalence of inducible nitric oxide synthase in atherosclerotic vessels of long-term cholesterol-fed rabbits. Atherosclerosis. 1999; 142: 335–344.[CrossRef][Medline] [Order article via Infotrieve]

4. Teng X, Zhang H, Snead C, Catravas JD. Molecular mechanisms of iNOS induction by IL-1 beta and IFN-gamma in rat aortic smooth muscle cells. Am J Physiol Cell Physiol. 2002; 282: C144–C152.[Abstract/Free Full Text]

5. Koide M, Kawahara Y, Tsuda T, Nakayama I, Yokoyama M. Expression of nitric oxide synthase by cytokines in vascular smooth muscle cells. Hypertension. 1994; 23: I45–I48.[Medline] [Order article via Infotrieve]

6. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med. 1994; 330: 1431–1438.[Free Full Text]

7. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002; 90: 251–262.[Abstract/Free Full Text]

8. Newby AC. Dual role of matrix metalloproteinases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Rev. 2005; 85: 1–31.[Abstract/Free Full Text]

9. Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T, Noonan DJ, Burka LT, McMorris T, Lamph WW, Evans RM, Weinberger C. Identification of a nuclear receptor that is activated by farnesol metabolites. Cell. 1995; 81: 687–693.[CrossRef][Medline] [Order article via Infotrieve]

10. Bishop-Bailey D, Walsh DT, Warner TD. Expression and activation of the farnesoid X receptor in the vasculature. Proc Natl Acad Sci U S A. 2004; 101: 3668–3673.[Abstract/Free Full Text]

11. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, Shan B. Identification of a nuclear receptor for bile acids. Science. 1999; 284: 1362–1365.[Abstract/Free Full Text]

12. Willson TM, Jones SA, Moore JT, Kliewer SA. Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism. Med Res Rev. 2001; 21: 513–522.[CrossRef][Medline] [Order article via Infotrieve]

13. Pellicciari R, Fiorucci S, Camaioni E, Clerici C, Costantino G, Maloney PR, Morelli A, Parks DJ, Willson TM. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J Med Chem. 2002; 45: 3569–3572.[CrossRef][Medline] [Order article via Infotrieve]

14. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell. 2000; 6: 517–526.[CrossRef][Medline] [Order article via Infotrieve]

15. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, Mangelsdorf DJ. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell. 2000; 6: 507–515.[CrossRef][Medline] [Order article via Infotrieve]

16. Bishop-Bailey D, Hla T, Warner TD. Intimal smooth muscle cells as a target for peroxisome proliferator-activated receptor-gamma ligand therapy. Circ Res. 2002; 91: 210–217.[Abstract/Free Full Text]

17. Snell JC, Colton CA, Chernyshev ON, Gilbert DL. Location-dependent artifact for NO measurement using multiwell plates. Free Radic Biol Med. 1996; 20: 361–363.[CrossRef][Medline] [Order article via Infotrieve]

18. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982; 126: 131–138.[CrossRef][Medline] [Order article via Infotrieve]

19. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55–63.[CrossRef][Medline] [Order article via Infotrieve]

20. Woods M, Wood EG, Bardswell SC, Bishop-Bailey D, Barker S, Wort SJ, Mitchell JA, Warner TD. Role for nuclear factor-kappaB and signal transducer and activator of transcription 1/interferon regulatory factor-1 in cytokine-induced endothelin-1 release in human vascular smooth muscle cells. Mol Pharmacol. 2003; 64: 923–931.[Abstract/Free Full Text]

21. Swales K, Plant N, Ayrton A, Hood S, Gibson G. Relative receptor expression is a determinant in xenobiotic-mediated CYP3A induction in rat and human cells. Xenobiotica. 2003; 33: 703–716.[CrossRef][Medline] [Order article via Infotrieve]

22. Bishop-Bailey D, Pepper JR, Larkin SW, Mitchell JA. Differential induction of cyclooxygenase-2 in human arterial and venous smooth muscle: role of endogenous prostanoids. Arterioscler Thromb Vasc Biol. 1998; 18: 1655–1661.[Abstract/Free Full Text]

23. Kocarek TA, Shenoy SD, Mercer-Haines NA, Runge-Morris M. Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes. J Pharmacol Toxicol Methods. 2002; 47: 177–187.[CrossRef][Medline] [Order article via Infotrieve]

24. Thomas GJ, Lewis MP, Whawell SA, Russell A, Sheppard D, Hart IR, Speight PM, Marshall JF. Expression of the alphavbeta6 integrin promotes migration and invasion in squamous carcinoma cells. J Invest Dermatol. 2001; 117: 67–73.[CrossRef][Medline] [Order article via Infotrieve]

25. Niesor EJ, Flach J, Lopes-Antoni I, Perez A, Bentzen CL. The nuclear receptors FXR and LXRalpha: potential targets for the development of drugs affecting lipid metabolism and neoplastic diseases. Curr Pharm Des. 2001; 7: 231–259.[CrossRef][Medline] [Order article via Infotrieve]

26. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM. Bile acids: natural ligands for an orphan nuclear receptor. Science. 1999; 284: 1365–1368.[Abstract/Free Full Text]

27. Swales KE, Korbonits M, Carpenter R, Walsh DT, Warner TD, Bishop-Bailey D. The farnesoid x receptor is expressed in breast cancer and regulates apoptosis and aromatase expression. Cancer Res. 2006; 66: 10120–10126.[Abstract/Free Full Text]

28. Koyama N, Hart CE, Clowes AW. Different functions of the platelet-derived growth factor-alpha and -beta receptors for the migration and proliferation of cultured baboon smooth muscle cells. Circ Res. 1994; 75: 682–691.[Abstract/Free Full Text]

29. Fiorucci S, Rizzo G, Antonelli E, Renga B, Mencarelli A, Riccardi L, Orlandi S, Pruzanski M, Morelli A, Pellicciari R. A farnesoid x receptor-small heterodimer partner regulatory cascade modulates tissue metalloproteinase inhibitor-1 and matrix metalloprotease expression in hepatic stellate cells and promotes resolution of liver fibrosis. J Pharmacol Exp Ther. 2005; 314: 584–595.[Abstract/Free Full Text]

30. Das A, Fernandez-Zapico ME, Cao S, Yao J, Fiorucci S, Hebbel RP, Urrutia R, Shah VH. Disruption of an SP2/KLF6 repression complex by SHP is required for farnesoid X receptor-induced endothelial cell migration. J Biol Chem. 2006; 281: 39105–39113.[Abstract/Free Full Text]

31. Delerive P, Gervois P, Fruchart JC, Staels B. Induction of IkappaBalpha expression as a mechanism contributing to the anti-inflammatory activities of peroxisome proliferator-activated receptor-alpha activators. J Biol Chem. 2000; 275: 36703–36707.[Abstract/Free Full Text]

32. Delerive P, De BK, Besnard S, Vanden BW, Peters JM, Gonzalez FJ, Fruchart JC, Tedgui A, Haegeman G, Staels B. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem. 1999; 274: 32048–32054.[Abstract/Free Full Text]

33. Pascual G, Fong AL, Ogawa S, Gamliel A, Li AC, Perissi V, Rose DW, Willson TM, Rosenfeld MG, Glass CK. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature. 2005; 437: 759–763.[CrossRef][Medline] [Order article via Infotrieve]

34. Taylor BS, de Vera ME, Ganster RW, Wang Q, Shapiro RA, Morris SM Jr, Billiar TR, Geller DA. Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J Biol Chem. 1998; 273: 15148–15156.[Abstract/Free Full Text]

35. Singer CA, Baker KJ, McCaffrey A, AuCoin DP, Dechert MA, Gerthoffer WT. p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L1087–L1098.[Abstract/Free Full Text]

36. Huang FM, Yang SF, Hsieh YS, Liu CM, Yang LC, Chang YC. Examination of the signal transduction pathways involved in matrix metalloproteinases-2 in human pulp cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004; 97: 398–403.[Medline] [Order article via Infotrieve]

37. He F, Li J, Mu Y, Kuruba R, Ma Z, Wilson A, Alber S, Jiang Y, Stevens T, Watkins S, Pitt B, Xie W, Li S. Downregulation of endothelin-1 by farnesoid X receptor in vascular endothelial cells. Circ Res. 2006; 98: 192–199.[Abstract/Free Full Text]

38. Hanniman EA, Lambert G, McCarthy TC, Sinal CJ. Loss of functional farnesoid X receptor increases atherosclerotic lesions in apolipoprotein E-deficient mice. J Lipid Res. 2005; 46: 2595–2604.[Abstract/Free Full Text]

39. Zhang Y, Wang X, Vales C, Ying LF, Lee H, Lusis AJ, Edwards PA FXR Deficiency Causes Reduced Atherosclerosis in Ldlr^–/– Mice. Arterioscler Thromb Vasc Biol. 2006.

40. Guo GL, Santamarina-Fojo S, Akiyama TE, Amar MJ, Paigen BJ, Brewer B Jr, Gonzalez FJ. Effects of FXR in foam-cell formation and atherosclerosis development. Biochim Biophys Acta. 2006; 1761: 1401–1409.[Medline] [Order article via Infotrieve]

41. Zhang Y, Lee FY, Barrera G, Lee H, Vales C, Gonzalez FJ, Willson TM, Edwards PA. Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice. Proc Natl Acad Sci U S A. 2006; 103: 1006–1011.[Abstract/Free Full Text]

42. Bilz S, Samuel V, Morino K, Savage D, Choi CS, Shulman GI. Activation of the farnesoid X receptor improves lipid metabolism in combined hyperlipidemic hamsters. Am J Physiol Endocrinol Metab. 2006; 290: E716–E722.[Abstract/Free Full Text]

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