Download Farnesoid X Receptor Ligands Inhibit Vascular Smooth Muscle Cell

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
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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)-1␤–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-1␤–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. (Arterioscler Thromb Vasc Biol. 2007;27:2606-2611.)
Key Words: FXR 䡲 vascular smooth muscle 䡲 iNOS 䡲 COX-2 䡲 cell migration
A
therosclerosis 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 ligandactivated transcription factors that binds and acts as a het-
erodimer 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
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.
Original received November 1, 2006; final version accepted September 3, 2007.
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 [email protected]
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
2606
DOI: 10.1161/ATVBAHA.107.152694
Li et al
FXR Inhibits VSMC Inflammation and Migration
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IL-1␤ was from R&D Systems. pNF-␬B-Luc was from BD Biosciences Clontech. FXR-responsive IR-1 reporter gene, pcDNArFXR, 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
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FXR ligands, 6ECDCA (0.1 to 30 ␮mol/L), GW4064 (0.1 to
30 ␮mol/L), or vehicle control(s) were added 1 hour prior to
incubation with IL-1␤ (0.01 to 10 ng/mL, 24 hour). In some
experiments, the selective iNOS inhibitor, 1400W (10 ␮mol/L) was
included 1 hour before IL-1␤ 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)-2,5diphenyltetrazolium 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 ␤-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–␤-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 dominantnegative-(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-1␤ (10
ng/mL). In some experiments 6ECDCA was added 1 hour before the
addition of IL-1␤. 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 (8␮ pore size, Transwell Corning), was
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 ␮mol/L) and
GW4064 (GW; 3 ␮mol/L) induce the FXR target gene, SHP,
in RASMCs as detected by RT-PCR. ␤-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.
essentially as previously described,24 using either IL-1␤ (10 ng/mL)
or PDGF-BB (25 ng/mL) as the stimulant for migration.
Results
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).
FXR Ligands Inhibit IL-1␤–Induced iNOS
Activity in RASMCs
Incubation of cultured RASMC with IL-1␤ (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-1␤–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-1␤–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-1␤–induced iNOS protein was inhibited by coincubation
with either GW4064 (3 ␮mol/L, a concentration that did not
significantly affect RASMC viability), 6ECDCA (30 ␮mol/L;
Figure 2d), or CDCA (supplemental Figure I). Similarly,
GW4064 and 6ECDCA also inhibited the induction of
COX-2 protein by IL-1␤ in RASMCs (Figure 2d).
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Arterioscler Thromb Vasc Biol
December 2007
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Figure 2. Synthetic FXR ligands inhibit
IL-1␤–induced inflammatory responses in
RASMCs. a, Nitrite release from
RASMCs induced by IL-1␤ (0.1 to 10
ng/mL; 24 hours) in the absence (filled
squares) or presence (unfilled squares) of
the selective iNOS inhibitor, 1400W
(10 ␮mol/L). Data represents mean⫾SEM
of 9 determinations from 3 experiments.
Selective FXR ligands, GW4064 (b) and
6ECDCA (c) inhibit basal and IL-1␤ (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-1␤–induced iNOS and COX-2 protein
in RASMCs. GW4064 (3 ␮mol/L) or
6ECDCA (30 ␮mol/L) inhibit basal and
IL-1␤–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-1␤ (10 ng/mL) stimulated
RASMC migration (Figure 3). Cell migration, basally and in
the presence of IL-1␤ or stimulated by PDGF-BB was
abolished in the presence of 6ECDCA (30 ␮mol/L; Figure 3).
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-1␤–induced COX-2
expression (Figure 4c and 4d), and abolished PDGF-BB
induced cell migration (Figure 4d).
NF-␬B Activation by IL-1␤ 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-1␤ (10 ng/mL)
induced NF␬B luciferase activity in transfected cells (Figure
5b). IL-1␤–induced NF␬B reporter gene activation was
reduced to baseline by 6ECDCA only when cells were
transfected with FXR. 6ECDCA had no inhibitory effects on
IL-1␤–induced NF␬B activation in cells without FXR or in
the presence of FXR cotransfected with DN-FXR. The
inhibitory effect of FXR activation on IL-1␤–induced NF␬B
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-1␤–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-1␤- induced iNOS activity (nitrite formation;
Figure 6d).
Discussion
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-1␤ and PDGF-BB are well established activators of
VSMCs. The induction of iNOS and COX-2 by IL-1␤ were
both inhibited by FXR ligands. Consistent with known
VSMC responses, PDGF-BB but not IL-1␤ induced VSMC
migration.28 RASMC migration under basal, IL-1␤, 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
cells 29 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
Li et al
FXR Inhibits VSMC Inflammation and Migration
Figure 3. FXR ligands reduce PDGF-BB–induced RASMC
migration. RASMC migration induced by either IL-1␤ (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 ␮mol/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.
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receptors. The peroxisome proliferator-activated receptors,
for example, inhibit vascular inflammation by a variety of
mechanisms including induction of the NF␬B inhibitor
(I␬B␣), inhibition of c-Jun or c-Fos binding to AP-1,31,32and
by direct interactions with AP-1, NF␬B, and STAT signaling
pathways via competition for essential cofactors.33 NF␬B can
regulate the expression of iNOS34 and COX-2.35 Moreover,
FXR has previously been shown to inhibit both AP-1 and
NF␬B activation in endothelial cells.36 In HEK293 cells the
FXR ligand 6ECDCA only induced an FXR-responsive
reporter gene activity, or inhibited IL-1␤–induced NF␬B
activity in the presence of active FXR. SHP overexpression in
HEK293 cells or RASMCs suppresses IL-1␤–induced NF␬B
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.
2609
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 athero-
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 ␮mol/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
␮mol/L) inhibits IL-1␤–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 ␮mol/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.
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Arterioscler Thromb Vasc Biol
December 2007
Figure 5. 6ECDCA inhibits IL-1␤–induced NF␬B activity in
HEK293 cells. a, Activation of an FXR reporter gene (fold
luciferase activity normalized to total protein) by 6ECDCA
(30 ␮mol/L) in cells transfected with or without wild-type FXR
or DN-FXR. b, Activation of an NF␬B reporter gene by IL-1␤
(10 ng/mL) in the presence or absence of 6ECDCA (30 ␮mol/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-1␤.
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sclerosis 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.
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.
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Downloaded from http://atvb.ahajournals.org/ by guest on May 13, 2017
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 and David
Bishop-Bailey
Arterioscler Thromb Vasc Biol. 2007;27:2606-2611
doi: 10.1161/ATVBAHA.107.152694
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Supplemental Figures
Figure I. Physiological FXR ligands inhibit IL-1β-induced iNOS activity and cell
death. Like the synthetic FXR ligands, “physiological” FXR ligands, (a) CDCA and
DCA inhibit IL-1β-induced nitrite release, (b) CDCA reduces IL-1β-induced iNOS
protein expression. As previously reported, CDCA and DCA weakly induce RASMC
death (c). CDCA and DCA inhibited nitrite release with greater potencies than they
induced cell death. Data represents the mean ± S.E.M. of 9 determinations from 3
separate experiments for graphs, and the Western blot is representative of 3 separate
experiments.
Supplemental figure I; Li et al.
a
CDCA
DCA
CDCA+IL-1β
DCA+IL-1β
Nitrite induction
(% of control)
125
100
75
50
25
0
0
10 30 100300
[FXR ligand] (μM)
b
iNOS
β-actin
IL-1β
-
+
-
+
CDCA
-
-
30
30
MTT (% of control)
c
125
100
75
50
25
CDCA
DCA
0
0
30
300
[FXR ligand] (μM)
10ng/ml