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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2991-2998
Fibrates Suppress Fibrinogen Gene Expression in Rodents Via
Activation of the Peroxisome Proliferator-Activated Receptor-
By
Maaike Kockx,
Philippe P. Gervois,
Philippe Poulain,
Bruno Derudas,
Jeffrey M. Peters,
Frank J. Gonzalez,
Hans M.G. Princen,
Teake Kooistra, and
Bart Staels
From Gaubius Laboratory, TNO-Prevention and Health, Leiden, The
Netherlands; U.325 INSERM, Département d'Athérosclerose,
Institut Pasteur, Lille; Faculté de Pharmacie, Université
de Lille II, Lille, France; and the Laboratory of Metabolism, National
Cancer Institute, National Institutes of Health, Bethesda,
MD.
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ABSTRACT |
Plasma fibrinogen levels have been identified as an important risk
factor for cardiovascular diseases. Among the few compounds known to
lower circulating fibrinogen levels in humans are certain fibrates. We
have studied the regulation of fibrinogen gene expression by fibrates
in rodents. Treatment of adult male rats with fenofibrate (0.5%
[wt/wt] in the diet) for 7 days decreased hepatic A -, B -, and
 -chain mRNA levels to 52% ± 7%, 46% ± 8%, and 81% ± 19%
of control values, respectively. In parallel, plasma fibrinogen
concentrations were decreased to 63% ± 7% of controls. The
suppression of fibrinogen expression was dose-dependent and was already
evident after 1 day at the highest dose of fenofibrate tested (0.5%
[wt/wt]). Nuclear run-on experiments showed that the decrease in
fibrinogen expression after fenofibrate occurred at the transcriptional
level, as exemplified for the gene for the A-chain. Other fibrates
tested showed similar effects on fibrinogen expression and
transcription. The effect of fibrates is specific for peroxisome
proliferator-activated receptor- (PPAR) because a high-affinity
ligand for PPAR, the thiazolidinedione BRL 49653, lowered
triglyceride levels, but was unable to suppress fibrinogen expression.
Direct evidence for the involvement of PPAR in the suppression of
fibrinogen by fibrates was obtained using PPAR-null ( /) mice.
Compared with (+/+) mice, plasma fibrinogen levels in (/)
mice were significantly higher (3.20 ± 0.48 v 2.67 ± 0.42 g/L). Also, hepatic fibrinogen A-chain mRNA levels were 25% ± 11% higher in the (/) mice. On treatment with 0.2% (wt/wt)
fenofibrate, a significant decrease in plasma fibrinogen to 77% ± 10% of control levels and in hepatic fibrinogen A-chain mRNA levels
to 65% ± 12% of control levels was seen in (+/+) mice, but not
in (/) mice. These studies show that PPAR regulates basal
levels of plasma fibrinogen and establish that fibrate-suppressed
expression of fibrinogen in rodents is mediated through PPAR.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MANY CROSS-SECTIONAL and case-control
studies and numerous prospective cohort studies have identified
elevated plasma fibrinogen levels as an independent risk factor for
coronary heart disease, stroke, and peripheral vascular disease. In
addition, several cardiovascular and metabolic risk factors such as
smoking, hypertension, hyperlipoproteinemia, and diabetes are also
associated with high plasma fibrinogen concentrations (for a review see
Handley and Hughes1 and references therein).
Interpretations of the relationship between fibrinogen and coronary
heart disease are interesting and unresolved, but most likely reflect
low grade inflammatory processes associated with
atherogenesis.2
The recognition that fibrinogen is an important factor in the promotion
of various disease states has led to the search for specific therapies
intended to reduce plasma fibrinogen levels. Although many different
pharmacologic approaches and strategies for therapeutic modulation of
fibrinogen have been tested, the efficacy of the different treatments
to lower plasma fibrinogen in humans is limited and the mode of action
unidentified.1,3 Among the few compounds that consistently
lower circulating fibrinogen levels are some, but not all of the
fibrates.1,4 Fibrates are widely used hypolipidemic drugs,
very effective in lowering elevated plasma triglyceride and cholesterol
levels.5 There is increasing evidence that at least part of
the action of fibrates on lipid metabolism is exerted via the
peroxisome proliferator-activated receptor- (PPAR).6
PPAR is a member of the nuclear receptor family of transcription
factors, a diverse group of proteins that mediate ligand-dependent
transcriptional activation and repression.7 Several studies
have shown a direct involvement of PPAR in the fibrate-modulated
gene expression of hepatic apo A-I and apo A-II, the major
apolipoproteins in high-density lipoprotein (HDL), of lipoprotein lipase and apo C-III, both major determinants of plasma triglyceride levels, and of several enzymes implicated in fatty acid
-oxidation such as acyl-CoA oxidase (ACO).6 The
importance of PPAR in these fibrate-induced changes in gene
expression and in lowering plasma triglyceride and cholesterol levels
was confirmed in experiments using PPAR-deficient
mice.8-10 More recently, activation of PPAR by fibrates
was also shown to inhibit the action of inflammatory cytokines by
antagonizing the activities of the transcription factor, nuclear
factor- B (NF-B).11
Given the reported suppressive effect of certain fibrates on plasma
fibrinogen levels in humans, the hypothesis that PPAR is involved in
regulating fibrinogen gene expression was tested. To that end, we first
established the effect of fibrates on fibrinogen expression in rats.
The fibrate-induced decrease of fibrinogen expression is regulated at
the transcriptional level, as shown by nuclear run-on experiments, and
is accompanied by a concomitant increase in ACO mRNA level and gene
transcription, indicating PPAR activation. To establish the role of
PPAR in fibrinogen gene expression, we studied the effect of
fibrates in PPAR-null mice. Plasma fibrinogen concentrations were
significantly higher in PPAR-null (/) mice than in
wild-type (+/+) mice. On treatment with fibrate, a significant decrease
in plasma fibrinogen levels and hepatic fibrinogen gene expression was
observed in (+/+) mice, but not in (/) mice. Our data
provide strong evidence for an important role of PPAR in the
suppression of fibrinogen gene expression and may explain
fibrate-induced reductions of fibrinogen in humans.
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MATERIALS AND METHODS |
Reagents.
Fenofibric acid, gemfibrozil, and ciprofibrate were kind gifts of Drs
A. Edgar (Laboratoires Fournier, Daix, France), B. Bierman (Warner-Lambert, Hoofddorp, The Netherlands), and M. Riteco (Sanofi Winthrop, Maassluis, The Netherlands), respectively. BRL 49653 was
generously provided by Dr J.-J. Berthelon (Lipha Merck,
Lyon, France). Bezafibrate was obtained from Boehringer Mannheim
(Almere, The Netherlands).
Animal studies.
Animal studies were performed in compliance with European Community
specifications regarding the use of laboratory animals. Details of
experimental conditions have been described previously.12 Briefly, male Wistar rats (3 months old) were divided in groups of four
animals each and treated for 7 days with fenofibrate mixed at the
indicated concentrations (by mass) in standard rat chow. The food
intake was recorded every 2 days throughout the treatment period. None
of the treatments caused major changes in the amount of food consumed
by the animals. Because each rat consumed approximately 20 g of chow
per day, doses of 0.5%, 0.05%, and 0.005% (wt/wt) corresponded to
320, 32, and 3 mg of fibrate/kg of body weight/day. In a subsequent
experiment, rats were treated with 0.5% (wt/wt) fenofibrate for
different time periods up to 14 days, followed by a wash-out period
varying from 1 to 14 days. In a second series of experiments, male
Sprague-Dawley rats (3 months old) were divided in groups of four
animals each and treated for 3 days with BRL 49653 (10 mg/kg of body
weight/d), fenofibrate (200 mg/kg of body weight/d), or 10% (wt/vol)
carboxymethylcellulose (vehicle for gavage) by gavage, twice a day. At
the end of the treatment period, rats were fasted overnight, weighed,
and killed by exsanguination under ether anesthesia between 8 and 10 am. Blood was collected by aortic puncture, and part of it was used for
serum preparation. The other portion was incubated with 0.1 vol of
trisodium citrate (3.8% [wt/vol]) to prevent coagulation, and
platelet-free plasma was prepared for determination of fibrinogen.
Livers were removed immediately, rinsed with 0.9% (wt/vol) NaCl,
weighed, frozen in liquid nitrogen, and stored at -70°C until RNA
preparation. In a third series of experiments, male Sv/129 homozygous
wild-type (+/+) and PPAR-null (/) mice8
(10 to 12 weeks of age) were fed for 17 days with either a standard
mouse chow or one containing 0.2% (wt/wt) fenofibrate. At the end of
the treatment period, the animals were fasted for 4 hours, weighed, and
killed by exsanguination under ether anesthesia. For determination of
plasma fibrinogen levels, blood was collected from a small tail-cut
using potassium-EDTA Microvette CB 300 tubes (Sarstedt,
Nümbrecht, Germany). Livers were removed immediately, weighed,
rinsed with 0.9% (wt/vol) NaCl, frozen in liquid nitrogen, and stored
at 70°C until RNA preparation.
Rat hepatocyte isolation and culture.
Rat hepatocytes were isolated and cultured as described
previously.13 Briefly, hepatocytes were isolated by
perfusion with 0.05% (wt/vol) collagenase and 0.005% (wt/vol) trypsin
inhibitor. After a 4-hour attachment period in Williams E medium
supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 135 nmol/L insulin, 50 nmol/L dexamethasone, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, the
nonadherent cells were washed from the plates and the remaining cells
refed. After 16 hours, the medium was changed to incubation medium in
which the amount of insulin was lowered to 10 nmol/L. Experiments were started 20 hours after isolation. Conditioned media were obtained by
incubating cells at 37°C for 72 hours with incubation medium containing the appropriate test compound or stock solvent (dimethyl sulfoxide [DMSO]; final concentration 0.1% [vol/vol]). The media were changed every 24 hours. Conditioned media were centrifuged for 4 minutes at 5,000g to remove cells and cellular debris, and the
samples were kept at 20°C until use. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and used for isolation of RNA.
Serum triglycerides and plasma fibrinogen measurements.
Serum triglycerides were determined using a commercially available kit
to measure total serum triglycerides (Boehringer Mannheim). Fibrinogen
concentrations in plasma and conditioned media were measured by an
enzyme-linked immunosorbent assay (ELISA) procedure using polyclonal
antibodies to rat fibrinogen both as catching and tagging
antibodies.14
RNA analysis.
RNA was isolated from liver and cultured cells by the acid guanidinium
thiocyanate/phenol/chloroform method.15 Northern and
dot-blot analysis of total cellular RNA was performed as
described.12 All probes were labeled with a Megaprime kit,
yielding an average activity of 0.5 µCi/ng DNA. Filters were
hybridized with 3 ng of [-32P] deoxycytidine
triphosphate (dCTP)-labeled probe per mL as
described.16 Mouse fibrinogen cDNA probes used were
provided by F. Razaee from our institute and were a 1.2-kb fragment of
the mouse fibrinogen A-chain cDNA; a 1.0-kb fragment of the mouse
fibrinogen B-chain cDNA; a 0.6-kb fragment of the mouse fibrinogen
-chain cDNA. Other cDNA probes used were a 2.0-kb Sac I
fragment of the rat ACO cDNA, provided by Dr T. Osumi,17
and a 3.8-megadalton (mDa) EcoRI fragment of the
human 18S ribosomal DNA.18 The intensities of the signals
were determined using a Fujix Bas 1000 phosphoimager (Fuji
Photo Film Co, Tokyo, Japan) and expressed relative to the signal of
the 18S ribosomal RNA band.
Isolation of nuclei and transcriptional rate assay.
Nuclei were prepared from livers of untreated rats and rats treated for
14 days with 0.5% (wt/wt) of different fibrates in rat chow, exactly
as described by Gorski et al.19 Transcription run-on assays
were performed as described by Nevins.20 Equivalent amounts
of labeled nuclear RNA were hybridized for 36 hours at 42°C to 5 µg of purified cDNA probes immobilized on Hybond C Extra filters
(Amersham, Arlington Heights, IL). The following cDNAs were spotted: a
mouse fibrinogen A-chain cDNA probe, a rat ACO cDNA probe, and a
chicken -actin cDNA probe. As a control, 5 µg of vector DNA was
applied to the filter. After hybridization, filters were washed at room
temperature for 10 minutes in 0.5 × SSC (1 × SSC being 0.15 mol/L NaCl, 0.015 mol/L Na3 citrate) and 0.1% (wt/vol)
sodium dodecyl sulfate (SDS), and twice for 30 minutes at 65°C, and
subsequently exposed to x-ray (X-OMAT-AR, Eastman-Kodak, Rochester,
NY) film. The intensities of the signals present were
determined by scanning densitometry (Bio-Rad GS670 densitometer;
Bio-Rad, Paris, France) and expressed relative to the
signal of the -actin mRNA band.
Statistical analysis.
The data are presented as means ± standard deviation (SD). The
significance of treatment was assessed by an unpaired Student's t-test, with exception of the dose-response and time-course
experiments in which analysis of variance was used to evaluate the
results. For comparison of the wild-type and PPAR-deficient mice, an
unpaired Student's t-test was also used. Differences were
considered significant at P < .05.
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RESULTS |
Fibrates decrease hepatic fibrinogen gene expression and plasma
fibrinogen concentrations.
Adult male rats were treated for 7 days with different concentrations
(0.005, 0.05, or 0.5% [wt/wt]) of fenofibrate mixed in standard rat
chow and analyzed for hepatic fibrinogen gene expression and plasma
fibrinogen levels (Fig 1). Fibrinogen is secreted as a fully assembled dimer, with each half composed of three
nonidentical polypeptide chains, A, B, and . Fenofibrate treatment decreased hepatic fibrinogen A-, B-, and -chain mRNA as well as plasma fibrinogen levels in a dose-dependent fashion. At the
highest dose (0.5%) of fenofibrate tested, fibrinogen mRNA levels were
reduced to 52% ± 7%, 46% ± 8%, and 81% ± 19% of
control values for the A-, B-, and -chain, respectively (Fig
1A and B). This weaker effect of fibrates on the -chain was
consistently found in all experiments performed. In parallel, plasma
fibrinogen concentrations were decreased to 63% ± 7% of control
values (Fig 1C). At the lowest dose (0.005%) of fenofibrate tested,
fibrinogen mRNAs in the liver and plasma fibrinogen levels were not
significantly affected. Hepatic mRNA levels of ACO, the rate-limiting
enzyme in peroxisomal -oxidation, the induction of which by fibrates is strictly PPAR-mediated,8 showed a dose-dependent
response to fenofibrate-treatment comparable to that of fibrinogen,
reaching an approximately sixfold increase at a fenofibrate dose of
0.5%.
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| Fig 1.
Dose-dependent effect of fenofibrate on hepatic
fibrinogen mRNAs and plasma fibrinogen levels. Adult male rats were
treated with 0.005%, 0.05%, or 0.5% of fenofibrate ([wt/wt] in rat
chow) for 7 days and compared with animals on rat chow only. Total RNA
was extracted from livers and analyzed by Northern blotting for
fibrinogen A-, B-, and -chain mRNA and ACO mRNA. Equal loading
was checked by hybridizing with an 18S rRNA cDNA probe. Plasma
fibrinogen levels were measured as described in Materials and Methods.
Data shown are from a representative experiment with four animals per
experimental group. (A) Representative Northern blot analysis of
fibrinogen (Fbg) A-, B-, and -chain mRNA, ACO mRNA, and 18S
rRNA. (B) Signals for the three fibrinogen chain mRNAs and ACO mRNA
were quantified by densitometry and adjusted for the corresponding rRNA
signals. Data are expressed relative to that found in untreated
animals. Results are means ± SD of four animals. (C) Plasma
fibrinogen data are means ± SD of four animals. Statistically
significant differences (P < .05) are indicated by an
asterisk; # P = .13.
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When rats were treated with 0.5% (wt/wt) fenofibrate for different
periods of time, the mRNA levels of fibrinogen A-chain, the presumed
rate-limiting chain in the assembly of the mature fibrinogen molecule
in rats,21 were found to be decreased to 55% ± 3% of
control levels after just 1 day of fenofibrate treatment (Fig 2). Fibrinogen A-chain mRNA
concentrations decreased only slightly on further prolonged treatment,
reaching 44% ± 9% and 41% ± 3% of control values after 7 and 14 days of treatment, respectively.
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| Fig 2.
Time-dependent effect of fenofibrate on fibrinogen
A-chain mRNA levels. Adult male rats were treated with 0.5% (wt/wt)
fenofibrate for different time periods. Total RNA was extracted from
livers and analyzed for fibrinogen A-chain mRNA levels by dot-blot
analysis as described in Materials and Methods. Equal loading was
checked by hybridizing with an 18S rRNA cDNA probe. Values are means ± SD of three animals and presented as percentage of control values.
Statistically significant differences (P < .05) are indicated
by an asterisk.
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To examine whether the observed downregulation of plasma fibrinogen and
hepatic fibrinogen gene expression is a general characteristic of
fibrates rather than a specific effect of fenofibrate, we also tested
the effect of other fibrates. In rats exposed for 14 days to 0.5%
(wt/wt) of gemfibrozil or bezafibrate, or 0.05% (wt/wt) of
ciprofibrate, fibrinogen A-chain mRNA levels were reduced to 74% ± 12%, 53% ± 3%, and 59% ± 2% of control values, respectively.
Downregulation of fibrinogen expression is due to a direct effect of
fibrates on hepatocytes.
Fibrates are known to cause extensive peroxisome proliferation and
hepatomegaly in rodents.6 In the present study, we found that treatment of rats with 0.05% (wt/wt) and 0.5% (wt/wt) of fenofibrate for 14 days increased liver weights 1.4-fold and 2.0-fold, respectively. To exclude the possibility that the suppressive effects
of fenofibrate on fibrinogen expression are due to changes in liver
structure and/or function, we performed wash-out experiments: male rats
were treated for 14 days with 0.5% (wt/wt) of fenofibrate, after which
the fibrate was withdrawn from the food. At the start of the wash-out
period, hepatic fibrinogen A-chain mRNA levels were 51% ± 9%
of control levels (Fig 3). Fibrinogen
A-chain mRNA levels increased to 76% ± 13% of control values
within 1 day after withdrawal of fenofibrate and reached control levels
after 4 days, staying constant thereafter. These findings indicate that
fibrates decrease fibrinogen expression reversibly and independent of
changes in liver structure and/or function.
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| Fig 3.
Effect of cessation of fenofibrate treatment on
fibrinogen A-chain mRNA levels. Adult male rats were treated with
0.5% (wt/wt) of fenofibrate for 14 days, after which fenofibrate was
withdrawn from the food. Total RNA was extracted from livers and
analyzed for fibrinogen A-chain mRNA at different time points after
cessation of fenofibrate treatment. Equal loading was checked by
hybridizing with an 18S rRNA cDNA probe. Values are means ± SD of
three animals per group and presented as percentage of control values
obtained from untreated animals (C). Statistically significant
differences (P < .05) are indicated by an asterisk.
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To find further evidence for a direct effect of fibrates on hepatic
fibrinogen expression, we investigated whether these effects are also
observed in primary cultures of rat hepatocytes. Treatment of rat
hepatocytes for 72 hours with ciprofibrate or the active form of
fenofibrate, fenofibric acid, reduced fibrinogen production dose-dependently to 62% ± 13% and 59% ± 2% of control
values, respectively, at the highest concentration of the fibrate
tested (1 mmol/L) (Fig 4). The reduction of
fibrinogen antigen levels was reflected in a reduction of fibrinogen
A-chain mRNA levels (data not shown), indicating that fibrates
suppress fibrinogen gene expression in a direct manner.
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| Fig 4.
Effect of ciprofibrate and fenofibric acid on fibrinogen
production in primary cultures of rat hepatocytes. Isolated rat
hepatocytes were incubated with 0.3 or 1 mmol/L ciprofibrate or
fenofibric acid or vehicle for three consecutive periods of 24 hours.
The conditioned media were analyzed for fibrinogen antigen as described
in Materials and Methods. Results are means ± SD of three independent
experiments performed in duplicate. The data are expressed as
percentage values of controls. Statistically significant differences
(P < .05) are indicated by an asterisk.
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Fibrates suppress fibrinogen gene transcription.
To assess the effects of various fibrates on fibrinogen gene
transcription rate, nuclear run-on transcription assays were performed
on nuclei prepared from livers of untreated (control) rats or rats
treated for 14 days with 0.5% (wt/wt) of fenofibrate or ciprofibrate.
Both fibrates decreased fibrinogen A-chain transcription rate (to
24% and 45% of control levels for fenofibrate and ciprofibrate, respectively) and increased ACO transcription rate (to 373% and 589%
of control levels for fenofibrate and ciprofibrate, respectively) (Fig 5A and B), reflecting
activation of PPAR.
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| Fig 5.
Effect of fibrates on fibrinogen and ACO gene
transcription. Nuclear run-on assays were performed on nuclei obtained
from livers of control rats and rats treated with 0.5% (wt/wt)
fenofibrate or 0.5% (wt/wt) ciprofibrate for 14 days as described in
Materials and Methods. The data shown are of a representative
experiment. (A) Autoradiogram showing vector (pSG5), -actin, ACO,
and fibrinogen A-chain (Fbg A) signals. (B) Signals were
quantified by densitometric scanning and adjusted for the corresponding
-actin signal. Data are expressed as percentage values relative to
that in control nuclei.
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Fibrinogen gene expression is suppressed by PPAR, but not by
PPAR activators.
In addition to their PPAR activating capacity, fibrates are also
known to activate, albeit much more weakly, PPAR.22 To verify that the suppressive effect of fibrates on fibrinogen is mediated via activation of PPAR rather than PPAR, we compared the
effects of fenofibrate with the effects of the antidiabetic drug
thiazolidinedione, BRL 49653, previously shown to be a high affinity
ligand for PPAR.23 Rats treated for 3 days with 400 mg/kg/d fenofibrate or 10 mg/kg/d BRL 49653 by gavage showed
significantly reduced plasma triglyceride levels (to 69% ± 12%
and 68% ± 8% of control levels for fenofibrate and BRL 49653, respectively). However, whereas treatment with fenofibrate reduced
fibrinogen A-chain mRNA levels to 55% ± 3% of control levels,
BRL 49653 did not affect fibrinogen expression (115% ± 11% of control levels) (Fig 6), indicating
that the suppressive effect of fibrates on fibrinogen levels requires
PPAR activation.
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| Fig 6.
Effect of BRL 49653 and fenofibrate on fibrinogen
A-chain mRNA levels in rats. Adult male rats were treated with 10 mg/kg of body weight/day BRL 49653 or 400 mg/kg of body weight/day
fenofibrate by gavage twice a day for 3 days. Plasma triglyceride
levels were determined as described in Materials and Methods. Total RNA
was extracted from livers and analyzed for fibrinogen A-chain mRNA
by Northern blotting. Equal loading was checked by hybridizing with an
18S rRNA cDNA probe. Values are means ± SD of two independent
experiments (with four animals per group) and presented as percentage
of control values. Statistically significant differences (P < .05) are indicated by an asterisk.
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PPAR-null mice are refractory to the suppressive effects of
fibrates on fibrinogen expression.
To establish a direct role of PPAR in the regulation of fibrinogen
gene expression, we studied fibrinogen expression and its response to
fenofibrate in PPAR-null (/) mice. Compared with
wild-type (+/+) mice, plasma fibrinogen levels were significantly higher in (/) mice, being 2.67 ± 0.42 g/L and 3.20 ± 0.48 g/L, respectively (Table 1).
Hepatic fibrinogen A-chain mRNA levels were 25% ± 11% higher
in the (/) mice (Fig 7). On
treatment with 0.2% (wt/wt) fenofibrate for 17 days, liver weights
were increased to 277% of controls in (+/+) mice, while no change in liver weights of (/) mice was observed (data not shown).
Fenofibrate significantly decreased plasma fibrinogen levels in (+/+)
mice (0.61 ± 0.31 g/L; P = .007), but not in
(/) mice (0.33 ± 0.36 g/L; P = .13)
(Table 1). Consistent with the antigen data, fibrinogen A-chain mRNA
levels were significantly decreased in (+/+) mice (35% ± 12%; P = .04), but not in the fibrate-treated
(/) mice (+1% ± 13%; P = .9) (Fig
7). These results indicate that PPAR is involved in the suppression
of basal levels of plasma fibrinogen, and that fibrate-suppressed
expression of fibrinogen in wild-type mice is dependent on PPAR
activation.
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| Fig 7.
Effect of fenofibrate on fibrinogen A-chain mRNA
levels in wild-type versus PPAR-null mice. Wild-type (+/+) and
PPAR-null (/) mice were treated with 0.2% (wt/wt) fenofibrate
mixed in chow for 17 days. Total RNA was extracted from livers and
analyzed for fibrinogen A-chain by Northern blotting. Equal loading
was checked by hybridizing with an 18S rRNA cDNA probe. Values are
means ± SD of seven animals per group and presented as percentage
values of control, untreated wild-type mice.
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DISCUSSION |
Fibrates reportedly lower plasma fibrinogen in humans, but the
regulatory mechanism of this effect remains to be clarified. Here, we
show that activation of the nuclear hormone receptor PPAR mediates
the suppression of fibrinogen gene transcription by fibrates in
rodents. A direct involvement of PPAR in fibrinogen gene expression
was provided using PPAR-null (/) mice. Basal levels of
plasma fibrinogen were significantly higher in the (/) mice than in (+/+) mice, and fibrates suppressed fibrinogen gene expression and plasma levels in (+/+) mice only. These observations clearly establish PPAR as a key regulatory factor in fibrinogen gene
expression in rodents and may explain the suppressive effect of
fibrates on plasma fibrinogen levels in humans.
The fibrinogen molecule is arranged as a dimer, with each monomer
composed of three nonidentical polypeptide chains: A, B, and
.24 The three fibrinogen chains are encoded by three
separate, closely linked genes situated on the same chromosome and
located in the sequence , A, and B, with the last one in
opposite transcriptional orientation to the first one.25 It
has been reported that in rat hepatocytes, the amount of A-chain
limits the rate of assembly of the fibrinogen molecule,21
whereas in human hepatoma cells, the amount of B-chain appears to
limit assembly.26,27 We found that, at least in rats, the
inhibition of gene expression by fibrates was evidently not confined to
the A-chain, but equally affected the B-chain and, albeit to a
lesser extent, the -chain. Recently, Binsack et al28
reported that in the human hepatoma cell line, HepG2, bezafibrate
suppressed A-, B-, and -chain mRNA levels. These findings
corroborate the concept of the coordinated expression of the three
fibrinogen chains in both rats and humans.27
Our results show that PPAR has an important role in mediating the
effects of fibrates on fibrinogen expression. Whereas several genes
involved in lipid metabolism, apo A-I, lipoprotein lipase, and acyl-CoA
synthetase, are positively regulated by PPAR,29-31 the
genes encoding the three fibrinogen chains are negatively regulated by
PPAR, like apo CIII.6 Although the coordinate suppression of gene transcription of the three fibrinogen chains by
fibrates would suggest a shared regulatory mechanism, the exact molecular mechanism by which PPAR acts is not understood. Further experiments, including functional analysis of the regulatory regions of
the genes encoding for the fibrinogen chains, will be necessary to
elucidate the precise mechanism of transcriptional repression of
fibrinogen gene expression by PPAR.
Fibrates are also implicated in suppressing elevated fibrinogen levels
under inflammatory conditions. Many reports link the inflammatory
mediator interleukin-6 (IL-6) to elevated fibrinogen expression,32,33 and indeed IL-6-responsive elements have
been identified in the promoters of the different rat and human
fibrinogen genes.34-36 Recent evidence indicates that
activated PPAR can interfere negatively with cytokine-induced
signaling pathways.11,37 It is thus conceivable that
PPAR also plays an important role in downregulating
cytokine-increased fibrinogen gene expression.
We found significantly higher basal plasma fibrinogen levels in
PPAR-null (/) mice than in wild-type (+/+) mice,
suggesting that PPAR is involved in modulating basal fibrinogen
expression. Several endogenous ligands have been identified such as
long chain fatty acids (palmitic acid, linoleic acid, arachidonic acid)
and eicosanoids [leukotriene B4, 8(S)-hydroxyeicosatetraenoic
acid],22,38 which could account for PPAR activation
under basal conditions. Therefore, changes in endogenous fatty acid
profiles as a result of changes in environmental and life-style factors
may explain reported intraindividual variation in fibrinogen levels of
about 10% to 15%.39-41 Similarly, the recent
identification of structural and functional polymorphisms in human
PPAR42 may be relevant for understanding regulation of
plasma fibrinogen levels. It would be interesting to delineate the role
of abnormal PPAR activity in patients with disturbed fibrinogen and
lipid levels by genetic linkage studies.
It is important to recognize that fibrates downregulate fibrinogen
expression via the same transcription factor as that identified for
reducing circulating triglyceride and cholesterol levels, ie, activated
PPAR. Other lipid lowering drugs such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) and
PPAR activators (thiazolidinediones) show no significant consistent effects on fibrinogen levels. For example, lovastatin therapy has
resulted in minor fibrinogen reductions in hypercholesterolemic patients41 or actually increased fibrinogen,43
while reductions were seen in a single reported study with pravastatin
therapy in familial hypercholesterolemia patients.44 In the
present study, we found no effect of the thiazolidinedione, BRL 49653, on plasma fibrinogen levels in rats, while triglyceride levels were
lowered to a similar extent as with fenofibrate. These results further
emphasize that the lowering effect of fibrates on fibrinogen are not
the result of lowered triglyceride levels. Because both elevated plasma
fibrinogen levels and elevated plasma lipids have been identified as
key risk factors for cardiovascular diseases,1 the
identification of a common, specific molecular target, PPAR, that is
suitable for application of modern drug discovery provides a new lead
for therapy. Such a novel compound specifically activating PPAR may
prove superior to existing fibrates, in the action of which other, as
yet unidentified molecular mechanisms are also involved.4,45
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Sabine Post for technical help with the rat
hepatocyte isolation and Karin Toet for technical assistance. We also
thank Farhad Rezaee for providing the mouse fibrinogen A-, B-,
and -chain cDNA probes.
| |
FOOTNOTES |
Submitted October 6, 1998; accepted December 29, 1998.
Supported by Grant No. NWO: 902-23-181 from the Netherlands
Organization for Scientific Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Teake Kooistra, PhD, Gaubius
Laboratory, TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, The
Netherlands.
| |
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ABSTRACT
INTRODUCTION