© 2000 American Heart Association, Inc.
Vascular Biology |
Differential Expression of Rat Frizzled-Related frzb-1 and Frizzled Receptor fz1 and fz2 Genes in the Rat Aorta After Balloon Injury
From INSERM U525, Hopital Saint-Louis (C.M., F.S.), and INSERM U460, Faculté Bichat (O.T.-B.M., M.E.P., P.G.S.), Paris, France. C.M. is presently at The Cleveland Clinic Foundation, Cell Biology Department, 9500 Euclid Ave, Cleveland, OH 44195.
Correspondence and reprint requests to Florent Soubrier, INSERM U525, Hopital Saint-Louis, 1 avenue Claude Vellefaux 75457 Paris cedex 10, France. E-mail soubrier{at}inserm.chu-stlouis.fr
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Abstract |
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Abstract—Frzb-1 is a secreted protein, presenting similarity with the Wnt-binding domain of the frizzled family of receptors, which acts as an antagonist of Wnt signaling. Using mRNA differential display in the rat aorta balloon injury model, we identified overexpression of Frzb-1 mRNA and determined its cDNA sequence. By quantitative reverse transcription–polymerase chain reaction and RNase protection assay, a biphasic upregulation of rFrzb-1 expression was observed, with significant peaks of a 1.7-fold increase at 4 days and a 1.5-fold increase at 3 weeks after aortic injury in vivo. In contrast, expression of the rat frizzled receptor genes rfz1 and rfz2 were transiently downregulated at 1 and 4 hours after balloon injury. rFrzb-1 was expressed predominantly in rat aortic smooth muscle cells (RASMCs) and barely in aortic fibroblasts and endothelial cells (RAECs), whereas rfz1 and rfz2 were expressed in all of these cells when stimulated with serum. Transient downregulation of rfz1 and rfz2 expression was reproduced by stimulation of quiescent RASMCs with serum, platelet-derived growth factor-BB, or fibroblast growth factor-2. In contrast, rFrzb-1 expression diminished slowly, to reach a 2-fold decrease 24 hours after growth factor stimulation, implying that quiescent RASMCs expressed higher levels of rFrzb-1 mRNA than did proliferative ones. Overexpression of rFrzb-1 in the aorta seemed to coincide with the arrest of RASMC proliferation occurring in the media 4 days and in the neointima 3 weeks after balloon injury. Our results demonstrate that rfrzb-1, rfz1, and rfz2 are differentially regulated in response to arterial injury and that this modulation seems to follow the proliferative state of RASMCs, suggesting that these Wnt-signaling components may be involved in intimal vascular disease.
Key Words: frizzled • vascular smooth muscle cells • vascular injury • gene expression • proliferation
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Introduction |
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Vascular smooth muscle cells (VSMCs), the predominant cell type in the tunica media of the mammalian vascular wall, maintain vascular tone by their contractile properties. Inappropriate VSMC activation plays a key role in the development of pathological processes characterized by intimal thickening, such as atherosclerosis, vascular rejection, and restenosis after angioplasty.1 2 In all of these vascular disorders, VSMCs display increased proliferation, migration, and synthesis of extracellular matrix (ECM) featuring different phenotypic "switches" mediated by changes in gene expression.3 4
In response to vascular injury, such as in the well-established model of rat carotid artery balloon injury,5 temporal and spatial changes in VSMC phenotype take place.6 7 Quiescent, contractile cells in the media of the vessel wall start proliferating and losing their differentiated, contractile phenotype 1 hour after injury and display a proliferative phenotype in the media for up to 3 days. From 3 to 14 days, VSMCs migrate from the media, through the internal elastic lamina, and into the intima, where a new proliferative wave is observed, as well as the synthesis of ECM components from 7 days up to at least 4 weeks after vascular injury. Although VSMCs with different phenotypes coexist in the injured artery, thus increasing the complexity of their analysis, in vivo models remain useful tools to study VSMC activation. To date, different growth factors, cytokines, chemokines, and ECM components have been identified that mediate or modify VSMC proliferation and/or migration in a paracrine or autocrine fashion.4 However, the factors and mechanisms controlling VSMC activation are not yet fully understood.
To identify genes that are associated with the modulation of VSMC phenotype after vascular injury and that are potentially causative, we applied mRNA differential display8 to the rat aorta balloon injury model.5 6 In the present study, we have identified the rat Frzb-1 because of its overexpression in the balloon-injured aorta. Frzb-1 is a secreted protein sharing high homology with the extracellular, cysteine-rich domain (CRD) of the frizzled family of proteins.9
The frizzled proteins are 7 transmembrane receptor types considered to be the Wnt receptor through binding with the CRD.9 10 11 12 With a similar CRD but without the membrane anchorage, Frzb-1 proteins are able to bind and inhibit Wnt in various models in vivo and in vitro.13 14 15 16 The Wnt pathway has been implicated in normal development through an effect on cell proliferation, migration, and differentiation.17 In view of the changes in Frzb-1 expression and the potential involvement of Wnt signaling in the cellular events occurring after arterial injury, we investigated the expression of the rat frizzled receptor genes, rfz1 and rfz2,18 in the balloon-injured rat aorta. To further determine the factors and cellular events that contribute to or are correlated with the modulation of rFrzb-1, rfz1, and rfz2 expression, we examined the effects of the mitogenic and/or chemotactic factors platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-22 in cultured rat aortic SMCs (RASMCs). Altogether, our results suggest that the Wnt-signaling components rFrzb-1, rfz1, and rfz2 are involved in the gene program of vascular wall remodeling after balloon injury.
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Methods |
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Materials
Radioactive products [
-32P]UTP
(800 Ci/mmol), [-35S]dATP (>1000
Ci/mmol), and [-33P]ddNTP
(1500 Ci/mmol) were obtained from Amersham.
Oligodeoxynucleotides and restriction enzymes were
purchased from Gibco-BRL. Recombinant human PDGF-BB and FGF-2 were
purchased from Sigma and Boehringer-Mannheim, respectively.
Aortic Balloon Injury
After undergoing pentobarbital anesthesia (0.06 mg/g
body weight, intraperitoneal), Wistar-Kyoto rats (Centre de
production animale, Olivet, France) weighing 
600 g underwent balloon
injury of the thoracic and abdominal aortas. A 2F Fogarty balloon
catheter (Baxter) was introduced via the carotid artery into the aorta
and then inflated and withdrawn 5 times. The rats were then killed by
pentobarbital overdose (10 mL 1% solution) at 0 (n=10), 1 hour (n=10),
4 hours (n=10), 2 days (n=10), 4 days (n=11), 7 days (n=10), 14 days
(n=10), 21 days (n=12), and 28 days (n=8) after balloon injury. Aortas
were immediately harvested and the adventitia removed before being
frozen in LN2.
Cell Fractionation and Culture
ASMCs, endothelial cells (ECs), and fibroblasts
were harvested from the aortas of male Wistar-Kyoto rats by enzymatic
dissociation as previously described.19 Primary cells were
grown on fibronectin-coated flasks for RAECs, on collagen I–coated
flasks for RASMCs, and on uncoated flasks for fibroblasts in
Dulbecco’s modified Eagle’s medium containing Glutamax supplemented
with 100 U/mL penicillin, 100 µg/mL streptomycin, 20 mmol/L
HEPES (pH 7.4; all from Gibco-BRL), and 10% FCS (Seromed). Purity of
cell preparations was confirmed by immunostaining with
specific antibodies against rat EC or SM -actin. Growth factor and
serum treatments were performed on subconfluent RASMCs at passage 2,
which were cultured in serum-free medium for 24 hours before further
treatment. For each treatment, 3 separate experiments were performed in
duplicate.
RNA Extraction
To minimize interanimal and procedural variability, 2 pools of
total RNA extracts (1 and 2) were prepared from 4 to 6 injured aortas
for each time point after balloon injury by
homogenization in TRIzol solution (Gibco-BRL).
Total RNAs from cultured RASMCs were isolated by direct lysis in
TRIzol. RNA concentration was calculated from the absorbance at 260 nm,
and RNA preparations were controlled by visualization of 28S and 18S
RNAs on agarose gel.
mRNA Differential Display Analysis
mRNA differential display was performed as previously
described8 on pool 1 RNA preparations from injured aortas
after elimination of DNA contamination by incubation with 5 U of
RQ1-DNase I (Promega). First-strand cDNA was synthesized from 200 ng of
DNase I–treated RNA, 20 pmol of oligo(dT) primer
5'-T12VG-3' (where V represent A, C, or
G), 25 µmol/L of each dNTP, and 200 U of SuperScript II reverse
transcriptase (Gibco-BRL) according to the manufacturer’s
recommendations. One microliter of such cDNA was used for polymerase
chain reaction (PCR) in the presence of 20 pmol of oligo(dT) primer
5'-T12VG-3', 10 pmol of SD4 decamer primer
5'-TTTTGGCTCC-3', 5 µmol/L dNTP, 3 µCi of
[-35S]dATP, and 2 U of AmpliTaq polymerase
(Perkin Elmer) for 40 cycles of the following steps: 94°C for 30
seconds, 40°C for 2 minutes, and 72°C for 1 minute. PCR
products were separated on a 6% DNA sequencing gel. After
autoradiography, differentially amplified bands were
excised and eluted from the gel and reamplified by using the same PCR
conditions and primers.
Cloning, Sequencing, and Homology Searching
PCR products were blunt-ended with 5 U of pfu polymerase,
ligated into the pCRScript cloning vector (Stratagene), and sequenced
in both directions by using universal -21M13 and REV ET dye primers
and the Thermosequenase dye-primer sequencing kit (Amersham) on an
ABI373 automatic sequencer. Sequence homology was obtained by BLAST
searching against the EMBL and GenBank databases.
Quantitative RT-PCR
Quantitative reverse transcription (RT)–PCR analysis
was done by coamplification of the tested cDNA and the internal cDNA
standard, rpL32. PCR primers were previously described for rat
gax20 and rat rpL3221 and were designed
according to the published sequences for rat fz1, rat
fz2,18 and the determined 4D4A sequence for rat
4D4A–rFrzb-1 (the Table
). RT-PCR was
applied to DNase I–treated RNA isolated from injured aortas or to the
total RNA isolated from RASMCs after various treatments. RNA (1 µg)
was subjected to RT in the presence of 200 U of SuperScript reverse
transcriptase and 10 pmol of oligo(dT)12–18
primer (Pharmacia). For each tested cDNA and rpL32 cDNA, initial
studies were done to determine the amount of cDNA, the annealing
temperature, and the number of cycles required to ensure that the
amplification was in the linear range. At the chosen annealing
temperatures (the Table), only 1 band was observed for each
amplified product, and its identity was confirmed by direct
sequencing. Standard conditions for PCR were performed with 1 µL of
cDNA (equivalent to 20 ng of RNA), 12 pmol of each sense and antisense
primer for the tested gene, 250 µmol/L dNTP, 3 µCi of
[-35S]dATP, and 2 U of Taq
polymerase (Gibco-BRL) with the following steps: 94°C for 30 seconds,
55°C to 58°C for 1 minute, and 72°C for 1 minute, repeated for 23
to 29 cycles as indicated in the Table. Primers for rpL32 (12
pmol each) were added during the PCR to maintain 21 PCR cycles. PCR
products were electrophoresed on native 7.5%
acrylamide gel and autoradiographed. Band intensities were
measured by using the Molecular Imager GS-525 system (Bio-Rad). The
ratios of the tested cDNA signal versus the rpL32 cDNA signal were
calculated for each coamplified sample and compared with the ratio
obtained for the control of the experiment (time 0=100%).
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RNase Protection Assay
For the generation of riboprobes, pCRScript vectors containing
the different PCR products, obtained with the primers described in
the Table
, were linearized, and these DNA templates were
transcribed and labeled in vitro with 50 µCi of
[-32P]UTP and either T3 or T7 RNA polymerase
(Promega). An RNase protection assay (RPA) was performed by using 5
µg of injured aorta total RNA and 2x105 counts
per minute of each labeled riboprobe as previously
described.22 Protected probes were separated on a 5%
acrylamide–8 mol/L urea acrylamide gel and
autoradiographed. Intensities of the protected bands were measured with
the Molecular Imager GS-525 system. For each time point, the intensity
ratio of the tested, protected band versus the rpL32 protected fragment
as the internal control was determined.
Statistical Analysis
Data from the semiquantitative RT-PCR and RPA are expressed as
mean±SE. Statistical comparisons were made by ANOVA (repeated measures
and Fischer’s protected least-squares difference), and values were
considered to be significant at P<0.05.
Cloning of Full-length 4D4A cDNA
The nucleotide sequence upstream from the initially
cloned 4D4A cDNA fragment was obtained by 2 cycles of rapid
amplification of the cDNA ends by using the 5'-RACE system (Gibco-BRL).
First-strand cDNA was synthesised from 1 µg of RASMC total RNA by
using the specific 4D4A antisense primer 5'-CTACTACCACATGGGTAAGC-3' and
the SuperScript II reverse transcriptase, and the full-length cDNA
4D4A, corresponding to the rat Frzb-1 cDNA, was obtained by 2 different
nested PCRs and sequenced. The primers used for this cloning are
available on request, and the sequence of Frzb-1 cDNA is available in
the EMBL database with the accession number AJ224337.
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Results |
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Identification of Differentially Expressed 4D4A cDNA Fragment After Aortic Balloon Injury
From the mRNA differential display performed with the SD4 decamer (5'-TTTTGGCTCC-3') and the oligo(dT) primer (5'-T12VG-3'), a band corresponding to the 4D4A DNA fragment with a marked increase of intensity at 4 days after injury was isolated (Figure 1A
). After
reamplification and cloning into the pCRScript vector, this cDNA
fragment of 331 bp was sequenced in both directions, and partial
homology with the 3' end of the murine frzb-1
cDNA13 was revealed by BLAST searching against
available databases. This partial homology was found as a 90%
nucleotide-sequence similarity on a 174-bp overlap (Figure 1B). This homology allowed us to orient the 4D4A DNA fragment in
the 5' to 3' direction (Figure 1B), and from this identified
sequence, 2 specific primers were designed (the Table and Figure 1B) that allowed measurement of 4D4A expression by quantitative
RT-PCR and RPA. The full-length cDNA corresponding to the 4D4A mRNA
species was cloned by 5'-RACE by using 2 nested PCRs, and a complete
4D4A cDNA sequence of 2512 bp was assembled, which corresponds to the
rat Frzb-1 (rFrzb-1) cDNA (EMBL accession number
AJ224337). Indeed, an overall 92% nucleotide-sequence
identity was observed with the murine Frzb-1 cDNA, and the
deduced 323–amino acid sequence represents 97.5% identity
with mouse Frzb-113 (not shown).
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Upregulation of 4D4A mRNA in Balloon-Injured Aortas
Quantitative RT-PCR analysis of 4D4A mRNA expression was
done by using specific primers (Figure 1B) and coamplification
of the standard rpL32 cDNA as an internal control in pools 1 and 2 of
DNase I–treated RNAs from rat aortas after balloon injury. In the
deendothelialized aorta (time 0), 4D4A mRNA was
already expressed, and upregulation of its expression was observed from
2 to 7 days after balloon injury, with a marked and significant
increase of 1.64-fold above baseline (P<0.01) at 4 days and
a 1.3-fold increase at 2 and 7 days after injury (Figures 2A and 2B). A second peak of increased
expression also appeared significant, with a 1.52-fold greater value 3
weeks after injury than at baseline (P<0.01). By RPA
(Figures 2C and 2D), which was performed with antisense
32P-labeled riboprobes to 4D4A and rpL32 mRNAs,
the overall curve of 4D4A mRNA expression at the various times after
arterial injury was similar to that obtained by
quantitative RT-PCR (Figures 2A and 2B). 4D4A mRNA expression
was upregulated 1.7-fold (P<0.01) 4 days after aortic
injury, and a second peak of increased expression, 1.55-fold above
baseline (P<0.06), was observed at 3 weeks.
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rfz1 and rfz2 Expression in
Balloon-Injured Aortas
To further investigate the involvement of Wnt-signaling components
in rat aorta balloon injury, expression of the frizzled rfz1
and rfz2 genes18 was analyzed.
Specific pairs of primers were designed for amplification of
rfz1 and rfz2 cDNA sequences (Table), and
RT-PCR products obtained from aortic RNA were sequenced to confirm
amplification specificity. As shown by the results of quantitative
RT-PCR analysis, deendothelialized
aortas expressed both rfz1 and rfz2 mRNAs, and
these basal levels were rapidly and transiently downregulated after
balloon injury (Figures 3A and 3B). The
nadir was reached 1 hour after injury, with a 1.67-fold
(P<0.05) decrease for rfz1 and a 1.6-fold
(P<0.01) decrease for rfz2 with respect to
baseline. At 4 hours after injury, recovery of the basal expression of
rfz1 and rfz2, respectively, was initiated with a
1.34-fold (P<0.05) and a 1.38-fold (P<0.05)
decrease below baseline. Of note, rapid and transient downregulation of
the growth-arrest gax gene after carotid injury has also
been described.23 Compared with the downregulation
observed for gax, with a 1.84-fold (P<0.01)
reduced level versus control, rfz1 and rfz2
downregulation displayed similar kinetics and levels (Figures 3A and 3B). From 2 days until 4 weeks after injury, rfz1 mRNA
expression was similar to that observed in the
deendothelialized aorta, whereas
rfz2 mRNA expression displayed a level slightly above
baseline, with a 1.4-fold increase (P<0.05) at 3 weeks
after injury. By RPA, rfz2 (Figure 3C) and
rfz1 (data not shown) variations in expression in the rat
aorta after balloon injury were confirmed.
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rFrzb-1, rfz1, and rfz2 Expression
in RASMCs
The pattern of rFrzb-1, rfz1, and
rfz2 expression in cells isolated from the aortic wall was
analyzed. Subconfluent RASMCs, fibroblasts, and RAECs were
serum deprived for 24 hours before stimulation with 10% serum. By
RT-PCR analysis, rFrzb-1 appeared to be expressed
predominantly in RASMCs, because its expression was undetectable in
fibroblasts and barely detectable in RAECs, even after increasing the
number of PCR cycles (30 instead of 26; Figure 4A). rfz1 and rfz2
were highly expressed in fibroblasts and RASMCs and to a lesser extent,
in serum-stimulated RAECs. In serum-stimulated RASMCs, expression of
rfz1 and rfz2 mRNAs first decreased at 8 hours
(3-fold, P<0.01) and then increased at 24 hours (1.5-fold,
P<0.01) compared with quiescent, serum-deprived cells
(Figures 4A and 4B), thereby reproducing the transient
downregulation observed in vivo (Figures 3A and 3B). In
contrast, rFrzb-1 expression remained downregulated 24 hours
after serum stimulation, with an expression level in quiescent,
serum-deprived RASMCs that was twice as high as in serum-stimulated
ones (Figure 4).
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Downregulation of rFrzb-1, rfz1, and
rfz2 Expression by PDGF-BB and FGF-2
To delineate more precisely which signals lead to the modulation
of expression of these genes, RASMCs were stimulated with either
PDGF-BB or FGF-2. Subconfluent RASMCs were serum deprived for 24 hours
before treatment, and rFrzb-1, rfz1, and rfz2
mRNA expression was determined by quantitative RT-PCR analysis
(Figure 5). With 20 ng/mL PDGF-BB, a
rapid and transient downregulation of rfz1 and
rfz2 expression was observed, with a maximum decrease of
2-fold (P<0.01) and 2.4-fold (P<0.01),
respectively, at 4 hours compared with serum-deprived cells. Similar
downregulation was observed for gax expression, with a
4.4-fold decrease induced by PDGF-BB at 4 hours. With 50 ng/mL FGF-2,
the extent of downregulation of rfz1 and rfz2
expression was different, with a 2.85-fold (P<0.01)
decrease for rfz2 and a slight 1.38-fold
(P<0.01) decrease for rfz1, 2 hours after
stimulation. gax expression was also less affected by FGF-2,
with only a 2.35-fold decrease at 4 hours (P<0.01). Slow
and progressive downregulation of rFrzb-1 mRNA expression
reached a 2-fold decrease at 24 hours with either PDGF-BB or FGF-2. No
significant difference in the regulation of rFrzb-1
expression was observed between the various factors used: serum,
PDGF-BB, and FGF-2.
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Discussion |
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In the present report, we describe the modulation of expression of genes belonging to the Wnt-signaling pathway in the rat arterial wall after in vivo injury and during proliferation of RASMCs in vitro: the frizzled-related rFrzb-1 gene and the frizzled rfz1 and rfz2 genes.18 The frizzled proteins are 7 transmembrane receptor types with an extracellular CRD,9 11 12 first identified as a regulator of hair polarity in Drosophila.10 Various studies have now demonstrated the ability of frizzled proteins to bind members of the Wnt family through the CRD and to elicit Wnt-dependent biological responses in vitro and in vivo.11 12 24 Frzb-1 proteins bearing a similar frizzled CRD are able to bind Wnt and to antagonize its biological activity in vitro and in vivo during Wnt-dependent ventral axis formation in the Xenopus embryo.13 14 The Wnt-signaling cascade has a crucial role in the control of cell-cell junctions and ECM-cell adhesion, probably via modulation of ß-catenin intracellular levels.9 17 Our results might suggest that at least part of the VSMC phenotypic changes occurring after vascular injury1 are regulated by components of the Wnt-signaling cascade.
Using mRNA differential display8 in the rat aorta balloon
injury model,5 we detected overexpression of the rat
Frzb-1 gene (Figure 1) and cloned its complete cDNA.
Upregulation of rFrzb-1 expression at 4 days and 3 weeks
after aortic balloon injury compared with the
deendothelialized aorta was confirmed by
quantitative RT-PCR and RPA (Figure 2). Analysis of
rFrzb1 expression in the different cell types isolated from
the aorta confirmed that rFrzb-1 expression in the
arterial wall reflects its expression in VSMCs. Indeed, in
contrast to its barely detectable expression in fibroblasts and RAECs
in culture, rFrzb-1 mRNA was found at relatively high levels
in RASMCs (Figure 4).
Cellular events known to occur in VSMCs after vascular injury have been
shown to be temporally and spatially regulated. At 4 days after injury,
medial VSMCs stop proliferating and start migrating toward the intima,
whereas at 3 weeks, most VSMCs in the neointima have
stopped proliferating except for those localized at the luminal surface
of the vessel wall.7 To distinguish whether
rFrzb-1 upregulation was correlated with cell arrest,
proliferation, or migration, in vitro studies were carried out on
RASMCs at a very early passage (passage 2) and cultured on collagen I.
In vitro, PDGF-BB has been shown to be a potent mitogen and chemotactic
factor for RASMCs, whereas FGF-2 displays mainly a
mitogenic effect.2 Regardless of the
stimulus used, a similar decrease of rFrzb-1 expression was
observed after treatment compared with quiescent, serum-deprived RASMCs
(Figures 4 and 5). Upregulation of rFrzb-1 in
the balloon-injured rat aorta was observed at 4 days and 3 weeks after
injury, when the arrest of VSMC proliferation is observed in the media
and in the neointima, respectively. Although these
observations were made at different time scales and under different
experimental conditions, they are both compatible with a decrease of
rFrzb-1 expression in proliferative RASMCs and an increase
of rFrzb-1 expression in more quiescent
cells.7
Although a 2-fold downregulation of rFrzb-1 was observed in
vitro when RASMCs become proliferative, a clear rFrzb-1
downregulation was not observed in vivo in the first hours after
injury, when proliferating VSMCs in the media
appeared.7 Only a faint and nonsignificant decrease
(20%) of rFrzb-1 expression was seen 1 hour after injury
(Figure 2). This finding can be explained by the proportion of
nonproliferating versus proliferating cells in the media after injury,
since only a subset of VSMCs undergoes
proliferation25 and thus, likely blunts the apparent
downregulation of rFrzb-1 expression. Alternatively, a
factor inducing rFrzb-1 expression might appear
concomitantly, and then the apparent level would reflect the
predominance of the inducer signal. These 2 explanations are not
mutually exclusive, and it will be of a great interest to identify such
rFrzb-1 inducer signals.
Frizzled-related proteins form an emerging family of secreted factors that already includes 8 members identified in different species.9 17 Frizzled-related proteins share relatively high homologies, with 40% to 50% identity in their amino acid sequence, and are involved in development, like Frzb-1,13 14 or in the control of cell proliferation, like DDC-426 and the secreted apoptosis-related proteins (SARPs).27 Expression of the rat DDC-4 gene was associated with apoptosis,26 and the murine SARP1, displaying higher expression in quiescent, mouse embryonic cells than in proliferating ones, has been shown to possess antiapoptotic activity by reducing cell sensitivity to tumor necrosis factor.27 SARP2, another member of the frizzled-related protein family, had an opposite effect.27 Evidence of VSMC apoptosis has been demonstrated in vivo in the rat carotid artery angioplasty model, in the media from 1 hour after injury,28 and in the neointima from 7 to 30 days after injury.29 30 Bennett et al31 have shown that human coronary VSMCs undergo apoptosis under serum-deprived culture conditions. In view of all these results and of our results on rFrzb-1 expression in RASMCs in vivo and in vitro, it will be of interest to look for its possible effects either on apoptosis or on the inhibition of cell proliferation.
To further investigate possible involvement of the Wnt-signaling
cascade in response to vascular injury, we studied the expression of
the rfz1 and rfz2 frizzled
genes.18 They are expressed in the
deendothelialized rat aorta and in cultured
RASMCs as well as in proliferating fibroblasts and RAECs, whereas
rFrzb-1 expression was more specific to RASMCs (Figures 3 and 4). In contrast to rFrzb-1, a rapid and
transient downregulation of rfz1 and rfz2
expression was observed during the first hours after balloon injury.
The decrease was 1.7-fold at the nadir, and recovery of the initial
levels of rfz1 and rfz2 expression was completed
2 days after injury (Figure 3). Interestingly, rfz1
and rfz2 represent a new set of genes whose
expression is rapidly downregulated after arterial trauma,
and which encode for receptors involved in the control of cell-cell and
ECM-cell contacts.9 17 The growth arrest gene
gax, which codes for a transcription factor containing a
homeobox domain,20 was the first gene identified that
displayed such regulation in a rat carotid injury model.23
Our results show that rfz1, rfz2, and
gax expression undergoes similar downregulation in the aorta
after balloon injury (Figure 4) and in RASMCs stimulated with
serum and PDGF-BB (Figure 5). This suggests that downregulation
of these genes is triggered by the same signals that induce aortic SMC
proliferation in vivo. Because rfz1 and rfz2 are
involved in the control of cell-cell and ECM-cell interactions,
distension of the vessel wall after balloon injury may be the common
signal for downregulation of rfz1, rfz2, and
gax expression.
Another intriguing feature is the secondary weak increase of
rfz2 but not of rfz1 expression observed after
arterial injury. Although an increase in rfz2
expression appeared significant only 3 weeks after injury, its overall
level of expression from 2 days to 4 weeks was slightly above basal
level, a phenomenon that was not observed for rfz1 (Figure 3). Proliferating RAECs also expressed rfz2 (Figure 4), but overexpression of rfz2 3 weeks after injury
is unlikely owing to endothelium regeneration, since
maximum endothelial proliferation is known to occur
beyond 4 weeks after injury.7 Recently,
rfz2 upregulation has been reported in
cardiomyocytes during development of left
ventricular hypertrophy32 and
in myofibroblasts in infarcted rat heart.33 The latter was
correlated with myofibroblast migration and proliferation around the
infarcted area, and in this case also, rfz1 expression was
not increased. After arterial wall injury, the
neointima displays some characteristics of chronic,
fibroproliferative lesions due to VSMCs.1 Our results
on rfz2 upregulation in the arterial wall after
injury are thus in agreement with those obtained in other
cardiovascular disorders involving fibroproliferative
repair, suggesting that rfz2, on the basis of frizzled
effects on polarity in Drosophila,10 34 may be
important for VSMC alignment during vascular remodeling.
Interestingly, our results show that rFrzb-1, rfz1, and rfz2 are differentially regulated in vivo after arterial injury and in subconfluent proliferating RASMCs in vitro, inducing modulations of the balance between factors displaying putative antagonist effects. Altogether, these expression studies, as well as the emerging evidence on the crucial role of the frizzled-related proteins and of the frizzled receptor families in the control of cell proliferation and/or apoptosis, strengthen the biological relevance of the differential modulation of rFrzb-1, rfz1, and rfz2 expression during arterial wall remodeling after injury. Future investigations will determine whether Wnt-signaling components play a key role in vascular wall pathology and morphogenesis and whether they may constitute novel therapeutic targets for vascular disorders.
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Acknowledgments |
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This work was supported by Institut National de la Recherche Medicale. O. Tahlil-Ben Malek was supported by a grant from Bioavenir (Universite Paris 7/Rhone-Poulenc Rorer-Gencell), and C. Mao received support from Merck-Sharp-Dohme-Chibret.
Received May 12, 1999; accepted July 8, 1999.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Ross R. The pathogenesis of atherosclerosis. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
2.
Schwartz SM, deBlois D, O’Brien ERM. The intima, soil
for atherosclerosis and restenosis. Circ
Res. 1995;77:445–465.
3.
Owens GK. Regulation of differentiation of vascular
smooth muscle cells. Physiol Rev. 1995;75:487–517.
4. Tanaka H, Suzuki A, Schwartz D, Sukhova G, Libby P. Activation of smooth muscle and endothelial cells following balloon injury. Ann N Y Acad Sci. 1995;748:526–529.[Medline] [Order article via Infotrieve]
5. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208–216.[Medline] [Order article via Infotrieve]
6. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
7. Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury. Lab Invest. 1985;52:611–616.[Medline] [Order article via Infotrieve]
8.
Liang P, Pardee AB. Differential display of
eukaryotic messenger RNA by means of the polymerase chain
reaction. Science. 1992;257:967–971.
9. Wordarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol. 1998;14:59–88.[Medline] [Order article via Infotrieve]
10. Vinson CR, Conover S, Adler PN. A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains. Nature. 1989;338:263–264.[Medline] [Order article via Infotrieve]
11. Bhanot P, Brink M, Harryman-Samos C, Hsieh JC, Wang Y, Macke JP, Andrew D, Nathans J, Nusse R. A new member of the frizzled family from Drosophila functions as a wingless receptor. Nature. 1996;382:225–230.[Medline] [Order article via Infotrieve]
12. Yang-Snyder J, Miller JR, Brown JD, Lai CJ, Moon RT. A frizzled homolog functions in a vertebrate Wnt-signaling pathway. Curr Biol. 1996;6:1302–1306.[Medline] [Order article via Infotrieve]
13. Leyens L, Bouwmeester T, Kim SH, Piccolo S, DeRobertis EM. Frzb-1 is a secreted antagonist of wnt-signaling expressed in the Spemann organizer. Cell. 1997;88:747–756.[Medline] [Order article via Infotrieve]
14. Wang W, Krinks M, Lin K, Luyten FP, Moos M Jr. Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell. 1997;88:757–766.[Medline] [Order article via Infotrieve]
15.
Finch PW, He X, Kelley MJ, Uren A, Schaudies RP,
Popescu NC, Rudikoff S, Aaranson SA, Varmus HE, Rubin JS. Purification
and molecular cloning of a secreted, Frizzled-related
antagonist of Wnt action. Proc Natl Acad Sci
U S A. 1997;94:6770–6775.
16.
Lin K, Wang S, Julius MA, Kitajewski J, Moos MJ, Luyten
FP. The cysteine-rich frizzled domain of Frzb-1 is required and
sufficient for modulation of Wnt-signaling. Proc Natl Acad Sci
U S A. 1997;94:11196–11200.
17. Dale TC. Signal transduction by the Wnt family of ligands. Biochem J. 1998;329:209–223.
18.
Chan SDH, Karpf DB, Fowlkes ME, Hooks M, Bradley MS,
Vuong V, Bambino T, Liu MYC, Arnaud CD, Strewler GJ, Nissenson RA. Two
homologs of the Drosophila polarity gene frizzled (fz) are
widely expressed in mammalian tissues. J Biol Chem. 1992;267:25202–25207.
19. Battle T, Arnal JF, Challah M, Michel JB. Selective isolation of rat aortic wall layers and their cell types in culture: application to converting enzyme activity measurement. Tissue Cell. 1994;26:943–955.[Medline] [Order article via Infotrieve]
20.
Gorski DH, LePage DF, Patel CV, Copeland NG, Jenkins
NA, Walsh K. Molecular cloning of a diverged homeobox that is rapidly
down-regulated during the G0/G1 transition in vascular smooth muscle
cells. Mol Cell Biol. 1993;13:3722–3733.
21. Wang X, Louden C, Ohlstein EH, Stadel JM, Gu JL, Yue TL. Osteopontin expression in platelet-derived-growth factor–stimulated vascular smooth muscle cells and carotid artery after balloon angioplasty. Circ Res. 1996;16:1365–1372.
22.
Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K,
Green MR. Efficient in vitro synthesis of biologically active RNA and
RNA hybridization probes from plasmids containing a bacteriophage SP6
promoter. Nucleic Acids Res. 1984;12:7035–7056.
23.
Weir L, Chen D, Pastore C, Isner JM, Walsh K.
Expression of gax, a growth arrest homeobox gene, is rapidly
down-regulated in the rat carotid artery during the proliferative
response to balloon injury. J Biol Chem. 1995;270:5457–5461.
24.
He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I,
Varmus HE. A member of the frizzled protein family mediating axis
induction by Wnt-5A. Science. 1997;275:1652–1654.
25.
Lindner V, Giachelli CM, Schwartz SM, Reidy MA. A
subpopulation of smooth muscle cells in injured rat arteries expresses
platelet-derived growth factor-B chain mRNA. Circ Res. 1995;76:951–957.
26. Wolf V, Ke G, Dharmarajan AM, Bielke W, Artuso L, Saurer S, Friis R. DDC-4, an apoptosis-associated gene, is a secreted frizzled relative. FEBS Lett. 1997;417:385–389.[Medline] [Order article via Infotrieve]
27.
Melkonyan HS, Chang WC, Shapiro JP, Mahadevappa M,
Fitzpatrick PA, Kiefer MC, Tomei LD, Umansky SR. SARPs: a family of
secreted apoptosis-related proteins. Proc Natl Acad Sci
U S A. 1997;94:13636–13641.
28.
Perlman H, Maillard L, Krasinski K, Walsh K. Evidence
for the rapid onset of apoptosis in medial smooth muscle cells
after balloon injury. Circulation. 1997;95:981–987.
29. Han DK, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau B. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995;147:267–277.[Abstract]
30. Bochaton-Piallat M, Gabbiani F, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1059–1064.[Abstract]
31.
Bennett MR, Evan GI, Newby AC. Deregulated
expression of the c-myc oncogene abolishes inhibition of
proliferation of rat vascular smooth muscle cells by serum reduction,
interferon-
, heparin and cyclic nucleotide analogues and
induces apoptosis. Circ Res. 1994;74:525–536.
32. Blankesteijn WM, Essers-Janssen YP, Ulrich MM, Smits JM. Increased expression of a homologue of Drosophila tissue polarity gene frizzled in the left ventricular hypertrophy in the rat as identified by subtractive hybridization. J Mol Cell Cardiol. 1996;28:1187–1191.[Medline] [Order article via Infotrieve]
33. Blankesteijn WM, Essers-Janssen YP, Verluyten MJA, Daemen MJAP, Smits JM. A homologue of Drosophila tissue polarity gene frizzled is expressed in migrating myofibroblasts in the infarcted rat heart. Nat Med. 1997;3:541–544.[Medline] [Order article via Infotrieve]
34. Adler, PN, Krasnow RE, Liu J. Tissue polarity points from cells that have higher frizzled levels towards cells that have lower frizzled levels. Curr Biol. 1997;7:940–949.[Medline] [Order article via Infotrieve]
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