© 1999 American Heart Association, Inc.
Original Contribution |
Tranilast Inhibits Vascular Smooth Muscle Cell Growth and Intimal Hyperplasia by Induction of p21waf1/cip1/sdi1 and p53
From the First Department of Internal Medicine (A.T., T.T., M.Y.), Kobe University School of Medicine, Chuo-ku, Kobe, and the Faculty of Health Science (Y.I.), Suma-ku, Kobe, Japan.
Correspondence to Takahiro Taniguchi, MD, PhD, First Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan. E-mail taniguch{at}med.kobe-u.ac.jp
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Abstract |
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Abstract—Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC50 value, evaluated as cell number, was 100 µmol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21waf1/cip1/sdi1 (p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G1/S transition. Tranilast suppressed their activities by >80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (P<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.
Key Words: angioplasty • restenosis • signal transduction • carotid arteries • smooth muscle
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Introduction |
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Proliferation and migration of vascular smooth muscle cells (SMCs) play a pivotal role in the development of restenosis and in the progression of atherosclerosis.1 Arterial injury results in the migration of SMCs into the intimal layer of the arterial wall, where they proliferate and synthesize extracellular matrix components. Many growth factors induce the proliferation of vascular SMCs in vitro and in vivo. Among them, platelet-derived growth factor (PDGF) and basic fibroblast growth factor are important regulators of SMC behavior through their well-defined actions as potent chemoattractants and strong mitogens. Administration of these growth factors enhances intimal thickening after angioplasty in the rat, whereas injection of antibodies or use of antisense technology to block signal transduction by either of these growth factors potently inhibits postinjury intimal hyperplasia in the rat and restenosis in the pig, suggesting that SMC growth plays an important role in these pathogenesis.
Progression through the mammalian mitotic cycle is controlled by multiple holoenzymes comprising a catalytic cyclin-dependent kinase (CDK) and a cyclin regulatory subunit. Functional CDK/cyclin holoenzymes are presumed to phosphorylate target protein substrates that facilitate cell cycle progression.2 3 4 Different CDK/cyclin complexes are activated in order at specific phases of the cell cycle. Progression through the first gap phase (G1) requires both cyclin D–dependent CDK4/CDK6 and CDK2/cyclin E holoenzymes.5 Functional CDK2/cyclin A and p34cdc2 kinase (CDC2)/cyclin B pairs are assembled and activated during the second gap (G2) and mitosis (M) phases, respectively. Recent evidence has suggested that CDK2 is required for entry into mitosis as a positive regulator of CDC2/cyclin B kinase activity.6 Moreover, cyclin D/CDK complexes can phosphorylate the retinoblastoma gene product (pRb) in vitro, which suggests that these complexes may directly regulate the phosphorylation of pRb in response to growth factor stimulation.7 The kinase activity of these cyclins/CDK complexes can be negatively regulated by CDK-inhibitory proteins, including p16INK (p16), p21waf1/cip1/sdi1 (p21), and p27kip1 (p27).8 9 It has become apparent that p21 is a universal inhibitor of cyclin/CDK catalytic activity.10 11 12 Several lines of evidence suggest that p21 expression is regulated by the p53 tumor-suppressor protein. The p21 gene has a p53 transcriptional regulatory motif, and cells lacking p53 express very low levels of p21. These findings have led to a model in which p21 serves as an effector of cell cycle arrest in response to activation of the p53 checkpoint pathway. However, p21 is also upregulated through p53-independent mechanisms as in the following situations: normal tissue development, cell differentiation, serum stimulation, and treatment with transforming growth factor-ß and prostaglandin A2.13 14 15 Although p21 monomers can associate with active cyclin D/CDK complexes in proliferating fibroblasts,16 overexpression of p21 has been shown to inhibit the kinase activities of these cyclin/CDK complexes and to arrest the cells in the G1 phase of the cell cycle.17 Therefore, p21 may inhibit cell cycle progression by inhibiting the cyclin/CDK–dependent phosphorylation of pRb.
Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid, is generally used as an antiallergic drug. This effect is thought to result from an inhibition of the release of chemical mediators from mast cells and basophils.18 Recently, it has been reported that tranilast markedly inhibits the proliferation and migration of SMCs as well as collagen synthesis by these cells.19 20 Furthermore, a double-blind, large-scale, multicenter trial demonstrated the potent preventive effect of tranilast on restenosis (restenosis rate 14.7% [tranilast 600 mg/day for 3 months, n=68] versus 46.5%, placebo, n=71; [P<0.001]) after percutaneous transluminal coronary angioplasty.21 Tranilast antagonizes angiotensin II,22 restores cytokine-induced NO production against PDGF,23 and inhibits calcium entry in SMC.24 Moreover, tranilast suppresses intimal hyperplasia after photochemically induced endothelial injury in the rat.25 However, the precise mechanisms by which this drug inhibits SMC growth remains obscure. The purpose of the present study was to determine the effect of tranilast on PDGF- and FBS-induced SMC growth, the mechanism of this effect, and intimal hyperplasia after balloon injury of the rat carotid artery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tranilast was obtained from Kissei Pharmaceutical Co Ltd and was dissolved in DMSO; recombinant human PDGF-BB; and insulin, transferrin, and selenium (ITS Premix) from Collaborative Biomedical Products. [
-32P]ATP (4500 Ci/mmol) was obtained from
ICN Biochemical. The concentration of protein in cell culture was
determined using the Bradford microassay procedure using a reagent from
Bio-Rad. The following antibodies were used in the present study.
Anti–cyclin D1 (monoclonal, 72-13G), anti–cyclin E (rabbit
polyclonal, M20), anti-CDK2 (rabbit polyclonal, M-2), anti-CDK4 (rabbit
polyclonal, C-22), anti-p21 (rabbit polyclonal, C-19), and anti-pRb
(rabbit polyclonal, C-15) antibodies were purchased from Santa Cruz
Biotechnology. Anti-human p21 mixed mouse monoclonal IgGs were obtained
from Upstate Biotechnology Inc. Anti-pRb (monoclonal, G-3-245) was from
Pharmingen. Anti-p53 antibodies (monoclonal, Ab-1, and sheep
polyclonal, Ab-7) from Calbiochem. Rabbit nonimmune IgG was purchased
from DAKO. Rabbit anti-mouse IgG antibody from Jackson ImmunoResearch
Laboratories, Inc, was coupled to protein A-Sepharose when monoclonal
antibody was used for immunoprecipitation. Histone
H1 was from Life Technologies, Inc. All other
chemicals and reagents were obtained from either Wako Pure Chemical
Industries or Sigma.
Cell Culture
DMEM and trypsin were obtained from Life Technologies, Inc, and
FBS and penicillin-streptomycin mixture from BioWhittaker. SMCs were
isolated from rat thoracic aorta by enzymatic dissociation as described
by Gunther et al.26 Cells were grown in DMEM supplemented
with 10% heat-inactivated FBS, 100 U/mL penicillin, and
100 µg/mL streptomycin. They were passaged twice weekly by harvesting
with trypsin/EDTA and seeding at a 1:5 ratio in flasks with a volume of
75 cm2 (Corning). Cells grown between
passages 8 and 15 were used for experiments.
Quantification of Cell Number
Cell number was measured using the crystal violet staining
method described before.27 Briefly, SMCs were suspended in
DMEM containing 0.5% FBS and seeded into each well of a 96-well plate
(Falcon, Becton Dickinson) at a density of
4x103/well. After cell attachment, the medium
was replaced with DMEM containing either 10% FBS or 2% FBS, 10 ng/mL
of PDGF, and ITS Premix in the presence or absence of known
concentrations of tranilast. After 3 days of incubation, the cells were
fixed with 10 µL of 10% glutaraldehyde, stained with
0.1% crystal violet solution at pH 6.0, and dissolved with 10% acetic
acid. The wells were read at 590 nm in a microplate reader (model 3550;
Bio-Rad). A calibration curve was drawn on the basis of a known number
of cells.
Immunological Analysis (Western Immunoblot
and Immunoprecipitation)
Cell lysates were prepared with ice-cold lysis buffer containing
(in mmol/L) Tris-HCl (pH 7.4) 50, NaCl 250, EDTA 5, NaF 10, DTT
0.1, phenylmethylsulfonyl fluoride 1, and leupeptin 0.1; 0.1%
NP-40; and 10 µg/mL aprotinin. Immunoprecipitation was performed by
incubating the cell lysates obtained from one 60-mm dish with 1 µg of
specific antibodies against pRb, p21, p53, cyclins, and CDKs. To
precipitate immune complexes, protein A-Sepharose (30 µL of 50%
slurry) was added and gently rotated for 1 hour at 4°C.
Immunoprecipitates were washed 4 times with lysis buffer and incubated
with 30 µL of SDS-PAGE sample buffer for 5 minutes at 100°C.
Samples were briefly spun down, and the protein in the supernatants was
electrophoresed on polyacrylamide gel and transferred to a PVDF
membrane (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat
dry milk in PBS containing 0.1% Tween-20, and
immunoblotting was performed with antibodies at 1
µg/mL. Positive antibody reactions were visualized using
peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cappel
Research Products). The peroxidase reaction was developed using an
enhanced chemiluminescence detection system (ECL, Amersham Corp).
Immune Complex Kinase Assays
Cell lysates were prepared with ice-cold lysis buffer
(containing, in mmol/L, HEPES [pH 7.5] 50, NaCl 150, EDTA 1,
EGTA 2.5, DTT 1, ß-glycerophosphate 10, NaF 1,
Na3VO4 0.1, and
phenylmethylsulfonyl fluoride 0.1 and 10% glycerol, 0.1%
Tween-20, 10 µg/mL of leupeptin, and 2 µg/mL of aprotinin) and
sonicated at 4°C (Micro ultrasonic cell disrupter [from
KONTES], 30% power, 2 times for 10 seconds each time). Lysates
were clarified by centrifugation at 10 000g
for 5 minutes, and the supernatants were precipitated for 2 hours at
4°C with protein A-Sepharose beads precoated with saturating amounts
of indicated antibodies. When monoclonal antibodies were used, protein
A-Sepharose was pretreated with rabbit anti-mouse immunoglobulin G
(Jackson ImmunoResearch Laboratories). Immunoprecipitated proteins on
beads were washed 4 times with 1 mL of lysis buffer and twice in a
kinase buffer (containing, in mmol/L, HEPES 50,
MgCl2 10, DTT 1, ß-glycerophosphate 10, NaF 1,
and sodium orthovanadate 0.1). The final pellet was resuspended in 25
µL of kinase buffer containing either 1 µg of glutathione
S-transferase (GST)–pRb C-terminal (pRb amino acids
769 to 921) fusion protein (Santa Cruz Biotechnology) or 5 µg of
histone H1 (Life Technologies, Inc.), 20
µmol/L ATP, and 5 µCi of [-32P]ATP (4500
Ci/mmol; ICN) and incubated for 20 minutes at 30°C with occasional
mixing. The reaction was stopped by addition of 25 µL of 2x
concentrated Laemmli sample buffer and separated on 10% or 12.5%
SDS-polyacrylamide gels. The migration of histone
H1 or GST-pRb were determined by Coomassie blue
staining. Phosphorylated pRb and histone
H1 were visualized and quantified with a BAS 2000
bioimaging analyzer.
Balloon Injury and Morphometric Analysis of Intimal
Thickening
All experiments were performed in accordance with a protocol
approved by the Guidelines for Animal Experimentation at Kobe
University School of Medicine. Male Sprague-Dawley rats weighing
200 to 250 g were divided into 2 groups (n=8 per group) and fed
with powdered chow. One group received 10 g of tranilast/kg chow
supplement, whereas the diet of the other group was unchanged
(control). After 3 days, all animals received balloon injury to the
left main carotid artery. After anesthetization with sodium
pentobarbital (50 mg/kg body weight IP; Abbott Laboratories), the right
iliac artery was exposed through a midline incision, a 2F embelectomy
catheter (Baxter Edwards Healthcare Corp) was introduced into the left
common carotid artery, and the balloon was inflated with saline and
drawn toward the arteriotomy site 5 times to produce a distending and
de-endothelializing injury.28 The right
uninjured carotid artery was used as control tissue. At the indicated
days after injury, rats were euthanized with sodium pentobarbital
(intraperitoneal injection, 100 mg/kg body weight)
and then perfused with saline followed by 10% formalin at
physiological pressure. For immunohistochemistry
and morphometric analysis, the arteries were fixed in 100%
methanol overnight, and then the middle one third of the common carotid
artery was cut into 4 segments and embedded in paraffin. The specimens
were cross-sectioned at a thickness of 3 µm and stained with
hematoxylin and eosin. For immunohistochemical staining, sections were
deparaffinized and rehydrated with PBS. Primary p21 antibody (Santa
Cruz Biotechnology) was diluted 1:500 and incubated for 20 minutes at
37°C. Staining was performed using a labeled streptavidin biotin kit
according to the recommendations of the manufacturer (DAKO Corp).
Sections were counterstained with hematoxylin. Intimal and medial
cross-sectional areas of 4 cross sections of the artery obtained from
each rat were measured. The intima/media cross-sectional area
ratios were determined using a computerized apparatus and
NIH Image software (version 1.57).
Statistics
The data were analyzed using either the Student
t test or 1-way ANOVA followed by the Fisher test.
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Results |
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Effect of Tranilast on Proliferation of Rat SMCs
We first investigated the effect of tranilast on SMC proliferation in the presence of 10% FBS. Tranilast inhibited FBS-induced SMC proliferation in a dose-dependent manner. The IC50 value, evaluated as cell number, was 100 µmol/L, and complete inhibition was observed at 300 µmol/L (data not shown). We then examined the effect of tranilast on the early signal-transduction pathway by PDGF-BB stimulation using the following parameters: active GTP-bound p21ras ratios and p42mapk, p44mapk, phosphatidylinositol 3-kinase, and p70 S6 kinase activities. However, all of these early signal-transduction pathways were not significantly affected by tranilast treatment (data not shown).
Effect of Tranilast on PDGF- and FBS-Induced
Phosphorylation of pRb
Next, we examined the effects of tranilast on the
phosphorylation status of pRb in SMCs. Since the
mobility shift of pRb to phosphorylated form began at
16 hours after stimulation and was sustained until 24 hours (data not
shown), we examined the effect of tranilast on pRb
phosphorylation at 18 hours after stimulation.
Tranilast (300 µmol/L) markedly inhibited the
phosphorylation of pRb by stimulation with both 10%
FBS and PDGF (Figure 1
),
suggesting that tranilast blocked a G1 event that
preceded pRb phosphorylation.
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Effect of Tranilast on FBS- and PDGF-Induced Activation of CDK2 and
CDK4 Activity
To elucidate the mechanism by which tranilast inhibits pRb
phosphorylation, we measured the activities of CDK2 and
CDK4, because pRb is the putative substrate for CDK2 and CDK4, which
are activated during G1 phase. We
measured the activities of CDK2 and CDK4 stimulated by PDGF, 2% FBS,
and ITS (Figure 2A). The CDK2 and CDK4
activities were low in the G0 and early
G1 phases. They began to increase at 12 hours
after stimulation, continued to increase until 24 hours, and declined
at 30 hours. Tranilast (200 µmol/L) added 1 hour before PDGF
stimulation suppressed the activities of both CDK2 and CDK4 measured at
18 and 24 hours. At 18 hours, tranilast (200 µmol/L) suppressed
CDK2 and CDK4 activities by 80% and 60%, respectively. To examine the
dose effect of tranilast on activities of CDK2, CDK4, and cyclin E–
and cyclin D1–associated kinases, we measured the kinase activities of
their immunoprecipitates at 18 hours after 10% FBS or PDGF
stimulation. Treatment of SMCs with 10% FBS or PDGF resulted in an
increase of CDK2 activity by 20- or 10-fold, respectively. Tranilast
(100 to 300 µmol/L) inhibited the kinase activities of both
anti-CDK2 and anti-CDK4 immunoprecipitates in a dose-dependent manner
(IC50 value was 
150 µmol/L). The cyclin
D1– and cyclin E–associated kinase activities were also inhibited by
tranilast treatment, although they were less marked than
anti-CDK2/anti-CDK4–immunoprecipitated kinase activities (Figure 2B).
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To examine the possibility that tranilast could affect CDK and cyclin
expression, we tested it by Western blotting (Figure 2C
).
Mitogenic stimulation induced an elevation of cyclin D1 but
did not alter the expression of CDK2, CDK4, and cyclin E. Tranilast
(100 to 300 µmol/L) did not alter the protein levels of CDK2,
CDK4, cyclin E, and cyclin D1 in cells exposed to 10% FBS or PDGF.
Effect of Tranilast on the Expression of p21 and p53
Because p21 inhibits the activities of cyclin E/CDK2 and cyclin
D1/CDK4 complexes, we investigated the effect of tranilast on p21
expression by PDGF. In control cells, Western blotting showed a
considerable amount of p21 protein in quiescent SMCs (serum depleted
for 2 days). The expression of p21 increased transiently at 16 hours
after PDGF stimulation and decreased at 24 hours. In the presence of
tranilast (300 µmol/L), there was little decrease in the amount
of protein, resulting in continuous and elevated expression throughout
the observed period; amount of p21 increased >4-fold (by densitometric
analysis) at 24 hours after PDGF stimulation compared with that
of control cells (Figure 3). To test
whether tranilast induces p21 through the induction of p53, we examined
the effect of tranilast on the expression of p53. The level of p53
increased after PDGF stimulation, but this increase was not so marked
compared with the increase of p21 by tranilast treatment (Figure 3).
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Association of p21 With CDK2, CDK4, and Cyclins
To further explore whether the observed inhibition of CDK2 and
CDK4 activities were attributable to the association of p21 with CDK2
and CDK4, we immunoprecipitated p21 from the lysates of cells at the
indicated times in the presence or absence of tranilast (300
µmol/L). Figure 4B reveals that CDK2
was coimmunoprecipitated by anti-p21 antibody, indicating the
association of them in vitro. In this experiment, a slightly faster
migrating band was detected. This band (faster migrating) was thought
to represent CDK2 phosphorylated on threonine
residue (Thr160 in CDK2).29 In control cells, p21/CDK2
complexes decreased at 24 hours after PDGF stimulation. However, in
tranilast-treated cells, the complexing of CDK2 with p21 was maintained
at high levels; this led to an inactivation of CDK2 activity. The
p21/CDK4 complexes were also increased in tranilast-treated cells at 24
hours after PDGF stimulation (Figure 4C), suggesting that not
only p21/CDK2 but also p21/CDK4 complex is important in
tranilast-mediated growth inhibition of SMCs.
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Next, we examined the amount of cyclin D1 and E remaining in the
supernatant after immunoprecipitating with anti-p21 antibody. We
immunoprecipitated p21 from the lysates of cells at 24 hours after 10%
FBS stimulation in the presence or absence of tranilast (300
µmol/L). Then, supernatant was subjected to
immunoblotting with either anti–cyclin D1 or
anti–cyclin E antibody. In tranilast-treated cells, the amount
of cyclin D1 in the supernatant was 50% less compared with the
control; however, we found no difference in the cyclin E levels (Figure 5A). Furthermore, there was no
difference in the amount of p27 with or without tranilast treatment. In
case of PDGF stimulation, we found that the amount of cyclin D1 in the
supernatant was also decreased, but we found no difference in the
amount of cyclin E (data not shown).
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To confirm the increased association of p21 with cyclin/CDK complex, we
immunoprecipitated cyclin D1 and cyclin E at 24 hours after 10% FBS
stimulation and then examined them with anti-p21 or anti-p27
antibody. As shown in Figure 5B, we found an increased
association of p21 with cyclin D1 by 2-fold compared with the control;
however, we could not detect p27 alteration in either cyclin D1 or
cyclin E immunoprecipitates (data not shown). These data suggest that
tranilast induces p21 without affecting p27 expression, and the
increased p21 makes more complexes with cyclin D1.
Tranilast Inhibits Neointimal Thickening of the Rat
Carotid Artery After Balloon Injury
Our in vitro experiments revealed that tranilast potently
inhibited both PDGF- and FBS-induced cell proliferation. To determine
whether these effects were applicable in vivo to inhibit
neointimal formation after arterial injury,
tranilast was administered as a dietary supplement before balloon
injury of the carotid artery. Plasma level of tranilast was
289±25 µmol/L when rats were euthanized. Tranilast
significantly inhibited the accumulation of neointimal SMCs
in injured vessel at 14 days (data not shown). Quantitative
analysis of the injured segments of arteries showed that
tranilast-fed animals had a 70% less neointima/media area
ratio than controls (0.443±0.071 versus 1.450±0.138 [n=8] for
tranilast-treated and normal chow controls, respectively,
P<0.001). Tranilast-fed animals exhibited similar weight
gain and exhibited no other evidence of toxicity compared with the
normal chow–fed rats.
Tranilast Induces p21 Expression in the Neointimal SMCs
After Balloon Injury of the Rat Carotid Artery
To determine whether these in vivo effects were associated
with changes in p21 expression, we examined the expression of p21 by
immunohistochemistry at 2, 7, and 14 days after balloon injury. p21
expression was undetectable in uninjured vessels and in sections
incubated with nonimmune rabbit IgG (data not shown).30 At
2 days after injury, many medial SMCs in the rat carotid artery died,
and p21 was detected mainly in the adventitia lesion (Figure 6A). The p21-positive adventitial
cells seemed to be reduced in the tranilast-treated group (Figure 6B). At day 7 after injury, a proliferative
neointimal lesion was starting to form in control rats
(Figure 6C). In the tranilast-treated group, cells in the
neointima were already stained with anti-p21 (Figure 6D), whereas p21 was not expressed in the control (Figure 6C). At day 14 after injury, when a prominent intimal lesion was
formed, p21 expression was seen predominantly on the luminal surface of
the intima, but it was very low or undetectable in the media (Figure 6E) in normal chow–fed rats. As shown in Figure 6F, in
tranilast-treated rats, p21 expression was detected throughout the
intima lesion and was also noted in the media and adventitia. Taken
together, these results indicate that p21 is expressed earlier in
tranilast-treated rats and may contribute to inhibition of
neointimal formation after balloon injury.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we showed that tranilast has a strong antiproliferative effect on SMCs both in vitro and in vivo without affecting the early signal-transduction pathway by PDGF stimulation. To elucidate this mechanism, we examined the effect of tranilast on pRb, cyclin, and CDK activity. Although tranilast completely inhibited the CDK2 activity (Figure 2B
, rows 7 and 11), a faint
phosphorylated pRb band was still observed (Figure 1). According to a recent report, pRb has multiple CDK consensus
phosphorylation sites, and each of them may have a
specific function of regulating the binding of pRb to various proteins,
such as E2F, c-Abl, and SV40 large T antigen.31 This
implies that CDK4 and other CDKs phosphorylate the specific
sites of pRb distinct from those phosphorylated by
CDK2. This may be the reason why pRb was phosphorylated
despite complete inhibition of CDK2 activity.
Cyclin levels have been shown to be rate-limiting factors for
G1 progression in mammalian cells, and many
studies show that synthesis of cyclins, especially D-type cyclins, may
be the target of physiological signals that control
cell proliferation. We found that tranilast did not reduce the protein
levels of these cyclins and CDKs (Figure 2C). Therefore, we
conclude that other mechanisms are involved in tranilast-induced
inhibition of CDK2 and CDK4 activities. In addition to cyclin binding,
complete CDK activation requires phosphorylation at a
conserved threonine residue (Thr160 in CDK2).29 Activation
of CDK2 during S phase is accompanied by an increase in its
electrophoretic mobility. In our results, tranilast did not affect the
mobility shift of CDK2 during S phase (data not shown); therefore
CDK-activating kinase was not responsible for the
inhibitory effect of tranilast.
p21, wild-type p53-activated fragment 1 (Waf1),32
CDK-inactivating protein 1 (Cip1), and senescent cell-derived
inhibitor 1 bind to cyclin-CDK2 complexes. The
catalytic activity of each member of the CDK family can be inhibited by
p21, although their relative affinities vary with each enzyme. In our
results, p21 protein was detectable in quiescent SMCs (Figure 3, control). In tranilast-treated cells, p21 remains persistently
upregulated at high levels and associates with cyclin/CDK2/CDK4 (Figure 4B and 4C) and inhibits their activities (Figure 2B),
whereas in control cells, the reduction of p21/CDK2/CDK4 complexes
permits cells to traverse the cell cycle and enter S phase. To
confirm this, when cyclin D1 was immunoprecipitated, we found an
increased association of p21 with cyclin D1. Furthermore, when p21 was
immunodepleted, the amount of cyclin D1 remaining in the supernatant
was reduced to the same level as in quiescent control cells (Figure 5A). Therefore, tranilast-induced p21 makes more complexes with
cyclin D1 and inhibits cyclin D1/CDK4 activities. It has been reported
that pRb is sequentially phosphorylated by CDK4/cyclin
D1 followed by CDK2/cyclin E complexes.33 Tranilast may
inhibit initial CDK4/cyclin D1 activities by complexing with p21 and
result in inactivation of subsequent CDK2/cyclin E activities.
The elevated levels of p53 protein at 24 hours after PDGF stimulation
were not as marked as those of p21 (Figure 3). These data
suggest that p21 induction by tranilast depends on p53 in part;
however, tranilast inhibited the growth of HL-60, a cell line that
lacks wild-type p53 (A. Takahashi, unpublished results, 1998).
Therefore, p53 is not a prerequisite for tranilast to inhibit cell
proliferation. Other CDK-inactivating proteins, p27 (Figure 5A)
and p16 (data not shown), were also examined, but neither of them were
induced by tranilast treatment.
More recently, it has been reported that CDK2, cyclin E, and cyclin A
are induced in balloon-injured rat carotid arteries.
Immunohistochemical staining of human restenotic legions also
revealed the expression of CDK2, cyclin E, and proliferating cell
nuclear antigen in 
–smooth muscle actin–immunoreactive cells, and
p21 expression was followed up to 60 hours after balloon
injury.34 According to that report, p21 is not involved in
the maintenance of the postmitotic state of SMC in vivo. On the
other hand, it is reported that p21 is induced in porcine arteries
after balloon catheter injury to play an important role in limiting
arterial cell proliferation in vivo.30 35
Moreover, adenovirus-mediated overexpression of p21 inhibits vascular
SMC proliferation and neointima formation in the rat
carotid artery model of balloon angioplasty,36 which
suggests that p21 functions to block cell cycle progression and to
promote growth arrest in vascular cells in vivo. The demonstration that
tranilast inhibits the development of neointima shows its
potential utility to inhibit human restenosis by upregulating
p21 expression. Plasma levels of tranilast in the rat, 3 days after
administration, were 250 to 300 µmol/L. These plasma levels seem
to be compatible with the concentrations of tranilast used in the vitro
study, which inhibited SMC proliferation.
The initial response to injury in the rat model is reported to be SMC
proliferation in the media.28 Within 24 hours after
balloon catheter injury, medial SMCs start to replicate, and by day 4,
these cells migrate into the intima. Medial SMC replication reaches a
peak between 2 and 3 days after injury, is reduced by 7 days, and is
undetectable at 14 days. In this study, at 2 days after injury, balloon
catheterization caused cell injury or death in the
media, and p21 immunoreactivity was detected mainly in the adventitia
(Figure 6A and 6B). Presently, we cannot explain why
adventitial cells were positive for p21 in both the control and the
tranilast-treated group. In addition, p21 immunoreactivity was not
observed in the media by day 7; therefore, it seems too late for p21 to
inhibit the replication of medial SMC. However, little is known as to
why this medial proliferation is so important. Further studies are
needed to evaluate the significance of medial and adventitial p21
expression.
If SMC replication in an injured artery is examined at a variety of times, the highest rates of replication are found in the intima, not in the media. This intimal replication, unlike that of the media, is sustained for several weeks.37 At 7 days after injury, p21 was already expressed in the neointima of carotid artery in tranilast-treated rats, but was not in normal chow–fed rats. These data suggest that tranilast inhibits intimal cell proliferation by expressing p21 and diminished the size of the final intimal area. Further studies that aim to elucidate mechanisms underlying the upregulation of p21 in the adventitial, medial, and intimal SMCs should not only provide insight into the pathophysiology of restenosis but may also have important implications for developing approaches to prevent restenosis after angioplasty.
Our findings demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 is one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.
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Acknowledgments |
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We thank Dr Syuuji Mikami for technical support with the immunostaining experiments.
Received December 2, 1997; accepted January 7, 1999.
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