0022-3565/07/3212-648–655$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 321:648–655, 2007
Vol. 321, No. 2
117622/3199104
Printed in U.S.A.
Epothilone B Inhibits Neointimal Formation after Rat Carotid
Injury through the Regulation of Cell Cycle-Related Proteins
Yong Lim, Tack-Joong Kim, Yong-Ri Jin, Dong-Woon Kim, Jin-Sook Kwon, Ju-Hee Son,
Jae-Chul Jung, Mitchell A. Avery, Dong Ju Son, Jin Tae Hong, and Yeo-Pyo Yun
College of Pharmacy, Research Center for Bioresource and Health (Y.L., T.-J.K., D.J.S., J.T.H., Y.-P.Y.), Research Institute of
Veterinary Medicine (Y.-R.J.), and Department of Internal Medicine, College of Medicine (D.-W.K., J.-S.K., J.-H.S.), Chungbuk
National University, Cheongju, Korea; and Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi,
Jackson, Mississippi (J.-C.J., M.A.A.)
Received November 24, 2006; accepted February 7, 2007
Proliferation and migration of vascular smooth muscle
cells (VSMCs) play a pivotal role in the development of restenosis and in the progression of atherosclerosis (Ross,
1993). Arterial injury results in the migration of VSMCs into
the intimal layer of the arterial wall, where they proliferate
and synthesize extracellular matrix components. Many
growth factors induce the proliferation of VSMCs in vitro and
This study was supported by the program of the Research Center for
Bioresource and Health, which is managed and funded by the Ministry of
Science and Technology and the Korean Science and Engineering Foundation,
and by a Korean Research Foundation Grant from the Korean Government
(Basic Research Promotion Fund KRF-2005-005-J15002).
Y.L. and T.-J.K. contributed equally to this work.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.117622.
lated kinases 1 and 2, Akt, phospholipase C ␥ 1, and PDGFreceptor ␤ chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant
decrease in the level of cyclin-dependent protein kinase (CDK)
2, whereas it caused no change in the levels of cyclin E and
down-regulated the phosphorylation of retinoblastoma, which
plays a critical role in cell cycle regulation. Furthermore, levels
of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating
that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of
VSMCs. These findings suggest that epothilone B can inhibit
neointimal formation via the cell cycle arrest by the regulation of
the cell cycle-related proteins in VSMCs.
in vivo. Among them, platelet-derived growth factor (PDGF)
and basic fibroblast growth factor are important regulators of
VSMC behavior through their well defined actions as potent
chemoattractants and strong mitogens. Administration of
these growth factors enhances intimal thickening after angioplasty in 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 rat and restenosis in pig, suggesting that VSMC
growth plays an important role in the pathogenesis of these
models.
Arterial injury during percutaneous transluminal coronary
angioplasty induces multiple signaling pathways that activate VSMC migration and proliferation. Immediately after
injury, VSMCs leave their quiescent state and enter the cell
ABBREVIATIONS: VSMCs, vascular smooth muscle cells; PDGF, platelet-derived growth factor; CDK, cyclin-dependent protein kinase; Rb,
retinoblastoma; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; PLC, phospholipase C; PDGF-R␤,
platelet-derived growth factor receptor ␤ chain; MLD, mean luminal diameter; BrdU, 5-bromo-2⬘-deoxyuridine; FBS, fetal bovine serum; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; PAGE, polyacrylamide gel electrophoresis; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; U73122,
1-[6-[[17␤-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; AU, arbitrary unit(s); AG1295, 6,7-dimethyl-2-phenylquinoxaline.
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ABSTRACT
The abnormal proliferation of vascular smooth muscle cells
(VSMCs) in arterial walls is an important pathogenetic factor of
vascular disorders such as atherosclerosis and restenosis after
angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury
model and examined the potential effects of epothilone B on
cardiovascular disease. We found that epothilone B potently
prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly
inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml plateletderived growth factor (PDGF)-BB-induced proliferation and cell
cycle progression in rat aortic VSMCs. Furthermore, FBS and
PDGF-BB induced the activations of extracellular signal-regu-
Epothilone B Inhibits Neointimal Formation
Materials and Methods
Materials. Epothilone B was synthesized and characterized as
described previously (Jung et al., 2004). It was dissolved in dimethylsulfoxide and added to Dulbecco’s modified Eagle’s medium
(DMEM) with a maximum final concentration of 0.05% dimethyl
sulfoxide. Cell culture materials were purchased from Invitrogen
(Carlsbad, CA). Anti-phospho-Rb, anti-p27, anti-phospho-ERK1/2,
anti-phospho-Akt, anti-phospho-PLC␥1, anti-phospho-PDGF-receptor ␤ chain (PDGF-R␤), anti-ERK1/2, anti-Akt, anti-PLC␥1, antiPDGF-R␤, and anti-␣-actin antibodies were from Cell Signaling
Technology Inc. (Beverly, MA). Anti-CDK2 and anti-cyclin E antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). PDGF-BB was obtained from Upstate Biotechnology (Lake
Placid, NY). Other chemicals were of analytical grade.
Preparation of Epothilone B-Containing Pluronic Gel.
Thirty percent (w/v) F-127 Pluronic gel solution was prepared and
kept at 4°C for 24 h. Two milligrams of epothilone B was dissolved in
1 ml of 100% ethanol, and 1 ml of this ethanolic solution was added
to 9 ml of the previously cooled 30% Pluronic gel solution. A 100-␮l
aliquot of this solution thus contained 20 ␮g of epothilone B. Pluronic
gel containing vehicle was formulated via the same method.
Surgical Procedures and Treatment Groups. Male SpragueDawley rats (Samtako Bio Korea Co., Ltd., Osan, Korea) weighing
around 280 g were fed a normal chow diet and given water ad
libitum. All protocols were approved by the Chungbuk National
University Animal Care and Use Committee. Animals were anesthe-
tized using an i.p. injection of ketamine (50 mg/kg) and xylazine (6.7
mg/kg), and the right carotid artery was surgically exposed. A 2F
Fogarty balloon embolectomy catheter (Baxter, McGraw Park, IL)
was advanced along the length of the common carotid artery and
retracted three times under mild balloon inflation pressure (Clowes
et al., 1983). The treatment groups were divided into two groups:
control (vehicle alone) and epothilone B. Immediately following balloon injury, 100 ␮l of Pluronic gel containing epothilone B (20 ␮g)
was applied to the exposed adventitial surface of approximately a
10-mm segment of the injured carotid artery, approximately 5 mm
proximal to the bifurcation of the internal and external carotid
arteries using a 1-ml syringe with an 18-gauge blunted-tip needle. In
control animals, 100 ␮l of Pluronic gel with vehicle alone was applied
to the carotid artery as described above.
Carotid Angiography. Carotid angiography was performed 14
days after balloon injury as described previously (Kim et al., 2004).
In brief, the rats were anesthetized using an i.p. injection of ketamine (50 mg/kg) and xylazine (6.7 mg/kg), and the abdominal aorta
of the rat was isolated by a midline abdominal incision. A 4F vascular cannula (Cook, Bloomington, IN) was introduced through the
abdominal aorta and advanced to the thoracic aorta. Carotid cineangiography was performed by injecting contrast agent (Visipaque;
Amersham Health, Cork, UK). Angiographic mean luminal diameters (MLDs) were measured using computerized coronary angiography (DCI Videodensitometry, Phillips, The Netherlands).
Morphometric Analysis. Immediately following the angiography, the rats were sacrificed, and morphometric analysis was performed. Carotid arteries were perfusion-fixed with 10% buffered
formalin. Carotid artery sections (5 ␮m) were stained with hematoxylin-eosin, and morphometric analysis was performed using three
individual sections from the middle of each Pluronic gel-applied
arterial segment by an investigator blind to the experimental procedure. Cross-sectional areas (Aintima and Amedia), area ratios (Aintima/Amedia), and percent area stenosis were analyzed and calculated using Image Inside (version 2002; Focus, Daejeon, Korea).
Measurement of in Situ VSMC Proliferation. The effect of
epothilone B on in situ VSMC proliferation was measured by BrdU
incorporation on 2 days after injury as described previously (Sollott
et al., 1995). In brief, epothilone B and vehicle-treated rats were
injected s.c. with BrdU (30 mg/kg) at 30, 38, and 46 h after injury.
The carotid artery sections were harvested at 48 h after injury, and
histologic sections were incubated with mouse anti-BrdU mAbs
(Roche Diagnostics, Indianapolis, IN). The fraction of BrdU-positive
medial VSMC nuclei per cross-section was compared between epothilone B and vehicle groups.
Rat Aortic VSMCs Isolation and Culture. Rat aortic VSMCs
were isolated by enzymatic dispersion as described previously (Kim
et al., 2002). Cells were cultured in DMEM supplemented with 10%
FBS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 8 mM HEPES,
and 2 mM L-glutamine at 37°C in a humidified 95% air and 5% CO2
incubator. The purity of rat VSMCs in culture was confirmed by the
Western blotting of ␣-smooth-muscle actin. VSMCs were used at
passages 4 to 8.
In Vitro VSMC Proliferation Assay. The VSMCs were precultured in serum-free medium in the presence or absence of epothilone
B (0.1–100 nM) for 24 h and then stimulated with 5% FBS or 50
ng/ml PDGF-BB for 24 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the medium, and the
cells were incubated for 1 h. The optical density was determined at
560 nm using a microplate reader.
Cell Cycle Analysis. Rat aortic VSMCs in 60-mm2 dishes were
incubated in DMEM without serum (Invitrogen), with/without
epothilone B (0.1–100 nM) for 24 h. The cells were then treated
with/without 5% FBS or 50 ng/ml PDGF-BB for 24 h and then were
trypsinized and centrifuged at 1500g for 7 min. Centrifuged pellets
were suspended in 1 ml of 1⫻ phosphate-buffered saline, washed
twice, and recentrifuged. Pellets were suspended in 70% ethanol and
fixed overnight at 4°C. The fixed cells were briefly vortexed and
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cycle, which is associated with the induction of early-response genes (Harper et al., 1993; Serrano et al., 1993;
Polyak et al., 1994). Cell division and growth are tightly
controlled by a series of positive and negative regulators that
act at sequential points throughout the cell cycle. The contribution of vascular proliferation to the pathophysiology of
in-stent restenosis, transplant vasculopathy, and vein bypass
graft failure is particularly important. Thus, an emerging
strategy for the treatment of those conditions is to inhibit cell
proliferation by targeting cell cycle regulation.
Cyclin-dependent protein kinases (CDKs) play central
roles in the cell cycle in mammalian cells (Morgan, 1995).
These proteins promote G1/S transition by phosphorylation of
retinoblastoma (Rb) protein (Weinberg, 1995). In addition,
the existence of intrinsic CDK inhibitors has been demonstrated, which apparently bind to the CDK/cyclin complex
and inhibit its kinase activity (Peter and Herskowitz, 1994).
Recently, it was reported that antisense oligonucleotides targeted to CDK2 or gene transfer of p27 into a balloon-injured
artery both led to inhibition of intimal thickening (Morishita
et al., 1994; Chang et al., 1995; Chen et al., 1997).
Epothilone B is a novel potential antitumor compound that
has been isolated from the myxobacterium Sorangium cellulosum. Epothilones show inhibitory effects on the cytoskeleton similar to those of taxol, an antineoplastic drug (Bollag et
al., 1995), but unlike taxol, epothilone B-mediated microtubule stabilization does not trigger endotoxin signaling pathways (Muhlradt and Sasse, 1997). Although epothilone B can
inhibit proliferation in multiple tumor cell lines (Altmann et
al., 2000), the effect of epothilone B on neointimal formation
after rat carotid injury has not been reported.
The present study was designed to investigate the inhibitory effects of epothilone B on proliferation of rat aortic
VSMCs in vitro and on neointimal formation in an in vivo
experimental rat model. Furthermore, the antiproliferative
mechanism of epothilone B was also investigated.
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Luminal narrowing was markedly reduced in the epothilone
B group (Fig. 1B). The MLD of the injured carotid artery was
compared with the luminal diameter of the uninjured left
carotid artery [set at 1 arbitrary unit (AU)]. The MLDs of the
epothilone B-treated groups (0.81 ⫾ 0.06 AU, n ⫽ 3) were
significantly larger than that of the control (0.58 ⫾ 0.02 AU,
n ⫽ 3). On morphometric analysis, the epothilone B group
(n ⫽ 10, 0.16 ⫾ 0.03 mm2) showed a significant reduction in
neointimal formation compared with the control (n ⫽ 8,
0.22 ⫾ 0.05 mm2). The neointima/media area ratios (1.51 ⫾
0.37 versus 2.06 ⫾ 0.46) and percent area stenosis (39.5 ⫾
13.2 versus 55.0 ⫾ 13.3%) of epothilone B group was also
reduced. However, the medial area (0.11 ⫾ 0.02 versus
0.11 ⫾ 0.01 mm2) was unchanged (Fig. 2A; Table 1). Pluronic
gel in this study was applied only to the quadrant of the
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centrifuged at 15,000g for 5 min. Ethanol was discarded, and pellets
were stained with 0.4 ml of propidium iodide (PI) solution (50 ␮g/ml
PI in buffer containing 100 ␮g/ml RNase A). Before analysis by flow
cytometry, samples were incubated for 1 h at room temperature. The
complex of DNA-PI in cell nuclei was measured by FACSCalibur (BD
Biosciences, San Jose, CA). Proportions of cells in the G0/G1, S, and
G2/M phases were determined using the ModFitLT program (Verity
House Soft-ware, Topsham, ME).
Immunoblotting. SDS-PAGE was performed on rat aortic
VSMCs lysates using 10% acrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Amersham Biosciences), and membranes were blocked overnight at 4°C in Trisbuffered saline containing 0.1% Tween 20 and 5% skim milk powder
and then incubated with a 1:1000 dilution of anti-phospho-ERK1/2,
anti-phospho-Akt, anti-phospho-PLC␥1, anti-ERK1/2, anti-Akt, anti-PLC␥1, anti-phospho-PDGF-R␤, anti-PDGF-R␤, anti-phospho-Rb,
anti-CDK2, anti-cyclin E, anti-p27, and anti-␣-actin antibodies.
Blots were then washed with Tris-buffered saline containing 0.1%
Tween 20 and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies
(New England Biolabs, Beverly, MA). The proteins were detected
using an enhanced chemiluminescence Western blotting detect reagent (Amersham Biosciences). The intensities of the bands were
quantified using the Scion Image for Windows program (Scion Corporation, Frederick, MD).
Statistical Analysis. The results are expressed as means ⫾ S.D.
Statistical comparisons between two groups were made using the
two-tailed, unpaired Student’s t test, whereas a one-way analysis of
variance was also used for multiple comparisons, and this was followed by Dunnett’s test. These data were considered significant with
a probability less than 0.05.
Results
Epothilone B Inhibits Neointimal Formation after
Rat Carotid Injury. In the control angiogram, a distinct
narrowing of the injured right carotid was evident (Fig. 1A).
Fig. 2. A, representative cross-sections of rat carotid arteries taken 14
days after balloon injury and in vivo medial VSMC proliferation. Epothilone B inhibited the accumulation of intimal smooth muscle cells 14 days
after balloon catheter injury of rat carotid artery. Note the eccentric
pattern of inhibition resulting from application of Pluronic gel over
roughly one fourth to one third of the circumference of the vessel wall. a,
control; b, epothilone B. Magnification, ⫻100 and ⫻400. B, epothilone B
inhibited in vivo medial VSMC proliferation. In situ VSMC proliferation
was assessed by BrdU labeling 2 days after injury. a, control; b, epothilone B.
TABLE 1
Histomorphometric indices of epothilone B group
Results are expressed as means ⫾ S.D.
Fig. 1. The representative carotid angiographic findings of the control (A)
and epothilone B-treated group (B) performed 14 days after rat carotid
artery injury. Diffuse and significant narrowing of the right carotid
artery was observed in the control group. The epothilone B group showed
significant reduction in the luminal narrowing of carotid artery compared
with the control group (A, MLD, 0.58 ⫾ 0.02 AU, n ⫽ 3; B, MLD, 0.81 ⫾
0.06 AUⴱ, n ⫽ 3). Results are expressed as mean ⫾ S.D. ⴱ, P ⬍ 0.05 versus
control.
Neointimal area (mm2)
Neointima/media area ratio
% Area stenosis
Media area (mm2)
* P ⬍ 0.05 vs. control.
Control
(n ⫽ 8)
Epothilone B
(n ⫽ 10)
0.22 ⫾ 0.05
2.06 ⫾ 0.46
55.0 ⫾ 13.3
0.11 ⫾ 0.01
0.16 ⫾ 0.03*
1.51 ⫾ 0.37*
39.5 ⫾ 13.2*
0.11 ⫾ 0.02
Epothilone B Inhibits Neointimal Formation
vessel exposed during the surgery. Comparison of the neointimal area of the entire vessel results in a significant underestimation of the degree of neointimal inhibition. Therefore,
we also analyzed the neointima/media area ratios of drugapplied and nonapplied quadrants. As expected, the neointima/media area ratios showed a significant difference between the drug-applied and nonapplied quadrants (Table 2).
All Pluronic gel-applied sections were examined grossly and
histologically for evidence of inflammation. Fourteen days
after the Pluronic gel was applied, they were completely
degraded, leaving no materials behind. At the time of sacrifice, there was no gross evidence of an inflammatory or foreign body response to either the drug- or vehicle-loaded Pluronic gel. This was confirmed by histological examination of
Results are expressed as means ⫾ S.D.
Pluronic Gel
Control (n ⫽ 8)
Epothilone B (n ⫽ 10)
Applied
Quadrant
Nonapplied
Quadrant
2.06 ⫾ 0.56
1.20 ⫾ 0.36*†
2.10 ⫾ 0.30
1.83 ⫾ 0.51
the injured vascular tissue, which was devoid of signs of
inflammation in the adventitia and media of treated vessels
(data not shown).
Epothilone B Inhibits in Vivo Medial VSMC Proliferation. The effect of epothilone B on in situ VSMC proliferation was measured by BrdU incorporation 2 days after
injury. In vivo medial VSMC proliferation was significantly
inhibited in the epothilone B-treated group at 2 days after
injury (control versus epothilone B, 25.5 ⫾ 4.8 versus 16.6 ⫾
1.2%, n ⫽ 3, per group, P ⬍ 0.05) (Fig. 2B).
Epothilone B Inhibits VSMC Proliferation and Cell
Cycle Progression in Vitro. Next, we tested the hypothesis
that its use would block the growth of VSMCs. Therefore, the
effect of epothilone B on in vitro VSMC proliferation was
measured by MTT assay. Compared with untreated cells,
pretreatment with epothilone B (0.1–100 nM) significantly
decreased in vitro VSMC proliferation in a concentrationdependent manner (Fig. 3A, inhibition percentages of FBSstimulated VSMC proliferation, 45, 63, 90, and 91%; Fig. 3B,
inhibition percentages of PDGF-BB-stimulated VSMC proliferation, 30, 47, 88, and 91%, at the concentrations of 0.1, 1,
10, and 100 nM, respectively). To test the generality of the
effects of epothilone B on cell cycle, we examined its effect on
* P ⬍ 0.05 vs. control, among the applied quadrants.
† P ⬍ 0.05, between the applied quadrant and non-applied quadrant.
Fig. 3. Effects of epothilone B on FBS- and PDGF-BB-stimulated VSMC
proliferation in vitro. The VSMCs were precultured in the presence or
absence of epothilone B (0.1–100 nM) in serum-depleted medium for 24 h,
and then VSMCs were stimulated by 5% FBS (A) or 50 ng/ml PDGF-BB
(B). After 24 h, MTT was added to the medium, and the cells were
incubated for 1 h. The optical density was determined at 560 nm using a
microplate reader. Each value represents the mean ⫾ S.D. from three
different sets of assay. ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01 compared with FBS or
PDGF-BB alone.
Fig. 4. Effects of epothilone B on FBS- and PDGF-BB-stimulated cell
cycle progression. The VSMCs were precultured in the presence or absence of epothilone B (0.1–100 nM) in serum-depleted medium for 24 h,
and then VSMCs were stimulated by 5% FBS (A) or 50 ng/ml PDGF-BB
(B). After 24 h, individual nuclear DNA content was reflected by fluorescence intensity of incorporated propidium iodide. Each item is derived
from a representative experiment, where data from at least 10,000 events
were obtained. Data are representative of at least three independent
experiments with similar results.
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TABLE 2
Neointima/media area ratios of Pluronic gel-applied quadrants and
non-applied quadrants
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Lim et al.
cell cycle progression elicited by FBS (5%) or PDGF-BB (50
ng/ml). Epothilone B (0.1–100 nM) potently blocked both
FBS- and PDGF-BB-induced cell cycle progressions in a concentration-dependent manner (Fig. 4). We also performed
cell counts and [3H]thymidine incorporation assay to confirm
that the effect of epothilone B on cell cycle progression in
VSMCs truly reflected changes in proliferation, and similar
results were obtained in both cell counting and [3H]thymidine incorporation assay experiments (data not shown).
Effects of Epothilone B on ERK1/2, Akt, PLC␥1, and
PDGF-R␤ Activations. By immunoblotting analysis, we
measured ERK1/2, Akt, and PLC␥1 activities as a number of
critical growth signaling pathways. We have used U0126,
LY294002, and U73122 as selective inhibitors for ERK1/2,
Akt, and PLC␥1 activation. Increased amounts of phosphorylated ERK1/2, Akt, and PLC␥1, compared with untreated
cells, were detected after stimulation of VSMCs with FBS
(5%) or PDGF-BB (50 ng/ml) for 5 to 15 min. Phosphorylations of ERK1/2, Akt, and PLC␥1 stimulated by FBS and
PDGF-BB were blocked by pretreatment with U0126,
LY294002, and U73122 but not with epothilone B (Fig. 5,
A–C). We also addressed whether epothilone B influenced
PDGF-BB-induced activation of PDGF-R␤ tyrosine kinase.
We used tyrphostin AG1295 as a selective inhibitor for
PDGF-R␤ tyrosine kinase. Increased amounts of phosphorylated PDGF-R␤ tyrosine kinase compared with untreated
cells were detected after stimulation of VSMCs with
PDGF-BB (50 ng/ml) for 1 min. Phosphorylation of PDGF-R␤
tyrosine kinase by PDGF-BB was blocked by pretreatment
with tyrphostin AG1295 but not with epothilone B (Fig. 5D).
These results provide evidence that the effect of epothilone B
on VSMC proliferation may be due to a specific arrest of the
cell-cycle machinery without affecting upstream mitogenic
signaling events.
Epothilone B Inhibits CDK2 Level and Rb Phosphorylation through p27 Protein Increase. To examine the
specific effect of epothilone B on the VSMC proliferation and
cell cycle machinery, we assessed cell cycle-related proteins
in cellular lysates from PDGF-BB-stimulated VSMCs. To
investigate the mechanism of epothilone B-induced cell cycle
checkpoint, VSMCs treated with epothilone B (100 nM) from
6 to 24 h were subjected to Western blot analysis (Fig. 6A).
Among several cell cycle-related proteins, CDK2 and cyclin E
protein levels were up-regulated in response to PDGF-BB
stimulation, whereas the increased CDK2 was significantly
blocked, but cyclin E level was not altered by pretreatment
with epothilone B. The level of p27 protein, a CDK inhibitor,
was also increased by the treatment of epothilone B (0.1–100
nM) in a concentration-dependent manner. These results
indicate that the epothilone B-induced cell cycle arrest can be
associated with the CDK2 protein at the G0/G1 phase. The
phosphorylation of Rb protein mediated by cyclin E/CDK2 is
necessary for cells to progress from G0/G1 to S phase. As a
final measurement of cell cycle-related proteins, the effect of
epothilone B on Rb phosphorylation was examined. The result showed that PDGF-BB-induced phosphorylation of Rb
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Fig. 5. Effects of epothilone B on the phosphorylations
of ERK1/2, Akt, PLC␥1, and PDGF-R␤ in FBS- and
PDGF-BB-stimulated VSMCs. The VSMCs were cultured in 12-well plates until confluence, and the medium was replaced with serum-free medium in the
presence or absence of epothilone B (0.1–100 nM),
U0126 (U0; 10 ␮M), LY294002 (LY; 50 ␮M), U73122
(U7; 10 ␮M), or tyrophostin AG1295 (AG; 10 ␮M) for
24 h. The cells were stimulated with 5% FBS or 50
ng/ml PDGF-BB for 5 (A, ERK1/2; B, PLC␥1), 15 (C,
Akt), or 1 (D, PDGF-R␤) min. The cells were lysed, and
proteins were subjected to 10% SDS-PAGE. Similar
results were obtained in three independent experiments. Quantitation of bands was done by densitometric analysis and is shown as -fold of basal compared
with vehicle control at the bottom of the bands.
Epothilone B Inhibits Neointimal Formation
was significantly decreased by the treatment of epothilone B
in a concentration-dependent manner (Fig. 6). Taken together, these results indicate that epothilone B influences
the activity of G0/G1 and S phase-related cell cycle proteins in
association with its antiproliferative effects in rat aortic
VSMCs.
Discussion
Cardiovascular diseases, which are a leading cause of mortality and morbidity in the Western world, are associated
with a multitude of pathophysiologic conditions, such as inflammation, pulmonary hypertension, coronary artery restenosis following balloon angioplasty, and VSMC proliferation
in response to vessel injury (Ross, 1993). Therefore, an un-
derstanding of its inhibition on VSMC proliferation is important in terms of developing methods of treating cardiovascular disease.
In the present study, the rat carotid artery injury model
was used to characterize the effect of epothilone B on the
vascular response to injury. The polymer used in this study
for local delivery of epothilone B was F-127 Pluronic gel as
described previously (Wu et al., 2005). This polymer has a
novel property of being soluble at 4°C, while solidifying on
contact with tissues at 37°C. This characteristic makes it
useful in pharmaceutical compounding because it can be
drawn into a syringe for accurate dose measurement when it
is cold. Pluronic gel was applied directly to the exposed adventitial surface over the site of injury with the effect on
neointimal growth as shown in Fig. 2A, a. The molar concentration of epothilone B was 0.394 mM in 100 ␮l of Pluronic gel
containing epothilone B, and local application of epothilone B
produced a highly eccentric pattern of inhibition of neointimal growth in the arterial cross-section (Fig. 2A, b). In the
current study, there was no gross evidence of an inflammatory or foreign body in response to either the drug- or vehicleloaded Pluronic gel. This eccentric response was correlated
with the site of Pluronic gel application, which was limited to
the surface of the vessel wall exposed during the surgical
procedure (approximately 25– 40%). As expected, the control
Pluronic gel had no effect, yielding a robust concentric neointimal growth (Fig. 2A; Table 2). The eccentric response to
epothilone B demonstrates its focal effect on neointimal
growth at the site of application. The ratio of the neointimal
to medial wall areas showed a significant difference between
the drug-applied and nonapplied areas. Thus, the inhibition
was only detected in that portion of the vessel in direct
contact with the epothilone B-containing Pluronic gel or in an
immediately adjacent region, indicating the site-specific, localized action of the drug. From the tissue sensitivity to
epothilone B and its pattern of inhibition, we would expect
that exposure of the entire adventitial surface to epothilone B
would lead to the profound inhibition of neointimal development in the injured vessel (Signore et al., 2001). Although the
perivascular administration of epothilone B is not a technically feasible approach for treating coronary restenosis after
percutaneous coronary intervention in humans, it may have
application in other surgical procedures in which stenosis
represents a significant complication. Procedures in which
the extravascular surface is accessible for application of the
drug include coronary artery bypass surgery and the creation
of graft anastomosis in hemodialysis patients with arteriovenous shunts. Epothilone B could also be a candidate agent
for drug-eluting stents (Heldman et al., 2001; Park et al.,
2003).
During atherosclerotic lesion progression, VSMC proliferation is of particular pathophysiologic importance (Gordon et al., 1990). In atherosclerotic lesions, VSMCs are
exposed to mitogenic substances such as PDGF (Cui et al.,
1998; Gutstein et al., 1999). Moreover, the association
between PDGF and VSMC proliferation has been demonstrated in animal experiments, in which increases and
augmentations of PDGF-BB after arterial injury were
found to be correlated with neointimal cellular proliferation (Uchida et al., 1996). In the present study, we investigated the inhibitory effect of epothilone B on FBS- and
PDGF-BB-induced VSMC proliferation and a possible sig-
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Fig. 6. Effects of epothilone B on cell cycle-related proteins in PDGF-BBstimulated VSMCs. A, VSMCs were cultured in 12-well plates until
confluence, and the medium was replaced with serum-free medium in the
presence or absence of epothilone B (100 nM) for 24 h. Then, the VSMCs
were stimulated with 50 ng/ml PDGF-BB for 6 to 24 h. B, VSMCs were
cultured in 12-well plates until confluence and replaced the medium with
serum-free medium in the presence or absence of epothilone B (0.1–100
nM) for 24 h. The cells were stimulated with 50 ng/ml PDGF-BB for 24 h.
The cells were lysed, and proteins were subjected to 10% SDS-PAGE.
Similar results were obtained in three independent experiments. Quantitation of bands was done by densitometric analysis and is shown as -fold
of basal compared with vehicle control at the bottom of the bands.
653
654
Lim et al.
Fig. 7. A proposed model for the inhibitory effect of epothilone B in the neointimal formation after carotid injury.
at 0.1 to 100 nM (Fig. 5D). Thus, we thought that epothilone B might directly target some species of the cell cycleregulated proteins to inhibit VSMC proliferation and cell
cycle.
Sriram and Patterson (2001) have demonstrated that a key
regulator of the G0/G1 to S phase transition in the cell cycle is
Rb, a tumor suppressor protein. The present study demonstrated that epothilone B down-regulated the phosphorylation
of Rb (Fig. 6). In addition, epothilone B treatment caused a
significant decrease in the level of CDK2 complex but no change
in the level of cyclin E. Furthermore, p27, an inhibitor of cyclin
E/CDK2 complex, plays a critical role in cell cycle regulation by
binding and inhibiting various CDK/cyclin complex activities
(Sherr, 1994; Morgan, 1995). The level of p27 was increased in
cells treated with epothilone B (Fig. 6B), indicating that this
might be a major molecular mechanism for the inhibitory effects of epothilone B on the growth and cell cycle of VSMCs.
In summary, we have demonstrated that epothilone B induces cell cycle arrest, inhibits VSMC proliferation in vitro,
and reduces neointimal formation in vivo after local epothilone B treatment. Our results indicate that epothilone B can
inhibit VSMC proliferation and neointimal formation via the
cell cycle arrest by the regulation of the cell cycle-related
proteins in VSMC (Fig. 7).
Acknowledgments
We thank No-Kwan Park, Il-Ha Jeung, and Yu-Kyung Kim for
technical assistance.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
naling pathway related to antiproliferative effect. Epothilone B was found to inhibit VSMC proliferation in vitro
(Fig. 3) and DNA synthesis (data not shown) induced by
FBS and PDGF-BB in a concentration-dependent manner,
and these results agreed with those of other studies, which
showed that taxol, a well known drug for cardiovascular
disease, inhibits VSMC proliferation (Blagosklonny et al.,
2006; Speck et al., 2006). Moreover, the present study
showed a concentration-dependent G0/G1 arrest in VSMCs
following treatment with epothilone B (Fig. 4). Next, we
aimed to elucidate the molecular mechanism of epothilone
B on VSMC proliferation. PDGF-induced mitogenesis and
proliferation are also known to be prerequisites for the
intimal thickening that is observed after angioplasty (Sachinidis et al., 1990). PDGF-BB binding to PDGF-R␤ leads
to its phosphorylation at multiple tyrosine residues. This
activated PDGF-R␤ is associated with signaling pathways,
such as ERK1/2, Akt, and PLC␥1 (Mulvany, 1990; Claesson-Welsh, 1994; Heldin et al., 1998). Moreover, PDGF-R␤
antagonist or protein kinase inhibitor reduced VSMC proliferation in vitro and prevented cardiovascular disorders
in several animal experiments (Waltenberger, 1997; Lipson et al., 1998; Myllärniemi et al., 1999). In this study, we
found that the inhibitory effects of epothilone B on cell
cycle progression and proliferation of VSMC were not mediated via the suppressions of the ERK1/2, Akt, and PLC␥1
signaling pathways (Fig. 5). Moreover, we observed that
PDGF-R␤ phosphorylation was not altered by epothilone B
Epothilone B Inhibits Neointimal Formation
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