Full text of DOI:10.1038/sj.bjp.0704281

British Journal of Pharmacology (2001) 134, 473 ± 483
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E€ects of local cytochalasin D delivery on smooth muscle cell
migration and on collar-induced intimal hyperplasia in the rabbit
carotid artery
1
,1
Richard H.J. Bruijns & * Hidde Bult
1
Laboratory of Pharmacology, University of Antwerp (UIA), Faculty of Medical and Pharmaceutical Sciences,
Universiteitsplein 1, B-2610 Wilrijk, Belgium
1
Smooth muscle cell (SMC) migration has been implicated in neointima formation after
angioplasty. Therefore, we investigated whether cytochalasin D, a fungal metabolite that inhibits
actin ®lament formation, suppressed SMC migration and collar-induced intimal hyperplasia in the
rabbit carotid artery.
2
To establish e€ective concentrations, contractions of carotid artery rings to phenylephrine were
8
determined after incubation with cytochalasin D (10 ± 10 M) for 30 min or 3 days. In vitro cell
7
76
migration was studied using carotid artery explants and a modi®ed Boyden chamber with SMCs
74
78
isolated from the rabbit aorta. The in vivo e€ect was tested after infusion of 10 ± 10
cytochalasin D into collars placed around the left carotid artery; collars placed around the right
artery served as controls.
M
77
76
3
Contractions to phenylephrine decreased after 30 min or 3 days exposure to 10 and 10
cytochalasin D; the e€ect was partly reversible. These concentrations also inhibited cellular
M
outgrowth and SMC migration in the in vitro assays.
75
74
4
Immunohistochemistry showed that local delivery of 10 or 10 M cytochalasin D for 2 weeks
suppressed collar-induced a-SMC actin expression in the intima by 68% and 84% respectively.
However, the cross-sectional area of the intima was not reduced due to an in¯ux of T-lymphocytes
and macrophages.
5
It is concluded that cytochalasin D suppressed SMC contractility and migration in vitro.
Although perivascular infusion of cytochalasin D inhibited collar-induced SMC migration from
media to intima in vivo as well, the intimal hyperplasia was not reduced due to concomitant
development of an in¯ammatory response.
British Journal of Pharmacology (2001) 134, 473 ± 483
Keywords: Smooth muscle cell migration; restenosis; local drug delivery; contractility; rabbit carotid artery; neointima;
cytochalasin D; actin ®laments; cytoskeleton
Abbreviations: ANOVA, one-way analysis of variance; DMEM, Dulbecco's modi®ed Eagle's medium; Emax, maximal contrac-
tion; Emin, initial tension, FBS, foetal bovine serum; SMC, smooth muscle cell; TUNEL, terminal
deoxynucleotide transferase-mediated deoxy-uridine-5'-triphosphate nick-end labelling; VCAM-1, vascular cell
adhesion molecule-1
Introduction
Actin ®laments form an important component of the
cytoskeleton and are key molecules in the regulation of cell
adhesion, spreading and shape and are critical for the
regulation of various cell functions including muscular
disease (Bauters et al., 1996; Topol & Serruys, 1998). During
balloon angioplasty, atherosclerotic plaque material is
ruptured and the vessel wall damaged, leading to the
exposure of medial SMCs to serum growth and chemotactic
factors. In animal models of vessel injury, the exposure to
these factors triggers vascular SMCs to migrate to the vessel
lumen where they cause intimal thickening due to prolifera-
tion and deposition of large amounts of extracellular matrix
(Strauss et al., 1994; Bauters et al., 1996). A plethora of
mitotic and chemotactic factors is activated at the angioplasty
sites and there is a redundancy of signalling mechanisms.
This may explain why therapies with proven ecacy in
neointima models have failed to limit restenosis in the clinic
contraction and locomotion (Burridge
1
& Chrzanowska,
996; Tobacman, 1996; Lau€enburger & Horwitz, 1996;
Schedlich et al., 1997). Migration of vascular smooth muscle
cells (SMCs) is important in angiogenesis, remodelling of
blood vessels and the formation of a physiological intima in
conduit arteries (Diglio et al., 1989).
Cell migration has also been implicated in the process of
late restenosis that occurs in 30 ± 60% of cases following
successful balloon angioplasty in patients with coronary
(
Bult, 2000). Therefore we investigated whether a mitogen-
independent inhibition of SMC migration might be achieved
by targeting the cytoskeleton as a common step essential for
cell locomotion.
*
Author for correspondence; E-mail: Hidde.Bult@ua.ac.be

474
R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
Cytochalasins are a group of fungal metabolites, which
becco's modi®ed Eagle's medium (DMEM, Gibco Ltd,
71
permeate cell membranes and cause cells to stop ru‚ing, stop
translocating and round up (Schliwa, 1982). Functionally,
cytochalasins inhibit micro®lament function and polymeriza-
tion by blocking actin monomer addition at the rapidly
growing end of the actin ®lament (Cooper, 1987). In this
study we ®rst determined the e€ect of cytochalasin D on
phenylephrine-induced contraction in the isolated rabbit
carotid artery and on SMC migration in vitro. After having
Paisley, U.K.) and antibiotics (penicillin 100 u ml , strep-
71
71
tomycin 100 mg ml , gentamycin 100 mg ml and polymix-
71
in
supplemented with either 10 M (n=6), 10 M (n=6) or
B sulphate 100 u ml ). Segments were separately
77
78
76
10 M (n=6) cytochalasin D and placed in a humidi®ed
5% CO2 incubator at 378C. Control segments (n=6) were
incubated without cytochalasin D. After
3
days of
incubation the vascular reactivity of the segments was
determined as described above.
established the concentrations of cytochalasin
D that
decreased contractility and SMC migration, the e€ect of
local, perivascular application of cytochalasin D on intimal
hyperplasia was investigated in the rabbit carotid artery using
the collar model.
Evaluation of cellular outgrowth from rabbit carotid
artery explants
Carotid artery segments (2 mm width) were incubated as
described above, but the DMEM was supplemented with
10% heat inactivated foetal bovine serum (FBS, Gibco Ltd,
Paisley, U.K.). Segments were separately exposed to 0
Methods
78
77
76
Animals and isolation of carotid arteries
(n=13), 10
cytochalasin D. After 10 days of incubation in a humidi®ed
5% CO incubator at 378C the carotid segments were
(n=13), 10
(n=13) or 10
(n=14) M
The ethical committee of the University of Antwerp approved
the animal studies. Male New Zealand White rabbits (2.2 ±
2
removed and the cells migrated from the explant were
harvested by trypsinization (Fluka, Switzerland). The number
of cells was determined after staining of the nuclei with
propidium iodide (Sigma, St. Louis, MO, U.S.A.) and
evaluated by ¯ow cytometry (FACSort, Becton-Dickinson)
(Vindelov & Christensen, 1990). In each experiment the
number of nuclei detected for 5 min by ¯ow cytometry was
also expressed as percentage of the events counted in the
control incubation. In a separate experiment, cells migrated
out of carotid artery explants were grown on culture slides
3.5 kg) were housed individually and fed standard rabbit
chow. For the isolation of the carotid artery the animals were
7
1
anaesthetized with sodium pentobarbital (30 mg kg body
weight, i.v.) and both carotid arteries were clamped, excised
and placed in cold Krebs ± Ringer solution. Thereafter the
rabbits were sacri®ced by a sodium pentobarbital overdose
7
1
(
60 mg kg , i.v.).
Acute contraction studies
Segments (2 mm width) from naõ
(Falcon, U.S.A.), ®xed with either methanol for immuno-
histochemistry or with acetone for micro®lament staining.
È
ve rabbit carotid arteries
were mounted in isolated organ baths ®lled with 5 ml of
Krebs ± Ringer solution (378C, continuously gassed with 95%
O ± 5% CO ) for force measurements at 6 g loading tension
Migration of aortic SMCs
2
2
as described (Geerts et al., 1999). Tension was measured
isometrically with a Statham UC force transducer (Gould,
Cleveland, U.S.A.) connected to a data acquisition system
Moise 3, EMKA Technologies, Paris, France). After an
equilibration period of 45 min the segments were exposed to
0 mM KCl. After washout, cumulative concentration
response curves of phenylephrine were constructed after
Migration of SMCs was assessed by a modi®ed Boyden
Chamber method using microchemotaxis chambers separated
by a polycarbonate ®lter with 8 mm pore size (Transwell,
Costar, Cambridge, MA, U.S.A.). Brie¯y, SMCs were
isolated from explants of the aorta by collagenase and
elastase digestion (Seye et al., 1997) and cultured in Ham F10
medium supplemented with 10% foetal bovine serum. More
than 95% of cells in the primary culture were a-SMC actin
positive. Cells between passage 3 and 5 were growth arrested
for 48 h, harvested by trypsinization and centrifuged. The
2
(
5
78
77
76
3
0 min incubation with 0, 10 , 10 or 10 M cytochalasin
D (n=7). At the end of the experiment, the segments were
either ®xed in a 4% formaldehyde solution and embedded in
paran for immunohistochemistry or snap-frozen in liquid
nitrogen for micro®lament staining.
5
71
cells were resuspended (5610 cells ml ) in Ham F10 (Gibco
Ltd., Paisley, U.K.). Then 100 ml of the SMC suspension was
placed in the upper chamber and 600 ml of Ham F10,
To determine whether the e€ects of cytochalasin D were
reversible responses to 50 mM KCl were measured before and
immediately after 30 min incubation with 0 (n=4, time
7
1
containing 10% FBS and 50 ng ml
factor as chemoattractants, was placed in the lower chamber.
hepatocyte growth
76
78
77
76
control) or 10 M (n=4) cytochalasin D. After washout
(
Cytochalasin D (0, 10 , 10 or 10 M, n=4) was added to
the lower chamber, and the chambers were incubated at 378C
3610 min) the segments were incubated in Krebs ± Ringer
solution without cytochalasin D for 30 min before measuring
the contraction to 50 mM KCl. This procedure was repeated
four times.
2
in 5% CO and 95% air for 6 h. Thereafter the cells attached
to the upper side of the ®lter were removed with a cotton-
wool swab; the ®lter was ®xed in methanol and stained with
haematoxylin. SMCs on the lower side were counted by light
microscopy in four high-power magni®cation ®elds (6400).
After correction for spontaneous migration in the absence of
FBS and hepatocyte growth factor, migration was expressed
as a percentage of the mean number of migrated cells relative
to control (0 M cytochalasin D).
Organ culture of rabbit carotid artery segments
Carotid artery segments (2 mm width) from naõ
were placed individually in 12 wells cell culture plates
Corning, Cambridge, MA, U.S.A.) containing 2 ml Dul-
È
ve rabbits
(
British Journal of Pharmacology vol 134 (3)

R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
475
Experimental model of intimal thickening
Male New Zealand white rabbits (2.5 ± 3.5 kg) were
anaesthetized with sodium pentobarbital (30 mg kg i.v.)
and both common carotid arteries were exposed surgically.
Detection of actin ®laments was performed by staining
cryostat sections (6 mm) or acetone ®xed cells (outgrowth)
with Oregon green 488-phalloidin (Molecular Probes, Leiden,
the Netherlands).
71
Apoptosis was detected using the terminal deoxynucleo-
tide transferase-mediated deoxy-uridine-5'-triphosphate nick-
end labelling (TUNEL) assay (ApopTag kit, Oncor) as
described (Kockx et al., 1998). After deparanization and
rehydration sections were incubated with 3% citric acid to
remove calcium-containing vesicles causing non-speci®c
nucleotide binding. Moreover the proteinase K step was
omitted to avoid non-speci®c TUNEL labelling due to RNA
synthesis/splicing. The labelled antibody was visualized using
3-amino-9-ethyl carbazole as chromogen. Sections were
lightly counterstained with haematoxylin and mounted in
glycerine-jelly. Negative controls included omission of
terminal deoxynucleotide transferase or the Klenow frag-
ment.
The cross-sectional area of intima and media was measured
on elastin (Verhoe€ ± van Giesson) stained cross-sections
using computer-assisted planimetry. For the quanti®cation
of the immunoreactive areas of a-smooth muscle actin,
RAM11 and CD43 the sections were superimposed with an
unbiased counting frame with a set of regularly spaced
points. The points hitting immunoreactive structures were
counted and expressed as percentage of the total number of
points in the intima or the media (Gundersen et al., 1988).
The number of TUNEL and Ki-67 positive nuclei in intima
and media was counted and expressed as percentage of the
total number of nuclei in each structure. In two sections
VCAM-1 expression was scored at eight randomly selected
sites along the perimeter of the lumen. The number of
positive hits was divided by eight to estimate the percentage
of VCAM-1 expressing endothelial cells; the average of both
sections was used for further analysis.
Non-occlusive, biologically inert, ¯exible silicone collars
(
3
inlet/outlet diameter 1.8 mm, interior volume 134 mm ,
Silicone MED-4211, Nusil Technology, CA, U.S.A.) were
placed around the carotid arteries and closed with silicone
glue (Kockx et al., 1992). The interior of the collar around
the left artery was connected to an osmotic minipump
(
thoracic region. Pumps delivered cytochalasin D in di€erent
Alzet, Charles River France) placed subdermally in the
7
8
77
76
concentrations (10 M, n=8; 10 M, n=8; 10 M, n=8;
74
75
1
0
M, n=4; 10 M, n=4). Collars placed around the
right carotid artery were not connected to a pump and
served as internal controls. The experiment and the
treatment lasted for 14 days. In a second study additional
74
groups of animals received 10 M cytochalasin D for 3
n=3), 5 (n=3) or 7 (n=3) days after collar placement. At
the end of the infusion period rabbits were anaesthetized
(
71
with sodium pentobarbital (30 mg kg
body weight, i.v.)
and both carotid arteries were excised. Two segments were
taken from the middle part of the collared section, and two
segments from the region proximal to the collar. From
each pair one ring was ®xed in 4% formaldehyde and
embedded in paran for determination of intimal hyper-
plasia and immunohistochemistry, the other ring was snap
frozen in liquid nitrogen for micro®lament staining.
Histological evaluation of potential hepatotoxicity was
performed
stained liver sections.
on
formaldehyde-®xed
haematoxylin-eosin
Immunohistochemistry and histology
Transverse paran sections (5 mm), cryostat sections (6 mm,
vascular cell adhesion molecule-1, VCAM-1) or methanol
Data analysis
®
xed cells on culture slides were stained with monoclonal
antibodies directed against rabbit macrophages (RAM-11,
: 1000), vimentin (1 : 10000) (Dako, Glostrup, Denmark),
SMCs and myo®broblasts (a-smooth muscle actin, 1A4,
: 2700, Sigma, St-Louis, MO, U.S.A.), contractile SMCs
smoothelin, 1 : 300, Monosan 2094, Sanbio, Uden, The
Data are expressed as mean+s.e.mean throughout, and
n=number of animals or cell lines. The concentration of
phenylephrine producing a half maximal response (EC50) was
determined by ®tting the individual dose response curves to a
four parameter logistic function using Graphpad Prism
version 3.0 (Graphpad Software, San Diego, CA, U.S.A.)
with the equation:
1
1
(
Netherlands) (van der Loop et al., 1996), desmin (1 : 1000,
Organon Teknika-Cappel, Durham, NC, U.S.A.), Ki-67
(
MIB-1, 1 : 500, Immunotech, Marseille, France), rabbit T-
E ˆ Emin ‡ ꢀEmax � Emin†ꢀ1 ‡ 10^‰ꢀlog EC50 � log A†nHŠ†�
1
lymphocytes (CD43, L11/35, 1 : 300, Serotec, Oxford, U.K.)
or rabbit VCAM-1 (Rb1/9, 1 : 300, gift of Dr M. Cybulski).
After inactivation of endogenous peroxidase activity with 3%
ꢀ1†
where E is the contraction (g), Emin the initial tension, Emax
the maximal contraction, A the phenylephrine concentration
and nH the Hill slope. The phenylephrine-induced contrac-
tions were expressed as percentage of the contraction induced
by 50 mM KCl. The in vitro data (Emax, 7log EC50, cellular
outgrowth and migration assay) were evaluated by means of
a one-way analysis of variance (ANOVA) and Dunnett's
multiple comparison post-hoc test. In the in vivo experiments
the treated artery was compared to the contralateral, collared
artery using Student's t-test, unless variances of the groups
were unequal. In the latter situation the non-parametric
Wilcoxon's test was used. Statistical signi®cance was assumed
at P50.05.
H
2
O
2
, primary antibodies against vimentin and a-SM actin
were detected with a goat anti-mouse-peroxidase antibody
Jackson, West Grave, PA, U.S.A.) (Kockx et al., 1992;
998), the other antibodies with the ABC technique (avidin
(
1
biotinylated horseradish peroxidase macromolecular complex,
Vectastain ABC kit, Vector Laboratories) as described
(
Matthys et al., 1997; Crauwels et al., 2000). For detection
of the complex 0.1% H was used as substrate and 3,3'
2
O
2
diaminobenzidine (Sigma, St. Louis, MO, U.S.A.) as the
chromogen and the sections were counterstained with
haematoxylin (Merck, Darmstadt, Germany). Controls from
which the primary antibody had been omitted were run for
each protocol. These consistently gave negative observations.
British Journal of Pharmacology vol 134 (3)

476
R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
Drugs
(n=6) vs. 5.6+1.3 g (n=7) in freshly isolated segments.
78
Though the e€ect of 10 M cytochalasin D seemed slightly
more pronounced, the Emax was not in¯uenced (76.6%,
p40.05) but force development decreased after 3 days
Phenylephrine was from Sigma (St. Louis, MO, U.S.A.),
cytochalasin D from Alexis (San Diego, CA, U.S.A.). The
Krebs-Ringer physiological salt solution contained (mM):
NaCl 118, KCl 4.7, CaCl2 2.5, KHPO4 1.2, MgSO4 1.2,
77
76
exposure to 10 M (720%) or 10 M cytochalasin D
(752%). In contrast to the acute experiment, the sensitivity
to phenylephrine declined after 3 days incubation with
NaHCO 25, CaEDTA 0.025 and glucose 11.1. The 50 mM
3
76
KCl solution was prepared by equimolar replacement of
sodium by potassium in the physiological salt solution.
10 M cytochalasin D, but not at the lower concentrations
(Table 1). Cytochalasin D did not change the expression
pattern of a-smooth muscle actin, vimentin, desmin and
Oregon green 488-phalloidin in the vascular segments in
either acute or `chronic' experiment (data not shown).
Results
Contractile responses of the isolated rabbit carotid artery
Cellular outgrowth and migration assay
The Emax of phenylephrine decreased by about 16% and 80%
76
Cellular outgrowth from carotid artery explants could be
detected microscopically after approximately 4 days. At 10
days the cells had almost reached con¯uence. Therefore, all
experiments were terminated after 10 days of organ culture.
77
after 30 min incubation with 10 M and 10 M cytochalasin
D respectively (Figure 1A). The sensitivity to phenylephrine
2
(7log EC50=pD ) was not in¯uenced by cytochalasin D at
77
76
any concentration (Table 1). The contractile force showed a
partial recovery after repeated washing. In the presence of
Incubation with 10 or 10 M cytochalasin D decreased the
cellular outgrowth by respectively 69+4% or 99.7+0.13%
76
78
1
0
M cytochalasin the contraction induced by 50 mM KCl
(Figure 2). Outgrowth was not in¯uenced by 10
M
was 32+1% of the preceding control response (4.1+0.05 g,
n=4). After repeated washing the force increased to 64+2%,
cytochalasin D (76.2+6.5%). Cells treated with cytochalasin
D were viable as shown by the exclusion of 0.4% Trypan
blue. Furthermore there were neither changes in the
frequencies of Ki-67 or TUNEL labelling, nor in the staining
patterns of a-smooth muscle actin, vimentin, desmin or
Oregon green 488-phalloidin at any concentration of
cytochalasin D (data not shown). The latter data indicate
that cells that do migrate possess intact actin micro®laments.
7
(
8+3%, 81+3% and 82+4%, respectively at 1, 2, 3 and 4 h
n=4).
Generally, the same results were obtained after 3 days of
exposure (`chronic') to cytochalasin D (Figure 1B). Force
development was smaller in control segments, but a similar
70% decrease was also seen for 50 mM KCl: 4.0+0.9 g
Table 1 E€ect of cytochalasin D on the sensitivity of the
rabbit carotid artery to phenlyephrine after in vitro exposure
to cytochalasin for 30 min (acute) or 3 days (chronic)
Cytochalasin D
M)
Acute
7log (EC50
Chronic
7log (EC50
(
)
n
)
n
0
1
1
1
6.44+0.10
6.46+0.08
6.43+0.04
6.33+0.10
7
6
7
7
6.56+0.17
6.35+0.16
6.36+0.18
5.89+0.20*
6
6
6
6
78
77
76
0
0
0
Data represent mean+s.e.mean. *Signi®cantly di€erent from
M cytochalasin D: P50.05, ANOVA, Dunnett's test.
0
Figure 1 Phenylephrine contractions in segments of the rabbit
carotid artery after in vitro exposure to cytochalasin for 30 min (A,
acute) or 3 days (B, chronic). Data represent mean+s.e.mean,
n=6 ± 7. *P50.05, **P50.01, di€erent from control (0 M), Dun-
nett's test.
Figure 2 Cytochalasin D inhibits cellular outgrowth from rabbit
carotid artery segments in culture. *P50.05, di€erent from control
(0 M), Dunnett's test.
British Journal of Pharmacology vol 134 (3)

R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
477
7
6
In the microchemotaxis chambers 10 M cytochalasin D
prevented SMC migration completely (P50.01), whereas
was largely con®ned to the intima. Relatively few CD43 or
RAM11 immunoreactive cells were found in the media, and
these were con®ned to the inner layers (Figure 3H,J). The
leukocyte in¯ux in the intima was seen as early as 3 days
77
78
10
Table 2).
M caused inhibition and 10 M was without e€ect
(
74
after infusion of 10 M cytochalasin D and still progressed
on day 5 (Figure 3I,J) and day 7 after collar implantation. In
contrast, the intima in the contralateral control (collar only)
developed from day 3 onward and consisted purely of a-
smooth muscle actin positive cells without signs of in¯am-
matory cells.
In vivo effects of cytochalasin D
To investigate whether cytochalasin D administered by
perivascular infusion had reached the inner media on day
14, the organization of actin ®laments was examined upon
staining with Oregon green 488-phalloidin. Disruptions of the
actin ®laments of the medial SMCs were evident in segments
The leukocyte in®ltration was associated with increased
expression of VCAM-1 by the endothelial cells. The
percentage of VCAM-1 expressing endothelial cells was low
(4+2%, n=24) on day 14 after collar placement and not
in¯uenced by low cytochalasin D concentrations (6+4,
75
74
treated with 10 M or 10 M cytochalasin D (Figure 3B),
but not at lower concentrations. In segments treated with
75
74
1
0
M and 10 M cytochalasin D a-smooth muscle actin
78
76
and smoothelin positive cells could still be detected in the
media (Figure 3D,F), but the staining pattern of both SMC
markers was di€erent from the contralateral controls (Figure
n=24, 10 710 M combined). However VCAM-1 expres-
75
sion was raised in segments exposed to 10 M (84+10%,
74
n=4) or 10
M
(71+6%, n=4) cytochalasin D when
3
C,E).
In the Central Segments the collar evoked intimal
compared to the contralateral controls (0+0%, n=4 and
20+14%, n=4 respectively, P50.05, Wilcoxon test).
thickening on day 14 (Figure 3, Table 3). This thickening
constisted of a-SMC actin and smoothelin-expressing cells
under a continuous layer of endothelial cells in control
segments (Figure 3C,E). In contrast, the myo®broblasts in the
perivascular granulation tissue stained for a-SMC actin, but
not for smoothelin. The expression of a-SMC actin and
smoothelin (Figure 3D,F) was strongly suppressed in the
Perivascular cytochalasin D infusion did not a€ect the
number of nuclei in cross sections of the media as compared
to the control (Table 4). Due to the in¯ammatory in®ltrate
the nuclear count increased in the intima. The e€ect was
74
statistically signi®cant at 10 M cytochalasin D. Expression
of the cell replication marker Ki-67 was low in both intima
and media of control arteries, con®rming earlier observations
75
74
75
intima of segments treated with 10 or 10 M cytochalasin
D. The a-SMC actin reduction was 69 and 84% respectively
in comparison to the contralateral control (Figure 4). Lower
on day 14 (Kockx et al., 1992). In segments treated with 10
74
or 10 M cytochalasin D the frequency of Ki-67 expressing
nuclei was augmented in the media and particularly in the
intima. Stringent TUNEL labelling of the cross sections
showed that apoptosis was rare in media and intima of
control segments and that it was not in¯uenced by
cytochalasin D (Table 4).
78
76
concentrations (10 to 10 M) were without e€ect on the
expression of a-SMC actin (Figure 4) or smoothelin (results
not shown) in the intima.
78
76
Cytochalasin D infusion (10 to 10 M) did not in¯uence
the cross sectional areas of intima or media (Table 3), but
both media and intima increased after exposure to the higher
Evaluation of segments taken proximally from the collared
sites showed no e€ect of cytochalasin D infusion on any
parameter (data not shown). This indicated that its e€ects
were con®ned exclusively to the segment enclosed by the
collar, irrespective of the concentration of cytochalasin D
that was used. Finally, microscopic inspection of the liver by
an experienced pathologist, who was unaware of the
treatments, did not reveal histological or cytological
abnormalities in any of the groups on day 14.
75
74
concentrations (10 M, 10 M). The intima ± media ratio did
not change at any cytochalasin D concentration. However, an
increase was noted in comparison to the contralateral control
75
74
when 10 M and 10 M cytochalasin D were combined
P50.05, n=8, Student's t-test). The intima had however, a
very di€erent cellular composition and both T-lymphocytes
CD43) and macrophages (RAM11) were very abundant.
These cell types were absent from the contralateral controls
Figure 3G ± J). Quanti®cation of the immunoreactive areas
(
(
(
con®rmed that the intima of control arteries was composed of
a-SMC actin expressing cells, whereas the intima of segments
treated with 10 or 10 M cytochalasin D consisted mainly
of T-lymphocytes (CD43) and macrophages (RAM11, Figure
Discussion
75
74
The major ®ndings in this study are that cytochalasin D: (i)
impaired phenylephrine induced contractions, (ii) inhibited in
vitro SMC migration, (iii) suppressed collar-induced SMC
migration in vivo, (iv) without reducing intimal thickening.
The study of the rabbit vessels con®rmed previous results in
which contractions of isolated rat and bovine arteries were
attenuated by cytochalasins without in¯uencing the sensitivity
to the agonist (Adler et al., 1983; Tseng et al., 1997). The
force measurements were mainly used as a quick bioassay to
determine biologically e€ective concentrations of cytochalasin
D. The concentration-e€ect relationship was rather steep. The
arteries gradually regained the capacity to constrict after
repeated washing. This indicated that the e€ect of cytocha-
lasin D was partly reversible. Upon prolonged exposure (3
days) the ecacy of cytochalasin D was not raised.
5
). Interestingly, the in¯ux of lymphocytes and macrophages
Table 2 In¯uence of cytochalasin D on SMC migration in
a microchemotaxis chamber
Cytochalasin D (M)
Cells (1 field)
%
0
25+6
25+7
20+6
0
100
101+10
77+8*
0*
78
77
76
10
10
10
Mean+s.e.mean, n=4. *Di€erent from 0 M, P50.05.
British Journal of Pharmacology vol 134 (3)

478
R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
Figure 3 Photomicrographs of cross-sections of rabbit carotid arteries infused for 14 days (B, D, F, H) or 5 days (J) with 1074
cytochalasin D (right panels) and their contra-lateral controls (A, C, E, G, I, left panels). Phalloidin: Oregon green 488-phalloidin;
M
a-actin: a-smooth muscle actin; RAM11: macrophage marker; CD43 T-lymphocyte marker. 6: internal elastic lamina. Bar=10 mm.
British Journal of Pharmacology vol 134 (3)

R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
479
Table 3 E€ect of perivascular cytochalasin D infusion (treated) on cross-sectional areas of intima and media of rabbit carotid arteries
surrounded by a collar for 14 days
Intima Media7
1
Intima
Media
Cytochalasin D
M)
Control
(mm )
Treated
(mm )
Control
(mm )
Treated
(mm )
Control
(ratio)
Treated
(ratio)
2
2
2
2
(
n
78
77
76
75
74
10
10
10
10
10
8
8
8
4
4
0.08+0.02
0.09+0.03
0.10+0.02
0.12+0.04
0.10+0.02
0.09+0.03
0.11+0.03
0.15+0.04
0.34+0.14
0.29+0.08*
0.60+0.03
0.63+0.02
0.54+0.02
0.65+0.02
0.52+0.04
0.58+0.04
0.60+0.03
0.60+0.03
0.79+0.06
0.70+0.05*
0.13+0.03
0.14+0.04
0.19+0.03
0.20+0.06
0.20+0.03
0.17+0.07
0.18+0.04
0.25+0.06
0.40+0.14
0.40+0.08
Data represent mean+s.e.mean. Control: contralateral artery not connected to an osmotic minipump. *Di€erent from control: P50.05,
Student's t-test.
Figure 5 E€ect of local cytochalasin D infusion for 14 days on a-
smooth muscle actin, RAM11 and CD43 immunoreactive areas in the
intima of rabbit carotid arteries. Data represent mean+s.e.mean,
n=4. Control: contralateral artery with a collar not connected to an
osmotic minipump.
Figure 4 E€ect of local cytochalasin D infusion for 14 days on
absolute (A) and relative (B) a-smooth muscle actin immunoreactive
of a `post mitotic' spindle and a telophase disc, a structure
templated by the mitotic spindle in anaphase that may
determine the position and timing of the cleavage furrow
(Martineau et al., 1995). In other words cytochalasins may
cause mammalian cells to fail in cell proliferation (Martineau
et al., 1995; Aubin et al., 1981) and this could explain the
decreased cellular outgrowth from the vessel segments.
However, no di€erences in the expression of the proliferation
marker Ki-67 were seen after cytochalasin D incubation as
compared to controls in this in vitro experiment, and
increased Ki-67 labelling rather than a decrease was noted
in the in vivo study.
In addition, increased apoptosis could explain the
decreased cell numbers found after stimulating cellular
outgrowth in the presence of cytochalasin D. An active
involvement of the cytoskeleton in the apoptotic process has
been suggested by several studies. A special role has been
proposed for the monomeric form, G-actin. G-actin binds to
and almost completely inhibits the nucleolytic activity of
DNase I (Kreuder et al., 1984), the endonuclease possibly
involved in DNA degradation during apoptosis (Peitsch et
78
areas in the intima. Data represent mean+s.e.mean, n=8 (10
±
M) or n=4 (10 ± 10 M). Control: contralateral artery with a
76
75
74
1
0
collar not connected to an osmotic minipump. * Di€erent from
control: P50.05, Student's t-test.
The same concentrations of cytochalasin D which impaired
77
vessel contractility also decreased (10 M) and even
76
abolished (10 M) cellular outgrowth from explants of the
rabbit carotid artery stimulated with 10% FBS. Evidence for
the importance of actin in cell migration originally came from
studies in living cells where ¯uorescently labelled actin
molecules were microinjected into migrating ®broblasts
(
Wang, 1984). The labelled actin was found to be
incorporated into the ®laments at the leading edge of the
cell. We were, however, not certain that the decreased
outgrowth of cells seen after cytochalasin D incubation was
entirely due to impaired migration. Dihydrocytochalasin B, a
related drug that interferes with actin assembly, retains the
machinery for cell cleavage for up to 1 h into G
cleavage cannot proceed. This is illustrated by the retention
1
and
British Journal of Pharmacology vol 134 (3)

480
R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
Table 4 E€ect of perivascular cytochalasin D infusion on nuclear count, K
Cytochalasin D Media
i
-67 and TUNEL reactive nuclei in media and intima
Intima
(
M)
Control
Treated
Control
Treated
3
75
Nuclei 610
10
1
0.96+0.03
0.77+0.07
0.9+0.2
0.7+0.3
0.10+0.04
0+0
1.13+0.10
1.72+0.08
2.9+1.2
5.8+1.5*
0.20+0.10
0.04+0.04
0.75+0.19
0.78+0.14
0.3+0.2
0.8+0.5
0.02+0.02
0+0
1.43+0.31
1.54+0.34*
11.1+2.7*
11.2+1.1*
0.15+0.05
0.03+0.03
74
75
74
75
4
0
10
0
10
1
Ki-67, %
1
TUNEL, %
07
Data represent mean+s.e.mean, n=4. *Di€erent from contralateral control, P50.05, Wilcoxon test.
al., 1993; Polzar et al., 1994). G-actin has also been shown to
be substrate for the proapoptotic cysteine protease
smoothelin. The latter ®ndings could suggest that the
epitopes recognized by the antibodies were still present but
that their distribution was di€erent due to the severing action
of cytochalasin D on actin ®laments. At those concentrations
cytochalasin D e€ectively inhibited SMC migration, since
cells staining for a-smooth muscle actin or smoothelin, a
cytoskeletal protein speci®c for contractile SMCs (van der
Loop et al., 1996), were virtually absent from the intima. As
cell proliferation (Ki-67) was not inhibited by cytochalasin D
and apoptosis (TUNEL) was not in¯uenced, these data
collectively indicated that cytochalasin D prevented in vivo
SMC migration towards the intima. The concentration-e€ect
relationship appeared to be steep, an unexplained ®nding that
was also seen our in vitro studies.
In spite of the reduced SMC migration, the cross-sectional
area and cell number of the intima increased rather than
decreased. The explanation was that the high cytochalasin D
concentrations elicited an in¯ammatory response. Indeed, the
intima of those segments consisted predominantly of T-
lymphocytes and macrophages. Leukocytes are rarely seen
after collar placement, in contrast to rabbit models employ-
ing more extensive injury, such as balloon denudation or
angioplasty (Kling et al., 1995; Golino et al., 1997; Welt et
al., 2000; Miller et al., 2001; Bosmans et al., 1997).
Immediately after collar placement neutrophils invade the
media (Kockx et al., 1992), but this brief, transient in¯ux has
little impact on intimal hyperplasia. Indeed, prevention of the
neutrophil extravasation by the administration of a mono-
clonal antibody against the adhesion molecule CD11/CD18
did not reduce collar-induced intimal thickening (van put et
al., 1998) in contrast to models with more pronounced
vascular injury (Kling et al., 1995; Golino et al., 1997; Welt et
al., 2000). However, perivascular application of oxidized low
density lipoproteins (Matthys et al., 1997) or advanced
glycation end products (Crauwels et al., 2000) elicits large
numbers of T-cells and macrophages to the collared segment.
In accordance with other models (Kling et al., 1995; Golino
et al., 1997; Welt et al., 2000; Miller et al., 2001), this marked
leukocyte in®ltrate caused adverse e€ects by promoting
intimal hyperplasia and collagen deposition (Crauwels et
al., 2000; Matthys et al., 1997).
a
interleukin-b-converting enzyme (Kayalar et al., 1996) and
other caspases (Mashima et al., 1995). Caspase-cleaved G-
actin can no longer inhibit DNase I activity and apoptosis
becomes eminent (Kayalar et al., 1996). However, no signs of
increased apoptosis (TUNEL-reactivity) were found in either
in vitro or in vivo experiments.
A drawback of the outgrowth assay as a model for cell
migration is the heterologous nature of the cell populations.
Although the cells migrating from the explants stained for a-
smooth muscle actin, they could be myo®broblasts (Shi et al.,
1
996) as well as SMCs. Therefore, we performed a migration
assay with a cell culture consisting solely of rabbit aortic
SMCs. These experiments gave identical results: no migration
76
77
78
at 10 M, decreased migration at 10 M whereas 10
cytochalasin D was without e€ect. Since the time window
6 h) of this assay was not sucient for the completion of cell
M
(
replication or apoptosis, this provides more direct proof that
the e€ect of cytochalasin D on SMCs is solely on migration.
To our knowledge this is the ®rst study describing that SMC
migration in vitro is impaired after cytochalasin D incuba-
tion.
To determine the in vivo e€ect of cytochalasin D on SMC
migration, collar-induced intimal thickening was studied in
the rabbit carotid artery. As early as 2 days after collar
placement SMCs start migrating from the media to the
intima (Kockx et al., 1992). This model has the advantage
that local application of substances at the site of intima
formation can be achieved by connecting the interior of the
collar to an osmotic minipump (Matthys et al., 1997;
Crauwels et al., 2000), thereby reducing systemic in¯uences,
such as potential hepatotoxic e€ects of cytochalasins.
76
Perivascular infusion of cytochalasin D up to 10 M did
not a€ect SMC migration or intima formation, but there
were also no indications of actin ®lament disruption upon
staining with Oregon green 488-phalloidin. Therefore, we
concluded that the concentration of cytochalasin D in the
arterial wall was insucient. This can be explained by
dilution of the drug in the perivascular space inside the collar
and by the marked rise of transmural ¯ow in response to
collar placement (Coleman et al., 1993; Bosmans et al., 1997).
In addition to causing further dilution, the latter factor will
also oppose its di€usional transfer across the arterial wall. In
view of this the concentration of cytochalasin D was raised
There was, however, a major di€erence with those previous
experiments, in which the outer media and adventitia
contained a dense in®ltrate of T-cells and macrophages
(Matthys et al., 1997; Crauwels et al., 2000). In striking
contrast, the deeper layers of the media and the adventitia
remained free of leukocytes during cytochalasin D treatment
(Figure 3b, J). This suggests that the drug caused paralysis of
the leukocytes that were in®ltrating the vessel wall from the
75
74
(
10 M and 10 M). Cytochalasin D then clearly reached
the inner media, as indicated by the disruption of the
lamentous-actin outline (phalloidin staining) and the
®
changed staining patterns for a-smooth muscle actin and
British Journal of Pharmacology vol 134 (3)

R.H.J. Bruijns & H. Bult
Cytochalasin D and smooth muscle cell migration
481
lumen. An indication for augmented cellular in¯ux from the
blood was the increased endothelial VCAM-1 expression
vascularization) and the 19% reduction in clinical restenosis
observed with cytochalasin B showed favourable tendencies.
The potential anti-restenotic activity of other drugs that
interfere with SMC migration and replication are currently
explored. The microtubule network constitutes another
important component of the cytoskeleton. The antineoplastic
compound paclitaxel (Taxol) causes an increased assembly of
extraordinarily stable microtubules, resulting in reduced
proliferation, migration and signal transduction in SMCs in
vitro (Axel et al., 1997). Local delivery of paclitaxel to the
rabbit carotid artery by use of microporous balloons resulted
in reduced neointima formation and enlargement in vessel
size after balloon angioplasty (Axel et al., 1997; Herdeg et al.,
2000). Sirolimus (rapamycin), a macrolide antibiotic known
to prevent allograft rejection, also inhibits SMC migration, in
addition to being a potent immunosuppressant and anti-
proliferative agent (Poon et al., 1996). Systemic administra-
tion of sirolimus has been shown to produce signi®cant
reductions in neointimal area and thickness of pig coronary
and carotid arteries subjected to balloon angioplasty when
compared with vehicle-treated animals (Burke et al., 1999;
Gallo et al., 1999). Furthermore, sirolimus-eluting stents
caused only minimal intimal hyperplasia and in-stent rest-
enosis was not observed in a small clinical trial (Sousa et al.,
2001), but it is unclear to which extent SMC paralysis
contributed to these bene®cial tendencies.
75
74
after infusion of 10 M and 10 M cytochalasin D. On day
4 VCAM-1 expression was infrequent in control segments or
1
upon infusion of lower concentrations of cytochalasin D.
From our experiments it is not clear whether cytochalasin D
raised endothelial VCAM-1 expression directly, or whether it
was the consequence of cytokines released from SMCs in the
media. The abundance of T-cells and macrophages could also
account for the increased labelling with the proliferation
marker Ki-67. Furthermore, oedema formation may form
another explanation for the increased intimal and medial
areas in addition to the increased cellularity.
The leukocytes could indirectly in¯uence the apparent
SMC accumulation in the intima, e.g. by suppressing a-actin
expression. However, the quanti®cation of the immunoreac-
tive areas in the intima did not show evidence for a loss of a-
actin expression in the SMCs, since T-cells and macrophages
accounted for the a-actin negative cells. In addition, it has
been reported that interferon-g secreted by T-lymphocytes
may inhibit SMC migration and proliferation (Tellides et al.,
2
000; Hansson, 1993). Yet, in previous studies the accumula-
tion of a-smooth muscle actin positive cells in the intima was
not reduced at all when large numbers of T-cells and
macrophages in®ltrated intima and media (Kling et al.,
1
995; Matthys et al., 1997; Crauwels et al., 2000). Those data
indicate that the mere presence of intramural leukocytes is
not sucient to reduce the number of a-smooth muscle actin
expressing cells. Though this does not exclude possible e€ects
of leukocyte-derived cytokines, both in vitro assays clearly
documented a direct, leukocyte-independent suppression of
SMC migration by cytochalasin D.
In a randomized clinical trial di€erent concentrations of
cytochalasin B and placebo were administered to the site of
balloon angioplasty using a microporous local delivery
infusion balloon (Lehmann et al., 2000). The minimal
stenosis diameter after the procedure was slightly better in
the active drug group, but the di€erence was not seen at 4 to
In conclusion, perivascular application of cytochalasin D
inhibited collar-induced SMC migration, but failed to
suppress the size of the intima due to the concurrent
development of an in¯ammatory response. Further studies
of cytochalasin D in models of balloon angioplasty, using
intra-luminal delivery by means of porous balloons or drug-
eluting stents, are required to test whether smooth muscle
paralysis is a potential approach against restenosis.
6
weeks. The inhibition of the transient vasoconstriction
The authors wish to thank Dr M. Cybulski for the gift of the
monoclonal antibody against VCAM-1, Prof Dr N. Buyssens for
evaluation of the hepatic morphology, Dr M.W.M. Knaapen for
the quanti®cation of the TUNEL data and Hermine Fret, Martine
De Bie and Rita Van den Bossche for excellent technical
assistance. The support by grants from the Special Research Fund
(BOF UA 1998) and the Fund for Scienti®c Research-Flanders
(FWO, Belgium, G.0079.98) is greatly appreciated.
immediately after balloon angioplasty reported by Lehman et
al. (2000) is in line with the reduced constrictor capacity of
cytochalasin D-treated arteries seen in the present study. This
small trial (n=43) was not powered to detect di€erences in
clinical outcomes. Yet, the 47% reduction in the combined
primary end point (death, non fatal infarction or repeat
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(Received April 23, 2001
Revised June 29, 2001
Accepted July 6, 2001)
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