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ATHEROSCLEROSIS

ELSEVIER SCIENCE
IRELAND Atherosclerosis 107 (1994) 179-185

Prominent inhibitory effects of tranilast on migration


and proliferation of and collagen synthesis by
vascular smooth muscle cells

Koichi Tanaka, Masaaki Honda*, Takehiko Kuramochi, Shigefumi Morioka


The 4th Department of Internal Medicine. Shimane Medical University. 89-l. Enya-rho.
Izumo. Shimane. Japan

(Received 6 August 1993; revision received 19 January 1994; accepted 15 February 1994)

Abstract

To obtain some ideas about prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA),
we examined the effects of tranilast (anti-allergic agent) on migration and proliferation of, and collagen synthesis by,
cultured vascular smooth muscle cells (VSMC) from the thoracic aorta of WKY rats. Tranilast was added to culture
medium containing 10% fetal calf serum (FCS). The cultures were pulse-labeled with ‘H-thymidine (TdR) or ‘H-
proline (Pro). TdR and Pro uptake into VSMC were measured. The effect of tranilast on migration of VSMC was
examined by using culture dishes of an original design. We also examined the inhibitory effects of various drugs, such
as a Ca antagonist, an angiotensin converting enzyme (ACE) inhibitor, a phosphodiesterase inhibitor, elastase, col-
chicine, and mitomycin C, on proliferation and migration of VSMC. Our data showed that the inhibitory effects of
tranilast on migration and proliferation of, and collagen synthesis by, VSMC were prominent. Maximal percentage
inhibition of proliferation, migration and collagen synthesis was 60.8 f 2.3%, 52.7 f 14.7% and 62.1 f 8.1X, respec-
tively. On the other hand, the inhibitory effects of other drugs, with the exception of colchicine and mitomycin C, on
proliferation and/or migration of VSMC were not very strong. Although the inhibitory effects of colchicine and mito-
mycin C were strong in vitro, their clinical usefulness may be limited by systemic side-effects. These results indicate
the potential usefulness of tranilast for prevention of restenosis of coronary arteries after PTCA.

Key words: Tranilast; Vascular smooth muscle cell; Proliferation; Migration; Collagen synthesis; PTCA

1. Introduction coronary artery disease. However, restenosis at the


angioplasty site is a major problem limiting the
Percutaneous transluminal coronary angiopla- long-term efficacy, as it occurs in 30%-400/o of
sty (PTCA) is widely used for the treatment of patients [l]. Several drugs have been tested for
their ability to prevent restenosis after PTCA.
* Corresponding author. Tel.: 0853-23-2111 (ext. 2548); Fax: However, none of the drugs has been proved to
I
0853-2 I-922 reduce the incidence of restenosis appreciably. Mi-

0021-9150~94607.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved


SSDI 002 l-91 50(94)05230-G
180 K. Tanaka er al. /Atherosclerosis 107 (1994) 179-185

gration of vascular smooth muscle cells (VSMC) modifications [4]. Briefly, a segment of the thor-
from the media to intima, and proliferation of and acic aorta was obtained from a rat under ether
collagen synthesis by VSMC in the intima are anesthesia. Connective tissue around the aorta was
thought to be major processes in the restenosis of removed with forceps and the aorta was opened
coronary arteries after PTCA. longitudinally with scissors. It was placed in a 35
Tranilast (N-(3,4-dimethoxycinnamoyl) anthr- mm dish containing collagenase (1 mg/ml) and
anilic acid), which inhibits the release of chemical elastase (0.5 mg/ml). After the incubation, the
mediators from mast cells, is known as an anti- adventitia and intima were carefully removed from
allergic agent and used clinically for the treatment the media with watchmaker forceps. The media
of patients with bronchial asthma, allergic rhinitis was cut into l-2 mm square pieces and explanted
and atopic dermatitis in Japan. Recently, it was into 35 mm dishes containing Dulbecco’s Modified
reported that tranilast is effective for the therapy Eagle Medium (DMEM) with 10 % FCS. The
of keloids [2]. The formation of keloids is thought dishes were incubated for approximately 2 weeks
to be associated with excessive proliferation of, in 5% C02-95% air at 37°C. After cells reached
and collagen synthesis by, fibroblasts. This report confluence, the dishes were treated with trypsin
led us to examine the usefulness of tranilast for (0.125%) for 5 min at 37°C and subcultured by re-
prevention of restenosis after PTCA. In the pre- peated passages. Cells of passages 3-7 were used
sent study, we examined the effects of tranilast on for the following experiments.
proliferation of and collagen synthesis by cultured
VSMC. We also examined the effects of tranilast 2.3. Drug effects on proliferation of and collagen
on migration of VSMC by using culture dishes of synthesis by VSMC
an original design. These effects of tranilast were Cells (5 x lo4 per well) were transferred to a
compared with those of other drugs. 24-well plate containing DMEM with 10% FCS.
After incubation for 24 h, tranilast and other
2. Materials and methods drugs were added to the culture medium at various
concentrations to examine the effects of these
2.1. Drugs drugs on proliferation of VSMC. Vehicle was also
We examined the effects of the following drugs: added to the medium as a control. Then the cul-
an anti-allergic agent (tranilast), a Ca-antagonist tures were pulse-labeled with 1 &i ‘H-thymidine
(nilvadipine), an ACE inhibitor (M-I, the active (TdR) for 8 h. After the cells were harvested by
metabolite of delapril-HCl), a phosphodiesterase using 10% trichloroacetic acid (TCA), the uptake
inhibitor (E-1020), elastase, colchicine and mito- of TdR into cells was measured by a liquid scin-
mycin C. Tranilast was kindly provided by Kissei tillation counter. The effect of tranilast on col-
Pharmaceutical Co., Nagano, Japan; Nilvadipine lagen synthesis by VSMC was also examined.
by Fujisawa Pharmaceutical Co., Osaka, Japan; Seven days after incubation, tranilast was added to
M-I by Takeda Chemical Industries, Tokyo, the culture medium. Then the cultures were pulse-
Japan; E-1020 and elastase (Elaszym@) by Eisai, labeled with 1 &i 3H-proline (Pro) for 16 h. The
Tokyo. Colchicine was purchased from Nacalai uptake of Pro into cells was measured in the same
Tesque, Kyoto, Japan. Mitomycin C, collagenase way.
and elastase were purchased from Sigma, St.
Louis, MO, and trypsin was from Difco Laborato- 2.4. Drug effects on migration of VSMC
ries, Detroit, MI. To measure the effects of tranilast and other
drugs on migration of VSMC, 2 x lo5 cells were
2.2. Cell culture transferred to 35 mm dishes which were sealed
Primary cultures of VSMC were obtained from with silicon disks (6 mm diameter) at the center of
the thoracic aorta of 15-week-old male WKY rats the bottoms and marked at the edges of the disks.
by the explantation method of Ross [3] with some After 7 days, cells reached confluence. Then,
K. Tanaka er al. /Atherosclerosis 107 (1994) 179-185 181

silicon disks were stripped off from the dishes and We also examined the inhibitory effects of
the drugs were added to the culture medium at nilvadipine (a calcium antagonist), M-I (an active
various concentrations. Vehicle was also added to metabolite of derapril-HCl), E-1020 (a phospho-
another dish as a control. After an additional 5- diesterase inhibitor), elastase, colchicine, and mito-
day culture, the dishes were photographed under a mycin C on the proliferation of cultured VSMC
light microscope at a magnification of x 2. Then (Fig. 2). Similarly, all these drugs showed concent-
the distances of migration from the edges of strip- ration-dependent inhibitory effects on VSMC pro-
ped silicon disks were measured at eight points and liferation. Except for colchicine and mitomycin C,
the mean distance of migration was calculated for these drugs showed less inhibitory effects than that
each dish. of tranilast. Maximal inhibitory effects of these
other drugs were about half that of tranilast.
2.5. Statistical analysis Migration of VSMC was also examined by using
All results are expressed as mean f S.D. or culture dishes of an original design. A prominent
mean f S.E. Differences between groups were inhibitory effect of 100 &ml tranilast is shown in
analyzed by the Student’s t-test. Fig. 3. Tranilast inhibited migration of VSMC in
a concentration-dependent manner (Fig. 4). Mi-
3. Results gration distance of VSMC on control dishes and
10, 50, 100 pg/ml tranilast-added dishes was 0.85
Tranilast markedly inhibited TdR uptake into f 0.16 mm, 0.81 f 0.12 mm, 0.63 f 0.18 mm
VSMC in a concentration-dependent manner (Fig. and 0.40 + 0.13 mm, respectively. Comparative
1). Tranilast at a concentration of 10 pg/rnl showed effects of drugs on migration of VSMC are sum-
weak inhibitory effects on the uptake of TdR, but marized in Table 1. The maximal inhibitory effects
50 and 100 &ml tranilast showed strong inhib- of tranilast and colchicine were 52.7 f 14.7% and
itory effects on TdR uptake into VSMC. Maximal 80.6 f 14.0%, respectively. However, maximal
percentage inhibition of TdR uptake into VSMC doses of nilvadipine, M-I, E-1020 and elastase
was 60.8 f 2.3%. These results suggest that prolif- used in this study did not show any inhibitory
eration of VSMC was prominently inhibited by effects of VSMC.
tranilast at concentrations of more than 50 &ml. Inhibitory effects of tranilast on collagen syn-
thesis were also examined (Fig. 5). Tranilast showed
w concentration-dependent inhibition of Pro uptake
70 into VSMC. Maximal percentage inhibition of Pro
1 uptake into VSMC was 62.1 f 8.1%. That is,
tranilast markedly inhibited collagen synthesis by
VSMC.

4. Discussion

Tranilast has been used clinically for patients


with bronchial asthma, allergic rhinitis or atopic
dermatitis. Its pharmacological properties are the
o+i 0 20
/
40
I r-.---T---f
60 60
1
100 inhibition of passive cutaneous anaphylaxis in
Tranilast C9’ml) vivo and chemical mediator release from mast cells
Fig. 1. Inhibitory effect of tranilast on proliferation of VSMC. in vitro [5]. It has been noted that the inhibitory
Percentage of inhibition was calculated by the following equa- mechanism of chemical mediator release is the in-
tion: % inhibition = (counts/min without drug - counts/min
with tranilast)/(counts/min without drug) x 100. Each experi-
hibition of the energy-requiring system and/or
ment was done in triplicate. Values of 10 different experiments Ca2+ influx at the time of mast cell degranulation
are expressed as mean f SE. WI.
182 K. Tanaka et al. /Atherosclerosis IO? (1994) 179-1M

(“4 w

1
70 70-

60 60.

01
-9 -0 -7

Nilvadipine (logM)

w (-4
70- 70-

60. 60-

50.
g 50.

F
B 40. 40.

z 30.

3
20.

f: [ 4
f lo;
0 --/' ,
OS I I
0 2 4 6 6 10 12
-7 -5

mw Elastase (pcmb

O-_i -6 Od 1.2

Colchicine (w4 Mitomycin C (pw-1)

Fig. 2. Inhibitory effects of various drugs on proliferation of VSMC. Percentage of inhibition was calculated as described in Fig. 1.
Each experiment was done in triplicate. Values of five different experiments are expressed as mean f SE.
K. Tanaka et al. /Atherosclerosis 107 (I 994) I79-185 183

mm)
(A) I
I
10 I I
1

Tranilast (riqiml)

Fig. 4. Inhibitory effect of tranilast on migration of VSMC.


Values of 10 different experiments are expressed as mean f
SE. *P < 0.05.

tion, the inhibitory effects of drugs other than col-


chicine were not comparable to those obtained
with tranilast. Although the inhibitory effects of
colchicine and mitomycin C on proliferation
and/or migration were strong in vitro, their clinical
Fig. 3. Tranilast inhibits migration of VSMC. (A) control; (B) usefulness may be limited by systemic side-effects.
tranilast (100 pg/ml). On the other hand, a standard clinical dose of
tranilast (300 mg/day) produced plasma concen-
trations of 20-40 &ml. Moreover, the effects of
tranilast on the cells were not cytotoxic, because
In the present study, we have shown that we confirmed that the cells showed recovery of
tranilast markedly inhibited proliferation and mi- proliferation, migration and collagen synthesis
gration of and collagen synthesis by VSMC. All after removal of tranilast, and the cell viability
other drugs also exerted inhibitory effects on pro- examined by trypan blue was not different between
liferation of VSMC, but except for colchicine and tranilast-treated and vehicle-treated cells.
mitomycin C, their inhibitory effects were weak Therefore, the effects of tranilast on VSMC are
and exerted by concentrations that cannot be specific pharmacological effects, and not
achieved by regular clinical doses. As for migra- nonspecific cytotoxic effects.

Table 1
Maximal inhibitory effects of drugs on migration of VSMC

Tranilast Nilvadipine M-I E-1020 Elastase Colchicine Mitomycin C


% Inhibition of 52.7 f 14.7 0 0 0 0 80.6 f 14.0 N.D.
migration (100 agW (lO-7 M) (10e5 g/ml) (10e5 M) (10 &ml) (5 x IO-’ M)
Drugs were added at the following concentrations. Tranilast, IO-100 &ml; Nilvadipine, 10-9-10-7 M; M-I, lO-7-lO-5 g/ml; E-
1020, lO-7-lO-s M; Elastase, l-10 &ml; Colchicine, IO-‘-5 x 10m7M.The concentration of drug that showed maximal inhibition
is shown in parentheses. N.D., not determined. Data show the maximal percentage inhibition by various drugs on migration of
VSMC. Values are expressed as mean f S.D.
184 K. Tanaka et al. /Atherosclerosis 107 (1994) 179-185

synthetic type, induced by growth factors such as


platelet derived growth factor (PDGF) which are
released from platelets, macrophages, damaged
endothelial cells and smooth muscle cells. Then
smooth muscle cells migrate from media to intima
and proliferate. Smooth muscle cells in intima
begin to produce an extracellular matrix such as
proteoglycan. The injured blood vessel thus
develops the histologic appearance of intimal hy-
10

0:
1 perplasia. Proliferation of smooth muscle cells
gradually diminishes, and their phenotype returns
0 20 40 60 80 100 to contractile type. These developments parallel
Tranilast G9’W
changes in the extracellular matrix: proteoglycan is
Fig. 5. Inhibitory effect of tranilast on collagen synthesis by gradually replaced by collagen. At this step, the
VSMC. Percentage of inhibition was calculated as described in restenotic response is probably largely complete.
Fig. 1. Each experiment was done in triplicate. Values of IO In the past, both experimental and clinical stud-
different experiments are expressed as mean f SE. ies of pharmacologic prevention of restenosis have
been reported. These drug therapies included an-
tiplatelet agents (aspirin, dipyridamole) [8,9], an-
We do not know in detail the mechanism by tithrombin agents (heparin) [lo- 141, calcium
which tranilast inhibits proliferation and migra- channel blockers (nifedipine, verapamil, diltiazem)
tion of VSMC, or collagen synthesis by VSMC. [ 151,ACE inhibitors (captopril, cilazapril) [ 16,171,
The following mechanisms are possible. (1) immunosuppressive agents (steroids [ 181, cyclo-
Tranilast may modify the autocrine secretion of sporine [ 19]), antimitogenic agents (colchicine and
growth factors by VSMC, or may influence the ac- others) [20], fish oils [21], and HMG-CoA reduc-
tivity of growth factors. (2) Intracellular Ca2+ tase inhibitors (lovastatin) [22-241. Some of these
concentration is closely related to cell proliferation drugs were effective in animal models. However,
and migration. Tranilast may change intracellular the majority of clinical studies have failed to iden-
Ca2+ concentration of VSMC, thereby inhibiting tify any single pharmacologic agent that reduced
proliferation and migration of VSMC. (3) Trani- the incidence of restenosis.
last may influence the function of cytoskeleton ele- In summary, our data show the marked inhib-
ments such as actin filament or microtubules. itory effects of tranilast on proliferation and mi-
Further studies are needed to define the exact gration of, and collagen synthesis by, VSMC in
mechanism by which tranilast inhibits prolifera- vitro. The maximal inhibitory effects of tranilast
tion and migration of VSMC and collagen syn- on proliferation of VSMC were about twice those
thesis by VSMC. of other drugs examined in this study. Only
Restenosis is a major problem of PTCA, tranilast showed marked inhibitory effects on
limiting the long-term efficacy. Although the migration of VSMC within the concentration ob-
detailed mechanisms of restenosis are not well- tained by clinical doses. Therefore, it is expected to
known, pathologic studies suggest that it develops be clinically useful. Moreover, it has been reported
by the following mechanisms [7]. Balloon inflation in a clinical study that tranilast at a dose of 600
denudes the endothelial surface, thereby exposing mg/day for 3 months reduced the rate of restenosis
myointima. Tearing often occurs at the junction after PTCA as compared with control (12.7% vs.
between normal and atherosclerotic tissue, and 38.0%) [25]. Further studies are necessary to define
atheroma itself develops fissures. Platelets ag- the mechanism of action and its clinical usefulness
gregate at these sites of vascular injury. Smooth for prevention of restenosis of coronary arteries
muscle cells in the media begin to proliferate and after PTCA. Clinical studies in multicenters are
change their phenotype from contractile type to now under way.
K. Tanaka et al. /Atherosclerosis 107 (1994) 179-185 I85

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