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CARDIOVASCULAR
Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka, Japan (S.T., H.S., K.F., D.J., M.S., M.M.); Environmental Biological Life Science Research Center (BILIS), Minakuchi-cho, Koka-gun, Shiga, Japan (H.S., K.F.); Research and Development Division, Nippon Kayaku Co., Ltd., Kita-ku, Tokyo, Japan (K.K., Y.S.)
Received August 1, 2002; accepted October 4, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Kim and Iwao, 2000). In the rat
model, neointima formation in vessels injured by a balloon catheter was
prevented by an angiotensin-converting enzyme (ACE) inhibitor
(Powell et al., 1989).
Clinical trials of ACE inhibitor for preventing restenosis after percutaneous
transluminal coronary angioplasty were unsuccessful, however
(MERCATOR Study Group, 1992).
Such species differences in the effects of ACE inhibitors on neointimal
formation may depend on species differences in the angiotensin II-forming
pathways. Rat vascular tissues contain ACE as the only angiotensin II-forming
enzyme, whereas vascular tissues of human and dog contain chymase in addition
to ACE as angiotensin II-forming enzymes
(Takai et al., 1999;
Jin et al., 2000). In general,
chymase is known to hydrolyze the C-terminal side of proteins after aromatic
amino acids such as Phe, Tyr, and Trp. Using the substrate angiotensin I,
however, human and dog chymases cleave the Phe8-His9
bond of angiotensin I to yield angiotensin II, whereas rat chymase cleaves the
Tyr4-Ile5 bond to form inactive fragments
(Wintroub et al., 1984;
Caughey et al., 2000).
Therefore, it is thought that ACE inhibitors could not suppress
chymase-dependent angiotensin II formation, resulting in vascular
proliferation in primate vessels despite the prevention of such proliferation
in rat. Previously, we reported that in dog vessels injured by a catheter, an
angiotensin II type 1 (AT1) receptor antagonist was effective in
preventing the intimal formation, but an ACE inhibitor was ineffective
(Miyazaki et al., 1999b). It
has been unclear, however, whether angiotensin II formation via the chymase
pathway plays an important role in developing vascular proliferation. In the
present study, we demonstrated for the first time whether a specific chymase
inhibitor could prevent the intimal hyperplasia in the balloon-injured
arteries. |
Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
Animal Treatment. Twelve beagle dogs weighing 9.4 to 11.4 kg were
obtained from Japan SLC (Shizuoka, Japan) and were divided into two groups, a
group treated with NK3201 and a placebo group. Each dog was given NK3201 (1
mg/kg per day, oral administration) or placebo beginning 5 days before balloon
injury and continuing through the experiments. Under pentobarbital anesthesia
(35 mg/kg, i.v.), a balloon catheter (4F; VERMED, Miami, FL) was inserted via
the right thyroid artery into the right common carotid artery
(Miyazaki et al., 1999a). The
balloon was filled with water to distend the artery, and the artery was
injured internally. The left common carotid artery was kept intact and used as
the control artery. The experimental procedures for animals were in accordance
with the Guide for the Care and Use of Laboratory Animals (Animal Research
Laboratory, Osaka Medical College).
Measurement of Blood Pressure and Preparation of Plasma and Vascular Tissue. Four weeks after the operation, the animals were anesthetized with sodium pentobarbital (35 mg/kg, i.v.). After anesthetizing, blood pressure was measured directly via a Suflo catheter (Termo, Tokyo, Japan) with a transducer (MP-4; Nihon Kohden, Tokyo, Japan). The plasma was separated from the blood samples by centrifugation at 3000 rpm for 15 min at 4°C, and the injured and the control arteries were removed. A small segment (5 mm long) was obtained and fixed with neutral buffered formalin and paraffin-embedded. The other segments were used for the measurements of the chymase and ACE activities.
Extraction for Enzyme Assay. The artery was homogenized in 10
volumes (w/v) of 20 mM Na-phosphate buffer, pH 7.4
(Miyazaki et al., 1999a). The
homogenate was centrifuged at 20,000 rpm for 30 min. The supernatant was
discarded, and the pellet was resuspended and homogenized in 5 volumes (w/v)
of 10 mM Na-phosphate buffer, pH 7.4, containing 2 M KCl and 0.1% Nonidet
P-40. The homogenate was centrifuged at 20,000 rpm for 30 min, and the
supernatant was used for measurement of the ACE and chymase activities.
Measurements of Enzyme Activities and Angiotensin II Concentration.
Plasma renin activity was determined by an SRL renin kit (TFB Co., Tokyo,
Japan). ACE activities were measured by incubating the plasma or the tissue
extracts for 1 h at 37°C with 5 mM hippuryl-His-Leu as a substrate in 100
mM phosphate buffer, pH 8.3, containing 800 mM NaCl
(Miyazaki et al., 1999a). The
enzyme reaction was terminated by the addition of 3% metaphosphoric acid
(w/v), and the reaction mixture was placed in ice water for 10 min. After
centrifugation of the mixture at 15,000 rpm for 5 min, we applied 50 µl of
the supernatant to an octadecyl silica reversed-phase column (4.6 mm x
25 cm; Tohso, Tokyo, Japan), which had been equilibrated with 10 mM
KH2PO4 and CH3OH (1:1, pH 3.0), and eluted it
with the same solution at a rate of 0.3 ml/min. Hippuric acid was detected by
ultraviolet absorbance at 228 nm. One unit of ACE activity was defined as the
amount of enzyme that cleaved 1 µmol of hippuric acid/min.
Chymase activities were measured by incubating the tissue extracts for 1 h
at 37°C with 4 mM angiotensin I in 150 mM boraxborate buffer, pH 8.5,
containing 5 mM EDTA, 8 mM dipyridyl, and 0.77 mM diisopropylfluorophosphate
(Miyazaki et al., 1999a). The
enzyme reaction was terminated by addition of 15% trichloroacetic acid (w/v),
and the reaction mixture was placed in ice water for 10 min. After
centrifugation of the reaction mixture at 15,000 rpm for 5 min, we applied 50
µl of the supernatant to an octadecyl silica reversed-phase column (4.6 mm
x 25 cm; Tohso), which had been equilibrated with 30% methanol in 10 mM
phosphoric acid and eluted it with a linear gradient of 30 to 90% methanol in
10 mM phosphoric acid at a rate of 1 ml/min. Angiotensin II was detected by
ultraviolet absorbance at 226 nm. One unit of chymase activity was defined as
the amount of enzyme that cleaved 1 µmol of angiotensin II/min.
Angiotensin II concentration in plasma was measured by using an enzyme immunoassay kit (Peninsula Laboratories). Protein concentration was assayed with BCA protein assay reagents (Pierce, Rockford, IL), using bovine serum albumin as a standard.
Histological Analysis. To minimize the variance of neointima formation in the arteries, the segment was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5 µm thick sections. The sections were stained with hematoxylin-eosin. The cross-sectional area of intima was quantified with an image analysis system (VM-30; Olympus Optical Co. Tokyo, Japan).
Statistical Analysis. All data indicated in the text are expressed as means ± S.E.M. Statistical analysis was conducted by analysis of variance followed by a Student's t test. Differences were considered statistically significant at a value of P < 0.05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Renin and ACE Activities and Angiotensin II Concentration in Plasma. Figure 1 shows the activities of renin and ACE and angiotensin II concentration in plasma 4 weeks after the operation. The plasma renin and ACE activities and were same levels between the placebo- and NK3201-treated groups. The angiotensin II concentration in plasma was not affected by treatment with NK3201.
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Chymase and ACE activities in Vascular Tissues. In the placebo-treated group, the chymase activity in the injured arteries was significantly increased about 2-fold compared with that in the control arteries (injured arteries, 0.68 ± 0.05 mU/mg of protein; control arteries, 0.34 ± 0.03 mU/mg of protein) (Fig. 2). In the NK3201-treated group, the chymase activity in the injured arteries was reduced significantly, and the percent inhibition of chymase activity by treatment with NK3201 was 66.4% (Fig. 2). On the other hand, the ACE activity tended to be increased in the injured arteries, whereas it was not affected by treatment with NK3201 (Fig. 2).
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Effects of NK3201 on Intimal Area in Injured Artery. As shown in Fig. 3 for typical sections, the neointimal formation was observed in the injured arteries, whereas NK3201 clearly suppressed this formation. The intimal area in the placebo- and NK3201-treated groups was 0.46 ± 0.06 and 0.24 ± 0.04 mm2, respectively, and this difference was significant (Fig. 4). The intimal area in the placebo-treated group was regarded as 100%, whereas in the group treated with NK3201 it was reduced to 52.2%. The ratio of intimal area to the medial area in the placebo-treated group was 18.4 ± 1.7%, whereas in the group treated with NK3201 it was 10.5 ± 1.8% (Fig. 4). The ratio in the placebo-treated group was regarded as 100%, whereas in the group treated with NK3201 it was reduced to 57.1%.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). NK3201
inhibited human, dog, and hamster chymases by an IC50 at
concentrations of 2.5, 1.2, and 28 nM, respectively
(Takai et al., 2001). In dog,
the concentration of NK3201 in plasma, heart, and aorta were about 470, 195,
and 78 nM 8 h after oral administration of the drug (1 mg/kg), respectively.
On the other hand, ACE activity was not inhibited by concentrations up to 100
µM of NK3201. In fact, in this study, plasma levels of renin and ACE
activities were not affected by treatment with NK3201. In the canine balloon
injury model, an AT1 receptor antagonist but not an ACE inhibitor
was effective in preventing canine intimal hyperplasia
(Miyazaki et al., 1999b). It
has been unclear, however, whether chymase plays an important role in the
intimal hyperplasia. Because this compound cannot be orally administered, and
we cannot investigate whether a chymase inhibitor suppresses the intimal
hyperplasia in vessels injured by a balloon catheter. In this study, we
demonstrated for the first time that a specific chymase inhibitor, NK3201,
significantly suppressed the development of intimal hyperplasia. These
findings suggest that chymase-dependent angiotensin II formation plays an
important role in the development of intimal hyperplasia after balloon
injury.
Chymase is a chymotrypsin-like serine protease contained in the secretory
granules of mast cells. It is reported that chymase is released immediately
from the granules upon strong stimulation such as balloon injury, binds to
extracellular matrix, and continues to function for several weeks
(Craig and Schwartz, 1990;
McEuen et al., 1995). In the
clinical study, Tamai et al.
(1999) reported that a mast
cell-stabilizer tranilast markedly reduced the restenosis rate after
percutaneous transluminal coronary angioplasty. Mast cells, however, release a
large number of factors such as chemotactic factors, cytokines, growth
factors, and serine proteases, and it has been unclear which factor plays an
important role in the development of intimal hyperplasia. We reported that
inhibition of the accumulation of mast cells by tranilast was effective in
preventing canine intimal hyperplasia after balloon injury
(Shiota et al., 1999).
Tranilast significantly reduced the chymase activity in the injured arteries,
although it does not directly affect chymase activity. Therefore, a preventive
effect of intimal hyperplasia by tranilast may depend on inhibition of chymase
released from mast cells. Our results indicate that the activation of chymase
plays an important role in the proliferation of the vessels injured by a
balloon catheter; further study is required for the elucidation of the
mechanism of the chymase inhibitor since chymase is involved in transforming
growth factor-
activation and increased production of collagen
(Taipale et al., 1995;
Kofford et al., 1997).
In this study, the renin and ACE activities and angiotensin II
concentration in plasma were same levels between the placebo- and
NK3201-treated groups. The blood pressure was also not affected by treatment
with NK3201. In general, ACE inhibitors and AT1 receptor
antagonists are known to increase renin and ACE activities and angiotensin II
concentration in plasma and to reduce blood pressure. Chymase-dependent
angiotensin II formation in vessels, however, may not affect systemic blood
pressure. In this model, the concentration of angiotensin II in plasma of dogs
treated with placebo had not changed
(Miyazaki et al., 1999b). This
result suggests that the significantly higher activity of chymase observed in
the injured vessels may not be high enough to increase the concentration of
angiotensin II in circulating blood. In the present study, in contrast,
administration of the chymase inhibitor failed to reduce the angiotensin II
concentration in plasma. The inhibition of chymase-dependent angiotensin-II
production only in the injured vessels may not influence blood pressure and
the concentration of angiotensin II in plasma. In the normal state, vascular
ACE regulates local angiotensin II formation and plays a crucial role in the
regulation of blood pressure, whereas chymase is stored in mast cells and has
no angiotensin II-forming activity. On the other hand, chymase is activated
immediately upon release into the extracellular matrix in vascular tissues
after mast cells have been activated by stimuli, such as vessel injury by a
balloon catheter, and the chymase-dependent angiotensin II formation occurs
only in injured vessels. In a dog model, treatment with a chymase inhibitor,
Suc-Val-Pro-Phep-(Oph)2, to the vein only once before
grafting was able to prevent the intimal hyperplasia in grafted veins
(Nishimoto et al., 2001). The
chymase inhibitor significantly suppressed the angiotensin II concentration
and the mRNA levels of fibronectin, collagen I, and collagen III in the
grafted veins, all of which are induced by an increase of angiotensin II
action. These findings suggest that chymase-dependent angiotensin II formation
plays an important role in the development of vascular proliferation.
Therefore, chymase is defined as an injury-induced angiotensin II-forming
enzyme in vascular tissues, and chymase inhibitor may be useful for preventing
vascular proliferation without affecting blood pressure.
In conclusion, a specific chymase inhibitor, NK3201, appears useful for preventing intimal hyperplasia after balloon injury, suggesting that chymase plays an important role in the intimal hyperplasia.
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Footnotes |
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ABBREVIATIONS: ACE, angiotensin-converting enzyme; AT1 receptor, angiotensin II type 1 receptor; NK3201, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide.
Address correspondence to: Dr. Shinji Takai, Department of Pharmacology, Osaka Medical College, Takatsuki City, Osaka 569-8686, Japan. E-mail: pha010{at}art.osaka-med.ac.jp
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, P < 0.05 versus injured arteries in
the placebo-treated group.
