(Circulation. 2001;103:284.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Role of Rho-Associated Kinase in Neointima Formation After Vascular Injury
From Internal Medicine III and Cardiovascular Research Institute (R.S., H.K., Y.S., T.I.) and Department of Pathology (S.K., M.M.), Kurume University School of Medicine, Kurume, and Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma (K.K.), Japan.
Correspondence to Hisashi Kai, MD, PhD, Cardiovascular Research Institute, Kurume University, 67 Asahimachi, Kurume 830-0011, Japan. E-mail naikai{at}med.kurume-u.ac.jp
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Background—The Rho/Rho-associated kinase (Rho-kinase) system is implicated in various cellular functions, including migration, proliferation, and apoptosis. Because a possible role of the system is suggested in neointima formation after vascular injury, we sought to examine whether a new specific Rho-kinase inhibitor, Y27632, prevents neointima formation of the balloon-injured rat carotid artery, and if so, to investigate the effects of Y27632 on migration, proliferation, and apoptosis of smooth muscle cells (SMCs) in the injured artery.
Methods and Results—Y27632 was administered intraperitoneally from 1 day before to 14 days after vascular injury. Treatment with Y27632 inhibited phenylephrine-induced Rho-kinase activation in the carotid artery on the basis of immunoblotting against the phosphorylated myosin-binding subunit of myosin phosphatase. Y27632 markedly prevented neointima formation at days 7 and 14. In controls, BrdU+ proliferating and TUNEL+ apoptotic SMCs were transiently and coincidentally increased in the neointima, with a peak at day 7. Y27632 significantly increased the neointimal TUNEL+ SMCs at days 7 and 14, but not BrdU+ SMCs. Y27642 significantly decreased the number of intimal SMCs at day 4, while not affecting the number of BrdU+ or TUNEL+ SMCs. Reendothelialization after balloon injury was not significantly affected by Y27632 at days 7 and 14.
Conclusions—Y27632 inhibited neointima formation by enhancing SMC apoptosis and probably by suppressing early SMC migration. Therefore, a role of Rho-kinase is suggested in neointima formation after vascular injury.
Key Words: kinases • neointima • apoptosis • migration
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Introduction |
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The small GTPase Rho is implicated in a variety of physiological functions associated with actomyosin-based cellular processes such as cell adhesion, cytokinesis, cell motility, migration, and contraction.1 2 Among several putative Rho effectors, the Rho-associated kinase (Rho-kinase/ROK/ROCK) family plays major roles in Rho-induced actin reorganization, such as focal adhesion and stress fiber formation.3 4 5 Also, Rho-kinase phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase, inhibits phosphatase activity, and thereby enhances the myosin light chain (MLC) phosphorylation level and subsequently myosin-based contractility in smooth muscle cells (SMCs).6 Furthermore, although the precise mechanism remains unclarified, there is increasing evidence that Rho has abilities to activate proliferation signals as well as signals that regulate apoptosis, either positively or negatively.7
Neointima formation after balloon injury in the rat carotid artery is the best-studied model of vascular remodeling after vascular injury. Balloon injury triggers medial SMC replication that peaks within a few days after injury, SMC migration into the intima beginning at day 4, and SMC proliferation in the neointima, with the cell number reaching a maximum at day 14.8 9 The SMC accumulation in the neointima is theoretically the sum of cell migration and proliferation as well as apoptotic cell death, and an alteration in any of these events could affect neointima formation.10 11 Therefore, it is plausible that the Rho/Rho-kinase system plays a role in neointima formation after vascular injury. However, little is known about the significance of the Rho/Rho-kinase system in vivo, because specific Rho inhibitors, such as botulinum C3 ectoenzyme and Rho–GDP dissociation inhibitor (Rho-GDI), are not permitted for in vivo use.
Y27632, a specific inhibitor for the Rho-kinase family, has been shown to suppress the Rho/Rho-kinase–mediated stress fiber formation in cultured SMCs and the phenylephrine-induced contraction of vascular strips.12 Furthermore, systemic administration of Y27632 induced significant and persistent decreases in blood pressure in various hypertensive rat models.12 These results suggest not only that a Rho-kinase–mediated mechanism contributes to blood pressure regulation but also that Y27632 is a valuable tool for investigating the Rho-kinase function of vascular cells in vivo and its pathophysiological implications. The aims of this study were to examine whether Y27632 prevents neointima formation after vascular injury and if so, to investigate the possible mechanisms of the inhibitory effect of Y27632.
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Methods |
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Vascular Injury Model and Morphometry
All procedures were approved by the Institutional Animal Care and Use Committee and were conducted in conformity with institutional guidelines. Male Wistar rats (400 to 450 g; Japan SLC Inc; Shizuoka, Japan) were anesthetized with sodium pentobarbital (50 mg/kg), and then balloon injury of the left carotid artery was performed as described previously.13 Sham-operated rats underwent the same operation, except that the balloon was not inserted. Y27632 was supplied by Yoshitomi Pharmaceutical Industries. Y27632 (15 mg/kg) or vehicle (saline) was administered intraperitoneally twice a day from 1 day before balloon injury for 4, 7, or 14 consecutive days. Blood pressure in conscious, unrestricted rats was measured by the tail-cuff method. After the rat was killed with an overdose of pentobarbital, the carotid artery was perfusion-fixed at 100 mm Hg with 4% paraformaldehyde, excised, and embedded in paraffin. Three individual sections (3 µm) from the middle of injured segments were stained with hematoxylin/eosin in each rat (n=5). The medial and intimal areas were measured with a computerized digital image analysis system and averaged in 3 independent sections.
Detection of DNA Synthesis In Vivo and
Apoptosis
In vivo bromodeoxyuridine (BrdU) labeling was
performed to identify replicating cells in balloon-injured artery by
detection of the DNA synthesis with a Cell Proliferation kit (Amersham
Pharmacia Biotech): 0.5 mg/kg BrdU, a thymidine analogue, was injected
intraperitoneally 24 hours before preparation of the artery, and BrdU
incorporated into replicating cells was detected immunohistochemically
with an anti-BrdU antibody. In each serial section, apoptotic cells
were detected by the terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end-labeling (TUNEL) method using Apop Tag
(Intergen). The counterstain was done with 0.5% methyl green. In each
experimental setup, rat small intestine and testis were used as
positive controls. A negative control was incubated without the TdT.
Double immunohistostaining with 
-smooth muscle actin showed that
these BrdU+ or
TUNEL+ cells were SMCs. The total,
BrdU+, and TUNEL+
nucleated SMCs were counted at a magnification of x400 by 3 observers
in a blinded manner, and the values obtained by the 3 observers were
averaged in each section. The quantitative analysis was performed in 5
independent sections in each rat (n=5). The numbers of
BrdU+ and TUNEL+
SMCs were expressed as BrdU labeling and TUNEL indexes
(BrdU+ and TUNEL+
SMCs/total nucleated SMCsx100), respectively.
Number of SMCs in Early Neointimal
Lesions
The number of SMCs in the injured artery was counted
at day 4 by the modified method of Prescott et
al.14 Briefly, the cross
sections were subjected to immunohistostaining against -smooth
muscle actin with a commercially available detection system (DAKO) and
counterstained with hematoxylin. The number of nuclei that were
accompanied by -smooth muscle actin–positive cytoplasm was counted
at a magnification of x400 in 10 independent sections from each rat
(n=5) by an observer in a blinded fashion.
Analysis of Reendothelialization
Planimetric analysis of reendothelialization after
balloon injury was performed with a computerized digital image analysis
system15 by an observer in a
blinded manner. Briefly, 30 minutes before rats (n=5) were killed, they
received an intravenous injection of 6 mL of 0.5% Evans blue dye
(Sigma) to identify areas of nonendothelialized artery with a blue
stain. After perfusion-fixation at 100 mm Hg with 100% methanol, the
injured artery was excised, incised longitudinally, and then
photographed with a dissecting microscope. The initially injured area
was defined as the total surface of the harvested arterial segment. The
total harvested segment corresponded to the total length of the injured
segment; in each case, this length was similarly defined proximally by
the carotid bifurcation and distally by the edge of the omohyoid
muscle. The reendothelialized area was defined as the area not stained
with Evans blue dye. Extent of reendothelialization (%
reendothelialization) was expressed as a percentage of the initially
injured area.
Measurement of MBS Phosphorylations
After Y27632 or saline had been administered to rats
twice a day for 4 days (n=4), the carotid artery was excised and cut
into 3-mm-wide strips. Equal amounts of the strips were incubated in
Hanks’ buffered salt solution with or without 10 µmol/L
phenylephrine for 5 minutes (37°C). The reaction was stopped by
immediately freezing the strips in dry ice/acetone. After tissue
homogenization, protein extract was separated by 7.5% SDS-PAGE and
subjected to immunoblotting with an anti–phosphorylated MBS
antibody,16 and the signals
were detected and analyzed with a chemifluorescence detection system
and FluoroImager (Amersham). The specificity of this antibody was
described
elsewhere.16
Statistical Analysis
Statistical analysis was performed by unpaired
Student’s t test or ANOVA
followed by Scheffé’s F test. A value of
P<0.05 was considered
significant. The interobserver or intraobserver variation was <5% in
each experiment.
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There were no significant differences in heart rate, blood pressure, or body weight between saline-treated control rats and Y27632-treated rats during the observation period, and apparently, no other evidence of an adverse effect of Y27632 was observed.
Neointima Formation After Vascular
Injury
After vascular injury, SMCs were first observed in the
intima at day 4, and thereafter, neointima formation progressed,
resulting in thick myointima at day 14
(Figure 1A
, top right). Y27632 remarkably prevented
the neointima formation
(Figure 1A, bottom left). The intima/media area ratio at days
7 and 14 was reduced by 43% and 74%, respectively, by Y27632
(P<0.05 and
P<0.001,
Figure 1B). The medial cross-sectional area did not differ
between them. The intima and media remained intact in the sham-operated
artery with Y27632 treatment.
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Cell Proliferation and Apoptosis in Injured
Artery
Serial arterial sections were subjected to in vivo BrdU
labeling and the TUNEL method
(Figures 2 and 3). In the intact artery, neither
BrdU+ nor TUNEL+
cells were detected in the media and intima
(Figures 2Aa and 3Aa). In controls,
BrdU+ SMCs were scarcely observed in the
intima at day 4, and a robust increase in neointimal
BrdU+ SMCs was observed at day 7
(Figure 2Ab), returning to lower levels at day 14
(Figure 2Ad). Y27632 had no effect on the number of
BrdU+ SMCs over the course of the study
(Figure 2B). In controls, very small numbers of
TUNEL+ SMCs were found in the intima at day
4, and TUNEL+ SMCs robustly increased in the
neointima at day 7
(Figure 3Ab) and decreased to lower levels at day 14
(Figure 3Ad). When Y27632 was administered, although the
TUNEL index at day 4 was similar to that of controls, the increase in
TUNEL+ SMCs was enhanced at day 7 versus
controls (P<0.01) and was
sustained at day 14 (P<0.001,
Figure 3B). SMC apoptosis was confirmed by typical electron
microscopic features of apoptosis and the DNA ladder formation on
genomic DNA gel electrophoresis (data not shown).
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, control; •, Y27632) and media (
, control; 
, Y27632). Bar=1 SD (n=5).
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In the media, smaller numbers of BrdU+ or TUNEL+ SMCs were observed after vascular injury than in the intima, and Y27632 had no effect on BrdU labeling and TUNEL indexes. In the sham-operated artery, BrdU+ or TUNEL+ SMCs were not found during the observation period, irrespective of Y27632 treatment.
Early Neointimal Lesion Formation
Consistent with earlier
studies,8 9 small
numbers of SMCs were sporadically observed in the intima at day 4
(Table).
Y27632 reduced the number of intimal SMCs at day 4 by 50%
(P<0.01), while not affecting
BrdU labeling and TUNEL indexes
(Table).
The number of medial SMCs did not differ between the
groups.
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Reendothelialization in Injured Artery
Effects of Y27632 on reendothelialization were
evaluated by planimetric analysis performed with Evans blue dye
staining
(Figure 4A). The initially injured area was equivalent in
Y27632-treated and control rats
(Figure 4Ba). Controls showed progressive
reendothelialization, with % reendothelialization of 20% and 43% at
days 7 and 14, respectively
(Figure 4Bb), consistent with the previous
studies.17 Y27632 had no
significant effects on reendothelialized area or %
reendothelialization at day 7 or 14
(Figure 4Bc).
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) or control (
) artery. % Reendothelialization at days 7 and 14 (c) was expressed as percentage of initially injured area. Bar=1 SD (n=5). *P<0.05 vs control.
MBS Phosphorylation Levels
Phosphorylation levels of MBS, a specific substrate of
activated Rho-kinase,6 were
examined to evaluate the Rho-kinase activity of the carotid artery.
Denoted doses of Y27632 or saline were administered to rats twice a day
for 4 days. In the vascular strips obtained from controls,
phosphorylated MBS was not detected at baseline, and ex vivo
phenylephrine stimulation markedly induced MBS phosphorylation
(Figure 5). In the vascular strips from Y27632-treated rats,
there was no baseline MBS phosphorylation, and the ex vivo
phenylephrine-induced MBS phosphorylation was dose-dependently
inhibited by Y27632.
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Discussion |
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The present study demonstrated that a Rho-kinase inhibitor, Y27632, prevented neointima formation after vascular injury. At day 4, Y27632 reduced the number of intimal SMCs but did not affect SMC replication and apoptosis. Thereafter, Y27632 enhanced neointimal SMC apoptosis. Reendothelialization after injury was not significantly affected by Y27632 at day 7 or 14.
Y27632, a pyrimidine derivative, is a new specific
inhibitor of the Rho-kinase
family.12 Y27632 is
competitive for the ATP-binding site of Rho-kinase and is very
selective for Rho-kinase; its affinity for Rho-kinase is >200 times
and >2000 times higher than that for protein kinase C or A and MLC
kinase, respectively.12
Y27632 inhibits the Rho-kinase–mediated processes, including MLC
phosphorylation, which enhances myosin-based contractility, in
SMCs.12 Accordingly, Y27632
can induce SMC relaxation and suppression of
hypertension12 and can
inhibit migration of cultured
SMCs.18 In contrast, Y27632
has no effects on other Rho-family GTPases, such as Rac and
Cdc42.12 As shown in
Figure 5
, the phenylephrine-induced MBS phosphorylation was
inhibited by Y27632, indicating that the arterial Rho-kinase activity
was inhibited by Y27632. In this study, the dose of Y27632 used was
similar to that showing antihypertensive effects in several
hypertensive rat models.12
Previous studies reported that Y27632 had little effect on blood
pressure in normotensive
rats.12 Consistent with
these studies, Y27632 did not change blood pressure in the
balloon-injured Wistar rats, indicating that the inhibitory effects of
Y27632 on neointima formation are independent of the effect on blood
pressure. Also, no apparent adverse effects were observed in
Y27632-treated rats, and Y27632 had no effects on the sham-operated
artery. Furthermore, the ability of endothelial cells to
reendothelialize was preserved in Y27632-treated rats
(Figure 4). Accordingly, the observed effects of Y27632 on
SMCs in the injured artery seem specifically, but not nonspecifically,
toxic.
A major process that leads to neointima formation after
vascular injury is the SMC accumulation in the neointima, which is the
sum of cell migration, cell proliferation, and apoptotic cell loss.
Consistent with earlier
studies,10 11 SMC
replication and apoptosis were transiently and coincidentally observed
after vascular injury in the neointima, with a peak at day 7, declining
to lower levels at day 14. Although the rates of replication and
apoptosis were increased after vascular injury, the rate of replication
must have predominated in the neointima, and subsequently, neointima
formation was produced. Although Y27632 had no effect on SMC
replication
(Figure 2), Y27632 significantly increased apoptotic SMCs in
the neointima at days 7 and 14
(Figure 3). Accordingly, enhanced apoptosis of the neointimal
SMCs is likely to be involved in the mechanisms of prevention of
neointima formation by Y27632. It is noteworthy that Y27632 enhanced
SMC apoptosis in the injured artery without any effect on cell
replication; in contrast, SMC replication is prevented by most other
inhibitors of neointima formation, including endothelin receptor
antagonist,19
cilostazol,20 and
tranilast.21 The precise
molecular mechanisms whereby Y27632 enhanced neointimal SMC apoptosis
remain to be determined. It is noteworthy that enhanced SMC apoptosis
by Y27632 was observed specifically in the neointima. Although the
mechanism remains unclarified, this may be related to the phenotypic
difference and the possible differential susceptibilities to apoptosis
between medial and neointimal SMCs, as suggested by Pollman et
al.22 23
Another possible mechanism of the inhibitory effect of
Y27632 would be the prevention of SMC migration from the media. There
is no available method to estimate SMC migration in vivo at the later
phase, when SMC replication has actively occurred. Thus, the effects of
Y27632 on early neointimal lesion formation were evaluated at day 4.
Intimal SMCs were first observed at day 4, as earlier studies
reported.8 9 The
intimal SMCs at day 4 are considered to derive primarily from medial
SMC migration into the intima and, to a lesser extent, from the initial
proliferation of migrated
SMCs.14 24 25
Y27632 reduced the number of intimal SMCs without any effects on SMC
replication and apoptosis at day 4
(Table).
Accordingly, it seems plausible that the reduction in neointimal SMCs
by Y27632 would be mainly due to decreased early SMC migration. This is
in accord with a recent observation that Y27632 suppressed
thrombin-induced migration of cultured
SMCs.18 At present, however,
it remains unclear not only how far the early suppression of SMC
migration contributed to the Y27632-induced prevention of neointima
formation observed at days 7 and 14 but also whether the inhibitory
effects of Y27632 on SMC migration continued up to the later phase of
neointima formation.
Reendothelialization is one of the important aspects of the
response to balloon
injury.8 17
Accordingly, we evaluated the effects of Y27632 on reendothelialization
at days 7 and 14. Y27632 did not affect the extent of
reendothelialization after vascular injury
(Figure 4). Thus, the inhibitory effects of Y27632 on
neointima formation were not attributed to reendothelialization at
least by day 14.
Recently, there have been increasing lines of evidence that Rho-kinase inhibitors may be a new category of agents that prevent a number of vascular responses to injury. A less specific Rho-kinase inhibitor than Y27632, fasudil, which is known to be an MLC kinase inhibitor, inhibited migration of cultured rabbit SMCs and reduced neointima formation by enhancing SMC loss without affecting cell proliferation in balloon-injured rabbit carotid artery.26 In porcine coronary artery showing the inflammatory/arteriosclerotic remodeling induced by interleukin-1ß treatment, increased Rho-kinase activity was documented, and hydroxyfasudil, a derivative of fasudil, suppressed serotonin-induced vasospasm at the remodeling site.27 This, taken together with the present study using a more specific Rho-kinase inhibitor, Y27632,12 indicates that the Rho-kinase–mediated pathway may play important roles in vascular responses to various kinds of vascular injury that is implicated in the pathogenesis of arteriosclerosis through the mechanisms of not only vascular remodeling but also vasomotion control.
Limitations of This Study
First, at present, no direct method is available
to assay Rho-kinase activity in the vascular samples. Also, no rescue
experiment was done on Y27632-treated rats. Thus, it remains possible
that inhibition of other serine-threonine protein kinases in addition
to Rho-kinase may have contributed to the effects of Y27632 observed in
the present study. Second, because Rho is not the only possible
activator of Rho-kinase,28
it is possible that upstream pathways other than Rho would activate
Rho-kinase in injured artery. Third, because Y27632 was systemically
administered for a long time in the present study, the possibility of
the involvement of indirect systemic effects, such as the neurohumoral
effects, cannot be excluded. Fourth, we used BrdU labeling and the
TUNEL method for the cell kinetic study, because these methods are
commonly accepted as the most reliable methods currently available in
vivo. However, quantitative analysis using these methods is potentially
limited, because both the methods are based on immunohistochemical
techniques. Furthermore, recent studies have suggested that the TUNEL
method does not necessarily provide a specific marker of apoptosis, and
the TUNEL phenomenon may also occur in other types of cell death,
including necrosis and
oncosis,29 and even in
nonapoptotic nuclei with abundant RNA transcription and
splicing30 or with DNA
repair synthesis.31 Thus, it
is conceivable that the present study based on the TUNEL method may
have overestimated the true incidence of apoptotic SMCs in injured
artery irrespective of Y27632 treatment. In addition, another
methodological limitation of the TUNEL method is the lack of
appropriate standardization. These issues should be addressed in future
studies. Finally, future studies should evaluate whether Y27632 remains
effective in the chronic phase of vascular remodeling later than day
14.
In conclusion, a Rho-kinase inhibitor, Y27632, inhibited early neointimal lesion formation, probably by suppressing early SMC migration into the intima and prevented neointima formation in the later phase by enhancing neointimal SMC apoptosis. Thus, a role of Rho-kinase in neointima formation is suggested by regulation of SMC migration and apoptosis. Finally, the present study may provide insight into the possible treatment strategy to prevent progression of atherosclerosis by inducing apoptosis in neointimal SMCs during the remodeling process after vascular injury.
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Acknowledgments |
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This study was supported in part by a Kimura Memorial Heart Foundation research grant. We thank Kaoru Moriyama for technical assistance.
Received June 9, 2000; revision received July 20, 2000; accepted July 21, 2000.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Takai Y, Sasaki T, Tanaka K, et al. Rho as a regulator of cytoskeleton. Trends Biochem Sci. 1995;20:227–231.[Medline] [Order article via Infotrieve]
2. Narumiya S, Ishizaki T, Watanabe N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 1997;410:68–72.[Medline] [Order article via Infotrieve]
3.
Leung T, Manser E,
Tan L, et al. A novel serine/threonine kinase binding the Ras-related
RhoA GTPase which translocates the kinase to peripheral membrane.
J Biol Chem. 1995;268:3813–3816.
4. Ishizaki T, Maekawa M, Fujisawa K, et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 1996;15:1885–1893.[Medline] [Order article via Infotrieve]
5. Matsui T, Amano M, Yamamoto T, et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 1996;15:2208–2216.[Medline] [Order article via Infotrieve]
6. Kimura K, Itoh M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase. Science. 1996;269:221–223.
7. Lacal JC. Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling. FEBS Lett. 1997;410:73–77.[Medline] [Order article via Infotrieve]
8. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]
9.
Clowes AW, Schwartz
SM. Significance of quiescent smooth muscle migration in the injured
rat carotid artery. Circ Res. 1985;56:139–145.
10. Han DKM, Haudenschild CC, Hong MK, et al. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995;147:267–277.[Abstract]
11. Bochaton-Piallat ML, Gabbiani F, Redard M, et al. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1059–1064.[Abstract]
12. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389:990–994.[Medline] [Order article via Infotrieve]
13.
Yasukawa H,
Imaizumi T, Matsuoka H, et al. Inhibition of intimal hyperplasia after
balloon injury by antibodies to ICAM-1.
Circulation. 1997;95:1515–1522.
14. Prescott M, Webb R, Reidy MA. Angiotensin-converting enzyme inhibitors versus angiotensin II, AT1 receptor antagonist: effects on smooth muscle cell migration and proliferation after balloon catheter injury. Am J Pathol. 1991;139:1291–1296.[Abstract]
15.
Asahara T, Chen
D, Tsurumi Y, et al. Accelerated restitution of endothelial integrity
and endothelium-dependent function after phVEGF165 gene transfer.
Circulation. 1996;94:3291–3302.
16.
Kawano Y, Fukata
Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit (MBS) of
myosin phosphatase by Rho-kinase during cell migration and cytokinesis.
J Cell Biol. 1999;147:1023–1038.
17.
Asahara T,
Beuters C, Pastore C, et al. Local delivery of vascular endothelial
growth factor accelerates reendothelialization and attenuates intimal
hyperplasia in balloon-injured rat carotid artery.
Circulation. 1995;91:2793–2801.
18. Seasholtz TM, Majumdar M, Kaplan DD, et al. Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res. 1999;84:1184–1193.
19. Ferrer P, Valentine M, Jenkins-West T, et al. Orally active endothelin receptor antagonist BMS-182874 suppresses neointimal development in balloon-injured rat carotid arteries. J Cardiovasc Pharmacol. 1995;26:908–915.[Medline] [Order article via Infotrieve]
20. Ishizaka N, Taguchi J, Kimura Y, et al. Effects of a single local administration of cilostazol on neointimal formation in balloon-injured rat carotid artery. Atherosclerosis. 1999;142:41–46.[Medline] [Order article via Infotrieve]
21.
Takahashi A,
Taniguchi T, Ishikawa Y, et al. Tranilast inhibits vascular smooth
muscle cell growth and intimal hyperplasia by induction of
p21(waf1/cip1/sdi1) and p53. Circ
Res. 1999;84:543–550.
22. Pollman MJ, Hall JL, Mann MJ, et al. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nat Med. 1998;4:222–227.[Medline] [Order article via Infotrieve]
23.
Pollman MJ, Hall
JL, Gibbons GH. Determinants of vascular smooth muscle cell apoptosis
after balloon angioplasty injury. Circ
Res. 1999;84:113–121.
24.
Bendeck MP, Zempo
N, Clowes AW, et al. Smooth muscle cell migration and matrix
metalloproteinase expression after arterial injury in the rat.
Circ Res. 1994;75:539–545.
25.
Furukawa Y,
Matusmori A, Ohashi N, et al. Anti-monocyte chemoattractant
protein-1/monocyte chemotactic and activating factor antibody inhibits
neointimal hyperplasia in injured rat carotid arteries.
Circ Res. 1999;84:306–314.
26. Negoro N, Hoshiga M, Seto M, et al. The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells. Biochem Biophys Res Commun. 1999;262:211–215.[Medline] [Order article via Infotrieve]
27.
Shimokawa H, Seto
M, Katsumata N, et al. Rho-kinase-mediated pathway induces enhanced
myosin light chain phosphorylations in a swine model of coronary spasm.
Cardiovasc Res. 1999;43:1029–1039.
28.
Feng J, Ito M,
Kureishi Y, et al. Rho-associated kinase of chicken gizzard smooth
muscle. J Biol Chem. 1999;274:3744–3752.
29. Grasl-Kraup B, Ruttkay-Nedecky B, Koudelka H, et al. In situ detection of fragmented DNA (TUNEL) assay fails to discriminate among apoptosis, necrosis and autolytic cell death: a caution note. Hepatology. 1995;21:1465–1468.[Medline] [Order article via Infotrieve]
30. Kockx MM, Muhring J, Knappen MWM, et al. RNA synthesis and splicing interferes with DNA in situ end labeling technique used to detect apoptosis. Am J Pathol. 1998;152:885–888.[Abstract]
31.
Kanoh M, Takemura
G, Misao J, et al. Significance of myocytes with positive DNA in situ
nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not
apoptosis but DNA repair.
Circulation. 1999;99:2757–2764.
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K. J. Ho, C. D. Owens, T. Longo, X. X. Sui, C. Ifantides, and M. S. Conte C-reactive protein and vein graft disease: evidence for a direct effect on smooth muscle cell phenotype via modulation of PDGF receptor-{beta} Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1132 - H1140. [Abstract] [Full Text] [PDF]
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T. Shimizu, T. Nakazawa, A. Cho, F. Dastvan, D. Shilling, G. Daum, and M. A. Reidy Sphingosine 1-Phosphate Receptor 2 Negatively Regulates Neointimal Formation in Mouse Arteries Circ. Res., November 9, 2007; 101(10): 995 - 1000. [Abstract] [Full Text] [PDF]
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 H. Ikeda, Y. Kume, K. Tejima, T. Tomiya, T. Nishikawa, N. Watanabe, N. Ohtomo, M. Arai, C. Arai, M. Omata, et al. Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G911 - G917. [Abstract] [Full Text] [PDF]
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 K. Nakamura, S.-i. Yamagishi, T. Matsui, T. Yoshida, K. Takenaka, Y. Jinnouchi, Y. Yoshida, S.-i. Ueda, H. Adachi, and T. Imaizumi Pigment Epithelium-Derived Factor Inhibits Neointimal Hyperplasia after Vascular Injury by Blocking NADPH Oxidase-Mediated Reactive Oxygen Species Generation Am. J. Pathol., June 1, 2007; 170(6): 2159 - 2170. [Abstract] [Full Text] [PDF]
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 K. Kusaba, H. Kai, M. Koga, N. Takayama, A. Ikeda, H. Yasukawa, Y. Seki, K. Egashira, and T. Imaizumi Inhibition of Intrinsic Interferon-{gamma} Function Prevents Neointima Formation After Balloon Injury Hypertension, April 1, 2007; 49(4): 909 - 915. [Abstract] [Full Text] [PDF]
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 E. Gurbanov and X. Shiliang The key role of apoptosis in the pathogenesis and treatment of pulmonary hypertension. Eur. J. Cardiothorac. Surg., September 1, 2006; 30(3): 499 - 507. [Abstract] [Full Text] [PDF]
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G. Loirand, P. Guerin, and P. Pacaud Rho Kinases in Cardiovascular Physiology and Pathophysiology Circ. Res., February 17, 2006; 98(3): 322 - 334. [Abstract] [Full Text] [PDF]
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I. Gorenne, L. Jin, T. Yoshida, J.M. Sanders, I.J. Sarembock, G.K. Owens, A.P. Somlyo, and A.V. Somlyo LPP Expression During In Vitro Smooth Muscle Differentiation and Stent-Induced Vascular Injury Circ. Res., February 17, 2006; 98(3): 378 - 385. [Abstract] [Full Text] [PDF]
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 H. Ono, T. Ichiki, H. Ohtsubo, K. Fukuyama, I. Imayama, Y. Hashiguchi, J. Sadoshima, and K. Sunagawa Critical Role of Mst1 in Vascular Remodeling After Injury Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1871 - 1876. [Abstract] [Full Text] [PDF]
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H. Shimokawa and A. Takeshita Rho-Kinase Is an Important Therapeutic Target in Cardiovascular Medicine Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1767 - 1775. [Abstract] [Full Text] [PDF]
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 Y.-X. Wang, B. Martin-McNulty, V. da Cunha, J. Vincelette, X. Lu, Q. Feng, M. Halks-Miller, M. Mahmoudi, M. Schroeder, B. Subramanyam, et al. Fasudil, a Rho-Kinase Inhibitor, Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm in Apolipoprotein E-Deficient Mice by Inhibiting Apoptosis and Proteolysis Circulation, May 3, 2005; 111(17): 2219 - 2226. [Abstract] [Full Text] [PDF]
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G. Loirand, M. Rolli-Derkinderen, and P. Pacaud RhoA and resistance artery remodeling Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1051 - H1056. [Abstract] [Full Text] [PDF]
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 T. M. Seasholtz and J. H. Brown RHO SIGNALING in Vascular Diseases Mol. Interv., December 1, 2004; 4(6): 348 - 357. [Abstract] [Full Text] [PDF]
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 M. Moore, B. A. Marroquin, W. Gugliotta, R. Tse, and S. R. White Rho Kinase Inhibition Initiates Apoptosis in Human Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2004; 30(3): 379 - 387. [Abstract] [Full Text] [PDF]
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H. Kawamura, K. Yokote, S. Asaumi, K. Kobayashi, M. Fujimoto, Y. Maezawa, Y. Saito, and S. Mori High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 276 - 281. [Abstract] [Full Text]
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K. Tokuda, H. Kai, F. Kuwahara, H. Yasukawa, N. Tahara, H. Kudo, K. Takemiya, M. Koga, T. Yamamoto, and T. Imaizumi Pressure-Independent Effects of Angiotensin II on Hypertensive Myocardial Fibrosis Hypertension, February 1, 2004; 43(2): 499 - 503. [Abstract] [Full Text] [PDF]
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T. Hattori, H. Shimokawa, M. Higashi, J. Hiroki, Y. Mukai, K. Kaibuchi, and A. Takeshita Long-Term Treatment With a Specific Rho-Kinase Inhibitor Suppresses Cardiac Allograft Vasculopathy in Mice Circ. Res., January 9, 2004; 94(1): 46 - 52. [Abstract] [Full Text] [PDF]
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 E. B. Okon, M. J. Millar, C. M. Crowley, J. G. Bashir, R. C. Cook, Y. N. Hsiang, B. McManus, and C. van Breemen Effect of moderate pressure distention on the human saphenous vein vasomotor function Ann. Thorac. Surg., January 1, 2004; 77(1): 108 - 114. [Abstract] [Full Text] [PDF]
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Y. Matsumoto, T. Uwatoku, K. Oi, K. Abe, T. Hattori, K. Morishige, Y. Eto, Y. Fukumoto, K.-i. Nakamura, Y. Shibata, et al. Long-Term Inhibition of Rho-Kinase Suppresses Neointimal Formation After Stent Implantation in Porcine Coronary Arteries: Involvement of Multiple Mechanisms Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 181 - 186. [Abstract] [Full Text] [PDF]
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 H. K. Surks, C. T. Richards, and M. E. Mendelsohn Myosin Phosphatase-Rho Interacting Protein: A NEW MEMBER OF THE MYOSIN PHOSPHATASE COMPLEX THAT DIRECTLY BINDS RhoA J. Biol. Chem., December 19, 2003; 278(51): 51484 - 51493. [Abstract] [Full Text] [PDF]
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A.-C. Tosello-Trampont, K. Nakada-Tsukui, and K. S. Ravichandran Engulfment of Apoptotic Cells Is Negatively Regulated by Rho-mediated Signaling J. Biol. Chem., December 12, 2003; 278(50): 49911 - 49919. [Abstract] [Full Text] [PDF]
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Y. Izumi, S. Kim, M. Yoshiyama, Y. Izumiya, K. Yoshida, A. Matsuzawa, H. Koyama, Y. Nishizawa, H. Ichijo, J. Yoshikawa, et al. Activation of Apoptosis Signal-Regulating Kinase 1 in Injured Artery and Its Critical Role in Neointimal Hyperplasia Circulation, December 2, 2003; 108(22): 2812 - 2818. [Abstract] [Full Text] [PDF]
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Z. Mallat, A. Gojova, V. Sauzeau, V. Brun, J.-S. Silvestre, B. Esposito, R. Merval, H. Groux, G. Loirand, and A. Tedgui Rho-Associated Protein Kinase Contributes to Early Atherosclerotic Lesion Formation in Mice Circ. Res., October 31, 2003; 93(9): 884 - 888. [Abstract] [Full Text] [PDF]
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V. Sauzeau, M. Rolli-Derkinderen, S. Lehoux, G. Loirand, and P. Pacaud Sildenafil Prevents Change in RhoA Expression Induced by Chronic Hypoxia in Rat Pulmonary Artery Circ. Res., October 3, 2003; 93(7): 630 - 637. [Abstract] [Full Text] [PDF]
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T. Tokunou, R. Shibata, H. Kai, T. Ichiki, T. Morisaki, K. Fukuyama, H. Ono, N. Iino, S. Masuda, H. Shimokawa, et al. Apoptosis Induced by Inhibition of Cyclic AMP Response Element-Binding Protein in Vascular Smooth Muscle Cells Circulation, September 9, 2003; 108(10): 1246 - 1252. [Abstract] [Full Text] [PDF]
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 P.-L. Tharaux, R. C. Bukoski, P. N. Rocha, S. D. Crowley, P. Ruiz, C. Nataraj, D. N. Howell, K. Kaibuchi, R. F. Spurney, and T. M. Coffman Rho Kinase Promotes Alloimmune Responses by Regulating the Proliferation and Structure of T Cells J. Immunol., July 1, 2003; 171(1): 96 - 105. [Abstract] [Full Text] [PDF]
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T. Uwatoku, H. Shimokawa, K. Abe, Y. Matsumoto, T. Hattori, K. Oi, T. Matsuda, K. Kataoka, and A. Takeshita Application of Nanoparticle Technology for the Prevention of Restenosis After Balloon Injury in Rats Circ. Res., April 18, 2003; 92 (7): e62 - e69. [Abstract] [Full Text] [PDF]
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F. Kuwahara, H. Kai, K. Tokuda, H. Niiyama, N. Tahara, K. Kusaba, K. Takemiya, A. Jalalidin, M. Koga, T. Nagata, et al. Roles of Intercellular Adhesion Molecule-1 in Hypertensive Cardiac Remodeling Hypertension, March 1, 2003; 41(3): 819 - 823. [Abstract] [Full Text] [PDF]
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Z. Chen, T. Fukutomi, A. C. Zago, R. Ehlers, P. A. Detmers, S. D. Wright, C. Rogers, and D. I. Simon Simvastatin Reduces Neointimal Thickening in Low-Density Lipoprotein Receptor-Deficient Mice After Experimental Angioplasty Without Changing Plasma Lipids Circulation, July 2, 2002; 106(1): 20 - 23. [Abstract] [Full Text] [PDF]
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F. Kuwahara, H. Kai, K. Tokuda, M. Kai, A. Takeshita, K. Egashira, and T. Imaizumi Transforming Growth Factor-{beta} Function Blocking Prevents Myocardial Fibrosis and Diastolic Dysfunction in Pressure-Overloaded Rats Circulation, July 2, 2002; 106(1): 130 - 135. [Abstract] [Full Text] [PDF]
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X. Li, L. Liu, J. C. Tupper, D. D. Bannerman, R. K. Winn, S. M. Sebti, A. D. Hamilton, and J. M. Harlan Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells J. Biol. Chem., May 3, 2002; 277(18): 15309 - 15316. [Abstract] [Full Text] [PDF]
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K. G. Lamping Enhanced Contractile Mechanisms in Vasospasm: Is Endothelial Dysfunction the Whole Story? Circulation, April 2, 2002; 105(13): 1520 - 1522. [Full Text] [PDF]
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C. Kataoka, K. Egashira, S. Inoue, M. Takemoto, W. Ni, M. Koyanagi, S. Kitamoto, M. Usui, K. Kaibuchi, H. Shimokawa, et al. Important Role of Rho-kinase in the Pathogenesis of Cardiovascular Inflammation and Remodeling Induced by Long-Term Blockade of Nitric Oxide Synthesis in Rats Hypertension, February 1, 2002; 39(2): 245 - 250. [Abstract] [Full Text] [PDF]
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H. Kai, F. Kuwahara, K. Tokuda, R. Shibata, K. Kusaba, H. Niiyama, N. Tahara, T. Nagata, and T. Imaizumi Coexistence of Hypercholesterolemia and Hypertension Impairs Adventitial Vascularization Hypertension, February 1, 2002; 39(2): 455 - 459. [Abstract] [Full Text] [PDF]
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N. L. Weintraub Nox Response to Injury Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 4 - 5. [Full Text] [PDF]
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K. Szocs, B. Lassegue, D. Sorescu, L. L. Hilenski, L. Valppu, T. L. Couse, J. N. Wilcox, M. T. Quinn, J.D. Lambeth, and K. K. Griendling Upregulation of Nox-Based NAD(P)H Oxidases in Restenosis After Carotid Injury Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 21 - 27. [Abstract] [Full Text] [PDF]
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F. Kuwahara, H. Kai, K. Tokuda, R. Shibata, K. Kusaba, N. Tahara, H. Niiyama, T. Nagata, and T. Imaizumi Hypoxia-Inducible Factor-1{alpha}/Vascular Endothelial Growth Factor Pathway for Adventitial Vasa Vasorum Formation in Hypertensive Rat Aorta Hypertension, January 1, 2002; 39(1): 46 - 50. [Abstract] [Full Text] [PDF]
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 E. McGregor, M. Gosling, D. K Beattie, D. M.P Ribbons, A. H Davies, and J. T Powell Circumferential stretching of saphenous vein smooth muscle enhances vasoconstrictor responses by Rho kinase-dependent pathways Cardiovasc Res, January 1, 2002; 53(1): 219 - 226. [Abstract] [Full Text] [PDF]
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F. Degraeve, M. Bolla, S. Blaie, C. Creminon, I. Quere, P. Boquet, S. Levy-Toledano, J. Bertoglio, and A. Habib Modulation of COX-2 Expression by Statins in Human Aortic Smooth Muscle Cells. INVOLVEMENT OF GERANYLGERANYLATED PROTEINS J. Biol. Chem., December 7, 2001; 276(50): 46849 - 46855. [Abstract] [Full Text] [PDF]
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V. Sauzeau, E. Le Mellionnec, J. Bertoglio, E. Scalbert, P. Pacaud, and G. Loirand Human Urotensin II-Induced Contraction and Arterial Smooth Muscle Cell Proliferation Are Mediated by RhoA and Rho-Kinase Circ. Res., June 8, 2001; 88(11): 1102 - 1104. [Abstract] [Full Text] [PDF]
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