© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo
From the Department of Cardiovascular Medicine (K. Morishige, H.S., Y.E., T.K., K. Miyata, Y.M., A.T.), Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; the Institute of Molecular Medicine (M.H.), University of California, San Diego; and the Department of Pharmacology (K.K.), Nagoya University Graduate School of Medicine, Nagoya, Japan.
Correspondence to Hiroaki Shimokawa, MD, PhD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail shimo{at}cardiol.med.kyushu-u.ac.jp
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Abstract |
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Abstract—Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1ß, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of ß-galactosidase, into the interleukin-1ß–treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRhoK. These results indicate that Rho-kinase is substantially involved in coronary constrictive remodeling and vasospastic responses, both of which can be reversed by the selective inhibition of the molecule in our porcine model in vivo.
Key Words: Rho • Rho-kinase • arteriosclerosis • vasospasm • remodeling
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Introduction |
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Arteriosclerosis is an inflammatory vascular disease that is caused by a series of processes, including endothelial activation, the migration of macrophages and other inflammatory cells, and proliferative responses of vascular smooth muscle cells.1
Recent studies in vitro have suggested that the small GTP-binding protein Rho and its target protein Rho-kinase/ROK/ROCK2 3 4 play an important role in various cellular functions, including smooth muscle contraction,5 6 actin cytoskeleton organization,7 8 cell adhesion and motility,9 cytokinesis,10 and gene expression,11 all of which may be involved in the pathogenesis of arteriosclerosis. We have recently demonstrated that Rho-kinase is functionally upregulated at the inflammatory coronary lesions and plays an important role in the pathogenesis of coronary hyperconstriction in our porcine model with interleukin (IL)-1ß.12 The IL-1ß–treated coronary segment is characterized by luminal reduction caused by neointimal formation and constrictive remodeling.13 Thus, our porcine model may be useful in the examination of the molecular mechanism of coronary arteriosclerosis in humans.
We have recently found that hydroxyfasudil, an active metabolite of fasudil after oral absorption, is a potent and specific inhibitor of Rho-kinase and markedly inhibits coronary hyperconstriction and macrophage migration.14 15 Thus, it is conceivable that Rho-kinase is involved not only in the pathogenesis of coronary artery spasm but also in that of coronary arteriosclerosis.
To test this hypothesis, we examined in the present study whether selective inhibition of Rho-kinase by the local adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) can effectively induce a regression of coronary arteriosclerotic lesions in our porcine model in vivo.
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Methods |
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This experiment was reviewed by the Committee of the Ethics on Animal Experiments at Kyushu University and was carried out under the control of the Guidelines for Animal Experiments at Kyushu University.
Animal Preparation
Domestic male pigs (2 to 3 months old, weighing 25 to
30 kg) were sedated with intramuscular ketamine hydrochloride
(1.5 mg/kg) and anesthetized with intravenous
sodium pentobarbital (30 mg/kg). The animals were then intubated and
ventilated with room air. Under aseptic conditions, the proximal
segments of the left anterior descending and the left circumflex
coronary arteries with a comparable external diameter were
applied with a recombinant human IL-1ß bead suspension (2.5 µg in
0.05 mL) as described.13 Two
weeks after the operation, each animal was again anesthetized
in the same manner, and then a 10F sheath was inserted through the
carotid artery. After systemic heparinization (10 000 U per body), a
preshaped 10F Judkins catheter was inserted into the carotid artery,
and coronary angiography (CAG) in a left oblique view was
performed. After the virus injection into the coronary artery
wall, the carotid artery was ligated, the skin was closed, and the
animal was allowed to recover from anesthesia. The animals
were euthanized with an excess dose of intravenous
pentobarbital (60 to 90 mg/kg), and coronary arteries were
excised 1, 2, and 3 weeks after the procedure for
immunostaining, organ chamber experiment and
immunoblot analysis, and
histological analysis,
respectively.
Construction of Adenoviral Vectors
Rho-binding domain (RB) and mutant
(NK1036
TT) RB with a pleckstrin homology
domain [RB/PH(TT)],which are DNRhoK mutants driven by the
cytomegalovirus promoter with a
c-myc tag, were prepared
through homologous recombination between cotransfected pJM17 and the
shuttle plasmids in 293 cells. Integration of the transgene into the
adenoviral genome was determined by a polymerase chain reaction and
restriction analysis. The titer of the virus stock was assessed
by a plaque formation assay that used the 293 cell and was expressed as
plaque-forming unit (pfu). AdLacZ was used as a
control.16 In the
present study, AdDNRhoK represents an adenoviral vector
encoding RB; however, we confirmed that an adenoviral vector encoding
RB/PH(TT) also caused the same inhibitory effect on the
constrictive vascular remodeling and vasospastic activity in the
present porcine model in vivo (data not
shown).
Adenovirus-Mediated Gene Transfer Into Porcine
Coronary Arteries In Vivo
Two weeks after the application of IL-1ß,
adenovirus-mediated gene transfer was performed at the coronary
segments previously treated with the cytokine. The
IL-1ß–treated coronary segment was easily recognized as a
stenotic lesion by CAG after intracoronary
administration of nitroglycerin (10 µg/kg) and
heparin (3000 U). Then, infiltrator angioplasty balloon catheter (IABC,
Interventional Technologies Inc) was advanced to the IL-1ß–treated
segment, followed by inflation of a 3.5-mm balloon at 2.0 atm for 30
seconds. This catheter has 21 small nipples in 3 lines located on the
surface of the balloon connected to the drug delivery
port,17 which is filled with
virus solution until droplets appear through the needles before use.
This catheter is useful for the direct delivery of fluid into the
vessel wall in vivo with >90% efficiency and minimal vascular
damage17 and also for in
vivo gene transfer into the coronary
artery.16 AdLacZ and
AdDNRhoK (final titer, 4x109 pfu in 0.4 mL
sorbitol-added lactated Ringer’s saline) were injected into the
coronary segment in a randomized manner, and then the catheter
was deflated and withdrawn.
CAG and Coronary Diameter
Measurement
CAG was performed before and every week until 3 weeks
after the gene transfer. The cineangiograms were
projected on a screen by using a cine projector, and an
end-diastolic frame was selected and printed.
Coronary stenosis at the IL-1ß–treated segments was
expressed as the percent decrease in the luminal diameter compared with
the mean diameter of adjacent proximal and distal normal
coronary segments after intracoronary administration of
nitroglycerin (10 µg/kg). Coronary
hyperconstrictions induced by intracoronary administration of
serotonin (10 µg/kg) and histamine (10 µg/kg) were
expressed as the percent decrease in luminal diameter from the control
level, as previously
described.13
Coronary Intravascular Ultrasound
Imaging
To assess the extent of constrictive remodeling of
the coronary artery in vivo, we performed intravascular
ultrasound (IVUS) imaging immediately before and 3 weeks after the gene
transfer. A 30-MHz 3.5F monorail ultrasound catheter interfaced with a
monitor and scanner (HP SONOS, Hewlett-Packard) was used. After
intracoronary administration of heparin (3000 U) and
nitroglycerin (10 µg/kg), the catheter was placed
over the guidewire beyond the lesion site. The catheter was then
withdrawn manually during continuous imaging recorded on a VHS
videotape. The target lesion and a proximal reference site were
selected for measurement. The target lesion was easily defined as the
site with marked hyperechoic density in the adventitial area due to the
chronic treatment with IL-1ß. The medial area at the IL-1ß–treated
site was relatively hypoechoic compared with the adventitia. At each
selected site, the intimal leading edge and the leading edge of the
adventitia were used to manually trace the lumen and external elastic
lamina (EEL) areas, respectively. Constrictive remodeling was assessed
by calculating the percent reduction of the EEL and the luminal area at
the IL-1ß–treated coronary segments to the EEL and the
luminal area of the proximal normal coronary segments,
respectively.
Immunostaining for
c-Myc
Immunostaining for
c-myc, a tag protein of DNRhoK,
was performed 1 and 3 weeks after the gene transfer to confirm the
efficacy of the procedure. After the coronary artery was
excised, it was quickly frozen in OCT compound (Tissue-Tek), sectioned
at 5 µm, and subjected to immunostaining with
polyclonal antibody against
c-myc (Peninsula Laboratory).
Intact arteries and nonimmune rabbit IgG were used as controls.
Immunoreactive materials were visualized by use of a biotinylated
anti-rabbit IgG antibody (Wako), peroxidase-labeled streptavidin, and
diaminobenzidine.
Organ Chamber Experiment
To examine the coronary reactivity in vitro,
we performed an organ chamber experiment 2 weeks after the gene
transfer, as previously
described.12 14 18
Serotonin (0.1, 0.3, and 1.0 µmol/L) induced contractions
of the isolated coronary rings without
endothelium, which rapidly developed and reached a
maximum in 5 to 8 minutes. The developed tension was expressed as a
percentage of that attained in the last precontraction with 62
mmol/L KCl.
Western Blot Analysis for Substrates of
Rho-Kinase
Isolated coronary rings without
endothelium and adventitial tissue were subjected to
SDS-PAGE immunoblot analysis 2 weeks after the gene
transfer.12
Phosphorylation of the myosin-binding subunit (MBS) of
myosin phosphatase was measured when the serotonin (1.0
µmol/L)–induced contraction reached a maximum.
Phosphorylation of ERM (ezrin/radixin/moesin)
and of adducin was measured without serotonin. The
antibodies used in the present study included rabbit anti-rat MBS
polyclonal antibody (pAb),19
rabbit anti-human moesin pAb (anti-phosphorylated
Thr558), which also binds to the phosphorylated
ezrin (Thr567) and radixin
(Thr564),20 and rabbit
anti-human 
-adducin pAb (anti-phosphorylated
Thr445).21
Histological
Examination
Three weeks after the gene transfer, the left
coronary arteries were subjected to
histological examinations. The medial area, percent
medial area (ratio of medial area to EEL area), the
neointimal area, and percent intimal area (ratio of intimal
area to internal elastic lamina [IEL] area) were also calculated, and
the degree of intimal and medial thickening was assessed. Constrictive
remodeling of the coronary artery was assessed by measuring the
ratio of the EEL, IEL, and luminal area at the IL-1ß–treated
coronary segments to those respective areas of the adjacent
proximal
segments.13
Data Analysis
All results are expressed as the mean±SEM. Multiple
comparisons were made by ANOVA for repeated examinations followed by
the Fisher post hoc test. Paired data were analyzed by the
Student t test. A value of
P<0.05 was considered to be
statistically significant.
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Results |
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Immunohistochemical Analysis for c-Myc
One week after the gene transfer, the expression of c-myc was noted throughout the media (smooth muscle cells) and partially at the adventitia (fibroblasts) at the AdDNRhoK-transfected site (online Figure
I; please
see http://www.atvb.ahajournals.org). The staining with nonimmune IgG
was negative, confirming the specificity of our
immunostaining. Three weeks after the gene transfer,
the immunoreactivity of c-myc
was markedly reduced (data not shown). No immunoreactivity of
c-myc was noted in the
coronary segment adjacent to the AdDNRhoK-transfected site
(data not shown).
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CAG and Coronary Diameter
Measurement
Coronary stenosis at the
IL-1ß–treated site significantly regressed, and vasospastic response
to serotonin (10 µg/kg) and histamine (10 µg/kg)
progressively decreased at the AdDNRhoK site, whereas those changes
persisted at the AdLacZ site
(Figures 1
and 2).
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Coronary IVUS
No significant difference in the extent of constrictive
remodeling was noted between the AdDNRhoK and the AdLacZ site before
the gene transfer
(Figure 3). However, 3 weeks after the gene transfer, the
constrictive remodeling induced by the IL-1ß application markedly
regressed at the AdDNRhoK site, whereas it persisted at the AdLacZ site
(Figure 3).
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Histological
Examination
The EEL, IEL, and luminal areas were all significantly
reduced by the IL-1ß application, whereas this constrictive
remodeling markedly regressed at the AdDNRhoK site but not at the
AdLacZ site (online Figure II; please see
http://www.atvb.ahajournals.org and
Figure 4). Because the neointimal formation at
the IL-1ß–treated site was minimal, the luminal reduction was caused
primarily by the constrictive remodeling. There was no significant
reduction in the media or the neointima at the AdDNRhoK
site compared with the AdLacZ site, indicating that the enlargement of
luminal area was caused primarily by the regression of constrictive
remodeling but not by the reduction in the media or the
neointima
(Figure 4).
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Organ Chamber Experiment
Serotonin (0.1, 0.3, and 1.0 µmol/L)
induced concentration-dependent contractions of isolated
coronary rings without endothelium. The
serotonin-induced contractions were augmented at the AdLacZ
site compared with the normal site, whereas they were significantly
suppressed at the AdDNRhoK site
(Figure 5). The extent of the hypercontractions at the AdLacZ
site was comparable to that of coronary segments treated with
IL-1ß
alone.12 14
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Measurement of Rho-Kinase Activity
Rho-kinase–dependent phosphorylation
of MBS on stimulation by serotonin (1.0 µmol/L) was
increased at the AdLacZ site compared with the normal site, whereas it
was suppressed at the AdDNRhoK site
(Figure 6A). Furthermore, the phosphorylation
of the ERM family and of adducin was also increased at the AdLacZ site
and was markedly suppressed at the AdDNRhoK site
(Figure 6B).
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Discussion |
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The novel findings of the present study were that (1) Rho-kinase and its substrates (ERM family and adducin) were functionally upregulated at the IL-1ß–treated arteriosclerotic coronary lesions in vivo, and (2) the selective inhibition of Rho-kinase by the gene transfer of its dominant-negative mutant caused a marked regression of the coronary constrictive remodeling and abolished the coronary vasospastic activity in vivo. To the best of our knowledge, this is the first report that demonstrates that selective inhibition of Rho-kinase induces a regression of coronary arteriosclerotic lesions in vivo.
Rho-Kinase and Coronary
Arteriosclerosis
We have recently demonstrated that Rho-kinase is
upregulated at the IL-1ß–treated coronary lesions in our
porcine model, causing coronary vasospastic responses through
the inhibition of myosin
phosphatase.12 The
present study further demonstrated that the Rho-kinase–mediated
phosphorylation of ERM and of
adducin20 21 is
also increased at the coronary lesions, further confirming the
upregulation of Rho-kinase. Recent studies in vitro have shown that
Rho-kinase (and thus its substrates) mediates actin cytoskeleton
organization,7 8
cell adhesion and migration,9
and cytokinesis,10
which may be regulated by adhesion complexes that consist of focal
adhesion, anchoring proteins, and actin
filament.9 It has also been
demonstrated that ERM and adducin are phosphorylated by
Rho-kinase and are involved in cell adhesion and cell
migration.20 21 22
We also have recently demonstrated that Rho-kinase is upregulated after
balloon injury in porcine femoral
arteries.23 Among the
cellular functions mediated by Rho-kinase and its substrates, cell
adhesion may regulate 3D structure of blood vessels, including vascular
remodeling, because it is a molecular basis of tissue
architecture.24
Although the molecular mechanism of the upregulation of Rho-kinase at the arteriosclerotic coronary lesions remains to be elucidated, it has recently been demonstrated that IL-1ß directly enhances the Rho-kinase activity in vitro.25 In our porcine model, a cytokine network is activated; this network involves platelet-derived growth factor,13 fibroblast growth factor-2,26 and inflammatory cytokines,27 all of which could cause upregulation of Rho-kinase. Furthermore, accumulating evidence indicates that Rho-kinase is involved in the vascular effects of various vasoactive factors, including angiotensin II,28 thrombin,29 endothelin-1,30 and serotonin.12 14 Thus, Rho-kinase may play an important role in the pathogenesis of arteriosclerosis directly (by enhancing the process through activation of its substrates) and indirectly (by mediating the signal transduction of various vasoactive mediators).
Rho-Kinase as a Novel Therapeutic Target in
Treatment of Arteriosclerosis
One of the novel findings of the present study is
that the selective and local inhibition of Rho-kinase, by the in vivo
transfer of its dominant-negative mutant, induces a marked
regression of the coronary constrictive remodeling in our
porcine model. This result indicates that Rho-kinase is involved in the
constrictive remodeling. It is now widely accepted that luminal
narrowing after coronary angioplasty is caused primarily by
constrictive remodeling, with a minimal contribution of
neointimal
formation.31 32
We have previously demonstrated that adventitial
inflammatory/proliferative responses play an important role in the
pathogenesis of the geometric remodeling in porcine coronary
arteries in vivo, where adventitial delivery of antiproliferative
agents (eg, tyrosine kinase inhibitors) is useful in
preventing the
process.33
Because the structure of the arteriosclerotic artery is affected by cell adhesion, cytoskeleton organization, and smooth muscle cell contractility, Rho-kinase might be involved in the vascular remodeling. Recent studies have indicated that collagen accumulation and endothelial dysfunction are associated with constrictive remodeling.34 Because endothelial vasodilator function is fairly preserved in our porcine model,35 inflammatory vascular changes associated with collagen accumulation (especially at the adventitia) may play a primary role in the vascular remodeling.
We have also recently demonstrated that in vivo transfer of DNRhoK inhibits the neointimal formation after balloon injury in porcine femoral arteries in vivo.23 In the present study, the inhibitory effect of DNRhoK on neointimal formation was not evident. This is probably because of the differences between the 2 models in terms of preexisting neointimal formation. In the present study, DNRhoK was transfected into the coronary artery with neointimal formation, whereas in the previous study, it was transfected immediately after balloon injury into the femoral artery without neointimal formation. It has been also reported that a Rho-kinase inhibitor exerts an inhibitory effect on the neointimal formation by balloon injury in rat carotid arteries.36 Thus, Rho-kinase could be regarded as a novel therapeutic target in the treatment of arteriosclerosis.
Rho-Kinase as a Novel Therapeutic Target in
Treatment of Vasospasm
Another novel finding of the present study was that
the in vivo transfer of DNRhoK abolished the vasospastic activity of
the coronary artery. We have previously demonstrated that acute
administration of Rho-kinase inhibitors suppresses the
coronary vasospastic responses in our porcine
model.12 14 We
confirmed that at 3 weeks after the transfer of DNRhoK, its
immunoreactivity was markedly reduced in the coronary artery.
Thus, the abolishment of the coronary vasospastic activity
might mainly be due to the marked regression of the coronary
remodeling (probably associated with the reduction in Rho-kinase
expression, per se) and partly be due to the direct
inhibitory effect of DNRhoK on the coronary
contraction. This finding suggests the potential usefulness of
Rho-kinase inhibitors in the treatment of coronary
artery spasm because they could abolish the coronary
vasospastic activity. This potential effect of Rho-kinase
inhibitors is in contrast to that of calcium channel
blockers, which cannot abolish the vasospastic activity, per se, as
evidenced by the rebound coronary spasm after abrupt
cessation.37
Limitations of the Present Study
Several limitations of the present study could be
raised. First, the present study was performed in a porcine model
of coronary arteriosclerosis but not in
humans. Thus, our observations should be confirmed in humans in a
future study. Second, although the DNRhoK used in the present study
potently inhibits Rho-kinase activity in vivo, it cannot be ruled out
that other Rho and Rho-kinase effectors (eg, p140mDia, protein kinase
N, and myosin light chain kinase) might also be involved in the
inhibitory effect of DNRhoK. Third, the adenoviral vector
may not be suitable for repeated gene transfer because of its intrinsic
immunity. An inhibitory method of immunity and a less
immunogenic vector remain to be developed for clinical use.
In summary, the present study has demonstrated that Rho-kinase is involved in the pathogenesis of constrictive remodeling and vasospastic responses of the coronary artery in our porcine model in vivo, suggesting that the molecule could be regarded as a novel therapeutic target in the treatment of arteriosclerotic vascular diseases.
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Acknowledgments |
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This work was supported in part by grants-in-aid from the Japanese Ministry of Education, Science, Sports, and Culture, Tokyo, Japan (Nos. 09470169, 10177223, 10357006, 12032215, and 12470158). The authors wish to thank M. Sonoda for excellent technical assistance.
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Footnotes |
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Guest Editor was Elizabeth Nabel, MD, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.
Received November 17, 2000; accepted January 9, 2001.
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