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1 Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Higashi-ku, Fukuoka 812-8582, 2 Division of Signal Transduction, Nara Institute of Science, and Technology, Nara 630-0121, Japan; and 3 Molecular Cardiology, University of California, San Diego, California 92093-0613
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Restenosis after angioplasty still remains a major
problem for which neointimal formation appears to play an important
role. Recent studies in vitro suggested that Rho kinase, a target
protein of Rho, is important in various cellular functions. We thus
examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after
balloon injury in both sides of the porcine femoral
arteries with adenoviral vector encoding either a dominant
negative form of Rho kinase (AdDNRhoK) or 
-galactosidase (AdLacZ) as
a control. One week after the transfer, immunohistochemistry confirmed
the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the
AdLacZ site where histological neointimal formation was noted, whereas
those changes were significantly suppressed at the AdDNRhoK site. These
results indicate that Rho kinase is involved in the pathogenesis of
neointimal formation after balloon injury in vivo.
restenosis; signal transduction; small guanosine 5'triphosphate-binding protein; vascular smooth muscle
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ALTHOUGH PERCUTANEOUS TRANSLUMINAL coronary angioplasty is an established and useful treatment for coronary artery disease, the restenosis, which occurs in 30-40% of patients after the procedure, still remains a major problem (21, 33). The reduction of luminal diameter is mainly due to neointimal formation (15, 20) and geometric (constrictive) remodeling (22). The former is caused by dedifferentiation, migration, and proliferation of medial vascular smooth muscle cells (VSMCs). Indeed, VSMCs are stimulated after balloon injury by growth factors such as platelet-derived growth factor (PDGF) (7, 24), fibroblast growth factors-2 (24), and insulin-like growth factor-I (2). However, the intracellular signal transduction initiated by those growth factors has not been fully elucidated.
Recent studies in vitro suggested that Rho kinase, a target protein of
small GTP-binding protein Rho (9), plays an important role for various
cellular functions, including focal adhesions (1), motility (4), smooth
muscle contraction (12), and cytokinesis (32). It has also been
demonstrated that Rho/Rho kinase pathway is involved in DNA synthesis
and migration in VSMCs of the rat aorta in vitro (25). We also recently
demonstrated that Rho kinase is functionally upregulated at the
inflammatory coronary lesions and plays an important role in the
pathogenesis of coronary hyperreactivity in our porcine model with
IL-1 (10, 29). Thus Rho kinase could be regarded as a novel
therapeutic target for arteriosclerotic vascular disease (28).
The present study was thus designed to examine whether or not the selective inhibition of Rho kinase by adenovirus-mediated gene transfer of its dominant negative form suppresses the vascular lesion formation after balloon injury in porcine femoral arteries in vivo.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was reviewed by the Ethics Committee on Animal Experiment at the Kyushu University Graduate School of Medical Sciences and was carried out in accordance with the Guidelines for Animal Experiment at the Kyushu University Graduate School of Medical Sciences and the Law (no. 105) and the Notification (no. 6) of the Japanese Government.
Adenovirus vector. As previously described (30),
replication-defective recombinant adenoviral vectors expressing
Escherichia coli -galactosidase (AdLacZ) were prepared under
the control of CA promotor composed of cytomegalovirus enhancer and
chicken -actin promotor. Dominant negative Rho kinase
(AdDNRhoK) (Rho-binding domain) (1) was prepared under the control of
pEF-BOS promotor. A recombinant adenovirus was constructed
by in vitro homologous recombinant in 293 cells. The desired
recombinant adenovirus, designated as AdDNRhoK, was purified by
ultracentrifugation through a CsCl2 gradient followed by
extensive dialysis. The titer of the virus stock was assessed by a
plaque formation assay that used the 293 cells and was expressed in
plaque-forming units. We also used adenovirus vector encoding
-galactosidase (AdLacZ) as a control (17).
Animal preparation. Domestic male pigs (Nihon Crea, Tokyo, 2-3 mo old, and weighing 25-30 kg) were used for this study. The animals were housed individually under a controlled room temperature. They were sedated with an intramuscular administration of ketamine hydrochloride (12.5 mg/kg) and anesthetized with an intravenous administration of pentobarbital sodium (20 mg/kg) (10, 17, 29). They were intubated and ventilated with room air while oxygen was supplemented via a positive pressure respirator (Shinano, Tokyo, Japan). An incision was performed on the inguinal skin and both sides of the femoral arteries (diameter 3 mm) were carefully isolated as long as 2 cm, and their branches were ligated. A 9-Fr sheath was then introduced into the right carotid artery, and an 8-Fr guiding catheter was introduced into each side of the common iliac artery. Angioplasty catheter (diameter 4.0 mm) with a diameter 1.3 times larger than the femoral diameter was inserted into the isolated site through a guiding catheter. Balloon injury was then performed at 10 atm for 30 s five times (5, 17). For gene transfer, a 3-Fr catheter was introduced through a major branch, both ends of isolated space were temporarily cramped, and virus solution (0.5 ml) was incubated for 30 min. AdDNRhoK was incubated in one of the balloon-injured femoral segments, whereas AdLacZ was incubated in the contralateral balloon-injured femoral segment. The side of the femoral segment for the gene delivery was randomized. After 30 min of incubation, the virus solution was collected and femoral blood flow was restored. The skin incisions were sutured, and the animals were allowed to recover.
In vivo experiment. Two weeks after the in vivo gene transfer, animals were again anesthetized and ventilated as described above, and femoral arteriography was performed after an intravenous administration of nitroglycerin (10 µg/kg, Nihon-Kayaku Pharmaceutical, Tokyo, Japan). A catheter was inserted into the left carotid artery, and heparin (3,000 units bolus) was administered intravenously. Femoral arteriography was performed in a posteroanterior view, using the Toshiba cineangiography system (DG-15GB/CAS-CA, Toshiba Medical, Tokyo, Japan). Electrocardiograms (leads I, II, III, V1, and V6), along with mean arterial pressure and heart rate, were continuously monitored and recorded during the experiments. The angiograms were recorded on 35-mm cinefilm (Varicath I; VARI-X) at 48 frames per second. The cineangiograms were projected on a screen using a cineprojector (ELK-35CB, Nishimoto Sangyo, Osaka, Japan), and the femoral luminal diameters were measured with a caliper (10, 17, 29). The degree of the stenosis was expressed as the percent decrease in the luminal diameter from the adjacent control site.
Histopathology and immunohistochemistry. After the angiographic
study, the animals were killed with a lethal dose of pentobarbital sodium, an 8-Fr tube was inserted from the thoracic aorta, and the vena
cava inferior was ligated. Femoral arteries were perfused via a
constant-pressure perfusion system (120 cmH2O) with saline (500 ml) and subsequently with 5% formaldehyde (1,000 ml). After the
fixation, both sides of the femoral arteries were removed. Each vessel
was cut transversely, dehydrated, embedded in paraffin, and cut into
5-µm-thick slices. These sections were stained with hematoxylin-eosin
and van Gieson's elastic staining for photomicroscopy. Three areas
[lumen, internal elastic lamina (IEL), and external elastic
lamina (EEL) areas] were measured by using a computer-assisted picture system (Genlocker System, Sony, Tokyo, Japan) (5, 17). The
intimal area (Ai) was calculated by the formula
Ai = Ae 
Al, where Ae and
Al are the area within the IEL and the lumen area,
respectively. The degree of neointimal formation was expressed by the
following two parameters, maximal intimal thickness (mm) measured with
a caliper and percent intima calculated by the following equation:
Ai/Ae × 100 (%) (5, 17).
The degree of vascular remodeling was expressed by the changes in the
three vessel areas (luminal, IEL, and EEL) by the following formula:
(ATx ACont)/ACont × 100, where
ATx and ACont are vessel areas
of the segments at the treated and the adjacent control site,
respectively (5, 17).
In some experiments, femoral arteries were removed 1 wk after the in vivo gene transfer to confirm the gene expression. These segments were embedded in the OCT compound (Tissue Tek) without being embedded in paraffin, frozen, and cut into 5-µm-thick slices. Serial cryosections were stained with a nonimmune IgG (Zymed Lab) or c-myc antibody, which was encoded into the vector as a tag.
Measurements of Rho kinase mRNA. Rho kinase was also identified
as ROK
(14) and as ROCK2 (18). ROK (13)/ROCK1 (8) is an isoform
of Rho kinase/ROK/ROCK2 and is thought to be functionally equivalent. Because it has been previously demonstrated that
ROK/ROCK1 is expressed to more extent than ROK/ROCK2 in porcine
VSMCs (19), the mRNA expression of the former was examined using RT-PCR
analysis in the present study.
Total RNA was isolated from the sections of the balloon-injured or
control femoral arteries. Possible contaminating genomic DNA was
digested by RNase-free DNase. The total RNA (1 µg) was incubated for
60 min at 37°C for RT reaction in a total volume of 33 µl. An
aliquot (5 µl) of RT product was used for PCR amplification in a
total volume of 100 µl. The thermal cycle profile used in this study
was denaturing for 30 s at 94°C, annealing primers for 90 s at 55°C, and extending the primers for 30 s at 72°C. The
sequence of the primer for RT-PCR analysis of porcine Rho kinase
(ROC/ROCK1) in this study has been previously reported (19). The PCR
amplification was performed for Rho kinase for 30 cycles and for
-actin (as an internal control) for 25 cycles. These amplifications
were performed in the linear relationship between signal cycle number
and intensity of RT-PCR products (data not shown). A portion (10 µl)
of the PCR mixture was electrophoresed in 2% agarose gel in
Tris-acetate-EDTA buffer. The gel was stained with ethidium bromide and
then photographed. For the quantitative analysis, the density of bands
was measured by an NIH image analyzer, and then the levels of PCR
products were normalized to PCR products for -actin (10).
Measurements of Rho kinase activity. To evaluate Rho kinase activity in vivo, the extent of phosphorylated myosin binding subunit (MBS) of myosin phosphatase, one of the substrates of Rho kinase in the vascular wall (9, 10), was measured by SDS-PAGE followed by electrophoretic transfer of the proteins to a nitrocellulose membrane. The amount of phosphorylated MBS in each sample was quantified by immunoblot procedures. Briefly, removed tissues were frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) cooled with dry ice. Frozen tissues were washed three times with acetone containing 10 mM dithiothreitol to remove the TCA, and the dried tissue was cut into small pieces and exposed to 200 µl of SDS-PAGE sample buffer for purposes of extraction. The supernatant of extracted samples were subjected to SDS-PAGE immunoblot analysis, using a specific antibody against phosphorylated MBS by an enhanced chemiluminescence Western blotting luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) (10).
Statistical analysis. All results are expressed as means ± SE. Throughout the text, n represents the number of animals tested. Statistical analysis was performed using paired t-test or one-way ANOVA followed by Fisher's test. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Immunohistochemistry. Sections from the AdDNRhoK
site were highly positive for the immunostaining of c-myc, a
tag of the transfected gene, mainly in the media, whereas
immunostaining with nonimmune IgG showed no staining in the vascular
wall (Fig. 1). No staining for
c-myc was observed in the distal segment of the transfected femoral artery or in any other arteries. Immunostaining for -actin in the adjacent section showed that cells positive for the
c-myc staining were mainly VSMCs in the media.
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RT-PCR and Western blot analysis. RT-PCR analysis for Rho
kinase performed 14 days after balloon injury demonstrated that mRNA
expression of Rho kinase was significantly upregulated at the
balloon-injured site compared with control site (Fig.
2). Western blot analysis also demonstrated
that the MBS phosphorylation was significantly increased at the
balloon-injured (AdLacZ) site, whereas it was significantly suppressed
to the control level at the AdDNRhoK site (Fig.
3).
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Femoral arteriography. Two weeks after the balloon injury and
the in vivo gene transfer, femoral arteriography showed the development
of stenotic lesion at the AdLacZ site, whereas the extent of the
stenosis was significantly suppressed in the AdDNRhoK site (Figs.
4 and 5).
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Histopathology. The extent of balloon injury (as expressed by
the degree of fractured IEL) (5) was comparable between the AdLacZ (7 ± 3%) and the AdDNRhoK (5 ± 2%) site. Neointimal formation (as
expressed by % intima and maximal intimal thickness) was observed at
the AdLacZ site, whereas those changes were significantly suppressed at
the AdDNRhoK site (Figs. 6 and
7). In contrast, the extent of geometric
remodeling was mild and comparable at both sites, indicating that the
preserved lumen area at the AdDNRhoK site was mainly due to
the inhibition of neointimal formation (Fig. 8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major findings of the present study were that 1) Rho kinase was functionally upregulated at the balloon-injured site of the porcine femoral artery in vivo, and 2) gene transfer of dominant negative Rho kinase significantly suppressed the development of neointimal formation after balloon injury. To the best of our knowledge, this is the first report that demonstrates the important role of Rho kinase in the molecular mechanism of neointimal formation by balloon injury in vivo.
Upregulation of Rho kinase by balloon injury. It was previously reported that small GTP-binding protein Ras (31) and its downstream extracellular signal-regulated kinase (11, 23) are involved in the vascular lesion formation by balloon injury in vivo. The present study provides the first evidence that balloon injury also causes the upregulation of Rho kinase at both the mRNA (RT-PCR analysis) and functional levels (Western blot for phosphorylated MBS). Because mRNA expression was upregulated at the balloon-injured site, one of the key mechanims for the upregulation of Rho kinase appears to be present at the transcriptional level. We have previously demonstrated that after balloon injury, a network of inflammatory cytokines and growth factors is upregulated, for which a specific blockade of tyrosine kinases is effective to suppress the vascular lesion formation (5). We are now working to identify the inflammatory stimuli to enhance the transcriptional regulation of Rho kinase in vitro.
Inhibitory effect of gene transfer of dominant negative Rho
kinase. The gene transfer technique employed in the present study (adenovirus vector and in vivo incubation with blood vessels from luminal surface) is widely and effectively used for in vivo gene transfer into the vascular wall (30, 31). In the present study, we also
confirmed that the gene encoded in the adenovirus vector was highly
expressed (by c-myc immunostaining) but only at the transfected
site and that Rho kinase was functionally suppressed by the gene
transfer of AdDNRhoK (by Western blotting for phosporylated MBS). The
cells transfected with the gene appeared to be medial VSMCs for the
following reasons. First, because we incubated the virus solution in
normal porcine femoral arteries immediately after the balloon injury
with massive endothelium removal, medial VSMCs were directly exposed to
the vector solution. Second, we observed the positive immunostaining
for c-myc mainly in medial cells where immunostaining for
-smooth muscle actin (a marker of VSMCs) was also positive. Because
neointimal formation was significantly suppressed at the AdDNRhoK site
compared with the AdLacZ site, the present result demonstrates that the
selective inhibition of Rho kinase at the medial VSMCs is effective in
suppressing the neointimal formation after balloon injury in vivo.
The present result is in contrast to our recent study in which we were able to induce a regression of vascular remodeling (caused by adventitial inflammation) through gene transfer of AdDNRhoK into the outer layer of medial VSMCs and the adventitial fibroblasts (16). In this study we used an infiltrator angioplasty balloon catheter for in vivo gene delivery into the porcine coronary artery, which enabled us to deliver a gene into the outer layer of medial VSMCs and adventitial fibroblasts when applied to the normal coronary arteries without intimal thickening (16). Indeed, we and others (6, 26) have confirmed the potential importance of the adventitia in the pathogenesis of the pathological remodeling. Taken together, it is thus conceivable that Rho kinase activation in the vessel wall may have a different effect; in the inner layer of VSMCs it may stimulate the neointimal formation, whereas in the outer layer of VSMCs or adventitial fibroblsts it may be involved in the development of pathological remodeling.
Mechanism of inhibitory effect of dominant negative Rho kinase. It is possible in the present study that the inhibitory effect of dominant negative Rho kinase on the neointimal formation after balloon injury was due to inhibition of VSMCs proliferation, enhancement of their apoptosis, or both. Although the present study did not directly address this issue, preliminary results from our laboratory (unpublished observations) and others (27) suggest that inhibition of Rho kinase in VSMCs induces thier apoptosis. However, this issue remains to be fully clarified in future studies.
There are several substrates of Rho kinase other than MBS, including the Ezrin, Radixin, and Moesin (ERM) family and adducin (9). Studies in vitro demonstrated the important role of adducin on cell motility and membrane ruffling (4), whereas the ERM family may play an important role in cytokinesis and microvilli formation (9). Thus it is conceivable that the inhibitory effect of dominant negative Rho kinase could also be mediated by those substrates of Rho kinase other than MBS.
Limitations of the present study. Several limitations can be raised regarding the present study. First, this study was performed in the normal porcine femoral artery without preexisting intimal thickening, whereas atherosclerotic intimal lesions preexisted in humans. It is conceivable that the vascular response to balloon injury is different between the normal and atherosclerotic arteries (15). Further studies are required in animal models with atherosclerotic lesions. Second, this study did not fully demonstrate the time course of the Rho kinase expression after balloon injury. Although we were able to demonstrate the upregulation of Rho kinase mRNA at 2 wk after the balloon injury, it is possible that the expression peaked earlier (e.g., 3-7 days) after the injury. This point remains to be fully elucidated in future studies. Third, the dominant negative Rho kinase (Rho-binding site) that we used potently inhibits Rho kinase activity as shown in this study, it cannot be completely ruled out that other Rho effectors might also be inhibited (1). Fourth, the adenoviral vector that we used may not be suitable for repeated gene transfer because of its intrinsic immunity (3). The establishement of the inhibitory method of the immunity as well as the development of a new vector with no or less immunity in vivo are thus required. Fifth, we studied the efficacy of in vivo gene transfer of dominant negative Rho kinase in the femoral artery rather than in the coronary artery of the pig. This was due to a technical reason because we needed to interrupt the blood flow for 30 min for the gene transfer into the medial VSMCs, which is impossible in the coronary circulation. However, a sophisticated use of IABC catheter might overcome this problem (16, 17).
In summary, the present study demonstrated that upregulation of Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury, suggesting that the molecule could be regarded as a novel therapeutic target to prevent restenosis after percutaneous transluminal coronary angioplasty.
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ACKNOWLEDGEMENTS |
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The authors thank T. Akiyama for cooperation in this study and M. Sonoda, S. Tomita and E. Gunshima for excellent technical assistance.
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FOOTNOTES |
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This study was supported in part by grants from the Japanese Ministry of Education, Science, Sports and Culture, Tokyo, Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Shimokawa, Dept. of Cardiovascular Medicine, Kyushu Univ. 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).
Received 29 December 1999; accepted in final form 16 February 2000.
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TOP
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RESULTS
DISCUSSION
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