(Circulation. 2000;101:1447.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Transfection of Antisense p53 Tumor Suppressor Gene Oligodeoxynucleotides Into Rat Carotid Artery Results in Abnormal Growth of Vascular Smooth Muscle Cells
From the Department of Geriatric Medicine, Osaka University Medical School, Suita 565, Japan (H.M., R.M., M.A., N.T., Y.T., H.N., J.H., T.O.); Kankyou Bilis, Shiga, Japan (T.S.); and the Division of Gene Therapy Science, Osaka University Medical School, Suita 565, Japan (R.M., Y.K.).
Correspondence to Ryuichi Morishita, MD, PhD, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. E-mail morishit{at}geriat.med.osaka-u.ac.jp
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Abstract |
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Background—Although loss of activity of an antioncogene, the p53 tumor suppressor gene product, has been postulated in the pathogenesis of human restenosis, little is known about the role of p53 in the regulation of vascular smooth muscle cell (VSMC) growth. In this study, to clarify the role of p53 in the pathogenesis of restenosis, we examined transfection of antisense p53 oligodeoxynucleotides (ODN) into VSMC in vitro and rat carotid artery in vivo.
Methods and Results—The specificity of antisense p53 ODN was confirmed by a significant decrease in p53 protein. Transfection of antisense p53 ODN into VSMC resulted in a significant increase in DNA synthesis and cell number as compared with sense and scrambled ODN (P<0.01). Importantly, transfection of antisense p53 ODN into rat intact carotid artery resulted in a significant increase in the ratio of neointima to medial area at 2 and 4 weeks after transfection, accompanied by a significant decrease in p53 protein (P<0.01). Moreover, cotransfection of wild-type p53 plasmid completely abolished neointimal formation induced by antisense p53 ODN. The sustained effect of a single antisense ODN administration was confirmed by the kinetics of ODN in the vessel wall with the use of FITC-labeled ODN.
Conclusions—Overall, the present study demonstrated that loss of p53 by antisense p53 ODN resulted in an abnormal VSMC growth in vitro and in vivo. These results demonstrated the potential contribution of p53 to the pathogenesis of restenosis.
Key Words: restenosis • muscle, smooth • apoptosis • cells • genes
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Introduction |
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Intimal hyperplasia is the pathological process that underlies restenosis after angioplasty, atherosclerosis, and vascular graft occlusion1 and develops in large part as a result of vascular smooth muscle cells (VSMC) proliferation and migration that are activated by vascular injury.1 The process of VSMC proliferation is dependent on the coordinated activation of a series of cell cycle–regulatory genes, which results in mitosis. Especially p53 (p53 tumor suppressor gene) has been postulated to negatively regulate the cell cycle in various cell types.2 3 4 However, little is known about the role of p53 in the regulation of VSMC. The presence of a functional p53 protein has been implicated as a critical determinant to regulate DNA replication, DNA repair, and programmed cell death.5 6 7 8 First, p53 has been shown to induce cell cycle arrest at the G1/S boundary border, related to its ability to induce expression of a cellular gene, WAF1/CIP1/SDI1, which encodes a 21-kDa inhibitor of G1 cyclin–dependent kinases.9 Second, p53 can induce apoptosis through bcl-2–dependent and bcl-2–independent pathways.10 11 Therefore the abnormal progression of the cell cycle seen in cancer is thought to result from the mutation of these negative cell cycle regulatory genes, especially p53.2 3
The importance of cell cycle regulation in VSMC is apparently great, since we and others have previously reported the successful prevention of restenosis after angioplasty by using antisense oligodeoxynucleotides (ODN) against cell cycle–regulatory genes, decoy cis element of E2F binding site, and gene transfer of nonphosphorylated Rb (retinoblastoma gene).12 13 14 Of importance, recent studies have suggested that loss of p53 activity by cytomegalovirus infection might be responsible for the pathogenesis of human restenosis.15 16 17 From this viewpoint, it is necessary to understand the negative regulation of the cell cycle by p53 in VSMC. This study examined the role of p53 in negative regulation of the cell cycle in the pathogenesis of restenosis, with the use of antisense strategy in vivo. The present study successfully demonstrated in vivo evidence of importance of p53 in the regulation of VSMC growth.
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Results |
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In Vitro Transfection of Antisense p53 ODN Into VSMC
Initially, we tested whether transfection of antisense p53 ODN into VSMC has an effect on VSMC growth. Transfection of antisense p53 ODN resulted in a significant increase in DNA synthesis assessed by 3H-labeled thymidine incorporation as compared with untreated and sense p53 ODN-treated VSMC (Figure 1a
, P<0.01). Of importance, transfection of antisense
p53 ODN also resulted in a significant increase in the
number of VSMC as compared with sense ODN-transfected VSMC
(P<0.01, Figure 1b). The specificity of antisense
p53 ODN was confirmed by the observation that a marked
decrease in p53 protein was only observed with transfection
of antisense p53 ODN but not sense p53 ODN or HVJ
complex alone (Figure 2).
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In Vivo Transfection of Antisense p53 ODN Into Rat
Intact Carotid Artery
As shown in Figure 3, successful
transfer of antisense p53 ODN by means of HVJ-liposome
complex was confirmed by Western blotting of p53 protein.
Five days after transfection, Western blots from untransfected intact
vessels and intact vessels transfected with sense ODN demonstrated a
single band corresponding to p53 protein. Administration of
antisense p53 ODN decreased p53 protein but did
not affect the level of tubulin protein (Figure 3). Transfection
of antisense p53 ODN into intact rat carotid artery resulted
in significant decrease in p53 expression as compared with
transfection of HVJ alone or sense ODN (P<0.01).
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As shown in Figure 4a, PCNA-stained
nuclei were observed in the medial layer of blood vessels transfected
with antisense p53 ODN at 4 days after transfection, whereas
few PCNA-stained nuclei were found in vessels transfected with sense
p53 ODN. In addition, untransfected intact carotid arteries
also exhibited few PCNA-stained nuclei. Consistent with in
vitro experiments, the ratio of PCNA-positive nuclei to total cells in
the blood vessels transfected with antisense p53 ODN was
significantly increased as compared with those transfected with sense
p53 ODN (P<0.01, Figure 4b). Next, we
examined the effects of antisense p53 ODN on
neointimal formation after transfection. As shown in Figure 5, a and b, the vessels transfected with
HVJ-liposome complex without ODN, sense p53 ODN, or
scrambled p53 ODN exhibited little neointimal
formation at 2 weeks after transfection. In contrast, a single
administration of antisense p53 ODN resulted in a
significant increase in the ratio of neointimal area to
medial area (Figure 6). There was no significant difference in medial
area among HVJ-transfected, sense p53 ODN-transfected,
scramble p53 ODN-transfected, and antisense p53
ODN-transfected vessels (Table 1).
Further evidence for the specificity of antisense p53 ODN
was provided by the observation that treatment with neither antisense
ACE ODN nor antisense thrombomodulin ODN affected
neointimal formation (antisense ACE 124±95
µm2 vs antisense thrombomodulin 156±101
µm2, P>0.05). In addition,
neointimal formation was due to the lack of p53
activity, as cotransfection of wild-type p53 plasmid and
antisense p53 ODN completely abolished
neointimal formation induced by antisense p53
ODN (Figure 7). Surprisingly,
neointimal formation was still observed 4 weeks after a
single transfection in blood vessels transfected with antisense
p53 ODN (P<0.01, Figure 8). There was no significant difference
in medial area between sense p53 ODN-transfected and
antisense p53 ODN-transfected vessels (Table 2).
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To confirm the sustained effect of a single intraluminal administration
of antisense p53 ODN, we performed in vivo transfer of
FITC-labeled ODN into intact carotid arteries by using the HVJ-liposome
method. Transfer of FITC-labeled phosphorothioate ODN by the HVJ method
resulted in widespread distribution of fluorescence in medial
vascular cells even 2 weeks after transfection (Figure 9). The fluorescence was
localized primarily in cell nuclei and persisted up to 2 weeks after
transfection. Untreated or HVJ complex without ODN-treated vessels
revealed no specific fluorescence in the elastic lamina.
Intraluminal incubation of free FITC did not result in specific
fluorescence in the vessel wall, demonstrating that this
fluorescence was specific for FITC-labeled ODN (also see Figure 9).
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Discussion |
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A fundamental pathological feature of vascular disease is the abnormal accumulation of cells within the intimal space, resulting in neointimal formation produced by alterations in the homeostatic balance between cell growth and cell death.18 Of importance, loss of p53 activity induced by cytomegaloviral infection might be related to the pathogenesis of human restenosis.15 16 17 However, there is no direct in vivo evidence that loss of p53 might induce restenosis. Thus it is important to understand the role of p53 in the regulation of VSMC. Our in vitro experiments demonstrated that transfection of antisense p53 ODN promoted DNA synthesis and growth of VSMC. Since regulation of the intimal cell population requires a delicate balance between cell influx, cell growth, and cell death,19 cells lacking p53 undergo cell cycle progression, resulting in abnormal cell growth. More importantly, in vivo transfection of antisense p53 ODN into intact carotid artery also resulted in a significant increase in neointimal area at 2 and 4 weeks after transfection. Several observations provide evidence that VSMC growth is induced by lacking p53: (1) PCNA-positive cells were only detected in blood vessels transfected with antisense p53 but not sense p53 ODN; (2) neointimal formation induced by antisense p53 ODN was observed both at 2 and 4 weeks after transfection; (3) neointimal formation was only observed in blood vessels transfected with antisense p53 ODN but not with HVJ-liposome alone, sense, or scrambled ODN; (4) neointimal formation was accompanied by a significant decrease in vascular p53 protein as compared with HVJ-liposome alone or sense ODN; (5) the increase in neointimal area was abolished by cotransfection of wild-type p53 plasmid. Because p53 plays an important role in the regulation of apoptosis,6 7 8 decrease in apoptosis by antisense p53 ODN also might be involved in the neointimal formation. The present study is consistent with the previous report that the atherosclerotic lesions in p53-/-/apoE-/- mice had a significant increase in cell proliferation rate compared with those in p53+/+/apoE-/- mice.20 p53 may play a pivotal role in restenotic as well as atherosclerotic lesion development associated with its function in cell replication control.
To confirm the sustained effect of a single administration of ODN, we
examined whether ODN persisted in the vessel wall by using the
HVJ-liposome method. In previous studies, the HVJ-liposome–mediated
transfer method for antisense ODN, which uses the fusion system
mediated by HVJ, results in intracellular ODN delivery bypassing
endocytosis.21 22 Consequently, this method increases the
effectiveness and prolongs the half-life of antisense ODN in
vitro.22 The present study demonstrates that the
HVJ-liposome method actually prolongs the in vivo half-life of ODN. In
fact, there is evidence that FITC-labeled ODN persist in the nucleus
for at least 2 weeks after a single transfection. This method is
clearly efficient for inducing sustained action of antisense ODN in
vivo. Indeed, a high transfection efficiency of the HVJ-liposome method
into blood vessels has also been reported.23 Yonemitsu et
al23 documented that HVJ-liposomes could achieve highly
efficient gene transfection into the medial smooth muscle cells of
intact arteries at 150 and 760 mm Hg of pressure (mean=85.3% and
93.5% of total VSMC) without any inflammatory reaction for 
14
days.
Overall, the present study demonstrated that loss of p53 by antisense p53 ODN resulted in abnormal VSMC growth in vitro as well as in vivo. The observation in the present study is extremely important for understanding the molecular mechanisms of restenosis. In contrast, researchers have focused on the inhibitory effect of p53 on VSMC growth as a potential tool in molecular therapy to treat restenosis. Indeed, overexpression of the p53 gene in the injured arterial wall inhibits the proliferation of VSMC in vivo.24 Alternatively, a single intraluminal incubation of human p21, identified as a downstream mediator of p53 gene, resulted in significant inhibition of neointimal formation after vein grafting, associated with a phenotypic change of VSMC from neonatal to adult type in a rabbit model.25 Further studies of p53 in the pathological conditions in vascular disease may provide new insights into therapeutic strategies.
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Methods |
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In Vitro Experiments
Cell Culture
Human aortic VSMC (passage 5) were obtained from Clonetics Corp and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL epidermal growth factor, 2 ng/mL bFGF, and 1 mmol/L dexamethasone in the standard fashion.26 These cells showed the specific characteristics of VSMC by immunohistochemical examination and morphological observation. All the cells were used within passage 5 to 7.
Synthesis of ODN and Selection of Target Sequences
The sequences of phosphorothioate ODN against
p53 were as previously reported, with the ATG initiation
codon or its complement CAT in the sense and antisense,
respectively19 : (p53-sense:
5'-ACTGCCATGGAGGAGCCG-3', p53-antisense:
5'-CGGCTCCTCCATGGCAGT-3'). These antisense ODN inhibit
p53 synthesis in human hematopoietic
cells.19 We also used scrambled p53 ODN
(5'-AGTGGCCTCCATCTCCTCCGG-3') as a negative control.
Western Blot
VSMC were seeded onto 10-cm dishes (Corning). VSMC were grown to
confluence and made quiescent by incubation in defined serum-free
medium, which was supplemented with insulin
(5x10-7 mol/L), transferrin (5 mg/mL), and
ascorbate (0.2 mmol/L)27 before transfection.
Seventy-two hours after transfection, the cells were fixed with 10%
trichloroacetic acid (TCA) in saline, followed by extraction of total
protein with urea-TX (9 mol/L urea, 2% Triton-X and 5%
2-mercaptoethanol). Samples containing 100 µg protein were run on
12.5% SDS polyacrylamide gels. Proteins were incubated with a
monoclonal antibody to p53 (1:20, Calbiochem) at 4°C
overnight. To quantify and compare levels of proteins, the density of
each band was measured by densitometry. Staining with Coomassie
Brilliant Blue R (Sigma) was used to confirm that identical protein
amounts were loaded in all samples for Western blotting. Western
blotting of tubulin with antitubulin antibody (anti-human mouse IgG;
1:100; Oncogene) was also performed to confirm equal amounts of loaded
proteins.
Effect of Antisense ODN on DNA Synthesis and VSMC Growth
VSMC were seeded onto 96-well tissue culture plates. At 80%
confluence, VSMC were rendered quiescent by incubation for 48 hours in
defined serum-free medium. Then, 10 µL HVJ-liposomes (containing
15 µmol/L ODN) was added to the wells. The cells were incubated
at 4°C for 10 minutes and then at 37°C for 30 minutes. The cells
were washed 3 times with balanced salt solution (BSS) containing 2
mmol/L CaCl2. Relative rates of DNA synthesis
were assessed by determination of 3H-thymidine
incorporation into TCA-precipitable material over the next 24 hours
after 16 hours of incubation. After 3 days, an index of cell
proliferation was determined with the use of a WST cell counting kit
(Wako).
Preparation of HVJ-Cationic Liposomes In Vitro
In this study, we used HJV-cationic liposome method as the
in vitro transfection method because this method demonstrated highly
effective transfection efficiency, especially in in vitro cultured
cells.28 In brief, a lipid mixture containing 6 mg
phosphatidylcholine, 3 mg cholesterol, and 0.75 mg
3ß-[N-(N'
N-dimethylaminoethane)carbamoyl]cholesterol was
dissolved in chloroform and evaporated with the use of a rotary
evaporator. The dried mixture was hydrated with 200 µL of BSS
(137 mmol/L NaCl, 5.4 mmol/L KCl, 10 mmol/L Tris-HCl, pH
7.6) containing ODN. Purified HVJ (Z strain) was
inactivated by UV irradiation (110
erg/mm2 per second) for 3 minutes just before
use. The liposome suspension was mixed with HVJ in a total volume of 1
mL BSS. Free HVJ was removed from the HVJ liposomes by sucrose density
gradient centrifugation. The top layer of the sucrose
gradient was collected for use.
In Vivo Experiments
Synthesis of ODN and Selection of Target Sequences
The sequences of phosphorothioate ODN against p53
used in in vivo study were changed to the identical sequence of rat
p53: (p53-sense:
5'-ACTGTCATGGAGGATTCA-3', p53-antisense:
5'-TGAATC-CTCCATGACAGT-3'). We also used scrambled
p53 ODN (5'-AGTGGCCTCCATCTTATCAAG-3') as a negative control.
Antisense thrombomodulin (5'-ACCCAGAAAGAAAATCCC-3'; 12) and antisense
ACE (5'-GCCCCCATGGCGCGGT-3')(position -8 to +8 of the rat
sequence) were used as additional negative controls.
In Vivo Transfer Into Intact Rat Carotid Artery
Male Sprague-Dawley rats (weight 250 to 300 g; Charles
River) were anesthetized with ketamine, and the left
common carotid artery was surgically exposed.12 13 A
cannula was introduced into the common carotid artery through the
external carotid artery. Then, 200 µL of HVJ-liposome complex was
infused into the segment and incubated for 10 minutes at room
temperature. After 10-minute incubation, the infusion cannula was
removed. After transfection, blood flow to the common carotid artery
was restored by release of the ligatures, and the wound was closed. No
adverse neurological or vascular effects were observed in any animal
undergoing this procedure.
Conventional HVJ-Liposome Preparation In Vivo
For in vivo experiments, we used the conventional anionic
HVJ-liposome method.12 13 29 Briefly,
phosphatidylserine, phosphatidylcholine, and
cholesterol were mixed in a weight ratio of 1:4.8:2. Dried
lipid was hydrated in BSS containing sense or antisense ODN. Liposomes
were prepared by shaking and sonication.
Western Blotting
For the measurement of vascular p53 content, rats
were killed 5 days after transfection. After infusion of PBS, the
carotid arteries were removed and dissected free of periadventitial
tissues and immediately frozen in liquid nitrogen. Western blotting was
performed for analysis of p53 protein, as described
above. Carotid arteries frozen in liquid nitrogen were physically
crushed with a Cryo-Press CP-100 (Microtech Nichion Corp), followed by
extraction of total protein with lysis buffer (50 mmol/L Tris-HCl,
pH 8.0, 20 mmol/L EDTA, 1% SDS, 100 mmol/L NaCl). Samples
containing 30 µg protein were run on 12.5% SDS
polyacrylamide gels. Proteins were incubated with a monoclonal
antibody to p53 (1:100; Calbiochem) at 4°C overnight.
Quantification of Cell Proliferation in Medial Lesions
Paraffin-embedded sections of carotid artery harvested 4 days
after transfection were used for quantification of cell
proliferation.12 13 Four sections of each vessel spaced at
0.4-mm intervals were measured by a computerized image analyzer
system (Image Command 5098, Olympus). Monoclonal antibody against
proliferating cell nuclear antigen (PCNA) (PC-10; 1:500, Neo Markers)
was used as specific markers for proliferating cells in
paraffin-embedded sections. The number of PCNA-positive nuclei was
counted in the media by the image analyzer system at x400
magnification. Frequency of cell proliferation was expressed as PCNA
index, defined as the ratio of the number of PCNA-positive nuclei to
that of all the nuclei in the media. Samples were coded so that the
analysis was performed without knowledge of which treatment
each individual vessel had received. Intraobserver variability was
determined from triplicate measurements performed by 1 observer for all
sections (2.4±0.3%).
Measurement of Neointimal Formation
At 2 and 4 weeks after transfection, each carotid artery was
processed for morphological study.12 13 For
histological analyses, a segment of each artery
was perfusion-fixed with 4% paraformaldehyde at
physiological pressure (110 mm Hg) and
subsequently processed. Medial and luminal areas were measured on a
digitizing tablet after staining with hematoxylin. The medial area of
the vessel was readily demarcated as the area between the internal and
external elastic laminae. At least 3 individual sections from the
middle of the transfected arterial segments were
analyzed. Animals were coded so that the analysis was
performed without knowledge of which treatment each individual animal
had received.
Cotransfection of wild-type p53 plasmid was also examined. The p53 expression vector is driven by cytomegalovirus promoter.24 As the control, we constructed a control expression vector driven by the same promoter without p53 plasmid.
In Vivo Transfection of FITC-Labeled ODN
FITC-labeled phosphorothioate ODN on the 3' and 5' ends were
provided by Nihon Seifun Inc. Transfer of phosphorothioate FITC-labeled
ODN was performed under the following protocol; HVJ complex with
FITC-labeled phosphorothioate ODN (3 µL) was incubated for 10
minutes. The vessels were harvested 2 weeks after transfection and
perfusion-fixed with 4% paraformaldehyde. Sections
were examined by fluorescence microscopy after staining in
Erichrome black T solution. Elastic fibers stained dark red and were
readily distinguishable from the specific FITC ODN by the treatment of
Erichrome black T solution.30
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent
Bonferroni’s test was used to determine the significance of
differences in multiple comparisons. A value of P<0.05 was
considered significant.
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Acknowledgments |
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This work was partially supported by grants from the Uehara Memorial Foundation, the Hoan-sya Foundation, the Japan Cardiovascular Research Foundation, a Japan Heart Foundation Research Grant, and a grant-in-aid from The Tokyo Biochemical Research Foundation.
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Footnotes |
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The Methods section of this article can be found at http://www.circulationaha.org
Received July 19, 1999; revision received September 24, 1999; accepted October 6, 1999.
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References |
|---|
|
TopAbstractIntroductionResultsDiscussionMethodsReferences |
|---|
-
Libby PD, Schwartz E, Brogi H, Tanaka S, Clinton
K. A cascade model for restenosis: a special case of
atherosclerosis progression. Circulation.
1992;86(suppl III):III-47–III-52.
-
Vogelstein B, Kinzler KW. P53 function and
dysfunction. Cell. 1992;70:523–526.[Medline]
[Order article via Infotrieve]
-
Hollstein M, Sidransky D, Vogelstein B, Harris CC. P53
mutations in human cancers. Science. 1991;253:49–53.
[Abstract/Free Full Text]
-
Lane DP. p53, guardian of the genome.
Nature. 1992;258:15–16.
-
Lu X, Lane DP. Differential induction of
transcriptionally active p53 following UV or ionizing radiation:
defects in chromosome instability syndromes. Cell. 1993;75:765–778.[Medline]
[Order article via Infotrieve]
-
Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe
S, Jacks T, Dyke TV. p53-dependent apoptosis suppresses tumor
growth and progression in vivo. Cell. 1994;78:703–711.[Medline]
[Order article via Infotrieve]
-
Shaw P, Bovey R, Tardy S, Sahli R, Sordat B, Costa
J. Induction of apoptosis by wild-type p53 in a human
colon tumor-derived cell line. Proc Natl Acad Sci U S A. 1992;89:4495–4499.
[Abstract/Free Full Text]
-
Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi
A, Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic
cells that is inhibited by interleukin-6. Nature.
1991;352.
-
El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons
R, Trent JM, Lin D, Mercer WE, Kinzeler KW, Vogelstein B. WAF 1, a
potential mediator of p53 tumor suppression. Cell. 1993;75:817–825.[Medline]
[Order article via Infotrieve]
-
Miyashita T, Harigai M, Hanada M, Reed JC.
Identification of a p53-dependent negative response element in the
bcl-2 gene. Cancer Res. 1994;54:3131–3135.
[Abstract/Free Full Text]
-
Miyashita T, Reed JC. Tumor suppressor p53 is a direct
transcriptional activator of the human bax gene.
Cell. 1995;80:293–299.[Medline]
[Order article via Infotrieve]
-
Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang
L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delivery of
antisense cdc 2 kinase and PCNA oligonucleotides
results in chronic inhibition of neointimal hyperplasia.
Proc Natl Acad Sci U S A. 1993;90:8474–8478.
[Abstract/Free Full Text]
-
Morishita R, Gibbons GH, Horiuchi M, Ellison KE,
Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. A novel molecular
strategy using cis element "decoy" of E2F binding site inhibits
smooth muscle proliferation in vivo. Proc Natl Acad Sci
U S A. 1995;92:5855–5859.
[Abstract/Free Full Text]
-
Chang MW, Barr E, Seltzer J, Jiang YQ, Nabel GJ, Nabel
EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular
proliferative disorders with a constitutively active form of the
retinoblastoma gene product. Science. 1995;287:518–522.
-
Speir E, Modali R, Huang ES, Leon MB, Shawl F, Finkel
T, Epstein SE. Potential role of human cytomegalovirus and p53
interaction in coronary restenosis. Science. 1994;265:391–394.
[Abstract/Free Full Text]
-
Tsai HL, Kou GH, Chen SC, Wu CW, Lin YS. Human
cytomegalovirus immediate-early protein IE2 tethers a transcriptional
repression domain to p53. J Biol Chem. 1996;271:3534–3540.
[Abstract/Free Full Text]
-
Zhou YF, Leon MB, Waclawiw MA, Popma JJ, Yu ZX, Finkel
T, Epstein SE. Association between prior cytomegalovirus infection and
the risk of restenosis after coronary atherectomy.
N Engl J Med. 1996;335:624–630.
[Abstract/Free Full Text]
-
Gibbons GH, Dzau VJ. The emerging concept of vascular
remodeling. N Engl J Med. 1994;30:1431–1438.
-
Bi S, Lanza F, Goldman JM. The involvement of "tumor
suppressor" p53 in normal and chronic myelogenous leukemia
hemopoiesis. Cancer Res. 1994;54:582–586.
[Abstract/Free Full Text]
-
Morishita R, Gibbons GH, Ellison KE, Nakajima M, Leyen
HVL, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Intimal hyperplasia
after vascular injury is inhibited by antisense cdk 2 kinase
oligonucleotides. J Clin Invest. 1994;93:1458–1464.
-
Guevara NV, Kim HS, Antonova EI, Chan L. The absence of
p53 accelerates atherosclerosis by increasing cell
proliferation in vivo. Nat Med. 1999;5:335–339.[Medline]
[Order article via Infotrieve]
-
Morishita R, Gibbons GH, Horiuchi M, Nakajima M,
Ellison KE, Lee W, Kaneda Y, Ogihara T, Dzau VJ. Molecular delivery
system for antisense oligonucleotides: enhanced
effectiveness of antisense oligonucleotides by
HVJ-liposome mediated transfer. J Cardiovasc Pharmacol
Ther. 1996;2:213–222.
-
Yonemitsu Y, Kaneda Y, Morishita R, Nakagawa K,
Nakashima Y, Sueishi K. Characterization of in vivo gene transfer into
the arterial wall mediated by the Sendai virus
(hemagglutinating virus of Japan) liposomes: an effective tool for the
in vivo study of arterial diseases. Lab Invest. 1996;75:313–323.[Medline]
[Order article via Infotrieve]
-
Yonemitsu Y, Kaneda Y, Tanaka S, Nakashima Y, Komori K,
Sugimachi K, Sueishi K. Transfer of wild-type p53 gene effectively
inhibits vascular smooth muscle cell proliferation in vitro and in
vivo. Circ Res. 1998;82:147–156.
[Abstract/Free Full Text]
-
Bai HZ, Morishita R, Kida I, Yamakawa T, Zhang WD, Aoki
M, Matsushita H, Noda A, Nagai R, Kaneda Y, Higaki J, Ogihara T,
Sawa Y, Matsuda H. Inhibition of intimal hyperplasia after vein
grafting by in vivo transfer of human senescent cell-derived
inhibitor-1 gene. Gene Ther. 1998;5:761–769.[Medline]
[Order article via Infotrieve]
-
Bonin PD, Leadley RJ, Erickson LA. Growth
factor-induced modulation of endothelial-1 binding to
human smooth muscle cells. J Cardiovasc Pharmacol. 1993;22:S125–S127.
-
Libby P, O’Brien KV. Culture of quiescent
arterial smooth muscle cells in a defined serum-free
medium. J Cell Physiol. 1983;115:217–223.[Medline]
[Order article via Infotrieve]
-
Yonemitsu Y, Kaneda Y, Muraishi A, Yoshizumi T,
Sugimachi K, Sueishi K. HVJ (Sendai virus)-cationic liposomes: a novel
and potentially effective liposome-mediated technique for gene transfer
to the airway epithelium. Gene Ther. 1997;4:631–638.[Medline]
[Order article via Infotrieve]
-
Kaneda Y, Iwai K, Uchida T. Increased expression of DNA
cointroduced with nuclear protein in adult rat liver.
Science. 1989;243:375–378.
[Abstract/Free Full Text]
-
Kittelberger R, Davis PF, Stehbens WE. An improved
immunofluorescence technique for the
histological examination of blood vessel tissue.
Acta Histochem. 1989;86:137–142.[Medline]
[Order article via Infotrieve]
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  Y. Takami, H. Nakagami, R. Morishita, T. Katsuya, T.-X. Cui, T. Ichikawa, Y. Saito, H. Hayashi, Y. Kikuchi, T. Nishikawa, et al. Ubiquitin Carboxyl-Terminal Hydrolase L1, a Novel Deubiquitinating Enzyme in the Vasculature, Attenuates NF-{kappa}B Activation Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2184 - 2190. [Abstract] [Full Text] [PDF]
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 S. M. Sanz-Gonzalez, L. Barquin, I. Garcia-Cao, M. Roque, J. M. Gonzalez, J. J. Fuster, M. T. Castells, J. M. Flores, M. Serrano, and V. Andres Increased p53 gene dosage reduces neointimal thickening induced by mechanical injury but has no effect on native atherosclerosis Cardiovasc Res, September 1, 2007; 75(4): 803 - 812. [Abstract] [Full Text] [PDF]
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 J. Mercer, N. Figg, V. Stoneman, D. Braganza, and M. R. Bennett Endogenous p53 Protects Vascular Smooth Muscle Cells From Apoptosis and Reduces Atherosclerosis in ApoE Knockout Mice Circ. Res., April 1, 2005; 96(6): 667 - 674. [Abstract] [Full Text] [PDF]
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 V. Andres Unexpected Proatherogenic Properties of p21: Beyond Cell Cycle Control? Circulation, December 21, 2004; 110(25): 3749 - 3752. [Full Text] [PDF]
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M. Sata, K. Tanaka, N. Ishizaka, Y. Hirata, and R. Nagai Absence of p53 Leads to Accelerated Neointimal Hyperplasia After Vascular Injury Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1548 - 1552. [Abstract] [Full Text] [PDF]
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S. Scott, M. O'Sullivan, S. Hafizi, L. M. Shapiro, and M. R. Bennett Human Vascular Smooth Muscle Cells From Restenosis or In-Stent Stenosis Sites Demonstrate Enhanced Responses to p53: Implications for Brachytherapy and Drug Treatment for Restenosis Circ. Res., March 8, 2002; 90(4): 398 - 404. [Abstract] [Full Text] [PDF]
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U. Mayr, M. Mayr, C. Li, F. Wernig, H. Dietrich, Y. Hu, and Q. Xu Loss of p53 Accelerates Neointimal Lesions of Vein Bypass Grafts in Mice Circ. Res., February 8, 2002; 90(2): 197 - 204. [Abstract] [Full Text] [PDF]
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 W. R. P. Agema, J. W. Jukema, S. N. Pimstone, and J. J. P. Kastelein Genetic aspects of restenosis after percutaneous coronary interventions;towards more tailored therapy Eur. Heart J., November 2, 2001; 22(22): 2058 - 2074. [PDF]
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 C. D. Mao, P. Hoang, and P. E. DiCorleto Lithium Inhibits Cell Cycle Progression and Induces Stabilization of p53 in Bovine Aortic Endothelial Cells J. Biol. Chem., July 6, 2001; 276(28): 26180 - 26188. [Abstract] [Full Text] [PDF]
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U. Mayr, M. Mayr, C. Li, F. Wernig, H. Dietrich, Y. Hu, and Q. Xu Loss of p53 Accelerates Neointimal Lesions of Vein Bypass Grafts in Mice Circ. Res., February 8, 2002; 90(2): 197 - 204. [Abstract] [Full Text] [PDF]
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S. Scott, M. O'Sullivan, S. Hafizi, L. M. Shapiro, and M. R. Bennett Human Vascular Smooth Muscle Cells From Restenosis or In-Stent Stenosis Sites Demonstrate Enhanced Responses to p53: Implications for Brachytherapy and Drug Treatment for Restenosis Circ. Res., March 8, 2002; 90(4): 398 - 404. [Abstract] [Full Text] [PDF]
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Y. Taniyama, K. Tachibana, K. Hiraoka, T. Namba, K. Yamasaki, N. Hashiya, M. Aoki, T. Ogihara, K. Yasufumi, and R. Morishita Local Delivery of Plasmid DNA Into Rat Carotid Artery Using Ultrasound Circulation, March 12, 2002; 105(10): 1233 - 1239. [Abstract] [Full Text] [PDF]
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