© 1997 American Heart Association, Inc.
Articles |
Increased Sensitivity of Human Vascular Smooth Muscle Cells From Atherosclerotic Plaques to p53-Mediated Apoptosis
From the Unit of Cardiovascular Medicine (M.R.B., P.L.W.), University of Cambridge (UK) Clinical School of Medicine, Department of Medicine, Addenbrooke's Hospital, and the Biochemistry of the Cell Nucleus Laboratory (T.D.L.), Imperial Cancer Research Fund, London, and the Department of Pathology (S.M.S.), University of Washington, Seattle.
Correspondence to Dr Martin R. Bennett, Unit of Cardiovascular Medicine, University of Cambridge Clinical School of Medicine, Department of Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK. E-mail mrb{at}mole.bio.cam.ac.uk © 1997 American Heart Association, Inc.
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Abstract The recent demonstration that apoptosis of vascular smooth muscle cells (VSMCs) occurs in human atherosclerotic plaques suggests that VSMC apoptosis may promote plaque rupture and subsequent myocardial infarction. In culture, human plaque VSMCs show higher rates of apoptosis than VSMCs from normal vessels, although the mechanism of this effect is unknown. In earlier studies, we have shown that the tumor suppressor gene p53 regulates apoptosis of rat VSMCs after deregulated cell cycle control. We therefore analyzed p53 function in cultured VSMCs derived from human coronary plaques or the media of normal coronary arteries. VSMCs with reduced or increased p53 activity were created by infecting VSMCs with retroviruses containing a dominant-negative p53 minigene or a chimeric p53 protein (p53TMER), which could be activated pharmacologically. Basal p53 protein expression and transcriptional activity were similar in plaque and normal VSMCs, and suppression of p53 activity blocked growth arrest in response to DNA damage in both VSMC types. In contrast, suppression of p53 activity failed to block apoptosis of plaque or normal VSMCs in low- or high-serum conditions or after DNA damage. Furthermore, in plaque VSMCs, p53 overexpression induced apoptosis in all conditions tested and also induced growth arrest. p53-mediated apoptosis was independent of new gene transcription or protein synthesis but was suppressed by prior growth arrest of cells, indicating that growth status can regulate sensitivity to p53-mediated apoptosis. No effect of increased p53 activity was seen in normal VSMCs. We conclude that VSMCs from human plaques have an increased sensitivity to p53-mediated apoptosis compared with normal VSMCs. Our data also suggest that the mechanism of p53-mediated apoptosis of plaque VSMCs may be distinct from that inducing growth arrest.
Key Words: p53 • apoptosis • atherosclerosis • restenosis • cell cycle
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Introduction |
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Apoptosis is a physiological mediator of cell death, regulated by the action and interaction of specific gene products. In the artery wall, apoptosis has been demonstrated after balloon catheter injury1 and during arterial remodeling following changes in blood flow after birth.2 However, the recent identification that VSMCs undergo apoptosis in the human atheromatous plaque, particularly at areas prone to rupture,3 4 5 suggests that VSMC apoptosis may promote plaque rupture and the subsequent thrombosis of the vessel leading to myocardial infarction. Although the pathways that control human VSMC apoptosis are unknown, the recent demonstration that VSMCs derived from plaques show increased susceptibility to apoptosis in culture6 suggests that stable changes in expression of proapoptotic or antiapoptotic genes control plaque VSMC apoptosis rather than local cell-cell interactions or the cytokine environment in the plaque.
We have previously demonstrated that specific gene products, in particular the proto-oncogene c-myc, the adenovirus gene product E1A, the proto-oncogene bcl-2, and the tumor suppressor gene p53, can regulate apoptosis in VSMCs in vitro.7 8 Deregulated expression of c-myc or E1A induces apoptosis in VSMCs, which is regulated and mediated in part by p53. In contrast, bcl-2 suppresses apoptosis induced by c-myc or E1A or, in otherwise normal smooth muscle cells, via a p53-independent pathway.8 Despite these observations, we have not been able to demonstrate differences in expression of bcl-2 or c-myc (Reference 66 and M.R. Bennett, unpublished data, 1996) in plaque versus normal VSMCs.
p53 has recently been implicated in the control of proliferation of human VSMCs in atheromatous plaques, in particular, after angioplasty restenosis. The study by Speir et al9 suggested that inactivation of p53, possibly via interaction with a cytomegalovirus gene product, IE84, induces aberrant proliferation of VSMCs. Against this hypothesis, cell proliferation indexes are low in primary plaques and at sites of angioplasty restenosis in humans.10 Moreover, the expression of high levels of wild-type or mutant-type p53 in normal rat VSMCs has little effect on cell proliferation.8 However, profound changes in vessel wall mass can be achieved by changes in apoptotic rates, without significant changes in cell proliferation.2 Moreover, in advanced plaques, cell death leading to rupture is more likely to be an important issue than plaque growth. If p53 regulates apoptosis of human VSMCs, changes in p53 expression or activity in plaques may induce changes in tissue mass without significant effects on cell proliferation.
The aim of the present study was to determine the role of p53 in human VSMC apoptosis. Human coronary plaque and normal medial VSMCs having reduced p53 activity were obtained by infecting cells with amphotropic retroviruses encoding a dominant-negative p53 minigene (DN-p53), containing just the p53 oligomerization domain; the protein produced from this construct oligomerizes with native p53 and, because the miniprotein lacks a DNA-binding domain, prevents DNA binding of p53.11 Expression of this miniprotein has been shown to inhibit p53-mediated apoptosis.12 13 To overexpress p53, cells derived from plaques or normal vessels were infected with full-length human p53, whose activity can be regulated pharmacologically. We demonstrate that apoptosis of VSMCs from human atheromatous plaques but not normal vessels is regulated by p53.
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Materials and Methods |
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Cell Isolation and Culture
Plaque cells were derived from primary coronary artery plaques by atherectomy, and normal medial VSMCs were from coronary arteries of patients undergoing cardiac transplantation for nonischemic cardiomyopathy, as part of the University of Washington Heart Transplantation Program. The isolation and characterization of these cells has already been described.6 Briefly, cultures were obtained from specimens from individual patients of both sexes (age range, between 34 and 62 years; mean age, 49.8 [plaque cells] and 47.8 [normal coronary artery VSMCs] years). Plaque cultures were obtained from patients with either stable or unstable angina; we have previously demonstrated that cells from both types of lesions undergo similar rates of apoptosis in culture.6 Characterization of VSMCs as intimal (from atherectomy tissues) and medial (from normal vessels) was performed by hematoxylin and eosin histology of the atherectomies or arteries removed (not shown). Cells were characterized as VSMCs by culture morphology and immunocytochemical staining pattern at passage 3 (
-actin positive, vimentin positive, von
Willebrand factor negative, desmin negative, and smooth muscle
myosin negative) (not shown). In primary isolates from atherosclerotic
plaques, both macrophages and T lymphocytes were present;
however, by passage 3, the only cells identifiable were VSMCs. Three
cultures each of plaque-derived or normal smooth muscle cells were used
for gene transfer. Cells from atherectomy specimens and from normal
vessels were cultured in medium 199 containing 10% FCS and 10
mmol/L HEPES (Sigma Chemical Co) and equilibrated with 95%
air/5% CO2. Cultures from individual patients were
maintained as separate cultures, and cells were not pooled.
Subconfluent cells were passaged by trypsinization in 0.05% trypsin in
PBS.
Generation of Human VSMCs Expressing Introduced p53 or
DN-p53
Human VSMCs from atheromatous plaques and from
normal coronary arteries expressing exogenous genes were
generated by retrovirus-mediated gene transfer. Full-length cDNAs
encoding human p53 or a dominant-negative p53 minigene (DN-p53)
encoding amino acids 302 to 39011 were cloned into the
retrovirus vector pBabe (neo/puro) using conventional techniques.
Ecotropic retroviruses containing these genes or the retrovirus vector
alone were generated by calcium phosphate transfection of the vector
containing the gene of interest into an ecotropic packaging cell line,
GP+E86.14 The medium was then harvested from these cells
after 48 hours and used to infect a second generation packaging cell
line, PA317, in medium containing polybrene (8 µg/mL).
Packaging cells were selected in 500 µg/mL of G418 (Geneticin,
GIBCO) or 2.5 µg/mL of puromycin (Sigma), until a confluent
10-cm-diameter dish of resistant cells was obtained.
Amphotropic virus was then harvested from the medium of this dish after
48 hours of incubation. This concentrated retrovirus stock was then
used to infect human VSMCs at passage 3 as previously
described.6 Resistant human VSMCs were selected in
750 µg/mL of G418 or 2.5 µg/mL of puromycin after at
least 4 weeks in antibiotic-containing medium. Pooled populations of
both plaque-derived and normal human VSMCs were used for experiments
rather than clones of antibiotic-resistant cells. All
experiments were performed on passage-5 cells.
Time-Lapse Videomicroscopy
Cells were prepared for videomicroscopy as previously
described.6 Briefly, cells were maintained in medium
containing 10% FCS, washed three times in medium containing 0% FCS,
and then cultured in this latter medium. This was supplemented with
selenite (30 nmol/L), transferrin (5 µg/mL),
fibronectin (1 µg/mL), and albumin (1 mg/mL).
For experiments involving etoposide (5 µmol/L), cells
were filmed between 24 and 48 hours after addition of this agent.
Flasks were gassed with 95% air/5% CO2 every 24 hours and
sealed. An Olympus OM-70 microscope was enclosed in a plastic
environment chamber and maintained at 37°C by an external heater. The
time-lapse equipment consisted of a Sony 92D CCD camera with a
Panasonic 6730 time-lapse video recorder. Films were
analyzed for morphology of apoptosis and cell death
rates as previously described,6 using an observer blind to
cell type and treatment conditions. Apoptotic cell death events
were scored midway between the last appearance of normality and the
point at which the cell became fully detached and fragmented, an
interval of typically 60 to 90 minutes. Cell division was scored at the
time at which septa appeared between two daughter cells. Each
individual cell culture was analyzed in duplicate as a minimum
(plaque VSMCs, n=8; normal coronary VSMCs, n=8).
Propidium Iodide Staining
To confirm that cell death was occurring by apoptosis,
cells were grown in eight-well tissue culture chamber slides
(Tissue-Tek, Nunc) and then transferred to low-serum conditions. After
24 hours, cells were incubated with 10 µg/mL of propidium
iodide and then fixed in 4% formaldehyde for 15 minutes. Cells were
then viewed by fluorescence microscopy.
Western Blotting
Western blots were prepared by lysis of cells cultured in medium
containing 10% FCS. Protein isolation, electrophoresis, and blotting
were performed as previously described,15 using a mouse
monoclonal anti-p53 antibody that recognizes only wild-type p53 (Pab1,
Oncogene Science). Protein concentrations were assessed by modified
Bradford assay (Bio-Rad) before loading.
Assay of p53 Transcriptional Activity
To assess p53 activity in native and infected cell lines, a 75%
confluent 10-cm dish of uninfected VSMCs from each patient or the cell
lines expressing DN-p53 or p53 TMER were transfected using 10 µg of
the p53 reporter plasmid, p53CAT, by calcium phosphate transfection and
glycerol shock.14 This plasmid contains multiple p53
consensus binding sites linked to the CAT gene.16 After 48
hours, cells were harvested and lysed, and CAT activity was determined
using standard assay conditions with
[14C]chloramphenicol.17 Transfection
efficiencies were standardized by cotransfection with a reporter
construct containing a ß-galactosidase gene, and percent infection of
cells was determined by immunocytochemistry using
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal).
Fold induction of the p53 reporter was calculated by scintillation
counting of the samples.
Flow Cytometry
Cell lines growing in 10% FCS for at least 48 hours or 48 hours
after the addition of etoposide (5 µmol/L) were prepared
for flow cytometry as previously described, using a 2-hour pulse of
5-bromo-2'-deoxyuridine (10 µmol/L) before
harvesting.7 Cells demonstrating less than the diploid
content of DNA were excluded from the measurement of the percentages of
cells in each cell cycle phase.
Statistical Analyses
The means of apoptotic deaths were analyzed
using ANOVA for multiple comparisons. Paired analysis between
two groups (eg, between plaque and normal VSMCs or between uninfected
and infected cell lines) was performed using Student's t
test where ANOVA indicated significance for the multiple
comparison.
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Results |
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To analyze p53 function in human VSMCs, we cultured cells from both normal human coronary artery media and atherosclerotic plaques. Retrovirus vectors were used to insert specific genes into plaque and normal VSMCs. A dominant-negative p53 minigene (DN-p53) was used to suppress p53 transcriptional activity. Initially, we infected human VSMCs with full-length human p53. However, despite obtaining antibiotic-resistant cells after infection, we were unable to expand the population of plaque p53 cells. In contrast, normal VSMCs expressing high levels of p53 could be obtained. In view of the possibility that high levels of p53 were arresting and/or inducing apoptosis of the plaque cells, we used a construct encoding a p53 protein that could be activated pharmacologically, p53TMER. p53TMER was constructed by fusing human p53 cDNA to a transactivation-defective form of the mouse estrogen receptor (ER).18 This construct encodes a chimeric p53-ER protein (p53-TMER), which is not activated by estradiol. However, the protein can be activated by coincubation with 100 nmol/L of 4-hydroxytamoxifen.19 By using this construct, resistant pools of human plaque and normal p53TMER cells were produced by culture in the absence of 4-hydroxytamoxifen. Cells infected with the retrovirus constructs were thus designated plaque DN-p53, p53-TMER, or vector, and normal VSMCs expressing these gene products were similarly designated. After all infections, expression of the inserted gene was confirmed by Northern and Southern blotting (not shown).
p53 Protein Expression by Normal or Plaque VSMCs
Although p53 activity was similar in uninfected normal or plaque
VSMCs, interaction of p53 with virus proteins, which has been proposed
to occur in human angioplasty restenosis, or the presence of
mutant p53 protein can elevate p53 protein levels 20- to 50-fold. This
is because mutant p53 or p53 complexed with certain virus proteins has
a markedly increased half-life. To analyze p53 content of
plaque and normal VSMCs, passage-5 cells were analyzed by
Western blotting of lysates from cells maintained in 10% FCS. Although
there was minor variability in p53 expression in cells derived from
either normal vessels or plaques, Fig 1
shows that p53 expression was low in all cells and that there was no
consistent difference in p53 expression in plaque versus normal
VSMCs. In particular, in both plaque and normal uninfected VSMCs, there
was no evidence of the high levels of p53 expression seen when cells
were infected with viruses such as simian virus 40 (Fig 1).
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p53 Transcriptional Activity in Normal or Plaque VSMCs
Because p53 protein levels do not always reflect underlying p53
activity and also to prove that infection with the specific virus
vectors produced the expected changes in p53 activity, we studied p53
transcriptional activity in VSMCs. We examined the ability of p53 in
VSMCs to transactivate a reporter construct, containing
multiple copies of the p53 consensus binding site, linked to the CAT
gene. Normal and plaque VSMCs were transfected with the p53 reporter
construct, and CAT activity was assayed after 48 hours. Fig 2 shows that, consistent with the
data from Western blots, uninfected normal and plaque VSMCs from three
separate patients displayed similar ability to transactivate a
p53 reporter construct. Cells expressing the retrovirus vector alone
had p53 activity similar to that of uninfected cells (not shown). In
contrast, cells expressing DN-p53 had markedly reduced CAT activity
(
20% of uninfected cells), whereas p53-TMER cells had increased CAT
activity only in the presence of 4-hydroxytamoxifen (Fig 2).
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To confirm that CAT expression correlated with biologically active p53,
we studied the ability of normal and plaque VSMCs to growth-arrest
after etoposide treatment. Etoposide is a topoisomerase II
inhibitor that induces DNA damage, and the p53-dependent
growth arrest after etoposide treatment is a well-characterized assay
for functional p53.20 21 Cell proliferation was
analyzed by time-lapse videomicroscopy and flow cytometry.
Cells were incubated in the presence or absence of 5
µmol/L etoposide and isolated 48 hours later for flow
cytometry or filmed between 24 and 48 hours after the addition of
etoposide. Etoposide treatment induced growth arrest in normal medial
and plaque VSMCs, as shown by the suppression of cell division on
time-lapse videomicroscopy, and a loss of cells in S phase, as shown by
flow cytometry (Tables 1 and 2). 4-Hydroxytamoxifen-treated p53TMER
cells or cells expressing the retrovirus vector alone also underwent
growth arrest after etoposide treatment. In contrast, cells with DN-p53
continued to proliferate after etoposide treatment. This confirms that
uninfected VSMCs, cells infected with the vector alone, and p53TMER
cells contain functional p53 protein. In contrast, cells expressing
DN-p53 have markedly reduced levels of p53 biological activity.
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Effect of p53 on Cell Proliferation
Because plaque-derived cells and cell lines show constant
spontaneous apoptosis in culture, cell proliferation of
uninfected human VSMCs and cell lines was analyzed by
time-lapse videomicroscopy and flow cytometry, methods that do not rely
on indexes of cell number. Plaque-derived cells proliferated more
slowly than did normal medial VSMCs, as assessed by S-phase percentage
and the number of divisions observed over 24 hours (Tables 1
and 2),
and expression of the retrovirus vector alone did not affect cell
proliferation. Abrogation of p53 by the DN-p53 minigene had no effect
on cell proliferation under baseline conditions. However, increased p53
activity in p53TMER cells in the presence of 4-hydroxytamoxifen slowed
proliferation of plaque VSMCs but not normal medial VSMCs. Thus, p53
appears to be involved in the regulation of proliferation of plaque
VSMCs but not cells from normal vessels under these experimental
conditions. Interestingly, plaque-derived cells always divided more
slowly than their normal medial counterparts expressing the same gene
product, and plaque VSMCs expressing DN-p53 or p53TMER underwent a
terminal senescence (characterized by no identifiable cell
proliferation on time lapse over 1 week) at four or five passages
earlier than normal VSMCs with similar levels of p53 activity.
Effects of p53 on Apoptosis
The effect of p53 activity on apoptosis of human VSMCs was
determined by time-lapse videomicroscopy (Fig 3A). We have previously shown that
apoptotic rates derived from videomicroscopy agree closely with
less direct measures of apoptosis, such as DNA fragmentation
and electron microscopic morphology.6 Time-lapse assays
have the advantage that they use small numbers of cells, an important
issue when few cells are available. Apoptotic cell death was
also confirmed by propidium iodide staining, showing condensation of
nuclear chromatin and nuclear fragmentation (Fig 3B). Apoptotic
rates were studied in cells in 10% FCS±5 µmol/L
etoposide or at 24 hours after serum withdrawal (0% FCS).
Plaque-derived cells showed some apoptotic death in high-serum
conditions (Fig 4A), but both plaque and
normal VSMCs showed evidence of apoptosis in low-serum
conditions (Fig 4B) or after etoposide treatment (Fig 4C).
Apoptotic rates of cells infected with the retrovirus vector
alone were similar to those of uninfected cells (not shown).
Suppression of p53 activity by the DN-p53 minigene did not affect
apoptosis under any of the conditions studied. In contrast,
expression of active p53 via the p53TMER construct increased
apoptosis of plaque but not normal VSMCs in 10% FCS or 0% FCS
or after DNA damage.
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High levels of p53 activity induced by 4-hydroxytamoxifen promoted
apoptosis and slowed cell proliferation of plaque VSMCs but had
little or no effect in normal VSMCs. To confirm that the
apoptosis and growth arrest induced in plaque p53TMER cells was
not due to the effects of 4-hydroxytamoxifen alone, both plaque and
normal p53TMER cells were compared with cells infected with the TMER
vector alone (Fig 5). 4-Hydroxytamoxifen
(100 nmol/L) did not affect either cell proliferation or
apoptosis of cells with this vector alone. In addition, there
was no difference in apoptotic rates in plaque TMER cells
compared with uninfected plaque cells in the absence of
4-hydroxytamoxifen (not shown).
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p53-Dependent and -Independent Apoptosis of Human VSMCs Is
Suppressed by Growth Arrest
We have previously shown that p53-mediated apoptosis is
most apparent in cells that are driven to proliferate, for example, by
deregulated expression of oncogenes such as c-myc or the
adenovirus E1A gene.8 To examine whether being in the cell
cycle per se determines the sensitivity of human plaque VSMCs to
apoptosis, we studied p53-mediated apoptosis in
proliferating plaque VSMCs or in plaque VSMCs after growth arrest by 48
hours of low-serum or etoposide treatment. Table 3 shows that when p53TMER cells in 10%
FCS were transferred to low serum with simultaneous
activation of p53 by 4-hydroxytamoxifen, 37% of cells underwent
apoptosis over 72 hours. To examine the effect of prior growth
arrest on p53-mediated apoptosis, plaque p53-TMER cells were
arrested by etoposide or 0% FCS for 48 hours. Cells were then placed
in medium containing 4-hydroxytamoxifen for 24 hours, with deaths
assayed over the whole 72 hours. If cells were growth-arrested with low
serum or etoposide before activation of p53 with 4-hydroxytamoxifen,
this reduced apoptosis to 12.6% and 13.2%, respectively.
Similarly, cells placed in 5 µmol/L etoposide with
simultaneous activation of p53 showed 10.4% death over 72
hours. Treatment with etoposide or low serum for 48 hours before
activation of p53 suppressed this level to 5.2% and 6.6%,
respectively (Table 3) (P<.05 versus 10% FCS). Thus,
growth arrest by etoposide suppressed the subsequent apoptosis
induced by low serum. Similarly, growth arrest due to serum removal
reduced apoptosis induced by DNA damage. Because etoposide
arrests cells in G2 and because serum removal arrests cells
in G0/G1, these data suggest that growth arrest
per se suppresses subsequent p53-mediated apoptosis of plaque
TMER cells.
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p53-Mediated Apoptosis of Human Plaque p53TMER Cells Does
Not Require New Protein Synthesis or Transcription
Because p53 induces apoptosis in plaque-derived cells and
because direct p53 transactivation of target genes by p53 is
responsible for many of its actions, including growth arrest, we
investigated whether p53-mediated killing of VSMCs was dependent on new
protein synthesis or transcription. Plaque p53-TMER cells were
incubated with either cycloheximide (100 µg/mL, a
concentration that completely suppresses new protein synthesis, as
determined by incorporation of S-methionine into protein [not shown])
or actinomycin D (2.5 µg/mL) for 1 hour, and then
4-hydroxytamoxifen was added 1 hour before serum removal. Activation of
p53 induced apoptosis of plaque TMER cells in low serum.
Neither cycloheximide nor actinomycin D suppressed this p53-mediated
killing of plaque cells (Fig 6A),
suggesting that transcriptional activation of p53 target genes is not
involved in p53-mediated killing of plaque VSMCs.
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To determine whether p53 induction of growth arrest in plaque TMER
cells was dependent on new gene transcription or protein synthesis,
cells were incubated in medium+10% FCS with either cycloheximide (100
µg/mL) or actinomycin D (2.5 µg/mL) for 1 hour and
were then p53-activated by 4-hydroxytamoxifen. In contrast to
apoptosis, p53-mediated growth arrest of plaque TMER cells
could be suppressed by actinomycin D (Fig 6B). Cycloheximide actually
induced growth arrest in cells in the presence or absence of
4-hydroxytamoxifen, and no additional effect of increasing p53 activity
by 4-hydroxytamoxifen could be demonstrated.
These data suggested that p53-mediated apoptosis of plaque
cells was not due to p53-induced transcription from target
genes. We therefore reassessed the use of the DN-p53 construct in
apoptosis of plaque and normal VSMCs expressing basal levels of
p53. Since this construct blocks DNA binding of endogenous
p53, if p53-induced plaque VSMC apoptosis does not require new
gene transcription, the DN-p53 construct might be expected to be an
incomplete suppressor of p53-mediated apoptosis (Fig 4). To
determine whether this is the case, we infected plaque and normal VSMCs
with a retrovirus containing the human papilloma virus type-16 E6 gene.
E6 binds to p53, blocking p53-mediated transactivation, but also
targets p53 for ubiquitin-mediated degradation.22 23 Thus,
cells containing E6 have very low levels of both p53 protein and p53
transcriptional activity. This was confirmed by transfection assay (Fig 2) and Western blot (Fig 1). Plaque VSMCs expressing E6 did not show
reduced levels of apoptosis in high- or low-serum conditions or
after etoposide treatment (Fig 7), and in
fact, E6 significantly increased apoptosis in all conditions
studied. These results confirm that apoptosis in plaque VSMCs
in high- or low-serum conditions or after etoposide treatment is not
blocked by suppression of basal p53 activity.
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Discussion |
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The role of p53 in the regulation of proliferation and apoptosis of nontransformed cells remains contentious. In contrast to tumor cells lacking p53, where reintroduction of p53 can induce growth arrest and apoptosis,24 25 many nontransformed cell lines show neither response to p53 overexpression,26 27 28 and the fact that p53-deficient mice develop normally indicates that p53 plays no essential role in the normal proliferation of most cells.29 Indeed, we have previously shown that a temperature-sensitive p53 mutant in either the wild-type or mutant conformation has minimal effect on normal rat VSMC proliferation or apoptosis.8 However, recent evidence implicates suppression of p53 in intimal VSMC accumulation seen in angioplasty restenosis,9 suggesting that p53 inactivation by a virus protein promotes cell proliferation of human VSMCs. Of interest, higher levels of apoptosis have been documented in restenosis lesions compared with primary atherosclerotic plaques.3 The present study sought to clarify the role of p53 in the control of apoptosis and proliferation in normal human VSMCs and in VSMCs from plaques.
We demonstrated that VSMCs derived from human coronary atherosclerotic plaques were very sensitive to p53-mediated apoptosis. High levels of p53 activity induced apoptosis in plaque VSMCs both in high- and low-serum conditions and also after DNA damage. In contrast, similar levels of p53 protein and activity had no effect on normal VSMC apoptosis. p53-mediated apoptosis of plaque VSMCs was affected by cell cycle transition, since cells that were growth-arrested via p53-dependent or -independent means showed reduced p53-induced death. Thus, plaque cells are intrinsically very sensitive to p53-mediated apoptosis, and the presence of cells in the cell cycle sensitizes plaque VSMCs to p53-dependent apoptosis. In addition, we found that p53-mediated apoptosis of plaque VSMCs was not dependent on new gene transcription or protein synthesis. We also found that basal levels of p53 were not proapoptotic in plaque or normal VSMCs in any of the conditions tested, since abrogation of p53 activity using DN-p53 (or E6) did not suppress apoptosis in either cell type. This observation suggests that the spontaneous apoptosis of plaque VSMCs in culture is not driven by p53.
Our data also help clarify the effect of p53 on VSMC cell proliferation. We found that abrogation of basal p53 activity by DN-p53 in either plaque or normal VSMCs did not increase cell proliferation of either cell type, although DN-p53 VSMCs did not arrest after DNA damage. Thus, the basal expression and activity of p53 seen in plaque and normal VSMCs in culture are not growth suppressive. In addition, overexpression of p53 had no effect on normal VSMC division, although there was slowing of plaque VSMC replication. In contrast to apoptosis, p53-mediated arrest of plaque VSMCs did require new gene transcription.
We found no evidence of mutant p53 activity in plaque or normal VSMCs.
In particular, only low levels of p53 protein were found in plaque or
normal VSMCs, and we did not observe the high levels of p53 protein
often seen when cells express mutant p53 or when p53 is stabilized by
some virus proteins. In addition, p53 activity, as assessed by
transfection assay and growth arrest after DNA damage, was not
different in passaged plaque versus normal VSMCs, suggesting that p53
function in our passaged plaque and normal VSMCs was intact. Our
antibody recognized only wild-type p53 (Fig 2), and blots using an
antibody to mutant p53 (Pab2, Oncogene Sci) failed to recognize p53 in
normal or plaque VSMCs (not shown). These data are consistent
with other studies showing that p53 in plaques is of the wild
type.30
Our data are in agreement with a number of recent studies. First, we showed that DNA damage–induced apoptosis may occur in the absence of functional p53.31 32 33 Second, we showed that the sensitivity to p53-mediated apoptosis is related to the presence of the cells in the cell cycle. Although we cannot extrapolate from our studies using growth arrest induced by serum removal or etoposide to other agents that induce arrest, our data are consistent with studies indicating that a stable p53-mediated or p53-independent growth arrest inhibits apoptosis due to other agents21 34 35 36 37 and that the regulatory role of p53 in apoptosis is influenced by the particular cellular context in which the gene is expressed.
In addition, we present two lines of evidence suggesting that the p53 activity that induces apoptosis is distinct mechanistically from that inducing growth arrest. First, after DNA damage, cells with reduced p53 activity did not undergo growth arrest but retained the ability to undergo apoptosis. Second, p53-mediated apoptosis was not reduced by suppression of new gene transcription or protein synthesis, whereas p53-mediated cell cycle arrest was blocked by actinomycin D. This is consistent with evidence indicating that p53-mediated growth arrest is due to the transcription of target genes. Although the transcriptional target genes responsible for p53-mediated growth arrest (p21, GADD45) are well established, the mechanism of p53-mediated apoptosis is unsure. Genes such as Bax, Bcl-2, and Fas/APO-1 have all been implicated in mediating the proapoptotic action of p53,38 39 40 41 42 43 and recent studies have indicated that the transactivation domain of p53 is necessary for its apoptotic function.44 However, p53-mediated cell death can occur without the induction of p21 or in Bax-deficient animals.44 45 46 47 We have previously demonstrated that IGF-1 is a potent survival factor in rat VSMCs whose proliferation is driven by c-myc48 or in human plaque or normal VSMCs.6 The recent observation that p53 induces the expression of IGF-binding protein 3,49 effectively blocking IGF-1–mediated signaling, may explain how p53 can induce apoptosis in plaque VSMCs. However, p53-mediated apoptosis may also be due to the suppression of labile cell survival signals, such as IGF-1,50 or direct protein-protein interactions, since, consistent with other studies,45 51 we have found that p53-mediated death does not need new protein synthesis or gene transcription. Indeed, recent studies have indicated that transactivation-defective p53 can still induce apoptosis,36 and agents that actually increase p53 transcriptional activity can block the apoptotic function of p53.52 Thus, there appears to be no direct relationship between the transcriptional activity of p53 and its ability to induce apoptosis. This makes it likely that several activities of p53 contribute to its ability to induce apoptosis, the importance of each being dependent on cell type and the presence of survival factors.
Recently, a model has been proposed connecting the p53-induced growth
arrest and apoptosis pathways (Fig 8).53 In this model,
activation of p53 may induce growth arrest via p21 and other target
genes. A stable growth arrest can then inhibit p53-mediated death.
However, in some cell types, an additional (unknown) factor, designated
factor D, possibly induced by p53 (Fig 8), can override the growth
arrest and induce p53-mediated apoptosis. This factor is not
p53 itself, since cells with no p53 activity have different
sensitivities to apoptosis induced by introduced
p53.53 In accordance with this model, we have observed in
the present study that a stable growth arrest inhibits p53-mediated
apoptosis of plaque cells and also that p53-mediated growth
arrest and apoptosis of plaque VSMCs may occur via different
pathways. In addition, we have previously found that inhibition of the
growth-arrest pathway sensitizes VSMCs to p53-mediated
apoptosis.8 In this proposed model, the relative
activity of factor D can determine the susceptibility to p53-mediated
apoptosis. If this model is a true representation of
p53-mediated apoptosis of human VSMCs, then plaque VSMCs may be
more sensitive to apoptosis because of a higher activity of
such a factor(s).
|
In conclusion, we have demonstrated that human plaque VSMCs show an increased sensitivity to p53-mediated growth arrest and apoptosis and that the sensitivity of plaque VSMCs to p53-mediated death can be changed by the presence or absence of the cell in the cell cycle. Our data also suggest that the proapoptotic action of p53 may be different mechanistically from its growth-arrest action. Moreover, although the increased susceptibility of plaque VSMCs to apoptosis is not due to increased p53 expression or transcriptional activity, the ability of p53 to induce apoptosis only in plaque VSMCs implies that some additional p53-sensitive activity is present in plaque VSMCs, which may promote plaque VSMC apoptosis and induce plaque breakdown and rupture.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by British Heart Foundation (BHF) grant number PG/95057 to Dr Bennett and National Institutes of Health grant HL-18641 to Dr Schwartz. Dr Bennett is supported by BHF Clinical Scientist Fellowship FS/00263, and Dr Weissberg is supported by a BHF chair grant (CH/94001).
Received January 21, 1997; accepted July 16, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Bochatonpiallat M, Gabbiani F, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1059-1064.[Abstract]
2.
Cho A, Courtman DW, Langille BL.
Apoptosis (programmed cell death) in arteries of the neonatal
lamb. Circ Res. 1995;76:168-175.
3.
Isner J, Kearney M, Bortman S, Passeri J.
Apoptosis in human atherosclerosis and
restenosis. Circulation. 1995;91:2703-2711.
4. Han D, Haudenschild C, Hong M, Tinkle B, Leon M, Liau G. Evidence for apoptosis in human atherosclerosis and in a rat vascular injury model. Am J Pathol. 1995;147:267-277.[Abstract]
5. Geng Y, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1ß converting enzyme. Am J Pathol. 1995;147:251-266.[Abstract]
6. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266-2274.
7.
Bennett MR, Evan GI, Newby AC. Deregulated
expression of the c-myc oncogene abolishes inhibition of
proliferation of rat vascular smooth muscle cells by serum reduction,
interferon-
, heparin, and cyclic nucleotide analogues
and induces apoptosis. Circ Res. 1994;74:525-536.
8.
Bennett MR, Evan GI, Schwartz SM.
Apoptosis of rat vascular smooth muscle cells is regulated by
p53-dependent and -independent pathways. Circ Res. 1995;77:266-273.
9.
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.
10.
O'Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran N,
Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM.
Proliferation in primary and restenotic coronary
atherectomy tissue: implications for antiproliferative therapy.
Circ Res. 1993;73:223-231.
11.
Shaulian E, Zauberman A, Ginsberg D, Oren M.
Identification of a minimal transforming domain of p53: negative
dominance through abrogation of sequence-specific DNA binding.
Mol Cell Biol. 1992;12:5581-5592.
12. Gottlieb E, Haffner R, von Rüden RT, Wagner EF, Oren M. Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal. EMBO J. 1994;13:1368-1374.[Medline] [Order article via Infotrieve]
13.
Bowman T, Symonds H, Gu L, Yin C, Oren M, Van Dyke
T. Tissue-specific inactivation of p53 tumor suppression in the
mouse. Genes Dev. 1996;10:826-835.
14. Morgenstern J, Land H. Choice and manipulation of retroviral vectors. In: Murray EJ, eds. Gene Transfer and Expression Protocols. Clifton, NJ: The Humana Press Inc; 1991:181-206.
15. Waters CM, Littlewood TD, Hancock DC, Moore JP, Evan GI. c-myc protein expression in untransformed fibroblasts. Oncogene. 1991;6:797-805.[Medline] [Order article via Infotrieve]
16.
Kern SE, Kinzler KW, Bruskin A, Jarosz D, Friedman P,
Prives C, Vogelstein B. Identification of p53 as a
sequence-specific DNA-binding protein. Science. 1991;252:1708-1711.
17. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989.
18.
Littlewood T, Hancock D, Danielian P, Parker M, Evan
G. A modified estrogen-receptor ligand-binding domain as an
improved switch for the regulation of heterologous proteins.
Nucleic Acids Res. 1995;23:1686-1690.
19.
Danielian PS, White R, Hoare SA, Fawell SE, Parker
MG. Identification of residues in the estrogen receptor that
confer differential sensitivity to estrogen and
4-hydroxytamoxifen. Mol Endocrinol. 1993;7:232-240.
20.
Fan S, el Deiry WS, Bae I, Freeman J, Jondle D, Bhatia
K, Fornace AJJ, Magrath I, Kohn KW, O'Connor PM. p53 gene
mutations are associated with decreased sensitivity of human lymphoma
cells to DNA damaging agents. Cancer Res. 1994;54:5824-5830.
21. Malcomson R, Oren M, Wyllie A, Harrison D. p53-independent death and p53-induced protection against apoptosis in fibroblasts treated with chemotherapeutic drugs. Br J Cancer.. 1995;72:952-957.[Medline] [Order article via Infotrieve]
22. Crook T, Tidy JA, Vousden KH. Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation. Cell. 1991;67:547-556.[Medline] [Order article via Infotrieve]
23. Lechner MS, Mack DH, Finicle AB, Crook T, Vousden KH, Laimins LA. Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription. EMBO J. 1992;11:3045-3052.[Medline] [Order article via Infotrieve]
24. 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;352:345-347.[Medline] [Order article via Infotrieve]
25.
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.
26. Chen YM, Chen PL, Arnaiz N, Goodrich D, Lee WH. Expression of wild-type p53 in human A673 cells suppresses tumorigenicity but not growth rate. Oncogene. 1991;6:1799-1805.[Medline] [Order article via Infotrieve]
27.
Baker SJ, Markowitz S, Fearon ER, Willson JK,
Vogelstein B. Suppression of human colorectal carcinoma cell
growth by wild-type p53. Science. 1990;249:912-915.
28. Casey G, Lo HM, Lopez ME, Vogelstein B, Stanbridge EJ. Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene. Oncogene. 1991;6:1791-1797.[Medline] [Order article via Infotrieve]
29. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CJ, Butel JS, Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992;356:215-221.[Medline] [Order article via Infotrieve]
30. Iacopetta B, Wysocki S, Norman P, House A. The p53 tumour suppressor gene is overexpressed but not mutated in human atherosclerotic tissue. Int J Oncol. 1995;7:399-402.
31.
Slichenmeyer WJ, Nelson WG, Slebos RJ, Kastan
MB. Loss of a p53-associated G1 checkpoint does not decrease
cell survival following DNA damage. Cancer Res. 1993;53:4164-4168.
32. Strasser A, Harris AW, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell. 1994;79:329-339.[Medline] [Order article via Infotrieve]
33.
Gartenhaus R, Wang P, Hoffmann P. Induction of
the waf1/cip1 protein and apoptosis in human T-cell
leukemia-virus type I-transformed lymphocytes after treatment with
adriamycin by using a p53-independent pathway.
Proc Natl Acad Sci U S A. 1996;93:265-268.
34.
Levy N, Yonish RE, Oren M, Kimchi A.
Complementation by wild-type p53 of interleukin-6 effects on M1 cells:
induction of cell cycle exit and cooperativity with c-myc
suppression. Mol Cell Biol. 1993;13:7942-7952.
35.
Guillouf C, Grana X, Selvakumaran M, Deluca A, Giordano
A, Hoffman B, Liebermann D. Dissection of the genetic programs
of p53-mediated G1 growth arrest and apoptosis: blocking
p53-induced apoptosis unmasks G1 arrest.
Blood. 1995;85:2691-2698.
36.
Haupt Y, Rowan S, Shaulian E, Vousden K, Oren M.
Induction of apoptosis in HELA cells by
transactivation-deficient p53. Genes Dev. 1995;9:2170-2183.
37. Rowan S, Ludwig R, Haupt Y, Bates S, Lu X, Oren M, Vousden K. Specific loss of apoptotic but not cell cycle arrest function in a human tumor-derived p53 mutant. EMBO J. 1996;15:827-838.[Medline] [Order article via Infotrieve]
38. Zhan Q, Fan S, Bae I, Guillouf C, Liebermann DA, O'Connor PM, Fornace AJJ. Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis. Oncogene. 1994;9:3743-3751.[Medline] [Order article via Infotrieve]
39. Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, Hoffman B, Reed JC. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994;9:1799-1805.[Medline] [Order article via Infotrieve]
40. Miyashita T, Reed J. Tumor suppressor gene p53 is a direct transcriptional activator of the human Bax gene. Cell. 1995;80:293-299.[Medline] [Order article via Infotrieve]
41.
Canman C, Gilmer T, Coutts S, Kastan M.
Growth-factor modulation of p53-mediated growth arrest versus
apoptosis. Genes Dev. 1995;9:600-611.
42.
Owen-Schaub L, Zhang W, Cusack J, Angelo L, Santee S,
Fujiwara T, Roth J, Deisseroth A, Zhang W, Kruzel E, Radinsky R.
Wild-type human p53 and a temperature sensitive mutant induce Fas/APO-1
expression. Mol Cell Biol. 1995;15:3032-3040.
43. Selvakumaran M, Lin HK, Miyashita T, Wang HG, Krajewski S, Reed JC, Hoffman B, Liebermann D. Immediate early up-regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways. Oncogene. 1994;9:1791-1798.[Medline] [Order article via Infotrieve]
44. Donatella Attardi L, Lowe S, Brugarolas J, Jacks T. Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene-mediated apoptosis. EMBO J. 1996;15:3693-3701.[Medline] [Order article via Infotrieve]
45.
Wagner AJ, Kokontis JM, Hay N. Myc-mediated
apoptosis requires wild-type p53 in a manner independent of
cell cycle arrest and the ability of p53 to induce
p21waf1/cip1. Genes Dev. 1994;8:2817-2830.
46. Lotem J, Sachs L. Interferon-gamma inhibits apoptosis induced by wild-type p53, cytotoxic anticancer agents and viability factor deprivation in myeloid cells. Leukemia. 1995;9:685-692.[Medline] [Order article via Infotrieve]
47.
Knudson C, Tung K, Tourtellotte W, Brown G, Korsmeyer
S. Bax-deficient mice with lymphoid hyperplasia and male
germ-cell death. Science. 1995;270:96-99.
48. Harrington EA, Bennett MR, Fanidi A, Evan GI. c-Myc induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J. 1994;13:3286-3295.[Medline] [Order article via Infotrieve]
49. Buckbinder L, Talbott R, Velascomiguel S, Takenaka I, Faha B, Seizinger B, Kley N. Induction of the growth inhibitor IGF-binding protein-3 by p53. Nature. 1995;377:646-649.[Medline] [Order article via Infotrieve]
50.
Werner H, Karnieli E, Rauscher F, Leroith D.
Wild-type and mutant p53 differentially regulate transcription of the
insulin-like growth factor-I receptor gene. Proc Natl
Acad Sci U S A. 1996;93:8318-8323.
51. Caelles C, Helmberg A, Karin M. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature. 1994;370:220-223.[Medline] [Order article via Infotrieve]
52.
Maheswaran S, Englert C, Bennett P, Heinrich G, Haber
D. The wt1 gene-product stabilizes p53 and inhibits
p53-mediated apoptosis. Genes Dev. 1995;9:2143-2156.
53.
Polyak K, Waldman T, He T, Kinzler K, Vogelstein
B. Genetic determinants of p53-induced apoptosis and
growth arrest. Genes Dev. 1996;10:1945-1952.
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|
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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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B. J.M. van Vlijmen, G. Gerritsen, A. L. Franken, L. S.M. Boesten, M. M. Kockx, M. J. Gijbels, M. P. Vierboom, M. van Eck, B. van de Water, T. J.C. van Berkel, et al. Macrophage p53 Deficiency Leads to Enhanced Atherosclerosis in APOE*3-Leiden Transgenic Mice Circ. Res., April 27, 2001; 88(8): 780 - 786. [Abstract] [Full Text] [PDF]
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V. A. Patel, Q.-J. Zhang, K. Siddle, M. A. Soos, M. Goddard, P. L. Weissberg, and M. R. Bennett Defect in Insulin-Like Growth Factor-1 Survival Mechanism in Atherosclerotic Plaque-Derived Vascular Smooth Muscle Cells Is Mediated by Reduced Surface Binding and Signaling Circ. Res., May 11, 2001; 88(9): 895 - 902. [Abstract] [Full Text] [PDF]
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J. H. von der Thusen, B. J.M. van Vlijmen, R. C. Hoeben, M. M. Kockx, L.M. Havekes, T. J.C. van Berkel, and E. A.L. Biessen Induction of Atherosclerotic Plaque Rupture in Apolipoprotein E-/- Mice After Adenovirus-Mediated Transfer of p53 Circulation, April 30, 2002; 105(17): 2064 - 2070. [Abstract] [Full Text] [PDF]
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