(Circulation. 2001;103:407.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Growth Suppression of Human Coronary Vascular Smooth Muscle Cells by Gene Transfer of the Transcription Factor E2F-1
From the Wafic Said Molecular Cardiology and Gene Therapy Research Laboratory, Texas Heart Institute (H.S.S., M.K.B., P.M.D., J.T.W., P.Z.); the Departments of Medicine (H.S.S., P.M.D., J.T.W., P.Z.), Pathology (D.L.H.-B.), and Integrative Biology (T.V.), University of Texas–Houston Medical School; and the Department of Neuro-Oncology, M.D. Anderson Cancer Center (T.-J.L.), Houston, Tex.
Correspondence to Pierre Zoldhelyi, Texas Heart Institute MC 2-255, 1101 Bates St, Houston, TX 77030. E-mail zoldhelyi{at}aol.com
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Abstract |
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Background—The transcription factor E2F-1 promotes S-phase entry and death in transformed cells and primary cardiomyocytes. We tested the hypothesis that overexpression of E2F-1 forces growth-arrested human coronary vascular smooth muscle cells (VSMCs) to enter the S phase, undergo apoptosis, and thereby regulate VSMC growth.
Methods and
Results—Early-passage (
5 passages) coronary
VSMCs were transduced at an MOI of 100 with a recombinant adenovirus
encoding human E2F-1. E2F-1 expression was observed by
immunohistochemistry as early as 6 to 8 hours after exposure of the
VSMCs to Ad.E2F-1 but not to the control vector Ad.RR. When cells were
kept in growth-arrest medium, 40% of Ad.E2F-1–treated VSMCs entered
the S phase by 96 hours, whereas the percentage remained <5% in
Ad.RR-treated cells. Transition to the S phase in the E2F-1–transduced
VSMCs was followed by apoptosis, as reflected by chromatin
condensation, membrane blebbing, cell detachment, and loss of
mitochondrial membrane integrity. E2F-1 overexpression resulted in
positive dUTP nick end-labeling mediated by terminal deoxynucleotidyl
transferase, associated with a robust increase in caspase 3–like
activity. Four days after infection with Ad.E2F-1, the fraction of
hypodiploid VSMCs in subG1 increased to 75%. At
7 days, gene transfer of E2F-1 had completely suppressed the growth of
VSMCs, whereas the number of Ad.RR-infected cells had increased >8
times.
Conclusions—Overexpression of the transcription factor E2F-1 regulates growth of human coronary VSMCs by forcing the cells to enter the S phase and then to die. Cell death appears to involve caspase 3–like activity, which, in the VSMCs, is markedly increased by overexpression of E2F-1.
Key Words: cells • genes • muscle, smooth • apoptosis • restenosis
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Introduction |
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Vascular injury during percutaneous revascularization interventions stimulates proliferation and migration of vascular smooth muscle cells (VSMCs), which accumulate in the intima of the injured vessel site.1 2 Gene transfer strategies that have attempted to reduce postinjury intimal hyperplasia have focused on the transfer of gene products that block cell cycle initiation or progression or induce apoptosis in VSMCs.3 4 5
E2F transcription factors function as initiators of cell growth in multicellular organisms and are tightly regulated by binding to members of the Rb family of pocket proteins. On phosphorylation of Rb by cyclin-dependent kinases, members of the E2F family are released and (with the exception of the transcriptional repressor E2F-6) transactivate gene expression of proteins that promote transition to the S phase and DNA replication, followed, in general, by completion of the cell cycle with mitosis of the cell.6 7
Early studies have concentrated on the growth-promoting properties of the E2F family of transcription factors and their putative role as oncogenes.8 9 In the vascular system, the introduction into balloon-injured rat carotid arteries of double-stranded DNA containing the consensus sequence that binds E2F with high affinity functioned as an E2F decoy and inhibited in vivo VSMC proliferation and formation of postinjury neointima.10 These studies, carried out before full recognition of the diversity of the E2F family, have promoted the notion that E2F transcription factors serve as universal initiators of cell proliferation and growth. More recently, however, mice with an inactivated E2F-1 gene were generated and, unexpectedly, exhibited features of hyperproliferation, suggesting that E2F-1 may function in vivo as a growth regulator and tumor suppressor gene.11 12 Further studies demonstrated that overexpression of E2F-1 in transformed rat fibroblasts,13 neonatal and postmitotic adult rat ventricular myocytes,14 and tumor cells15 16 promoted S-phase entry, followed by apoptosis of the cells.
Because E2F-1 may have divergent growth-regulatory functions, depending on the tissue type, developmental stage, and coexistence of dormant oncogenes,6 17 18 we studied the effect of overexpressed E2F-1 on early-passage human coronary VSMCs. We found that in human coronary VSMCs, E2F-1 induces caspase 3–like activity, initiates apoptosis, and markedly suppresses proliferation of these cells. Overexpressed E2F-1 in VSMCs appears to regulate growth by forcing vascular smooth muscle to enter the S phase and then to die.
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Methods |
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Preparation of Recombinant Adenoviral Vectors
Construction of the recombinant adenovirus encoding human E2F-1 has been described.16 In brief, the full-length human E2F-1 cDNA, cloned from ML-1 cells, was inserted into the shuttle plasmid pXCJL-1 containing the human cytomegalovirus promoter and the bovine growth hormone polyadenylation signal. Recombinants were generated by cotransfection into 293 cells of pXCJL-1-E2F-1 and pJM17. Replication-defective adenovirus containing a human cytomegalovirus promoter expression cassette but no foreign gene (Ad.RR) was used as a control vector.
Viral stocks of Ad.E2F-1 and Ad.RR were propagated in 293 cells and purified as previously described.19 The concentration of infectious viral particles was determined in 293 cells by plaque assay. Viral preparations were found to be endotoxin-free (<0.125 EU/mL) when tested with a limulus amoebocyte lysate assay (BioWhittaker, Inc). When the lysate of VSMCs infected with Ad.E2F-1 at a multiplicity of infection (MOI, viral plaques/cell) of 100 to 500 was applied undiluted to 293 cells, no cytopathic effect or viral plaques were observed after 10 days.
Cell Culture
Passage-2 human coronary VSMCs were purchased from
Cascade Biologics, Inc, and were not used after passage 5. For cell
growth experiments, VSMCs were seeded in triplicate at a density of
10x103/cm2
and were growth-arrested for 60 hours in DMEM with added 0.1% BSA.
Recombinant adenovirus was suspended in growth-arrest medium, and the
cells were incubated for 6 hours with either Ad.E2F-1, Ad.RR, or
growth-arrest medium alone (mock). After removal of the viral
suspension, the cells were washed with DMEM and cultured in DMEM or
Medium 231 (Cascade Biologics) supplemented with 10% FBS or the growth
supplement SMGS (Cascade Biologics), respectively. At intervals, the
cells were trypsinized and counted in a Coulter counter (Model Z1,
Coulter Inc).
E2F-1 Immunohistochemistry
VSMCs were plated into chamber slides (Nalgen-Nunc
Intl) and infected for 6 hours with Ad.E2F-1, Ad.RR, or mock, followed
by removal of the virus suspension. The cells were washed with DMEM and
fed fresh growth medium. At intervals beginning at the completion of
the 6-hour infection, the cells were washed, fixed for 10 minutes at
-20°C in methanol/acetone 3:1, and immunostained for E2F-1 with a
mouse monoclonal antibody recognizing human E2F-1 (Santa Cruz
Biotechnology). Antibody binding was visualized with DAB, with a
biotinylated secondary antibody and a streptavidin-biotin–horseradish
peroxidase kit (Vector). PBS with 0.1% Triton X-100 was used to
suspend the antibodies and for all washing steps. Cells were
counterstained with Alcian blue/methyl green in PBS, dehydrated in
ethanol, and coverslipped with Cytoseal 60 mounting medium (Stephens
Scientific).
Cell Cycle Analyses by DNA Flow
Cytometry
The coronary VSMCs were plated at
3.5x105 cells/60-mm dish and
growth-arrested for 60 hours, followed by 6-hour treatment with
Ad.E2F-1, Ad.RR, or mock control. Cells were either stimulated with
10% FBS or kept in growth-arrest medium (see Results) and were
harvested daily for cell cycle analysis by DNA flow cytometry, pooling
detached cells and cells removed with trypsin.
Samples were centrifuged for 10 minutes at 1800 rpm and resuspended in 0.2 mL of PBS, followed by dropwise addition of 5 mL of ice-cold 85% ethanol with gentle vortexing. Fixed cells were stored at -20°C. On the day of analysis, samples were centrifuged at 2500 rpm for 10 minutes and washed with PBS before resuspension in 400 µL of 100 µg/mL propidium iodide and 50 µg/mL RNAse and incubation for 20 minutes at 37°C. At least 4x103 cells were analyzed on a Coulter EPICS Profile instrument (Coulter Inc). Histograms were analyzed with the Multicycle program from Phoenix Flow Systems.
Microscopic Evaluation of Cell Death
Cell morphology was evaluated by combined
epifluorescence and differential interference contrast (DIC)
microscopy. Briefly, the VSMCs were kept in serum-free medium for 60
hours, treated with Ad.E2F-1, Ad.RR, or growth-arrest medium alone, and
stimulated by addition of DMEM with 10% FBS. Thirty hours after
infection, the cells were incubated in the dark at 37°C with 10
µg/mL of Hoechst 33342 and
4-(4-(dimethylamino)styryl)-N-methylpyridinium
iodide (DASPMI) for staining of nuclear DNA and mitochondrial
membranes, respectively. DNA staining and mitochondrial staining were
detected with DAPI and FITC filter sets, respectively. All micrographs
were digitally captured on a Zeiss Axioskop epifluorescence microscope
with an Optronics DEI-750 CCD color camera with Adobe Premiere software
(Adobe Systems), a TARGA 2000 video board (Truevision, Inc), and a
PowerPC Macintosh 9500 (Apple Computer). Images were edited with Adobe
Photoshop software (Adobe Systems).
Terminal Deoxynucleotidyl Transferase–Mediated
dUTP Nick End-Labeling
Terminal deoxynucleotidyl transferase (TdT)–mediated
dUTP nick end-labeling (TUNEL) staining was carried out according to a
recently modified
procedure,20 in which TUNEL
is performed immediately after the cells are labeled with trypan blue.
The human VSMCs were infected at an MOI of 100 with Ad.E2F-1 or the
Ad.RR-control and kept in either serum-supplemented or serum-free
culture medium for an additional 44 hours. The VSMCs were then
harvested and incubated for 2 minutes in trypan blue. After 2 washing
steps in PBS, the cells were subjected to cytospin, and TUNEL staining
was performed with a kit from Intergen. New fuchsin chromagen (Dako), a
red horseradish peroxidase substrate, was used to visualize nick
end-labeled DNA.
Caspase Fluorogenic Activity Assay
Human coronary VSMCs were plated at
1.2x106 cells/60-mm plate. After growth
arrest as described above, the cells were treated for 6 hours with
Ad.E2F-1, Ad.RR, or mock. After removal of the virus, the cells were
cultured in serum-supplemented growth medium. At intervals, both
detached and adherent cells were harvested in 200 µL ICE lysis buffer
(12.5 mmol/L Tris [pH 7.0], 125 mmol/L EDTA, 5% glycerol),
snap-frozen on dry ice, and stored at -80°C until analysis of all
samples. On the day of analysis, samples were freeze-thawed 3 times,
vortexed briefly, and centrifuged at 13 000 rpm for 10 minutes. The
cleared supernatant was transferred to a new microcentrifuge tube for
measurement of total protein and caspase 3–like activities. For
measurement of caspase 3–like activity, 50 µL of the lysate from
each sample was transferred in triplicate to a 96-well plate. Caspase
3–like activity was measured by monitoring the cleavage of the
specific fluorescent substrate, Ac-DEVD-AMC (Calbiochem-Novabiochem),
over a period of 3 hours in a fluorescent plate reader (PEBiosystems),
as previously described.21
The rate of fluorescence change, an average of 3 replicate
measurements, was normalized to the protein concentration, as
determined by BCA protein reagent (Pierce Chemical Corp). In some
experiments, the cell lysates were incubated with the caspase 3
inhibitor Z-DEVD-FMK (Calbiochem-Novabiochem) before assessment of
caspase
activity.22
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Results |
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Transduction of Human Coronary VSMCs by Ad E2F-1
Growth-arrested human coronary VSMCs were incubated at an MOI of 50 with Ad.E2F-1, Ad.RR, and mock control and after various time periods were processed for E2F-1 immunohistochemistry. Predominantly nuclear E2F-1 expression was detected as early as 6 to 8 hours after infection in the transduced VSMCs, whereas no staining was seen by 20 hours in cells treated with the Ad.RR control or mock (Figure 1
). A faint signal of endogenous E2F-1 was detected
by immunoblotting of the lysate from Ad.RR- and mock-treated VSMCs,
when the immunoblots were exposed for several hours to a
chemoluminescent enhancing solution (Amersham Pharmacia Biotech),
greatly exceeding the recommended exposure time (data not
shown).
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Overexpression of E2F-1 Forces Serum-Deprived
Coronary VSMCs to Enter the S Phase
After demonstrating relatively early overexpression of
E2F-1 in the transduced VSMCs, we examined whether E2F-1 gene transfer
promotes S-phase entry of VSMCs rendered quiescent by a 60-hour growth
arrest. Serum-deprived coronary VSMCs were infected with Ad.E2F-1,
Ad.RR, or mock control and kept in serum-free medium for an additional
4 days. DNA flow cytometry on the cells harvested daily demonstrated
that despite prolonged serum starvation, transduction with E2F-1
promoted the transition from G1 to S in the
VSMCs that had been made quiescent
(Figure 2). In contrast, the percentage of VSMCs in the S
phase remained consistently <5% in the control-treated
cells.
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S-Phase Entry of E2F-1–Transduced Coronary
VSMCs Is Associated With Induction of Apoptosis
Within 24 to 36 hours after gene transfer of E2F-1 to
VSMCs, we observed the development of apoptotic features, including
membrane blebbing, cell shrinkage, and cleavage and condensation of
chromatin
(Figures 3 and 4). Chromatin condensation was apparent as
intensified epifluorescence of nuclear fragments after staining with
the intercalating DNA dye Hoechst 33342
(Figure 4B). In addition, E2F-1 overexpression resulted in
mitochondrial membrane disarray, as visualized with DASPMI, a
mitochondrial membrane–specific dye
(Figure 4C). Ad.E2F-1–infected VSMCs studied 44 hours after
infection stained positive for TUNEL, irrespective of the presence of
serum before or after transduction of the cells
(Figure 5).
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In an individual cell, the development of apoptotic changes
induced by overexpression of E2F-1 appeared to proceed in a relatively
short time.
Figure 6 shows the changes observed with video time-lapse
microscopy in a single VSMC from a sample of cells infected with
Ad.E2F-1. Surface blebbing and loss of membrane integrity with
extrusion of cellular contents were nearly complete within 2 hours
after the first changes were observed at 30 hours after infection
(Figure 6: frame 1, 30 hours; frame 9, 32 hours).
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To further evaluate the time course and magnitude of VSMC
apoptosis induced by E2F-1, the cells were infected with Ad.E2F-1,
followed by growth stimulation in 10% FBS. Using DNA flow cytometry on
samples harvested at 24-hour intervals, we observed a dose-dependent
induction of apoptosis by Ad.E2F-1, reflected by an increase in the
hypodiploid cell population (subG1 cells)
containing cleaved DNA and observed at an MOI of Ad.E2F-1 as low as 5
(Figure 7A). With MOIs of 100 and 200, the fraction of
apoptotic cells reached a plateau of 75% on day 4. With Ad.E2F-1 at an
MOI of 10, only 17% of cells were in subG1 on
day 3, whereas the subG1 fraction was <5% in
control cells
(Figure 7B).
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Activation of Caspase 3 by E2F-1
We examined whether E2F-1 induced caspase 3–like
activity in coronary VSMCs. With Ad.E2F-1 at an MOI of 100, caspase
3–like activity increased 2- and >6-fold compared with the Ad-RR at
24 and 30 hours after infection of the cells, respectively
(Figure 8). In contrast, with an MOI of 10, caspase 3–like
activity above baseline was detected only 30 hours after infection of
the VSMCs with Ad.E2F-1. The E2F-1–induced increase in caspase 3–like
activity was suppressed completely by addition to the VSMC lysate of
the caspase–3 inhibitor Z-DEVD-FMK
(Figure 8), indicating specificity of the caspase
measurement.22
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Growth Suppression of Coronary VSMCs After
Adenovirus-Mediated E2F-1 Gene Transfer
After we had observed that E2F-1–transduced VSMCs
undergo cell death at a rate commensurate with the amount of gene
vector used, we assessed whether Ad.E2F-1–induced cell death
translated into growth suppression of the VSMCs. Growth curves were
established for the early-passage human coronary VSMCs after a 6-hour
treatment with Ad.E2F-1, Ad.RR, or mock control. Daily cell counts
demonstrate that gene transfer of E2F-1 suppressed VSMC proliferation
after infection at an MOI as low as 10. Growth of the VSMCs was
abolished after a single application of Ad.E2F-1 at an MOI of 100
(Figure 9).
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Discussion |
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We report that overexpression of E2F-1 forces growth-arrested, early-passage human coronary VSMCs into the S phase, followed by apoptosis of the VSMCs. Overexpression of E2F-1 was detected as early as 6 to 8 hours after infection, and the growth of VSMCs infected with Ad.E2F-1 was markedly suppressed for up to 7 days. Another novel observation is that E2F-1 induces caspase 3–like activity in VSMCs. Both caspase activation and apoptosis correlated with the viral dose.
Induction of the S phase and apoptotic cell death after overexpression of exogenous E2F-1 has been observed previously in cardiac myocytes and tumor cells.14 16 23 24 In early-passage human coronary VSMCs, overexpressed E2F-1 led to cell detachment, cytoplasmic membrane blebbing, nuclear fragmentation, and loss of mitochondrial integrity, which, in combination with other markers, serve to distinguish apoptosis from inflammatory-necrotic modes of death.25 26 One of these is TUNEL.27 TUNEL, performed 44 hours after infection with Ad.E2F-1, was occasionally accompanied by VSMC uptake of trypan blue, indicating loss of cell membrane integrity. Thus, after a longer interval, E2F-1–induced apoptosis can progress to cell lysis, representative of the late stage of "aponecrosis," combining features of apoptotic and necrotic death.26 28 29 In contrast, treatment of VSMCs with the empty control virus Ad.RR at an MOI >500 caused predominantly necrosis of cultured VSMCs, as judged by the presence of marked trypan blue positivity, extensive cell lysis, and sparse TUNEL-positivity (Shelat et al, unpublished observations, 2000).
Neither conventional TUNEL staining nor the demonstration of internucleosomally cleaved DNA on gel electrophoresis is an absolute criterion for apoptosis, and, conversely, apoptosis may occur without the characteristic laddering pattern observed on electrophoretically separated DNA.29 30 To further substantiate the induction of apoptosis by E2F-1 and investigate whether overexpressed E2F-1 in coronary VSMCs activates the death cascade common to other apoptosis initiators, we examined caspase 3–like activity in VSMCs after transduction with E2F-1. Consistent with a recent report of activation-cleavage of the execution caspase, caspase 3 (CPP32), in E2F-1–transduced glioma cells,15 we found that overexpression of E2F-1 in VSMCs induced a dose-dependent increase in caspase 3–like activity. Thus, in coronary VSMCs, as in tumor cells, overexpressed E2F-1 appears to activate common downstream effectors of apoptotic death. Of note, E2F-1 expression by immunohistochemistry was observed as early as 6 to 8 hours after VSMC infection with Ad.E2F-1, whereas activation of caspases and cell death became apparent only after 24 to 32 hours. This time lag argues for the requirement for the arrested VSMCs to reenter G1 and transit to the S phase before engaging in the apoptosis signaling and execution cascade.
How overexpressed E2F-1 initiates the apoptotic cascade is unclear and may be influenced, in principle, by the cell type, the presence of cycling versus growth-arrested cells, and the status of endogenous p53 and other proapoptotic genes.31 Cell cycle stage and p53 status, however, do not appear to pose absolute restraints on the ability of overexpressed E2F-1 to induce cell death. Indeed, apoptosis was observed in transduced coronary VSMCs whether the cells were maintained in continuous serum deprivation or allowed to cycle before and after infection with Ad.E2F-1. In addition, induction of cell death by E2F-1 was previously observed in p53 -/- cardiac myocytes19 as well as in breast cancer, melanoma, and squamous cell carcinoma cells.16 23 24 Thus, our findings in VSMCs enhance the notion that E2F-1, if present in sufficient amounts, exerts proapoptotic effects in many transformed and nontransformed lineages. Of note, however, cell lines harboring the Harvey (H)-ras gene undergo tumorigenic transformation in the presence of overexpressed E2F-1,9 18 indicating that overexpressed E2F-1 may have profoundly different effects in the presence of dormant oncogenes. The tumorigenicity of E2F-1 in cells that do not harbor oncogenes has not been demonstrated conclusively.9
Whether endothelial cells constitute an exception to the
proapoptotic effect of overexpressed E2F-1 remains to be confirmed. In
a recent report, adenovirus-mediated E2F-1 overexpression was reported
to protect cultured bovine endothelial cells from tumor necrosis factor
(TNF)-
–mediated
apoptosis.32 In preliminary
studies, in contrast, we have observed that gene transfer of E2F-1
resulted in marked S-phase induction, apoptosis, and growth suppression
of human umbilical vein endothelial cells (Zoldhelyi et al, unpublished
observations, 1999). These observations suggest that the effects of
E2F-1 may be modulated by the presence of cytokines (such as TNF-)
and growth factors, including insulin-like growth factor-1, which was
reported to rescue cells from E2F-1–mediated
death.33
Our observation of marked growth suppression of VSMCs by overexpressed E2F-1 requires reconciliation with those showing a similar effect by inhibiting E2F.10 34 This apparent paradox may be resolved by considering the mechanism of growth control exerted by these approaches. By using an oligonucleotide decoy strategy of binding all E2F transcription factors to a consensus sequence, Morishita et al and Mann et al appear to have achieved a favorable shift in the balance between inhibition of E2F transcription factors that promote (E2F-2 to E2F-4) and those that limit (E2F-1 and E2F-6) proliferation of cells. The endogenous levels of individual transcription factors are critical determinants of the response to an inhibitory decoy strategy but may have little relevance when a potent growth suppressor gene is overexpressed. Consistent with this hypothesis is the observation that endogenous levels of E2F-1 in coronary VSMC cultures were low (see Results).
In conclusion, our observations indicate that gene transfer
of E2F-1 leads to S-phase entry of growth-arrested human coronary
VSMCs, followed by dose-dependent caspase activation and apoptotic
death. Given the critical role of VSMC proliferation, migration, and
synthesis of extracellular matrix and growth factors, E2F-1–induced
apoptosis may play a role in future strategies to prevent neointima
formation after vascular injury. Potentially undesirable effects of
E2F-1 gene transfer on cells other than VSMCs could be circumvented in
vivo by the use of vectors with smooth muscle–specific promoters, such
as SM22-.35 Use of
tissue-specific promoters and other strategies, including the use of
vectors with extensive deletions of viral genes, may provide an
opportunity to evaluate an E2F-1–based strategy of VSMC apoptosis for
the prevention of postinjury vascular stenosis and vein graft
disease.
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Acknowledgments |
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This work was supported in part by the Keith Meadows Foundation, Dallas, Tex; NIH grants 1RO1-HL-50179-01 and 1RO1-HL-54839-01; an American Heart Association Scientist Development grant; and a grant from the Texas Higher Education Coordinating Board. We thank Dr F.L. Graham, McMaster University, Hamilton, Ont, Canada, for the gift of pXCJL-1 and pJM17, and Dr Robert D. Gerard, University of Michigan, Ann Arbor, for the gift of Ad.RR.
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Footnotes |
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Guest Editor for this article was Elizabeth G. Nabel, MD, National Institutes of Health, NHLBI, Bethesda, Md.
Received July 21, 2000; revision received September 6, 2000; accepted September 11, 2000.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
O’Brien
ER, Alpers CE, Stewart DK, et al. Proliferation in primary and
restenotic coronary atherectomy tissue: implications for
antiproliferative therapy. Circ
Res. 1993;73:223–231.
[Abstract/Free Full Text]
-
Schwartz SM,
deBlois D, O’Brien ERM. The intima: soil for atherosclerosis and
restenosis. Circ Res. 1995;77:445–465.
[Free Full Text]
-
Chang MW, Barr E,
Seltzer J, et al. Cytostatic gene therapy for vascular proliferative
disorders with a constitutively active form of the retinoblastoma gene
product. Science. 1995;267:518–522.
[Abstract/Free Full Text]
-
Speir E, Epstein
SE. Inhibition of smooth muscle cell proliferation by an antisense
oligodeoxynucleotide targeting the messenger RNA encoding proliferating
cell nuclear antigen.
Circulation. 1992;86:2:538–547.
-
Sata M, Perlman H,
Muruves DA, et al. Fas ligand gene transfer to the vessel wall inhibits
neointima formation and overrides the adenovirus-mediated T-cell
response. Proc Natl Acad Sci
U S A. 1998;95:1213–1217.
[Abstract/Free Full Text]
-
Helin K. Regulation
of cell proliferation by the E2F transcription factors.
Curr Opin Genet Dev. 1998;8:28–35.[Medline]
[Order article via Infotrieve]
-
Mittnacht S.
Control of pRb phosphorylation. Curr Opin
Genet Dev. 1998;8:21–27.[Medline]
[Order article via Infotrieve]
-
Nevins JR. E2F: a
link between the Rb tumor suppressor protein and viral oncoproteins.
Science. 1992;258:424–429.
[Abstract/Free Full Text]
-
Johnson DG, Cress
WD, Jakoi L, et al. Oncogenic capacity of the E2F1 gene.
Proc Natl Acad Sci
U S A. 1994;91:12823–12827.
[Abstract/Free Full Text]
-
Morishita
R, Gibbons GH, Horiuchi M, et al. A gene therapy strategy using a
transcription factor decoy of the E2F binding site inhibits smooth
muscle proliferation in vivo. Proc Natl
Acad Sci
U S A. 1995;92:5855–5859.
[Abstract/Free Full Text]
-
Yamasaki L, Jacks
T, Bronson R, et al. Tumor induction and tissue atrophy in mice lacking
E2F-1. Cell. 1996;85:537–548.[Medline]
[Order article via Infotrieve]
-
Field SJ, Tsai F,
Kuo F, et al. E2F-1 functions in mice to promote apoptosis and suppress
proliferation. Cell. 1996;85:549–561.[Medline]
[Order article via Infotrieve]
-
DeGregori J,
Leone G, Alexander M, et al. Distinct roles for E2F proteins in cell
growth control and apoptosis. Proc Natl
Acad Sci
U S A. 1997;94:7245–7250.
[Abstract/Free Full Text]
-
Agah R,
Kirshenbaum LA, Abdellatif M, et al. Adenoviral delivery of E2F-1
directs cell cycle reentry and p53-independent apoptosis in
post-mitotic adult myocardium in vivo.
J Clin Invest. 1998;100:2722–2728.[Medline]
[Order article via Infotrieve]
-
Fueyo J,
Gomez-Manzano C, Yung WK, et al. Overexpression of E2F-1 in glioma
triggers apoptosis and suppresses tumor growth in vitro and in vivo.
Nat Med. 1998;6:685–690.
-
Hunt KK, Deng J,
Liu T-J, et al. Adenovirus-mediated overexpression of the transcription
factor E2F-1 induces apoptosis in human breast and ovarian carcinoma
cell lines and does not require p53.
Cancer Res. 1997;57:4722–4726.
[Abstract/Free Full Text]
-
Pierce AM,
Gimenez-Conti IB, Schneider-Broussard R, et al. Increased E2F-1
activity induces skin tumors in mice heterozygous and nullizygous for
p53. Proc Natl Acad Sci
U S A. 1998;95:8858–8863.
[Abstract/Free Full Text]
-
Pierce AM, Fisher
SM, Conti CJ, et al. Deregulated expression of E2F1 induces hyperplasia
and cooperates with ras in skin tumor development.
Oncogene. 1998;16:1267–1276.[Medline]
[Order article via Infotrieve]
-
Zoldhelyi P,
McNatt JM, Shelat HS, et al. Thromboresistance of balloon-injured
porcine carotid arteries after local gene transfer of human tissue
factor pathway inhibitor.
Circulation. 2000;101:289–295.
[Abstract/Free Full Text]
-
Perry SW, Epstein
LG, Gelbard HA. Simultaneous in situ detection of apoptosis and
necrosis in monolayer cultures by TUNEL and trypan blue staining.
Biotechniques. 1997;22:1102–1106.[Medline]
[Order article via Infotrieve]
-
Higuchi M,
Aggarwal BB, Yeh ETH. Activation of CPP32-like protease in tumor
necrosis factor-induced apoptosis is dependent on mitochondrial
function. J Clin Invest. 1997;99:1751–1758.[Medline]
[Order article via Infotrieve]
-
Masuda Y, Nakaya
M, Nakajo S, et al. Geranylgeraniol potently induces caspase-3-like
activity during apoptosis in human leukemia U937 cells.
Biochem Biophys Res Commun. 1997;234:641–645.[Medline]
[Order article via Infotrieve]
-
Dong YB, Yang HL,
Jane M, et al. Adenovirus-mediated E2F-1 gene transfer efficiently
induces apoptosis in melanoma cells.
Cancer. 1999;86:2021–2033.[Medline]
[Order article via Infotrieve]
-
Liu TJ, Wang M,
Breau RL, et al. Apoptosis induction by E2F-1 via adenoviral-mediated
gene transfer results in growth suppression of head and neck squamous
cell carcinoma cell lines. Cancer Gene
Ther. 1999;6:163–171.[Medline]
[Order article via Infotrieve]
-
Searle J, Kerr
JF, Bishop CJ. Necrosis and apoptosis: distinct modes of cell death
with fundamentally different significance.
Pathol Annu. 1982;17:229–259.
-
Walker NI, Harmon
BV, Gobe GC, et al. Patterns of cell death.
Methods Achiev Exp Pathol. 1988;13:18–54.[Medline]
[Order article via Infotrieve]
-
Gavrieli Y,
Sherman Y, Ben-Sasson SA. Identification of programmed cell death in
situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol. 1992;119:493–501.
[Abstract/Free Full Text]
-
Willingham MC.
Cytochemical methods for the detection of apoptosis.
J Histochem Cytochem. 1999;47:1101–1109.
[Abstract/Free Full Text]
-
Buja LM, Entman
ML. Modes of myocardial cell injury and cell death in ischemic heart
disease. Circulation. 1998;98:1355–1357.
[Free Full Text]
-
van Lookeren
Campagne M, Lucassen PJ, Vermeulen JP, et al. NMDA and kainate induce
internucleosomal DNA cleavage associated with both apoptotic and
necrotic cell death in the neonatal rat brain.
Eur J Neurosci. 1995;7:1627–1640.[Medline]
[Order article via Infotrieve]
-
Bates S, Vousden
KH. Mechanisms of p53-mediated apoptosis.
Cell Mol Life Sci. 1999;55:28–37.[Medline]
[Order article via Infotrieve]
-
Spyridopoulos IN,
Principe KL, Krasinski S, et al. Restoration of E2F expression rescues
vascular endothelial cells from tumor necrosis factor-–induced
apoptosis. Circulation. 1998;98:2883–2890.
[Abstract/Free Full Text]
-
von Harsdorf R,
Hauck L, Mehrhof F, et al. E2F-1 overexpression in cardiomyocytes
induces downregulation of p21CIP1 and p27KIP1 and release of active
cyclin-dependent kinases in the presence of insulin-like growth factor
I. Circ Res. 1999;85:128–136.
[Abstract/Free Full Text]
-
Mann MJ, Gibbons
GH, Kernoff RS, et al. Genetic engineering of vein grafts resistant to
atherosclerosis. Proc Natl Acad Sci
U S A. 1995;92:4502–4506.
[Abstract/Free Full Text]
-
Kim S, Lin H,
Barr E, et al. Transcriptional targeting of replication-defective
adenovirus transgene expression to smooth muscle cells in vivo.
J Clin Invest. 1997;100:1006–1014.[Medline]
[Order article via Infotrieve]
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