Cardiovascular Research 59 (2003) 512–519
www.elsevier.com / locate / cardiores
Mechanism of E2F1-induced apoptosis in primary vascular smooth
muscle cells
3
¨
¨
Jens Stanelle 1 , Thorsten Stiewe 1,2 , Florian Rodicker,
Karin Kohler
, Carmen Theseling,
*
¨
Brigitte M. Putzer
Center for Cancer Research and Cancer Therapy, Institute of Molecular Biology, University of Essen Medical School, Hufelandstrasse 55,
45122 Essen, Germany
Received 15 October 2002; accepted 1 April 2003
Abstract
Objective: The transcription factor E2F1 serves as a major regulator of the cell-cycle by controlling G1-S phase transition. However,
apart from its proliferative function high levels of deregulated E2F1 are capable of inducing apoptosis depending on the cellular context.
In particular the tumor suppressor p53 and its homologue p73 are implicated in this proapoptotic function. Methods: Here, we
investigated the mechanistic basis for E2F1-mediated apoptosis in vascular smooth muscle cells (VSMCs) which have previously been
shown to be E2F1-responsive. Results: Interestingly, E2F1-expression in these cells induced clear signs of apoptosis in the absence of any
proliferative activity. Although cell-cycle regulated genes such as CCNE1 and CDC25 A were activated, BrdU-staining revealed no
S-phase entry. Instead, a rapid loss of cell viability by induction of apoptosis was observed. Using a transactivation-defective
E2F1-mutant, we show that apoptosis induction is independent of the transactivation function and therefore independent of ARF and p73.
However, this mutant retains its ability to stabilize and phosphorylate p53, suggesting that p53 is sufficient for the effect of E2F1.
Conclusion: VSMCs therefore represent a cellular system in which the transactivation-independent, proapoptotic activity of E2F1 is the
primary cellular function. Ectopic expression of E2F1 might therefore be a suitable therapy to prevent VSMC hyperproliferation.
 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Apoptosis; Gene expression; Gene therapy; Protein phosphorylation; Restenosis
1. Introduction
Over the past decade, a large number of studies revealed
the central role of the RB-pathway in the regulation of
Abbreviations: Ad, adenovirus; ARF, ADP-ribosylation factor; MEF,
mouse embryo fibroblast; OHT, hydroxytamoxifen; FACS, fluorescenceactivated cell sorting; RT-PCR, reverse transcription polymerase chain
reaction
*Corresponding author. Tel.: 149-201-723-3687; fax: 149-201-7235974.
¨
E-mail address: [email protected] (B.M. Putzer).
1
Both authors have contributed equally to the data presented.
2
¨
Present address: Rudolf-Virchow-Zentrum, University of Wurzburg,
¨
Versbacher Strasse 9, 97078 Wurzburg,
Germany.
3
Present address: Department of Internal Medicine, West German
Cancer Center, University of Essen Medical School, Hufelandstrasse 55,
45122 Essen, Germany.
G1 / S transition and the control of cell proliferation by
modulating the activity of the transcription factor E2F.
From these studies it has become clear that E2F determines
whether or not a cell will divide by controlling the
expression of S-phase genes that encode cell cycle regulatory functions and DNA replication activities [1,2]. Despite
the clear importance in allowing cell cycle progression,
several studies have shown that particularly E2F1 promotes apoptosis in several systems [3–7]. Induction of
apoptosis by either the loss of RB or the deregulation of
E2F activity occurs both in association with p53 and
independent of p53.
Ectopic expression of E2F1 has been shown to lead to
increased levels of p53 [8], as a result of E2F1-mediated
induction of the CDKN2 A transcript p14ARF that in turn
blocks MDM2-associated degradation of p53 [9]. MoreTime for primary review 24 days.
0008-6363 / 03 / $ – see front matter  2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016 / S0008-6363(03)00392-4
J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
over, E2F1 can signal p53 phosphorylation that is coincident with p53 accumulation and apoptosis in the
absence of ARF [10], similar to the observed stimulation
of the apoptotic function of p53 in response to DNA
damage by direct binding to E2F1 [11]. E2F1-induced
apoptosis occurs also independent of p53 in tissue culture
and transgenic mice [6,7,12,13], and RB has been shown
to protect p53-null cells from apoptosis in an E2F1-binding
dependent manner [14]. Mapping studies revealed that this
ability of E2F1 requires its DNA-binding domain but not
its transactivation function [6,7,15], suggesting that
proapoptotic E2F1 target genes are activated by removal of
E2F1 / RB repression rather than direct transactivation
[6,7,16]. However, we and others have recently identified
the p53-homologue p73 as a target of p53-independent
apoptosis [17,18]. E2F1 regulates p73 levels directly,
through recognition and transactivation of the TP73 promoter. Recently, the gene for apoptosis protease-activating
factor 1, Apaf-1, has been identified as another target by
which E2F1 can induce apoptosis directly and independently of p53 induction [19]. Moreover, it is known that
E2F1 expression can lead to the sensitization of cells to
apoptosis independently of p53 by a death receptor-dependent mechanism in response to tumor necrosis factor a
(TNFa) by downregulating TRAF2 protein levels and
inhibiting antiapoptotic signaling such as NF-kB [20].
In the vascular system, E2F decoy oligonucleotides
inhibit in vivo proliferation of human coronary vascular
smooth muscle cells (VSMCs) and formation of postinjury
neointima in balloon-injured rat carotid arteries [21].
Shelat et al. reported that overexpression of E2F1 in
VSMCs leads to S-phase entry [22], followed by caspase
3-activation and apoptotic cell death. In contrast, restoration of E2F expression rescues vascular endothelial cells
from TNF-a induced apoptosis [23]. In view of the central
role of E2F1 in the regulation of cell growth and death in
primary vascular cells, we analyzed the mechanism of
E2F1-induced apoptosis in VSMCs.
2. Methods
2.1. Cell culture and virus construction
Passage-2 of human coronary VSMCs were purchased
from Cascade Biologics, and were not used after passage
5. Cells were maintained in Dulbecco’s modified Eagle
medium (DMEM) or Medium 231 (Cascade Biologics)
supplemented with 10% fetal bovine serum (FBS) or
growth supplement SMGS (Cascade Biologics), respectively. All adenoviruses were grown in 293 cells (Ad5
E1-transformed human embryonic kidney cells) maintained
in modified Eagle’s medium (MEM) F-11 with 10% fetal
bovine serum. Media were supplemented with 2 mM Lglutamine, 100 mg / ml penicillin, and 100 U / ml streptomycin. Adenoviruses encoding ER-E2F1, green fluores-
513
cent protein (GFP), and p53 have been described previously [24,25]. For generation of AdER-E(-TA), the cDNA
fragment encoding the influenca hemagglutinin (HA) / estrogen receptor ligand binding domain (ER) chimaeric
protein fused to the N-terminus (amino acid 1 to 374) of
the E2F1 cDNA which lacks the transactivation domain
between was cloned into pMH4. Recombinant adenovirus
was produced as described [24].
2.2. Western blotting
For Western blot analysis, cells were infected at a
multiplicity of infection (MOI) of 100 plaque forming
units (PFU) per cell. At 72 h after infection, cell lysates
were prepared and protein levels were analyzed essentially
as described [17]. Samples were probed with mouse antihuman E2F1 monoclonal antibody (KH95, Santa Cruz
Biotechnology), murine anti-p53 monoclonal antibody
(DO-1, Calbiochem) or phospho-p53 (Ser15) antibody
(Cell Signaling Technology). Full-length caspase-3 (35
kDa) and its large cleavage product (17 kDa) was detected
using antibodies directed against Caspase-3 (9662) and
Cleaved Caspase-3 (9661; Cell Signaling Technology).
Antibody binding sites were visualized using appropriate
horseradish-peroxidase conjugated secondary antibodies
according to the enhanced chemiluminescence (ECL)
protocol (Amersham).
2.3. Cell viability assay
For MTT cell viability assays, serum-starved cells were
infected and cell viability was determined in the presence
and absence of OHT at a final concentration of 1 mM over
3 days after infection. Triplicate wells of each treatment
were assayed for cell viability by the CellTiter96  AQ ueous
One Solution Cell Proliferation Assay (Promega).
2.4. Apoptosis assay
To visualize apoptosis in unfixed monolayer cultures, 72
h after infection the cells were incubated at 37 8C in the
presence of 1 mg / ml Hoechst 33342. 15 min later propidium iodide (PI) solution was included to 5 mg / ml and
monolayers were observed by epifluorescence microscopy
[26]. Cells dissociating from the monolayer exhibited
bright blue, fluorescing masses of chromatin that abutted at
the nuclear membrane or, at later stages showed bright
blue, fluorescing spherical bodies and were therefore
identified as apoptotic.
2.5. DNA fragmentation assay
For low-molecular-weight DNA extraction, VSMCs
were infected with AdER-E2F1 or AdER-E(-TA) in the
presence of OHT for 72 and 96 h. Cells were lysed in 50
mM Tris, pH 7.8, 10 mM EDTA, 1% sodium dodecyl
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J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
sulfate (SDS), and 0.5 mg / ml proteinase K. After overnight incubation at 37 8C, lysates were digested with 5
mg / ml RNase A for 3 h at 37 8C, extracted twice with
phenol–chloroform–isoamyl alcohol and DNA was precipitated overnight. Samples were analyzed by electrophoresis in 1.5% agarose gels in TBE buffer (90 mM Tris,
90 mM Boric acid, 2 mM EDTA (pH 8.0)). Analysis was
performed with DNAs extracted from equal numbers of
cells.
2.6. RT-PCR analysis
Semiquantitative RT-PCR was performed on total RNA
from serum-starved cells infected with Ad vectors and
prepared by RNeasy Mini Kit (Qiagen, Hilden, Germany).
PCR amplification was performed as described [17]. A
minimum amount of cycles was carried out to stay within
the linear amplification process. Primer sequences are
available upon request.
2.7. Flow cytometry
To quantitate apoptosis, serum-starved cells were infected at 60–80% confluence and further incubated in the
absence or presence of 1 mM OHT. Cells were harvested
72 h after infection, fixed in 70% ethanol and stained for
DNA content with propidium iodide. For analysis of Sphase entry, VSMCs were labeled with 5-bromo-29-desoxyuridine (BrdU) 24 h after infection using the In Situ Cell
Proliferation Kit FLUOS (Roche). BrdU incorporation was
detected using fluorescein isothiocyanate (FITC)-linked
anti-BrdU according to the manufacturer’s protocol. Flow
cytometric measurements were performed in a FACSVantage sorter (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson).
ence of OHT and measured the expression of three known
direct E2F target genes by semiquantitative RT-PCR.
Activation of E2F1 led to increased mRNA levels of
CCNE1 (encoding cyclin E) [30], CDC25 A encoding
CDC25A phosphatase, shown to be required for efficient
E2F1-induced S-phase [31], and TP73 which encodes the
proapoptotic p53-homologue p73 [17] at 8 h after induction (Fig. 1). In contrast, the transactivation-defective
mutant E(-TA) did not induce target gene expression. Also
no effect was detectable in virus-infected VSMCs without
OHT and in the GFP control. These data demonstrate that
E2F1 is competent as a transcriptional activator in VSMCs,
whereas mutant E2F1 is not.
3.2. Block of S-phase entry
Next, we examined whether E2F1 gene transfer promotes S-phase entry in growth arrested VSMCs. Following
2 days of serum starvation, VSMCs were infected with
adenovirus expressing either E2F1 or E(-TA) and labeled
with BrdU at 24 h after infection. BrdU incorporation was
determined by immunofluorescence. In response to serum,
cells showed a rapid entry into S-phase (Fig. 2, left panel).
Interestingly, however, very little S-phase entry was
observed in VSMCs infected with AdER-E2F1 or AdERE(-TA) in the presence of OHT where the percentage of
cells in the S-phase remained consistently,5% (Fig. 2,
middle and right panel). Thus, E2F1 does not lead to
S-phase entry in VSMCs.
3. Results
3.1. E2 F1 -mediated transactivation of target genes
There is evidence that ectopic expression of the E2F
transcription factor can induce both S-phase progression
and apoptosis, depending on the cell type context [6–
8,27,28]. We have previously developed an inducible Ad
vector system, in which regulation of potentially cytotoxic
gene products such as E2F1 is achieved by fusion of the
transgene to the OHT regulatable ER domain [24]. By
using this vector in the presence of OHT, we have shown
that E2F1 can be efficiently activated in quiescent fibroblasts and several human tumor cell lines, resulting in
the induction of genes involved in cell cycle progression
and apoptosis [24,29]. To analyze the transactivation
function of E2F1 in primary VSMCs, we infected serumstarved cells with AdER-E2F1 or a transactivation-defective E2F1-mutant, AdER-E(-TA) in the absence or pres-
Fig. 1. Transactivation of E2F1 target genes. Semiquantitative RT-PCR
analysis on total RNA from VSMCs infected with AdER-E2F1, AdER-E(TA), and AdGFP in the absence or presence of OHT for CCNE1,
CDC25 A, TP73, and GAPDH expression was carried out under linear
amplification conditions. GAPDH expression is shown as a control. One
representative experiment of three is shown.
J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
515
Fig. 2. E2F1 does not lead to S-phase entry in VSMCs. Growth-arrested cells were infected with Ad vector expressing E2F1 or E(-TA) in the presence of
OHT. At 24 hours post infection cells were labeled with BrdU and DNA synthesis was determined by flow cytometry. Compared to serum-induced cells
showing S-phase entry (left panel), the percentage of cells in the S-phase after AdER-E2F1 (middle panel) or AdER-E(-TA) (right panel) infection
remained consistently,5%. One representative experiment of three is shown.
3.3. Apoptosis induction is independent of the
transactivation function
To directly assess the cytotoxic effect of E2F1 in
VSMCs, growth-arrested cells were incubated with AdERE2F1, AdER-E(-TA), and AdGFP control virus in the
presence of OHT over 3 days. Cytotoxicity was analyzed
by quantitating viable cells using the MTT assay. As
shown in Fig. 3A, overexpression of both E2F1 and the
mutant E2F1 protein [E(-TA)] resulted in a substantial loss
of cell viability by more than 90% at day 3, compared to
control vector (mock) infected VSMCs (|40%). Within 36
to 72 h after Ad vector-mediated gene transfer of E2F1 and
E(-TA) to VSMCs, morphological changes characteristic
for cells undergoing apoptosis were observed with cells
rounding up, membrane blebbing, cell shrinkage, and
condensation of chromatin (Fig. 3B). In support, flow
cytometry analysis of serum-starved VSMCs infected
either with AdER-E2F1 or AdER-E(-TA) in the presence
of OHT revealed a significant increase of sub-G1 cells on
day 3 (Fig. 4AIII, V) of 43.5% (E2F1) and 49.9% [E(TA)], respectively, indicative of apoptosis. In contrast, in
the absence of tamoxifen or in mock infected cells, no
significant increase in the sub-G1 population was observed
(Fig. 4A, I, II and IV). Induction of apoptosis in VSMCs
by E2F1 and E(-TA) was also accompanied by processing
of caspase-3, as revealed by the appearance of a |17 kDa
product which corresponds to the 17 kDa subunit of
activated caspase-3 (Fig. 4B). As shown in Fig. 4C, a
typical DNA laddering pattern consistent with apoptosis
was evident at 72 and 96 h after infection with AdERE2F1 (lanes 1 and 3) as well as AdER-E(-TA) (lanes 2 and
4). These data indicate that E2F1 clearly induces apoptosis
in VSMCs which is independent of the transactivation
function.
3.4. p53 accumulation and phosphorylation by E2 F1
We and others have previously shown that part of the
p53-independent apoptotic activity of E2F1 reflects its
ability to induce p73 expression [17,18] and that the
mechanism of induction depends on the transactivation
domain ([18], unpublished data). As shown in Fig. 1, the
E2F mutant which lacks the transactivation domain is
unable to stimulate p73 expression in VSMCs. Because the
E2F mutant is capable of inducing apoptosis at levels
similar to the wild-type transcription factor, it appears that
apoptosis induction by E2F1 is independent of p73. We
therefore examined the contribution of p53 to E2F1-induced apoptosis in these cells. VSMCs were infected with
AdER vector expressing E2F1 or E(-TA), respectively, in
the absence or presence of OHT, and cell extracts were
subsequently analyzed for p53 protein levels. Consistent
with the apoptotic rate measured by flow cytometry, both
E2F1 and the transactivation-defective mutant induced
equally high levels of p53 compared to the controls (Fig.
5), indicating that the E(-TA) mutant retains its ability to
stabilize p53. Based on previous findings, there are different pathways for E2F1 to signal p53-mediated apoptosis
and p53 accumulation. Since E2F1-mediated activation of
p53 via the ARF /Mdm2 pathway requires the transactivation domain of E2F1, it is likely that p53 stimulation
occurs through a ARF-independent mechanism. Therefore,
we tested the ability of E2F1 to induce a change in p53
phosphorylation. Importantly, we found that ectopic expression of both E2F1 and E(-TA) in VSMCs resulted in
increased p53 phosphorylation of serine 15, which has
recently been shown to contribute to E2F1-mediated
apoptosis in the absence of ARF [10] (Fig. 5). These data
suggest that this covalent modification of p53 is sufficient
for E2F1-mediated apoptosis in VSMCs.
4. Discussion
During normal cell proliferation, E2F modulates the
expression of many genes involved in G1-S phase transition and DNA replication [2]. In addition to the well-
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J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
Fig. 3. Cytotoxic effect of E2F1 in VSMCs. VSMCs infected with AdER-E2F1 or AdER-E(-TA) and induced by OHT show a substantial loss of cell
viability within 3 days after infection. (A) MTT cell viability assay. AdER-E2F1 (j), AdER-E(-TA) (d) and AdGFP (s). Shown is the mean of three
independent experiments6S.D. Significant differences (P,0.001; paired, two-sided t-test) between AdER-E2F1, AdER-E(-TA), or AdGFP and untreated
cells (set as 1) are labelled with *. (B) Morphologic changes of VSMC expressing ER-E2F1 or ER-E(-TA) compared to Mock-infection are shown by laser
scan microscopy (upper panel) and Hoechst 33342-fluorescence micrographs (lower panel). Infected cells dissociating from the monolayer exhibited bright
blue fluorescing masses of chromatin that abutted at the nuclear membrane and were therefore identified as apoptotic (marked by arrows).
J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
517
Fig. 4. E2F1-mediated apoptosis is independent of the transactivation function. (A) Serum-starved primary vascular smooth muscle cells (I) were infected
with AdER-E2F1 (II, III) or the transactivation-defective E2F1 mutant (IV, V) in the absence (II, IV) or presence (III, V) of OHT. At 72 hpi cells were
harvested and processed for PI staining and flow cytometry. A significant cell population in sub-G1 was seen in both AdER-E2F1 (43.5%) and
AdER-E(-TA) (49.9%) infected cells with OHT. One representative experiment of three is shown. (B) Activation of caspase-3 in cells infected with
AdER-E2F1 or AdER-E(-TA) over 5 days was analyzed by Western blotting. Full-length caspase-3 (35 kDa) and the cleaved 17 kDa subunit are indicated.
(C) DNA fragmentation in VSMCs overexpressing E2F1 and E(-TA) detected by gel electrophoresis. DNA fragmentation in the form of oligonucleosomal
DNA ladders occurred in growing VSMCs in the presence of OHT at 72 (lanes 1 and 2) and 96 h after infection (lanes 3 and 4) with AdER-E2F1 (lanes 1
and 3) and AdER-E(-TA) (lanes 2 and 4, respectively). Lane M, small fragment DNA ladder; lane 0, untreated cells at 96 h.
established proliferative effect, particularly E2F1 has also
been implicated in the induction of apoptosis [5] by both
p53-dependent
and
p53-independent
pathways
[6,7,13,27,28,32,33]. In agreement with these studies, it
has been shown that E2F1 overexpression mediates growth
suppression in coronary VSMCs which involves caspase
3-like activity [22].
Here, we have investigated the mechanism of E2F1induced cell death in primary vascular smooth muscle
cells. We found that increased E2F1 activity results in a
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J. Stanelle et al. / Cardiovascular Research 59 (2003) 512–519
Fig. 5. p53 expression and phosphorylation by E2F1. Western blot
analysis for endogenous p53, the phospho-Ser 15 form of p53, and E2F1 in
lysates of human VSMCs. Cells were infected with AdER-E2F1 and
AdER-E(-TA), respectively, in the absence or presence of OHT, or the
control vector AdGFP. Uninfected cells are shown as a control (mock).
One representative experiment of two is shown.
rapid loss of cell viability by the induction of apoptosis in
VSMCs. In contrast to a previous work [22], however,
suggesting that E2F1 regulates growth of VSMCs by
forcing the cells to enter the S-phase and then to die, we
show that E2F1 is unable to induce S-phase entry in
growth-arrested cells. Thus, the consequence of increased
E2F1 activity in VSMCs is apoptosis, and not DNA
replication. Nevertheless, E2F1 activation leads to increased expression of cell cycle regulated target genes (as
shown here for CCNE1 and CDC25 A) in the absence of
S-phase entry, demonstrating that E2F1 is competent as a
transcriptional activator in vascular smooth muscle cells.
Our results agree with previous findings indicating that
E2F1 is not sufficient to induce S-phase progression in
quiescent human diploid fibroblasts unless other genetic
alterations (transformed cells) occur [34,35]. In the absence of proliferative E2F activity, Lomarri et al. also
observed increased expression of S-phase relevant genes
such as CCNE1 after E2F1 activation [35]. Based on their
findings, E2F1-induced S-phase entry requires suppression
of the RB- or p53-regulated G1 checkpoint, which is
consistent with the observation that p53 and RB negatively
regulate the cell cycle of primary VSMCs [36]. By
contrast, E2F1 efficiently induced cell cycle progression in
cells that are impaired in RB- or p53 function [37]. At the
G1 checkpoint, RB acts as a transcriptional repressor of
E2F1 by preventing it from S-phase entry [38].
Our results, however, indicate that both E2F1 and the
mutant lacking the transactivation domain induce similar
levels of apoptotic cells, suggesting that apoptosis induction by E2F1 is independent of the transactivation function. Since E2F1-mediated activation of p53 via the ARF /
Mdm2 pathway requires the transactivation domain of
E2F1, it is therefore likely that p53 stimulation in vascular
smooth muscle cells occurs through a ARF-independent
mechanism. Interestingly, we have seen increased p53
phosphorylation by ectopic expression of both E2F1 and
the transactivation-deficient mutant. This result seems
mechanistically similar to a previous work showing that
E2F1 can signal p53 phosphorylation in the absence of
ARF in mouse embryo fibroblasts [10]. From these data, it
appears that the covalent modification of p53 which is
coincident with p53 accumulation contributes to E2F1mediated apoptosis in VSMCs.
We and others have previously shown that activation of
p73 provides a means for E2F1 to induce cell death
independent of p53 [17,18]. Disruption of p73 function
inhibits E2F1-induced apoptosis in p53 2 / 2 MEFs. Whereas activation of p53 in response to E2F1 is indirect
involving ARF, E2F1 regulates p73 levels directly through
recognition and transactivation of the TP73 promoter
[17,18]. E2F1-mediated transactivation of p73 then results
in the activation of p53-responsive target genes and
apoptosis. This is again demonstrated in VSMCs, where
the E2F mutant which lacks the transactivation domain is
unable to stimulate p73 expression, but is capable of
inducing apoptosis at levels similar to the wild-type
transcription factor. From these data, we conclude that
apoptosis by E2F1 in VSMCs is also independent of p73.
VSMCs therefore seem to represent a cellular system,
where the transactivation-independent, apoptotic activity of
E2F1 is the primary cellular function.
5. Conclusion
Increased E2F1 activity can induce apoptotic cell death
in VSMCs, in the absence of S-phase entry. Apoptosis
induction by E2F1 is independent of its transactivation
domain and does therefore not require ARF or p73,
suggesting that VSMCs represent a cellular system in
which the transactivation-independent, proapoptotic activity of E2F1 is the primary cellular function. Instead,
increased p53 expression and phosphorylation appear to be
crucial for E2F1-mediated effect in VSMCs. Thus, overexpression of E2F1 may provide a suitable gene therapeutic strategy to prevent VSMC hyperproliferation in atherosclerosis, hypertension, and restenosis after injury.
Acknowledgements
This work was supported by the IFORES program of the
Medical Faculty of the University of Essen.
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