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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2936-2944
E1 E4+ Adenoviral Gene Transfer Vectors
Function as a "Pro-Life" Signal to Promote Survival of
Primary Human Endothelial Cells
By
Ramachandran Ramalingam,
Shahin Rafii,
Stefan Worgall,
Douglas E. Brough, and
Ronald G. Crystal
From Division of Pulmonary and Critical Care Medicine, and Division
of Hematology-Oncology, The New York Hospital-Cornell Medical Center,
New York, NY, and GenVec, Inc, Rockville, MD.
 |
ABSTRACT |
Although endothelial cells are quiescent and long-lived in vivo,
when they are removed from blood vessels and cultured in vitro they die
within days to weeks. In studies of the interaction of
E1 E4+ replication-deficient adenovirus
(Ad) vectors and human endothelium, the cells remained quiescent and
were viable for prolonged periods. Evaluation of these cultures showed
that E1E4+ Ad vectors provide an
"antiapoptotic" signal that, in association with an increase in
the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter
a state of "suspended animation," remaining viable for at least
30 days, even in the absence of serum and growth factors. Although the
mechanisms initiating these events are unclear, the antiapoptoic signal
requires the presence of E4 genes in the vector genome, suggesting that
one or more E4 open reading frames of subgroup C Ad initiate a
"pro-life" program that modifies cultured endothelial cells to
survive for prolonged periods.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE VASCULAR SYSTEM is covered by a
single layer of endothelium, cells derived from the
mesoderm.1,2 Although endothelial cells replicate in
response to angiogenic mediators in association with hypoxia, wound
healing, inflammation, and tumor growth,2-5 the normal
adult endothelium is quiescent and remarkably long-lived, with an
average half-life of years.5 Endothelial cells removed from
the vasculature and cultured in vitro in the presence of serum and
growth factors, such as vascular endothelial growth factor (VEGF) and
basic fibroblast growth factor (bFGF), proliferate to reach
confluence.6-10 Confluent monolayers of endothelial cells
are contact inhibited for growth if they are not
passaged,11 and, even in the presence of serum and growth factors, the cells detach and die.12 In the absence of
serum and growth factors, endothelial cells rapidly undergo
apoptosis.12-14
As part of studies focused on adenovirus (Ad)-mediated gene transfer to
human endothelium, we noted that endothelial cells infected by
E1E4+ replication-deficient Ad vectors
remained viable for prolonged periods, even in the absence of serum and
growth factors and independent of the transgene carried by the Ad
vector. This study presents evidence that
E1E4+ Ad vectors function to provide an
"antiapoptotic" signal that, accompanied by an increase in the
ratio of Bcl2 to Bax levels in the cells, puts cultured primary human
endothelial cells in growth factor-free and serum-free conditions into
a state of "suspended animation," maintaining cell viability for
at least 30 days. Although the mechanisms of induction of this state
are unclear, they require the presence of the Ad E4 genes, suggesting
that the one or more open reading frames (ORF) of E4 may provide a
signal to induce these cells to revert to a more in vivo-like phenotype.
| |
MATERIALS AND METHODS |
Endothelial cells.
Primary human endothelial cells were isolated from freshly obtained
umbilical cords by collagenase treatment6 and grown in
"growth factor medium" (M199 medium, 20% fetal calf serum, 10 ng/mL VEGF [Peprotech, Piscataway, NJ], 5 ng/mL bFGF [Peprotech], and 1 unit/mL heparin sulfate) at 37°. Cells from passage 2 to 5 were
used in all the experiments. Where indicated, studies were carried out
using "growth factor-free medium" (X-vivo; Bio-Whitaker, Walkersville, MD; contains no serum and no growth factors).
Adenovirus vectors.
The Ad vectors used in this study included: (1)
E1E4+ AdNull (E1,
E3, E4+, cytomegalovirus early/intermediate
promoter/enhancer [CMV], no transgene in the expression
cassette)15; (2) E1E4+ Ad gal
(E1, E3, E4+; CMV promoter
driving the Escherichia coli -galactosidase [gal] gene15); (3) E1E4+ AdGFP
(identical to E1E4+ Adgal, but with a
modified form of the Aeguorea victoria green fluorescent
protein cDNA [GFP] in place of gal)16,17; (4) E1E4 Adgal (same as Adgal, but with
a complete deletion of the E4 gene, using the E coli
-glucuronidase gene as a spacer in the E4 region)18; and
(5) AdZ.11E4ORF6 (E1E3; expresses only
E4ORF6 from the E4 promoter and all other E4ORFs deleted; CMV promoter
driving the E coli gal). Ad vector stocks were purified
by cesium chloride centrifugation and dialysis and quantified by plaque
forming units (pfu) in 293 cells, as previously described.19 All Ad vectors had a particle/pfu ratio of
approximately 100, and all vectors were determined to be free of
replication-competent Ad.20,21
Growth curves and cell survival.
To evaluate the growth response of the endothelial cells to Ad vector
infection, the cells were cultured in 6 well plates (for quantification
of viable cell number) or poly-D-lysine-coated glass coverslips with
grids to allow for identification of the specific locations (for
studies of growth curves; Brinkman Instruments, Westbury, NY). The
cells were washed, and the Ad vectors (50 pfu/cell) were then added in
"growth factor-free medium" (X-vivo; Bio-Whitaker) with gentle
shaking. Control cells were maintained in growth factor-free medium
alone. After 90 minutes, the cells were washed, and for the first 24 hours, the cells were cultured in growth factor medium. Cells were then
cultured for up to 30 days in growth factor medium or growth
factor-free medium, as indicated. At various times, the total cell
number and percentage of viable cells (trypan blue exclusion) were determined.
Endothelial markers.
To insure that endothelial cells infected with Ad vectors maintained
their differentiation with respect to an endothelial-specific marker,
endothelial cells were infected with E1E4+
AdNull as described above in growth factor medium or growth
factor-free medium in 6 well plates. After 30 days in culture, the
cells were evaluated for expression of von Willebrand factor using a
mouse monoclonal antibody (Dako Corp, Carpenteria, CA). Alkaline
phosphatase-labeled goat antimouse antibody (Jackson Immunoresearch
Laboratories, Westgrove, PA) was used to identify the primary antibody,
and the enzyme reaction product (red) was visualized using a new
fuchsin substrate (Dako Corp). Cellular structures were visualized by hematoxylin counter staining. Samples were analyzed by microscopy. Irrelevant isotype-matched monoclonals were used as controls. Expression of the VEGF receptor KDR was evaluated by flow cytometry in
the Ad vector-infected endothelial cells maintained as above, using a
mouse monoclonal antibody (gift from Imclone Systems, New York, NY) and
a flourscine-conjugated secondary goat antimouse IgG (Sigma
Immunochemicals, St Louis, MO). Irrelevant isotype-matched monoclonals
were used as controls. Expression of E-selectin in endothelial cells
was assessed after treatment of interleukin-1 (IL-1)
(10 units/mL; R&D Systems, Minneapolis, MN) for 18 hours and by flow
cytometry using a rabbit polyclonal antibody (R&D Systems).
Persistence of the Ad genome.
To evaluate the endothelial cells for the presence of the Ad genome
over time, E1E4+ AdNull vector-infected
cells were collected after 0, 24, and 48 hours, 7 and 30 days after
infection. Mock-infected cells were used as a control. Total DNA was
extracted using overnight digestion of the cell pellet with Proteinase
K (100 µg/mL) (Sigma) in digestion buffer (100 mmol/L NaCl, 10 mmol/L
Tris-HCl, 25 mmol/L EDTA, pH 8.0, and 0.5% sodium dodecyl sulfate
[SDS]) and subsequent phenol/chloroform extraction and
ethanol precipitation. Total DNA (500 µg) was used as template for
Ad5-specific polymerase chain reaction (PCR) using Ad5 E4 primers
(sense: 5'-GTAGAGTCATAATCGTGCATCAGG; antisense: 5'-TTTATATGGTACCGGGAGGTGGTG). As a control, the DNA was also
evaluated using E1-specific primers (sense:
5'-GAGACATATTATCTGCCACGGAGG; antisense:
5'-TTGGCATAGAAACCGGACCCAAGG). The cycling parameters were 94°, 30 seconds; 65°, 30 seconds; and 72°, 30 seconds, for a total of 40 cycles. The PCR products were resolved in a 1% agarose gel containing
ethidium bromide.
DNA synthesis.
To quantify DNA synthesis in endothelial cells after Ad vector
infection, cells cultured (104/well) in a 96-well plate
were either mock treated with growth factor-free medium alone or
infected with either E1E4+ Adgal or
E1E4 gal (50 pfu/cell) for 90 minutes
in growth factor-free medium. The medium was changed to growth factor
medium. After 24 hours, fresh growth factor medium or growth
factor-free medium containing [3H]thymidine (0.5 µCi/well; 85 mCi/mmol; Amersham Life Science, Arlington Heights, IL)
was added, and cells were incubated for an additional 24 hours. Cells
were harvested by trypsin and EDTA treatment and collected on
glass-fiber filters. Filter-bound radioactivity was measured in a
liquid scintillation counter. Each analysis was done in triplicate.
DNA analysis.
To assess the content of the DNA in the endothelial cells after Ad
vector infection, adherent cells were harvested together with floating
cells, washed with phosphate buffered saline (PBS), pH 7.4, and
resuspended in low ionic strength buffer (0.1% sodium citrate, 0.1%
Triton-X100, 50 µg/mL propidium iodide [Sigma], and 20 µg/mL
DNase free RNaseA [Boeghringer Mannheim, Indianapolis, IN]). After
overnight incubation at 4°, the DNA content of the cells was analyzed
in a flow cytometer (Elite Profile; Coulter, Miami, FL). The results
are presented in graphic format showing cell count on the y-axis and
fluorescence intensity on the x-axis. To show the proportion of cells
showing <2n complement of DNA (including the apoptosis fraction), the
x-axis was displayed in log format.22
DNA fragmentation in apoptotic endothelial cells was also assessed by
incorporating fluorescein-12-dUTP at the 3'-OH DNA ends using the
enzyme terminal deoxynucleotidyl transferase-mediated dUTP nick-end
labeling assay.23 Endothelial cells were grown in
poly-D-lysine-coated glass coverslips, the subconfluent cells were
infected with E1E4+ or
E1E4 Ad vectors, and the cells were
maintained in growth factor medium or growth factor-free medium.
Mock-infected cells were used as a control and were maintained as
above. Cells maintained in growth factor medium were analyzed on day
10, and cells maintained in growth factor-free medium were evaluated
on day 1 postinfection using the Apoptosis Detection System (Promega,
Madison, WI), following the manufacturer's protocol. Samples were
analyzed under fluorescence microscope, and the number of green
fluorescent nuclei, characteristic of apoptosis, were counted.
Propidium iodide counter stain was used to visualize the nucleus and
cytoplasm. The percentage of apoptotic cells was calculated from
triplicate measurements.
Western analysis.
To evaluate the endothelial cells for levels of Bcl2 and Bax protein,
the cells were harvested by trypsin and EDTA treatment, washed once
with PBS, and lysed with boiled lysis solution (10 mmol/L Tris-HCl, pH
7.4; 1% SDS; and 1 mmol/L sodium vanadate). The lysate was boiled for
an additional 5 minutes and cleared by centrifugation (14,000 rpm,
4°, 10 minutes). Protein content was estimated using Pierce reagents
(Rockford, IL). The cellular proteins (50 µg) were resolved in a 12%
SDS-polyacrylamide gel and transferred to nitrocellulose membranes by
semidry blotting. Rabbit polyclonal or mouse monoclonal primary
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used at
1:250 dilutions to detect human Bax and Bcl2, respectively. Rabbit
polyclonal antibodies were used at 1:100 dilution to detect human
-actin (Sigma Immunochemicals). Immunoreactive bands were visualized by a secondary antibody conjugated to horseradish peroxidase and enhanced chemiluminescence (Amersham, Little Chalfont, Buckinghamshire, UK). Additional controls (not shown) included nonimmune serum (for
Bcl2) and irrelevant isotype-matched monoclonals (for Bax and
-actin).
Statistical analysis.
All data are presented as mean ± standard error of the mean. All
comparisons were made using the two-tailed Student's t-test.
| |
RESULTS |
To evaluate the effect of an E1E4+ Ad vector
on the growth and viability of primary human endothelial cells, growth
curves and viability of E1E4+
AdNull-infected and control cells cultured in growth factor medium and
in growth factor-free medium were compared (Fig
1). As is typical for human endothelial
cells cultured in the presence of growth factors and serum, control
subconfluent cells cultured in growth factor medium became confluent by
day 8; thereafter, the number of adherent cells declined progressively,
such that by day 30, no cells remained adherent (Fig 1A). In contrast,
under the same conditions, AdNull-infected cells were growth inhibited, and the number of adherent cells remained constant for 30 days, the
duration of the experiment. Endothelial cells infected with E1E4+ Adgal or with
E1E4+ AdGFP under the same conditions with
growth factor medium showed similar growth inhibition as with the
AdNull vector (not shown). Strikingly, when subconfluent endothelial
cells cultured in growth factor-free medium were exposed to the
E1E4+ AdNull vector, the control cells died
by day 12, whereas the AdNull-infected endothelial cells remained
adherent for at least 30 days (the duration of the study) without a
loss of cell number (Fig 1B).
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| Fig 1.
Effect of an E1E4+
replication-deficient Ad vector on the growth and viability of primary
human endothelial cells. (Panels A, B) Subconfluent endothelial cells
cultured in poly-D-lysine-coated glass coverslips. (Panels C, D)
Confluent endothelial cell cultured in 6 well plates. (A) Number of
endothelial cells over time cultured in growth factor medium after
exposure to the E1E4+ AdNull vector (50 pfu/cell) or to no vector ("control"). (B) Number of endothelial
cells over time cultured in growth factor-free medium after exposure
to AdNull compared with uninfected cells ("control"). (C)
Viability of confluent endothelial cells over time cultured in growth
factor medium after infection with E1E4+
AdNull vector (50 pfu/cell) compared with no vector ("control").
(D) Viability of confluent endothelial cells over time cultured in
growth factor-free medium after infection with the AdNull vector
compared with control. For panels C and D, viability was assessed by
trypan blue dye exclusion. For all panels, the data represents the mean ± standard error of the mean of triplicate measurements.
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To examine the effect of an E1E4+ Ad vector
on the viability of confluent endothelial cultures, confluent
endothelial monolayers cultured in growth factor medium or in growth
factor-free medium were infected with E1E4+
AdNull vector, and viability was assessed by trypan blue dye exclusion.
In growth factor medium, the control cells lost viability progressively
and died by day 12 (Fig 1C). In marked contrast, 90% of
AdNull-infected cells remained viable for 30 days, the duration of the
experiment. In the growth factor-free medium, the control cells lost
viability quickly and died by day 5 (Fig 1D). In marked contrast, 80%
of the AdNull-infected cells were still viable at day 30. Endothelial
cells infected with E1E4+ Adgal or with
E1E4+ AdGFP under the same condition with
growth factor medium or growth factor-free medium showed maintenance
of viability similar to that with the AdNull vector over the 30 days of
the experiment (not shown). Assessment of the endothelial cultures 30 days after exposure to the E1E4+ Ad vector
showed that the cells maintained the endothelial cell marker von
Willebrand factor and VEGF receptor KDR. E-selectin, another unique
endothelial cell marker, was expressed in control as well as
E1E4+ Ad vector-infected cells after
IL-1 stimulation. This was observed in cultures maintained in growth
factor medium or growth factor-free medium, ie, infection with an
E1E4+ Ad vector did not cause the cells to
lose their endothelial cell-specific characteristics (not shown).
Evaluation of the effects of an E1E4+ Ad
vector on cellular DNA synthesis by the cultured endothelium showed
that infection with the vector suppressed the synthesis of cellular
DNA, independent of the culture conditions. In this context,
subconfluent cultures of primary endothelial cells cultured in growth
factor medium had a threefold reduction in DNA synthesis when infected
with the E1E4+ AdNull vector
(P < .0001; Fig
2A).
Likewise, subconfluent cultures of endothelial cells cultured in growth
factor-free medium had a threefold reduction in DNA synthesis when
infected with the E1E4+ AdNull vector
(P < 0.0003).
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| Fig 2.
Evaluation of the status of the DNA in the endothelial
cells after exposure to the E1E4+ AdNull
vector or no vector ("control"). (A) Effect of AdNull on DNA
synthesis of subconfluent endothelial cells. Cells were exposed to the
AdNull vector (50 pfu/cell) or no vector ("control") and were
cultured in 96 well plates in growth factor medium or growth
factor-free medium. After 24 hours, [3H]thymidine was
added, and the incubation was continued for 24 hours. The
data represents the mean ± standard error of the mean of triplicate
measurements. (B) Effect of AdNull infection on the status of cellular
DNA. Endothelial cells cultured in 6 well plates were exposed to the
AdNull vector as per panel A and were evaluated by flow cytometry after
propidium iodide staining. Shown are data for the confluent cultures of
endothelial cells infected with AdNull in growth factor medium or
growth factor-free medium. The data for day 1 is displayed with a
linear abscissa to best show the >2n peaks; the data for day 5 and
day 12 is displayed with a log scale to best show the <2n fragmented
DNA. The <2n, 2n, and 4n DNA peaks are indicated. (C) Evaluation of
apoptosis in cells infected with E1E4+
AdNull vector. Subconfluent endothelial cells cultured in
poly-D-lysine-coated glass coverslips were infected with
E1E4+ AdNull vector as described in the
Fig 1 legend. After vector infection, cells were maintained in growth
factor medium for 10 days or in growth factor-free medium for 1 day,
and apoptotic cells were identified by assessment of free DNA 3'-OH
ends.
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Flow cytometry analysis using propidium iodide staining showed that the
control endothelial cells cultured in growth factor medium showed a
sub-G0/G1 DNA peak beginning by day 5, and by day 12, 100% of the cells were in this peak (Fig 2B). Strikingly, E1E4+ AdNull-infected cells cultured in
growth factor medium did not show a sub-G0/G1
DNA peak. Likewise, when the confluent endothelial cells were cultured
in growth factor-free medium, the control cells showed a
sub-G0/G1 DNA peak starting by day 5, and by
day 12, 100% of the cells were in this peak (Fig 2B). In contrast, 85% of AdNull-infected cells had a  2n complement of DNA at day 12, with only 15% showing a sub-G0/G1 peak. Based
on the likelihood that the sub-G0/G1 peaks
corresponded to 180 to 200 base pairs of nucleosomal DNA fragments
(<2n complement) characteristic of programmed cell death
(apoptosis),22 the flow cytometry experiments were repeated
with an assay to quantify free DNA 3'-OH ends, a characteristic of
apoptosis.23 When cultured in growth factor medium, 15.0 ± 3.0% of control cells were undergoing apoptosis by day 10. In
contrast, 5.0 ± 0.5% of the E1E4+ AdNull
vector-infected cells showed free DNA 3'-OH ends
(P < 0.05; Fig 2C). Because about 30% of control cells
died by day 10 (Fig 1A), it is likely that control cell death in growth
factor medium resulted from a combination of apoptosis and possibly
necrosis. When cultured in growth factor-free medium, 26.0 ± 0.5%
of control cells were undergoing apoptosis by day 1, whereas only 1.0 ± 1.0% of E1E4+ AdNull vector-infected
cells were undergoing apoptosis (P < 0.0001). Together with
the growth curve data (Fig 1), this data is consistent with the concept
that death of control cells in growth factor-free medium was primarily
through apoptotic pathways, and suggests that
E1E4+ AdNull infection protected endothelial
cells from apoptosis in the absence or presence of growth factors.
Vector genome-specific PCR assessment of the cultures of
E1E4+ AdNull-infected cells showed that at
30 days, the vector DNA was present in the subconfluent cells cultured
in growth factor medium, as well as confluent cells cultured in growth
factor-free medium (not shown). As expected, E1A genes were not
detected in the DNA of the E1E4+-infected
cells (not shown).
Bcl2, a mediator that promotes cell survival, and Bax, an initiator of
cell death, have been implicated in the control of apoptotic pathways
initiated by growth factor withdrawal,24-26 with an
increase in the relative amounts of Bcl2 compared with Bax protein
levels favoring cell survival.26,27 Evaluation of Bcl2 and
Bax protein levels in control confluent endothelial cells cultured in
growth factor-free medium showed that Bcl2 protein levels did not
change significantly, with a small decrease in Bax protein levels
observed over time (Fig 3). In contrast, in the E1E4+ AdNull-infected cells, Bcl2
protein levels increased significantly beginning at 24 hours, whereas
Bax protein levels decreased progressively over time. The control
-actin levels remained constant in both control and
E1E4+ AdNull-infected endothelial cells.
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| Fig 3.
Bcl2 and Bax protein levels in primary human endothelial
cells after infection with an E1E4+ Ad
vector. Confluent endothelial cells were infected with the
E1E4+ AdNull vector or no vector
("control"), cultured in growth factor-free medium for 0 to 48 hours and assessed by Western analysis for human Bcl2 and Bax using
rabbit polyclonal and mouse monoclonal antibodies, respectively. Equal
protein loading was confirmed with a control antibody to detect human
-actin detected by a rabbit polyclonal antibody. Blots were
developed by enhanced chemiluminescence.
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Based on the knowledge that E1E4+ Ad vectors
express low levels of E4 ORF,28-31 and that several
E4+ ORF have functions that may be linked to the cell
cycle,32-34 we hypothesized that E4 region gene products
may play a role in enhancing endothelial cell survival after infection.
To evaluate this hypothesis, cell growth and survival, DNA synthesis,
cell cycle analysis, analysis of free DNA 3'-OH ends, and the levels of
Bcl2 and Bax were compared in endothelial cells after infection with
E1E4+ or E1E4
Ad vectors. In contrast to the control cells that became confluent by
day 8, E1E4 Ad-infected subconfluent
cells cultured in growth factor medium grew slowly and never became
confluent (Fig
4A). Like the
control uninfected cells, the endothelial cells infected with the
E1E4 vector gradually lost viability,
such that by day 18, no cells had survived. In contrast, as observed
with the E1E4+ AdNull vector (Fig 1A), the
E1E4+ Adgal vector-infected cells did
not grow, but the number of viable cells remained constant for the
30-day duration of the experiment. When confluent cells were cultured
in growth factor-free medium, both the control and
E1E4 Ad vector-infected cells lost
viability quickly, such that by day 5 all the cells had died (Fig 4B).
In contrast, the cells infected with E1E4+
Ad vector remained viable throughout the 30-day duration of the experiment.
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| Fig 4.
Effect of an E1E4 Ad vector
on growth and survival of primary human endothelial cells. (A) Growth
of endothelial cells in response to growth factors after exposure to an
E1E4 vector. Subconfluent primary human
endothelial cells were cultured in growth factor medium, as in Fig 1A,
after exposure to E1E4 Adgal (50 pfu/cell) compared with E1E4+ Adgal (50 pfu/cell) or no vector ("control"). Shown is the number of viable
cells as assessed by trypan blue dye exclusion. (B) Survival of
endothelial cells cultured in growth factor-free medium after exposure
to an E1E4 Ad vector and
E1E4 ORF6 Ad vector. Confluent primary
human endothelial cells were cultured in growth factor-free medium
after exposure to E1E4 Adgal,
E1E4 ORF6 Ad vector,
E1E4+ Adgal, or control, as in panel A. (C) Effect of an E1E4 Ad vector on
cellular DNA synthesis of primary endothelial cells. Cells were exposed
to the E1E4 Adgal vector or no vector
("control") and cultured in growth factor medium or growth
factor-free medium, with [3H]thymidine uptake quantified
as in Fig 2A. The data for panels A through C represent the mean ± standard error of triplicate measurements. (D) Evaluation of the status
of the cellular DNA in cultured endothelial cells after exposure to an
E1E4 Ad vector. Cells exposed to the
E1E4 Adgal vector or no vector
("control") were cultured in growth factor medium or growth
factor-free medium as described in Fig 2B, and evaluated by propidium
iodide staining and flow cytometry. The data for day 1 is displayed
with a linear abscissa to best show the >2n peaks; the data for day 5 and day 12 is displayed with log scale to best show the <2n
fragmented DNA. The <2n, 2n, and 4n DNA peaks are indicated. (E)
Levels of Bcl2 and Bax protein in human endothelial cells after
exposure to E1E4 Adgal vector.
Confluent endothelial cells were exposed to the vector or no vector
("control") and cultured in growth factor-free medium. The
analysis was identical to that in Fig 3. (F) (see page 2939) Evaluation
of apoptosis in cells infected with E1E4
Ad vector. Subconfluent endothelial cells cultured in
poly-D-lysine-coated glass coverslips were infected with
E1E4 Ad vector as described in the Fig 1
legend. After vector infection, cells were maintained in growth factor
medium for 10 days or in growth factor-free medium for 1 day, and
apoptotic cells were identified by assessment of free DNA 3'-OH ends.
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The differences in the effect of E1E4 and
E1E4+ Ad vectors on endothelial cells were
not linked to differences in suppression of DNA synthesis, in that the
E1E4 Ad vector- induced
inhibition of endothelial cell DNA synthesis in growth factor medium
and in growth factor-free medium in a fashion similar to that of the
E1E4+ vector (Fig 4C;
E1E4 Ad vector compared with control in
growth factor medium, P < 0.0001;
E1E4 Ad vector compared with control in
growth factor-free medium, P < 0.0001; compare Fig 4C with
Fig 2A). Interestingly, the endothelial cells infected with
E1E4 Ad vector showed increasing amounts
of fragmented <2n DNA over time in both growth factor-free medium
and growth factor medium, similar to that of the control cells (Fig
4D). Furthermore, assessment of free DNA 3'-OH ends showed that,
similar to control cells, 12.0 ± 2.5% of
E1E4 Ad vector-infected cells exhibited
free DNA 3'-OH ends by day 10 in growth factor medium
(P > 0.4; Fig 4F). In growth factor-free medium, by day 1, 28.0 ± 4.0% of E1E4 Ad
vector-infected cells was undergoing apoptosis, similar to that
observed in control cells (P > 0.6), ie, infection with the E1E4 Ad vector did not suppress
apoptosis. Consistent with this observation, Bcl2 and Bax protein
levels did not change in the E1E4 Ad
vector-infected endothelial cells compared with the control cells (Fig
4E). Taken together, these results suggest that Ad E4 gene products
play a role in the ability of the E1E4+ Ad
vectors to induce the survival of primary endothelial cells in vitro.
To begin to identify the E4ORF(s) that is required for the prolonged
survival of endothelial cells, the effect of E4ORF6 protein on the
survival of endothelial cells after E1E4ORF6 Ad vector
infection was assessed. We chose to evaluate E4ORF6 because of the
known interaction of E4ORF6 protein and P53 that has impact on cell
cycle regulation and survival.33 However, when confluent
cells were cultured in growth factor-free medium, endothelial cells
infected with E1E4ORF6 Ad vector died by day 5, similar
to the control and the E1E4 Ad
vector-infected cells (Fig 4B). These results suggest that AdE4ORF6
protein alone was not sufficient for the prolonged survival of
endothelial cells in growth factor-free medium.
| |
DISCUSSION |
In vivo, uninjured endothelial cells are quiescent and long-lived, with
very low levels of DNA synthesis and proliferation.5,9 When
subconfluent endothelial cells are cultured in the presence of growth
factors and serum, the cells initially proliferate, but then rapidly
lose viability if they are not passaged. When cultured in the absence
of growth factors, the cells fail to grow, and they rapidly enter
apoptotic pathways and die.13,14 In the present study, we
have observed that infection of cultured primary human endothelial
cells with E1E4+ Ad vectors evokes a
phenotype characterized by minimal growth, longevity, suppression of
DNA synthesis, and suppression of apoptotic pathways, regardless of the
presence or absence of growth factors. The
E1E4+ Ad vector seems to freeze the cells in
a phenotype that has at least some characteristics shown by uninjured,
resting endothelium in vivo. The remarkable sustenance of viability of
endothelial cells by E1E4+ Ad vectors in the
absence of growth factors has never been described for primary
endothelial cells.
Possible mechanisms of Ad vector-mediated endothelial cell survival.
Although the mechanisms by which the E1E4+
Ad vectors accomplish the prolonged survival of Ad vector-infected
endothelial cells in vitro are unknown, they seem to be linked, at
least in part, to inducing the Bcl2 and Bax pathways to favor cell
survival, and they seem to be initiated by low level expression of E4
gene products in the E1E4+ Ad vectors.
Consistent with this concept, retrovirus-mediated transfer and
expression of Bcl2 in cultured endothelial cells have been shown to
reduce apoptosis in the absence of bFGF.35 The increased
ratio of Bcl2 and Bax, and the requirement of E4 gene products
associated with the prolonged survival of Ad vector-infected endothelial cells provide clues to mechanisms that may be involved.
First, wild-type Ad encodes several proteins that promote infected host
cell survival to prevent premature cell death.34 The E1A
12S gene product is antiapoptotic and capable of immortalizing various
cell types.34,36 The E1B 19-kD protein is analogous to Bcl2
in blocking apoptosis initiated through several cell death pathways.37,38 However, the possibility of
E1E4+ Ad vector-mediated endothelial
survival being mediated through E1A or E1B gene products is not
possible because the E1E4+ Ad vectors used
in this study are completely E1A deleted and partially E1B deleted, ie,
the vectors cannot possibly express the 12S- or 19-kD genes.
Second, the E2 gene encodes the 72-kD single-stranded DNA binding
protein, the terminal protein precursor, and the Ad DNA polymerase, all
of which are essential for viral DNA replication.39 Although a low level expression of E2 gene is possible in the absence
of the E1A gene, E2 gene products are not known to be involved in host
cell growth and survival functions.28,33 Ad E3 region
encodes 19- and 14.7-kD proteins that effectively inhibit cellular
immune response to Ad virus infection and Fas ligand-induced apoptosis.40-42 However, the
E1E4+ Ad vectors used in this study have a
large deletion in the E3 region that eliminates both the 19- and
14.7-kD genes.19,43 Ad late genes that encode hexon,
penton, and fiber are not likely to be involved in enhanced survival of
endothelial cells infected with AdNull.34
Third, because the E1E4+ Ad vectors, but not
the E1E4 vectors, induced the prolonged
survival of infected cells, it is logical to conclude that Ad E4 gene
products may play a role in the E1E4 Ad
vector-infected endothelial cell survival. The E4 region of Ad
contains seven ORFs that encode a variety of regulatory
functions.34,44-51 The E4ORF6 and E4ORF3 seem to encode
redundant functions involved in host cell shutoff and accumulation of
late viral mRNAs.46 The E4ORF6 protein interacts with the
tumor suppressor p53 and inhibits p53-mediated transcriptional
activation.33 E4ORF6/7 protein binds as a homodimer to the
cellular transcription factor E2F to promote E2 promoter
activity.45 E4ORF4 protein binds and activates protein
phosphatase 2A and may have a role in the regulation of DNA synthesis
and AP-1 transcription factor activity.49,50 E4ORF1,
E4ORF2, and E4ORF3/4 proteins encoded by Ad9 serotype cooperate with
E1A in cell transformation.48,51 Although E4 gene
expression is downregulated in the absence of E1A
gene,28,51-53 a basal level expression of E4 gene is still
detected in the cells infected with E1-deleted Ad
vectors,28,30,54,55 and this low level expression of E4
proteins seems to be sufficient to function to modify gene expression
from the vector.29-31,55 Any of the E4 products could be
linked to the antiapoptotic function of the
E1E4+ Ad vector infection. However, the
increase in the ratio of Bcl2 to Bax protein levels in
E1E4+ Ad vector-infected endothelial cells
is consistent with a role for E4ORF6 protein in blocking p53-mediated
transcriptional activation,33,56 possibly by relieving p53
repression of Bcl233,56 and activation of Bax gene
expression.57,58
Finally, it is possible that low level expression of one or more E4ORF
promotes cellular gene(s) that play a role in cell survival, and that one relevant to the in vivo phenotype of endothelial cells. It is known that infection of cells with Ad vectors upregulates cell signaling molecules such as MAP kinase,59 as well as
cytokines, such as interleukin-1 and interleukin-6.60-62
Consistent with this concept, E4ORF6 has been shown to block
p53-mediated apoptosis.63 However, E4ORF6 alone was not
sufficient for prolonged survival of endothelial cells in the absence
of serum and growth factors, ie, it is highly likely that other E4ORFs
in combination with E4ORF6 may be required for the antiapoptotic role
of AdE4 gene. Ongoing studies are designed to identify those E4ORFs.
| |
ACKNOWLEDGMENT |
The authors thank Philip Leopold and Barbara Ferris (Weill Medical
College, New York, NY) for assisting in fluorescence microscopy, and N. Mohamed (Weill Medical College, New York, NY) for help in preparing
this manuscript.
| |
FOOTNOTES |
Submitted July 30, 1998; accepted December 14, 1998.
Supported, in part, by the National Institutes of Health/National
Heart, Lung and Blood Institute P01 HL51746, P01 HL59312, the Cystic
Fibrosis Foundation, Bethesda, MD; Will Rogers Memorial Fund, Los
Angeles, CA; and GenVec, Inc, Rockville, MD (R.R., S.W., and R.G.C.).
S.R. is supported by NIH/NHLBI R01 HL58707 and the New York Heart
Association Grant-in-Aid, New York, NY.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ronald G. Crystal, MD, Division of
Pulmonary and Critical Care Medicine, The New York
Hospital-Cornell Medical Center, 520 E 70th St, ST505, New York, NY
10021; e-mail: geneticmedicine{at}mail.med.cornell.edu.
| |
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ABSTRACT
INTRODUCTION