 |
INTRODUCTION |
The survival of endothelial cells is critical for angiogenesis and
the maintenance of blood vessel integrity.
VEGF1 is an endothelial
cell-specific mitogen that functions to induce blood vessel formation
during normal development and various pathological processes (1). VEGF
biological activity is mediated through its binding to at least two
endothelial cell-specific receptors, fms-like tyrosine kinase (flt-1)
and fetal liver kinase (flk-1) (2, 3). Heterozygous deletion of the
VEGF gene results in the embryonic lethality due to the
abnormal blood vessel development (4, 5). VEGF also functions in adult
organisms to promote angiogenesis in ischemic tissue (6) and during
wound healing (7). Augmented expression of VEGF by gene transfer has
been shown to promote the formation of collateral arteries in patients with peripheral artery disease (8), and VEGF is also associated with
pathological angiogenesis such as in tumors that express high levels of
this factor (9, 10). VEGF activates receptor tyrosine kinases leading
to the activation of various signal transducers including
phosphoinositide 3-kinase (PI3-kinase) (11, 12). In addition to its
mitogenic effects, VEGF acts to promote endothelial cell survival both
in vitro and in vivo (13-15). Consistent with its function as a survival factor, VEGF withdrawal is associated with
vascular regression in both developing retina and tumors (13, 16,
17).
Endothelial cells are also dependent on adhesion to extracellular
matrix for their survival (18). Cell adhesion to matrix is mediated by
integrins and disruption of proper integrin-ligand interactions leads
to a phenomenon termed anoikis, apoptosis induced by the disruption of
cell-matrix interactions (19). Endothelial cell adhesion to matrix is
mediated, at least in part, by integrin
v
3, which transmits an "outside-in"
survival signal that is essential for angiogenesis (20). Therefore,
agents that induce endothelial cell apoptosis, either by antagonizing
integrin binding or perturbing adhesion plaque formation, are currently
being considered for cancer therapy by virtue of their ability to
inhibit tumor neovascularization (21-23).
The serine/threonine protein kinase Akt/PKB is recognized increasingly
as a key regulator of cell viability in a number of systems (24-27).
Activation of Akt occurs through the direct binding of the
phosphoinositide products of the PI3-kinase reaction to its pleckstrin
homology domain (28). Phosphoinositol lipids also activate a protein
kinase that phosphorylates Akt, resulting in further activation (29,
30). Here, we examined the involvement of Akt in the survival of
cultured human umbilical vein endothelial cells (HUVEC). VEGF-mediated
HUVEC survival correlated with the wortmannin-sensitive induction of
Akt activity. Adenovirus-mediated expression of a dominant-negative
mutant of Akt reduced HUVEC viability in the presence of VEGF. Forced
expression of wild-type Akt promoted cell survival in response to low
doses of VEGF, whereas constitutive overexpression of Akt was
sufficient for endothelial cell survival in the absence of VEGF. VEGF
did not promote Akt activation in suspension culture nor did it inhibit
anoikis. However, expression of constitutively active Akt was
sufficient for survival in the absence of attachment. These data
suggest that growth factor and attachment-mediated signaling pathways
converge on Akt to control endothelial cell survival under conditions
of angiogenesis and vessel regression.
 |
MATERIALS AND METHODS |
Reagents, Plasmids, and Cell Culture--
Anti-Akt antibody was
purchased from Santa-Cruz. Wortmannin was purchased from Sigma. Akt
plasmids were a gift from Dr. J. Testa. Recombinant human VEGF was the
165 amino acid isoform. HUVECs were cultured in monolayers in
Dulbecco's modified Eagle medium (DMEM) under conditions of serum
deprivation or stimulation with VEGF as described by Spyridopoulos
et al. (14). For suspension cultures, HUVEC monolayers were
washed with phosphate-buffered saline (PBS) twice and incubated with
PBS containing 0.5% EDTA. Cells were collected with centrifugation
(2,000 rpm for 5 min) and washed with serum-depleted media. For
suspension culture, bacterial culture dishes were precoated with 20 mg/ml bovine serum albumin overnight to prevent the cells from
attaching to the substratum.
Akt Kinase Assay--
Cells were washed twice with PBS and lysed
with cell lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 20 mM NaF, 2 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride). Lysates were precleaned with protein
G-agarose for 30 min at 4 °C, and immunoprecipitated for 2 h
with anti-Akt antibodies in the presence of 2 mg/ml bovine serum
albumin with or without 16 µg/ml competitor peptides (Santa Cruz).
Immunoprecipitates were washed twice with cell lysis buffer, once with
water, and once with kinase buffer (20 mM HEPES, pH 7.2, 10 mM MgCl2, 10 mM MnCl2).
Immunoprecipitated proteins were incubated in 50 µl of kinase buffer
containing 2 µg of histone H2B (Roche Molecular Biochemicals) and
[
-32P]ATP (5 µM, 10 µCi) for 30 min at
room temperature. Kinase reactions were stopped by the addition of SDS
sample buffer and subjected to SDS-polyacrylamide gel electrophoresis
on 15% polyacrylamide gels before autoradiography.
Cell Viability Assay--
Cells were plated on 24-well dishes at
a density of 4 × 104 cells/well. After overnight
incubation, the media was changed to DMEM containing 20% fetal bovine
serum or the indicated concentrations of VEGF for 21 h. Cells were
then washed with PBS, harvested by trypsinization, and viability was
determined by the trypan blue exclusion assay (31, 32). Alternatively
cells were analyzed for the appearance of pyknotic nuclei. After
removal of culture media, 3.7% formaldehyde in PBS was added to the
culture. After fixation for 20 min, cells were washed in PBS twice and
stained with Hoechst 33342. Cells were examined with a Nikon Diaphot
microscope. Cell viability was also assessed by the MTS assay. Cells
were cultured in 96-well dish precoated with bovine serum albumin in the presence or absence of VEGF (100 ng/ml) for 21 h. MTS assay was performed according to the manufacturer's protocol (Promega). Cells were incubated with MTS reaction solution for 2 h.
Adenoviral Constructs and
Transfection--
Replication-defective adenovirus vectors expressing
mouse Akt proteins fused in-frame to the hemagglutinin (HA) epitope
under the control of the cytomegalovirus (CMV) promoter were
constructed according to the method of Becker (33). In brief, HA-Akt
fragment from pcDNA3-HA-Akt plasmid was inserted into the
multicloning site of pACCMVpLpA plasmid. pACCMVpLpA-HA-Akt was
co-transfected with pJM17 plasmid into 293 cells to allow for
homologous recombination. The mutant Akt and constitutively active Akt
adenoviral vectors were constructed by the same protocol. The
dominant-negative mutant Akt (T308A,S473A) protein cannot be activated
by phosphorylation (34) and it functions in a dominant-negative fashion
(35). The constitutively active Akt construct has the c-src
myristoylation sequence fused in-frame to the N terminus of the HA-Akt
(wild-type) coding sequence. Adenoviral Akt constructs were amplified
in 293 cells and purified by ultracentrifugation through a CsCl
gradient. Adeno-
-gal expresses the bacterial
-galactosidase gene
from the CMV promoter (36). HUVEC cultures were infected for 24 h with Ad-Akt or Ad-
-galactosidase at a multiplicity of infection of
50, achieving a 90% transduction efficiency (data not shown).
Electrophoretic Gel Mobility Analysis of DNA
Fragmentation--
Cells were cultured under the indicated conditions.
Floating and attached cells were collected and resuspended in 200 µl
of PBS containing proteinase K (0.5 mg/ml), RNase A (0.5 mg/ml), and
1% SDS. After 30 min incubation at 37 °C, 300 µl of NaI solution (6 M NaI, 13 mM EDTA, 0.5% SDS, 10 mg/ml
glycogen, 26 nM Tris-HCl, pH 8.0) was added. Cell lysates
were incubated at 60 °C for 15 min and 500 µl of isopropanol was
added. DNA was precipitated by centrifugation at 15,000 rpm for 15 min.
After washing once with 50% isopropanol and once with 100%
isopropanol, the DNA was dried. DNA (15 µg/lane) was examined
following electrophoresis on a 1.5% agarose gel.
Western Immunoblot Analyses--
Cells were treated as described
in the figure legends, and cell lysates were prepared as described for
the Akt kinase assays. 20 µg of protein was separated on
SDS-polyacrylamide gel electrophoresis gel and transferred onto a
polyvinylidene difluoride membrane (Millipore). After blocking with
T-PBS (PBS containing 0.2% Tween 20) containing 5% milk for 1 h,
the membrane was incubated with anti-Bcl-2 antibody (Transduction
Labs), anti-Akt antibody (Santa Cruz), phospho-specific Akt antibody
(New England Biolabs), or tubulin (Calbiochem). ECL (Amersham) was used
for detection.
 |
RESULTS |
VEGF Promotes HUVEC Survival--
Consistent with previous reports
(15, 37, 38), serum deprivation induces endothelial cell death (Fig.
1). Serum deprivation for 21 h
resulted in 50-80% decreases in HUVEC viability depending on the
preparation and passage number, where higher passage cells (passage 4 or 5) were more sensitive to death than lower passage cells (not
shown). It has been reported that VEGF inhibits HUVEC apoptosis induced
by serum deprivation (15). A dose dependence of VEGF in preventing
HUVEC death is shown in Fig. 1A. Under the conditions of our
assays, 100 ng/ml VEGF reversed cell loss induced by serum deprivation.
Consistent with an anti-apoptotic effect, treatment with VEGF also
diminished the frequency of endothelial cells exhibiting pyknotic
nuclei (Fig. 1B). Inclusion of 100 ng/ml VEGF diminished the
frequency of pyknotic nuclei from 19.6 ± 2.6 to 10.7 ± 0.8 in cultures that were incubated in the absence of serum for 21 h.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
VEGF protects HUVEC cultures against cell
death induced by the serum depletion. A, dose
dependence of VEGF cytoprotection. Cells were plated in 24-well dishes
overnight and then cultured in DMEM containing 20% fetal bovine serum
(open bar) or the indicated concentrations of VEGF
(closed bars). After 21 h in culture, cell viability
was determined by the trypan blue exclusion assay (31, 32) performed in
quadruplicate. B, VEGF reduces the frequencies of pyknotic
nuclei in HUVECs cultured under serum depletion conditions. HUVECs were
incubated in the presence (+) or absence ( ) of VEGF (100 ng/ml) for
21 h. Cells were stained with Hoechst 33342 as described under
"Materials and Methods."
|
|
The cell survival effects of VEGF were blocked by the PI3-kinase
inhibitor wortmannin. Inhibition of VEGF-mediated cell viability by
wortmannin was dose-dependent (Fig.
2A). Partial inhibition of
VEGF-mediated HUVEC survival occurred when cultures were incubated with
10 or 50 nM wortmannin, whereas 200 nM
wortmannin completely abrogated the survival effects of VEGF. 200 nM wortmannin also abrogated the VEGF-mediated diminution
of pyknotic nuclei in the serum-deprived cultures (Fig.
2B).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Wortmannin abrogates the cytoprotection of
VEGF. Cells were plated overnight and then cultured in DMEM
containing the indicated concentrations of wortmannin in the presence
(closed bars) or absence (open bar) of VEGF (100 ng/ml). A, after a 21-h culture, viability was determined by
the trypan blue exclusion assay performed in quadruplicate. Data are
shown as the mean ± S.E. (n = 4). B,
effects of wortmannin on VEGF-mediated reduction in the frequency of
HUVECs with pyknotic nuclei. Data are shown as the mean ± S.E.
(n = 4). Cells were cultured in the presence or absence
of VEGF with or without wortmannin for 21 h. Cells were stained
with Hoechst 33342, and cells with pyknotic nuclei were counted.
|
|
VEGF Activates Akt in HUVEC Cultures--
To determine whether
VEGF regulates the activity of Akt family proteins in endothelial
cells, HUVEC cultures were incubated in serum-free DMEM with or without
VEGF (100 ng/ml). The kinase activity of Akt was determined in lysates
immunoprecipitated with specific anti-Akt antibody. As shown in Fig.
3, VEGF activated Akt kinase activity in
mitogen-deprived HUVEC cultures. Because Akt is regulated by PI3-kinase
in other cell types (39), we investigated the effects of wortmannin on
Akt activation by VEGF. A 30-min preincubation period with 200 nM wortmannin blocked VEGF-induced Akt activity (Fig. 3).
Incubation with anti-Akt antibody competitor peptide diminished the H2B
phosphorylation signal to levels seen in cultures incubated with
wortmannin. Collectively, these data show that HUVEC survival
correlates with Akt activity.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
VEGF activates Akt in HUVEC cultures in a
wortmannin-dependent manner. HUVEC cultures were
preincubated in serum-free media in the presence (lane 3) or
absence (lanes 1, 2, and 4) of
wortmannin for 30 min. Cells were then stimulated with VEGF (100 ng/ml)
(lanes 2, 3, and 4) for 15 min. Cell
lysates were prepared and immunoprecipitated with anti-Akt antibody
with (lane 4) or without (lanes 1, 2,
and 3) competitor peptide. Kinase activity was measured with
histone H2B as a substrate.
|
|
Properties of Adenoviral Transgenes Expressing Wild-type,
Dominant-Negative, and Constitutively Active Akt--
To explore the
functional significance of VEGF-induced Akt activity in endothelial
cell survival, replication-defective adenoviral vectors expressing
wild-type (Adeno-wtAkt), dominant-negative (Adeno-dnAkt), or
constitutively active Akt (Adeno-myrAkt) were constructed (Fig.
4A). All adenovirus-encoded
transgenes were fused with the HA epitope to distinguish exogenous from
endogenous Akt. Control cultures were infected with an adenoviral
vector expressing
-galactosidase (Ad-
-gal), which does not affect
endothelial cell viability under the conditions of our assay (not
shown). Western blot analyses, using anti-Akt antibodies on anti-HA
immunoprecipitated material, revealed that Adeno-wtAkt, Adeno-dnAkt,
and Adeno-myrAkt expressed comparable levels of protein (Fig.
4B). Akt protein kinase activity was detected in the anti-HA
immunoprecipitates from cells infected with Adeno-wtAkt in the
presence, but not the absence, of VEGF (Fig. 4B) In
contrast, no kinase activity was detected in the cultures infected with
the Adeno-dnAkt vector in the presence or absence of VEGF. Cultures
infected with Adeno-myrAkt contained high levels of HA-associated Akt
activity that was not influenced by VEGF stimulation.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Adenovirus constructs expressing wild-type
Akt and its mutants. A, structures of adenovirus
vectors expressing wild-type, dominant-negative, and constitutively
active Akt are indicated. B, HUVECs were infected
with adenovirus vector expressing Akt or -galactosidase at
multiplicity of infection 50 as described under "Materials and
Methods." After 30 min of serum starvation, cells were stimulated
with VEGF (100 ng/ml) for 15 min. Cell lysates were immunoprecipitated
with anti-HA antibody. Kinase activity was measured with histone H2B as
a substrate (top panel). Anti-HA-immunoprecipitated protein
was Western blotted with anti-Akt antibody (bottom
panel).
|
|
Akt Is Essential for VEGF-mediated Cell Survival--
HUVEC
cultures were infected with Adeno-dnAkt to test whether Akt is
essential for VEGF-mediated survival. In the absence of growth factor,
the dominant-negative Akt construct did not affect endothelial cell
survival, but it inhibited the cytoprotective action of VEGF (Fig.
5A). DNA prepared from the
serum-deprived HUVEC cultures displayed the typical nucleosome spacing
ladder upon agarose gel electrophoresis that is indicative of apoptosis (Fig. 5B). The DNA ladder was diminished by the inclusion of
VEGF in the culture media. Consistent with the data from the trypan blue exclusion assays, infection with Adeno-dnAkt blocked the ability
of VEGF to inhibit DNA fragmentation.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 5.
Dominant-negative Akt abrogates
VEGF-mediated cell survival. HUVEC cultures were infected with
adenovirus expressing -galactosidase ( -gal) or
dominant-negative Akt (dnAkt). Cells were incubated with (+)
or without ( ) VEGF (100 ng/ml) for 21 h (A). Viable
cells were counted using a trypan blue exclusion assay. Data are shown
as the mean ± S.E. (n = 4) (B).
Floating and attached cells were collected, and DNA fragmentation was
analyzed as described under "Materials and Methods."
|
|
Akt Promotes Endothelial Cell Survival--
Adenoviral
transduction of wild-type Akt markedly augmented VEGF-induced
endothelial cell survival (Fig.
6A). Cultures infected with
Ad-Akt displayed 75 and 65% less cell death than control cultures at 1 and 10 ng/ml, respectively, whereas no decrease in cell death was
detected in the cultures exposed to serum-free media in the absence of
VEGF. These data show that forced expression of wild-type can enhance
the sensitivity of endothelial cells to VEGF survival signals at
subsaturating levels of VEGF.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Akt promotes endothelial cell survival.
A, HUVEC cultures were transfected with adenovirus
expressing -galactosidase ( -gal) or wild-type Akt
(wtAkt) at a multiplicity of infection of 50. Cells were
cultured in the indicated concentrations of VEGF for 21 h. Viable
cells were determined by trypan blue exclusion assay. Data are shown as
the mean ± S.E. (n = 4). B, HUVEC
cultures were transfected with adenovirus expressing -galactosidase
( -gal) or constitutively active Akt (myrAkt).
Cells were cultured in the serum-depleted condition for 21 h.
Cells were fixed and stained with Hoechst 33342. The percentage of
cells with pyknotic nuclei is demonstrated. Data are shown as the
mean ± S.E. (n = 4).
|
|
In contrast to infection with Adeno-wtAkt, infection of cultures with
Adeno-myrAkt promoted endothelial cell survival in the absence of
growth factor. Analyses of serum-deprived HUVEC cultures revealed that infection with Adeno-myrAkt significantly decreases the
frequency of pyknotic nuclei (Fig. 6B) to levels similar to that observed in the presence of VEGF (Fig. 2B).
Akt Activation Is Essential but not Sufficient for VEGF-mediated
Induction of Bcl-2--
A previous study showed that VEGF induces the
anti-apoptotic protein Bcl-2 in HUVECs and that forced expression of
Bcl-2 is sufficient to prevent apoptosis in the absence of VEGF (15). Thus, we used the adenoviral Akt vectors to test for the potential involvement of Akt activation in VEGF-mediated Bcl-2 induction. As
shown in Fig. 7, VEGF stimulation led to
a modest induction of Bcl-2. This up-regulation was blocked by
infection with Adeno-dnAkt, demonstrating that Akt is essential for
VEGF-mediated induction of Bcl-2. However, infection with Adeno-myrAkt
was not sufficient to induce Bcl-2, suggesting that the endothelial
cell survival conferred by Akt under these conditions involves other
apoptosis-regulatory proteins.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Akt activation is essential but not
sufficient for VEGF-mediated induction of Bcl-2. HUVECs were
transfected with adenovirus expressing -galactosidase
( -gal), dominant-negative Akt (dnAkt) or
constitutively active Akt (myrAkt). Cells were cultured in
media containing 2% fetal calf serum in the presence or absence of
VEGF (100 ng/ml) for 24 h. Cells were harvested, and cell lysates
were analyzed by Western blot with anti-Bcl-2 or anti- -tubulin
antibody.
|
|
Akt-mediated Anchorage-dependent Survival
Signals--
To examine the role of Akt in
anchorage-dependent endothelial cell survival, HUVECs were
cultured in suspension under serum-deprivation conditions in
nonadhesive bacteriological dishes. Under these conditions, endothelial
cells rapidly underwent anoikis (19) as indicated by cell shrinkage and
membrane blebbling, characteristic of the apoptotic morphology (Fig.
8A). Inclusion of VEGF in the suspension culture media had no effect on cell morphology. However, cells pre-infected with the adenoviral construct expressing
constitutively active Akt appeared viable in suspension culture (Fig.
8A). To confirm that constitutively active Akt construct
promotes endothelial cell viability under these conditions, MTS assays
were performed on cells cultured under these conditions (Fig.
8B). Consistent with observations of cell morphology,
infection with Adeno-myrAkt preserved mitochondrial function, an
indicator of cellular viability (40). These data show that constitutive
Akt activity, but not stimulation with VEGF, is sufficient to confer
survival in endothelial cells in the absence of matrix
attachment.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 8.
Constitutively active Akt confers resistance
to apoptosis induced by cell detachment. Cells were transfected
with adenovirus expressing -galactosidase ( -gal) or
constitutively active Akt (myrAkt). Cells were collected
with EDTA treatment and cultured in a suspension system with (+) or
without ( ) VEGF (100 ng/ml) as described under "Materials and
Methods." A, cells were cultured for 12 h in
suspension system and examined with a phase contrast microscope.
B, cells were cultured in suspension system for 21 h
and then MTS assays were performed. Data are shown as the mean ± S.E. (n = 5).
|
|
To further study the role of Akt in anchorage-dependent
endothelial cell survival, activated Akt levels were assessed in HUVEC monolayer and suspension cultures using an antibody that is specific for Akt phosphorylated at residue 473 and indicative of the status of
Akt activation (34). Consistent with measurements of Akt-associated histone H2B-kinase activity (Fig. 3A), VEGF stimulation of
HUVEC monolayers increased the level of phosphorylated (activated) Akt with no detectable change in the total level of Akt protein (Fig. 9A). However, VEGF stimulation
had no effect on the level of Akt phosphorylation within 1 h of
incubation in suspension culture (Fig. 9A). Moreover, these
suspension culture cells (<1 h) displayed lower levels of basal Akt
phosphorylation though levels of Akt protein were comparable between
attached cells and cells in suspension. VEGF stimulation of HUVECs in
suspension for 21 h also did not induce Akt phosphorylation. At
this time, HUVEC death is prevalent (Fig. 8) and Akt protein levels are
reduced relative to tubulin (Fig. 9B).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 9.
VEGF cannot activate Akt in suspension
culture. Cells were cultured in either monolayer (M) or
suspension (S) conditions for 15 min (A) or
21 h (B). After 45 min of serum deprivation, cells were
cultured with (+) or without ( ) VEGF (100 ng/ml) for 15 min
(A) or 21 h (B). Cell lysates were prepared
and immunoblotted with anti-phosphospecific Akt (p-Akt),
anti-Akt (Akt), or anti-tubulin antibody.
|
|
 |
DISCUSSION |
Exposure of endothelial cells to survival factors and proper
cell-matrix attachments are required for the formation of new blood
vessels as well as the maintenance of existing vessels. Consistent with
its function as a survival factor, VEGF withdrawal is associated with
vascular regression in both developing retina and tumors (13, 16, 17,
41). In this study, we explored the role of the Akt protein kinase in
mediating the survival functions of VEGF and matrix attachment in
endothelial cells. We have shown that VEGF-induced survival in
monolayer cultures correlates with the wortmannin-sensitive activation
of the Akt protein. The functional significance of Akt activation is
indicated by the finding that adenovirus-mediated expression of a
dominant-negative Akt mutant inhibited the cell survival effect of
VEGF. We also showed that forced expression of constitutively active
Akt is sufficient to confer survival to serum-deprived endothelial
cells. Following the initial submission of this paper, Gerber et
al. (42) reported that VEGF activates Akt and that
plasmid-mediated overexpression of constitutively active Akt protects
endothelial cells from apoptosis, whereas a dominant-negative Akt
construct inhibits the cytoprotection conferred by VEGF. The combined
results of these two studies demonstrate that Akt activation is
essential for VEGF-induced cytoprotection and that plasmid- or
adenovirus-mediated gene transfer of constitutively active Akt can
suffice for VEGF with regard to cell survival. Here, we have also shown
that adenovirus-mediated transfer of wild-type Akt does not promote
endothelial cell survival in the absence of VEGF, but it could
potentiate the survival effects of subsaturating levels of VEGF. Thus,
these data indicate that mitogen-induced activation of Akt can be a
limiting event in the signaling cascade that controls endothelial cell viability.
Though growth factors initiate angiogenic process, it has been proposed
that proper matrix associations are essential for endothelial cell
survival during neovascularization as cells migrate toward the
angiogenic source (20). Thus, it was of interest to test whether Akt
was also involved in attachment-mediated endothelial cell survival and,
if so, how VEGF and matrix survival signals were coordinated with
regard to Akt. Consistent with reports in endothelial cells and other
cell types (18), HUVECs rapidly underwent apoptosis when incubated in
suspension culture. Though VEGF stimulation did not inhibit apoptosis
under these conditions, transduction of constitutively active Akt was
sufficient to promote cell viability in the absence of matrix
attachment. Immediately following cell detachment, VEGF was unable to
stimulate Akt activation via phosphorylation of serine 473 suggesting
that matrix attachment is essential for signal transduction events
upstream of Akt. At later time points in suspension culture, Akt
protein was selectively lost suggesting that reductions in Akt protein
might also contribute to this reduction in cell viability. However,
adenovirus-mediated overexpression of wild-type Akt in either the
presence or absence of VEGF stimulation had no effect on endothelial
cell viability in suspension culture (data not shown). Therefore, it
appears that the loss of Akt protein is a consequence of the apoptotic process, as has been found in Jurkat and U937 cell death (43), and that
apoptosis initiates from a failure of VEGF to promote Akt activation in
the absence of matrix attachment.
As noted above, attachment appears to be required for VEGF-initiated
signal transduction events upstream of Akt. It has recently been shown
that
v
3 occupancy on smooth muscle cells
stimulates insulin-like growth factor-1 signal transduction by
enhancing the tyrosine kinase activity of the receptor (44), and
similar mechanisms may be operating in endothelial cells with regard to VEGF activation of the Flk-1 receptor. An alternative hypothesis is
that VEGF may function by modulating attachment-mediated survival signals. Of note, in the absence of VEGF stimulation, cell attachment has a marked influence on the basal level of Akt activation (see Fig.
9A). These data suggest that growth factor-independent
mechanisms of Akt activation exist within endothelial cells, and it is
possible that VEGF stimulation serves to facilitate these
anti-apoptotic signals from integrins and the extracellular matrix, the
production of which are also regulated by VEGF (14).
In endothelial cells, VEGF promotes tyrosine phosphorylation of various
signal transducers, including PI3-kinase, Ras GTPase-activating protein, and phospholipase C-
(11). Here, we have shown that VEGF-induced Akt activation and cytoprotection in endothelial cells was
suppressed by wortmannin, a PI3-kinase inhibitor, indicating that Akt
acts downstream of PI3-kinase in the VEGF signaling pathway. In
contrast, VEGF mitogenic functions are transduced through protein kinase C, which is not inhibited by wortmannin (12). Taken together, these data suggest that the cytoprotective and mitogenic signals of
VEGF are transduced by independent pathways in endothelial cells.
In summary, the findings of this study show that Akt is essential in
regulating both VEGF- and attachment-mediated survival signals in
endothelial cells. In particular, these data document the importance of
cross-talk between these two signaling pathways in promoting Akt
activation with consequences on endothelial cell survival. Because
endothelial cell viability is essential for the maintenance of new
blood vessels, further analysis of Akt may provide new insights about
the regulatory control of angiogenesis during normal development and tumorigenesis.