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Vascular Endothelial Growth Factor Regulates Endothelial Cell
Survival through the Phosphatidylinositol 3*-Kinase/Akt Signal
Transduction Pathway
REQUIREMENT FOR Flk-1/KDR ACTIVATION*
(Received for publication, March 26, 1998, and in revised form, July 28, 1998)
Hans-Peter Gerber‡, Amy McMurtrey‡, Joe Kowalski‡, Minhong Yan§, Bruce A. Keyt‡,
Vishva Dixit§, and Napoleone Ferrara‡¶
From the Departments of ‡Cardiovascular Research and §Molecular Oncology, Genentech, Inc.,
South San Francisco, California 94080
Vascular endothelial growth factor (VEGF) has been
found to have various functions on endothelial cells, the
most prominent of which is the induction of prolifera-
tion and differentiation. In this report we demonstrate
that VEGF or a mutant, selectively binding to the Flk-1/
KDR receptor, displayed high levels of survival activity,
whereas Flt-1-specific ligands failed to promote survival
of serum-starved primary human endothelial cells. This
activity was blocked by the phosphatidylinositol 3*-ki-
nase (PI3-kinase)-specific inhibitors wortmannin and
LY294002. Endothelial cells cultured in the presence of
VEGF and the Flk-1/KDR-selective VEGF mutant in-
duced phosphorylation of the serine-threonine kinase
Akt in a PI3-kinase-dependent manner. Akt activation
was not detected in response to stimulation with pla-
centa growth factor or an Flt-1-selective VEGF mutant.
Furthermore, a constitutively active Akt was sufficient
to promote survival of serum-starved endothelial cells
in transient transfection experiments. In contrast, over-
expression of a dominant-negative form of Akt blocked
the survival effect of VEGF. These findings identify the
Flk-1/KDR receptor and the PI3-kinase/Akt signal trans-
duction pathway as crucial elements in the processes
leading to endothelial cell survival induced by VEGF.
Inhibition of apoptosis may represent a major aspect of
the regulatory activity of VEGF on the vascular
endothelium.
Angiogenesis and vascular remodeling occur throughout
growth and development and involve both proliferation and
regression of vascular endothelial cells. Although extensive
research has been dedicated to the elucidation of the factors
that induce endothelial cell proliferation and differentiation
(for review, see Refs. 1, 2), surprisingly little is known about
the mechanisms that regulate regression of blood vessels.
Rapid degeneration of vascular structures occurs under various
physiological or pathological circumstances. Apoptosis of endo-
thelial cells probably contributes to the maturation of vascular
granulation tissue into avascular scar tissue (3) and to the
pruning of retinal vessels during development (4). Apoptosis-
like changes in endothelial cells have been also observed in the
involuting corpus luteum, breast (5), parotid glands undergo-
ing pressure atrophy (6), and fibrotic lung lesions (7). However,
experiments designed to study blood vessel regression in vivo
have not been very successful so far at detecting apoptotic
endothelial cells lining the blood vessels. This may be attrib-
utable, at least in part, to the fact that endothelial cells under-
going apoptosis tend to lose their attachment to the basement
membrane. In addition, apoptotic endothelial cell may become
the target of the immune system and thus are eliminated by
phagocytes as soon as they manifest early signs of apoptosis (8).
In vitro, apoptosis often follows withdrawal of a critical trophic
factor. Accordingly, human umbilical vein endothelial (HUVE)
1
cells undergo apoptosis after loss of adhesion or serum depri-
vation (9, 10). Fibroblast growth factor and phorbol esters
reduce the apoptosis of serum-deprived HUVE cells (11), and
heat shock and endotoxin act together to increase apoptosis in
porcine endothelial cells (12).
It has recently been shown that the survival signal mediated
by various growth factors and cytokines may be dependent on
the phosphatidylinositol 39-kinase (PI3-kinase)/Akt signal
transduction pathway (13–19). However, not all survival sig-
nals require PI3-kinase activity, and Akt (also referred as
protein kinase B
a
or Rac
a
)-independent survival pathways
exist (20–22). It was shown previously that vascular endothe-
lial growth factor (VEGF) can induce PI3-kinase activity in a
variety of endothelial cell (23–25), but so far a specific biological
function of PI3-kinase in endothelial cells has not been
demonstrated.
The endothelial cell-specific mitogen VEGF has been shown
to be a key positive regulator of normal and abnormal angio-
genesis (2, 26). The critical role of VEGF in the development of
the vascular system is emphasized by embryonic lethality after
loss of a single VEGF allele. A growing body of evidence indi-
cates that VEGF may also act as a survival factor for newly
formed blood vessels. In the developing retina, vascular regres-
sion in response to hyperoxia has been correlated with inhibi-
tion of VEGF release by glial cells (4). Furthermore, adminis-
tration of anti-VEGF monoclonal antibodies results in
regression of already established tumor-associated vasculature
in xenograft models (27). More recently, using a tetracycline-
regulated VEGF expression system in xenografted C6 glioma
cells, it has been shown that decreased levels of VEGF produc-
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
¶To whom correspondence should be addressed: Dept. of Cardiovas-
cular Research, Genentech, Inc., 1 DNA Way, South San Francisco, CA
94080. Tel.: 650-225-2968; Fax: 650-225-6327.
1
The abbreviations used are: HUVE, human umbilical vein endothe-
lial; PI3-kinase, phosphatidylinositol 39-kinase; VEGF, vascular endo-
thelial growth factor; rh, recombinant human; PlGF, placenta growth
factor; PBS, phosphate-buffered saline; PI, propidium iodide; bFGF,
basic fibroblast growth factor; sel, selective; KDR, kinase domain
region.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 46, Issue of November 13, pp. 30336–30343, 1998
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org30336
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tion lead to detachment of endothelial cells from the walls of
preformed vessels in the tumor, and detached apoptotic endo-
thelial cells were identified by means of double staining for Van
Willebrand factor and terminal deoxynucleotidyl transferase-
mediated biotinylated UTP nick end labeling staining (28).
VEGF exerts its biological effects by binding to its respective
transmembrane receptors VEGF receptor 1 (Flt-1) and VEGF
receptor 2 (Flk-1/KDR), both of which are expressed on endo-
thelial cells specifically and contain a cytoplasmic tyrosine
kinase domain. Knockout studies in mice revealed that both
receptors are essential for the development of the embryonic
vasculature, and mouse embryos null for either receptor died in
utero between days 8.5 and 9.5 (29, 30). Stimulation of endo-
thelial cells with VEGF demonstrated that VEGF induces
phosphorylation of a variety of proteins, including phosphati-
dylinositol 3-kinase, Ras GTPase-activating protein, p190-
rhoGAP, p62, phospholipase C-
g
, the oncogenic adaptor protein
NcK, p125 focal adhesion kinase, paxilin, and several others
(23, 31–37; for review, see Ref. 38). Strong experimental evi-
dence links Flk-1/KDR activation to VEGF-induced mitogene-
sis and angiogenesis (2). In contrast, the maintenance and
survival function of VEGF has been associated with the Flt-1
receptor, because the Flt-1 mRNA is highly expressed in qui-
escent endothelium (39), whereas Flk-1/KDR is primarily ex-
pressed in proliferating vessels (40).
In the present study, we investigated the survival role of
VEGF in HUVE cells cultured in serum-free conditions. We
found that VEGF potently prevents apoptosis and promotes
survival in this system. Using several approaches, we demon-
strate the critical role of the PI3-kinase-Akt pathway in such
effects. We also show that Flk-1/KDR, not Flt-1, is the primary
mediator of such a signal.
EXPERIMENTAL PROCEDURES
Materials—Primary cultures of HUVE cells and human dermal mi-
crovascular endothelial cells were purchased from Cell Systems (Kirk-
land, WA). Wortmannin and dimethylsulfoxide were purchased from
Sigma. ZVAD-fmk was purchased from Enzyme Systems Products
(Dublin, CA). Cytomegalovirus-luciferase was purchased from Pro-
mega, and pGREEN LANTERN was from Life Technologies, Inc..
The KDR receptor-selective VEGF
165
mutant D63A, E64A, E67A has
been previously described (41). The triple mutant R82E, K84E, H86E is
a derivative of the Flt-1-selective R82A, K84A, H86A VEGF mutant
(41), with ;10-fold higher selectivity, which results in an overall 1000-
fold selective binding of Flt-1 over KDR. Wild-type recombinant human
(rh) VEGF
165
and receptor-selective VEGF mutants were expressed in
Escherichia coli and purified to homogeneity as described previously.
The endotoxin content of the final purified material did not exceed 0.3
endotoxin units/mg of protein. rhPlGF
152
was expressed and purified as
described previously (42). Recombinant proteins were quantified by
amino acid analysis. Medium 199 was purchased from Life Technolo-
gies, Inc. CS-C medium was from Cell Systems. EB medium was from
Clonetics (San Diego, CA). Flag-Akt179 was generated by mutation of
the lysine 179, which is critical for ATP binding to alanine, which
resulted in a kinase death Akt construct (43). Flag-Akt 2D was gener-
ated by mutating threonine 308 and serene 473 to asparagine, which
results in a constitutively active Akt form (44). PlGF used in the Akt
phosphorylation assay was purchased fromR&DSystems, Inc (Min-
neapolis, MN).
Cell Culture—HUVE cells were maintained in CS-C complete me-
dium containing 10% fetal bovine serum and mitogens, according to the
instructions of the supplier (Cell Systems). Twenty-four hours before
serum starvation, cells were trypsinized and plated to a density of
20–25 310
3
cells/cm
2
in 10- or 6-cm dishes for fluorescence-activated
cell sorting analysis and in 6-cm dishes for biolistic transfection exper-
iments. Immediately before the experiment, cells were washed twice
with phosphate-buffered saline (PBS). For serum starvation, CS-C me-
dium without serum and without mitogen, complemented with 0.1%
bovine serum albumin, was added during the indicated length of time.
After biolostic transfection, cells were allowed to recover for 24 h in
medium containing 10% serum. After washing the cells twice with
Tris-buffered saline, minimum medium (without mitogen and serum)
was added, and the levels of apoptosis were analyzed 9 h after induction
of serum starvation.
For survival assays, HUVE cells were seeded at the density of 2 310
4
cells per well in 48-multiwell plates in the presence of EB medium plus
10% FBS in 0.5 ml of M199 medium containing 10% serum. After 8–12
h, cells were washed twice with serum-free M199, and cells were incu-
bated in serum-free M199 complemented with 0.1% bovine serum al-
bumin, with or without growth factors. After 4 or 5 days, 25
m
l of alamar
blue was added to each well. After a 2-h incubation time, plates were
read at 405 nm in an enzyme-linked immunosorbent assay plate reader.
Alternatively, cells were dissociated by exposure to trypsin, and the cell
number was determined.
Gene Transfer of HUVE Cells by Microprojectile Bombardment—
Gene transfer was performed using the Bio-Rad 500 optimization kit in
the Biolistic PDS-1000/He* particle delivery system (Bio-Rad). In a
series of pilot experiments using luciferase reporter gene DNA, param-
eters were established to achieve the highest levels of luciferase activity
in primary HUVE cell cultures (data not shown): particle size, 1.6
m
m;
rupture disc, 1100 p.s.i. bombardment chamber vacuum, 25 mm Hg; the
microcarrier launch assembly was positioned in shelf position 1, the
target shelf was at position 3 (from the top). Gold particles were pre-
pared according to the manufacturer’s recommendations and stored as
50-
m
l aliquots for 3–4 months at 220 °C. Aliquots were coated with
DNA immediately before gene transfer following the manufacturer’s
recommendations. Briefly, for five samples, a 50-
m
l aliquot of gold
particles was vortexed for 3 min. and 15
m
l of plasmid DNA (1 mg/ml)
was added. Vortexing was continued for 2 min. Thereafter, 50
m
lof2.5
MCaCl
2
solution was added to the mix. After 2 min of additional
vortexing, 20
m
l of a freshly prepared 0.1 Mspermidine solution was
added, followed by 2 min of constant vortexing. The mixture was al-
lowed to settle for 1 min before tubes were centrifuged for5sina
table-top microcentrifuge. The supernatant was removed, and the gold
pellet was washed first with 140
m
l of 70% ethanol followed by 140
m
lof
100% ethanol. After removing the supernatant, the gold particles were
taken up in 55
m
l of 100% ethanol, and 10
m
l was used per biolistic
transfection. Immediately after bombardment, 5 ml of complemented
medium was added.
Analysis of Endothelial Cell Apoptosis—For fluorescence-activated
cell sorting analysis, cells were stained by fluorescein isothiocyanate-
conjugated annexin V and by the fluorescent dye propidium iodide (PI).
Cells negative for both PI and annexin V staining are live cells; PI-
negative, annexin V-positive staining cells are early apoptotic cells;
PI-positive, annexin V-positive staining cells are primarily cells in a
late stage of apoptosis (45). Cells of one 6- or 10-cm dish were harvested
and incubated on ice in annexin- and PI-containing binding buffer for 10
min and further processed as described previously (46).
Time lapse video recording was done with an Optronics DEI-470T
charge-coupled device video camera hooked up to a Panasonic AG-6730
time lapse cassette recorder and a Nikon Diaphot-300 inverted micro-
scope at a magnification of 203. The graphics program Adobe Photo-
shop 4.0 was used to generate the picture.
Akt Phosphorylation Assay—HUVE cells were grown essentially as
described in cell culture. Cells were split to a density of 14 310
3
cells/cm
2
24 h before start of serum starvation. Cells were washed twice
with PBS, and 5 ml of CS medium without serum and mitogen (Cell
Systems), complemented with 0.1% bovine serum albumin, was added.
18–20 h after start of serum starvation, cells were washed with PBS,
and 5 ml of fresh starvation medium was added, containing wortman-
nin (30 nM) where indicated. 2–3 h later, growth factors were added at
the indicated amounts, and incubation occurred for 15 min in a tissue
culture incubator before lysing the cells with 100
m
l of SDS gel loading
buffer. For detection of the levels of Akt phosphorylation, whole-cell
extracts were treated essentially as described in the instruction manual
included in the PhophoPlus Akt (Ser
473
) antibody kit (New England
BioLabs, Beverly, MA). Blocking of the membrane was done by incuba-
tion overnight at 4 °C; incubation with the phospho-specific Akt (Ser
473
)
antibody (rabbit polyclonal IgG, affinity purified) occurred at room
temperature for 3 h; and the incubation with the secondary antibody
(anti-rabbit secondary antibody conjugated to horseradish peroxidase)
wasfor1hatroom temperature. The immune complexes were detected
with the enhanced chemiluminescence detection system. The chemilu-
minescent detection was provided by the manufacturer (New England
Biolabs, Beverly, MA).
RESULTS
Growth Factor Removal Induces Apoptotic Endothelial Cell
Death—To investigate the survival activity of VEGF relative to
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that of basic fibroblast growth factor (bFGF), which was previ-
ously found to be a survival factor for endothelial cells (11), we
incubated HUVE cells in serum-free medium in the presence of
VEGF (100 ng/ml) or bFGF (100 ng/ml). Control cells were
cultured in the presence of 10% serum, which stimulated pro-
liferation. As illustrated in Fig. 1A, the number of cells de-
creased significantly after 24 h of serum starvation, whereas
cell numbers remained relatively constant over 48 h of incuba-
tion in the presence of VEGF or bFGF. These findings indicated
that VEGF can induce a survival response in HUVE cells to
similar levels as bFGF. In the absence of VEGF, extensive
membrane blebbing, loss of contacts with neighboring cells,
shrinking of the cytoplasm, disintegration into small vesicles,
also called apoptotic bodies, and ultimately detachment of cells
from the culture dish were observed by means of time lapse
video recording (Fig. 1B). These features are indicative of apo-
ptotic cell death. Under such conditions, we did not observe any
obvious morphological differences between VEGF-treated cells
and cells exposed to 10% serum (data not shown). To determine
whether the endothelial cell loss was a consequence of apop-
totic cell death, we analyzed HUVE cells for binding to annexin
V, which serves as a marker for early apoptosis (46). As early as
16 h after growth factor removal, we observed a significant
increase in the number of apoptotic, annexin V-positive cells,
which continued over time, and after 48 h, 50–80% of the cells
had undergone apoptotic cell death (Fig. 2Band data not
shown).
To test whether the observed induction of endothelial cell
apoptosis was mediated by the universal cell death machinery,
we added ZVAD-fmk, an inhibitor of caspases I (interleukin-1
b
-converting enzyme) (ICE) and 3 (CPP32) (48) to HUVE cells.
Apoptosis induced by serum starvation was completely blocked
after 24 h by ZVAD-fmk at concentrations as low as 2
m
M. These
findings indicated that endothelial cells grown in culture un-
derwent caspase-dependent, programmed cell death on serum
withdrawal.
VEGF Is a Survival Factor for Primary Human Endothelial
Cells: Requirement for Flk-1/KDR Activation—VEGF reduced
the serum starvation-induced apoptosis of HUVE cell with a
bell-shaped dose-response curve, with a maximal effect be-
tween 10 and 100 ng/ml (Fig. 2A). Higher concentrations re-
sulted in lower reduction of apoptosis. Human dermal micro-
vascular endothelial cells responded at even lower VEGF levels
(0.3 ng/ml; data not shown). Similar bell-shaped dose-response
curves in response to VEGF in cell-based assays have been
reported before (49). Although the cause of this effect is not
entirely clear, a current thought is that a vast excess of ligand
over receptor results in a reduced effectiveness of receptor
dimerization and signaling, such that an increasing number of
receptors remain in a monomeric form after ligand binding.
However, the percentage of apoptotic cells detected in the pres-
ence of 10% serum was usually lower compared with cells
grown in the presence of VEGF, at all concentrations tested,
suggesting that additional factors present in the serum render
the cells more resistant to apoptosis (9). Whether this is a
consequence of increased survival activity or is attributable to
the ongoing proliferation, or both, remains to be analyzed.
We next attempted to determine the role of the two VEGF
receptors in mediating the survival activity induced by VEGF.
We first tested placenta growth factor (PlGF), a member of the
VEGF family of proteins, which binds Flt-1 with high affinity
but fails to bind Flk-1/KDR. Previous studies have shown that
FIG.1.Primary endothelial cells in culture undergo apoptotic
cell death after serum starvation. A, HUVE cells were plated at the
density of 1.5 310
5
cells per well in six-well dishes and cultured for 24 h
in the presence of 10% serum. Cells were then washed twice with PBS
before adding serum-free medium containing 0.1% bovine serum albumin
complemented with either VEGF (100 ng/ml) or bFGF (100 ng/ml). 10%
serum was added to control wells. 24 or 48 h after changing to serum-free
medium, cells were trypsinized and counted in a cell counter. Error bars
indicate the S.D. of triplicate plates. B, HUVE cells were plated at the
density of 20,000 cells/cm
2
and cultured for 24 h in medium containing
10% serum. Immediately after changing to serum-free medium, rh-
VEGF
165
was added (100 ng/ml), and a photograph was taken after 24 h.
Photographs were taken with a computerized video imaging system. C,
HUVE cells were plated at the density of 200,000 cells/cm
2
and grown for
24 h in the presence of 10% serum. Two hours before switching to serum-
free conditions, dimethylsulfoxide (control) or ZVAD was added. Cells
were washed twice in PBS, and medium containing 10 serum, 0% serum,
dimethylsulfoxide (DMSO), or ZVAD in the indicated amounts was added
24 h after initiation of serum starvation. Cells were then harvested, and
apoptotic cell death was determined by flow cytometry by annexin V
binding and propidium iodide staining, as described under “Experimen-
tal Procedures.” Bars represent means 6S.E. of two independent ex-
periments conducted with different preparations of endothelial cells.
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PlGF induces little or no mitogenic activity on HUVE cells, nor
does it induce tyrosine phosphorylation of Flt-1 (42). In our
survival assay, PlGF did not reduce the number of apoptotic
HUVE cells at all concentrations tested, as assessed by binding
to annexin V (Fig. 2, Band D) or alamar blue staining of cells
(Fig. 2C). To further confirm these findings, we tested receptor-
selective VEGF mutants, which bind preferentially to either
one of the VEGF receptors, KDR/Flk or Flt-1, respectively.
Previous studies have shown that mutants defective in KDR
binding lack the ability to induce proliferation, whereas Flt-1
binding-deficient mutants are effective mitogens (41). We
found that the KDR-selective VEGF
165
mutant (KDR-sel) ex-
FIG.2.VEGF is a survival factor for primary HUVE cells in culture. A, HUVE cells were grown for 24 h in the presence of 10% serum and
rhVEGF
165
was added immediately after changing to serum-free conditions. Apoptotic cell death was determined after 24 h by flow cytometry for
annexin V and propidium iodide, as described under “Experimental Procedures.” Bars represent means 6S.E. of two independent experiments
conducted with different preparations of endothelial cells. B, HUVE cells were cultured for 48 h in the presence of the indicated amounts of
rhVEGF
165
or PlGF. Apoptotic cell death was determined as described above. C, After 8–12 h in serum-containing media, HUVE cells were washed
twice and incubated in 0% serum in the presence of rhVEGF
165
(100 ng/ml), Flt-1-sel (100 ng/ml), which is an Flt-1-selective mutant VEGF
165
form,
or KDR-sel (100 ng/ml), which binds Flk-1/KDR 100-fold more selective than VEGF (for descriptions see “Materials” under “Experimental
Procedures”). The relative number of cells was quantified after 4 days in culture by alamar blue staining, as described under “Experimental
Procedures.” Error bars represent the S.E. of triplicate analysis of one representative experiment. D, HUVE cells were cultured for 36–48 h in the
presence of the indicated concentrations of VEGF, PlGF, and the receptor-selective mutant VEGF forms Flt-1-sel and KDR-sel. The number of
apoptotic cells was determined as described in A. Data points represent the average of three independent experiments with different HUVE cell
preparations.
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erted a similar survival activity as wild-type VEGF in the 4-day
survival assay. However, we could not detect any significant
survival effect by the Flt-sel mutant (Fig. 2C).
In agreement with these findings, neither PlGF nor the
Flt-sel VEGF mutant led to a reduction in the amount of
apoptotic HUVE cells after 36–48 h, as assessed by annexin V
binding (Fig. 2D). In contrast, the KDR-sel mutant was as
potent as wild-type VEGF
165
. From these findings we conclude
that activation of Flk-1/KDR is necessary to mediate the sur-
vival-promoting activity of VEGF.
The Antiapoptotic Activity of VEGF Is Mediated by PI3-
kinase—The PI3-kinase-specific inhibitor wortmannin is
known to block the protective action of nerve growth factor (50)
and platelet-derived growth factor in serum-deprived PC12
cells (14). These findings implicated an important role of the
PI3-kinase pathway in apoptosis prevention by growth factors.
To examine the involvement of PI3-kinase in VEGF-mediated
protection, we incubated HUVE cells with the two specific
inhibitors of PI3-kinase, wortmannin and the structurally un-
related synthetic compound LY294002 (51). As assessed by the
numbers of apoptotic cells, wortmannin (30 nM) and LY294002
(100 nM) almost completely blocked the protective effect of
VEGF (Fig. 3, Aand B). Both agents slightly enhanced the
degree of apoptosis observed in the absence of VEGF, possibly
resulting from the inhibition of basal PI3-kinase activity pres-
ent in serum-starved cells, similarly to what has been observed
in other systems (16, 18, 52). Our results support the conclu-
sion that the antiapoptotic activity of VEGF on endothelial
cells is mediated via PI3-kinase.
The Flk-1/KDR-selective VEGF Mutant but Not Flt-1-selec-
tive Ligands Triggers Akt Phosphorylation—To test whether
the PI3-kinase effector protein Akt is involved in the survival
response, we analyzed whole-cell lysates of HUVE cells treated
with VEGF for activation of Akt by phosphorylation at serine
473 by means of a phospho-specific antibody. Akt is activated
by phospholipid binding, and phosphorylation occurs within
the activation loop at threonine 308 and within the carboxyl
terminus at serine 473. In initial time course experiments we
found Akt to be maximally activated 15–30 min after exposure
to VEGF. The response gradually decreased after prolonged
incubation (data not shown). Incubation of HUVE cells with
increasing concentrations of VEGF led to a dose-dependent
activation of Akt, with highest levels between 1 and 10 ng/ml
(Fig. 4A). Densitometric analysis of the signals displayed in
Fig. 4 revealed a maximal 5.7-fold stimulation by VEGF at 1
ng/ml. At the same concentration, KDR-sel-stimulated cells
displayed a 3.6-fold stimulation of Akt phosphorylation. Nei-
ther PlGF nor Flt-sel led to any increase in Akt activity at all
concentrations tested (Fig. 4, Cand D). Interestingly, the PI3-
kinase inhibitor wortmannin (30 nM) completely abolished the
Akt activation in response to VEGF or KDR-sel. In fact, Akt
levels fell below unstimulated background. This latter finding
correlates well with the slight increase in the amount of apo-
pototic cells observed in the presence of wortmannin alone, as
shown in Fig. 3A. The reduction of background Akt activity in
the presence of wortmannin or LY has been observed in other
systems (17, 53) and is probably attributable to the low consti-
tutive PI3-kinase background activity in HUVE cells cultured
in serum-free conditions.
The Serine-Threonine Kinase Akt/Protein Kinase B Is Nec-
essary and Sufficient to Transduce the Survival Signal Induced
by VEGF—To verify that Akt, the downstream effector of PI3-
kinase, is responsible for the survival response, we transiently
transfected HUVE cells with expression vectors coding for wild-
type Akt (Flag-Akt) or two mutant forms. Flag-Akt 179A rep-
resents a catalytically inactive mutant that contains a point
mutation in the ATP binding site (43) and Flag-Akt 2D encodes
a constitutively active Akt mutant (44). Transfected endothe-
lial cells were identified after co-transfection with an expres-
sion vector for green fluorescent protein in a fluorescence mi-
croscope and classified by their morphology. Healthy
FIG.3.The survival activity of VEGF is inhibited by the PI3-
kinase inhibitors wortmannin and LY294002. A2 h before serum
starvation, wortmannin (WM) was added to HUVE cells grown in the
presence of 10% serum for 24 h. After washing the cells twice with PBS,
media containing 0% serum and the indicated amounts of VEGF and
wortmannin were added. Cells were harvested after 24–36 h, and
apoptotic cell death was determined by flow cytometry for annexin V
and propidium iodide. Bars represent means 6S.E. of two independent
experiments conducted with different preparations of endothelial cells.
B, immediately after exposing HUVE cells to serum starvation condi-
tions, the PI3-kinase inhibitor LY294002 (LY) was added to the cells.
Two hours later, VEGF was added to the medium, and cells were
cultured for additional 24–36 h before harvest. Apoptotic cell death was
determined as described above. Bars represent means 6S.E. of two
independent experiments conducted with different preparations of en-
dothelial cells.
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endothelial cells appear flat and well attached to the plate,
forming the classical cobblestone pattern. Apoptotic endothe-
lial cells are rounded, and some are fragmenting with small
cytoplasmic blebs (Fig. 1B; Refs. 54, 55). When we stained cells
with the DNA dye bisbenzimide (Hoechst 33258), apoptotic
endothelial cells could be easily identified by the pronounced
nuclear condensations (data not shown). We first examined
whether the constitutively active Flag-Akt 2D form was suffi-
cient to mediate survival of serum-starved endothelial cells. As
shown in Fig. 5, biolistic transfection of the Flag-Akt 2D con-
struct prevented endothelial cell apoptosis in the absence of
VEGF and promoted survival to similar levels as seen when
cells were grown in the presence of VEGF or 10% serum.
Moreover, transfection of endothelial cells with the dominant-
negative Flag-Akt 179A construct blocked VEGF survival ac-
tivity completely. In the absence of VEGF, a weak increase in
apoptosis was observed in cells transfected with Flag-Akt
179A, which probably reflects inhibition of constitutive Akt
activity in serum-starved cells. Our findings indicate that Akt
is critically involved in mediating the VEGF effects on endo-
thelial cell survival.
DISCUSSION
The elucidation of the mechanisms responsible for blood
vessel regression is critical to address several fundamental
biological questions. This process is prominent not only in the
course of organogenesis and organ remodeling during embry-
onic life but also in a variety of physiological situations in the
adult, such as corpus luteum regression (56) and uterine or
breast involution after delivery (5). Over the last few years,
several inhibitors of angiogenesis have been identified, includ-
ing angiostatin (57) and endostatin (58). These agents are able
to inhibit tumor growth by suppressing angiogenesis in xe-
nograft models. However, it is presently unknown whether
these molecules participate in the physiological regulation of
blood vessel regression. Recently, members of the family of
Tie-2 ligands such as angiopoietin-2 have been shown to be
involved in blood vessel remodeling and pruning, at least dur-
ing embryonic development (59).
Increasing evidence supports the concept that the with-
drawal of a positive effector such as VEGF is sufficient to result
in blood vessel regression in various in vivo systems, including
tumors (60) and developing organs (4, 28, 56). These studies
demonstrated the presence of an inverse correlation between
VEGF expression and the levels of vessel regression, implicat-
ing VEGF as an important survival factor for endothelial cells
present in newly formed vasculature.
Using an in vitro model system of serum-starved HUVE
cells, we attempted to elucidate the signal transduction path-
ways resulting in VEGF-mediated survival. The HUVE cells
used in our experiments consisted of pooled primary isolates
from 300 individual donor umbilical veins and thus are likely to
represent an average population of endothelial cells. Both
VEGF receptors, Flk-1/KDR and Flt-1, were previously found
to be expressed on HUVE cells by cell binding and cross-linking
studies with VEGF or the Flt-1-specific ligand PlGF (35, 42) or
by real-time reverse transcription-polymerase chain reaction
analysis (61). We first verified that serum removal indeed
resulted in apoptotic cell death. This was confirmed by a series
of morphological changes consistent with apoptosis, as evi-
denced by time lapse video recording. Furthermore, the finding
that the caspase inhibitor ZVAD-fmk completely prevented
apoptosis and mimicked VEGF activity demonstrates that this
process is mediated by the universal cell death machinery.
In our survival assay, we found decreased levels of VEGF-
dependent survival when cells were exposed to wortmannin or
LY 294002, two potent inhibitors of PI3-kinase. Activation of
PI3-kinase increases the intracellular amounts of phosphatidy-
linositol-3,4,5-bisphosphate and phosphatidylinositol-3,4,5-
triphosphate, which positively regulates Akt by binding to the
pleckstrin homology domain of Akt. Akt activation by growth
factors requires PI3-kinase activity (62). In our experiments we
found that Akt becomes phosphorylated after VEGF stimula-
tion, and such activation could be inhibited by the PI3-kinase-
FIG.4.Akt is activated by Flk-1/KDR but not by Flt-1-specific
ligands. Akt activation assay was performed as described under “Ex-
perimental Procedures.” A, blot of HUVE cell extracts treated with
various amounts of rhVEGF
165
for 15 min (lanes 2–7). In lane 8, cells
were exposed to 10% serum, reflecting maximal Akt activation. In lane
9, HUVE cells were exposed to VEGF (30 ng/ml) in presence of wort-
mannin (30 nM). B, blot of HUVE cell extracts treated with various
concentrations of KDR-sel VEGF mutant for 15 min (lanes 2–7). In lane
9, HUVE cells were exposed to a KDR-specific VEGF mutant (KDR sel,
30 ng/ml), in the presence of wortmannin (30 nM). C, blot of HUVE cell
extracts treated with various concentrations of Flt-selective VEGF mu-
tant (Flt sel), for 15 min (lanes 2–7). In lane 9, HUVE cells were exposed
to Flt-sel (30 ng/ml) in the presence of wortmannin (30 nM). In lane 9,
HUVE cells were exposed to PlGF (30 ng/ml) in the presence of wort-
mannin (30 nM). D, Western blot of HUVE cell extracts treated with
various concentrations of PlGF for the duration of 15 min (lanes 2–7).
NA, cells grown in the absence of serum and growth factors.
FIG.5.Akt is necessary and sufficient to mediate the survival
signal induced by VEGF. HUVE cells were plated at the density of
7310
5
per 6-cm culture dish and cultured in the presence of 10% serum
24 h before biolistic transfection. Per dish, 3
m
g of expression vector
encoding cytomegalovirus-driven green fluorescent protein (pGREEN
LANTERN) and 3
m
g of plasmid DNA encoding the indicated Akt form
or empty vector (cytomegalovirus-luciferase) were used per dish. Cells
were allowed to recover for 24 h in presence of 10% serum after trans-
fection. Serum starvation was induced by changing the medium to 0%
serum, and cells were then cultured in the presence of VEGF (100
ng/ml) or in 10% serum for 12 h. The number of green fluorescent
protein-positive and apoptotic cells was assessed phenotypically by the
characteristic nuclear condensation and membrane morphology in a
defined area of the culture dish (54). The percentage of apoptotic endo-
thelial cells was calculated by dividing the total amount of green fluo-
rescent protein-positive cells by the amount of apoptotic, green fluores-
cent protein-positive cells. Data shown are the means 6S.E. of two
independent experiments using different passage number endothelial
cells. NA, cells grown in the absence of serum and growth factors.
VEGF Inhibits Apoptosis by Akt Activation 30341
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specific inhibitor wortmannin. In a previous report, PI3-kinase
was found to be tyrosine phosphorylated after exposure of
endothelial cells to VEGF (23). However, another report failed
to detect induction of PI3-kinase after stimulation of HUVE
cells by VEGF (34). These latter findings do not necessarily
conflict with our data, because those experiments were con-
ducted in presence of 2% fetal bovine serum, which may induce
the PI3-kinase/Akt pathway to high levels, which cannot be
further increased by VEGF. Interestingly, Xia et al. (24) re-
cently reported that PI3-kinase is activated 2.1-fold by VEGF
in cultured endothelial cells but is not required for VEGF-de-
pendent proliferation.
The serine-threonine protein kinase Akt (63) is one of the
major targets of PI3-kinase-generated signals. The ability of
activated Akt to promote survival was found in fibroblasts (18)
and PC12 pheochromocytoma cells (64) and neuronal cells (16).
A number of different growth factors have been shown to rap-
idly activate Akt via PI3-kinase activation, such as platelet-
derived growth factor, epidermal growth factor, bFGF, insulin,
and insulin-like growth factor 1 (43, 65–69). In our experi-
ments, VEGF and a mutant VEGF form, which binds to the
Flk-1/KDR receptor specifically, could strongly induce Akt
phosphorylation, and this induction was PI3-kinase-depend-
ent. Moreover, in transient transfection experiments, the ki-
nase-inactive Akt mutant, which has been suggested to act in a
dominant-negative manner (43), was found to block VEGF
survival activity on endothelial cells. These findings constitute,
to the best of our knowledge, the first direct demonstration of a
role played by the PI3-kinase/Akt pathway in mediating a
VEGF biological activity.
Although the significance of VEGF and its endothelial cell-
specific receptors in angiogenesis is well established, the signal
transduction cascades initiated by the two known VEGF recep-
tors resulting either in proliferation or in survival of endothe-
lial cells are incompletely characterized.
Based on gene knockout experiments in mice and several
other studies, it can be deduced that Flk-1/KDR is critical for
VEGF-mediated angiogenesis, both in the developing and in
the adult animal (29, 70). In contrast, Flt-1 appears to be
primarily involved in endothelial cell morphogenesis, at least
during embryonic development (30). Although the role of Flt-1
in the adult animal is less clearly defined, there is increasing
evidence that Flt-1 is able to transduce a signal and mediate a
biological response. Flt-1, but not Flk-1/KDR, has been impli-
cated in monocyte migration in response to VEGF or PlGF via
a GTP-dependent signaling pathway (71, 72). Furthermore, the
finding that Flt-1 mRNA is expressed in both proliferating and
quiescent endothelial cells suggests a role for this receptor in
the maintenance of endothelial cells (39).
The motif YXXM required for binding of the SH2-domain of
the p85 regulatory subunit of PI3-kinase is absent from the
noncatalytic regions of the intracellular domain of Flt-1 as well
as from Flk-1/KDR. Using the yeast two-hybrid system, two
groups reported independently an association between tyrosine
1213 of the Flt-1 carboxyl terminus, present within the tyrosine
kinase domain and the p85 subunit of phosphatidylinositol
3-kinase. (47, 73). However, when we tested PlGF or an Flt-1-
specific VEGF mutant form in different assay systems, we
could not detect any significant survival effect. The same mu-
tants did not induce Akt phosphorylation, as assessed by West-
ern blotting using an antibody that specifically recognizes the
phosphorylated Akt. Likewise, we were unable to detect any
change in the percentage of annexin V-positive, apoptotic en-
dothelial cells in response to the Flt-1-selective ligands. These
findings lead us to the conclusion that the Flt-1 receptor, at
least when present in the homodimeric form, is not sufficient to
mediate survival activity. In contrast, a KDR-selective VEGF
mutant completely mimicked the antiapoptotic and survival-
promoting effects of VEGF and led to strong activation of Akt
via PI3-kinase. Given the absence of a consensus binding site
on the cytoplasmic domain of Flk-1/KDR, we speculate on the
existence of an adaptor molecule mediating the activation of
p85 by Flk-1/KDR. We consider the possibility that only het-
erodimeric VEGF receptors consisting of Flt-1 and Flk-1 sub-
units would allow for p85 binding and activation to be very
unlikely, because the KDR-selective mutant was sufficient to
induce survival and Akt activation.
In conclusion, our findings provide evidence that VEGF sur-
vival signals in endothelial cells are mediated by the Flk-1/
KDR receptor through the PI3-kinase/Akt signal transduction
pathway. Further delineation of this signaling pathway may
yield attractive targets for cancer therapy aimed to induce
endothelial cell apoptosis and blood vessel regression.
Acknowledgments—We thank the fluorescence-activated cell sorting
lab at Genentech for their excellent support, H. Nguyen for cell culture,
M. Vasser, P. Ng, and P. Jhurani for oligonucleotide synthesis, and M.
Winkler and T. Yee for purification of VEGF mutants. We also thank
Hendrik Gille for valuable discussion and David Wood and Charles
Hoffman for excellent graphic art work.
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VEGF Inhibits Apoptosis by Akt Activation 30343
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Vishva Dixit and Napoleone Ferrara
Kowalski, Minhong Yan, Bruce A. Keyt,
Hans-Peter Gerber, Amy McMurtrey, Joe
ACTIVATION
REQUIREMENT FOR Flk-1/KDR
-Kinase/Akt Signal Transduction Pathway:
′through the Phosphatidylinositol 3
Regulates Endothelial Cell Survival
Vascular Endothelial Growth Factor
CELL BIOLOGY AND METABOLISM:
doi: 10.1074/jbc.273.46.30336
1998, 273:30336-30343.J. Biol. Chem.
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