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J. Biol. Chem., Vol. 277, Issue 13, 11410-11415, March 29, 2002
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§,§,§,
,**, and
From the Departments of Pathology,
§ Pediatrics, ¶ Neurobiology, Surgery, and
Neurology, Yale University School of
Medicine, New Haven, Connecticut 06510
Received for publication, November 19, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Recent data have demonstrated that vascular
endothelial growth factor (VEGF) is expressed by subsets of neurons,
coincident with angiogenesis within the developing cerebral cortex.
Here we investigate the characteristics of VEGF expression by neurons and test the hypothesis that VEGF may serve both paracrine and autocrine functions in the developing central nervous system. To begin
to address these questions, we assayed expression of VEGF and one of
its potential receptors, Flk-1 (VEGFR-2), in the embryonic mouse
forebrain and embryonic cortical neurons grown in vitro.
Both VEGF and Flk-1 are present in subsets of post-mitotic neurons
in vivo and in vitro. Moreover, VEGF levels are
up-regulated in neuronal cultures subjected to hypoxia, consistent with
our previous results in vivo. While the abundance of Flk-1
is unaffected by hypoxia, the receptor exhibits a higher level of
tyrosine phosphorylation, as do downstream signaling kinases, including
extracellular signal-regulated protein kinase, p90RSK and
STAT3a, demonstrating activation of the VEGF pathway. These same
signaling components also exhibited higher tyrosine phosphorylation
levels in response to exogenous addition of rVEGFA165. This
activation was diminished in the presence of specific inhibitors of
Flk-1 function and agents that sequester VEGF, resulting in a
dose-dependent increase in apoptosis in these neuronal
cultures. Further, inhibition of MEK resulted in increased apoptosis,
while inhibition of phosphatidylinositol 3-kinase had no appreciable
affect. In addition to the novel function for VEGF that we describe in
neuronal survival, neuronal VEGF also affected the organization and
differentiation of brain endothelial cells in a three-dimensional
culture paradigm, consistent with its more traditional role as a
vascular agent. Thus, our in vitro data support a role for
neuronal VEGF in both paracrine and autocrine signaling in the
maintenance of neurons and endothelia in the central nervous system.
VEGF,1 a
hypoxia-inducible endothelial cell mitogen, has been characterized
as a potent vascular permeability factor and a critical factor in
vasculo- and angiogenesis (1-3). VEGF is known to exert its effects
via two high affinity receptors, feline sarcoma virus-like
tyrosine kinase (Flt-1, VEGFR-1) and fetal liver kinase receptor
(Flk-1, VEGFR-2) (4-9). Both receptors are critical for the proper
differentiation and organization of endothelial cells into vascular
beds. In the central nervous system, a cellular response to hypoxic
exposure is increased VEGF production by glial cells that invest
cerebral vessels (10-12), which results in increased angiogenesis and
changes in vessel homeostasis.
More recently, in the peripheral nervous system VEGF has been shown to
be a neurotropic factor, which stimulates axonal outgrowth, enhances
cell survival, and increases Schwann cell proliferation in cultured
superior cervical and dorsal root ganglia from adult mice.
Co-expression of VEGF and Flk-1 in many neurons in these superior
cervical ganglia cultures was also noted (13-15).
In addition, VEGF expression has been localized to subpopulations of
neurons in the developing and mature central nervous system (14-18).
Jin et al. demonstrated VEGF gene and protein expression in
neurons of the cortex and hippocampus following a model for global
cerebral ischemia in the adult rat brain, and the application of
exogenous VEGF was shown to promote survival of rat cerebral neurons in
culture and rescue HN33 hippocampal cells from death by serum
withdrawal (18). Furthermore, our previous work has demonstrated
that VEGF expression by neurons of the developing cerebral cortex
coincides both spatially and temporally with angiogenesis and that
levels of VEGF in cortical neurons increase following hypoxic exposure
(11). Thus, this previously unknown source of endogenous VEGF
production in the brain presents a novel paradigm for examining VEGF function.
Interest in VEGF as a therapeutic molecule in a wide number of
pathological conditions such as stroke and peripheral nerve damage led
us to test the hypothesis that neuronal VEGF secretion may have
specific signaling functions in the central nervous system (17,
19-21). Here we report that neuronal VEGF and its receptor Flk-1 are
expressed by cortical neurons of E15 embryos in vitro and
in vivo. Moreover, use of an in vitro culture
model demonstrates that neuronal VEGF expression is hypoxia-inducible,
with the resulting VEGF capable of supporting tube formation in a brain
microvascular endothelial cell line. Co-localization of VEGF and its
Flk-1 receptor, as well as changes in tyrosine phosphorylation of both
Flk-1 and downstream signaling molecules, including mitogen-activated
protein kinase, p90RSK, and STAT family members, suggests that VEGF
mediates both auto- and paracrine signaling functions in central
nervous system neurons. Finally, inhibition of either VEGF function or Flk-1 activity results in increased cell apoptosis, indicating that endogenously produced VEGF acts as a neuronal survival factor.
Immunohistochemistry--
Sections of embryonic day 15 (E15)
mouse brains or cortical neurons derived from them and grown in
vitro (11) were fixed and subjected to immunohistochemistry. The
antibodies used follow: polyclonal anti-VEGF (Neomarkers Inc., 1:250),
anti-Flk-1 (Santa Cruz Biotechnology Inc., 1:250), monoclonal
anti-neuron-specific class III Primary Cortical Neuronal Cultures--
The dorsal telencephalon
was dissected from E15 mice (Charles River Laboratories), as previously
described (22). Cells were plated on either glass coverslips or plastic
Petri dishes coated with poly-L-ornithine and laminin.
Cells were incubated for 6 days in normal atmosphere with 5%
CO2 or in hypoxic conditions consisting of a mixture of 5%
CO2, 10% O2, 85% N2 (BOC Gases, North Haven, CT). In some experiments cultures were incubated with the
following:
( Western Blotting--
Western blotting was carried out on
lysates of E15 neurons and E15-conditioned media as previously
described. (11) Antisera directed against VEGF, Flk-1, PI 3-kinase,
Akt, pAkt, Erk-2, pErk, p90RSK, PY-p90RSK, STAT3, pSTAT3, and STAT1 at
1:500 dilution were used. Detection was carried out using Pierce
supersignal detection reagent (Pierce, Milwaukee, WI) with membrane
exposure to Hyperfilm reagent (Amersham Biosciences, Inc.).
Quantitation was carried out on scanned images (Agfa Arcus II Scanner
and Adobe PhotoShop 4.0, Adobe Systems, San Jose, CA) using the
BioMaxTM Program (Kodak, Rochester, NY) on a MacIntosh G3 computer.
All experiments were performed at least three times. Statistical
analysis was performed using Student's t test.
Immunoprecipitation--
Cell cultures were washed with cold
phosphate-buffered saline containing 1 mM sodium
orthovanadate and 50 mM NaF and scraped into lysis buffer
(see above). Immunoprecipitation was carried out as previously
described (26). All experiments were performed at least three times.
Three-dimensional RBE4 Culture and Microscopy--
Rat
brain microvascular endothelial (RBE4) cells (27) were cultured as
previously described (28). The droplets were cultured in unconditioned
media, in either normoxic or hypoxic E15 neuron-conditioned media for 6 days in 5% CO2 with media replenishment on day 3. After 6 days the cultures were fixed and mounted on slides for light
microscopy. Controls were carried out using unconditioned media.
Experiments were performed at least three times. Images were taken
using a Zeiss research microscope equipped with epifluorescence and a
SPOTTM camera before being transferred electronically to Adobe
PhotoShop without image editing.
VEGF and Flk-1 Are Expressed by E15 Mouse Cortical Neurons in Vivo
and in Vitro--
We performed immunohistochemistry to localize VEGF
and Flk-1 in E15 forebrain sections using a post mitotic neuronal
marker (Tuj-1) to identify neuronal cells. We found that VEGF was
expressed by neurons throughout the wall of the developing cerebrum
(Fig. 1a). Flk-1 was also
expressed by neurons of the E15 forebrain and widely distributed and at
low levels (Fig. 1b). Additionally as expected, both VEGF
and Flk-1 were present within vascular structures superficial to the
cortical plate (Fig. 1, a and b).
To determine whether cells in vitro expressed VEGF and
Flk-1, we prepared primary cultures of E15 cortical neurons and
performed immunohistochemistry. Staining revealed that ~30% of
Tuj-1-positive cells in culture expressed VEGF (Fig. 1, c
and d); about 25% of Tuj-1-positive cells expressed Flk-1
(Fig. 1f). Furthermore, ~90% of cells that expressed VEGF
also expressed Flk-1 (Fig. 1g). To determine whether
expression changed when cells were grown in vitro, we
assayed levels of VEGF and Flk-1 in freshly dissociated but unplated
neurons, as well as cells grown in culture. Both populations of cells
expressed VEGF (Fig. 1e, lanes 1 and
2) and Flk-1 (Fig. 1h, lanes 1 and
2), and furthermore, the VEGF produced by these neurons was
secreted into the media (Fig. 1e, lane 3). Similarly, E15 cortical neurons express Flk-1 as dissociated cells or
cells grown in vitro (Fig. 1h, lanes 1 and 2). These results demonstrated that neurons in culture
mimic neurons in the brain in their ability to synthesize and secrete
VEGF. In addition, they have a receptor with which they could respond
to this factor.
Flk-1 and Its Downstream Targets Are Phosphorylated in Cortical
Neurons--
Next, we examined the activation state of Flk-1 in
cortical neuronal cultures and found that the Flk-1 was
tyrosine-phosphorylated, indicating that a portion of the Flk-1
receptors is in an activated state (Fig.
2a). Furthermore,
immunoprecipitation using an anti-Flk-1 antisera co-precipitated
proteins of 85, 65, and 45 kDa (Fig. 2b), consistent with
these bands representing p85 PI 3-kinase, Shc, and Nck, respectively,
known signaling partners of Flk-1 (29).
To determine whether VEGF and Flk-1 levels are modulated by hypoxia, we
performed Western blot analysis on neuronal culture lysates derived
from normoxic and hypoxic cultures. While overall protein levels of
Flk-1 remained unchanged (Fig. 2c), levels of VEGF in both
lysates (data not shown) and conditioned media (Fig. 2d)
were up-regulated ~30% as expected by hypoxia. Interestingly, although Flk-1 protein levels did not change appreciably in response to
hypoxia, Flk-1 tyrosine phosphorylation levels were observed to
significantly increase in response to hypoxia (Fig. 2e).
Further, to determine whether signaling cascade components related to
Flk-1 activation are responsive to hypoxic conditions, we compared
levels of total protein and activated (tyrosine-phosphorylated) protein for ERK, p90RSK, STAT-3a, STAT1, Akt, and p85 PI 3-kinase. Levels of
total ERK, p90RSK, Akt, and STAT3a protein did not vary between cells
grown in normoxic versus hypoxic conditions. In contrast, levels of tyrosine-phosphorylated ERK, p90RSK, and STAT3a were significantly increased following hypoxia, indicating that they are
being activated (Fig. 2f, fold change 1.9 ± 0.59, 2.5 ± 0.69, and 2.5 ± 0.58, respectively). Notably, Akt
phosphorylation levels did not change appreciably after hypoxic
exposure. Expression of STAT1 and PI 3-kinase were also not
significantly increased by hypoxia.
Since hypoxia is a known stimulus for VEGF induction we were interested
in determining whether addition of exogenous rVEGF-A165 could mimic the changes in signaling pathway components that we observed following sustained culture in hypoxia. Following daily administration of 10 ng/ml rVEGF-A165 for 6 days, we
observed that levels of total ERK, p90RSK, Akt, and STAT3a protein did not vary greatly among cells grown under normoxic, hypoxic, and normoxic + rVEGF-A165 conditions. However, comparable with
results with hypoxic cultures, levels of tyrosine-phosphorylated ERK, p90RSK, and STAT3a were significantly increased following addition of
rVEGF-A165, indicating activation of a VEGF-inducible
signaling pathway (Fig. 2f, fold change 2.0 ± 0.16, 4.2 ± 0.9, and 2.0 ± 0.41, respectively). Notably, as in
hypoxic cultures, Akt phosphorylation levels and STAT1 and PI 3-kinase
expression levels were not significantly changed by VEGF treatment.
Inhibition of VEGF Signaling Induces Apoptosis in E15 Neuronal
Cultures--
To examine the effects of blocking VEGF/Flk-1 signaling
in cortical neurons two approaches were taken. First, we inhibited VEGF
receptor tyrosine kinase activity using SU1498, a potent and selective
Flk-1 tyrosine kinase inhibitor (23), or added CB676475, a broad range
VEGF receptor tyrosine kinase inhibitor (24). SU1498 was found to
elicit increased apoptosis (as determined by TUNEL labeling) in a
dose-dependent manner when added to E15 neuronal cultures
(Fig. 3a). CB676475 exhibited
similar properties (Fig. 3b, upper two
panels).
The second approach taken was to sequester endogenously expressed VEGF
with either a neutralizing VEGF antibody or a recombinant soluble
chimeric Flt-1 molecule. In both instances, cultures containing either
of these agents exhibited increased apoptosis as evaluated by TUNEL
labeling (Fig. 3b, lower three panels).
Inhibition of PI 3-Kinase and MEK Signaling Induces
Differential Apoptosis in E15 Neuronal Cultures--
To further
examine the specific signaling pathways downstream of the VEGF-induced
inhibition of apoptosis a pharmacological approach was taken. Namely,
chemical inhibitors of either MEK (PD98059, 20 µM) and PI
3-kinase (Wortmannin and LY24002, 20 µM) were added daily
from day one to day six of culture of the E15 neurons. As illustrated
in Fig. 4, only occasional apoptotic
cells were identified by caspase 3 staining of normoxic cultures (Fig. 4A) and normoxic cultures incubated with either 20 µM of the PI 3-kinase inhibitors LY (Fig. 4B)
or Wortmannin (data not shown). In contrast, normoxic cultures
incubated with 20 µM of the MEK inhibitor PD98059
exhibited markedly increased apoptosis as evidenced by the increase in
caspase 3 staining (Fig. 4C).
Neuronal VEGF Supports the Organization of Endothelial Cells into
Tubes--
To determine whether neuronal VEGF is capable of promoting
endothelial cell organization and differentiation, we assessed the
effects of media conditioned by cortical cultures on brain microvascular endothelial cells cultured in three-dimensional collagen
gels.2 We found that
neuronal-conditioned media supports rearrangement and differentiation
of rat brain endothelial (RBE4) cells, resulting in the formation of
tube-like structures (Fig. 5). The
ability to affect tube formation correlated with levels of VEGF in the media, as no tubes form in cultures treated with unconditioned media
(Fig. 5, panel 1). Modest tube formation is observed in cultures supplemented with media from normoxic neuronal cultures (Fig.
5, panel 2), and robust tube formation occurs in cultures that were supplemented with media from hypoxic neuronal cultures (Fig.
5, panel 3). Thus, although produced by cortical neurons in
culture, neuronal VEGF has the ability to influence endothelial function and subsequent angiogenesis.
We show here that embryonic cortical neurons, in vivo
and in vitro, express VEGF and one of its receptors, Flk-1
(Flt-1 was not detected in these cultures). Moreover, Flk-1 is
activated in cultured neurons and appears to initiate signaling via a
cascade that likely includes PI-3 kinase, Akt, Shc, Nck, and the
kinases ERK, p90RSK, and STAT3a. Under hypoxic conditions, cortical
neurons express elevated levels of VEGF and Flk-1 activation is
increased, supporting the concept that a primary response to hypoxia by
cortical neurons is to increase VEGF production and subsequent Flk-1
signaling. An identical response was obtained using exogenous
rVEGF-A165 confirming that neurons are capable of
responding to both endogenous and exogenous VEGF production. Inhibition
of VEGF signaling, using either pharmacological agents to block Flk-1
kinase activity or reagents that sequester endogenous VEGF, results in
increased apoptosis, suggesting that VEGF may function as an autocrine
and paracrine neuronal survival factor. Further, utilizing inhibitors of PI 3-kinase and MEK we generated data consistent with the concept that the MEK-ERK-p90RSK pathway is responsible, in part, for mediating neuronal survival in this culture model. Interestingly, the PI 3-kinase-Akt pathway does not appear to be involved in the modulation of neuronal apoptosis in our in vitro model (Figs. 2, 4, and
6B). Finally, media conditioned by both normoxic and hypoxic
cortical neurons promoted in vitro angiogenesis, with levels
of VEGF correlating with the extent of tube formation.
Our previous findings that neurons in the brain express VEGF are
extended by these studies (11).2 Indeed, the maintenance of
expression of VEGF by neurons in culture demonstrates that this
expression is intrinsic to these neurons, since it exists in the
absence of proper cellular contact and connectivity. Furthermore, the
observation that VEGF and Flk-1 are co-localized supports the notion
that neuronal VEGF in the brain can act as an autocrine as well as a
paracrine factor for neurons (Fig. 6,
A and B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-tubulin Tuj-1 (Berkeley Antibody
Co., Richmond, CA., 1:500), anti-Erk-2 (Santa Cruz Biotechnology Inc.,
1:1000), anti-phospho-Erk (Santa Cruz Biotechnology Inc., 1:1000),
anti-STAT1, anti-STAT3a, and anti-p-STAT3a, (Chemicon, International,
Inc., Temecula, CA; 1:1000), anti-PI 3-kinase (Upstate Biotechnology,
Lake Placid, NY; 1:1000), anti-p90RSK and anti-p90RSK PY (Cell
Signaling Technology, Inc., New England Biolabs, Beverly, MA, 1:1000),
and anti Cleaved Caspase 3 (D175) (Cell Signaling, Beverly, MA, 1:500).
Wortmannin, LY294002, and PD98059 were purchased from Sigma.
TUNEL labeling was performed as recommended by the manufacturer (Roche
Diagnostics, Indianapolis, IN).
)-3(3,5-diisoproply-4-hydroxyphenyl)-2-[3-phenyl-n-propyl)amino-carbonyl]acrylonitrile (SU1498) (23), a potent and selective inhibitor of Flk-1 kinase; 4-[94'-chloro-2'-fluoro)phenylamino]-6,7-dimethoxyquinazoline (CB676475) (24), a potent and selective inhibitor of VEGFR1 and 2 tyrosine kinase activity; an inhibitor of the Flk-1 kinase or a
neutralizing antibody directed against VEGF-A (R&D Systems, Minneapolis, MN; recombinant VEGF-A165 (Vendor); a
recombinant soluble Flt-1 (Flt (1-3)-IgG, a truncated Flt 1-3 Fc
fusion protein), a generous gift of Dr. Napoleon Ferrara (Genentech;
San Francisco, CA) (25); or PD98059, a MEK inhibitor, or Wortmannin and
LY24002, inhibitors of PI 3-K, (Sigma).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
VEGF and Flk-1 localization in mouse
E15 brain and cultured neurons. Panels a and b,
fluorescent immunohistochemistry of mouse embryo sections stained with
either anti-VEGF (red) and Tuj-1 (green;
panel a) or anti-Flk-1 (red) and Tuj-1
(green; panel b). Double labeled Tuj-1 and VEGF
and Tuj-1 and Flk-1 positive neurons (yellow, big
arrows) are seen from the ventricular zone (VZ) toward
the cortical plate (CP) of the developing brain. Both
vascular structures (red) and neurons (green and
yellow) are apparent in the cortical plate. Panel b
inset is a low power image for orientation with the small
arrow denoting the enlarged areas in panels a and
b. Scale bars for panels a,
b and the inset in panel b all equal
250 microns. Panels c and d, fluorescent
immunohistochemistry showing co-localization of VEGF and Tuj-1 in
subsets of neurons (yellow fluorescence) using anti-VEGF
(red) and anti-Tuj-1 (green) antibodies
(panel c, low power; panel d,
high power). An elaborate network is evident with ~50% of
neurons positive for both VEGF and Tuj-1 (yellow).
Scale bars = 50 microns. Panel e, Western
blot analyses of E15 neuronal cell lysates shows that endogenous VEGF
production can be detected in unplated (lane 1) and plated
(lane 2) cells as well as neuron-conditioned
media (lane 3). Panels f and g,
fluorescent photomicrographs of cultured E15 neurons double-labeled
with anti-Flk-1 (red) and anti-Tuj-1 (green)
antibodies (panel f). Not all Tuj-1-positive neurons express
Flk-1. Co-localization results in yellow fluorescence illustrated in
two of several neurons in this field (panel f). Double
labeling with anti-Flk-1 (red) and anti-VEGF
(green) antibodies revealed co-localization in large subsets
of neurons (yellow), with some neurons positive for only
VEGF (green) (panel g). Scale
bars = 50 microns. Panel h, Western blot analyses
of E15 neuronal lysates showing that expression of Flk-1 can be
detected in both unplated (lane 1) and plated (lane
2) cortical neurons.
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Fig. 2.
Phosphorylation state of neuronal Flk-1 is
modulated by endogenous VEGF levels and affects signaling cascades.
Panels a and b, representative
immunoprecipitation (IP) with anti-PY antibodies followed by
Western blotting with anti-Flk-1 demonstrates tyrosine phosphorylation
(activation) of the receptor in cortical neuronal cultures (panel
a). The reciprocal experiment, immunoprecipitation anti-Flk-1
followed by Western blotting with anti-PY, confirms this (panel
b). A number of putative tyrosine-phosphorylated
adapter/signaling-associated proteins also precipitate with Flk-1 as
indicated by the arrows (panel b). Panel
c, representative Western blot of normoxic (Nx) and
hypoxic (Hx) neuronal lysates with anti-Flk-1 antibody,
illustrating no appreciable change in protein expression levels.
Panel d, representative Western blot analysis of conditioned
media from cultures exposed to normoxic (Nx) and hypoxic
(Hx) conditions shows that neuronal VEGF is
hypoxia-inducible with an increase of 30% over normoxic cultures
observed. Panel e, representative immunoprecipitation of
normoxic (Nx) and hypoxic (Hx) neuronal lysates
with anti-Flk-1 followed by Western blotting with anti-PY illustrates
increased tyrosine phosphorylation of Flk-1 under hypoxic conditions.
Panel f, representative Western blot analyses of normoxic
(Nx), hypoxic (Hx), and normoxic + rVEGF
(VEGF) neuronal lysates using anti-ERK, p-ERK, p90RSK, PY
p90RSK, STAT3a, p-STAT3, Akt, pAkt, STAT1, and PI 3-kinase antibodies
reveal significant (p > 0.05) increases in p-ERK,
p90RSK, and p-STAT3a tyrosine phosphorylation levels in response to
hypoxic and VEGF treatment culture conditions. All experiments were
performed at least three times with similar results.
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Fig. 3.
Inhibition of Flk-1 activation or
sequestration of VEGF elicits neuronal apoptosis. Panel a:
The Flk-1 phosphorylation inhibitor SU1498 induces apoptosis in E15
neuronal cultures. E15 neurons were cultured in the presence of 70 nM, 350 nM, 700 nM and 3.5 µM SU1498. Cultures were also exposed to control
conditions; no inhibitor or vehicle only (Me2SO). TUNEL
labeling was used to observe apoptotic neurons in the cultures. The
left panel of micrographs illustrates the cellular
morphology (phase) of control, vehicle and inhibitor treated cultures.
Right hand panels illustrate the corresponding fluorescent TUNEL images
from a representative experiment. The number of TUNEL-positive cells
increase with increasing concentrations of the inhibitor in a dose
dependent manner. Scale bar = 100 microns. Panel b,
similar results were obtained with VEGFR tyrosine Kinase Inhibitor CB
676475, which elicited increased apoptosis in the 10-µM
range (panel b, second row). In addition, VEGF
was sequestered by adding 1.0 µg/ml neutralizing antibody directed
against VEGF-A or 1.0 µg/ml recombinant soluble Flt-1
(mFlt(1-3)-IgG) to the cultures daily. As illustrated in panel
b, fourth and fifth rows, addition of either
reagent elicited increased apoptosis in the neuronal cultures compared
with controls (panel b, third row). Scale
bar = 100 microns.
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Fig. 4.
Inhibition of MEK, but not PI 3-kinase
elicits neuronal apoptosis. The MEK inhibitor PD98059 (20 µM) induces markedly increased apoptosis in cultured E15
neurons (panel C) compared with cultured E15 neurons in
control normoxic conditions (panel A) and E15 neurons
cultured in the presence of the PI 3-kinase inhibitor LY294002
(panel B). Tuj-1 labeling (green fluorescence)
was utilized to identify neurons, while red fluorescence was
used to mark apoptotic cells. The orange color denotes
apoptotic neurons. Scale bar = 100 microns.
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Fig. 5.
Representative photomicrographs illustrating
the effect of normoxic and hypoxic neuron-conditioned media on RBE4
cell differentiation and tube formation in three-dimensional collagen
gel cultures. In unconditioned media the endothelial cells do not
form any significant tube-like structures (panel a).
Normoxic neuron-conditioned media causes modest tube formation as
evidenced by the occasional sprout formations (arrow)
emanating from the multicellular endothelial cysts (panel
b). In contrast, hypoxic neuron-conditioned media results in
robust tube formation with networks of tubes (arrows)
connecting cysts of RBE4 cells clearly visible (panel c).
Thus it appears that secreted VEGF is bioactive and can signal to
adjacent cells. All experiments were performed at least three times
with similar results. Scale bar = 200 microns.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Working models of the autocrine and
paracrine effects of neuronal VEGF (A) and the
signaling pathways involved (B). A,
VEGF expressed by selected murine E15 neurons is capable of promoting
paracrine endothelial survival and angiogenesis as well as autocrine
and paracrine neuronal survival in the central nervous system
(synthesized from the data presented and data in Ref. 11.2
B, signaling pathways: neuronal VEGFR-2 engagement by VEGF
initiates a signaling cascade that results in the activation of ERK,
p90RSK, and STAT3 (green arrows), that in turn, inactivate
Bad (red line) and activate Bcl-2 and Bcl-XL (green
arrows), which results in an inhibition of apoptosis (red
lines). Additionally, inhibition of VEGFR-2 and MEK increase E15
neuronal apoptosis and is consistent with this concept. In contrast,
although the persistent stable activation of Akt (evidenced by its
stable phosphorylation state, black arrows) suggests that
the PI 3-kinase pathway is also likely involved in modulating neuronal
behaviors in this model, PI 3-kinase inhibitors fail to affect
apoptosis, suggesting that this pathway is not involved in modulating
apoptosis in this model.
In addition to its more widely recognized role in angiogenesis, VEGF is involved in regulation of the early developmental program of retinal neurogenesis (31). In the peripheral nervous system, the neurotrophic and mitogenic effects of exogenous VEGF result in stimulation of axon outgrowth through the Flk-1 receptor as well as increased survival of peripheral neurons in vitro, suggesting that further functions may exist (14, 15). In the central nervous system, exogenous VEGF reduced neuronal cell death in an in vitro model of cerebral ischemia (18, 30, 32). In this study we attribute a role to the endogenous production of VEGF by neuronal cells and suggest that not only VEGF from exogenous sources (such as astrocytes and endothelial cells) is important for neuronal survival. Our findings add to the complexity of biological roles that have been attributed to VEGF.
We show that increased cell death results from a blockade of endogenous VEGF signaling indicating the importance of both VEGF and the Flk-1 receptor in neuronal survival. Our studies illustrate that complex signaling cascades are susceptible to elevation of endogenous VEGF levels in cortical neurons exposed to hypoxic conditions in vitro. Interestingly, contrary to findings by Jin et al., which demonstrate the importance of PI 3-kinase/Akt signal transduction system in VEGF-mediated neuroprotection in HN33 cells (30, 32), we find that the MEK-ERK pathway is crucial to the survival of cortical neurons in vitro. This discrepancy could be due to differences in experimental design as well as differences in the cell populations tested. Potentially acting through both autocrine and paracrine Flk-1 activation, ERK, p90RSK, and STAT 3a phosphorylation levels are up-regulated, either in abundance or in activity in cortical neurons under hypoxic conditions or after exposure to rVEGF. These findings, along with the observation of persistent stable Akt phosphorylation, highlight the fact that VEGF can modulate a number of downstream pathways some of which have not yet been identified. Furthermore our data suggests that neurons themselves provide a source of VEGF to aid their survival under adverse conditions, a response that will presumably depend on the severity of the insult. Perhaps this initial release of VEGF into the local environment may prolong survival of cells that are not irreversibly damaged until angiogenesis is initiated. Additional functional and novel responses of neurons to changes in local VEGF expression and their impact are still to be identified.
We show that neuron-conditioned media supports differentiation and tube formation in a three-dimensional model of angiogenesis. Although we cannot exclude the presence of other pro-angiogenic factors in the conditioned media, our study suggests that neuronal VEGF is bioactive, can signal to surrounding endothelial cells, and can potentially produce a gradient to direct angiogenesis during development and in response to injury and trauma. Furthermore, hypoxia-conditioned media augments tube formation, consistent with our previous finding that neuronal VEGF can produce a gradient to direct angiogenesis in the developing cortex.
Since cortical neurons are diverse and because we know that this
diversity is at least in part recapitulated in vitro, it will be important in the future to understand the effects of VEGF stimulation on distinct cortical neuron subtypes. It is also tempting to speculate that there may be a neuronal-specific form of VEGF. Interestingly, not all cortical neurons express VEGF and/or flk-1 receptors, which may be a reason why some cells are more susceptible to
injury and insult. In addition, the issue of whether modulating levels
of Flk-1 signaling under hypoxic conditions will protect cells from the
apoptosis they are destined to undergo is an interesting and pertinent
question, as it may have profound implications for the treatment of
cortical injury. Finally, our results suggest that there appears to be
a coordinated process in which neurogenesis and angiogenesis share
common molecular triggers. In the future, we will examine the roles
that VEGF plays in neuronal maturation and assess the potential roles
of VEGF in modulating neuronal plasticity within the developing brain
and the coordination that exists between neurons and the blood vessels
that supply them.
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ACKNOWLEDGEMENTS |
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We thank Dr. Napoleon Ferrara, Genentech, South San Francisco, CA for the generous gift of sFlt, truncated Flt 1-3 Fc fusion protein, and Dr. F. Roux, Hospital F. Widal, Paris, France for the generous gift of transformed rat brain endothelial (RBE4) cells.
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FOOTNOTES |
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* This work is supported by United States Public Health Service Grants PO1-NS 35476 (to L. R. M.) and PO1-DK38979 (to J. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 Pathology, Yale Univ. School of Medicine, 310 Cedar St., P.O. Box 208023, New Haven, CT 06520-8023. Tel.: 203-785-2763; Dept. Fax: 203-785-7303; Office Fax: 203-785-7213; E-mail: joseph.madri@yale.edu.
Published, JBC Papers in Press, January 3, 2002, DOI 10.1074/jbc.M111085200
2 Chow, J., Ogunshola, O. O., Fan, S. Y., Li, Y., Ment, L. R., and Madri, J. A. (2001) Brain Res. Dev. Brain Res. 130, 123-132.
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; STAT, signal transducers and activators of transcription; E, embryonic day; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling: MEK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; PI, phosphatidylinositol.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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