paracrine and autocrine functions of bdnf and ngf in brain ...transformed rat brain endothelial...
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. 1 Kim, Li, Hempstead, Madri
Paracrine and Autocrine Functions of BDNF and NGF in Brain-derivedEndothelial Cells*
Hyun Kim1,3, Qi Li1, Barbara L. Hempstead2, and Joseph A. Madri1,4
Department of Pathology, Yale University School of Medicine1 and theDepartment of Medicine, Weill Medical College of Cornell University2
Running Title: Neurotrophin modulation of endothelial behavior
Keywords: BDNF, Angiogenesis, Apoptosis, proNGF, TrkB, p75NTR, Caspase,Akt, ERK, VEGFR2
* Supported, In part, by USPHS grants # PO1-NS-035476 and PO1-DK-55389to JAM and HL-58130, HL-59312 and the Burroughs Wellcome Fund to BLH
References: 52Figures: 10
3Current Address:Department of AnatomySeonam University, Medical School720, Kwang Chi Dong, NamwonChonbuk, 590-711, KoreaTel : +82-63-620-0312Fax : +82-63-620-0315e-mail : [email protected]
4All correspondence to:Joseph A. Madri, Ph.D., M.D.Pathology DepartmentYale University School of Medicine310 Cedar StreetP.O. Box 208023New Haven, CT 06520-8023Tel: 203-785-2763FAX: 203-785-7303 DeptE-mail: [email protected]
JBC Papers in Press. Published on May 28, 2004 as Manuscript M404115200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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. 2 Kim, Li, Hempstead, Madri
Abstract
Brain derived neurotrophic factor (BDNF) is expressed by endothelial
cells. We investigated the characteristics of BDNF expression by brain-derived
endothelial cells and tested the hypothesis that BDNF serves paracrine and
autocrine functions affecting the vasculature of the central nervous system. In
addition to expressing TrkB and p75NTR and BDNF under normoxic conditions,
these cells increased their expression of BDNF under hypoxia. While the
expression of TrkB is unaffected by hypoxia, TrkB exhibits a baseline
phosphorylation under normoxic conditions and an increased phosphorylation
when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF
is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis
and survival in three-dimensional cultures of these endothelial cells, while
sequestration of endogenous BDNF caused significant apoptosis. The effects of
BDNF engagement of TrkB appears to be mediated via the PI-3-kinase - Akt
pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation
and inhibitors of PI-3 kinase abrogate the BDNF responses. BDNF mediated
effects on endothelial cell survival/apoptosis correlated directly with activation
of Caspase 3. These endothelial cells also express p75NTR and respond to its
preferred ligand, proNGF by undergoing apoptosis. These data support a role
for neurotrophins signaling in the dynamic maintenance/differentiation of
central nervous system endothelia.
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. 3 Kim, Li, Hempstead, Madri
Introduction
Angiogenesis is a tightly controlled process in which new vessels form
from those pre-existing. This process occurs in a regulated fashion during
development and growth as well as in response to physiological and pathological
stimuli. Angiogenesis as been shown to be a receptor- and ligand-regulated
process, with a still-growing, diverse number of soluble factors and their
cognate receptors being involved in the different phases of the angiogenic
process (1). In the developing brain, angiogenesis has been shown to be
regulated by factors secreted by neuronal and glial cell populations in an
orderly, spatiotemporal fashion (2). In recent studies we and others have
shown that selected angiogenic factors, VEGF in particular, are capable of not
only affecting a variety of endothelial behaviors, but also are capable of
affecting neuronal behavior in a receptor-specific fashion (3,4). Interestingly,
recent studies have demonstrated that neurotrophins expressed by endothelia
and are capable of influencing several endothelial cell functions including
endothelial cell survival and vessel stabilization (5-7) and that endothelial cells
may express neurotrophin receptors (8).
Neurotrophins form a large family of dimeric polypeptides that include
nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), NT-4/5, NT-6 and NT-7 (9-12). They are known to promote the
growth, survival, and differentiation of developing neurons in the central and
peripheral nervous systems (13-18). BDNF, given peripherally, accelerates the
regenerative sprouting of injured adult spinal motor neurons and axotomized
retinal ganglion cells (19). Therefore, BDNF appears to be involved in peripheral
sensory and motor neuron regeneration at the site of nerve injury.
Neurotrophins mediate their action on responsive neurons by binding to
two classes of cell surface receptor (20). TrkA, TrkB and TrkC selectively bind
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. 4 Kim, Li, Hempstead, Madri
BGF, BDNF and NT-3 (21). In addition, the neurotrophins can interact with
another low-affinity neurotrophin receptor, p75NTR, which has been shown to
initiate an apoptotic signal in neurons when engaged by proNGF (22,23). TrkB
and BDNF are expressed at high levels not only in central and peripheral nervous
tissue (24-26), but also in several nonneuronal tissues, including muscle, heart
and the vasculature at levels comparable to those of the brain (27-30). In
pathologic states, BDNF and TrkB expression are induced in neointimal vascular
smooth muscle cells of the adult rodent and human aorta following vascular
injury (31). These studies suggest that there may be a complex and
dynamically regulated cross-talk between neuronal cells and endothelial cells
during development, growth and in response to pathological stimuli in the brain
and prompted us to investigate these potential interactions.
In this report we have demonstrated the expression of BDNF by brain-
derived endothelial cells and the expression and activation of the neurotrophin
receptors TrkB and p75NTR in these brain-derived endothelial cells. In addition,
we have shown that engagement of either TrkB or p75NTR (by BDNF and
proNGF respectively) results in distinct endothelial behaviors, survival &
angiogenesis in the case of BDNF activation of TrkB and apoptosis in the case
of proNGF activation of p75NTR. Further, the importance of these findings in
the control of neurovascular development and responses to chronic sublethal
hypoxic injury is discussed.
Materials and Methods
Recombinant NGF and proNGF
Cleavage resistant proNGF was purified from the media of cells stably
expressing the construct, using Ni-chromatography and imidazole elution as
described (23). Mature NGF or media from cells expressing the plasmid were
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. 5 Kim, Li, Hempstead, Madri
used in parallel (23).
Cell Culture: RBE4 and bEnd-WT Cell Cultur
Transformed rat brain endothelial (RBE4) cells were obtained from F.
Roux (Hospital F. Widal, Paris, France). The RBE4 cells were cultured from
passages 16-25 as previously described (32). Immortalized mouse brain
endothelial cells (bEnd-WT) were obtained from Dr. Britta Engelhardt (Max-
Planck Institute for Vascular Biology, Münster, Germany) and were cultured and
passaged as described (33). For three-dimensional culture experiments, acid-
soluble calf dermis type I collagen (ASC I) was prepared and solubilized in 10mM
acetic acid (2.5mg/ml) as previously described (32). RBE4 or bEnd-WT cells
were added to the collagen to a final concentration of 2x105 cells /ml. Droplets
of the cell-collagen suspension were spotted onto petri dishes. Following
polymerization, the droplets were overlaid with media (alpha-MEM, and F10
Nutrient Mixture with glutamine, bFGF, Geneticine, and 10% FBS) and incubated
in 5% CO2 at 37oC. For RBE4 and bEnd-WT culture experiments, recombinant
BDNF at concentrations of 10ng.ml and 50ng/ml, soluble, recombinant TrkB
receptor bodies (R&D systems, Minneapolis, MN) at a concentration of 2µg/ml,
proNGF at concentrations of 1ng/ml, 5 ng/ml and 10ng/ml, mature NGF at a
concentration of 50ng/ml were added. Wortmannin, LY294002, and PD98059
were purchased from Sigma (St Louis, MO).
Cells were cultured for 6 days. Media was changed and recombinant
proteins were added every 24 hours.
All hypoxia experiments were performed with cells incubated in a sealed,
humidified chamber gassed with 10% O2,, 5% CO2, 85% N2 at 37oC as
described(32).
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. 6 Kim, Li, Hempstead, Madri
Transfection of bEnd-WT cells
bEnd-WT cells were infected with recombinant adenoviruses at ~ 90%
confluency. Cells were infected with adenovirus containing HA-tagged dominant
negative Akt (Akt-AAA) with a marker of green fluorescent protein (GFP) (a
generous gift of Dr. William Sessa, Yale University) in serum-free DMEM medium
for 1 hour and then incubated for 24 hours in complete growth medium as
described(34-37) before the start of expereiments. Recombinant adenovirus
encoding β-galactosidase (Ad- β-gal) was used as a control. Infection efficiency
of bEnd-WT cells with recombinant adenoviruses at 40 multiplicity of infection
(m.o.i.) was close to 100% as determined by the green fluorescent color
observed in the cells and immunohistochemical staining of β-galactosidase. The
expression and relative levels of endogenous and recombinant adenoviral Akt
were confirmed by Western blot.
Matrigel AssayBD Matrigel™ matrix was used to coat tissue culture dishes according to
the manufacturer’s instructions (BD Biosciences, San Jose, CA). Cells were
plated onto the matrix at a density of 5x105 cells per 30 mm plate, and allowed
to grow for various times. At specific time points, light microscopy images
were taken and analyzed and cell lysates were prepared as described(38).
Vessel counting
Vessel counts were performed with 3 samples per condition. Three
random fields were photographed per sample. Random digital images of cultures
were taken using an inverted research microscope (IMT) (Olympus Co.)
equipped with Nikon coolpix 995 digital camera using Photoshop 5.0 software
on a Macintosh G4 computer. NIH Image 1.62 or IP LAbSpectrum software was
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. 7 Kim, Li, Hempstead, Madri
used to select, measure, and analyze the images to determine aggregate tube
length. Data was expressed in terms of pixel change (NIH Image) or microns (IP
LabSpectrum) compared to normoxic (5% CO2 & room air [20% O2]) controls.
Statistics (Student’s t-test and standard error) were calculated and graphically
presented using Excel 2000 on Macintosh G4 computer. Statistical significance
was assumed for p values < 0.05.
Immunoprecipitation and Western Blotting
Cell lysates and subsequent immunoprecipitation with anti-VEGFR-2/Flt-1
and Western blotting with anti-VEGFR-2/Flt-1 and anti-PY (PY 99) antibodies
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were performed as
described(39).
Western blots were performed on lysates of RBE4 and bEnd-WT cells as
previously described (3,32). Lysates were made with Modified RIPA buffer
(50mM Tris-HCl, pH7.4, 1% NP-40, 0.25% Na-deoxycholate, 150mM NaCl, 1mM
EDTA, 1mM PMSF, 1mg/ml Aprotinin, leupeptin, 1mM Na3VO4, 1mM NaF).
Antisera directed against BDNF (Santa Cruz Biotechnology Inc, SC-546 at
1:200), TrkB (BD bioscience, 610101 at 1:1,000 and Santa Cruz, SC-8316 at
1:1,000), pTrkB (Cell signaling technology, Inc., 9141 at 1:1,000), p75NTR
(Santa Cruz technology Inc., SC-8317 at 1:200), Flk-1 (VEGFR2) (Santa Cruz
technology, SC-504 at 1:200), pERK (Cell signaling technology, Inc., 3191 at
1:1,000), ERK2 (Santa Cruz Technology Inc., SC-1647 at 1:10,000), pAkt (Cell
signaling technology, Inc., 9171 at 1:1,000), Akt (Cell signaling technology,
Inc., 9272 at 1:1,000), Cleaved caspase 3 (Cell signaling technology, Inc., 9664
at 1:1,000) were used. Detection was carried out using Pierce supersignal
detection reagent (Pierce, Milwakee, WI) with membrane exposure to Hyperfilm
reagent (Amersham Biosciences, Inc). Quantitation was performed on scanned
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images (Agfa Arcus II Scanner using Adobe Photoshop 5.0, Adobe systems, CA)
using the BioMax Program (Kodak, Rochester, NY) on a Macintosh G4 computer.
All experiments were performed at least three times. Statistical analysis was
performed using Student t-test (p<0.05).
Immunocytochemistry
Cultured RBE4 cells were fixed with 4% paraformaldehyde in PBS (pH
7.4) and blocked with PBS in 3% BSA, 10% normal donkey serum, 0.1% triton
X-100. The primary antibodies used were anti-BDNF (Santa Cruz Biotechnology,
Inc., SC-546 at 1:200), and anti-TrkB (Santa Cruz Biotechnology, Inc., SC-8316
at 1:200), anti-p75NTR (Santa Cruz Biotechnology, Inc., SC-8317 at 1:200).
Primary antibodies were incubated overnight at 4oC. CY3-conjugated donkey
anti-rabbit IgG (Jackson Immunoresearch Laboratories Inc., Bar harbor, ME) was
used as secondary antibody. Images were taken using a Zeiss research
microscope equipped with SPOT camera. Images were collected using
Photoshop 5.0 on a Macintosh G4 computer.
FACS analysis of apoptosis
FACS analysis of cultured, transfected bEnd-WT cells was performed as
previously described(33). Propridium Iodide and Annexin V (BD Biosciences,
San Diego, CA) were used to assess apoptotic cells. BD FACStation Software
for Mac OSX was used to analyze the results and Statview Software was used to
determine significance.
Results
BDNF expression is induced by hypoxia in vivo in the microvasculature of the
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CNS and is expressed by RBE4 cells and induced in these cells by hypoxia in
vitro
Imunohistochemical staining of cortical sections of pups reared in
normoxia (Nx) and hypoxia (Hx) revealed increased expression of BDNF protein,
primarily in the microvasculature Figure 1A-D). We then performed
immunofluorescence microscopy to assess the expression of BDNF on RBE4
cells. We also performed Western blotting on RBE4 cell lysates. We determined
that RBE4 cells expressed BDNF under baseline (Nx - normoxic) culture
conditions. Interestingly, under hypoxic conditions (Hx - hypoxic) BDNF
expression was found to be increased in both RBE4 cells and astrocytes (data
not shown) using both immunofluorescence and Western blotting methods (n =
5; p<0.03) (Figure 1E-H). BDNF was localized in essentially all RBE4 cells and
its expression was noted to be significantly increased under hypoxic culture
conditions.
RBE4 cells form tubes and sprouts in collagen gels which is enhanced by
recombinant BDNF but blocked by soluble, recombinant TrkB.
RBE4 cells placed in three-dimensional (3D) matrices of collagen type I
gel and cultured for 6 days cluster to form multicellular cysts, from which
elongated tube-like processes extend (Chow et al., 2001). Addition of
exogenous recombinant BDNF stimulated increased tube formation in cultures
(compare Figures 2A & B); while addition of recombinant, soluble TrkB (which
sequesters BDNF), acted to inhibit the cyst and tube formation of RBE4 cells
(compare Figure 2C with 2A & 2B).
When cultured under hypoxic conditions, tube formation of RBE4 cells
was reduced compared with that of normoxic cultures (compare Figures 2D &
2A). As noted in normoxic cultures, treatment with exogenous BDNF
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stimulated tube formation (Figure 2E) and treatment with soluble, recombinant
TrkB significantly reduced cyst and tube formation (Figure 2F). Panel 2G
represents a quantitation analysis of these studies. Similar results were
obtained when bEnd-WT cells were used. Representative fields of bEnd-WT cells
cultured on Matrigel coatings under normoxic conditions in the absence or
presence of 10 ng/ml rBDNF or 2.0 µg/ml rTrkB are illustrated in panels 2H-J.
Note the increased amount of tube formation in the presence of rBDNF and the
decreased amount of tube formation in the presence of rTrkB. Quantitation of
bEnd-WT cell tube formation (panel 2I) in normoxic conditions in the absence or
presence of rBDNF or rTrkB revealed a robust increase in tube formation in the
presence of rBDNF and a marked decrease in tube formation when rTrkB was
added to the cultures.
TrkB is expressed by and activated by BDNF in RBE4 cells.
To determine whether TrkB is expressed on RBE4 cells we performed
Western blot analyses of lysates of RBE4 cells derived from normoxic (Nx) and
hypoxic (Hx) cultures. Western blotting illustrated the presence of TrkB in RBE4
cell lysates and overall, protein levels of TrkB remained unchanged in response
to hypoxic stimulation (Figure 3A).
To determine if the TrkB present in these brain-derive endothelial cells is
activated, we performed Western bolts using anti-pTrkB followed by Western
blotting analysis using anti-TrkB. We found that a fraction of TrkB was tyrosine
phosphorylated under baseline culture conditions and phosphorylated TrkB
levels were increased following the addition of exogenous BDNF (Figure 3B).
Additionally, treatment of RBE4 cultures with recombinant, soluble TrkB resulted
in a significant reduction of phosphorylated TrkB compared to the control
(normoxic) cultures. Similar results were observed under hypoxic conditions.
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These results suggest that TrkB expressed on RBE4 cells is activated by
endogenous and exogenous BDNF.
Akt, but not MAPK activation is associated with BDNF-mediated RBE4 cell
survival in vitro.
To elucidate the mechanisms involved in this BDNF-mediated endothelial
cell survival and tube formation in this culture system, we assessed the
activation states of Akt and ERK1/2, members of two signaling pathways
known to be involved in mediating endothelial survival and tube formation
(32,40). The level of phosphorylated ERK was significantly increased by hypoxia
but not following treatment of exogenous BDNF or soluble, recombinant TrkB
(Figure 4B). In contrast, the level of serine-phosphorylated Akt was significantly
increased following treatment with BDNF under normoxic and hypoxic conditions
(Figure 4A). In addition, treatment with soluble, recombinant TrkB reduced the
levels of phosphorylated Akt in normoxic and hypoxic cultures (Figure 4A).
These results suggest that Akt is activated following BDNF engagement and
activation of TrkB and this pathway may be, in part, responsible for mediating
the survival of these endothelial cells.
To confirm the role of Akt activation in mediating brain-derived
endothelial survival bEnd-WT cells were infected with a dominant negative HA-
tagged Akt construct (Akt-AAA) or an β-galactosidase containing vector and
apoptotic levels determined following normal culture conditions, serum
starvation and BDNF treatment. While a baseline low apoptotic level was noted
in cells infected with the β-galactosidase containing vector with and without
addition of exogenous BDNF (approx 10.6% +/- 1.3%), high apoptotic levels
were observed in the β-galactosidase vector infected cells cultured under serum
starvation (17.3%). In contrast, cells infected with the dominant negative Akt-
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AAA construct exhibited high apoptotic rates in the absence (approx 17.3% +/-
2%) and presence (approx 20.5% +/- 2%) of exogenous BDNF (Figure 4C).
These results lend additional support to the concept that Akt is activated
following BDNF engagement and activation of TrkB and this pathway may be, in
part, responsible for mediating the survival of these endothelial cells.
Exogenous BDNF blunted activation of caspase 3; while soluble, recombinant
TrkB induced activation of caspase 3 in RBE4 cells
Additional studies revealed that BDNF modulated caspase 3 cleavage.
Exogenous BDNF induced tube formation and rescued the cells from hypoxic
insult. Under normoxic conditions, cleaved caspase 3 expression was decreased
significantly by addition of exogenous BDNF and was increased following
treatment with recombinant soluble TrkB (Figure 5). Culture of RBE4 cells
under hypoxic conditions induced activation of caspase 3; however, cleaved
caspase 3 levels were significantly decreased following the addition of
exogenous BDNF to hypoxic cultures. As noted above, soluble, recombinant
TrkB further increased the activation of caspase 3 in hypoxic cultures (Figure
5). These results suggest that BDNF modulates apoptosis in these brain-
derived endothelial cells, in part, by regulating caspase 3 activity.
Modulation of cleaved Caspase 3 expression by PI3K & MEK inhibitors on BDNF-
treated endothelial cells under hypoxia
To further determine the specific signaling pathways involved in the
BDNF-induced inhibition of apoptosis and inhibition of caspase 3 activation, a
pharmacological approach was taken. Namely, chemical inhibitors of either MEK
(PD98059, 20 µM), or PI3-kinase (LY240002 and Wortmannin, 20 µM [not
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shown]) were added daily for six days to RBE4 cultures. Under normoxic
conditions, 20 µM of LY240002, but not PD98059, significantly increased the
levels of cleaved caspase 3 in cultures treated with BDNF (Figure 6). Under
hypoxic conditions, these PI3-kinase inhibitors, but not the MEK inhibitor, also
increased the levels of cleaved caspase 3 significantly in cultures treated with
BDNF (Figure 6). These data suggest that the PI-3 kinase signaling pathway is
involved in the BDNF-mediated modulation of RBE4 cell caspase activation and
survival behavior.
BDNF treatment increases VEGFR2 expression on RBE4 cells cultured under
normoxic and hypoxic conditions.
As we and others have reported, VEGF is a potent angiogenic and
survival factor in the CNS, affecting both endothelial cells and neurons (3,32).
In previous studies we determined that chronic hypoxia induces VEGF expression
in rodent cerebral cortex, specifically by neurons and glia and by astrocytes and
cortical neurons in culture (2,3,41,42). Considering that BDNF has been
reported to have a survival role in neurons and endothelial cells, we explored the
possibility that there may be interactions between the BDNF and VEGF signaling
pathways. To elucidate potential interactions between BDNF and VEGF signaling
pathways, we performed Western blotting on lysates of RBE4 cells incubated
with exogenous recombinant BDNF.
In addition to its modulation of caspase activation (Figures 5 & 6), BDNF
was found to modulate the expression levels of VEGFR2 in RBE4 cells as well as
in bEnd-Wt cells cultured under normoxic and hypoxic conditions (Figure 7).
Specifically, addition of exogenous recombinant BDNF elicited significant
increases in VEGFR2 expression of 50% and 100% in normoxic and hypoxic
cultures of RBE4 cells respectively (Figure 7A). Similar results were obtained
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using bEnd-WT cells as illustrated in Figure 7B. These results indicate that
exogenous BDNF can modulate VEGF-mediated activities in these brain-derived
endothelial cells by altering VEGF receptor expression.
bEnd-WT cells exhibit increased VEGFR2 phosphorylation, proliferation and tube
formation in response to VEGF following pre-treatment with rBDNF.
The findings of increased VEGFR2 expression following BDNF treatment
prompted us to determine VEGF-induced VEGFR2 phosphorylation levels
following BDNF-induced VEGFR2 expression and the potential functional
significance of increased VEGFR2 expression and activation. Determination of
phospho-VEGFR2 revealed increased VEGF-induced phosphorylation of the
receptor following BDNF pretreatment and the fraction of VEGFR2
phosphorylated was also significantly increased compared to that determined in
control and TrkB treated cultures (Figure 8A). bEnd-WT cell cultures pre-
treated with 10 ng/ml rBDNF for 72 hr followed by treatment with 10 ng/ml
VEGF for 24 hr also exhibited a marked increase in tube length and number,
consistent with the increase in VEGFR2 expression and phosphorylation (Figure
8B & C). Additionally, pre-treatment with rTrkB resulted in a modest decrease in
VEGFR2 expression (Figurer 7B) and both tube length and number compared to
control cultures (Figure 8B & C).
RBE4 cells express p75NTR and when engaged by proNGF, apoptosis is induced.
Interestingly, in addition to expressing TrkB, RBE4 cells were found to
express p75NTR (Figure 9A-C), a common low affinity receptor of pro-
neurotrophins, including proNGF (20,22). As observed for TrkB expression
(Figure 3A), expression of p75NTR was not altered by hypoxic culture
conditions. Since neurotrophins can be secreted as propeptides, proNGF would
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be capable of binding to RBE4 p75NTR and initiating signal transduction.
Engagement and activation of p75NTR has been demonstrated to induce
apoptosis in neuronal, vascular and smooth muscle cell populations that express
p75NTR (20,22). Thus, we hypothesized that proNGF may bind to p75NTR on
RBE4 cells and initiate a signal transduction pathway distinct from that noted
following BDNF mediated TrkB stimulation. To determine the effects of proNGF
on RBE4 cells, we incubated three-dimensional cultures of RBE4 cells with
proNGF and mature NGF. Addition of proNGF elicited a significant apoptosis of
RBE4 cells as evidenced by increased Annexin V staining (Figure 10A) and
reduction of endothelial cell sprout and cyst formation and maintenance (Figure
10B). In contrast, the vehicle alone and mature NGF treatment groups
experienced no appreciable apoptosis.
These results suggest a complex, receptor-mediated regulation of
endothelial cell behavior by the expression and processing of neurotrophins and
the expression of their cognate receptors.
Discussion
Angiogenesis is a tightly-controlled process, dependent upon the finely
integrated and orchestrated expression, availability and activities of a variety of
soluble factors, solid phase components including several extracellular matrix
proteins, glycoproteins, proteoglycans and glycosaminoglycans, their cellular
cognate receptors and several proteases. Angiogenesis occurs in a multicellular
environment in which there is direct contact as well as juxtacrine, paracrine and
endocrine interactions among a variety of cell types, dependent upon which
tissue/organ is involved. In previous studies we have determined that astrocyte
– endothelial cell interactions (via glial end-foot apposition and secretion of
VEGF) and neuronal – endothelial cell interactions (via secretion of VEGF) are
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critical for normal neurovascular and neuronal development (2,3,20,41,42).
However, given the variety of soluble factors known to be expressed during
neuronal and neurovascular development and recent reports of specific
neurotrophins playing roles in endothelial cell survival and vessel stabilization (6-
8,31), we reasoned that particular neurotrophins might elicit specific receptor-
mediated responses in endothelial cells derived from the brain.
Neurotrophins (BDNF) have been shown to be expressed by some, but
not all endothelial cells in culture (6,8,43). Using brain-derived, immortalized
endothelial cells (RBE4 cells) we determined that these cells indeed express
BDNF in our three-dimensional culture system and that expression is increased
following hypoxic treatment (Figure 1), consistent with our
immunohistochemical localization data. In previous studies we have shown that
these cells form cysts with linear angiogenic sprouts emanating from them
when placed in a three-dimensional collagen type I gel (3,32). Upon treatment
of these cultures with recombinant BDNF, a significant increase in angiogenic
sprouts was noted. In contrast, treatment with recombinant, soluble TrkB
(which sequesters BDNF) resulted in a marked loss of both angiogenic sprouts
and cysts (Figure 2A-C & G). Interestingly, under hypoxic culture conditions
addition of exogenous BDNF promoted cell survival and robust angiogenic
sprout formation (Figure 2 D-F & G), similar to that noted previously for VEGF
(32). These data are consistent with the presence of a BDNF receptor on these
cells. Indeed, Figure 3A illustrates the presence of TrkB. Activation (tyrosine
phosphorylation) of this receptor in response to its ligand (BDNF) is required if
we are to ascribe the survival/angiogenic response to a receptor-mediated
process. Figure 3B illustrates the required increase in TrkB phosphorylation in
response to exogenous BDNF and the reduction of TrkB phosphorylation
following sequestration of endogenous BDNF. This BDNF response appears to
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signal changes in endothelial cell survival via a PI-3 Kinase/Akt pathway as
addition of BDNF results in increased Akt phosphorylation, sequestration of
BDNF causes a reduction of Akt phosphorylation while ERK is not appreciably
affected and over-expression of a dominant negative Akt renders the cells
insensitive to exogenous BDNF (Figures 4 & 5).
Apoptosis can be assessed by determination of caspase activation
(cleavage). We found a correlation between the level of RBE4 cell apoptosis,
the level of BDNF, the phosphorylation state of Akt and the level of cleaved
caspase 3 suggesting that activation of TrkB results in signaling cell survival via
the PI-3 Kinase/Akt pathway (Figure 5). This was confirmed using synthetic
inhibitors of PI-3 Kinase (LY294002 and Wortmannin) and MEK (PD98059)
(Figure 6).
Endothelial apoptosis is known to be modulated by several soluble
factors and engagement of their cognate receptors (32,44). Our data suggests
that engagement of TrkB results in a survival signal. However, TrkB-induced
endothelial cell survival may also be mediated via indirect signaling pathways.
Since it is known that VEGF signaling appears to signal survival in these cells
(32), we assessed the effects of TrkB activation on expression of VEGFR-2
expression. Interestingly, in both normoxic and hypoxic conditions, addition of
BDNF resulted in increased expression of RBE4 VEGFR-2 (Figure 7). This data is
consistent with the notion that in addition to its direct effects on apoptosis,
engagement and activation of TrkB may exert some of its anti-apoptotic and
angiogenic effects via up-regulation of VEGFR-2, a known major modulator of
endothelial survival, proliferation and angiogenesis (Figures 7 & 8).
Neurotrophin expression is known to be upregulated during injury and
stress to the central nervous system (Hicks et al., 1999), resulting in significant
changes in the levels and states of the neurotrophins (45,46), presumably
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affecting extent of injury and subsequent repair (47-50). Neurotrophins can be
secreted as pro-peptides which are cleaved by specific metalloproteinases,
producing active, mature neurotrophins which, in turn, are capable of binding to
their cognate Trk receptors with high affinities (22). In contrast, the pro-
neurotrophins can interact with another neurotrophin receptor, p75NTR, which
has been shown to initiate an apoptotic signal in neurons when engaged
(22,51). Considering that neurotrophin levels and their state (pro- vs. mature-)
can initiate diverse signaling pathways (23), we investigated whether RBE4 cells
also expressed p75NTR. As illustrated in Figure 9, RBE4 cells indeed do express
p75NTR and appear to respond to its engagement with proNGF. Figure 10
illustrates the effects of engagement of p75NTR on the survival of cultured
RBE4 cells. Engagement of p75NTR by proNGF resulted in a marked increase in
Annexin V staining (Figure 9A) and a dramatic loss of cell viability as evidenced
by quantitation of the numbers of endothelial cysts and sprouts remaining after
24 and 48 hours of treatment (Figure 9B). As expected, mature NGF and
vehicle alone had no appreciable effects on cell viability.
Given our findings that “vascular” ligands and their receptors (VEGF and
VEGFRs ) (3,32) and neurotrophins and their receptors (BDNF, NGF, TrkB and
p75NTR) are differentially expressed in CNS-derived endothelial cells, glia and
neurons, it is likely that these cell types (as well as other cell types in the CNS)
interact with each other via soluble factors, resulting in a dynamic modulation of
cellular behaviors during normal development and maintenance of the CNS as
well as in response to noxious stimuli. During chronic sublethal hypoxia in vivo
(2,42,52), and as modeled in our culture system (2,3,32,42), increased glial
and neuronal VEGF expression is noted, which, in turn, initiates and maintains
cerebral angiogenesis and loss of permeability function and alters neuronal
apoptosis and differentiation. These perturbations and others involving
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neurotrophins (BDNF, NGF), their proteolytic processing and their receptors
(TrkB and p75NTR) would then affect subsequent CNS cell behaviors including
cell survival, proliferation, migration and differentiation, with in turn, would have
dramatic effects on the genes involved with synaptic maturation, post-synaptic
function, neuro-transmission, glial maturation and angiogenesis. Thus, a more
complete understanding of these complex, dynamic interactions among these
cells and their soluble factors appears warranted if we are to develop rational
therapeutic agents to beneficially affect the brain’s responses to injury and
reparative processes.
Acknowledgements: The authors would like to thank Ramee Lee for
generating the purified, recombinant proNGF.
Figure Legends
Figure 1. Cortical tissue, cortical microvasculature and RBE4 cells express
BDNF and exhibit induction of BDNF following hypoxia. Panels A and B are
representative sections of cortex from normoxia-reared pups revealing modest
BDNF expression. Panels C and D are representative sections of cortex from
hypoxia-reared pups revealing robust microvascular BDNF expression. Panels E
and F are representative immunofluorescence micrographs of cultured RBE4
cells stained with anti-BDNF illustrating modest expression in normoxic (Nx) and
increased expression in hypoxic (Hx) conditions. Panel G is a representative
Western blot of lysates of RBE4 cells cultured in normoxic and hypoxic
conditions for 6 days illustrating BDNF expression (13 kDa) (normalized for
actin expression) in both culture conditions, being increased in hypoxic
conditions. Panel H represents the average of 5 Western blotting experiments,
illustrating the expression of BDNF in RBE4 cells and its increased expression in
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hypoxic conditions. (n = 5; * = p < 0.05; vertical lines represent standard
deviations).
Figure 2. BDNF mediates RBE4 and bEnd-WT cell survival and angiogenesis.
Panels A & D: Representative fields of RBE4 cells cultured under normoxic (A)
and hypoxic (D) conditions. Note the loss of cystic and tubular structures in D.
Panels B & E: Representative fields of RBE4 cells under normoxic (B) and
hypoxic (E) conditions in the presence of 50 ng/ml rBDNF. Note the increased
numbers of cysts and sprouts in both panels.
Panels C & F: Representative fields of RBE4 cells under normoxic (C) and
hypoxic (F) conditions in the presence of 50 ng/ml rTrkB. Note the decreased
numbers of cysts and sprouts in both panels. Panel G: Quantitation of RBE4
cell survival and sprout formation/survival in normoxic and hypoxic conditions in
the absence or presence of 50 ng/ml rBDNF or rTrkB. (n = 5; * = p < 0.05;
vertical lines = standard deviations).
• Panels H-J: Representative fields of bEnd-WT cells cultured on Matrigel coatings
under normoxic conditions in the absence presence of 10 ng/ml rBDNF or 2.0
µg/ml rTrkB. Note the increased amount of tube formation in the presence of
rBDNF and the decreased amount of tube formation in the presence of rTrkB.
Panel K: Quantitation of bEnd-WT cell tube formation in normoxic conditions in
the absence or presence of rBDNF or rTrkB. (n = 5; * = p < 0.05; vertical lines
= standard deviations).
Figure 3. RBE4 cells express TrkB, which is activated by BDNF.
A, upper panel: Representative Western blots for TrkB (140 kDa) in lysates of
normoxic (Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of 50
ng/ml BDNF normalized for ERK2 expression. A, lower panel: Quantitation of
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TrkB expression in RBE4 cells as described above. Note that there are no
statistically significant changes in TrkB expression in any of the conditions
tested. (n = 5; p > 0.05; vertical lines = standard deviations).
B, upper panel: Representative Western blots for pTrkB in lysates of normoxic
(Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of 50 ng/ml
BDNF or rTrkB, normalized for TrkB expression. Note the increases in band
intensity in the cultures treated with BDNF and the decreases in band intensity
in the cultures treated with rTrkB. B, lower panel: Quantitation of pTrkB
expression in RBE4 cells as described above. Note that there are statistically
significant changes (*) in pTrkB expression in the BDNF treated and the rTrkB
treated cultures. (n = 5; p < 0.05; vertical lines = standard deviations).
Figure 4. BDNF induces Akt phosphorylation in RBE4 cells. Representative
Western blots for pAkt (A) and pERK (B) in lysates of normoxic (Nx) and
hypoxic (Hx) RBE4 cultures in the presence or absence of 50 ng/ml BDNF or
rTrkB, normalized for Akt and ERK2 respectively.
A. Upper panel: Representative Western blots for pAkt in lysates of normoxic
(Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of 50 ng/ml
BDNF or rTrkB, normalized for Akt. Note the increases in band intensity in the
cultures treated with BDNF and the decreases in band intensity in the cultures
treated with rTrkB. Lower panel: Quantitation of pAkt expression in RBE4 cells
as described above. Note that there are statistically significant changes (*) in
pAkt expression in the BDNF treated and the rTrkB treated cultures. (n = 5; p <
0.05; vertical lines = standard deviations).
B. Upper panel: Representative Western blots for pERK in lysates of normoxic
(Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of 50 ng/ml
BDNF or rTrkB, normalized for ERK2. Note there are no statistically significant
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changes in band intensities in any of the cultures. Lower panel: Quantitation of
pERK expression in RBE4 cells as described above. Note that there are no
statistically significant changes in pTrkB expression in the BDNF treated and the
rTrkB treated cultures. (n = 5; p > 0.05; vertical lines = standard deviations).
C. % apoptosis determined by Annexin V FACS analysis of bEnd cultures
transfected with β-galactiosidase or β-galactosidase and Akt-AAA implicates the
Akt pathway. Upper panel: Representative Western blots illustrating Akt and
HA expression in β-galactosidease infected (β-gal) and HA-tagged dominant
negative Akt-AAA infected (Akt-AAA) bEnd WT cells. Middle panel:
Representative FACS analyses of β-galactosidease infected and Akt-AAA
infected bEnd WT cells under control and serum starvation conditions in the
absence and presence of 10 ng/ml BDNF. Lower Panel: Quantitation of the
FACS analyses illustrating that infection with dominant negative Akt-AAA
increased apoptosis significantly compared to infection with β-galactosidease
alone. As expected, treatment with BDNF did not blunt the level of apoptosis
observed in the presence of Akt-AAA. (SS = Serum Starvation; n = 3; p >
0.001; vertical lines = standard deviations)
Figure 5. BDNF levels modulate Caspase 3 cleavage in RBE4 cells. Upper
panel: Representative Western blots for cleaved caspase 3 expression in lysates
of normoxic (Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of
50 ng/ml BDNF or rTrkB, normalized for ERK2. Note the decreases in band
intensity in the cultures treated with BDNF and the increases in band intensity in
the cultures treated with rTrkB. Lower panel: Quantitation of cleaved caspase
3 expression in RBE4 cells as described above. Note that there are statistically
significant changes in cleaved caspase 3 expression in the rTrkB treated
cultures compared to normoxic cultures (*), as well as in comparing normoxic
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and hypoxic conditions (**) and in comparing hypoxic and hypoxic + rBDNF
conditions (***). (n = 5; *, **, *** = p < 0.05; vertical lines = standard
deviations).
Figure 6. PI-3 kinase inhibitor modulates Caspase 3 cleavage in RBE4 cells.
Upper panel: Representative Western blots for cleaved caspase 3 expression in
lysates of normoxic (Nx) and hypoxic (Hx) RBE4 cultures in the presence or
absence of 50 ng/ml BDNF, 20 µg/ml LY294002 or 20 µg/ml PD98059,
normalized for ERK2. Note the decreases in band intensity in the cultures
treated with BDNF and the increases in band intensity in the cultures treated
with LY294002, but not PD98059. Lower panel: Quantitation of cleaved
caspase 3 expression in RBE4 cells as described above. Note that there are
statistically significant changes in cleaved caspase 3 expression in the BDNF
treated and the LY294002 treated cultures in both normoxic (*) and hypoxic
(**) conditions. (n = 5; * & ** = p < 0.05; vertical lines = standard deviations).
Figure 7. BDNF induces VEGFR-2 expression in RBE4 and bEnd-WT cells.
A. Upper Panel: Representative Western blots for VEGFR-2 expression in lysates
of normoxic (Nx) and hypoxic (Hx) RBE4 cultures in the presence or absence of
50 ng/ml BDNF, normalized for ERK2. Note the increases in band intensity in
the cultures treated with BDNF in both normoxic and hypoxic conditions. Lower
panel: Quantitation of VEGFR-2 expression in RBE4 cells as described above.
Note that there are statistically significant changes in VEGFR-2 expression in
the BDNF treated cultures in both normoxic and hypoxic conditions. (n = 5; * =p
< 0.05; vertical lines = standard deviations).
B. Upper Panel: Representative Western blots for VEGFR-2 expression in lysates
of normoxic bEnd-WT cultures in the absence (Cont) or presence of 2.0 µg/ml
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sTrkB (TrkB) or 10 ng/ml BDNF (BDNF), normalized for ERK2. Note the
increases in band intensity in the cultures treated with BDNF and the decreased
band intensity in the cultures treated with sTrkB. Lower panel: Quantitation of
VEGFR-2 expression in bEnd-WT cells as described above. Note that there are
statistically significant changes in VEGFR-2 expression in the BDNF- and sTrkB-
treated cultures. (n = 6; * =p < 0.05; vertical lines = standard deviations).
Figure 8. BDNF pretreatment induces increased VEGFR-2 phosphorylation and
VEGF-induced angiogenesis in bEnd-WT cells.
A. Determination of phospho-VEGFR2 revealed increased phosphorylation of
the VEGFR-2 following BDNF pretreatment of bEnd-WT cells. When normalized
to the amount of VEGFR-2 expressed, the fraction of phosphorylated VEGFR2
was also significantly increased compared to that determined in control and
TrkB treated cultures. The upper panel is a representative series of
immunoblots used for the quantitation that is illustrated in the lower panel. (n =
6; * = p < 0.05; vertical lines = standard deviations).
B & C. bEnd-WT cell cultures pre-treated with 10 ng/ml rBDNF for 72 hr
followed by no treatment (open boxes) or treatment with 10 ng/ml VEGF
(shaded boxes) for 24 hr exhibited a marked increases in tube length (B) and
aggregate tube number (C), consistent with the increases in VEGFR2 expression
and phosphorylation following BDNF pretreatment. As expected pre-treatment
with rTrkB resulted in decreased tube length and number compared to control
cultures. (n = 6; * =p < 0.05; vertical lines = standard deviations).
Figure 9 . RBE4 cells express p75NTR. Panels A & B: Representative
immunofluorescence micrographs of cultured RBE4 cells stained with anti-
p75NTR illustrating unchanged expression in normoxic (Nx) and hypoxic (Hx)
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conditions. Panel C, upper panel: Representative Western blots for p75NTR in
lysates of normoxic (Nx) and hypoxic (Hx) RBE4 cultures in the presence or
absence of 50 ng/ml BDNF normalized for ERK2 expression. Panel C, lower
panel: Quantitation of p75NTR expression in RBE4 cells as described above.
Note that there are no statistically significant changes in p75nntr expression in
any of the conditions tested. (n = 5; p > 0.05; vertical lines = standard
deviations).
Figure 10. ProNGF induces RBE4 cell apoptosis. Panel A: Representative
immunofluorescence micrographs of RBE4 cells cultured in the presence of
vehicle alone, (5 ng/ml mature NGF – not shown) 50 ng/ml mature NGF or 5
ng/ml proNGF and stained for Annexin V. Note the increased staining in the
cultures treated with proNGF, but not with mature NGF. Panel B: Quantitation
of RBE4 cell survival and sprout formation/survival in normoxic conditions in the
presence of vehicle alone, 50 ng/ml mature NGF or 5 ng/ml proNGF. Note the
decreased numbers of cysts in the cultures treated with proNGF (n = 5; * = p <
0.05; vertical lines = standard deviations).
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BDNF
Hx BD
NF
+ LY29
4002Hx
Hx BD
NF
+ PD98
059
Fo
ld C
han
ge
inC
leav
ed C
asp
ase
3
CleavedCaspase 3
ERK2
Nx BD
NF
Nx BD
NF + LY
2940
02
Nx Nx BD
NF + P
D9805
9
Hx BD
NF
Hx BD
NF + LY
2940
02
Hx Hx B
DNF +
PD98
059
**
Kim et al., Figure 6
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Kim et al., Figure 7B
0.0
5.0
10.0
TreatmentBDNF TrkBCont
Fo
ld C
han
ge
inV
EG
FR
-2 E
xpre
ssio
n *
*
VEGFR2
ERK
TrkB Cont BDNF
Flk-1
ERK2
Contro
l
BDNF
Contro
l
BDNF
RBE4 Nx RBE4 Hx
00.51.01.52.02.53.03.54.0
Contro
l
BDNF
RBE4 NxCon
trol
BDNF
RBE4 Hx
Fo
ld C
han
ge
inF
lk-1
Exp
ress
ion
*
*
A
B
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Kim et al., Figure 8A - C
PY-VEGFR-2VEGFR-2
β-Actin
BDNF TrkBCont
0
100
200
300
Control TrkB BDNF
No VEGF TXS/P VEGF
*
*
*
**
*
0
20
40
60
80
100
120
140
No VEGF TXS/P VEGF
Control Trk.B BDNF
Tub
e L
eng
th (µ
X 1
0 )3
0.0
2.5
5.0
7.5
BDNF
Fo
ld C
han
ge
inF
ract
ion
of
PY
-VE
GF
R-2
TrkBCont
*
A
B
Tub
e N
um
ber
/2.4
mm
2
C
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Kim et al., Figure 9
0
0.2
0.4
0.6
0.8
1.0
1.2
Nx NxBDNF
Hx HxBDNF
Fo
ld C
han
ge i
np
75
NTR
Exp
ress
ion
p75NTR
ERK2
NxNx
BDNF
Hx Hx BD
NF
Nx HxRBE4 Cells
A B
C
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Kim et al., Figure 10
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Hyun Kim, Qi Li, Barbara L. Hempstead and Joseph A. Madricells
Paracrine and autocrine functions of BDNF and NGF in brain-derived endothelial
published online May 28, 2004J. Biol. Chem.
10.1074/jbc.M404115200Access the most updated version of this article at doi:
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