paracrine and autocrine functions of bdnf and ngf in brain ...transformed rat brain endothelial...

. 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.

by guest on March 4, 2020

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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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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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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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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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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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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


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


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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Kim et al., Figure 8A - C

PY-VEGFR-2VEGFR-2

β-Actin

BDNF TrkBCont

0

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Control TrkB BDNF

No VEGF TXS/P VEGF

*

*

*

**

*

0

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No VEGF TXS/P VEGF

Control Trk.B BDNF

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0

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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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paracrine and autocrine functions of bdnf and ngf in brain ...transformed rat brain endothelial...

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