VEGF disrupts the neonatal blood–brain barrier and increases life span after non-ablative BMT in a...

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Experimental Neurology 188 (2004) 104–114

VEGF disrupts the neonatal blood–brain barrier and increases life span

after non-ablative BMT in a murine model of congenital

neurodegeneration caused by a lysosomal enzyme deficiency

Pampee P. Young,a,* Corinne R. Fantz,b and Mark S. Sandsc

aDepartment of Pathology, Vanderbilt University Medical Center, Nashville, TN 37232, USAbDepartment of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA

cDepartment of Internal Medicine and Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA

Received 14 November 2003; revised 25 February 2004; accepted 3 March 2004

Available online 8 May 2004

Abstract

The course of certain congenital neurodegenerative diseases like lysosomal storage diseases (LSDs) begins shortly after birth and can

progress quickly. Ideally, therapeutic interventions for LSDs, which include bone marrow transplantation (BMT), recombinant enzyme

replacement, or systemic viral-mediated gene therapy, should be initiated at birth. However, the blood–brain barrier (BBB) remains an

obstacle to effective therapy even when these strategies are initiated at birth. We studied whether VEGF, an endothelial cell mitogen and

permeability factor, can open the BBB in newborn mice for therapeutic purposes. Intravenous (IV) administration of VEGF at birth increased

BBB permeability within 2 h. The increased permeability persisted for at least 24 h, became undetectable 48 h after injection, and was

restricted to newborns. Systemic VEGF treatment before BMT or administration of recombinant lentivirus resulted in increased numbers of

both donor cells and virus-transduced cells, respectively, in the recipient brain. Administration of VEGF before BMT in newborn mice with a

neurodegenerative LSD, globoid-cell leukodystrophy, resulted in a significant increase in life span compared to affected animals that were

injected with saline before BMT.

D 2004 Elsevier Inc. All rights reserved.

Keywords: VEGF; Blood–brain barrier; BMT

Introduction Studies to determine VEGF’s effect in vivo on the blood–

VEGF is a heparin-binding growth factor specific for

endothelial cells. Although, it is well studied for its angio-

genic properties, it is also known for causing substantial

vascular leakage and is 50,000 times more potent than

histamine (Bates et al., 1999; Roberts and Palade, 1995).

In studies on the effect on microvascular permeability of

skin and cremastaric muscle, it was shown that capillary

endothelial cells become fenestrated within 10 min of

VEGF application. Studies suggest that the mechanisms

by which VEGF causes vascular permeability may be tissue

dependent and, thus far, are not well understood (Bates et

al., 1999; Dvorak et al., 1995).

0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.expneurol.2004.03.007

* Corresponding author. Department of Pathology, Vanderbilt Univer-

sity School of Medicine, 1161 21st Avenue South, U5211 MCN, Nashville,

TN 37232. Fax: +1-615-343-7023.

E-mail address: [email protected] (P.P. Young).

brain barrier (BBB) have produced conflicting results. Direct

infusion of VEGF into the cerebral cortex of adult mice

resulted in reversible local changes in endothelial cells and

an increase in permeability to plasma proteins (Dobrogowska

et al., 1998). By contrast, IV administration of the highest

tolerable doses of VEGF to adult rats resulted in increased

permeability to tracer protein dye only in the ischemic brain

but not in the contralateral normal brain (Zhang et al., 2000).

In vitro studies have supported the finding that the perme-

ability-enhancing effect of VEGF is limited to hypoxic con-

ditions on CNS-derived endothelial cells (Fisher et al., 1999).

Lysosomal storage diseases (LSDs) are a group of con-

genital disorders usually characterized by deficiencies of

specific acid hydrolases. These deficiencies result in the

accumulation of undegraded macromolecules in many cell

types, often including neurons and glial cells of the central

nervous system (CNS) (Neufeld and Muenzer, 1989). These

diseases are progressive in nature with usually little evidence

P.P. Young et al. / Experimental Neurology 188 (2004) 104–114 105

of disease at birth. Hence, early intervention is critical to

prevent neurologic sequelae (Sands et al., 1994, 1997).

Animal homologues of various LSDs serve as models for

testing the effectiveness of multiple therapeutic strategies for

treating congenital neurodegenerative diseases, including

enzyme replacement, bone marrow transplantation (BMT),

and somatic gene transfer approaches. While these strategies

have been successful to treat visceral disease, they have been

relatively unsuccessful towards CNS disease lesions due to

the BBB (Sands et al., 1997; Suzuki and Tanike, 1995). The

BBB significantly impedes entry from blood to brain of most

molecules, except those that are small and lipophilic (Miller,

2002; Neuwelt, 1989). The use of preconditioning regimens

such as irradiation, known to disrupt the BBB, results in

increased CNS levels of therapeutic enzymes (Monje et al.,

2002; Rubin et al., 1994; Sands et al., 1993; Soper et al.,

2001). However, the damaging effects of irradiation on the

brain are often long lasting and can result in permanent

neurologic impairment, especially when administered during

infancy or early childhood (Sands et al., 1993).

There is growing evidence that endothelial cells in the

neonatal period respond distinctly to VEGF (Gerber et al.,

1999; Young et al., 2002). In this study, we hypothesized that

VEGF may have distinct effects on the neonatal BBB to

proteins and other macromolecules. Therefore, we also

determined whether VEGF-mediated BBB disruption would

allow viral particles or even exogenous cells to enter the brain

during the neonatal period. We also determined whether

intravenous (IV) administration of VEGF to neonatal animals

could enhance the efficacy of a therapeutic approach

designed to treat congenital neurodegenerative diseases.

Methods

MPS VII and twitcher mice

h-Glucuronidase (GUSB)-deficient homozygous mutant

mice (mps/mps) were obtained from a B6.C-H-2bm1/ByBir-

gusmps/+ colony maintained by M.S.S. at Washington Uni-

versity (St. Louis, MO). Homozygous GUSB-deficient mice

were identified at birth by the absence of GUSB activity using

a fluorometric assay on a small sample of tissue (Freeman et

al., 1999) and confirmed by genotyping through a PCR assay

(Wolfe and Sands, 1996). The twitcher mice were originally

obtained from the Jackson Laboratory (Bar Harbor, ME), and

a colony has been maintained by MSS. The genotypes of

twitcher (twi/twi) and wild-type littermate (+/+) mice were

identified by PCR using genomic DNA tissue sample

obtained on the day of birth.

Administration of rVEGF164

rVEGF164 (R&D Systems, Minneapolis, MN) was in-

jected IV (1.7 ng) through the superficial right temporal vein

in 1-day-old mice (weight approximately 1 g) (Sands and

Barker, 1999). The dose of VEGF was chosen based on

studies to determine the greatest dose tolerated by newborn

mice. An equal volume of 0.9% saline was administered IV in

control animals. Adult animals were injected with 50 or 1000

ng of VEGF (2 or 50 ng/g weight) through the lateral tail vein.

Protein permeability experiments

Evans blue (EB, 2%; 0.2 mg/100 g body weight) was

administered simultaneously with or 2, 6, 24, or 48 h after

administration of VEGF or saline in newborn animals.

Four-week-old mice were administered EB either 30 min

or 2 h after VEGF administration. Thirty-four 1-day-old

wild-type mice and twelve 4-week-old wild-type mice were

utilized for this study, with at least three animals in each

time point for each condition. Mice were sacrificed 30 min

after EB administration and one sagittal half of each brain

was homogenized and EB levels were quantified. EB was

extracted and determined spectrophotometrically as de-

scribed previously (Belayev et al., 1996).

Mannitol-induced hyperosmolality

To compare the effect of VEGF to an agent that is known

to disrupt the BBB, an additional group of animals was used

for hyperosmotic opening of the BBB. Eight wild-type

C57BL/6 28- to 30-day-old and 15 newborn mice (from five

different litters) received mannitol as a single intraperitoneal

injection 2 h before injection with EB dye. Three control mice

received intraperitoneal saline injection. The dose of manni-

tol was 0.6 ml/g body weight of 1Mmannitol in 0.34MNaCl

(Ghodsi et al., 1999). Water was withheld in 4-week-old mice

after mannitol injection, whereas the newborn mice were

returned to their mothers immediately after injection. From

each litter of newborns, only half were injected with mannitol

and the other half were injected with equal volume of saline.

The mice were sacrificed 30 min after EB administration.

Cellular permeability experiments

BMs from sex-matched littermates were injected IV into

newborn mice through the superficial temporal vein (Sands

and Barker, 1999) at 12–16 h after IV administration of

rVEGF or saline. Fourteen 1-day-old GUSB-deficient mice

were used for this experiment. Briefly, unfractionated BM

cells were obtained by flushing the femurs of donor mice

with PBS. BM cells (5 � 106) were injected IV. For the

purpose of comparison to nonablated BM recipients, four

newborn GUSB-deficient recipients received a sublethal

dose of 300 rad of g-irradiation from a 137Cs source just

before BM administration.

Viral permeability experiment

Eight 1-day-old GUSB-deficient mice were utilized for

this experiment. The virus was injected into the superficial

P.P. Young et al. / Experimental Neurology 188 (2004) 104–114106

temporal vein of newborn mice. Each mouse received a total

volume of 0.1 ml to deliver a virus dose of 1 � 106

infectious units. Two weeks after recombinant GUSBHIV

(see below) administration, the mice were sacrificed, and

one sagittal half of each brain was used for biochemical

analysis and the other sagittal half was embedded in OCT

and examined by histochemistry. Biochemical analysis for

GUSB activity of murine tissues was performed as de-

scribed (Glaser and Sly, 1973; Soper et al., 1999).

Twitcher longevity experiments

A total of forty-four 1-day-old twitcher mice were used

for this experiment. Homozygous twitcher mice received

sex-matched BM 12–16 h after receiving IV injection of

VEGF or saline. A subset of the mice received 300 rad of

preconditioning g-irradiation before BM without VEGF or

saline pretreatment. As a control, some mice received VEGF

alone or were left untreated. The twitcher mice were

checked daily to determine date of death.

Analysis of hematopoietic engraftment and GUSB

quantitative biochemistry

BM and spleen hematopoietic engraftment was assessed

40–50 days after administration of BM cells by flow

cytometry using FACScan (Becton Dickinson). Marrow

isolated from transplanted recipients was resuspended at

1 � 106 cells/100 Al in IMDM/2% fetal calf serum and

analyzed as previously (Soper et al., 2001) described using

a FITC-conjugated GUSB substrate (Molecular Probes,

Eugene OR) and side scatter.

Histochemistry, immunofluorescence, and GUSB

biochemical assay

Brain sections from GUSB-negative recipients were

harvested 2 weeks after either BM or recombinant virus

administration and processed as previously described (Sands

et al., 1993). Histochemical analysis for GUSB activity was

performed as described by using naphthol-AS-BI-h-D-glu-curonide (ASBI) as the substrate (Sands et al., 1993). For

immunofluorescence, sections were fixed for 20 min at 4jCin 100% acetone and then incubated with PBS-blocking

buffer (0.01 g/ml BSA, 2 Ag powdered milk/ml, 3 Al/ml

triton X-100) and 10% horse serum for 1–2 h. The slides

were washed with PBS and incubated 12–16 h with either:

(1) mouse anti-NeuN (Chemicon, Temecula, CA) (1:20) and

goat antihuman GUSB (kind gift from W. Sly, St. Louis

University) (1:50), (2) rabbit anti-glial fibrillary acidic

protein (GFAP; Incstar Corp., Stillwater, MN) (1:50) and

goat antihuman GUSB, or (3) rabbit antihuman vWF (Dako,

Carpinteria, CA) (1:250) and goat antihuman GUSB. Slides

were washed and then incubated with fluorescein-conjugat-

ed swine anti-goat IgG (Boehringer Mannheim Corp.,

Indianapolis, IN) and one of the following, rhodamine-

conjugated donkey anti-mouse or donkey anti-rabbit IgG.

For immunohistochemistry, tissues were fixed and pro-

cessed as described above and anti-vWF was used at

1:100. Slides were washed with 0.05% Triton X-100 in

PBS and incubated for 1 h with an alkaline phosphatase-

conjugated goat anti-rabbit antibody (Sigma). After addi-

tional washes with PBS and detection with black substrate

(Vector Laboratories, Burlingame, CA), the pH was adjusted

to 4.5 and GUSB histochemistry was performed on the same

sections. Negative controls where the tissues were incubated

with the labeled secondary antibody alone in the absence of

primary antibody were performed in parallel with all

experiments.

Morphometry

To determine the number of GUSB-positive cells after

cellular transplant, the frozen sections of brain and eye

obtained from the experimental animals were examined at�10 to count the number of GUSB-positive cells distributed

over the entire area on each intact sagittal section. Anatom-

ically similar sagittal sections were examined and each

GUSB-positive area recorded. At least 12 sections repre-

senting a cross-section of the organ were examined from

two different experimental animals. For morphometry after

immunofluorescent analysis, slides were viewed on Nikon

Microphot-SA fluorescence microscope and images cap-

tured using a Colorview camera and analysis software (Soft

Imaging System, Lakewood, CO). Slides co-stained for

vWF and GUSB were utilized. Ten representative �20

fields from at least two animals were manually counted

and the result averaged for each animal. Areas of staining

without discrete breaks were counted as a single event.

Virus construction and production

An HIV-based lentiviral expression vector containing the

CMV promoter driving the human GUSB cDNA (gift from

Cell Genesys, Inc.) was produced by transient calcium

phosphate co-transfection in 293T cells of four HIV pack-

aging constructs, RSVREV, pMDLgpRRE, pRRLsinCMV-

GUSBppt, and pMDG (VSV-G) (Dull et al., 1998; Zufferey

et al., 1998). Viral supernatants were filtered through 0.45

Am pore filters, concentrated by ultracentrifugation at

19,500 � g for 2.2 h, resuspended in Tris-buffered saline,

pH 7.0, and stored at �80jC. Virus titer for the CMVGUSB

lentivirus was determined using serial dilutions of stock by

infection of GUSB-negative 3521 cells (Sands and Birken-

meier, 1993) and followed 2 days later by FACS analysis

using FDG12Gluc substrate (Molecular Probes, Inc.).

Statistical analysis

Student’s t tests were performed to compare different

data sets. All data are presented as mean plus or minus

SEM. Data used to construct the survival curve were


analyzed statistically using the Logrank sum test. A P value

of <0.05 was interpreted to denote statistical significance.

Results

VEGF reversibly increases BBB permeability to Evans blue

albumin

We first determined if VEGF increased neonatal BBB

permeability to proteins. Intravenous (IV) administration of

VEGF is poorly tolerated due to the known effects of VEGF

to cause hypotension (Li et al., 2002). The highest dose

tolerated by newborn mice (days 1–2) was determined to be

approximately 1.7 ng/g weight in 100 Al volume (data not

shown). BBB function was assessed by introducing a

cationic dye, Evans blue (EB), which binds albumin in the

circulation. VEGF (1.7 ng) was injected IV into newborn

mice (approximately 1 g) followed by EB dye at 0, 2, 6, 24,

and 48 h following VEGF injection. Mice were sacrificed

30 min after EB administration, and a semiquantitative

assay was utilized to assess EB content in the brain.

Increased levels of EB could be seen in the brain when

the tracer dye was injected simultaneously with VEGF, but

the results were not statistically significant compared to

saline controls. However, by 2 h after VEGF administration,

there was a significant increase in EB/albumin content into

the neonatal brain over saline controls (Fig. 1A). The effect

of VEGF was undetectable after 48 h. Injection of a much

greater dose/per body weight of EB (up to 50 ng/g weight,

approximately 30-fold of newborn dose) at either 30 min

(data not shown) or 2 h (Fig. 1B) after VEGF administration

to 4-week-old juvenile mice did not result in increased

permeability of EB over saline-pretreated controls (Fig.

1B). The mice receiving the higher dose (50 ng/g) exhibited

signs of acute VEGF toxicity. Bleeding from the site of

injection in the lateral tail vein continued for >2 h. Also, the

mice had profoundly decreased motor activity, likely result-

ing from VEGF-mediated hypotension, and essentially

crouched in one location for the remaining 2.5 h, suggesting

significant dose-related toxicity. Mice receiving doses of

VEGF lower than 30 ng/g weight had only mildly prolonged

bleeding and no changes in motor activity from their non-

injected littermates. In contrast, mannitol, known to cause

osmotic disruption of BBB, resulted in dramatically in-

creased EB-albumin flux in the brain of adult mice (Fig.

1B). However, administration of a weight-adjusted volume

of 20% mannitol to newborn mice (days 1–4) resulted in

lethality (data not shown). Hence, doses of VEGF that were

sufficient to reversibly increase the neonatal BBB perme-

ability to EB-albumin failed to change the barrier perme-

ability to the protein tracer when given to 4-week-old mice.

Because VEGF has been linked to vascular tumors

(Carmeliet, 2000) and relatively little information is avail-

able on the long-term effects of in vivo use of VEGF in

neonatal animals, we administered 1.7 ng of VEGF IV to

five wild-type newborn mice and observed them for 5

months. The mice were normal in size and were fertile.

The average brain weights of the mice were not significantly

different from saline-injected wild-type control littermates,

0.37 F 0.03 vs. 0.40 F 0.04 g, respectively (P = 0.48).

Gross examination of serial sections from brain, liver,

spleen, lung, and heart from these animals did not reveal

any macroscopic tumors (data not shown).

VEGF increased the number of donor cells in the brain after

BM administration

Thus far, there are few reports describing the permeabil-

ity of moieties as large as cells (>5 Am) across the BBB

(Miller, 2002; Neuwelt, 1989). This was of particular

interest to us because of our long-term goal of treating

LSDs using strategies such as gene and cellular therapies.

As discussed before, current therapeutic strategies of LSDs

include allogeneic BMT/hematopoietic stem cell transplant

and gene-modified autologous hematopoietic stem cells.

Success of these strategies is dependent on efficient ingress

of corrected marrow-derived cells into the CNS.

To study the fate of donor BM cells in vivo and quantify

their numbers, we utilized the h-glucuronidase (GUSB)-

deficient mouse as a recipient to track wild-type (GUSB-

positive) donor BM cells from a syngeneic mouse. This

mouse is a model of the human LSD mucopolysaccharidosis

type VII (MPS VII) and is completely deficient in GUSB

activity. In addition to being a bona fide model for human

disease, the GUSB-deficient mouse has also been utilized

extensively by our laboratory and others to track in vivo

GUSB-positive cells including highly purified BM or he-

matopoietic cells (Freeman et al., 1999; Hofling et al., 2003;

Soper et al., 1999; Young et al., 2003). Sensitive biochem-

ical, immunohistochemical, and histochemical assays are

available to identify GUSB-positive cells with single-cell

sensitivity. BM cells (5 � 106) from syngeneic wild-type

mice were injected IV into newborn GUSB-deficient recip-

ients 16–20 h after receiving an IV injection of 1.7 ng of

VEGF. The cells were administered to animals pretreated

with VEGF or saline (as control) without any radioablative

or chemical preconditioning. As a comparison, a group of

animals was also pretreated with 300 rad of g irradiation

before administration of the same number of BM cells. The

number of donor cells in the brain was evaluated by

measuring GUSB enzyme activity and by determining the

distribution of donor-derived cells in both the brain and eyes

of recipient mice 2 weeks after administration of BM.

GUSB specific activity was measured in the brain of

animals that had received BM cells after pretreatment with

either VEGF or saline as a means to quantify the level of

donor-derived (GUSB-positive) cells in the brain. VEGF

resulted in approximately 4-fold increase of GUSB activity

in the brain over saline-injected controls (Fig. 2a). GUSB

activity in the brain in the saline-treated (control) animals

was 1.1 F 0.01% of wild-type brain GUSB activity. The

Fig. 1. VEGF disrupts BBB to protein flux only in newborn mice. (A) Newborn mice were injected IV with VEGF. Time course of barrier disruption was

investigated by staggered injections of tracer dye EB following systemic VEGF (filled bars) or saline control (open bars) injections. The x-axis indicates the

time (h) of EB injection after VEGF. EB was quantified spectrophotometrically and expressed as Ag EB/g tissue. (B) Four-week-old mice were administered a

weight-adjusted dose (2 ng/g) and a high (50 ng/g) dose of VEGF or saline control. EB was injected 2 h after VEGF administration. Hyperosmolar disruption

was induced with mannitol (striped bar) as a positive control. EB was quantified on homogenized sagittal section of recipient brain and expressed as Ag of

protein/g brain weight. Statistical significance was denoted with asterisk.


enzyme activity of animals receiving VEGF or irradiation

preconditioning was 4.0 F 1.2% and 7.0 F 4.6%, respec-

tively, of wild-type enzyme levels. GUSB activity in the

eyes of both VEGF- and saline-treated animals was too low

to detect (data not shown).

The increase in GUSB activity determined by quantita-

tive assay correlated with an increase in the number of

donor-derived cells observed on histologic analysis. A

histochemical technique was utilized to identify any cell

expressing GUSB on frozen sections of brain tissues. This

analysis showed that donor-derived cells were found in

varying amounts in newborn recipients 2 weeks after

administration in all areas of the brain, including the

cerebellum, neocortex, and cortex (Fig. 2b). The average

numbers of GUSB-positive cells were higher in VEGF-

treated animals than in saline controls (Table 1). At least 12

anatomically comparable cross-sections were analyzed from

the brains of at least two animals for each condition.

Importantly, no donor-derived cells were identified in the

cerebellum of saline-pretreated animals. There was greater

variation among the experimental animals pretreated with

irradiation in the numbers of GUSB-positive cells identified

in the brain sections compared to VEGF- or saline-pre-

treated animals. One irradiated animal had greater numbers

Table 1

Number of cellsa

Cellular therapyb

Saline + BM cells Eye 1.2 F 1.1 N/A

Brain 1.4 F 1.0 N/A

VEGF + BM cells Eye 1.8 F 1.7 *P = 0.5

Brain 8.5 F 8.0 P = 0.01

300 rad + BM cells Eye 2.6 F 1.9 P = 0.12

Brain 7.1 F 7.5 P = 0.02

Recombinant virus therapyc

Saline + recombinant virus Eye 3.25 F 2.6 N/A

Brain 22.3 F 13 N/A

VEGF + recombinant virus Eye 5.75 F 3.7 P = 0.08

Brain 105 F 94 P = 0.01

N/A: not applicable.a Number of cells counted over entire sagittal sections. Equal numbers of

anatomically similar sections were counted for saline- and VEGF-treated

animals.b Animals were injected IV with 5 � 106 unfractionated BM cells at birth.c Animals were injected IV with recombinant lentivirus at birth.

* The effect of VEGF and irradiation are statistically compared to control

(saline) by Student’s t test; P < 0.05 indicates statistical significance.

Fig. 2. (a) VEGF resulted in BBB disruption to BM-derived cells in

newborn mice. Newborn GUSB-deficient mice were injected with

unfractionated syngeneic BM from GUSB wild-type mice approximately

16 h after IV VEGF (filled bar) or saline (open bar) administration. A

portion of mice received 300 rad of irradiation (striped bar) just before BM

administration. Mice were sacrificed 2 weeks after BM injection. Results

are expressed as GUSB specific activity (units/h/mg total protein). GUSB

activity in the brains of irradiated animals was not statistically significant

over VEGF-pretreated animals. Statistical significance denoted with

asterisk. (b) Donor BM cells with GUSB enzyme activity (red) in brain

sections from GUSB-deficient animals that received syngeneic GUSB-

positive BM cells after BBB disruption with VEGF. Frozen tissue sections

stained with napthal-AS-BI-h-D-glucuronide and counterstained with

methyl green. (a) Cerebellum, (b) cortex, (c) hippocampus, (d) meninges

(�20).


of cells than the VEGF-treated group in most regions of the

brain and also in the eyes (data not shown). However, in two

of the animals, only rare cells were identified in the brain

and none in the eye. This high level of variation observed in

histologic examination was consistent with the degree of

variation reflected in the standard error of the mean calcu-

lated from the quantitative data in 2a (Table 1). Examination

of the eyes of all experimental animals showed only rare

donor-derived cells, and the numbers of cells identified were

not significantly different between the VEGF and saline-

pretreated control animals (Table 1). To determine if the

cells identified in the VEGF-pretreated animals were either

within vessels or adjacent to vessels, we performed immu-

nohistochemistry to co-localize GUSB enzyme activity with

anti-vWF, which specifically labels blood vessels. We

analyzed 43 sagittal sections from two different animals

and found only 12% (7/60) of the GUSB-positive cells to

co-localize with vWF-positive cells associated with vascular

structures. The remaining enzyme-positive cells appeared to

have extravasated into the brain parenchyma.

VEGF increased the number of transduced cells in the brain

after recombinant lentivirus administration

We further utilized the GUSB-deficient mouse as a

system in which to determine if VEGF increased the

permeability of retroviral particles (approximately 70–100

nm) (Coffin, 1991) through the BBB or the blood–eye

barrier. We utilized a recombinant HIV-based gene transfer

vector expressing the human GUSB cDNA. The recombi-

nant lentivirus was psuedotyped with VSVG envelope

protein to enable it to transduce multiple cell types. Infec-

tious units (1 � 106) in 100 Al volume were injected IV into

the neonate 16 h following IV injection of either 1.7 ng of

VEGF or saline (as control). Analysis 2 weeks after treat-

ment showed a greater than 2-fold increase in GUSB

activity in the brains of VEGF-treated animals over saline

controls (Fig. 3a). Lentivirus-transduced cells were found

throughout the brain, the majority in the neocortex,

meninges, and cerebellum (Fig. 3b). Within the cerebellum,

transduced cells expressing GUSB were found in the

purkinje cell layer, internal granular layer, and in white

matter tracts within the molecular layer (data not shown).

The distribution pattern in saline-injected recipients was

similar, but dramatically fewer GUSB-positive areas were

observed (Table 1, Fig. 3b). The GUSB activity in the eye

was higher in VEGF-treated animals, but this difference was

Fig. 3. (a) VEGF results in BBB and blood–eye barrier disruption to recombinant lentivirus in newborn mice. Newborn GUSB-deficient mice were injected

with recombinant GUSB lentivirus after pretreatment approximately 16 h prior with VEGF or saline control. GUSB specific activity (units/h/mg total protein)

was measured in one eye and one sagittal half of brain. Statistical significance was denoted with an asterisk. (b) GUSB activity (red) was detected

histochemically throughout the brain after administration of recombinant lentivirus expressing GUSB. Animals were treated as above (a, �20). (a) Meninges,

(b) cerebellum, (c) cortex, (d) hippocampus, (e) vascular structure in cortex, (f) white matter tracts.


not statistically significant from the levels in the control

mice. Histologic examination of one eye from at least three

different VEGF-treated animals reflected slightly higher

numbers of GUSB-expressing cells compared to saline-

treated controls (Table 1). However, this difference was

not statistically significant.

Based on morphologic criteria, recombinant lentivirus

appeared to transduce neurons, glial, and endothelial cells.

To confirm this, immunolocalization studies were performed

using antibodies against the glial marker, GFAP, a neuronal

marker, Neu-N, and a endothelial cell marker, vWF, of the

brains obtained from animals pre-treated with VEGF before

recombinant virus. We were able to co-localize GUSB with

cells of all three types in immunofluorescent studies (Fig. 4).

Both morphologic evidence (Fig. 3b) and co-immuno-

fluorescent data (Fig. 4) provided evidence for lentivirus-

transduced vascular structures. To determine if the increased

VEGF-induced CNS lentiviral transduction levels were a

result of primarily vascular transduction, we performed

morphometry to quantify the number of GUSB-positive

cells co-localizing with vWF-positive cells by co-immuno-

fluorescence in both VEGF- and saline-pretreated newborns

treated with recombinant lentivirus. The level of vascular

cells transduced was 34 F 24% and 24 F 14% in VEGF-

and saline-pretreated newborns, respectively. This differ-

ence was not statistically significant (P = 0.07).

VEGF pretreatment before BMT increases the life span in a

mouse model of LSD

Our data indicate that VEGF disrupted the BBB to

moieties as large as cells and viruses. However, thus far,

no one has reported amelioration of CNS storage disease

following BBB disruption in newborns accompanied by

systemic cellular therapy. To study if BBB disruption can

enhance delivery of corrected cells following BMT, we

utilized an authentic model of LSD, the twitcher mouse.

This mouse is deficient in galactosylceramide h-galactosi-dase activity and represents a model of the LSD, globoid-

cell leukodystrophy (Krabbe’s disease) (Kolodny, 1996;

Suzaki et al., 1995). The CNS manifestations of the mouse

disease mirror that in humans in that they begin early (by

day 20 in mice), and if untreated, the disease progresses

rapidly to death (by 40–45 of age in mice) (Suzaki et al.,

Fig. 4. Systemic administration of recombinant lentivirus results in transduction of multiple CNS cell types. Photomicrographs showing immunofluorescent

localization of GUSB-positive cells (b, e, and h) and cells expressing GFAP (�40) (a), NeuN (�20) (d), or vWF (�10) (g). Co-localization was demonstrated

by overlay (c, f, and i).

Fig. 5. BBB disruption with VEGF before BMT ameliorates lethality in

twitcher mice treated in the newborn period. Newborn twitcher mice were

treated with BMT at birth with (VEGF + cells) or without (saline + cells)

VEGF pretreatment. Matched groups of untreated (untreated) and VEGF

alone (VEGF only) animals are used as controls. Also shown is the

longevity of newborn animals that received 300 rad (irrad + cells) before

BMT. Mice who received VEGF or irradiation pretreatment before BMT

had significant prolongation of life over untreated controls ( P < 0.001 and

P < 0.003, respectively).


1995). To determine if VEGF treatment before BM admin-

istration can prolong life in this model of LSD, either

VEGF or saline was administered 16 h before administra-

tion of BM cells from syngeneic wild-type donors in the

absence of any ablative radiation. Additional control groups

were studied that received VEGF pretreatment without BM

cells, no treatment, both without ablative radiation, or BM

cells with 300 rad of irradiation. BBB disruption with

VEGF <24 h before IV administration of 5 � 106 syngeneic

wild-type BM cells resulted in a significant prolongation of

life over saline and BM cell controls with median survival

48.5 vs. 41.0 days, P < 0.001 (Fig. 5). The cohort of

animals that received pretreatment with irradiation also

survived significantly longer than untreated controls with

median survival of 49.5 vs. 41.0 days, P < 0.003. There

was no statistically significant difference in survival be-

tween VEGF or irradiation pretreatments. Those animals

receiving saline pretreatments before BM cells fared no

better than untreated animals or those that received VEGF

without any BM cells.

Our data suggested that VEGF resulted in transient

disruption of the newborn BBB to both cellular and viral

particles. We have utilized this window of BBB disruption

to administer unfractionated BM cells to result in modest

amelioration of disease. Another alternative may be that the

observed in vivo therapeutic benefit of VEGF was second-

ary to increased BM engraftment. To test this hypothesis, we

administered unfractionated syngeneic wild-type (GUSB-

positive) BM to GUSB-deficient newborn mice 16 h after

IV VEGF or saline administration. The BM and spleen cells

were analyzed by FACS in the recipients 90 days after non-

ablative BMT to determine donor (GUSB-positive) engraft-

ment. Donor (GUSB) engraftment in BM of the VEGF- and

saline-pretreated recipient animals was 10.8 F 2.3% and

13.7 F 4.0%, respectively (P = 0.17).


Discussion

The BBB controls the composition of extracellular fluid

in the brain and restricts the access of the immune system,

invading microbes, and systemically administered drugs.

This structural and functional barrier is essential for normal

neural function, yet also presents an important obstacle

against achieving therapeutic levels of agents designed to

treat CNS diseases. Thus far, strategies for reversibly

opening the BBB have been directed largely towards small

molecules and are targeted primarily towards treating CNS

malignancies or infections (Edwards, 2001; Miller, 2002).

We have shown that VEGF can reversibly change BBB

permeability to Evans blue/albumin, an indirect marker of

protein permeability. We also show that VEGF reversibly

enhances the permeability of larger moieties such as recom-

binant viruses and cells in newborn recipients. These types

of therapeutic approaches may apply to the treatment of

neurodegenerative diseases and CNS tumors.

Significantly greater numbers of either BM-derived cells

or virus-transduced cells were detected in the CNS of

recipients after VEGF-induced BBB disruption. These cells

were distributed throughout the brain, including the cortex,

cerebellum, and olfactory bulb. Enzyme activity achieved

by mild irradiation at birth before BMT was approximately

7-fold higher than after saline-pretreated non-ablative BMT.

VEGF pretreatment before non-ablative BMT resulted in

enzyme levels approximately 4-fold higher than control.

The higher enzyme activity observed with radiation pre-

conditioning may be a result of more significant disruption

of BBB to cellular traffic. Alternatively, whereas the BBB

disruption after VEGF pretreatment appears to be transient,

cerebral endothelial and parenchymal injury after irradiation

is longer lasting and may continue to enable entry of

transplanted BM-derived cells long after administration

(Cicciarello et al., 1996; Remler et al., 1986).

Vascular localization or staining, and therefore no BBB

disruption, could possibly account for the distribution of

GUSB-positive cells observed in this study. Our finding that

only 12% of donor cells co-localized with vascular cells or

structures suggests that this is unlikely. We also showed that

VEGF did not significantly increase transduction of endo-

thelial cells after administration of recombinant lentivirus

over control. This is supported by our finding that the virally

encoded marker, GUSB, was found in neurons and glial

cells. This finding also suggests that a pseudotyped lenti-

viral vector was able to enter the parenchyma and transduce

multiple cell types. Because VEGF is not known to prolong

the continuous vascular circulation of administered viruses

or BM cells, vascular localization would not explain the

difference observed between VEGF- and saline-pretreated

animals.

In contrast to previously published studies, we chose to

administer intravenous VEGF. The level of VEGF that can

be administered as an IV bolus is limited by VEGF-

mediated hemodynamic alterations (Li et al., 2002). Al-

though direct intracranial or intraocular infusion of VEGF

does appear to reversibly disrupt the BBB, even in adult

animals, such an approach results in only localized disrup-

tion and carries significant risk of mechanical damage

(Belayev et al., 1996; Eliceiri et al., 1999; Hofman et al.,

2000). In previous reports, co-administration of mannitol (to

increase systemic osmolality) with intraventricular recom-

binant virus dramatically increased numbers of cells in the

CNS that were transduced. However, the increased activity

was limited to adjacent tissues and widespread cortical

distribution was not observed (Belayev et al., 1996). The

existence of a blood–eye barrier has also been described

(Neuwelt, 1989). Our studies suggest a trend towards

increased disruption of blood–eye barrier, particularly to

viral therapy, but the differences were not statistically

significant.

VEGF pretreatment to disrupt the BBB was applied in an

authentic model of the LSD, Krabbe’s disease, also known

as the twitcher mouse. VEGF pretreatment prolonged the

life of mice treated with syngeneic wild-type BM cells even

in the absence of preconditioning irradiation. We demon-

strated that this effect is not secondary to increased VEGF-

mediated BM engraftment. Our data support a model in

which VEGF, by transiently disrupting the BBB, enables

increased ingress of administered protein, BM cells, or

recombinant virus into the CNS, thereby providing greater

therapeutic levels of enzyme.

VEGF pretreatment, however, had only a modest effect,

increasing the life span approximately 9 days over control.

This is not surprising because previous reports indicate that

BMT performed between days 9 and 12 with an extremely

large dose of conditioning irradiation (900 rad) that was

associated with high early mortality resulted in a mean

increase in life span by only 40 days (Hoogerbrugge et

al., 1988; Yeager et al., 1984). These reports have also

demonstrated greater numbers of donor-derived cells over

time in the brains of the irradiated animals receiving BMT

(Cicciarello et al., 1996; Remler et al., 1986). Other studies

have shown that even low-level preconditioning irradiation

(200 rad) to newborn mice can result in 10-fold increase in

CNS levels of donor-derived enzyme following BMT over

non-ablative administration of donor cells as early as 2

weeks after administration (Sands et al., 1993; Soper et al.,

2001). The data in this study support these reports by

demonstrating that preconditioning irradiation treatment of

only 300 rad resulted in an overall disruption of the BBB to

donor-derived GUSB-positive cells with an approximately

7-fold increase in CNS enzyme activity over saline-injected

controls. We hypothesize that the gradual increase in CNS

enzyme levels after irradiation preconditioning is a result of

long-term disruption of the BBB. Irradiation is known to

cause not only long-lasting disruption of the BBB but also

direct CNS toxicity, particularly in young animals, by

mechanisms that are not understood (Cicciarello et al.,

1996; Remler et al., 1986; Rubin et al., 1994; Sands et al.,

1993). Furthermore, the extent of toxicity and length of


disruption is variable from animal to animal as also ob-

served in our study (Cicciarello et al., 1996). Irradiation-

induced barrier disruption has been detected for greater than

300 days in one model (Cicciarello et al., 1996). Hence,

there are two aspects of VEGF-mediated barrier disruption

that are important to highlight. First, BBB disruption is

transient and is not detectable at 48 h. Second, the extent of

barrier disruption appeared relatively uniform among differ-

ent experimental animals. Although we did not detect any

long-term sequelae in mice observed for an extended period

of time, more rigorous anatomic and physiologic studies are

necessary to establish the long-term safety.

The failure of VEGF to result in BBB disruption to EB or

albumin after IV administration to juvenile (4-week-old)

mice supports both previous findings by others and the

hypothesis that VEGF has distinct effects on newborn

endothelial cells (Gerber et al., 1999; Young et al., 2002).

Although the mechanism of VEGF’s effects on the BBB

permeability is not known, it has been shown that the

newborn BBB is unique in several aspects. In studies using

endothelial cells isolated from neonatal brain, it was shown

that specialized structures such as vesiculotubular structures,

vesiculovacular organelles, and junctional complexes were

incompletely sealed (Vorbrodt et al., 2001). There is also

evidence to suggest that newborn endothelial cells have a

higher level of ICAM expression that may allow for greater

trafficking of circulating cells (Vorbrodt et al., 2001).

Ultrastructural examination of intra-endothelial junctions

has shown tight junction protein levels increase between

birth and 14 days (Lossinsky et al., 1999; Vorbrodt and

Dobrogowska, 2003).

Our studies suggest that VEGF-mediated barrier disrup-

tion enables the entry of large moieties, including cells.

Interestingly, studies after intracerebral infusion of VEGF

have shown the formation of endothelial gaps. These gaps

may represent cell fragmentation followed by formation of

fenestrae and basement membrane degeneration (Chen et

al., 2002; Dobrogowska et al., 1998; Kusumanto et al.,

2003; Mayhan, 1999; Roberts and Palade, 1995). It is not

known if these alterations occur in newborn mice and if

they can account for the movement of viral particles or

cells. It is known that the BBB of mice and rats, although

intact, is less mature than the BBB of humans at birth

(Neuwelt, 1989; Vorbrodt and Dobrogowska, 2003; Vor-

brodt et al., 2001). Hence, it is possible that the molecular

properties of neonatal mice brain capillary endothelial cells

that enable transient disruption by VEGF are absent in

humans at birth. There is little known about the molecular

basis of developmental differences of blood vessels. Further

studies to understand these developmental endothelial cell

properties and to identify the molecular mechanism of

VEGF-mediated BBB disruption will be important towards

determining if the BBB can be similarly manipulated during

human neonatal life. Importantly, more studies are needed

to assess the biological impact of neonatal exposure to

exogenous VEGF, including the effects on angiogenesis,

growth, tumorigenesis, and promoting atherosclerosis

(Wang et al., 2001). In an earlier study, we have shown

that VEGF administration to neonates resulted in increased

vascular density in both heart and liver (Young et al., 2002).

Additionally, the effect of VEGF-induced hemodynamic

changes has not been studied in neonatal or infant animal

models.

In most congenital neurodegenerative diseases, efforts to

ameliorate symptoms by various therapies are thwarted by

the BBB. Our findings demonstrating transient BBB dis-

ruption in neonatal mice to protein, cellular, and gene

therapy may renew efforts to develop more effective ther-

apies for these diseases.

Acknowledgments

This work was supported in part by Vanderbilt Institu-

tional Funds (P.P.Y.), NIH training grant T32HL07088

(P.P.Y.), NIH grants DK057586 and NS44520 (M.S.S.), and

Hunter’s Hope Foundation (C.R.F.).

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