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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: pampee.young@vanderbilt.edu (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
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114 107
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.
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114108
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).
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114 109
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.
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114110
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).
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114 111
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).
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114112
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
P.P. Young et al. / Experimental Neurology 188 (2004) 104–114 113
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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