Department of Pharmacology, The University of Arizona College of Medicine, Tucson, AZ, USA
* Author for correspondence (e-mail: davistp{at}u.arizona.edu)
Accepted 8 November 2002
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Summary |
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Key words: Basic fibroblast growth factor, Vascular endothelial growth factor, Claudin-1, Actin, Hypoxic stress, NFB
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Introduction |
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The restrictive nature of the BBB is due in part to tight junctions (TJ)
formed between adjacent endothelial cells
(Reese and Karnovsky, 1967;
Kneisel and Wolburg, 2000
). TJ
allow for regulation of ion flux and paracellular diffusion through the
development of high transendothelial electrical resistances (TEER), in the
range of 1500-2000
·cm2
(Butt et al., 1990
). A number
of TJ protein components have been identified and extensively characterized,
including the claudin family (Furuse et
al., 1998
), occludin (Furuse
et al., 1993
) and zonula occludens-1, -2 and -3 (ZO-1, ZO-2 and
ZO-3), which interact with claudins and occludin
(Itoh et al., 1999
;
Mitic et al., 1999
). Claudin-1
and occludin have been found at the BBB
(Huber et al., 2001b
;
Mark and Davis, 2002
). Both of
these proteins have multiple transmembrane domains and form homodimeric
bridges with adjacent cells, creating a physical blockade to paracellular
diffusion (Tsukita and Furuse,
1999
). Stabilization of the TJ complex involves a network of
occludin and claudins linked to the actin cytoskeleton via the ZO proteins. ZO
proteins mediate this linkage by binding actin to the cytoplasmic tails of
occludin and claudin (Huber et al.,
2001a
), in a manner similar to the cadherin-catenins-actin
interaction at the adherens junction (Brown
and Davis, 2002
). ZO proteins are members of the
membrane-associated guanylate kinase (MAGUK) family; they have a conserved
guanylate kinase domain, an SH3 domain and multiple PDZ domains
(Huber et al., 2001a
),
suggesting that ZO proteins participate in signal transduction cascades.
In order to examine mechanistic and molecular events in the BBB that occur
under different pathological conditions and to assess drug delivery to the
CNS, in vitro models have been developed using primary cultures of brain
microvessel endothelial cells (BMEC). There is a great deal of evidence that
astrocytes are important for inducing and maintaining certain BBB
characteristics in vitro. The characteristics affected by astrocytes or
astrocyte products include high TEER, low paracellular diffusion and low
transcellular endocytosis (Wolburg et al.,
1994). Co-culture studies show that astrocytes induce BBB-like
characteristics in endothelial cells isolated from various sources, including
bovine aortic endothelial cells (Isobe et
al., 1996
), immortalized bovine BMEC
(Sobue et al., 1999
) and
immortalized rat BMEC (El Hafny et al.,
1997
). The influence of astrocytes on barrier permeability is
mediated by factors released from astroglioma cells
(Raub et al., 1992
) or mixed
cultures of rat astrocytes (Dehouck et
al., 1994
). Several factors have been identified that may account
for these BBB-inducing properties of astrocytes, including basic fibroblast
growth factor (bFGF) (Sobue et al.,
1999
) and glia-derived neurotrophic factor
(Igarashi et al., 1999
).
Furthermore, glial co-culture protects in vitro BBB models against breakdown
induced by hypoxic stress (Kondo et al.,
1996
; Fischer et al.,
2000
). However, there is little information as to how co-culture
with different glial cell types affects expression levels of TJ proteins under
normal and pathological conditions. In this study, we compared the effects of
conditioned media generated from C6 glioma and primary rat astrocytes on
permeability and TJ protein expression, both under normoxic conditions and
after a 24 hour hypoxic stress, to determine if the previously described
protective effect of glial cell co-culture may be due to changes in the
expression of key TJ components.
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Materials and Methods |
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Astrocytes were derived from two sources. C6 glioma cells (American Type
Culture Collection, Rockville, MD) from passages 6-10 were grown in MEM/F12
with 50 µg/ml gentamicin, 10% equine serum and 2.5% fetal bovine serum.
Primary rat astrocytes (RA) were isolated from newborn rat brains by the
method of Frangakis and Kimelberg
(Frangakis and Kimelberg,
1984). Briefly, newborn rat pups (<24 hours old) were
anesthetized and their brains removed. Cerebral cortices were minced and
incubated with trypsin/EDTA for 30 minutes at 37°C. The supernatant was
removed, and cells were seeded in tissue culture flasks coated with 0.01%
poly-L-lysine. Cells were allowed to grow for 4 days in astrocyte growth media
(MEM with 10% fetal bovine serum, 20 mM glucose, 0.5 mM L-glutamine, 12 mg/ml
penicillin, 12 mg/ml streptomycin and 5 mg/ml insulin). After 4 days, the
flasks were shaken at 120 oscillations/min at 37°C for 8 hours to remove
attached cells. Cells still attached to the plasticware (astrocytes) were
trypsinized and frozen in liquid nitrogen until used for experiments.
Conditioned media (CM) was generated by plating either C6 or RA cells at a
density of 40,000 cells/cm2 and harvesting media after 3 days.
BBMECs were exposed to conditioned media as previously described
(Abbruscato and Davis, 1999).
C6-conditioned media (C6-CM) or RA-conditioned media (RA-CM) was added to the
basolateral side of the Transwell filters for 1-3 days before further
experiments. Media was changed every 2 days to provide adequate nutrition, and
there was no evidence of detaching or dying cells with this feeding regimen.
On the day of the experiment, BBMEC monolayers were incubated in assay buffer
(122 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 25
mM NaHCO3, 10 mM HEPES and 0.4 mM K2HPO4) for
permeability studies or dissolved in TRI® reagent (Sigma, St Louis, MO)
for protein isolation and western blot analyses.
Permeability studies
Permeability studies with [14C]-sucrose were used to determine
paracellular flux across confluent BBMEC monolayers. Apical-to-basolateral
flux was determined by dividing the pmoles of radioactive marker appearing in
the receiver chamber by the time in minutes. The apparent permeability
coefficient was calculated using the equation:
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Western blot protein analyses
After coculturing, protein was isolated from cell cultures using TRI®
reagent (Sigma, St Louis, MO). Protein was separated from RNA and DNA by
chloroform and ethanol extraction and precipitated using isopropanol. Protein
pellets were washed with guanidinium chloride/95% ethanol and dissolved in 1%
SDS. Protein levels were measured using the bicinchonic acid method (Pierce,
Indianapolis, IN) using bovine serum albumin (BSA) as a standard. Protein
samples (10-20 µg) were separated on precast 4-12% Tris-glycine gels
(Invitrogen, Carlsbad, CA) at 125 V for 75-90 minutes. Proteins were
transferred to polyvinylidene fluoride membranes at 240 mA at 4°C for 30
minutes. Membranes and/or gels were stained to control for variability in
protein loading prior to blocking. Membranes were incubated with primary
antibody (anti-claudin-1, 1:1000; anti-occludin, 1:2000; anti-ZO-1, 1:2000;
anti-NFB, from Zymed Laboratories, anti-actin, 1:1000, from Sigma,
anti-HSP90
, 1:1000, from Calbiochem) in 0.5% BSA/PBS.
Horseradish-peroxidase-conjugated secondary antibody in 0.5% BSA/PBS was
applied for 30 minutes at room temperature. Protein bands were visualized
using the enhanced chemiluminescent method (ECLplus, Amersham,
Piscataway, NJ). Quantification of band density was done using Scion Image
(NIH, Bethesda, MD); band intensity was normalized to protein loading band
density and expressed as a percentage of control values.
Growth factor immunoassays
Samples of cell culture media were assayed for levels of vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).
Briefly, samples of growth media, C6-CM and RA-CM were incubated in
microplates coated with anti-VEGF or anti-bFGF antibodies (R&D Systems,
Minneapolis, MN). Antibody conjugate was added and incubated. A colorimetric
assay was developed and absorbance was read at 450 nm, with correction for
interference by subtracting readings at 540 nm. VEGF and bFGF levels in
different media were determined by calculation from standard curves. The
sensitivity limit of the assays was 3 pg/ml for both VEGF and bFGF.
Immunoprecipitation
Immunoprecipitation of NFB was performed using the µMACS
Microbeads protocol (Miltenyi Biotech, Auburn, CA). In brief, 4 µg of
antibody was incubated with 50 µg of total protein isolated from control
and co-cultured cells and 50 µl of Protein G microbeads on ice for 30
minutes and then run through columns. Columns were rinsed, and
immunoprecipitated proteins were eluted by applying 95°C SDS loading
buffer. Samples were run on precast 8% Trisglycine gels (Invitrogen, Carlsbad,
CA) and transferred electrophoretically to PVDF membranes. Membranes were
blocked in 5% milk in Tris buffer and incubated with
horseradish-peroxidase-linked anti-rabbit secondary antibody. Blots were
developed as previously described for western blot analyses.
Statistics
Data are presented as means±s.e.m. for n=4-16 permeability
studies or n=3-8 western blot analyses, growth factor assays or
immunoprecipitations. Growth factor levels were measured in duplicate from
three separate experiments. Data were analyzed by one-way (growth factor
assays) or two-way (permeability studies, western blot analyses and
immunoprecipitation) analysis of variance (ANOVA) followed by Tukeys posthoc
test, with significance defined as P<0.05.
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Results |
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BBMEC permeability studies showed a significant effect of both glia-conditioned media (F2,90=11.527, P<0.001) and hypoxic stress (F1,90=109.822, P<0.001). Glia-conditioned media had no significant effect on basal permeability of BBMEC monolayers in the normoxic groups (Fig. 1). After 24 hours of hypoxic stress, the permeability of BBMEC monolayers in MEM/F12 increased 2.2-fold (Fig. 1, P<0.001). Incubating monolayers with C6-CM partially blocked the hypoxia-induced increase in permeability (P<0.001 versus MEM/F12, P<0.05 versus RA-CM), although permeability in the hypoxic C6-CM monolayers was significantly higher then normoxic C6-CM monolayers (P<0.001). By contrast, RA-CM had no effect on hypoxia-induced increase in permeability when compared to hypoxic MEM/F12 monolayers. Statistical analysis showed a significant interaction between culture condition and hypoxic stress (F2,90=3.554, P=0.033).
|
Glia-conditioned media does not affect basal expression of TJ proteins (Table 1). Analysis of western blot expression of claudin-1 shows a significant effect owing to hypoxic stress (F1,42=4.306, P=0.044). After 24 hours of hypoxic stress, there is a significant increase in claudin-1 expression, although there is no significant effect from glia-conditioned media, nor is there an interaction between hypoxic stress and glia-conditioned media.
|
Hypoxic treatment also significantly increased actin expression (Table 1, F1,30=14.184, P<0.001). Similar to claudin-1 expression changes, there was no significant effect from culture conditions, although in both cases, C6-CM-treated monolayers had the largest increase in actin expression after hypoxic stress. No interaction between hypoxic stress and glia-conditioned media was observed. ZO-1 and occludin expression levels were not significantly affected by hypoxic stress or culture condition (Table 1).
We hypothesized that a glia-secreted factor might be involved in mediating
the C6-CM protective effect, and we measured levels of bFGF and VEGF in
glia-conditioned media. bFGF and VEGF were chosen as important factors in this
protective scenario because of evidence in the literature that these factors
are released from C6 glioma cells (Okumura
et al., 1989; Plate et al.,
1993
) and can modulate endothelial cell and BBB properties
(Dobrogowska et al., 1998
;
Sobue et al., 1999
). C6-CM had
20% higher levels of bFGF (Table
2) compared to MEM/F12 and RA-CM (F2,25=17.836,
P<0.001). Levels of VEGF were also significantly elevated in C6-CM
(Table 2) compared with either
MEM/F12 or RA-CM (F2,35=229.879, P<0.001).
|
We hypothesized that some secreted factor in C6-CM might be involved in mediating the C6-CM protective effect. We first hypothesized, owing to the very high levels of VEGF in the C6-CM, that VEGF might be acting in the initial stages of the co-culture as a preconditioning stress. To determine if this was the case, we examined permeability of BBMEC monolayers after one and three days of co-culture with C6-CM. Statistical analysis shows a significant effect of both C6-CM (F2,38=21.508, P<0.001) and hypoxic stress (F1,38=62.881, P<0.001) and a significant interaction between C6-CM and hypoxic stress (F2,38=9.804, P<0.001). Under normoxic conditions, there was no effect of C6-CM on basal permeability after either one or three days of co-culture (Fig. 2). After 24 hours of hypoxic stress, both one and three day co-culture with C6-CM protected against hypoxia-induced permeability increases, indicating that any preconditioning due to C6-CM exposure is probably occurring within a smaller time window.
|
In an attempt to elucidate cellular processes that might be triggered by
C6-CM, we analyzed expression of heat shock protein 90 (HSP90
),
a protein regulated by VEGF (Brouet et al.,
2001
) and by bFGF (Jerome et
al., 1991
). Glia-conditioned media does not affect HSP90
expression under normoxic conditions (Table
3). After 24 hours of hypoxic stress, control monolayers express
significantly higher levels of HSP90
(Table 3, P<0.01).
This increase was blocked by both types of glial conditioned media.
C6-CM-treated monolayers showed no change in HSP90
expression.
RA-CM-treated monolayers had significantly less HSP90
expression after
hypoxic stress compared with both MEM/F12 and C6-CM-treated monolayers.
Two-way ANOVA analysis indicates that levels of HSP90
in hypoxic C6-CM
and RA-CM monolayers were not significantly different from their respective
normoxic levels (P=0.498 for C6-CM-treated monolayers and
P=0.065 for RA-CM-treated monolayers). Two-way ANOVA indicates a
significant effect of glial conditioned media (F2,12=16.860,
P<0.001) and a significant interaction between glial conditioned
media and hypoxic stress (F2,12=9.219, P=0.004). These
results indicate that heat shock protein expression can be influenced by
glia-conditioned media, but this effect is probably not involved in protecting
the integrity of the BBMEC monolayers.
|
Growth factor modulation of BBB endothelial cell function can occur through
signaling cascades resulting in activation of transcription factors such as
NFB (Selzman et al.,
1999
; Sasaki et al.,
2000
). Immunoprecipitation with anti-NF
B antibody reveals
that both C6-CM and RA-CM treatment significantly increased NF
B
expression under normoxic conditions (Fig.
3). After 24 hours of hypoxia, the levels of NF
B in MEM/F12
and RA-CM-treated samples significantly increased, whereas levels in
C6-CM-treated samples did not change. There was a significant effect of
hypoxic stress (F1,30=12.633, P=0.001), as well as a
significant interaction between culturing condition and hypoxic stress
(F2,30=3.967, P=0.03).
|
![]() |
Discussion |
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We examined expression levels of four TJ proteins to determine if glial
conditioned media alone had any direct effect on TJ, which are critical for
maintaining BBB properties. Glial conditioned media had no effect on basal
levels of TJ protein expression. After hypoxic stress, there was no
significant change in protein expression levels of ZO-1 or occludin. Claudin-1
expression was increased by hypoxic stress, and co-culture with C6-CM caused a
greater increase in claudin-1 expression then MEM/F12 or RA-CM. Actin levels
were also increased by hypoxic stress, with the greatest increase seen with
C6-CM treatment. Together, these results suggest the protection of monolayer
integrity is potentially due to strengthening of existing TJ or formation of
new TJ complexes (Fig. 4). It
remains to be seen if the higher expression levels of claudin-1 and actin are
due to newly synthesized proteins or to the removal of already existing
protein components from other subcellular locations. Previously, we have found
that claudin-1 expression and localization is unlikely to be altered after
insult, in both in vivo (Huber et al.,
2001b) and in vitro (Mark and
Davis, 2002
) BBB model systems, although paracellular permeability
is significantly disrupted. Claudin-1 is considered to be the critical
transmembrane protein required for TJ structure and function, and the dramatic
changes seen in this system seem to indicate an alteration of the TJ protein
regulation. We have recently demonstrated that 24 hour hypoxic stress does
cause a dissociation of other TJ components, with the removal of occludin and
ZO-1 from their normal membrane localization and the formation of actin stress
fibers (Mark and Davis, 2002
).
It may be that under our C6-CM-treated conditions, ZO-1 and occludin released
initially from the dissolution of TJ are able to associate with increased
levels of claudin-1 and actin and make new TJ to prevent increased
paracellular permeability (Fig.
4). However, we can not rule out a mechanism by which C6-CM
protects already existing TJ during hypoxic stress.
|
To identify factors that may be involved in the C6-CM protective effect and
account for the interaction between culture condition and hypoxic stress seen
in the permeability data, we assayed two angiogenic growth factors known to
have effects on the BBB. Basic fibroblast growth factor (bFGF) has been shown
to tighten in vitro BBB models (Sobue et
al., 1999) and may be important for promoting proliferation in
hypoxic endothelial cells (Kuwabara et
al., 1995
). Vascular endothelial growth factor (VEGF) is a well
characterized angiogenic factor that increases BBB permeability
(Nag et al., 1997
;
Dobrogowska et al., 1998
) and
may be directly involved in increasing BBB permeability by modulating TJ after
hypoxia (Fischer et al.,
2002
). We found that C6-CM had significantly higher concentrations
of both bFGF and VEGF compared with MEM/F12 alone and RA-CM, which were not
significantly different from each other.
A possible mechanism by which C6-CM might be protective is a
`preconditioning' effect. Previous studies show that brief insults or
stressors can be protective against later, more sustained insults. These
preconditioning stressors are typically brief hypoxic exposures or heat shock
(Gobbel et al., 1995;
Pohlman and Harlan, 2000
),
which can prevent later hypoxia-induced endothelial cell damage. We
hypothesize that high levels of VEGF present in C6-CM potentially act as a
preconditioning stress, causing an initial breakdown of the BBB in the early
stages of co-culture. After three days exposure to high VEGF levels, BBMEC
monolayers adapt to the stress and the barrier retightens. However, when we
examined permeability of BBMEC monolayers at one and three days after exposure
to C6-CM, there was no increase in permeability at one day, as was expected.
Furthermore, after 24 hours pf hypoxic stress, both one and three days of
exposure to C6-CM were protective, with three days showing the greatest degree
of protection. As there is an increase in NF
B levels seen with C6-CM
exposure, indicating that the cells have been stressed, this suggests that any
transient increase in permeability that may occur upon initial exposure to
C6-CM has been recovered from by the time points examined. Previous studies
have shown relatively quick transient increases in BBB permeability ranging
from 15 minutes with bradykinin analogues
(Mackic et al., 1999
), to 2-4
hours after exposure to TNF
(Mark
et al., 2001
) or inflammatory pain
(Huber et al., 2001b
).
In support of this data ruling out a preconditioning effect of VEGF in
C6-CM-mediated protection, we found no correlation between HSP90 levels
and glial conditioned media treatment, although there was a significant effect
of culture condition and a significant interaction between culturing condition
and hypoxic stress. HSP90
is important in mediating VEGF activity on
endothelial cells, primarily by interacting with endothelial nitric oxide
synthase and increasing nitric oxide production
(Garcia-Cardena et al., 1998
;
Brouet et al., 2001
). Although
C6-CM did prevent the hypoxia-induced increase in HSP90
, RA-CM
significantly lowered HSP90
levels after hypoxia. It may be that
treatment with RA-CM causes a downregulation of HSP90
under hypoxic
conditions that prevents activation of a pathway necessary for TJ maintenance,
but further studies are required to determine the contribution of HSP90
to TJ integrity.
Another potential mediator of growth factor effects on endothelial cells is
the transcription factor NFB. NF
B is a major transcription
factor involved in the inflammatory process and is linked to cell adhesion
molecule expression in endothelial cells
(Kupatt et al., 1997
).
NF
B is also activated by hypoxic stress. The finding that C6-CM and
RA-CM increased basal levels of NF
B in our BBMEC monolayers suggests
that the co-culture conditions alone constitute a stressor. However, when
subjected to hypoxic stress, the levels of NF
B in the C6-CM treated
cells did not change significantly, whereas increased expression was observed
in both the MEM/F12- and RA-CM-treated cultures. The fact that NF
B
expression did not increase after hypoxic stress, whereas expression in the
other two treatment groups did increase, suggests that the C6-CM-treated
monolayers are not further stressed by the removal of oxygen or that they are
unable to respond above a maximum level of NF
B response. Although we
can not directly link NF
B activation to protection of paracellular
permeability, it may be an important trigger for downstream events involved in
this protection. NF
B has been linked to the expression of cell-cell
adhesion molecules involved in inflammatory processes, such as ICAM-1
(Kupatt et al., 1997
), as well
as to the expression of VEGF (Sasaki et
al., 2000
), various cytokines and immunoreceptors
(Li and Stark, 2002
) and
important proteins involved in reactive oxygen species homeostasis
(Chiarugi et al., 1999
), but it
may also alter TJ protein expression, and this remains to be determined.
In conclusion, we have demonstrated that the protective effect of culturing
BBMEC monolayers with C6-CM occurs in a time-dependent manner, with as little
as one day of exposure to C6-CM causing a significant protection against
hypoxic stress. This protection may be due to an enhancement of TJ protein
expression (claudin-1 and actin) after hypoxic stress. C6-CM has significantly
higher levels of VEGF and bFGF then MEM/F12 or RA-CM, but, surprisingly, VEGF
does not appear to be acting as a preconditioning stress under the conditions
examined. The C6-CM-mediated protection does appear to involve NFB
signaling. Although not a direct physiological paradigm except in the case of
hypoxic stress and/or brain tumors, this model system may be useful for
examining (1) changes in TJ protein expression at the BBB under various
stressors or pathological incidents and (2) may provide a system in which the
exact interactions between astrocyte-secreted growth factors and the
endothelial cells of the BBB may be better studied.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbruscato, T. J., Thomas, S. A., Hruby, V. J. and Davis, T.
P. (1997). Blood-brain barrier permeability and
bioavailability of a highly potent and mu-selective opioid receptor
antagonist, CTAP: comparison with morphine. J. Pharmacol. Exp.
Ther. 280,402
-409.
Abbruscato, T. J. and Davis, T. P. (1999).
Combination of hypoxia/aglycemia compromises in vitro blood-brain barrier
integrity. J. Pharmacol. Exp. Ther.
289,668
-675.
Banks, W. A. (1999). Physiology and pathology of the blood-brain barrier: implications for microbial pathogenesis, drug delivery and neurodegenerative disorders. J. Neurovirol. 5,538 -555.[Medline]
Banks, W. A., Jaspan, J. B. and Kastin, A. J. (1997). Effect of diabetes mellitus on the permeability of the blood-brain barrier to insulin. Peptides 18,1577 -1584.[CrossRef][Medline]
Brouet, A., Sonveaux, P., Dessy, C., Balligand, J. L. and Feron,
O. (2001). Hsp90 ensures the transition from the early
Ca2+-dependent to the late phosphorylation-dependent activation of
the endothelial nitric-oxide synthase in vascular endothelial growth
factor-exposed endothelial cells. J. Biol. Chem.
276,32663
-32669.
Brown, R. C. and Davis, T. P. (2002). Calcium
modulation of adherens and tight junction function: A potential mechanism for
blood-brain barrier disruption after stroke. Stroke
33,1706
-1711.
Brownson, E. A., Abbruscato, T. J., Gillespie, T. J., Hruby, V. J. and Davis, T. P. (1994). Effect of peptidases at the blood brain barrier on the permeability of enkephalin. J. Pharmacol. Exp. Ther. 270,675 -680.[Abstract]
Butt, A. M., Jones, H. C. and Abbott, N. J. (1990). Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J. Physiol. 429, 47-62.[Abstract]
Chiarugi, V., Magnelli, L., Chiarugi, A. and Gallo, O. (1999). Hypoxia induces pivotal tumor angiogenesis control factors including p53, vascular endothelial growth factor and the NFkappaB-dependent inducible nitric oxide synthase and cyclooxygenase-2. J. Cancer Res. Clin. Oncol. 125,525 -528.[CrossRef][Medline]
Dehouck, B., Dehouck, M. P., Fruchart, J. C. and Cecchelli, R. (1994). Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes. J. Cell Biol. 126,465 -473.[Abstract]
Dobrogowska, D. H., Lossinsky, A. S., Tarnawski, M. and Vorbrodt, A. W. (1998). Increased blood-brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor. J. Neurocytol. 27,163 -173.[CrossRef][Medline]
El Hafny, B., Chappey, O., Piciotti, M., Debray, M., Boval, B. and Roux, F. (1997). Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line. Neurosci. Lett. 236,107 -111.[CrossRef][Medline]
Fischer, S., Wobben, M., Kleinstuck, J., Renz, D. and Schaper,
W. (2000). Effect of astroglial cells on hypoxia-induced
permeability in PBMEC cells. Am. J. Physiol. Cell
Physiol. 279,C935
-C944.
Fischer, S., Wobben, M., Marti, H. H., Renz, D. and Schaper, W. (2002). Hypoxia-induced hyperpermeability in brain microvessel endothelial cells involves VEGF-mediated changes in the expression of zonula occludens-1. Microvasc. Res. 63, 70-80.[CrossRef][Medline]
Frangakis, M. V. and Kimelberg, H. K. (1984). Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures. Neurochem. Res. 9,1689 -1698.[Medline]
Furuse, M., Hirase, T., Itoh, M., Nagafuchi, A., Yonemura, S. and Tsukita, S. (1993). Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol. 123,1777 -1788.[Abstract]
Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K. and Tsukita,
S. (1998). Claudin-1 and -2: novel integral membrane proteins
localizing at tight junctions with no sequence similarity to occludin.
J. Cell Biol. 141,1539
-1550.
Garcia-Cardena, G., Fan, R., Shah, V., Sorrentino, R., Cirino, G., Papapetropoulos, A. and Sessa, W. C. (1998). Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 392,821 -824.[CrossRef][Medline]
Gobbel, G. T., Chan, T. Y. and Chan, P. H. (1995). Amelioration of hypoxic and hypoglycemic damage to cerebral endothelial cells. Effects of heat shock pretreatment. Mol. Chem. Neuropathol. 24,107 -120.[Medline]
Hawkins, C. P., Mackenzie, F., Tofts, P., du Boulay, E. P. and McDonald, W. I. (1991). Patterns of blood-brain barrier breakdown in inflammatory demyelination. Brain Res. 114,801 -810.
Huber, J. D., Egleton, R. D. and Davis, T. P. (2001a). Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends Neurosci. 24,719 -725.[CrossRef][Medline]
Huber, J. D., Witt, K. A., Hom, S., Egleton, R. D., Mark, K. S.
and Davis, T. P. (2001b). Inflammatory pain alters
blood-brain barrier permeability and tight junctional protein expression.
Am. J. Physiol. Heart. Circ. Physiol.
280,H1241
-H1248.
Igarashi, Y., Utsumi, H., Chiba, H., Yamada-Sasamori, Y., Tobioka, H., Kamimura, Y., Furuuchi, K., Kokai, Y., Nakagawa, T., Mori, M. and Sawada, N. (1999). Glial cell line-derived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier. Biochem. Biophys. Res. Commun. 261,108 -112.[CrossRef][Medline]
Isobe, I., Watanabe, T., Yotsuyanagi, T., Hazemoto, N., Yamagata, K., Ueki, T., Nakanishi, K., Asai, K. and Kato, T. (1996). Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: a possible use of aortic endothelial cell for in vitro BBB model. Neurochem. Int. 28,523 -533.[CrossRef][Medline]
Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M. and
Tsukita, S. (1999). Direct binding of three tight
junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of
claudins. J. Cell Biol.
147,1351
-1363.
Jerome, V., Leger, J., Devin, J., Baulieu, E. E. and Catelli, M. G. (1991). Growth factors acting via tyrosine kinase receptors induce HSP90 alpha gene expression. Growth Factors 4,317 -327.[Medline]
Kalaria, R. N. (1999). The blood-brain barrier
and cerebrovascular pathology in Alzheimer's disease. Ann. N Y
Acad. Sci. 893,113
-125.
Kneisel, U. and Wolburg, H. (2000). Tight junctions of the blood-brain barrier. Cell. Mol. Neurobiol. 20,57 -76.[CrossRef][Medline]
Kondo, T., Kinouchi, H., Kawase, M. and Yoshimoto, T. (1996). Astroglial cells inhibit the increasing permeability of brain endothelial cell monolayer following hypoxia/reoxygenation. Neurosci. Lett. 208,101 -104.[CrossRef][Medline]
Kupatt, C., Weber, C., Wolf, D. A., Becker, B. F., Smith, T. W. and Kelly, R. A. (1997). Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NFkappaB. J. Mol. Cell. Cardiol. 29,2599 -2609.[CrossRef][Medline]
Kuwabara, K., Ogawa, S., Matsumoto, M., Koga, S., Clauss, M., Pinsky, D. J., Lyn, P., Leavy, J., Witte, L., Joseph-Silverstein, J. et al. (1995). Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. Proc. Natl. Acad. Sci. USA 92,4606 -4610.[Abstract]
Li, X. and Stark, G. R. (2002). NFkappaB-dependent signaling pathways. Exp. Hematol. 30,285 -296.[CrossRef][Medline]
Mackic, J. B., Stins, M., Jovanovic, S., Kim, K. S., Bartus, R. T. and Zlokovic, B. V. (1999). Cereport (RMP-7) increases the permeability of human brain microvascular endothelial cell monolayers. Pharm. Res. 16,1360 -1365.[Medline]
Mark, K. S., Trickler, W. J. and Miller, D. W.
(2001). Tumor necrosis factor-alpha induces cyclooxygenase-2
expression and prostaglandin release in brain microvessel endothelial cells.
J. Pharmacol. Exp. Ther.
297,1051
-1058.
Mark, K. S. and Davis, T. P. (2002). Cerebral
microvascular changes in permeability and tight junctions induced by
hypoxia-reoxygenation. Am. J. Physiol. Heart Circ.
Physiol. 282,H1485
-H1494.
Mitic, L. L., Schneeberger, E. E., Fanning, A. S. and Anderson,
J. M. (1999). Connexin-occludin chimeras containing the
ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell
contacts. J. Cell Biol.
146,683
-693.
Nag, S., Takahashi, J. L. and Kilty, D. W. (1997). Role of vascular endothelial growth factor in blood-brain barrier breakdown and angiogenesis in brain trauma. J. Neuropathol. Exp. Neurol. 56,912 -921.[Medline]
Okumura, N., Takimoto, K., Okada, M. and Nakagawa, H. (1989). C6 glioma cells produce basic fibroblast growth factor that can stimulate their own proliferation. J. Biochem. (Tokyo) 106,904 -909.[Abstract]
Plate, K. H., Breier, G., Millauer, B., Ullrich, A. and Risau, W. (1993). Upregulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res 53,5822 -5827.[Abstract]
Pohlman, T. H. and Harlan, J. M. (2000). Adaptive responses of the endothelium to stress. J. Surg. Res. 89,85 -119.[CrossRef][Medline]
Raub, T. J., Kuentzel, S. L. and Sawada, G. A. (1992). Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells. Exp. Cell Res. 199,330 -340.[Medline]
Reese, T. S. and Karnovsky, M. J. (1967). Fine
structural localization of a blood-brain barrier to exogenous peroxidase.
J. Cell Biol. 34,207
-217.
Sasaki, H., Ray, P. S., Zhu, L., Galang, N. and Maulik, N. (2000). Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium: possible role of NFkappaB. Toxicology 155,27 -35.[CrossRef][Medline]
Selzman, C. H., Shames, B. D., McIntyre, R. C., Jr, Banerjee, A.
and Harken, A. H. (1999). The NFkappaB inhibitory peptide,
IkappaBalpha, prevents human vascular smooth muscle proliferation.
Ann. Thorac. Surg. 67,1227
-1231.
Sobue, K., Yamamoto, N., Yoneda, K., Hodgson, M. E., Yamashiro, K., Tsuruoka, N., Tsuda, T., Katsuya, H., Miura, Y., Asai, K. and Kato, T. (1999). Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors. Neurosci. Res. 35,155 -164.[CrossRef][Medline]
Takakura, Y., Audus, K. L. and Borchardt, R. T. (1991). Blood-brain barrier: transport studies in isolated brain capillaries and in cultured brain endothelial cells. Adv. Pharmacol. 22,137 -165.[Medline]
Tsukita, S. and Furuse, M. (1999). Occludin and claudins in tight-junction strands: leading or supporting players? Trends Cell Biol. 9,268 -273.[CrossRef][Medline]
Weber, S. J., Abbruscato, T. J., Brownson, E. A., Lipkowski, A. W., Polt, R., Misicka, A., Haaseth, R. C., Bartosz, H., Hruby, V. J. and Davis, T. P. (1993). Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs. J. Pharmacol. Exp. Ther. 266,1649 -1655.[Abstract]
Wolburg, H., Neuhaus, J., Kniesel, U., Krauss, B., Schmid, E.
M., Ocalan, M., Farrell, C. and Risau, W. (1994). Modulation
of tight junction structure in blood-brain barrier endothelial cells. Effects
of tissue culture, second messengers and cocultured astrocytes. J.
Cell Sci. 107,1347
-1357.