From the Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 1, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Vascular endothelial growth
factor (VEGF), also known as vascular permeability factor (VPF), has
been shown to increase potently the permeability of endothelium and is
highly expressed in breast cancer cells. In this study, we investigated
the role of VEGF/VPF in breast cancer metastasis to the brain. Very
little is known about the role of endothelial integrity in the
extravasation of breast cancer cells to the brain. We hypothesized that
VEGF/VPF, having potent vascular permeability activity, may support
tumor cell penetration across blood vessels by inducing vascular
leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels.
We observed that VEGF/VPF significantly increased the
penetration of the highly metastatic MDA-MB-231 breast cancer cells
across the HBMEC monolayer. We found that the increased
transendothelial migration (TM) of MDA-MB-231 cells resulted from the
increased adhesion of tumor cells onto the HBMEC monolayer. These
effects (TM and adhesion of tumor cells) were inhibited by the
pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor
(KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator
1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also
inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can
modulate the TM of tumor cells by regulating the integrity of the HBMEC
monolayer. Taken together, these findings indicate that VEGF/VPF might
contribute to breast cancer metastasis by enhancing the TM of tumor
cells through the down-regulation of endothelial integrity.
Brain metastasis, which is an important cause of cancer morbidity
and mortality, occurs in at least 30% of patients with breast cancer.
A key event of brain metastasis is the migration of cancer cells
through the blood-brain barrier
(BBB),1 which constitutes the
endothelium and the surrounding cells (1, 2). To metastasize to the
brain, malignant tumor cells must enter into the circulatory system
through the endothelium (intravasation) and then attach to microvessel
endothelial cells to invade the BBB (extravasation) (1). The precise
molecular mechanism of extravasation of tumor cells penetrating the BBB
is poorly defined. A widely supported hypothesis is that tumor cell
adhesion to endothelium induces the retraction of endothelial cells,
which exposes their basement membrane to the tumor cells (3). Tumor
cells recognize and bind to components in the vascular membrane,
thereby initiating extravasation and the beginning of new growth at
secondary organ sites (4). This suggests that the intact endothelium
can serve as a "defensive barrier" to the extravasation of
tumor cells.
Tumor-bearing blood vessels, however, do not seem to be effective in
acting as a defensive barrier for the prevention of tumor cell
intravasation, because these blood vessels display high leakage and
disrupted integrity (5, 6). Hypoxia is believed to contribute to the
leakage of tumor blood vessels by inducing increased vascular permeability (7, 8). Hypoxia is also a strong inducer of VEGF/VPF (9,
10). VEGF/VPF has potent mitotic activity specific to vascular
endothelial cells and significant vascular permeable activity (11, 12).
VEGF/VPF binds to its cognate receptors, Flt-1, KDR/Flk-1, and
neuropilin-1. Among them, KDR/Flk-1 is responsible for the initiation
of signal transduction pathways within the cells (11, 12). VEGF/VPF has
an essential role in promoting new blood vessel formation
(angiogenesis) during tumor development, and inhibition of its function
effectively prevents tumor growth through incomplete blood vessel
formation (13, 14). Indeed, VEGF/VPF expression has been reported in a
number of cancer cell lines and in several clinical specimens derived
from breast, brain, and ovarian cancers (15-18). Although the role of
VEGF/VPF as an angiogenic factor in primary tumor growth and secondary
tumor growth (metastatic tumors) is well studied, its role as a
vascular permeability factor in metastatic processes is not well
elucidated. Tumor cells may more easily penetrate a retracted
endothelial monolayer caused by VEGF/VPF than a tightly arranged
monolayer, suggesting that the vascular permeability activity of
VEGF/VPF contributes an "offensive ability" to the tumor
cells, allowing them to penetrate blood vessels. To examine this
hypothesis, we constructed a Transwell culture system of the human
brain endothelial monolayer as an in vitro model for the
BBB. We then evaluated the inter-relationship between the integrity of
the endothelial monolayer and the vascular permeability of VEGF/VPF and
their effect on the TM of the highly metastatic MDA-MB-231 breast
cancer cells, which are known to express high amounts of VEGF/VPF
(19).
Materials--
Human recombinant VEGF165/VPF
(VEGF165 consists of 165 amino acid residues) and
anti-human VEGF/VPF monoclonal antibody were obtained from Genentech,
Inc. (San Francisco, CA). Human recombinant bFGF was purchased from R & D Systems, Inc. (Minneapolis, MN). Rhodamine-phalloidin, DiI,
and BCECF-AM were from Molecular Probes, Inc. (Eugene, OR), and
PD98059, BAPTA-AM, Wortmannin, and SU-1498 were purchased from
Calbiochem. Anti-human VE-cadherin monoclonal antibody
was from Chemicon International, Inc. (Temecula, CA). Anti-human
VEGF/VPF polyclonal antibody and anti-human Csk antibody were from
Santa Cruz Biotechnology, Inc. (San Diego, CA).
Cell Culture--
Human brain microvascular endothelial cells
(HBMECs) were purchased from Cell Systems Inc. (Kirkland, WA). The
cells were seeded onto attachment factor-coated culture plates and
maintained in CSC complete medium according to the protocol of the
manufacturer. The HBMECs formed tubular-like networks on matrigel and
produced the endothelial-specific marker, von Willebrand factor,
indicating that these cells maintained the general properties of
endothelial cells (20). During the course of the experiment, the cells
were used until passages three to seven and checked routinely for
expression of von Willebrand factor. Human umbilical vein endothelial
cells (HUVEC) were from Clonetics (San Diego, CA) and were cultured in
EGM complete medium (Clonetics). MDA-MB-231, MDA-MB-453, and MCF-7 cells were obtained from ATCC and maintained in culture medium
(DMEM containing 10% fetal bovine serum, 2 mM
L-glutamine). HBL-100 cells (ATCC) were cultured in
McCoy's 5A medium containing 10% fetal bovine serum. T47D cells
(ATCC) were cultured in RPMI 1640 medium containing 10% fetal bovine
serum. All cell lines were incubated in 5% CO2 at
37 °C.
Northern Blot Analysis of KDR/Flk-1 Expression in
Breast Cancer Cell Lines--
mRNAs of breast cancer cell lines
were isolated from cellular extracts by using an oligo(dT) column
(Invitrogen), according to the manufacturer's protocol. The mRNAs
were separated on an agarose gel and transferred to a Hybond N membrane
(Amersham Biosciences). The membrane was hybridized with a
32P-labeled probe for KDR/Flk-1 (a generous gift of Dr.
Michael Klagsbrun, Children's Hospital, Boston, MA), and after
washing, the blot was exposed to x-ray film. The blot was also stripped and reprobed for actin mRNA.
Fluorescent Labeling of MDA-MB-231 Cells--
MDA-MB-231 cells
were incubated with 200 nM DiI for 30 min and then washed
twice with PBS. DiI-labeled cells were dispersed in 0.05% trypsin
solution and resuspended in culture medium. Alternatively, tumor cells
were dispersed in 0.05% trypsin solution and incubated with 1 µM BCECF-AM for 15 min and then centrifuged three times to remove free BCECF-AM.
TM Assay of MDA-MB-231 Cells--
HBMECs grown on attachment
factor-coated culture plates were dispersed in 0.05% trypsin solution
and resuspended in CSC complete medium. Approximately 100,000 cells
were added to the fibronectin-coated 24-well Transculture inserts with
pore sizes of 8 µm (Costar Corp.) and grown for 5 days in 5%
CO2 at 37 °C. The medium was replaced every day with
fresh medium. Prior to the assays, the monolayers were washed once with
CSC medium without growth factor and then 40,000 DiI-labeled MDA-MB-231
cells in 100 µl of the same medium were added to the apical chamber.
To exclude the chemoattractant effect of the added growth factor,
VEGF/VPF or bFGF was added evenly to the apical and basolateral
chambers. In the case of inhibitor treatment, the monolayers were
pre-treated for 30 min, and all synthetic inhibitors were removed from
the monolayers by washing twice with culture medium. After incubation
for 6 h, the apical chambers were fixed with 3.7% formaldehyde
and washed extensively with PBS. To remove non-migrating cells, the
apical side of the apical chamber was scraped gently with cotton wool, and only the migrating tumor cells were observed under a fluorescent microscope. Migrating cells were counted from 10 random fields of ×200 magnification.
TM Assay of Adenovirus-infected MDA-MB-231 Cells--
MDA-MB-231
cells were infected with adenovirus-encoding VEGF/VPF (VEGF-Ad; a
generous gift of Dr. Harold Dvorak, Beth Israel Deaconess Medical
Center, Boston, MA) or control adenovirus (CTL-Ad) at a multiplicity of
infection of 1,000 for 48 h. After labeling with DiI, the cells
were dispersed in trypsin solution and resuspended in CSC medium
without growth factor. 5,000 DiI-labeled cells were treated with
VEGF/VPF monoclonal antibody and were then added to the apical chamber
containing the HBMEC monolayer. After incubation for 18 h, the
apical chambers were fixed with 3.7% formaldehyde, and the migrating
tumor cells were observed according to the method described above.
Invasion Assay of MDA-MB-231 Cells--
24-well Transculture
inserts with pore sizes of 8 µm (Costar Corp.) were coated by air
drying with 50 ng of matrigel. MDA-MB-231 cells were trypsinized and
suspended at 1.5 × 105 in 100 µl of DMEM containing
1% BSA. The cells were added to the apical chamber and then VEGF/VPF
was added to the basolateral chamber containing 600 µl of DMEM with
1% BSA. After incubation for 6 h, the apical chambers were
stained with HEMA-3 solution (Fisher), and the apical side of the
apical chamber was scraped gently with cotton wool. The migrating tumor
cells were observed under a light microscope and were counted from 10 random fields of ×200 magnification.
Adhesion Assay of MDA-MB-231 Cells--
HBMECs were added to
attachment factor-coated 24-well culture plates and grown for 5 days in
5% CO2 at 37 °C. The medium was replaced every day with
fresh medium. Prior to the assays, the monolayers were washed once with
CSC medium without growth factor and then 100,000 of DiI-labeled
MDA-MB-231 cells in 500 µl of the same medium were added to each well
with or without test samples. In the case of inhibitor treatment, the
monolayers were pre-treated for 30 min, and all synthetic inhibitors
were removed from the monolayers by washing twice with culture medium.
After incubation for 2 h, the wells were fixed with 3.7%
formaldehyde and washed extensively with PBS to remove floating tumor
cells. Attached tumor cells were observed under a fluorescent
microscope and counted from 10 random fields of ×200 magnification.
To detach the endothelial monolayer and prepare the sub-endothelial
basement (SEB) membrane components, the monolayers were treated with 50 mM NH4OH solution for 5 min as indicated
previously (21) and washed extensively with PBS before adding the
DiI-labeled MDA-MB-231 cells.
Retraction Assay of HBMECs--
To monitor the extent of
endothelial cell retraction, the amount of [3H]inulin
(Amersham Biosciences) passing across an endothelial monolayer was
measured as described (14). Briefly, ~100,000 HBMECs were added to
fibronectin-coated 24-well Transculture inserts with pore sizes of 0.4 µm (Falcon Corp.) and grown for 5 days in 5% CO2 at
37 °C. The medium was replaced every day with fresh medium. After
the removal of culture medium, 0.4 ml of the fresh culture medium
containing [3H]inulin (1 µCi) was added to the apical
chamber. The basolateral chamber was filled with 0.6 ml of the same
medium without [3H]inulin and then 30 ng/ml of VEGF/VPF
were added to the apical and basolateral chambers. In the case of
inhibitor treatment, the monolayers were pre-treated for 30 min before
VEGF/VPF treatment. After incubation for 2 h, 30 µl of medium
from the basolateral chamber was collected, and the amount of
[3H]inulin across the monolayers was determined by
scintillation counting.
F-actin Staining of HBMECs--
HBMECs were added to
fibronectin-coated 24-well Transculture inserts with pore sizes of 0.4 µm and grown for 5 days in 5% CO2 at 37 °C. After the
assays, the cells were fixed with 3.7% formaldehyde in PBS for 20 min
and then permeabilized with 0.5% Triton X-100 for 10 min. Following a
PBS washing, the cells were blocked with 20% goat serum in PBS and
then incubated at room temperature with rhodamine-phalloidin (diluted
1:40) in PBS for 1 h. After washing by three changes of PBS, the
polycarbonate membranes were separated carefully from the apical
chamber and mounted on a slide, and F-actin staining was observed under
a fluorescent microscope.
VE-Cadherin staining of HBMECs--
HBMECs were added to
fibronectin-coated 24-well Transculture inserts with pore sizes of 0.4 µm and grown for 5 days in 5% CO2 at 37 °C. After the
assays, the cells were fixed with 3.7% formaldehyde in PBS for 20 min
and then permeabilized with 0.5% Triton X-100 for 10 min.
Alternatively, 1,000 BCECF-AM-labeled MDA-MB-231 cells were added to
each well with or without VEGF/VPF (30 ng/ml). After incubation for
2 h, the wells were fixed with 3.7% formaldehyde, washed
extensively with PBS to remove floating tumor cells, and then
permeabilized with 0.5% Triton X-100 for 10 min. Following a PBS
washing, the cells were blocked with 20% goat serum in PBS for 1 h before overnight incubation at 4 °C with anti-human VE-cadherin
monoclonal antibody (2 µg/ml) diluted by calcium containing
serum-free DMEM. After washing by three changes of PBS, the cells were
incubated for 1 h with goat anti-mouse IgG Texas-red (diluted
1:200) in PBS. After washing by three changes of PBS, the polycarbonate
membranes were separated carefully from the apical chamber, mounted on
a slide, and viewed under a confocal microscope.
Western Blot Analysis--
MDA-MB-231 cells were lysed in kinase
lysis buffer (New England Biolabs). Proteins were separated by SDS-PAGE
under reducing conditions and transferred onto nitrocellulose membranes
(Millipore, Boston, MA). The membranes were blocked with 5% bovine
serum albumin in PBS and subsequently incubated with primary antibody
for overnight incubation at 4 °C. Bound antibodies were detected by
horseradish peroxidase-conjugated secondary antibody and enhanced
chemiluminescence (Amersham Biosciences).
MDA-MB-231 Cells Express mRNA of the VEGF/VPF
Receptor, KDR/Flk-1--
It was reported that, in nearly
50% of breast tumors, there was significant expression of KDR/Flk-1 in
the tumor epithelial cells, which correlated with the expression of
VEGF/VPF by these cells (22). To examine whether various breast cancer
cell lines express the VEGF/VPF receptor, KDR/Flk-1, which is
responsible for most VEGF/VPF activity, we performed Northern blot
analysis with a specific probe for KDR/Flk-1. As shown in Fig.
1A, breast cancer cell lines,
such as MDA-MB-231 cells, expressed KDR/Flk-1 mRNA at a low
level as compared with HUVEC.
Effect of VEGF/VPF on the Invasion of MDA-MB-231
Cells--
Next, to test whether MDA-MB-231 cells respond to
exogenously added VEGF/VPF, an invasion assay was performed for 6 h under serum-free conditions. MDA-MB-231 cells were added to a
matrigel-coated polycarbonate filter and then invading cells were
assessed under a light microscope. When VEGF/VPF was added to the
basolateral chambers, the invasion of MDA-MB-231 cells was not
significantly increased, irrespective of KDR/Flk-1 expression (Fig.
1B).
VEGF/VPF Increases Penetration of MDA-MB-231 Cells
across an HBMEC Monolayer--
To test whether VEGF/VPF increases
tumor cell penetration, DiI-labeled MDA-MB-231 cells were added to an
HBMEC monolayer cultured onto a Transwell apical chamber and then
penetrating MDA-MB-231 cells were assessed under a fluorescent
microscope. To exclude the chemoattractant effect of the added growth
factor for the HBMEC monolayer or tumor cells, VEGF/VPF was added
evenly to the apical and basolateral chambers. VEGF/VPF treatment led
to a dose-dependent increase in the penetration of
MDA-MB-231 cells across the HBMEC monolayer as compared with the
untreated control (Fig. 2). bFGF is also known as a potent endothelial cell growth factor but does not
increase vascular permeability (23). When bFGF was added evenly to the
apical and basolateral chambers, it failed to significantly increase
the TM of the cells. These data indicate the possibility that the
increased TM of the MDA-MB-231 cells is because of the endothelial cell
retraction induced by VEGF/VPF. To further characterize whether
VEGF/VPF is related directly to the increased TM of MDA-MB-231 cells,
the HBMEC monolayer was treated with VEGF/VPF monoclonal antibody (20 µg/ml) and with SU-1498 (50 µM), an antagonist of KDR/Flk-1. As shown in Fig. 4, both treatments against VEGF/VPF significantly inhibited the TM of the MDA-MB-231 cells.
VEGF/VPF Increases the Adhesion of MDA-MB-231 Cells to
an HBMEC Monolayer--
Metastatic tumor cells attach more
preferentially to SEB membrane components than to the apical surface of
an intact endothelial monolayer (24). The same phenomenon was observed
in this study with MDA-MB-231 cells that attached preferentially to
areas where the SEB of the endothelial cell was exposed (Fig.
3, right panel). Therefore,
the increased TM of the MDA-MB-231 cells induced by VEGF/VPF might
result from the increased adhesion of the cells to the SEB membrane
components of the endothelial cells that were exposed. To test this
possibility, DiI-labeled MDA-MB-231 cells were added to an HBMEC
monolayer cultured onto 24-well plates with or without VEGF/VPF, and
cell adhesion was then assessed under a fluorescent microscope. At a
concentration of 30 ng/ml, VEGF/VPF increased the adhesion of
MDA-MB-231 cells to the HBMEC monolayer 3-fold as compared with the
untreated control (Fig. 3, left panel), and this effect was
blocked by VEGF/VPF monoclonal antibody and SU-1498 (see Fig. 5).
However, bFGF failed to significantly increase the adhesion of tumor
cells to the monolayer as compared with the untreated control (Fig. 3).
These results indicate that the increased TM of MDA-MB-231 cells
induced by VEGF/VPF was at least in part derived from enhanced tumor
cell adhesion onto the exposed SEB membrane components.
VEGF/VPF Increases the TM and Adhesion of MDA-MB-231
Cells through Calcium Signaling--
VEGF/VPF stimulates several
molecules mediating intracellular signals in endothelial cells,
including mitogen-activated protein/extracellular signal-regulated
kinase (ERK) kinase (MEK), phosphatidylinositol 3-kinase, and calcium
(11). To examine which signaling pathways of VEGF/VPF in endothelial
cells are responsible for the increased TM and adhesion of MDA-MB-231
cells, the effects of specific inhibitors for various VEGF/VPF
signaling pathways were tested. As shown in Figs.
4 and 5,
the intracellular calcium chelator (BAPTA-AM) slightly inhibited the
increased TM and adhesion of MDA-MB-231 cells stimulated by VEGF/VPF
whereas the MEK inhibitor (PD98059) and the phosphatidylinositol
3-kinase inhibitor (Wortmannin) had no effect, indicating that VEGF/VPF
increases the TM and adhesion of MDA-MB-231 cells through activation of
endothelial calcium signaling.
VEGF/VPF Increases the Permeability of the HBMEC
Monolayer--
Endothelial cell retraction induces the breakdown of
intercellular junctions and leads to an increase in vascular
permeability. Therefore, we measured the extent of endothelial cell
retraction induced by VEGF/VPF as the degree of permeability change of
[3H]inulin through the HBMEC monolayer. As expected,
VEGF/VPF meaningfully increased the permeability of the monolayer as
compared with the untreated control, and this effect was blocked by
VEGF/VPF monoclonal antibody and SU-1498 (Fig.
6). Furthermore, the increased vascular permeability caused by VEGF/VPF was abolished by BAPTA-AM but not by
PD98059 and Wortmannin (Fig. 6), indicating that calcium signaling
mediates the increased permeability induced by VEGF/VPF.
VEGF/VPF Induces Cytoskeletal Rearrangement of
HBMECs--
We then assessed the mechanism that leads to increased
endothelial cell permeability. One important regulatory mechanism for the integrity of endothelial cell junction maintenance is the distribution of actin to a cortical pattern, precluding stress fiber
formation. As shown in Fig.
7A, VEGF/VPF caused a marked redistribution of actin fibers that condensed toward the center of the
cell, with resulting stress fiber formation. The actin condensation in
the endothelial cells occurred within 15 min after VEGF/VPF treatment.
Actin redistribution induced by VEGF/VPF was substantially reversed by
co-incubation with VEGF/VPF monoclonal antibody and SU-1498 (data not
shown). These data indicated that VEGF/VPF was responsible for the
architectural change within the endothelial cell, leading to increased
vascular permeability. Because we found that calcium signaling
contributed to the increased permeability induced by VEGF/VPF, we
assessed its contribution to the redistribution of actin. We found that
BAPTA-AM potently blocked the effect of VEGF/VPF in stimulating actin
redistribution (Fig. 7A), suggesting a molecular mechanism
for the role of calcium in modulating the permeability of the HBMEC
monolayer.
We also examined adherens junction protein alignment at the HBMEC
monolayer (Fig. 7B). The specific endothelial adherens
junctional protein, VE-cadherin, has been shown to maintain and perhaps
regulate endothelial barrier properties (25). We found that the
VE-cadherin of the HBMEC monolayer was disrupted to a zigzag form
within 15 min after treatment with VEGF/VPF (Fig. 7B).
Furthermore, VE-cadherin was seen to be discontinuous under these
conditions, as indicated by the arrows. However, in the
presence of VEGF/VPF plus BAPTA-AM, VE-cadherin staining was
continuous, as indicated. By 120 min, VEGF/VPF induced gap formation at
the corner of the triendothelial cells, as indicated by the
arrows, whereas BAPTA-AM inhibited the VEGF/VPF-induced gap
formation at this corner (Fig. 7B). This indicates that
calcium signaling governs the disorganization of the endothelial
junction induced by VEGF/VPF. These data suggest that the
increased TM of MDA-MB-231 cells induced by VEGF/VPF occurs through the
loss of junctional proteins, with concomitant gap formation in the
endothelial monolayer, as shown in Fig. 7C.
VEGF/VPF Overexpression Increases the TM of
MDA- MB-231 Cells--
To examine whether endogenously secreting
VEGF/VPF modulates the TM of tumor cells, MDA-MB-231 cells were
infected with VEGF-Ad, and their TM was examined. As shown in Fig.
8A, VEGF-Ad-infected cells
expressed a high amount of VEGF/VPF as compared with the cells infected
with the CTL-Ad. Furthermore, VEGF-Ad-infected cells showed increased
TM as compared with the cells infected with CTL-Ad. The increased TM of
the VEGF-Ad-infected cells was significantly abolished in the presence
of VEGF/VPF monoclonal antibody but not control antibody (Fig.
8B). These data indicate that the endogenous expression of
VEGF/VPF modulates the TM of tumor cells.
A key event in cancer metastasis is the TM of tumor cells.
Metastasizing tumor cells should penetrate blood vessels twice, by
intravasation and extravasation. These tumor cells may possess an
offensive ability to penetrate blood vessels. It is well known that
increased endothelial cell retraction is closely associated with the
enhanced adhesion of tumor cells and their invasion into the
endothelial monolayer. Metastasizing tumor cells can induce endothelial
cell retraction through the secretion of soluble factors (26, 27)
and/or through direct adhesion to the endothelial monolayer where
intracellular signals are transduced to induce morphological changes
(28). For example, Honn et al. (26) reported that
12(S)-hydroxyeicosatetraenoic acid produced by tumor cells
induces retraction of endothelial cells and can enhance tumor cell
adhesion to the SEB membrane components of the exposed endothelial
monolayer. Kusama et al. (27) also reported that endothelial
cell retraction factor secreted by tumor cells increases the TM of
tumor cells through enhanced tumor cell adhesion.
In this report, we examined whether VEGF/VPF, which is expressed highly
in breast cancer cells, enhances the TM of MDA-MB-231 cells across a
monolayer of brain microvascular endothelial cells. VEGF/VPF has been
shown to potently induce the retraction of endothelial cells. As
expected, VEGF/VPF significantly increased the TM of MDA-MB-231 cells
across a monolayer of HBMECs. Our data indicate that the enhanced TM by
VEGF/VPF, at least in part, is derived from the increased adhesion of
these cells onto exposed SEB membrane components of the monolayer. The
mitogenic effect of VEGF/VPF on endothelial cells does not seem to be
related to the increased TM and adhesion of the MDA-MB-231 cells,
because bFGF and the MEK inhibitor PD98059 failed to stimulate the TM
and adhesion of these cells. bFGF has been shown to stimulate the
growth of endothelial cells through the ERK pathway as does VEGF/VPF
but does not increase vascular permeability (23). However, the
permeability effect of VEGF/VPF seems to be related to the increased TM
and adhesion of MDA-MB-231 cells, because inhibition of
VEGF/VPF-induced vascular permeability by the calcium chelator BAPTA-AM
prevented the TM and adhesion of these cells.
Calcium, as a multifunctional modulator, also regulates permeability in
the vascular system (29). Many inflammatory agents including histamine
and thrombin, as well as VEGF/VPF, are known to increase vascular
permeability through calcium up-regulation in endothelial cells
(30-32). Although the mechanism of how this transient increase in
calcium concentration alters vascular permeability has not been fully
elucidated, increased calcium in endothelial cells can induce the
activation of myosin light chain kinase, and phosphorylated myosin
light chain can contribute to the contraction of endothelial cells upon
the increased actin-myosin interaction (31). Our data, as well as other
studies, have shown that VEGF-induced actin rearrangement and gap
formation in interendothelial junctions result in increased
permeability (33) and that these effects were significantly blocked by
the intracellular calcium chelator BAPTA-AM (Fig. 7, A and
B) (33). Interestingly, after adhesion to the endothelial
monolayer, malignant tumor cells can transiently increase the calcium
concentration of endothelial cells in the contact area and stimulate
endothelial cell retraction by breaking the intercellular junctions
(28). In the TM of leukocytes, leukocytes migrate across the
endothelial monolayer by inducing an enhanced intracellular calcium
concentration of the endothelial cells (34-37), and pre-treatment of
the endothelial monolayer with BAPTA-AM potently blocked the TM of the
leukocytes (37). These studies suggest the possibility that cells
penetrating through blood vessels have the ability to elicit changes in
intracellular calcium concentration in endothelial cells.
VEGF/VPF may also enhance the TM of MDA-MB-231 cells through increased
adhesion onto the apical surface of endothelial cells, as well as onto
exposed SEB membrane components. Recently, Kim et al. (38,
39) reported that VEGF/VPF significantly increased the expression of
E-selectin in HUVEC. Endothelial E-selectin has been shown to mediate
increased adhesion onto an endothelial monolayer and the TM of some
tumor cells across the layer (40, 41). These studies suggest the
possibility that VEGF/VPF increases the TM and adhesion of MDA-MB-231
cells through up-regulation of adhesion molecules such as E-selectin in
endothelial cells. However, tumor cells have been reported to attach
with higher affinity to SEB membrane components than to the apical
surface of an intact endothelial monolayer (24). Thus, we suggest that the vascular permeability of VEGF/VPF might contribute predominantly to
the TM of tumor cells as compared with the up-regulation of endothelial
adhesion molecules by VEGF/VPF.
The hepatoprotective agent Malotilate intensifies the cell-to-cell
contact of endothelial cells and increases the integrity of the
endothelial monolayer (42, 43). Interestingly, this compound inhibits
significantly the in vitro TM and in vivo
metastasis of some carcinoma cells (42, 43). Furthermore, some peptides and antibodies that are able to block the adhesion of tumor cells to
SEB membrane components have exerted anti-metastatic effects (44-46).
Taken together, our data, as well as other studies, indicate that the
integrity of blood vessels and the progression of tumor metastasis are
closely related.
In addition to its vascular permeability activity in endothelial cells,
VEGF/VPF induces intracellular signaling-mediated proliferation and
invasion in VEGF/VPF receptor-expressing breast cancer cells (47, 48).
Thus, it is possible that exogenously added VEGF/VPF
affects the TM of breast cancer cells through the altered invasive
property of the tumor cells. However, this possibility can be excluded
for the following reasons. First, VEGF/VPF (or bFGF) was added evenly
to the apical and basolateral chambers to exclude the chemoattractant
effect of the added growth factor. Second, VEGF/VPF failed to
significantly increase the invasion of MDA-MB-231 cells across a
matrigel-coated polycarbonate filter (Fig. 1B). Third,
VEGF/VPF monoclonal antibody did not have any effect on the survival
and invasion of MDA-MB-231 cells (data not shown). Last, the expression
of KDR/Flk-1 in MDA-MB-231 cells was very weak as compared with its
expression level in endothelial cells (Fig. 1A).
In this study, we also examined whether endogenous expression of
VEGF/VPF modulates the TM of tumor cells. MDA-MB-231 cells were
infected with VEGF-Ad to overexpress VEGF/VPF, and then their TM was
examined. As expected, VEGF-Ad-infected cells showed increased TM as
compared with the cells infected with the CTL-Ad. Furthermore, the
increased TM of the VEGF-Ad-infected cells was significantly abolished
in the presence of VEGF/VPF monoclonal antibody but not control
antibody (Fig. 8B). These data indicate that the endogenous expression of VEGF/VPF modulates the TM of tumor cells.
Next, to reduce VEGF/VPF expression, we transfected MDA-MB-231 cells
with antisense VEGF/VPF cDNA and selected the stable MDA-MB-231
cell clones. Interestingly, antisense VEGF/VPF stable-transfected MDA-MB-231 cell clones showed reduced VEGF/VPF expression and increased
apoptosis as compared with the parental
cells.2 Similar to our
observations, the reduction in endogenous VEGF/VPF expression has been
reported to induce apoptosis in hematopoietic stem cells, although
exogenously added VEGF/VPF or soluble VEGF/VPF receptor did not exert
phenotypically any effects in the cells (49). Taken together, it is
possible that KDR/Flk-1 in hematopoietic stem cells and breast cancer
cells might be bound to the endogenous VEGF/VPF, leading to its
desensitization to VEGF when the exogenous VEGF/VPF is added. Thus, the
survival mechanism of VEGF/VPF in both cell types (hematopoietic stem
cells and breast cancer cells) might be different from that of VEGF/VPF
in the endothelial cells.
To date, VEGF/VPF has been shown to exert an essential role in tumor
angiogenesis. However, its role as a vascular permeability factor in
metastatic processes, such as TM, has not been defined. Melnyk et
al. (50) reported that neutralization of the function of VEGF/VPF
potently blocked tumor metastasis irrespective of its angiogenic
property. However, their study did not demonstrate the
inter-relationship between tumor metastasis and the functions of
VEGF/VPF in blood vessels. Thus, functional blocking of VEGF/VPF is a
potentially effective therapeutic approach to delay or prevent tumor
metastasis through the inhibition of multiple steps of tumor progression, such as neovascularization (angiogenesis) in tumor-bearing tissue and the TM of tumor cells. In conclusion, we report that VEGF/VPF modulates the TM of MDA-MB-231 breast cancer cells through the
down-regulation of brain microvascular endothelial cell permeability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
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Fig. 1.
KDR/Flk-1 expression in various breast cancer
cells and the effect of VEGF/VPF on the invasion of MDA-MB-231
cells. Panel A, the isolated mRNAs of the various
breast cancer cell lines (as indicated) were separated on an agarose
gel and transferred to a Hybond N membrane. The membrane was hybridized
with a probe for KDR/Flk-1, and after washing, the blot was exposed to
x-ray film. HUVEC were used as a positive control for the KDR/Flk-1
mRNA. Actin mRNA is shown as an internal control. Panel
B, MDA-MB-231 cells were trypsinized and suspended at 1.5 × 105 in 100 µl of DMEM containing 1% BSA. The cells were
added to the apical chamber of the matrigel-coated Transculture inserts
and then VEGF/VPF was added to the basolateral chambers containing 600 µl of DMEM with 1% BSA. After incubation for 6 h, the apical
chambers were stained with HEMA-3 solution, and the apical side of the
apical chamber was scraped gently with cotton wool. The migrating tumor
cells were observed under a light microscope and counted from 10 random
fields of ×200 magnification. The results are presented as the
mean ± S.D. of triplicate samples. CTL, control.
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Fig. 2.
VEGF/VPF increases the TM of MDA-MB-231 cells
across an HBMEC monolayer. HBMECs were added to
fibronectin-coated 24-well Transculture inserts with pore sizes of 8 µm (Costar Corp.) and grown for 5 days in 5% CO2 at
37 °C. 40,000 DiI-labeled MDA-MB-231 cells were added to the apical
chamber. To exclude the chemoattractant effect of the added growth
factor, bFGF or VEGF/VPF was added evenly to the apical and basolateral
chambers. After incubation for 6 h, the apical chamber was fixed
with 3.7% formaldehyde and washed extensively with PBS. The apical
side of the apical chamber was scraped gently with cotton wool. Only
the migrating tumor cells were observed by a fluorescent microscope and
counted from 10 random fields of ×200 magnification. The results are
presented as the mean ± S.D. of duplicate samples and are
representative of five individual studies. CTL,
control.
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Fig. 3.
VEGF/VPF increases the adhesion of MDA-MB-231
cells onto the HBMEC monolayer. HBMECs were added to attachment
factor-coated 24-well culture plates and grown for 5 days in 5%
CO2 at 37 °C. 100,000 DiI-labeled MDA-MB-231 cells in
500 µl of the same medium were added to each well with or without
test samples. After incubation for 2 h, the wells were fixed with
3.7% formaldehyde and washed extensively with PBS to remove floating
tumor cells. Attached tumor cells were observed by a fluorescent
microscope and counted from 10 random fields of ×200 magnification.
Alternatively, to detach the endothelial monolayer, the monolayers were
treated with 50 mM NH4OH solution for 5 min and
washed extensively with PBS before adding the DiI-labeled MDA-MB-231
cells. The results are presented as the mean ± S.D. of triplicate
samples. CTL, control; EC, endothelial
cells.
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Fig. 4.
BAPTA/AM inhibits the TM of MDA-MB-231 cells
across an HBMEC monolayer. HBMECs were added to fibronectin-coated
24-well Transculture inserts with pore sizes of 8 µm and grown for 5 days in 5% CO2 at 37 °C. The monolayers were
pre-treated for 30 min with the indicated synthetic inhibitors and
washed twice with culture medium. 40,000 DiI-labeled MDA-MB-231 cells
were added to the apical chamber. To exclude the chemoattractant effect
of the added growth factor, VEGF/VPF was added evenly to the apical and
basolateral chambers. After incubation for 6 h, the apical chamber
was fixed with 3.7% formaldehyde and washed extensively with PBS. The
apical side of the apical chamber was scraped gently with cotton wool.
Only the migrating tumor cells were observed by a fluorescent
microscope and counted from 10 random fields of ×200 magnification.
The results are presented as the mean ± S.D. of triplicate
samples. CTL, control; VEGF-Ab, VEGF/VPF
monoclonal antibody (20 µg/ml); SU, SU-1498 (50 µM); PD, PD98059 (10 µM);
WM, Wortmannin (1 µM); BAPTA,
BAPTA-AM (10 µM).
View larger version (23K):
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Fig. 5.
BAPTA/AM inhibits the adhesion of MDA-MB-231
cells onto the HBMEC monolayer. HBMECs were added to attachment
factor-coated 24-well culture plates and grown for 5 days in 5%
CO2 at 37 °C. The monolayers were pre-treated for 30 min
with the indicated inhibitors, and all synthetic inhibitors except
VEGF/VPF monoclonal antibody were removed from the monolayers by
washing twice with culture medium. 100,000 DiI-labeled MDA-MB-231 cells
in 500 µl of the same medium were added to each well with or without
VEGF/VPF. After incubation for 2 h, the monolayers were fixed with
3.7% formaldehyde and washed extensively with PBS to remove floating
tumor cells. Attached tumor cells were observed by a fluorescent
microscope and counted from 10 random fields of ×200 magnification.
The results are presented as the mean ± S.D. of triplicate
samples. CTL, control; VEGF-Ab, VEGF/VPF
monoclonal antibody (20 µg/ml); SU, SU-1498 (50 µM); PD, PD98059 (10 µM);
WM, Wortmannin (1 µM); BAPTA,
BAPTA-AM (10 µM).
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Fig. 6.
VEGF/VPF increases the permeability of the
HBMEC monolayer. Approximately 100,000 HBMECs were added to
fibronectin-coated 24-well Transculture inserts with pore sizes of 0.4 µm and grown for 5 days in 5% CO2 at 37 °C. After the
removal of culture medium, 0.4 ml of the fresh culture medium
containing [3H]inulin (1 µCi) was added to the apical
chamber. The basolateral chamber was filled with 0.6 ml of the same
medium without [3H]inulin and then VEGF/VPF (30 ng/ml)
was added to the apical and basolateral chambers. Prior to the addition
of VEGF/VPF, the monolayers were pre-treated for 30 min with various
inhibitors. After the incubation for 2 h, 30 ml of medium from the
basolateral chamber were collected, and the amount of
[3H]inulin across the monolayers was determined by
scintillation counting. The data represent the mean values of total cpm
of three separate experiments. CTL, control;
VEGF-Ab, VEGF/VPF antibody (20 µg/ml); SU,
SU-1498 (50 µM); PD, PD98059 (10 µM); WM, Wortmannin (1 µM);
BAPTA, BAPTA-AM (10 µM).
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[in a new window]
Fig. 7.
VEGF/VPF induces actin redistribution and
VE-cadherin disruption in HBMECs. Panel A, HBMECs were
added to fibronectin-coated 24-well Transculture inserts with pore
sizes of 0.4 µm and grown for 5 days in 5% CO2 at
37 °C. After assaying for 2 h, the HBMEC monolayer was fixed
with 3.7% formaldehyde in PBS and then permeabilized with 0.5% Triton
X-100. The F-actin in the cells was stained according to the method
described under "Experimental Procedures" and observed by a
fluorescent microscope. Panel B, after assaying for the
indicated times, the HBMEC monolayer was fixed with 3.7% formaldehyde
in PBS and then permeabilized with 0.5% Triton X-100. The VE-cadherin
of the cells was stained according to the method described under
"Experimental Procedures" and viewed under a confocal microscope.
Panel C, 1,000 BCECF-AM-labeled MDA-MB-231 cells were added
to each HBMEC monolayer with or without VEGF/VPF. After incubation for
2 h, the cells were fixed with 3.7% formaldehyde in PBS.
VE-cadherin in the HBMECs was stained as described under
"Experimental Procedures" and viewed under a confocal microscope.
MDA-MB-231 cells are visualized by green color, whereas
VE-cadherin is viewed as red color, respectively.
CTL, control; VEGF/VPF, 30 ng/ml;
BAPTA-AM, 10 µM.
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Fig. 8.
Overexpression of VEGF/VPF increased the TM
of MDA-MB-231 cells. Panel A, MDA-MB-231 cells were
infected with VEGF-Ad or CTL-Ad for 48 h. The VEGF/VPF expression
of these cells was analyzed by Western blotting using an anti-VEGF/VPF
polyclonal antibody (Santa Cruz Biotechnology, Inc.). Total protein
extracts were analyzed by Western blotting using anti-Csk antibody as
an internal control. Panel B, after labeling with DiI,
adenovirus-infected MDA-MB-231 cells were dispersed in trypsin solution
and resuspended in CSC medium without growth factor. 5,000 DiI-labeled
cells were treated with VEGF/VPF monoclonal antibody and were then
added to the apical chamber containing the HBMEC monolayer. After
incubation for 18 h, the apical chambers were fixed with 3.7%
formaldehyde, and the migrating tumor cells were observed as described
under "Experimental Procedures." The results are presented as the
mean ± S.D. of triplicate samples. rh-VEGF,
recombinant human VEGF/VPF (25 ng); VEGF-Ab, VEGF/VPF
monoclonal antibody (20 µg/ml); CTL-IgG, control mouse
IgG; WB, Western blotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Harold Dvorak for providing the adenoviral construct encoding VEGF/VPF and Dr. Michael Klagsbrun for providing the KDR/Flk-1 probe. We also thank Dr. Tae Kim for valuable advice during this study, Janet Delahanty for editing, Daniel Kelley for preparation of the figures, and Mikyung Kim-Park for typing the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HL55445 (to S. A.), CA87200 (to H. K. A.), DAMD17-99-1-9078 (to H. K. A.), CA76226 (to H. K. A.), Experienced Breast Cancer Research Grant 34080057089 (to H. K. A.), a Massachusetts Department of Public Health grant (to H. K. A.), the Milheim Foundation (to S. A.), a Claudia Sargent Breast Cancer fellowship, and a Diana Michelis fellowship (to T.-H. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Ronald Ansin and Charlene Engelhard for continuing friendship and support for our research program.
Established investigator of the American Heart Association.
§ To whom correspondence should be addressed: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Inst. of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0063; Fax: 617-975-6373; E-mail: savraham@caregroup.harvard.edu.
Published, JBC Papers in Press, November 20, 2002, DOI 10.1074/jbc.M210063200
2 T.-H. Lee, H. K. Avraham, S. Jiang, and S. Avraham, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: BBB, blood-brain barrier; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; HBMEC, human brain microvascular endothelial cell; HUVEC, human umbilical vein endothelial cell; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; TM, transendothelial migration; VEGF, vascular endothelial growth factor; VPF, vascular permeability factor; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; VEGF-Ad, adenovirus-encoding VEGF/VPF; CTL-Ad, control adenovirus; SEB, sub-endothelial basement; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboxyanine perchlorate; BCEGF-AM, 2,7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein acetoxymethylester; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester.
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