From the Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, California 94080
Received for publication, August 31, 2000
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ABSTRACT |
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Vascular endothelial growth factor (VEGF)
and tumor necrosis factor- Vascular endothelial growth factor
(VEGF)1 is a potent
endothelial cell (EC)-specific mitogen that promotes the proliferation and migration of EC, remodeling of the extracellular matrix, formation of capillary tubules, and vascular leakage (1-6). As a key angiogenic growth factor, VEGF plays a critical role in the development of the
fetal cardiovascular system, as well as a significant role in the
physiological and pathological angiogenesis (7-10). Although it is
well accepted that Flt-1 and KDR are both high affinity receptors for
VEGF, it is not clear which receptor activates the downstream signaling
pathways responsible for the diverse biological responses of VEGF.
Transgenic knockout studies in mice showed that both receptors are
essential for animal survival, because mouse embryos null for KDR or
Flt-1 die in utero (11-12). Experiments using
receptor-specific binding variants or receptor-specific inhibitors have
associated KDR receptor activity with EC proliferation, migration,
vascular permeability, cell survival, and angiogenesis (13-17). The
role of Flt-1 receptor signaling in VEGF biology is less clear. Studies
indicate that Flt-1 may mediate, at least in part, chemotaxis and
procoagulatant activity in macrophages and up-regulation of matrix
metalloproteinases in vascular smooth muscle cells (18-19). Upon VEGF
stimulation, KDR receptor is strongly tyrosine-phosphorylated, but
little or no autophosphorylation of Flt-1 occurs (19-21). Rahimi
et al. (19) recently proposed that KDR activation plays a
dominant role in angiogenesis by promoting EC proliferation, whereas
Flt-1 binding plays a stationary role by antagonizing the interaction
of VEGF with KDR. A novel VEGF receptor with a sequence identical to
that of neuropilin was recently identified by Soker et al.
(22). This receptor binds to VEGF via an interaction with C-terminal
heparin binding domain. The role of neuropilin in VEGF signaling is
currently under active investigation (22-24).
VEGF has previously been demonstrated to regulate tissue factor (TF)
expression in monocytes and ECs (25-27). Although VEGF alone induced
only a moderate increase in TF expression, it significantly enhanced
TNF- Materials--
Recombinant human VEGF165
(rhVEGF165) was produced in Escherichia
coli (Genentech, Inc., South San Francisco, CA). The
VEGF109, an heparin binding domain (HBD)-deficient variant,
was prepared as described previously (28). The VEGF receptor-selective
variants were prepared as described by Li et al. (14).
Briefly, a Flt-1-selective variant (Flt-sel) was created
based on a comprehensive mutational analysis of the receptor binding
site of VEGF. This variant contains four substitutions (Ile-43
Cell Culture and Drug Treatment--
HUVEC were purchased from
Cell Systems (Kirkland, WA) and maintained as instructed by the
manufacturer. Briefly, cells were first grown in a T-75 flask. When
HUVEC cells reached 70-80% confluency, they were subcultured onto
6-well tissue culture plates for Western blot (WB) analysis and
fluorescence-activated cell sorting (FACS). All flasks and culture
plates were precoated with an attachment factor provided by the
manufacturer. HUVEC cells were used between passages 3 and 8. Fresh
medium was replaced every 24 h. For drug treatment, subconfluent
cells were switched to a GF/serum-free medium overnight and then
treated with VEGF or other drugs as specified.
Western Blot Detection of Tissue Factor--
The methods for
cell lysis and WB are described in detail elsewhere (30). Briefly,
cells were lysed in a buffer containing 50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1%
sodium deoxycholate, 1 mM sodium vanadate, 50 mM sodium fluoride, 2 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin/pepstatin A/aprotinin for 15 min on ice. Cell lysates were
clarified by centrifugation. Protein concentration in the supernatants
was determined using a BCA assay (Pierce). An equal amount of protein
was denatured and separated using SDS polyacrylamide gel
electrophoresis (Novex, San Diego, CA), transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and blocked in 5% milk
overnight. A monoclonal anti-TF antibody (3D1) was used at a 1:2500
dilution to probe TF protein. A secondary antibody conjugated with
horseradish peroxidase (Zymed Laboratories Inc., South
San Francisco, CA) and enhanced chemiluminescent kit (Amersham Phamacia Biotech) were used to visualize the TF immunoreactive bands. Multiple exposures of films were obtained to determine the optimal exposure time. In general, the blots for samples that were treated in the presence of TNF- FACS Analysis--
The treated HUVEC cells were washed with a
FACS buffer (phosphate-buffered saline, 0.5% bovine serum albumin) and
dissociated with versene (Life Technologies, Inc.) or Accutase
(Innovative Cell Technologies, La Jolla, CA) for 10 min at 37 °C,
harvested, pelleted at 1050 rpm for 5 min, and then resuspended in cold
FACS buffer (0.1% bovine serum albumin in phosphate-buffered saline, filtered) at 3-5 million cells/ml. Cells (0.3-0.5 million) were incubated with 167 nM of 3D1 or anti-GP120 for 1 h at
4 °C on a shaker. When the incubation was complete, cells were
washed twice with FACS buffer and further incubated with 100 nM fluorescein isothiocyanate goat anti-mouse IgG for
1 h at 4 °C on a shaker. Cells were washed and resuspended in
500 µl of FACS buffer for FACS analysis.
FACS analysis was carried out with a FACS Calibur flow cytometer
(Becton Dickinson, Immunocytometry Systems, San Jose, CA). Data were
collected in FL-1 with a 530-nm band pass filter. For each
analysis, 10,000 events were recorded. The data were gated on the main
population identified on the forward scatter versus side
scatter dot plot (80-85% of total events) and analyzed with CellQuest
software (Becton Dickinson, Immunocytometry Systems, San Jose,
CA) for relative fluorescence intensity.
Statistics Analysis--
The results are reported as means ± S.D. Statistical analysis was conducted following comparison of
results from the VEGF variants in the absence of TNF to the TNF minus
control (no VEGF or TNF); similarly, results obtained in the presence
of TNF/VEGF variants were compared with the TNF plus control (with TNF
but no VEGF). An unpaired Student's t test was used to
determine significant differences. A value of p < 0.05 was considered significant.
To investigate the effect of VEGF on TF expression and whether
VEGF interacts with TNF in regulating TF expression, subconfluent HUVEC
cells were treated with VEGF, TNF- (TNF-
) have been shown to
synergistically increase tissue factor (TF) expression in endothelial
cells; however, the role of the VEGF receptors (KDR, Flt-1, and
neuropilin) in this process is unclear. Here we report that VEGF
binding to the KDR receptor is necessary and sufficient for the
potentiation of TNF-induced TF expression in human umbilical vein
endothelial cells. TF expression was evaluated by Western blot analysis
and fluorescence-activated cell sorting. In the absence of TNF-
,
wild-type VEGF- or KDR receptor-selective variants induced an
approximate 7-fold increase in total TF expression. Treatment with TNF
alone produced an approximate 110-fold increase in total TF expression,
whereas coincubation of TNF-
with wild-type VEGF- or KDR-selective
variants resulted in an approximate 250-fold increase in TF expression.
VEGF lacking the heparin binding domain was also able to potentiate TF
expression, indicating that heparin-sulfate proteoglycan or neuropilin
binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF
expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity
significantly blocked the TNF/VEGF potentiation of TF up-regulation,
whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced TF up-regulation through a synergistic mechanism in EC
(26-27). The role that the two TNF receptors, TNFR60 and TNFR80, play
in the synergy between VEGF and TNF has been studied. Clauss et
al. (27) reported that stimulation of the 60-kDa TNF receptor by a
mutant of TNF specific for TNFR60 induced a TF up-regulation comparable
with wild-type TNF. In contrast, stimulation of TNFR80 by a
TNFR80-specific TNF mutant did not enhance TF expression. Thus, TNFR60
is the principle receptor involved in the synergistic up-regulation of
TF induced by TNF and VEGF. The role that the VEGF receptors, KDR and
Flt-1, play in this process is unknown. Therefore, in the present study
we have investigated the role of KDR and Flt-1 receptor signaling in TF
up-regulation and in its synergy with TNF by using VEGF receptor-selective variants. VEGF and TNF work in concert to
synergistically up-regulate TF expression in human umbilical vein
endothelial (HUVEC) cells. Here we demonstrate that KDR receptor
signaling is necessary and sufficient for the potentiation of
TNF-induced TF up-regulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ala, Ile-46
Ala, Gln-79
Ala, and Ile-83
Ala) from the
wild-type protein and is heparin binding domain-deficient.
Flt-sel binds to Flt-1 with wild-type affinity and has
470-fold reduced affinity for KDR binding. In addition, Flt-sel had no effect on autophosphorylation of KDR but
significantly induced the secretion of matrix metalloprotease-9 in
human aorta smooth muscle cells that express Flt-1 only. Two
KDR-selective variants (KDR-sel1 and KDR-sel2)
were created by using a competitive phage display strategy. The
KDR-sel1 has the HBD, whereas KDR-sel2 is
HBD-deficient. These variants have approximately wild-type affinity for
KDR but 2000-fold reduced affinity for binding to Flt-1. KDR variants
showed activity comparable with the wild-type VEGF in KDR
autophosphorylation and EC proliferation assays but had no effect on
the secretion of matrix metalloprotease-9. The heterodimeric form of
recombinant human hepatocyte growth factor (HGF) was produced in and
isolated from Chinese hamster ovary cells as described previously (29).
Recombinant human TNF-
, fibroblast growth factor basic (FGF),
transforming growth factor (TGF)-
1, TGF-
2, and epidermal growth
factor (EGF) were purchased from R & D Systems (Minneapolis, MN).
TNF-
and anti-TF antibody (3D1) were from Genentech (South San
Francisco, CA). Genistein, staurosporine, phorbol 12-myristate
13-acetate (PMA), PD98059, PP1, and wortmannin were purchased from
BIOMOL (Plymouth Meeting, PA). All other chemicals were purchased from
Sigma. All reagents were prepared as 1000× stock solutions
unless otherwise specified.
were exposed to x-ray films for ~2-5 min,
whereas the blots for samples that were treated in the absence of
TNF-
were exposed to x-ray films for ~30-50 min because of lower
TF signal. The protein bands were scanned by a densitometer, and the
relative intensities were quantified using ImageQuant software (Molecular Dynamics). The relative TF expression was always normalized to controls (no VEGF or TNF) on the same blot with the same exposure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, or a combination of VEGF and
TNF-
for 0 to 9 h. The VEGF concentration was selected based on
a dose-response study that showed that 1000 pM of
VEGF induced a maximal increase in TF expression (data not shown). The
optimal TNF-
concentration (5 ng/ml) was selected based on previously reported studies (26-27). Total TF expression in whole cell
lysates was evaluated by WB using a specific anti-human TF antibody. In
the absence of TNF-
, VEGF induced a modest
time-dependent increase in TF expression (Fig.
1, A and B). TF
up-regulation was noted at 3 h and reached a peak (7-fold over
control) at 6 h post-VEGF exposure. TNF-
treatment resulted in
a more rapid and greater increase in TF expression relative to VEGF.
The maximal TF expression (110-fold) was observed at 3 h
post-TNF-
exposure (Fig. 1, A and B).
Coincubation of VEGF with TNF-
produced an even more striking
increase in total TF expression. The maximum 270-fold increase occurred
at 3 h post-exposure to both agonists (Fig. 1, A and
B). The TF up-regulation persisted for at least 9 h. We
also investigated the effect of TNF-
, another member of TNF family
cytokine, on TF expression in this cell culture model. As shown in Fig.
1, C and D, treatment with TNF-
alone also
resulted in a time-dependent increase in TF expression,
although to a lesser degree than TNF-
(35-fold for TNF-
versus 110-fold TNF-
). When VEGF was coincubated with
TNF-
, a maximal of 130-fold increase in TF expression was
observed at 9 h post-exposure (Fig. 1, C and
D). Taken together, these data suggest that VEGF is capable of interacting with either TNF-
or TNF-
in a synergistic manner to increase TF expression.
View larger version (17K):
[in a new window]
Fig. 1.
VEGF potentiates
TNF- - and
TNF-
-induced TF expression. Subconfluent
HUVEC cells were starved in a serum/GF-free medium overnight and then
treated with VEGF (1000 pM), TNF-
(5 ng/ml), TNF-
(5 ng/ml), or a combination of VEGF and TNF-
, VEGF, and TNF-
for 0, 3, 6, and 9 h. TF expression in whole cells lysates was evaluated
by WB as described under "Experimental Procedures." A,
representative WB of cell lysates following treatment with VEGF,
TNF-
alone, or the combination of VEGF and TNF. B,
densitometeric quantification of WB from three independent experiments.
C, representative WB of cell lysates following treatment
with VEGF, TNF-
, or VEGF + TNF-
. D, densitometeric quantification of WB
from three independent experiments. -Fold increase of TF expression in
treated groups was normalized to control cells that were not exposed to
VEGF or TNF. Data shown are means ± S.D.
To elucidate the role of VEGF receptors in the synergistic
up-regulation of TF induced by VEGF and TNF concomitant treatment, we
treated HUVEC cells with equimolar concentrations of wild-type VEGF-,
KDR-, or Flt-1 receptor-selective variants in the presence or absence
of TNF- for 6 h. The VEGF HBD-deficient variants
(VEGF109, KDR-sel2) were also used to address
the role of the HBD in TF up-regulation. Fig.
2, A and B shows a
representative WB result and quantification by densitometry of three
independent experiments. In the absence of TNF-
, KDR-selective
variants induced an increase in total TF expression similar to that
induced by wild-type VEGF (both ~7-fold); in the presence of TNF-
,
a more pronounced increase (~250-fold) in total TF expression was
induced by wild-type VEGF- and KDR-selective variants (Fig. 2,
A and B). In contrast, the Flt-1-selective
variant had no effect on TF expression in either the presence or
absence of TNF-
(Fig. 2, A and B). The HBD did not appear to play a significant role in TF up-regulation, as VEGF
variants without the HBD (VEGF109 and KDR-sel2)
induced an increase in TF expression similar to that induced by
VEGF variants with the HBD (VEGF165 and
KDR-sel1) (Fig. 2).
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FACS analysis was used to confirm these observations and to further
characterize the subcellular localization of the newly synthesized TF.
In the absence of TNF-, KDR receptor variants induced an increase in
cell surface TF expression similar to that induced by wild-type VEGF
(~3-fold) (Fig. 3), and the
Flt-1-selective variant had no significant effect (Fig. 3). In a
separate experiment, treatment with TNF-
alone induced an ~10-fold
maximal increase in TF fluorescence density (data not shown). When
coincubated with TNF-
, wild-type VEGF- and KDR-selective variants
both induced an ~20-fold maximal increase in the cell surface TF
expression (Fig. 3). In contrast, Flt-selective variant did not
potentiate TNF-
-induced TF expression on the cell surface (Fig. 3).
Once again, the HBD did not seem to play a role in up-regulating cell surface TF expression as VEGF variants without HBD (VEGF109
and KDR-sel2) induced a similar increase in TF expression
as VEGF variants with HBD (VEGF165 and
KDR-sel1) (Fig. 3).
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To further address the role of the downstream signaling pathway
following KDR binding and activation in TF up-regulation, several
inhibitors to tyrosine kinases, Src kinase, PKC, or
phosphatidylinositol 3-kinase (PI3K) were coincubated with VEGF
and TNF. Fig. 4 shows that inhibition of
protein-tyrosine kinase (PTK) with genistein, a potent tyrosine kinase
inhibitor, significantly blocked VEGF/TNF-induced TF up-regulation.
Similarly, inhibition of PKC with staurosporine also blocked
VEGF/TNF-induced TF up-regulation. In contrast, treatment of HUVECs
with only PMA, a PKC activator, resulted in an increase in TF
expression similar to that induced by VEGF/TNF. Inhibition of
phospholipase C- with PP1 or inhibition of mitogen-activated protein kinase with PD-98059 only partially blocked VEGF/TNF-induced TF
up-regulation. Meanwhile, inhibition of PI3K with wortmannin resulted
in an increase in TF expression. These data suggest that both PTK and
PKC are involved in VEGF/TNF-induced TF up-regulation and that PI3K may
negatively regulate TF expression in ECs.
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The effect of VEGF on TF expression was also compared with several
other GFs including FGF, HGF, EGF, TGF-1, and TGF-
2. HUVEC cells
were treated with equimolar concentrations of VEGF, FGF, HGF, EGF,
or TGF-
in either the presence or absence of TNF-
for
6 h. Fig. 5 shows that only VEGF
treatment increased TF expression and potentiated TNF-
-induced TF
up-regulation, whereas FGF, HGF, EGF, or TGF had little or no
significant effect.
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DISCUSSION |
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Tissue factor is a 47-kDa transmembrane glycoprotein that plays an important role in coagulation by serving as a cofactor for coagulation factor VII activation. It is also involved in early wound healing, angiogenesis, and tumor metastasis (31-33). Normally, TF is not expressed on the surface of cells that are in direct contact with blood such as the endothelium lining vessels and circulating monocytes; however, growth factors and cytokines may induce TF expression in these cells (25-27). HUVEC cells were selected as our model system, because they have been shown to express both KDR and Flt-1 receptors and have been previously used to assess TF regulation (26-27). VEGF and TNF have been shown to synergistically up-regulate TF expression in endothelial cells, but the role of VEGF receptors in this synergy has not been studied. Therefore, we have 1) investigated the role of the VEGF receptor(s) in TF up-regulation by using VEGF receptor-selective binding variants; 2) determined the contribution of PTK, PKC, and PI3K to VEGF/TNF-induced TF up-regulation; and 3) evaluated the specificity for the effect by comparing the ability of VEGF with various other GFs to up-regulate TF. Our data demonstrate that wild-type VEGF- or KDR-selective variants alone induce a modest increase in both total and cell surface TF expression relative to TNF. In the presence of TNF, wild-type VEGF- and KDR-selective variants both significantly potentiate TNF-induced TF up-regulation. Furthermore, we demonstrate that PTK and PKC signaling pathways are required for this effect. In comparison with other angiogenic growth factors, VEGF is unique in its ability to induce TF up-regulation and synergistically interact with TNF to produce TF.
TNF- and TNF-
are structurally similar, but TNF-
binds to the
TNFR60 receptor with higher affinity (31). Our data are consistent with
this observation in that VEGF/TNF-
induced a higher TF expression
relative to VEGF/TNF-
. Previously, Clauss et al. (27) and
Camera et al. (26) reported that VEGF or TNF-
alone had
little effect on TF expression in HUVEC, but cells treated with both
agonists showed a significant increase in TF expression. Our
observations are consistent with these previously published reports in
that VEGF synergistically up-regulates TNF-induced TF expression;
however, we were able to demonstrate an increase in TF expression with
either VEGF or TNF alone.
It is well accepted that VEGF exerts its biological effects through the
two high affinity receptor tyrosine kinases, KDR and Flt-1,
predominately expressed on the vascular endothelial cells (2, 11).
However, it is unclear which VEGF receptor(s) mediates the synergy
between VEGF and TNF with respect to TF up-regulation. To address this
question, we have used VEGF receptor-selective variants recently
developed by Li et al. (14). These receptor-selective binding variants have been demonstrated to be highly selective and
bioactive (14). Treatment of HUVEC cells with KDR-selective variants
resulted in an increase in both total and cell surface TF expression
similar to that induced by wild-type VEGF and significantly enhanced
TNF--induced TF up-regulation (Figs. 2 and 3), whereas the
Flt-1-selective binding variant had no significant effect on TF
expression in either the presence or absence of TNF (Figs. 2 and 3).
The dose (1000 pM) of KDR-selective variants used in the
study was about 5-10-fold higher than the Kd for KDR but well below the Kd for Flt-1 considering the
2000-fold affinity reduction for Flt-1 binding. The dose (1000 pM) of Flt-1-selective variant was about 50-fold higher
than the Kd for Flt-1, yet still well below the
Kd of the KDR receptor. The HBD did not play a
significant role in mediating TF up-regulation as VEGF variants without
the HBD (VEGF109 and KDR-sel2) induced an
increase in TF expression similar to that induced by VEGF variants with
HBD (Figs. 2 and 3). These results rule out the requirement for heparin
sulfate proteoglycans or neuropilin binding in TF up-regulation. Taken
together, our data strongly indicate that the KDR receptor signaling
mediates TF up-regulation and governs the synergy between VEGF and
TNF.
Consistent with this observation, Meyer et al. (32) reported that VEGF-E, a novel VEGF encoded by Orf virus and bound with high affinity to KDR but not to Flt-1, induced the production of TF in HUVEC cells. Others have reported that the Flt-1 receptor may also play a role in TF up-regulation in monocytes and ECs (25, 33). Addition of placental growth factor, which binds to Flt-1 but not to KDR, resulted in a relatively small increase in TF expression in monocytes and ECs (25). In the present study, we were unable to demonstrate any increase in TF expression with the Flt-1-selective variant or placental growth factor in HUVEC cells (data not shown). Thus, our data strongly imply that KDR is the dominant receptor signaling governing TF up-regulation and mediating the synergy between VEGF and TNF in EC. Our observation adds to the growing list of actions attributed to KDR receptor signaling, which includes cell proliferation, endothelial nitric oxide synthase, and KDR up-regulation, cell survival, and vascular permeability (13, 16, 30, 34-36).
Many studies have shown the importance of protein-tyrosine kinases and
PKC in VEGF signaling (21, 30, 34-35). KDR itself and many downstream
molecules are protein-tyrosine kinases or regulated by tyrosine kinase
phosphorylation (21). To address the role of these signaling molecules
in VEGF/TNF-induced TF up-regulation, cells were treated in medium
containing VEGF/TNF, as well as inhibitors to PTK, PKC, PI3K,
phospholipase C-, or mitogen-activated protein kinase. As expected,
inhibition of PTK with genistein significantly blocked the synergistic
up-regulation of TF. Similarly, inhibition of PKC with staurosporine
also significantly attenuated the synergistic up-regulation of TF.
Stimulation with PMA alone resulted in a striking increase in TF
expression, indicating that PKC plays a central role in
VEGF/TNF-induced up-regulation of TF. Previously, several isoforms of
PKC such as PKC-
, -
, -
, -
, and -
have been shown to be
activated by VEGF stimulation (34, 37-38). Because TNF has also been
shown to activate PKC (39), the synergistic up-regulation of TF induced
by VEGF and TNF may occur at the level of PKC. More studies will be
required to elucidate the specific role of PKC isoform(s) in TF
up-regulation. Inhibition of Src kinase and
mitogen-activated protein kinase partially blocked TF up-regulation,
indicating that these signaling molecules may also contribute to the
signaling mechanism involved in TF up-regulation. Interestingly, we
found that inhibition of PI3K with wortmannin resulted in an even
higher induction of TF expression than VEGF/TNF, suggesting that PI3K
may negatively regulate TF production.
Other mechanisms may also contribute to the synergy between TNF and
VEGF. For example, TNF- has been reported to modulate VEGF action by
promoting VEGF production and regulating KDR and neuropilin-1 receptor
expression (40-42). It is unlikely that TNF-induced expression of VEGF
contributed to the TF up-regulation in our model system as endothelial
cells express little or no VEGF, and saturating concentrations of
exogenous VEGF (1000 pM) were used in the study. It is also
unlikely that TNF-induced KDR expression augmented the biological
response as it took ~24 h for TNF to significantly increase KDR
expression (40), and we observed TF expression occurring as early as
3 h post-VEGF treatment.
Up-regulation of TF induced by the interplay between VEGF and TNF may
be critical in physiological angiogenesis, as well as tumor
angiogenesis and metastasis (43-47). TNF- has been found to
cooperate with VEGF and other GFs to induce capillary-like tubular
structure of human microvascular endothelial cell growth in a
three-dimensional gel of extracellular matrix proteins (47). Increased
TF expression may facilitate cell migration, an important step in tube
formation (48). In addition to its role in angiogenesis, TF may also
play a role in cancer progression, atherosclerosis, and thrombotic
complications (43, 49-52). Both VEGF and TNF levels have been found to
be increased under these pathophysiological conditions (53-54). The
elevated TNF and VEGF levels in these conditions may contribute to the
increased TF expression in vivo, resulting in increased
angiogenesis, thrombosis, and tumor metastasis.
In summary, we have investigated the role of VEGF receptors in
mediating the synergistic up-regulation of TF induced by VEGF and TNF
in human endothelial cells. Our data indicate that KDR receptor
signaling governs the synergistic up-regulation of TF induced by VEGF
and TNF. These data provide new insights into the role of the KDR
receptor in mediating both physiological and pathophysiological processes.
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ACKNOWLEDGEMENT |
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We thank Bing Li and Abraham M. de Vos for providing VEGF-selective variants, Glibert Keller for critical review of the manuscript, Julie Snider for skillful editorial assistance, and PeterHaughney for providing excellent tissue culture support.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of
Pharmacokinetics and Metabolism, Genentech, Inc., MS #70, 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-3269; Fax: 650-225-6452; E-mail: zion@gene.com.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M007969200
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; GF, growth factor; TNF, tumor necrosis factor; TF, tissue factor; EC(s), endothelial cell(s); HUVEC, human umbilical vein endothelial cell; HBD, heparin binding domain; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; FACS, fluorescence-activated cell sorting; PTK, protein-tyrosine kinase; TNFR, TNF receptor; HGF, hepatocyte growth factor; FGF, fibroblast growth factor basic; TGF, transforming growth factor; EGF, epidermal growth factor; WB, Western blot.
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