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INTRODUCTION |
The extrinsic pathway of blood coagulation is initiated when
factor VII (fVII)1 binds to
its cellular receptor, tissue factor (TF). The fVIIa·TF complex then
functions as a potent enzyme, activating factor X which leads to
thrombin generation. In addition to initiating coagulation, recent
research suggests that fVIIa·TF complexes play an important role in
angiogenesis (1-3). Zhang et al. (1) correlated TF
expression in tumor cells with the ability of the tumors to secrete
vascular endothelial growth factor (VEGF) and, in turn, to induce an
angiogenic response when implanted in immunodeficient mice. Although TF
is not normally expressed on the surface of vascular endothelial cells,
in situ hybridization studies have detected TF mRNA in
tumor-associated endothelial cells from patients with invasive breast
cancer (2). Finally, in a TF-dependent metastasis model,
the binding and proteolytic activity of VIIa was shown to be
necessary for the initial steps of tumor metastasis (3).
The tissue factor pathway is regulated by a potent inhibitor termed
tissue factor pathway inhibitor (TFPI). TFPI forms a tight complex with
both fXa and fVIIa leading to their inhibition. The TFPI molecule
contains three Kunitz-type domains and a basic carboxyl-terminal region. By employing site-directed mutagenesis, Girard et
al. (4) demonstrated that the second Kunitz domain is required for
efficient binding and inhibition of fXa, and both Kunitz domains 1 and
2 are required for the inhibition of fVIIa/TF activity. Mutation of the
predicted inhibitory residues of the third Kunitz domain had no
significant effect on either function of TFPI. Rather, this portion of
the molecule binds to the cell surfaces by interacting with
cell-surface glycosaminoglycans (5). TFPI can also bind to the low
density lipoprotein receptor-related protein (LRP) (6) and, in doing
so, mediates the permanent down-regulation of cell-surface TF in
monocytic cells via LRP-dependent internalization and
degradation (7).
LRP is a member of the LDL receptor family, which includes the LDL
receptor, LRP-1b, gp330/megalin, apoER2, and the very low density
lipoprotein receptor (VLDL receptor). LRP is widely expressed in
fibroblasts, macrophages, smooth muscle cells, neurons, and activated
glial cells (8) but does not appear to be highly expressed in vascular
endothelium (9). In contrast, VLDL receptor is expressed in HUVECs, in
the endothelium of capillaries and small arterioles (10), and on the
endothelial surface of bovine arteries (11). This receptor was
originally identified by homology cloning (12) and initially was
thought to function in the delivery of triglyceride-rich lipoproteins
to peripheral tissues (12, 13). However, studies in homozygous knockout
mice that lack the VLDL receptor gene revealed that the animals had
normal levels of plasma triglyceride and cholesterol indicating that in
mice the VLDL receptor is not likely a major receptor involved in
lipoprotein catabolism (14).
Since fVIIa·TF complexes seem to play a role in the pathological
proliferation of endothelial cells, we sought to determine whether
TFPI, the natural inhibitor of fVIIa/TF, was capable of regulating this
process. Our initial studies focused on determining the effect of TFPI
on basic fibroblast growth factor (bFGF)-stimulated endothelial cell
growth. We report here that TFPI is a potent inhibitor of the growth of
bFGF-stimulated endothelial cells. Furthermore, we show that the
antiproliferative activity of TFPI is independent of its fVIIa/TF
inhibitory activity but rather results from association with the VLDL receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
Human umbilical vein endothelial cells (HUVECs),
human microvascular dermal endothelial cells, media for their
proliferation (Endothelial Cell Basal Medium (EBM), and Endothelial
Cell Growth Medium (EGM) were purchased from Clonetics (San Diego, CA).
Rat C6 glioma cells were obtained from ATCC (Manassas, VA). B16BL6 melanoma cell line is a variant cell line derived from B16F10 melanoma
cells obtained from the National Cancer Institute-Central Repository
(Frederick, MD) certified free of Mycoplasma and other pathogenic murine viruses. Dulbecco's modified Eagle's medium (DMEM),
fetal calf serum (FCS), and L-glutamine were obtained from
BioWhittaker (Walkerville, MD). TFPI purified from human plasma or
HepG2 cells, recombinant full-length TFPI, recombinant truncated TFPI
(1-160 a.a), and a rabbit anti-human TFPI were purchased from American
Diagnostica Inc. (Greenwich, CT). bFGF was purchased from R & D Systems
(Minneapolis, MN). 2ME2, heparin (16-17 kDa), gelatin, and
hematoxylin were from Sigma. Cell proliferation enzyme-linked
immunosorbent assay BrdUrd kit was from Roche Molecular Biochemicals.
Antibodies--
Rabbit polyclonal antibodies against a synthetic
peptide corresponding to the human VLDL receptor carboxyl terminus
(R2623) has been described (15), and the antibody was affinity-purified on peptide-Sepharose as described. Rabbit polyclonal antibodies prepared against the purified VLDL receptor (R4522) have been described
(16) and were purified by chromatography on protein G-Sepharose as
described previously (16). Polyclonal antibodies to human LRP (R777)
were developed as described (17).
Proliferation Assay of Endothelial Cells--
HUVECs were
routinely cultured to confluence in EGM. The cells were trypsinized and
plated in a 96-well plate at 5,000 cells per well per 100 µl of EBM
supplemented with 2% FCS and antibiotics. The cells were allowed to
adhere to the plate for at least 2 h. Then, bFGF at 10 ng/ml and
various concentrations of TFPI or 2ME2 were added to the
wells. The cells were cultured for 48 h at 37 °C in a 5%
CO2 atmosphere. Cell proliferation was determined using a
uridine incorporation method as described by the manufacturer and
confirmed by cell counting (Cell Counter model Z1, Coulter Incorporation, Miami, FL). Inhibition of cell proliferation, as assessed by uridine incorporation, was calculated using Equation 1.
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(Eq. 1)
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Additional experiments were performed using VLDL
receptor-specific antibodies to block the antiproliferative activity of TFPI. In these experiments, either fixed or increasing concentrations of the rabbit polyclonal anti-VLDL receptor IgG (R4522) or the control
anti-VLDL receptor IgG (R2623) were preincubated with HUVECs for 1 h at 37 °C. In other experiments, the VLDL receptor antagonist, RAP,
was preincubated with endothelial cells for 24-36 h. After addition of
VLDL receptor antagonists, TFPI and bFGF were added, and HUVEC
proliferation was determined as above.
Proliferation of Tumor Cells--
Rat C6 glioma and B16BL6
melanoma tumor cells were routinely cultured to confluence in DMEM
supplemented with 10% FCS. B16BL6 melanoma cells were used between 6 and 18 passages. For the proliferation assay, tumor cells were plated
in 96-well plate at 2,500 cells per well per 100 µl of media and were
allowed to adhere to the plate for at least 2 h. Various
concentrations of TFPI or 2ME2 were then added to the
wells. The cells were cultured for 48 h at 37 °C in a 5%
CO2 atmosphere. Cell proliferation was determined using a
uridine incorporation method. Inhibition of cell proliferation was
defined as described above.
Generation of a Soluble VLDL Receptor Domain--
A fragment of
the human VLDL receptor was generated as a glutathione
S-transferase fusion protein. The polymerase chain reaction primers used are sense, AGTTGGATCCGGGAGAAAAGCCAAATGTGAA, and antisense, AGTTGAATTCTTAGTAGTAACACTCTTTCAGGGGCTCATCACT. These primers generated a
989-bp fragment corresponding to the 8 ligand binding repeats of the
VLDL receptor. Two tyrosine residues were added after the final
cysteine of repeat 8 so the fragment could be radioiodinated. The
polymerase chain reaction fragment was digested with BamHI and EcoRI, and the resulting fragment was ligated into the
pGEX-2T vector. The entire insert of this final pGEX-VLDLR1-8
construct was sequenced.
Escherichia coli BL21 cells were transformed with
pGEX-2T-VLDLr1-8 and grown in a 10-liter fermenter. When cells were in
mid-log phase isopropyl-1-thio-
-D-galactopyranoside was
added to induce fusion protein production. After 3 more hours, cells
were centrifuged, and the pellets were resuspended in 10 mM
Tris, 1 mM EDTA, pH 7.4, and lysed by treatment with 1 mg/ml lysozyme and sonication. After centrifugation, the supernatant
was incubated with 50 µM MgCl2, 25 µg/ml
DNase I for 1 h at 4 °C. The digest was diluted 2-fold into GSH
wash buffer (20 mM HEPES, 0.1 M KCl, 1 mM dithiothreitol, 0.5 mM EDTA, pH 7.5) and
applied to the GSH-Sepharose column. Following washing, the column was
eluted with wash buffer containing 25 mM GSH.
The GST-VLDLr1-8 fusion protein was digested with 20 nM
thrombin for 1.5 h at 25 °C, and the reaction was stopped with
200 nM D-Phe-Pro-Arg-CH2Cl. The
digestion reaction was re-applied to the GSH-Sepharose, and the
flow-through containing free VLDLr1-8 was recovered. This was dialyzed
against 5% acetic acid overnight and lyophilized. The fragment was
then resuspended in nitrogenated 6 M guanidinium HCl, 50 mM Tris, 1 mM dithiothreitol, pH 8.5, and
refolded according to the method of Dolmer et al. (18) by diluting 10-fold into nitrogenated 50 mM Tris, 10 mM CaCl2, 1 mM GSH, 0.5 mM GSSG, pH 8.5, and then dialyzing into 200× volume of
this same buffer overnight at 25 °C. The refolded VLDLr1-8 was
subjected to RAP affinity chromatography, and bound VLDLr1-8 was
eluted from RAP-Sepharose with 20 mM EDTA.
For the solid phase binding assays, purified VLDLr1-8 was
immobilized on microtiter wells at a concentration of 4 µg/ml. The microtiter wells were then blocked with 3% bovine serum albumin. RAP,
uPA, TFPI, and truncated TFPI were radioiodinated using IODO-GEN-coated tubes, whereas VLDL was iodinated by the iodine monochloride method as
described (16). To form the uPA·PAI-1 complex, a 3-fold molar excess
of PAI-1 was incubated with 125I-labeled uPA. To determine
binding affinities, radiolabeled ligand at the indicated concentration
was added to the initial well, and a 3-fold serial dilution was
performed, and ligand binding was allowed to continue 1 h at
37 °C. Specific binding was determined using 100-fold molar excess
of RAP. After binding, wells were washed 3 times and counted in a
counter.
VLDL Receptor-mediated Degradation of TFPI--
Mouse PEA13
cells, an LRP-negative cell line, were infected with one of two
adenovirus constructs essentially as described (16): Ad-VLDLr, which
encodes for the VLDL receptor, and Ad-LacZ, a control virus which
encodes for the
-galactosidase gene. Briefly, either Ad-LacZ or
Ad-VLDLr was added to PEA-13 cells at a concentration of 50,000 particles/cell and incubated overnight. After incubation, cells were
rinsed and split into 12-well dishes. TFPI binding, internalization,
and degradation studies were performed as described (16). 25 nM 125I-TFPI in binding buffer (DMEM, 2%
bovine serum albumin, 10 mM HEPES, Neutridoma®-NS media
supplement (Roche Molecular Biochemicals)) was incubated with infected
PEA13 cells for various lengths of time. Control wells included excess
cold RAP, which inhibits ligand binding to the VLDL receptor, or 1 µg/ml chloroquine, which inhibits lysosomal degradation of
internalized ligands. At various time points the media were removed and
mixed with 50% trichloroacetic (1:5 v/v). The pellet was collected by
centrifugation and contained intact ligand, whereas the supernatant
contained degraded ligand. Adherent cells were then treated with
trypsin, EDTA, 0.5 mg/ml proteinase K (Sigma) for 10 min at 4 °C.
Cells are gently rinsed off the dish and centrifuged. After
centrifugation, surface-bound TFPI was localized in the supernatant,
whereas the cell pellet contained internalized TFPI. The radioactivity
associated with each fraction was determined in a
counter.
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RESULTS |
TFPI Inhibits Endothelial Cell Proliferation--
To
determine the effect that TFPI has on endothelial cell growth,
bFGF-stimulated endothelial cells were grown in the absence or presence
of increasing concentrations of TFPI. As shown in Fig.
1A, TFPI had a dramatic effect
on bFGF-stimulated endothelial cell growth, inhibiting proliferation of
endothelial cells in a dose-dependent fashion with an
IC50 value of 350 nM. In several additional
experiments, the IC50 value for TFPI ranged from 150 to 400 nM. In contrast to its effect on endothelial cells, TFPI had no effect on the growth of B16BL6 melanoma cells (Fig.
1B), Lewis lung carcinoma (LLC), or rat C6 glioma cells
(data not shown). As a control in each experiment, 2ME2, a
naturally occurring antiproliferative agent (19), was used and
inhibited the proliferation of both endothelial cells (Fig.
1A) and B16BL6 cells (Fig. 1B). Inhibition of
cell proliferation by TFPI was reversible and was restored by removal
of TFPI and addition of fresh media containing bFGF. TFPI derived from
nonrecombinant sources exhibited a similar antiproliferative activity.
Full-length protein purified from human plasma or HepG2 cells inhibited
bFGF-promoted proliferation of HUVECs in a dose-dependent manner with a similar IC50 value (data not shown).

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Fig. 1.
TFPI inhibits the proliferation of
HUVEC but not B16BL6 melanoma cells. Indicated concentrations of
TFPI (opened circles) and 2ME2 (solid
squares) were added to subconfluent cultures of B16BL6 cells
(A) and HUVECs (B). After incubation at 37 °C
for 48 h, cell proliferation was assessed by measuring uridine
incorporation, and data were expressed as percent inhibition as
described under "Experimental Procedures." Each point represents
the average of three wells, and the error for each point is less than
15%.
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The Antiproliferative Activity of TFPI Is Lost When the Kunitz 3 Domain of TFPI Is Deleted--
TFPI is composed of three Kunitz-type
proteinase inhibitor domains. Domains 1 and 2 are required for factor
VIIa and Xa binding (4), whereas domain 3, along with the carboxyl
terminus of TFPI, can bind to heparin (20). To identify the region of
TFPI responsible for this antiproliferative activity, proliferation studies were performed with both full-length TFPI and a truncated TFPI
containing only domains 1 and 2 (1-160 a.a.). The results of this
experiment (Fig. 2) demonstrated that
truncated TFPI exerts a diminished inhibitory action, suggesting that
Kunitz-3 and the carboxyl terminus of TFPI play an important role in
the antiproliferative action of this protein.

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Fig. 2.
Comparison of the antiproliferative activity
of full-length (1-276 a.a.) and truncated (1-160 a.a.) TFPI
molecules. Full-length TFPI (closed circles) and
truncated TFPI (open circles) were added to subconfluent
cultures of HUVECs. After incubation at 37 °C for 48 h, cell
proliferation was assessed by uridine incorporation and expressed as
percent inhibition as described under "Experimental Procedures."
Each point represents the average of three wells. For each point the
error is less than 15%.
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Receptor-associated Protein (RAP) Inhibits the Antiproliferative
Activity of TFPI--
We next sought to determine the mechanism of the
antiproliferative activity of TFPI. TFPI is known to bind and inhibit
the activity of fVIIa·TF complexes. However, we were unable to detect tissue factor antigen on proliferating endothelial cells using immunoblotting experiments, suggesting that another receptor is likely
responsible for the antiproliferative activity of TFPI. Previous
studies have shown that TFPI binds to the LRP (6), and thus we employed
RAP to determine whether TFPI is mediating its effect via an LDL
receptor family member. RAP was initially identified when it was noted
to copurify with LRP during ligand affinity chromatography (21). This
molecule binds with high affinity to LRP, VLDL receptor, and
gp330/megalin and antagonizes the binding of all ligands to these
receptors (22). In the current study, a fixed amount of RAP was first
preincubated with cells, and 24-36 h later increasing amounts of TFPI
were added, such that the molar excess of RAP over TFPI varied from 2- to 8-fold. The results (Fig. 3)
demonstrate that RAP inhibits the TFPI antiproliferative effect by
shifting the IC50 value of TFPI from 300 to 700 nM. Webb et al. (23) demonstrated that
incubation of cells with RAP for up to 5 days had no effect on the cell
surface levels of the VLDL receptor nor on its biological activity.
Thus, we conclude that RAP acts as a competitive inhibitor of TFPI,
preventing it from binding to a member of the LDL receptor family. RAP
alone had no effect on bFGF stimulation of endothelial cell growth and does not affect the binding of 125I-labeled TFPI to heparin
sulfate proteoglycans (HSPG) (data not shown), confirming the results
of Warshawsky et al. (6).

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Fig. 3.
Effect of RAP on TFPI-mediated
antiproliferative activity. HUVECs were preincubated with 2 µM RAP for 36 h, and then various concentrations of
TFPI were added in the presence of 10 ng/ml bFGF. In control cells, RAP
pretreatment was omitted. After 48 h, cell proliferation was
measured as described under "Experimental Procedures." Each point
is the average of three wells ± S.E.
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VLDL Receptor-specific Antibodies Inhibit the Antiproliferative
Activity of TFPI--
Whereas LRP has been previously reported to bind
TFPI, this receptor could not be detected in HUVEC cell extracts by
immunoblotting or RAP ligand blotting (9). In contrast, the
structurally related VLDL receptor is expressed in HUVECs in the
endothelium of capillaries and small arterioles and on the endothelial
surface of bovine arteries, suggesting that the VLDL receptor may be
responsible for the effect of TFPI. To test this, we employed specific
antibodies to LRP and the VLDL receptor. Although previous studies have
determined that the anti-VLDL receptor IgG blocks ligand uptake by this
receptor (16), the specificity of this antibody on endothelial cells has not been demonstrated. To investigate the specificity of the anti-VLDL receptor antibody, immunoblotting experiments on membrane preparations from endothelial cells were performed. The results of this
experiment (Fig. 4) confirm that the
anti-VLDL receptor antibody is highly specific and only reacts with two
polypeptides with estimated molecular weights of 130,000 and 95,000. Multiple forms of the VLDL receptor exist (13) resulting from alternate splicing of the pre-mRNA; type I receptors contain the
O-linked sugar domains and have an apparent molecular weight
of ~130,000 under nonreducing conditions, whereas type II receptors
lack the O-linked sugar domain and have an apparent
molecular weight of 95,000-100,000 under nonreducing conditions. VLDL
receptor mRNA that lacks exon 4 (encoding for L3) has also been
detected in the brain (24).

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Fig. 4.
Immunoblotting of endothelial cell membrane
extracts by anti-VLDL receptor IgG. HUVEC membrane preparations
were extracted into SDS sample buffer and subjected to SDS-PAGE on
4-20% gels. Following electrophoresis, the membrane proteins were
transferred to nitrocellulose and immunoblotted with 1 µg/ml of
anti-VLDL receptor IgG (R4522).
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LRP antibodies had no effect on TFPI inhibition of endothelial cell
proliferation (data not shown). In contrast, anti-VLDL receptor IgG did
partially neutralize the effect of TFPI on HUVEC proliferation (Fig.
5A), whereas a control IgG
prepared against the VLDL receptor cytoplasmic domain had no effect in
this assay (Fig. 5A). In the absence of TFPI, anti-VLDL
receptor IgG had no effect on bFGF-stimulated HUVEC proliferation. Fig.
5B demonstrates the effect of increasing antibody
concentration on HUVEC proliferation while maintaining a constant
concentration of TFPI. Anti-VLDL receptor IgG significantly reduced the
effect of TFPI on endothelial cell growth, although it did not
completely restore the growth to levels seen in the absence of TFPI.
These experiments confirm that the VLDL receptor is involved in this
effect. Fig. 5C shows an experiment in which fixed levels of
anti-VLDL receptor IgG were incubated in the presence of increasing
amounts of TFPI, demonstrating that high concentrations of TFPI can
overcome the blocking effects of anti-VLDL receptor IgG. This apparent
competition between TFPI and VLDL receptor antibody further supports
our hypothesis that the VLDL receptor plays an important role in the
TFPI-mediated inhibition of endothelial cell growth.

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Fig. 5.
Anti-VLDL receptor IgG block TFPI-mediated
antiproliferative activity. A, HUVECs were incubated
with bFGF (10 ng/ml) (control) in the presence of anti-VLDL
receptor IgG (R4522, 1 µM), control IgG made against the
VLDL receptor cytoplasmic tail (1 µM), TFPI (20 µg/ml),
TFPI (20 µg/ml) in the presence of anti-VLDL receptor IgG (R2623, 1 µM), or TFPI (20 µg/ml) in the presence of control IgG
(1 µM). After 48 h, cell proliferation was measured
as described under "Experimental Procedures." B, various
concentrations of anti-VLDL receptor IgG R4522 were preincubated with
HUVEC for 1 h at 37 °C. Then 10 ng/ml bFGF was added in the
absence or presence of 375 nM TFPI. After 48 h, cell
proliferation was measured as described under "Experimental
Procedures." B, HUVECs were preincubated in the presence
(open circles) or absence (closed circles) of 1 µM anti-VLDL receptor R4522 IgG for 1 h at 37 °C
before adding 10 ng/ml bFGF in the presence of increasing amounts of
TFPI. After 48 h, cell proliferation was measured as described
under "Experimental Procedures." For each pair the error is less
than 15%.
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Identification of the VLDL Receptor as a Novel TFPI
Receptor--
The ability of RAP and anti-VLDL receptor IgG to inhibit
the antiproliferative activity of TFPI on endothelial cells suggests that the VLDL receptor is involved in this response. We next sought to
determine whether TFPI can directly bind to the VLDL receptor. To
measure this, a soluble form of the VLDL receptor (sVLDLr1-8) containing the ligand binding portion of the VLDL receptor (12, 25) was
expressed in E. coli. Following refolding and affinity purification, the sVLDLr1-8 was analyzed by SDS-PAGE which revealed that the purified receptor migrated as a single band under reducing (Fig. 6A, lane 1) and
nonreducing conditions (Fig. 6A, lane 2). The faster
electrophoretic mobility for sVLDLr1-8 under nonreducing conditions is
consistent with the notion of a compactly folded structure arising from
the formation of intra-chain disulfide bonds. sVLDLr1-8 was recognized
by the anti-VLDLR IgG (Fig. 6B, lanes 3 and 4)
confirming that this antibody is capable of binding to the VLDL
receptor ligand binding region.

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Fig. 6.
Analysis of sVLDLr1-8 by SDS-PAGE
(A) or immunoblotting with anti-VLDLr IgG
(B). Purified sVLDLr1-8 was subjected to
SDS-PAGE under reducing (lanes 1 and 3) or
nonreducing (lanes 2 and 4) conditions. Following
electrophoresis, the bands were visualized with Coomassie Blue
(A), or samples were transferred to nitrocellulose and
immunoblotted with 1 µg/ml anti-VLDL receptor IgG (R4522)
(B).
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The functional properties of the recombinant sVLDLr1-8 were examined
by solid phase binding assays. In these experiments, sVLDLr1-8 was
coated on microtiter wells, and the ability of 125I-labeled
RAP, VLDL, and uPA·PAI-1 complexes to bind to this molecule was
measured. The results (Fig. 7) indicate
high affinity binding of RAP (KD = 1.4 nM) and uPA·PAI-1 complexes (KD = 7.3 nM) to the immobilized receptor. The KD
values measured in these experiments are virtually identical to the
KD of 0.7 and 15 nM measured for the
binding of RAP and uPA·PAI-1 complexes to the intact VLDLr,
respectively (9, 15). Immobilized sVLDLr1-8 also recognized
125I-labeled VLDL particles, and its binding to immobilized
sVLDLr1-8 was inhibited by excess RAP (Fig. 7C). These data
confirm that the sVLDLr1-8 is correctly folded and capable of binding
known VLDLr ligands with appropriate affinity.

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Fig. 7.
Binding of 125I-RAP
(A), 125I-uPA·PAI-1 complexes
(B), or 125I-VLDL (C) to
immobilized sVLDLr1-8. Soluble VLDLr1-8 (4 µg/ml) was
immobilized on microtiter plates (100 µl/well). Indicated
concentrations of 125I-labeled ligand (filled
circles) or 125I-labeled ligand plus excess unlabeled
RAP (filled squares) was added to the wells. Following
incubation, the wells were washed, and bound counts/min were
determined. A, 125I-RAP; B,
125I-uPA·PAI-1 complexes; C,
125I-VLDL.
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We next employed the solid phase assay to measure the ability of
125I-TFPI and 125I-TFPI-(1-160) to bind
to immobilized sVLDLr1-8. Specific binding was determined by
subtracting background binding observed in the presence of a large
molar excess of RAP. Full-length TFPI bound in a specific and saturable
manner to the sVLDLr1-8 with an apparent KD of 80 nM determined by nonlinear regression analysis (Fig.
8). A 100-fold molar excess of RAP
blocked more than 95% of TFPI binding to VLDL receptor. In contrast to
the ability of full-length TFPI to bind sVLDLr1-8, no specific binding
of the truncated TFPI to the sVLDLr1-8 was detected under these
conditions. Taken together, these data indicate that TFPI binds to the
VLDL receptor and that this binding requires the third Kunitz-type domain and/or the flanking sequences. It is interesting to note that
the truncated form of TFPI, which does not bind to the VLDL receptor,
is also much less effective in endothelial proliferation studies.

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Fig. 8.
Binding of full-length TFPI (1-276 a.a.) and
truncated TFPI (1-160 a.a.) to immobilized sVLDLr1-8. Soluble
VLDLr1-8 (4 µg/ml) was immobilized on microtiter plates (100 µl/well). Indicated concentrations of 125I-labeled TFPI
(filled circles) or truncated TFPI (filled
squares) was added to the wells. Following incubation, the wells
were washed, and bound counts/min were determined. Specific binding was
determined by 100-fold molar excess of RAP.
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LDL receptor family members were initially characterized as endocytic
receptors that target ligands to lysosomes for proteolytic degradation.
By having demonstrated specific binding of TFPI to the VLDL receptor
in vitro, we next sought to determine whether the VLDL
receptor was also able to mediate the cellular internalization and
degradation of TFPI. These experiments were performed in mouse embryonic fibroblasts (PEA-13) known to be genetically deficient in
LRP, since LRP is known to bind, internalize, and degrade TFPI. These
fibroblasts also express very little VLDL receptor, thus the VLDL
receptor gene was introduced into these cells using an adenovirus-VLDL
receptor construct. This experimental system has been previously used
to identify and characterize VLDL receptor ligands (16). In this way,
the contribution of the VLDL receptor to TFPI internalization and
degradation can be readily measured. Cells expressing the VLDL receptor
are able to internalize and degrade TFPI (Fig.
9) in a RAP-sensitive fashion, confirming
the role of the VLDL receptor in this process. In contrast, parental cells, or cells infected with an adenovirus construct expressing the
lacZ gene, are unable to internalize
125I-labeled TFPI.

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Fig. 9.
VLDL receptor-dependent
degradation of TFPI. PEA13 cells infected with either
adenovirus-VLDL receptor (circles) or adenovirus-LacZ
(inverted triangles) were used to measure degradation of
TFPI. 35 nM 125I-labeled TFPI was incubated
with the cells for the indicated times, and the extent of cellular
mediated degradation was measured as described under "Experimental
Procedures." TFPI degradation is only seen with Ad-VLDL
receptor-infected cells (filled circles). No degradation is
seen in LacZ-infected cells (filled triangles), whereas
excess RAP (open circles) dramatically reduces VLDL
receptor-mediated TFPI degradation.
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DISCUSSION |
An association between components of the coagulation pathway and
tumor growth has been recognized for more than a century (26). Multiple
studies have implicated fVIIa·TF complexes in the progression of
tumor growth and metastases by promoting fibrin deposition and
angiogenesis. Additional coagulation proteins, including thrombin (27)
and protein C (28, 29), are also able to regulate endothelial cell
growth. In light of these observations, we sought to investigate
whether TFPI could regulate the growth of endothelial cells. Our
studies focused on TFPI for two reasons. First, the ability of TFPI to
block fVIIa/TF activity suggested that it might regulate the signaling
activity of this complex, and second, previous reports suggested that
high concentrations of TPFI might have antiproliferative or apoptotic
effects on smooth muscle cells and endothelial cells (30, 31).
The antiproliferative activity of TFPI appears specific for endothelial
cells as no inhibition was observed in any tumor cell line tested. Our
initial hypothesis was that the antiproliferative activity of TFPI was
mediated through fVIIa·TF complexes. However, we were unable to
detect any expression of tissue factor on HUVECs. Thus, we speculated
that a different receptor was responsible for the observed activity. It
is unlikely that TFPI binding to HSPGs was responsible for the
antiproliferative activity of TFPI, since RAP was able to inhibit the
TFPI antiproliferative effect. As RAP does not affect the binding of
TFPI to HSPGs, we concluded the mechanism involved association of TFPI
with a member of the LDL receptor family. Since the VLDL receptor is
expressed on endothelial cells, we examined the potential involvement
of the VLDL receptor in this activity. Several lines of evidence
substantiate that TFPI binding to the VLDL receptor is a necessary
component of the antiproliferative activity of TFPI. First, TFPI binds
to the VLDL receptor with an apparent KD of 80 nM, which is close to the IC50 value for the
inhibition of endothelial cell proliferation (150-400 nM).
Second, RAP inhibits both the binding of TFPI to the VLDL receptor as
well as its antiproliferative activity. Third, a specific polyclonal
antibody to the VLDL receptor, which is known to block ligand
recognition by this receptor (16), also blocked the antiproliferative
effect of TFPI on endothelial cells. Finally, a deletion mutant of TFPI
that failed to bind to the VLDL receptor in vitro was also
much less active in inhibiting endothelial cell proliferation. All of
these data corroborate the role of the VLDL receptor in this process
and indicate that TFPI and the VLDL receptor are capable of modulating
endothelial cell growth.
It should be noted that a severalfold molar excess of VLDL receptor
antagonist was unable to block completely the TFPI antiproliferative activity, suggesting that the VLDL receptor may not be completely responsible for this effect. There are several possible explanations for this result. First, the concentrations of inhibitor used may not
have been high enough to block completely the VLDL receptor-mediated TFPI effect. Second, the binding of TFPI to HSPGs may enhance the
ability of TFPI to associate with the VLDL receptor and inhibit proliferation. A number of other VLDL receptor ligands have high affinity for HSPGs, including lipoprotein lipase and thrombospondin. The binding of these ligands to LRP is promoted when prior HSPG binding
occurs (32, 33). Finally, we cannot exclude the possibility that
another mechanism also contributes to the TFPI activity that is
insensitive to VLDL receptor-binding antagonists.
The mechanism by which TFPI binding to the VLDL receptor blocks
bFGF-stimulated endothelial cell proliferation remains to be determined
at this time and may involve signaling events. The VLDL receptor can
participate in signal transduction pathways as shown in studies
demonstrating that knockout mice lacking both the VLDL receptor and the
closely related apoE receptor 2 have a defect in neuronal migration
(34). Mice lacking the neuronal ECM protein Reelin, the
disabled gene product (Dab1), or VLDL receptor/ApoER2 have
identical phenotypes. Dab1 is an adaptor protein that interacts with
the cytoplasmic domains of LDL receptor family members including the
VLDL receptor (35, 36) and appears to function in a tyrosine kinase
signaling pathway. These findings suggest that the VLDL receptor
and apoER2 participate in transmitting the extracellular Reelin
signal through intracellular signaling processes mediated by mDab1. The
hypothesis that the VLDL receptor signals in response to Reelin binding
was recently supported by studies (37) showing direct binding of Reelin
to the VLDL receptor and changes in tyrosine phosphorylation in
response to Reelin-VLDL receptor association.
We propose that similar mechanisms occur in endothelial cells in
response to TFPI binding to the VLDL receptor. TFPI binding could
initiate signal transduction by cytoplasmic adaptor proteins (possibly
a member of the disabled family of genes) in association with the VLDL receptor cytoplasmic domain, which would ultimately result in the inhibition of HUVEC proliferation. Alternatively, it is
possible that the VLDL receptor does not signal directly but that it is
involved in the formation of a multiprotein signaling complex induced
by TFPI binding. Currently, the function of the VLDL receptor is not
known. This molecule is most abundant in skeletal muscle, heart,
adipose tissue, and brain (13, 38). Subsequent studies confirmed that
this receptor is expressed in the endothelium (10, 11) and in
macrophages present in human atherosclerotic lesions (11, 16). The role
of the VLDL receptor in neuronal development (34) and recent findings
by Webb et al. (23) indicating a major role for the VLDL
receptor in breast cancer cell migration suggest an important role for
this receptor in cellular signaling events.
Several ligands for the VLDL receptor have been identified in addition
to TFPI. These include apolipoprotein E (apoE), thrombospondin-1 (TSP-1), urokinase complexed to its inhibitor, plasminogen inhibitor type I (PAI-1), and Lp(a) (9, 16). It is interesting to note that apoE
(39), TSP-1 (40), and PAI-1 (41) have been reported to inhibit
endothelial cell proliferation and angiogenesis, whereas urokinase (42)
and Lp(a) (43) have been reported to enhance angiogenesis. It is
unknown whether these molecules require binding to the VLDL receptor to
exert effects on endothelial cells. However, it is compelling to
speculate that the endocytic activity of the VLDL receptor could
regulate the local concentrations of these various molecules, thus
controlling the local balance of pro- and anti-angiogenic molecules.
Therefore, the VLDL receptor may play a pivotal role in regulating
endothelial cell growth and angiogenesis, directly through TFPI binding
and indirectly through its effect on the turnover of pro- and
anti-angiogenic molecules.
Based upon these in vitro results, it will be important to
determine whether TFPI has anti-angiogenic and antitumor activity in vivo, and at present it is unknown whether TFPI/VLDL
receptor interaction has an antiproliferative effect on endothelial
cells in vivo. Studies are currently underway with VLDL
receptor knockout mice to determine if the VLDL receptor and its
interaction with TFPI plays a role in pathological angiogenesis.
In conclusion, our data identify TFPI as an inhibitor of endothelial
cell proliferation. We show that the VLDL receptor is a novel TFPI
receptor on endothelial cells and, remarkably, that the VLDL receptor
mediates the antiproliferative effect of TPFI. Together these data
demonstrate the ability of the VLDL receptor to regulate the normal and
pathological growth of endothelial cells and to position these families
of molecules as potential targets for the development of new therapies
of angiogenesis-based diseases.