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INTRODUCTION |
Glypican-1 is the prototype member of the
glycosylphosphatidilinositol anchored cell surface heparan sulfate
proteoglycans (HSPGs)1
implicated in cell adhesion and migration, lipoprotein metabolism, anticoagulation, and modulation of growth factor activities (1, 2). The
glypican family includes the human and rat glypican (3, 4), rat
cerebroglycan (5), rat OCI-5 (6) and its human homologue glypican-3
(7), K-glypican (8), glypican-5 (9), and the Drosophila
dally (10). Mutations in dally affect cell
division in the Drosophila visual system and lead to
morphological defects in the eyes, antenna, genitalia, and wings (10).
In addition, mutations in glypican-3 are responsible for the
Simpson-Golabi-Behmel syndrome, a disease that is associated with
specific congenital malformations and a predisposition to tumors (2,
7). Recent studies in mammalian cells indicated that glypican-1 binds
to members of the fibroblast growth factor (FGF) family. Thus, OCI-5 binds to FGF-2, and glypican-1 interacts with at least three FGFs: FGF-1, FGF-2, and FGF-7 (11-13). Furthermore, glypican-1 modulates the
biological activities of these growth factors, and this modulation can
be stimulatory or inhibitory depending on the FGF type (11, 12). Taken
together, these findings indicate that the glypicans play an important
regulatory role in the control of cellular growth, differentiation, and morphogenesis.
Vascular endothelial growth factors (VEGFs) are mitogens for
endothelial cells and are potent angiogenic factors in vivo. Five VEGF isoforms, designated VEGF121,
VEGF145, VEGF165, VEGF189, and
VEGF206, are generated via an alternative splicing
mechanism from a unique gene (14-16). The active form of the VEGFs is
a homodimer, but active heterodimers have also been observed. Two VEGF
tyrosine-kinase receptor types have been characterized. These
tyrosine-kinase receptors do not differentiate between the various VEGF
forms. The KDR/flk-1 receptor mediates the mitogenic
activity of VEGF, whereas flt-1 stimulates VEGF-induced cell
migration (17). In addition, endothelial cells express
VEGF165-specific receptors of unknown function. These
receptors do not bind VEGF121 or VEGF145 (16,
18).
The best characterized VEGF forms are VEGF121 and
VEGF165 (165- and 121-amino acid-long polypeptide,
respectively). VEGF165 contains the peptide encoded by
exon-7 of the VEGF gene, whereas VEGF121 lacks this
peptide. The presence of exon-7 confers on VEGF165 the
ability to bind heparin-like molecules. Removal of heparan sulfates
(HS) from the surface of endothelial cells by heparinase digestion
reduces the binding of 125I-VEGF165 to its
receptors, and addition of heparin restores
125I-VEGF165 binding. We have recently shown
that part of the effect of heparin involves restoration of
125I-VEGF165 binding to KDR/flk-1 by
a chaperone-like effect that recuperates the biological activity of
VEGF165 that was damaged by oxidizing agents (18). Such a
role of heparin-like molecules may be important under conditions like
wound repair, hypoxia-induced angiogenesis, or inflammation, in which
oxidants or free radicals are produced and may damage VEGFs (19-21).
Unlike VEGF165, VEGF121 is irreversibly
inactivated by oxidizing reagents, and heparin cannot restore its
receptor binding ability (18).
Recent evidence strongly suggests that VEGFs play a critical role in
the process of tumor angiogenesis. This process is essential for tumor
progression and for the subsequent process of tumor metastasis. A
number of molecules that display some structural similarity to heparin
such as suramin and pentosan sulfate exert an anti-angiogenic effect.
These observations, together with the findings that HS-degrading
enzymes can inhibit tumor angiogenesis (22), suggest that HSPGs play an
important role in the angiogenic process. So far, HSPGs that can bind
VEGFs and modulate their biological activities have not been
identified. Among glypicans, glypican-1 is the only member expressed in
the vascular system (23, 24). We have therefore examined whether
glypican-1 can interact with VEGFs and modulate their interaction with
VEGF receptors. We show here that purified glypican-1 binds
VEGF165 with high affinity and supports its binding to
heparinase-treated endothelial cells. Furthermore, glypican-1 can also
restore the receptor binding ability of oxidized VEGF165
and can therefore be viewed as a native, cell surface-localized
proteoglycan that displays a chaperone-like activity.
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EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant VEGF165 and
VEGF121 were produced and purified from Sf-9 insect cells
as described previously (25-27). Human recombinant FGF-2, FGF-1, PF4,
and FGF-7 were produced in bacteria and purified as described
previously (28-30). Bovine brain FGF-1 was purchased from R & D.
Heparinase type I and III was kindly provided by IBEX Technologies
(Montreal, Canada). Intestinal mucosa-derived heparin and chondroitin
sulfates A and C and hyaluronic acid were from Sigma. Heparan sulfate
from bovine lung and heparin-sepharose were obtained from Amersham
Pharmacia Biotech. Carrier-free Na125I and
[35S]Na2SO4 were purchased from
NEN Life Science Products. Microtiter ELISA plates were from Corning.
Tissue culture media, sera, and cell culture supplements were from
Beth-Haemek Biological Industries (Israel) or from Life Technologies,
Inc. Disuccinimidyl suberate was obtained from Pierce. All other
chemicals were purchased from Sigma.
Cells--
Human umbilical vein-derived endothelial cells
(HUVEC) were isolated and cultured in M199 medium containing 20% fetal
calf serum, as described previously (18). Rat myoblast cells (L6E9) were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum as described previously (31).
Purification of Glypican, Enzymatic Deglycosylation, and
Isolation of Glypican-1-associated GAG Side Chains--
Glypican-1 was
purified from salt extracts of subconfluent cultures of the rat
myoblast cell line L6E9 by anion exchange chromatography with
DEAE-Sephacel followed by FGF affinity purification as described previously (11). Enzymatic deglycosylation was carried out in PBS
containing 125I-glypican-1 and 0.5 units/ml of heparinase
I+III. Incubation was for 2 h at 37 °C. Degradation of the core
protein of glypican-1 was carried out overnight with proteinase K (0.5 mg/ml). To ensure that digestion was complete, a parallel incubation
was carried out in the presence of radioiodinated glypican-1, and
digestion was monitored by SDS-polyacrylamide gel electrophoresis and
autoradiography. The GAG side chains were then separated from the
protease and degradation products by DEAE-Sephacel chromatography as
described (11).
Quantitation of GAG and HSPG--
The Safranin O dye that reacts
with carboxyl and sulfate groups and can detect ng amounts of sulfated
GAGs (32) was used to estimate GAG content.
The concentrations of glypican-1 are relative to its HS concentration,
which accounts to about two-thirds of the total PG concentration (11).
Prior to performing the study we examined the biological activity of
glypican-1 by testing its ability to enhance binding of FGF-1 and FGF-2
to FGF receptor 1 and to modulate the biological activities of FGF-7
and FGF-1 as described previously (11).
Radioiodination of Glypican-1 and VEGFs--
Radioiodination was
carried out utilizing the chloramine T method as described previously
(11, 33, 34). 125I-VEGF165 was separated from
free iodine using a heparin-Sepharose column, and
125I-VEGF121 was separated from free iodine
using size exclusion chromatography on Sephadex-G25 as described (34).
The specific activities of the 125I-VEGF165 and
the 125I-VEGF121 were about 105
cpm/ng. Radiolabeled glypican-1 was separated from free iodine by
chromatography on DEAE-Sephacel (11). Specific activities of iodinated
glypican-1 were in range of 6-12 × 106 cpm/µg.
Binding of 125I-Glypican-1 to VEGF-coated
Wells--
Binding of radioiodinated glypican-1 to wells coated with
VEGF165 and various other proteins was done essentially as
described (35). Briefly, 0.2 µg of each protein in 100 µl of
coating buffer were adsorbed to 96-well ELISA plates for 2 h at
room temperature, and binding was performed with
125I-glypican-1 for 2 h at room temperature. Free
125I-glypican-1 was removed by three washes with wash
buffer (35). Estimation of bound 125I-glypican-1 was done
following solubilization with 0.2 M NaOH. All the
experiments were done in triplicate and were repeated at least three
times. Nonspecific binding was estimated as described under
"Results," and it was less than 10% of the total binding. Standard
deviation between replicates in all the experiments was less than 10%.
Reverse Immunodot Assay on Nitrocellulose
Filters--
35S-HSPGs were partially purified from
conditioned medium of metabolically labeled HUVEC cells as described
(11). Various proteins were spotted onto nitrocellulose filters at 2 µg/spot. The filters were blocked with coating buffer (35) containing 1% BSA, washed three times with coating buffer, and incubated with
binding buffer (35) containing HUVEC-derived 35S-HSPGs
(15,000 cpm/cm2) for 2 h at room temperature.
Following extensive washes, one filter was air dried and taken for
autoradiography to confirm binding of radiolabeled HSPGs, and the
remaining filters were incubated with a monoclonal antibody directed
against human glypican-1 (monoclonal antibody S1) or with a
anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology Inc.)
as described (36). Detection of bound antibodies was performed with
enhanced chemiluminescence reagents.
Heparinase Digestion and Receptor Binding Assays--
Binding
and cross-linking of VEGFs to a soluble extracellular domain of
flk-1 fused to secreted human alkaline phosphatase (designated flk/SEAP) was done as described previously (18, 26). Binding and cross-linking to HUVEC cells was essentially performed
as described previously, except that instead of using adherent cells,
the binding was performed using cells in suspension. The cells were
first treated with heparinase (0.5 units/ml) for 1 h at 37 °C
and then detached from the tissue culture dishes using PBS solution
containing 2 mM EDTA. The cells were collected, washed
twice with PBS and once with binding buffer, and then subjected to the
binding assay. Following binding the cells were washed extensively with
PBS, and cross-linking was performed in solution. Equal amounts of
total cell lysates were loaded onto each lane.
Inactivation of VEGF165 by Oxidation--
Oxidation
of VEGF165 was performed essentially as described (18).
Briefly, 2 µg of growth factor in 100 mM sodium phosphate buffer (pH 7.2) were incubated for 1 min with 0.1% of
H2O2 in a final reaction volume of 40 µl. The
reaction was terminated by sodium metabisulfite, and the growth factor
was separated from the H2O2 and sodium
metabisulfite using heparin-Sepharose affinity chromatography (recovery
was over 90%).
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RESULTS |
Glypican-1 Binds to VEGF165--
A solid phase assay
was utilized to investigate the ability of glypican-1 to bind
VEGF165 and VEGF121. Increasing concentrations of radioiodinated glypican-1 were bound to microtiter plates precoated with 0.2 µg/well of VEGF165 or VEGF121 as
well as FGF-7, which was previously reported to bind glypican (11). As
shown in Fig. 1, only VEGF165
and FGF-7 bind similarly to 125I-glypican, whereas
VEGF121 or BSA did not exhibit any binding capacity.
Binding was saturable and Scatchard analysis yielded an apparent
dissociation constant of 1.2 × 10
10 M
for VEGF165 and 3 × 10
10 M
for FGF-7 (Fig. 1B).

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Fig. 1.
Binding of glypican-1 to VEGF.
A, binding saturation of 125I-glypican-1 to
VEGF. 125I-Labeled glypican-1 (specific activity of 12 × 106 cpm/µg) was bound to VEGF165-coated
( ), VEGF121-coated ( ), and FGF-7-coated ( ) wells
as described under "Experimental Procedures." After 2 h of
incubation at room temperature, the wells were extensively washed, and
quantification of bound 125I-glypican-1 was done as
described under "Experimental Procedures." B, Scatchard
analysis of glypican-1 binding to VEGF165 ( ) and FGF-7
( ). The Ligand program was used for the analysis (55).
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To test the specificity of this interaction we measured the ability of
a panel of proteins to compete with 125I-glypican for
binding to VEGF165. As shown in Fig.
2, all three FGF members inhibited
binding. This finding is with accordance with the previously reported
capacity of these growth factors to bind glypican (11). By contrast,
VEGF121, insulin, transferrin, and BSA could not compete
with 125I-glypican for binding to VEGF165.

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Fig. 2.
Inhibition of the binding of
125I-glypican-1 to VEGF165 by heparin-binding
growth factors. 125I-Glypican-1 (5 ng/well) was bound
to VEGF165-coated wells in the presence of the above
indicated growth factors and proteins (1 µg/ml of each). Binding and
subsequent quantification of bound 125I-glypican-1 was done
as described under "Experimental Procedures." V165,
VEGF165; V121, VEGF121;
INS, insulin; TR, transferrin.
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Binding of Glypican-1 to VEGF165 Is Mediated by Its HS
Chain--
To determine whether glypican-1-derived GAGs are involved
in VEGF165 binding, we prepared heparinase-digested or
protease-digested glypican. Heparinase treatment abolished the ability
of glypican-1 to compete with intact 125I-glypican-1 for
binding to VEGF165, whereas the free glypican-1-derived GAGs competed with 125I-glypican-1 as efficiently as intact
glypican-1 (Fig. 3A). These findings suggest that the interaction of glypican-1 with VEGF is
mediated by its GAG side chains. To further verify these findings we
compared the ability of intact glypican, glypican-1-derived GAGs,
heparin, commercial HS, and non-HS GAGs to inhibit the binding of
glypican-1 to VEGF165. Glypican-1-derived HS inhibited the binding in a concentration-dependent manner (Fig.
3B). Half-maximal inhibition was obtained at a concentration
of about 180 ng/ml glypican-1-derived HS. This concentration was 8-fold
higher as compared with the heparin concentration required for
half-maximal inhibition but about 10-fold lower than that of commercial
HS. By contrast neither chondroitin sulfate nor hyaluronic acid were able to compete with glypican-1 for binding to VEGF165.
These results establish that the HS side chains of glypican-1 mediate binding to VEGF165.

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Fig. 3.
Binding of glypican-1 to VEGF165
is mediated by its HS side chains. A, VEGF does not
interact with glypican-1 core protein. 125I-Glypican-1 (5 ng/well) was bound to VEGF165-coated wells in the absence
or presence of 1 µg/ml of unlabeled intact glypican-1
(Gly), glypican-1-free GAGs (Gly-HS) obtained by
proteinase K digestion, or glypican-1 core protein
(Gly-core) obtained by heparinase digestion. B,
inhibition of the binding of 125I-glypican-1 to
VEGF165 by glycosaminoglycans. Binding was performed in the
presence of the indicated concentrations of heparin ( ),
glypican-1-derived HS ( ), commercial HS ( ), chondroitin sulfate
( ), and hyaluronic-acid ( ). The wells were washed, and the amount
of bound radioactivity was quantified.
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Glypican-1 Enhances the Binding of VEGF165 to VEGF
Receptors--
VEGF165 binds to three VEGF receptors on
HUVEC-derived endothelial cells (28). The larger of these receptors is
KDR/flk-1, whereas the identity of the other two is not yet
known (18). Heparin enhances the binding of VEGF165 to all
three receptors (28). Heparin is not a normal constituent of
endothelial cells, whereas glypican-1 is known to be expressed in these
cells (24).2 We have
therefore examined whether glypican-1 can modulate the binding of
VEGF165 to cell surface receptors in untreated and in
heparinase-digested endothelial cells. Digestion of the endothelial cells by heparinase inhibited almost completely the binding of 125I-VEGF165 to the two smaller receptors,
whereas the binding to KDR/flk-1 (18) was inhibited by about
80% (Fig. 4A, lane
3). Exogenously added glypican-1 restored the binding to all three receptors (Fig. 4A, lane 4). Glypican-1 also
potentiated the binding of 125I-VEGF165 to the
two smaller receptors in untreated cells (Fig. 4A,
lane 2).

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Fig. 4.
Effect of glypican-1 on the binding of
125I-VEGF165 to its receptors.
A, glypican-1 restores binding of VEGF165 to
heparinase-treated HUVEC cells. Cells were treated with heparinase and
detached from the plate using PBS solution containing EDTA as described
under "Experimental Procedures." Cells were allowed to bind
125I-VEGF165 (10 ng/ml) for 2 h at
4 °C, the cells were then washed, and bound
125I-VEGF165 was cross-linked to cell surface
receptors. Cross-linked complexes were visualized following
SDS-polyacrylamide gel electrophoresis and autoradiography. Lane
1, untreated cells; lanes 2, untreated cells bound to
VEGF in the presence of 3 µg/ml of glypican; lane 3,
heparinase-treated cells; lane 4, heparinase-treated cells
bound to VEGF in the presence of 3 µg/ml of glypican. B,
glypican-1 potentiates the binding of VEGF165 to a soluble
form of KDR/flk-1. 125I-VEGF165 (20 ng/ml) was bound to flk-1/SEAP-coated wells in the presence
of increasing concentrations of glypican-1 ( ) or heparin ( ), with
or without 1 µg/ml unlabeled VEGF165, as described under
"Experimental Procedures." Specific binding was determined by
subtracting normalized cpm of samples incubated with 1 µg/ml
unlabeled VEGF165 from the normalized cpm bound in the
absence of unlabeled ligand.
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The ability of glypican-1 to potentiate the binding of
VEGF165 to KDR/flk-1 was also confirmed
utilizing a soluble extracellular domain of this receptor
(flk-1/SEAP) in a cell-free assay (26). The potentiation of
125I-VEGF165 binding to the soluble receptor
was concentration-dependent and was maximal at about 1 µg/ml of glypican-1 (Fig. 4B). These findings were also
confirmed in cross-linking experiments (data not shown).
The ability of myoblast-derived glypican-1 to restore binding of
VEGF165 to heparinase-treated HUVEC to a level that is
higher than the binding observed in untreated cells strongly suggests that glypican-1 from endothelial cells also fulfills a similar function. We have therefore partially purified glypican-1 from HUVEC
cells and have tested its ability to interact with VEGF165. VEGF165 was bound to nitrocellulose membranes and incubated
with partially purified 35S-HSPGs. As shown in Fig.
5A, VEGF165 and
FGF-2 bound 35S-HSPGs, whereas VEGF121, BSA,
transferrin, and insulin did not exhibit any HSPG binding ability.
Moreover, a monoclonal antibody directed against human glypican-1
interacted specifically with the VEGF165 and FGF-2-bound
HSPGs (Fig. 5B). These observations strongly suggest that
HUVEC-associated glypican-1 is a candidate modulator of
VEGF165 activity in these cells.

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Fig. 5.
Specific binding of HUVEC-derived glypican-1
to VEGF165. VEGF165
(V165), FGF-2, BSA, VEGF121 (V121),
transferrin (TR), and insulin (INS) at
concentrations of 2 µg/spot were dot-blotted on to a nitrocellulose
filter. Binding of soluble 35S-HSPGs from HUVEC cells to
the filters and immunodetection of bound glypican were carried out as
described under "Experimental Procedures." A, specific
binding of soluble 35S-HSPGs to nitrocellulose filters.
B and C, immunodetection of bound glypican by
monoclonal antibody S1 (B) or with a monoclonal
anti-phosphotyrosine antibody (C). The filters were also
autoradiographed at the end of the assay to confirm that bound
35S-HSPGs are detectable in both filters. D, a
scheme indicating the positions of the various proteins on the
filters.
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Glypican-1 Restores the Activity of Oxidized
VEGF165--
We have previously shown that oxidation of
VEGF165 with agents such as H2O2
impairs its ability to bind to the KDR/flk-1 receptor and
that heparin restores the receptor binding capacity of
VEGF165 (18). Because glypican-1 binds efficiently to
VEGF165, we examined whether it displays a similar
restorative capacity. As shown in Fig. 6,
H2O2-treated VEGF165 lost the
ability to efficiently compete with VEGF121 for binding to
flk-1/SEAP, and addition of glypican-1 restored the receptor
binding capacity of the oxidized VEGF165.

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Fig. 6.
Glypican-1 restores the receptor binding
ability of oxidized VEGF165.
125I-VEGF121 (20 ng/ml) was bound to ELISA
dishes coated with flk-1/SEAP. The binding was performed in
the absence or presence of 0.5 µg/ml of unlabeled VEGF121
(V121), VEGF165 (V165),
H2O2-treated VEGF165
(*V165), and H2O2-treated
VEGF165 plus 3 µg/ml of glypican
(*V165+Gly).
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PF4 Inhibits the Binding of Glypican-1 to
125I-VEGF165 and Abrogates the Stimulatory
Effect of Glypican-1 on 125I-VEGF165 Receptor
Binding--
PF4 is a heparin-binding protein that is synthesized by
megakaryocytes, sequestered in platelets, and released from
-granules as a complex with chondroitin 4-sulfate proteoglycan (37,
38). PF4 displays an anti-angiogenic activity in vivo that
is attributed in part to its heparin binding capacity. Thus, a peptide
derived from the heparin-binding carboxyl-terminal domain of PF4
possesses anti-angiogenic properties (29). The receptor binding ability of VEGF165 and its mitogenic activity are inhibited by PF4
(35). Our results indicate that glypican may be one of the endothelial cell-associated heparan sulfate proteoglycans that bind
VEGF165 and mediate its biological activity. We reasoned
that PF4 may inhibit angiogenesis by preventing the interaction between
VEGF and glypican. We have therefore examined whether PF4 can inhibit the binding of VEGF165 to glypican-1 and abrogate the
stimulatory effect of glypican-1 on VEGF receptor binding. As shown in
Fig. 7A, PF4 efficiently
inhibited the binding of glypican-1 to VEGF165-coated wells. Half-maximal inhibition was obtained at a PF4 concentration of
100 ng/ml. Moreover, PF4 nullifies the stimulatory effect of glypican-1
on the binding of 125I-VEGF165 to
flk-1/SEAP (Fig. 7B).

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Fig. 7.
PF4 inhibits binding of glypican-1 to
VEGF165 and abrogates the stimulatory effect of glypican-1
on binding of VEGF to flk-1/SEAP. A,
effect of PF4 on the binding of glypican-1 to VEGF165.
125I-Labeled glypican-1 (specific activity of 6 × 106 cpm/µg) was bound to ELISA dishes coated with
VEGF165 in the presence of increasing concentrations of
PF4. Binding was quantified as described under "Experimental
Procedures." B, PF4 abrogates the stimulatory effect of
glypican-1 on VEGF165 binding to flk-1/SEAP
receptor. Binding of 125I-VEGF165 (20 ng/ml) to
flk-1/SEAP-coated wells was performed in the absence
(open bars) or presence (shaded bars) of 2 µg/ml of PF4. When indicated, 1 µg/ml of glypican-1 (Gly) or
heparin (Hep) were added.
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DISCUSSION |
Heparin had been previously shown to bind to VEGF165
and to act as an accessory receptor that enhances the interaction of VEGF165 with its signaling receptors. Furthermore, heparin
restores the bioactivity of damaged VEGF165 (18). However,
because cells express several types of HSPGs on their surfaces but not
heparin, it was important to identify native HSPGs that bind VEGF and
modulate its activity. In the present study we have demonstrated for
the first time an interaction of VEGF with the lipid anchored cell surface heparan sulfate proteoglycan glypican-1. Glypican-1 was found
to interact with VEGF165 and to modulate its receptor
binding properties. We show that glypican-1 binds to
VEGF165 via its heparan sulfate chains and that the binding
is specific and saturable. The affinity of glypican-1 to
VEGF165 is high and has an apparent dissociation constant
of 1.2 × 10
10 M. Glypican-1 not only
binds to VEGF165 but is also capable of enhancing the
interaction of VEGF165 with its signaling receptors both in
cell-free binding assays and in heparinase-treated endothelial cells.
VEGF165 is a major angiogenic factor that is active in
processes such as wound repair, hypoxia-induced angiogenesis, and
inflammation (15), processes associated with the generation of
oxidizing agents and free radicals. VEGF165 is inactivated
by these agents (18), and therefore their presence may inhibit
VEGF-induced angiogenesis. Therefore, repair mechanisms allowing
restoration of VEGF activity could fulfill an important biological
role. Glypican-1 restores the biological activity of oxidized VEGF and
is expressed on the cell surface of the endothelial cells that are the
natural target for VEGF (24). The presence of glypican on the surface of endothelial cells could therefore be a fail-safe mechanism ensuring
that every VEGF165 molecule that reaches the endothelial cells will be eventually able to interact and activate signaling VEGF
receptors such as the KDR receptor. This function of glypican-1 may be
critical under conditions in which the concentrations of VEGF are
limited or under conditions in which the activity of VEGF is compromised.
Studies in recent years have strongly indicated that HSPGs are
important modulators of the activity of heparin-binding growth factors,
an issue that is particularly well studied for FGFs (39, 40). FGFs bind
to cell surface and extracellular matrix-associated HSPGs. The
mechanism by which these HSPGs regulate FGF activity is complex and can
be manifested at several levels. These molecules protect FGFs from
protease digestion or from heat/acid inactivation, and it was shown
that they can restore the activity of heat inactivated FGF (41). HSPGs
are also thought to provide a reservoir from which FGFs can be rapidly
released in response to specific triggering events (42). In addition to
these effects, cell-associated HSPGs can increase the affinity of FGFs
to their signaling receptors by stabilizing ligand-receptor complexes
(43-45). VEGFs interact with several receptor types, some of which may
also be able to interact directly with heparan sulfates (46-48). It is
therefore possible that in addition to its chaperone-like role,
glypican-1 may be able to directly modulate the interaction between
VEGF and these receptors using similar mechanisms.
The defined structure of the HSPGs side chains is regulated in a cell
type-dependent manner. For example, human lung
fibroblast-derived glypican-1 cannot enhance the biological activity of
FGF-2 (49). On the other hand, glypican-1 derived from rat myoblasts or
expressed ectopically in K562 cells can stimulate the binding of FGF-2
to FGF-receptor 1 and potentiates cellular responses to FGF-2 (11, 12).
The ability of glypican-1 from rat myoblasts and from human endothelial
cells to interact with VEGF165, together with the finding
that rat myoblast glypican-1 can restore VEGF receptor binding in
heparinase-treated HUVEC cells, strongly suggests that the HS side
chains attached to glypican-1 in both cell types contain similar
VEGF165-binding domains. Glypican-1 exists on the surface of cells both as a lipid anchored form and as a peripheral membrane proteoglycan. The peripheral form is generated following cleavage of
the lipid anchored form by a specific phospholipase and can be shed
from cell surfaces as a soluble proteoglycan (50). The fact that
soluble myoblast-derived glypican-1 can bind and modulate VEGF165 activity suggests that HSPGs derived from one cell
type can act in a paracrine manner to affect biological responses to heparin-binding growth factors.
Beside glypican-1, endothelial cells express other HSPGs such as
fibroglycan, perlecan, and syndecan (24, 51, 52). Studies conducted
with growth factors of the FGF family revealed that distinct core
proteins can bear HS side chains with similar function (12, 49). A
similar situation might exist for growth factors of the VEGF family.
The ability of glypican-1 to fully restore VEGF receptor binding to
heparinase-treated endothelial cells indicates that even if endothelial
cells do express more than one type of stimulatory HSPG species for
VEGF, it is likely that they bear HS with a fine structure similar to
that of glypican-1 side chains. In addition, in heparinase-digested
endothelial cells, glypican-1 enhances VEGF165 binding to its receptors
to a level that is higher than that observed in untreated cells
incubated with glypican-1 (compare lanes 2 and 4,
Fig. 4A). These findings could also imply that endothelial
cells express HSPG species that inhibit the binding of VEGF to its receptors.
Heparin-like molecules have long been implicated in the control of
angiogenesis. The earliest indication that heparin may be involved in
regulation of the angiogenic process was the finding that mast cells
accumulate at the site of tumor angiogenesis before capillary ingrowth,
that conditioned medium from mast cells induces angiogenesis in
vitro, and that this activity can be abolished by protamine and
heparan sulfate degrading enzymes (53, 54). It was subsequently found
that many heparin-binding growth factors, including members of the FGF
and VEGF family, are highly angiogenic. It soon became apparent that
heparin and HS play an important role in modulating the biological
activity of the growth factors which bind to them. However, only
recently were efforts taken to identify native HSPGs that bind and
modulate the activity of these growth factors (11-13, 49). Our
previous studies have shown that glypican-1 modulates the biological
activity of FGF-2, suggesting a role for glypican-1 in the regulation
of angiogenesis (11). The present study lends support to this
hypothesis and further suggests that glypican-1 may play a more general
modulatory role in angiogenesis by regulating the stability and
activity of VEGFs.