From the
The 121-amino acid form of vascular endothelial growth factor
(VEGF
Vascular endothelial growth factors (VEGFs)
The development of solid tumors is dependent
upon the process of tumor angiogenesis(11) . Several studies
have recently indicated that the induction of VEGF expression may play
a major role in tumor
angiogenesis(12, 13, 14, 15, 16, 17) .
Several substances with anti-angiogenic activity have been described in
the past 2
decades(18, 19, 20, 21, 22, 23, 24) .
The mechanisms by which the anti-angiogenic effects are produced are
unclear in most cases, but several of these angiogenesis inhibitors
were found to be either heparin binding substances such as
protamine(19) , subfractions of heparin(25, 26) ,
or molecules bearing some structural resemblance to heparin such as
suramin or pentosan sulfate(22, 27, 28) . These
observations, and recent observations which indicate that heparin
degrading enzymes can inhibit tumor angiogenesis(29) , suggest
that heparin and heparan sulfates play an important regulatory role in
the angiogenic process.
Platelet factor-4 (PF4) is synthesized by
megakaryocytes and sequestered normally in platelets(30) . It is
released from
In the present work
we show that PF4 inhibits the binding of VEGF
The binding
and the cross-linking of
PF4
inhibited efficiently VEGF
Angiogenesis is almost always correlated with the
proliferation of vascular endothelial cells, and angiogenesis-promoting
factors are usually mitogenic to endothelial cells. The various VEGF
species conform to this principle and induce the proliferation of
endothelial cells grown in cell culture. We have shown here that the
heparin binding anti-angiogenic factor PF4 inhibits VEGF
Our experiments indicate
that PF4 can block the activity of a heparin binding growth factor like
VEGF
Because PF4 also inhibited the mitogenic activity of
VEGF
How does
PF4 inhibit the mitogenic activity of VEGF
In conclusion, we have
shown here that PF4 inhibits the mitogenic activity of VEGF
We thank Dr. Dina Ron for critically reading this
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) and the 165-amino acid form (VEGF
)
are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF
possesses a heparin binding
ability and in the absence of heparin-like molecules does not bind
efficiently to the VEGF receptors of vascular endothelial cells. The
binding of
I-VEGF
to the VEGF receptors of
endothelial cells, and the heparin-dependent binding of
I-VEGF
to a soluble extracellular domain of
the VEGF receptor KDR/flk-1, were inhibited by the
angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was
not able to inhibit the binding of VEGF
, a VEGF isoform
which lacks a heparin binding capacity, to the VEGF receptors of the
cells or to KDR/flk-1. These results indicate that PF4 may
inhibit VEGF
binding to VEGF receptors by disrupting the
interaction of VEGF
with cell surface heparan sulfates.
Since PF4 mutants lacking a heparin binding ability retain their
anti-angiogenic activity, alternative inhibitory mechanisms were also
examined.
I-PF4 bound with high affinity (K
5
10
M) to VEGF
-coated wells. The binding of
I-PF4 to the VEGF
-coated wells was
inhibited by several types of heparin binding proteins, including
unlabeled PF4 and unlabeled VEGF
. The binding was not
inhibited by proteins which lack a heparin binding capacity, nor was it
inhibited by VEGF
. Heparinase did not inhibit the binding
of
I-PF4 to VEGF
, indicating that
heparin-like molecules are not required. These experiments suggest that
PF4 can bind to heparin binding proteins such as VEGF
leading to an inhibition of their receptor binding ability. In
agreement with these results, we have observed that PF4 inhibits
efficiently the VEGF
induced proliferation of vascular
endothelial cells. Unexpectedly, PF4 also inhibited efficiently the
VEGF
-induced proliferation of the cells, indicating that
PF4 can disrupt VEGF receptor mediated signal transduction using an
unknown mechanism which does not interfere with VEGF
binding.
(
)are mitogenic for vascular endothelial cells. In
vivo the VEGFs act as potent angiogenic factors and as blood
vessel permeabilizing agents and are therefore also known as vascular
permeability
factors(1, 2, 3, 4, 5, 6, 7) .
Four forms of human VEGF containing 121, 165, 189, and 206 amino acids
are produced from a single gene as a result of alternative
splicing(6) . The active forms of the VEGFs are homodimers, and
the best characterized VEGF species is the heparin binding 165-amino
acid-long form (VEGF
)(6, 8) . The binding
of VEGF
to the VEGF receptors of vascular endothelial
cells is modulated by the addition of exogenous heparin and is
inhibited when cell surface heparan sulfates are removed by heparinase
digestion(9) . The 121-amino acid form of VEGF
(VEGF
) induces the proliferation of endothelial cells,
but in contrast to VEGF
lacks heparin binding
ability(10) .
-granules of platelets as a complex with chondroitin
4-sulfate proteoglycan(31) . PF4 displays an anti-angiogenic
activity in vivo and can inhibit the growth of tumors without
affecting the proliferation rate of the cancerous
cells(32, 33) . PF4 is a heparin-binding protein, and it
was shown that peptides derived from its heparin binding
carboxyl-terminal domain possess anti-angiogenic properties. However,
high concentrations of these peptides are required for the
anti-angiogenic activity as compared with the concentration of intact
PF4 required for a similar effect(32) . It was also shown, that
PF4 could inhibit the binding of the angiogenic factor bFGF to its
receptors on vascular endothelial cells (34) and that this
property was related, at least in part, to its heparin binding
ability(35) . Nevertheless, binding to heparin-like molecules
cannot be the only mechanism by which PF4 inhibits angiogenesis, since
a PF4 mutant protein lacking the heparin binding C-terminal domain
retained anti-angiogenic properties(36) .
to the
three VEGF receptor species found in vascular endothelial cells, but
not the binding of VEGF
. We also present evidence
indicating that PF4 is able to bind to heparin-binding proteins such as
VEGF
. However, PF4 inhibited the mitogenic activity of
both VEGF
and VEGF
, indicating that PF4 can
disrupt VEGF receptor signaling without affecting VEGF binding and that
the inhibition of VEGF induced proliferation of vascular endothelial
cells by PF4 may be achieved by several concomitant mechanisms.
Materials
Human recombinant VEGF was produced and purified from Sf-9 insect cells as described
previously (37, 38). VEGF
was produced similarly (39) and purified using phenyl-Sepharose hydrophobic
chromatography followed by anion-exchange chromatography. Reduced
VEGF's were obtained following a 3-min incubation of VEGF at 100
°C in the presence of 0.1 M dithiothreitol. Human
recombinant bFGF, aFGF, and PF4 were produced in bacteria as described
previously(9, 32) . Keratinocyte growth factor was
kindly provided by Dr. Dina Ron (Department of Biology, Technion,
Haifa, Israel). Heparinase type 1 was kindly provided by IBEX
Technologies (Montreal, Canada). Intestinal mucosa-derived heparin and
condroitin sulfates A and C were from Sigma. Heparan sulfate from
bovine lung and heparin-Sepharose were from Pharmacia.
Na
I was from DuPont NEN. Anti-alkaline phosphatase
antibody was from Dako. The flk-1/SEAP-soluble receptor was
produced as described(38) . Tissue culture plasticware was
obtained from Nunc. Tissue culture media, sera, and cell culture
supplements were from Beth-Haemek Biological Industries, Kibbutz Beth
Haemek, Israel. Endothelial SFM medium was from Life Technologies, Inc.
Prestained high molecular weight size markers were purchased from
Bio-Rad. Disuccinimidyl suberate was obtained from Pierce. All other
chemicals were purchased from Sigma.
Cell Culture
Bovine aortic arch-derived
endothelial cells (ABAE) were cultured as described
previously(40) . Human umbilical vein-derived endothelial cells
(HUE) were grown in gelatin-coated dishes in M199 medium supplemented
with 20% fetal calf serum, 4 mM glutamine, antibiotics, and 1
ng/ml bFGF which was added to the cells every other day. For cell
proliferation assays, HUE cells were seeded in 24-well dishes (20,000
cells/well). The medium was changed after cell adhesion to endothelial
SFM medium. Growth factors and other substances were added every other
day. The cells were counted in a ZM model Coulter counter after 4 days.
Binding and Cross-linking Experiments
Iodination
of human recombinant VEGF or VEGF
was
performed using either the chloramine-T method or the iodogen method
with similar results as described previously for
I-VEGF
(41, 42) . However,
whereas
I-VEGF
was separated from free
iodine using a heparin-Sepharose column as described(42) ,
I-VEGF
was separated from free iodine using
size exclusion chromatography on Sephadex-G25. The specific activities
of the
I-VEGF
and the
I-VEGF
were about 10
cpm/ng.
Iodination of recombinant PF4 was performed by a similar procedure.
I-PF4 was separated from the free iodine using
heparin-sepharose affinity chromatography and was eluted from the
column with a buffer containing 20 mM sodium phosphate buffer,
pH 7.2, and 1.2 M NaCl. The specific activity of the
I-PF4 ranged from 30,000 to 50,000 cpm/ng.
I-VEGF
to
endothelial cells was done as described
previously(9, 42) , and the binding of
I-VEGF
was done similarly. Nonspecific
binding was determined in the presence of 0.5-1 µg/ml
unlabeled VEGF. The level of nonspecific binding ranged between 10 and
20% of the total binding. To bind VEGF to a flk-1/SEAP fusion
protein containing the extracellular domain of the flk-1 receptor, conditioned medium from flk-1/SEAP producing
cells containing the fusion protein (38) was adsorbed to 96-well
enzyme-linked immunosorbent assay dishes coated with an antibody
directed against alkaline phosphatase. Following adsorption, the wells
were washed five times with wash buffer containing 10 mM Tris-HCl, pH 7.2, 0.1 M NaCl, and 0.1% Tween 20.
I-VEGF (20 ng/ml) in 100 µl of binding buffer (10
mM HEPES, pH 7.2, 150 mM NaCl, 0.1% bovine serum
albumin) was bound to the wells for two hours at room temperature. At
the end of the reaction wells were washed three times with wash buffer,
bound
I-VEGF was solubilized using 0.2 N NaOH,
and aliquots counted in a
-counter.
Binding of
Coating buffer (50 µl of 20 mM KI-PF4 to VEGF-coated
Wells
HPO4, 10 mM KH
PO
, 1
mM EDTA, O.8% NaCl) containing 20 ng of VEGF
was
added to each well of a 96-well enzyme-linked immunosorbent assay dish.
The plates were incubated for 2 h at room temperature and subsequently
washed five times with wash buffer (10 mM Tris-HCl, pH 7.2,
150 mM NaCl, 0.1% Tween 20). The wells were then incubated for
another hour at room temperature in coating buffer containing in
addition 1% bovine serum albumin or 0.1% gelatin and were subsequently
washed again five times with wash buffer. The VEGF-coated wells were
then incubated with
I-PF4 in coating buffer containing
0.1% gelatin for 1 h at 37 °C. Unbound
I-PF4 was
removed by three washes with wash buffer at room temperature. Bound
I-PF4 was solubilized using 200 µl of 0.2 M NaOH. Aliquots of 100 µl were taken for counting in a
-counter. All the experiments were done in triplicate and were
repeated at least three times.
PF4 Inhibits the Binding of
VEGF is an important inducer of
angiogenesis and seems to be an important contributor to the process of
tumor angiogenesis(16, 17, 43) . It is therefore
possible that some angiogenesis inhibitors may function by inhibiting
VEGF function. We have found that PF4, a known anti-angiogenic
substance(32) , is able to inhibit the specific binding of I-VEGF
to the VEGF Receptors of
Vascular Endothelial Cells
I-VEGF
to endothelial cells (Fig. 1A). Half-maximal inhibition of
I-VEGF
binding was achieved using about 0.3
µg/ml PF4, and the inhibition was complete when 10 µg/ml PF4
were used (Fig. 1A). PF4 inhibited the formation of the
225-, 195-, and 175-kDa
I-VEGF
-receptor
complexes usually seen when
I-VEGF
is bound
and cross-linked to endothelial cells (Fig. 1B)(9, 42) . The 225-kDa complex
apparently contains the KDR/flk-1 VEGF receptor, since
antibodies directed against this VEGF receptor specifically
immunoprecipitate this labeled complex.
(
)In
addition PF4 inhibited the formation of a
450-kDa complex which
may represent a dimer of the 225-kDa complexes (Fig. 1B). The binding of
I-VEGF
to the two smaller VEGF receptor types was also inhibited by PF4,
but the PF4 concentration required for complete inhibition to the two
smaller receptors was about 1 µg/ml, whereas complete inhibition of
I-VEGF
binding to the larger receptor
species required somewhat higher PF4 concentrations (Fig. 1B).
Figure 1:
Binding
of I-VEGF
and
I-VEGF
to endothelial cells in the presence of increasing concentrations
of PF4. A,
I-VEGF
(10 ng/ml,
) or
I-VEGF
(5 ng/ml,
) were
bound to confluent HUE cells grown in 24-well dishes in the presence of
increasing concentrations of PF4 for 2 h at 4 °C. Nonspecific
binding of
I-VEGF
was determined in the
presence of 1 µg/ml VEGF
, and the nonspecific binding
of
I-VEGF
was measured in the presence of 2
µg/ml VEGF
. At the end of the binding the cells were
washed as described under ``Experimental Procedures'' and
lysed using 0.5 N NaOH. Samples were counted in a
-counter. Shown is the specific binding which was calculated by
subtracting the nonspecific binding from the total binding. The
experiment was repeated three times with similar results. B,
HUE cells were grown to confluence in 6-cm dishes. The cells were
washed once with cold PBS and incubated with
I-VEGF
(5 ng/ml) for 2 h at 4 °C in the
presence of increasing concentrations of PF4. The PF4 concentrations
used were (in µg/ml): 0 (lane 1), 0.25 (lane 2),
1 (lane 3), and 10 (lane 4). At the end of the
incubation, the cells were washed twice with cold PBS, and bound
I-VEGF
was cross-linked to the VEGF
receptors using disuccinimidyl suberate as described. The cells were
then lysed, and samples were separated on a 6% SDS-polyacrylamide gel
electrophoresis gel under reducing conditions. The
I-VEGF
-receptor complexes were visualized
using autoradiography. The numbers on the left side of the gel represent the molecular mass of size markers in kDa. C,
I-VEGF
(20 ng/ml) was bound to flk-1/SEAP-coated wells in the absence (
) or in the
presence (
) of 5 µg/ml PF4 and in the presence of increasing
concentrations of heparin. Shown is the specific binding which was
calculated after substraction of nonspecific binding values from the
total binding. Nonspecific binding was determined in the presence of 1
µg/ml of unlabeled VEGF
for each point. Error
bars represent the deviation from the mean of
triplicates.
The binding of I-VEGF
to the VEGF receptors of vascular
endothelial cells is inhibited when cell surface heparan sulfates are
removed by heparinase digestion(9) . Since PF4 binds to heparin
with high affinity, we thought that PF4 may compete with
I-VEGF
for binding to cell surface heparan
sulfate chains and that the masking of these heparan sulfates by PF4
may lead to inhibition of
I-VEGF
binding.
This mechanism predicts that PF4 should not be able to inhibit the
binding of VEGF
, a VEGF splice variant with no heparin
binding capacity(10) . Indeed, as expected, PF4 failed to
inhibit the binding of
I-VEGF
to the VEGF
receptors of the endothelial cells (Fig. 1A).
Furthermore, PF4 failed to inhibit the binding of
I-VEGF
to a soluble extracellular domain of
the VEGF receptor flk-1 (not shown). In contrast, PF4
inhibited efficiently the heparin-dependent binding of
I-VEGF
to the extracellular domain of the flk-1 VEGF receptor (Fig. 1C)(38) ,
lending further support to the above mentioned inhibitory mechanism.
The Effect of Heparin on the Inhibition of
The binding of I-VEGF
Binding by
PF4
I-VEGF
to
the VEGF receptors of HUE cells is potentiated in the presence of
0.1-1 µg/ml heparin (compare in Fig. 2A, lane 1 with lanes 3 and 5), whereas heparin
concentrations higher than 100 µg/ml inhibit
I-VEGF
binding(9) . Heparin is also
known to inhibit the anti-angiogenic effect of PF4(32) . To find
out how heparin influences PF4 inhibition of
I-VEGF
binding,
I-VEGF
was bound and cross-linked to
endothelial cells in the presence or absence of 2 µg/ml PF4 and
increasing concentrations of heparin (Fig. 2).
Figure 2:
Cross-linking and binding of I-VEGF
to endothelial cells in the presence
of PF4 and heparin. A, ABAE cells were grown to subconfluence
in 6-cm dishes. The cells were washed with cold PBS and incubated with
I-VEGF
(1 ng/ml) for 2 h at 4 °C in the
absence of PF4 (lanes 1, 3, and 5), or in the
presence of 2 µg/ml PF4 (lanes 2, 4, and 6), in
the presence of increasing concentrations of heparin. The heparin
concentrations used in µg/ml were: 0 (lanes 1 and 2), 0.1 (lanes 3 and 4), and 1 (lanes 5 and 6). At the end of the incubation, the cells were
washed twice with cold PBS, and bound
I-VEGF
was cross-linked to the VEGF receptors using disuccinimidyl
suberate as described under ``Experimental Procedures.'' The
cells were lysed, and aliquots were separated on a 6%
SDS-polyacrylamide gel electrophoresis gel under reducing conditions.
I-VEGF
-receptor complexes were visualized
using autoradiography following a week of exposure. B, ABAE
cells were grown in 24-well dishes to a concentration of 200,000
cells/well. The cells were washed with cold PBS and incubated with
I-VEGF
(5 ng/ml) and either 0, 1, or 10
µg/ml of heparin (Hep) or heparan sulfate (Hp-Sulf) in the absence (empty columns) or presence (hatched columns) of 2 µg/ml PF4. After 2 h of incubation
at 4 °C, bound
I-VEGF
was measured as
described under ``Experimental Procedures.'' Nonspecific
binding was determined for each binding reaction in the presence of 1
µg/ml of unlabeled VEGF
as described. Nonspecific
binding values were subtracted from the total binding to yield specific
binding values. The figure shows the inhibition of
I-VEGF
binding by PF4 as a function of the
concentration of added heparin. Error bars represent the
deviation from the mean of triplicate
measurements.
The binding of I-VEGF
to the VEGF receptors was already
potentiated by 0.1 µg/ml heparin (compare Fig. 2A, lanes
1 and 3)(9) , but this concentration of heparin
had no noticeable effect on the inhibitory effect of 2 µg/ml PF4 (Fig. 2A, lane 4). When 1 µg/ml heparin was used,
the potentiation of
I-VEGF
binding was
maximal, but at this heparin concentration the activity of PF4 was
already partially inhibited (Fig. 2B). At this
concentration of heparin the formation of the two smaller
I-VEGF
-receptor complexes was completely
inhibited, whereas the formation of the 225-kDa
I-VEGF
-receptor complex was only partially
inhibited by PF4 (compare Fig. 2A, lane 5 with lane
6). In the presence of 10 µg/ml heparin the inhibitory effect
of PF4 was completely nullified, whereas the potentiation of
I-VEGF
binding remained almost maximal (Fig. 2B). These experiments indicate that heparin
inhibits the inhibitory effects of PF4 and that it independently
potentiates
I-VEGF
binding to the VEGF
receptors of the cells. Heparan sulfate had effects which resembled
those of heparin, except that the potentiation of
I-VEGF
binding was smaller, and the
abolishment of the PF4 inhibitory activity was likewise less efficient (Fig. 2B). Other glycosaminoglycans such as chondroitin
sulfate had no effects on the inhibitory activity of PF4, nor did they
affect the binding of
I-VEGF
to VEGF
receptors (not shown)(45) .
The anti-angiogenic
activity of PF4 may not depend upon the heparin binding capacity of
PF4, because truncated PF4 lacking heparin binding ability retains
anti-angiogenic properties(36) . We therefore examined
alternative mechanisms by which PF4 may inhibit VEGF activity. To find
out if PF4 is able to bind to VEGFI-PF4 Binds to
VEGF
-coated Wells
,
I-PF4
was bound to VEGF
-coated 96-well dishes.
I-PF4 did not bind to uncoated wells (Fig. 3, column 3), or to wells coated with inactive reduced monomers
of VEGF
(not shown), but VEGF
-coated wells
bound
I-PF4 efficiently (Fig. 3, column
1). The binding of
I-PF4 to the
VEGF
-coated dishes was saturable, and Scatchard analysis
done with the aid of the LIGAND program (46) showed that the
dissociation constant of
I-PF4 was 5
10
M. The binding was inhibited by
unlabeled PF4, by unlabeled VEGF
, and by an antiserum
directed against VEGF
(Fig. 3, columns 2,
4, and 6, respectively). In contrast, pre-immune serum
(not shown) and inactive monomers of reduced VEGF
(Fig. 3, column 5) were not able to inhibit the
binding.
Figure 3:
I-PF4 binding to
VEGF
-coated wells. Wells of 96-well dishes were incubated
with coating buffer in the absence (column 3) or presence (columns 1, 2, 4-9) of 20 ng/well VEGF
for
2 h at room temperature as described under ``Experimental
Procedures.'' The VEGF
containing solution was
aspirated, and the wells were incubated for 1 h in coating buffer
containing 0.1% gelatin. The wells were then preincubated for another
hour at 37 °C with 50 µl of binding buffer (20 mM HEPES, pH 7.3, 0.1% gelatin in Dulbecco's modified
Eagle's medium) in the absence of any additions (columns
1-6), in the presence of 0.05 unit/ml heparinase (column
7), in the presence of 200 ng/ml heparin (column 8), or
in the presence of both heparinase and heparin (column 9).
Following preincubation, coating buffer (50 µl) containing
I-PF4 was added to give a final concentration of 100
ng/ml
I-PF4. Some wells received at that time the
following substances in addition to give the indicated final
concentrations: 10 µg/ml PF4 (column 2), 10 µg/ml
VEGF
(column 4), 10 µg/ml reduced
VEGF
(column 5), anti-VEGF antiserum (1:50) (column 6). The wells were incubated for 1 h at 37 °C and
were subsequently washed three times with wash buffer. Bound
I-PF4 was then extracted from the wells using 100 µl
of 0.5 N NaOH. Aliquots were counted in a
-counter. 100%
equals 10,000 cpm/well.
Both VEGF and PF4 were purified using
heparin-Sepharose affinity chromatography, and low concentrations of
heparin which may have been released from the column could therefore be
present as contaminants in our PF4 and in our VEGF
. Such
heparin molecules could conceivably form a link between the immobilized
VEGF
and
I-PF4 and mediate the binding of
I-PF4 to VEGF
-coated wells. To exclude this
possibility,
I-PF4 was bound to VEGF
-coated
wells in the presence of the heparin degrading enzyme heparinase-1 (Fig. 3, column 7). Heparinase-1 did not inhibit the
binding of PF4 to the immobilized VEGF
, indicating that
heparin is not involved in the binding mechanism. Similar results were
obtained when VEGF
or
I-PF4 were digested
separately with heparinase before the binding reaction. Addition of
heparin to the binding reaction inhibited the binding completely (Fig. 3, column 8). The failure of heparinase-1 to
inhibit
I-PF4 binding to VEGF
was not due
to inactivation of the heparinase-1, because the same heparinase-1
prevented efficiently the heparin-induced inhibition of
I-PF4 binding to VEGF
-coated wells (Fig. 3, column 9).
The Binding of
To find out if I-PF4 to
VEGF
-coated Wells Is Inhibited by Various
Heparin-binding Proteins
I-PF4
is also able to bind to VEGF
, the binding of
I-PF4 to VEGF
was done in the presence of
10 µg/ml VEGF
. VEGF
was not able to
inhibit the binding of
I-PF4 to VEGF
-coated
wells (Fig. 4, column 9), and subsequent experiments
showed that
I-PF4 is not able to bind to VEGF
coated wells (not shown). The only difference between
VEGF
and VEGF
is the presence of the domain
encoded by exon 7 of the VEGF gene, containing the putative heparin
binding domain of VEGF
(47) . This experiment,
therefore, indicated that the binding of
I-PF4 to
VEGF
may depend upon the presence of a heparin binding
domain.
Figure 4:
Inhibition of I-PF4 binding
to VEGF
by heparin-binding proteins. VEGF
was adsorbed (columns 1-16) or not (column
17) to the wells of 96-well plates as described under
``Experimental Procedures.''
I-PF4 (100 ng/ml)
was bound to the VEGF
-coated wells in the absence (column 1) or presence of the following additions (10
µg/ml): insulin (column 2), soybean trypsin inhibitor (column 3), thyroglobulin (column 4), transferrin
(column 5), protein A (column 6), bovine serum albumin (column 7), cytochrome c (column 8),
VEGF
(column 9), lysosyme (column 10),
bFGF (column 11), keratinocyte growth factor (column
12), protamine (column 13), PF4 (column 14), VEGF
(column 15), acidic fibroblast growth factor (column
16). The binding and the subsequent quantification of bound
I-PF4 were done as described under ``Experimental
Procedures.'' 100% represents 6000 cpm bound per
well.
To further test this hypothesis, a series of heparin-binding
proteins were examined for their ability to inhibit the binding of I-PF4 to VEGF
-coated wells.
I-PF4 binding to VEGF
-coated wells was
inhibited by all of the heparin-binding proteins tested (Fig. 4, columns 11-16). These included bFGF, keratinocyte growth
factor, protamine, PF4, VEGF
, and aFGF. The heparin
binding cytokine interleukin-8 also inhibited PF4 binding to
VEGF
(not shown). In contrast, a series of proteins
lacking heparin binding ability (Fig. 4, columns
2-10) and 1% fetal calf serum (not shown) were not able to
inhibit
I-PF4 binding. The heparin binding ability of the
proteins was probably more significant than their general charge, since
a basic protein such as cytochrome c was unable to inhibit the
binding (Fig. 4, column 7). However, very basic
synthetic polymers such as polylysine and polyarginine were able to
inhibit the binding. It therefore seems that PF4 may be able to bind to
several types of proteins provided that they contain a basic heparin
binding domain. This tentative conclusion was also supported by
experiments showing that
I-PF4 is able to bind to wells
coated with bFGF or protamine, but not to wells coated with cytochrome c or bovine serum albumin (not shown).
PF4 Inhibits the Mitogenic Effects of Both VEGF
The previous experiments
indicated that PF4 may be able to inhibit the interaction of
VEGFand VEGF
with the VEGF receptors by inhibiting the
interaction of VEGF
with cell surface heparan sulfates or
by directly binding to VEGF
. However, the binding of
VEGF
to the VEGF receptors was not affected by PF4, and
in addition PF4 was not able to bind to VEGF
. We
therefore expected that PF4 would prove to be an efficient inhibitor of
VEGF
-induced cell proliferation but that it will not be
able to inhibit the mitogenic activity of VEGF
.
-induced proliferation of HUE
cells (Fig. 5). The mitogenic effect of 5 ng/ml VEGF
was completely inhibited in the presence of 5 µg/ml PF4,
although at this concentration the basal proliferation rate of the
cells (in the absence of added growth factor) was also significantly
inhibited. However, when a 0.5 µg/ml PF4 was used, the specific
VEGF
-induced proliferation of the cells was already
inhibited by about 75% (Fig. 5A), whereas the basal
proliferation of the cells remained almost unaltered. Contrary to our
expectations, PF4 also inhibited efficiently the mitogenic activity of
VEGF
(Fig. 5, A and B). The
mechanism of the inhibition seems to be noncompetitive, since
increasing the VEGF
or VEGF
concentration
did not alleviate the inhibition caused by a fixed concentration of PF4 (Fig. 5B). Inclusion of heparin (10 µg/ml) prevented
the PF4-induced inhibition of VEGF
or VEGF
cell proliferation (not shown). It therefore seems that PF4 is
also able to inhibit VEGF-induced cell proliferation using an unknown
mechanism that does not interfere with the binding of the various VEGF
forms to VEGF receptors.
Figure 5:
PF4
inhibits both VEGF- and VEGF
-induced
proliferation of vascular endothelial cells. A, HUE cells were
seeded in 24-well dishes at a concentration of 20,000 cells/well in
M199 medium containing 10% fetal calf serum and antibiotics. After cell
attachment, the medium was exchanged for endothelial serum-free medium.
The cells were cultured with 5 ng/ml VEGF
(
), with
10 ng/ml VEGF
(
), or in the absence of added growth
factors (
) and in the presence of the indicated concentrations
of PF4. Additions of growth factors and PF4 were done every other day.
The cells were counted in a Coulter counter after 4 days. Points
represent the average of triplicate wells. The deviation from the mean
within points did not exceed 10%. The experiment was repeated three
times with similar results. B, HUE cells were seeded in
24-well dishes (15,000 cells/well) in the presence of increasing
concentrations of VEGF
(
,
) or VEGF
(
,
) in the absence (
,
) or presence
(
,
) of 3 µg/ml PF4. VEGF and PF4 were added every
other day. Cells were detached using trypsin and counted in a Coulter
counter on the 5th day. Results represent the average of triplicate
wells, and the variation among wells was less than
10%.
-
or VEGF
-induced proliferation of endothelial cells. The
binding of VEGF
to the VEGF receptors of the cells was
inhibited efficiently by PF4, but the binding of VEGF
to
the VEGF receptors of the cells was not affected by PF4. This result is
surprising, since both VEGF
and VEGF
bind
to the KDR/flk-1 VEGF receptor which was shown to transduce
VEGF
mitogenic signals in endothelial
cells(17, 48) .
by at least two concurrent mechanisms. PF4 can
probably compete with VEGF
for binding to the cell
surface heparan sulfate chains that are required for the efficient
binding of VEGF
to VEGF receptors(9) . Using this
mechanism PF4 would produce an inhibitory effect similar to that
produced by the digestion of cell surface heparan sulfates by
heparinase(9) . PF4 also inhibited specifically the
heparin-dependent binding of VEGF
to soluble flk-1/SEAP receptors. Since flk-1/SEAP does not bind
efficiently to heparin(38) , and since VEGF
binding to soluble flk-1/SEAP receptors is not inhibited
by PF4, it seems likely that the inhibition of VEGF
binding is produced by competition for available heparin-like
molecules. This is also the mechanism that was suggested for PF4
inhibition of bFGF-induced cell proliferation, as the activity of bFGF
also seems to depend upon the availability of cell surface heparin-like
molecules(34, 49, 50, 51) . This
hypothesis is also supported by experiments indicating that PF4 derived
C-terminal peptides containing the heparin binding domain of PF4 retain
an anti-angiogenic activity and inhibit the binding of bFGF to the bFGF
receptors of vascular endothelial cells(32, 35) .
, competition for cell surface heparan sulfate
residues cannot be the only mechanism by which PF4 can inhibit the
mitogenic activity of VEGF
. Unlike VEGF
,
VEGF
does not interact with the VEGF receptors in a
heparan sulfate-dependent manner,
and its binding to the
VEGF receptors of the endothelial cells or to soluble flk-1 is
not inhibited by PF4. It therefore seems that PF4 can block
VEGF
- or VEGF
-induced cell proliferation by
an additional mechanism that interferes with VEGF-induced signal
transduction. This conclusion is also supported by experiments
indicating that PF4 mutants which have lost their heparin binding
ability are still able to inhibit angiogenesis(36) .
? In searching
for alternative mechanisms by which PF4 mutants deficient in heparin
binding ability may function to inhibit angiogenesis, we have noticed
that PF4 can bind directly to a variety of heparin-binding proteins.
The binding was not dependent upon the presence of heparin and could
perhaps be mediated by the highly acidic free N-terminal domain of
PF4(52) . The ability of PF4 to bind to heparin binding growth
factors such as VEGF
and bFGF could contribute to the
inhibitory effects that PF4 exerts on the receptor binding ability of
such heparin binding growth factors and may constitute another
potential inhibitory mechanism(32, 35) . This property
of PF4 may also enable it to bind to cell surface heparin receptors.
Vascular endothelial cells contain cell surface heparin binding sites
of unknown function(9, 53, 54) , and it is
likely that several types of cell surface proteins may function as
heparin receptors. Some of these heparin receptors may correspond to
known proteins such as the FGFR-1 receptor for FGF which was reported
to contain a heparin binding domain in its extracellular
part(55) . Others may perhaps be able to regulate cell cycle
progression and the binding of heparin-like molecules could perhaps
modulate this activity(44, 56) . If this supposition is
correct, PF4 may be able to bind to such cell surface heparin-binding
proteins and disrupt their normal function.
and of VEGF
. PF4 apparently inhibits the activity
of the various VEGF forms using several concurrent mechanisms. One such
mechanism could involve binding to cell surface heparin-like molecules
required for the receptor binding ability of heparin binding growth
factors like VEGF
, whereas another mechanism may involve
direct binding of PF4 to heparin binding growth factors. A third
mechanism by which PF4 inhibits VEGF signal transduction also seems to
exist, but its nature remains to be elucidated.
, 165-amino acid form of vascular endothelial growth
factor; VEGF
, 121-amino acid form of vascular endothelial
growth factor; ABAE, bovine aortic arch-derived endothelial cells;
aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth
factor; HUE, human umbilical vein-derived endothelial cells; PBS,
Dulbecco's phosphate-buffered saline; PF4, platelet factor 4.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.