©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Platelet Factor-4 Inhibits the Mitogenic Activity of VEGF and VEGF Using Several Concurrent Mechanisms (*)

Stela Gengrinovitch (1), Sheryl M. Greenberg (2), Tzafra Cohen (1) (3), Hela Gitay-Goren (1), Patricia Rockwell (4), Theodore E. Maione (2), Ben-Zion Levi (3), Gera Neufeld (1)(§)

From the (1)Department of Biology, Technion, Israel Institute of Technology, Haifa, 32000, Israel, the (2)Repligen Corporation, Cambridge, Massachusetts 02139, the (3)Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa, 32000, Israel, and (4)ImClone Systems Inc., New York, New York 10014

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 121-amino acid form of vascular endothelial growth factor (VEGF) 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 10M) 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.


INTRODUCTION

Vascular endothelial growth factors (VEGFs)()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) .

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 -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) .

In the present work we show that PF4 inhibits the binding of VEGF 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.


EXPERIMENTAL PROCEDURES

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. NaI 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.

The binding and the cross-linking of 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 I-PF4 to VEGF-coated Wells

Coating buffer (50 µl of 20 mM KHPO4, 10 mM KHPO, 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.


RESULTS

PF4 Inhibits the Binding of I-VEGFto the VEGF Receptors of Vascular Endothelial Cells

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 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 I-VEGFBinding by PF4

The binding of 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) .

I-PF4 Binds to VEGF-coated Wells

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 VEGF, 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 10M. 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 I-PF4 to VEGF-coated Wells Is Inhibited by Various Heparin-binding Proteins

To find out if 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 VEGFand VEGF

The previous experiments indicated that PF4 may be able to inhibit the interaction of 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.

PF4 inhibited efficiently 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%.




DISCUSSION

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- 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) .

Our experiments indicate that PF4 can block the activity of a heparin binding growth factor like VEGF 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) .

Because PF4 also inhibited the mitogenic activity of VEGF, 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) .

How does PF4 inhibit the mitogenic activity of VEGF? 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.

In conclusion, we have shown here that PF4 inhibits the mitogenic activity of VEGF 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.


FOOTNOTES

*
This work was supported by grants from the Israel Ministry of Science, joint programs with Germany (DKFZ and GSF), by a grant from the Israel Academy of Sciences and Humanities, and by a grant from the Israel Cancer Research Fund (ICRF) (to G. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-4294216; Fax: 972-4225153; E-mail: gera@techunix.technion.ac.il.

The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF, 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.

H. Gitay-Goren, T. Cohen, S. Tessler, S. Soker, S. Gengrinovitch, P. Rockwell, M. Klagsbrun, B.-Z. Levi, and G. Neufeld, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Dina Ron for critically reading this manuscript.


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