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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 984-993
Inhibition of In Vitro Angiogenesis by Platelet Factor-4-Derived
Peptides and Mechanism of Action
By
Valérie Jouan,
Xavier Canron,
Monica Alemany,
Jacques P. Caen,
Gérard Quentin,
Jean Plouet, and
Andreas Bikfalvi
From the Growth Factor and Cell Differentiation Laboratory,
University Bordeaux I, Talence; Institut des Vaisseaux et du Sang,
Paris; SERBIO, Gennevilliers; and CNRS UPR 9006, Toulouse, France.
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ABSTRACT |
In this study, we examined in detail the interaction of platelet
factor-4 (PF-4) with fibroblast growth factor-2 (FGF-2) and vascular
endothelial growth factor (VEGF) and the effect of PF-4-derived synthetic peptides. We show that a peptide between amino acids 47 and
70 that contains the heparin-binding lysine-rich site inhibits FGF-2 or
VEGF function. This is based on the following observations: PF-4
peptide 47-70 inhibited FGF-2 or VEGF binding to endothelial cells; it
inhibited FGF-2 or VEGF binding to FGFRs or VEGFRs in heparan
sulfate-deficient CHO cells transfected with FGFR1 (CHOFGFR1) or
VEGFR2 (CHOmVEGFR2) cDNA; it blocked proliferation or tube formation in
three-dimensional angiogenesis assays; and, finally, it competed with
the direct association of 125I-PF-4 with FGF-2 or VEGF,
respectively, and inhibited heparin-induced FGF-2 dimerization. A
shorter C-terminal peptide (peptide 58-70), which still
contained the heparin-binding lysin-rich site, had no effect. Peptide
17-58, which is located in the central part of the molecule, although
it does not inhibit FGF-2 or VEGF binding or biologic activity in
endothelial cells, inhibited heparin-dependent binding of
125I-FGF-2 or 125I-VEGF to CHOmFGFR1 or
CHOmVEGFR2 cells, respectively. Shorter peptides (peptides 34-58 and
47-58) did not show any of these effects.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ANGIOGENESIS IS CONTROLLED by a balance
of positive and negative regulators. Fibroblast growth factor-2 (FGF-2)
and vascular endothelial growth factor (VEGF) are among the principal positive regulators.1,2 Negative regulators include
inhibitory molecules, such as angiostatin,3,4
endostatin,5 thrombospondin-1 (TSP-1),6 the
16-kD human prolactin fragment (16-kD PRL),7 and platelet
factor-4 (PF-4) 8.
FGF-2 and VEGF mediate their biologic activity by binding to specific
cell surface receptors. The interaction of these ligands with their
receptors is modulated by heparan sulfate proteoglycans (HSPGs). For
example, HSPG stabilizes the FGF-2/FGF receptor complex, protects FGF-2
from degradation, and facilitates FGF-2 dimerization.1 HSPGs also modulate the binding of VEGF to VEGFR1 or
VEGFR2.9 This indicates that angiogenesis inhibitors may
possibly interfere with FGF-2 or VEGF activity by disrupting
HSPG/FGF-2/FGF receptor or HSPG/VEGF/VEGF receptor interactions.
PF-4 is a member of the C-X-C chemokine family.10 PF-4 is a
7.8-kD protein of 70-amino acid length11 that shares
homologies in particular with  -thromboglobulin and interleukin-8
(IL-8).10,12 The crystal structure of human PF-4 has been
solved to a resolution of 2.4 A.13 The major
heparin-binding site forms a  helicoidal structure and is located at
the C-terminus between 61 and 70. In contrast, the central core forms
antiparallel -sheet-like structures. Furthermore, additonal
potential heparin-binding sites exist as a positively charged ring of
lysine and arginine side chains that encircles the PF-4
molecule.14
A number of observations indicate that PF-4 is an inhibitor of
angiogenesis. First, PF-4 inhibits endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo.8,15
Second, PF-4 is targeted in vivo to endothelial cells that undergo
active angiogenesis.16 Third, tumor growth in vivo is
inhibited by PF-4 through an angiogenesis-dependent
mechanism.17,18 Recombinant human PF-4 inhibits tumor
angiogenesis and the growth of melanoma cells or HCT 116 colon
carcinoma cells.17 In addition, human glioma cells infected
with a secretable PF-4 cDNA grew slowly in vivo and formed only
hypovascular tumors in vivo.19 Megakaryocytes and platelets
are the major source of PF-4. Indeed, activated platelets release an
inhibitor of FGF-2 activity that was shown to be identical to
PF-4.20 Thus, PF-4 may counteract excessive angiogenic
factor activity at sites of platelet activation.
In a series of systematic studies, we have recently partially
elucidated the mechanism of inhibition by which PF-4 inhibits FGF-2
activity.21 We have demonstrated that PF-4 inhibits FGF-2 binding to high-affinity receptors and inhibits FGF-2 internalization. Furthermore, PF-4 is able to bind surface-immobilized or soluble FGF-2
and inhibited endogenous and heparin-induced FGF-2 dimerization. Similarily, PF-4 also binds surface-immobilized VEGF.22
In the present study, we sought to determine the inhibitory capacity of
various peptides that correspond to different PF-4 domains.
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MATERIALS AND METHODS |
Synthetic peptides.
Peptides were synthesized using standard solid-phase methodology and
purified by high-performance liquid chromatography (HPLC) using a C18
column and a 0% to 80% linear acetonitrile gradient in 0.1%
trifluorouracetic acid. The following synthetic peptides were used for
the study. Peptide 47-70: NGRKICLDLQAPLYKKIIKKLLES; peptide 58-70:
PLYKKIIKKLLES; peptide 47-58: NGRKICLDLQAP; peptide 34-58:
PHCPTAQLIATLKNGRKICLDLQAP; peptide 17-58:
SQVRPRHITSLEVIKAGPHCPTAQLIATLKNG RKICLDLQAP. Recombinant human PF-4 or
platelet-purified PF-4 (Serbio, Gennevilliers, France) was used as
control in the different experiments.
Cells.
Murine lung microvascular endothelial cells (LEII cells; (kindly
donated by Dr Thomas Maciag, American Red Cross, Rockville, MD) were
grown in Dulbecco's modifed Eagle's medium (DMEM; GIBCO, Life
Technologies, Gaithersburg, MD) containing 10 % fetal calf serum (FCS;
GIBCO), 1 g/L glucose, 1% glutamin, and 50 IU/mL penicillin-50 µg/mL streptomycin at 37°C in a 5% CO 2 atmosphere.
Adrenal cortex capillary endothelial cells (ACE cells; kindly donated
by Dr Jean-Jacques Feige, CENG, Grenoble, France) were grown in the
same medium as LEII cells, except that FCS newborn calf serum (NCS) was
used instead of FCS. Heparan sulfate-deficient chinese hamster ovary cells (CHOm-FGFR1 cells, 745-flg; kindly donated by Dr Avner Yayon, The
Weizmann Institute, Rehovot, Israel) were grown in DMEM containing 10%
FCS, 1 g/L glucose, and 1% nonessential amino acids at 37°C in a
5% CO 2 atmosphere. Heparan sulfate-deficient CHO cells
were transfected with a human VEGFR2 cDNA (CHOmVEGFR2 cells) in a pSV7d vector as already described.23
Cell proliferation experiments.
Proliferation assays were performed as described.24
Briefly, cells were seeded at 20,000 cells on 3.5-cm2
dishes in DMEM containing 10% FCS, 1% glutamin, and
antibiotics, or into wells of 24-well plates at 7,000 cells/well. After
overnight attachment, the cells were washed once with serum-free DMEM
and test medium that contained 1% FCS or 1% NCS and subsequently
incubated with the indicated concentrations of recombinant human FGF-2, recombinant human baculovirus-derived VEGF 165 (165-amino acid splicing variant of VEGF), PF-4, or PF-4 peptides. Cells were counted
at specified days with a Coulter counter (Coultronics, Margency, France).
In vitro angiogenesis assays.
In vitro angiogenesis assays were performed according to Montesano et
al25 using collagen type I as the three-dimensional matrix.
Briefly, 20,000 cells were plated on the top of collagen type I gels
into wells of 96-well plates. Twenty-four hours after plating, 10 ng/mL
FGF-2 or VEGF was added with or without the indicated peptide
concentrations. The experiments were performed over a period of 7 days,
after which photographs were taken from each well using a 10X objective
(Zeiss, Wetzlar, Germany). Alternatively, the cells were fixed with
methanol, stained with methylene blue, and photographed. Endothelial
tube length was quantified in each well using a Biocom
image analyzer (Biocom, les Ulis, France). The results were expressed
as the mean ± SD of the total tube length from experiments run in duplicates.
Binding studies and cross-linking to receptors.
FGF-2 and PF-4 were labeled with 125I-Na using iodogen
(Pierce, Rockford, IL) as coupling agent according to the
manufacturer's indications and according to Moscatelli.26
VEGF was labeled with 125I-Na using iodobeads (Pierce). The
specific activities of 125I-FGF-2 , 125I-PF-4
and 125I-VEGF were 80,000 to 200,000 cpm/ng, 35,000 to
100,000 cpm/ng, and 150,000 to 200,000 cpm/ng, respectively. FGF-2
binding experiments to high- and low-affinity sites were performed
essentially as described by Moscatelli.26 Cells were seeded
at 2.5 × 105/cm2 and cultured in complete
medium into 3.5-cm diameter dishes for 2 days. Cells were washed twice
with ice-cold phosphate-buffered saline (PBS) and incubated with the
indicated concentrations of 125I-FGF-2 in DMEM, which
contained 20 mmol/L HEPES (pH 7.4), 0.15% gelatin, for 2 hours at
4°C. At the end of the incubation period, cells were washed three
times with ice-cold PBS. 125I-FGF-2 was dissociated from
its cellular low-affinity binding sites by two 20-second washes with
ice-cold 20 mmol/L HEPES (pH 7.4), 2 mol/L NaCl, and from its
high-affinity sites by two 20-second washes with ice-cold 20 mmol/L
NaAc (pH 4.0), 2 mol/L NaCl. Bound 125I-FGF-2 was
quantified using a Kontron MR 250  -counter (Saint-Quentin-Yvelines, France). Nonspecific binding was determined by incubating separate dishes with 125I-FGF-2 and a 100-fold excess of unlabeled
ligand. Specific binding was determined by substracting nonspecific
binding from total binding.
Cross-linking of 125I-FGF-2 or 125I-VEGF to
receptors was performed and analyzed as described by Bikfalvi et
al,21 using 0.2 mmol/L Bis(sulfosuccinyl) suberate
(BS3; Pierce) in PBS as a coupling agent. The quantity of
protein used in each experiment was normalized according to cell number
or protein. Samples were run on a 7% sodium dodecyl sulfate
(SDS)-polyacrylamide gel. Autoradiographies were performed with X-OMAT
AR films (Eastman Kodak, Rochester, NY) at  80°C in the
presence of an intensifying screen or analyzed by PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
125I-PF-4 binding experiments with surface-immobilized
FGF-2 or VEGF.
Binding of 125I-PF-4 to FGF-2 or VEGF 165-coated wells was
performed as follows. Ninety-six-well enzyme-linked immunosorbent assay (ELISA) plates were coated with FGF-2 or VEGF in buffer A (1 mmol/L EDTA, 20 mmol/L K2HPO4, 10 mmol/L
KH2PO4, 150 mmol/L NaCl) in a volume of 50 µL. After a 2-hour incubation at room temperature, the plates were
washed five times with buffer B (10 mmol/L Tris-HCl pH 7.2, 150 mmol/L
NaCl, 0.1% Tween 20). The wells were subsequently incubated with
buffer A with an additional 0.1% gelatin and washed five times after 1 hour with buffer B. The FGF-2- or VEGF-coated wells were then
incubated in buffer A with 10 ng/well 125I-PF-4,
competitors, or polyclonal anti-PF-4 antibodies at 37°C for 1 hour. Rabbit polyclonal anti-PF-4 antibodies were prepared as
previously reported.27 At the end of the incubation period, the wells were washed five times with buffer B. Surface-associated 125I-PF-4 was then extracted with 200 µL of 0.2 mol/L
NaOH and counted in a -counter. At the end of the preincubation
period, the wells were washed twice before the addition of
125I-PF-4. To determine nonspecific binding, separate wells
were not coated with FGF-2 and only preincubated with buffer A before the initiation of binding. Specific binding was determined by substracting nonspecific binding from total binding.
FGF-2 dimerization experiments.
FGF-2 dimerization was studied according to the method described by
Ornitz et al.28 Briefly, 5 ng 125I-FGF-2 and
500 ng unlabeled FGF-2 were incubated for 1 hour at room temperature
with or without the indicated concentrations of heparin and PF-4 or
peptides in PBS in a final volume of 45 µL. At the end of the
incubation period, 5 µL BS3 (0.1 mmol/L final
concentration) was added for another 30-minute incubation. The reaction
was stopped by adding SDS-sample buffer from a five-times concentrated
stock solution. The samples were boiled and run on a 12% or 15%
SDS-polyacrylamide gel. The gels were analyzed by PhosphorImager (Molecular Dynamics, Bondoufle, France) or
autoradiography. For all of the experiments outlined here,
autoradiograms or PhosphoImager results were analyzed by a public
domain National Institutes of Health (NIH) Image Program developed at
the US NIH and available over the internet by anonymous FTP from
zippy.nimh.nih.gov or by using a Bio Profil V 6.0 scanner with Bio 1 D
software (Vilber Lourmat, Paris, France).
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RESULTS |
Effect of PF-4 peptides on FGF-2 or VEGF-induced endothelial cell
proliferation or angiogenesis in vitro.
We examined the effect of the different peptides on the biologic
activity induced by FGF-2 or VEGF. We first performed proliferation assays using bovine ACE cells or murine LEII cells. Inhibition of
FGF-2-induced endothelial cell proliferation was only observed with 10 µmol/L of peptide 47-70, but not with peptide 47-70, 58-70, 17-58, 34-58, or 47-58 (Fig 1A through D). Inhibition of
endothelial cell proliferation was observed for both ACE cells and LEII
cells and was consistent during the whole time course of incubation.
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| Fig 1.
Effect of PF-4-derived peptides on endothelial cell
proliferation. (A) ACE cells or (B) LEII cells are incubated in DMEM
containing 1% bovine serum (FCS for LEII cells and NCS for ACE cells)
with or without 10 ng/mL FGF-2 and 10 µmol/L PF-4-derived peptides.
1% serum without FGF-2 ( ), 1% serum with FGF-2 ( ), 1% serum
with FGF-2 and peptide 47-70 ( ), 1% serum with FGF-2 and peptide
34-58 ( ). Cells are counted every other day. (C) ACE cells or (D)
LEII cells are incubated with 10 ng/mL FGF-2 and 10 µmol/L of various
peptides and counted after 6 days.
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To ensure that sprouting and tubulogenesis were also impaired by
peptide 47-70, we performed in vitro angiogenesis assays according to
Montesano et al.25 In the absence of peptide, FGF-2 or VEGF
induced a total tube length of 3.5 mm ± 0.25/well (mean ± SD)
and 2.1 mm ± 0.65/well. respectively. A 10-µmol/L quantity of
peptide 47-70 completely inhibited tube formation induced by FGF-2 or
VEGF (Fig 2A and B). On the contrary,
peptides 17-58, 47-58, 34-58, or 58-70 were unable to inhibit tube
formation induced by FGF-2 or VEGF (Fig 2A and B). However, peptide
47-58 was able to increase FGF-2-stimulated tube formation to 3.5 mm ± 0.25/well to 6 mm ± 0.65/well (mean ± SD; 175% in
comparison to control). This effect was not seen when tube formation
was induced with VEGF.
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| Fig 2.
Effect of PF-4-derived peptides on angiogenesis in
vitro. ACE cells are grown onto 3-dimensional collagen type I gels in
the presence of 10 ng/mL FGF-2 (A) or VEGF (B) in the presence or
absence of 10 µmol/L of the different peptides. Photomicrographs were
taken after 7 days incubation. Results were quantified as indicated in
Materials and Methods and expressed as the mean ± SD of experiments
done in duplicates.
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These experiments indicate that FGF-2 or VEGF activities are inhibited
by peptide 47-70, but not by peptides 58-70, 17-58, 47-58, or 34-58. In
addition, peptide 47-58 appears to increase FGF-2-stimulated tube formation.
Effect of PF-4 peptides on 125I-FGF-2 or
125I-VEGF binding on endothelial cells.
We next investigated the effects of peptides 17-58, 34-58, 47-58, 47-70, or 58-70 on the inhibition of 125I-FGF-2 or
125I-VEGF binding to endothelial cells. We used LEII cells
and ACE cells for these studies, because both cell types express FGF or VEGF receptors. PF-4 inhibited 125I-FGF-2 binding to low-
and high-affinity binding sites in endothelial cells with a
half-maximum inhibition (IC50) of 0.6 µg/mL for the inhibition of
FGF-2 binding to the high-affinity binding sites.21 Binding
of 125I-VEGF to endothelial cells was also inhibited by
PF-4 with an IC50 of 0.25 µmol/L (data not shown). When the peptides
indicated above were used in these binding experiments, inhibition was
only observed with peptide 47-70 (Fig 3A
through C). High-affinity binding of 125I-FGF-2 to FGF
receptors on LEII or ACE cells was inhibited maximally at 5 µmol/L
and half maximally at 1 µmol/L peptide concentration (Fig 3A and data
not shown). Peptides 58-70, 17-58, 34-58, and 47-58 were without effect
(Fig 3B and C).
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| Fig 3.
Effect of PF-4-derived peptides on binding of
125I-FGF-2 to endothelial cells. LEII cells or ACE cells
are incubated with 10 ng/mL 125I-FGF-2 in the presence or
absence of 2 µg/mL unlabeled ligand (nonspecific binding, NS), 10 µg/mL PF-4 or PF-4-derived peptides. Binding experiments are
performed as indicated in Materials and Methods. Binding experiments on
LEII cells with increasing concentrations (A) of peptides 47-70 ()
or 34-58 () or fixed concentrations (B, 10 µmol/L) of the
indicated peptides; binding experiments on ACE cells with fixed
concentrations (C, 10 µmol/L) of indicated peptides. Results are
expressed as the mean± SD of experiments done in duplicates.
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We then examined the effect of the different peptides on the binding of
125I-VEGF to LEII or ACE cells (Fig
4A through D). Peptide 47-70 at 5 to 10 µmol/L completely inhibited 125I-VEGF binding to both
endothelial cell types with a IC50 of 1.5 and 2 µmol/L for LEII and
ACE cells, respectively (Fig 4B and D). Peptides 58-70, 17-58, 34-58, and 47-58 did not show inhibitory activity (Fig 4 A and C).
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| Fig 4.
Effect of PF-4±derived peptides on binding of
125I-VEGF to endothelial cells. LEII cells or ACE cells are
incubated with 5 ng/mL 125I-VEGF in the presence or absence
of 500 ng/mL unlabeled ligand (nonspecific binding, NS), or
PF-4-derived peptides. Binding experiments are performed as indicated
in Materials and Methods. Binding experiments on LEII cells with 10 µmol/L of indicated peptides (A) or increasing concentrations (B) of
peptide 47-70; binding experiments on ACE cells with fixed (10 µmol/L) concentrations (C) of the different peptides or increasing
concentrations (D) of peptide 47-70. Results are expressed as the mean ± SD of experiments done in duplicates.
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These results indicate that peptide 47-70 not only interferes with the
binding of FGF-2, but also with the binding of VEGF to endothelial cells.
Effect of PF-4-derived peptides on 125I-FGF-2 or
125I-VEGF binding on heparan sulfate-deficient CHO cells
expressing FGFR1 or VEGFR2.
We next set out to examine the inhibitory activity of the various
peptides by using heparan sulfate-deficient CHO cells that were
transfected with either expression vectors that contained FGFR1(CHOmFGFR1) or VEGFR2 (CHOmVEGFR2) cDNAs. We have previously shown
that PF-4 inhibits the heparin-dependent and -independent binding of
125I-FGF-2 in CHOmFGFR1.21 As depicted in Fig
5A, peptide 47-70 did only slightly inhibit
125I-FGF-2 binding to CHOmFGFR1 cells in the absence of
heparin ( 20% at 10 µmol/L in comparison to control). However, in
the presence of heparin (100 ng/mL), heparin-induced binding was
strongly impaired by peptide 47-70 (Fig 5B and C). Maximum inhibition
of 125I-FGF-2 binding by peptide 47-70 was attained at 2 µmol/L (IC50 at 0.35 µmol/L). This is also reinforced by
cross-linking of 125I-FGF-2 to FGFR1 in the presence of
peptide 47-70 (Fig 5D). These experiments show that increasing
concentrations of peptide 47-70 (0.6 to 4 µmol/L) progressively
decreased the intensity of the signal that corresponds to
125I-FGF-2/FGFR1 complexes (Fig 5D). None of the other
peptides inhibited 125I-FGF-2 binding in the absence of
heparin (Fig 5A and data not shown). However, in the presence of
heparin, peptide 17-58, like peptide 47-70, inhibited
125I-FGF-2 binding (maximum inhibition of 50% to 60%
of binding, Fig 5C). Maximum inhibition was reached at 2 µmol/L and
the IC50 at 0.7 µmol/L. Peptides 34-58, 47-58, and 58-70 did not
inhibit heparin-induced 125I-FGF-2 binding to CHOmFGFR1
cells.
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| Fig 5.
Effect of PF-4-derived peptides on binding and
cross-linking of 125I-FGF-2 to CHOmFGFR1 cells. CHOmFGFR1
cells are incubated with 10 ng/mL 125I-FGF-2 with or
without 2 µg/mL unlabeled ligand, 10 µg/mL PF-4 or PF-4-derived
peptides in the absence (A) or presence (B, C) of 100 ng/mL heparin.
47-70 (); 34-58 (); 17-58 (). (A and C) Concentration
dependency; (B) 10 µmol/L PF-4-derived peptides. Cross-linking (D)
is done with the different peptides in the presence of 100 ng/mL
heparin. Peptide 47-58 (5.3 µmol/L), 34-58 (5.3 µmol/L), 47-70 (1, 0.65 µmol/L; 2, 1.3 µmol/L; 3, 2.6 µmol/L; 4, 4.1 µmol/L).
Binding or cross-linking experiments are performed as indicated in
Materials and Methods. Results (A-C) are expressed as the mean± SD of
experiments done in duplicates.
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In the absence of heparin, 125I-VEGF only weakly bound
CHOmVEGFR2 (data not shown). Heparin increased the binding of
125I-VEGF to these cells by fourfold to fivefold (data not
shown). We therefore investigated the effect of the different peptides on 125I-VEGF binding to CHOmVEGFR2 cells in the presence of
100 ng/mL heparin (Fig 6).
125I-VEGF binding to CHOmVEGFR2 was also inhibited by
peptides 47-70 and 17-58. Peptide 47-70 inhibited maximally and half
maximally 125I-VEGF binding at 1 and 0.5 µmol/L,
respectively. For peptide 17-58, maximum inhibition was attained at 1 µmol/L and the IC50 at 0.17 µmol/L. None of the other peptides
interfered with 125I-VEGF binding to CHOmVEGFR2 cells.
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| Fig 6.
Effect of PF-4-derived peptides on binding of
125I-VEGF to CHOmVEGFR2 cells. CHOmVEGFR2 cells are
incubated with 5 ng/mL 125I-VEGF with or without 500 ng/mL
unlabeled ligand (NS) or PF-4-derived peptides in the presence of 100 ng/mL heparin. Binding experiments are performed as indicated in
Materials and Methods. Peptide 47-70 (), 34-58 (); 17-58 (),
47-58 ( ). Results are expressed as the mean ± SD of experiments
done in duplicates.
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Effect of PF-4 peptides on the interaction between PF-4 and FGF-2 or
VEGF.
We next analyzed the effect of the different peptides on the direct
interaction of PF-4 with FGF-2 or VEGF. These experiments were
performed using a solid-phase binding assay. We have previously demonstrated that PF-4 directly binds to FGF-2 and inhibits FGF-2 dimerization.21 In the solid-phase binding assay, unlabeled PF-4 competed with 125I-PF-4 binding to FGF-2 or VEGF
nearly completely at 5 µmol/L and half maximally at 0.1 µmol/L (Fig
7A and C; and data not shown). Furthermore,
125I-PF-4 binding to FGF-2 or VEGF was also nearly
completely blocked by 10 µg/mL anti-PF-4 antibodies (Fig 7A and C).
At 10 µmol/L, peptide 47-70 inhibited the association of
125I-PF-4 to FGF-2 or VEGF by 75% in comparison to control
(Fig 7B and D; and data not shown). When increasing concentrations of peptide 47-70 were added, maximum inhibition (95% inhibition) was
reached between 20 to 30 µmol/L and the IC50 at approximately 0.5 µmol/L (Fig 7D and data not shown). Peptide 17-58 was also able to
compete with the association of 125I-PF-4 to
surface-immobilized growth factors (42% at 10 µmol/L of peptide
17-58), but the other peptides were unable to do so (Fig 7B and data
not shown).
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| Fig 7.
Effect of PF-4-derived peptides on the direct
association of 125I-PF-4 with FGF-2 or VEGF. Wells of
96-well plates are coated with 15 ng FGF-2 (A, B) or 35 ng VEGF (C, D)
and incubated with 10 ng 125I-PF-4 with or without 2 µg
unlabeled PF-4, 10 µg anti-PF-4 antibodies, or peptides in 50 µL
incubation volume. Solid-phase binding experiments are performed as
indicated Materials and Methods with fixed (10 µmol/L) or increasing
peptide concentrations. Peptide 47-70 ( ), 34-58 (). Results are
expressed as the mean ± SD of experiments done in triplicates.
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We then tested the effect of the different PF-4 peptides on FGF-2
dimerization (Fig 8A through C). PF-4 by
itself severely decreased multimerization and simultaneously promoted
the appearance of a intermediate band of 25 kD corresponding to
PF-4/FGF-2 complexes.21 Five nanograms of
125I-FGF-2 and 500 ng unlabeled FGF-2 were incubated with
various concentrations of peptides in a 50-µL incubation volume. The
experiments were performed in the absence and presence of heparin. When
PF-4 peptide 47-70 was used in these experiments, a modulation of FGF-2 dimer formation was observed. Surprisingly, in the absence of heparin,
FGF-2 dimers were increased by peptide 47-70 with a maximum increase at
10.8 µmol/L (Fig 8A). At higher peptide concentrations (21 to 134 µmol/L), FGF-2 dimers progressively decreased. To the contrary, in
the presence of 50 ng/50 µL heparin, dimer formation was already
severely inhibited at 5.4 µmol/L of peptide 47-70 and remained low
with increasing peptide concentrations (Fig 8B). We also investigated
the effects of peptides 17-58, 34-58, 47-58, and 58-70 on FGF-2
dimerization (Fig 8C). A 10-µmol/L quantity of each peptide was added
to the incubation mixture. In the presence of 50 ng/50 µL heparin,
FGF-2 dimerization was slightly increased by peptide 17-58 and an
additional band of approximately 20 kD was observed. This signal may
correspond to cross-linked 125I-FGF-2-peptide 17-58 complexes. Peptides 34-58, 47-58, and 58-70 were unable to modulate
FGF-2 dimer formation (Fig 8C).
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| Fig 8.
Effect of PF-4-derived peptides on FGF-2 dimerization.
10 ng 125I-FGF-2, 500 ng unlabeled FGF-2 are incubated in
the presence or absence of 50 ng heparin with fixed (10 µmol/L) or
increasing concentrations of the different PF-4-derived peptides.
Dimerization experiments were performed as indicated in Materials and
Methods. (A) Increasing concentrations of peptide 47-70 in the absence
of heparin (lane 1: control, lane 2: 2.6 µmol/L, lane 3: 5.4 µmol/L, lane 4: 10.8 µmol/L, lane 5: 21.4 µmol/L, lane 6: 42.7 µmol/L, lane 7: 87 µmol/L, lane 8: 134.5 µmol/L); (B) increasing
concentrations of peptide 47-70 in the presence of heparin (lane 1:
control with heparin, lane 2: 5.4 µmol/L, lane 3: 10.8 µmol/L, lane
4: 21.4 µmol/L, lane 5: 42.7 µmol/L, lane 6: 87 µmol/L, lane 7:
134.5 µmol/L) ; C, fixed concentrations (10 µmol/L) of various
PF-4-derived peptides (lane 1: control without heparin, lane 2:
control with heparin, lane 3: peptide 34-58, lane 4: peptide 47-58, lane 5: peptide 58-70, lane 6: peptide 17-58).
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DISCUSSION |
PF-4 has several interesting structural features. Three major clusters
of basic amino acids are found within the PF-4 sequence. One cluster is
localized near the N-terminus (Arg20, Arg22, His23), another in the
middle of the molecule (Arg49, Lys50), and a third at the C-terminus
(Lys61, Lys62, Lys65, Lys66). The cluster found at the C-terminus
constitutes the major heparin-binding domain of PF-4. This sequence is
located within an -helicoidal structure that protrudes from the
whole PF-4 molecule. Furthermore, PF-4 has two motifs with the amino
acids AspLeuGln (DLQ) at position 7 to 9 and 54 to 56. This sequence
seems to be implicated in the inhibitory activity of PF-4 on
colony-forming unit-granulocyte, macrophage (CFU-GM) progenitor
cells.29 Finally, four cystein residues are found in this
chemokine at position 10, 12, 36, and 52 that form disulfite bridges
between Cys10 and Cys 36 and between Cys 12 and Cys 52. To identify
antiangiogenic PF-4-derived peptides, we synthesized a number of
overlaping peptides that corresponded to different domains of PF-4 and
investigated their effect in different biological assays. These
comprise two peptides that map PF-4 from position 17 at the N-terminus
to two thirds of the molecule (peptide 17-58 and peptide 34-58) and
three other peptides that map PF-4 from the middle of the molecule to
the carboxy terminus (peptides 47-58, 47-70, and 58-70). Peptide 17-58 has two clusters of basic amino acids and peptide 34-58 contains only
one cluster of basic amino acids. However, both peptides contain a DLQ
motif. Therefore, peptide 17-58 but not peptide 34-58 contains the
first cluster of basic amino acids. Peptide 47-70 contains two clusters of basic amino acids (including the major heparin-binding domain) and
the DLQ motif. Peptide 47-58 only possess the DLQ motive and one
cluster of basic amino acids, but not the major heparin-binding domain.
Peptide 58-70 only contains the major heparin-binding domain, but not
the DLQ sequence or the second cluster of basic amino acids at position
49 and 50.
We chose to compare the activity of the different PF-4-derived
peptides towards FGF-2 and VEGF for the following reasons. FGF-2 exist
as monomers and dimerizes for receptor activation.30 VEGF,
on the contrary, exists already as a natural dimer.2,9 Dimerization seems not to be required for VEGF receptor
activation.9 Nevertheless, HSPGs regulate the activity of
both growth factors.9,30 Furthermore, FGF-2 and VEGF are
inhibited by PF-4.20,21 It was therefore of interest to
examine whether similar PF-4-derived peptides are involved in the
inhibition of the activity of both growth factors.
Peptide 47-70, but not the other peptides, completely inhibited
proliferation and angiogenesis in vitro induced by FGF-2 or VEGF. This
is in agreement with Sato et al,31 who have reported that a
carboxyl-terminal peptide that corresponds to the heparin-binding fragment of PF-4 retained the blocking effect on FGF-2 activity in
endothelial cells. Peptides 47-70 and 47-58 have also been shown to
inhibit the proliferation of leukemic cell lines and megakaryocytopoiesis.32, 33 However, myelopoiesis (CFU-GM,
burst-forming unit-erthrocyte [BFU-E], and CFU-megakaryocyte
[CFU-MK]) is also inhibited by peptide 34-58 and this at much lower
concentrations when compared with native PF-4 or peptide
47-70.27 The inhibition of megakaryocytopoiesis by peptide
34-58 does not exceed 30% to 40% at 20 nmol/L peptide concentration.
To the contrary, peptide 47-70 nearly completely inhibited endothelial
cell function, albeit at higher peptide concentrations.
Binding of 125I-FGF-2 or 125I-VEGF to
endothelial cell FGF or VEGF receptors was inhibited by peptide 47-70, but not by the other peptides. Peptide 47-70 inhibited binding of
125I-FGF-2 to FGF receptors and to proteoheparan sulfates.
Proteoheparan sulfates also modulate VEGF binding.9 This
may indicate that peptide 47-70 interferes directly with FGF-2/FGF or
VEGF/VEGF receptor interactions or indirectly via proteoheparan sulfates.
To address the requirement of proteoheparan sulfates in the effect of
peptide 47-70 on FGF-2 or VEGF binding, binding studies were performed
with heparan sulfate-deficient CHO cells transfected with a vector
that contained a FGFR1 or VEGFR2 cDNA. Peptide 47-70 inhibited
heparin-induced binding of 125I-FGF-2 or
125I-VEGF to FGFR1 or VEGFR2 in CHOm cells, respectively.
However, binding was not inhibited in the absence of heparin. We have
previously shown that the whole PF-4 molecule inhibits FGF-2 binding to
CHOmFGFR1 in the absence of heparin by 50%.21 This would
indicate that other PF-4 domains are required for full inhibitory
activity. We also found that a peptide derived from the central part of PF-4 between amino acids 17 to 58 interfered with the heparin-induced binding of FGF-2 or VEGF to CHOmFGFR1 or CHOmVEGFR2 cells. Peptide 17-58 contains at position 20, 22, 23 49, and 50 basic amino acids with
potential heparin-binding activity. This might explain the effect of
the peptide on heparin-dependent binding of FGF-2 or VEGF to CHOmFGFR1
or CHOmVEGFR2 cells.
PF-4 associates directly with FGF-2 or VEGF.21,22 We
therefore examined whether the different peptides were able to compete with the direct binding of PF-4 to FGF-2 or VEGF. As expected, peptide
47-70 competed with the association of PF-4 with FGF-2 or VEGF. This
may indicates that the PF-4 domain 47-70 is essential for direct
association of PF-4 to FGF-2 or VEGF. We investigated the effect of
peptides on the formation of FGF-2 dimers because PF-4 inhibits
endogenous or heparin-induced FGF-2 dimerization.21 In the
absence of heparin, peptide 47-70 modulated FGF-2 dimer formation. With
increasing concentrations of peptide, FGF-2 dimers increased first and
subsequently declined. However, in the presence of heparin, FGF-2
dimerization is severely inhibited. What is the explanation for these
findings? The stimulation of FGF-2 dimer formation may be explained by
direct binding of peptide 47-70 to FGF-2, thus inducing inactive FGF-2
dimers. Through an elegant series of structural studies, it has
recently been shown that FGF-2 undergoes two types of dimerizations
that lead to active or inactive FGF-2 dimers.34 Active
FGF-2 dimers are "side by side" dimers and induced with heparan
sulfate oligomers, dodecamers, dimers, or trimers, but not by hexamers
or septamers. Inactive dimers are "head to head" dimers and are
induced by compounds such as sucralsulfate. These studies suggest that
peptide 47-70 behaves to a certain extent similarily to sucralsulfate
and induces head to head dimers in a heparin-free context. Structural
biology studies should establish the type of FGF-2 dimer that occurs in the presence of peptide 47-70. Peptide 17-58 was also able to interfere
to some extent with the association of PF-4 to the angiogenic growth
factors. However, peptide 17-58 did not inhibit heparin-dependent dimer
formation of FGF-2. This may account for the lack of inhibitory activity of this peptide in the biological assays and in the binding experiments of FGF-2 or VEGF to endothelial cells.
It was surprising that peptide 17-58 inhibited to some extent the
heparin-dependent binding of FGF-2 or VEGF to heparan
sulfate-deficient cells that expressed FGF or VEGF receptors, but did
not inhibit the binding of these growth factors to endothelial cells.
The reasons for these differences are not understood. Peptide 17-58 is
alone perhaps insufficient to disturb the interaction of FGF-2 or VEGF
and their receptors with heparan sulfates bound to proteoglycans at the
cell surface.
Another observation is the stimulatory activity of peptide 47-58 on
FGF-2 induced tube formation in in vitro angiogenesis, but not in any
other assays we performed. The reasons for this stimulatory activity
remain to be elucidated.
What are the implications of these results for the structure and
function of PF-4? The fact that peptide 47-70 but not 47-58 or 58-70 showed inhibitory activity would indicate that the heparin-binding domain is not sufficient for full antiangiogenic activity. An additional sequence that contains the second basic amino acid cluster
and/or the DLQ motif (Asp54, Leu55, Gln56) is possibly required. On the
other hand, peptide 17-58, but not peptide 34-58, showed inhibitory
activity in CHOm cells transfected with FGFR1 or VEGFR2 cDNA. This
indicates that a sequence that comprises amino acid 17-34 is required
for this inhibitory activity. As mentioned earlier, a cluster of basic
amino acid residues is localized within this sequence. It is possible
that this sequence is implicated in the inhibitory activity of peptide
17-58.
Our results are in apparent conflict with those reported by Maione et
al.35 Maione et al35 have reported that a
mutated PF-4 molecule that had the C-terminal heparin-binding domain
mutated was still able to inhibit angiogenesis. The reasons for this
difference are not known. In our hands, it is unlikely that PF-4
domains found outside the 47-70 sequence are essential for the
inhibitory activity of PF-4 in angiogenesis. However, other domains may
contribute to enhance the inhibitory effect of domain 47-70 in vivo.
This may also be supported by the intriguing finding that a peptide derived from the central part of PF-4 that contains a potential heparin-binding motif at positions 20, 22, and 23 only showed inhibitory activity on heparin-induced FGF-2 or VEGF binding to FGF or
VEGF receptors to heparan sulfate-deficient cells, but not in binding
assays performed with capillary endothelial cells and not on biologic activity.
Taken together, our results indicate that peptide 47-70 derived from
the C-terminus of PF-4 interferes with both FGF-2 and VEGF function.
Furthermore, a peptide derived from the central part of PF-4 that
contains a potential heparin-binding domain interferes with
heparin-dependent binding of FGF-2 or VEGF to FGF or VEGF receptors in
heparan sulfate-deficient cells. These observations are of
significance for PF-4's mechanisms of angiogenesis inhibition.
| |
ACKNOWLEDGMENT |
The authors thank Dr H. Prats and Dr D.B Rifkin for providing
recombinant human FGF-2, Dr Avner Yayon for providing CHOmFGFR1 cells,
and Dr I. Kramer (Growth Factor and Cell Differentiation Laboratory)
for critically reading the manuscript.
| |
FOOTNOTES |
Submitted December 22, 1998; accepted April 6, 1999.
Supported by grants from the Fondation de France, The Association de la
Recherche sur le Cancer (ARC), and the Ministère de la Science et
de la Recherche (MSR) to A.B. and from the Del Duca foundation to M.A.
and J.P.C.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Andreas Bikfalvi, MD, PhD, Laboratoire des
Facteurs de Croissance et de la Differenciation Cellulaire,
Bâtiment de Recherche Biologie Animale, Avenue des
Facultés, 33405 Talence, France; e-mail:
a.bikfalvi{at}croissance.u-bordeaux.fr.
| |
REFERENCES |
1.
Bikfalvi A, Klein S, Pintucci G, Rifkin DB:
Biological roles of fibroblast growth factor-2.
Endocr Rev
18:26, 1997[Abstract/Free Full Text]
2.
Ferrara N, Davis-Smyth T:
New insights in the biology of vascular endothelial growth factor.
Endocr Rev
18:4, 1997[Abstract/Free Full Text]
3.
Hanahan D, Folkman J:
Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell
86:353, 1996[Medline]
[Order article via Infotrieve]
4.
O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J:
Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.
Cell
79:315, 1994[Medline]
[Order article via Infotrieve]
5.
O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J:
Endostatin: An endogenous inhibitor of angiogenesis and tumor growth.
Cell
88:277, 1997[Medline]
[Order article via Infotrieve]
6.
Dameron KM, Volpert OV, Tainsky MA, Bouck N:
Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1.
Science
265:1582, 1994[Abstract/Free Full Text]
7.
Ferrara N, Clapp C, Weiner R:
The 16K fragment of prolactin specifically inhibits basal or fibroblast growth factor stimulated growth of capillary endothelial cells.
Endocrinology
129:896, 1991[Abstract/Free Full Text]
8.
Maione TE, Gray GS, Petro J, Hunt AJ, Donner AL, Bauer SI, Carson HF, Sharpe RJ:
Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides.
Science
247:77, 1990[Abstract/Free Full Text]
9.
Tessler S, Rockwell P, Hicklin D, Cohen T, Levi BZ, Witte L, Lemischka IR, Neufeld G:
Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors.
J Biol Chem
269:12456, 1994[Abstract/Free Full Text]
10.
Strieter RM, Polverini PJ, Arenberg DA, Kunkel SL:
The role of CXC chemokines as regulators of angiogenesis.
Shock
4:155, 1995[Medline]
[Order article via Infotrieve]
11.
Deuel TF, Keim PF, Farmer M, Heinrickson RL:
Amino acid sequence of human platelet factor 4.
Proc Natl Acad Sci USA
74:2256, 1977[Abstract/Free Full Text]
12.
Miller MD, Krangel MS:
Biology and biochemistry of the chemokines: A family of chemotactic and inflammatory cytokines.
Crit Rev Immunol
12:17, 1992[Medline]
[Order article via Infotrieve]
13.
Zhang X, Chen L, Bancroft DL, Lai CK, Maione TM:
Crystal structure of recombinant human platelet factor 4.
Biochemistry
33:8361, 1994[Medline]
[Order article via Infotrieve]
14.
Stuckey JA, St Charles R, Edwards B:
A model of the platelet factor-4 complex with heparin.
Proteins
14:277, 1992[Medline]
[Order article via Infotrieve]
15.
Gupta SK, Singh JP:
Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression.
J Cell Biol
127:1121, 1994[Abstract/Free Full Text]
16.
Hansell P, Maione TE, Borgstrom P:
Selective binding of platelet factor-4 to regions of active angiogenesis in vivo.
Am J Physiol
269:829, 1995
17.
Sharpe RJ, Byers HR, Scott CF, Bauer SI, Maione TE:
Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor-4.
J Natl Cancer Inst
82:848, 1990[Abstract/Free Full Text]
18.
Kolber DL, Knisely TL, Maione TE:
Inhibition of development of murine melanoma lung metastases by systemic administration of recombinant platelet factor-4.
J Natl Cancer Inst
87:304, 1995[Abstract/Free Full Text]
19.
Tanaka T, Manome Y, Wen P, Kufe D, Fine HA:
Viral vector-mediated transduction of a modified platelet factor-4 cDNA inhibits angiogenesis and tumor growth.
Nature Med
3:437, 1997[Medline]
[Order article via Infotrieve]
20.
Watson JB, Getzler SB, Mosher DF:
Platelet factor-4 modulates the mitogenic activity of basic fibroblast growth factor.
J Clin Invest
94:261, 1994
21.
Perollet C, Han ZC, Savona C, Caen JP, Bikfalvi A:
Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization.
Blood
91:3289, 1998[Abstract/Free Full Text]
22.
Gengrinovitch S, Greenberg SM, Cohen T, Gitay-Goren H, Rockwell P, Maione TE, Levi BZ, Neufeld G:
Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.
J Biol Chem
270:15059, 1995[Abstract/Free Full Text]
23.
Jonca F, Ortega N, Gleizes PE, Bertrand N, Plouet J:
Cell release of bioactive fibroblast growth factor 2 by exon 6-encoded sequence of vascular endothelial growth factor.
J Biol Chem
272:24203, 1997[Abstract/Free Full Text]
24.
Bikfalvi A, Klein S, Pintucci G, Quarto N, Mignatti P, Rifkin DB:
Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms.
J Cell Biol
129:233, 1995[Abstract/Free Full Text]
25.
Montesano R, Vassalli JD, Baird A, Guillemin R, Orci L:
Basic fibroblast growth factor induces angiogenesis in vitro.
Proc Natl Acad Sci USA
83:7297, 1986[Abstract/Free Full Text]
26.
Moscatelli D:
High and low affinity binding sites for basic fibroblast growth factor on cultured cells: Absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells.
J Cell Physiol
131:123, 1987[Medline]
[Order article via Infotrieve]
27.
Lecomte-Raclet L, Alemany M, Sequiera-Le Grand A, Amiral J, Quentin G, Vissac AM, Caen JP, Han ZC:
New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides.
Blood
91:2772, 1998[Abstract/Free Full Text]
28.
Ornitz DM, Yayon A, Flanagan JG, Svan CM, Levi E, Leder P:
Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.
Mol Cell Biol
12:240, 1992[Abstract/Free Full Text]
29.
Daly T, La Rosa G, Dolich S, Maione T, Cooper S, Broxmeyer H:
High activity suppression of myeloid progenitor proliferation by chimeric mutants of interleukin-8 and platelet factor-4.
J Biol Chem
270:23282, 1995[Abstract/Free Full Text]
30.
Schlessinger J, Lax I, Lemmon M:
Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors?
Cell
83:357, 1995[Medline]
[Order article via Infotrieve]
31.
Sato Y, Waki M, Kuwano M, Sakata T:
Carboxyl-terminal heparin-binding fragment of platelet factor-4 retain the blocking effect of the receptor binding of basic fibroblast growth factor.
Jpn J Cancer Res
84:485, 1993[Medline]
[Order article via Infotrieve]
32.
Gewirtz AM, Calabretta B, Rucinski B, Niewiarowski S, Xu WY:
Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.
J Clin Invest
83:1477, 1989
33.
Lebeurier I, Raclet L, Amiral J, Caen JP, Han ZC:
Carboxyterminal peptides with the dimeric form of PF4 retain the inhibitory effect on the growth of human megakaryoblastic cell lines.
J Lab Clin Med
127:179, 1996[Medline]
[Order article via Infotrieve]
34.
Venkataraman G, Sasisekharan V, Herr AB, Ornitz DM, Waksman G, Cooney CL, Langer R, Sasisekharan R:
Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.
Proc Natl Acad Sci USA
93:845, 1996[Abstract/Free Full Text]
35.
Maione TE, Gray GS, Hunt AJ, Sharpe RJ:
Inhibition of tumor growth in mice by an analog of platelet factor-4 that lacks affinity for heparin and retains potent angiostatic activity.
Cancer Res
51:2077, 1991[Abstract/Free Full Text]
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|
|
J. A. Belperio, M. P. Keane, D. A. Arenberg, C. L. Addison, J. E. Ehlert, M. D. Burdick, and R. M. Strieter
CXC chemokines in angiogenesis
J. Leukoc. Biol.,
July 1, 2000;
68(1):
1 - 8.
[Abstract]
[Full Text]
|
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|
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M. I. Koukourakis, A. Giatromanolaki, P. E. Thorpe, R. A. Brekken, E. Sivridis, S. Kakolyris, V. Georgoulias, K. C. Gatter, and A. L. Harris
Vascular Endothelial Growth Factor/KDR Activated Microvessel Density versus CD31 Standard Microvessel Density in Non-Small Cell Lung Cancer
Cancer Res.,
June 1, 2000;
60(11):
3088 - 3095.
[Abstract]
[Full Text]
[PDF]
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R. M. Lozano, M. Redondo-Horcajo, M. A. Jimenez, L. Zilberberg, P. Cuevas, A. Bikfalvi, M. Rico, and G. Gimenez-Gallego
Solution Structure and Interaction with Basic and Acidic Fibroblast Growth Factor of a 3-kDa Human Platelet Factor-4 Fragment with Antiangiogenic Activity
J. Biol. Chem.,
September 14, 2001;
276(38):
35723 - 35734.
[Abstract]
[Full Text]
[PDF]
|
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TOP
ABSTRACT
INTRODUCTION