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J. Biol. Chem., Vol. 278, Issue 37, 35564-35573, September 12, 2003
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**
From the
Molecular Angiogenesis Laboratory (INSERM
E 0113), Université de Bordeaux 1, 33405 Talence,
¶Groupe de Chimie Bio-Organique (INSERM U 577),
Université Victor Segalen Bordeaux 2, 33076 Bordeaux Cedex,
||Structure et Fonction de Molécules
Bioactives (UMR 7613 CNRS-Université Paris 6), Université Pierre
et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France, and
Neurosurgery, Department of
Neurological Sciences, University of Milano, Ospedale Maggiore Policlinico,
Istituto di Ricovero e Cura a Carattere Scientifico, 20122 Milan, Italy
Received for publication, April 29, 2003 , and in revised form, June 30, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-sheet
structures or random coil conformations have been observed for macrocyclic
peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to
endothelial cells, endothelial cells proliferation, migration, and signaling
induced by VEGF165. This peptide also exhibits anti-angiogenic
activity in vivo on the differentiated chicken chorioallantoic
membrane. Furthermore, cyclo-VEGI significantly blocks the growth of
established intracranial glioma in nude and syngeneic mice and improves
survival without side effects. Taken together, these results suggest that
cyclo-VEGI is an attractive candidate for the development of novel
angiogenesis inhibitor molecules useful for the treatment of cancer and other
angiogenesis-related diseases. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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VEGFs are endothelial cell mitogens in vitro and also stimulate angiogenesis in vivo (2, 5, 6). VEGF exerts its biological effects through high affinity binding to two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (kinase domain receptor), which are expressed in most vascular endothelial cells (6–8). VEGF is expressed in a large number of human tumor types (9, 10). Clinical studies have documented the importance of VEGF in human cancers. As an example, VEGF is increased in the plasma of cancer patients and is correlated with response to chemotherapy (11, 12). In growing tumors, VEGF expression is up-regulated by hypoxia, growth factors, and oncogenes (6, 13).
Antagonizing VEGF has shown to inhibit tumor development in vivo. Different strategies have been designed to inhibit VEGF function. These include monoclonal neutralizing antibodies (14), dominant-negative mutants of the VEGF receptor-2 (VEGFR2) (15), antisense oligonucleotides (16), anti-VEGFR2 antibodies (17), blockers of VEGFR2 tyrosine phosphorylation (18), VEGF-toxin conjugates (19), antagonistic VEGF mutants (20), peptides that interfere with VEGF/VEGFR interactions (21–25), and decoy-soluble receptors (26).
Relevant regions of VEGF important for its binding on VEGFR2 and VEGFR1
have been investigated by structural and mutagenesis studies
(27–29).
Some of the residues important for the interaction between VEGF and kinase
domain receptor are clustered within region 79–93 of VEGF which forms a
-hairpin, Arg82, Lys84, and His86 being
key residues (27). Based on
these structural and mutagenesis studies, a number of cyclic and linear
peptides have been synthesized. One of the most potent compound was the cyclic
peptide CBO-P11 (D-Phe-Pro79–93) designated herein
cyclopeptidic vascular endothelial growth inhibitor
(cyclo-VEGI).2 In the
present report, we determined the structural features of this peptide and
characterized its biological properties. We show that this peptide adopts an
unexpected three-dimensional structure and is able to inhibit critical steps
of angiogenesis. Moreover, this peptide inhibits the growth of both
established human intracranial and syngeneic glioma in mice.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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/3 shifted square sinebell function and zero-filled.
Base-line distortions were corrected with a fifth-order polynomial function.
1H-13C HSQC experiments were acquired using pulsed field
gradients for coherence selection
(33). The chemical shift
deviations of Ha and Ca were calculated using random
coil values reported in water
(34). The temperature
gradients of the amide proton chemical shifts were derived from a series of
TOCSY spectra recorded at different temperatures (from 283 to 303 K). The
resonances assignments are listed in Tables S1–S3 in the Supplemental
Material.
Structure Calculation—Interproton distance restraints were
derived from the analysis of NOESY spectra. NOE cross-peaks were categorized
as strong, medium, and weak and converted into distance ranges of
0.18–0.28, 0.18–0.38, and 0.18–0.5 nm, accordingly.
Pseudoatoms were introduced for distances involving methyl protons and
non-resolved methylene protons, and upper limits were corrected appropriately
(35). The 
angle of
L-amino acid residues was restrained to negative values because all
these residues did not exhibit strong intraresidual d
N NOEs. Structures
were calculated using InsightII version 98.0 and Discover (Accelrys, San
Diego, Inc.), running on SGI O2 R10000
[GenBank]
workstations, and AMBER forcefield
(36). The simulated annealing
protocol first comprised 15 ps of dynamics at 1000 K during which the force
constants of the distance and dihedral restraint terms were gradually
increased. The non-bond interaction, defined by a simple quartic repulsive
potential, was slowly increased during the next 10 ps of dynamics. Finally,
the structures were cooled from 1000 to 0 K over 25 ps. The structures were
then minimized by steepest descent and conjugated gradient algorithms, using a
Lennard-Jones potential for the van der Waals interaction and a
distance-dependent dielectric screening of 4 ·r for the
electrostatic term.
Growth Factors and Peptides Synthesis
Recombinant human FGF-2 was kindly provided by Dr. Hervé Prats
(INSERM U397, Toulouse, France) and stored in sterile, double-distilled water
at –80 °C. Recombinant human VEGF165 was produced in
insect cells and purified as described elsewhere
(37,
38). Human
VEGF165-encoding baculovirus was a kind gift of Dr. Jean
Plouët (Institut de Pharmacologie et de Biologie Stucturale, UMR 5089,
Toulouse, France). CBO-P11 (cyclo-VEGI) (DFPQIMRIKPHQGQHIGE) and
linear control CBO-P14 (PQIMRIKPHQGQHIGE) peptides were synthesized by
Fmoc/t-butyl batch solid phase synthesis on an Applied Biosystems
430A automated peptide synthesizer. For preparation of protected peptide
fragments, pre-loaded acid-labile 2-chlorotrityl resins
(H-His(Trt)2-ClTrt and H-Ile2-ClTrt resin) or HMPB-BHA
resins based on BHA polystyrene functionalized with Rink's
4-hydroxymethyl-3-methoxyphenoxybutanoic acid linker (Fmoc-Gly-HMPB-BHA resin)
were utilized. Subsequent Fmoc amino acids were coupled using a 4-fold excess
of amino acids activated as HOBt ester by means of DCC. The crude linear
protected peptides were analyzed by reverse phase high pressure liquid
chromatography on a Lichrosorb RP-18 column (Merck) and purified on
semi-preparative high pressure liquid chromatography. Cyclization of the
protected linear precursor (head-to-tail) was performed by
Fmoc/t-butyl/allyl
strategy.2
Cell Culture
Bovine capillary endothelial (BCE) cells were a kind gift of Dr. Daniel B.
Rifkin (New York University Medical Center, New York). Bovine aortic
endothelial (BAE) cells were from Dr. Georg Breier (Department of Molecular
Biology, Max-Planck-Institut für physiologische und Klinische Forschung,
Bad Nauheim, Germany). All endothelial cells were grown at 37 °C, 5%
CO2 in DMEM containing 10% newborn calf serum, 2 mM
L-glutamine, and 50 IU/ml penicillin, 50 IU/ml streptomycin
antibiotics and were used up to passage 25. BCE cells were grown in the
presence of 2 ng/ml FGF-2. CHO-VEGFR2 and CHO-VEGFR1 cells were a kind gift of
Dr. J. Plouët (GDR CNRS 1927 "Angiogenèse," Toulouse,
France). CHO-VEGFR2 and CHO-VEGFR1 were grown in DMEM supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 1% non-essential
amino acids and antibiotics (Invitrogen). Balb3T3 were grown in DMEM
supplemented with 10% FBS, 2 mM L-glutamine, and
antibiotics. U87 human glioma cells (ATCC) were grown in minimum Eagle's
-medium, 10% FBS, 2 mM L-glutamine, and
antibiotics. GL261 murine glioblastoma cells (a gift from Dr. David Zagzag,
New York University, New York) were cultured in DMEM supplemented with 10%
FBS, 2 mM L-glutamine, 1% non-essential amino acids, and
antibiotics.
Binding Assays
VEGF165 was labeled with Na125I using IODO-GEN
(Pierce) as coupling agent according to the manufacturer's instructions. BAE,
CHO-VEGFR2, and CHO-VEGFR1 cells were seeded at 2.5 x 105
density in gelatin-coated 6-well plates and cultured in complete medium for 2
days. Cells were washed twice with ice-cold PBS and incubated with 10 ng/ml
125I-VEGF and peptides at indicated concentrations in binding
medium (DMEM; 20 mM Hepes, pH 7.4; 0.15% gelatin) on a shaker at 4
°C. After 2 h, cells were washed 3 times with PBS and solubilized by the
addition of 2% Triton, 10% glycerol, and 1 mg/ml bovine serum albumin prior to
-counting. Each condition was tested in duplicate and repeated at least
two times. Data are expressed as percentage of total radioactivity.
Proliferation Assays
BCE and BAE cells were seeded in 24-well culture plates overnight in 10%
newborn calf serum at 7500 cells per well. Medium was changed to 1% newborn
calf serum, 10 ng/ml growth factor, and peptides at indicated concentrations
were added to duplicate wells. After 48 h, medium was changed, and stimulation
with growth factor and peptide treatment were repeated. One day later, cells
were counted on a Coulter counter. Balb3T3 were seeded at a density of 10,000
cells/well in complete medium. Medium was changed to 0.5% fetal calf serum and
20 ng/ml human recombinant PDGF-BB (Sigma), and peptides at indicated
concentrations were added to duplicate wells. Cells were counted 2 days later.
U87 and GL261 cells were seeded at 10,000 cells/well in 1% FBS, treated with
peptides at the same concentrations as ECs, and counted 72 h later. Each
condition was tested in duplicate and repeated at least two times.
ERK-1 (p44)/ERK-2 (p42) Phosphorylation Assay
BAE cells were plated in 35-mm plates. Subconfluent cultures were
serum-deprived for 24 h. Peptides were then added for 6 min in the presence of
10 ng/ml VEGF165. Cells were scraped off the dish and lysed for 10
min on ice in Nonidet P-40/SDS lysis buffer (50 mM Hepes, pH 7.4;
75 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 0.1% SDS)
containing a mixture of protease inhibitors. The insoluble material was
removed by centrifugation for 20 min at 12,000 x g at 4 °C.
The cleared supernatant was stored at –80 °C. Protein concentration
was measured by using the Bradford method (Bio-Rad). The cytoplasmic extracts
were resolved by SDS-PAGE on 12% gels under reducing conditions and
electrotransferred onto a nitrocellulose membrane. The blocked membranes were
then incubated with primary antibodies (phospho-specific mitogen-activated
protein kinase p42/p44 or mitogen-activated protein kinase p42/p44 were
obtained from New England Biolabs, Ozyme, France), washed, and incubated with
secondary horseradish peroxidase-conjugated mouse or rabbit antibodies (Dako
SA, Trappes, France). Detection of antibodies was performed using the
ECLplus Western blot detection system (Amersham Biosciences). Each
condition was tested at least two times.
Migration Assay
Migration test with BAE cells was performed using a method described
earlier (39). In brief, ECs
were seeded in 350-mm culture plates and were allowed to grow to confluence.
Complete medium was replaced with serum-free DMEM, and incubation was
continued overnight. One linear scar was drawn in the monolayer and divided
into seven equal fields. A set of digital photos was taken of each scar, and
the denuded area was marked using digital image analysis software (Lucia G,
www.lim.cz).
The dishes were washed and incubated with fresh serum-free medium containing
0.1% bovine serum albumin, 10 ng/ml VEGF165, and peptides at
indicated concentrations. After 16 h, cells were fixed in 1% glutaraldehyde
and counterstained (Giemsa), and a second set of photos was taken. Photos were
superposed, and endothelial cells having migrated across the line drawn at the
border of the scar in the first photo set were counted. Each condition was
tested in two independent experiments. Means for all fields of each group were
calculated; data are expressed in percentage of the mean number of migrated
cells over VEGF165-induced cell migration.
CAM Assay
Fertilized chicken eggs (Gallus gallus) (E.A.R.L. Morizeau,
Dangers, France) were incubated at 37 °C and 80% humidified atmosphere. On
day 4 of development, a window was made in the eggshell and sealed with
Durapore® tape. On day 13, plastic rings (made from Nunc Thermanox®
coverslips) were put on the CAM. 3 µg of VEGF165 was premixed
with 50 µg of peptides or with the equivalent volume of sterile water alone
and deposed in the center of the plastic ring as described previously
(21). Treatment was repeated
the following day. On day 17, the CAMs were fixed in vivo with 4%
paraformaldehyde for 30 min at room temperature, and the area containing the
ring was cut out for further analysis. Photos of each CAM were taken under a
stereomicroscope (Nikon SMZ800) using a digital camera (Nikon Coolpix 950).
Two observers scored the angiogenic response from 0 to 3 (0 = none, 1 =
medium, 2 = medium/high, and 3 = high). The angiogenic score was indicated as
the mean number from the two observers. Statistical analysis was carried out
using Student's two-tailed t test. For confocal studies, 6 CAM
samples for each condition were processed. Each sample was washed by
incubation with 0.1% Triton in PBS. Fluorescein-Sambucus nigra lectin
(M0928 Vector Laboratories) at 40 µg/ml was added, and incubation was
performed in 0.1% Triton/PBS for half an hour at room temperature. Samples
were washed 3 times with 0.1% Triton/PBS, 3 times with PBS, and rinsed 3 times
with water. The samples were finally mounted and embedded with Antifading
Prolong (Molecular Probes, Eugene, OR) and viewed by confocal fluorescent
microscopy at x600 magnification equipped with an anti-bleed through
control system based on sequential capture of the green signal (Nikon,
PCM2000).
Animal Experiments
Short-term Experiments—Groups of 10 6-week-old nude mice
(Charles River Italia, Italy) were implanted intracranially with 50,000 U87
glioma cells using an open window technique. For the syngeneic model, groups
of 10 6-week-old BALB/c mice (Charles River Italia) were implanted
intracranially with 50,000 GL261 glioma cells using the same technique.
In a first group of experiments, 12 days after tumor cell injection, groups of 10 nude or BALB/c mice each were treated intraperitoneally with 2 mg/kg/day of cyclo-VEGI or saline in a single shot. Treatment was continued for 28 days. Afterward, animals were sacrificed, and their brains were removed, embedded in OCT, and stored at –70 °C.
In a second group of experiments, 12 days after tumor cell injection, two groups of 10 nude mice each were implanted subcutaneously with 2004 Alzet osmotic minipumps. The pumps used in these experiments afford a constant period of work of 28 days. In the first group, the pump reservoir was filled with 0.45 mg (total amount) of the cyclo-VEGI, which corresponds to 0.45 mg/kg/day. The second group of 10 animals was implanted with pumps containing PBS, and a third additional group did not receive any pumps. The last two groups served as the controls for the experiment. Animals were sacrificed when signs of any distress were evident and after 29 days from implantation of the pumps. Then the brains were removed, embedded in OCT, and stored at –70 °C.
Long-term Experiments—For the long term experiments, groups of 10 nude mice each were implanted intracranially with 50,000 U87 glioblastoma cells using the same procedure described previously. Twelve days after tumor cell injection, a first group of animals was implanted subcutaneously with Alzet minipumps, and the reservoir was filled with 0.45 mg (total amount) of cyclo-VEGI (0.45 mg/kg/day). A second group of nude mice received subcutaneous minipumps containing saline and served as a control. Twenty-eight days later, the pump was changed with a new one containing the same amount of the inhibitor, in order to afford a 58-day period of treatment. Animals were carefully monitored for the occurrence of any side effects and immediately sacrificed at the occurrence of any neurological deficits. Seventy days after tumor cell implantation, all the remaining animals were sacrificed. All animal experiments were repeated at least two times.
All brains were fixed in 5% paraformaldehyde in PBS for 24 h at 4 °C, dehydrated in 30% sucrose in PBS for 24 h at 4 °C, embedded in OCT, and stored at –70 °C. The brains were then sectioned, and a portion of them submitted to routine histological examination with hematoxylin and eosin staining. Tumor volume was calculated and expressed as a mean ± S.E. Tumor volume was estimated using the formula for ellipsoid (width2 x length)/2. The remaining slides were used for the immunohistochemistry analysis as described below. Kaplan-Meyer survival curves were statistically analyzed using repeated measures analysis of variance.
Immunohistochemistry
Immunohistochemistry was performed on 5-µm sections.
Immunohistochemistry on 5-µm sections was carried using the Vectastain
Elite kit (Vector Laboratories). Primary antibodies include anti-CD31 (1:100
dilution, BD Biosciences) and anti-Ki-67 (1:100 dilution, Dako). Detection was
carried out using DAB chromogen. Sections were counterstained with
hematoxylin. Negative control slides were obtained by omitting the primary
antibody. Ki-67 staining was quantified by counting the number of positively
stained cells of 100 nuclei in 20 randomly chosen fields
(40,
41). Microvessel count and
density were scored as reported previously
(40,
41). Apoptotic cells were
detected with ApopTagTM plus kit (Genenco International, New York) with
1% methyl green as a counterstain. Apoptosis and proliferative indices were
quantified by determining the percentage of positively stained cells for all
nuclei in 20 randomly chosen fields per section at x200 magnification
(40).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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carbons were assigned from
13C-1H HSQC spectra. Weak additional resonances could be
observed in the NMR spectra recorded in water and also in TFE/water mixtures,
albeit to a lesser extent. In the absence of chemical heterogeneity, a likely
explanation of the observed chemical shift heterogeneity would be
cis-trans isomerism of peptide bonds preceding proline. The major
form corresponds to a trans conformation of the peptide bonds
preceding the two Pro residues, as evidenced by the observation of strong
(i – 1,i) sequential NOEs. The
proportion of minor forms was too weak to give rise to NOE cross-peaks.
However, the presence of alternative spin systems of Gln/Glu, Phe, His, and
Lys in TOCSY spectra, i.e. amino acids around Pro residues, suggests
that both D-Phe1–Pro2 and
Lys8–Pro9 peptide bonds are likely to isomerize.
The lower proportion of minor species in mixtures of TFE/water indicates that
the stabilization of folded structures (see below) decreases the amount of
cis isomers, as observed for cyclic gramicidin S.
The chemical shift deviations (CSDs) of H and
C resonances, calculated as the difference between observed
chemical shifts and corresponding random coil values, carry information about
the secondary structure (43).
A stretch of residues exhibiting upfield shifts of H protons
and downfield shifts of C carbons is characteristic of a
helix, whereas the observation of downfield shifts of H and
upfield shifts of C indicates the presence of -sheet
structures. Fig. 1 shows the
H and C CSDs in water and in TFE/water
mixtures. The peptide in water displays very weak CSDs along its whole
sequence, indicating that it is largely unstructured in aqueous solution. The
addition of TFE (from 15 to 30%) progressively induces an upfield shift of
H and a downfield shift of C resonances
for residues 1–6, supporting the formation of a helical structure.
Because the corresponding H CSDs are slightly negative in
water, this suggests that the 1–6 segment has a small helical propensity
in water and that helical conformations explored in this region are stabilized
by the addition of TFE. Strong upfield shifts of amide protons chemical shifts
are also induced by the addition of TFE for residues 3–8. Interestingly,
these variations are opposite to those observed for cyclic gramicidin S under
the same conditions and are correlated to the formation of a -sheet
structure. Furthermore, residues 3–8 exhibit weak temperature gradients
of their amide proton chemical shifts
(
NH/T). Altogether, these chemical
shift data are consistent with the formation of a helical structure in segment
1–8. No significant CSD variations could be observed in the other parts
of the sequence, suggesting that the peptide remains unstructured, apart from
the 1–8 region.
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Three-dimensional Structure—The cyclic peptide shows few
NOEs in water, besides intraresidual and sequential connectivities. In
contrast, the peptide in 30% TFE exhibits NOEs characteristic of helical
structure in segment 1–8, including strong dNN (i,i + 1) and
medium dN (i,i + 1) sequential connectivities, together with
several dN(i,i + 2), dN(i,i + 3), and
dN(i,i + 4) medium range connectivities. The structure of the
cyclic peptide in 30% TFE was calculated by restrained molecular dynamics,
using a set of 17 intraresidual, 63 sequential, and 20 medium range distance
restraints. The best 20 calculated structures are shown in
Fig. 2. They have low energies
and few distance violations, indicating that they are in good agreement with
NMR experimental restraints. The overall structure of the cyclic peptide is
poorly defined, as evidenced by the large root mean square deviation of
backbone atoms (3.4 Å). However, the superimposition of structures using
backbone atoms of residues 1–8 or 1–6 shows that the backbone is
better defined in this region, with corresponding root mean square deviations
of 1.6 and 1.1 Å, respectively. Segment 1–8 adopts a helical
conformation. Unexpectedly, residues
D-Phe1–Pro2 do not form a type
II' -turn but are rather part of the helical 1–8 segment, as
shown by medium range correlations between
D-Phe1–Ile4 and
Pro2–Met5. Similarly, residues
Pro9–His10 do not form a stable -turn. The
conformations of other residues are not well defined but are not completely
random coil due to the cyclic topological constraint.
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Inhibition of VEGF Binding to Its Receptors
We first looked at the ability of cyclo-VEGI to interfere with binding of
125I-labeled VEGF165 to Chinese hamster ovary (CHO)
cells expressing VEGFR2. Cyclo-VEGI inhibited 125I-labeled
VEGF165 binding to VEGFR2 in a dose-dependent manner
(Fig. 3A) with a
half-maximal inhibition (IC50) of 1.3 µM. The linear
control peptide P14 with the same amino acid sequence as cyclo-VEGI did not
compete for receptor binding even at the highest concentration tested
(Fig. 3A). To
investigate whether cyclo-VEGI inhibits binding of VEGF165 to
VEGFR2 specifically, we tested the effect of cyclo-VEGI on the binding of
125I-labeled VEGF165 to CHO cells expressing VEGFR1.
Interestingly cyclo-VEGI also inhibited binding of VEGF to VEGFR1
(IC50 value of 0.7 µM,
Fig. 3B). We next
evaluated its effect on 125I-labeled VEGF165 binding to
high affinity VEGF receptors in BAE cells. As shown in
Fig. 3C, cyclo-VEGI
effectively inhibits the binding of 125I-labeled VEGF165
to high affinity receptors with an IC50 of 12 µM.
Linear control P14 at the highest concentration tested (50 µM)
had no effect (Fig.
3C).
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Inhibition of the Proliferation of Endothelial Cells but Not of
Glioma Cells
We next investigated whether cyclo-VEGI inhibits VEGF-induced proliferation
of BAE cells. When BAE cells were stimulated by 10 ng/ml of
VEGF165, cyclo-VEGI showed a dose-dependent inhibitory effect with
an IC50 of 5.8 µM, whereas the linear control P14 had
no effect at the highest concentration tested (100 µM)
(Fig. 4A).
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VEGF is a member of the cystine-knot family of growth factors, with the highest sequence identity and structural similarity to PDGF-BB (44, 45). Moreover, endothelial cell proliferation is induced by FGF-2 (46). Thus, to investigate whether the effect of cyclo-VEGI on endothelial cells proliferation was specific for VEGF165, we tested the activity of cyclo-VEGI on PDGF- or FGF-induced cell proliferation. When Balb3T3 cells were stimulated with 20 ng/ml PDGF-BB, cyclo-VEGI did not show any effect even at the highest concentration tested (50 µM) (Fig. 4B). Surprisingly, cyclo-VEGI decreased FGF-induced BCE proliferation, however three times less potently (IC50 of 18 µM in comparison to 5.8 µM for VEGF-induced endothelial cell proliferation) (Fig. 4B). Proliferation of two glioma cell lines, human U87 and mouse GL261 cells, was not inhibited by cyclo-VEGI peptides at all the concentrations tested (Fig. 4C).
Inhibition of VEG165 Signal Transduction
We next investigated the effect of cyclo-VEGI on ERK1 and ERK2
phosphorylation. As expected, activation of VEGFR2 with 10 ng/ml
VEGF165 in BAE cells induced a strong phosphorylation of ERK1 and
ERK2 (Fig. 4D, 2nd
lane). Cyclo-VEGI, at 20 µM, completely suppressed
VEGF-induced ERK1 and ERK2 phosphorylation
(Fig. 4D, 3rd
lane). The linear control P14 did not show any inhibitory effect on
VEGF165-induced phosphorylation of ERK1 and ERK2
(Fig. 4D, 4th
lane). Similar results were also observed on human umbilical vein
endothelial cells stimulated with 10 ng/ml of VEGF165 (data not
shown).
Inhibition of Endothelial Cell Migration
We then investigated whether endothelial cell motility, an important
pre-requisite for angiogenesis, was affected by cyclo-VEGI. The wounding assay
was used to determine the effect of cyclo-VEGI on VEGF-induced bovine aortic
endothelial cells migration in vitro. In the absence of VEGF, some
random background migration occurred (Fig.
5A). When stimulated with VEGF165, an
increased number of BAE cells migrated into the denuded scar area
(Fig. 5B). Migration
was not affected when the linear control peptide (P14) was added
(Fig. 5, D and
E). On the contrary, cyclo-VEGI inhibited VEGF-induced
migration of endothelial cells in a dose-dependent manner with an
IC50 of 8.2 µM
(Fig. 5, C and
E). Inhibition of migration of the endothelial cells
below base-line levels was also observed
(Fig. 5E).
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Inhibition of VEGF-induced Angiogenesis on the CAM
Peptides were tested in the differentiated CAM assay, where capillary
angiogenesis is induced by VEGF165
(21). For this experiment, a
plastic ring is filled in the center with 3 µg of VEGF165 alone
or with VEGF165 plus either the vehicle alone (water), 50 µg of
cyclo-VEGI, or 50 µg of the control peptide P14. Recombinant human
VEGF165 alone induced significant angiogenesis
(Fig. 6, B and
I). Some stimulation is also present around the site of
application because of diffusion of the growth factor. Vehicle alone (water)
in the plastic ring had no effect (Fig. 6,
A and I). When premixed with VEGF165
and deposed on the CAM, a clear anti-angiogenic effect of cyclo-VEGI was
visible inside the ring (Fig. 6, C
and I). The linear control P14 did not inhibit
VEGF-induced capillary growth in the CAM
(Fig. 6, D and
I). 50 µg of cyclo-VEGI applied alone on the CAM
without VEGF165 did not show any effect or toxicity (data not
shown).
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The capillary structure in the stroma was also examined by immunohistology
and confocal microscopy. Vessels were visualized by staining endothelial cells
with fluorescein isothiocyanate (FITC) conjugated to S. nigra lectin.
This lectin interacts preferentially to sialic acid attached to terminal
galactose by -2,6-link and was shown to bind endothelial cells in the
CAM. The extensive characterization of the CAM vasculature using this method
will be described
elsewhere.3
VEGF165 induced a strong increase in vessel density, a reduction of
the inter-capillary space, and structures suggestive of ongoing fusion events
between small vessels (Fig.
6F). These chaotic vascular structures were not present
when cyclo-VEGI was added together with VEGF165 in the plastic ring
(Fig. 6G). However,
some neovessels remained, as evidenced by a slightly higher microvessel
density compared with control CAM treated with water alone
(Fig. 6, G and
E). CAMs treated with control peptide P14 with
VEGF165 had a similar morphology than CAMs treated with
VEGF165 alone (Fig.
6H).
Inhibition of Glioma Growth in Vivo
The ability of cyclo-VEGI to reduce glioma growth in vivo was
initially investigated by short term studies. In these experiments, nude mice
implanted intracranially with human glioblastoma cells were treated
intraperitoneally with 2 mg/kg/day of cyclo-VEGI. Treatment was started 12
days after tumor cell implantation in order to treat well established tumors.
Twenty eight days later, the treatment was stopped, and the animals were
sacrificed. Histological analysis of the brains from treated and untreated
animals showed that treatment with cyclo-VEGI was associated with a 70%
reduction of U87 tumor growth (Fig.
7A, lower panel). A similar significant
inhibition (78%) was observed in immunocompetent BALB/c mice implanted with
GL261 murine glioblastoma cells (Fig.
7A, top panel).
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In a second set of experiments we investigated the ability of cyclo-VEGI to reduce U87 glioma growth when administered continuously and systemically by subcutaneous minipumps in nude mice. In these experiments, the pump reservoirs were filled with 0.45 mg/kg/day of the inhibitor. Analysis of tumor volumes showed that continuous systemic administration of peptide cyclo-VEGI resulted in a 72% inhibition of intracranial glioma growth (Fig. 7B, top panel).
The same experimental design was used to perform term experiments. In these studies, the pump reservoirs changed with a new one containing the same amount of the inhibitor 28 days after implantation. Animals belonging to the control group showed a 50% survival of 32 days. Animals treated with cyclo-VEGI had a 50% survival of 75 days. No side effects were registered during the entire duration of the treatment (Fig. 7B, lower panel).
Histological Analysis
We next performed histological analysis of U87 glioma sections from the
short-term and long-term experiments (using the subcutaneous minipumps). To
quantify angiogenesis in these tumors, we stained endothelial cells for the
expression of CD31. Treatment of U87 tumors with cyclo-VEGI reduced
microvessel density by 68 and 62% when compared with PBS-treated controls for
short-term and long-term experiments, respectively
(Fig. 7C, top
panel). A more detailed analysis revealed that, in control tumors, 55%
capillary-like structures, 35% teleangiectatic or dilated vessels, and 10%
glomeruloid structures were present. In cyclo-VEGI-treated tumors, 85%
capillary structures, 15% teleangiectatic or dilated vessels, and no
glomeruloid structures were observed. This indicates that vessels tends to be
normalized by cyclo-VEGI treatment. Apoptotic indices quantified in
situ by labeling fragmented DNA using the terminal
deoxynucleotidyltransferase-mediated nick end labeling method were increased
in tumors treated with cyclo-VEGI compared with PBS-treated controls
(Fig. 7C, middle
panel). An examination of proliferating cells within the tumor using
Ki-67 nuclear antigen staining revealed no differences in the proliferating
indices between PBS-treated controls and any cyclo-VEGI-treated groups
(Fig. 7C, lower
panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-sheet structure formed by two anti-parallel strands
(5 and 6) and connected by a type II -turn
(27–29,
47). We designed linear and
cyclic peptides based on this sequence and tested them for inhibition of
VEGF165 binding to VEGFR2. It appears that a 17-amino acid cyclic
peptide (CBO-P11), herein designated cyclo-VEGI, inhibits very efficiently the
binding of VEGF to its receptor VEGFR2.2 In this article, we
undertook a series of systematic studies to determine the structural and
biological properties of this peptide.
In aqueous solution, the cyclic peptide presents a weak propensity to adopt
a helix structure in the 1–8 domain. The addition of TFE stabilizes
helical conformations in this region, without any noticeable effect on the
structure of the other parts of this peptide. TFE is known to strengthen
intramolecular hydrogen bonds in peptides. The stabilization of the secondary
structure by TFE has been observed for cyclic analogs of gramicidin S
containing 4n + 2 residues, but intramolecular hydrogen bonding
occurs between two strands in this latter case, resulting in the stabilization
of -sheet structures
(48). The property of this
cyclic peptide to adopt helical conformations was largely unexpected because
mostly -sheet structures or random coil conformations have been observed
for macrocyclic peptides
(49).
Pro has two opposite roles as inducer of helical structure when located at
the N terminus or helix breaker or bender when found in the middle of a
helical domain. In this cyclic peptide, Pro2 and Pro9
have these two opposite effects. Indeed, Pro2 induces the formation
of helix, whereas Pro9 breaks the 1–8 helical domain. The
heterochiral D-Phe-Pro sequence is known to adopt either a type
II' -turn or a -turn in small cyclic peptides. In this
macrocyclic peptide, Pro2 is not part of a type II'
-turn and initiates helix formation. Thus, despite the presence of a
motif known to stabilize a type II' -turn in -hairpins, the
peptide does not adopt a -sheet structure. However, it is not in a
random coil state, because region 1–8 of the peptide explores helical
conformations. This topology could open new possibilities in the design of
scaffolds for protein engineering, because further chemical modifications,
such as C methylation, could be used to stabilize the
-helical domain. The residues Arg82, Lys84, and
His86 localized in the -hairpin 79–93 of VEGF are
implicated in the binding to the VEGFR2 receptor. In this VEGF cyclic peptide,
they are found at the end of the helical domain. Their side chains are not
well defined, and it is likely that they retain enough conformational
flexibility in the cyclic peptide to explore the orientation required for
binding to the VEGF receptors and to prevent VEGF binding.
Although VEGFR1 binds VEGF with 50-fold higher affinity than VEGFR2, most
of the VEGF angiogenic properties like mitogenicity and migration of
endothelial cells are mediated by interaction with VEGFR2
(6,
50). Thus, our initial goal
was to design peptides that specifically interfere with binding of VEGF to its
receptor VEGFR2. Indeed, cyclo-VEGI did interfere with VEGF165
binding on CHO-VEGFR2 in a dose-dependent manner. The linear control peptide
P14 with the same amino acid sequence as cyclo-VEGI did not compete for
receptor binding even at the highest concentration tested
(Fig. 3A). This
indicates that the inhibitory effect of cyclo-VEGI is structure-dependent.
This result is in agreement with the results of other groups who showed that
residues 82–90 in the loop between -strands 5 and 6 of VEGF are
critical for binding to and activation of VEGFR2
(51,
52). This loop is part of the
cyclo-VEGI peptide we have designed. Surprisingly, our peptide also inhibited
the binding of VEGF165 to VEGFR1. One possible explanation for this
finding might be that the receptor binding domain determinants of
VEGF165 for VEGFR1 and VEGFR2 share similar residues. Indeed,
crystallographic studies of VEGF bound to the second Ig-like domain of VEGFR1
and mutagenesis analysis of the binding surface of VEGF for VEGFR2 revealed
that some but not all residues in this loop domain are important for the
binding to both receptors, VEGFR1 and VEGFR2
(27,
28).
Treatment of BAE cells with micromolar concentration of cyclo-VEGI strongly inhibited VEGF165-induced cell proliferation. Although selectivity was observed for inhibition of VEGF-induced proliferation of endothelial cells in comparison to the structurally related growth factor PDGF-BB, cyclo-VEGI was also found to impair FGF-2-induced endothelial cell proliferation. Nevertheless, the activity of cyclo-VEGI appears to be severalfold lower toward FGF-2 than toward VEGF. This effect of cyclo-VEGI on FGF-2-induced proliferation was rather unexpected but may constitute an advantage for several reasons. First, it has been shown previously that FGF-2 can induce production of VEGF via an autocrine feedback loop and that both growth factors can synergize with respect to their ability to induce angiogenesis (53, 54). Second, this might prove beneficial in vivo, because tumor cells can produce both VEGF and FGF-2 and their inhibition has a synergistic effect in the impairment of tumor growth (55, 56). This is also illustrated by the observation that a modified peptide derived from platelet factor-4, which inhibits FGF-2 and VEGF165-induced endothelial proliferation, had a strong inhibitory effect on human intracranial established glioma growth (21). The reasons for the effect of cyclo-VEGI on FGF-2 activity are not clearly understood. HSPGs are molecules found on the cell surface of almost all mammalian cells and have been shown to modulate the biological activity of growth factors (57). FGF-2 and VEGF165 function in concert with cell surface-bound HSPGs to promote binding to their specific receptors and to induce their biological responses (58). We cannot rule out the possibility for cyclo-VEGI to bind to the cell surface heparan sulfate proteoglycans (HSPGs) because of its basic residues, thus preventing the binding of VEGF165 and FGF-2 to their receptors and subsequently inhibiting proliferation of endothelial cells. However, the linear control peptide P14, which has the same sequence as cyclo-VEGI, does not have any antagonist effect on FGF-2 or VEGF165-induced proliferation even at the highest dose of 100 µM. This finding suggests that cyclo-VEGI inhibits FGF-2-induced endothelial cell proliferation by another mechanism, probably involving specific structural properties of this cyclic peptide. Preliminary experiments indicate that cyclo-VEGI also impairs FGF-2 binding to endothelial cells, although at higher cyclo-VEGI concentrations. Further studies are underway to solve this issue.
To substantiate further the antagonist activity of cyclo-VEGI, we tested its activity on VEGF-induced signaling and migration. MAP kinases play a central role in controlling growth signals from growth factor receptor tyrosine kinases such as VEGFR2 (59). In agreement with the effect of cyclo-VEGI on VEGF-induced endothelial cell proliferation, we also observed an inhibition of MAP kinase activation by cyclo-VEGI in endothelial cells upon VEGF stimulation. Migration of endothelial cells, an important pre-requisite for angiogenesis, was also affected by cyclo-VEGI in a dose-dependent manner. Cyclo-VEGI inhibited EC migration below 0% serum control levels which suggests that residual VEGF was still present in the media. This may be due to the presence of residual VEGF165 attached to HSPGs on endothelial cells or of VEGF produced by endothelial cells themselves (60, 61).
We next tested effects of cyclo-VEGI for its ability to interfere with
VEGF165-induced angiogenesis on the differentiated day 13 CAM
(62). The typical brush-like
formation of capillaries in the stroma of the CAM induced by human recombinant
VEGF165 is strongly reduced by cyclo-VEGI peptides at 50 µg and
not by linear control peptide P14. Cyclic peptide antagonists for
V3 integrin showed strong anti-angiogenic
effects in the day 10 CAM at 300 µg. Angiogenesis was also induced by VEGF
at lower doses than in our assay (1 versus 6 µg)
(63). These comparisons
indicate that cyclo-VEGI inhibits VEGF-induced angiogenesis in vivo
in a very efficient way.
Gliomas constitute the most frequent class of primary brain tumors and are
among the most malignant cancers, often resulting in the death of affected
patients within months (64).
In our model, established human intracranial glioma in nude mice decreased
significantly by 
70% in size when treated with cyclo-VEGI in short and
long term experiments. Moreover tumor-bearing mice treated with cyclo-VEGI
lived significantly longer than those in the control group. Highly
vascularized brain tumors such as gliomas produce high levels of VEGF in
culture (56,
65). The decreased number of
vessels for the cyclo-VEGI-treated tumors is consistent with a direct
inhibition of VEGF signaling (as observed in our in vitro
experiments) which in turn impairs tumor growth. This is also in agreement
with an indirect (i.e. anti-angiogenic) antitumor effect rather than
a direct antiproliferative effect on the tumor cells as evidenced by the
absence of effect of cyclo-VEGI on the proliferation of tumor cells in
vitro and in vivo. Histological analysis of cyclo-VEGI-treated
gliomas showed characteristic findings observed in tumors treated with other
potent anti-angiogenic compounds like endostatin and angiostatin. Although the
proliferation index remained unchanged in tumor tissue irrespective of
treatment, cyclo-VEGI decreased microvessel density which was accompanied by
an increase in the apoptotic index as observed previously for other
angiogenesis inhibitors (66,
67).
Taken together, these results indicate that cyclo-VEGI exhibits unique structural features and inhibits angiogenesis and tumor development in vivo. Furthermore, its inhibitor activity affects multiple angiogenesis pathways. Cyclo-VEGI is a promising candidate for the development of new cyclic angiogenesis inhibitor molecules useful for the treatment of cancer or other angiogenesis-related diseases.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains Tables S1–S3.
Supported by the "Association pour la Recherche Contre le
Cancer."
** Supported by the "Fédération des Avaugles et
Handicapés Visuels de France."
To whom correspondence should be addressed: INSERM E 0113, Molecular
Angiogenesis Laboratory, Université de Bordeaux 1, 33405 Talence,
France. Fax: 33-5-40-00-87-05; E-mail:
a.bikfalvi{at}croissance.u-bordeaux.fr.
1 The abbreviations used are: VEGF, vascular endothelial growth factor;
FGF-2, fibroblast growth factor-2; PDGF-BB, platelet-derived growth factor-BB;
cyclo-VEGI, cyclo-vascular endothelial growth inhibitor; CAM, chorioallantoic
membrane; VEGFR, vascular endothelial growth factor receptor; BAE, bovine
aortic endothelial; BCE, bovine capillary endothelial; CHO, Chinese hamster
ovary; ERK1,2, extracellular signal-regulated kinases 1 and 2; HUVEC, human
umbilical vein endothelial cell; PBS, phosphate-buffered saline; CSD, chemical
shift deviation; HSQC, heteronuclear single quantum correlation spectroscopy;
TFE, 2,2,2-trifluoroethanol; DMEM, Dulbecco's modified Eagle's medium; FBS,
fetal bovine serum; MAP, mitogen-activated protein; HSPG, heparan sulfate
proteoglycan; Fmoc, N-(9-fluorenyl)methoxycarbonyl; NOE, nuclear
Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; EC,
endothelial cell; TOCSY, total correlation spectroscopy; HMPB, total
correlation spectroscopy; BHA, butylated hydroxyanisole; HSPGs, heparan
sulfate proteoglycans.
2 S. Shinkaruk, L. Zilberberg, X. Canron, C. Chauseau, M. Bayle, B. Desbas,
A. Bikfalvi, and G. Deleris, manuscript in preparation.
3 M. Hagedorn, M. Balke, A. Schmidt, W. Bloch, H. Kurz, S. Javerzat, B.
Rousseau, J. Wieting, and A. Bikfalvi, submitted for publication.
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