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Complete Structure of an Increasing Capillary Permeability Protein
(ICPP) Purified from Vipera lebetina Venom
ICPP IS ANGIOGENIC VIA VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR SIGNALING*
Received for publication, March 6, 2002, and in revised form, May 13, 2002
Published, JBC Papers in Press, May 20, 2002, DOI 10.1074/jbc.M202202200
Ammar Gasmi‡
储
, Christine Bourcier§, Zohra Aloui‡, Najet Srairi‡, Sandrine Marchetti§,
Clotilde Gimond§, Stephen R. Wedge
¶
, Laurent Hennequin
¶
, and Jacques Pouysse´ gur§**
From the ‡Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, B. P. 74, 1002 Tunis-Belvede`re, Tunisia,
¶
Cancer and Infection Research, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom,
and the §Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre A. Lacassagne,
33 Avenue Valombrose, 06189 Nice, France
The partial sequence of the increasing capillary per-
meability protein (ICPP) purified from Vipera lebetina
venom revealed a strong homology to vascular endothe-
lial growth factor (VEGF)-A. We now report its complete
amino acid sequence determined by Edman degradation
and its biological effects on mouse and human vascular
endothelial cells. ICPP is a homodimeric protein linked
by cysteine disulfide bonds of 25115 Da revealed by mass
spectrometry. Each monomer is composed of 110 amino
acids including eight cysteine residues and a pyroglu-
tamic acid at the N-terminal extremity. ICPP shares 52%
sequence identity with human VEGF but lacks the hep-
arin binding domain and Asn glycosylation site. Besides
its strong capillary permeability activity, ICPP was
found to be a potent in vitro angiogenic factor when
added to mouse embryonic stem cells or human umbili-
cal vein endothelial cells. ICPP was found to be as po-
tent as human VEGF165 in activating p42/p44 MAPK, in
reinitiation of DNA synthesis in human umbilical vein
endothelial cells, and in promoting in vitro angiogenesis
of mouse embryonic stem cells. All these biological ac-
tions, including capillary permeability in mice, were
fully inhibited by 1
M of a new specific VEGF receptor
tyrosine kinase inhibitor (ZM317450) from AstraZeneca
that belongs to the anilinocinnoline family of com-
pounds. Indeed, up to a 30 times higher concentration of
inhibitor did not affect platelet-derived growth factor,
epidermal growth factor, FGF-2, insulin,
␣
-thrombin, or
fetal calf serum-induced p42/p44 MAPK and reinitiation
of DNA synthesis. Therefore, we conclude that this
venom-derived ICPP exerts its biological action (perme-
ability and angiogenesis) through activation of VEGF
receptor signaling (VEGF-R2 and possibly VEGF-R1).
Angiogenesis is a tightly regulated process occurring physi-
ologically during embryonic development, during the men-
strual cycle, and in wound healing. It is also associated with a
number of pathological situations including diabetic retino-
pathy, inflammation, brain edema following ischemic stroke,
solid tumor growth, and metastasis. A number of polypeptide
growth factors have been demonstrated to induce and regulate
angiogenesis in vivo, among them fibroblast growth factor
(FGF),
1
platelet-derived growth factor (PDGF), epidermal
growth factor (EGF), and vascular endothelial growth factor
(VEGF)-A (1, 2). Although the mode of activation at the recep-
tor level differs, all these mitogens activate the ubiquitously
expressed isoforms of mitogen-activated protein kinases re-
ferred to as p42/p44 MAPK or Erk, two essential transducers of
growth, survival, and differentiation signals.
VEGF was the first mitogenic growth factor proven to have
endothelial cell specificity and to be critical for blood vessel
formation. The vascular endothelium-specific growth factors
are now known to include five members of the VEGF family,
four members of the angiopoietin family, and at least one
member of the large ephrin family (3). VEGF is a multifunc-
tional cytokine that is produced by virtually every tissue and
overexpressed upon hypoxic stress and oncogenic transforma-
tion (4). It is a homodimeric glycoprotein, expressed as several
spliced variants; the major forms contain 121, 165, 189, and
206 amino acids. VEGF121 differs from the larger VEGF iso-
forms in that it is the only VEGF type that does not possess
heparin binding ability (5). These isoforms act in a coordinate
fashion to recruit and expand the tumor vasculature (6). The
main receptors that seem to be involved in initiating signal
transduction cascades in response to VEGFs comprise a family
of closely related receptor tyrosine kinases that are expressed
almost exclusively on neovasculature and on the tumor endo-
thelium. They consist of three members now termed VEGFR-1
(Fms-like tyrosine kinase, Flt-1), VEGFR-2 (kinase insert
domain-containing receptor (KDR)), and the VEGF-C and -D
receptor, VEGFR-3 (known previously as Flt-3). Some other
accessory receptors (neuropilins) that seem to be involved pri-
marily in modulating binding to the main receptors have also
been reported (7). Their roles in signaling have not yet been
fully elucidated (3). VEGFR-2, however, via activation of in-
trinsic tyrosine kinase activity, appears to mediate all the
* This work was supported by grants from the Centre National de la
Recherche Scientifique, Le Ministe`re de l’Education, de la Recherche et
de la Technologie, La Ligue Nationale Contre le Cancer (e´quipe labe-
lise´e J. P.). The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
储
To whom correspondence may be addressed. Fax: 216-71-791833;
E-mail: ammar.gasmi@pasteur.rns.tn.
** To whom correspondence may be addressed. Fax: 33-492-03-1225;
pouysseg@unice.fr.
1
The abbreviations used are: FGF, fibroblast growth factor; VEGF,
vascular endothelial growth factor; svVEGF, snake venom VEGF;
VEGFR, VEGF receptor; VTKI, VEGF receptor tyrosine kinase inhibi-
tor; PDGF, platelet-derived growth factor; ICPP, increasing capillary
permeability protein; KDR, kinase insert domain-containing receptor;
MAPK, mitogen-activated protein kinase; ERK, extracellular signal-
regulated kinase; MEK, MAPK/ERK kinase; ES, embryonic stem; HF,
hypotensive factor; SFM, serum-free media; PBS, phosphate-buffered
saline; HPLC, high pressure liquid chromatography.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 33, Issue of August 16, pp. 29992–29998, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org29992
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major actions of VEGF: capillary permeability, chemotaxis, cell
survival, and cell division (8).
The possibility that vascular growth factors may help pre-
vent or repair damaged and leaky vessels offers therapeutic
hope for ischemic diseases, diabetic retinopathy, or inflamma-
tory setting (9, 10). In opposition, new antitumoral approaches
targeting the tumor vasculature via inhibition of VEGF signal-
ing are actively being developed; they include neutralizing
anti-VEGF antibodies, anti-VEGF receptor antibodies, soluble
VEGF receptors, antisense VEGF techniques, and VEGF re-
ceptor tyrosine kinase inhibitors (11).
We have previously isolated a protein from Vipera lebetina
venom based on its potent ability to increase capillary perme-
ability. The partial sequence of this protein, referred to as
increasing capillary permeability protein (ICPP), revealed a
VEGF-like structure (12). In this report, we present the com-
plete amino acid sequence of ICPP and demonstrate that this
venom related-VEGF is capable of inducing p42/p44 MAPK
activity and DNA synthesis in human umbilical vein endothe-
lial cells (HUVEC) and of promoting in vitro angiogenesis.
Interestingly, all these ICPP-induced biological actions are
fully inhibited by a new VEGF receptor tyrosine kinase inhib-
itor, as presented here.
EXPERIMENTAL PROCEDURES
Materials—ICPP was purified from V. lebetina venom (12). Reverse
phase analytical columns C8 (5
m, 4.6 ⫻ 250 mm) were purchased
from Beckman Instruments. Endoproteinases Asp-N, Arg-C, and Lys-C
were of sequencing grade and were obtained from Roche Molecular
Biochemicals. Recombinant human FGF-2 and VEGF165 were pro-
duced in our laboratory from Escherichia coli and Pichia pastoris,
respectively, after purification on heparin binding affinity columns,
whereas human recombinant PDGF

and EGF were from Sigma. All
other reagents used were of analytical grade from commercial sources.
The AstraZeneca compound 4-fluro-5-{[6-methoxy-7-(2-methoxye-
thoxy) cinnolin-4-yl]amino}-2-methylphenol (ZM317450), referred to
here as VEGF receptor tyrosine kinase inhibitor (VTKI), belongs to the
anilinocinnoline family of compounds. It was prepared according to the
protocol presented in the patent WO 9734876 by A. P. Thomas and
L. F. Hennequin.
Reduction and Alkylation of ICPP—Reduction was performed by
incubating ICPP for1hat37°Cin6
M guanidine-HCl, 0.5 M Tris-HCl,
2m
M EDTA, 1.4
M DTT (dithiothreitol), pH 7.5. Then, alkylation
occurred following addition of 4-vinylpyridine (9
mol final concentra-
tion) and terminated after 5 min by addition of DTT to a final concen-
tration of 14
M. The mixture was desalted on a reverse phase HPLC on
a C8 column. Solvents A and B were 0.1% trifluoroacetic acid (v/v) and
0.1% trifluoroacetic acid (v/v) in 100% acetonitrile, respectively. Protein
was eluted from the column by a linear gradient of 10 – 80% of solvent
B in 60 min at a flow rate of 1 ml/min and monitored at 214 nm.
Enzymatic Digestions of ICPP—Digestions of reduced and alkylated
ICPP were carried out by the endoproteinases Arg-C, Lys-C, or Asp-N.
The denatured protein was incubated in appropriate buffer medium.
The suitable time, temperature, enzyme/substrate ratio, and termina-
tion of the reactions were performed according to the manufacturer’s
instructions. Urea (2
M) was added to the reaction mixture to ensure
solubility. The resulting peptides were subjected to a reverse phase
chromatography on C8 column and eluted by an increasing gradient of
10 – 60% of solvent B in 60 min. Effluent was continuously monitored at
214 nm, and peaks were collected manually and subjected to sequence
analysis.
Amino Acid Analysis and Sequence Comparison—The sequences of
N-terminal subunits were determined after chemically unblocking with
HCl in anhydrous methanol (13) by Edman degradation with an Ap-
plied Biosystems 470A liquid-phase sequencer equipped with on-line
phenylthiohydantoin reverse HPLC using an RP18 column. The se-
quences of peptides obtained from enzymatic digestions of reduced and
alkylated ICPP were performed as described previously. A search for
similar proteins was performed following computer analysis with the
BLAST data base search program.
Mass Spectral Analysis—Determination of the molecular mass of
native ICPP was carried out on a Voyager DE-RP matrix-assisted laser
desorption ionization time-of-flight mass spectrometer (PerSeptive Bio-
systems, Inc., Framingham, MA). A sinapinic acid matrix at 10 mg/ml
in 50% acetonitrile/50% H
2
O/0.1% trifluoroacetic acid was used.
Culture, MAPK Activity, and DNA Replication of HUVECs—
HUVECs were isolated from umbilical cord veins by collagenase perfu-
sion as described previously (14) and cultivated in SFM (Invitrogen)
supplemented with 20% fetal calf serum, 20 ng of FGF-2, and 10 ng of
EGF/ml (EGF provided by Sigma). For reinitiation of DNA synthesis,
HUVEC were serum-starved for 24 h, trypsinized, and replated in
24-well plates coated with gelatin at 50,000 cells/well in 0.5 ml of SFM.
Six h later, cells were incubated in 0.5 ml of SFM containing 1
Ci of
tritiated thymidine and stimulated with growth factors or ICPP in the
presence or absence of various concentrations of VTKI. After 24 h,
TCA-insoluble material was collected, and radioactivity was counted.
For the measurement of p42/p44 MAPK activation, HUVEC were
serum-starved for 24 h, trypsinized, and replated in 12-well plates at
10
5
cells in 1 ml of SFM. After 6 h, cells were stimulated with growth
factors or ICPP with or without VTKI; VTKI was preincubated 15 min
prior to stimulation. Stimulation was arrested after 10 min of stimula-
tion with cold PBS washing, and cells were harvested in 100
lof
Laemmli sample buffer followed by protein separation on SDS-10%
polyacrylamide gel electrophoresis (15). Western blotting was per-
formed as described previously (16). The blots were incubated with a
1/5000 dilution of the anti-phospho p42/p44 MAPK monoclonal anti-
body (Sigma).
Receptor Tyrosine Kinase Assays—The ability of VTKI to inhibit the
kinase activity associated with the VEGF receptors R1 (Flt-1) and R2
(KDR), the FGF receptor FGFR1, and the EGF receptor was determined
using a previously described enzyme-linked immunosorbent assay (17).
Receptor tyrosine kinases used in isolated enzyme assays were gener-
ated as insect cell lysates following cell infection with recombinant
baculoviruses containing kinase domains.
Briefly, compounds were incubated with enzyme, 10 m
M MnCl
2
, and
2m
M ATP in 96-well plates coated with a poly(Glu,Ala,Tyr) 6:3:1
random copolymer substrate (Sigma). The ATP concentration used was
at, or just below, the respective K
m
value. Phosphorylated tyrosine was
detected by sequential incubation with mouse IgG anti-phosphotyrosine
antibody (Upstate Biotechnology Inc., Lake Placid, NY), a horse radish
peroxidase-linked sheep anti-mouse Ig antibody (Amersham Bio-
sciences), and 2,2⬘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid
(Roche Molecular Biochemicals). Microcal Origin software (Version
3.78, Microcal Software Inc., Northhampton, MA) was used to interpo-
late IC
50
values by non-linear regression (four-parameter logistic
equation).
Protein Determination—Protein concentration was determined by
the procedure of Lowry et al. (18) with the folin phenol reagent and with
bovine serum albumin as a standard.
In Vitro Angiogenesis—Culture and differentiation of embryonic
stem (ES) cells was used as an angiogenesis test in vitro. Mouse 129/
OLA ES cells (E14Tg2A.IV clone, initially provided by Dr. M. Hooper,
Edinburgh, UK and subcloned by Dr. A. Smith, Edinburgh, UK) were
grown in Dulbecco’s modified Eagle’s medium with Glutamax-1 and
sodium pyruvate (Invitrogen) containing 10% fetal calf serum
(Dutscher, Brumath, France), 50 units/ml penicillin, 50
g/ml strepto-
mycin, 0.1 m
M

-mercaptoethanol, and non-essential amino acids (all
reagents from Invitrogen). They were kept undifferentiated by the
addition of either 10
3
units/ml recombinant leukemia inhibitory factor
purchased from Sigma or 100 units/ml leukemia inhibitory factor pro-
duced in COS cells as described previously (19).
For differentiation, ES cells were cultured in hanging drops as de-
scribed previously (20) with some modifications. Briefly, ES cells were
detached in trypsin/EDTA and aggregated into embryoid bodies in the
above described Dulbecco’s modified Eagle’s medium lacking supple-
mental leukemia inhibitory factor. Aggregation was performed in 20-
l
drops hanging from the lids of bacteriological Petri dishes and contain-
ing 800 cells. The lids were then placed over PBS-filled dishes and
incubated at 37 °C. This was designated as day 0. At day 3, the result-
ing embryoid bodies were then transferred to gelatin-coated 24-well
tissue culture plates. When indicated, the medium was supplemented
with human 10 ng/ml rVEGF165 (Sigma) or various concentrations of
purified ICPP. After 12 days of differentiation, embryoid bodies were
fixed in 4% paraformaldehyde (in PBS) for 20 min at room temperature,
permeabilized with 0.2% Triton X-100 for 5 min, and blocked in PBS
containing 10% fetal calf serum for 2 h. Cells were then incubated with
a rat anti-CD31 antibody (clone MEC13.3; BD PharMingen) for1hat
room temperature. Staining was revealed by incubating first with a
biotin-conjugated donkey anti-rat antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA), and second with Alexa Fluor-conjugated
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streptavidin (Molecular Probes, Eugene, OR). Preparations were
mounted in PBS:glycerol (1:9) and viewed under a Leica microscope.
Vascular Permeability Assays—Capillary permeability activities
were tested using slight modifications of the Miles permeability assay
(21) in female MF1 mice (Harlan France; 3–4 weeks of age). Prior to the
assay, mice were injected intravenously (tail vein) with 50
lofa1%
Evan’s Blue solution. Immediately afterward, 50
l of PBS, or an
equivalent volume of various concentrations of VEGF165 or ICPP in
PBS, were injected intradermally on the back of mice. To evaluate the
inhibitory action of VTKI, the compound (50
lat10
M in PBS) was
preinjected (intradermally), 60 min prior to administration of a cyto-
kine, at the same site. Animals were sacrificed 30 min after cytokine
injection, and the dorsal epithelium was dissected and photographed.
To quantify the leakage of the dye, the skin patches were eluted at 56 °C
with formalin and measured with a spectrophotometer (A
600
).
RESULTS
Amino Acid Sequence and Molecular Mass Determination of
ICPP—Reduced and alkylated ICPP was subjected to separate
enzymatic digestions by Asp-N, Lys-C, and Arg-C. These diges-
tions yielded many peptides designated N, L, and R, respec-
tively. The resulting fragments were purified by reverse phase
chromatography on a C8 column (data not shown), numbered
according to their order of elution from the column, and sub-
jected to sequencing by Edman degradation. Unblocking of the
N-terminal amino acid sequence was performed as described
under “Experimental Procedures,” and 52 residues were clearly
identified. ICPP was found to be a homodimeric protein, each
monomer consists of 110 residues with 8 half-cystines/mol, and
the entire sequence and the sequence of the peptide fragments
determined are presented in Fig. 1. The amino acid data were
corroborated with mass spectrometric data. The molecular
mass of ICPP calculated from the sequence is 25099 Da and is
in good agreement with that determined by mass spectrometry,
which is 25115 Da.
Sequence Comparison of ICPP with Hypotensive Factor (HF),
Snake Venom VEGFs (svVEGFs), and Human VEGF—The
search for similar proteins by computer analysis of the se-
quence data of ICPP revealed that this protein had a high
similarity with the VEGF of many animal species and some
similarity with PDGF. Fig. 2 shows the alignment of amino
acid sequences of ICPP with those of human VEGF (22), an HF
purified from Vipera aspis aspis (23), and svVEGF derived from
Bothrops insularis (Fig. 2, Bins svVEGF) and Bothrops jara-
raca (Fig. 2, Bjar svVEGF) venoms (24). Based on a BLAST
search, ICPP shares sequence identities with these proteins
with rates of 52, 95, 65, and 67%, respectively. Sequence data
of ICPP exhibit many residues identified by site-directed mu-
tagenesis of human VEGF as being important for receptor
binding (25). It is interesting to note that most of the residues
implicated as being important for VEGF binding to VEGFR-1/
R-2 were also conserved (26). The heparin binding site in hu-
man VEGF has been localized to the C-terminal 55 residues of
the VEGF165 spliced form (27). Interestingly, ICPP lacks the
heparin binding domain, and unlike all VEGF isoforms, it does
not contain any Asn-linked glycosylation site defined by the
consensus sequence: NX(S/T). Perhaps this subtle difference
with VEGF may result in distinct biological activities.
ICPP Stimulates p42/p44 MAP Kinase Activity in HUVEC,
Implication of VEGF Receptors—Since ICPP and human VEGF
have a high sequence similarity, it was appropriate to compare
the effect of these proteins on signaling and angiogenic poten-
tial using HUVEC. The Raf-1⬎MEK⬎p42/p44 MAPK has been
demonstrated to rapidly convey growth and survival signals
from a variety of receptor tyrosine kinases. We therefore tested
the biological action of ICPP on HUVEC that are responsive to
VEGF and FGF for growth and differentiation. Fig. 4A shows
that like human VEGF165 and FGF-2, V. lebetina ICPP stim-
ulates p42/p44 MAP kinases in HUVEC. This action is rapid,
detected within 2 min, peaks around 10–15 min, and decreases
to basal level after4hofstimulation (data not shown). This
temporal action as well as the maximal intensity of MAPK
activation parallel that observed with human VEGF165. In the
same cells, however, p42/p44 MAPK activation could reach 3–5
times higher levels in response to FGF-2. We then compared
the potency of ICPP and human VEGF165 using MAP kinase
activation as a reporter system. On a molar basis, ICPP puri-
fied to homogeneity is at least 1.5 times more potent than
human recombinant VEGF165 obtained from Sigma or freshly
prepared from P. pastoris and purified on heparin affinity
columns (data not shown).
We then sought to determine whether ICPP can signal
through endogenous VEGF receptors. To answer this point, we
exploited the specificity of tyrosine kinase inhibitors developed
by AstraZeneca. These compounds attain selectivity by compet-
ing with the non-conserved hydrophobic pocket of the ATP
binding site in kinases. The inhibitor used in this study
(ZM317450) is a new molecule of the anilinocinnoline family of
compounds (Fig. 3), referred to here as VTKI. In isolated en-
zyme assays, VTKI is a potent inhibitor of VEGFR-2 tyrosine
kinase (IC
50
⫽ 50 nM) with submicromolar activity versus the
kinase activity of VEGFR-1 (Table I). In comparison with its
inhibitory activity versus VEGFR-2 tyrosine kinase, VTKI
demonstrated ⬎2000-fold selectivity versus that associated
with FGFR1 and EGFR (Table I). Selectivity is also conserved
in endothelial cells (HUVEC); 10
M VTKI ablates fully ICPP-
and VEGF-induced MAPK activation but does not affect that
stimulated by FGF-2 (Fig. 4A). Inhibition of ICPP-stimulated
MAPK activation was even found to be complete at 1
M. The
dose response of VTKI was explored on HUVEC stimulated
with equally potent growth factors: VEGF (30 ng/ml) or ICPP
(10 ng/ml). The VTKI concentration inhibiting half of the re-
sponse (EC
50
) is 100 nM for both VEGF (Fig. 4B) and ICPP
(data not shown). Next we explored the specificity of VTKI by
evaluating its action on the ER22, a derivative of the fibroblas-
tic cell line CCL39 that express several receptor tyrosine
kinases, PDGF-R, FGF-R, and EGF-R (28). Fig. 5 shows that
MAPK stimulation by all agonists remains unaffected by VTKI
at a concentration 300 times greater than the EC
50
for inhibi
-
tion MAPK stimulated by VEGF. In addition, we showed that
VTKI did not affect MAP kinase activation in response to the G
protein-coupled receptor agonist,
␣
-thrombin, or fetal calf
serum (Fig. 5). These results demonstrate that VTKI is not a
FIG.1. Complete amino acid se-
quence of ICPP. The N-terminal amino
acid sequence was determined after
chemical unblocking of the intact protein.
Peptides obtained from enzymatic diges-
tions with Asp-N, Lys-C and Arg-C were
designated A, L, and R, respectively.
Structure and Biology of Vipera lebetina Venom VEGF29994
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general blocker of MAPK signaling and highlight the specificity
toward VEGF receptor kinase. Collectively, these data suggest
that ICPP mediates its effect on MAPK signaling via VEGF
receptor signaling.
ICPP Is a Potent Angiogenic Factor through VEGF Receptor
Signaling—Since we demonstrated in the previous section that
ICPP can signal through VEGF receptors, the next question
was to investigate whether, besides MAPK signaling, ICPP is
capable of mimicking all the biological actions of VEGF. We
first tested its capacity to induce angiogenesis in two in vitro
systems, DNA proliferation in HUVEC and growth and differ-
entiation of mouse vascular endothelial cells. Fig. 6A shows
that serum-starved HUVEC are able to reinitiate DNA synthe-
sis in response to VEGF165 or ICPP. FGF-2, a more potent
agonist for stimulation of MAPK signaling, is also a much more
potent mitogen for HUVEC (Fig. 6A). In our assays, DNA
synthesis was stimulated 3-fold above basal with ICPP and
5–10-fold above basal with FGF-2. Interestingly the mitogenic
action of ICPP or VEGF is fully suppressed by VTKI, whereas the
activity of FGF is practically unaffected. We regularly observed a
10 –15% inhibition of FGF-induced mitogenic action that we at-
tributed to the autocrine production of VEGF in response to FGF,
a proposal consistent with a previous observation (29). This bio-
logical assay was then used to compare the sensitivity of ICPP
and VEGF toward the VTKI. As shown in Fig. 6B, reinitiation of
DNA synthesis was fully suppressed at 100 n
M of VTKI, indicat-
ing that reinitiation of DNA synthesis appears slightly more
sensitive than the MAPK inhibition. The VTKI dose-response
curves for ICPP and VEGF are identical, suggesting that ICPP
and VEGF are using the same receptor system for in vitro angio-
genesis. In addition, we concluded that ICPP triggers MAPK
activation and reinitiation of DNA synthesis exclusively via
VEGF-R2 signaling. Indeed, expression of VEGF-R2 alone in the
fibroblastic cell line CCL39, devoid of VEGF-R, was sufficient to
elicit MAPK activation and DNA synthesis in response to ICPP.
This action was fully abolished by VTKI (data not shown).
The second in vitro system exploited to assess ICPP-induced
angiogenesis is more demanding since it records the capacity of
a cytokine to promote the proliferation of embryonic vascular
endothelial cell precursors and their capacity to differentiate
into a vascular network. In comparison with FGF-2, VEGF has
been reported to be an extremely potent cytokine in this bio-
logical assay (30). Interestingly, as shown in Fig. 6C, ICPP is a
potent molecule that, like VEGF, can induce a vascular-like
network positive for the vascular specific marker CD31. Al-
though this assay does not allow an accurate quantitation,
ICPP has always been more potent than VEGF165 in estab-
lishing a vascular differentiated network. This ICPP-induced
growth and differentiation is again fully prevented by the
addition of VTKI (Fig. 6C).
ICPP-stimulated Increased Capillary Permeability Is Medi-
ated through VEGF Receptor Signaling—ICPP was isolated by
its capacity to stimulate capillary permeability on mice (12),
and indeed, when compared with VEGF, the best permeability
factor known to date, ICPP was found to be an extremely potent
permeability factor (Fig. 7A). This biological activity was ex-
amined using the Miles assay (21), which involved intradermal
injection of purified ICPP and VEGF165 in mice and measure-
ment of the leakage of Evan’s Blue dye into the extravascular
space (Fig. 7B). Intradermal injection of PBS was used as a
control. For this biological action, ICPP was found to be as
potent if not more potent than VEGF165. The ICPP- and
VEGF-induced permeability was inhibited by VTKI when the
inhibitor was preinjected 60 min before the peptide growth
factor (Fig. 7). This inhibition reflects VEGF receptor targeting
since VTKI is unable to prevent increased permeability trig-
FIG.2.Sequence alignment of ICPP (Vleb ICPP) with the hypotensive factor (Vasp HF) purified from V. aspis aspis venom (23),
the deduced svVEGF amino acid sequences characterized from B. insularis (Bins svVEGF) and B. jararaca (Bjar svVEGF) venoms
(24), respectively, and human VEGF (22). Human VEGF residues identified by site-directed mutagenesis as being important for receptor
binding (25) are in bold. Residues implicated in VEGF R2 (Flk-1 (fetal liver kinase-1)/KDR) binding along with the VEGF crystal structure are
underlined (26). The heparin binding domain (55 residues) is indicated by italics. The box indicates the N-glycosylation site, and the asterisks
indicate identical residues.
FIG.3.Structure of VTKI. See “Experimental Procedures” for the
preparation of the AstraZeneca compound (ZM317450).
TABLE I
Inhibition of isolated receptor tyrosine kinase activity
(IC
50
,
M) by VTKI
The ability of VTKI to inhibit recombinant kinase activity was exam-
ined using a 96-well ELISA assay with 2
M ATP. Data represent the
mean ⫾ S.E. of at least three separate determinations.
VEGF-R1 VEGF-R2 EGFR FGFR1
0.50 ⫾ 0.04 0.05 ⫾ 0.01 ⬎100 ⬎100
Structure and Biology of Vipera lebetina Venom VEGF 29995
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gered via the
␣
-thrombin receptor peptide (31) activating
PAR-1.
2
DISCUSSION
Snake venoms represent an extraordinary source of biologi-
cal molecules that have been invaluable to our knowledge and
understanding of basic biological processes. Besides the toxins
acting directly in the nervous system, many venom proteins
target blood capillaries, preventing blood coagulation via dis-
integrins and enzymes degrading fibrinogens, whereas others
increase the permeability of blood capillaries. This is the case
for bradykinin potentiating factors purified from B. jararaca
that convert angiotensin I into angiotensin II and phospho-
lipase A2 purified from Trimeresurus mucrosquamatus, which
induces release of histamine from mast cell degranulation (32).
In this context, it is remarkable to see the emergence of a new
set of selected molecules with permeability capacity in snake
venoms. ICPP, an increasing capillary permeability protein of
V. lebetina venom, is indeed a VEGF-related molecule highly
homologous to the HF with vascular permeability activity (23).
During the preparation of this manuscript, another report
demonstrated the expression of related VEGF molecules in the
venom of Bothrops jararaca (pit viper) (24), a result extending
the notion that many snakes have evolved to specifically ex-
press in their venoms VEGF-like molecules, the most potent
permeability factors described so far in vertebrates. During
evolution, the co-selection in snake venoms of potent toxins
together with the most effective permeability factors was cer-
tainly crucial for rapid dissemination of the toxins in the gen-
eral circulation of the prey.
In the present study, we determined the complete amino acid
sequence of ICPP, confirming that it is structurally related to
the PDGF family. This protein consists of two homodimers of
110 amino acids having a molecular mass of 25115 Da. ICPP
shares the highest sequence identity with HF, the hypotensive
factor purified from V. aspis aspis (23), and the recently iden-
tified svVEGF from B. insularis pit viper venom (24). ICPP,
together with the other snake svVEGFs, shares about 50%
identity with human VEGF-A (22). ICPP, like HF and svVEGF,
differs in length from VEGF165, lacking a heparin binding
domain and a potential N-linked glycosylation site. The recom-
binant protein svVEGF from the B. insularis pit viper was
biologically characterized only by its ability to increase vascu-
lar permeability (24). However, in addition to increasing per-
meability, HF has been shown to also exert a strong hypoten-
sive effect and have a mitogenic effect on endothelial cells. The
mitogenic activity of HF was 5–10 times lower than that of
VEGF and was inhibited by cycloheximide. The authors spec-
ulated that this protein may induce a signaling response and
increase protein synthesis in endothelial cells, but studies ex-
amining the mechanism of action have not been reported (23).
VEGF functions by interacting with two well characterized
high affinity tyrosine kinase receptors, VEGF-R1 and VEGF-
R2, that are selectively expressed on endothelium. VEGF-R2
2
V. Vouret-Cravieri, unpublished results.
FIG.4. ICPP, like VEGF, activates
p42/p44 MAP kinases in HUVEC; the
dose response of the VTKI on VEGF-in-
duced MAP kinase activation is shown.
Serum-starved HUVEC were stimulated
for 15 min with 10 ng/ml VEGF165, ICPP
or FGF-2 in the absence (0) or presence
(10
M) of VTKI (A). The kinase inhibitor
(dissolved in Me
2
SO) was added 15 min
prior to stimulation, and Me
2
SO alone
was added to the media of cells that did
not receive VTKI (1% final concentration).
Total extracts (50
g of proteins) were
separated on SDS-polyacrylamide gel
electrophoresis and immunoblotted with
a specific anti-phospho p42/p44 MAP ki-
nase monoclonal antibody as described
under “Experimental Procedures.” The
doublet represents both ERK isoforms. C,
control. HUVEC were stimulated for 15
min with 10 ng/ml VEGF165 (⫹) and pre-
incubated with various concentrations of
VTKI (B). Me
2
SO was kept constant at
1%. Activation of p42/p44 MAPK was
monitored as indicated in “Experimental
Procedures.”
FIG.5. VTKI is a specific VEGF re-
ceptor antagonist. ER22 fibroblastic
cells, a derivative of CCL39 expressing
800,000 EGF receptors (28), were serum-
starved for 24 h and stimulated with five
different agonists: human PDGFb (10 and
30 ng/ml), human FGF-2 (10 and 30 ng/ml),
human EGF (10 and 30 ng/ml), 5% fetal
calf serum (FCS), or human
␣
-thrombin
(THR) at 1 unit/ml. Where indicated, cells
were preincubated with either 1 or 10
M of
VTKI. Activation of p42/p44 MAPK was
monitored as described under “Experimen-
tal Procedures.”
Structure and Biology of Vipera lebetina Venom VEGF29996
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appears to be the dominant signaling receptor in VEGF-
induced mitogenesis, and permeability increases (33, 34),
whereas the role of VEGF-R1 in endothelial cell function is
much less clear. Recent findings suggest that VEGF-R1 may
have a negative role, either by acting as a decoy receptor or by
suppressing signaling through VEGF-R2 (35).
Plasmin cleavage of VEGF165 generates a 110-residue long
N-terminal fragment (VEGF110) that lacks the heparin bind-
ing domains. VEGF110 is thought to maintain the ability to
bind to the VEGF receptors, but its endothelial cell mitogenic
potency is decreased substantially (100-fold) relative to
VEGF165, indicating that the heparin binding domains are
critical for stimulating endothelial cell proliferation (36). This
finding also concurs with experiments using VEGF121, which
does not bind to either heparan sulfates or to the extracellular
matrix. VEGF121 has been described as being 10–100-fold less
potent than VEGF165 at inducing biological responses in en-
dothelial cells (37). The effect of glycosylation of VEGF165 on
receptor binding has also been studied by Keyt et al. (25) using
a constructed unglycosylated form of VEGF. They showed that
N-linked carbohydrate at Asn-75 does not appear to have an
effect in mediating VEGF receptor binding and the precise role
of glycosylation remains to be elucidated.
ICPP and VEGF165 were tested in parallel in various bioas-
says including MAP kinase activation, in vitro angiogenesis,
and capillary permeability. In this regard, our studies have
shown that ICPP, like VEGF165 and FGF-2, stimulates p42/
p44 MAP kinases in HUVEC. The activity of ICPP was found to
FIG.6. ICPP is angiogenic; it stimulates reinitiation of DNA
synthesis in HUVEC and promotes cell proliferation and differ-
entiation of mouse embryonic stem cells. In A and B, for reinitia-
tion of DNA synthesis, confluent and serum-starved HUVEC were
stimulated for 24 h (see “Experimental Procedures”) in SFM in the
presence of an agonist (VEGF165, ICPP, or FGF-2) and tritiated thy-
midine (1
Ci/ml). Where indicated (gray bars), cells were preincubated
with 10
M VTKI. Tritiated thymidine incorporation into acid-insoluble
material was measured in triplicate wells, and the average (less than
15% variation for each condition) was plotted. In B, the stimulation
conditions were identical to those in panel A, except that cells received
various concentrations of VTKI during the course of the stimulation. In
C, mouse ES cells were cultivated and differentiated as described under
“Experimental Procedures.” At day 3, the resulting embryo bodies were
then transferred to gelatin-coated 24-well tissue culture plates. Where
indicated, the medium was supplemented with 10 ng/ml VEGF165, 10
ng/ml ICPP, or no supplement (Control). After 12 days of differentia-
tion, embryoid bodies were fixed and revealed with a rat anti-CD31
antibody as indicated under “Experimental Procedures.”
FIG.7. Capillary permeability stimulated by ICPP is antago-
nized by VTKI. ICPP, VEGF165, or PBS was injected intradermally
into mice within a dorsal region (n ⫽ 5 per group). To evaluate the
inhibitory action of VTKI, the compound (50
lat10
M in PBS) was
preinjected (60 min prior to administration) at the intended site of
cytokine administration. Animals were sacrificed 30 min after cytokine
injection, and the dorsal epithelium was dissected and photographed
(A). The leakage of the Evan’s Blue dye into the extravascular space was
quantified as indicated under “Experimental Procedures” (B). The val-
ues represent the mean of three independent experiments ⫾ S.D. In the
picture shown, 50
l of VEGF165 at 30 ng/ml and ICPP at 10 ng/ml
were injected intradermally (see “Experimental Procedures”).
Structure and Biology of Vipera lebetina Venom VEGF 29997
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be at least 1.5 times more potent than human recombinant
VEGF165 and mediated via signaling through endogenous
VEGF receptors. The requirement for VEGF receptor signaling
in transduction of a biological response to ICPP was demon-
strated using VTKI, an antagonist that can selectively inhibit
the kinase activity associated with VEGF-R2 and VEGF-R1.
This inhibitor ablates fully ICPP- and VEGF-induced MAPK
activation (IC
50
of ⬃100 nM versus both) but does not affect that
induced by FGF-2. The specificity of VTKI has been further
demonstrated in ER22 cells, where it was found to not affect
PDGF-, EGF-, or FGF-2-mediated MAPK activation at a con-
centration of 30
M. Furthermore, VTKI did not inhibit the
MAP kinase activation in response to the G protein-coupled
receptor agonist,
␣
-thrombin, or fetal calf serum.
When the mitogenic action of ICPP was studied, it was found
to be as potent if not slightly more potent than VEGF165 at
reinitiating DNA synthesis in HUVEC. This ICPP-induced re-
sponse was fully suppressed by VTKI. In addition, we have
observed that ICPP promotes DNA proliferation in HUVEC
and induces growth and differentiation of mouse embryonic
endothelial cells. VTKI can also inhibit these ICPP-induced
angiogenic and permeability responses, indicating an involve-
ment of VEGF receptor signaling. In each biological response
examined, ICPP was found to possess remarkably similar and
at least three times more potent bioactivity than VEGF165
despite the absence of a heparin binding domain. The struc-
tural differences between ICPP and VEGF, in particular the
absence in ICPP of heparin binding domains and consensus
N-glycosylation sites, suggest that some of their biological
activities might be different.
In conclusion, we have shown that the amino acid sequence
of ICPP displays a high similarity to that of VEGF and that
capillary permeability, angiogenesis, endothelial cell mitoge-
nicity, and MAP kinase activation induced by ICPP were
mediated through VEGF receptor signaling. These findings
provide the first evidence that ICPP is a novel member of the
family of VEGF-like growth factors. Moreover, considering the
therapeutic impact of VEGF-A in the treatment of coronary
heart disease or critical limb ischemia (38, 39), ICPP, which
appears more potent than VEGF, may represent a novel can-
didate for therapeutic angiogenic approaches aimed at growing
new vasculature.
Acknowledgments—We thank Dr. Z. Ben Lasfar (Veterinary Labora-
tory, Pasteur Institute of Tunis) for providing snake venom, P. Y.
Haumont from PerkinElmer Life Sciences for the mass determination
of ICPP by mass spectrometry, J. Kendrew (AstraZeneca, Alderley
Park, UK) for generating tyrosine kinase data, D. Roux and M. Hattab
(CNRS, Nice, France) for the CCL39 cell line expressing VEGF-R2 and
for the purification of recombinant FGF and VEGF.
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Structure and Biology of Vipera lebetina Venom VEGF29998
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Hennequin and Jacques Pouysségur
Clotilde Gimond, Stephen R. Wedge, Laurent
Aloui, Najet Srairi, Sandrine Marchetti,
Ammar Gasmi, Christine Bourcier, Zohra
RECEPTOR SIGNALING
ENDOTHELIAL GROWTH FACTOR
IS ANGIOGENIC VIA VASCULAR
Venom: ICPPVipera lebetinaPurified from
Capillary Permeability Protein (ICPP)
Complete Structure of an Increasing
TRANSDUCTION:
MECHANISMS OF SIGNAL
doi: 10.1074/jbc.M202202200 originally published online May 20, 2002
2002, 277:29992-29998.J. Biol. Chem.
10.1074/jbc.M202202200Access the most updated version of this article at doi:
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