1 Molecular and Cell Biology Laboratory, IDI-IRCCS, via Monti di Creta 104,
00167 Rome, Italy
2 Pharmacology Laboratory, IDI-IRCCS, via Monti di Creta 104, 00167 Rome,
Italy
3 Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH,
UK, and CNR Center of Molecular Biology, c/o Department of Biochemical
Sciences, University of Rome 'La Sapienza', P.le A. Moro 5, 00185 Rome,
Italy
* Author for correspondence (e-mail: a.orecchia{at}idi.it)
Accepted 16 May 2003
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Summary |
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Key words: Angiogenesis, Soluble receptor, Integrin, VEGFR-1
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Introduction |
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Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is one of the
tyrosine kinase transmembrane receptors for VEGF
(De Vries et al., 1992), and
the only known high affinity receptor for VEGF-B and placenta growth factor
(PlGF) (Korpelainen and Alitalo,
1998
). Despite being closely related to the type III tyrosine
kinase-Fms/Kit/platelet derived growth factor (PDGF), VEGFR-1 is classified
into a distinct class of receptors composed of seven immunoglobulin (Ig)-like
domains in the extracellular region
(Shibuya et al., 1990
). The
VEGFR-1 II Ig-like domain comprises the regions that are principally involved
in VEGF and PlGF binding and in the activation of the signal transduction
cascade (Davis-Smyth et al.,
1996
).
VEGFR-1 is mainly expressed on endothelial cells, but other cell types,
such as monocytes, macrophages (Sawano et
al., 2001) and tumour cells
(Bellamy et al., 1999
;
Lacal et al., 2000
;
Masood et al., 2001
), have
been shown to express this receptor on the cell surface. In endothelial cells,
VEGF-mediated activation of VEGFR-1 has been reported not to induce cell
proliferation efficiently (Landgren et
al., 1998
; Rahimi et al.,
2000
; Seetharam et al.,
1995
), whereas it plays a prominent role in cell migration
(Barleon et al., 1996
;
Clauss et al., 1996
).
A differently spliced form of VEGFR-1 mRNA encoding a soluble receptor
variant (sVEGFR-1/sFlt-1) has been isolated in cultured endothelial cells
(Kendall and Thomas, 1993) and
different cell lines (Inoue et al.,
2000
). sVEGFR-1 is thought to be a naturally produced VEGF
antagonist that inhibits the mitogenic effects of this cytokine by functioning
as a dominant-negative trapping protein
(Inoue et al., 2000
) or by
forming non-signalling complexes with VEGFR-2
(Kendall et al., 1996
), but
the physiological role of this soluble variant has not been fully
characterised yet.
Gene knockout studies have demonstrated that VEGFR-1 is essential for
development and differentiation of the embryonic vasculature
(Fong et al., 1995). Mouse
embryos homozygous for a targeted mutation in the VEGFR-1 locus die in utero
at day 8.5 to 9.0 (Fong et al.,
1995
). In these animals, EC develop in both embryonic and
extraembryonic sites, but fail to organise in normal vascular channels,
suggesting that VEGFR-1 is primarily involved in vascular morphogenesis.
Moreover, embryos lacking VEGFR-1 display an increased outgrowth of EC and
hemangioblast commitment (Fong et al.,
1999
). The excess of EC in these animals inhibits the proper
organisation of vascular structures. In contrast, mice carrying a homozygous
deletion limited to the intracellular kinase domain of VEGFR-1 show a correct
development of blood vessels (Hiratsuka et
al., 1998
). This selective knockout is still able to produce the
soluble form of the receptor and displays a truncated form comprising only the
receptor extracellular domain on the cell surface. The phenotype of these
animals suggests that VEGFR-1 has a role that is independent of its tyrosine
kinase activity.
In this study, we localised the soluble VEGFR-1 within the extracellular
matrix deposited by EC in culture, and demonstrated that it is able to support
EC adhesion and migration through the interaction with the 5ß1
integrin.
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Materials and Methods |
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The anti-VEGFR-1 polyclonal antibody was tested for VEGFR-1 specificity in ELISA and western blotting. No cross reaction was observed against either VEGFR-2/Fc or VEGFR-3/Fc chimeras.
Rabbit polyclonal antibody (pAb) H-225, C-17 (anti-VEGFR-1), and C-20
(anti-VEGFR-2) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), while
the goat pAb recognising the N terminus of VEGFR-1 (AF321) was from the
R&D Systems (Minneapolis, MN). The monoclonal antibody (mAb) anti-human
fibronectin FN 15 was from ICN Biomedicals Inc. (Costa Mesa, CA), whereas the
mAb anti-fibronectin cell attachment fragment 3E3 was from Chemicon (Temecula,
CA). Function blocking antibodies against various integrins were as follows:
mouse mAb JBS5 (anti-5ß1), mouse mAb Jß1a (anti-ß1),
mouse mAb LM609 (anti-
vß3), and goat pAb anti-
5ß1 (all
purchased from Chemicon). The rat mAb GoH3 (anti-
6) was kindly provided
by Dr A. Sonnenberg (The Netherlands Cancer Institute, Amsterdam, The
Netherlands) and the mAb NKI-SAM-1 (anti-
5) was from Immunotech
(Marseille, France). Recombinant human VEGFR-1/Fc, PDGFRß/Fc, VEGFR-3/Fc,
VEGFR-2/Fc chimeras, and recombinant human VEGF and placenta growth factor
were purchased from R&D Systems. Human IgG1 were from Calbiochem (La
Jolla, CA). Vitronectin, fibronectin, cycloheximide and monensin were obtained
from Sigma-Aldrich (St Louis, MO). Trypsin was from ICN Biomedicals Inc. The
GRGDTP and GRGESP peptides were from Invitrogen-Life Technologies (Paisley,
UK), and the purified
5ß1 integrin, octyl-ß-D-glucopyranoside
formulation, was from Chemicon.
Cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated from freshly
delivered umbilical cords, as previously described
(Gimbrone, 1976), and cultured
in Endothelial Cell Growth Medium-2 Kit from Clonetics (BioWhittaker Inc,
Walkersville, MD). The human microvascular endothelial cell line (HMEC-1) was
a generous gift of Dr F. J. Candal (Center of Disease Control and Prevention,
Atlanta, GA) (Ades et al.,
1992
) and was cultured in MCDB 131 medium (Sigma-Aldrich)
supplemented with 10% foetal bovine serum (Hyclone Laboratories, Logan, UT)
plus hydrocortisone (Sigma-Aldrich) and epidermal growth factor (Austral
Biological, San Ramon, CA). Normal human fibroblasts were isolated from human
skin biopsies and cultured as previously described
(Wirtz et al., 1987
).
Preparation and analysis of the extracellular matrix (ECM)
The analysis of the ECM components was carried out according to previously
described protocols (Delwel et al.,
1993; Gagnoux-Palacios et al.,
2001
; Owensby et al.,
1989
), with minor modifications. Briefly, HUVEC were grown to
confluence on 96-multiwell culture plates. Cell monolayer was then incubated
overnight at 4°C with PBS/20 mM EDTA and washed with PBS/1% Triton X-100.
This treatment leaves the ECM intact, free of cell debris and firmly attached
to the well surface.
For the detection of the soluble VEGFR-1, the matrix was blocked with 1% BSA/PBS for 3 hours and then incubated for 2 hours with 10 µg/ml mAb against VEGFR-1 (H-225) or fibronectin (FN-15). After five washes with PBS/0.1% Tween 20, plates were incubated with a secondary biotinylated antibody (Vector Laboratories Inc., Burlingame, CA) for 2 hours at room temperature. Streptavidin-alkaline phosphatase conjugate and the appropriated substrate (4-nitrophenylphosphat, Roche Diagnostic, Basel, Switzerland) were used for detection. Absorbance was determined at 405 nm using a Microplate reader 3550-UV (Bio-Rad, Hercules, CA).
Immunoblot analysis of the ECM was also performed. Cell, ECM or total (containing both ECM and cells) samples were collected in the same final volume of SDS sample buffer (50 mM Tris-HCl pH 7.5, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and boiled for 5 minutes. Polypeptides were separated on 6% SDS-polyacrylamide gels and then transferred to supported nitrocellulose membranes (Hybond-C; Amersham Biosciences, Buckinghamshire, UK), using a Transphor TE 50X unit (Hoefer Scientific Instruments, San Francisco, CA). Membranes were blocked in 2% blocking solution (Roche Diagnostics)/TBS pH 7.5 for 1 hour, and incubated overnight at 4°C either with the goat pAb AF321 recognising the N terminus of VEGFR-1 (0.5 µg/ml), or with the rabbit pAbs recognising the C terminus of VEGFR-1 (C-17) and VEGFR-2 (C-20), diluted 1:200. After two washes with 0.1% Tween 20/TBS pH 7.5, membranes were incubated with the appropriated secondary antibody, diluted 1:5000 in 1% blocking solution/TBS, for 1 hour at room temperature and then washed four times with 0.1% Tween 20/TBS. Detection was carried out using the ECLTM western blotting detection reagents from Amersham Biosciences.
For immunofluorescence experiments, EC were seeded on untreated glass coverslips and left to reach confluence. Matrix was prepared as described above, and fixed with 4% paraformaldehyde in PBS for 5 minutes at room temperature. The anti-sVEGFR-1 polyclonal antibody or the anti-FN (FN-15) monoclonal antibody (1:100 dilution) were layered on the fixed matrix for 1 hour at 37°C. After several washes, secondary anti-rabbit or anti-mouse biotinylated antibodies (Vector Laboratories Inc.) were added, and then a streptavidin-FITC conjugate (Amersham Biosciences) was used. Matrix, on coverslips, were then mounted on slides and observed with a fluorescence microscope (Zeiss-Axiophot, Oberkochen, Germany). To test the specificity of the signal obtained with the anti-sVEGFR-1 pAb, the antibody was incubated with the corresponding blocking peptide and then used in the assay as described above.
Cell adhesion assay
Solid support was prepared by incubating immunological 96-multiwell plates
with various concentrations of the receptor/Fc chimeras, fibronectin or
vitronectin solubilised in PBS. After 2 hours, the coating solution was
removed, and the well surface was blocked with 3% BSA in PBS for 18 hours
before plating EC in serum-free medium at 3104 cells per well.
After incubation at 37°C for the indicated time, the wells were washed
with PBS, attached cells were fixed with 3% formaldehyde and stained with 0.5%
crystal violet. The attachment efficiency was determined by quantitative dye
extraction and spectrophotometric measurement of the absorbance at 540 nm
using a Microplate reader 3550-UV (Bio-Rad). Results represent the mean of
triplicate samples ± s.d. All experiments were repeated at least three
times. In competition experiments, cells were preincubated with 10 µg/ml
antibodies, 20 µg/ml VEGFR-1/Fc chimera, 0.4 mM RGD or RGE peptides for 15
minutes at room temperature before plating and were then left to adhere for 30
minutes at 37°C in the presence of the compounds. VEGF or placenta growth
factor (20 µg/ml), EGTA (10 mM), EDTA (10 mM), MgSO4 (5 mM),
MnCl2 (5 mM), CaCl2 (5 mM) or trypsin (0,05% w/v) were
added at the time of cell plating.
Solid-phase binding assay
The solid-phase binding assay was performed as previously described
(Mould et al., 1998).
Immunological 96-multiwell plates were coated overnight at 4°C with 1
µg/ml purified
5ß1 integrin (Chemicon). Plates were then
blocked for 2 hours at room temperature with 1% BSA/PBS. VEGFR-1/Fc was
diluted at the indicated concentrations in 25 mM Tris-HCl, 150 mM NaCl, 1 mM
MgCl2 (buffer A), and overlaid on plates for 1 hour at 37°C.
After three washes with buffer A, plates were incubated with a 1:10000
dilution of an anti-human IgG (Fc specific) alkaline phosphatase-conjugated
antibody (Sigma-Aldrich) for 1 hour at 37°C. The appropriate substrate
(4-nitrophenylphosphat, Roche Diagnostic) was then used for detection.
Absorbance was determined at 405 nm using a Microplate reader 3550-UV
(Bio-Rad). Where indicated, 1 mM EDTA or 10 µg/ml anti-
5ß1
blocking antibody (mAb JBS5) were added during the binding assay. When EDTA
was used, buffer A was prepared without MgCl2. VEGFR-2/Fc (20
µg/ml) was also used as a negative control.
Cell migration
The migration assays were performed in Boyden chambers, as previously
described (Mensing et al.,
1984). Polycarbonate filters (8 µm pore diameter, Nuclepore,
Whatman Incorporated, Clifton, NJ) were coated with 5 µg/ml gelatine
solution. The stimuli for chemotaxis were added to the lower chamber at the
indicated concentrations and HUVEC (1.5x105) were loaded into
the upper chamber. Chemokinesis was tested by including the VEGFR-1/Fc chimera
only in the upper chamber, together with the cells, and, in selected
experiments, the chimera was included in both the upper and the lower
chambers. For haptotactic assays, the under surface of membrane filters,
precoated in the upper surface with gelatine, was coated with 10 µg/ml
VEGFR-1/Fc or vitronectin, as described
(Nasreen et al., 2000
).
Background migration was measured by using filters coated with 10 µg/ml
BSA. Migration medium (1 µg/ml heparin/0.1% BSA in EBM-2) was always used
to prepare the solutions with the stimuli and as a negative control. After 2
hours incubation (5% CO2, 37°C), the filter was removed and
cells were fixed in 3% paraformaldehyde in PBS and stained in 0.5% crystal
violet. Cells from the upper surface were removed by wiping with a cotton
swab. The chemotactic and chemokinetic responses were determined by counting
the migrating cells attached to the lower surface of the filter in 12 randomly
selected microscopic fields (x200 magnification) per experimental
condition. Blocking of the
5ß1 integrin was performed by
preincubating the cells for 45 minutes with antibodies specific for this
integrin (mAb JBS5), or with unrelated antibodies as controls (mAb GoH3), at
room temperature and under constant shaking. Afterwards, cells were loaded
into the Boyden chambers in the presence of the antibodies, and the migration
assay was carried out as described above.
Cell spreading assays
Polystyrene Petri dishes, 100 mm diameter, were coated with 10 µg/ml
VEGFR-1/Fc chimera or 10 µg/ml fibronectin, washed and blocked, as
described above. EC (2.5x106/dish) were plated and left to
adhere for 1 to 12 hours. Cell spreading was monitored using an inverted
microscope. To show the F-actin distribution and microfilament organisation,
glass coverslips were coated with 10 µg/ml VEGFR-1/Fc chimera or 10
µg/ml fibronectin for 2 hours at room temperature, then saturated by
further incubation with 3% BSA/PBS. Coverslips were washed with PBS and cells
seeded at 2.5x 104cells/cm2. Cells were fixed at
different times with 4% paraformaldehyde for 5 minutes, washed twice with PBS
and stained with fluorescein-labelled phalloidin (Sigma-Aldrich) for 30
minutes at room temperature. To detect fibronectin fibrils deposited by EC,
coverslips were coated with 10 µg/ml VEGFR-1/Fc or 10 µg/ml vitronectin
and treated as described above. Three hours after cell seeding, matrix was
prepared as described above and then immunostained with an anti-fibronectin
antibody (FN-15). After several washes, secondary anti-mouse biotinylated
antibody was added, and then the streptavidin-FITC conjugate was used.
Coverslips were then mounted on slides, and the preparations observed with a
fluorescence microscope (Zeiss-Axiophot). In selected experiments,
cycloheximide was added at 100 µg/ml before plating and monensin was added
at 1 µM, 18 hours before plating. Experiments with RGD or RGE peptides (0.4
mM) were carried out by adding them to confluent EC monolayers and evaluating
cell detachment after 1 hour incubation at 37°C.
Sequence analysis
The sequence of the soluble VEGFR-1 was compared to those of the proteins
of known structure from the PDB (Berman et
al., 2000) and to those in non-redundant databases of protein
sequences
[http://www.ncbi.nlm.nih.gov/
and (Holm and Sander, 1998
)]
by using the BLAST (Altschul et al.,
1997
) and SUPERFAMILY (Gough
et al., 2001
) servers available through the World Wide Web.
Sequence homologues collected with BLAST were aligned using CLUSTALW
(Thompson et al., 1994
),
whereas SUPERFAMILY provides multiple sequence alignments. Domain analysis was
performed using the Pfam (Bateman et al.,
2002
) and SMART (Schultz et
al., 1998
) servers available via the World Wide Web.
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Results |
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EC and human fibroblasts were cultured for 72 hours and then detached from
the plates using a method that leaves the extracellular matrix intact
(Delwel et al., 1993;
Gagnoux-Palacios et al., 2001
;
Owensby et al., 1989
). Human
fibroblasts do not express either the membrane-bound or the soluble form of
VEGFR-1, and were thus used as a negative control. The presence of VEGFR-1
within the matrix was detected by using an anti-VEGFR-1 antibody (H-225)
against the extracellular region of the receptor. VEGFR-1 could be detected in
the matrix produced by both HMEC-1 and HUVEC whereas no signal was obtained in
the matrix deposited by human fibroblasts
(Fig. 1A). The EC matrix was
also analysed using a rabbit polyclonal antibody raised against a peptide
mapping at the C terminus of soluble VEGFR-1 (anti-sVEGFR-1). The C-terminal
region of the soluble receptor differs from that of the transmembrane VEGFR-1
since it is encoded by an alternatively-spliced RNA. As shown in
Fig. 1B, soluble VEGFR-1 was
detected in the EC matrix. As a positive control, the matrix was labelled with
an anti-fibronectin antibody (Fig.
1B). Consistent with the ELISA and immunofluorescence findings, a
western blotting analysis showed that the VEGFR-1 variant present within the
ECM has a molecular weight that corresponds to the soluble form and is
specifically recognised by an antibody directed towards the extracellular
region of the receptor (Fig.
1C). Moreover, this polypeptide was not revealed by using an
antibody directed towards the intracellular domain of the receptor
(Fig. 1D). Both antibodies
detected the presence of the transmembrane variant of VEGFR-1 in the cell
extract, but not in the ECM fraction (Fig.
1C,D). Transmembrane VEGFR-1 appeared as a doublet, probably
because of different glycosylation forms
(Seetharam et al., 1995
).
VEGFR-2, analysed as a negative control, was only detected in the cell extract
sample (data not shown). As a positive control and to further validate the ECM
fraction obtained, the presence of fibronectin within this fraction was
confirmed by immunoblotting (Fig. 1C and
D). Altogether, these data demonstrate the presence of
polypeptides from the extracellular domain of VEGFR-1 in the matrix produced
by human endothelial cells.
|
VEGFR-1 is directly involved in human EC adhesion
The possible role of the VEGFR-1 extracellular region as a direct mediator
of EC/matrix interactions was then tested using an in vitro cell adhesion
assay. Ninety-six-multiwell plates were coated with increasing amounts of a
chimeric protein comprising the extracellular region of VEGFR-1 fused to the
human immunoglobulin Fc domain (VEGFR-1/Fc) or with fibronectin as a positive
control. Wells were also coated with BSA as a negative control. The relative
number of adherent HMEC-1 or HUVEC was quantified 1 hour after plating. As
expected, EC effectively adhered to fibronectin, but also to VEGFR-1/Fc-coated
surfaces at similar levels (Fig.
2A). A dose-dependent increase in cell adhesion was observed for
both substrates as early as 30 minutes after plating. Comparable data were
obtained using the HMEC-1 cell line or HUVEC primary cultures. To exclude
non-specific interactions involving the Fc domain present in the fusion
protein, or contaminants in the commercial preparation, PDGFRß/Fc and
VEGFR-2/Fc chimeric proteins or human IgG1 were used as controls. None of
these substrates supported EC adhesion
(Fig. 2B). Addition of
VEGFR-1/Fc chimera to the cell suspensions abrogated cell attachment
(Fig. 2C). Adhesion of HMEC-1
and HUVEC to VEGFR-1/Fc-coated surfaces was also specifically inhibited by a
rabbit polyclonal anti-VEGFR-1 antibody, but not by the preimmune serum
(Fig. 2C). The same antibody
did not affect cell attachment on fibronectin-coated surfaces.
|
Integrin subunits mediate cell attachment to VEGFR-1
The capability of VEGFR-1/Fc to support EC adhesion was almost completely
abolished by pretreatment of the cells with trypsin, indicating that the cell
interaction with the matrix-associated VEGFR-1 is mediated by a protein
(Fig. 3A). To assess whether
divalent cations affect EC/VEGFR-1 interactions, HMEC-1 and HUVEC adhesion on
VEGFR-1/Fc was assayed in the presence of different cations at a concentration
of 5 mM. VEGFR-1/Fc-mediated EC attachment was supported by the addition of
Mg2+ to the medium. Mn2+ further enhanced EC attachment,
while Ca2+ inhibited it (Fig.
3A). Addition of 10 mM EGTA or EDTA reduced adhesion levels to
those obtained in the presence of Ca2+. These data indicate that
soluble VEGFR-1 binds to a divalent cation-dependent protein on the cell
surface. Since cells interact with the extracellular matrix in a divalent
cation-dependent way mainly through receptors of the integrin family
(Mould et al., 1995;
Smith et al., 1994
), we
investigated whether integrin subunits could be involved in the binding of the
EC to VEGFR-1. The effect of anti-integrin antibodies on this interaction was
therefore analysed. Considering the critical role played by the
5ß1 and
vß3 integrins in angiogenesis both in vitro
and in vivo (Brooks et al.,
1994
; Kim et al.,
2000
), we used blocking antibodies directed against these two
integrins. Adherence of EC to VEGFR-1/Fc was greatly reduced in the presence
of blocking antibodies directed either towards the
5ß1 integrin or
the ß1 or
5 integrin subunits. An anti-
vß3 antibody
did not affect EC adhesion on VEGFR-1/Fc
(Fig. 3B). As expected, EC
binding to fibronectin was decreased by the anti-
5ß1 blocking
antibody and, to a lesser extent, by the anti-
vß3 antibody
(Fig. 3B). Simultaneous
incubation of EC with the anti-
5ß1 and anti-
vß3
antibodies resulted in a greater inhibitory effect on adhesion to fibronectin,
whereas no further inhibition could be observed on VEGFR-1/Fc
(Fig. 3B). Indeed, the
combination of the anti-
5ß1 and anti-
vß3 antibodies
appeared slightly less effective in inhibiting cell adhesion on VEGFR-1/Fc
than the anti-
5ß1 antibody alone. This attenuated blocking might
be due to steric hindrance between the two antibodies, as previously reported
(Leong et al., 2002
). As
expected, the anti-
vß3 antibody alone could almost completely
block EC adhesion on vitronectin (Fig.
3B).
|
VEGFR-1 induces EC migration through the interaction with the
5ß1 integrin
The role of soluble VEGFR-1 in supporting EC adhesion and its localisation
in the ECM suggested a possible involvement of this form in inducing EC
migration, similarly to that already shown for other components of the ECM
(Clark et al., 1988;
Mensing et al., 1984
). We
therefore evaluated the ability of the VEGFR-1/Fc chimera to induce EC
migration. As shown in Fig. 4A,
VEGFR-1/Fc-stimulated EC chemotaxis, and this effect was dose-dependent and
detectable when the protein was present at 1 µg/ml, reaching maximal
activity at 5 µg/ml. At this concentration, VEGFR-1/Fc showed an effect
comparable to that of the epidermal growth factor (EGF), a known
chemoattractant for EC (Fig.
4A) (Chen et al.,
1993
). To determine whether VEGFR-1/Fc also stimulated
chemokinesis (random cell movement), VEGFR-1/Fc was placed in the lower and/or
upper chambers of the Boyden chamber as indicated in
Fig. 4A. The presence of
VEGFR-1/Fc in the upper chamber, together with the cells, induced a slight
increase in cell motility, that was not concentration dependent
(Fig. 4A). Modest chemokinetic
activity was observed at lower VEGFR-1/Fc concentrations (1 µg/ml). In this
condition, the chemokinetic response was slightly more relevant than the
chemotactic one. At VEGFR-1 concentrations in which chemotaxis was substantial
(5 µg/ml), the simultaneous presence of the chimera in the upper chamber
abrogated the capability of EC to migrate through the filter
(Fig. 4A). This result shows
that EC are sensitive to a concentration gradient of VEGFR-1/Fc between the
two chambers, and indicates that the chemotactic response is preponderant with
respect to chemokinesis. Since ECM components usually stimulate cell motility
through haptotactic mechanisms, the capability of VEGFR-1/Fc molecules
immobilised on polycarbonate filters to induce haptotaxis of EC was analysed
(Fig. 4C). An increased
migration of EC to the under surface of the filters was observed (70% increase
with respect to the background controls,
Fig. 4C). Control filters
coated with vitronectin showed a 100% increase in EC migration with respect to
the background control (Fig.
4C). The role of the VEGFR-1/
5ß1 interaction in both
chemotaxis and haptotaxis was investigated by incubation of the EC with
anti-
5ß1 integrin blocking antibodies before the assay. An
inhibition of almost 70% of the VEGFR-1/Fc-induced chemotaxis and 100% of the
VEGFR-1/Fc-induced haptotaxis was observed when compared to EC pretreated with
an unrelated antibody (Fig.
4B,D). In contrast, neither EGF-induced chemotaxis nor
vitronectin-induced haptotaxis were significantly affected by the
anti-
5ß1 antibodies (Fig.
4B,D).
|
Characterisation of VEGFR-1 binding to the 5ß1
integrin
To confirm the capability of VEGFR-1 to interact with 5ß1, we
analysed direct VEGFR-1/Fc-integrin interaction in vitro by using a
solid-phase binding assay (Rehn et al.,
2001
). VEGFR-1/Fc specifically bound to immobilised
5ß1 integrin, whereas direct binding of related proteins, such as
VEGFR-2/Fc, was not detected (Fig.
5B). No significant binding was observed in control BSA-coated
wells. Binding was concentration dependent
(Fig. 5A), and the specificity
of the interaction was confirmed by binding inhibition using the
anti-
5ß1 blocking antibody or EDTA
(Fig. 5B).
|
These findings strongly support the assumption that cell adhesion to
VEGFR-1 and VEGFR-1-induced cell migration are mediated by the interaction of
this receptor with 5ß1.
Since VEGFR-1 and fibronectin seem to bind the same integrin receptor, it
could be hypothesised that VEGFR-1 interaction with the integrin could be
mediated by fibronectin. In order to exclude this possibility, cell adhesion
assays were performed in the presence of RGD peptides that should compete with
integrin for binding to fibronectin. When EC were treated with the RGD peptide
prior to plating on VEGFR-1/Fc, or with the peptide containing the RGE-related
sequence, used as a control, no inhibitory effect was observed
(Fig. 5C). As expected,
treatment with the same concentration of RGD peptides blocked cell binding to
fibronectin (Fig. 5C). These
data confirmed that the EC interaction with VEGFR-1 is independent of the
presence of other matrix proteins that bind through the RGD sequence. As
further evidence, an anti-fibronectin antibody, reported to significantly
block cell adhesion on fibronectin
(Pierschbacher et al., 1981),
was also used. In this assay, no significant inhibition of cell adhesion on
VEGFR-1 was seen (data not shown).
In addition, neither VEGF nor placenta growth factor, another ligand of VEGFR-1, competed with EC adhesion to VEGFR-1/Fc (Fig. 5C), suggesting that different receptor sites are involved in growth factor or integrin binding.
VEGFR-1 induces EC spreading
To investigate whether the interaction between VEGFR-1 and the
5ß1 integrin that supports cell adhesion could also activate EC
spreading, cells were seeded on VEGFR-1/Fc or fibronectin and analysed at
different times. Cells plated on VEGFR-1/Fc spread and organised actin
microfilaments in a similar manner to cells plated on fibronectin. However,
cell spreading on VEGFR-1/Fc was slower than on fibronectin. On the latter
substratum, EC were fully spread after 1 hour
(Fig. 6A), whereas on
VEGFR-1/Fc spreading was not detectable 1 hour after plating
(Fig. 6B), and was completed
only after 6 hours (Fig. 6C).
Extracellular matrix proteins, newly produced by EC attached to VEGFR-1, might
be required to achieve complete cell spreading after the primary interaction
with VEGFR-1. We therefore tested whether de novo protein synthesis and
secretion were required. Cells treated with cycloheximide, an inhibitor of
protein translation, or monensin, which blocks protein secretion, still
adhered to VEGFR-1/Fc, but only a few cells spread
(Fig. 7A). The same treatment
did not significantly affect spreading on fibronectin
(Fig. 7A). Since EC bind to
different extracellular matrix proteins mainly by recognising the RGD
sequence, we tested the effect of such a peptide on VEGFR-1-induced cell
spreading. The exposure to the RGD-containing peptide (but not to the peptide
containing the RGE-related sequence, used as a control) of confluent EC
monolayers seeded on VEGFR-1/Fc induced a significant rounding of cells
(Fig. 7B), suggesting that EC
spreading on this substratum could be dependent upon the cell secretion of
other extracellular matrix proteins. We finally tested the secretion and
organisation of endogenous fibronectin by EC adherent on VEGFR-1/Fc and on
vitronectin, as a control. After 3 hours, cells seeded on VEGFR-1 began to
spread and showed organised fibronectin fibrils in the newly deposited matrix,
whereas cells plated on vitronectin were completely spread on this substratum,
and no fibronectin fibrils could be detected
(Fig. 7C).
|
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Discussion |
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The presence of the soluble VEGFR-1 within the matrix has therefore
directed our research towards further investigation of the role of this
receptor variant, in addition to that of growth factor binder. In our assays a
VEGFR-1 polypeptide, which corresponds to the soluble variant, has been
attached on a solid surface, mimicking the ECM deposited by the cells. In such
an assay, this protein directly supported EC adhesion. We have demonstrated
that antibodies directed towards the 5 or the ß1 integrin subunits
specifically inhibit the adhesion of EC to VEGFR-1. In addition, we have shown
a direct interaction between VEGFR-1 and the
5ß1 integrin in
vitro, which strongly suggests that the
5ß1 integrin is an EC
ligand for the matrix-associated VEGFR-1. The
5ß1 integrin has
already been implicated in the regulation of several aspects of EC growth and
differentiation.
5ß1 binding to fibronectin results in the
accumulation of signalling molecules and cytoskeletal components at focal
adhesion sites and in the stimulation of tyrosine phosphorylation of proteins
associated with focal adhesions (Hocking
et al., 1998
). An important role for
5ß1 integrin in
angiogenesis has been clearly established, since antagonists of this integrin
block tumour-induced angiogenesis (Kim et
al., 2000
). Moreover,
5-null teratocarcinomas are poorly
vascularised and
5-null embryoid bodies show delayed and reduced
formation of tubular endothelial structures
(Taverna and Hynes, 2001
).
Although a functional role for
5ß1 integrin in vasculogenesis and
embryonic angiogenesis has never been directly confirmed, the loss of the gene
encoding the
5 integrin subunit is lethal during embryogenesis and
associated with vascular and cardiac defects
(Yang et al., 1993
).
5ß1 integrin interacts with more than one ligand during
angiogenesis, and our results raise the possibility that the
VEGFR-1/
5ß1 interaction also could play a role in the correct
development of the primary vasculature or in the modulation of angiogenesis in
the adult. Further studies are required to characterise the biological
relevance of such an interaction in vivo.
Neither RGD nor other sequence motifs commonly recognised by integrin
5ß1 on different ligands could be mapped on the VEGFR-1 sequence
(Koivunen et al., 1993
).
However, several ligands bind integrins through motifs that include structural
features as well as specific residues. For example, the integrin binding motif
of the vascular cell adhesion molecule-1 (VCAM-1) is a single aspartate
residue at the end of a relatively long loop, and integrin binding residues in
the intercellular adhesion molecules (ICAMs) are involved in long range
interactions (Clothia and Jones,
1997
). We are currently analysing the presence of such structural
determinants in the VEGFR-1 protein.
When administered in solution to the cells, VEGFR-1 was capable of
modulating EC migration in a chemotactic manner. In addition, when VEGFR-1 was
immobilised on the lower surface of the filter, it sustained a haptotactic
response in the EC. Both chemotaxis and haptotaxis appear to be mediated by
the 5ß1 integrin, since they were impaired by blocking antibodies
directed against this adhesion molecule. The induction of EC motility implies
the activation of a cellular response, probably mediated by the
5ß1 integrin itself as a consequence of VEGFR-1 binding. We had
further indication of the initiation of such a response when we analysed EC
spreading on VEGFR-1-coated surfaces. In this case, VEGFR-1 alone was not
sufficient to support EC spreading. However, cell adhesion on VEGFR-1
triggered molecular activation mechanisms that resulted into new ECM protein
secretion, finally leading to cell spreading. Such a process was blocked by
both monensin and cycloheximide, supporting the hypothesis that EC spreading
on VEGFR-1 requires protein synthesis and secretion. Moreover, we have shown
that EC, left to adhere on VEGFR-1, secrete and organise a fibronectin matrix
on which they subsequently spread. Therefore, in promoting EC spreading,
VEGFR-1 acts differently from vitronectin, which induces direct EC spreading,
and shows a behaviour similar to other proteins, such as fibrinogen
(Dejana et al., 1990
) that
promote EC spreading via the release of newly synthesised matrix proteins.
To date the role of the soluble VEGFR-1 in angiogenesis has not been
thoroughly characterised. Soluble VEGFR-1 has been proposed to be a negative
regulator of VEGF-mediated signalling, acting as a decoy molecule or a soluble
competitor. In addition to this function, our findings indicate that the
soluble VEGFR-1 might be actively involved in other aspects of angiogenesis by
playing a role in cell-matrix interactions. This new property of VEGFR-1 and
the previously implied negative regulation of VEGF signalling need not be
mutually exclusive. Indeed, neither VEGF nor placenta growth factor competed
EC adhesion to VEGFR-1. Such a dual mechanism of action has already been shown
for other molecules implicated in angiogenesis. Endostatin, for example,
functions as an angiogenesis inhibitor when present in a soluble form, and as
a mediator of EC adhesion and migration when bound to a solid support
(Rehn et al., 2001).
The finding that a VEGF receptor may be directly involved in cell adhesion
is completely novel. Previous reports demonstrated only an indirect role in
cell adhesion explicated through the up-regulation of integrin expression.
VEGF treatment of EC, in fact, enhances the expression of vß3
(Senger et al., 1996
),
1ß1 and
2ß1
(Senger et al., 1997
)
integrins. Direct interactions between integrins and transmembrane growth
factor receptors on the surface of the same cell have also been described.
VEGFR-2 binds to the
vß3 integrin after activation by VEGF
(Borges et al., 2000
;
Soldi et al., 1999
), and this
interaction seems to contribute to enhancing VEGFR-2 phosphorylation. Similar
data is not yet available for VEGFR-1.
In addition to EC, several tumour cells express VEGFR-1
(Bellamy et al., 1999;
Lacal et al., 2000
;
Masood et al., 2001
), both as
transmembrane and soluble protein. The VEGFR-1/integrin interaction described
here might therefore also play a role in tumour cell adhesion to the
extracellular matrix and could thus represent a new target for the development
of compounds aimed at limiting tumour-induced angiogenesis and tumour
metastatization.
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