JCS ePress online publication date 15 July 2003
Research Article
3479
Vascular endothelial growth factor receptor-1 is
deposited in the extracellular matrix by endothelial
cells and is a ligand for the α5β1 integrin
Angela Orecchia1,*, Pedro Miguel Lacal2, Cataldo Schietroma1, Veronica Morea3, Giovanna Zambruno1 and
Cristina Maria Failla1
1Molecular and Cell Biology Laboratory, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy
2Pharmacology Laboratory, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy
3Laboratory 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: [email protected])
Accepted 16 May 2003
Journal of Cell Science 116, 3479-3489 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00673
Summary
Vascular endothelial growth factor receptor-1 (VEGFR-1)
is a tyrosine kinase receptor for several growth factors of
the VEGF family. Endothelial cells express a membranespanning form of VEGFR-1 and secrete a soluble variant
of the receptor comprising only the extracellular region.
The role of this variant has not yet been completely defined.
In this study, we report that the secreted VEGFR-1 is
present within the extracellular matrix deposited by
endothelial cells in culture, suggesting a possible
involvement in endothelial cell adhesion and migration. In
adhesion assays, VEGFR-1 extracellular region specifically
promoted endothelial cell attachment. VEGFR-1-mediated
cell adhesion was divalent cation-dependent, and inhibited
by antibodies directed against the α5β1 integrin. Moreover,
VEGFR-1 promoted endothelial cell migration, and this
effect was inhibited by anti-α5β1 antibodies. Direct binding
of VEGFR-1 to the α5β1 integrin was also detected. Finally,
binding to VEGFR-1 initiated endothelial cell spreading.
Altogether these results indicate that the soluble VEGFR1 secreted by endothelial cells becomes a matrix-associated
protein that is able to interact with the α5β1 integrin,
suggesting a new role of VEGFR-1 in angiogenesis, in
addition to growth factor binding.
Introduction
The assembly of endothelial cells (EC) into vascular structures
requires the establishment of cell-cell interactions as well as
the activation of cell membrane transducing receptors by
soluble ligands and by components of the extracellular matrix.
Vascular endothelial growth factor receptor-1 (VEGFR1/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, VEGFmediated 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 VEGFR1 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
Key words: Angiogenesis, Soluble receptor, Integrin, VEGFR-1
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Journal of Cell Science 116 (17)
deletion limited to the intracellular kinase domain of VEGFR1 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 VEGFR1 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 α5β1 integrin.
Materials and Methods
Antibodies and reagents
Two polyclonal sera were generated, one against a peptide mapping
in the second Ig-like domain of the receptor extracellular region (antiVEGFR-1), and the other against a peptide mapping in the unique C
terminus of the soluble form of VEGFR-1 (anti-sVEGFR-1). Rabbit
immunisation was carried out by PRIMM, Milan, Italy.
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 VEGFR1 (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 antifibronectin cell attachment fragment 3E3 was from Chemicon
(Temecula, CA). Function blocking antibodies against various integrins
were as follows: mouse mAb JBS5 (anti-α5β1), mouse mAb JΒ1a (antiβ1), mouse mAb LM609 (anti-αvβ3), and goat pAb anti-α5β1 (all
purchased from Chemicon). The rat mAb GoH3 (anti-α6) was kindly
provided by Dr A. Sonnenberg (The Netherlands Cancer Institute,
Amsterdam, The Netherlands) and the mAb NKI-SAM-1 (anti-α5) was
from Immunotech (Marseille, France). Recombinant human VEGFR1/Fc, PDGFRβ/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 α5β1
integrin, octyl-β-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 (SigmaAldrich) 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. Streptavidinalkaline phosphatase conjugate and the appropriated substrate (4nitrophenylphosphat, 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 96multiwell 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
VEGFR-1 is a ligand for α5β1 integrin
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 α5β1 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 (SigmaAldrich) for 1 hour at 37°C. The appropriate substrate (4nitrophenylphosphat, 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 antiα5β1 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 (1.5×105) 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 (×200
magnification) per experimental condition. Blocking of the α5β1
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 (2.5×106/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%
3481
BSA/PBS. Coverslips were washed with PBS and cells seeded at 2.5×
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.
Results
VEGFR-1 is a component of the endothelial cell
extracellular matrix
Previously reported data have shown that the soluble variant of
VEGFR-1 is secreted by endothelial cells in culture (Kendall
and Thomas, 1993). From a structural point of view, this
receptor form is predicted to be formed by six domains
belonging to the immunoglobulin superfamily, which produces
a fold very commonly found in proteins involved in ECM-cell
adhesion (Clothia and Jones, 1997; Hohenester and Engel,
2002), indicating that the structure of soluble VEGFR-1 is
compatible with a role as an ECM protein. We therefore
investigated whether the soluble VEGFR-1 could become a
component of the extracellular matrix (ECM).
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; GagnouxPalacios 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
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Journal of Cell Science 116 (17)
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 VEGFR1 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, PDGFRβ/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
Fig. 1. VEGFR-1 is a component of the extracellular matrix deposited by EC. (A) HUVEC or
normal human fibroblasts (Fb) were seeded into 96-multiwell culture plates and incubated for 72
hours. Cells were mechanically detached and the amount of VEGFR-1 or fibronectin (FN) deposited
in each well was evaluated by ELISA using anti-VEGFR-1 or anti-fibronectin antibodies.
Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. The
experiment was repeated at least three times with comparable results. (B) Extracellular matrix
deposited by HUVEC was immunostained after cell detachment with an anti-sVEGFR-1 antibody or
with an anti-fibronectin antibody. Both sVEGFR-1 and fibronectin were detectable in the matrix
deposited by the EC. Scale bar: 5 µm. (C,D) Immunoblotting analysis of VEGFR-1 polypeptides in HUVEC extracts and the ECM produced by
these cells, using antibodies recognizing (B) the extracellular (AF327) or (C) the intracellular (C-17) region of VEGFR-1. Equal volumes of
cell extract, ECM material or total samples (in which cells and ECM were collected together), prepared as described in Materials and Methods,
were analysed. Upper panels show the immunoblot analysis of the samples using the anti-human fibronectin antibody FN-15. The molecular
weight markers in kDa are indicated in C.
VEGFR-1 is a ligand for α5β1 integrin
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
Fig. 2. VEGFR-1 supports EC attachment. (A) Dose-dependent cell
attachment on wells coated with the indicated concentrations of
VEGFR-1/Fc or fibronectin (FN). The relative number of attached
cells was assessed by staining with crystal violet and determining the
A540 1 hour after plating. Absorbance resulting from non-specific
cell adhesion was measured on BSA-coated wells. (B,C) VEGFR-1specific promotion of EC adhesion. (B) EC were plated on 10 µg/ml
VEGFR-1/Fc-, VEGFR-2/Fc-, PDGFRβ/Fc-, IgG-, or BSA-coated
wells and cell adhesion was measured as described for A. (C) EC
were plated on 10 µg/ml FN-, VEGFR-1/Fc-, or BSA-coated wells.
Before cell seeding, wells were incubated for 30 minutes with
undiluted preimmune serum or anti-VEGFR-1 rabbit serum.
Alternatively, cells were treated with 20 µg/ml of VEGFR-1/Fc for
15 minutes before plating. Histograms represent the mean
absorbance value of medium from triplicate wells ± s.e.m. The
experiments were repeated at least five times.
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trypsin, indicating that the cell interaction with the matrixassociated 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 cationdependent 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 VEGFR1. The effect of anti-integrin antibodies on this interaction was
Fig. 3. α5β1 integrin mediates EC attachment to VEGFR-1.
(A) Effect of divalent cations and trypsin treatment on EC adhesion
to VEGFR-1. Cells were plated on fibronectin (FN), BSA or
VEGFR-1/Fc in the presence of EGTA, EDTA, Ca2+, Mg2+, Mn2+ or
trypsin, and after checking cell viability by trypan-blue dye exclusion
in control wells, the relative number of attached cells was estimated
as described in Fig. 2. Histograms represent the mean absorbance
value of medium from triplicate wells ± s.e.m. (B) Cells were
incubated with mAbs against β1, α5, α6 (unrelated Ab), α5β1 and
αvβ3 15 minutes before plating on VEGFR-1/Fc, fibronectin (FN) or
vitronectin (VN). Histograms represent the percentage of inhibition,
calculated relative to the adhesion of cells on the same substrates in
the absence of blocking antibodies, ± s.e.m. The experiments in A
and B were repeated at least three times.
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Journal of Cell Science 116 (17)
therefore analysed. Considering the critical role played by the
α5β1 and αvβ3 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 α5β1 integrin
or the β1 or α5 integrin subunits. An anti-αvβ3 antibody did
not affect EC adhesion on VEGFR-1/Fc (Fig. 3B). As
expected, EC binding to fibronectin was decreased by the antiα5β1 blocking antibody and, to a lesser extent, by the antiαvβ3 antibody (Fig. 3B). Simultaneous incubation of EC with
the anti-α5β1 and anti-αvβ3 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-α5β1 and anti-αvβ3
antibodies appeared slightly less effective in inhibiting cell
adhesion on VEGFR-1/Fc than the anti-α5β1 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-αvβ3 antibody alone could
almost completely block EC adhesion on vitronectin (Fig. 3B).
VEGFR-1 induces EC migration through the interaction
with the α5β1 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 VEGFR1/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
Fig. 4. VEGFR-1 is involved in EC migration. (A) VEGFR-1/Fc was included in the lower chamber (chemotaxis), in the upper chamber
(chemokinesis) or in both, at the indicated concentrations (µg/ml). EGF (100 ng/ml) was used as a positive control, and 0 indicates the presence
of only the basal migration medium in the chamber. (B) Specific inhibition of VEGFR-1-induced migration by anti-α5β1 antibodies. Cells were
preincubated for 45 minutes at room temperature with the anti-α5β1 mAb, or with an unrelated anti-α6 integrin mAb (unrelated Ab), before
seeding. Histograms represent the percentage of inhibition of the chemotactic response induced by either VEGFR-1/Fc (5 µg/ml) or EGF (100
ng/ml). (C) Haptotaxis assay was performed on filters in which the underside was coated with 10 µg/ml BSA, VEGFR-1/Fc (VEGFR-1) or
vitronectin (VN). (D) Specific inhibition of VEGFR-1 induced haptotaxis by anti-α5β1 antibodies. Cells were preincubated for 45 minutes at
room temperature with the anti-α5β1 mAb, or with an unrelated anti-α6 integrin mAb (unrelated Ab), before seeding. Histograms represent the
percentage of inhibition of the haptotactic response induced by either VEGFR-1/Fc or vitronectin (VN). In every experiments, migration was
monitored in a Boyden chamber assay by counting 12 high-power fields for each condition. Histograms represent the mean value ± s.d.
VEGFR-1 is a ligand for α5β1 integrin
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/α5β1
interaction in both chemotaxis and haptotaxis was investigated
by incubation of the EC with anti-α5β1 integrin blocking
antibodies before the assay. An inhibition of almost 70% of the
VEGFR-1/Fc-induced chemotaxis and 100% of the VEGFR1/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-α5β1
antibodies (Fig. 4B,D).
Characterisation of VEGFR-1 binding to the α5β1
integrin
To confirm the capability of VEGFR-1 to interact with α5β1,
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 α5β1 integrin,
whereas direct binding of related proteins, such as VEGFR2/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 antiα5β1 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 α5β1.
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 VEGFR1/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 antifibronectin 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 α5β1 integrin that supports cell adhesion could also
3485
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
Fig. 5. Characterisation of the VEGFR-1/α5β1 integrin interaction.
(A) Dose-dependent VEGFR-1 binding to purified α5β1-coated
wells. Different concentrations (10–1-104 ng/ml) of VEGFR-1/Fc
were added to α5β1 coated wells (1 µg/ml) and bound molecules
were detected using an anti-human IgG (Fc specific)-alkaline
phosphatase conjugated antibody. Results represent the mean
absorbance value of medium from triplicate wells ± s.e.m.
(B) VEGFR-1/Fc and VEGFR-2/Fc (20 µg/ml) were added to α5β1coated wells. In competition experiments, EDTA (1 mM) or a
blocking anti-α5β1 antibody (10 µg/ml) was added during the assay.
Absorbance resulting from non-specific cell adhesion was measured
on BSA-coated wells. Histograms represent the mean absorbance
value of medium from triplicate wells ± s.e.m. (C) Effect of RGD
peptides or VEGFR-1 growth factor ligands on cell attachment. Cells
were pretreated with 0.4 mM of RGD or RGE peptides, before
plating on fibronectin (FN) or VEGFR-1/Fc (5 µg/ml), or the
adhesion assays were performed in the presence of 20 µg/ml VEGF
and placenta growth factor (PlGF). Results are expressed as the
percentage of adherent cells compared to untreated controls.
3486
Journal of Cell Science 116 (17)
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
Fig. 6. EC spread on VEGFR-1-coated plates. Cells were plated on
Petri dishes coated with 10 µg/ml fibronectin (FN) (A) or 10 µg/ml
VEGFR-1/Fc (B,C), and F-actin was stained with fluoresceinlabelled phalloidin 1 hour (A,B) or 6 hours after plating (C). Scale
bars: 5 µm.
Fig. 7. VEGFR-1 induces EC spreading. (A) Cells were plated on 10
µg/ml fibronectin (FN) or VEGFR-1/Fc in the presence of monensin
(mn) or cycloheximide (chx), and the percentage of adherent cells
with respect to untreated samples was measured. Histograms
represent the mean absorbance value of medium from triplicate wells
± s.e.m. (B) Effect of RGD peptides on the spreading of EC. Cells
were plated on 1 µg/ml vitronectin (VN), or 10 µg/ml VEGFR-1/Fc
and left to adhere. RGD peptides (0.4 mM) were then added to the
cell monolayer. Results are expressed as the percentage of spread
cells compared to controls without peptide addition ± s.e.m.
(C) Immunofluorescence staining for fibronectin in the matrix
deposited by EC. Cells were allowed to adhere on VEGFR-1/Fc or
vitronectin (VN), for 3 hours and the deposited matrix was analysed
with an anti-fibronectin antibody. Arrowheads indicate organised
fibronectin fibrils. Scale bar: 5 µm.
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
VEGFR-1 is a ligand for α5β1 integrin
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).
Discussion
The identification and characterisation of molecules that
mediate cell-matrix interactions is of great importance in the
understanding of multicellular tissue development. These
interactions are extremely relevant in the creation of vascular
structures, and vessel formation may be facilitated by shifting
the predominance of cell-matrix contacts towards cell-cell
contacts. Our data show, for the first time, that the soluble form
of VEGFR-1 in the matrix deposited by EC in culture,
suggesting that this molecule could function as an extracellular
matrix protein. Sequence analysis shows that the domain
architecture of the secreted VEGFR-1 is compatible with a role
as a matrix protein involved in cell adhesion. Tandem repeats
of domains, often with very similar structure, which probably
arose by gene-duplication events, seem to be a common feature
of both cell membrane and extracellular proteins involved in
adhesion (Clothia and Jones, 1997; Hohenester and Engel,
2002). Moreover, our finding is in keeping with a recent work
of Witmer and colleagues (Witmer et al., 2002), who
characterised the expression pattern of the three VEGFR in
human retina and suggested that VEGFR-1 could be present in
the endothelial cell extracellular matrix in vivo.
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 α5 or the β1
integrin subunits specifically inhibit the adhesion of EC to
VEGFR-1. In addition, we have shown a direct interaction
between VEGFR-1 and the α5β1 integrin in vitro, which
strongly suggests that the α5β1 integrin is an EC ligand for the
matrix-associated VEGFR-1. The α5β1 integrin has already
been implicated in the regulation of several aspects of EC
growth and differentiation. α5β1 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 α5β1
integrin in angiogenesis has been clearly established, since
antagonists of this integrin block tumour-induced angiogenesis
(Kim et al., 2000). Moreover, α5-null teratocarcinomas are
poorly vascularised and α5-null embryoid bodies show delayed
and reduced formation of tubular endothelial structures
(Taverna and Hynes, 2001). Although a functional role for
α5β1 integrin in vasculogenesis and embryonic angiogenesis
has never been directly confirmed, the loss of the gene
3487
encoding the α5 integrin subunit is lethal during
embryogenesis and associated with vascular and cardiac
defects (Yang et al., 1993). α5β1 integrin interacts with more
than one ligand during angiogenesis, and our results raise the
possibility that the VEGFR-1/α5β1 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 α5β1 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 α5β1 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 α5β1 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, VEGFR1 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,
3488
Journal of Cell Science 116 (17)
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 αvβ3
(Senger et al., 1996), α1β1 and α2β1 (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
αvβ3 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 VEGFR1/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.
We wish to thank Prof. J. Jiricny, Prof. K. Ballmer-Hofer, and Prof.
A. Sonnenberg for helpful scientific discussion, and Dr T. Odorisio
for critical review of our data. We would also like to thank N. De Luca
and M. Teson for skilful technical assistance and M. Inzillo for
artwork. This work was supported by grants from CNR, Progetto
Finalizzato Biotecnologie (P.n.115.23287), from Ministero della
Sanità, Italy and from the TMR Marie Curie Research Training
scheme for funding (contract n° B104CT985056).
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