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Originally published In Press as doi:10.1074/jbc.M609323200 on March 14, 2007

J. Biol. Chem., Vol. 282, Issue 20, 15187-15196, May 18, 2007
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Integrin {alpha}9beta1 Directly Binds to Vascular Endothelial Growth Factor (VEGF)-A and Contributes to VEGF-A-induced Angiogenesis*

Nicholas E. Vlahakis{ddagger}1, Bradford A. Young§, Amha Atakilit§, Anne E. Hawkridge{ddagger}, Rachel B. Issaka{ddagger}, Nancy Boudreau§, and Dean Sheppard§2

From the §Lung Biology Center, University of California, San Francisco, California 94143-2922 and {ddagger}Thoracic Disease Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Received for publication, October 2, 2006 , and in revised form, February 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin {alpha}9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from {alpha}9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike {alpha}3beta1or {alpha}vbeta3 integrins, {alpha}9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because {alpha}9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that {alpha}9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin {alpha}9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis requires the coordinated function of both cellular and extracellular regulatory and effector proteins to ensure optimal new vessel formation and function in both non-pathogenic and pathogenic settings (1). The identified molecular regulators of angiogenesis include the VEGF3 family of proteins and receptors, fibroblast growth factors, and integrins (2-4). VEGF-A is a potent pro-angiogenic stimulus and acts by potentiation of endothelial cell adhesion, migration, and proliferation (5, 6). Although VEGF-induced angiogenesis is principally mediated by interactions with its cognate receptor, VEGF-R2, its angiogenic effect may also be modulated through non-VEGF-R2 pathways, including neuropillin-2 (7, 8), heparin sulfate proteoglycans (8), and integrins (9-13).

Integrins are heterodimeric transmembrane proteins that can mediate cell adhesion, migration, and proliferation. Following activation by their respective ligands, integrins can also modulate these cell functions through coordinated cross-talk with growth factor receptors, including VEGF receptors (14, 15) often utilizing signaling proteins common to both receptor pathways (13, 16, 17). Indirect evidence suggests that inhibition of another beta1 integrin, {alpha}3beta1, can inhibit cell adhesion to VEGF-A, suggesting that VEGF-A could serve as an {alpha}3 ligand (18). We have previously shown that the integrin {alpha}9beta1 directly binds to the growth factors, VEGF-C and -D (19), a finding that may help explain the abnormal lymphatic phenotype of mice expressing a null mutation of the {alpha}9 subunit. Because the VEGF homology domain of VEGF-C and -D shares 40% homology with VEGF-A, we hypothesized that VEGF-A might also be a ligand for {alpha}9beta1 and could potentially modulate VEGF-A-induced angiogenesis.

VEGF-A can be synthesized in a variety of forms based on alternative splicing. A previous study has shown that the integrins {alpha}3beta1 and {alpha}vbeta3 specifically modulate cellular interactions with the 165-kDa form of VEGF-A but not the 121-kDa form (18). These results suggest that any association between these integrins and VEGF-A likely does not involve interaction with sequences encoded by exon 6. To determine whether interactions between {alpha}9beta1 and VEGF-A involved similar sites in VEGF-A, we incorporated both splice variants into our studies.

Here we report that {alpha}9beta1 does bind directly to VEGF-A and does cooperate with VEGF-R2 to modulate in vitro endothelial cell adhesion and migration on both VEGF-A165 and VEGF-A121. We also show that VEGF-A (but not bFGF)-induced angiogenesis in chick CAMs can be inhibited by antibody to {alpha}9beta1, suggesting that this interaction could have in vivo relevance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human VEGF-A165, VEGF-A121, bFGF, and phycoerythin-conjugated mouse monoclonal antibody to VEGF-R2 were purchased from R & D Systems (Minneapolis, MN). Production of Y9A2 (blocking antibody to {alpha}9beta1), A9A1 (nonblocking antibody to {alpha}9beta1) (19), CSbeta6 (20), and purification of the integrins {alpha}9beta1 (19) and {alpha}vbeta6 (20) have been described previously. Antibodies were purchased from the following companies as noted. Rabbit polyclonal antibody to VEGF-R2 (M-20) and goat polyclonal antibody to VEGF-R2pY951 (sc-16628) were from Santa Cruz Biotechnology, Santa Cruz, CA; VEGF-R2 kinase inhibitor (SU1498) was from Calbiochem; rabbit polyclonal anti-paxillin pY31, anti-ERKTpY185/187, and VEGF-R2pY1054/59 were from BIOSOURCE; mouse monoclonal antibody to {alpha}3 and {alpha}vbeta3 (LM609) integrins was from Chemicon; and anti-phosphotyrosine (clone 4G10) and mouse monoclonal anti-beta-actin antibodies were from Sigma. Peroxidase-conjugated goat anti-rabbit, goat anti-mouse IgG, and donkey anti-goat IgG were from Jackson ImmunoResearch (West Grove, PA).


Figure 1
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  		FIGURE 1.
{alpha}9beta1 integrin mediates cell adhesion on VEGF-A165. A, flow cytometry analysis of mock-(left) and {alpha}9-transfected (right) MEF used in cell adhesion assays. B, VEGF-A was used as substrate for cell adhesion assays with mock-(diagonal bars) or {alpha}9-transfected (black bars) MEF in the absence or presence of {alpha}9beta1-blocking Ab. Cells were allowed to adhere to wells coated with a range of VEGF-A concentrations and then fixed and stained with crystal violet. Adhesion is expressed as absorbance at 595 nm. C, flow cytometry analysis of mock-(left) and {alpha}9-transfected (right) SW-480 cells used in cell adhesion assays. D, VEGF-A was used as substrate for cell adhesion assays with SW-480 mock cells in the absence of inhibitors (black bars) or the presence of {alpha}3 (white bars), {alpha}9 (diagonal bars), or both (brick bars) blocking Ab or VEGF-R2 inhibitor (diamond bars). E, similar adhesion assay to D but with {alpha}9-transfected SW-480 cells.

 
Cells and Cell Culture—{alpha}9 and mock-transfected mouse embryonic fibroblasts (MEF) and SW-480 cells were made and grown as described previously (21, 22). Primary adult human microvascular endothelial cells (HMVEC, Cambrex, East Rutherford, NJ) were grown in cell-specific growth factor-supplemented nutrient media (Cambrex, EBM-2).

Immunoprecipitation, SDS-PAGE, and Western Blot Analysis—For immunoprecipitation of VEGF-R2, HMVEC were grown in 6-well plates with full growth media until 70% confluent and subsequently in basal media with 0.1% BSA for 4 h. Cells were then added to 12-well dishes coated with VEGF-A (23, 24). Cells were exposed to VEGF-A or medium alone for 5-30 min, washed with phosphate-buffered saline/sodium orthovanadate (NaV 10 mM), and then lysed with buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X, 0.5% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaV, and protease inhibitors (Complete Mini EDTA-free, Roche Applied Science). Pre-cleared lysates were immunoprecipitated with 2 µg of antibody bound to protein A-Sepharose beads (Amersham Biosciences). The beads were washed with lysis buffer, resuspended in Laemmli sample buffer, boiled, resolved on 8% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Billerica, MA). The membrane was blocked in 5% milk/Tris-buffered saline with 0.1% Tween (TBST) for 1 h at room temperature and then probed with anti-VEGF-R2 antibody.

For immunoblotting of paxillin pY31, ERKTpY185/187, 4G10, and beta-actin, proteins were suspended in Laemmli sample buffer and resolved on 4 -15% gradient SDS-PAGE, before transfer to polyvinylidene difluoride membrane. The membrane was blocked in 5% BSA, 0.1% Tween, probed with the appropriate specific primary antibodies, washed three times with TBST, and subsequently probed with horseradish peroxidase-conjugated secondary antibodies and developed using chemiluminescence (ECL, Amersham Biosciences).

Flow Cytometry—Cultured cells were trypsinized, washed with phosphate-buffered saline, and incubated with the appropriate primary antibodies and then appropriate phycoerythrin-conjugated secondary antibody. Phycoerythin-conjugated VEGF-R2 was used to detect VEGF-R2. Fluorescence of labeled cells was determined with a flow cytometer (BD Biosciences).

Adhesion Assay—Assays were performed as described previously (19) with some minor modifications. After coating 96-well microtiter plates (ICN, Linbro/Titertek, Aurora, OH) with VEGF-A at 4 °C overnight, wells were blocked with 3% bovine serum albumin (BSA, Sigma) for 30 min at 37 °C. After trypsinization, cells were incubated with or without relevant antibodies (20 µg/ml) for 30 min on ice, and 5 x 104 cells/well were seeded. Adherent cells were fixed and stained with 1% formaldehyde, 0.5% crystal violet, 20% methanol for 30 min, and the number of adherent cells was evaluated by measuring absorbance at 595 nm in a microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA).


Figure 2
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  		FIGURE 2.
Endothelial cell adhesion and migration on VEGF-A are {alpha}9beta1-dependent. A, flow cytometry analysis of HMVEC showing expression of both {alpha}9 integrin (left) and VEGF-R2 (right). B, cell adhesion assays using HMVEC plated on various concentrations of VEGF-A165 in the presence of {alpha}9 blocking (squares) or nonblocking antibody (triangles). C, flow cytometry analysis of HMVEC showing expression of both {alpha}3 (left) and {alpha}vbeta3 (right) integrins. D, cell adhesion assays using HMVEC plated on various concentrations of VEGF-A in the presence of {alpha}9, {alpha}3, or {alpha}vbeta3 integrin antibodies.

 
Migration Assay—Assays were performed as described previously (19) using 8-µm Transwell plates (Corning Costar, Cambridge, MA), either uncoated or coated, at 4 °C overnight with relevant ligand (VEGF-A) or 1% BSA as a binding control. After trypsinization, 5 x 104 cells were incubated with or without relevant inhibitors for 30 min on ice and seeded into the top chamber of the Transwell (22). 1% fetal calf serum or soluble VEGF-A was added to the bottom well, to serve as a chemoattractant, and the plates were incubated at 37 °C for 3 h. Cells that migrated and adhered to the bottom surface of the Transwell membrane were fixed, stained (DiffQuik, Pierce), and counted in 10 high power (x25) fields for each condition.

Binding Assay—Purified {alpha}9beta1 (19) and {alpha}vbeta6 (20) integrins were used in solid phase binding assays with VEGF-A, as described previously (19). Recombinant VEGF-A (5 µg/ml) was coated on 96-well microtiter plates (Nunc ImmunoPlate, Naperville, IL), and purified {alpha}9beta1or {alpha}vbeta6 at various concentrations was added for 2 h at room temperature in the presence or absence of {alpha}9 blocking antibody or 10 mM EDTA. The extent of {alpha}9beta1 binding was detected using A9A1 antibody (20 µg/ml, 1 h at 37 °C). Following labeling with horseradish peroxidase (BD Biosciences), binding was quantified by measuring absorbance at 450 nm with a microplate reader (Molecular Devices).

CAM Assay—Chick eggs were maintained in a humidified 39 °C incubator (Lyon Electric, Chula Vista, CA). Pellets containing 0.5% methylcellulose plus recombinant human VEGF-A (50 ng) or bFGF (150 ng) were placed onto the CAM of 10-day-old chick pathogen-free embryos (SPAFAS; Charles River Breeding Laboratories, Wilmington, MA). The CAMs were exposed by cutting a small window in the egg shell to facilitate application of the pellet. Relevant antibodies or agonist/antagonist compounds were applied to the site 24 h after stimulation with VEGF protein. CAMs were imaged on day 13, both following fixation and excision or with real time live imaging, using a digital camera (Canon Supershot6) attached to a Zeiss stereomicroscope. Angiogenesis was quantified by counting branch points arising from tertiary vessels from a minimum of 10 specimens from three separate experiments.

Statistical Methods—Data are presented as mean values ± S.D. from at least three separate experiments unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Integrin {alpha}9beta1 Mediates Cell Adhesion and Migration on VEGF-A—To determine whether cells use integrin {alpha}9beta1to adhere to VEGF-A, adhesion assays were performed using two different cell lines, {alpha}9- and mock-transfected MEF and SW-480 cells (Fig. 1). Flow cytometry with the anti-{alpha}9beta1 antibody, Y9A2, showed robust expression of {alpha}9beta1inthe {alpha}9-transfected cell types and no expression in mock transfectants (Fig. 1, A and C). {alpha}9-Transfected MEF demonstrated concentration-dependent adhesion to VEGF-A (Fig. 1B) that was abolished by the {alpha}9beta1 blocking antibody Y9A2. In contrast, mock-transfected MEF did not adhere to VEGF-A above background levels of attachment to BSA-coated wells. Because cells have also been shown to adhere to VEGF-A in an {alpha}3beta1 integrin-dependent manner, we performed assays in a second {alpha}9-transfected cell line (SW-480) known to express {alpha}3beta1 (Fig. 1C). {alpha}9beta1-dependent adhesion on VEGF-A was also demonstrated in these cells. In mock-transfected cells, blocking {alpha}3beta1 inhibited cell adhesion, whereas blockade of {alpha}9beta1 or VEGF-R2, neither of which are expressed in these cells, had no effect (Fig. 1D). In {alpha}9, compared with mock-transfected cells, adhesion to VEGF-A in the absence of blocking antibodies was substantially enhanced, as demonstrated by higher absorbance values. In these cells, blockade of {alpha}9beta1 caused substantial inhibition of adhesion, whereas blocking antibody to {alpha}3beta1 had no detectable effect (Fig. 1E), perhaps because {alpha}9beta1 was expressed at substantially higher levels than {alpha}3beta1 (Fig. 1C). Because these transfected cells do not express VEGF-R2, the cognate receptor for VEGF (25, 26), these findings suggest that {alpha}9beta1 might interact directly with VEGF-A.


Figure 3
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  		FIGURE 3.
VEGF-A165 and -121 isoforms bind directly to integrin {alpha}9beta1. A, VEGF-A165 was used for solid phase binding assays with purified {alpha}9beta1 (diamonds) or {alpha}vbeta6 (x), an irrelevant integrin, at various concentrations in the absence or presence (squares) of {alpha}9 inhibiting antibody or 10 mM EDTA (triangles). B, schematic demonstrating the exons of VEGF-A and its 165 and 121 isoforms. C, adhesion assays with mock-(top) or {alpha}9-transfected (bottom) SW-480 cells on VEGF-A121 in the absence (diamonds) or presence of {alpha}3 (squares), {alpha}9 (circles), or both (x) blocking antibodies or VEGF-R2 inhibitor (triangles). D, HMVEC adhesion assay at various concentrations of VEGF-A165 and VEGF-A121 in the absence (squares or triangles) or presence (diamonds and circles) of {alpha}9beta1 integrin blocking antibody. E, HMVEC migration assay on VEGF-A165 (top panel) or VEGF-A121 (bottom panel) in the absence (squares) or presence of {alpha}9beta1 (circles), {alpha}3beta1 (triangles), or {alpha}vbeta3 (diamonds) integrin blocking antibody. F, HMVEC migration assay as in E but instead using VEGF-A165 (top panel) or VEGF-A121 (bottom panel) as soluble chemotaxis agents.Figure 3

 
To confirm these findings in a physiologically relevant cell type, separate experiments were performed using HMVEC that express both VEGF-R2 and {alpha}9beta1 (Fig.2A). Concentration-dependent adhesion of HMVEC to VEGF-A165 was inhibited by antibody to {alpha}9beta1 (Fig. 2B). Because {alpha}3beta1 and {alpha}vbeta3 also interact with VEGF-A (18), we performed adhesion assays using HMVEC expressing these two integrins and {alpha}9beta1 (Fig. 2, A and C) in the presence of blocking antibodies to each of the three integrins. Fig. 2D shows that inhibition of any single integrin inhibited adhesion of these cells to VEGF-A, with nearly complete inhibition caused by antibodies to {alpha}9beta1or {alpha}vbeta3 and partial inhibition with antibody to {alpha}3beta1. This was not because of artifactual effects of antibody binding to integrins on these cells, because a nonblocking antibody to {alpha}9beta1 had no effect.

The Integrin {alpha}9beta1 Directly Binds VEGF-A—To determine whether the findings from adhesion assays were a result of direct binding of VEGF-A165 to {alpha}9beta1, we performed solid phase protein-protein binding assays. {alpha}9beta1 bound to VEGF-A in a concentration-dependent fashion, and binding was inhibited by blocking antibody to {alpha}9beta1 or by chelating divalent cations (10 mM EDTA), as expected for authentic integrin-ligand interactions (Fig. 3A). In contrast, the irrelevant integrin, {alpha}vbeta6, showed no binding to VEGF-A.

Because we have shown that the homologous proteins VEGF-C and -D also bind to {alpha}9beta1 integrin, we wished to better localize potential {alpha}9-binding sites for VEGF, using splice variants of VEGF-A. In contrast to VEGF-A165, which is missing exon 6, the 121 isoform is missing both exons 6 and 7 (Fig. 3B). Mock-transfected SW-480 cells, which express {alpha}3beta1 but not {alpha}9beta1 or VEGF-R2, adhered to VEGF-A121, and this binding was unaffected by antibody to {alpha}3beta1, confirming previous reports (18) that {alpha}3beta1 specifically mediates adhesion to the 165-kDa splice variant (Fig. 3C, top panel). In contrast, {alpha}9-transfected SW-480 cells, which also demonstrated increased adhesion to VEGF-A121, was specifically inhibited by antibody to {alpha}9beta1 (Fig. 3C, bottom panel). Fig. 3D shows that in physiologically relevant HMVEC, cell adhesion to either VEGF-A165 or -121 isoform was partially {alpha}9-dependent.

To investigate the relative role of {alpha}9beta1, {alpha}3beta1, and {alpha}vbeta3 integrins in cell migration, we performed HMVEC migration assays on either immobilized VEGF-A165 (Fig. 3E, top panel) or VEGF-A121 (Fig. 3E, bottom panel) in the absence or presence of inhibitory antibodies to either of the three integrins. Consistent with the specialized role of {alpha}9beta1 in facilitating accelerated cell migration, inhibition of {alpha}9 resulted in the greatest reduction in cell migration on VEGF-A165. HMVEC migration on VEGF-A121 was also {alpha}9 integrin-dependent and, as expected, not dependent on integrins {alpha}3beta1or {alpha}vbeta3. Because VEGF-A121 and VEGF-A165 are physiologically active in a soluble form, we tested whether soluble VEGF-A-induced cell migration was also {alpha}9beta1-dependent. Fig. 3F (top and bottom panels) shows that both soluble VEGF-A165- and -121-induced endothelial cell migration was inhibited by {alpha}9beta1 blocking antibody. As for responses to immobilized VEGFA isoforms, inhibition of {alpha}3beta1 or {alpha}vbeta3 integrins decreased migration induced by VEGF-A165 (Fig. 3F, top panel) but not VEGF-A121 (bottom panel).


Figure 4
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  		FIGURE 4.
{alpha}9beta1 and VEGF-R2 act coordinately to mediate cell adhesion and migration on VEGF-A. A, HMVEC adhesion assay at various concentrations of VEGF-A165 in the presence (squares) or absence (diamonds) of {alpha}9 inhibiting antibody or VEGF-R2 tyrosine kinase inhibitor (triangles) or both inhibitors (circles). B, HMVEC adhesion assay on collagen-coated dishes. C, HMVEC migration assay on various concentrations of VEGF-A165 in the presence (squares) or absence (diamonds) of {alpha}9 inhibiting antibody or VEGF-R2 tyrosine kinase inhibitor (triangles) or both inhibitors (circles). D, HMVEC migration assay as in C but instead using VEGF-A165 (top panel) or VEGF-A121 (bottom panel) as soluble chemotaxis agents.

 
These findings taken together with our previous report showing that the homologous proteins VEGF-C and -D also bind to {alpha}9beta1 (19) suggest that the {alpha}9beta1 binding domain resides within the VEGF homology domain (exons 1-5). However, a binding site in exon 8, which is present in both VEGF165 and VEGF121, cannot be excluded.

{alpha}9beta1 and VEGF-R2 Act Coordinately to Mediate Endothelial Cell Adhesion and Migration—To assess the relative contribution of VEGF-R2 and {alpha}9beta1 to HMVEC adhesion and migration on VEGF-A, cells were treated with {alpha}9beta1 blocking antibody and/or the VEGF-R2 tyrosine kinase inhibitor SU1498 (27). Inhibition of either {alpha}9beta1 or VEGF-R2 significantly decreased endothelial cell adhesion to VEGF-A165 (Fig. 4A), and the combination of both inhibitors caused maximal inhibition. Neither inhibitor affected adhesion to the irrelevant ligand, collagen (Fig. 4B). Inhibition of {alpha}9beta1 or VEGF-R2 also decreased endothelial cell migration on VEGF-A (Fig. 4C), and again inhibition was maximal by the combination of both inhibitors. Inhibition of either {alpha}9beta1 or VEGF-R2 also decreased endothelial cell migration in response to soluble VEGF-A165 or -121 (Fig. 4D), although in this case the effects of both inhibitors together were not different from the effects of each individual inhibitor. Taken together, these findings suggest that both {alpha}9beta1 and VEGF-R2 are required for maximal cell adhesion and migration on immobilized VEGF-A and in response to soluble VEGF-A.


Figure 5
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  		FIGURE 5.
Following binding of VEGF-A, ERK and paxillin are activated by coordinate signaling through {alpha}9beta1 and VEGF-R2. A, protein lysates from HMVEC exposed to immobilized VEGF-A in the presence or absence of {alpha}9 inhibiting antibody after 5, 15, or 30 min were immunoblotted for phospho-ERK or phosphopaxillin. B, in separate experiments, protein lysates exposed to immobilized VEGF-A165 for 15 min in the presence or absence of {alpha}9 blocking antibody or VEGF-R2 tyrosine kinase inhibitor or both inhibitors were immunoblotted for phospho-ERK (top panel) or paxillin (bottom panel). C, immunoblot as in B above, but protein lysates were from cells exposed to soluble VEF-A165 (top panel) or VEF-A121 (bottom panel). D, HMVEC lysates from separate experiments were protein immunoprecipitated (IP) using {alpha}9beta1 antibody or the irrelevant antibody to {alpha}vbeta6 and immunoblotted (IB) with anti-VEGF-R2.

 
In Response to Immobilized VEGF-A, {alpha}9beta1 and VEGF-R2 Become Physically Associated and Both Contribute to VEGF-A-induced Phosphorylation of the Signaling Intermediates ERK and Paxillin—To determine whether VEGF-R2 and {alpha}9beta1 coordinately signal in response to VEGF-A, we examined the effects of VEGF-A165 on phosphorylation of the downstream signaling intermediates ERK and paxillin, in the presence or absence of inhibitors of either receptor (Fig. 5). In the absence of VEGF-A, tyrosine phosphorylation of each protein was minimal. When these cells were plated on immobilized VEGF-A, both ERK1/2 and paxillin (Fig. 5A, top panel) were phosphorylated maximally after 15 min. Phosphorylation of each protein was inhibited by {alpha}9beta1 blocking antibody at all time points. Following HMVEC binding to immobilized VEGF-A, ERK and paxillin phosphorylation was also inhibited by the VEGF-R2 kinase inhibitor (Fig. 5B). When cells were exposed to soluble VEGF-A (Fig. 5C), paxillin was maximally phosphorylated after 5 min, an effect that was inhibited by either blocking {alpha}9beta1 or VEGF-R2. However, ERK phosphorylation in response to soluble VEGF-A was not affected by inhibition of {alpha}9beta1 (data not shown).

We also wished to determine whether co-ligation of VEGF-R2 and {alpha}9beta1 by VEGF-A leads to physical association of these two receptors. By co-immunoprecipitation, we could detect minimal association of VEGF-R2 and {alpha}9beta1in the absence of VEGF-A, and this association was substantially increased when plated on immobilized VEGF-A (Fig. 5D). Using irrelevant antibody, VEGF-R2 was unable to be immunoprecipitated in the absence or presence of VEGF-A. Also, we were not able to co-immunoprecipitate VEGF-R2 and {alpha}9beta1 from cells exposed to soluble VEGF-A (data not shown). Furthermore, using either VEGF-R2-phosphospecific antibodies (pY951, 1054/1059) or immunoprecipitation, we found that in the presence of VEGF-A or the {alpha}9beta1-specific ligand Tnfn3RAA (where Tnfn3RAA is {alpha}9-specific ligand, recombinant third fibronectin repeat of tenascin C in which arginine-glycine-as-partic acid is mutated to RAA), VEGF-R2 phosphorylation was not {alpha}9beta1-dependent (data not shown).

VEGF-A-induced Angiogenesis Is {alpha}9beta1-Dependent—To determine the in vivo relevance of our in vitro findings, we performed chick CAM assays to measure VEGF-A-induced angiogenesis, in the presence or absence of {alpha}9beta1 blocking or nonblocking antibody. VEGF-A induced significant angiogenesis that was inhibited by the {alpha}9beta1 blocking antibody, Y9A2, but not by the nonblocking antibody, A9A1 (Fig. 6, A and B), or by isotype-matched control antibody (data not shown). Also, as expected, VEGF-induced angiogenesis was inhibited by blocking VEGF-R2 (SU1498). Further inhibition was achieved by blocking both VEGF-R2 and {alpha}9beta1. In separate experiments, similar results were found when the CAMs were stimulated with VEGF-A121 (Fig. 6C). Consistent with previous reports and our in vitro work, inhibition of {alpha}3beta1 and {alpha}vbeta3 did not result in inhibition of VEGF-A121-induced angiogenesis. In contrast, induction of angiogenesis by bFGF (Fig. 6D) was not inhibited by the {alpha}9 blocking antibody. These results suggest that the interaction between {alpha}9beta1 and VEGF-A is relevant to in vivo angiogenesis, and the role of {alpha}9beta1 in this process may be specific for angiogenesis induced by VEGF-A.


Figure 6
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  		FIGURE 6.
VEGF-A- and not bFGF-induced angiogenesis is {alpha}9beta1-dependent. A and B, chick CAMs were exposed to 50 ng of VEGF-A165 alone (A, left panel, and B, black bar) or in the presence of either {alpha}9 inhibiting (A, center panel, and B, diamond bar) or noninhibiting Ab (A, right panel, and B, diagonal bar) or VEGF-R2 inhibitor alone (B, brick bar) or VEGF-R2 inhibitor and{alpha}9 inhibiting Ab (B, hatched bar) on incubation day 10 and imaged 3 days later for quantification of angiogenesis. C, in separate CAMs under the same conditions angiogenesis was induced by VEGF-A121 in the absence (white bar) or presence of blocking Ab for the integrins {alpha}9beta1 (diagonal bar), {alpha}3beta1 (diamond bar), or {alpha}vbeta3 (brick bar). D, in separate CAM experiments bFGF (150 ng) was used to induce angiogenesis, in the absence (black bars) or presence of {alpha}9 blocking (white bar) or nonblocking (diagonal bar) Ab or {alpha}vbeta3 blocking Ab (diamond bar). Values are reported as mean ± S.E.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have identified the angiogenic growth factor VEGF-A as a ligand for the integrin {alpha}9beta1. Experiments with {alpha}9-transfected cells and primary human endothelial cells demonstrated that they adhere to and migrate on VEGF-A using {alpha}9beta1. In response to immobilized VEGF-A, VEGF-R2 and {alpha}9beta1 assemble into a macromolecular complex to cooperatively signal in an additive manner through phosphorylation of the downstream signaling intermediates ERK and paxillin. In vivo, endothelial cells respond to both immobilized VEGF-A, bound to the extracellular matrix, and to soluble VEGF-A, which may either be secreted from cells or released from the matrix through the action of matrix-degrading proteases. It appears that endothelial cell responses to soluble VEGF-A also depend on signals from {alpha}9beta1. In contrast to immobilized VEGF-A, we could not demonstrate critical roles for ERK phosphorylation or the formation of a physical complex between VEGF-R2 and {alpha}9beta1 following soluble VEGF-A stimulation. Using an in vivo angiogenesis assay, we provide evidence that the interactions between both immobilized and/or soluble VEGF-A and {alpha}9beta1 are likely to be biologically significant.

{alpha}vbeta5, {alpha}5beta1, and {alpha}4beta1 (9-11, 28-30) are at least three other integrins that have been implicated in angiogenesis. One could thus argue that the findings reported here are not novel or unexpected. However, each of the integrins thus far shown to contribute to angiogenesis appears to do so by distinct mechanisms (10, 11, 28, 29). One such novel mechanism is through direct binding of growth factor to the integrin. Hutchings et al. (18) showed in protein-protein binding assays that VEGF-A165 bound directly to {alpha}vbeta3. However, there has been only indirect evidence that growth factors may directly bind to beta1 integrins. In the same study by Hutchings et al. (18), human umbilical artery endothelial cells were reported to adhere to immobilized VEGF-A in an {alpha}3beta1-dependent fashion. The authors suggested this adhesion was independent of VEGF-R2 based on the results of presumably down-regulating the receptor following cell pre-treatment with VEGF-A. However, the authors did not provide biochemical evidence of direct binding in cell-free experiments. In this study we show that purified {alpha}9beta1 directly binds to recombinant VEGF-A and that cell adhesion to VEGF-A is not {alpha}3-dependent in the presence of {alpha}9beta1, suggesting the interaction with {alpha}9beta1 is more robust than with {alpha}3beta1.

Although both {alpha}3beta1 and {alpha}vbeta3 integrins were found to bind the 165 isoform of VEGF-A, they did not bind VEGF-A121, suggesting the binding site for these integrins is encoded within exons 6-8. In contrast, {alpha}9beta1 does bind to VEGF-A121 and, as we have shown previously, binds the homologous VEGF-C and -D growth factors (19), suggesting that a unique {alpha}9beta1-binding site is encoded within the VEGF homology domain (exons 1-5). Not only is the interaction of the {alpha}9beta1 integrin with VEGF-A121 unique for beta1 integrins but also biologically significant as demonstrated by inhibition of cell migration and in vivo angiogenesis following blockade of {alpha}9beta1 activity.

There have been numerous previous reports of "cross-talk" between tyrosine kinase growth factor receptors and integrins (9, 13, 16, 17). In some cases, this cross-talk has been associated with either growth factor or integrin ligand-induced co-association of both receptors (31). In the case of cross-talk between integrin {alpha}5beta1and epidermal growth factor receptor, ligation of the integrin induces phosphorylation of the receptor that appears to be independent of binding of the epidermal growth factor receptor itself (32, 33). In contrast, activation of {alpha}9beta1by its specific ligand, Tnfn3RAA, does not appear to lead to phosphorylation of VEGF-R2, and blocking {alpha}9beta1 has no effect on VEGF-A-induced phosphorylation of VEGF-R2. It thus appears that the signaling pathways activated by these two receptors intersect downstream of activation of VEGF-R2.

The role of {alpha}9beta1 in signaling responses to immobilized and soluble VEGF-A appears to be dissimilar. In response to immobilized VEGF-A, {alpha}9beta1 seems to play an important role in downstream phosphorylation of both paxillin and ERK and leads to the formation of a macromolecular complex containing both the integrin and VEGFR-2. In response to soluble VEGF-A, {alpha}9beta1 also contributes to phosphorylation of paxillin, but it does not appear to enhance ERK phosphorylation or induce the formation of a dual receptor complex. These differences might reflect the influence of the extracellular matrix and its associated receptors such as heparin sulfate proteoglycans (34) that facilitate immobilization of a high density of VEGF-A, which in turn may stimulate an increase in both {alpha}9beta1 and VEGFR-2 clustering. Nonetheless, the findings suggest that although important for full signaling through VEGF-R2 the integrin is not necessary and that paxillin phosphorylation may be the most relevant end point leading to enhanced cell migration in response to VEGF-A.

Our findings that blocking {alpha}9beta1 specifically inhibited CAM angiogenesis induced by the 165 and 121 isoforms of VEGF-A and not bFGF suggest that the selective interaction of VEGF-A with this integrin might be the mechanism by which {alpha}9beta1 enhances in vivo angiogenesis. This is supported by our findings that blocking both {alpha}9beta1 integrin and VEGF-R2 resulted in greater inhibition of angiogenesis than blocking either receptor alone. Of course, in vivo angiogenesis is a complex process, and we cannot be certain that inhibition of {alpha}9beta1 only inhibits angiogenesis by interfering with binding to VEGF-A. For example, the closely related integrin, {alpha}4beta1, was recently shown to contribute to angiogenesis following binding to one of its ligands, VCAM-1 (10). We have previously shown that {alpha}9beta1is also a receptor for VCAM-1 (35), so it is conceivable that a similar mechanism contributes to the in vivo role of {alpha}9beta1in angiogenesis. However, in contrast to results for {alpha}9beta1, blockade of {alpha}4beta1 inhibits angiogenesis induced by bFGF, so this effect is clearly not specific for VEGF-A. Furthermore, mice lacking VCAM-1 or the integrin {alpha}4 subunit (36, 37) develop similar defects in vascular development, whereas development of the nonlymphatic vasculature appears to be normal in {alpha}9 knock-out mice (38). These results are most consistent with the hypothesis that the mechanisms by which {alpha}9beta1 and {alpha}4beta1 contribute to angiogenesis are distinct.

The absence of major vascular defects in {alpha}9 knock-out mice might suggest that the {alpha}9beta1 integrin is not essential for normal vascular development. However, this should not be taken as evidence that {alpha}9beta1 would not contribute to pathologic angiogenesis (39) as has been clearly demonstrated in the case of the {alpha}v integrins. Antagonists of {alpha}vbeta3or {alpha}vbeta5 inhibit pathologic angiogenesis, but mice lacking beta3 and/or beta5 integrins have normal vascular development and even demonstrate enhanced tumor and ischemic angiogenesis (12). Additional experiments will be required using inhibitors of {alpha}9beta1 that are effective in mammalian disease models of angiogenesis such as tumor growth and metastasis to more definitively address this question. The results presented here, in our previous reports (19, 38) and by others (40), demonstrate the unique role of {alpha}9beta1innot only lymphatic development but also VEGF-induced angiogenesis and lymphangiogenesis and suggest that inhibition of this integrin could affect pathologic vasculogenesis supporting cancer cell growth and metastasis.


   FOOTNOTES
 
* This work was supported by a Mayo Foundation scholarship (to N. E. V.), American Lung Association Research Grant RG-1018-N (to N. E. V.), and by NHLBI Grant R01 HL64353 from National Institutes of Health (to D. S.). 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. Back

1 To whom correspondence may be addressed: Thoracic Disease Research Unit, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-2957; Fax: 507-284-4521; E-mail: vlahakis.nicholas{at}mayo.edu.

2 To whom correspondence may be addressed: Lung Biology Center, University of California, P. O. Box 2922, San Francisco, CA 94143-2922. Tel.: 415-514-4269; Fax: 415-514-4278; E-mail: dean.sheppard{at}ucsf.edu.

3 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor; MEF, mouse embryonic fibroblasts; HMVEC, human microvascular endothelial cells; BSA, bovine serum albumin; bFGF, basis fibroblast growth factor; CAM, chorioallantoic membrane; Ab, antibody; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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