Novel Vascular Endothelial Growth Factor Binding Domains of Fibronectin Enhance Vascular Endothelial Growth Factor Biological Activity
Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, α5β1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to α5β1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.
The growth, repair, and regeneration of blood vessels are complex processes that involve coordinated regulation of endothelial cell proliferation, migration, and differentiation.1 One of the most important vascular morphogens is vascular endothelial growth factor (VEGF). VEGF has been shown to play a major role in vasculogenesis and angiogenesis by gene deletion studies.2,3⇓ Targeted disruption of the VEGF receptor Flk-1 (VEGFR-2) in mice resulted in failure of blood-island formation and endothelial differentiation.4 Flk-1 is also the first endothelial receptor tyrosine kinase to be expressed in the hemangioblast.5 We and others recently demonstrated that the hematopoietic progenitor cell CD34+ can differentiate into endothelial cells, and that VEGF was one of the critical factors promoting this differentiation.6,7⇓ Interactions between cells and their extracellular matrix (ECM) play an integral role in blood vessel development. The earliest ECM protein expressed in the embryo during vasculogenesis is fibronectin (FN).8 Gene deletion studies have demonstrated that both FN and its major integrin receptor, α5β1, are critical for vasculogenesis and angiogenesis in the developing embryo.9–11⇓⇓ Collectively, these observations suggest important roles for FN and its integrin receptor, α5β1, in vasculogenesis and angiogenesis.
In this study, we show that novel VEGF binding domains of FN are required for promoting the specific association of the FN receptor integrin α5β1 with the VEGF receptor, Flk-1. This association between VEGF and FN is required for the full effects of VEGF-induced endothelial cell migration and proliferation. This study demonstrates that FN can profoundly affect VEGF biological activity and consequently the behavior of endothelial cells through their coordinated effects on Flk-1 and α5β1.
Materials and Methods
Solid-Phase VEGF Binding Assay
ECM proteins and FN peptides were purchased from Sigma and Gibco and were purified further by gel filtration and ion exchange chromatography. Microtiter plates were coated with the appropriate ECM proteins (50 μL; 10 μg/mL) in 100 mmol/L bicarbonate buffer (pH 9) overnight at 4°C. 125I-VEGF165 (NEN) in binding buffer (PBS containing 2% BSA) was added to the microtiter plates and incubated for 30 minutes at room temperature. After washing, radioactivity was eluted with 100 mmol/L NaOH and determined using a gamma counter. To determine nonspecific binding, 100-fold excess of cold VEGF was added to the binding buffer and counts subtracted from the total binding.
Slot Blot Assay
ECM protein or FN peptides (2.5 μg of each ) were immobilized on nitrocellulose membranes and incubated with 50 ng/mL VEGF165 (R&D Systems) for 1 hour at 37°C in 20 mmol/L Tris, pH 7.5/0.15 mmol/L NaCl/0.1% BSA. The membranes were incubated with mouse anti-human VEGF antibody (R&D Systems), followed by goat anti-mouse HRP-conjugated in binding buffer for 30 minutes each. All blots were visualized by chemiluminescence (Pierce).
Surface Plasmon Resonance Analysis (SPR)
SPR analysis was performed on the BIAcore X (Biacore). FN fragments were coupled to CM5 dextran chips by amine coupling chemistry according to the manufacturer’s protocols. The reference cell had immobilized mouse IgG. In competition experiments with the FN 40-kDa fragment, 1.3 μmol/L VEGF was incubated with increasing amounts of FN 40-kDa (0.5 to 8.0 μmol/L) for 30 minutes at 37°C in a reaction volume of 30 μL. Samples were then injected across the FN 70-kDa biosensor chip and sensograms recorded. The value for IC50 was determined using the ASSAY program (Biosoft). Assuming a simple competition between the FN fragments for binding to VEGF, an estimate of the Kd for VEGF binding to the FN 40-kDa fragment was determined using the equation; Kdcomp=IC50/(1+Lt/KdLig), where Kdcomp is the Kd value for the competitor (FN-40 kDa fragment), Lt is the ligand concentration, and KdLig is the dissociation constant for VEGF binding to the 70-kDa fragment.
Immunoprecipitation of VEGF/FN Complex From Platelet Supernatants
Washed platelets were prepared as previously described.12 Platelets were resuspended in the presence of 1.5 mmol/L calcium and at a count of 30×108/mL. One milliliter of platelets was stimulated with either saline (resting) or thrombin (1 U/mL) for 10 minutes. Supernatants were immunoprecipitated with an antibody to FN (Chemicon). After SDS-PAGE and electrotransfer to PVDF membranes, VEGF was detected with a polyclonal antibody (Santa Cruz) by immunoblotting and chemiluminesescence detection.
Human microvessel endothelial cells (HMVECs) in serum-free MCDB-131 medium (BioWhittaker) supplemented with 0.1% BSA were plated on polylysine (1 mg/mL), FN (10 μg), vitronectin (VN; 10 μg/mL), or FN peptide (50 μg/mL) coated plates containing VEGF (50 ng/mL) for 1 hour. Cells were lysed with lysis buffer (20 mmol/L HEPES, pH 7.5, 0.5% Brij 35, 0.5% NP-40, 100 mmol/L NaCl, 5% glycerol, 0.1% BSA and protease inhibitors) and immunoprecipitated with antibodies to α5β1, αvβ1, αvβ3, or αvβ5 integrin (Chemicon). After SDS-PAGE and protein transfer, membranes were immunoblotted with antibodies to Flk-1 (Santa Cruz). Bands were detected by chemiluminescence.
Migration studies were carried out using 6.5-mm Transwells (Costar). VEGF (50 ng/mL) and ECM (10 μg/mL) or FN fragment (50 μg/mL) mixtures in MCDB 131 medium containing 0.5% bovine serum albumin were added to the bottom chambers of the Transwell and incubated at 37°C for 30 minutes. HMVECs (5×104) were then placed in the upper chamber in the same medium and the Transwells were incubated for a further 6 hours at 37°C. Transwells were processed as described previously.13 For studies on the effect of antibodies to integrins, HMVECs were preincubated for 30 minutes with the indicated antibodies before addition to the upper chamber of Transwells. The lower chamber contained VEGF (50 ng/mL), FN, and VN (10 μg/mL).
MAPK In Vitro Kinase Assay
HMVECs were lysed, and MAPK was immunoprecipitated using a pan MAPK antibody (Pharmigen), washed in 50 mmol/L Tris-HCL, pH 7.4, containing 120 mmol/L NaCl, 0.1% Triton-X-100, and 10% glycerol. After washing in kinase buffer (50 mmol/L Tris-HCL, pH 7.4, 0.5 mmol/L DTT, 10 mmol/L MgCl2, 10 mmol/L MnCl2, 120 mmol/L NaCl, and 10% glycerol), the immunoprecipitates were incubated for 15 minutes at 30°C in 30 μL of kinase buffer containing 2.5 μg myelin basic protein, 20 μmol/L ATP, and 10 μCi/nmol [γ-32P]ATP (3000 Ci/mmol). Reactions were stopped with 4× SDS-PAGE sample buffer, resolved by 10% SDS-PAGE. Radioactivity incorporated into the myelin basic protein bands were determined by Cerenkov counting.
Intact FN Promotes VEGF-Induced Endothelial Cell Migration
Endothelial cell migration was slightly enhanced by either VEGF or FN alone. But the combination of VEGF/FN increased migration by 2.5-fold over VEGF alone or VEGF/vitronectin (VN) and VEGF/collagen mixtures (Figure 1). Because FN has proteolytic fragments that are chemotatic for endothelial cells,14 we tested the effect of FN fragments combined with VEGF on VEGF-induced endothelial cell migration (see map of fragments, Figure 5B). When endothelial cells were exposed to VEGF and only the FN 120-kDa cell-binding domain peptide, no enhancement of endothelial cell migration was observed (Figure 1). Adding both the 70-kDa N-terminal and the 40-kDa C-terminal FN peptides to the VEGF/120-kDa FN peptide mixture failed to restore endothelial migration to levels observed with the VEGF/intact FN combination (Figure 1). To determine whether intact FN was enhancing VEGF-induced migration by protecting VEGF from proteolysis, we compared the rates of VEGF degradation by plasmin in the absence or presence of FN and VN. VEGF alone was completely degraded within 15 minutes. Both FN and VN did significantly protect VEGF from degradation, but VN was as effective as FN (≈50% degraded after 4 hours in the presence of FN or VN; data not shown).
α5β1 Integrin Mediates VEGF/FN-Induced Migration
Although α5β1 is the key receptor for FN, other integrin receptors such as αvβ3, the major receptor for VN, can also bind FN. To determine the integrin responsible for the enhanced migration, endothelial cells were exposed to a mixture of VEGF/FN/VN, and migration measured across a combined FN/VN substrate. By using specific integrin-blocking antibodies, the integrin responsible for migration could be identified. In the presence of antibodies to α5β1, VEGF-induced cell migration across the mixed FN/VN substrate was suppressed, whereas antibodies to αvβ1, αvβ3, or αvβ5 had no effect (Figure 2). This inhibition of migration by antibodies to α5β1 was not due to suppression of cell adhesion because endothelial cells were still able to attach to the FN/VN through αvβ3 (data not shown). In addition, soluble flt-1, a high-affinity receptor for VEGF, blocked migration by over 70%.
VEGF Receptor Flk-1 Associates With Integrin α5β1
We next studied how the VEGF/FN mixtures might influence the association of their respective receptors. Incubating endothelial cells on VEGF/FN-coated plates promoted the association of Flk-1 receptor (VEGFR-2) with α5β1 as demonstrated by immunoprecipitation and Western blotting (Figure 3A). Flt-1 receptor (VEGFR-1) did not coprecipitate with α5β1 (data not shown). When endothelial cells were incubated on VEGF/VN-coated plates, we observed only modest coprecipitation of Flk-1 with the αvβ3 integrin (Figure 3A), consistent with previous reports.15,16⇓ Association of Flk-1 with α5β1 integrin was not observed when endothelial cells were incubated on FN alone without VEGF. Similarly, coprecipitation of Flk-1 with α5β1 integrin was not observed when endothelial cells were either incubated on plates coated with VEGF and the FN 120-kDa, 70-kDa, and 40-kDa fragments or polylysine (Figure 3B). Coprecipitation of Flk-1 with α5β1 was only observed when endothelial cells were incubated with VEGF and intact FN.
Association of Flk-1 With the α5β1 Integrin Promotes Prolonged MAP Kinase Activation
Endothelial cells incubated on VEGF/FN-coated plates demonstrated sustained MAP kinase activation compared with cells incubated on VEGF/120-kDa FN-coated plates or VEGF/VN. In addition, an intact FN molecule is required to mediate the VEGF-induced activation of MAP kinase because the 120-kDa FN peptide failed to promote VEGF induced MAP kinase activation (Figure 4A). Figure 4B shows that U0126, a specific MAP kinase inhibitor, blocked endothelial cell migration by 90%, whereas wortmannin, a PI3-kinase inhibitor, suppressed migration by 20%.
FN Contains Two VEGF Binding Sites
Using the solid-phase assay, the binding of VEGF to a variety of ECM proteins was tested. 125I-VEGF165 bound mainly to FN (Figure 5A). Binding of VEGF was also observed with fibrin and fibrinogen (recently reported17). VEGF did not bind to vitronectin or collagen I, III, or IV. To locate the VEGF binding site on the FN molecule, slot blot assays were performed using purified proteolytically cleaved FN fragments immobilized onto nitrocellulose membranes. VEGF bound strongly to the 70-kDa N-terminal fragment (70-kDa FN peptide) and the 40-kDa C-terminal fragment (40 kDa FN peptide; Figure 5B). Binding was not observed with the 120-kDa internal cell binding domain fragment (120-kDa peptide; data not shown). Equivalent binding to FN was also observed with VEGF121 (data not shown).
To confirm the observations of the slot blot assays, the equilibrium binding of VEGF to the 70-kDa FN peptide was quantified over a range of concentrations using surface plasma resonance analysis (SPR). As shown in Figure 6, VEGF bound to the 70-kDa FN peptide immobilized on the sensor chip in a specific and saturable manner. The estimated Kd was 2 μmol/L. VEGF binding to the 40-kDa FN peptide could not be measured directly by SPR because immobilization of the 40-kDa FN peptide appeared to mask the VEGF binding site. Accordingly, the Kd for VEGF binding to the 40-kDa FN peptide was determined by competition experiments in which increasing amounts of the 40-kDa FN peptide were preincubated with VEGF before injection across the SPR sensor chip coated with the 70-kDa FN peptide surface (Figure 6C and 6D). These experiments showed that the 40-kDa FN peptide blocked binding of VEGF to the immobilized 70-kDa FN peptide with an IC50 of 1 μmol/L and a calculated Kd of 200 nmol/L for the 40-kDa fragment.
VEGF/FN Complex Formation In Vivo
To determine whether VEGF/FN complexes were spontaneously formed after platelet activation, we immunoprecipitated the supernatants from resting and thrombin-activated platelets with an antibody to FN. Precipitation of FN from supernatant of thrombin-activated platelets caused significant coprecipitation of VEGF compared with resting platelets or to a negative control antibody (Figure 7A). To determine whether the VEGF/FN complex was biologically active, supernatants from thrombin-activated platelets were filtered through Amicon filters (105-kDa cut-off) to obtain VEGF/FN complexes. The presence of these complexes was confirmed by immunoprecipitation. Filtered supernatants from thrombin-stimulated platelets promoted endothelial cell migration (Figure 7B). Addition of soluble Flt-1, a high-affinity receptor for VEGF, inhibited endothelial cell migration by 25%. Blocking antibody to α5β1 inhibited migration by 45%. The combination of both soluble Flt-1 and anti-α5β1 inhibited migration by more than 60%.
In this study, we demonstrate that VEGF binding to FN serves to amplify the biological effects of VEGF. Specific VEGF binding domains within FN were required to promote sustained MAP kinase activation and endothelial cell migration. The observation that both VEGF-165 and VEGF-121 can bind FN suggests that their FN binding ability is contained within exons 1 to 5 or 8. The VEGF binding domains on FN were mapped to the 70-kDa N-terminal and 40-kDa C-terminal ends of the FN molecule. Although the 40- and 70-kDa FN fragments bound VEGF, these fragments on their own were not sufficient to exert the full biological effect of enhanced migration seen with the intact FN molecule. One possible explanation is that the 70- and 40-kDa fragments do not fully support α5β1 adhesion and that an intact FN molecule containing both the cell binding and VEGF binding domains is required to facilitate the association of α5β1 with Flk-1 to promote enhanced cell migration. These findings identify the VEGF binding domains on intact FN as important cofactors for initiating a signaling pathway mediated by the α5β1/Flk-1 complex (Figure 8).
The integration of signals from integrins and receptor tyrosine kinases is essential in mediating cellular events such as cell proliferation, migration, and differentiation.18–22⇓⇓⇓⇓ Several recent studies have demonstrated that integrins and growth factor receptors can interact to form functional complexes although the mechanism(s) by which they associate to integrate their signals is unclear. For example, recent reports have demonstrated association of the β1 integrin with VEGFR-3 (Flt4) that is required for cell migration23 and that platelet-derived growth factor (PDGF) in the presence of vitronectin induces the association of the PDGF-β receptor with the αvβ3 integrin to enhance PDGF-BB induced proliferation and migration of fibroblasts.16,24–26⇓⇓⇓ The α6β4 and α6β1 integrins have been shown to associate with ErB-2 in human carcinoma cell lines after stimulation with epidermal growth factor or insulin.27 In addition, binding of tenascin-C to αvβ3 was shown to promote epidermal growth factor (EGF) receptor recruitment to focal adhesions, which resulted in increased smooth muscle cell proliferation, and that sustained activation of MAP kinase by EGF required αvβ3 aggregation.28,29⇓ Recently, it was demonstrated that PDGF-β and Flk-1 associated with the β3 integrin through its extracellular domain. However, it is still unclear how growth factors complexed to ECM proteins mediate the association of growth factor receptors and integrins. Indeed, several recent reports have demonstrated binding of soluble growth factors to ECM proteins. Fibrinogen was shown to bind both bFGF and VEGF and promote the proliferative effects of bFGF,17,30⇓ whereas tenascin-X was shown to bind VEGF-B.31 Vitronectin was also shown to bind VEGF.32 The binding of VEGF to heparan sulfate proteoglycan in ECM protects VEGF from proteolytic degradation. Bound VEGF can be released in a soluble and bioactive form by heparin and plasmin.33
Although a strong association between Flk-1 and α5β1 was observed when endothelial cells were plated on FN/VEGF-coated plates, we also, consistent with two other reports, observed a weak physical association between Flk-1 and αvβ3 when VN was the substrate.15,16⇓ When compared with the Flk-1/α5β1 association, Flk-1/αvβ3 association did not translate into prolonged MAP kinase activity or endothelial cell migration. One plausible explanation for VEGF/FN promotion of MAP kinase activation and endothelial cell migration versus VEGF/VN is that the VEGF binding domains on FN may help bridge the Flk-1 and α5β1 receptors for signal amplification. This is supported by the observation that the synergistic effects of VEGF/FN on endothelial cell migration, VEGF/Flk-1 association, and MAP kinase activation requires intact FN molecules. In support of our observations, it was shown recently that breast cancer cells had a higher rate of proliferation and migration in response to VEGF when cultured on a FN substrate.34 The enhanced biological effects of the VEGF/FN complexes observed in this study were not due to the protective effects of FN on VEGF because both FN and VN equally protected VEGF degradation by plasmin. Further work will be needed to elucidate the structure-function relationship important for the coordinate actions of VEGF/FN.
Using specific inhibitors to MAP kinase and PI3-kinase, we demonstrate that MAP kinase activation is important for VEGF/FN-induced endothelial cell migration. MAP kinase inhibition resulted in almost total suppression of endothelial cell migration, whereas PI3-kinase inhibition only resulted in ≈20% inhibition. This observation, in contrast to a recent study,35 may be due to the presentation to the cell of VEGF as a complex with FN. Consistent with our studies, fibroblasts obtained from MEK-1-deficient mice failed to migrate on FN. Re-expression of functional MEK-1 in the mutant fibroblasts restored their ability to migrate on FN,36 suggesting that MAP kinase may play an important role in α5β1-mediated migration. Indeed, several other studies have provided evidence that sustained activation of MAP kinase may play a role in cell migration37 by phosphorylation and activation of myosin light chain kinase as well as regulating focal adhesion assembly.38,39⇓
It is known that platelets are a major source of VEGF, and activated platelets release VEGF.40 This present study shows that platelets release VEGF complexed to FN. This is a significant finding with regard to the process of neovascularization in wound healing and tumor angiogenesis. This suggests that growth factors in general are released from activated platelets complexed to ECM. The formation of a complex may serve to protect the growth factor from degradation and also to integrate the signals generated by integrins and receptor tyrosine kinases.
In summary, we have identified two novel VEGF binding domains on the 70-kDa N-terminal and 40-kDa C-terminal FN molecule, which we propose are necessary for promoting the physical association of α5β1 and Flk-1. This association of integrin and receptor tyrosine kinase enhances the amplification of signals required for sustained activation of MAP kinase and subsequent endothelial cell migration. These present data provide further insights into mechanisms by which growth factors and ECM cooperate to influence cellular behavior.
This work was supported by grants from the Murdock Charitable Foundation and Patterson Foundation (E.S.W., J.M., K.S.), Grupo Grifols and Centeon (S. Rahman, Y.P.), and National Institutes of Health Grant HL39903 (M.S.).
↵*These authors contributed equally to this work.
Original received June 5, 2001; resubmission received April 1, 2002; revised resubmission received June 4, 2002; accepted June 5, 2002.
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