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The FASEB Journal express article10.1096/fj.02-0691fje. Published online April 8, 2003. Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation Helen Hutchings, Nathalie Ortega, and Jean Plouët GDR CNRS 1927 – Angiogenese and UMR CNRS 5089, Institut de Pharmacologie et Biologie Structurale, Toulouse, France Corresponding author: Jean Plouët, Institut de Pharmacologie et Biologie Structurale, UMR CNRS 5089, 205 route de Narbonne, 31077 Toulouse cedex, France. E-mail: firstname.lastname@example.org ABSTRACT Vascular endothelial growth factor (VEGF), a major factor mediating endothelial cell survival, migration, and proliferation during angiogenesis, is expressed as five splice variants (121, 145, 165, 189, and 206 aminoacids) encoded by a single gene. Although the three shorter isoforms are mainly diffusible, the two longer ones are sequestered in cell membranes after secretion. However, their potential role as true components of the extracellular matrix has not been investigated. We determined that endothelial cells could adhere and spread on VEGF189 and VEGF165, but not on VEGF121. Adhesion was mediated by the α3β1 and αvβ3 integrins and other αv integrins but not by the cognate VEGF receptors. Cells migrated on VEGF165 and VEGF189 and displayed a stellate morphology with numerous lamellopodia and FAK staining but no actin stress fibers. Tumstatin, an antiangiogenic peptide that interacts with the αvβ3 integrin, could inhibit adhesion on VEGF, and this effect was potentiated by anti-αvβ3 blocking antibody. Immobilized VEGF almost totally abolished endothelial cell apoptosis through interactions with integrins. The inhibition of αvβ3 engagement with immobilized VEGF by tumstatin inhibited most of its survival activity. We have thus determined a new VEGF receptor-independent role for immobilized VEGF in supporting cell adhesion and survival through interactions with integrins. Key words: angiogenesis • VEGF • tumstatin A ngiogenesis, the formation of new blood vessels from the preexisting vasculature, is a critical process contributing to embryonic vessel development, wound healing, corpus luteum formation, and pathological processes such as tumor development, diabetic retinopathies, and rheumatoid arthritis. The process of angiogenesis can be divided into several overlapping phases. It begins with an increase of local blood vessel permeability and extravasation of plasma proteins such as fibrinogen at the site where the new blood vessel is to sprout. Degradation of the basement membrane by proteases of the metalloproteinase family allows for proliferation and migration of endothelial cells toward the angiogenic stimulus. Finally, during the maturation stage, endothelial cells differentiate, form a lumen and a new basement membrane, and recruit pericytes to support the structure of the new vessel. Vascular endothelial growth factor (VEGF) is a key regulator of both physiological and pathological angiogenesis (1). VEGF was first described as an endothelial cell-specific mitogen (2, 3), but it is now known to act on certain other cell types (4, 5). VEGF exists in five isoforms-- 121, 145, 165, 189, and 206 amino acids--which are generated by alternative splicing of the pre- mRNA, and these isoforms can be further matured by proteolytic cleavage. VEGF165, a 46-kD homodimeric glycoprotein, is the most abundant and most intensely studied of the five isoforms. Although the five isoforms can be cleaved by plasmin and generate a 110-amino acid factor, which does not bind to heparin, only exon 6-containing isoforms are cleaved by urokinase (6). However, little is known about the specific biological responses they might induce. Cellular responses to VEGF165 are mediated by two high-affinity type III tyrosine kinase receptors, KDR/Flk1 (7) and Flt (8), and two receptors of the semaphorin receptor family, neuropilin-1 (9) and neuropilin-2 (10). Gene targeting studies have shown that VEGF, KDR, and Flt1 are essential for fetal angiogenesis (11–13). The loss of even a single allele of the VEGF gene is lethal, underlining the critical role of this factor. In VEGF null mice embryos, most steps of early vascular development are impaired: endothelial cell differentiation, sprouting from preexisting vessels, lumen formation, formation of vessel interconnections, and spatial organization (11). The phenotype of VEGF null mice illustrates the multiple roles that VEGF could have during vascular development: as a differentiation factor for angioblasts, as a mitogen for endothelial cells, and as a mediator of cell-cell and cell-matrix interactions. Little is known about VEGF’s role in mediating cell-matrix interactions. The extracellular matrix (ECM) gluttonously sequesters VEGF soon after its secretion on heparan-sulfates (14) and probably another binding site specific for exon 6 (6). Accordingly, systemic injections of VEGF have very little effect because all the protein is absorbed by the ECM at the site of injection or in the vascular wall. It has been shown recently that cells could adhere directly on cytokines involved in angiogenesis. For instance, Carlson and coworkers demonstrated that cells could adhere both on angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) but only spread and migrate on Ang-1 (15). Several integrins were shown to be involved in adhesion on Ang-1 and Ang-2, and the α5 integrin subunit had a particulary important role. Latent forms of transforming growth factor β (TGF-β) have also been shown to be a substrate for cell adhesion, and this adhesion was, as for the angiopoietins, mediated by integrins (16). Here, we demonstrate that endothelial cells can adhere directly and spread on two immobilized VEGF isoforms: VEGF165 and VEGF189. Endothelial cells clearly show a preference for VEGF189 as an adhesion substrate because adhesion reaches higher levels and occurs at a faster rate than on VEGF165. We propose the existence of additional cell binding sites for the immobilized forms of VEGF, which include the α3β1 and αvβ3 integrins, and we have determined that immobilized VEGF induces endothelial cell migration and survival through direct αvβ3 ligation. Moreover, tumstatin, a fragment of collagen IV, reversed the effect of VEGF on endothelial survival. MATERIALS AND METHODS Materials Human umbilical arterial endothelial cells (HUAEC) were isolated from dissected umbilical arteria perfused with collagenase (Sigma, St. Louis, MO) to digest the basal membrane. HUAEC were maintained in SFM (Life Sciences) supplemented with 20% heat-inactivated fetal calf serum (FCS). Stock cultures received 1 ng/ml VEGF every other day. Human bone marrow endothelial cells (HBMEC) were a kind gift from L. Pelletier and were cultured in the same medium. Bovine retinal endothelial cells (BREC) and fetal aorta (FBAE) endothelial cells were isolated from organs obtained from a local slaughterhouse as described previously (17). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) glutamax (Life Sciences) supplemented with 10% heat-inactivated newborn calf serum (NBCS), 100 µg/ml penicillin, and 100 µg/ml streptomycin at 37°C in 10% CO2. Rhodamine-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). rhVEGF121, rhVEGF165, and rhVEGF189 were produced using a baculoviral expression system and iodinated as described previously (6). rhVEGF110 was produced by plasmin digestion of rhVEGF165 as described previously (18). Fibronectin, collagen I, poly-lysine, heparin, and bovine serum albumin (BSA) were from Sigma. αvβ3 recombinant protein was purchased from Chemicon (Temecula, CA). The RGD peptides and RAD control peptides were also purchased from Sigma. The α3β1 integrin blocking peptide, p678 (FQGVLQNVRFVF), and its control peptide, p690 (FQGVLQNVAFVF) (19), as well as the tumstatin peptide, (TMPFLFCNVNDVCNFASRNDYSYWL), were synthesized by Eurogentec (Philadelphia, PA). The peptides corresponding to the VEGF amino acid sequences 119–134 (P2: RGKGKGQKRKRKKSRY) and 126–134 (P1: KRKRKKSRY) were synthesized by a solid-phase technique. The anti-β1 (6S6), anti-αv subunit, and anti-αvβ5 (P1F6) blocking antibodies were purchased from Chemicon, the αvβ3 blocking antibody was produced from the HB-11029 hybridoma (ATCC), and the control monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-β3 (RM13) antibody was purchased from Bioline (Milan, Italy). The anti-FAK (sc 517) antibody was from Santa Cruz Biotechnology, and the FITC conjugated anti-rabbit secondary antibody was from Sigma. Cell adhesion assays Ninety-six-well ELISA plates (Nunc, Rochester, NY) were coated with protein diluted in carbonate buffer, 0.05 M, pH 9.6, overnight at 4°C. Nonspecific binding sites were blocked for 1 h at 37°C with 5 mg/ml BSA in carbonate buffer and washed twice with DMEM before experiments. Cells were trypsinized, washed, and resuspended in 5 ml of DMEM with 10% FCS in an untreated plastic tube and incubated for 1 h at 37°C with 10% CO2. Cells were then pelleted and resuspended in DMEM and 0.2% BSA without serum, and the cell suspension was treated for 20 min (37°C, 10% CO2) with the proteins, peptides, or antibodies used to modulate adhesion. Cells (40,000 per well; three wells per condition) were distributed into the wells in a volume of 100 µl DMEM and 0.2% BSA. Cells were allowed to adhere at 37°C, 10% CO2, for the desired time. Wells were washed gently three times with DMEM to remove nonadherent, cells and adherent cells were fixed with 1% glutaraldehyde for 20 min at room temperature. Fixed cells were quantified by crystal violet incorporation (20): Cells were incubated with 0.1% crystal violet (Sigma) diluted in 0.2 M borate buffer, pH 9.5, for 20 min at room temperature, nonincorporated dye was eliminated by thoroughly washing the wells with large amounts of water, and incorporated crystal violet dye was then solubilized by 100 µl of 10% acetic acid per well. Optical density readings were performed at 595 nm. T1/2 is the time for which cell adhesion has attained its half-maximal level. Solid-phase αvβ3 binding assay Ninety-six-well ELISA plates (Nunc) were coated with VEGF isoforms, BSA, or fibronectin at 4 µg/ml diluted in carbonate buffer, 0.05 M, pH 9.6, overnight at 4°C. Nonspecific binding sites were blocked for 1 h at 37°C with 5 mg/ml BSA in carbonate buffer. Wells were washed and incubated for 2 h with different concentrations of αvβ3 recombinant protein in PBS, 5 mg/ml BSA, and 0.05% Tween 20. Binding of αvβ3 to VEGF isoforms was revealed by incubation with an anti-β3 antibody (1/1000), followed by an horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody. Plates were developed with Sigma Fast OPD tablets, and optical densities were read at 492 nm. Immunofluorescence For all experiments, glass Labtek slides (Nunc) were coated with protein diluted in carbonate buffer, 0.05 M, pH 9.6, overnight at 4°C. Nonspecific binding sites were blocked for 1 h at 37°C with 5 mg/ml BSA in carbonate buffer. Cells were harvested by trypsinization, washed, and resuspended in DMEM with 10% NBCS for bovine cells or FCS for human endothelial cells in an untreated plastic tube and incubated for 1 h at 37°C with 10% CO2. Cells were then pelleted, resuspended in DMEM and 2% BSA, and seeded at ~25% confluency in the Labtek wells. After 2 h at 37°C, cells were fixed with 3.7% paraformaldehyde and 15 mM sucrose in PBS for 20 min at room temperature and permeabilized with 0.3% Triton-X100 in PBS for 5 min at room temperature. Nonspecific binding sites were blocked by incubation with 5 mg/ml BSA for 2 h at room temperature. For F-actin labeling, cells were incubated for 20 min at room temperature with rhodamine-conjugated phalloidin. For anti-FAK labeling, cells were first incubated with an anti-FAK antibody (1/100) for 1 h at 37°C and then with an anti-rabbit FITC-conjugated secondary antibody (1/100) for 45 min at 37°C. The plastic wells of the Labtek slides were then removed, and slides were mounted with Paramount (Dako, Glostrup, Denmark) and analyzed with a Zeiss fluorescence microscope. Migration assays Cells were allowed to adhere for 2 h in plastic dishes coated with either 4 µg/ml VEGF165, VEGF189, fibronectin, or BSA, as described previously. After this incubation period, dishes were washed thoroughly with DMEM to eliminate nonadherent cells. Cell movement was monitored by successive photography of cells at a given spot of the dish. Image overlay was used to evaluate the migration patterns of individual cells. Annexin V-fluorescein isothiocyanate assay Annexin V, a calcium-dependent phospholipid binding protein with a high affinity for phosphatidylserine was used to detect apoptosis. In brief, HUAEC were seeded in 12-well culture plates previously coated with VEGF165, VEGF189, or collagen I (0.5 × 106 cells/well) in DMEM and 2% FCS in the presence or absence of anti-αvβ3 antibody (1/3). Cells were left to adhere for 2 h, and then 10 µg/ml tumstatin was added. Three hours later, floating and attached cells were harvested and processed as described previously (21). Statistical analysis Data are mean ±SD. Statistical comparisons were made by ANOVA followed by an unpaired Student’s t test. Data were considered significantly different if values of P<0.05 were observed. RESULTS Endothelial cell adhesion to VEGF165 and VEGF189 To determine whether endothelial cells could adhere to VEGF, HUAEC were plated on plastic wells that had been coated with BSA, fibronectin, VEGF121, VEGF165, or the full-length or cleaved forms of VEGF189. As shown in Figure 1A, HUAEC adhered well to the VEGF189 isoforms (cleaved or full-length) compared with BSA within 20 min after plating. Cells adhered moderately well to VEGF165 (60% of maximum adhesion reached on VEGF189) and not at all to VEGF121. The amount of cells adhered to VEGF189 was similar to that on fibronectin, which is one of the best adhesion substrates for endothelial cells. Differences were shown not to be due to variable absorption rates between the VEGF isoforms, becauses we determined that 12% and 9% of VEGF165 and VEGF189, respectively, bound to plastic. VEGF121 adhered as well as the other isoforms to plastic and inhibited the binding of iodinated VEGF165 and VEGF189 with a similar efficacy (IC50 = 7 µg/ml) when it was coincubated with 4 µg/ml of either long isoform (data not shown). This decrease of the amount of longer isoforms immobilized on plastic achieved by coincubation with VEGF121 reduced HUAEC adhesion in a dose-response manner (IC50 = 8µg/ml for VEGF165 and 15 µg/ml for VEGF189). HUAEC adhesion on VEGF was comparable, on a molar basis, to that on other ECM proteins (data not shown). Maximal adhesion levels on VEGF165 and VEGF189 were attained at a coating concentration of 100 nM (parallel experiments showed that maximal absorption of VEGF on the plastic was not achieved at this concentration and therefore that the amount of VEGF bound to the plastic was not a limiting step for cell adhesion). Microscopic observation of the cells during adhesion assays revealed that HUAEC adhered extremely rapidly to VEGF189. We therefore decided to study adhesion in a time-dependent manner. As expected, HUAEC adhered much faster to VEGF189 or to fibronectin than to VEGF165 (Fig. 1B) (T1/2 = 7.5 min). In a second set of experiments, we evaluated the involvement of heparan-sulfate proteoglycans in cell adhesion on VEGF. Soluble heparin (at concentrations >50 nM) inhibited HUAEC adhesion to VEGF189; maximal inhibition represented ~50% total adhesion (Fig. 2A). On VEGF165, heparin increased rather than inhibited cell adhesion. Heparin had no effect on adhesion to fibronectin. Because HUAECs did not adhere to VEGF121 (which is encoded by exons 1–5 and 8), we investigated whether peptides derived from exon 6 of VEGF could inhibit cell adhesion. High concentrations of peptides P1 and P2 (20 µg/ml) significantly delayed HUAEC adhesion to VEGF (Fig. 2B). The longer peptide, P2, was a better inhibitor than P1. Neither P1 nor P2 had any effect on HUAEC adhesion to fibronectin. The inhibitory activity of these two peptides seems to be mostly due to their basic charge, because they both inhibit adhesion to VEGF165, which does not contain the exon 6 from which they are derived. Strong concentrations of these peptides were needed to obtain any effect; this could be due to a difference in the conformations of soluble and immobilized VEGF domains. Integrin blocking agents inhibit adhesion We next determined which cell surface binding sites were involved in adhesion to the two VEGF isoforms. Adhesion assays showed that pretreatment of HUAEC with soluble VEGF165 or VEGF189 had no significative effect on cell adhesion (Fig. 3). Because soluble VEGF should induce internalization of known VEGF receptors or at least block access to them, we conclude that the molecules that interact with the immobilized VEGF and mediate cell adhesion differ from known VEGF receptors. To test whether or not integrins may serve as receptors for immobilized VEGF, adhesion experiments were performed in the presence of the calcium chelator EDTA, a universal inhibitor of integrins; RGD peptides, which inhibit integrins binding this sequence; and p678, a specific α3β1 blocking peptide. Blocking peptides for RGD integrins and α3β1 were used at maximal concentrations effective for blocking adhesion--500 µM (15, 22) and 20 µM (19), respectively. As shown in Figure 3, addition of these inhibitors revealed different adhesion profiles for HUAEC on VEGF165 and VEGF189. Whereas EDTA inhibits all adhesion on both VEGF isoforms, the α3β1 blocking peptide, p678, completely blocks adhesion on VEGF165 but only ~60% of that on VEGF189. RGD peptides had little effect alone, but when used together with p678, all adhesion was inhibited on VEGF189. Time-course experiments with HUAEC showed that adhesion was not completely abolished on VEGF165 or VEGF189 by addition of p678 or RGD peptides (Fig. 4). However, all adhesion was inhibited on both VEGF isoforms by addition of a combination of p678 and RGD peptides. These results demonstrate that HUAEC adhere to VEGF165 and VEGF189 uniquely via integrins: the α3β1 integrin and one or more of the RGD sequence binding integrins. Similar results were obtained with aortic endothelial cells and two capillary-derived endothelial strains cultured from bone marrow or retina (Table 1). These findings underline that the observations were not a particular feature of artery endothelial cells. Next, we used blocking antibodies against specific integrins. Figure 5 shows time-course adhesion experiments in the presence of antibodies directed against the β1 integrin subunit, the αv integrin subunit, and the αvβ3 and αvβ5 integrins. β1 and αvβ3 blocking antibodies both partially inhibit adhesion on VEGF165. The αv blocking antibody strongly inhibits adhesion on VEGF165 (from 100% inhibition after 7.5 min to ∼65% inhibition after 40 min). Adhesion is dependent on integrins β1 and αvβ3 on VEGF165. Because a combination of these antibodies inhibits practically all adhesion (Fig. 5A), the small remaining fraction of adhesion seems to be due to another αv-subunit integrin (but not αvβ5, data not shown). On VEGF189, initial adhesion can be significantly blocked with the anti-αvβ3 antibody (Fig. 5B). The β1-subunit antibody only very slightly inhibits adhesion but enhances inhibition by the αvβ3 and αv subunit blocking antibodies. About 50% of adhesion on VEGF189 could not be attributed to any of the tested integrins and must depend on other RGD integrins that do not possess a αv subunit. αvβ3 binds directly to immobilized VEGF In a solid-phase binding assay, we investigated whether recombinant αvβ3 could bind directly to VEGF isoforms absorbed on plastic. As shown in Figure 6, we observed that αvβ3 bound to immobilized VEGF165 and VEGF189 isoforms but not to VEGF121. αvβ3 bound to VEGF165 and VEGF189 as strongly as it did to fibronectin, a well-characterized αvβ3 ligand. Control experiments with a nonspecific primary antibody showed optical densities comparable to background signaling on BSA (data not shown). These results demonstrate that VEGF121 is not a ligand for αvβ3 and therefore confirms that the interaction of αvβ3 and VEGF occurs through the basic domain of VEGF corresponding to exons 6 and 7. Endothelial cells spread on VEGF but display no actin stress fibers Endothelial cell adhesion to ECM proteins usually induces reorganization of the actin cytoskeleton with a great number of filaments of polymerized actin that attach the cells to their substrate via focal adhesions at the abluminal membrane. We examined the cell morphology induced by VEGF. As seen in Figure 7A, HUAEC displayed a classical well-organized actin cytoskeleton with many actin stress fibers on fibronectin. On VEGF165 and VEGF189, cells spread well compared with cells attached to poly-lysine but presented a very different morphology. Cells adhering to VEGF display a stellate morphology with numerous lamellopodia (HUAEC) and filipodia. Although polymerized actin could be discerned in these membrane protrusions, no actin stress fibers were observed and the filamentous actin appeared to be organized into large bundles in the cytoplasm. Because spreading of cells on ECM components requires integrin clustering and subsequent activation of downstream signaling molecules, we investigated whether focal adhesion kinase (FAK) localized at focal adhesions in cells adhering to VEGF. FAK immunofluorescence experiments revealed that cells adhering to both VEGF165 and VEGF189 showed typical FAK localization at focal adhesions (Fig. 7B). On VEGF, FAK clustering was mostly concentrated at the edges of the cells, whereas FAK clusters were observed all over the abluminal membrane of cells on fibronectin. VEGF supports cell migration The ability of cells to migrate on VEGF165 and VEGF189 was tested next. After plating, cells were observed over a 4-h period, and successive photography was used to analyze movement. VEGF189 supported strong HUAEC migration in the absence of any other ECM components or serum. This migration was comparable to that seen on fibronectin (Fig. 8). Migration on VEGF189 could not be attributed to its basic charge because endothelial cells plated on poly- lysine, a highly basic substrate, did not migrate at all. Migration of cells on VEGF165 was significantly lower than on VEGF189 over the same time period but was however statistically significant compared with cells on poly-lysine. Tumstatin inhibits the αvβ3-dependent effects of immobilized VEGF on adhesion and survival Because tumstatin is known to interact with αvβ3 and induce endothelial cell apoptosis, we tested its activity on cells plated on VEGF165 or VEGF189. Tumstatin inhibited HUAEC adhesion to both VEGF isoforms in a dose-dependent manner (Fig. 9). This effect was enhanced when cells were treated simultaneously with an anti-αvβ3 blocking antibody. Next, we compared the survival rates of HUAEC adhered to VEGF or to collagen I. Cells were plated on VEGF165, VEGF189, or collagen I and treated with either an anti-αvβ3 blocking antibody, tumstatin, or both. Apoptosis levels were analyzed by FACS after Annexin-V isothiocyanate and propidium iodide staining. Table 2 shows that VEGF165 and VEGF189 protect HUAEC from apoptosis at a level similar to that observed in the presence of collagen I. Tumstatin and an anti-αvβ3 antibody induced a synergistic effect on apoptosis of HUAEC adhering to either VEGF isoform, whereas they had no combined action on HUAEC seeded on collagen I. This sugests that ligation of αvβ3 to immobilized VEGF generates a survival signal that opposes the proapoptotic action of tumstatin. However, all the survival effecst of VEGF could not be accounted for by αvβ3 because none of the treatments could induce more than 60% of apoptosis, thus demonstrating that other integrins involved in adhesion could also be involved in survival signals. DISCUSSION Our results show for the first time that endothelial cells adhere directly to immobilized VEGF isoforms VEGF165 and VEGF189 and that this adhesion is mediated by integrins. We find that the basic moieties of VEGF trigger adhesion mechanisms, because the anionic form did not promote cell adhesion, whereas VEGF165 and VEGF189 supported endothelial cell spreading, migration, and survival. Cell surface VEGF binding sites have been identified as two tyrosine kinase receptors (namely, VEGFR1 and VEGFR2), two semaphorin receptors (neuropilin-1 and neuropilin-2), and heparan-sulfate proteoglycans. It is unlikely that adhesion on VEGF is mediated by its tyrosine kinase receptors because the binding domains of VEGF to its receptors are present in VEGF121, which cannot mediate adhesion. VEGF121 does not interact directly with αvβ3 either, one of the integrins largely responsible for adhesion on VEGF165 and VEGF189. Additional evidence comes from the fact that no difference was observed between adhesion on VEGF189, which does not bind to VEGFR2, and its cleaved counterpart uPA-VEGF189, which does (6). These results are again strengthened by the fact that peptides derived from exon 6, which does not bind either VEGFR1 or VEGFR2, partially inhibit cell adhesion. Addition of soluble heparin partially inhibited cell adhesion on VEGF189, but not on VEGF165, which suggests that nonspecific electrostatic interactions between the anionic charges of heparin and the basic sequence encoded by exon 6 partially mask VEGF189 binding sites for endothelial cells. On VEGF165, soluble heparin slightly enhanced cell adhesion, which suggests that as for it’s binding to VEGFR2, soluble heparin might help to give recombinant VEGF165 a more active conformation and thus enhance adhesion. VEGF145 and VEGF206 that contain exons 6a or 6b and 7 bind strongly to the ECM, and therefore they could be expected to act as substrates for cell adhesion as well. However, cell pretreatment with chlorate, which inhibits the sulfatation of proteoglycans and reduces their anionic charge, had no effect on adhesion (data not shown). These results contrast with the observation that heparan-sulfate proteoglycans could serve as coreceptors for cell adhesion on Cyr61 mediated by the α6β1 integrin (23). We observed an important difference in the way that endothelial cells adhere to VEGF165 and VEGF189, which suggests that neuropilins, which interact with the basic sequence of VEGF encoded by exon 7 (9), are not involved in endothelial cell adhesion to VEGF. Little is known about the putative distinct functions of VEGF189 compared with VEGF165. VEGF189, in contrast to VEGF165, is rapidly sequestered by heparan-sulfate proteoglycans of the ECM (14) and another as yet unidentified binding site (6). We show here that all cell adhesion on VEGF165 can be attributed to the α3β1 and αvβ3 integrins and another αv subunit containing integrin (but not αvβ5). On VEGF189, the same integrins are involved (α3β1, αvβ3, and another αv integrin); however, ~50% of adhesion is mediated by a yet unidentified receptor. If the results shown in Figure 4 are to be taken into account, this receptor should be an RGD integrin, because a combination of α3β1 blocking peptides and RGD peptides inhibit all adhesion on VEGF189. The only known RGD integrin that does not contain either a β1 subunit or a αv subunit is αIIbβ3. However, it could be possible that the RGD peptides might nonspecifically block other nonintegrin cell surface receptors. Several hypotheses could explain the differences (maximal levels and rapidity) between adhesion on VEGF165 and VEGF189. First, a simple explanation could be that α3β1 and αvβ3 integrins bind with a greater affinity to VEGF189 than to VEGF165. It has been shown that in some cases, integrins must be activated to be able to induce a functional response, so it is plausible that contact with immobilized VEGF189 could activate this integrin much faster than immobilized VEGF165. It has been shown that soluble VEGF can induce activation of several integrins (24). In our case, immobilized VEGF could act in the same way and the efficiency of integrin activation could depend on the VEGF isoform. Another hypothesis is that the yet unidentified receptors for VEGF189 are responsible for the observed differences. This would be a very interesting finding because it would mean that VEGF189 possesses a specific cell surface receptor that could mediate isoform-specific responses (such as cell migration). The clear preference that endothelial cells show for adhesion to VEGF189 compared with VEGF165 is not translated by different cell morphology. In contrast to cells plated on fibronectin, which display prominent stress fibers, cells on VEGF possess numerous lamellopodia and filopodia but no actin stress fibers. The cell shape on VEGF can be compared with that seen by Waltenberger and coworkers who observed the appearence of lamellopodia and cytoskeletal reorganization when soluble VEGF was added to endothelial cultures (25). However, the morphology of cells attached to VEGF and those treated by soluble VEGF is quite distinct because soluble VEGF did not cause stress fibers to disappear. Actin dynamics appear to be coordinated in time and space by intracellular signaling pathways, and it has been shown that two separate signaling pathways acting on actin reorganization can be initiated by integrins (26, 27). Engagement of integrins with the ECM leads to activation of Cdc42, which subsequently leads to activation of Rac, and together, these GTPases mediate cell spreading and formation of lamellopodia and filopodia. Integrins can also activate Rho independently of Rac, and this leads to formation of stress fibers. After observation of endothelial morphology on VEGF, one could imagine that only one of these signaling pathways has been activated--the Cdc42-Rac pathway, leading to formation of lamellopodia and filopodia. The functionality of this morphology is yet to be determined, but the absence of stress fibers does not weaken the cells because they survive on VEGF189 in serum-deprived conditions just as long as their counterparts plated on fibronectin. Cells on VEGF may not display actin stress fibers, but they show signs of active integrin signaling. FAK, a major integrin signaling molecule, is localized at focal adhesions in cells adhering to VEGF. Integrin clustering and recruitment of several adaptator molecules and kinases is known to be a key step in the process of cell spreading and migration. Our results show the presence of focal adhesions in cells adhering to VEGF, and it is therefore possible that integrins clustered at these focal adhesions induce active signal transduction. Note that focal adhesions are found mostly near the edges of cells on VEGF, whereas they can be observed throughout cells on fibronectin. This particularity could be related to the cell morphology observed on VEGF, where actin is not organized into stress fibers. This underlines the fact that the role of VEGF immobilized in the ECM is probably not to induce the formation of an actin stress fiber network but to provide the cell with a specific growth factor-like stimulus (e.g., migration or antiapoptosis). Although cell morphology on VEGF165 and VEGF189 is very similar, a functional distinction appears when cells are tested for their capacity to migrate on a surface coated with the two isoforms. After 4–6 h in the absence of serum, cells migrate on VEGF189 as much as on fibronectin. However, although migration on VEGF165 is significant compared with that on poly- lysine, it is much lower than on VEGF189. This experiment describes a distinct role for VEGF189, which does not involve, at least in the initial stages, known VEGF receptors. This VEGF189- specific activity could be due to release of FGF2, because we have already shown that soluble VEGF189 added to corneal endothelial cells that do not express VEGF receptors induces cell proliferation and migration through FGF2 release from the cell membranes (28). To our knowledge, this is the first work demonstrating that VEGF can be used by endothelial cells as an adhesion substrate but not the first that shows that cytokines can support cell adhesion. Carlson and coworkers recently showed that angiopoietins Ang-1 and Ang-2, two others factors involved in angiogenesis, could serve as substrates for cell adhesion (15). Latent forms of TGF-β can also support cell adhesion via integrins (16, 29), and factor XIIIa can support microvascular endothelial cell adhesion (30). In the last part of our work, we observed the action of tumstatin on cells attaching to immobilized VEGF. Both immobilized VEGF and tumstatin interact directly with the αvβ3 integrin: VEGF promotes endothelial cell adhesion, whereas tumstatin induces apoptosis (31, 32). In our experiments, tumstatin inhibits adhesion to VEGF (both VEGF165 and VEGF189) in a dose-dependent fashion. This inhibition is potentiated by an anti-αvβ3 blocking antibody, but both treatments cannot fully reverse the survival activity of VEGF, thus demonstrating that other integrins such as α3β1 involved in adhesion may also play a role in apoptosis protection. However, tumstatin and anti-αvβ3 blocking antibodies do not have a combined effect on inducing apoptosis in cells attached on collagen I, which is not a substrate for αvβ3. This suggests that immobilized VEGF and tumstatin compete for signaling through αvβ3. Tumstatin and VEGF do not interact directly (data not shown) and probably do not share the same binding site on αvβ3: Tumstatin has been shown to bind to the β3 subunit, whereas VEGF seems to bind the classical RGD binding site (even if no RGD sequence is present in VEGF). This could mean that both molecules might bind αvβ3 simultaneously, implying that the αvβ3 integrin is the major regulation point leading to either survival or death of the endothelial cell (see Fig. 10). The role of αvβ3 in angiogenesis is still controversial. Pioneer work has focused attention on the proangiogenic role of αvβ3 (33), showing that disruption of αvβ3 anchorage with the ECM promoted apoptosis, whereas other work has shown that disruption of either the αv or β3 genes leads to enhanced angiogenesis (34). However, these demonstrations address functional relationships between αvβ3 and soluble VEGF, which in turn activates VEGF receptors. The recent demonstration that ligation of αvβ3 with tumstatin induces apoptosis specifically in endothelial cells allowed the postulation that it might bind simultaneously to other substrates that have not been yet identified. We propose that this missing link for αvβ3-dependent survival is immobilized VEGF. Therefore, the balance between the amounts of matrix-bound VEGF and tumstatin could be an essential mechanism used by the organism to control the quiescence of endothelial cells or the fate of angiogenic endothelial cells (death or proliferation). The finding that VEGF serves as an adhesive protein in the ECM helps to explain many other previous results. It is well known that VEGF is not a factor that circulates well in the blood stream; it is rapidly sequestered by the proteoglycans of the ECM (14, 35). This results in VEGF being present and available only close to its production site. One of the hypotheses concerning the role of VEGF trapped in the matrix is that it constitutes a natural reserve of growth factor that can be freed by proteolytic digestion when necessary. Here, we have determined another biological function for these sequestered molecules of VEGF, especially for the 189-amino acid isoform. Immobilized VEGF189 remains functional and could serve in vivo as a provisional matrix and participate in directing migrating endothelial cells at the site of angiogenesis. This correlates with results from Pat D'Amore’s team, which showed that the basic ECM binding VEGF isoforms are necessary in retinal vascular development to allow vessels to branch out from the center of the retina (36). It has been shown that VEGF189 expression is up-regulated more than other VEGF isoforms in esophageal xenografts (37) and by some types of tumor cells, for example, in human colon and in non-small-cell lung cancer (38). In these cases, VEGF189 in the tumor ECM or associated with the cell surface could be a support for endothelial cell migration into the tumor mass. It has also been reported that VEGF189, and not VEGF165, expression is up-regulated in osteoartritic cartilage (39), where uncontrolled angiogenesis contributes to the pathology. Another hypothesis as to the function of VEGF in the ECM could be that the interaction between immobilized VEGF and integrins may not be strictly necessary for cell adhesion but might influence VEGF receptor activity. It has previously been shown that αvβ3 interacts and modulates the activity of VEGFR2 (40). 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(1999) Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 18, 882–892 Received August 5, 2002; accepted February 14, 2003. Table 1 Adhesion of four endothelial cell strains on VEGF isoformsa Cell type (optical density, 595 nm) Adhesion substrate HUAEC FBAE BREC HBMEC BSA 0.08 0.02 0.10 0.08 VEGF121 0.05 0.03 0.11 0.08 VEGF165 0.42 0.224 0.41 0.43 + heparin 0.46 nd 0.32 nd + p678 0.08 0.067 0.06 0.14 + RGD 0.37 0.09 0.40 0.32 + p678 + RGD 0.11 0.02 0.04 0.09 + soluble VEGF165 0.33 0.16 0.44 nd VEGF189 0.92 0.38 0.51 0.66 + heparin 0.44 nd 0.25 nd + p678 0.44 0.21 0.42 0.48 + RGD 0.75 0.20 0.49 0.55 + p678 + RGD 0.16 0.11 0.31 0.27 + soluble VEGF189 0.87 0.19 0.51 nd a ELISA plates were coated with concentrations of 4 µg/ml of all proteins. Cells were trypsinized, washed, and resuspended in DMEM with 10% FCS and incubated for 1 h at 37°C. Cells were then pelleted and resuspended in DMEM without serum in the presence of heparin (50 nM), p678 (20 µM), RGD peptides (500 µM), or soluble VEGF (200 nM) for an additioanl 20 min. Cells were then distributed (40,000 cells per well) and allowed to adhere for 20 min. Nonadherent cells were washed away, and the amount of cell adhesion was quantified by crystal violet incorporation. All conditions were performed in triplicate, and results are representative of at least two experiments. Nd, not determined. Values are given as optical density readings at 595 nm. Table 2 Cooperation between integrin avß3 and tumstatin to induce apoptosis of HUAEC plated on VEGF isoformsa Substratum (apoptosis, %) Modulator BSA VEGF165 VEGF189 Col I Blank 100 22.8 31.6 27.7 IgG Ctrl — 27.8 35.8 28.4 Anti-αvβ3 — 39.9 34.2 26.9 Tumstatin — 34.9 49.9 36.0 Anti-αvβ3 + Tumstatin — 61.3 62.3 33.9 a HUAEC cells were trypsinized and plated in wells coated with either BSA, VEGF165, VEGF189, or collagen I (Col I) in the presence or absence of anti-αvβ3. Cells were left to adhere for 2 h and were then treated with 10 µg/ml tumstatin. Annexin V-fluorescein isothiocyanate staining and propidium iodide (PI) incorporation were performed after 3 h treatment. FACS analysis was done to quantitate the percentage of cells undergoing apoptosis (annexin V-positive, PI-negative cells). Values are expressed as percentages of apoptotic cells. Results of one out of three similar experiments are shown. Fig. 1 Figure 1. HUAEC adhesion on VEGF. A) Adhesion of HUAEC to fibronectin, VEGF121, VEGF165, VEGF189, and uPA- VEGF189. Coating concentrations were 4 µg/ml for all proteins. Cells were allowed to adhere for 20 min at 37°C. B) Time-course adhesion of HUAEC to VEGF165, VEGF189, and fibronectin. Fig. 2 Figure 2. Effect of heparin and exon 6-derived peptides on endothelial cell adhesion to VEGF isoforms. A) HUAEC were pretreated with various concentrations of heparin before adhesion in wells coated with either VEGF165, VEGF189, or fibronectin. B) Time-course HUAEC adhesion to VEGF165, VEGF189, or fibronectin in the presence of 20 µg/ml P1 and P2 peptides. i) Adhesion on VEGF165, ii) adhesion on VEGF189, and iii) adhesion on fibronectin. Fig. 3 Figure 3. Effect of integrin blocking agents on HUAEC adhesion to VEGF. HUAEC adhesion was observed on VEGF165 (A), VEGF189 (B), and fibronectin (C). Ninty-six-well ELISA plates were coated with concentrations of 4 µg/ml for all proteins. Cells were trypsinized, washed, and resuspended in DMEM with 10% FCS and incubated for 1 h at 37°C. Cells were then pelleted and resuspended in DMEM without serum in the presence of EDTA (10 mM), RGD peptides (500 µM), RAD peptides (500 µM), p678 (20 µM), p690 (20 µM), or soluble VEGF (200 nM) for an additional 20 min. Cells were then distributed and allowed to adhere for 20 min. All conditions were performed in triplicate. Shown values are the mean ±SD for each condition. (*P<0.05, **P<0.005, and ***P<0.0005 compared with control nontreated cells). Fig. 4 Figure 4. Time-course adhesion of HUAEC on VEGF isoforms VEGF165 and VEGF189. A) Time-dependent adhesion on VEGF165. B) Time-dependent adhesion on VEGF189. Before distribution in wells coated with either substrate, cells were treated by RGD peptides (500 µM) or p678 (20 µM) for 30 mon. Cells were allowed to adhere for up to 60 min. All conditions were performed in triplicate. Fig. 5 Figure 5. Time-course HUAEC adhesion on and VEGF165 and VEGF189 in the presence of anti-integrin antibodies. Before distribution in wells coated with either substrate, cells were treated by anti-β1 6S6 (2 µg/ml), anti-αvβ3 (1/3), or anti-αv (1/10) antibodies for 20 min. Cells were then allowed to adhere for various lengths of time. All conditions were performed in triplicate. Shown values are the mean ±SD for each condition. Fig. 6 Figure 6. Solid-phase αvβ3 binding assay. VEGF 165, VEGF189, fibronectin, or BSA was immobilized on plastic. αvβ3 recombinant protein was incubated with the immobilized protein, and interaction between the two proteins was revealed by the ELISA technique with an anti-β3 antibody followed by a HRP-conjugated secondary antibody. Three concentrations of αvβ3 recombinant protein were used to observe a dose response. All conditions were performed in triplicate. Shown values are the mean ±SD for each condition. Fig. 7 Figure 7. HUAEC and BREC actin morphology on VEGF165 and VEGF189. Glass Labtek slides precoated with VEGF165, VEGF189, fibronectin, or poly-lysine at 4 µg/ml. A) F-actin labeling. B) FAK staining. Arrows point to FAK localizing to focal adhesions. Fig. 8 Figure 8. BREC and HUAEC migrate on VEGF. Cells were seeded at ∼25% confluency on plastic bacteriological dishes coated with 4 µg/ml of VEGF165, VEGF189, fibronectin, or poly-lysine as previously described. Cells were allowed to adhere for 2 h at 37°C. Nonadherent cells were removed, and cell movement was monitored by successive photography of cells at a given spot of the dish. Image overlay was used to evaluate the migration patterns of individual cells. A) Mean values ±SD of the trajectories of individual BREC (n=10) over a 5-h migration period. B) Mean values ±SD of the trajectories of individual HUAEC (n=10) over a 5-h migration period. Distances in arbitrary units (AU). Results are representative of three separate experiments. ***P<0.0005 compared with migration distance on VEGF189. Fig. 9 Figure 9. HUAEC adhesion on and VEGF165 and VEGF189 in the presence of tumstatin and anti-αvβ3 integrin antibodies. Ninety-six-well ELISA plates were coated with either VEGF165 or VEGF189 at 4 µg/ml. Cells were trypsinized, washed, and resuspended in DMEM with 10% FCS and incubated for 1 h at 37°C. Cells were then pelleted and resuspended in DMEM without serum in the presence of either anti-αvβ3 (1/3), tumstatin, or both for an additional 20 min. Cells were then distributed at 40,000 cells per well and allowed to adhere for 20 min. Nonadherent cells were washed away, and the amount of cell adhesion was quantified by crystal violet as described in Materials and Methods. All conditions were performed in triplicate. Shown values are the mean ±SD for each condition. (*P<0.05, **P<0.005, and ***P<0.0005 compared with control nontreated cells). Fig. 10 Figure 10. Schematic representation of the opposite effects of tumstatin and matrix-bound VEGF on endothelial cell survival. Endothelial cells adhere to matrix-bound VEGF by integrins such as αvβ3. A) Cells attached to immobilized VEGF survive, whereas floating cells do not. B) If the αvβ3 integrin is engaged with matrix-bound VEGF, tumstatin can induce a certain amount of endothelial cell apoptosis. C) If the interaction between matrix-bound VEGF and αvβ3 is blocked by an anti-αvβ3 antibody, tumstatin-induced apoptosis is significantly increased.
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