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Extracellular matrix-bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation ¹

GDR CNRS 1927-Angiogenese and UMR CNRS 5089, Institut de Pharmacologie et Biologie Structurale, Toulouse, France

²Correspondence: Institut de Pharmacologie et Biologie Structurale, UMR CNRS 5089, 205 route de Narbonne, 31077 Toulouse cedex, France. E-mail: jean.plouet{at}ipbs.fr

SPECIFIC AIMS

Although alternative splicing of vascular endothelial growth factor (VEGF) pre-mRNA regulates the binding of the isoforms to the extracellular matrix (ECM) and cell membranes, little is known about their specific biological responses as diffusible growth factors and nothing about their potential role as matrix-bound factors. In this report we examined the role of various immobilized VEGF isoforms on cell adhesion and survival of four endothelial strains.

PRINCIPAL FINDINGS

1. Endothelial cells adhere to VEGF independent of VEGF receptors
Human umbilical artery endothelial cells (HUAEC) were plated in plastic wells that had been coated with BSA, fibronectin, VEGF₁₂₁, VEGF₁₆₅, or the full-length or cleaved forms of VEGF₁₈₉. At various intervals, the wells were rinsed and cell adhesion was assessed by crystal violet staining. HUAEC adhered well to the VEGF₁₈₉ isoforms (cleaved or full-length), moderately well to VEGF₁₆₅, but not at all to VEGF₁₂₁. The amount of cells adhered to VEGF₁₈₉ was similar to that on fibronectin. Pretreatment of HUAEC with soluble VEGF₁₆₅ or VEGF₁₈₉, which induces internalization of VEGF receptors, had no significant effect on cell adhesion. We concluded that VEGF receptors are not involved in the adhesion of HUAEC to immobilized VEGF and searched for cellular binding sites for matrix-bound VEGF.

2. Integrins 3ß1 and vß3 mediate HUAEC adhesion to immobilized VEGF
To test whether integrins may serve as receptors for immobilized VEGF, adhesion experiments were performed in the presence of the universal inhibitor of integrin EDTA, specific blocking peptides, and antibodies. Whereas EDTA inhibits all adhesion on both VEGF isoforms, the 3ß1 blocking peptide p678 completely blocks adhesion on VEGF₁₆₅ but only 60% of that on VEGF₁₈₉. RGD peptides had little effect alone, but all adhesion was inhibited on both VEGF isoforms by the addition of a combination of p678 and RGD peptides. These results demonstrate that HUAEC adhere to VEGF₁₆₅ and VEGF₁₈₉ uniquely via integrins: the 3ß1 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. The use of blocking antibodies confirmed that ß1 integrins and the vß3 integrin are responsible for adhesion to VEGF₁₆₅ (Fig. 1 ); however, the same antibodies inhibit only 15 to 25% of adhesion to VEGF₁₈₉. The anti-v subunit antibody completely blocked adhesion on VEGF₁₆₅ and blocked 50% adhesion on VEGF₁₈₉. 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 an v subunit.

View larger version (25K): [in this window] [in a new window]   Figure 1. Time course HUAEC adhesion on and VEGF165 and VEGF189 in the presence of anti-integrin antibodies. 96-well ELISA plates were coated with 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 pelleted and resuspended in DME medium without serum in the presence of anti-ß1 6S6 (2 µg/mL), anti-vß3 (1/3), and anti-v (1/10) antibodies for a further 20 min. Cells were then distributed at 40,000 cells per well and allowed to adhere for various lengths of time. Nonadherent cells were washed away and the amount of cell adhesion was quantified by crystal violet. All conditions were performed in triplicate. Values shown are the mean ±SD for each condition.

Solid-phase binding assays with recombinant vß3 protein demonstrated that vß3 could bind directly to immobilized VEGF₁₆₅ and VEGF₁₈₉.

3. Endothelial cells spread and migrate 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. As seen in Fig. 2 , HUAEC displayed a classical well-organized actin cytoskeleton with many actin stress fibers on fibronectin. On VEGF₁₆₅ and VEGF₁₈₉, cells spread as well but display a stellate morphology with numerous lamellipodia and filopodia. 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. Immunostaining showed that FAK was localized at focal adhesions in both cells adhering to fibronectin and VEGF isoforms. Therefore, it seems probable that integrins interacting with immobilized VEGF induce active signal transduction.

View larger version (124K): [in this window] [in a new window]   Figure 2. HUAEC and BREC actin morphology on VEGF165 and VEGF189. Glass Labtek slides precoated with VEGF165, VEGF189, fibronectin or poly-lysine at 4 µg/mL. Cells were harvested by trypsinization, washed, and resuspended in DMEM with 10% serum and incubated for 1 h at 37°C with 10% CO2. Cells were then pelleted, resuspended in DMEM + 2% BSA, and seeded at 25% confluency in the Labtek wells. After 2 h at 37°C, adherent cells were fixed with 3.7% paraformaldehyde + 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. For F-actin labeling, cells were incubated for 20 min at room temperature with rhodamine-conjugated phalloidin. For anti-FAK labeling, cells were incubated with an anti-FAK antibody (1/100) for 1 h at 37°C, then with an anti-rabbit FITC-conjugated secondary antibody (1/100) for 45 min at 37°C.

Migration assays showed that VEGF₁₈₉, and to a lower extent VEGF₁₆₅, could support strong HUAEC migration in the absence of any other ECM components or serum.

4. vß3 is required for HUAEC survival on immobilized VEGF
Since tumstatin is known to interact with vß3, we tested its activity on cells plated on VEGF₁₆₅ or VEGF₁₈₉. Tumstatin inhibited HUAEC adhesion to both VEGF isoforms in a dose-dependent manner. This effect was enhanced when cells were treated simultaneously with an anti-vß3 blocking antibody. HUAEC apoptosis, analyzed by FACS after annexin-V isothiocyanate and propidium iodide staining was almost totally abrogated by adhesion to immobilized VEGF₁₆₅ and VEGF₁₈₉ or collagen I. Tumstatin and an anti-vß3 antibody induced a synergistic effect on apoptosis of HUAEC adhering to either VEGF isoform but had no combined action on HUAEC seeded on collagen I. This suggests that ligation of vß3 to immobilized VEGF generates a survival signal that opposes the proapoptotic action of tumstatin. However, ligation to vß3 could not account for all of the survival effect of VEGF since treatment with tumstatin and the anti-vß3 antibody did not induce > 60% of apoptosis, thus demonstrating that other integrins involved in adhesion could also be involved in survival signals.

CONCLUSIONS AND SIGNIFICANCE

Our findings constitute the first report demonstrating that at least some functions of VEGF can be exerted through a VEGF receptor-independent pathway. We show here that all cell adhesion on VEGF₁₆₅ can be attributed to integrins: 3ß1, vß3, and another v subunit containing integrin (but not vß5). On VEGF₁₈₉, the same integrins are involved (3ß1, vß3, and another v integrin); however, 50% of adhesion is mediated by a yet unidentified receptor. The clear preference that endothelial cells show for adhesion and migration on VEGF₁₈₉ compared with VEGF₁₆₅ supports the hypothesis that yet unidentified receptors for VEGF₁₈₉ might be responsible for the differences observed. In contrast to cells plated on fibronectin, which display prominent stress fibers, cells on VEGF possess numerous lamellipodia and filopodia but no actin stress fibers. The morphology of cells attached to VEGF is quite different from that observed after VEGFR2 activation, which does not cause stress fibers to disappear.

Here we show that immobilized VEGF supports cell migration and survival. After degradation of the basement membrane, endothelial cells migrate onto a provisional matrix between the blood vessel and the hypoxic cells; here their fate must be decided: survival or cell death. VEGF might be accumulated in the provisional matrix during initial steps of angiogenesis and contribute to the survival of migrating endothelial cells.

Matrix-bound VEGF promotes endothelial survival through a vß3-dependent pathway. Accordingly, tumstatin, the potent endothelial-specific inducer of apoptosis, which does not compete with soluble VEGF binding to VEGF receptors, inhibited adhesion and survival of endothelial cells. The role of vß3 in angiogenesis is still controversial. Pioneer work focused on the proangiogenic role of vß3 and showed that disruption of vß3 anchorage to the ECM promoted apoptosis, whereas other work has shown that disruption of the v or ß3 genes leads to enhanced angiogenesis However, these demonstrations address functional relationships between vß3 and soluble VEGF which in turn activates VEGF receptors. The recent demonstration that ligation of vß3 with tumstatin induces apoptosis specifically in endothelial cells allowed us to postulate that it might bind simultaneously to other substrates that have not been identified so far. We propose that this missing link for vß3-dependent survival is immobilized VEGF. Therefore, the balance between the amount 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).

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized role of matrix-bound VEGF. During tumor angiogenesis, tumor cells secrete VEGF isoforms: VEGF165 diffuses toward the target blood vessels whereas VEGF189 is sequestered in the tumor ECM. Endothelial cells adhere on matrix-bound VEGF isoforms via v, ß1, and vß3 integrins (and another unidentified receptor for VEGF189). Matrix-bound VEGF and tumstatin bind the vß3 integrin and exert opposite effects on endothelial cell survival.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0691fje; doi: 10.1096/fj.02-0691fje

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