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First published online 15 July 2003
doi: 10.1242/jcs.00673

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Vascular endothelial growth factor receptor-1 is deposited in the extracellular matrix by endothelial cells and is a ligand for the 5ß1 integrin

¹ Molecular and Cell Biology Laboratory, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy
² Pharmacology Laboratory, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy
³ Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK, and CNR Center of Molecular Biology, c/o Department of Biochemical Sciences, University of Rome 'La Sapienza', P.le A. Moro 5, 00185 Rome, Italy

View larger version (47K): [in a new window]   Fig. 1. VEGFR-1 is a component of the extracellular matrix deposited by EC. (A) HUVEC or normal human fibroblasts (Fb) were seeded into 96-multiwell culture plates and incubated for 72 hours. Cells were mechanically detached and the amount of VEGFR-1 or fibronectin (FN) deposited in each well was evaluated by ELISA using anti-VEGFR-1 or anti-fibronectin antibodies. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. The experiment was repeated at least three times with comparable results. (B) Extracellular matrix deposited by HUVEC was immunostained after cell detachment with an anti-sVEGFR-1 antibody or with an anti-fibronectin antibody. Both sVEGFR-1 and fibronectin were detectable in the matrix deposited by the EC. Scale bar: 5 µm. (C,D) Immunoblotting analysis of VEGFR-1 polypeptides in HUVEC extracts and the ECM produced by these cells, using antibodies recognizing (B) the extracellular (AF327) or (C) the intracellular (C-17) region of VEGFR-1. Equal volumes of cell extract, ECM material or total samples (in which cells and ECM were collected together), prepared as described in Materials and Methods, were analysed. Upper panels show the immunoblot analysis of the samples using the anti-human fibronectin antibody FN-15. The molecular weight markers in kDa are indicated in C.

View larger version (17K): [in a new window]   Fig. 2. VEGFR-1 supports EC attachment. (A) Dose-dependent cell attachment on wells coated with the indicated concentrations of VEGFR-1/Fc or fibronectin (FN). The relative number of attached cells was assessed by staining with crystal violet and determining the A540 1 hour after plating. Absorbance resulting from non-specific cell adhesion was measured on BSA-coated wells. (B,C) VEGFR-1-specific promotion of EC adhesion. (B) EC were plated on 10 µg/ml VEGFR-1/Fc-, VEGFR-2/Fc-, PDGFRß/Fc-, IgG-, or BSA-coated wells and cell adhesion was measured as described for A. (C) EC were plated on 10 µg/ml FN-, VEGFR-1/Fc-, or BSA-coated wells. Before cell seeding, wells were incubated for 30 minutes with undiluted preimmune serum or anti-VEGFR-1 rabbit serum. Alternatively, cells were treated with 20 µg/ml of VEGFR-1/Fc for 15 minutes before plating. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. The experiments were repeated at least five times.

View larger version (23K): [in a new window]   Fig. 3. 5ß1 integrin mediates EC attachment to VEGFR-1. (A) Effect of divalent cations and trypsin treatment on EC adhesion to VEGFR-1. Cells were plated on fibronectin (FN), BSA or VEGFR-1/Fc in the presence of EGTA, EDTA, Ca2+, Mg2+, Mn2+ or trypsin, and after checking cell viability by trypan-blue dye exclusion in control wells, the relative number of attached cells was estimated as described in Fig. 2. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. (B) Cells were incubated with mAbs against ß1, 5, 6 (unrelated Ab), 5ß1 and vß3 15 minutes before plating on VEGFR-1/Fc, fibronectin (FN) or vitronectin (VN). Histograms represent the percentage of inhibition, calculated relative to the adhesion of cells on the same substrates in the absence of blocking antibodies, ± s.e.m. The experiments in A and B were repeated at least three times.

View larger version (21K): [in a new window]   Fig. 4. VEGFR-1 is involved in EC migration. (A) VEGFR-1/Fc was included in the lower chamber (chemotaxis), in the upper chamber (chemokinesis) or in both, at the indicated concentrations (µg/ml). EGF (100 ng/ml) was used as a positive control, and 0 indicates the presence of only the basal migration medium in the chamber. (B) Specific inhibition of VEGFR-1-induced migration by anti-5ß1 antibodies. Cells were preincubated for 45 minutes at room temperature with the anti-5ß1 mAb, or with an unrelated anti-6 integrin mAb (unrelated Ab), before seeding. Histograms represent the percentage of inhibition of the chemotactic response induced by either VEGFR-1/Fc (5 µg/ml) or EGF (100 ng/ml). (C) Haptotaxis assay was performed on filters in which the underside was coated with 10 µg/ml BSA, VEGFR-1/Fc (VEGFR-1) or vitronectin (VN). (D) Specific inhibition of VEGFR-1 induced haptotaxis by anti-5ß1 antibodies. Cells were preincubated for 45 minutes at room temperature with the anti-5ß1 mAb, or with an unrelated anti-6 integrin mAb (unrelated Ab), before seeding. Histograms represent the percentage of inhibition of the haptotactic response induced by either VEGFR-1/Fc or vitronectin (VN). In every experiments, migration was monitored in a Boyden chamber assay by counting 12 high-power fields for each condition. Histograms represent the mean value ± s.d.

View larger version (15K): [in a new window]   Fig. 5. Characterisation of the VEGFR-1/5ß1 integrin interaction. (A) Dose-dependent VEGFR-1 binding to purified 5ß1-coated wells. Different concentrations (10-1-104 ng/ml) of VEGFR-1/Fc were added to 5ß1 coated wells (1 µg/ml) and bound molecules were detected using an anti-human IgG (Fc specific)-alkaline phosphatase conjugated antibody. Results represent the mean absorbance value of medium from triplicate wells ± s.e.m. (B) VEGFR-1/Fc and VEGFR-2/Fc (20 µg/ml) were added to 5ß1-coated wells. In competition experiments, EDTA (1 mM) or a blocking anti-5ß1 antibody (10 µg/ml) was added during the assay. Absorbance resulting from non-specific cell adhesion was measured on BSA-coated wells. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. (C) Effect of RGD peptides or VEGFR-1 growth factor ligands on cell attachment. Cells were pretreated with 0.4 mM of RGD or RGE peptides, before plating on fibronectin (FN) or VEGFR-1/Fc (5 µg/ml), or the adhesion assays were performed in the presence of 20 µg/ml VEGF and placenta growth factor (PlGF). Results are expressed as the percentage of adherent cells compared to untreated controls.

View larger version (70K): [in a new window]   Fig. 6. EC spread on VEGFR-1-coated plates. Cells were plated on Petri dishes coated with 10 µg/ml fibronectin (FN) (A) or 10 µg/ml VEGFR-1/Fc (B,C), and F-actin was stained with fluorescein-labelled phalloidin 1 hour (A,B) or 6 hours after plating (C). Scale bars: 5 µm.

View larger version (231K): [in a new window]   Fig. 7. VEGFR-1 induces EC spreading. (A) Cells were plated on 10 µg/ml fibronectin (FN) or VEGFR-1/Fc in the presence of monensin (mn) or cycloheximide (chx), and the percentage of adherent cells with respect to untreated samples was measured. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. (B) Effect of RGD peptides on the spreading of EC. Cells were plated on 1 µg/ml vitronectin (VN), or 10 µg/ml VEGFR-1/Fc and left to adhere. RGD peptides (0.4 mM) were then added to the cell monolayer. Results are expressed as the percentage of spread cells compared to controls without peptide addition ± s.e.m. (C) Immunofluorescence staining for fibronectin in the matrix deposited by EC. Cells were allowed to adhere on VEGFR-1/Fc or vitronectin (VN), for 3 hours and the deposited matrix was analysed with an anti-fibronectin antibody. Arrowheads indicate organised fibronectin fibrils. Scale bar: 5 µm.