1. A proangiogenic peptide derived from vascular endothelial growth factor receptor-1 acts through α5β1 integrin
by Simonetta Soro, Angela Orecchia, Lucia Morbidelli, Pedro Miguel Lacal, Veronica Morea, Kurt Ballmer-Hofer, Federica Ruffini, Marina Ziche, Stefania D'Atri, Giovanna Zambruno, Anna Tramontano, and Cristina Maria Failla
Blood
Volume 111(7):
April 1, 2008
©2008 by American Society of Hematology

2. VEGFR-1 dII mediates cell adhesion and interaction with α5β1 integrin.
VEGFR-1 dII mediates cell adhesion and interaction with α5β1 integrin. (A) In a solid-phase binding assay, plates coated with VEGFR-1/Fc were treated with anti–VEGFR-1 antibodies and incubated with purified α5β1 integrin. The amount of bound integrin was quantified by incubation with an anti-α5β1 antibody and colorimetric detection of this antibody. (B) HUVECs were incubated on wells coated with recombinant dII. VEGFR-1/Fc and fibronectin (FN) were used as positive controls, bovine serum albumin (BSA) as a negative control. The relative number of attached cells was assessed by staining with crystal violet and determining A540 1 hour after plating. Absorbance resulting from nonspecific cell adhesion was measured on BSA-coated wells. (C) Purified VEGFR-1/Fc or dII were added at the indicated concentrations to wells coated with α5β1 integrin. Bound molecules were detected using either an anti-Fc alkaline phosphatase (AP)-conjugate or an anti–VEGFR-1 antibody and a secondary anti-rabbit antibody AP-conjugate. Representative experiments performed in triplicate are shown; data are mean plus or minus SEM.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

3. Peptide 12 inhibits endothelial cell adhesion to VEGFR-1 and directly supports cell adhesion.
Peptide 12 inhibits endothelial cell adhesion to VEGFR-1 and directly supports cell adhesion. (A) Ribbon representation of the alpha carbon trace of VEGFR-1 dII structure determined by X-ray crystallography (PDB ID: 1flt). Peptides 1, 3, 5, 7, 9, and 11 (left panel) and 2, 4, 6, 8, 10, and 12 (right panel) are colored red, orange, yellow, blue, purple and magenta, respectively. Residues belonging to both peptide 5 and 7 (left panel) or 6 and 8 (right panel) are colored green. (B) Microtiter plates were coated with VEGFR-1/Fc or fibronectin (FN). HUVECs were preincubated with each peptide and then plated on the coated wells. Data are reported as percentage of adhesion, calculated relative to the number of cells attached on the same substrate in the absence of the peptides. (C) Different VEGFR-1 peptides and RGD peptide, as a positive control, were covalently linked to microtiter plates. HUVECs were then added to the coated plates and allowed to adhere. Nonspecific cell adhesion was measured by the absorbance in BSA-coated wells. Representative experiments performed in triplicate are shown; data are mean plus or minus SEM.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

4. α5β1 integrin mediates the biologic activity of peptide 12.
α5β1 integrin mediates the biologic activity of peptide 12. (A) Peptide 12 or BSA, as a nonspecific adhesion control, was coated onto microtiter plates, and HUVECs were added in the presence of chelating agents (EDTA or EGTA). Alternatively, HUVECs were preincubated with an antibody against α5β1 integrin, against α5 or β1 integrin subunits, or against α6 integrin subunit and allowed to adhere on coated wells. Data are mean plus or minus SEM; a representative experiment performed in triplicate is shown. (B) Fibronectin-adherent HUVECs were incubated with magnetic beads coated with RGE peptide (i), RGD peptide (ii,iv), or peptide 12 (iii,v). HUVECs were then immunostained for α5β1 integrin. Integrin clusters around the beads are indicated (arrowheads). Bars represent 10 μm.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

5. Alanine substitution in peptide 12.
Alanine substitution in peptide 12. (A) Peptides whose sequence differs from that of peptide 12 for the substitution of one residue with Ala were covalently linked to microtiter plates. A “scrambled” version of peptide 12 (sP12) and BSA, as a negative control, were also tested. HUVECs were added to the plates and allowed to adhere. Results are expressed as percentage of cell adhesion observed on peptide 12-coated plates. (B) Purified α5β1 integrin was added to wells coated with the same peptides as in panel A, and bound integrin was detected by using an anti-α5β1 antibody and a colorimetric assay. Data are expressed as percentage of protein binding on peptide 12-coated plates. (C) Biotinylated VEGF-A was added to wells coated with peptide 12, 1, 7, VEGFR-1/Fc, as a positive control, or BSA, as a negative control. The amount of attached VEGF-A was quantified by incubation with streptavidin alkaline phosphatase-conjugated and a colorimetric assay. Representative experiments performed in triplicate are shown; data are mean plus or minus SEM.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

6. Peptide 12 sustains vessel formation in vitro.
Peptide 12 sustains vessel formation in vitro. HUVECs were incubated on 24-well plates precoated with a type I collagen gel mixture, in the absence (A) or presence of peptide 12 (B), peptide Q225A (D), or peptide Y220A (E). VEGF-A was used as a reference control (C). To assess the specificity of peptide 12 activity, an antibody against peptide 12 was also tested in combination with peptide 12 (F). Results are representative of 3 independent experiments. Bars represent 25 μm in C, 50 μm in the others.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

7. Peptide 12 sustains neoangiogenesis in vivo.
Peptide 12 sustains neoangiogenesis in vivo. (A) The effect of 100 μg/pellet of peptide 12 or Y220A peptide was compared with that of 400 ng/pellet VEGF-A. *Pellet implants. (B) Quantification of the data (means ± SEM) shown in panel A reported as angiogenic score during time (days). Numbers are the means of 6 implants for each experimental point. One-way analysis of variance (P < .001). *P < .05 peptide 12 versus Y220A peptide (Student-Newman-Keuls multiple comparison test).
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology

8. Effect of peptide 12 on α5β1 integrin-mediated downstream signaling.
Effect of peptide 12 on α5β1 integrin-mediated downstream signaling. HUVECs were starved and treated with cycloheximide as described in “Western blotting analysis” and then plated on peptide 12-, or fibronectin- (FN)-coated dishes in the presence or absence of VEGF-A. (A) Immunoblotting analysis for the presence of phosphorylated-FAK or phosphorylated-ERK1/2 polypeptides after 1-hour adhesion. Actin staining was performed to normalize the results (ACT). (B) Histograms showing the relative OD of the bands in panel A, normalized using absorbance of the actin bands for each sample, after subtraction of the background (phosphorylation levels in cells plated on BSA-coated dishes). (C) Immunoblotting analysis of phosphorylated-Shc and total Shc polypeptide after 30 minutes of adhesion. The histogram shows the OD ratio between the phosphorylated band and the total protein. Data represent the results of a representative experiment.
Simonetta Soro et al. Blood 2008;111:
©2008 by American Society of Hematology