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Blood, Vol. 112, Issue 9, 3638-3649, November 1, 2008
Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization
Blood Kawamura et al.
112: 3638
Supplemental materials for: Kawamura et al
Files in this Data Supplement:
- Figure S1. Characterization of PAE cell HS expression and heparin-binding by VEGF ligands (JPG, 93.6 KB)
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(A) Heparan/chondroitin sulfate analysis. PAE cells were metabolically labeled for 20 h with 0.1 mCi/ml 35S-sulfate in 15 ml of culture medium. After incubation, 35S-labeled glycosaminoglycans were isolated from the medium as previously described.1 After treatment with 0.5M NaOH for 18 h at 4°C followed by neutralization, the labeled glycosaminoglycans were analyzed by gel chromatography on Sephadex G50 superfine (0.5 × 100 cm) before and after deamination with HNO2 at pH 1.5 or CSas ABC, to digest HS and CS, respectively. The column was eluted in 0.2 M NH4HCO3 and fractions of 0.5 ml were collected and analyzed for 35S-radioactivity. The analysis shows similar relative expression of HS and CS in the different PAE cell lines. (B) Heparan sulfate compositional analysis. The disaccharide composition of HS isolated from the different PAE cell lines was determined after heparinase digestion by RPIP-HPLC as previously described.2 Nac, ΔHexA-GlcNAc; NS, ΔHexA-GlcNS; 2S, ΔHexA2S-GlcNAc; NS2S, ΔHexA2S-GlcNS, 6S, ΔHexA-GlcNAc6S; NS6S, ΔHexA-GlcNS6S; NS2S6S, ΔHexA2S-GlcNS6S. (C) Heparin-binding assay. Triplicate samples of ligands (26.2 nmole) were incubated with 7.1 × 103 cpm 3Hheparin in 200 µl PBS, and passed through a nitrocellulose filter using a multiwell vacuum-assisted filtration apparatus. After washing away unbound 3Hheparin, filters were incubated in 2 M NaCl to dissociate the heparin. The radioactivity of protein-bound 3Hheparin retained on the filter was determined using a Wallac 1414 liquid scintillation counter (PerkinElmer). The analysis shows retention of heparin after binding to VEGF-A165 but not VEGF-A121 or VEGF-E-NZ2.
- Figure S2. Competitive binding assay (JPG, 60.9 KB)
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Competition assays were performed using 125I-VEGF-A165 in the presence and absence of unlabeled VEGF-A165, VEGF-A121 and VEGF-E-NZ2, which competed for binding to PAE/VEGFR-2 cells (A), PAE/VEGFR-2, NRP1 cells (B) or PAE/NRP1 cells (C). The ability of VEGF-A121 to compete with 125I-VEGF-A165 was determined by expression of VEGFR-2 alone. The ability of VEGF-E-NZ2 to compete for binding of VEGF-A165 was determined by expression of NRP1. In cells expressing VEGFR-2 alone (PAE/VEGFR-2), VEGF-E-NZ2 competition was inefficient, indicating lower affinity for binding to the receptor than for the VEGF-A ligands. The reduction in 125I-VEGF-A165–binding by a 300-fold molar excess of unlabeled VEGF-E-NZ2 was 30% on PAE/NRP1 cells and 63% on PAE/VEGFR-2, NRP1 cells. These data indicate the formation of a high affinity ligand-binding VEGFR-2/NRP1 complex by VEGF-A165 and VEGF-E-NZ2 in coexpressing cells.
- Figure S3. Flow cytometry of embryoid bodies treated with VEGF-A165, VEGF-A121 and VEGF-E-NZ2 (JPG, 62.7 KB)
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Embryoid bodies in two-dimensional cultures at day 14 of differentiation treated with 30 ng/ul VEGF-A165, VEGF-A121 or VEGF-E-NZ2 from day 4, were dissociated at 37°C by incubation in 2.5 mg/ml collagenase (Sigma Aldrich, St. Louis, MO) for 30 min. This was followed by incubation and careful pipetting in dissociation solution (Sigma Aldrich) to obtain a single cell suspension. Complete dispersion was obtained by passing cells through a 40 µm strainer. The cleared cell suspension was incubated with mouse vascular endothelial (VE)-cadherin antibody (R&D, Minneapolis, MN) and secondary Alexa donkey anti-goat cy5 (Molecular Probes, Eugene, OR). DAPI-negative viable cells were analyzed on a FACSVantageSE machine using FACSDiVa software (BD Biosciences). Green shows gated VE-cadherin positive cells and figures indicate the percentage VE-cadherin positive cells in relation to the total pool.
- Figure S4. Analysis of the contribution of NRP1 and VEGFR-2 Y1214F to p38MAPK activation (JPG, 55.5 KB)
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(A) PAE cells expressing VEGFR-2 alone were treated with 1 nM of VEGF-A165 or VEGF-A121 for different time periods, followed by analysis of p38MAPK using an exogenous substrate (ATF-2). Immunoblotting (IB) for p38MAPK shows equal amount of protein in the samples. Activation of receptors was determined by immunoblotting for phosphoVEGFR-2. Immunoblotting for VEGFR-2 showed equal amounts of protein in the samples. (B) PAE/VEGFR-2 Y1214F cells, expressing a mutant VEGFR-2 in which tyrosine residue Y1212 was replaced with a phenylalanine residue, were treated with VEGF-A165 and analyzed for p38MAPK activation as described in A. Control analyses were performed in the form of immunoblotting for p38MAPK, phosphoY1173VEGFR-2 (i.e. phosphorylation of VEGFR-2 at Y1173/Y1175), phosphoY1214VEGFR-2 (i.e. phosphorylation of VEGFR-2 at Y1212/Y1214), VEGFR-2 and NRP1.
- Figure S5. VEGFR-2 activation in embryoid bodies treated with the p38MAPK inhibitor SB203580 (JPG, 38.1 KB)
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(A) Embryoid bodies at day 10 of differentiation were treated with SB203580 or the intert analogue SB202474 at different concentrations, followed by immunoprecipitation (IP) of VEGFR-2 and immunoblotting (IB) with phosphotyrosine antibody (4G10). This analysis shows similar extent of VEGF-A165 induced VEGFR-2 tyrosine phosphorylation in the presence and absence of the p38MAPK inhibitor. Immunoblotting for VEGFR-2 shows equal amount of protein in the samples. (B) Quantification of the immunoblots in A.
- Figure S6. Effect of Sema3A on vascular development (JPG, 34.5 KB)
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2D embryoid bodies cultures were treated with VEGF-A165 (0.79 nM) from day 6–10 with and without 500-fold molar excess of purified Sema3A,3 from day 4 to day 10. Cultures were processed for immunohistochemical staining to detect CD31 expression in endothelial cells. Arrows indicate the peripheral capillary plexus. Scale bar; 100 µm.
REFERENCES
1. Holmborn K, Ledin J, Smeds E, Eriksson I, Kusche-Gullberg M, Kjellen L. Heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is 6-O-sulfated but contains no N-sulfate groups. J Biol Chem. 2004;279:42355–42358.
2. Ledin J, Staatz W, Li JP, et al. Heparan sulfate structure in mice with genetically modified heparan sulfate production. J Biol Chem. 2004;279:42732–42741.
3. Miao HQ, Soker S, Feiner L, Alonso JL, Raper JA, Klagsbrun M. Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165. J Cell Biol. 1999;146:233–242.
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