Fig 1: VEGF-C increased SiHa cell migration and invasion and induced actin cytoskeleton remodeling. (A) Cells were treated with VEGF-C (100 ng/mL) for 24 or 48 h and cell migration was assayed. SiHa cells were scraped out of the cell culture dish and the extent of migration of the remaining cells was assayed. The upper black lines indicate the starting line and the lower black lines indicate the mean migration distance. (B) Migrating cell numbers were measured and data representing the migration cell numbers from the starting line are expressed as mean ± SD. * = P < 0.05 vs. 24 h control; ** = P < 0.01 vs. 48 h control. (C) Cells were treated with VEGF-C (100 ng/mL) for 24 or 48 h. Cell invasion was assayed using invasion chambers. Invading cells were counted in three different central fields of triplicate membranes and invasion indexes are shown. ** = P < 0.01 vs. control. (D) SiHa cells were treated with VEGF-C (100 ng/mL) for 6 h, 12 h, 24 h or 48 h alone, or treated with VEGF-C for 48 h in the presence of Flt-4/IgG (100 ng/mL). Immunofluorescent analysis of Texas Red-phalloidin (in red) revealed the spatial modifications of actin fibres and the formation of specialized cell membrane structures. Green, white and yellow arrows indicate lamellipodia, pseudopodia and focal adhesion complexes, respectively. Nuclei were counterstained in blue. All the experiments were repeated three times with consistent results, and a representative result is shown.
Fig 2: VEGF-C activated RhoA/ROCK-2 pathway. (A-B) RhoA expression and its activity were assayed in cells treated with VEGF-C (100 ng/mL) for 24 or 48 h alone, or treated with VEGF-C for 48 h in the presence of Flt-4/IgG (100 ng/mL). Active, GTP-bound RhoA was immunoprecipitated with Rhoteckin and subsequently assayed with western analysis with an anti-RhoA Ab. ** = P < 0.01 vs. corresponding control; *** = P < 0.001 vs. corresponding control; ## = P < 0.01 vs VEGF-C 48 h. (C-D) ROCK-2 expression and its activity were assayed in cells treated as indicated. ROCK-2 was immunoprecipitated with a specific Ab and the IPs were used to phosphorylate the bait protein, myelin basic protein (MBP). ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). ** = P < 0.01 vs. corresponding control; ## = P < 0.01 vs VEGF-C 48 h. All the above experiments were performed in triplicates and representative images are shown.
Fig 3: Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A, the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 (left) or VEGFR-3 (right) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC (39). B, peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph, amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph, examples of peptides used in mapping (mAb 286 binding site is in a rectangle). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C, detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 (top) or M2 anti-FLAG mAb as a positive control (bottom). Each well contained 30 ng of purified protein. VEGF-D, VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left. The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D, analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A, B, and D, assays were conducted three times. Columns, mean; error bars, S.D.
Fig 4: VEGF-C led to activation of the actin-regulatory protein, moesin. (A-B) SiHa cells were treated with VEGF-C (100 ng/mL) for 24 or 48 h alone, or treated with VEGF-C for 48 h in the presence of Flt-4/IgG (100 ng/mL). Total cell amount of wild-type (Moesin) or Thr558-phosphorylated moesin (P-Moesin) or β-actin (Actin) are shown with western blot analysis. Densitometry values were adjusted to β-actin intensity and then normalized to expression from the control sample. ** = P < 0.01 vs. corresponding control. ## = P < 0.01 vs VEGF-C 48 h. (C) SiHa cells were transfected with scrambled siRNA (-S) or moesin targeted siRNA (-M) for 48 h. After that the level of moesin expression was detected by western blot as indicated. β-actin was used as the loading control. (D) SiHa cells were treated with VEGF-C (100 ng/mL) for 48 h, in the presence or absence of scrambled siRNA or Moesin siRNA. Cells were stained and analyzed by immunofluorescence. Green, white and yellow arrows indicate lamellipodia, pseudopodia and focal adhesion complexes, respectively. All the experiments were repeated three times with consistent results, and a representative result is shown.
Fig 5: Analyses of the role of N-terminal α-helices of mature VEGF-D and VEGF-C for proliferation and migration by LECs. A, LEC proliferation assays. Adult LECs were treated with VEGF-DΔNΔC (VEGF-D), VEGF-CΔNΔC (VEGF-C), or their variants or left untreated (No GF). VEGF-D+286, combination of VEGF-DΔNΔC and a 10-fold molar excess of mAb 286. y axes represent proliferation by LECs stimulated with growth factor relative to that of unstimulated cells. x axes denote VEGF-D variants (left) and VEGF-C variants (right) used in assays. B, LEC migration assay. The capacity of variant proteins to induce cell migration was assessed in a scratch wound assay. Neonatal LECs were wounded, and the amount of wound closure was calculated for each variant as described under “Experimental Procedures.” y axes show migration of cells stimulated with growth factor relative to that of unstimulated cells. x axes denote VEGF-D variants (left) and VEGF-C variants (right) used in assays. C, images of selected scratch wounds. Wounds were imaged immediately post-wounding (T0, two examples) and after 24-h treatment with VEGF-DΔNΔC, VEGF-CΔNΔC, or the 3Ala variant of each (D3Ala and C3Ala, respectively). No GF, two results after 24 h with no growth factor. White lines, edges of the wounds. In A and B, the capacity of variants to activate VEGFR-2 (R2) or VEGFR-3 (R3) is indicated above the graphs, and asterisks indicate that results differ from No GF in a statistically significant fashion, as assessed by one-way analysis of variance with Tukey's post hoc test. The amounts of VEGF-D or VEGF-C variants were matched in each assay.
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