Fig 2: Effects of mutating residues in N-terminal α-helices of VEGF-DΔNΔC or VEGF-CΔNΔC. A, sequences within the N-terminal α-helices of human VEGF-DΔNΔC (VEGF-D) and VEGF-CΔNΔC (VEGF-C) (top, with identical residues underlined) with variants in which multiple residues were altered to alanine shown below. B and C, blots show analyses of receptor phosphorylation by variants of VEGF-DΔNΔC and VEGF-CΔNΔC, respectively. D, blots show analyses of receptor phosphorylation induced by VEGF-CΔNΔC and mutants of VEGF-CΔNΔC lacking residues 113–115 (designated Δ3), 113–118 (Δ6), and 113–121 (Δ9). Graphs below blots show the results of bioassays of binding and cross-linking of VEGFR-2 and VEGFR-3 extracellular domains by VEGF-C variants (data are mean percentage of fluorescence relative to VEGF-CΔNΔC ± S.D.). For blots in B–D, adult LECs were stimulated with VEGF-DΔNΔC, VEGF-CΔNΔC or their variants or left unstimulated (No GF). Lysates were immunoprecipitated with antibody against VEGFR-2 (left-hand blots) or VEGFR-3 (right-hand blots) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine to assess receptor activation (top blots) or with antibody against VEGFR-2 (bottom left blots) or VEGFR-3 (bottom right blots) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the blots. The amounts of VEGF-D or VEGF-C variants were matched in each experiment. Dotted lines indicate where irrelevant tracks have been excised from the images. In C and D, numbers under the lanes of blots represent the ratios of the intensities of phosphorylated receptor signals to intensities of total receptor signals ([PO4]/[total]) for each ligand treatment as determined by calculating the mean ratios from two independent experiments. The ratios for VEGFR-2 were derived by combining the intensities of the signals for bands in the size range of 188–230 kDa (note that the lower band of ∼125 kDa in the top left blot of C was not used because it probably represents co-immunoprecipitated VEGFR-3 arising from receptor heterodimers, as reported previously (68)), whereas those for VEGFR-3 are based on combining the intensities of the ∼125-, ∼175-, and ∼195-kDa forms of this receptor. pY, phosphotyrosine; IP, immunoprecipitation.

Fig 3: Pharmacological blockade of ccRCC-induced HUVEC sprouting. (A and B) Confocal z-stack volume projections (400 μm total thickness) of HUVEC vessels grown for 5 days in the presence of ccRCC tumor clusters with flow containing EGM media plus vehicle (0.0002% BSA) or 200 ng/ml recombinant VEGFR2-Fc chimera. Scale bar, 100 μm. (C) Quantification of sprouting. Error bars represent the average deviation. The P value is < .01 for the comparison of sprouting between vehicle versus VEGFR2-Fc across both patients.

Fig 4: Interaction of VEGFR-2 and VEGFR-3 with VEGF-DΔNΔC variants. A, representation of structure for part of the N-terminal α-helix (93FYDIETLKVIDEEWQ107) in human mature VEGF-D with the mAb 286 binding site shown in red. B, analysis of binding of VEGF-DΔNΔC variants to VEGFR-2 (left) and VEGFR-3 (right) by ELISA (see “Experimental Procedures”). y axes show binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100%), and x axes define the mutated amino acid in each variant. The same amount of each VEGF-DΔNΔC variant was used. VEGF-D, VEGF-DΔNΔC. Assays were conducted three times. Columns, mean; error bars, S.D. C, bioassays for binding and cross-linking of the extracellular domains of VEGFR-2 (left) and VEGFR-3 (right) by VEGF-DΔNΔC variants. The same amount of each VEGF-DΔNΔC variant was used in each assay. Results are expressed as a percentage of fluorescence units generated by VEGF-DΔNΔC variants relative to VEGF-DΔNΔC (y axes). x axes define the mutated amino acid in each variant. Assays were conducted five times. Columns, mean; error bars, S.D. D, receptor phosphorylation induced by selected VEGF-DΔNΔC variants. Adult LECs were stimulated with matched quantities of VEGF-DΔNΔC or its variants or left unstimulated (No GF). Lysates were immunoprecipitated (IP) with an antibody against VEGFR-2 (left) or VEGFR-3 (right) and analyzed by reducing SDS-PAGE and Western blotting with an antibody against phosphotyrosine (pY) to assess activation of receptors (top blot in each pair) or with an antibody against VEGFR-2 (bottom blot in each pair on the left) or VEGFR-3 (right bottom blot) to confirm the presence of each receptor. VEGFR-2 migrated predominantly at ∼230 kDa, whereas VEGFR-3 migrated as three bands, a ∼125-kDa cleaved form and two uncleaved forms of ∼175 and ∼195 kDa that differed in degree of glycosylation. Sizes of molecular mass markers (in kDa) are shown to the left of the panels. Dotted lines indicate where irrelevant tracks have been excised from images.

Fig 5: Assessment of the D103A variant and VEGF-DΔNΔC for receptor interactions, stimulation of COX-2 expression, and sprouting lymphangiogenesis. A, bioassays for binding and cross-linking of extracellular domains of VEGFR-2 (left) and VEGFR-3 (right) with VEGF-DΔNΔC (VEGF-D) and the D103A variant of VEGF-DΔNΔC. Data points, mean; error bars, S.D. B, effect of VEGF-DΔNΔC, the D103A variant, and other selected variants of VEGF-DΔNΔC (gray bars) and VEGF-CΔNΔC (VEGF-C) and the 3Ala variant of VEGF-C (C3Ala) (black bars) on the level of COX-2 mRNA in adult LECs as assessed by quantitative RT-PCR (D3Ala denotes the 3Ala variant of VEGF-DΔNΔC). Cells were exposed to 100 ng/ml ligands for 30 min before lysis for RNA preparation, as described under “Experimental Procedures.” COX-2 mRNA levels were normalized to β-actin and are expressed relative to the level in cells that were not treated with ligand (No GF). Columns, mean; error bars, S.D. C, titrations of VEGF-DΔNΔC and VEGF-CΔNΔC in the VEGFR-3 bioassay (left) and for the capacity to increase COX-2 mRNA levels in LECs (right). -Fold increases of COX-2 mRNA are relative to cells that were not treated with growth factor. In both graphs, data points indicate the mean, and error bars denote S.D. D, VEGF-DΔNΔC and the D103A variant (1 μg) were subcutaneously injected into ears of mice every 24 h for 3 days, as described under “Experimental Procedures”; PBS was the negative control. Ears were harvested and stained for lymphatics using antibody to LYVE-1 (green); the vessels shown are predominantly initial lymphatics. A high power image of the region within the white rectangle in the D103A image, showing three lymphatic sprouts, is shown below the lower power D103A image. Red arrows indicate lymphatic sprouts, which are quantitated in the left-hand graph; scale bars, 50 μm. HPF, high powered field. The width of LYVE-1-positive lymphatics is quantified in the right-hand graph. In both graphs, columns show mean and error bars denote S.E. In B and D, asterisks indicate statistically significant differences, as assessed by one-way analysis of variance with Tukey's post hoc test.