The effects of VEGF Trap, ranibizumab and bevacizumab on luciferase activation induced by VEGF-A₁₂₁, VEGF-A₁₆₅, human PlGF-2 (hPlGF-2) or mouse PlGF-2 (mPLGF-2) in HEK293/VEGFR1 cells. a Dose response curves for VEGF-A₁₂₁, VEGF-A₁₆₅ and hPlGF-2 yielded EC₅₀ values of 13, 17, and 29 pM, respectively. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 20 pM of VEGF-A₁₂₁. c Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 20 pM of VEGF-A₁₆₅. d Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 40 pM of human PlGF-2. e Dose response curve for mPlGF-2 yielded an EC₅₀ value of 10 pM (f). Serial dilutions of VEGF Trap were added to HEK293/VEGFR1 cells along with 20 pM of mPlGF-2. The cells were incubated for 6 h and OneGlo luciferase substrate was then added to each well. The plates were read on a luminometer and the data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents a replica of 3 wells at each concentration

The effects of VEGF Trap, ranibizumab and bevacizumab on luciferase activation induced by VEGF-A₁₂₁ and VEGF-A₁₆₅ in HEK293/VEGFR2 cells. a Dose response curves for VEGF-A₁₂₁ and VEGF-A₁₆₅ with EC₅₀ values of 70 and 30 pM, respectively. PlGF-2 was not active in this assay. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A₁₂₁. c Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A₁₆₅. The cells were incubated for 6 h and OneGlo luciferase substrate was then added to each well. The plates were read on a luminometer and the data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents a replica of 3 wells at each concentration

The effects of VEGF Trap, ranibizumab and bevacizumab on calcium mobilization induced byVEGF-A₁₆₅ in HUVEC. a A dose–response curve generated using serial dilutions of VEGF-A₁₆₅ (4.0 nM–0.023 pM) resulted in an EC₅₀ value of 5 pM. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HUVEC along with 20 pM of VEGF-A₁₆₅. The VEGF-A₁₆₅ was preincubated with the inhibitors for 10 min at 25°C. The solution was added to HUVEC preloaded with fluo-4 and the fluorescence of the well was determined on a FLIPR instrument. The data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents duplicate wells at each concentration

The effects of VEGF Trap, ranibizumab and bevacizumab on HUVEC migration. a HUVEC were placed in the upper compartment of the Boyden chamber and allowed to migrate towards basal media containing 0.1% fetal bovine serum with or without VEGF-A₁₆₅ or VEGF-A₁₆₅ mixed with four concentrations each of VEGF Trap (circles, solid line), ranibizumab (triangles, dotted line) or bevacizumab (squares, dashed line) ranging from 0.013 to 13 nM. The percentage of total migration (y-axis) was calculated as (F_Drug − F_Basal)/(F_Total− F_Basal) × 100; where F_Total is fluorescence in the presence of VEGF-A₁₆₅, F_Basal is fluorescence in the absence of VEGF-A₁₆₅, and F_Drug is fluorescence in the presence of VEGF-A₁₆₅ mixed with drug at a specific molar ratio (x-axis). b HUVEC migration was assessed in the absence and presence of human PLGF-2 (hPLGF-2) or mouse PLGF-2 (mPLGF-2) with and without a 100-fold molar excess of VEGF Trap (VGT), ranibizumab (RAN) or bevacizumab (BEV). Fold migration (y-axis) was calculated as the ratio F/F_Basal; where F is the total fluorescence measured for the indicated condition (x-axis) and F_Basal is the fluorescence in the absence of either hPLGF-2 or mPLGF-2. Statistical significance: *P < 0.05; **P < 0.01; ns, no significance. Values and error bars represent the average value and standard error of the mean from at least three independent experiments with each experiment containing four biological replicates per condition (total n = 12–16 per condition) for all conditions tested. AU arbitrary units