(a)
(b)
(c)

Figure 2: Uptake and degradation of [125I]-VEGF-A by LSECs. (a) Time course of [125I]-VEGF-A uptake and degradation: LSECs (4 × 106/ml) were incubated at 37°C in medium containing [125I]-VEGF-A (40 pM). At the indicated times, duplicate aliquots were removed, and cells and medium were assayed for TCA-soluble and TCA-precipitable radioactivity as described in “Materials and Methods.” Degradation was calculated as the sum of the cell-associated and released TCA-soluble radioactivity at each time point. The specific uptake (per 4 × 106 cells) at each time point was determined by adding up the released TCA-soluble radioactivity to the total cell-associated radioactivity and then subtracting the cell-associated radioactivity at the zero-time and expressed as a percentage of total TCA-precipitable (TCA-ptt.) radioactivity initially added to the cell suspension. Data represent the mean ± SD (n = 3). (b) Effects of the endocytic pathway inhibitors on uptake and degradation of [125I]-VEGF-A. LSECs (4 × 106/ml) were pretreated with inhibitors or vehicle controls [DMSO for dynasore and concanamycin A (Con-A), PBS for leupeptin] at 37°C for 10 min prior to the addition of [125I]-VEGF-A (40 pM). The cells were then further incubated at 37°C for 90 min in the continued presence of inhibitor or vehicle. The specific uptake was determined as described for (a). Data are expressed as the percentage inhibition relative to control (vehicle-treated) cells. Concentrations of inhibitors employed were as follows: dynasore (40 μM), Con-A (0.1 μM), and leupeptin (50 μg/ml). Data represent the mean ± SD (n = 3). Asterisk () denotes statistically different from control (p < 0.001 by one-way ANOVA and Dunnett’s post hoc test among control and treated groups with various inhibitors). (c) Effect of the VEGFR1-binding peptide on [125I]-VEGF-A uptake. A preincubation step of 5 min at 37°C was performed in the presence or absence of the VEGFR1-binding peptide (400 μM in DMSO) prior to addition of [125I]-VEGF-A (1 nM). The cells (4 × 106/ml) were then further incubated at 37°C for the indicated times in the continued presence or absence of the peptide. The specific uptake (per 4 ×106 cells) at each time point was determined as described for (a). For the sake of simplicity, only the specific uptake is shown. Data represent the mean ± SD (n = 3). p < 0.03 vs. control by unpaired t-test. No statistical difference was found between conditions at 30 min using unpaired t-test.