sG18 inhibits VEGF165 and Sema3A binding to HUVECs and VEGF165-induced HUVEC proliferation. (A) sG18 inhibits VEGF165 and Sema3A binding to HUVECs. The cells were incubated with sG18 (0, 4, or 16 μg/mL) at 37°C for 1 hour, followed by addition of biotin-VEGF165 or Sema3A/Fc. Bound biotin-VEGF165 or Sema3A/Fc was detected by flow cytometry. (B) sG18 (16 μg/mL) inhibits VEGF165-induced phosphorylation of VEGFR-2 in HUVECs. After incubation with sG18 (0 or 16 μg/mL) at 37°C for 1 hour, the cells were treated with VEGF165 (100 ng/mL) or IL-6 (100 ng/mL) in conjunction with sIL-6R (100 ng/mL) at 37°C for 5 minutes. Phosphorylated (p-Y) VEGFR-2 and p-Y STAT3 was detected by immunoblotting with specific antibodies; after stripping, the membranes were reprobed for total VEGFR-2 and STAT3. Molecular weights (kDa) are indicated on the left of the panel. (C) sG18 inhibits VEGF165-induced phosphorylation of VEGFR-2 in HUVECs, as detected by confocal microscopy. Conditions for cell incubation with sG18 and VEGF165-induced activation of VEGFR-2 are those described in panel B. (D) sG18 specifically inhibits VEGF165 (50 ng/mL)-induced transwell migration of endothelial cells. HUVECs (5 × 105 cells/well) were preincubated for 30 minutes with sG18 or sA18 (1, 4, or 16 μg/mL) added to the upper chamber and incubated at 37°C for 18 hours. The results reflect the mean fold increase (± SEM) in cell migration compared with medium alone in 3 experiments performed in duplicate. (E) HUVECs were cultured for 24 hours with or without sA18, sT18, sG18, or sC18 at varying concentrations (1, 2, 4, 8, 16, or 32 μg/mL) in the presence of VEGF165 (25 ng/mL); proliferation was measured by Cell Counting Kit-8. Results are expressed as mean ± SD optical density at 450 nm of triplicate cultures.
