IL-33 induces migration, tube formation, and permeability of ECs. (A) Proliferative indices of HUVECs treated with various concentrations of IL-33 were accessed by [³H]thymidine incorporation assay. (B) Chemotactic motility of HUVECs induced by various concentrations of IL-33. HUVECs were placed in the upper chamber, and M199 (1% FBS) with various concentrations of IL-33 was placed in the lower wells of chemotaxis chamber. After 4 hours of incubation, chemotaxis was quantified by counting the cells that migrated to the lower side of the filter by optical microscopy at ×200 magnification. (C) HUVECs were plated on Matrigel-coated wells at a density of 1.5 × 10⁵ cells/well with various concentrations of IL-33. After 20 hours, microphotographs were taken (×200). Capillary-like networks were quantified with ImageJ software. (D) HUVECs were incubated with various concentrations of IL-33 for 1 hour. Three independent experiments were performed in duplicate. (E) In vivo Miles vascular permeability assay. IL-33 or PBS was injected intradermally into the skin of C57BL/6 mice after intravenous injection of Evans blue. Data are means ± SDs; *P < .05, **P < .01 vs untreated control. (F) Confluent HUVECs were stained for VE-cadherin after cells were treated with IL-33 or VEGF for 1 hour. (G) VE-cadherin phosphorylation was assayed. HUVECs were grown to confluence and treated with 20 ng/mL IL-33 for the indicated time. Immunoprecipitated phosphotyrosine proteins were analyzed by SDS–polyacrylamide gel electrophoresis followed by immunoblotting with antibody to VE-cadherin.