Impairment of IL-33–induced angiogenesis and vascular hyperpermeability in eNOS-deficient mice. (A-C) HUVECs were preincubated for 30 minutes with or without 100 nmol/L wortmannin or 1 mmol/L NMA before stimulation with IL-33 (20 ng/mL). (A) After 4 hours of incubation, chemotaxis was quantified by counting the cells that had migrated to the lower side of the filter by optical microscopy at ×200 magnification. (B) Cells were collected and replated on Matrigel-coated plates at a concentration of 1.5 × 10⁵ cells/well. Microphotographs were taken after 20 hours (×200). (C) HUVECs were preincubated for 30 minutes with or without NMA (1 mmol/L) and then treated with 20 ng/mL IL-33 for 1 hour. A [¹⁴C]sucrose permeability assay was then performed. Three independent experiments were performed in duplicate. Data are means ± SDs; *P < .05, **P < .01 vs IL-33 alone. (D) In vivo Miles vascular permeability assay was performed in WT and eNOS KO mice. IL-33 (500 ng) and PBS were injected intradermally into the skin of mice after intravenous injection of Evans blue. Data are means ± SDs; **P < .01 vs IL-33 in eNOS KO.