IL-33 stimulates NO production in ECs via Akt/eNOS signaling pathway in ECs. (A-C) Phosphorylation of Akt and eNOS by IL-33 were determined by Western blotting. (A) HUVECs were stimulated with various concentrations of IL-33 for 30 minutes. (B) HUVECs were stimulated with 20 ng/mL IL-33 for the indicated times. (C) HUVECs were pretreated with 5 μmol/L PP1 (P) or 100 nmol/L wortmannin (W) for 30 minutes and then stimulated with 20 ng/mL IL-33 for 30 minutes. Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of P-Akt to Akt and P-eNOS to eNOS. The relative ratio in untreated control is arbitrarily presented as 100 (bottom). (D) HUVECs were pretreated for 30 minutes with inhibitors and then treated with 20 ng/mL IL-33 for 4 hours. ECs were incubated with DAF-FM diacetate for 1 hour at 37°C, and fluorescence images were captured with microscope. The relative levels of intracellular NO were quantified with Metamorph software (Molecular Devices). Three independent experiments were performed in duplicate. Data are means ± SDs; **P < .01 vs untreated control.