1. Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production
by Yeon-Sook Choi, Hyun-Jung Choi, Jeong-Ki Min, Bo-Jeong Pyun, Yong-Sun Maeng, Hongryeol Park, Jihye Kim, Young-Myeong Kim, and Young-Guen Kwon
Blood
Volume 114(14):
October 1, 2009
©2009 by American Society of Hematology

2. IL-33 induces migration, tube formation, and permeability of ECs
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.
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.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology

3. IL-33 induces vessel sprouting ex vivo and angiogenesis in vivo.
IL-33 induces vessel sprouting ex vivo and angiogenesis in vivo. (A) Aortic segments were harvested from C57BL/6 mice. Aortic segments in Matrigel were treated with IL-33 (100 ng/mL) or VEGF (50 ng/mL) for 2 weeks (n = 5 per group). (B) Sprouting were classified from 0 (least positive) to 5 (most positive) as described in “Aortic ring assay.” (C-F) C57BL/6 mice were injected with 0.6 mL of Matrigel containing IL-33 (n = 6 per group). After 6 days, the mice were killed, and the Matrigel plugs were excised. (C) Representative Matrigel plugs were photographed. (D) Quantification of neovessel formation by measuring hemoglobin in the Matrigel. (E) Plugs were stained for infiltrating ECs by the use of anti-CD31 antibody. (F) Quantitative assessment of CD31+ ECs. Data are means ± SDs; *P < .05, **P < .01 vs untreated control.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology

4. IL-33–induced angiogenesis and vascular hyperpermeability is mediated by the ST2 receptor.
IL-33–induced angiogenesis and vascular hyperpermeability is mediated by the ST2 receptor. (A-C) HUVECs were pretransfected with control siRNA (Con siRNA) or ST2 siRNA before IL-33 treatment. Cells were harvested for assay at 40 hours after transfection. (A) Expression levels of ST2 were determined by reverse-transcription polymerase chain reaction and Western blotting. (B) After IL-33 stimulation, chemotaxis was performed. (C) Cells were collected and replated on Matrigel-coated plates at a density of 1.5 × 105 cells/well and incubated with 20 ng/mL IL-33. Microphotographs were taken after 20 hours (×200). Tube networks were quantified with ImageJ software. (D) HUVECs were stimulated with IL-33 (20 ng/mL) for 1 hour. A [14C]sucrose permeability assay was then performed. Three independent experiments were performed in duplicate. Data are means ± SDs; *P < .05 vs control cell without IL-33; #P < .05 vs control cell with IL-33.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology

5. 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.
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.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology

6. IL-33 induces Akt/eNOS activation and NO production via ST2/TRAF6.
IL-33 induces Akt/eNOS activation and NO production via ST2/TRAF6. (A-D) HUVECs were pretransfected with ST2 siRNA (40 nmol/L) and TRAF6 siRNA (40 nmol/L) before IL-33 treatment. Cells were harvested for assay at 40 hours after transfection. (A-B) HUVECs were treated with 20 ng/mL IL-33 for 30 minutes. Cells were harvested and phosphorylation of Akt and eNOS by IL-33 were detected by Western blotting. 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). (C-D) After transfection, HUVECs were treated with 20 ng/mL IL-33 for 4 hours and incubated with DAF-FM diacetate for 1 hour. The relative levels of intracellular NO were determined from the fluorescence intensity of DAF-FM. Three independent experiments were performed in duplicate. Data are means ± SDs; *P < .01 vs control cell without IL-33; #P < .01 vs control cell with IL-33.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Impairment of IL-33–induced angiogenesis and vascular hyperpermeability in eNOS-deficient mice.
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.
Yeon-Sook Choi et al. Blood 2009;114:
©2009 by American Society of Hematology