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Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn

Yasushi Mochizuki1, Takao Nakamura1, Hiroshi Kanetake1 and Shigeru Kanda1,2,*

1 Department of Urology and
2 Department of Molecular Microbiology and Immunology, Division of Endothelial Cell Biology, Nagasaki University Graduate School of Medicine, 1-7-1, Sakamoto, Nagasaki 852-8501, Japan



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  		Fig. 1. Ang2 stimulates autophosphorylation of Tie2. (A) Expression of Tie2 on parental IBE cells was examined by immunoprecipitation with or without immunized peptide, followed by immunoblotting. About 135 kDa single protein was observed. (B) Tie2 was immunoprecipitated from cells (15 cm dishes) either stimulated or left unstimulated with 2 µg/ml Ang2 for 15 minutes and immune complex kinase assay was performed using 90% of cell extracts. Tie2 was immunoprecipitated followed by immunoblotting from the remaining 10% of cell extracts to examine whether a similar amount of Tie2 protein was present in the immune complex kinase assay.

 


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  		Fig. 2. Ang2 induces chemotaxis, but not proliferation of IBE cells. (A) Cells were inoculated into fibronectin-coated wells and cultured in Ham’s F-12 medium containing 10% FBS. On the following day, medium was replaced with Ham’s F-12 medium containing 0.25% BSA with (closed bar) or without (open bar) 5 ng/ml FGF-2 in the presence or absence of Ang2 and the culture continued for 3 days. Data are expressed as means±s.d. for triplicated wells and similar results were obtained from two independent experiments. The number of cells counted in untreated samples was set as 100%. (B) Cells were suspended in Ham’s F-12 medium containing 0.25% BSA and seeded onto the upper surface of a fibronectin-coated Traswell membrane. In the lower wells, 50 ng/ml FGF-2 was added to the same medium (indicated as closed bars) in combination with Ang2. Four hours later, cells attached on the lower surface of the membranes were counted. Data are expressed as means±s.d. for quadruplicated wells and similar results were obtained from two independent experiments. The number of cells counted in untreated samples was set as 100%.

 


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  		Fig. 3. Ang2 induces tube-like structure formation by IBE cells. Cells with or without 10 ng/ml FGF-2 in the absence or presence of 1 µg/ml Ang2 were cultured between two layers of collagen gels. Representative data were obtained from two independent experiments. Magnification is 100x.

 


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  		Fig. 4. Ang2 activates a panel of non-receptor protein tyrosine kinases. (A,B) IBE cells (6 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and c-Fyn (A) or c-Src (B) was immunoprecipitated from 90% of lysate and used for in vitro kinase assay. Acid-denatured enolase was used as a substrate. The remaining 10% total lysate was examined by immunoblotting to determine the amount of loaded proteins. (C) IBE cells expressing FLAG-tagged wild-type c-Fes (FesWT6-8 cells; 10 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and FLAG-tagged c-Fes was immunoprecipitated followed by imunoblotting. Between two probings, membranes were stripped. Representative data were obtained from two independent experiments.

 


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  		Fig. 5. Ang2 activates PI 3-kinase in a manner depenedent on c-Fes. In vitro PI 3-kinase assay was performed in immunoprecipitates with anti-p85 subunit (A,B,E), anti-phosphotyrosine (PY99; C) or anti-Tie2 (D) antibodies. Phosphatidylinositol was separated by thin layer chromatography and phosphorylation of phosphatidylinositol was measured by the BioImager BAS 5000. The activity of phosphatidylinositol triphosphates (PIP) in untransfected cells was set as 1.00. (A,C,D) IBE cells (6 cm dishes) were either stimulated or left unstimulated with 1 µg/ml Ang2 for 15 minutes and PI 3-kinase activity was examined. (B) Ang2 (1 µg) was preincubated with extracellular domain of either Tie1/Fc chimera or Tie2/Fc chimera (25 µg) at room temperature for 10 minutes, then added to the cells. (E) IBE cells expressing either FLAG-tagged kinase-inactive c-Fes (FesKE5-8 cells) or FLAG-tagged wild-type c-Fes (FesWT6-8 cells) were either stimulated or left unstimulated with 1 µg/ml Ang2, and PI 3-kinase activity was examined. Representative data were obtained from two independent experiments.

 


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  		Fig. 6. Stable cell lines expressing either kinase-inactive c-Src (denoted SrcKD-2 cells) or c-Fyn (denoted FynKD-8 cells) migrate toward Ang2. Chemotaxis assay was performed as described for Fig. 2B. Data are expressed as means±s.d. for quadruplicated wells. Representative data were obtained from two independent experiments.

 


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  		Fig. 7. Ang2-mediated chemotaxis is PI 3-kinase-dependent. (A) IBE cells were preincubated with either 0.1% DMSO (vehicle) or 50 µM LY294002 for 30 minutes and assayed for chemotaxis as described for Fig. 2B. (B) FesKE5-8 cells and FesWT6-8 cells were examined for chemotaxis towards 1 µg/ml Ang2. Data are expressed as means±s.d. for quadruplicated wells. Representative data were obtained from two independent experiments.

 


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  		Fig. 8. Ang2-mediated tube formation is not dependent on PI 3-kinase. Treatment of IBE cells with PI 3-kinase inhibitor LY294002 (A) or FesKE5-8 cells, which have a dominant-negative effect on PI 3-kinase activation by Ang2 (B) form tube-like structures in the presence of 1 µg/ml Ang2. Representative data were obtained from two independent experiments.

 


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  		Fig. 9. Ang2-mediated tube-like structure formation was c-Fyn dependent. IBE cells expressing kinase-inactive c-Fyn (K299M), denoted FynKD-8 cells, were examined by in vitro kinase assay to determine the dominant-negative effect on Ang2 (A). Phosphorylation of acid-denatured enolase was measured by the use of BAS5000 Image Analyzer. FynKD-8 cells were examined for their ability to form tube-like structures mediated by Ang2 as described for Fig. 3B. Representative data were obtained from two independent experiments.

 

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