Effect of angiogenic factors on induction of Notch and Delta genes. Total RNA was extracted from various endothelial cells transduced with recombinant adenoviruses and subjected to reverse transcription-PCR. The specific PCR bands were separated in 2% agarose gels and stained with ethidium bromide. β-Actin mRNA was amplified as a control. Results are from a representative experiment of three performed.

Requirement for VEGFR1/R2 in Notch1/Dll4 expression. (a) Expression of VEGFR1, VEGFR2, but not VEGFR3 mRNA in HIAECs. RNA from HIAECs and 293 cells was subjected to Northern blot analyses. Membranes were hybridized with ³²P-labeled VEGFR1, -R2, and -R3 probes at an activity of 10⁶ cpm/ml. Data for R1 was obtained by PhosphoImager scanning, while those for R2 and R3 were obtained by exposure to Kodak film for 4 days. (b) Both VEGFR1 and -R2 are involved in VEGF-induced Notch1/Dll4 expression. HIAECs were transduced with the different recombinant adenoviruses indicated and harvested at 48 h, and RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed.

Involvement of Akt in phosphatidylinositol 3-kinase-mediated signaling. (a) Activation of endogenous Akt by activated phosphatidylinositol 3-kinase. Cell lysates used in Fig. 4d, which contained equal amounts of proteins, were subjected to Western blot analysis with anti-phospho-Akt antibody. (b) Enforced expression of Myr-Akt in HIAECs. Cells transduced with Myr-Akt/Ad5 for 48 h or left untreated were harvested, lysed, and analyzed by Western blotting. Membranes were first immunoblotted with anti-Akt antibody to demonstrate overexpression of Myr-Akt and then reblotted with anti-phospho-Akt antibody to detect Akt phosphorylation. (c) Effect of Myr-Akt on VEGF-induced Notch1/Dll4 expression. HIAECs transduced with Myr-Akt/Ad5 for 48 h were either stimulated with recombinant human VEGF₁₆₅ or left untreated. The experiment was performed as described for Fig. 4e. Results are from a representative experiment of three performed.

Enhancement of network and cord formation in HIAECs by NICD and HES-1. (a) Stabilization of HIAEC network formation on Matrigel. Transduced HIAECs were seeded on Matrigel and photographed in representative areas at the indicated times. Experiments were repeated three times. Magnification, ×10. (b) Enhancement of HIAEC network and cord formation in an in vitro three-dimensional model. Fluorescent staining of endothelial cells in whole-mount collagen gels is shown for VEGF/FF plus LacZ/HIAECs (panel 1), VEGF/FF plus NICD/HIAECs (panel 2), VEGF/FF plus HES/HIAECs (panel 3), LacZ/FF plus LacZ/HIAECs (panel 4), LacZ/FF plus NICD/HIAECs (panel 5), and LacZ/FF plus HES/HIAECs (panel 6). Magnification, ×10. The scale bar is shown in panel 4.

Induction of Notch1/Dll4 by recombinant human VEGF₁₆₅ in HIAECs. (a) Dose effect of VEGF on Notch1/Dll4 induction. Cells were harvested 24 h after stimulation with recombinant human VEGF₁₆₅ at the indicated doses, and total RNA was extracted and subjected to reverse transcription-PCR analyses. Intensity of Notch1/Dll4 bands obtained from cells stimulated with 100 ng of recombinant human VEGF₁₆₅ per ml was normalized as 100, and amounts of Notch1/Dll4 mRNA are given relative to this value. Data are means ± standard deviations of three independent experiments. (b) Specific effect of VEGF on Notch1/Dll4 induction. Cells were harvested 24 h after stimulation with recombinant human VEGF₁₆₅, bFGF, and acidic FGF at the indicated doses, and total RNA was extracted and subjected to reverse transcription-PCR analyses. Results are from a representative experiment of two performed. (c) Kinetics of Notch1/Dll4 induction by recombinant human VEGF₁₆₅. Subconfluent HIAECs were stimulated with 100 ng of recombinant human VEGF₁₆₅ per ml, harvested at the indicated times, and analyzed for Notch1/Dll4 expression by reverse transcription-PCR. Results are from a representative experiment of two performed. (d) Kinetics of Notch1/Dll4 induction by recombinant human VEGF₁₆₅. The same samples obtained as in panel c were analyzed for Notch1/Dll4 expression by Northern blotting. 28s rRNA was stained with methylene blue. Results are from a representative experiment of two performed. (e) Specificities of VEGF induction of Notch1/Dll4. Cells were transduced with VEGF-Trap/Ad5 and Fc/Ad5 for 48 h and stimulated with 100 ng of recombinant human VEGF₁₆₅ per ml for 24 h. RNA was isolated and subjected to reverse transcription-PCR. β-Actin mRNA was amplified as a control. Results are from a representative experiment of three performed.

Phosphatidylinositol 3-kinase is involved in VEGF-induced Notch1/Dll4 expression. (a) Effect of VEGF and PD98059 on MAPK activation. HIAECs were left untreated or treated with PD98059 for 5 min before addition of recombinant human VEGF₁₆₅. Cells were lysed 15 min later and analyzed by Western blotting. Specific bands were immunoblotted with anti-phospho-MAPK antibody, stripped, and reblotted with anti-MAPK antibody. (b) Effect of VEGF and exogenous phosphatidylinositol 3-kinase mutants on phosphatidylinositol 3-kinase activation. HIAECs were transduced with adenoviruses for 48 h and then either stimulated with recombinant human VEGF₁₆₅ for 15 min or left unstimulated. The phosphatidylinositol 3-kinase assay was performed as described in the text. The PIP3 product, separated by thin-layer chromatography, and the origin are indicated. (c) Effect of specific kinase inhibitors on VEGF-induced Notch1/Dll4 expression. HIAECs were treated for 5 min with various inhibitors before addition of recombinant human VEGF₁₆₅. Cells were harvested 24 h later, and extracted RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed. (d) Enforced expression of DN-Δp85 in HIAECs. Cells transduced with adenoviruses as indicated for 48 h or left untreated were lysed, and whole-cell lysates were subjected to Western blot (IB) analysis. The endogenous wild-type p85 (Wp85) and exogenous mutant of p85 (Δp85) are indicated. (e) Effect of phosphatidylinositol 3-kinase mutants on VEGF-induced Notch1/Dll4 expression. HIAECs transduced with adenoviruses for 48 h were either stimulated with recombinant human VEGF₁₆₅ or left untreated. Cells were harvested at 24 h, and extracted total RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed.

Effects of NICD and HES1 on cell proliferation and survival. (a) [³H]thymidine uptake by HIAECs. Cells were transduced with NICD/Ad5, HES1/Ad5, or LacZ/Ad5. The assay was performed as described in the text. Results are means ± standard deviations of three independent experiments. *, P < 0.05, Student's t test. (b) Prolongation of cell survival by NICD and HES1. Adenovirus-transduced cells were cultured in serum-free medium, and live cells were counted. Results are means ± standard deviations of three independent experiments. Inset: Western blot assay of NICD and HES1 (exogenous HES1 in HES/HIAECs and induced endogenous HES1 in NICD/HIAECs) expression in HIAECs. β-Actin was used as a control for equal loading of proteins.

Suppressive effect of RBP-Jκ (R218H) on VEGF-driven network and cord formation in the three-dimensional model. (a) Exogenous expression of RBP-Jκ (R218H) in HIAECs. Equal numbers (10⁶) of cells transfected with either pEFBOS-RBP-Jκ (R218H)-Myc-tag or pEFBOS for 48 h were harvested, lysed, and analyzed by blotting (IB) with anti-Myc tag antibody. (b) Inhibition of HIAEC network and cord formation in an in vitro three-dimensional model. Fluorescent staining of endothelial cells in whole-mount collagen gels was done with anti-vWF. Magnification, ×10. (c) Quantitative representation of network and cord formation in an in vitro three-dimensional model. The numbers of networks and cords were counted in at least three low-power fields (LPF) per sample. Magnification, ×10. Experiments were performed in quadruplicate, and results are means ± standard deviations of three independent experiments. *, P < 0.05, Student's t test.