FIGURE 1.

Ang1 induces Dll4 expression leading to activation of Notch signaling in confluent endothelial cells. A, sparse and confluent HUVECs were starved in medium 199 containing 1% BSA for 12 h, and stimulated with vehicle (−) or COMP-Ang1 at 400 ng/ml (C-Ang1) for 1 h. (COMP-Ang1 was used at the concentration of 400 ng/ml throughout the following experiments.) After stimulation, total RNA was extracted and subjected to real time RT-PCR analysis to determine the expression of Dll4 mRNA as described under “Experimental Procedures.” Bar graphs show relative mRNA levels of Dll4 mRNA normalized to that of GAPDH. Data are expressed as fold-induction relative to that in the vehicle-treated sparse cells, and shown as mean ± S.D. of three independent experiments. B, confluent HUVECs starved for 12 h were stimulated with COMP-Ang1 for the periods indicated at the bottom (h). Dll4 mRNA levels were analyzed by real time RT-PCR as described in A. Values are expressed as fold-induction relative to that in the unstimulated cells, and shown as mean ± S.D. of five independent experiments. C, confluent and sparse HUVECs were starved in medium 199 containing 1% BSA for 12 h, and stimulated with COMP-Ang1 for the periods indicated at the top (h). Cell lysates were subjected to Western blot analysis with anti-Dll4 (Dll4) and anti-tubulin (tubulin) antibodies. D, the relative expression of Dll4 observed in C are quantified by normalizing the expression of Dll4 by that of tubulin. Values are expressed as fold-induction relative to that observed in the confluent unstimulated cells, and shown as mean ± S.D. of three independent experiments. E, confluent HUVECs starved for 12 h were stimulated with vehicle (control), 600 ng/ml of Ang1 (Ang1), 600 ng/ml of Ang2 (Ang2) and COMP-Ang1 (C-Ang1) for 1 h. Cell lysates were subjected to Western blot analysis with anti-Dll4 (Dll4) and anti-tubulin (tubulin) antibodies. F, expression of Dll4 protein observed in E are quantified as described in D. Values are expressed as fold-induction relative to that observed in the control cells, and shown as mean ± S.D. of four independent experiments. G, confluent and sparse HUVECs were starved in medium 199 containing 1% BSA for 12 h, and stimulated with COMP-Ang1 for the periods indicated at the top (h). Cell lysates were subjected to Western blot analysis with anti-NICD (NICD), anti-Dll4 (Dll4), and anti-tubulin (tubulin) antibodies. H, confluent HUVECs transfected without (−) or with either control siRNA (control) or two independent siRNAs targeting Dll4 (Dll4#1 and Dll4#2) were stimulated with COMP-Ang1 as described in A. Cell lysates were subjected to Western blot analysis with anti-NICD (NICD), anti-Dll4 (Dll4), and anti-tubulin (tubulin) antibodies. Significant differences between two groups (A) or from the control (B, D, and F) are indicated as: *, p < 0.05; **, p < 0.01; or ***, p < 0.001. n.s. indicates no significance between two groups or from the control.