Development of endothelial cell colonies is regulated by angiopoietin-1. (A, left panel) Tyrosine phosphorylation of Tie-2 in HMECs is specifically induced by angiopoietin-1–conditioned medium (CM) from HEK293 transfectants (∼450 ng/mL angiopoietin-1) but not control CM from mock HEK293 transfectants. This phosphorylation is effectively blocked by the antiangiopoietin-1 antibody SC no. 6319 (15 μg/mL) but not by control goat IgGs. The bottom panel shows the same blot reprobed with an anti-Tie-2 antibody to demonstrate equal protein loading. (A, right panel) Detection of Akt phosphorylation induced by angiopoietin-2 (800 ng/mL) in HUVECs. Addition of the antiangiopoietin-2 antibody SC no. 7016 to the cultures at a concentration of 60 μg/mL significantly reduces Akt phosphorylation, indicating a function-blocking activity of this antibody. The bottom panel shows the same blot reprobed with a pan anti-Akt antibody to verify equal protein loading in each lane. (B) Inhibition of endothelial colony formation by antiangiopoietin-1–, but not antiangiopoietin-2–blocking antibodies. Numbers of colonies generated over 7 days in culture of CD34⁺ cells in the presence of angiopoietin-specific antibodies (15 μg/mL) are expressed as percentage of those developing in control cultures without IgGs. Data represent the mean ± SE of n = 7, P < .0005 in antiangiopoietin-1 antibody cultures versus control. (C, left panel) Coculture of CD34⁺ cells with conditioned medium (CM) from angiopoietin-1 HEK293 transfectants leads to an increased number of endothelial cell colonies as compared to control cocultures using mock-transfected HEK293 cells. Data represent the mean ± SE of n = 3, P < .05 in angiopoietin-1–treated cultures versus control. (C, right panel) Similarly, addition of increasing concentration of purified recombinant angiopoietin-1 to the cultures results in the development of an increased number of endothelial colonies. Graphed data are expressed as mean ± SE of 3 independent experiments (P < .05 at all concentrations tested versus control). (D) Similar to the inhibitory effects of antiangiopoietin-1 antibodies, high concentrations of angiopoietin-2 (800 ng/mL), a known antagonist of angiopoietin-1/Tie-2 interactions, inhibit the formation of endothelial cell colonies. Graphed data are expressed as mean ± SE of 6 independent experiments (P < .0006 in cultures treated with 800 ng/mL angiopoietin-2 versus control).