Expansion of endothelial cell colonies is regulated by angiopoietin-2. (A-C) Representative microscopic fields showing the decreased size of endothelial cell colonies developing in the presence of antiangiopoietin-2–blocking antibodies (B) as compared to control cultures, containing control IgGs (A). Conversely, significantly larger colonies are formed in cultures treated with low concentrations of angiopoietin-2 (200 ng/mL) as shown in panel C. (D) Morphometric assessment of the number of endothelial cells per colony developing in cultures treated with increasing concentrations of angiopoietin-2 (left) or in the presence of antiangiopoietin-1, antiangiopoietin-2, or control antibodies (right). Addition of angiopoietin-2 up to 200 ng/mL significantly increases the number of cells per colony. Conversely, blocking signaling of endogenous angiopoietin-2 using antiangiopoietin-2–specific antibodies decreases the number of cells per colony. Graphed data represent the number of cells per colony expressed as percentage of control cultures and plotted as mean ± SE of 3 to 4 independent experiments (P < .04 at 50 ng/mL and P = .008 at 200 ng/mL angiopoietin-2 versus control). (E) BrdU incorporation at day 7 of culture in endothelial colonies derived from cord blood CD34⁺ cells, treated with recombinant angiopoietin-1 or angiopoietin-2 at 200 ng/mL. Data are presented as the mean ± SE from 7 independent experiments. Angiopoietin-2 versus control P = .0017; angiopoietin-1 versus control, not significant. (F) Morphometric analysis of functional human blood vessels developing from cord blood–derived endothelial cells in Matrigel plugs, transplanted in vivo in the presence or absence of angiopoietin-2–loaded alginate beads. Cryostat sections of Matrigel grafts were collected at 100-μm intervals over a 4-mm range and scored for the presence of human VWF⁺/ISB4⁺ vascular structures. Data are expressed as mean ± SE of 100 to 396 microscopic fields per graft (n = 4; P < .0001; one microscopic field = 645 μm2).