Cell surface–bound endogenous angiopoietins and expression of angiopoietin-1 and angiopoietin-2 transcripts in a CD34⁺CD11b⁺ subset. (A) Purified cord blood CD34⁺ cells were cultured for 48 hours in EBM and then analyzed by 3-color flow cytometry for the expression of CD34, CD11b, and either surface-bound endogenous angiopoietin-1 or angiopoietin-2. Gating on CD34⁺CD11b⁺ (R1) and CD34⁺CD11b^– (R2) subsets reveals that the CD34⁺CD11b⁺ but not the CD34⁺CD11b^– subset comprises a significant proportion of angiopoietin-1–bearing cells (left histograms). A smaller fraction of CD34⁺CD11b⁺ also binds angiopoietin-2, although dimly (right histograms). Quadrants in the dot-plot have been set to comprise background fluorescence of isotype control antibodies in the bottom left quadrant. Vertical bars within histograms indicate the upper limit of the negative isotype control. Numbers within the histograms plots identify the percentage of cells bearing angiopoietin-1– and angiopoietin-2–specific immune reactivity at the cell surface and the MIF of the angiopoietin-positive populations. Data are representative of 3 independent experiments. (B) Detection of endogenous angiopoietin-1 and angiopoietin-2 bound to the cell surface of endothelial cells derived from cord blood CD34⁺ cells at day 7 of culture. Numbers within the histogram plots identify the percentage of cells bearing angiopoietin-1–specific (top panel) and angiopoietin-2–specific (bottom panel) immune reactivity at the cell surface and the MIF of the angiopoietin-positive populations. Data are representative of 3 independent experiments. (C) RT-PCR analysis of angiopoietin-1 (top panels), angiopoietin-2 (middle panels), and GAPDH (bottom panels) mRNA expressed in HUVECs, purified CD34⁺, CD34⁺CD11b⁺, and CD34⁺CD11b^– subsets. Densitometric quantifications of angiopoietin-1 and angiopoietin-2 PCR products normalized to GAPDH are shown at the bottom.