CXCR4 neutralization inhibits PGE₂-mediated human endothelial cell organization and angiogenesis in vivo. (A-E) Confocal microscopy of human microvascular endothelial cells expressing CXCR4 and organizing into tubes in PGE₂-containing (10^–8 M final) Matrigel, 48 hours after in vivo implantation into SCID mice. Cells were stained with PE-conjugated mouse antihuman CD34, as a marker for human endothelial cells (red; A); CXCR4 expression was detected by using fluorescein-conjugated mouse antihuman CXCR4 (green; B); the nuclei were visualized by DAPI staining (blue; C); panel D is superimposed panels A-C. CXCR4 neutralization inhibits PGE₂-induced endothelial cell organization in vivo (E). (F) Fluorometric quantitative analysis of the effect of CXCR4 neutralization on PGE₂-mediated angiogenesis at the fifth day after implantation of human endothelial cells into SCID mice. FITC-dextran was used to assess the level of vascularity in Matrigel plugs, as described in “Materials and methods.” Fluorometric quantification of the dextran fluorescence intensity obtained per 100 mg Matrigel implants. The mean + SEM of a representative of 2 independent experiments is shown. *P < .01. Original magnification A-E, × 300.