Dll4-containing exosomes increase vessel branching. (A) Normal dermal fibroblasts were seeded onto 24-well plates at a density of 2 × 104/well, and HUVECs were seeded onto the fibroblasts at a density of 4 × 103/well. The cells were cultured for 10 days in EGM2 with either control exosomes (50 μg/mL) or Dll4-containing exosomes (50 μg/mL), with the media changed every 2 days. Cells were fixed and stained as described in “Methods,” and pictures were taken with a Zeiss Axiovert 135 microscope at 5× magnification. (A) Three batches of control exosomes and 3 batches of Dll4-containing exosomes were tested, each in triplicate. Ten images were captured from each well for analysis (a total of 90 pictures for control and 90 for Dll4-containing exosome–treated cells). (B) Representative images were analyzed with ImageJ software for vessel length, size, and density. The number of tips and nodes was also counted. (C) U87 xenografts were grown and injected 3 times per week for 5 weeks with either PBS, control exosomes (Control exo), or Dll4 exosomes (Dll4 exo, isolated from U87 cells) at a concentration of 50 μg/mL. The animals were then killed and the tumors sectioned for vessel staining. A Chalkley vessel count (CVC) was performed for vessel density, and the vessel length and lumen size were estimated with ImageJ. The number of branches was counted manually and normalized to the CVC. Data were analyzed by t test or 1-way analysis of variance with a Tukey multiple-comparison post hoc test with GraphPad Prism 4.0b software and shown as mean with standard deviation. (D) Representative images taken on a Nikon Eclipse E800 microscope at 10× magnification.
