Effect of angiogenic growth factors on endothelial cell invasion and vessel formation.

Collagen implants and underlying tissues were harvested from the CAM at 24 hours (A,B), 48 hours (C,D), or 66 hours (E,F), cryosectioned, and processed for immunohistochemistry. Endothelial markers (red) were detected by immunofluorescence in sections from implants that had been treated with buffer alone (A,C,E) or bFGF/VEGF (B,D,F). Sections were counterstained for nuclei (green). Immunostaining was performed using anti-VEGFR2/QUEK-1 IgG in conjunction with an amplification system, as described in “Materials and methods.” The subectodermal vascular plexus across the top of the CAM was apparent at 24 hours (asterisks in A,B) and was no longer discernible by 66 hours. New vascular sprouts (red endothelial staining) began to appear within the implants by 48 hours (C,D), with a clear angiogenic response seen at 66 hours (E,F). Arrows indicate some of the vascular sprouts that have entered the grids of the upper nylon mesh (clear circles) of the collagen implant. Large CAM blood vessels (lu) were frequently packed with nucleated red blood cells. Bar indicates 200 μm. Inset in panel F shows both blood vessels (lu) clearly containing nucleated red blood cells (green) and also endothelial staining that is not associated with an obvious lumen (arrowheads).