Sema4D displays angiogenic activity in vivo. (A) CAM assays were performed to test the angiogenic effect of Sema4D. Mock (i), Sema4D (1.2 pmol/sponge; ii), HGF (1.8 pmol/sponge; iii), or bFGF (30 pmol/sponge; iv) were loaded on sponges and implanted into 8-day-old chicken embryos. Seventy-two hours after factor soaking, the angiogenic activity of the different factors was evaluated. Numerous newly formed capillaries, growing straight toward the angiogenic source, were observed in the presence of Sema4D. In size and number, they were comparable to those observed in the presence of HGF and bFGF. (B) Matrigel plug assay in mice. Subcutaneously injected Matrigel plugs containing either PBS (Control), bFGF, or Sema4D were isolated after 7 days and analyzed histologically. When compared with control, bFGF and Sema4D elicited strong angiogenic invasion of Matrigel plugs. Images were captured with a 10×/0.40 air objective lens. Mean ± SD blood vessel area (square micrometers) per observed field is shown in the graph.