Soluble Sema4D shows proangiogenic activity on ECs. (A) Mitogenic ability of Sema4D on HUVECs was evaluated in Transwell assay. Cells that underwent migration in response to the different chemoattractants were stained and solubilized, and the absorbance was calculated (see “Materials and methods”). The obtained values for stimulation with mock were normalized to 100, and migration in response to different factors was calculated as a percentage increase with respect to the mock. VEGF-A₁₆₅ (4 nM) and HGF (4 nM) were used as positive controls. Data represent the mean of at least 6 independent experiments. As shown, Sema4D (6 nM) is endowed with mitogenic activity on HUVECs, comparable to those of VEGF and HGF. Data are shown as mean ± SD. (B) To evaluate the in vitro angiogenic activity, spheroids of ECs were grown in a collagen matrix in the absence or in the presence of angiogenic molecules. HUVECs-Sema4D are HUVECs infected with a lentivirus containing the Sema4D full-length cDNA. Infected cells overexpressed Sema4D full length, as observed in Western blot analysis (not shown). As shown, soluble Sema4D (3.5 nM) and the positive controls HGF (4 nM) and VEGF-A₁₆₅ (2 nM) elicited strong morphogenetic responses. Moreover, HUVECs expressing Sema4D full-length underwent spontaneous sprouting, without requiring any other stimulating factors. Images were captured with a 10×/0.40 NA air objective lens. (C) Spheroid assay on microendothelial cells grown as in panel B, in the presence of the indicated growth factors. These cells show an angiogenic response in the presence of Sema4D. Images were captured with a 20×/0.70 NA air objective lens.