Figure 4. Exogenous SEMA3B mimicked the effects of sPE on CTBs and endothelial cells and inhibited angiogenesis.

(A) The addition of anti-VEGF or SEMA3B protein significantly inhibited CTB invasion as compared with the addition of a control protein, CD6-Fc. The removal of both ligands (anti–VEGF/NRP1-Fc and anti–VEGF/NRP-2–Fc) restored invasion to control levels. (B) The variables tested in A had the opposite effects on CTB apoptosis, suggesting that increased programmed cell death contributed to decreased invasion. (C) Exogenous VEGF stimulated the migration of UtMVECs, which was inhibited by SEMA3B. (D) The results in C were quantified relative to the addition of CD6-Fc. (E) In UtMVECs, VEGF promoted survival and SEMA3B increased apoptosis relative to control levels. (F) In the chick chorioallantoic membrane angiogenesis assay, VEGF promoted angiogenesis by approximately 3 fold and SEMA3B inhibited this process approximately 5 fold relative to the effects of CD6-Fc. Arrows mark the edge of the filter paper used to apply the protein. The area of the CAM beneath the filter paper is shown in the bottom row. Scale bar: 200 μm (top row); 100 μm (bottom row). n = 6 replicates (A–D); n = 3 replicates (E and F). Mean ± SEM; 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.