Semaphorin-3A and semaphorin-3F work together to repel endothelial cells and to inhibit their survival by induction of apoptosis.

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF(165)) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF(165) do not inhibit VEGF(165)-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF(121), a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF(165) activity does not depend on competition with VEGF(165) for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.