|Ph.D Thesis||Department of Medicine|
|Supervisor:||Prof. Neufeld Gera|
Neuropilin-1 (Np1) and neuropilin-2 (Np2) function as receptors for heparin binding forms of vascular endothelial growth factor (VEGF) such as VEGF165. Np1 functions as an enhancer of VEGF165 signaling via the VEGFR-2 receptor of VEGF. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF165 mutant (VEGF165KF) that binds to neuropilins but displays a much lower affinity towards VEGFR-1 and VEGFR-2. VEGF165KF failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of Np1, VEGF165KF bound weakly to VEGFR-2, induced submaximal VEGFR-2 phosphorylation and activated ERK1/2. Interestingly, VEGF165KF did not promote formation of VEGFR-2/Np1 complexes, nor did high concentrations of VEGF165KF inhibit VEGF165 induced formation of such complexes, suggesting that VEGF165 does not stabilize VEGFR-2/Np1 complexes by forming bridges spanning VEGFR-2 and Np1. VEGF121 is a VEGF form that does not bind to neuropilins. Surprisingly, Np1 and Np2 enhanced VEGF165 and VEGF121 induced phosphorylation of VEGFR-2 and proliferation of endothelial cells. The enhancement of VEGF121 activity by Np1 was not associated with the formation of new VEGFR-2/Np1 complexes above the basal level observed in non-stimulated cells. In contrast, both VEGF165 and VEGF121 induced low levels of VEGFR-2/Np2 complex formation.
The Np1 and Np2 receptors also form complexes with type-A plexins. These complexes serve as signaling receptors for specific class-3 semaphorins. human umbilical vein derived endothelial cells (HUVEC) express tyrosine-kinase receptors for VEGF and bFGF, as well as Np1, Np2, and several type-A plexins. We found that semaphorin-3F (sema3F), which signals through Np2, and semaphorin-3A (sema3A), which signals through Np1, were able to inhibit VEGF andbFGF induced proliferation of HUVEC cells and phosphorylation of ERK-1/2. Both semaphorins inhibit the activities of VEGF and bFGF by a mechanism that requires active semaphorin signal transduction rather than by competition for shared neuropilin receptors.
Sema3A and sema3F expressing HEK293 cells repulse HUVEC cells and induce endothelial cell apoptosis. The anti-proliferative effects of sema3A and sema3F are additive and at low concentrations appear to be synergistic. Sema3F also inhibited VEGF165 as well as bFGF induced in-vivo angiogenesis. Over-expression of both semaphorins in tumorigenic human HEK293 cells inhibited their tumor forming ability but not their proliferation in cell culture. Tumors that developed from semaphorin expressing cells were smaller and contained lower concentrations of blood vessels, indicating that both semaphorins are inhibitors of tumor angiogenesis and can cooperate efficiently.