Survival, growth and spread of tumors depend upon an adequate blood supply ensured by neovascularization, cooption of existing vessels and stem cell differentiation into endothelial cells. Our laboratory is interested in studying tumor angiogenesis and in manipulating this process to reduce tumor growth. We have focused on 3 related areas. First, we have explored the role of neuropilin-1 (Npn-1) as a receptor shared by heparin-binding forms of vascular endothelial growth factor (VEGF) and class 3 semaphorins, protein families that regulate endothelial and neuronal function, respectively. Previous studies have shown that ligand binding to Npn-1 dictates the choice of signal transduction; plexins tranduce semaphorin signaling and VEGF receptors transduce VEGF signaling. We have explored the mechanisms underlying Npn-1 binding to VEGF or Sema3A, and how the engagement of Npn-1 by Sema3A affects endothelial cell function. We have identified Sema 3A as an inhibitor of endothelial cell adhesion, survival and proliferation and formation of vascular-like structures. Furthermore, we have found that Npn-1-binding forms of VEGF block all these activities of Sema3A. We found that VEGF-A can compete with Sema3A for endothelial cell binding, and can promote Npn-1 internalization from the cell surface. Biochemical analysis of VEGF-A binding to endothelial cells revealed that Npn-1 internalization requires ligand bridging of Npn-1 and VEGF receptors. We also found that Sema3A can promote Npn-1 internalization, but requires a significantly higher concentration than VEGF-A. Thus, our results unveil an essential role for Npn-1 as a sensor and priority setter for endothelial cell responses to conflicting signals. Second, we have explored a role for Delta4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4, which is selectively expressed in the developing endothelium and is required for normal vascular development, continues to be expressed in angiogenic endothelium postnatally. We generated primary endothelial cells overexpressing Dll4 protein, and found that Dll4 overexpression reduces endothelial cell proliferative and migratory responses selectively in response to VEGF-A. We identified reduced VEGF receptor 2 and Npn-1 expression in Dll4-overexpressing endothelial cells as the reasons underlying their defective responses to VEGF-A. Consistent with Dll4 signaling through Notch, expression of the transcription factor HEY2 was significantly induced in Dll4-overexpressing endothelial cells, and a gamma secretase inhibitor significantly reconstituted endothelial cell proliferation inhibited by Dll4. Thus, these studies have identified the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating the principal VEGF-A signaling receptor, VEGFR-2 and co-receptor Npn-1. Third, we have studied how ephrinB and SDF-1 orchestrate endothelial cell movement and morphogenesis into capillary-like structures. Eph receptors are tyrosine kinases interacting with their membrane-anchored ephrin ligands. In our previous studies, we have demonstrated that the G-protein-coupled receptor CXCR4 interacts with its unique SDF-1 chemokine ligand to regulate endothelial cell movement and morphogenic changes mostly through soluble gradients. By using biochemical and functional approaches, we found that in primary human endothelial cells endogenous EphB2 and EphB4 signaling is required for the formation of extracellular matrix-dependent capillary-like structures. We also found that EphB2 and EphB4 activation enhances SDF-1-induced signaling and chemotaxis that are also required for extracellular matrix-dependent endothelial cell clustering. These results support a model in which SDF-1 gradients first promote endothelial cell clustering, and EphB2 and EphB4 critically contribute to subsequent cell movement and alignment into cord-like structures.
