We have focused on 3 related areas. 1) We have explored the role for Delta4 (Dll4), an endothelial specific membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4 is a cell-surface ligand of Notch that is selectively expressed in the developing endothelium and is required for normal vascular development. Post-natally, Dll4 is expressed in the angiogenic endothelium, particularly in the tumor vasculature. 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 responsible for 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. In additional experiments utilizing pre-clinical cancer models, we have explored the possibility of utilizing Dll4 as an activator of Notch signaling in endothelial cells to inhibit angiogenesis and tumor growth. In xenogeneic and syngeneic tumor models established in mice, we have documented tha Dll4 can markedly reduce tumor angiogenesis and tumor growth, particularly in tumors of lymphoid origin. Studies of the mechanisms for the anti-tumor effects of Dll4 have shown that these are attributable at least in part, to Notch activation in the tumor microenvironment and in the tumor vasculature resulting in reduced VEGFR2 expression and reduced tumor blood perfusion. Current studies are focused in further defining the role of Dll4 in reduced tumor neovascularization, with a particular focus on the effects of Dll4 on extracellular matrix deposition and interactions with pericytes. 2) 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 now examined 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. In additional studies, we have explored the possibility of targeting Npn-1 for internalization as a tool to regulate endothelial cell responses to VEGF. In so doing, we have identified a group of polysaccharides and other hybrid molecules that can induce Npn-1 internalization and can thus serve as inhibitors of angiogenesis. We have named these compounds as """"""""internalization inducers"""""""". We have explored a variety of internalization-inducing compounds that could be useful as therapeutics to reduce angiogenesis. One such synthetic compound has shown clear efficacy in an in vivo model of retinal neovascularization. 3) We have studied how ephrinB ligands and their EphB receptors orchestrate endothelial/pericyte assembly in newly-formed vessels. EphrinB ligands are surface-bound;in addition to activating their cognate EphB receptors, they can function as signaling molecules when engaged by the receptor through """"""""reverse signaling"""""""". Eph receptors are tyrosine kinases interacting with their membrane-anchored ephrin ligands. In our previous studies, we have demonstrated that signaling by Eph B receptors in endothelial cells is critical to assembly into vascular structures. We have now investigated the potential role of Eph/ephrin signaling in the regulation of endothelial/pericytes assembly. A critical step in angiogenesis consists of the recruitment of pericytes to the outer vessel wall, a process that stabilizes and fortifies vessels structure. Mesenchymal stem cells (MSC) can differentiate into pericytes upon interaction with endothelial cells. Using bone marrow-derived MSC, we have established that MSC interact with endothelial cells during extracellular matrix-dependent tube formation in vitro and matrigel angiogenesis assay in vivo, such that MSC establish contact with endothelial cells in a time-dependent and spatially-constrained manner. P-ephrinB is activated in the course of endothelial cell morphogenic processes leading to capillary network stabilization and new vessel formation in vivo, including physiological retinal vessel formation, pathological retinal neovascularization, wound healing and tumorigenesis. This activation is inhibited by specific EphB peptide inhibitors, by a soluble recombinant protein ephrinB2-Fc, by silencing expression of EphrinB2 and by inhibiting Jak2 activity. The importance of EphrinB2 intracellular signaling has emerged from mutagenesis studies in which all the putative phosphorylation sites within the EphrinB2 intracellular domain were mutated, whereas the extracellular domain was intact and capable of signaling through the EphB receptors. In vivo experiments, in which MSC-GFP were injected subcutaneously into immuno-compromised mice in the context of extracellular matrix, show that P-ephrinB reverse signaling is present at those sites where endothelial cells/MSC physically interact. These experiments establish a role for ephrinB reverse signaling in endothelial/pericyte interactions, and suggest that ephrinB signaling is a potential therapeutic target for modulation of physiologic and pathologic angiogenesis. In summary, Dll4 and its target molecules neuropilin1 and ephrinB are critical regulators of angiogenesis, including tumor angiogenesis. We have explored their activities and relative roles during the angiogenic process, have identified them as potential targets for modulation of tumor angiogenesis, and developed compounds that could be useful as angiogenesis inhibitors.
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