We have focused on 3 related areas. 1) We have continued to explore the role of Delta4 (Dll4), an endothelial-specific membrane-bound ligand for Notch1 and Notch4, as a regulator of endothelial cell function. Dll4 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 reduces endothelial cell proliferative and migratory responses in response to VEGF-A. We identified reduced VEGF receptor 2 and Npn-1 expression in Dll4-overexpressing endothelial cells as responsible for reduced biological responses to VEGF-A. Consistent with Dll4 signaling through Notch, we found that 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 that Dll4 can markedly reduce tumor angiogenesis and the growth of 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. We have observed that a number of experimental carcinomas and other tumor types are unresponsive to the inhibitor effects of Dll4/Notch signaling in the tumor vasculature. Current studies are focused on characterizing the role of tumor-associated Dll4, and other Notch ligands, as well as the roles of tumor-associated Notch receptors. In these experiments, we are analyzing the effects of Notch ligand expression by tumor cells on angiogenic sprouting of tumor vessels. In addition, we are exploring the pro-angiogenic effects of Notch activation in the tumor cells induced by environmentally-expressed Notch ligands. Conversely, we are analyzing the pro-angiogenic effects of Notch signaling in tumor-infiltrating myeloid cells induced by tumor-derived Notch ligands. Our goal is to define the mechanisms of responsiveness and resistance to anti-tumor therapeutic approaches based on Dll4/Notch signaling in the tumor vasculature. 2) We have explored the role of neuropilin-1 (Npn1) 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 Npn1 dictates the choice of signal transduction;plexins tranduce semaphorin signaling and VEGF receptors transduce VEGF signaling. We have now examined the mechanisms underlying Npn1 binding to VEGF or Sema3A, and how the engagement of Npn1 by Sema3A affects endothelial cell function. We have identified Sema3A as an inhibitor of endothelial cell adhesion, survival and proliferation and formation of vascular-like structures. Furthermore, we have found that Npn1-binding forms of VEGF block all these activities of Sema3A. 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 Npn1 internalization requires ligand bridging of Npn1 and VEGF receptors, and that Sema3A can promote Npn1 internalization, but requires a significantly higher concentration than VEGF-A. Thus, our results unveil an essential role for Npn1 as a sensor and priority setter for endothelial cell responses to conflicting signals. In additional studies, we have explored the possibility of targeting Npn1 for internalization as a tool to regulate endothelial cell responses to VEGF. In so doing, we have identified a group of polysaccharides, oligonucleotides, and other hybrid molecules that can induce Npn1 internalization and can thus serve as inhibitors of angiogenesis. We have named these compounds as """"""""internalization inducers"""""""". Such internalization-inducing compounds could be useful as therapeutics to reduce angiogenesis. One such synthetic compound,an oligoguanosine nucleotide, has shown clear efficacy both in vitro and in an in vivo model of retinal neovascularization. 3) We have continued investigations on how ephrinB ligands and their EphB receptors orchestrate endothelial/endothelial/pericyte assembly in newly-formed vessels. EphrinB ligands are surface-bound;thus receptor-ligand interactions in the B-type Eph/Ephrin interactions involve adjacent cells. In addition to activating their cognate EphB receptors, B Ephrins 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 cells survival. We have found that silencing EphrinB expression or expression of a tyrosine-phosphorylation-deficient mutant EphrinB (contains substitutions of all tyrosine residues that prevent tail phosphorylation and acts as a dominant-negative inhibitor of endogenous WT ephrin) causes endothelial cell death. This outcome cannot be prevented by the addition of exogenous VEGFA or FGF2. Biochemical and genetic experiments have revealed that such death is mediated by JNK3/MAPK10, and that EphrinB2 tyrosine phosphorylation-dependent signaling serves as a modulator of MAPK10/JNK3 expression. Thus, the silencing of JNK3 prevents cell death in endothelial cells, which are EphrinB signaling-deficient. Consistent with these results, the retinal vasculature in mice genetically-deficient of EphrinB2 undergoes cell death in association with JNK3 activation, and JNK3-deficient mice display ocular vascular defects that mirror those of EphrinB2 signaling deficiency. These results provide additional evidence supporting a role for EphrinB as a therapeutic target for inhibition of angiogenesis. 4)We have pursued earlier observations on the potential activities of semaphorin6A (Sema6A) in the vascular endothelium. We now found that transmembrane Sema6A is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating VEGFR2 signaling in response to exogenous and endogenous VEGF, which contributes to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of pro-survival signaling from endogenous VEGF. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, Matrigel and choroidal angiogenesis compared to controls. Prior to these studies, Sema6A was known to play important roles in development of the nervous system. We have now discovered that it also regulates vascular development and adult angiogenesis.

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Chen, Inn-Inn; Caprioli, Arianna; Ohnuki, Hidetaka et al. (2016) EphrinB2 regulates the emergence of a hemogenic endothelium from the aorta. Sci Rep 6:27195
Espígol-Frigolé, Georgina; Planas-Rigol, Ester; Ohnuki, Hidetaka et al. (2016) Identification of IL-23p19 as an endothelial proinflammatory peptide that promotes gp130-STAT3 signaling. Sci Signal 9:ra28
Salvucci, Ombretta; Ohnuki, Hidetaka; Maric, Dragan et al. (2015) EphrinB2 controls vessel pruning through STAT1-JNK3 signalling. Nat Commun 6:6576
Ohnuki, Hidetaka; Tosato, Giovanna (2014) Notch and TGFβ: Functional partners facilitating tumor progression. Oncoimmunology 3:e29029
Segarra, Marta; Ohnuki, Hidetaka; Maric, Dragan et al. (2012) Semaphorin 6A regulates angiogenesis by modulating VEGF signaling. Blood 120:4104-15
Salvucci, Ombretta; Tosato, Giovanna (2012) Essential roles of EphB receptors and EphrinB ligands in endothelial cell function and angiogenesis. Adv Cancer Res 114:21-57
Noberini, Roberta; Mitra, Sayantan; Salvucci, Ombretta et al. (2011) PEGylation potentiates the effectiveness of an antagonistic peptide that targets the EphB4 receptor with nanomolar affinity. PLoS One 6:e28611
Ciraci, Elisa; Della Bella, Silvia; Salvucci, Ombretta et al. (2011) Adult human circulating CD34ýýýLinýýýCD45ýýýCD133ýýý cells can differentiate into hematopoietic and endothelial cells. Blood 118:2105-15
Narazaki, Masashi; Segarra, Marta; Hou, Xu et al. (2010) Oligo-guanosine nucleotide induces neuropilin-1 internalization in endothelial cells and inhibits angiogenesis. Blood 116:3099-107
Tosato, Giovanna; Segarra, Marta; Salvucci, Ombretta (2010) Cytosolic phospholipase A2{alpha} and cancer: a role in tumor angiogenesis. J Natl Cancer Inst 102:1377-9

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