A study of Notch signalling in developmental angiogenesis

Blood vessel patterning during angiogenesis is a guided process. Endothelial tip cells located at the end of vascular sprouts are important for vessel guidance. One of the aims of my studies is to determine how endothelial tip cell formation is regulated. Previous studies have shown that the Notch ligand, Delta-like 4 (Dll4), is prominently expressed in tip cells, therefore suggesting a role of Notch signalling in its regulation. In the mouse, suppression of Notch signalling by pharmacological inhibition of γ-secretase or genetic deletion of one Dll4 allele resulted in excessive endothelial tip cell formation and a poorly-patterned, hyperdense vessel network. Induction of Notch signalling by Jagged1 peptide treatment led to a decrease in tip cell formation and a sparse vessel network. Thus, the Notch pathway negatively regulates tip cell formation to promote a non-sprouting endothelial stalk cell. The Notch-regulated ankyrin repeat protein, Nrarp, is a Notch target gene that is highly expressed in stalk cells and endothelial cells located at vessel branchpoints. Deletion of Nrarp in mouse and morpholino knockdown of nrarp-a and nrarp-b in zebrafish resulted in decreased endothelial cell proliferation and junctional instability, leading to ectopic vessel regression and the formation of a sparse vessel network. In endothelial cells, loss of Nrarp function increased Notch signalling but decreased Wnt signalling, thereby confirming its role as a negative and positive modulator of Notch and Wnt signalling, respectively. Mice null for Lymphoid enhancer factor 1 (Lef1) or deficient in endothelial Ctnnb1 also displayed increased vessel regression and decreased vessel density, suggesting a role of canonical Wnt/β-catenin signalling in maintaining vessel stability. In conclusion, my studies show that Notch signalling regulates endothelial tip cell formation and vessel stability to fine-tune vessel patterning. Furthermore, I demonstrate that Nrarp provides a molecular link to Wnt signalling to regulate vessel stability

A study of Notch signalling in development angiogenesis

Blood vessel patterning during angiogenesis is a guided process. Endothelial tip cells located at the end of vascular sprouts are important for vessel guidance. One of the aims of my studies is to determine how endothelial tip cell formation is regulated. Previous studies have shown that the Notch ligand, Delta-like 4 (D114), is prominently expressed in tip cells, therefore suggesting a role of Notch signalling in its regulation. In the mouse, suppression of Notch signalling by pharmacological inhibition of y-secretase or genetic deletion of one DU4 allele resulted in excessive endothelial tip cell formation and a poorly-patterned, hyperdense vessel network. Induction of Notch signalling by Jagged 1 peptide treatment led to a decrease in tip cell formation and a sparse vessel network. Thus, the Notch pathway negatively regulates tip cell formation to promote a non-sprouting endothelial stalk cell. The Notch-regulated ankyrin repeat protein, Nrarp, is a Notch target gene that is highly expressed in stalk cells and endothelial cells located at vessel branchpoints. Deletion of Nrarp in mouse and morpholino knockdown of nrarp-a and nrarp-b in zebrafish resulted in decreased endothelial cell proliferation and junctional instability, leading to ectopic vessel regression and the formation of a sparse vessel network. In endothelial cells, loss of Nrarp function increased Notch signalling but decreased Wnt signalling, thereby confirming its role as a negative and positive modulator of Notch and Wnt signalling, respectively. Mice null for Lymphoid enhancer factor 1 (Lefl) or deficient in endothelial Ctnnbl also displayed increased vessel regression and decreased vessel density, suggesting a role of canonical Wnt/p-catenin signalling in maintaining vessel stability. In conclusion, my studies show that Notch signalling regulates endothelial tip cell formation and vessel stability to fine-tune vessel patterning. Furthermore, I demonstrate that Nrarp provides a molecular link to Wnt signalling to regulate vessel stability

Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo

How vascular tubes build, maintain and adapt continuously perfused lumens to meet local metabolic needs remains poorly understood. Recent studies showed that blood flow itself plays a critical role in the remodelling of vascular networks, and suggested it is also required for the lumenization of new vascular connections. However, it is still unknown how haemodynamic forces contribute to the formation of new vascular lumens during blood vessel morphogenesis. Here we report that blood flow drives lumen expansion during sprouting angiogenesis in vivo by inducing spherical deformations of the apical membrane of endothelial cells, in a process that we have termed inverse blebbing. We show that endothelial cells react to these membrane intrusions by local and transient recruitment and contraction of actomyosin, and that this mechanism is required for single, unidirectional lumen expansion in angiogenic sprouts. Our work identifies inverse membrane blebbing as a cellular response to high external pressure. We show that in the case of blood vessels such membrane dynamics can drive local cell shape changes required for global tissue morphogenesis, shedding light on a pressure-driven mechanism of lumen formation in vertebrates

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis

During angiogenesis, Rho GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4. DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis

Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation

The Lack of ADAM17 Activity during Embryonic Development Causes Hemorrhage and Impairs Vessel Formation

Background: ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development. Methodology/Principal Findings: Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5. Conclusions/Significance: These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function

Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis

The transcription factor Prox1 plays a crucial role in intermediate progenitor maintenance and hippocampal neuron differentiation during adult neurogenesis in the dentate gyrus region of the hippocampus

Low-Dosage Inhibition of DII4 Signaling Promotes Wound Healing by Inducing Functional Neo-Angiogenesis

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing

A core human primary tumor angiogenesis signature identifies the endothelial orphan receptor ELTD1 as a key regulator of angiogenesis.

Limited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation in vitro and in vivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation