Tissue guidance without filopodia

Filopodia are highly dynamic, rod-like protrusions that are found in abundance at the leading edge of migrating cells such as endothelial tip cells and at axonal growth cones of developing neurons. One proposed function of filopodia is that of an environmental probe, which serves to sense guidance cues during neuronal pathfinding and blood vessel patterning. However, recent studies show that tissue guidance occurs unhindered in the absence of filopodia, suggesting a dispensability of filopodia in this process. Here, we discuss evidence that support as well as dispute the role of filopodia in guiding the formation of stereotypic neuronal and blood vessel patterns

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

Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth

Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by in situ repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences

Angiogenesis: a team effort coordinated by notch

The past two decades of angiogenesis research have identified a wealth of pro- and antiangiogenic signals originating from the tissue environment, which control blood vessel density and function. Understanding when and how blood vessels respond to the combination of signals they encounter to achieve a balanced cellular response is a major challenge for the field of developmental and tumor angiogenesis. This review focuses on how endothelial cell-cell communication via the Notch pathway contributes to this signal integration and is essential for functional vessel patterning

Filopodia are dispensable for endothelial tip cell guidance

Actin filaments are instrumental in driving processes such as migration, cytokinesis and endocytosis and provide cells with mechanical support. During angiogenesis, actin-rich filopodia protrusions have been proposed to drive endothelial tip cell functions by translating guidance cues into directional migration and mediating new contacts during anastomosis. To investigate the structural organisation, dynamics and functional importance of F-actin in endothelial cells (ECs) during angiogenesis in vivo, we generated a transgenic zebrafish line expressing Lifeact-EGFP in ECs. Live imaging identifies dynamic and transient F-actin-based structures, such as filopodia, contractile ring and cell cortex, and more persistent F-actin-based structures, such as cell junctions. For functional analysis, we used low concentrations of Latrunculin B that preferentially inhibited F-actin polymerisation in filopodia. In the absence of filopodia, ECs continued to migrate, albeit at reduced velocity. Detailed morphological analysis reveals that ECs generate lamellipodia that are sufficient to drive EC migration when filopodia formation is inhibited. Vessel guidance continues unperturbed during intersegmental vessel development in the absence of filopodia. Additionally, hypersprouting induced by loss of Dll4 and attraction of aberrant vessels towards ectopic sources of Vegfa165 can occur in the absence of endothelial filopodia protrusion. These results reveal that the induction of tip cells and the integration of endothelial guidance cues do not require filopodia. Anastomosis, however, shows regional variations in filopodia requirement, suggesting that ECs might rely on different protrusive structures depending on the nature of the environment or of angiogenic cues

Nrarp coordinates endothelial Notch and Wnt signaling to control vessel density in angiogenesis

When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections

Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis

In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. VEGF-A is also essential for the induction of endothelial tip cells, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)-Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using gamma-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4-Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene, and indicates that modulators of Dll4 or Notch signalling, such as gamma-secretase inhibitors developed for Alzheimer's disease, might find usage as pharmacological regulators of angiogenesis