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Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.

Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning. Throughout life, blood vessels are constantly remodelled to ensure that oxygen and nutrients reach every part of the body where they are needed. If a tissue is not receiving an adequate blood flow, existing blood vessels may widen or new blood vessels may sprout from their walls. In certain diseases, such as cancer, blood vessels may grow excessively to form disorganized networks, and preventing this growth may help to treat these conditions. However, we do not fully understand how the body controls the size, shape and branching pattern of blood vessels. For a new blood vessel to sprout out of an existing vessel, the tip of the new branch must first develop. The tip forms when the endothelial cells that line the blood vessel are activated by a protein called vascular endothelial growth factor A (Vegfa), which is produced by the surrounding tissue. The activated endothelial cells respond to Vegfa stimulation by producing the protein Dll4, which talks to neighboring endothelial cells to prevent them from also forming new tips. In a way, this process bears all the signs of a competition between cells, as they fight for which one is allowed to take the lead. The losers of this competition, when forced into subordination by the tips, also serve an important function, as they will help to form and elongate the base of the new sprout. Although it is known that changes in the levels of Vegfa in tissues can cause blood vessel branching to alter dramatically, the mechanisms that enable this to occur are not well understood. Computer simulations of the process predicted that an unexpected synchronization of Dll4 dynamics would be triggered when Vegfa levels increased; however, this remained to be observed in real cells. Ubezio, Blanco et al. have now used fluorescent markers to observe the Dll4 production in lab-grown mouse endothelial cells as they formed new vessel sprouts in response to Vegfa. This revealed that the levels of Dll4 fluctuate widely in individual cells. Time-lapse movies of the cells showed that as a new sprout forms, the levels of Dll4 in neighbouring cells fluctuate in an uncoordinated manner. However, increasing the amount of Vegfa in the cells indeed synchronizes these fluctuations. This causes the new sprout to retract and allows the original blood vessel to widen. Increasing the levels of Dll4 had the same effect. Further experiments confirmed that increasing the amount of Vegfa also reduces blood vessel branching in tumours in mice by synchronizing the fluctuations in the levels of Dll4 in neighbouring endothelial cells. In the future, these results could help refine anti-cancer treatments that work by blocking the activity of Vegfa and Dll4.

The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis. This study identifies a crucial role for fatty acid oxidation (FAO) in endothelial cells during angiogenesis, and reveals that fatty-acid-derived carbons are used for the de novo synthesis of nucleotides, and hence FAO stimulates vessel sprouting by increasing endothelial cell proliferation. Fatty acids a carbon source for angiogenesis Peter Carmeliet and colleagues identify a crucial role for the oxidation of fatty acids in endothelial cells during angiogenesis. They show that fatty acids provide the carbons for the de novo synthesis of nucleotides, and hence fatty acid oxidation stimulates vessel sprouting by increasing endothelial cell proliferation. Pharmacological blockade of fatty acid oxidation can reduce pathological angiogenesis in a mouse model of retinopathy of prematurity.

Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies. Endothelial cells undergo rearrangements during angiogenic sprouting. Gerhardt and colleagues show that the flux in Notch signalling levels in individual cells of sprouting vessels results in differential dynamics of VE-cadherin at junctions to drive functional endothelial cell rearrangements during sprouting. They also find that differential VE-cadherin dynamics are affected in retinopathy and tumour vessels.

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus. Blood vessels play an essential role in growth and development as they transport many important molecules that help cells to survive. Throughout life, the forces that act on the blood vessels help to remodel the vessel network to ensure that blood gets to the parts of the body that need it. For example, the movement of blood across the surface of the endothelial cells that line the inside of the blood vessels applies a force called “shear stress” to the cells. The endothelial cells respond and adapt to the stress by altering their shape, patterns of gene activity and internal organization (known as their polarity). It was not fully understood exactly how the forces acting on endothelial cells help to remodel the blood vessel network. Franco et al. have now investigated how a signalling pathway known as non-canonical Wnt signalling affects the remodelling of blood vessels in mice, and found that this pathway stabilizes existing connections between vessels. Disrupting non-canonical Wnt signalling, by genetically engineering mice to lack proteins called Wnt5a and Wnt11, increased the sensitivity of endothelial cells to shear stress. Franco et al. then built a computer model that simulates blood flow and endothelial cell polarity in a network of blood vessels; this enabled them to measure the endothelial cells’ response to blood flow in complex vascular networks. The model was then used to show that endothelial cells lacking non-canonical Wnt signalling are able to reorient and become polarized against the direction of blood flow at lower levels of shear stress. Thus, non-canonical Wnt signalling helps to raise the threshold of shear stress above which endothelial cells change their properties. Further work is now needed to identify how non-canonical Wnt signalling interferes with the ability of the endothelial cells to sense shear stress levels.

It takes two to make blood vessels-endothelial cells and pericytes. While the endothelial cells are the better characterized of the two, pericytes are now coming into focus as important regulators of angiogenesis and blood vessel function, and as potential drug targets. However, pericytes are still surrounded by much controversy. They are difficult to define, they constitute a heterogeneous population of cells, and their ontogeny is not well understood. They are plastic and have the capacity to differentiate into other mesenchymal cell types, such as smooth muscle cells, fibroblasts and osteoblasts. Recent interest in pericytes also stems from their potential involvement in diseases such as diabetic microangiopathy, tissue fibrosis, cancer, atherosclerosis and Alzheimer's disease. The present review focuses on the role of pericytes in physiological angiogenesis. The currently favored view states that the initial endothelial tubes form without pericyte contact, and that subsequent acquisition of pericyte coverage leads to vessel remodeling, maturation and stabilization. Improved means of identifying and visualizing pericytes now challenge this view and show that high numbers of pericytes invest in actively sprouting and remodeling vessels. Genetic data demonstrate the critical importance of pericytes for vascular morphogenesis and function, and imply specific roles for the cell type in various aspects of angiogenesis.

Cell polarity involves the asymmetric distribution of cellular components such as signalling molecules and organelles within a cell, alterations in cell morphology and cell-cell contacts. Advances in fluorescence microscopy and deep learning algorithms open up a wealth of unprecedented opportunities to characterise various aspects of cell polarity, but also create new challenges for comprehensible and interpretable image data analysis workflows to fully exploit these new opportunities. Here we present Polarity-JaM, an open source package for reproducible exploratory image analysis that provides versatile methods for single cell segmentation, feature extraction and statistical analysis. We demonstrate our analysis using fluorescence image data of endothelial cells and their collective behaviour, which has been shown to be essential for vascular development and disease. The general architecture of the software allows its application to other cell types and imaging modalities, as well as seamless integration into common image analysis workflows, see https://polarityjam.readthedocs.io . We also provide a web application for circular statistics and data visualisation, available at www.polarityjam.com , and a Napari plug-in, each with a graphical user interface to facilitate exploratory analysis. We propose a holistic image analysis workflow that is accessible to the end user in bench science, enabling comprehensive analysis of image data. Studying cell polarity is often limited by the complexity and variety of image data. Polarity-JaM is a software suite streamlining cell polarity analysis with easy-to-use tools and interfaces demonstrated for endothelial cell behaviour.

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

Sprouting angiogenesis plays a key role during bone regeneration. For example, insufficient early revascularization of the injured site can lead to delayed or non-healing. During sprouting, endothelial cells are known to be mechano-sensitive and respond to local mechanical stimuli. Endothelial cells interact and communicate mechanically with their surroundings, such as outer-vascular stromal cells, through cell-induced traction forces. In addition, external physiological loads act at the healing site, resulting in tissue deformations and impacting cellular arrangements. How these two distinct mechanical cues (cell-induced and external) impact angiogenesis and sprout patterning in early bone healing remains however largely unknown. Therefore, the aim of this study was to investigate the relative role of externally applied and cell-induced mechanical signals in driving sprout patterning at the onset of bone healing. To investigate cellular self-organisation in early bone healing, an in silico model accounting for the mechano-regulation of sprouting angiogenesis and stromal cell organization was developed. Computer model predictions were compared to in vivo experiments of a mouse osteotomy model stabilized with a rigid or a semirigid fixation system. We found that the magnitude and orientation of principal strains within the healing region can explain experimentally observed sprout patterning, under both fixation conditions. Furthermore, upon simulating the selective inhibition of either cell-induced or externally applied mechanical cues, external mechanical signals appear to overrule the mechanical communication acting on a cell-cell interaction level. Such findings illustrate the relevance of external mechanical signals over the local cell-mediated mechanical cues and could be used in the design of fracture treatment strategies for bone regeneration.

In this study, we evaluated the consequences of reducing Galectin-1 (Gal-1) in the tumor micro-environment (TME) of glioblastoma multiforme (GBM), via nose-to-brain transport. Gal-1 is overexpressed in GBM and drives chemo- and immunotherapy resistance. To promote nose-to-brain transport, we designed siRNA targeting Gal-1 (siGal-1) loaded chitosan nanoparticles that silence Gal-1 in the TME. Intranasal siGal-1 delivery induces a remarkable switch in the TME composition, with reduced myeloid suppressor cells and regulatory T cells, and increased CD4+ and CD8+ T cells. Gal-1 knock-down reduces macrophages’ polarization switch from M1 (pro-inflammatory) to M2 (anti-inflammatory) during GBM progression. These changes are accompanied by normalization of the tumor vasculature and increased survival for tumor bearing mice. The combination of siGal-1 treatment with temozolomide or immunotherapy (dendritic cell vaccination and PD-1 blocking) displays synergistic effects, increasing the survival of tumor bearing mice. Moreover, we could confirm the role of Gal-1 on lymphocytes in GBM patients by matching the Gal-1 expression and their T cell signatures. These findings indicate that intranasal siGal-1 nanoparticle delivery could be a valuable adjuvant treatment to increase the efficiency of immune-checkpoint blockade and chemotherapy.