Vinculin strengthens the endothelial barrier during vascular development.

Remodelling of cell-cell junctions is crucial for proper tissue development and barrier function. The cadherin-based adherens junctions anchor via ß-catenin and α-catenin to the actomyosin cytoskeleton, together forming a junctional mechanotransduction complex. Tension-induced conformational changes in the mechanosensitive α-catenin protein induce junctional vinculin recruitment. In endothelial cells, vinculin protects the remodelling of VE-cadherin junctions. In this study, we have addressed the role of vinculin in endothelial barrier function in the developing vasculature. In vitro experiments, using endothelial cells in which α-catenin was replaced by a vinculin-binding-deficient mutant, showed that junctional recruitment of vinculin promotes endothelial barrier function. To assess the role of vinculin within blood vessels in vivo, we next investigated barrier function in the vasculature of vcl knockout zebrafish. In the absence of vinculin, sprouting angiogenesis and vessel perfusion still occurred. Intriguingly, the absence of vinculin made the blood vessels more permeable for 10 kDa dextran molecules but not for larger tracers. Taken together, our findings demonstrate that vinculin strengthens the endothelial barrier and prevents vascular leakage in developing vessels.

Interferon-γ drives macrophage reprogramming, cerebrovascular remodelling, and cognitive dysfunction in a zebrafish and a mouse model of ion imbalance and pressure overload.

AIMS: Dysregulated immune response contributes to inefficiency of treatment strategies to control hypertension and reduce the risk of end-organ damage. Uncovering the immune pathways driving the transition from the onset of hypertensive stimulus to the manifestation of multi-organ dysfunction are much-needed insights for immune targeted therapy. METHODS AND RESULTS: To aid visualization of cellular events orchestrating multi-organ pathogenesis, we modelled hypertensive cardiovascular remodelling in zebrafish. Zebrafish larvae exposed to ion-poor environment exhibited rapid angiotensinogen up-regulation, followed by manifestation of arterial hypertension and cardiac remodelling that recapitulates key characteristics of incipient heart failure with preserved ejection fraction. In the brain, time-lapse imaging revealed the occurrence of cerebrovascular regression through endothelial retraction and migration in response to the ion-poor treatment. This phenomenon is associated with macrophage/microglia-endothelial contacts and endothelial junctional retraction. Cytokine and transcriptomic profiling identified systemic up-regulation of interferon-γ and interleukin 1ß and revealed altered macrophage/microglia transcriptional programme characterized by suppression of innate immunity and vasculo/neuroprotective gene expression. Both zebrafish and a murine model of pressure overload-induced brain damage demonstrated that the brain pathology and macrophage/microglia phenotypic alteration are dependent on interferon-γ signalling. In zebrafish, interferon-γ receptor 1 mutation prevents cerebrovascular remodelling and dysregulation of macrophage/microglia transcriptomic profile. Supplementation of bone morphogenetic protein 5 identified from the transcriptomic approach as a down-regulated gene in ion-poor-treated macrophages/microglia that is rescued by interferon-γ blockage, mitigated cerebral microvessel loss. In mice subjected to transverse aortic constriction-induced pressure overload, typically developing cerebrovascular injury, neuroinflammation, and cognitive dysfunction, interferon-γ neutralization protected them from blood-brain barrier disruption, cerebrovascular rarefaction, and cognitive decline. CONCLUSIONS: These findings uncover cellular and molecular players of an immune pathway communicating hypertensive stimulus to structural and functional remodelling of the brain and identify anti-interferon-γ treatment as a promising intervention strategy capable of preventing pressure overload-induced damage of the cerebrovascular and nervous systems.

Vinculin controls endothelial cell junction dynamics during vascular lumen formation.

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

Endothelial cell mechanics and blood flow forces in vascular morphogenesis.

The vertebrate cardiovascular system is made up by a hierarchically structured network of highly specialised blood vessels. This network emerges during early embryogenesis and evolves in size and complexity concomitant with embryonic growth and organ formation. Underlying this plasticity are actin-driven endothelial cell behaviours, which allow endothelial cells to change their shape and move within the vascular network. In this review, we discuss the cellular and molecular mechanisms involved in vascular network formation and how these intrinsic mechanisms are influenced by haemodynamic forces provided by pressurized blood flow. While most of this review focusses on in vivo evidence from zebrafish embryos, we also mention complementary findings obtained in other experimental systems.

Building the complex architectures of vascular networks: Where to branch, where to connect and where to remodel?

The cardiovascular system is the first organ to become functional during vertebrate embryogenesis and is responsible for the distribution of oxygen and nutrients to all cells of the body. The cardiovascular system constitutes a circulatory loop in which blood flows from the heart through arteries into the microvasculature and back through veins to the heart. The vasculature is characterized by the heterogeneity of blood vessels with respect to size, cellular architecture and function, including both larger vessels that are found at defined positions within the body and smaller vessels or vascular beds that are organized in a less stereotyped manner. Recent studies have shed light on how the vascular tree is formed and how the interconnection of all branches is elaborated and maintained. In contrast to many other branched organs such as the lung or the kidney, vessel connection (also called anastomosis) is a key process underlying the formation of vascular networks; each outgrowing angiogenic sprout must anastomose in order to allow blood flow in the newly formed vessel segment. It turns out that during this "sprouting and anastomosis" process, too many vessels are generated, and that blood flow is subsequently optimized through the removal (pruning) of low flow segments. Here, we reflect on the cellular and molecular mechanisms involved in forming the complex architecture of the vasculature through sprouting, anastomosis and pruning, and raise some questions that remain to be addressed in future studies.

The tight junction protein Claudin-5 limits endothelial cell motility.

Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells. In vitro and in vivo studies demonstrate that loss of claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development.

ErbB4 tyrosine kinase inhibition impairs neuromuscular development in zebrafish embryos.

Tyrosine kinase inhibitors are widely used in the clinic, but limited information is available about their toxicity in developing organisms. Here, we tested the effect of tyrosine kinase inhibitors targeting the ErbB receptors for their effects on developing zebrafish ( Danio rerio) embryos. Embryos treated with wide-spectrum pan-ErbB inhibitors or erbb4a-targeting antisense oligonucleotides demonstrated reduced locomotion, reduced diameter of skeletal muscle fibers, and reduced expression of muscle-specific genes, as well as reduced motoneuron length. The phenotypes in the skeletal muscle, as well as the defect in motility, were rescued both by microinjection of human ERBB4 mRNA and by transposon-mediated muscle-specific ERBB4 overexpression. The role of ErbB4 in regulating motility was further controlled by targeted mutation of the endogenous erbb4a locus in the zebrafish genome by CRISPR/Cas9. These observations demonstrate a potential for the ErbB tyrosine kinase inhibitors to induce neuromuscular toxicity in a developing organism via a mechanism involving inhibition of ErbB4 function.

Sprouting and anastomosis in the Drosophila trachea and the vertebrate vasculature: Similarities and differences in cell behaviour.

Branching morphogenesis is a fascinating process whereby a simple network of biological tubes increases its complexity by adding new branches to existing ones, generating an enlarged structure of interconnected tubes. Branching morphogenesis has been studied extensively in animals and much has been learned about the regulation of branching at the cellular and molecular level. Here, we discuss studies of the Drosophila trachea and of the vertebrate vasculature, which have revealed how new branches are formed and connect (anastomose), leading to the establishment of complex tubular networks. We briefly describe the cell behaviour underlying tracheal and vascular branching. Although similar at many levels, the branching and anastomosis processes characterized thus far show a number of differences in cell behaviour, resulting in somewhat different tube architectures in these two organs. We describe the similarities and the differences and discuss them in the context of their possible developmental significance. We finish by highlighting some old and new data, which suggest that live imaging of the development of capillary beds in adult animals might reveal yet unexplored endothelial behaviour of endothelial cells.

Wnt/ß-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling.

Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility.

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction.

Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements.

Live imaging molecular changes in junctional tension upon VE-cadherin in zebrafish.

Forces play diverse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development.

Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis.

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and re-establish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell-specific adhesion proteins, VE-cadherin (VE-cad/Cdh5) and Esama (endothelial cell-selective adhesion molecule a), during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appears similar to wild type, whereas esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, and display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis, and both adhesion proteins are central to endothelial cell recognition during anastomosis.

Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance.

Endothelial cells (ECs) line the inside of blood vessels and respond to mechanical cues generated by blood flow. Mechanical stimuli regulate the localization of YAP by reorganizing the actin cytoskeleton. Here we demonstrate blood-flow-mediated regulation of endothelial YAP in vivo. We indirectly monitored transcriptional activity of Yap1 (zebrafish YAP) and its spatiotemporal localization in living zebrafish and found that Yap1 entered the nucleus and promoted transcription in response to blood flow. In cultured human ECs, laminar shear stress induced nuclear import of YAP and its transcriptional activity in a manner independent of Hippo signaling. We uncovered a molecular mechanism by which flow induced the nuclear translocation of YAP through the regulation of filamentous actin and angiomotin. Yap1 mutant zebrafish showed a defect in vascular stability, indicating an essential role for Yap1 in blood vessels. Our data imply that endothelial Yap1 functions in response to flow to maintain blood vessels.

Absence of 11-keto reduction of cortisone and 11-ketotestosterone in the model organism zebrafish.

Zebrafish are widely used as model organism. Their suitability for endocrine studies, drug screening and toxicity assessements depends on the extent of conservation of specific genes and biochemical pathways between zebrafish and human. Glucocorticoids consist of inactive 11-keto (cortisone and 11-dehydrocorticosterone) and active 11ß-hydroxyl forms (cortisol and corticosterone). In mammals, two 11ß-hydroxysteroid dehydrogenases (11ß-HSD1 and 11ß-HSD2) interconvert active and inactive glucocorticoids, allowing tissue-specific regulation of glucocorticoid action. Furthermore, 11ß-HSDs are involved in the metabolism of 11-oxy androgens. As zebrafish and other teleost fish lack a direct homologue of 11ß-HSD1, we investigated whether they can reduce 11-ketosteroids. We compared glucocorticoid and androgen metabolism between human and zebrafish using recombinant enzymes, microsomal preparations and zebrafish larvae. Our results provide strong evidence for the absence of 11-ketosteroid reduction in zebrafish. Neither human 11ß-HSD3 nor the two zebrafish 11ß-HSD3 homologues, previously hypothesized to reduce 11-ketosteroids, converted cortisone and 11-ketotestosterone (11KT) to their 11ß-hydroxyl forms. Furthermore, zebrafish microsomes were unable to reduce 11-ketosteroids, and exposure of larvae to cortisone or the synthetic analogue prednisone did not affect glucocorticoid-dependent gene expression. Additionally, a dual-role of 11ß-HSD2 by inactivating glucocorticoids and generating the main fish androgen 11KT was supported. Thus, due to the lack of 11-ketosteroid reduction, zebrafish and other teleost fish exhibit a limited tissue-specific regulation of glucocorticoid action, and their androgen production pathway is characterized by sustained 11KT production. These findings are of particular significance when using zebrafish as a model to study endocrine functions, stress responses and effects of pharmaceuticals.

Cell behaviors and dynamics during angiogenesis.

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo This Review summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo.

Mapping the molecular steps of secretory-lysosome-driven tracheal tube fusion.

During development, tubular networks form through the joining of lumenized branches. Further insights into tracheal tube fusion in Drosophila melanogaster now reveal the molecular steps that promote the connection of two apical membrane compartments within a single cell through secretory lysosomes.

Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. METHODOLOGY/PRINCIPAL FINDINGS: We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.

Endothelial cell division in angiogenic sprouts of differing cellular architecture.

The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network.