Koji Ando

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization

Rap1 potentiates endothelial cell junctions by spatially controlling non-muscle myosin II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.

Cdc42 Mediates Bmp-Induced Sprouting Angiogenesis through Fmnl3-Driven Assembly of Endothelial Filopodia in Zebrafish

During angiogenesis in vivo, endothelial cells (ECs) at the tips of vascular sprouts actively extend filopodia that are filled with bundles of linear actin filaments. To date, signaling pathways involved in the formation of endothelial filopodia have been studied using in-vitro-cultured ECs that behave differently from those in vivo. Herein, we have delineated a signaling pathway that governs the assembly of endothelial filopodia during angiogenic sprouting of the caudal vein plexus (CVP) in zebrafish. During CVP formation, bone morphogenetic protein induces the extension of endothelial filopodia and their migration via Arhgef9b-mediated activation of Cdc42. Active Cdc42 binds to and stimulates Formin-like 3, an actin-regulatory protein of the formin family, which, in turn, promotes the extension of endothelial filopodia to facilitate angiogenic sprouting of the CVP. Thus, this study has elucidated molecular mechanisms underlying the formation of endothelial filopodia and their role in angiogenesis in vivo.

RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock.

Histamine-induced vascular leakage is an integral component of many highly prevalent human diseases, including allergies, asthma, and anaphylaxis. Yet, how histamine induces the disruption of the endothelial barrier is not well defined. By using genetically modified animal models, pharmacologic inhibitors, and a synthetic biology approach, here we show that the small GTPase RhoA mediates histamine-induced vascular leakage. Histamine causes the rapid formation of focal adherens junctions, disrupting the endothelial barrier by acting on H1R Gαq-coupled receptors, which is blunted in endothelial Gαq/11 KO mice. Interfering with RhoA and ROCK function abolishes endothelial permeability, while phospholipase Cβ plays a limited role. Moreover, endothelial-specific RhoA gene deletion prevents vascular leakage and passive cutaneous anaphylaxis in vivo, and ROCK inhibitors protect from lethal systemic anaphylaxis. This study supports a key role for the RhoA signaling circuitry in vascular permeability, thereby identifying novel pharmacological targets for many human diseases characterized by aberrant vascular leakage.

Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish

Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo. To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs. Highlighted article: Live imaging of mural cell dynamics in zebrafish shows how the mural cells develop and cover the endothelial cells during vascular development.

β-Catenin-dependent transcription is central to Bmp-mediated formation of venous vessels.

β-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that β-catenin is required for vascular development. However, the precise function of β-catenin-mediated gene regulation in vascular development is not well understood, since β-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of β-catenin transcriptional activity specifically in ECs and discovered that β-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel–Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates β-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes β-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of β-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, β-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of β-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of β-catenin-mediated gene expression in the development of venous vessels. Highlighted article: In the developing fish caudal vein, β-catenin is activated by BMP in a Wnt-independent manner and cooperates with Aggf1 to control venous endothelial cell differentiation.

Visualizing the cell-cycle progression of endothelial cells in zebrafish.

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.

The parallel growth of motoneuron axons with the dorsal aorta depends on Vegfc/Vegfr3 signaling in zebrafish.

Blood vessels and neurons grow often side by side. However, the molecular and cellular mechanisms underlying their parallel development remain unclear. Here, we report that a subpopulation of secondary motoneurons extends axons ventrally outside of the neural tubes and rostrocaudally as a fascicle beneath the dorsal aorta (DA) in zebrafish. We tried to clarify the mechanism by which these motoneuron axons grow beneath the DA and found that Vegfc in the DA and Vegfr3 in the motoneurons were essential for the axon growth. Forced expression of either Vegfc in arteries or Vegfr3 in motoneurons resulted in enhanced axon growth of motoneurons over the DA. Both vegfr3 morphants and vegfc morphants lost the alignment of motoneuron axons with DA. In addition, forced expression of two mutant forms of Vegfr3 in motoneurons, potentially trapping endogenous Vegfc, resulted in failure of growth of motoneuron axons beneath the DA. Finally, a vegfr3 mutant fish lacked the motoneuron axons beneath the DA. Collectively, Vegfc from the preformed DA guides the axon growth of secondary motoneurons.

P2Y2 receptor-Gq/11 signaling at lipid rafts is required for UTP-induced cell migration in NG 108-15 cells

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y2 receptors are known to couple with Gq family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca2+ ([Ca2+]i) levels. In the present study, we investigated the involvement of lipid rafts in P2Y2 receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Gαq/11 and a part of P2Y2 receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-β-cyclodextrin (CD) disrupted not only lipid raft markers but also Gαq/11 and P2Y2 receptors in this fraction. In the presence of CD, P2Y2 receptor-mediated phosphoinositide hydrolysis and [Ca2+]i elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the Gq/11-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,–20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115–regulator of G protein signaling, which inhibits the G12/13 signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the Gq/11 signaling pathway. These results suggest that lipid rafts are critical for P2Y2 receptor-mediated Gq/11–PLC–Ca2+ signaling and this cascade is important for cell migration in NG 108-15 cells.

Neuronal sFlt1 and Vegfaa determine venous sprouting and spinal cord vascularization

Formation of organ-specific vasculatures requires cross-talk between developing tissue and specialized endothelial cells. Here we show how developing zebrafish spinal cord neurons coordinate vessel growth through balancing of neuron-derived Vegfaa, with neuronal sFlt1 restricting Vegfaa-Kdrl mediated angiogenesis at the neurovascular interface. Neuron-specific loss of flt1 or increased neuronal vegfaa expression promotes angiogenesis and peri-neural tube vascular network formation. Combining loss of neuronal flt1 with gain of vegfaa promotes sprout invasion into the neural tube. On loss of neuronal flt1, ectopic sprouts emanate from veins involving special angiogenic cell behaviours including nuclear positioning and a molecular signature distinct from primary arterial or secondary venous sprouting. Manipulation of arteriovenous identity or Notch signalling established that ectopic sprouting in flt1 mutants requires venous endothelium. Conceptually, our data suggest that spinal cord vascularization proceeds from veins involving two-tiered regulation of neuronal sFlt1 and Vegfaa via a novel sprouting mode.

Dynamic Regulation of Vascular Permeability by Vascular Endothelial Cadherin-Mediated Endothelial Cell-Cell Junctions.

Endothelial cells lining blood vessels regulate vascular barrier function, which controls the passage of plasma proteins and circulating cells across the endothelium. In most normal adult tissues, endothelial cells preserve basal vascular permeability at a low level, while they increase permeability in response to inflammation. Therefore, vascular permeability is tightly controlled by a number of extracellular stimuli and mediators to maintain tissue homeostasis. Accordingly, impaired regulation of endothelial permeability causes various diseases, including chronic inflammation, asthma, edema, sepsis, acute respiratory distress syndrome, anaphylaxis, tumor angiogenesis, and diabetic retinopathy. Vascular endothelial (VE)-cadherin, a member of the classical cadherin superfamily, is a component of cell-to-cell adherens junctions in endothelial cells and plays an important role in regulating vascular permeability. VE-cadherin mediates intercellular adhesion through trans-interactions formed by its extracellular domain, while its cytoplasmic domain is anchored to the actin cytoskeleton via α- and β-catenins, leading to stabilization of VE-cadherin at cell-cell junctions. VE-cadherin-mediated cell adhesions are dynamically, but tightly, controlled by mechanisms that involve protein phosphorylation and reorganization of the actomyosin cytoskeleton. Phosphorylation of VE-cadherin, and its associated-catenins, results in dissociation of the VE-cadherin/catenin complex and internalization of VE-cadherin, leading to increased vascular permeability. Furthermore, reorganization of the actomyosin cytoskeleton by Rap1, a small GTPase that belongs to the Ras subfamily, and Rho family small GTPases, regulates VE-cadherin-mediated cell adhesions to control vascular permeability. In this review, we describe recent progress in understanding the signaling mechanisms that enable dynamic regulation of VE-cadherin adhesions and vascular permeability. In addition, we discuss the possibility of novel therapeutic approaches targeting the signaling pathways controlling VE-cadherin-mediated cell adhesion in diseases associated with vascular hyper-permeability.