Intersegmental Vessels Research Articles

Fine-Tuning of Pten Localization and Phosphatase Activity Is Essential for Zebrafish Angiogenesis

The lipid- and protein phosphatase PTEN is an essential tumor suppressor that is highly conserved among all higher eukaryotes. As an antagonist of the PI3K/Akt cell survival and proliferation pathway, it exerts its most prominent function at the cell membrane, but (PIP3-independent) functions of nuclear PTEN have been discovered as well. PTEN subcellular localization is tightly controlled by its protein conformation. In the closed conformation, PTEN localizes predominantly to the cytoplasm. Opening up of the conformation of PTEN exposes N-terminal and C-terminal regions of the protein that are required for both interaction with the cell membrane and translocation to the nucleus. Lack of Pten leads to hyperbranching of the intersegmental vessels during zebrafish embryogenesis, which is rescued by expression of exogenous Pten. Here, we observed that expression of mutant PTEN with an open conformation rescued the hyperbranching phenotype in pten double homozygous embryos and suppressed the increased p-Akt levels that are characteristic for embryos lacking Pten. In addition, in pten mutant and wild type embryos alike, open conformation PTEN induced stalled intersegmental vessels, which fail to connect with the dorsal longitudinal anastomotic vessel. Functional hyperactivity of open conformation PTEN in comparison to wild type PTEN seems to result predominantly from its enhanced recruitment to the cell membrane. Enhanced recruitment of phosphatase inactive mutants to the membrane did not induce the stalled vessel phenotype nor did it rescue the hyperbranching phenotype in pten double homozygous embryos, indicating that PTEN phosphatase activity is indispensable for its regulatory function during angiogenesis. Taken together, our data suggest that PTEN phosphatase activity needs to be carefully fine-tuned for normal embryogenesis and that the control of its subcellular localization is a key mechanism in this process.

Spatiotemporal expression pattern of the zebrafish aquaporin 8 family during early developmental stages

Aquaporin 8 (Aqp8) is a transmembrane protein that is selectively permeated by water and some small solutes, and physiologically contributes to acid-base equilibrium in the gastrointestinal tract. Here, we described the characterization and spatiotemporal expression pattern of zebrafish aqp8 (zaqp8) gene family, including zaqp8a.1, zaqp8a.2, and zaqp8b, during the early developmental stages. The expression of zaqp8a.1 started first in the lateral plate mesoderm at the 12-somite stage (ss) and then expanded sequentially to the dorsal aorta, intersegmental blood vessels and then to the dorsal longitudinal anastomotic vessel at 24 h post fertilization (hpf). At 28 hpf, expression of zaqp8a.1 was also detected in the embryonic heart tube. Four days post fertilization (dpf), strong zaqp8a.1 expression was detected in the gastrointestinal tract and liver. By 72 hpf, the expression of zaqp8a.2 was first detected in the primitive gut region but not detected in the liver. The expression of zaqp8b was first detected in the intermediate mesoderm at 10 ss. From 24 hpf to 6 dpf, the proximal convoluted segment of the embryonic kidney was marked by zaqp8b expression Overall, these differential expression patterns of aqp8a.1, aqp8a.2, and aqp8b suggest that they possibly play distinct roles throughout the embryonic development by controlling or maintaining organ-specific cellular water homeostasis. Our study provides new evidence that organogenesis requires differential roles of Aqp8 proteins in zebrafish.

Mutation of rnf213a by TALEN causes abnormal angiogenesis and circulation defects in zebrafish

Moyamoya disease (MMD) is characterized by a stenosis at the terminal of the internal carotid artery and an abnormal vascular network at the base of the brain. RNF213 is a susceptibility gene for MMD in East Asians. The role of RNF213 in the etiology of MMD remains unknown. Here we generated rnf213a mutant zebrafish using transcription activator-like effector nuclease (TALEN) technique and described the characteristics of a zebrafish embryonic model of MMD. rnf213a mutant zebrafish developed abnormal angiogenesis in intersegmental vessels and cranial secondary vessels. Endothelial cells exhibited the defects in morphogenesis and formation of vascular tubes despite normal cell to cell contacts under electron microscope. Circulatory disorder was induced by abnormal sprouts in the trunk and head. Reduced circulation in the abnormal vessels was revealed by microangiography. No blood flow permeated across the vessels wall despite the extremely abnormal structure. rnf213a mutant showed lower erythrocyte velocity in dorsal aorta than that in wild-type siblings. In this study, we provided a promising in vivo model for MMD, and this model would aid to understand the function of rnf213a in angiogenesis.

Closantel Suppresses Angiogenesis and Cancer Growth in Zebrafish Models.

Angiogenesis has emerged as an important therapeutic target in several major diseases, including cancer and age-related macular degeneration. The zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. In this study, we have taken advantage of the transgenic Tg (fli1a:EGFP) zebrafish line to screen the U.S. Drug Collection Library and identified 11 old drugs with antiangiogenic activity, including Closantel, an FDA-approved broad-spectrum salicylanilide antiparasitic drug for a variety of types of animals. Closantel was confirmed to have antiangiogenic activity in zebrafish with a half-inhibitory concentration (IC50) at 1.69 μM on the intersegmental vessels and 1.45 μM on the subintestinal vessels. Closantel also markedly suppressed cancer growth in zebrafish xenotransplanted with human lymphoma, cervical cancer, pancreatic cancer, and liver cancer cells, generally in a dose-dependent manner. These data reveal that Closantel has antiangiogenesis and anticancer effects and could be a potential drug candidate for animal and human cancer treatments. Further study is needed to clarify the mechanisms involved in the antiangiogenesis and anticancer effects of Closantel.

Anti-angiogenic Activity and Mechanism of Sesquiterpene Lactones from Centipeda minima

Centipeda minima is a Chinese herbal medicine used in the treatment of various diseases including cancer. An ethanol extract of the herb, its four fractions with different polarities, and two volatile oils prepared by steam distillation (SD) and supercritical fluid extraction (SFE) were investigated for their anti-angiogenic activity in a wild-type zebrafish model using a quantitative endogenous alkaline phosphatase (EAP) assay. The SFE oil displayed potent anti-angiogenic activity. Fifteen sesquiterpene lactones (SLs; compounds 1-15) isolated from the SFE oil were evaluated for their anti-angiogenic effect. Results revealed that pseudoguaianolide type SLs (1-8) inhibited vessel formation in the zebrafish embryos while guaianolide type SLs (9-15) showed little effect. Among the active ones, 6-O-angeloylenolin (1), a major component of SFE oil, possessed the strongest effect by reducing vessel formation in zebrafish embryos to 40% of the control value at 29.7 µM. Further study using the Tg (fli1a:EGFP) y1-type zebrafish model revealed that it blocked both intersegmental blood vessels (ISVs) and subintestinal vessels plexus (SIVs) formation in zebrafish embryos. Real-time polymerase chain reaction assay on the wild-type zebrafish embryos suggested that 6-O-angeloylenolin affected multiple molecular targets related to angiogenesis including VEGF receptor, angiopoietin, and its receptors. Taken together, our findings demonstrate that C. minima possesses anti-angiogenic activity, and 6-O-angeloylenolin is a promising candidate for the development of an anti-angiogenic agent.

Noncanonical function of threonyl-tRNA synthetase regulates vascular development in zebrafish

The canonical functions of Aminoacyl-tRNA synthetases (AARSs) are indispensable for protein synthesis. However, recent evidence indicates that some AARSs possess additional biological functions (noncanonical functions) related to immune responses and vascular development. Here, we identified a zebrafish cq16 mutant presenting the disorganized vessels with abnormal branching of the established intersegmental vessels (ISVs) as well as aberrant patterning of the brain vascular network after 50 h post fertilization. The cq16 mutant gene is responsible for encoding threonyl-tRNA synthetase (tars) with a missense mutation. The abnormal branching of ISVs was caused by the increased expression of vascular endothelial growth factor A (vegfa) in tarscq16 mutant. Inhibition of Vegf signaling suppresses the abnormal vascular branching observed in tarscq16 mutant. Furthermore, injection of human TARS mRNA potently reduced the vascular aberrant branching in tarscq16 mutant, indicating a conserved function of tars in regulating angiogenesis between zebrafish and human. Therefore, we conclude that noncanonical function of tars regulates vascular development presumably by modulating the expression of vegfa.

Preclinical Evaluation of a Novel Orally Available SRC/Raf/VEGFR2 Inhibitor, SKLB646, in the Treatment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive and deadly breast cancer subtype. To date, chemotherapy is the only systemic therapy and prognosis remains poor. Herein, we report the preclinical evaluation of SKLB646 in the treatment of TNBC; SKLB646 is a novel multiple kinase inhibitor developed by us recently. This compound potently inhibited SRC and VEGFR2 with IC50 values of 0.002 μmol/L and 0.012 μmol/L, respectively. It also considerably inhibited B-Raf and C-Raf with IC50 values of 0.022 and 0.019 μmol/L, respectively. It exhibited significant antiproliferation and antiviability activities against TNBC cell lines. Studies of mechanism of action indicated that SKLB646 inhibited the activation of SRC signaling and blocked the MAPK signaling through inhibiting the Raf kinases. Interestingly, SKLB646 dose dependently downregulated the expression of Fra1, a transcriptional factor that plays a critical role in the epithelial-to-mesenchymal transition. In addition, SKLB646 could inhibit HUVEC proliferation, migration, and invasion. It effectively blocked the formation of intersegmental vessels in zebrafish embryos and displayed considerable antiangiogenic effects in the tumor-induced neovascularization zebrafish model. In TNBC xenograft models, SKLB646 suppressed the tumor growth in a dose-dependent manner. Moreover, SKLB646 could remarkably inhibit TNBC cell migration and invasion in vitro. Furthermore, in an experimental lung metastasis model, the overall survival time of groups treated with SKLB646 was much longer compared with the control-, dasatinib-, and paclitaxel-treated groups. In a preliminary pharmacokinetic study, SKLB646 showed good pharmacokinetic properties. Taken together, the preclinical data show that SKLB646 could be a promising lead compound for the treatment of TNBC.

Expression of zTOR-associated microRNAs in zebrafish embryo treated with rapamycin

AimsMicroRNAs (miRNAs) are vital in modulating lifespan and various biological processes including vascular function. The pivotal roles of mammalian target of rapamycin (mTOR) in regulating senescence and angiogenesis have been extensively described. However, the roles of its orthologue, zebrafish target of rapamycin (zTOR) in senescence and angiogenesis remain to be unravelled. In the present study, we aimed to investigate the role of zTOR and identify miRNAs associated with senescence and angiogenesis. Main methodsZebrafish embryos were treated with rapamycin and the inhibition of zTOR and its downstream proteins were validated by immunoblotting. Following the treatment, melanocyte density was quantitated, and senescence and angiogenic responses were determined by senescence-associated beta-galactosidase (SA-β-gal) and endogenous alkaline phosphatase (ALP) staining, respectively. Relative expression of microRNAs were determined by quantitative RT-PCR. Key findingsRapamycin (400nM) suppressed zTOR pathway by down-regulating the phosphorylation of zTOR-associated proteins such as P70S6K and S6K at both 4h post-fertilisation (hpf) and 8hpf while 4E-BP1 was only down-regulated at 8hpf when compared to their respective vehicle controls. Treatment with rapamycin also resulted in significant suppression of melanocyte development and senescence-associated beta-galactosidase (SA-β-gal) activity, and perturbed the development of intersegmental vessels (ISVs) of zebrafish embryos. In addition, the expressions of dre-miR-9-5p and -3p, dre-miR-25-3p and dre-miR-124-3p were significantly up-regulated in embryos treated with rapamycin from 4hpf. SignificanceOur findings suggest the involvement of zTOR in embryonic senescence and angiogenesis which could be potentially mediated by selected miRNAs.

Haploinsufficiency of RCBTB1 is associated with Coats disease and familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) belongs to a group of genetically and clinically heterogeneous disorders in retinal vascular development. To date, in approximately 50% of patients with FEVR, pathogenic mutations have been detected in FZD4, LRP5, TSPAN12, NDP and ZNF408. In this study, we identified two heterozygous frameshift mutations in RCBTB1 from three Taiwanese cases through exome sequencing. In patient-derived lymphoblastoid cell lines (LCLs), the protein level of RCBTB1 is approximately half that of unaffected control LCLs, which is indicative of a haploinsufficiency mechanism. By employing transient transfection and reporter assays for the transcriptional activity of β-catenin, we demonstrated that RCBTB1 participates in the Norrin/FZD4 signaling pathway and that knockdown of RCBTB1 by shRNA significantly reduced nuclear accumulation of β-catenin under Norrin and Wnt3a treatments. Furthermore, transgenic fli1:EGFP zebrafish with rcbtb1 knockdown exhibited anomalies in intersegmental and intraocular vessels. These results strongly support that reduced RCBTB1 expression may lead to defects in angiogenesis through the Norrin-dependent Wnt pathway, and that RCBTB1 is a putative genetic cause of vitreoretinopathies.

Differential Requirement for Pten Lipid and Protein Phosphatase Activity during Zebrafish Embryonic Development.

The lipid- and protein phosphatase PTEN is one of the most frequently mutated tumor suppressor genes in human cancers and many mutations found in tumor samples directly affect PTEN phosphatase activity. In order to understand the functional consequences of these mutations in vivo, the aim of our study was to dissect the role of Pten phosphatase activities during zebrafish embryonic development. As in other model organisms, zebrafish mutants lacking functional Pten are embryonically lethal. Zebrafish have two pten genes and pten double homozygous zebrafish embryos develop a severe pleiotropic phenotype around 4 days post fertilization, which can be largely rescued by re-introduction of pten mRNA at the one-cell stage. We used this assay to characterize the rescue-capacity of Pten and variants with mutations that disrupt lipid, protein or both phosphatase activities. The pleiotropic phenotype at 4dpf could only be rescued by wild type Pten, indicating that both phosphatase activities are required for normal zebrafish embryonic development. An earlier aspect of the phenotype, hyperbranching of intersegmental vessels, however, was rescued by Pten that retained lipid phosphatase activity, independent of protein phosphatase activity. Lipid phosphatase activity was also required for moderating pAkt levels at 4 dpf. We propose that the role of Pten during angiogenesis mainly consists of suppressing PI3K signaling via its lipid phosphatase activity, whereas the complex process of embryonic development requires lipid and protein phosphatase of Pten.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis

Ethnopharmacological relevanceThe root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing “congealed” blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. Material and methodsIn this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. ResultsOur results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25–50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. ConclusionsThe pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Effect of schisantherin A inhibit liver sinusoid endothelial cell function and action against liver fibrosis relating to angiogenesis

To investigate the effect of schisantherin A on liver sinusoid endothelial cell function and angiogenesis. Different dosages (0-40 μmol•L⁻¹) of schisantherin A were incubated 24 h with SK-HEP-1 cells, and the toxicity of SK-HEP-1 cells was assayed by MTT method. The proliferation of SK-HEP-1 cells were induced by the vascular endothelial growth factor (VEGF), with receptor tyrosine kinase inhibitor sorafenib as the control, at the same time, set up the control group, 2, 20 μmol•L⁻¹ schisantherin A were incubated with SK-HEP-1 cells, cell proliferation was analyzed by EdU DNA cell proliferation kit. Fluorescence probe method was used to assay the intracellular NO levels and NOS activity. Tube formation was observed using cell migration and a matrigel tube formation assay. Rat aortic ring assay was performed to observe the sprouting vessels from aortic ring. The fluorescence vessels, the number of functional blood vessels, and intersegmental vessel changes of transgenic zebrafish were also observed. Compared with control group, the proliferation of SK-HEP-1 cells induced by VEGF increased and and the level of NO and NOS activity induced; compared with model group, 2, 20 μmol•L⁻¹ schisantherin A and sorafenib inhibited the proliferation of SK-Hep-1 cells induced by VEGF, and reduced the level of NO and NOS activity. At the dosage of 20 μmol•L⁻¹, schisantherin A attenuated the migration and tube formation of SK-HEP-1 cells induced by VEGF, and also inhibition the formation of rat aortic rings and intersegmental vessel changes of transgenic zebrafish, and significantly reduce the number of vessels in zebrafish. Schisantherin A has potential effects on function of endothelial cell proliferation and angiogenesis.

HYPOXIA-INDUCIBLE FACTOR 2-ALPHA PARTICIPATES IN THE REGULATION OF INTERSEGMENTAL VESSELS ANGIOGENESIS AND NEUROMAST DEPOSITION

Subtle morphogenesis begins during trunk development after the segmentation period in zebrafish. Sprouting angiogenesis forms the trunk vascular system after the extension of the dorsal aorta and cardinal veins. The posterior lateral line primordium migrates from head to tail, forming the sensory organs on the skin. It is still unclear whether this morphogenesis requires several master regulators to control the ongoing process. Hypoxia- inducible factor 2-alpha (Hif2-alpha) plays an essential role in the maturation of both central neural system and digestive system during embryogenesis. We hypothesized that Hif2-alpha also regulates trunk development. Through loss-of-function experiments, we found that Hif2-alpha, but not Hif1-alpha, is necessary for angiogenesis and the formation of neuromasts in the trunk. Hif2-alpha regulates the expression of spleen tyrosine kinase (Syk), maintaining sprouting in the trunk. Furthermore, Hif2-alpha also regulates lymphoid enhancer factor 1 (Lef1), moderating the deposition of neuromasts. The regulation of syk and lef1 expression by Hif2-alpha occurs through the direct binding of Hif2-alpha to the hypoxia- responsive elements (HRE) of syk and lef1. This study provides the first evidence that Hif2-alpha plays a pivotal role in the vertebrate embryonic development of intersegmental vessels and the mechanosensory system in the zebrafish trunk.

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.

Development and characterization of functionalized self-assembling nanopeptide on the effect of angiogenesis for injured brain tissue regeneration

Event Abstract Back to Event Development and characterization of functionalized self-assembling nanopeptide on the effect of angiogenesis for injured brain tissue regeneration Kai-Chiech Chang1, Yung-Jen Chuang2, Wen-Han Chang3, Ming-Yuan Huang3 and Tzu-Wei Wang1 1 National Tsing Hua University, Department of Materials Science and Engineering, Taiwan 2 National Tsing Hua University, Department of Medical Science & Institute of Bioinformatics and Structural Biology, Taiwan 3 Mackay Memorial Hospital, Department of Emergency, Taiwan Introduction: Tissue and organ regeneration has been regarded as an optimal therapeutic strategy for traumatic brain injury. In this study, we proposed a three-dimensional (3D) hydrogel matrix to facilitate neoangiogenesis around damaged brain wound region[1]-[3]. A functionalized peptide, RADA16-SVVYGLR (AcN-RADARADARADARADASVVYGLR-CONH2), is designed, synthesized, and self-assembles into nanofibers network to form hydrogel with fiber entanglements[4]. The hydrogel with mechanical stiffness provides a suitable substrate for encapsulated neural stem cells adhesion and differentiation. The functional peptide, SVVYGLR, had been shown to regulate growth of hemopoietic stem cells and to induce angiogenesis[5],[6]. From previous in vitro and in vivo study, the hydrogel made of RADA16-SVVYGLR was compatible for cells proliferation in zebrafish embryos development. We expect that functionalized peptide hydrogel would create a favorable microenvironment for neural tissue repair and serve as a potential therapeutic application in traumatic brain damages. Method: Peptide solution: All aqueous peptide solutions were prepared by dissolving in Milli-Q water, stored at 4°C. Physiochemical properties analyses: Peptide secondary structure, nanofibers morphology and hydrogel rheology were analyzed by circular dichroism (CD), transmission electron microscopy (TEM), and rheometer, respectively. In vivo study: Zebrafish line (Tg(kdr:EGFP)) were used to perform zebrafish embryo toxicity test (zFET) including analysis of the survival rate and the development of intersegmental vessels (ISVs). Results and Discussion: The CD spectrum showed RADA16 and RADA16-SVVYGLR were in the form of beta sheet with minimum at 216 nm and maximum at 196 nm (Fig. 1). Morphology of both peptides were nanofibers network, developing scaffolds favorable to cellular processes and molecules transportation (Fig. 2). As the CD and TEM results, the self-assembling mechanism was not changed dramatically after linking the motif on the terminal of RADA16. The rheology of hydrogels showed that G’ was higher than G’’, indicating the gel-like property. (Fig. 3) The survival rate of fish embryos treated with peptide hydrogel was similar to that in the control group (Fig. 4). After 72 hours post-fertilization, the phenotypic traits of ISVs growth were identical in fish treated with hydrogel and that in control group, showing the normal physiological growth of ISVs to the DLAVs and PCVs. (Fig. 5) The zFET showed toxicity was not observed. Conclusion: The self-assembling peptides in our study, characterized as beta sheet secondary structure with angiogenic sequence, formed into a gel-like 3D scaffold with nanofibers network which was a suitable matrix for NSCs proliferation and a biocompatible biomaterial by zebrafish animal model. The hydrogel scaffold by functionalized self-assembling peptide has a potential role in clinical application to improve tissue regeneration. Dr. Wen-Han Chang; Dr. Ming-Yuan Huang; Prof. Yung-Jen Chuang; NTHU-Mackay Memorial Hospital Joint Research Program (103N2770E1); Research funding support of Ministry of Science and Technology, R.O.C. (104-2628-E-007-002-MY3)

Toxicity effects of the environmental hormone 4-tert-octylphenol in zebrafish (<i>Danio rerio</i>)

4-tert-octylphenol (4-t-OP), an environmental exogenous estrogen is produced by microbial degradation of alkylphenol polyethoxylates (APEOs). Although it is well known that 4-t-OP can cause the feminization of male, sterility and deficiency of gonad development of aquatic animals by disrupting the endocrine reproductive signaling, less is known about the effects of 4-t-OP on embryonic development. Moreover, the presence of 4-t-OP were detected in umbilical cord blood samples of newborns suggesting infants during development may expose to the risk of 4-t-OP contaminant, hence to investigate the effect of 4-t-OP on physiological function during embryonic development is necessary. In the present study, zebrafish embryos exposed to 4-t-OP were used to evaluate the toxicity of 4-t-OP. The 50% lethal dose (LD50) for wild type zebrafish embryos exposure to 4-t-OP for 3 days is approximately 1.0 mM, and a high ratio of cardiovascular defects were showed in survival embryos. To observe the cardiovascular defects more efficiently, Tg(fil-1:EGFP) zebrafish embryos was used in 4-t-OP exposure treatment. Following exposure Tg(fil-1:EGFP) zebrafish embryos to 4-t-OP at 1.0 mM for 4 days, a highly proportion of defects revealed in cardiovascular system, including pericardical edema, irregular shape or incomplete looping of ventricle and atrium, the absence of intersegmental vessel in the tail of notochord, unformed parachordal vessel and kinks in the caudal vein. The phenotype of cardiovascular defects was accompanied by reduced heart rate and impaired blood circulation. The mRNA expression levels of transcription factors, which are critical for zebrafish heart chamber formation and blood vessel development, were analyzed by RT-PCR. The results showed that the presence of 4-t-OP significantly induce expression level of ERα and ERβ2 , and caused cardiovascular defects by suppressing transcription factor Nkx2.7 , Hand2 , Tbx2a , Tbx2b , Tbx5a , FGF1a , GATA-4 , -5 and -6 in zebrafish. The present study suggests that 4-t-OP affects the cardiovascular development in zebrafish and elucidated that early life exposure to 4-t-OP potentially may take a risk of impaired cardiovascular function.

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

BackgroundPlasminogen 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 FindingsWe 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/SignificanceOur 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.

Factor X Mutant Zebrafish Tolerate a Severe Hemostatic Defect in Early Development Yet Develop Lethal Hemorrhage in Adulthood

Factor X (F10) deficiency is a rare inherited bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted knockout of F10 and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering further studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in development. The zebrafish model is characterized by external development, optical transparency, ability to generate thousands of offspring at low cost, and a highly characterized vasculature. We have used these advantages for more in depth study of the role of the coagulation cascade in developmental regulation of hemostasis and vasculogenesis.We generated a 17 base pair deletion in the zebrafish f10 locus by genome editing with TALENs. Although indistinguishable morphologically from f10+/+ and f10+/- siblings at early stages, f10-/- mutants demonstrated progressive lethality between 1 and 5 months of age. Extensive hemorrhage was identified in multiple tissues starting at 3-4 weeks of age, particularly the brain. Notably, intracranial hemorrhage is a common feature in multiple zebrafish mutants with various vascular defects, including anomalies of endothelial differentiation and specification, apoptosis, and vessel integrity/permeability. Gross inspection of f10 mutant embryos and larvae in the first week of life revealed no apparent defects in circulation or vascular development. Expression of arterial and venous endothelial markers were examined by in situ hybridization at 24 and 72 hours post fertilization, the time period during which axial and intersegmental vessels form, along with early establishment of the vascular network. Markers included ephb2a, cdh5, flk1, flt4, and ephb4, and expression patterns were indistinguishable between mutants and wild type siblings. Acridine orange staining at 5 days post fertilization (dpf) did not detect any dysregulation of apoptosis. Hemoglobin staining found no specific hemorrhage in vehicle or warfarin treated mutants. However, 3 dpf f10-/- mutants did not develop occlusive thrombi in response to laser-mediated venous endothelial injury, indicating that F10 is required for hemostasis. We used quantitative PCR to measure transcription of f10 and downstream coagulation factors in 3 dpf larvae. f10 mRNA was undetectable in homozygous mutants, presumably due to nonsense-mediated decay as a consequence of the TALEN-induced frameshift mutation. fga (fibrinogen alpha) and at3 (antithrombin III) mRNAs were increased by 1.8 and 2.3-fold, respectively (p<0.05), when compared to wild type siblings. Prothrombin (f2) mRNA transcription was slightly decreased, although the effect was not statistically significant.In summary, we have produced a zebrafish model of human F10 deficiency that exhibits a spontaneous adult lethal bleeding phenotype, although early embryonic/larval survival is unaffected despite an underlying severe hemostatic defect. Further study of this mutant may identify species specific factors enabling this early survival. qPCR analysis in mutant larvae suggests that the level of F10 mRNA or protein indirectly regulates other coagulation factors during development. We have also used this model to evaluate the longstanding hypothesis that coagulation cascade mediated thrombin signaling is required for embryonic vasculogenesis. Thus far we have not found evidence for defects in vessel development or integrity, apoptosis, or endothelial differentiation/specification. Taken together, these data suggest that the effects of F10 loss are restricted to hemostasis, and thus therapies should continue to be focused on this in deficient patients. Further study as to why mutant embryos and larvae are able to tolerate what would likely cause lethal hemorrhage in mammals, as well as small molecule screens using this model, could potentially lead to innovative therapeutic modalities for patients with bleeding disorders. DisclosuresJoung:Horizon Discovery: Consultancy; Editas Medicine: Other: Financial interest; Poseida Therapeutics: Other: Financial interest; Hera Testing Laboratories: Other: Financial interest; Transposagen Biopharmaceuticals: Other: Financial interest. Shavit:CSL Behring: Consultancy; Octapharma: Consultancy; Bayer: Consultancy; Baxter: Consultancy.

Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development.

BackgroundThe specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein identity in mice. In this study, we show that nr2f1b is important for vein and tip cell identity during zebrafish development.ResultsNr2f1b mRNA is expressed in ventral lateral mesoderm at 15S stage and in vessels at 24 hpf consistent with a role in early vascular specification. Morpholino knockdown of nr2f1b results in a decrease in both vein cell number and expression of the vein specific marker flt4 and mrc1, suggested its role in venous specification. We also show loss of nr2f1b reduced ISV cell number and impairs ISV growth, which is likely due to the impairment of angiogenic cells migration and/or proliferation by time-lapse imaging. Consequently, nr2f1b morphants showed pericardial edema and circulation defects. Overexpression of nr2f1b under the fli promoter increases the number of venous cells and ISV endothelial cells indicated the function of nr2f1b is required and necessary for vascular development. We further showed that nr2f1b likely interact with Notch signalling. nr2f1b expression is increased in rbpsuh morphants and DAPT-treatment embryos suggested nr2f1b is negatively regulated by Notch activity.ConclusionsWe show nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development, which is mediated by Notch signalings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0209-0) contains supplementary material, which is available to authorized users.

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways.

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products.