Caudal Vein Plexus Research Articles

ADAMTS18 Metalloproteinase Regulates Angiogenesis In Vivo: Evidence from ADAMTS18 Deficient Mice and Zebrafish

Background: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) members play crucial role in development, inflammation, cancer, and vascular biology. ADAMTS18 is an orphan ADAMTS for which substrates have yet to be discovered. Using ADAMTS18 knock-out (KO) mice, we demonstrated that ADAMTS18 deficiency in mice generated abnormal vascular phenotypes in particular in common carotid artery that is associated with aggravated thrombosis and ischemic cerebral stroke (Blood 122: 31, 2013). Methods and Results: Here, we further investigated the role of ADAMTS18 in angiogenesis. In an established in vivo angiogenesis model (Matrigel plug assay), ADAMTS18 KO mice showed significantly reduced neovessel formation compared to WT mice (Hemoglobin: WT vs. KO, 76.25 ± 28.8 vs. 50.5 ± 17.4, P = 0.048). To systematically evaluate the effect of ADAMTS18 on embryo angiogenesis, we developed zebrafish ADAMTS18 morpholino (MO) knock-down model. Images of trunk regions were taken at 48-hpf (hours post fertilization) with the vascular structures visualized by eGFP fluorescence. Labeled ISV (intersegmental vessel) and DLAV (dorsal longitudinal anastomotic vessel) showed regular development in the embryo injected with control MO. However, embryos injected with ADAMTS18-e3i3-MO presented a lower number of incomplete and thinner ISVs and ectopic sprouts of dorsal aorta. Additionally, ADAMTS18 knock down impaired formation of the CVP (caudal vein plexus) in zebrafish. In control embryos, CVP were formed honeycomb-like structures at the tail around 48-hpf. In contrast, ADAMTS18 knock down resulted in specific defects in CVP formation. Notably, poor blood circulation in the CV (caudal vein) and CCV (common cardinal vein) was observed in ADAMTS18-e3i3-MO injected zebrafish compared to that of control zebrafish. To disclose the potential signaling molecules involved in vascular abnormities, the proteins extracted from common carotid artery (CCA) of WT and ADAMTS18 KO mice were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. A total of 510 proteins in CCA were identified, and 77 of these proteins (~15%) of ADAMTS18 KO mice were differentially expressed compared to WT mice. 22 of the 77 (~29%) proteins are responsible for the regulation of vascular morphology and angiogenesis. Ingenuity® Pathway Analysis (IPA) predicated that TGFβ signaling (z-score = 2.358) was significantly activated. The key molecules involved in TGFβ signaling were then examined by Western blotting. Compared to WT mice, the expression of ALK-1 (a type of TGFβ receptor I, TβRI), smad-1, -5, phosphorylated smad-1, -5 were significantly decreased in the carotid artery of ADAMTS18 KO mice, while the expression of ALK-5 (another type of TGF-β receptor I) smad-2, -3, phosphorylated smad-2, -3 were significantly increased, which may result in the reduced endothelial cell proliferation and migration in ADAMTS18 KO mice. Conclusions: These data imply crucial roles of ADAMTS18 in the regulation of angiogenesis in vivo. DisclosuresNo relevant conflicts of interest to declare.

ADAMTS13-/-zebrafish Model Created By Crispr/Cas Reveals a Novel Role of ADAMTS13 in Vascular Development and Prothrombotic Phenotype

Background: Deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), a von Willebrand factor (VWF)-cleaving enzyme, in humans results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome. Previous studies have demonstrated that ADAMTS13, particularly the C-terminal domains of ADAMTS13 may stimulate or inhibit angiogenesis, depending on the growth environment. However, the role of ADAMTS13 in vascular development in vivo is not known.Objective: To identify the novel function of ADAMTS13 beyond the proteolysis of VWF, we generated ADAMTS13 knockdown and knockout zebrafish to determine the vascular development and propensity for development of thrombosis.Methods: Morpholino anti-sense RNA was microinjected to the embryos of a double transgenic Tg (gata1:dsRed; fli1:eGFP)zebrafish to transiently block ADAMTS13 translation. Also, CRSIPR/Cas9 system was employed to generate ADAMTS13 knockout zebrafish. The vascular development and thrombus formation were determined by fluorescent microscopy.Results: Approximately 30% of ADAMTS13 knockdown zebrafish exhibited vascular abnormalities in the dwarf intersegmental vessels and narrow caudal vein plexus (Fig. 1A and B). To confirm the phenotype, ADAMTS13 knockout using CRSIPR/Cas system was performed. Approximately 85% of the F0 fish carried insertion or deletion mutations in the targeted region. The founder fish were outcrossed to wild type fish to generate heterozygous F1 fish. Fourteen different mutation sequences with germ line transmission were identified in 33 F1 mutants. The siblings carrying an 8-bp deletion mutation, which created a premature stop codon in the signal peptide region of ADAMTS13, were then crossed to produce F2 progeny. The genotypes and protein expression were further confirmed by sequencing and by Western blot, respectively. As shown, a significant higher rate of vascular development defects, particularly in intersegmental vessels and caudal vein, was observed in homozygous ADAMTS13 knockout fish compared to the heterozygous and wild type fish (Fig. 1C). Thrombosis was triggered by incubation of larvae with various low concentrations (0.06%-0.25%) of FeCl3 and determined by the real-time change of fluorescence intensity within 10 min in the head region of larvae under a fluorescent microscope. When compared with wild type fish, ADAMTS13-/- fish demonstrated dramatically decreased fluorescence signal (Fig.1D), indicative of the cessation of blood flow elsewhere in the body after FeCl3injury.Conclusion: Our results demonstrate that severe deficiency of ADAMTS13 in zebrafish results in abnormalities of vascular development and increases the propensity of thrombosis after oxidative injury in fish larvae. These results suggest that ADAMTS13 may have other substrates than VWF. The zebrafish model may turn out to be a powerful tool for identifying a novel pro-angiogenic or anti-thrombotic agent. [Display omitted] DisclosuresZheng:Alexion: Research Funding; Ablynx: Consultancy.

Modeling the Behavior of Red Blood Cells within the Caudal Vein Plexus of Zebrafish.

Due to the important biological role of red blood cells (RBCs) in vertebrates, the analysis of reshaping and dynamics of RBCs motion is a critical issue in physiology and biomechanics. In this paper the behavior of RBCs within the immature capillary plexus during embryonic development of zebrafish has been analyzed. Relying on the fact that zebrafish embryos are small and optically transparent, it is possible to image the blood flow. In this way the anatomy of blood vessels is monitored along with the circulation throughout their development. Numerical simulations were performed using a specific numerical model that combines fluid flow simulation, modeling of the interaction of individual RBCs immersed in blood plasma with the surrounding fluid and modeling the deformation of individual cells. The results of numerical simulations are in accordance with the in vivo observed region of interest within the caudal vein plexus of the zebrafish embryo. Good agreement of results demonstrates the capabilities of the developed numerical model to predict and analyze the motion and deformation of RBCs in complex geometries. The proposed model (methodology) will help to elucidate different rheological and hematological related pathologies and finally to design better treatment strategies.

Looking back and moving forward: recent advances in understanding of cardiovascular development by imaging of zebrafish.

Development requires cell proliferation, migration, differentiation, apoptosis, and many kinds of cell responses. Cells prepare intracellular conditions to respond to extracellular cues from neighboring cells. We have studied the development of the cardiovascular system (CVS) by visualizing morphology and signaling simultaneously using zebrafish, which express probes for both. Endodermal sheet is required for the bilateral cardiac precursor cell (CPC) migration toward the midline. Endothelial cells (ECs) proliferate specifically in the certain regions of blood vessels. Bone morphogenetic proteins (BMP) induce the remodeling of the caudal vein plexus (CVP) to form the caudal vein (CV). Our findings point to the pre-existing neighboring cells as the cells exhibiting certain responses during the development of CVS. In this review, we introduce recent results of our research on angiogenesis and cardiogenesis by spotlighting the mechanism by which ECs and CPCs are regulated by the cells next to themselves. In addition, we discuss the unanswered questions that should be clarified in the future in the field of CVS development.

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.

Nuclear receptor subfamily 2 group F member 1a (nr2f1a) is required for vascular development in zebrafish.

Genetic regulators and signaling pathways are important for the formation of blood vessels. Transcription factors controlling vein identity, intersegmental vessels (ISV) growth and caudal vein plexus (CVP) formation in zebrafish are little understood as yet. Here, we show the importance of the nuclear receptor subfamily member 1A (nr2f1a) in zebrafish vascular development. Amino acid sequence alignment and phylogenetic analysis of nr2f1a is highly conserved among the vertebrates. Our in situ hybridization results showed nr2f1a mRNA is expressed in the lateral plate mesoderm at 18 somite stage and in vessels at 24–30 hpf, suggesting its roles in vasculization. Consistent with this morpholino-based knockdown of nr2fla impaired ISV growth and failed to develop fenestrated vascular structure in CVP, suggesting that nr2f1a has important roles in controlling ISV and CVP growth. Consequently, nr2f1a morphants showed pericardial edema and circulation defects. We further demonstrated reduced ISV cells and decreased CVP endothelial cells sprouting in nr2f1a morphants, indicating the growth impairment of ISV and CVP is due to a decrease of cell proliferation and migration, but not results from cell death in endothelial cells after morpholino knockdown. To test molecular mechanisms and signals that are associated with nr2f1a, we examined the expression of vascular markers. We found that a loss of nr2f1a results in a decreased expression of vein/ISV specific markers, flt4, mrc1, vascular markers stabilin and ephrinb2. This indicates the regulatory role of nr2f1a in controlling vascular development. We further showed that nr2f1a likely interact with Notch signaling by examining nr2f1a expression in rbpsuh morphants and DAPT-treatment embryos. Together, we show nr2f1a plays a critical role for vascular development in zebrafish.

Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish

Exposure to arsenic in its inorganic form, arsenite, causes adverse effects to many different organs and tissues. Here, we have investigated arsenite-induced adverse effects on vascular tissues in the model organism zebrafish, Danio rerio. Zebrafish embryos were exposed to arsenite at different exposure windows and the susceptibility to vascular tissue damage was recorded at 72hours post fertilization (hpf). Intersegmental vessel sprouting and growth was most perturbed by exposure to arsenite during the 24–48hpf window, while disruption in the condensation of the caudal vein plexus was more often observed at the 48–72hpf exposure window, reflecting when these structures develop during normal embryogenesis. The vascular growth rate was decreased by arsenite exposure, and deviated from that of control embryos at around 24–26.5hpf. We further mapped changes in expression of key regulators of angiogenesis and vasculogenesis. Downregulation of vascular endothelial growth factor receptor 1/fms-related tyrosine kinase 1 (vegfr1/flt1) expression was evident already at 24hpf, coinciding with the decreased vascular growth rate. At later time points, matrix metalloproteinase 9 (mmp9) expression was upregulated, suggesting that arsenite affects the composition of the extracellular matrix. In total, the expression of eight key factors involved in different aspects of vascularization was significantly altered by arsenic exposure. In conclusion, our results show that arsenite is a potent vascular disruptor in the developing zebrafish embryo, a finding that calls for an evaluation of arsenite as a developmental vascular toxicant in mammalian model systems.

MicroRNA-26a Regulates Pathological and Physiological Angiogenesis by Targeting BMP/SMAD1 Signaling

The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3'-untranslated region, an effect that decreased expression of Id1 and increased p21(WAF/CIP) and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti-miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Sphingosine 1-Phosphate Receptor Signaling Regulates Proper Embryonic Vascular Patterning

Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P(1-5)) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development.

Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF-induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH(-/-) mice. Similarly, sEH(-/-) mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

12,13‐dihydroxyoctadecenoic acid regulates hematopoietic stem cell and progenitor cell function in zebrafish and mouse

The soluble epoxide hydrolase (sEH) is a fatty acid epoxide metabolizing enzyme and its deletion and/or inhibition have been linked with altered cardiovascular homeostasis. Epoxyeicosatrienoic acids, main substrates of the sEH are known to promote angiogenesis. The morpholino (MO) mediated downregulation/inhibition of the sEH in zebrafish results in a defective caudal vein plexus (CVP). Beside the vascular defects a reduced number of hematopoietic stem and progenitor cells (HSPC) could be observed. A screen of fatty acids revealed that the sEH metabolite 12,13‐dihydroxyoctadecenoic acid (12,13‐ DiHOME) was able to rescue these HSPC defects. In a CFU‐S12 assay a reduced number of colonies could be detected in spleens of those mice that received bone marrow cells from sEH−/− mice in comparison to cells derived from wild type mice, whereas 12,13‐ DiHOME was able to rescue these phenotype. Furthermore sEH−/− mice showed less HSPC mobilization after G‐CSF treatment. Summarized, the sEH activity regulates vascular sprouting of the caudal vein plexus in the zebrafish and its product 12,13‐DiHOME influences hematopoietic stem and progenitor cell function in zebrafish and mouse.

Abstract 17179: Tissue Factor Pathway Inhibitor Regulates Vascular Patterning in the Developing Zebrafish

Background: The tissue factor pathway regulates vascular structure in the adult through regulation of thrombin generation and signaling. Deletion of tissue factor pathway inhibitor (TFPI) in the mouse results in early intrauterine lethality, precluding study of vascular development. In addition, the carboxyl terminus of TFPI may have a TF-independent effect on vascular structure. We sought to determine the role of TFPI and its carboxyl terminus in vascular development of zebrafish. Methods and results: TFPI mRNA localization studies demonstrated expression in the developing kidney, gut, and vasculature of the zebrafish. We further assessed the role of TFPI in zebrafish angiogenesis, by injecting translational and splice-inhibiting morpholino (MO) oligonucleotides directed against zebrafish TFPI mRNA into embryos from the double transgenic zebrafish Tg(Fli1GFP); Tg(GATA1dsRed) expressing GFP in vascular endothelium and dsRed in erythroid cells. Knockdown of TFPI protein expression was subsequently confirmed by immunoblotting. In a dose-dependent manner, multiple non-overlapping MOs targeting the translation initiation site of TFPI independently and synergistically led to the formation of abnormally targeted intersegmental arteries which crossed somite boundaries by 48 hours post fertilization (hpf). Additionally, a plethoric caudal vein plexus with increased honeycombing was present by 24 hpf. This latter aspect of the phenotype was most striking after a MO targeted against a splice site involving the Kunitz 3 and carboxyl terminus domains of zebrafish TFPI was injected alone. Conclusions: TFPI is expressed during zebrafish development. Morpholino knockdown of TFPI expression alters both arterial and venous patterning in the developing zebrafish, indicating a fundamental role for TFPI in targeting of newly-formed blood vessels. Moreover, different domains of the TFPI protein may preferentially act on distinct vascular networks.

Mutation in utp15 Disrupts Vascular Patterning in a p53-Dependent Manner in Zebrafish Embryos

BackgroundAngiogenesis is the process by which the highly branched and functional vasculature arises from the major vessels, providing developing tissues with nutrients, oxygen, and removing metabolic waste. During embryogenesis, vascular patterning is dependent on a tightly regulated balance between pro- and anti-angiogenic signals, and failure of angiogenesis leads to embryonic lethality. Using the zebrafish as a model organism, we sought to identify genes that influence normal vascular patterning.Methodology and Principal FindingsIn a forward genetic screen, we identified mutant LA1908, which manifests massive apoptosis during early embryogenesis, abnormal expression of several markers of arterial-venous specification, delayed angiogenic sprouting of the intersegmental vessels (ISV), and malformation of the caudal vein plexus (CVP), indicating a critical role for LA1908 in cell survival and angiogenesis. Genetic mapping and sequencing identified a G to A transition in the splice site preceding exon 11 of utp15 in LA1908 mutant embryos. Overexpression of wild type utp15 mRNA suppresses all observed mutant phenotypes, demonstrating a causative relationship between utp15 and LA1908. Furthermore, we found that injecting morpholino oligonucleotides inhibiting p53 translation prevents cell death and rescues the vascular abnormalities, indicating that p53 is downstream of Utp15 deficiency in mediating the LA1908 phenotypes.Conclusions and SignificanceTaken together, our data demonstrate an early embryonic effect of Utp15 deficiency on cell survival and the normal patterning of the vasculature and highlight an anti-angiogenic role of p53 in developing embryos.

Aplexone targets the HMG-CoA reductase pathway and differentially regulates arteriovenous angiogenesis

Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we have identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells.

Critical roles of CD146 in zebrafish vascular development

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial-specific expression of CD146 was verified by whole-mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by-pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in-frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish.