Search

Angiogenesis is target for antineoplastic and chemopreventive therapies. The natural phytoalexin resveratrol is found in grapes and red wine as cis and trans stereoisomers. trans-Resveratrol shows antiangiogenic activity, but its mechanism of action is not fully elucidated. Recently, trans-resveratrol has been shown to interact with the β3 integrin subunit, raising the possibility that inhibition of endothelial αvβ3 integrin function may concur to its angiosuppressive activity. To get novel insights about the antiangiogenic activity of resveratrol, we compared cis- and trans-resveratrol stereoisomers for their effect on the angiogenesis process and endothelial αvβ3 integrin function. trans-Resveratrol inhibits endothelial cell proliferation and the repair of mechanically wounded endothelial cell monolayers. Also, it prevents endothelial cell sprouting in fibrin gel, collagen gel invasion, and morphogenesis on Matrigel. In vivo, trans-resveratrol inhibits vascularization of the chick embryo area vasculosa and murine melanoma B16 tumor growth and neovascularization. In all the assays, cis-resveratrol exerts a limited, if any, effect. In keeping with these observations, trans-resveratrol, but not cis-resveratrol, inhibits αvβ3 integrin-dependent endothelial cell adhesion and the recruitment of enhanced green fluorescent protein-tagged β3 integrin in focal adhesion contacts. In conclusion, stereoisomery affects the antiangiogenic activity of resveratrol, the trans isomer being significantly more potent than the cis isoform. The different antiangiogenic potential of resveratrol stereoisomers is related, at least in part, to their different capacity to affect αvβ3 integrin function. This may have profound implications for the design of synthetic antiangiogenic/angiopreventive phytoalexin derivatives. [Mol Cancer Ther 2008;7(12):3761–70]

Supplementary Fig. S1 from αvβ3 Integrin-dependent antiangiogenic activity of resveratrol stereoisomers

Supplementary Figure 1 from Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein–positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis. [Cancer Res 2007;67(7):2927–31]

Supplementary Figure 2 from Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and severe congenital brain arteriovenous malformation, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-functionde novovariants (p=4.79×10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (p=1.22×10-5), which cooperates with p120 RasGAP to limit Ras activation. Other probands had pathogenic variants inACVRL1,NOTCH1,ITGB1, andPTPN11.ACVRL1variants were also identified in a multi-generational VOGM pedigree. Integrative genomics defined developing endothelial cells as a key spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specificEPHB4kinase-domain missense variant exhibited constitutive endothelial Ras/ERK/MAPK activation and impaired hierarchical development of angiogenesis-regulated arterial-capillary-venous networks, but only when carrying a “second-hit” allele. These results illuminate human arterio-venous development and VOGM pathobiology and have clinical implications.

Multipotent hematopoietic stem/progenitor cells (HSPCs) generate all mature blood cells in the erythroid, lymphoid, and myeloid lineages. HSPCs are initially produced in the embryo, via transdifferentiation of hemogenic endothelial cells (hemECs) in the aorta-gonad mesonephros (AGM). HSPCs in the AGM are functionally heterogenous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. This knowledge could inform approaches to overcome the dysregulation of HSPC heterogeneity associated with poor outcomes of autologous transplants. Here we discovered that loss of microRNA (miR)-128 (miR-128Δ/Δ) in zebrafish leads to an expansion of hemECs forming replicative HSPCs in the AGM, and a skew towards the erythroid and lymphoid lineages in larval and adult stages. Furthermore, we found that inhibiting miR-128 during the differentiation of human pluripotent stem cells into hemECs, but not during the endothelial-to-hematopoietic transition, recapitulated the lineage skewing. In vivo, expression of wild-type miR-128 in endothelium restored the blood lineage distribution in miR-128Δ/Δ zebrafish. We found that miR-128 represses the expression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b, and thus promotes Wnt and Notch signaling in hemECs. De-repression of cskn1a1 resulted in hemECs generating replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in hemECs forming lymphoid-biased HSPCs in the AGM and relative mature blood cells in adult. We propose that HSPC heterogeneity is established in hemogenic endothelium prior to transdifferentiation and is programmed in part by Wnt and Notch signaling modulation.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

(1) Background: Whole Exome Sequencing of patients with thoracic aortic aneurysm often identifies “Variants of Uncertain Significance” (VUS), leading to uncertainty in clinical management. We assess a novel mechanism for potential routine assessment of these genes in TAA patients. Zebrafish are increasingly used as experimental models of disease. Advantages include low cost, rapid maturation, and physical transparency, permitting direct microscopic assessment. (2) Methods: Zebrafish loss of function mutations were generated using a CRISPRC/CAS9 approach for EMILIN1 and MIB1 genes similar to VUSs identified in clinical testing. Additionally, “positive control” mutants were constructed for known deleterious variants in FBN1 (Marfan’s) and COL1A2, COL5A1, COL5A2 (Ehlers-Danlos). Zebrafish embryos were followed to six days post-fertilization. Embryos were studied by brightfield and confocal microscopy to ascertain any vascular, cardiac, and skeletal abnormalities. (3) Results: A dramatic pattern of cardiac, cerebral, aortic, and skeletal abnormalities was identified for the known pathogenic FBN1 and COL1A2, COL5A1, and COL5A2 mutants, as well as for the EMILIN1 and MIB1 mutants of prior unknown significance. Visualized abnormalities included hemorrhage (peri-aortic and cranial), cardiomegaly, reduced diameter of the aorta and intersegmental vessels, lower aortic cell counts, and scoliosis (often extremely severe). (4) Conclusion: This pilot study suggests that candidate genes arising in clinical practice may be rapidly assessed via zebrafish mutants—thus permitting evidence-based decisions about pathogenicity. Thus, years-long delays to clinically demonstrate pathogenicity may be obviated. Zebrafish data would represent only one segment of analysis, which would also include frequency of the variant in the general population, in silico genetic analysis, and degree of preservation in phylogeny.

Messenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellular structures responsible for mediating cell adhesion and response to changes in the extracellular matrix (ECM). Using high-throughput single molecule imaging and genomic profiling approaches, we find that mRNAs with distinct sequence characteristics localize to FAs in different human cell types. Notably, ∼85% of FA-mRNAs are not translationally active at steady state or under conditions of FA dissolution or activation. Untranslated mRNA sequences are anchored to FA based on their functional states by the RNA binding protein, G3BP1, forming biomolecular granules. Removing RNA or G3BP1, but not blocking new polypeptide synthesis, dramatically changes FA protein composition and organization, resulting in loss of cell contractility and cellular ability to adapt to changing ECM. We have therefor uncovered a novel, non-coding role for mRNAs as scaffolds to maintain FA structure and function, broadening our understating of noncanonical mRNA functions.

SummaryTissue homeostasis and repair are orchestrated by resident and newly recruited macrophages that alter their gene expression program in response to changes in tissue microenvironment. Embryonic macrophages, such as fetal liver derived macrophages (FLDM) seed the organs, including heart and lung during embryonic development and persist throughout the adult lifetime, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation. Transcriptome analyses of FLDM and BMDM identified differences between them at the level of RNA expression, which correlates imperfectly with protein levels. Post-transcriptional regulation by microRNAs (miRNAs) and RNA-binding proteins determines mRNA stability and translation rate and may override transcriptional cues in response to environmental changes, such as hypoxia. To identify distinct features of FLDM and BMDM response to hypoxia at the level of translation, we employed translating ribosome affinity purification (TRAP) to isolate polysomal RNA. RNA-seq profiling of translated RNA identified distinct hypoxia-induced translational signature of BMDM (Ly6e, vimentin and glycolysis-associated enzymes Pgk1, Tpi1, Aldoa, Ldha) and FLDM (chemokines Ccl7 and Ccl2). By translational profiling of BMDM and FLDM with deletion of the RNA-binding protein HuR, we identified transcripts that were dependent on HuR. These findings highlight the importance of HuR and identify its distinct targets for post-transcriptional regulation of gene expression in embryonic vs. adult-derived macrophages.

SUMMARYOne of the first events in the development of the cardiovascular system is morphogenesis of the main embryonic artery, the dorsal aorta (DA). The DA forms via a conserved genetic process mediated by the migration, specification, and organization of endothelial progenitor cells into a distinct arterial lineage and vessel type. Several angiogenic factors activate different signaling pathways to control DA formation, however the physiological relevance of distinct kinases in this complex process remains unclear. Here, we identify the role of Akt during early vascular development by generating mutant zebrafish lines that lack expression of akt isoforms. Live cell imaging coupled with single cell RNA sequencing of akt mutants reveal that Akt is required for proper development of the DA by sustaining arterial cell progenitor specification and segregation. Mechanistically, inhibition of active FOXO in akt mutants rescues impaired arterial development but not the expression of arterial markers, whereas Notch activation rescues arterial marker expression. Our work suggests that Akt activity is critical for early artery development, in part via FOXO and Notch-mediated regulation.

Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity; however, the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between “epi”-machineries. Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Endothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα’s, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.

Hematopoietic stem and progenitor cells (HSPCs) that establish and maintain the blood system in adult vertebrates arise from the transdifferentiation of hemogenic endothelial cells (hemECs) during embryogenesis. This endothelial-to-hematopoietic transition (EHT) is tightly regulated, but the mechanisms are poorly understood. Here, we show that microRNA (miR)-223-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. Single cell RNA-sequencing revealed that miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid/myeloid lineages by suppressing the expression of mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in N-linked protein glycosylation. High-throughput glycomics of ECs lacking miR-223 showed a decrease of high mannose versus sialylated complex/hybrid sugars on N-glycoproteins involved in EHT such as the metalloprotease Adam10. Endothelial-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied the aberrant HSPC production of miR-223 mutants. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of their hematopoietic fate.One Sentence SummaryThe N-glycan “sugar code” governs the hematopoietic fate of endothelial cells and regulates blood stem cell production in vivo.

Differentiation of hematopoietic stem cells into distinct cell types was thought to occur through a series of discrete, stable progenitor states. Work from Velten et al. (2017) now shows that hematopoietic cells differentiate via a mechanism of continuous lineage priming and thus represent a CLOUD-HSPC.

Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis., info:eu-repo/semantics/published

PURPOSE OF THE REVIEW: Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. RECENT FINDINGS: Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between ‘epi’-machineries. SUMMARY: Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Summary Dicer controls the biogenesis of microRNAs (miRNAs) and is essential for neurogenesis. Recent reports show that the levels and substrate selectivity of DICER result in the preferential biogenesis of specific miRNAs in vitro. However, how dicer expression levels and miRNA biogenesis are regulated in vivo and how this affects neurogenesis is incompletely understood. Here we show that during zebrafish hindbrain development dicer expression levels are controlled by miR-107 to tune the biogenesis of specific miRNAs, such as miR-9, whose levels regulate neurogenesis. Loss of miR-107 function stabilizes dicer levels and miR-9 biogenesis across the ventricular hindbrain zone, resulting in an increase of both proliferating progenitors and postmitotic neurons. miR-9 ectopic accumulation in differentiating neuronal cells recapitulated the excessive neurogenesis phenotype. We propose that miR-107 modulation of dicer levels in differentiating neuronal cells is required to maintain the homeostatic levels of specific miRNAs, whose precise accumulation is essential for neurogenesis.

Anaplastic lymphoma kinase (Alk) and leucocyte tyrosine kinase (Ltk) were identified as "orphan" receptor tyrosine kinases (RTKs) with oncogenic potential. Recently ALKAL1 and ALKAL2 (also named "augmentor-β" and "augmentor-α" or "FAM150A" and "FAM150B," respectively) were discovered as physiological ligands of Alk and Ltk. Here, we employ zebrafish as a model system to explore the physiological function and to characterize in vivo links between Alk and Ltk with their ligands. Unlike the two ligands encoded by mammalian genomes, the zebrafish genome contains three genes: aug-α1, aug-α2, and aug-β Our experiments demonstrate that these ligands play an important role in zebrafish pigment development. Deficiency in aug-α1, aug-α2, and aug-β results in strong impairment in iridophore patterning of embryonic and adult zebrafish that is phenocopied in zebrafish deficient in Ltk. We show that aug-α1 and aug-α2 are essential for embryonic iridophore development and adult body coloration. In contrast, aug-α2 and aug-β are essential for iridophore formation in the adult eye. Importantly, these processes are entirely mediated by Ltk and not by Alk. These experiments establish a physiological link between augmentor ligands and Ltk and demonstrate that particular augmentors activate Ltk in a tissue-specific context to induce iridophore differentiation from neural crest-derived cells and pigment progenitor cells.

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

microRNAs (miRNAs) are small noncoding RNAs that act as posttranscriptional regulators of gene expression by triggering target mRNAs degradation or translational repression. Several studies show the importance of miRNAs in many developmental processes including neurogenesis. In multicellular organisms, most miRNAs are expressed in neural tissue and their dynamic spatiotemporal expression plays a key role in regulating multiple steps of neurogenesis. miRNAs are integrated into neural gene regulatory pathways and control processes such as neural stem cell proliferation, cell fate determination, and neuronal differentiation. In this chapter, we discuss the contribution of highly evolutionary conserved, neural-enriched miRNAs in shaping the central nervous systems of invertebrate and vertebrate model organisms focusing on their conserved and novel roles in controlling neurogenesis.

A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

SummaryAngiogenesis requires coordination of distinct cell behaviors between tip and stalk cells. Although this process is governed by regulatory interactions between the vascular endothelial growth factor (Vegf) and Notch signaling pathways, little is known about the potential role of microRNAs. Through deep sequencing and functional screening in zebrafish, we find that miR-221 is essential for angiogenesis. miR-221 knockdown phenocopied defects associated with loss of the tip cell-expressed Flt4 receptor. Furthermore, miR-221 was required for tip cell proliferation and migration, as well as tip cell potential in mosaic blood vessels. miR-221 knockdown also prevented “hyper-angiogenesis” defects associated with Notch deficiency and miR-221 expression was inhibited by Notch signaling. Finally, miR-221 promoted tip cell behavior through repression of two targets: cyclin dependent kinase inhibitor 1b (cdkn1b) and phosphoinositide-3-kinase regulatory subunit 1 (pik3r1). These results identify miR-221 as an important regulatory node through which tip cell migration and proliferation are controlled during angiogenesis.

Objective— Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P 1 ) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P 1 ortholog in vascular development in zebrafish. Methods and Results— zS1P 1 is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P 1 downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P 1 morphants are characterized by downregulation of vascular endothelial cadherin ( VE-cadherin ) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P 1 morphants. Finally, S1P 1 small interfering RNA transfection and the S1P 1 antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion— The data demonstrate a nonredundant role of zS1P 1 in the regulation of venous endothelial barrier in zebrafish and identify a S1P 1 / VE-cadherin / EphB4a genetic pathway that controls venous vascular integrity.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

Angiogenesis plays a key role in tumour growth and metastasis. The teleost zebrafish (Danio rerio) represents a promising alternative model in cancer research. Here, we describe a zebrafish yolk membrane (ZFYM) angiogenesis assays based on the injection of 1–30 ng of human recombinant FGF2 (rFGF2) in the perivitelline space of zebrafish embryos in the proximity of developing subintestinal vein vessels (SIVs) at 48 hrs after fertilization. The rFGF2 induces a rapid and dose‐dependent angiogenic response from the SIV basket, characterized by the ectopic growth of newly formed, alkaline phosphatase‐positive blood vessels. These vessels are formed by proliferating cells that incorporate bromodeoxyuridine and express the endothelial cell markers vegfr2/kdr and fli1. Microangiography shows that rFGF2‐induced vessels are patent and connected to the systemic circulation of the embryo. In keeping with these observations, fli1:EGFP(+) cells isolated from transgenic tg(fli1:EGFP)(y1) zebrafish embryos express the tyrosine kinase (TK) FGF receptor‐1 (FGFR1) and activate extracellular signal‐regulated kinase signalling when stimulated in vitro by rFGF2. The low molecular weight TK‐FGFR1 inhibitor SU5402 and the high molecular weight FGF2 antagonist long‐pentraxin 3 inhibit the angiogenic activity of rFGF2 when added to fish water or when co‐injected with the growth factor, respectively. Moreover, similar to rFGF2, injection of the zebrafish form of vascular endothelial growth factor‐A (VEGF‐A) induces a significant angiogenic response in the ZFYM assay that is suppressed by the VEGF receptor‐2/KDR TK inhibitor SU5416. The ZFYM assay represents a novel tool for testing the activity of low and high molecular weight inhibitors targeting a defined angiogenic growth factor in zebrafish. The assay may offer significant advantages when compared to other animal models.

The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog–vegf-notch signaling cascade that controls arterial/venous fate.

Xenopus laevis Gremlin has been isolated as a novel dorsalizing factor, belonging to a family of secreted proteins with axial patterning activity [Hsu, D.R., Economides, A.N., Wang, X., Eimon, M., Harland, R.M., 1998. The Xenopus dorsalizing factor gremlin identified a novel family of secreted proteins that antagonize BMP activities. Mol. Cell 1, 673–683] . In a search for genes that control development in zebrafish (Danio rerio), we have identified a sequence homologous to Xenopus gremlin. This paper describes the cloning of zebrafish gremlin (grm) and its expression pattern during development. Our results show that grm encodes a maternal transcript, and the zygotic transcription is turned on at the mid-blastula transition (MBT), when grm is detected in the entire blastoderm. In the gastrula grm becomes restricted to the dorsolateral region of the embryo, and during somitogenesis it is strongly expressed in the presomitic mesoderm and developing somites, and in the ventral neural tube. From 24 hpf to 48 hpf, we show that grm transcription is downregulated in the whole embryo, even though Grm protein is still present and localized into the entire myotome at 48–72 hpf. Finally, grm transcript is strongly downregulated in fibroblast growth factor-8 (fgf8) and sonic hedgehog (shh) mutants, thus implicating a putative role of Fgf/Shh signalling loop in grm expression regulation.

Neovascularization plays an important role in neoplasia and angioproliferative diseases. Two major modalities have been developed so far to affect neovascularization: its prevention by antiangiogenic compounds, and immature vessel disruption by vascular-targeting agents. trans-Resveratrol, found in grapes and wine, exerts antioxidant, antineoplastic, and antiangiogenic activities. Here, among various synthetic trans-resveratrol derivatives tested, 3,5,4'-trimethoxystilbene was an antiangiogenic agent 30 to 100 times more potent than parent compound in inhibiting endothelial cell proliferation, sprouting, collagen gel invasion, and morphogenesis (ID50 = 0.3-3.0 microM). In addition, 3,5,4'-trimethoxystilbene acts as a vascular-targeting agent by causing microtubule disassembling and tubulin depolymerization and by impairing the repositioning of the microtubule organization center and the formation of membrane ruffles in migrating endothelial cells. In keeping with a vascular-targeting ability, 3,5,4'-trimethoxystilbene induced apoptosis only in subconfluent endothelial cells and apoptotic regression of immature vessels in the ex vivo rat aorta ring assay. In vivo, 3,5,4'-trimethoxystilbene caused the rapid stasis of blood flow and regression of intersegmental vessels in the trunk of zebrafish embryos. In addition, it inhibited blood vessel growth and caused the disappearance of pre-existing blood vessels in the area vasculosa of the chick embryo. In conclusion, 3,5,4'-trimethoxystilbene associates an antiangiogenic profile to a significant vascular-targeting activity.

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot grow as a mass above few mm unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.

In this chapter we will describe in detail a method to identify the expression profile of miRNAs in developing vessels in zebrafish embryonic development using Illumina deep sequencing strategy. We will describe how to obtain RNA from FACS-sorted primary endothelial cells from growing vessels at early stages of development and how to prepare high-quality small RNA libraries using the TruSeq small RNA strategy for Illumina Hi-Seq machine. This methodology can be applied to discover and profile all forms of small noncoding RNA, including novel miRNA and sequence variants as well as quantification of miRNAs differentially expressed in endothelial cells during angiogenesis.

In this chapter we will describe in detail a method to identify the expression profile of miRNAs in developing vessels in zebrafish embryonic development using Illumina deep sequencing strategy. We will describe how to obtain RNA from FACS-sorted primary endothelial cells from growing vessels at early stages of development and how to prepare high-quality small RNA libraries using the TruSeq small RNA strategy for Illumina Hi-Seq machine. This methodology can be applied to discover and profile all forms of small noncoding RNA, including novel miRNA and sequence variants as well as quantification of miRNAs differentially expressed in endothelial cells during angiogenesis.

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2 , one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

SummaryThe sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

The regulation of gene expression that determines stem cell fate determination is tightly controlled by both epigenetic and posttranscriptional mechanisms. Indeed, small non-coding RNAs such as microRNAs (miRNAs) are able to regulate neural stem cell fate by targeting chromatin-remodeling pathways. Here, we aim to summarize the latest findings regarding the feedback network of epigenetics and miRNAs during embryonic and adult neurogenesis.

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfcum18 could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

microRNAs (miRNAs) have a pivotal role during the formation and function of the cardiovascular system. More than 300 miRNAs have been currently found within the mammalian genome, however only few specific miRNAs, named endomiRNAs, showed conseved endothelial cell expression and function. In this review we present an overview of the currently known endomiRNAs, focusing on their genome localization, processing and target gene repression during vasculogenesis and angiogenesis.