Reactive oxygen species driven angiogenesis by inorganic nanorods.

The exact mechanism of angiogenesis by europium hydroxide nanorods was unclear. In this study we have showed that formation of reactive oxygen species (H(2)O(2) and O(2)·-) is involved in redox signaling pathways during angiogenesis, important for cardiovascular and ischemic diseases. Here we used single-walled carbon nanotube sensor array to measure the single-molecule efflux of H(2)O(2) and a HPLC method for the determination of O(2)·- from endothelial cells in response to proangiogenic factors. Additionally, reactive oxygen species-mediated angiogenesis using inorganic nanorods was observed in transgenic (fli1a:EGFP) zebrafish embryos.

Sox factors transcriptionally regulate ROBO4 gene expression in developing vasculature in zebrafish.

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish.

BACKGROUND: Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp(+) cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli(+)etsrp(+) hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp(+) cell numbers at the LPM. CONCLUSIONS/SIGNIFICANCE: This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.

Nogo-B receptor is essential for angiogenesis in zebrafish via Akt pathway.

Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B-stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)-stimulated phosphorylation of Akt and vascular endothelial growth factor-induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B- and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity.

Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.

A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo.

Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.

Heat-shock inducible Cre strains to study organogenesis in transgenic Xenopus laevis.

The frog Xenopus is a well established vertebrate model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a whole series of methods. We have expanded these approaches by establishing two transgenic Xenopus strains that allow specific interference with the activity of defined genes using a heat-shock inducible Cre recombinase that can induce upon heat-shock expression of a reporter gene in crossings to a corresponding reporter strain. We have applied this binary technique of gene interference in Xenopus development to overexpress the mutated HNF1 beta transcription factor at distinct developmental stages. Induction of HNF1 beta P328L329del by heat-shock at the gastrula stage resulted in a dramatic phenotype including malformation of the pronephros, gut, stomach, abnormal tail development and massive edemas indicative for kidney dysfunction. Thus, we have established the first binary inducible gene expression system in Xenopus laevis that can be used to study organogenesis.

Dusp-5 and Snrk-1 coordinately function during vascular development and disease.

Mitogen-activated protein kinases play an integral role in several cellular processes. To regulate mitogen-activated protein kinases, cells express members of a counteracting group of proteins called phosphatases. In this study, we have identified a specific role that one member of this family of phosphatases, dual-specific phosphatase-5 (Dusp-5) plays in vascular development in vivo. We have determined that dusp-5 is expressed in angioblasts and in established vasculature and that it counteracts the function of a serine threonine kinase, Snrk-1, which also plays a functional role in angioblast development. Together, Dusp-5 and Snrk-1 control angioblast populations in the lateral plate mesoderm with Dusp-5 functioning downstream of Snrk-1. Importantly, mutations in dusp-5 and snrk-1 have been identified in affected tissues of patients with vascular anomalies, implicating the Snrk-1-Dusp-5 signaling pathway in human disease.

Snrk-1 is involved in multiple steps of angioblast development and acts via notch signaling pathway in artery-vein specification in vertebrates.

In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain- and loss-of-function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance, and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.

A whole-animal microplate assay for metabolic rate using zebrafish.

Regulation of whole-body metabolism and energy homeostasis has been shown to require signaling between multiple organs. To identify genetic programs that determine metabolic rate, and compounds that can modify it, a whole-animal assay amenable to large-scale screening was developed. The direct correlation of acid production with metabolic rate was exploited to use a noninvasive colorimetric assay for acid secretion by individual zebrafish larvae in a 96-well plate format. A 3-fold increase in metabolic rate was detected that accompanied development between 24 and 96 h postfertilization. Dynamic changes in metabolic rate were also detected in response to different conditions such as temperature and drug treatments, in general agreement with the rate of oxygen consumption measured concomitantly. This assay was used to measure metabolic rate in the progeny of fish known to carry a recessive mutation in a gene required for ribosome biogenesis ( npo(fW07-g)), which would be expected to reduce energy consumption. A strong correlation was found (p < 10(-6) ) between reduced metabolic rate and genotype even before the developmental defect was visually evident. These studies support the conclusion that whole-animal acid secretion can be used as a readout for energy metabolism, thus enabling large-scale screening for genetic and chemical regulators of metabolic rate in a vertebrate.

Silencing of directional migration in roundabout4 knockdown endothelial cells.

BACKGROUND: Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4), the predominant Robo in endothelial cells using small interfering RNA technology in vitro. RESULTS: Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. CONCLUSION: This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.

The aorta-gonad-mesonephros-derived stroma cell line DAS104-4 induces differentiation of leukemic cells.

While critical steps in the regulation of leukemia cell development have been intensively studied in recent years, less is known about the interactions of leukemic cells with their stroma. Previously, we have shown that human acute myeloid leukemia (AML) cells differentiate upon injection into murine blastocysts. We here describe that human AML Kasumi-1 cells, cocultured with murine aorta-gonad-mesonephros (AGM) region-derived DAS104-4 stromal cells, decrease proliferation and colony formation efficiency; and up-regulate myelo-monocytic cell surface markers. Gene expression analysis showed decreased transcription of the AML1-ETO fusion gene and increased transcription of p16 (INK4A), p21 (WAF1) and C/EBPalpha genes. Coculture can induce myeloid differentiation also in patient-derived AML cells. Our findings strengthen the notion that the embryonic milieu can regulate the proliferation and differentiation of leukemic cells.