Silencing of directional migration in roundabout4 knockdown endothelial cells

<p>Abstract</p> <p>Background</p> <p>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 <it>roundabout 4 </it>(<it>robo4</it>), the predominant Robo in endothelial cells using small interfering RNA technology <it>in vitro</it>.</p> <p>Results</p> <p><it>Robo1 and Robo4 </it>knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of <it>robo4 </it>abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while <it>robo1 </it>knockdown cells do not display chemotactic response to serum or VEGF. <it>Robo4 </it>knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in <it>robo4 </it>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.</p> <p>Conclusion</p> <p>This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.</p

Stroma-leukaemic cell interactions : Analysis of stroma environment-induced effect on human acute myeloid leukaemic cells

In spite of the progress made in deciphering regulatory networks of cancer cells on the molecular level, the interaction of tumour cells with their stroma has not been adequately analyzed. Earlier, we have addressed the hypothesis that the murine embryonic microenvironment can induce the differentiation of human tumour cells. To examine such interactions, human leukaemic AML cells were injected into pre-implantation murine blastocysts at embryonic day 3.5 of gestation. Analysis of developing mice revealed the presence of human AML cells in chimaeric embryos and adults and the appearance of haematopoietic differentiation markers on progeny of injected human AML cells. This finding strengthens the notion that the embryonic microenvironment is capable of regulating the proliferation and differentiation of leukaemic AML cells. Based on these results, I embarked to analyse the consequences of stromal environment-induced changes in human AML cells upon in vitro coculture with selected haematopoietic stromal cell lines in terms of changes in differentiation and proliferation properties of AML cells. For this purpose, established human AML cell lines were cocultured on a variety of mitotically inactivated stromal cell lines derived from different murine embryonic/foetal haematopoietic sites such as yolk sac, aorta-gonad-mesonephros (AGM) region and foetal liver. To score for coculture-induced changes, I compared the morphology, histo-chemical properties, immunophenotype, proliferation rate, and gene expression profile in cocultured and non-cocultured AML cells. Results show that, upon coculture of Kasumi-1 cells- a cell line established from a FAB class M2 patient - with AGM-derived DAS 104-4, but not with other stromal cell lines, Kasumi-1 AML cells exibit decreased proliferation and colony formation capabilities and acquire differentiated morphologies. Along this line, coculturing of Kasumi-1 cells resulted in the up-regulation of the myelo-monocytic lineage cell surface markers CD11b and CD14. Coculture also resulted in increase in lysosomal marker CD68, a hallmark of myeloid differentiation. Interestingly, apart from cell lines, coculture on DAS 104-4 stroma was also efficient in inducing myeloid differentiation of patient derived primary M2-AML cells. Moreover, cocultivation of KG-1 cell line on DAS 104-4 showed activation of &#61538;-globin transcription and up-regulation of Glycophorin A on its surface, which indicate DAS 104-4 coculture-induced erythroid differentiation of KG-1 cells. Analysis of the proliferation rate of Kasumi-1 cells using the CFSE retention assay revealed that upon cocultivation on DAS 104-4, but not on NIH 3T3 cells, there is a decrease both in the proliferation rate and in the frequency of colony forming cells in clonogenic methyl cellulose cultures. Cell cycle analysis revealed the coculture-induced accumulation of G1-G0 stage cells. Gene-expression analysis by quantitative RT-PCR revealed a substantial decrease in the amount of AML1 and AML1-ETO fusion transcripts in parallel with an increase in p16, p21, C/EBP&#61537; and PU.1 transcription levels. Interestingly, AML1-ETO transcription down-regulation of AML cells needs direct contact with DAS 104-4 cells. Knocking down AML1-ETO expression by siRNA strategy led to reduction in proliferation and depletion of colony forming cells in Kasumi1 cell population. siRNA-mediated AML1-ETO knock-down Kasumi-1 cells showed increased susceptibility to stroma-induced myeloid differentiation. However, on its own, AML1-ETO down-regulation was not sufficient to induce myeloid differentiation. This indicates that AML1-ETO down-regulation may have an active role on the coculture-induced effect but in addition to AML1-ETO down-regulation, further stimuli are required for the coculture-induced myeloid differentiation in the AML cells. In summary, in the present study I established and characterised a coculture-based in vitro system, which is capable of reducing the proliferation while inducing differentiation of human AML cells. The concept emerging from the studies indicates that the stroma environment can affect leukaemic cell proliferation and differentiation in contact-dependent and CD44 activation-independent manner. Furthermore, this study emphasizes the role of AML1-ETO in AML and indicates that AML1-ETO down-regulation is involved in the stroma-induced differentiation of Kasumi-1 cells. The result described here encourages further investigation into the mechanistic details of molecular and cellular interactions between the leukaemic cells and their stroma, which in turn may lead to the identification of new paradigms for a knowledge-based control and reprogramming of leukaemic cells.Neuere Erkenntnisse in der Tumorforschung belegen, dass die Mikroumgebung neben anderen Faktoren eine bedeutende Komponente im komplexen regulatorischen Netzwerk von Tumorzellen darstellt. Doch obwohl in den letzten Jahren große Fortschritte hinsichtlich des molekularen Verständnisses der Tumorzell-Regulation gemacht wurden, wurden die Interaktionen zwischen Tumor- und Stromazellen bislang nur unzureichend analysiert. In früheren Untersuchungen stellten wir die Hypothese auf, dass die murine embryonale Mikroumgebung humane Tumorzellen zur Differenzierung anregen kann. Um diese Wechselwirkungen zu untersuchen, wurden humane AML-Zellen in murine Blastozysten injiziert und diese in scheinträchtige Ammentiere implantiert. Humane Zellen konnten sowohl in den sich entwickelnden Embryonen als auch in daraus hervorgegangenen adulten Tieren nachgewiesen werden. Außerdem exprimierten die Nachkommen der injizierten AML-Zellen hämatopoetische Differenzierungsmarker. Diese Beobachtungen unterstützen die Hypothese, dass die embryonale Mikroumgebung in der Lage ist, die Proliferation und Differenzierung humaner Leukämiezellen zu beeinflussen. Aufbauend auf diesen Ergebnissen habe ich damit begonnen, die Stroma-vermittelten Veränderungen in humanen AML-Zellen hinsichtlich ihres Proliferations- und Differenzierungsverhaltens in einem in vitro Kokultur-System zu untersuchen. Hierzu wurden etablierte humane AML-Zelllinien mit verschiedenen murinen Stromazelllinien kokultiviert, die embryonalen hämatopoetisch aktiven Geweben (Dottersack, Aorta-Gonaden-Mesonephros-Region, foetale Leber) entstammen. Anschließend wurden verschiedene Parameter, wie Morphology, Histochemie, Immunphänotyp, Proliferationsrate und Expression bestimmter Gene, kokultivierter und nicht kokultivierter AML-Zellen verglichen. Die Ergebnisse zeigen für Kasumi-1 Zellen, eine etablierte humane Leukämie-Zelllinie vom AML FAB-Typ M2, dass die Kokultur mit DAS 104-4, einer murinen Sromazelllinie, die der AGM-Region entstammt, eine Reduzierung der Proliferations- und Koloniebildungs-Fähigkeit hervorruft und sich die Morphologie der AML-Zellen in Richtung eines differenzierteren Zelltyps ändert. Übereinstimmend damit können nach Kokultur die myelo-monozytären Differenzierungsmarker CD11b und CD14 auf der Oberfläche der Kasumi-1-Zellen nachgewiesen werden. Die Kokultur führte ebenfalls zu einer Zunahme des lysosomalen Markers CD68, der ebenfalls eine myeloide Differenzierung kennzeichnet. Bemerkenswert ist, dass DAS 104-4 Stromazellen in der Lage sind, myeloide Differenzierung auch in primären M2-AML-Zellen aus einem leukämischen Patienten zu induzieren. Außerdem wurde in KG-1 AML Zellen nach Kokultur mit DAS 104-4 eine Aktivierung der &#61538;-Globin-Transkription und eine verstärkte Glycophorin-A-Expression beobachtet, was auf eine Differenzierung der KG-1-Zellen in Richtung erythroide Linie hindeutet. Untersuchungen zur Proliferationsfähigkeit von Kasumi-1-Zellen mittels CFSE-Retentions-Messungen ergaben, dass nach Kokultur mit DAS 104-4 - nicht aber mit NIH 3T3-Kontrollzellen - die Zellteilungsrate vermindert ist. Gleiches gilt für die Koloniebildungs-Kapazität in Methylzellulose-Kulturen. Zellzyklus-Analysen zeigen eine kokulturinduzierte Akkumulation der AML-Zellen im G1-G0 Stadium. Genexpressionsanalysen mit Hilfe quantitativer RT-PCR verweisen auf eine deutlich herabgesetzte Transkription von AML1 und dem AML1-ETO-Fusionsgen, verbunden mit einem Anstieg der p16-, p21-, C/EBP&#61537;&#61485; und PU.1-Transkription. Interessanterweise ist die Abnahme von AML1-ETO Transkripten abhängig vom direkten Zellkontakt zwischen AML- und DAS 104-4-Zellen. Wird die AML1-ETO-Expression nach Einsatz spezifischer siRNA herunter reguliert, führt dies zu einer verminderten Proliferation und zur Depletion koloniebildender Zellen innerhalb der Kasumi-1-Population. Außerdem bewirkt der siRNA-vermittelte knockdown von AML1-ETO eine höhere Empfänglichkeit der Kasumi-1-Zellen für die Stroma-induzierte myeloide Differenzierung. Die Verringerung von AML1-ETO Transkripten allein hat allerdings keinen differenzierenden Effekt. Diese Beobachtungen sprechen dafür, dass AML1-ETO zwar aktiv an der kokultur-vermittelten Reaktion beteiligt ist, dass aber zusätzliche Stimuli nötig sind, um myeloide Differenzierung in den AML-Zellen auszulösen. Zusammenfassend lässt sich feststellen, dass in der vorliegenden Arbeit ein Kokultur-basiertes in vitro System entwickelt und charakterisiert wurde, das in der Lage ist, die Proliferationsfähigkeit von humanen AML-Zellen zu senken und ihre Differenzierung in die myeloide Linie zu induzieren. Aus den dargestellten Ergebnissen lässt sich schließen, dass das umgebende embryonale Stroma die Proliferation und Differenzierung leukämischer Zellen beeinflussen kann. Die zugrunde liegenden Mechanismen sind abhängig vom direkten Kontakt zwischen Stroma- und AML-Zellen. Eine CD44-Aktivierung konnte nicht beobachtet werden. Weiterhin liefert die vorliegende Arbeit Hinweise darauf, dass die Verminderung der AML1-ETO-Transkription ein bedeutendes, jedoch nicht das allein auslösende, Ereignis der stroma-induzierten Differenzierung von Kasumi-1-Zellen darstellt. Die hier beschriebenen Resultate regen zu weiterführenden Untersuchungen an, die Aufschluss über zelluläre und molekulare Details der Interaktionen zwischen Leukämischen und Stromazellen geben sollen. Neue Erkenntnisse über die beteiligten Mechanismen könnten den Ansatz bieten, der es erlaubt, leukämische Zellen aktiv zu kontrollieren und zu reprogrammieren

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

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

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 (H2O2 and O2·−) 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 H2O2 and a HPLC method for the determination of O2·– 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.National Institutes of Health (U.S.) (CA150190)National Institutes of Health (U.S.) (grant HL70567)India. Dept. of Science and Technology (Ramanujan Fellowship grant (SR/S2/RJN-04/2010, GAP 0305\DST\CP))National Institutes of Health (U.S.) (HL090712)Children's Research Institute (Washington, D.C.

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

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

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

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

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