a-simple-bioassay-for-evaluation-vascular-endothelial-growth

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Peter MacCallum Cancer Centre

Two important new reports identify endocytosis of EphB-ephrinB complexes as a mechanism for switching between cell-cell adhesion and repulsion following plasma membrane contact. Together with the previously described shedding of ephrinA following EphA engagement, these findings resolve the paradox of how an adhesive receptor-ligand interaction generates a repulsive cellular response.

Ephective endocytosis.

Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.

Vascular endothelial growth factor D is dispensable for development of the lymphatic system.

Vascular endothelial growth factor receptor 3 (Vegfr-3) est une kinase de tyrosine qui s&#39;exprime sur l&#39;endoth&eacute;lium lymphatique et que des signaux pour la croissance des vaisseaux lymphatiques (lymphangiogen&egrave;se). VEGF-d, une glycoprot&eacute;ine s&eacute;cr&eacute;t&eacute;e, est l&#39;un des deux ligands activation connus pour Vegfr-3, l&#39;autre &eacute;tant le Vegf-c. VEGF-d stimule la lymphangiogen&egrave;se dans les tissus et les tumeurs ; Cependant, son r&ocirc;le dans le d&eacute;veloppement embryonnaire &eacute;tait inconnue. Ici, nous rapportons la g&eacute;n&eacute;ration et l&#39;analyse des souris mutantes d&eacute;ficientes pour le Vegf-d. Souris d&eacute;ficientes en VEGF-d &eacute;taient en bonne sant&eacute; et fertile, avaient masse corporelle normale et n&#39;affichent aucun changement pathologique coh&eacute;rente avec un d&eacute;faut dans la fonction lymphatique. Les poumons, les sites de forte expression du g&egrave;ne Vegf-d au cours de l&#39;embryogen&egrave;se chez les souris de type sauvage, &eacute;taient normaux chez les souris d&eacute;ficientes en Vegf-d en ce qui concerne les tissus masse et morphologie, sauf que l&#39;abondance des vaisseaux lymphatiques adjacents aux bronchioles a &eacute;t&eacute; l&eacute;g&egrave;rement r&eacute;duite. Exp&eacute;riences d&#39;absorption de teinture a indiqu&eacute; que gros vaisseaux lymphatiques sous la peau &eacute;taient pr&eacute;sents dans des endroits normales &eacute;taient fonctionnels. Petits cutan&eacute;es lymphatics ressemblaient en nombre, l&#39;emplacement et fonction &agrave; ceux dans les contr&ocirc;les de type sauvage. L&#39;absence d&#39;un ph&eacute;notype lymphatique profond chez les souris d&eacute;ficientes en Vegf-d sugg&egrave;re que le Vegf-d ne joue pas un r&ocirc;le majeur dans le d&eacute;veloppement lymphatique ou que Vegf-c ou un autre, comme-encore-inconnu activant Vegfr-3 ligand peut compenser de Vegf-d au cours du d&eacute;veloppement.

Facteur de croissance endoth&eacute;lial vasculaire D est indispensable pour le d&eacute;veloppement du syst&egrave;me lymphatique.

Vascular endothelial growth factor receptor 3 (Vegfr-3) &egrave; una tirosina chinasi che segnali per la crescita dei vasi linfatici (linfangiogenesi) e che si esprime sull&#39;endotelio linfatico. VEGF-d, una glicoproteina secreta, &egrave; uno dei due ligandi attivante noti per Vegfr-3, l&#39;altro &egrave; il Vegf-c. VEGF-d stimola linfangiogenesi nei tessuti e tumori; Tuttavia, il ruolo dello sviluppo embrionale era precedentemente sconosciuto. Qui riportiamo la generazione e l&#39;analisi dei topi mutanti carenti per Vegf-d. VEGF-d-deficient mice erano sani e fertili, avevano massa corporea normale e non visualizzato nessun cambiamento patologico coerenza con un difetto nella funzione linfatica. I polmoni, i siti di forte espressione del gene Vegf-d durante l&#39;embriogenesi nei topi wild-type, erano normali nei topi Vegf-d-carenti rispetto al tessuto massa e morfologia, ad eccezione del fatto che l&#39;abbondanza dei vasi linfatici adiacenti a bronchioli &egrave; stata leggermente ridotta. Esperimenti di assorbimento di colorante indicato che grandi vasi linfatici sotto la pelle erano presenti nelle posizioni normali e sono stati funzionali. Piccoli vasi linfatici dermici erano simili in numero, posizione e funzione a quelli in controlli di tipo selvatico. La mancanza di un fenotipo linfatico profondo nei topi Vegf-d-carenti suggerisce che il Vegf-d non gioca un ruolo importante nello sviluppo linfatico o che Vegf-c o un altro, non ancora sconosciuto ligando Vegfr-3 attivazione pu&ograve; compensare Vegf-d durante lo sviluppo.

Fattore di crescita endoteliale vascolare D &egrave; superfluo per lo sviluppo del sistema linfatico.

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Recently, Ryk-Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, in Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk(-/-) mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk(-/-) embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.

The Wnt receptor Ryk is required for Wnt5a-mediated axon guidance on the contralateral side of the corpus callosum.

More than 10(10) cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.

EphB receptors coordinate migration and proliferation in the intestinal stem cell niche.

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression.

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.

The Wnt receptor Ryk plays a role in mammalian planar cell polarity signaling.

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.

RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain-specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies.

A fully human inhibitory monoclonal antibody to the Wnt receptor RYK.

The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin source of bioactive recombinant human vitronectin (rhVTN) is highly desirable for in vitro use in diverse cell culture systems ranging from basic research settings to clinical-grade production of human cells. We describe modifications to a previously reported heparin-based affinity chromatography procedure that improve yield and achieve efficient removal of endotoxin from washed and urea-solubilized human VTN inclusion bodies following standard autoinduction of expression in Escherichia coli. This simple procedure makes accessible the low-cost expression and purification of large quantities of bioactive rhVTN using basic equipment and facilitates its use in a spectrum of endotoxin-sensitive applications.

Expression and purification of bioactive, low-endotoxin recombinant human vitronectin.

A chronic hyperactivated angiogenic state in cancer plays an important role in tumour growth and metastasis and has been identified as one of the hallmarks of cancer. Inhibition of this process has been associated with tumour suppression in many pre-clinical contexts using different animal tumour models. Anti-angiogenic therapeutics were subsequently developed and used to treat several prevalent types of human cancer. However, recent clinical experience has revealed limitations of this approach in treating cancer as patient response varies over a wide range. Given that there are complex underlying molecular and cellular changes provoked by anti-angiogenic treatment within the tumour microenvironment (TME), it is not surprising that modest effectiveness and resistance have been observed in the clinical setting. This article discusses these issues in the context of VEGF-A-targeted anti-angiogenic treatment of cancer and provides insight into the importance of tumour endothelium for understanding the tumour response to anti-angiogenic therapy. Special consideration is also given to possible approaches for investigating how endothelium contributes to the tumour response to anti-angiogenic agents and for exploring the therapeutic and biomarker potential of targeting tumour endothelium.

Michael M. Halford

Tumour Angiogenesis Program

Peter MacCallum Cancer Centre

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Michael M. Halford

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Peter MacCallum Cancer Centre

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Tumour Angiogenesis Program