RESUMEN
Glioblastoma (GBM) is the most common and aggressive adult brain cancer with an average survival rate of around 15 months in patients receiving standard treatment. Oncolytic adenovirus expressing therapeutic transgenes represent a promising alternative treatment for GBM. Of the many human adenoviral serotypes described to date, adenovirus 5 (HAdV-C5) has been the most utilised clinically and experimentally. However, the use of Ad5 as an anti-cancer agent may be hampered by naturally high seroprevalence rates to HAdV-C5 coupled with the infection of healthy cells via native receptors. To explore whether alternative natural adenoviral tropisms are better suited to GBM therapeutics, we pseudotyped an HAdV-C5-based platform using the fibre knob protein from alternative serotypes. We demonstrate that the adenoviral entry receptor coxsackie, adenovirus receptor (CAR) and CD46 are highly expressed by both GBM and healthy brain tissue, whereas Desmoglein 2 (DSG2) is expressed at a low level in GBM. We demonstrate that adenoviral pseudotypes, engaging CAR, CD46 and DSG2, effectively transduce GBM cells. However, the presence of these receptors on non-transformed cells presents the possibility of off-target effects and therapeutic transgene expression in healthy cells. To enhance the specificity of transgene expression to GBM, we assessed the potential for tumour-specific promoters hTERT and survivin to drive reporter gene expression selectively in GBM cell lines. We demonstrate tight GBM-specific transgene expression using these constructs, indicating that the combination of pseudotyping and tumour-specific promoter approaches may enable the development of efficacious therapies better suited to GBM.
Asunto(s)
Infecciones por Adenoviridae , Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/terapia , Estudios Seroepidemiológicos , Línea Celular Tumoral , Receptores Virales/genética , Adenoviridae/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Vectores Genéticos/genéticaRESUMEN
Blood vessels form vast networks in all vertebrate organs to sustain tissue growth, repair and homeostatic metabolism, but they also contribute to a range of diseases with neovascularisation. It is, therefore, important to define the molecular mechanisms that underpin blood vessel growth. The receptor tyrosine kinase KIT is required for the normal expansion of hematopoietic progenitors that arise during embryogenesis from hemogenic endothelium in the yolk sac and dorsal aorta. Additionally, KIT has been reported to be expressed in endothelial cells during embryonic brain vascularisation and has been implicated in pathological angiogenesis. However, it is neither known whether KIT expression is widespread in normal organ endothelium nor whether it promotes blood vessel growth in developing organs. Here, we have used single-cell analyses to show that KIT is expressed in endothelial cell subsets of several organs, both in the adult and in the developing embryo. Knockout mouse analyses revealed that KIT is dispensable for vascularisation of growing organs in the midgestation embryo, including the lung, liver and brain. By contrast, vascular changes emerged during late-stage embryogenesis in these organs from KIT-deficient embryos, concurrent with severe erythrocyte deficiency and growth retardation. These findings suggest that KIT is not required for developmental tissue vascularisation in physiological conditions, but that KIT deficiency causes foetal anaemia at late gestation and thereby pathological vascular remodelling.
Asunto(s)
Células Endoteliales , Neovascularización Fisiológica , Animales , Embrión de Mamíferos , Femenino , Ratones , Ratones Noqueados , Neovascularización Patológica , Neovascularización Fisiológica/genética , Embarazo , Saco Vitelino/irrigación sanguíneaRESUMEN
The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.
Asunto(s)
Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Linaje de la Célula , Células Endoteliales/citología , Eritrocitos/citología , Células Progenitoras Mieloides/citología , Envejecimiento , Animales , Linaje de la Célula/genética , Proliferación Celular , Células Endoteliales/metabolismo , Eritrocitos/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Integrasas/genética , Integrasas/metabolismo , Hígado/citología , Hígado/embriología , Ratones , Células Progenitoras Mieloides/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Rombencéfalo/irrigación sanguínea , Rombencéfalo/citología , Rombencéfalo/embriología , Transcripción Genética , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
The effects of endogenous and synthetic glucocorticoids on fetal lung maturation are well-established, although the role of leptin in lung development before birth is unclear. This study examined mRNA and protein levels of the signalling long-form leptin receptor (Ob-Rb) in fetal ovine lungs towards term, and after experimental manipulation of glucocorticoid levels in utero by fetal cortisol infusion or maternal dexamethasone treatment. In fetal ovine lungs, Ob-Rb protein was localised to bronchiolar epithelium, bronchial cartilage, vascular endothelium, alveolar macrophages and type II pneumocytes. Pulmonary Ob-Rb mRNA abundance increased between 100 (0.69 fractional gestational age) and 144 days (0.99) of gestation, and by 2-4-fold in response to fetal cortisol infusion and maternal dexamethasone treatment. In contrast, pulmonary Ob-Rb protein levels decreased near term and were halved by glucocorticoid treatment, without any significant change in phosphorylated signal transducer and activator of transcription-3 (pSTAT3) at Ser727, total STAT3 or the pulmonary pSTAT3:STAT3 ratio. Leptin mRNA was undetectable in fetal ovine lungs at the gestational ages studied. These findings demonstrate differential control of pulmonary Ob-Rb transcript abundance and protein translation, and/or post-translational processing, by glucocorticoids in utero. Localisation of Ob-Rb in the fetal ovine lungs, including alveolar type II pneumocytes, suggests a role for leptin signalling in the control of lung growth and maturation before birth.
Asunto(s)
Pulmón/embriología , Pulmón/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Células Epiteliales Alveolares/metabolismo , Animales , Dexametasona/farmacología , Femenino , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Hidrocortisona/sangre , Hidrocortisona/farmacología , Pulmón/efectos de los fármacos , Fosforilación , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Oveja Doméstica , Transducción de SeñalRESUMEN
In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest-derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.
Asunto(s)
Tabiques Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Cresta Neural/metabolismo , Neuropilina-1/metabolismo , Semaforinas/metabolismo , Animales , Apoptosis , Proliferación Celular , Endotelio Vascular/citología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Femenino , Tabiques Cardíacos/citología , Tabiques Cardíacos/metabolismo , Ventrículos Cardíacos/embriología , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Cresta Neural/embriología , Neuropilina-1/deficiencia , Neuropilina-1/genética , Embarazo , Semaforinas/deficiencia , Semaforinas/genética , Transducción de Señal , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular/deficiencia , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cardiac neural crest cells (NCCs) are a transient, migratory cell population exclusive to vertebrate embryos. Ablation, transplantation, and lineage-tracing experiments in chick and mouse have demonstrated their essential role in the remodeling of the initially bilateral and symmetric pharyngeal artery pairs into an aortic arch and for the septation of the cardiac outflow tract into the base of the pulmonary artery and aorta. Accordingly, defective cardiac NCC function is a common cause of congenital birth defects. Here, we review our current understanding of cardiac NCC-mediated vascular remodeling and signaling pathways important for this process. We additionally discuss their contribution to the cardiac valves as well as the still contentious role of cardiac NCCs in the development of the myocardium and conductive system of the heart.
Asunto(s)
Región Branquial/embriología , Sistema Cardiovascular/embriología , Modelos Cardiovasculares , Cresta Neural/embriología , Transducción de Señal/fisiología , Remodelación Vascular/fisiología , Animales , Región Branquial/irrigación sanguínea , Válvulas Cardíacas/embriología , RatonesRESUMEN
Angiogenesis, defined as the sprouting of new blood vessels from preexisting ones, is a biological process of great clinical relevance due to its involvement in disease as well as its therapeutic potential for revascularizing ischemic tissues. The embryonic mouse hindbrain provides an excellent model to study the molecular and cellular mechanisms of angiogenesis in vivo due the simple geometry of the hindbrain vasculature and its easy accessibility for fluorescent or histochemical staining, and for image capture and quantitation. This chapter outlines protocols for dissection, staining, and analysis of the mouse embryo hindbrain vasculature.
Asunto(s)
Neovascularización Fisiológica , Rombencéfalo/irrigación sanguínea , Rombencéfalo/embriología , Animales , Disección , Femenino , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Embarazo , Rombencéfalo/citología , Rombencéfalo/efectos de los fármacos , Coloración y Etiquetado , Tamoxifeno/farmacología , Fijación del TejidoRESUMEN
The formation of the cardiovasculature, consisting of both the heart and blood vessels, is a critical step in embryonic development and relies on three processes termed vasculogenesis, angiogenesis, and vascular remodeling. The transmembrane protein NRP1 is an essential modulator of embryonic angiogenesis with additional roles in vessel remodeling and arteriogenesis. NRP1 also enhances arteriogenesis in adults to alleviate pathological tissue ischemia. However, in certain circumstances, vascular NRP1 signaling can be detrimental, as it may promote cancer by enhancing tumor angiogenesis or contribute to tissue edema by increasing vascular permeability. Understanding the mechanisms of NRP1 signaling is, therefore, of profound importance for the design of therapies aiming to control vascular functions. Previous work has shown that vascular NRP1 can variably serve as a receptor for two secreted glycoproteins, the VEGF-A and SEMA3A, but it also has a poorly understood role as an adhesion receptor. Here, we review current knowledge of NRP1 function during blood vessel growth and homeostasis, with special emphasis on the vascular roles of its multiple ligands and signaling partners.
Asunto(s)
Permeabilidad Capilar , Proteínas de Neoplasias/metabolismo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Neuropilina-1/metabolismo , Remodelación Vascular , Adulto , Animales , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/patología , Semaforina-3A/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1(Y297A/Y297A) mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1(Y297A/Y297A) mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.
Asunto(s)
Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Neuropilina-1/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Peso Corporal/genética , Carcinogénesis/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Miocardio/metabolismo , Miocardio/patología , Neovascularización Patológica/embriología , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Neuropilina-1/metabolismo , Oxígeno , Unión Proteica , Arteria Retiniana/patología , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Rombencéfalo/patología , Análisis de SupervivenciaRESUMEN
The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiogenesis or Matrigel implants, there is no requirement for the breeding of conditional knockout mice. The unique architecture of the hindbrain vasculature allows whole-mount immunolabeling of blood vessels and high-resolution imaging, as well as easy quantification of angiogenic sprouting, network density and vessel caliber. The hindbrain model also permits the visualization of ligand binding to blood vessels in situ and the analysis of blood vessel growth within a natural multicellular microenvironment in which endothelial cells (ECs) interact with non-ECs to refine the 3D organ architecture. The entire procedure, from embryo isolation to imaging and through to results analysis, takes approximately 4 d.
Asunto(s)
Diagnóstico por Imagen/métodos , Embrión de Mamíferos/embriología , Modelos Animales , Neovascularización Fisiológica/fisiología , Rombencéfalo/embriología , Animales , Anticuerpos Monoclonales , Ligandos , Ratones , Rombencéfalo/irrigación sanguíneaRESUMEN
Neuropilin (NRP) 1 is a receptor for the vascular endothelial growth factor (VEGF)-A and is essential for normal angiogenesis. Previous in vitro experiments identified NRP1 interactions with VEGF-A's main signaling receptor VEGFR2 within endothelial cells, but also between nonendothelial NRP1 and endothelial VEGFR2. Consistent with an endothelial role for NRP1 in angiogenesis, we found that VEGFR2 and NRP1 were coexpressed in endothelial tip and stalk cells in the developing brain. In addition, NRP1 was expressed on two cell types that interact with growing brain vessels-the neural progenitors that secrete VEGF-A to stimulate tip cell activity and the pro-angiogenic macrophages that promote tip cell anastomosis. Selective targeting of Nrp1 in each of these cell types demonstrated that neural progenitor- and macrophage-derived NRP1 were dispensable, whereas endothelial NRP1 was essential for normal brain vessel growth. NRP1 therefore promotes brain angiogenesis cell autonomously in endothelium, independently of heterotypic interactions with nonendothelial cells. Genetic mosaic analyses demonstrated a key role for NRP1 in endothelial tip rather than stalk cells during vessel sprouting. Thus, NRP1-expressing endothelial cells attained the tip cell position when competing with NRP1-negative endothelial cells in chimeric vessel sprouts. Taken together, these findings demonstrate that NRP1 promotes endothelial tip cell function during angiogenesis.