Asunto(s)
Factores de Crecimiento Endotelial , Sistema Linfático/embriología , Animales , Hibridación in Situ , Ratones , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas Receptoras , Receptores de Superficie Celular , Factor C de Crecimiento Endotelial Vascular , Factor D de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells.
Asunto(s)
Sistema Linfático/fisiopatología , Sarcoma Experimental/fisiopatología , Animales , Factores de Crecimiento Endotelial/metabolismo , Endotelio Linfático/metabolismo , Endotelio Linfático/patología , Endotelio Linfático/fisiopatología , Femenino , Fluorescencia , Hibridación in Situ , Sistema Linfático/anatomía & histología , Sistema Linfático/metabolismo , Linfografía/métodos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Sarcoma Experimental/patología , Factor C de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) is a key modulator of angiogenesis during development and in adult tissues, whereas the related VEGF-C has been shown to induce both lymphangiogenesis and angiogenesis. To better understand the specific functions of these growth factors, we have here analyzed their binding to sections of mouse embryonic and adult tissues and compared the distribution of the bound growth factors with the expression patterns of the 3 known members of the VEGF receptor family as well as with neuropilin-1, a coreceptor for VEGF(165). Partially overlapping patterns of VEGF and VEGF-C binding were obtained in embryonic tissues, consistent with the expression of all known VEGF receptors by vascular endothelial cells. However, the most striking differences of binding were observed in the developing and adult heart, in which VEGF decorated all vessels, whereas strong VEGF-C signals were obtained only from epicardial vessels. In the lymph nodes, VEGF and VEGF-C showed distinct binding patterns in agreement with the differential location of their specific receptors. These results show that both VEGF-C and VEGF target embryonic blood vessels, whereas a more selective binding of VEGF-C occurs to its lymphatic vascular receptor in certain adult tissues. Our results suggest that VEGF and VEGF-C have both overlapping and distinct activities via their endothelial receptors.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/metabolismo , Proteínas Fetales/metabolismo , Linfocinas/metabolismo , Proteínas Musculares/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Animales , Sitios de Unión , Vasos Coronarios/metabolismo , Endotelio Vascular/embriología , Corazón Fetal/metabolismo , Regulación de la Expresión Génica , Edad Gestacional , Corazón/crecimiento & desarrollo , Humanos , Hibridación in Situ , Ganglios Linfáticos/crecimiento & desarrollo , Ganglios Linfáticos/metabolismo , Ratones , Miocardio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1 , Especificidad de Órganos , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factor C de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor B (VEGF-B) is structurally closely related to VEGF and binds one of its receptors, VEGFR-1. In situ hybridization and immunohistochemistry were used to localize VEGF-B mRNA and protein in embryonic mouse tissues. In 8.5-17.5 day embryos, VEGF-B was most prominently expressed in the developing myocardium, but not in the cardiac cushion tissue. The strong expression in the heart persisted at later developmental stages, while weaker signals were obtained from several other tissues, including developing muscle, bone, pancreas, adrenal gland, and from the smooth muscle cell layer of several larger vessels, but not from endothelial cells. VEGF-B is likely to act in a paracrine fashion, as its receptor is almost exclusively present in endothelial cells. VEGF-B may have a role in vascularization of the heart, skeletal muscles and developing bones, and in paracrine interactions between endothelial and surrounding muscle cells.
Asunto(s)
Sistema Cardiovascular/embriología , Factores de Crecimiento Endotelial/análisis , Factores de Crecimiento Endotelial/fisiología , Comunicación Paracrina , Animales , Baculoviridae/metabolismo , Células Cultivadas , Embrión de Mamíferos/anatomía & histología , Corazón/anatomía & histología , Corazón/embriología , Proteínas de Insectos/metabolismo , Ratones , Miocardio/metabolismo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Distribución Tisular , Factor B de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel development in embryos and angiogenesis in adult tissues. Unlike VEGF, the related VEGF-C stimulates the growth of lymphatic vessels through its specific lymphatic endothelial receptor VEGFR-3. Here it is shown that targeted inactivation of the gene encoding VEGFR-3 resulted in defective blood vessel development in early mouse embryos. Vasculogenesis and angiogenesis occurred, but large vessels became abnormally organized with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at embryonic day 9.5. Thus, VEGFR-3 has an essential role in the development of the embryonic cardiovascular system before the emergence of the lymphatic vessels.
Asunto(s)
Vasos Sanguíneos/embriología , Sistema Cardiovascular/embriología , Endotelio Vascular/embriología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Superficie Celular/fisiología , Animales , Vasos Sanguíneos/química , Sistema Cardiovascular/química , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/química , Desarrollo Embrionario y Fetal , Factores de Crecimiento Endotelial/análisis , Endotelio Vascular/química , Marcación de Gen , Hematopoyesis , Heterocigoto , Homocigoto , Inmunohistoquímica , Hibridación in Situ , Ligandos , Ratones , Neovascularización Fisiológica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Transducción de Señal , Factor C de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.
Asunto(s)
Endotelio Linfático/metabolismo , Neoplasias de Tejido Vascular/metabolismo , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/biosíntesis , Adulto , Feto/metabolismo , Hemangioma/metabolismo , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Laminina/metabolismo , Linfangioma/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/análisis , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Superficie Celular/genética , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Factor de von Willebrand/metabolismoRESUMEN
The growth of solid tumors is dependent on angiogenesis, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a secreted endothelial-cell-specific mitogen. We have recently characterized two novel endothelial growth factors with structural homology to VEGF and named them VEGF-B and VEGF-C. To further define the roles of VEGF-B and VEGF-C, we have studied their expression in a variety of human tumors, both malignant and benign. VEGF-B mRNA was detected in most of the tumor samples studied, and the mRNA and the protein product were localized to tumor cells. Endothelial cells of tumor vessels were also immunoreactive for VEGF-B, probably representing the binding sites of the VEGF-B polypeptide secreted by adjacent tumor cells. VEGF-C mRNA was detected in approximately one-half of the cancers analyzed. Via in situ hybridization, VEGF-C mRNA was also localized to tumor cells. All lymphomas studied contained low levels of VEGF-C mRNA, possibly reflecting the cell-specific pattern of expression of the VEGF-C gene in the corresponding normal cells. The expression of VEGF-C is associated with the development of lymphatic vessels, and VEGF-C could be an important factor regulating the mutual paracrine relationships between tumor cells and lymphatic endothelial cells. Furthermore, VEGF-C and VEGF-B can, similarly to VEGF, be involved in tumor angiogenesis.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Neoplasias/metabolismo , Adenocarcinoma/metabolismo , Northern Blotting , Neoplasias de la Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Linfoma no Hodgkin/metabolismo , Melanoma/metabolismo , ARN Mensajero/análisis , Sarcoma/metabolismo , Factor B de Crecimiento Endotelial Vascular , Factor C de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: The growth and differentiation of endothelial cells are regulated by signal transduction through tyrosine protein kinases. Recently, a novel cytoplasmic tyrosine kinase gene, Bmx (Bone Marrow tyrosine kinase gene in chromosome X), was identified in human bone marrow RNA and found to be expressed predominantly in myeloid hematopoietic cell lineages. Our preliminary analyses indicated that the Bmx gene was also highly expressed in human heart. METHODS AND RESULTS: Mouse Bmx cDNA was isolated, sequenced, and found to encode a polypeptide approximately 91% identical to the human Bmx tyrosine kinase. Northern blotting and in situ hybridization of tissue sections indicated that Bmx mRNA is specifically expressed in the endocardium of the developing heart as well as in the endocardium of the left ventricle and in the endothelium of large arteries in adult mice. A weak signal was seen also in coronary arterial endothelium. CONCLUSIONS: Bmx shows a unique specificity of expression among tyrosine kinase genes and may be involved in signal transduction in endocardial and arterial endothelial cells. The results suggest that specific signal transduction mechanisms are present in such endothelia.
Asunto(s)
Endocardio/enzimología , Endotelio Vascular/enzimología , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Arterias/enzimología , Northern Blotting , Médula Ósea/enzimología , Clonación Molecular , ADN Complementario/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/química , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Dominios Homologos srcRESUMEN
Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Expresión Génica , Linfocinas/genética , Placenta/química , Proteínas Gestacionales/genética , Northern Blotting , Factores de Crecimiento Endotelial/análisis , Endotelio Vascular/química , Femenino , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Linfocinas/análisis , Factor de Crecimiento Placentario , Embarazo , Proteínas Gestacionales/análisis , ARN Mensajero/análisis , Trofoblastos/química , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The vascular endothelial growth factor family has recently been expanded by the isolation of two new VEGF-related factors, VEGF-B and VEGF-C. The physiological functions of these factors are largely unknown. Here we report the cloning and characterization of mouse VEGF-C, which is produced as a disulfide-linked dimer of 415 amino acid residue polypeptides, sharing an 85% identity with the human VEGF-C amino acid sequence. The recombinant mouse VEGF-C protein was secreted from transfected cells as VEGFR-3 (Flt4) binding polypeptides of 30-32x10(3) Mr and 22-23x10(3) Mr which preferentially stimulated the autophosphorylation of VEGFR-3 in comparison with VEGFR-2 (KDR). In in situ hybridization, mouse VEGF-C mRNA expression was detected in mesenchymal cells of postimplantation mouse embryos, particularly in the regions where the lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary and jugular regions. In addition, the developing mesenterium, which is rich in lymphatic vessels, showed strong VEGF-C expression. VEGF-C was also highly expressed in adult mouse lung, heart and kidney, where VEGFR-3 was also prominent. The pattern of expression of VEGF-C in relation to its major receptor VEGFR-3 during the sprouting of the lymphatic endothelium in embryos suggests a paracrine mode of action and that one of the functions of VEGF-C may be in the regulation of angiogenesis of the lymphatic vasculature.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Sistema Linfático/embriología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Hibridación in Situ , Ligandos , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , ARN Mensajero/análisis , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Factor C de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
The Myc oncoprotein is associated with cell proliferation and is often down-regulated during cell differentiation. The related Mad transcription factor, which antagonises Myc activity, is highly expressed in epidermal keratinocytes. Mad also inhibits cell proliferation in vitro. To study Mad expression in keratinocyte proliferation and differentiation, we have analysed Mad RNA expression in regenerating and hyperproliferative epidermal lesions and epidermal tumours of varying degrees of differentiation using the RNA in situ hybridisation and RNAase protection techniques. Mad was strongly expressed in differentiating suprabasal keratinocytes in healing dermal wounds and in benign hyperproliferative conditions, but also in squamous cell carcinomas, in which the keratinocytes retain their differentiation potential. However, Mad expression was lost in palisading basal carcinoma cells and poorly differentiated squamous cell carcinomas, which lacked the epithelial differentiation marker syndecan-1. We therefore suggest that Mad expression is closely associated with epithelial cell differentiation, and that this association is retained in epithelial tumours of the skin.