RESUMEN
Prominent among molecules that control neovascular processes is vascular endothelial growth factor (VEGF). The VEGF ligands comprise a family of well-studied mitogens/permeability factors that bind cell surface receptor tyrosine kinases. Targets include VEGF receptor-1/Flt1 and VEGF receptor-2/Flk1. Mice lacking genes for VEGF ligand or VEGF receptor-2 die early in gestation, making it difficult to determine the precise nature of underlying endothelial cellular behavior(s). To examine the effect(s) of VEGF signaling on cell behavior in detail, we conducted loss-of-function studies using avian embryos. Injection of soluble VEGFR-1 results in malformed vascular networks and the absence of large vessels. In the most severe cases embryos exhibited vascular atresia. Closely associated with the altered phenotype was a clear endothelial cell response-a marked decrease in cell protrusive activity. Further, we demonstrate that VEGF gain of function strikingly increased cell protrusive activity. Together, our data show that VEGF/VEGF receptor signaling regulates endothelial cell protrusive activity, a key determinant of blood vessel morphogenesis. We propose that VEGF functions as an instructive molecule during de novo blood vessel morphogenesis.
Asunto(s)
Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/embriología , Linfocinas/fisiología , Animales , Factores de Crecimiento Endotelial/metabolismo , Linfocinas/metabolismo , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Codorniz/embriología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Thyrotropin (TSH), free thyroxine (fT4) and testosterone assays have been used as a probe to evaluate the performances of a new modular chemiluminescence (CL) immunoassay analyser, the Abbott Architect 2000. The evaluation was run in parallel on other systems that use CL as the detection reaction: DPC Immulite, Chiron Diagnostics ACS-180 and ACS Centaur (TSH functional sensitivity only). TSH functional sensitivity was 0.0012, 0.009, 0.033 and 0.039 mU/I for the Architect, Immulite, ACS Centaur and ACS-180, respectively. Testosterone functional sensitivity was 0.38, 3.7 and 2.0 nmol/l for Architect, Immulite and ACS-180, respectively. Good correlation was obtained between the ACS-180 and Architect for all assays. The Immulite correlation did not agree well with the Architect or ACS-180 for fT4 and testosterone but was in good agreement for TSH. Regarding fT4 and testosterone, equilibrium dialysis and isotopic dilution gas-chromatography mass-spectrometry (GC-MS) respectively were used as reference methods. For both within- and between-run precision, the Architect showed the best reproducibility for all three analytes (CV < 6%).
Asunto(s)
Inmunoensayo/instrumentación , Inmunoensayo/métodos , Mediciones Luminiscentes , Automatización , Calibración , Estudios de Casos y Controles , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hipertiroidismo/sangre , Hipotiroidismo/sangre , Fallo Renal Crónico/sangre , Masculino , Embarazo , Reproducibilidad de los Resultados , Factor Reumatoide/sangre , Sensibilidad y Especificidad , Testosterona/sangre , Tirotropina/sangre , Tiroxina/sangre , Factores de TiempoRESUMEN
The process of vasculogenesis was characterized in the 6.5- to 9.5-day mouse embryo and in allantoic culture by analysis of spatial and temporal expression patterns of the endothelial or hematopoietic lineage-associated proteins, TAL1, Flk1, platelet/endothelial cell adhesion molecule (PECAM), CD34, VE-cadherin, and Tie2. The study establishes that: (1) TAL1 and Flk1 are coexpressed in isolated mesodermal cells that give rise to endothelial cells and thus can be defined as angioblasts; (2) hematopoietic cells of blood islands express TAL1, but not Flk1; (3) vasculogenesis in the embryo proper is initiated by mesoderm fated to give rise to the endocardium; (4) the maturation/morphogenesis of blood vessels can be defined in terms of a sequential pattern of expression in which TAL1 and Flk1 are expressed first followed by PECAM, CD34, VE-cadherin, and later Tie2; and (5) TAL1 expression is down-regulated in endothelial cells of mature vessels. (Blood. 2000;95:1671-1679)
Asunto(s)
Ratones/embriología , Neovascularización Fisiológica , Proteínas Proto-Oncogénicas , Factores de Transcripción , Alantoides/citología , Animales , Antígenos CD , Antígenos CD34/biosíntesis , Antígenos CD34/genética , Aorta/embriología , Aorta/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cadherinas/biosíntesis , Cadherinas/genética , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/irrigación sanguínea , Endocardio/embriología , Endocardio/metabolismo , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Corazón Fetal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Mesodermo/citología , Morfogénesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor TIE-2 , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Proteína 1 de la Leucemia Linfocítica T AgudaRESUMEN
The avian embryo is well suited for the study of blood vessel morphogenesis. This is especially true of investigations that focus on the de novo formation of blood vessels from mesoderm, a process referred to as vasculogenesis. To examine the cellular and molecular mechanisms regulating vasculogenesis, we developed a bioassay that employs intact avian embryos. Among the many bioactive molecules we have examined, vascular epithelial growth factor (VEGF) stands out for its ability to affect vasculogenesis. Using the whole-embryo assay, we discovered that VEGF induces a vascular malformation we refer to as hyperfusion. Our studies showed that microinjection of recombinant VEGF165 converted the normally discrete network of embryonic blood vessels into enlarged endothelial sinuses. Depending on the amount of VEGF injected and the time of postinjection incubation, the misbehavior of the primordial endothelial cells can become so exaggerated that for all practical purposes the embryo contains a single enormous vascular sinus; all normal vessels are subsumed into a composite vascular structure. This morphology is reminiscent of the abnormal vascular sinuses characteristic of certain neovascular pathologies. (J Histochem Cytochem 47:1351-1355, 1999)
Asunto(s)
Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/fisiología , Linfocinas/fisiología , Mesodermo/fisiología , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Animales , Embrión de Pollo , Embrión de Mamíferos , Endotelio Vascular/fisiopatología , Humanos , Fusión de Membrana , Ratones , Modelos Cardiovasculares , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.
Asunto(s)
Proteínas Portadoras , Endocitosis/fisiología , Endodermo/fisiología , Túbulos Renales Proximales/fisiología , Lipoproteínas HDL/metabolismo , Proteínas de Unión al ARN , Receptores de Superficie Celular/fisiología , Receptores de Lipoproteína/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/metabolismo , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Cromatografía de Afinidad , Humanos , Cinética , Glicoproteínas de Membrana/fisiología , Ratones , Microscopía Confocal , Receptores de Lipoproteína/aislamiento & purificación , Saco Vitelino/fisiologíaRESUMEN
The MEF2 family of transcription factors has been implicated in transcriptional regulation in a number of different cell types. Targeted deletion of the MEF2C gene, in particular, revealed its importance for early cardiogenesis (Q. Lin et al., 1997, Science 276, 1404-1407). We report here that this deletion also resulted in vascular anomalies characterized by extreme variability in lumen size and defects in remodeling. While primary vascular networks formed in the yolk sac of the mutants, they failed to remodel into more complex vascular structures. Likewise, although the primordia of the dorsal aortae formed normally, anomalies were observed in these vessels later in development. Dorsal and anterior to the heart, the aortae exhibited abnormally small lumens, as did the anterior cardinal veins and intersegmental arteries. In contrast, the dorsal aortae and intersegmental arteries caudal to the heart were grossly enlarged. Cranial vessels were also enlarged and less branched than normal. Endocardiogenesis in the mutant was abnormal with the endothelial cells exhibiting a number of aberrant phenotypes. These endocardial defects were accompanied by a notable reduction in angiopoietin 1 and VEGF mRNA production by the myocardium, indicating that MEF2C is required for myocardial expression of these important endothelial-directed cytokines and thus for correct endocardial morphogenesis.
Asunto(s)
Malformaciones Arteriovenosas/genética , Factores de Crecimiento Endotelial/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Corazón/fisiología , Linfocinas/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Factores Reguladores Miogénicos/fisiología , Angiopoyetina 1 , Animales , Vasos Sanguíneos/embriología , Vasos Sanguíneos/fisiología , Eliminación de Gen , Corazón/embriología , Factores de Transcripción MEF2 , Ratones , Ratones Noqueados , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Due to the availability of the endothelial cell marker QH1, experiments using quail embryos have traditionally been used to trace the endothelial cell lineage and describe the morphologic events of vessel formation. A comparable marker in the chicken has not been available. Here we report that antibodies raised against the extracellular domain of the chicken type II TGFbeta receptor (TBRII) preferentially identify endothelial cells in the chick. Endothelial cells can first be identified in the 6-somite chick embryo by TBRII expression. TBRII expression in 12- and 22-somite chick and quail embryos was found to directly correlate with the endothelial QH1 staining pattern in quail. This preferential labeling of endothelial cells persists until at least embryonic day 10 in the chick. These data indicate that antibodies to TBRII are an effective marker of endothelial cells in chick and provide useful reagents for the evaluation of vascular patterning.
Asunto(s)
Endotelio/embriología , Codorniz/embriología , Factor de Crecimiento Transformador beta/inmunología , Animales , Western Blotting , Embrión de Pollo , Endotelio/inmunología , Codorniz/inmunología , Factor de Crecimiento Transformador beta/análisisRESUMEN
The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart-forming regions and septation-stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3-immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping distribution pattern with fibrillin-1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol. 165:585-601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3-immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin-1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin-2. Live, whole-mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin-2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observed, suggesting a developmental progression of fibrillin-2 fibril assembly across the somite-forming region of avian embryos. Extensive filaments of fibrillin-2 connect somites to the notochord. Similarly, fibrillin-2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin-2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial-to-caudal segmentation, and regression of the anterior intestinal portal. Fibrillin-2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan.
Asunto(s)
Antígenos/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina , Animales , Biomarcadores , Embrión de Pollo , Cromatografía de Afinidad , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Immunoblotting , Espectrometría de Masas , Pruebas de PrecipitinaRESUMEN
The initial phase of vessel formation is the establishment of nascent endothelial tubes from mesodermal precursor cells. Development of the vascular epithelium is examined using the transcription factor TAL1 as a marker of endothelial precursor cells (angioblasts), and a functional assay based on intact, whole-mounted quail embryos. Experimental studies examining the role(s) of integrins and vascular endothelial growth factor (VEGF) establish that integrin-mediated cell adhesion is necessary for normal endothelial tube formation and that stimulation of embryonic endothelial cells with exogenous VEGF results in a massive "fusion" of vessels and the obliteration of normally avascular zones. The second phase of vessel morphogenesis is assembly of the vessel wall. To understand the process by which mesenchyme gives rise to vascular smooth muscle, a novel monoclonal antibody, 1E12, that recognizes smooth muscle precursor cells was used. Additionally, development of the vessel wall was examined using the expression fo extracellular matrix proteins as markers. Comparison of labeling patterns of 1E12 and the extracellular matrix molecules fibulin-1 and fibrillin-2 indicate vessel wall heterogeneity at the earliest stages of development; thus smooth muscle cell diversity is manifested during the differentiation and assembly of the vessel wall. From these studies it is postulated that the extracellular matrix composition of the vessel wall may prove to be the best marker of smooth muscle diversity. The data are discussed in the context of recent work by others, especially provocative new studies suggesting an endothelial origin for vascular smooth muscle cells. Also discussed is recent work that provides clues to the mechanism of vascular smooth muscle induction and recruitment. Based on these findings, vascular smooth muscle cells can be thought of as existing along a continuum of phenotypes. This spectrum varies from mainly matrix-producing cells to primarily contractile cells; thus no one cell type typifies vascular smooth muscle. This view of the smooth muscle cell is considered in terms of a contrasting opinion that views smooth muscle cell as existing in either a synthetic or proliferative state.
Asunto(s)
Vasos Sanguíneos/embriología , Inducción Embrionaria , Endotelio Vascular/embriología , Animales , Adhesión Celular , Embrión no Mamífero/fisiología , Desarrollo Embrionario y Fetal , Factores de Crecimiento Endotelial/fisiología , Humanos , Integrinas/fisiología , Linfocinas/fisiología , Morfogénesis , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
In this study we establish that TAL1/SCL, a member of the helix-loop-helix family of transcription factors, and an important regulator of the hematopoietic lineage in mice, is expressed in the endothelial lineage of avians. The earliest events of vascular development were examined using antibodies to TAL1/SCL, and the QH1 antibody, an established marker of quail endothelial cells. Analyses using double immunofluorescence confocal microscopy show that: (i) TAL1/SCL is expressed by both quail and chicken endothelial cells; (ii) TAL1/SCL expression precedes that of the QH1 epitope; and (iii) TAL1/SCL, but not QH1, expression defines a subpopulation of primordial cells within the splanchnic mesoderm. Collectively these data suggest that TAL1/SCL-positive/QH1-negative cells are angioblasts. Further, using TAL1/SCL expression as a marker of the endothelial lineage, we demonstrate that in addition to the previously described cranial-to-caudal gradient, there is a dorsal-to-ventral progression of vasculogenesis.
Asunto(s)
Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biomarcadores , Embrión de Pollo , Coturnix , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Endotelio Vascular/citología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice/genética , Hematopoyesis , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Microscopía Fluorescente , Células Madre/citología , Células Madre/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: The epicardium is the site of initial cardiac neovascularization and formation of the coronary circulatory system. Recent evidence indicates that vascular progenitor cells are influenced by the connective tissue proteins of their extracellular environment, yet little is known about the composition or function of the embryonic epicardial extracellular matrix (ECM). This study examines the distribution of ECM proteins during the migration, growth and maturation of epicardial cells and also during the development of the coronary vascular network. METHODS: Immunofluorescence microscopy was used to determine the distributions of vitronectin, fibronectin and a newly described fibrillin-like protein, the JB3 antigen, in the embryonic chicken heart. Immunoblot analysis was performed to compare the relative electrophoretic mobilities of the JB3 antigen and fibrillin-1. RESULTS: The data show that vitronectin and fibronectin are present at sites of initial migration of the epicardial cells. The expression of vitronectin (and also fibronectin) becomes more pronounced as the epicardium thickens, undergoes remodeling and differentiates. The JB3 antigen is prominently expressed in the coronary arteries, allowing visualization of their connection to the systemic circulation and to the heart muscle, as well as vessel wall formation and organization. Immunoblot analysis suggests that the JB3 antibody recognizes a fibrillin-like polypeptide that is distinct from fibrillin-1. CONCLUSIONS: The observed distributions of vitronectin and fibronectin are consistent with roles in migration of epicardial cells, in remodeling of the epicardium and as substratum components during blood vessel formation. The observed distribution of the JB3 antigen indicates a structural/organizational role in coronary arterial wall assembly and suggests that the JB3 antibody be considered an early marker for maturing coronary arteries.
Asunto(s)
Vasos Coronarios/embriología , Proteínas de la Matriz Extracelular/metabolismo , Pericardio/fisiología , Animales , Movimiento Celular/fisiología , Embrión de Pollo , Vasos Coronarios/química , Vasos Coronarios/metabolismo , Técnicas de Cultivo , Proteínas de la Matriz Extracelular/análisis , Fibronectinas/análisis , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Immunoblotting , Vitronectina/análisis , Vitronectina/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is a potent and specific endothelial mitogen that is able to induce angiogenesis in vivo [Leung, D. W., Cachianes, G., Kuang, W.-J., Goeddel, D. V. & Ferrara, N. (1989) Science 246 1306-1309]. To determine if VEGF also influences the behavior of primordial endothelial cells, we used an in vivo vascular assay based on the de novo formation of vessels. Japanese quail embryos injected with nanomolar quantities of the 165-residue form of VEGF at the onset of vasculogenesis exhibited profoundly altered vessel development. In fact, the overall patterning of the vascular network was abnormal in all VEGF-injected embryos. The malformations were attributable to two specific endothelial cell activities: (i) inappropriate neovascularization in normally avascular areas and (ii) the unregulated, excessive fusion of vessels. In the first instance, supernumerary vessels directly linked the inflow channel of the heart to the aortic outflow channel. The second aberrant activity led to the formation of vessels with abnormally large lumens. Ultimately, unregulated vessel fusion generated massive vascular sacs that obliterated the identity of individual vessels. These observations show that exogenous VEGF has an impact on the behavior of primordial endothelial cells engaged in vasculogenesis, and they strongly suggest that endogenous VEGF is important in vascular patterning and regulation of vessel size (lumen formation).
Asunto(s)
Vasos Sanguíneos/anomalías , Embrión no Mamífero/efectos de los fármacos , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/embriología , Linfocinas/farmacología , Neovascularización Patológica , Animales , Arterias/anomalías , Arterias/efectos de los fármacos , Arterias/embriología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/embriología , Coturnix , Embrión no Mamífero/citología , Embrión no Mamífero/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Microscopía Confocal , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/fisiología , Receptores Mitogénicos/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/farmacología , Valores de Referencia , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Experimental data in this study demonstrate that integrin alpha v beta 3 is fundamentally involved in the maturation of blood vessels during embryonic neovascularization (vasculogenesis). Integrin alpha v beta 3 was specifically expressed on the surface of angioblasts during vessel development in quail embryos and vitronectin, a ligand for alpha v beta 3, localized to the basal surface of these cells. More importantly, microinjection of the anti-alpha v beta 3 monoclonal antibody, LM609, disrupted the normal pattern of vascular development. After exposure to LM609 the angioblasts in experimental embryos appeared as clusters of rounded cells lacking normal cellular protrusions. This led to disruption of lumen formation and abnormal vessel patterning. These findings demonstrate that during vasculogenesis ligation of integrin alpha v beta 3 on the surface of primordial endothelial cells is critical for the differentiation and maturation of blood vessels. Similar studies on chicken chorioallantoic membrane showed that LM609 blocks angiogenesis. Together the two studies suggest that integrin alpha v beta 3 plays a role in neovascularization of tissues.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Vasos Sanguíneos/fisiología , Embrión no Mamífero/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/embriología , Coturnix , Embrión no Mamífero/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Microinyecciones , Microscopía Confocal , Receptores de Vitronectina/inmunologíaRESUMEN
Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.
Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Colágeno/fisiología , Endotelio Vascular/citología , Neovascularización Patológica , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Matriz Extracelular/ultraestructura , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Modelos BiológicosRESUMEN
Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.
Asunto(s)
Adhesión Celular/fisiología , Sistema Nervioso Central/embriología , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Mesodermo/fisiología , Notocorda/embriología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Sistema Nervioso Central/ultraestructura , Coturnix/embriología , Relación Dosis-Respuesta a Droga , Integrina beta1 , Integrinas/inmunología , Mesodermo/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Notocorda/ultraestructuraRESUMEN
Vasculogenesis is the de novo formation of blood vessels from mesoderm. This process occurs very early in development and provides a convenient system for studying morphogenesis in higher vertebrates. The cell-extracellular matrix (ECM) interactions that occur during dorsal aortic vasculogenesis were examined using the monoclonal antibody, CSAT, a reagent known to neutralize the ligand-binding activity of avian beta 1-integrins. We injected CSAT into quail embryos during a period of active vasculogenesis (4-10 somites). The CSAT antibodies, but not controls, had a marked and reproducible effect on aortic vessel formation. Vasculogenesis appeared to be arrested at the stage when slender cord-like assemblies of angioblasts rearrange to form tubules. Indeed, aortic primordia near the site of CSAT injection did not form patent vessels.
Asunto(s)
Aorta/embriología , Integrinas/fisiología , Animales , Anticuerpos Monoclonales , Aorta/anomalías , Coturnix , Endodermo/fisiología , Inmunoglobulina G , Integrinas/inmunología , Mesodermo/fisiología , MorfogénesisRESUMEN
Integrins are proteins that mediate cell adhesion, mainly to the extracellular matrix. One of the first integrins discovered belongs to the beta 1 class of avian integrins and is defined by a monoclonal antibody, CSAT. Using a whole-embryo culture system we injected nanoliter quantities of CSAT caudolateral to the last somite of early quail embryos. The CSAT antibodies, but not control antibodies, resulted in a striking lateral translocation of somites. Relatively higher doses or longer incubation times increased the severity of the effect. We conclude that somite segmentation per se is not influenced by CSAT, but that somite adhesion to axial structures requires integrin-mediated ECM adhesions.
Asunto(s)
Coturnix/embriología , Integrinas/fisiología , Mesodermo/citología , Animales , Anticuerpos Monoclonales , Adhesión Celular , Matriz Extracelular/fisiología , Integrinas/inmunologíaRESUMEN
The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.
Asunto(s)
Colágeno/análisis , Matriz Extracelular/análisis , Corazón/embriología , Animales , Membrana Basal/fisiología , Movimiento Celular , Embrión de Pollo , Endotelio , Matriz Extracelular/fisiología , Técnica del Anticuerpo Fluorescente , Laminina/metabolismo , MesodermoRESUMEN
Light microscopic immunolabeling studies were designed to identify and locate structural components within the cell-free extracellular matrix which lies between the embryonic endocardial and myocardial tubes. Affinity-purified antibodies were used to examine stage 15-22 embryonic chicken hearts. Specimens were immunolabeled by using three different methodologies: 1) postembedding labeling of 10 microns cryostat sections, 2) preembedding labeling (en bloc) of whole hearts, and 3) postembedding labeling of ethanol/acetic acid-fixed paraffin sections. Our results establish the spatial distribution of collagen type I and demonstrate for the first time the presence of collagen type IV and laminin in the myocardial-basement-membrane/cardiac jelly.