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  • 1
    Electronic Resource
    Electronic Resource
    Influence of intercellular junctions on endothelin secretion of human umbilical vein endothelial cells in vitro (2000)
    Springer
    Basic research in cardiology 95 (2000), S. 299-307 
    Details
    ISSN: 1435-1803
    Keywords: Key words Endothelial junctions – calcium – endothelin – cadherins – von Willebrand factor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The endothelium plays a pivotal role in the theological regulation of blood flow by the secretion of vasoactive factors. The interaction between shear forces and the endothelium is determined by the mechanical properties of the endothelial cell layer which are associated with intercellular junctions. Cell-cell contacts could therefore modulate the secretion of vasocative factors in response to theological stimuli. We investigated the relationship between intercellular junctions and the secretion of the vasoconstrictor peptide endothelin and the coagulation co-factor von Willebrand factor (vWF). Human umbilical vein endothelial cells (HUVECs) were used as in vitro endothelial model system. Intercellular junctions were reversibly disrupted by calcium chelation or hypertonic stress; alternatively, the formation of intercellular junctions was inhibited by culturing the cells in suspension or by plating them in the presence of an inhibitory anti-VE-cadherin antibody. The opening of intercellular junctions was verified by assessing transmonolayer electrical resistance (TMR) and immunofluorescence morphology. The concentration of endothelin and vWF was measured in the cell culture supernatants using specific ELISAs. The secretion of endothelin was inhibited by EGTA (5 mM) and stimulated by incubation with tumor necrosis factor α (TNFα, 40 ng/ml). Treatment with hypertonic medium (glycerol, 1200 mosmol/l) for 10 minutes opened intercellular junctions and markedly reduced the secretion of endothelin. HUVECs in suspension culture did not secrete endothelin and failed to respond to TNFα, but readily resumed these functions upon forming a new monolayer on plastic. The reconstitution of intercellular junctions after suspension culture could be inhibited using a specific anti-VE-cadherin antibody. This antibody, but not a non-specific anti-humanIgG antibody reduced endothelin secretion. The secretion of von Willebrand Factor was less dependent on intercellular junctions. The opening of intercellular junctions did not induce cell death, since the cells continued to exclude trypan blue. The results of this study suggest a novel and potentially pathophysiologically/clinically relevant correlation between intercellular junctions and the secretion of endothelin in endothelial cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s003950070049
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  • 2
    Electronic Resource
    Electronic Resource
    Assessment of heterologous membrane protein polarity in transiently transfected MDCK cells (1995)
    Springer
    Cytotechnology 17 (1995), S. 71-82 
    Details
    ISSN: 1573-0778
    Keywords: MDCK cells ; epithelial polarity ; transient transfection ; CD7 ; CD55 ; Decay Accelerating Factor ; Vesicular Stomatitis Virus G protein ; chloroquine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We have evaluated transient transfection of MDCK cells by the DEAE-dextran/chloroquine method as a rapid method for study of heterologous plasma membrane protein polarity. Transiently transfected cells reseeded onto permeable supports formed confluent monolayers with normal tight junctions and normal distribution of endogenous apical and basolateral surface markers. Transfected monolayers reseeded onto opaque polycarbonate filters attained cell heights 3 times greater than on transparent filters. Conventional and confocal immunofluorescence microscopy were used to assess polarity of transient expression of heterologous proteins previously defined in stably transfected cell lines as apical (DAF-CD55), basolateral (VSV-G), and nonpolarized (CD7) in distribution. Through each transiently expressed protein exhibited a polarity phenotype in most cells which resembled the stable phenotype, consistency of polarized localization was less than in stably transfected cells. Similar results were obtained by lipofection. We conclude that transient transfection of MDCK cells may be useful as a rapid screen, but is not sufficiently reliable for definitive assessment of heterologous membrane proein polarity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749394
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  • 3
    Electronic Resource
    Electronic Resource
    Erratum (1995)
    Springer
    Cytotechnology 18 (1995), S. 235-248 
    Details
    ISSN: 1573-0778
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00767771
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  • 4
    Electronic Resource
    Electronic Resource
    Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics (1999)
    Springer
    Cell & tissue research 297 (1999), S. 131-140 
    Details
    ISSN: 1432-0878
    Keywords: Key words Polarity ; Adherens junctions ; Tight junctions ; Desmosomes ; Transmembrane resistance ; Endothelial permeability ; Cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, β-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410051340
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