POLARIZED EXPRESSION OF HETEROLOGOUS MEMBRANE-PROTEINS TRANSFECTED INA HUMAN ENDOTHELIAL-DERIVED CELL-LINE

The generation and maintenance of cell polarity in endothelial cells i s poorly understood, partly because of a lack of a permanent endotheli al in vitro model system, Here we evaluated the spontaneously immortal ized human endothelial-derived cell line ECV304 as an in vitro model s ystem for the study of the polarized expression of heterologous membra ne proteins. Several stable ECV304 clones were generated by calcium ph osphate transfection/G418 selection with cDNAs encoding membrane prote ins of known cell surface distribution in the epithelial Madin Darby c anine kidney (MDCK) cell line: influenza hemagglutinin and uvomorulin/ E-cadherin were used as markers for the apical, respectively lateral c ell membrane, the human lymphocyte surface marker CD7 served as an exa mple of a circumferentially distributed membrane protein. Analysis of the transfected ECV304 clones using conventional and confocal immunofl uorescence microscopy and immunoelectron microscopy revealed the same membrane distribution of the heterologous proteins in ECV304 cells as in MDCK cells. This polarized expression of heterologous membrane prot eins in the endothelial-derived ECV304 cell line indicates efficient p rotein sorting/membrane trafficking mechanisms. The epical, lateral an d basal cell membrane domains could be distinguished in ECV304 cells b y confocal immunofluorescence microscopy. The permanent endothelial-de rived ECV304 cell line may be a useful in vitro model system for the s tudy of endothelial cell polarity.