MAINTENANCE OF VASCULAR ENDOTHELIAL CELL-SPECIFIC PROPERTIES AFTER IMMORTALIZATION WITH AN AMPHOTROPHIC REPLICATION-DEFICIENT RETROVIRUS CONTAINING HUMAN PAPILLOMA-VIRUS-16 E6 E7 DNA/

Primary human vascular endothelial cells were immortalized by the inte gration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To da te, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more than 1 year. The cell lines have retained a diploid karyotype, display no abnormalities, and are able to grow in a polar mode. Analysis of the EC-RF cell lines by indirect immunofluorescence, using an extensive panel of monoclonal antibodies, showed expression of endothelial cell-specific soluble (von Willebrand factor) and surfa ce-bound antigens (endoglin, PCAM-1) indistinguishable from that of pr imary cells. In addition, the expression of the markers CD9, 13, 14, 2 9, 36, 40, 51, and 55 that are not restricted to endothelial cells was also similar for the immortalized and the primary endothelial cells. Immortalization did not alter the expression of the surface adhesion m olecules E-selectin, VCAM-1, and ICAM-1 nor transmigration of neutroph ils. The regulation of extracellular proteolytic activity by EC-RF24 w as established by measuring both the induction of functional tissue fa ctor (promotion of Factor Xa generation) and the functional deposition of plasminogen activator inhibitor 1 in the subendothelial matrix (SD S-resistant complex formation with thrombin). Finally, the biosynthesi s of the endothelial cell-specific von Willebrand factor was studied i n detail in the EC-RF24 cell line and the results were compared with t hose of primary endothelial cells. (C) 1995 Academic Press, Inc.