ESTABLISHMENT AND CHARACTERIZATION OF A LINE OF ADIPOSE-DERIVED HUMAN MICROVASCULAR ENDOTHELIAL CELLS (HADMEC-5) TRANSFORMED BY SIMIAN VIRUS 40 LARGE T ANTIGEN EXPRESSION

This paper reports the establishment and initial characterization of an immortalized line of human, adipose tissue-derived microvascular endothelial cells. Transfection of primary endothelial cell cultures was accomplished by the introduction of a plasmid, which contained simian virus 40 large T antigen DNA as well as a Rous sarcoma viral promoter region. One emergent colony, termed HADMEC-5, was isolated and has been passaged 45 times to date. The cells express simian virus 40 large T antigen protein, are immunohistochemically positive for factor VIII-related antigen, bind Ulex europaeus lectin, and accumulate Dil-labeled acetylated low density lipoprotein. The HADMEC-5 line demonstrates a highly proliferative growth rate in the absence of supplemental growth factors, when compared with primary cultures of nontransformed endothelial cells. HADMEC-5 growth remains serum dependent, but exhibits a lower serum requirement than nontransformed cells. The transformed cells grow well upon a variety of matrix compounds and in a variety of growth media. When grown upon Matrigel, the HADMEC-5 cells form three dimensional tube-like structures. The HADMEC-5 line was also tested for its ability to produce eicosanoids in response to bacterial lipopolysaccharide. The transformed cells, tested at passages 7 and 45, displayed a dose-dependent production of prostaglandin E2 in response to lipopolysaccharide in a manner similar to that seen in primary cell cultures. Threshold sensitivity to lipopolysaccharide was 10 pg/mL of media. The HADMEC-5 cell line represents a unique model in which to investigate lipopolysaccharide interactions with microvessel-derived endothelial cells and is of potential value in the study of other aspects of endothelial cell physiology.