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Human vascular endothelial cells with extended life spans: in vitro cell response,protein expression,and angiogenesis
Authors:Gagnon Edith  Cattaruzzi Paola  Griffith May  Muzakare Lea  LeFlao Katell  Faure Robert  Béliveau Richard  Hussain Sabah N  Koutsilieris Michael  Doillon Charles J
Affiliation:(1) Oncology and Molecular Endocrinology Research Center, CHUL, Québec, Canada;(2) University Ottawa Eye Institute, Ottawa, Ontario, Canada;(3) Oncology Molecular Laboratory, Hopital Ste Justine/UQAM, Montreal, Québec, Canada;(4) Critical Care and Respiratory Division, Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, Québec, Canada;(5) Department of Experimental Physiology, Medical School, University of Athens, Athens, Greece;(6) Oncology and Molecular Endocrinology Research Center, CHUL Research Center, 2705 boul, Laurier, Québec, Canada, G1V 4G2
Abstract:
An in vitro angiogenesis system was designed for screening angiogenic agonists and antagonists. In order to obtain large quantities of cells and reproducibility, human endothelial cells with extended life spans were developed by retroviral transfection. The resulting cells grown in a serum-free medium containing endothelial cell growth supplement (ECGS) have a telomerase activity, extended life spans of at least 21 passages, and an endothelial cell phenotype (diI-acetylated-LDL upake, factor VIII-related antigen, VEGFR-1 and R-2, and tissue-type plasminogen activator (tPA)) that resembled that of unaltered primary endothelial cells. Exceptions were (i) a higher expression of tPA, and (ii) a non-significant growth response to FGF-2 or VEGF stimulation. Within three-dimensional fibrin gels, specific cell clones rapidly formed tubular structures in a more reproducible manner than those observed with low-passage primary cells. Tube formation by primary endothelial cells and those with extended life spans was dependent upon FGF-2 and ECGS, respectively. Both cell types produced FGF-2 and VEGF cytokines. Increasing doses of suramin significantly decreased the size of microvessels formed by both cell lines. These functional results indicate that a vascular matrix system containing human cells with extended life spans can be successfully utilized as an in vitro assay for antiangiogenic compounds.
Keywords:angiogenesis  endothelial cells with extended life spans  fibrin matrix  FGF  human endothelial cells  immortalization  three-dimensional cell culture  VEGF  VEGF receptors
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