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BACKGROUND: Fas ligand expression by cells of the vessel wall has been proposed to play a role in normal and pathologic conditions. Genetic engineering of vascularized organs for endothelial cell (EC) expression of FasL could protect the endothelium and underlying tissues from infiltrating Fas+ leukocytes. Nevertheless, the endogenous expression of FasL by ECs of different species and the potential deleterious effects of enforced FasL expression by ECs are largely unknown. In human ECs, levels of FLICE/caspase 8-inhibitory protein (FLIP) have been shown to control apoptosis mediated by Fas. MATERIALS AND METHODS: Cell surface expression of FasL in rat, mouse, human, and pig ECs was obtained using recombinant adenoviruses or transient plasmid transfection assays. FasL expression was evaluated by FACS analysis and cytotoxicity assays. Apoptosis was evaluated using annexin V, TUNEL, and cytotoxicity assays. FLIP levels were evaluated by Western blot analysis and overexpression was obtained by transient transfection. RESULTS: Analysis of ECs from different species showed that FasL was predominantly present in the cytoplasm, and depending on the species, little or no cell surface expression was detected. Enforced cell surface expression of FasL on rat or mouse ECs, either in culture or within the vessel wall resulted in massive apoptosis. In contrast, porcine or human ECs were completely resistant to apoptosis mediated by Fas-FasL interaction. Markedly reduced FLIP levels were observed in rat and mouse ECs compared to human and porcine ECs. Overexpression of FLIP in rat ECs conferred protection against cell surface expression of FasL. CONCLUSIONS: The consequences of FasL overexpression depend on the subcellular compartment and species in which FasL enforced expression is targeted and this is at least partially related to FLIP levels.