Activation by C5a of Endothelial Cell Caspase 8 and cFLIP
Molecular and Cellular Biology
Objectives and design: In this study, we examine the relationship between C5a and activation of cysteine aspartic acid protease 8 (caspase 8) in human umbilical vein endothelial cells (HUVEC).
Materials or subjects: Primary cultures of HUVEC were used.
Treatments: Recombinant human C5a (50 ng/ml) was used in the presence or absence of 10 μg/ml cycloheximide (CHX).
Methods: HUVEC were treated with C5a alone and in the presence of CHX, then monitored for cell viability, poly-ADP-ribose 1 (PARP-1) and caspase 8 activities. Gene and protein expressions were assessed for caspase 8 and the caspase 8 homologue, FLICE –inhibitory protein (cFLIP).
Results: We found a 43.1 ± 6.9 percent reduction in viability of HUVEC stimulated for 18 h with 50 ng/ml C5a in the presence of 10 μg/ml CHX (p < 0.05). In contrast, the cell viability of cells stimulated for 18 h with 50 ng/ml C5a or 10 μg/ml CHX alone was not significantly different compared to the non-stimulated control. Treatment of HUVEC with C5a induced an increase in caspase 8 activity but did not significantly affect cFLIP levels.
Conclusions: These data suggest caspase 8 activation induced by C5a leads to cell death if protein synthesis of anti-apoptotic protein(s) is blocked.
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