Activation by C5a of endothelial cell caspase 8 and cFLIP
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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.
Recombinant human C5a (50 ng/ml) was used in the presence or absence of 10 μg/ml cycloheximide (CHX).
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).
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.
These data suggest caspase 8 activation induced by C5a leads to cell death if protein synthesis of antiapoptotic protein(s) is blocked.
Keywords:Complement factor 5a (C5a) Cycloheximide (CHX) Caspase 8 Caspase 8 homologue FLICE Inhibitory protein (cFLIP)
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