Activation by C5a of endothelial cell caspase 8 and cFLIP.
OBJECTIVE
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).


METHODS
Primary cultures of HUVEC were used.


METHODS
Recombinant human C5a (50 ng/ml) was used in the presence or absence of 10 microg/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 microg/ml CHX (p < 0.05). In contrast, the cell viability of cells stimulated for 18 h with 50 ng/ml C5a or 10 microg/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 antiapoptotic protein(s) is blocked.
Albrecht
E A
EA
Department of Biology and Physics, Kennesaw State University, Kennesaw, GA 30144, USA.
Sarma
J V
JV
Ward
P A
PA
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D002478:Cells, Cultured; D015936:Complement C5a / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D015936:Complement C5a; D003513:Cycloheximide; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D053181:Caspase 8