Differential effect of storage on molecular and ultra-molecular dilutions of histamine

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 antiapoptotic protein(s) is blocked. Received 23 July 2008; returned for revision 10 September 2008; received for final revision 29 September 2008; accepted by M. Parnham 18 September 2008