Silencing of caspase-8 and caspase-3 by RNA interference prevents vascular endothelial cell injury in mice with endotoxic shock.
OBJECTIVE
Septic shock and sequential multiple organ failure remain the cause of death in septic patients. Vascular endothelial cell apoptosis may play a role in the pathogenesis of the septic syndrome. Caspase-8 is presumed to be the apex of the death receptor-mediated apoptosis pathway, whereas caspase-3 belongs to the "effector" protease in the apoptosis cascade. Synthetic small interfering RNAs (siRNAs) specifically suppress gene expression by RNA interference. Therefore, we evaluated the therapeutic efficacy of caspase-8/caspase-3 siRNAs in a murine model of polymicrobial endotoxic shock.


METHODS
Polymicrobial endotoxic shock was induced by cecal ligation and puncture (CLP) in BALB/c mice. In vivo delivery of siRNAs was performed by using a transfection reagent (Lipofectamine 2000) at 10 h after CLP. As a negative control, animals received non-sense (scrambled) siRNA.


RESULTS
Marked increases in caspase-8 and caspase-3 protein expression in CLP aortic tissues were strongly suppressed by treatment with caspase-8/caspase-3 siRNAs. This siRNA treatment prevented DNA ladder formation and less phosphorylation of the pro-apoptotic protein Bad seen in CLP aortic tissues. Transferase-mediated dUTP nick end labeling (TUNEL) revealed that the appearance of apoptosis in aortic endothelium after CLP was eliminated by this siRNA treatment. Although all of the control animals subjected to CLP died within 2 days, administration of caspase-8/caspase-3 siRNAs indefinitely (>7 days) improved the survival of CLP mice.


CONCLUSIONS
Gene silencing of caspase-8 and caspase-3 with siRNAs provided profound protection against polymicrobial endotoxic shock. The prevention of vascular endothelial cell apoptosis appears to be, at least in part, responsible for their beneficial effects in endotoxic shock.
Matsuda
Naoyuki
N
Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Takano
Yasuo
Y
Kageyama
Shun-Ichiro
S
Hatakeyama
Noboru
N
Shakunaga
Kiyoshi
K
Kitajima
Isao
I
Yamazaki
Mitsuaki
M
Hattori
Yuichi
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3 / Q000235:genetics*; D053181:Caspase 8 / Q000235:genetics*; D042783:Endothelial Cells / Q000201:enzymology / Q000473:pathology; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D008055:Lipids / Q000008:administration & dosage; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage; D012772:Shock, Septic / Q000201:enzymology / Q000473:pathology / Q000628:therapy*; D014162:Transfection / Q000379:methods; D051025:bcl-Associated Death Protein / Q000378:metabolism
C494821:Bad protein, rat; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D051025:bcl-Associated Death Protein; D053148:Caspase 3; D053181:Caspase 8