| Budget Amount *help |
¥3,840,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥240,000)
Fiscal Year 2007: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2006: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Rationale: Not only better understanding of the molecular mechanisms involved in the pathogenesis of sepsis and its resultant organ failure, but also new therapeutic approaches and targets are urgently needed. Accumulating evidence suggests that apoptosis plays an important role in the pathophysiology of sepsis, and apoptosis may be potentially detrimental in septic acute lung injury (ALI). Also, vascular endothelial cell apoptosis may play a role in the pathogenesis of the septic syndrome.
Objectives: We tested the hypothesis that systemic administration of small interfering RNA (siRNA) targeting Fas-associated death domain (FADD), which recruits procaspase-8 into the death-inducing signaling complex, may be protective in septic ALI and mortality We also evaluated the therapeutic efficacy of caspase-8/caspase-3 siRNAs in a murine model of polymicrobial endotoxic shock.
Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. In vivo delivery of siRN
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As was performed by using a transfection reagent (Lipofectamine^(TM) RNAiMAX) at 10 hours after CLP. As a negative control, animals received non-sense (scrambled) siRNA.
Measurements and Main Results: In CLP-induced septic mice, surface expression of death receptors were up-regulated and FADD was highly expressed and activated. DNA fragmentation ladder and TUNEL (transferase-mediated dUTP nick end labeling) assays showed that treatment with siRNAs suppressed apoptosis induction in lungs and vascular tissues following sepsis. Moreover, these siRNA treatments prevented the development of ALI in CLP mice, as indicated by great improvements of blood-gas derangements, histologic lung damage, and increased pulmonary inflammatory cells. Furthermore, hematoxylin/eosin-stained sections of septic aorta displayed partial detachment of endothelial cells from the basal membrane. These histopathologic changes in endothelial cells were drastically prevented by systemic treatment with FADD siRNA or caspase-8/-3 siRNAs. Finally, administration of FADD siRNA or caspase-8/-3 dramatically improved the survival of CLP mice.
Conclusions: These results indicate the pathophysiological significance of the death receptor apoptotic pathway, including FADD, in septic ALI and the potential usefulness of FADD siRNA for gene therapy of the septic syndrome. In addition, 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. Less
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