The development of sepsis in critically ill patients is an ominous event that frequently leads to multiple organ failure and death. Increased release of cytokines has been implicated in the pathophysiology of these events, although the mechanisms underlying this relationship are not fully understood. Tissue hypoxia may also develop during sepsis, due to microvascular dysfunction and a failure to distribute capillary blood flow in accordance with tissue O2 need. A central hypothesis of this application is that tissue hypoxia will amplify the intracellular response to sepsis, by activating signaling pathways that regulate gene expression and subsequent release of cytokines and other inflammatory mediators. In sepsis, endotoxin (LPS) released from gram-negative bacteria complexes with binding proteins, which then bind to the CD14 receptor on cells. This initiates an intracellular signaling cascade leading to the activation of transcription factors that stimulate expression of genes including the cytokine TNFalpha and the inducible isoform of nitric oxide synthase (iNOS). TNFalpha can amplify the inflammatory cascade and may contribute to the pathophysiological state, while iNOS can result in unregulated release of nitric oxide, which may contribute to vascular dysfunction, organ failure and cell death. Activation of the transcription factor nuclear regulatory factor kappa B (NF-kappaB) is an important event in the expression of TNFalpah and iNOS. Previous studies demonstrate that cellular hypoxia (PO2 less than 40 torr) elicits an increase in release of reactive oxygen species (ROS) from mitochondria, which then activate transcription factors such as Hypoxia Inducible Factor-1 (HIF-1). This project will test the hypothesis that these ROS produced during hypoxia amplify the response to sepsis by independently activating NF-kappaB, thereby augmenting the TNFa and iNOS expression. Hypoxia may also heighten the cytotoxic effects of TNFa and NO, by augmenting oxidant stress and by contributing to the dissipation of mitochondrial potential. Proposed studies will clarify the role of mitochondrial ROS in amplifying NF-kappaB activation and TNFalpha and iNOS mRNA expression in a cellular model of sepsis. Additional studies will determine the significance of these events for cell death pathways in that model. Collectively, this work will shed new light on the intracellular signaling events during hypoxia and sepsis involving ROS, NF-kappaB and expression of TNFalpha and iNOS.
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