Ischemic liver injury is a major clinical problem in shock, liver surgery and transplantation. Our initial results allow us to implicate apoptosis of sinusoidal endothelial cells (SEC) - an active process of cell suicide - as a central feature of reperfusion injury in rat models of hepatic ischemia. We have also discovered that calpains, a family of intracytoplasmic calcium dependent cysteine proteases, are activated during periods of ischemia and reperfusion (I/Rp) and in part mediate SEC apoptosis. We HYPOTHESIZE that ischemia sensitizes SEC to the effects of pro- apoptotic mediators released at the time of reperfusion, resulting in the apoptosis of SEC, even when the direct ischemic damage is reversible. The overall objective of this project is therefore to elucidate in vivo mechanisms of SEC apoptosis in rodent models of hepatic I/Rp, and design protective strategies to improve viability of the ischemic liver. The first objective is to characterize the temporal dynamics of SEC apoptosis in hepatic injury by monitoring characteristic microscopic, histomorphologic and biochemical markers of apoptotic cell death at successive time points during I/Rp. Second, we will determine the cellular mechanisms of calpain- mediated SEC apoptosis by identifying tissue localization of calpain activity and the relationship between calpain activation and other possible calpain related cellular effectors of apoptosis (caspase and ceramide) in a hepatic I/Rp whole-organ model. Third, we will design protective strategies by inhibiting mediators of SEC apoptosis or by manipulations designed to favor anti-apoptotic activities. We have shown that calpain inhibition is effective in protecting SEC in normothermic I/Rp. Unfortunately, calpain inhibitors are not as effective in cold I/Rp injury, relevant for transplantation, and are not water soluble reagents, limiting their clinical applicability. Because many interacting pathways lead to apoptosis, we anticipate that interventions at multiple levels will be necessary. In addition to using new water soluble calpain inhibitors, we will block FAS and TNFalpha, well characterized extracellular pro-apoptotic mediators that may be active in the I/Rp-injured liver. A potential regulatory role for the Bcl-2 gene, whose product is inhibitory at various levels of the apoptotic process, will be investigated using transgenic mice overexpressing Bcl-2. Our whole-organ model is uniquely suited to these studies, since - if known precedents hold - signaling for apoptosis most likely involves complex interactions among several cell types. Identification of mechanisms of SEC death in hepatic I/Rp injury and the ability to protect these cells may have significant clinical impact by allowing longer and safer preservation prior to transplantation and by lessening ischemic liver damage during non- transplant surgery and shock.
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