Acute liver failure (ALF), a devastating condition that affects patients with and without prior liver disease, has a mortality rate of 80% (>20,000 deaths/year in the USA). Although the causal factors for the "irreversible" ALF are generally known, due to the lack of effective pharmacologic treatment, liver transplantation continues to be the only life-saving choice. However, organ shortage, increasing costs, as well as associated complications that cause delay or even rapid failure of the graft function are major limitations. Therefore, the knowledge of the precise mechanisms of ALF becomes very critical to develop therapeutic interventions since the failed liver may revive if given proper support in a timely manner. There is compelling evidence for an association between increased circulating levels of endotoxin (lipopolysaccharide, LPS) and ALF. Although mediators produced in the hepatic microenvironment by Kupffer cells and infiltrating leukocytes have been postulated to cause liver failure, hepatic stellate cells (HSCs), which are situated in a location that allows them to affect hepatocyte survival both by direct contact and via soluble mediators can have significant influence in ALF. We found that in addition to TNF-1, IL-6 and nitric oxide, LPS also stimulates the expression of interferon-2 (IFN-2) in HSCs, which appears to cause death of hepatocytes via a mechanism involving nuclear expression of IRF-1. LPS also increases expression of FasL on HSCs, which causes death of adjacent hepatocytes by interacting with Fas. Interestingly, LPS-induced production of the factors by HSCs that cause hepatocyte apoptosis is not mediated by the known pathways involving CD14, LPS-binding protein (LBP) and Toll-like receptor 4 (TLR4), and is very novel. CD14/TLR4 pathway is essential for the actions of LPS on Kupffer cells, thus exemplifying the unique aspect of HSC-LPS interactions in hepatic pathobiology of endotoxemia. Therefore, we will test the hypothesis that IFN-2 and FasL derived from LPS- stimulated HSCs play a major role in ALF directly or by sensitizing hepatocytes to the "second hit" by mediators released in the liver's micoenvironment.
Our Specific Aims are:
Aim 1 : To investigate mechanisms of LPS-stimulated IFN-2 and FasL synthesis in HSCs. We will determine the molecular mechanisms of LPS-induced synthesis of IFN-2 and FasL in HSCs in primary culture. The cells will be isolated from CD14- and TLR4-deficient mice to demonstrate existence of a novel pathway of LPS actions on HSCs.
Aim 2 : To investigate the mechanisms of hepatocyte injury elicited by HSC-derived mediators. Experiments of this Aim will delineate the specific intracellular pathways by which the mediators derived from LPS-stimulated HSCs cause injury to hepatocytes. Specifically, we will assess how IRF-1 nuclear translocation initiates and propagates endoplasmic reticulum stress and apoptosis. Both wild type and IRF-1-deficient hepatocytes will be used for these delineations.
Aim 3 : To investigate the role of HSCs in liver injury in vivo via their deletion. We have developed, for the first time, a mouse model in which HSCs are selectively deleted. Using this mouse, we propose to unequivocally demonstrate the role of HSCs in liver injury. We believe that successful completion of the experiments of these Aims will provide novel and important information in regard to the mechanisms by which HSCs contribute to liver failure. Such understanding is expected to highlight future avenues in developing effective treatment options, particularly in blocking the responses of HSCs to LPS or the specific signaling pathways responsible for hepatocyte injury.
NARRATIVE Acute liver failure (ALF), a devastating condition that affects patients with and without prior liver disease, has a mortality rate of 80% (>20,000 deaths/year in the USA). This statistics also applies to the veteran patients served by the Veterans Administration. Although the causal factors for the "irreversible" ALF are generally known, due to the lack of effective pharmacologic treatment, liver transplantation continues to be the only life- saving choice. Using specific rodent models, this investigation will obtain critically important mechanistic information in regard to ALF. Such understanding is expected to highlight future avenues in developing effective treatment options, particularly in blocking the specific biological pathways responsible for liver injury.