Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis.

OBJECTIVE: Caspase-2 is an initiator caspase involved in multiple apoptotic pathways, particularly in response to specific intracellular stressors (eg, DNA damage, ER stress). We recently reported that caspase-2 was pivotal for the induction of cell death triggered by excessive intracellular accumulation of long-chain fatty acids, a response known as lipoapoptosis. The liver is particularly susceptible to lipid-induced damage, explaining the pandemic status of non-alcoholic fatty liver disease (NAFLD). Progression from NAFLD to non-alcoholic steatohepatitis (NASH) results, in part, from hepatocyte apoptosis and consequential paracrine-mediated fibrogenesis. We evaluated the hypothesis that caspase-2 promotes NASH-related cirrhosis. DESIGN: Caspase-2 was localised in liver biopsies from patients with NASH. Its expression was evaluated in different mouse models of NASH, and outcomes of diet-induced NASH were compared in wild-type (WT) and caspase-2-deficient mice. Lipotoxicity was modelled in vitro using hepatocytes derived from WT and caspase-2-deficient mice. RESULTS: We showed that caspase-2 is integral to the pathogenesis of NASH-related cirrhosis. Caspase-2 is localised in injured hepatocytes and its expression was markedly upregulated in patients and animal models of NASH. During lipotoxic stress, caspase-2 deficiency reduced apoptosis, inhibited induction of profibrogenic hedgehog target genes in mice and blocked production of hedgehog ligands in cultured hepatocytes. CONCLUSIONS: These data point to a critical role for caspase-2 in lipid-induced hepatocyte apoptosis in vivo for the production of apoptosis-associated fibrogenic factors and in the progression of lipid-induced liver fibrosis. This raises the intriguing possibility that caspase-2 may be a promising therapeutic target to prevent progression to NASH.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice.

BACKGROUND: Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. METHODS: Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-ß, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. RESULTS: OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-ß signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. CONCLUSIONS: OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

Myofibroblastic cells function as progenitors to regenerate murine livers after partial hepatectomy.

OBJECTIVE: Smoothened (SMO), a coreceptor of the Hedgehog (Hh) pathway, promotes fibrogenic repair of chronic liver injury. We investigated the roles of SMO+ myofibroblast (MF) in liver regeneration by conditional deletion of SMO in α smooth muscle actin (αSMA)+ cells after partial hepatectomy (PH). DESIGN: αSMA-Cre-ER(T2)×SMO/flox mice were treated with vehicle (VEH) or tamoxifen (TMX), and sacrificed 24-96 h post-PH. Regenerating livers were analysed for proliferation, progenitors and fibrosis by qRT-PCR and quantitative immunohistochemistry (IHC). Results were normalised to liver segments resected at PH. For lineage-tracing studies, αSMA-Cre-ER(T2)×ROSA-Stop-flox-yellow fluorescent protein (YFP) mice were treated with VEH or TMX; livers were stained for YFP, and hepatocytes isolated 48 and 72 h post-PH were analysed for YFP by flow cytometric analysis (FACS). RESULTS: Post-PH, VEH-αSMA-SMO mice increased expression of Hh-genes, transiently accumulated MF, fibrosis and liver progenitors, and ultimately exhibited proliferation of hepatocytes and cholangiocytes. In contrast, TMX-αSMA-SMO mice showed loss of whole liver SMO expression, repression of Hh-genes, enhanced accumulation of quiescent HSC but reduced accumulation of MF, fibrosis and progenitors, as well as inhibition of hepatocyte and cholangiocyte proliferation, and reduced recovery of liver weight. In TMX-αSMA-YFP mice, many progenitors, cholangiocytes and up to 25% of hepatocytes were YFP+ by 48-72 h after PH, indicating that liver epithelial cells were derived from αSMA-YFP+ cells. CONCLUSIONS: Hh signalling promotes transition of quiescent hepatic stellate cells to fibrogenic MF, some of which become progenitors that regenerate the liver epithelial compartment after PH. Hence, scarring is a component of successful liver regeneration.