Guanhua Xie

Smoothened is a master regulator of adult liver repair

When regenerative processes cannot keep pace with cell death, functional epithelia are replaced by scar. Scarring is characterized by both excessive accumulation of fibrous matrix and persistent outgrowth of cell types that accumulate transiently during successful wound healing, including myofibroblasts (MFs) and progenitors. This suggests that signaling that normally directs these cells to repair injured epithelia is deregulated. To evaluate this possibility, we examined liver repair during different types of liver injury after Smoothened (SMO), an obligate intermediate in the Hedgehog (Hh) signaling pathway, was conditionally deleted in cells expressing the MF-associated gene, αSMA. Surprisingly, blocking canonical Hh signaling in MFs not only inhibited liver fibrosis but also prevented accumulation of liver progenitors. Hh-sensitive, hepatic stellate cells (HSCs) were identified as the source of both MFs and progenitors by lineage-tracing studies in 3 other strains of mice, coupled with analysis of highly pure HSC preparations using flow cytometry, immunofluorescence confocal microscopy, RT-PCR, and in situ hybridization. The results identify SMO as a master regulator of hepatic epithelial regeneration based on its ability to promote mesenchymal-to-epithelial transitions in a subpopulation of HSC-derived MFs with features of multipotent progenitors.

NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease

Objective Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. Design The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. Results When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18‒/‒ and CD1d‒/‒ mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. Conclusion Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.

Hedgehog Controls Hepatic Stellate Cell Fate by Regulating Metabolism

BACKGROUND & AIMS The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.

Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease

Background and aims Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our time. Although there are several animal models of NASH, consensus regarding the optimal model is lacking. We aimed to compare features of NASH in the two most widely-used mouse models: methionine-choline deficient (MCD) diet and Western diet. Methods Mice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared. Results The metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation. Conclusion Feeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH.

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-ERT2×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-ERT2×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.

Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice

Liver repair involves phenotypic changes in hepatic stellate cells (HSCs) and reactivation of morphogenic signaling pathways that modulate epithelial‐to‐mesenchymal/mesenchymal‐to‐epithelial transitions, such as Notch and Hedgehog (Hh). Hh stimulates HSCs to become myofibroblasts (MFs). Recent lineage tracing studies in adult mice with injured livers showed that some MFs became multipotent progenitors to regenerate hepatocytes, cholangiocytes, and HSCs. We studied primary HSC cultures and two different animal models of fibrosis to evaluate the hypothesis that activating the Notch pathway in HSCs stimulates them to become (and remain) MFs through a mechanism that involves an epithelial‐to‐mesenchymal–like transition and requires cross‐talk with the canonical Hh pathway. We found that when cultured HSCs transitioned into MFs, they activated Hh signaling, underwent an epithelial‐to‐mesenchymal–like transition, and increased Notch signaling. Blocking Notch signaling in MFs/HSCs suppressed Hh activity and caused a mesenchymal‐to‐epithelial–like transition. Inhibiting the Hh pathway suppressed Notch signaling and also induced a mesenchymal‐to‐epithelial–like transition. Manipulating Hh and Notch signaling in a mouse multipotent progenitor cell line evoked similar responses. In mice, liver injury increased Notch activity in MFs and Hh‐responsive MF progeny (i.e., HSCs and ductular cells). Conditionally disrupting Hh signaling in MFs of bile‐duct–ligated mice inhibited Notch signaling and blocked accumulation of both MF and ductular cells. Conclusions: The Notch and Hedgehog pathways interact to control the fate of key cell types involved in adult liver repair by modulating epithelial‐to‐mesenchymal–like/mesenchymal‐to‐epithelial–like transitions. (Hepatology 2013;58:1801–1813)

Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation

Objective Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. Design Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. Results Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from SmoloxP/loxP transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. Conclusions LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.

Evidence for and against epithelial-to-mesenchymal transition in the liver

The outcome of liver injury is determined by the success of repair. Liver repair involves replacement of damaged liver tissue with healthy liver epithelial cells (including both hepatocytes and cholangiocytes) and reconstruction of normal liver structure and function. Current dogma posits that replication of surviving mature hepatocytes and cholangiocytes drives the regeneration of liver epithelium after injury, whereas failure of liver repair commonly leads to fibrosis, a scarring condition in which hepatic stellate cells, the main liver-resident mesenchymal cells, play the major role. The present review discusses other mechanisms that might be responsible for the regeneration of new liver epithelial cells and outgrowth of matrix-producing mesenchymal cells during hepatic injury. This theory proposes that, during liver injury, some epithelial cells undergo epithelial-to-mesenchymal transition (EMT), acquire myofibroblastic phenotypes/features, and contribute to fibrogenesis, whereas certain mesenchymal cells (namely hepatic stellate cells and stellate cell-derived myofibroblasts) undergo mesenchymal-to-epithelial transition (MET), revert to epithelial cells, and ultimately differentiate into either hepatocytes or cholangiocytes. Although this theory is highly controversial, it suggests that the balance between EMT and MET modulates the outcome of liver injury. This review summarizes recent advances that support or refute the concept that certain types of liver cells are capable of phenotype transition (i.e., EMT and MET) during both culture conditions and chronic liver injury.

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. Methods To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. Results In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12–8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. Conclusions TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.

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.