The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.

BACKGROUND & AIMS: In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. METHODS: C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro. RESULTS: In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses. CONCLUSIONS: In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.

Reply to "Comments on the Editor Re: The Relationship of Obesity, Nutritional Status and Muscle Wasting in Patients Assessed for Liver Transplantation, Nutrients 2019, 11, 2097."

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The Relationship of Obesity, Nutritional Status and Muscle Wasting in Patients Assessed for Liver Transplantation.

INTRODUCTION: Obesity co-exists with malnutrition and muscle atrophy in patients with cirrhosis. Muscle wasting is a feature of sarcopenia, a known determinant of patient outcomes. This is the first description of a relationship between obesity, subjective global assessment (SGA) of nutritional status and muscle wasting in patients with cirrhosis. METHODS: The relationship between body mass index (BMI with obesity defined as ≥ 30 kg/m2), nutritional status (assessed by liver-specific subjective global assessment-SGA) and muscle wasting (assessed by corrected total cross-sectional psoas muscle area-cTPA) was analysed in patients with cirrhosis considered for liver transplantation between 1 January 2012 and 31 December 2014. RESULTS: There were 205 patients, of whom 70% were males. The mean age was 52 ± 0.7 years and the Model for End-Stage Liver Disease (MELD) score was 16.8 ± 0.5. Overall, 31% of patients were obese and 56% of well-nourished (SGA A) individuals were obese. Muscle wasting was identified in 86% of all patients, irrespective of their nutritional status (A, B, C). All obese males classified as well-nourished (SGA A) were sarcopenic and 62% of obese females classified as SGA A were sarcopenic. Muscle wasting was worse in obese individuals (cTPA 230.9 mm2/m2 ± 12.9, p < 0.0001) and more likely to be associated with hepatic encephalopathy (p = 0.03). Univariate and multivariate analysis demonstrated testosterone deficiency was significantly associated with muscle wasting (p = 0.007) but not obesity (p = 0.8). CONCLUSION: Obesity combined with muscle wasting is common in patients with cirrhosis. Muscle wasting is common in well-nourished (SGA A) obese patients. Consequently, all patients assessed for liver transplantation should undergo additional screening for malnutrition and muscle wasting irrespective of BMI.

Sequence analysis of integrated hepatitis B virus DNA during HBeAg-seroconversion.

Hepatitis B virus (HBV) integration into the host cell genome occurs early on in infection and reportedly induces pro-oncogenic changes in hepatocytes that drive HCC initiation. However, it remains unclear when these changes occur during hepatocarcinogenesis. Extensive expansion of hepatocyte clones with a selective advantage was shown to occur prior to cancer formation during the HBeAg-seroconversion phase of chronic HBV infection. We hypothesized that since integrations occur during the early stages of infection, cell phenotype could be altered and induce a selection advantage (e.g., through insertional mutagenesis or cis-mediated activation of downstream genes). Here, we analyzed the enrichment of genomic and functional patterns in the cellular host sequence adjacent to HBV DNA integration events. We examined 717 unique integration events detected in patients who have and have not undergone HBeAg-seroconversion (n = 41) or in an in vitro model system. We also used an in silico model to control for detection biases. We showed that the sites of HBV DNA integration were distributed throughout the entire host genome without obvious enrichment of specific structural or functional genomic features in the adjacent cellular genome during HBeAg-seroconversion. Currently, this is the most comprehensive characterization of HBV DNA integration events prior to hepatocarcinogenesis. Our results suggest no significant selection for (or against) specific cellular sites of HBV DNA integration occur during the clonal expansion phase of chronic HBV infection. Thus, HBV DNA integration events likely represent passenger events rather than active drivers of liver cancer, which was previously suggested.

Randomised clinical trial: oral taurine supplementation versus placebo reduces muscle cramps in patients with chronic liver disease.

BACKGROUND: Painful muscle cramps occur in the majority of patients with cirrhosis impacting significantly on quality of life and sleep patterns. They are frequently unrecognised or overlooked. Current management is based on anecdotal evidence or case study reports. AIM: To investigate the effect of oral taurine supplementation on frequency, duration, and intensity of muscle cramps in patients with chronic liver disease. METHODS: Patients with chronic liver disease who experienced three or more muscle cramps/week were enrolled in a double-blinded, randomised control, crossover, taurine dose-variable study. Each participant received either taurine supplementation or placebo for 4 weeks then crossed to the alternative arm. Primary outcome data for frequency, duration, and intensity of muscle cramps was recorded by participants. Participants recorded frequency, duration, and location of muscle cramps. Biochemical parameters, including serum taurine and methionine levels, were measured at each time point. Linear mixed models were used to analyse outcomes. RESULTS: Forty-nine patients were enrolled in the study and 30 patients completed the protocol. Participants who were unable to complete the protocol were not included in the final analysis due to the absence of outcome data. The mean age of participants was 54.7 years and 70% were males. Oral taurine supplementation increased serum taurine levels (P < 0.001). There were no adverse side effects associated with taurine supplementation. Participants receiving 2 g taurine/d experienced a reduction in cramp frequency (seven cramps fewer/fortnight, P = 0.03), duration (89 minutes less/fortnight P = 0.03), and severity (1.4 units less on a Likert scale P < 0.004) compared to placebo. CONCLUSIONS: Oral supplementation with 2 g taurine/d results in a clinically significant reduction in the frequency, duration, and intensity of muscle cramps in patients with chronic liver disease. Taurine should be considered as a safe and effective intervention in the management of muscle cramps in individuals with chronic liver disease. This study was registered with the Australian New Zealand Clinical Trials Register: ACTRN12612000289819.

Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease.

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.