Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells
Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.
Apolipoprotein B100 is required for hepatitis C infectivity and Mipomersen inhibits hepatitis C
AIM/OBJECTIVE:To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection. METHODS:cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method. RESULTS:cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus. CONCLUSION/CONCLUSIONS:ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.
Long noncoding RNAs expressed in human hepatic stellate cells form networks with extracellular matrix proteins
BACKGROUND: Hepatic fibrosis is the underlying cause of cirrhosis and liver failure in nearly every form of chronic liver disease, and hepatic stellate cells (HSCs) are the primary cell type responsible for fibrosis. Long noncoding RNAs (lncRNAs) are increasingly recognized as regulators of development and disease; however, little is known about their expression in human HSCs and their function in hepatic fibrosis. METHODS: We performed RNA sequencing and ab initio assembly of RNA transcripts to define the lncRNAs expressed in human HSC myofibroblasts. We analyzed chromatin immunoprecipitation data and expression data to identify lncRNAs that were regulated by transforming growth factor beta (TGF-beta) signaling, associated with super-enhancers and restricted in expression to HSCs compared with 43 human tissues and cell types. Co-expression network analyses were performed to discover functional modules of lncRNAs, and principle component analysis and K-mean clustering were used to compare lncRNA expression in HSCs with other myofibroblast cell types. RESULTS: We identified over 3600 lncRNAs that are expressed in human HSC myofibroblasts. Many are regulated by TGF-beta, a major fibrotic signal, and form networks with genes encoding key components of the extracellular matrix (ECM), which is the substrate of the fibrotic scar. The lncRNAs directly regulated by TGF-beta signaling are also enriched at super-enhancers. More than 400 of the lncRNAs identified in HSCs are uniquely expressed in HSCs compared with 43 other human tissues and cell types and HSC myofibroblasts demonstrate different patterns of lncRNA expression compared with myofibroblasts originating from other tissues. Co-expression analyses identified a subset of lncRNAs that are tightly linked to collagen genes and numerous proteins that regulate the ECM during formation of the fibrotic scar. Finally, we identified lncRNAs that are induced during progression of human liver disease. CONCLUSIONS: lncRNAs are likely key contributors to the formation and progression of fibrosis in human liver disease.
Hepatic Injury in Nonalcoholic Steatohepatitis Contributes to Altered Intestinal Permeability
BACKGROUND & AIMS: Emerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism. METHODS: A meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells. RESULTS: Nonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-alpha (TNFalpha) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNFalpha production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNFalpha in altering intestinal permeability, we found that mice genetically resistant to TNFalpha-myosin light chain kinase (MLCK)-induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury. CONCLUSIONS: Our clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNFalpha and MLCK.
Noninvasive Biomarkers of Liver Fibrosis: Clinical Applications and Future Directions
Chronic liver disease is a significant cause of morbidity and mortality worldwide. Current strategies for assessing prognosis and treatment rely on accurate assessment of disease stage. Liver biopsy is the gold standard for assessing fibrosis stage but has many limitations. Noninvasive biomarkers of liver fibrosis have been extensively designed, studied, and validated in a variety of liver diseases. With the advent of direct acting antivirals and the rise in obesity-related liver disease, there is a growing need to establish these noninvasive methods in the clinic. In addition, it has become increasingly clear over the last few years that noninvasive biomarkers can also be used to monitor response to antifibrotic therapies and predict liver outcomes, including hepatocellular carcinoma development. This review highlights the most well-established noninvasive biomarkers to-date, with a particular emphasis on serum and imaging-based methodologies.
Hepatic Gap Junction Inhibition Protects Against Liver Injury and Inflammation in a Murine Model of Alcoholic Steatohepatitis [Meeting Abstract]
A TALEN genome-editing system for generating human stem cell-based disease models
Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
[Association between overweight, glucocorticoids and metabolic syndrome in cancer patients under chemotherapy]
Publisher: Abstract available from the publisher. OABL- spa
Structural conservation of ligand binding reveals a bile acid-like signaling pathway in nematodes
Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways.
The Rieske oxygenase DAF-36 functions as a cholesterol 7-desaturase in steroidogenic pathways governing longevity
Bile acids are cholesterol-derived signaling molecules that regulate mammalian metabolism through sterol-sensing nuclear receptor transcription factors. In C. elegans, bile acid-like steroids called dafachronic acids (DAs) control developmental timing and longevity by activating the nuclear receptor DAF-12. However, little is known about the biosynthesis of these molecules. Here, we show that the DAF-36/Rieske oxygenase works at the first committed step, converting cholesterol to 7-dehydrocholesterol. Its elucidation as a cholesterol 7-desaturase provides crucial biochemical evidence that such oxygenases are key steroidogenic enzymes. By controlling DA production, DAF-36 regulates DAF-12 activities for reproductive development and longevity and may illuminate related pathways in metazoans.