We asked if ancestral liver damage leads to heritable reprogramming of hepatic wound-healing. We discovered that male rats with a history of liver damage transmit epigenetic suppressive adaptation of the fibrogenic component of wound-healing through male F1 and F2 generations. Underlying this adaptation was reduced generation of liver myofibroblasts, increased hepatic expression of antifibrogenic PPAR-γ and decreased expression of profibrogenic TGF-β1. Remodelling of DNA methylation and histone acetylation underpinned these alterations in gene expression. Sperm from rats with liver fibrosis were enriched for H2A.Z and H3K27me3 at PPAR-γ chromatin. These sperm chromatin modifications were transmittable by adaptive serum transfer from fibrotic rats and were induced in stem cells exposed to myofibroblast-conditioned media. A myofibroblast secreted soluble factor therefore stimulates heritable epigenetic signatures to sperm so as to adapt fibrogenesis in offspring. Humans with mild liver fibrosis display PPAR-γ promoter hypomethylation compared with severe fibrotics, thus lending support for epigenetic regulation of fibrosis.

Objective Liver biopsy is currently the most reliable way of evaluating liver fibrosis in patients with Non-Alcoholic Fatty Liver Disease (NAFLD). Its inherent risks limit its widespread use. Differential liver DNA methylation of PPARγ gene promoter has recently been shown to stratify patients in terms of fibrosis severity but requires access to liver tissue. The aim of this study was to assess whether DNA methylation of circulating DNA could be detected in human plasma and potentially used to stratify liver fibrosis severity in patients with NAFLD. Design Patients with biopsy- proven NAFLD, and age-matched controls were recruited from the Liver and Gastroenterology Clinics at the Newcastle upon Tyne Hospitals NHS Foundation Trust. Plasma cell free circulating DNA methylation of PPARγ was quantitatively assessed by pyrosequencing. Liver DNA methylation was quantitatively assessed by pyrosequencing NAFLD explant tissue, subjected to laser capture microdissection (LCM). Patients with ALD were also subjected to plasma DNA and LCM pyrosequencing. Results 26 patients with biopsy-proven NAFLD were included. Quantitative Plasma DNA methylation of PPARγ stratified patients into mild (Kleiner 1-2) and severe (Kleiner 3-4) fibrosis (CpG1: 63% vs 86%, p<0.05; CpG2: 51% vs 65% p>0.05). Hypermethylation at the PPARγ promoter of plasma DNA correlated with changes in hepatocellular rather than myofibroblast DNA methylation. Similar results were demonstrated in patients with ALD cirrhosis. Conclusions Differential DNA methylation at the PPARγ promoter can be detected within the pool of cell-free DNA of human plasma. With further validation, plasma DNA methylation of PPARγ could potentially be used to noninvasively stratify liver fibrosis severity in patients with NAFLD. Plasma DNA methylation signatures reflect the molecular pathology associated with fibrotic liver disease.

BackgroundChronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patients. Currently, it is not clear which factors determine progression to fibrosis. We investigated whether DNA\methylation profile as determined by pyrosequencing can distinguish patients with mild from those with advanced/severe fibrosis in non-alcoholic liver disease (NAFLD) and alcoholic liver disease (ALD). To this end, paraffin-embedded liver biopsies were collected from patients with biopsy-proven NAFLD or ALD, as well as paraffin-embedded normal liver resections, genomic DNA isolated, bisulfite converted and pyrosequencing assays used to quantify DNA methylation at specific CpGs within PPARα, PPARα, TGFβ1, Collagen 1A1 and PDGFα genes. Furthermore, we assessed the impact of age, gender and anatomical location within the liver on patterns of DNA methylation in the same panel of genes.ResultsDNA methylation at specific CpGs within genes known to affect fibrogenesis distinguishes between patients with mild from those with severe fibrosis in both NAFLD and ALD, although same CpGs are not equally represented in both etiologies. In normal liver, age, gender or anatomical location had no significant impact on DNA methylation patterns in the liver.ConclusionsDNA methylation status at specific CpGs may be useful as part of a wider set of patient data for predicting progression to liver fibrosis.

Acute alcoholic hepatitis is characterized by disproportionate macrophage inflammatory cytokine responses to bacterial lipopolysaccharide. Lack of knowledge of the underlying mechanism has limited progress toward effective therapy. We postulated a novel mechanism by which ethanol increases histone acetylation, increasing proinflammatory gene transcription and cytokine synthesis. Cytokine responses to lipopolysaccharide in a human macrophage cell line cultured in 86 mM ethanol, 1 mM acetate, and normal media were measured by multiplex immunoassay. Changes in histone acetylation were determined by immunofluorescence microscopy and chromatin immunoprecipitation on presentation. The effect of ethanol and acetate on acetyl-coenzyme A (acetyl-coA) synthetases, which convert acetate to acetyl-coA, the substrate for histone acetylation, was determined by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Knockdown of acetyl-coA synthetases by short hairpin RNA (shRNA) was used to determine their role in ethanol's enhancement of the inflammatory cytokine response. Ethanolexposed macrophages developed enhanced interleukin 6 (IL6), IL8, and tumor necrosis factor alpha responses to lipopolysaccharide with time-dependent increases in histone acetylation that could be prevented by inhibition of ethanol metabolism. Chromatin immunoprecipitation confirmed increased histone acetylation at promoter regions of specific cytokine genes. The effect of ethanol was reproduced by incubation with acetate, the principal hepatic metabolite of ethanol, and both ethanol and acetate reduced histone deacetylase activity and up-regulated acetyl-coA synthetases. Knockdown of the acetyl-coA synthetases abrogated the effect of ethanol on cytokine production. Conclusion: Synthesis of metabolically available acetyl-coA from acetate is critical to the increased acetylation of proinflammatory gene histones and consequent enhancement of the inflammatory response in ethanol-exposed macrophages. This mechanism is a potential therapeutic target in acute alcoholic hepatitis. (HEPATOLOGY 2010; 51:1988-1997 A lcoholic liver disease (ALD) is a significant and growing global health problem. Clinical liver failure in ALD can result from chronic hepatocyte injury producing cirrhosis or from rapid, acute hepatocellular dysfunction secondary to inflammation in acute alcoholic hepatitis. AAH This acute inflammatory form of ALD carries a mortality of up to 35% on first presentation, killing patients before they have the opportunity to reap the benefits of appropriate health education and subsequent abstinence from alcohol. 1

Transdifferentiation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in liver fibrosis. The dramatic change in phenotype associated with transdifferentiation is underpinned by a global change in gene expression. Orchestrated changes in gene expression take place at the level of chromatin packaging which is regulated by enzymatic activity of epigenetic regulators that in turn affect histone modifications. Using expression profiling of epigenetic regulators in quiescent and activated primary HSCs we found a number of histone methyltranferases including MLL1, MLL5, Set1 and ASH1 to be highly up-regulated during transdifferentiation of HSCs. All of these histone methyltranferases regulate methylation of lysine 4 of histone H3, which is a signature of actively transcribed genes. We therefore postulated that one or more of these enzymes may be involved in positively influencing expression of profibrogenic genes. Conclusion: We find that ASH1 directly binds to the regulatory regions of alpha smooth muscle actin (αSMA), collagen I, tissue inhibitor of metalloproteinase-1 (TIMP1) and transforming growth factor beta1 (TGFβ1) in activated HSCs while depletion of ASH1 caused broad suppression of fibrogenic gene expression. We also discovered that MeCP2 positively regulates ASH1 expression and therefore identify ASH1 as a key transcriptional activator component of the MeCP2 epigenetic relay pathway that orchestrates coordinated induction of multiple profibrogenic genes. (Hepatology 2012;56:1129–1139)