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.

Background and aims DNA methylation is an epigenetic mark that is an established regulator of transcriptional repression with an important role in liver fibrosis. Currently, there is very little knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cell (HSC), the key cell type responsible for onset and progression of liver fibrosis. Moreover, recently discovered DNA hydroxymethylation is involved in transcriptional activation and its patterns are often altered in human diseases. The aim of this study is to investigate the role of DNA methylation/hydroxymethylation in liver fibrosis. Methods Levels of 5-mC and 5-hmC were assessed by slot blot in a range of animal liver fibrosis models and human liver diseases. Expression levels of TET and DNMT enzymes were measured by qRT-PCR and western blotting. Reduced representation bisulfite sequencing method was used to examine 5-mC and 5-hmC patterns in quiescent and in vivo activated rat HSC. Results We demonstrate global alteration in 5-mC and 5-hmC and their regulatory enzymes that accompany liver fibrosis and HSC transdifferentiation. Using RRBS, we show exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. In addition, we demonstrate that reduction in DNMT3a expression leads to attenuation of pro-fibrogenic phenotype in activated HSC. Conclusions Our data suggest that DNA methylation/hydroxymethylation is a crucial step in HSC activation and therefore fibrogenesis. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.

In high-risk patients with necrotising pancreatitis, minimally invasive surgical and endoscopic necrosectomy are associated with reduced death rates compared with open necrosectomy.

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.

Background and aims Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSC are responsive to direct stimulation by alcohol. Methods HSC undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from mouse alcohol model, ALD patient liver and precision cut liver slices. Results HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced altered expression of multiple epigenetic regulators indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. Conclusions Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure resulting in increased expression of ECM proteins. The ability of alcohol to remodel epigenome during HSC transdifferentiation provides mechanisms for it to act as a comorbidity factor in liver disease.