Abstract
Background and AimsHepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.Methods and ResultsThe analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.ConclusionsIn summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.
Highlights
Hepatic stellate cells (HSC) have recently been identified as liver-resident mesenchymal stem cells and are thought to contribute to liver repair and fibrogenesis [1,2,3]
Global DNA demethylation during HSC activation was investigated by 5bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism
These results indicate that epigenetic mechanisms are important for the control of early HSC activation
Summary
Hepatic stellate cells (HSC) have recently been identified as liver-resident mesenchymal stem cells and are thought to contribute to liver repair and fibrogenesis [1,2,3]. Activated HSC start to express α-smooth muscle actin (αSma) and extracellular matrix proteins such as collagen type I, a process involved in liver fibrosis. Despite their role during fibrogenesis the true function of HSC in the normal, uninjured liver remained unknown. HSC have been described as mesenchymal stem cells due to their potential to differentiate into adipocytes and osteocytes and their ability to support hematopoiesis [1,8]. HSC are important players during liver regeneration, where they can either support regeneration through the secretion of mitogenic factors like hepatocyte growth factor (Hgf) [9] or even participate directly in regeneration by differentiating into hepatocytes as shown in a stem-cell based liver regeneration model in rat [2]
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