The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl 4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent transmethylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl 4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis.

A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies. Dynamic changes in chromatin organization and DNA modifications participate in the establishment of cell identity in normal human biology and disease 1. Epigenetic marks such as DNA methylation are characteristic of a particular cell type as they help define the associated transcriptome 2,3. DNA methyl-transferases (DNMTs) establish 5-methylcytosine (5mC) from S-adenosylmethionine (SAM), the principal methylating agent in the body derived from the methionine cycle 4. In humans, the liver is the organ with the highest turnover of SAM and

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Hepatic fibrosis occurs in response to persistent liver damage and is characterized by an excessive accumulation of extracellular matrix. When the damage is prolonged, there is a chronic inflammation and persistent hepatic fibrosis eventually leads to cirrhosis, where in addition to the scar, there is an important vascular remodeling associated with portal hypertension and, if decompensated, leads to death or can develop hepatocellular carcinoma. We have been studying the pharmacologic functions of adenosine, finding that a derivative of this nucleoside, IFC-305, shows hepatoprotective effects in a CCl 4-induced rat cirrhosis model where it reverses liver fibrosis through modulation of fibrosis-related genes and by ameliorating hepatic function. Furthermore, this compound has the property to rescue cell cycle inhibition in vivo, prevents hepatic stellate cell activation, modulates antiinflammatory macrophage polarization, and favors a chromatin context that could decrease the genomic instability and characteristics of cirrhosis, enabling the recovery of gene expression profile. Here we show results that contribute to the comprehension of molecular and cellular mechanism of cirrhosis, give the opportunity to suggest biomarkers to the early diagnostic of this pathology, and constitute the fundaments to suggest IFC-305 as a coadjuvant for treatment of this disease.

ObjectiveTo evaluate the relationship between pro-atherogenic biomarkers and epicardial adipose tissue (EAT) thickness in patients with cardiovascular risk factors.MethodsPlasma nitric oxide (NO), soluble intercellular adhesion molecule-1 and malondialdehyde (MDA) levels, EAT thickness, flow-mediated dilation (FMD) and carotid intima media thickness (CIMT) were determined in patients aged >18 years who were referred for echocardiography for heart ischemia or non-ischemic diseases. Cardiovascular risk factors (Framingham score [FS] ≥ 20) were weighted.ResultsHypertension, dyslipidaemia and type 2 diabetes mellitus were prevalent (≥55% of 40 patients). Patients with FS ≥ 20 (n = 21) showed significantly higher EAT and CIMT values. Globally, MDA, CIMT, age, waist circumference, high-density lipoprotein cholesterol (HDL-C) and FS were associated with EAT thickness. EAT was significantly associated with NO in patients with FS ≥ 20. Significant differences in EAT thickness were found between patients stratified by NO value, FMD, age, smoking status, dyslipidaemia, type 2 diabetes mellitus and FS. An EAT-associated atherogenic risk (CIMT ≥ 1 mm) model was statistically significant when MDA and type 2 diabetes mellitus were included.ConclusionEAT thickness was associated with MDA, CIMT, age, waist circumference, HDL-C and FS globally, but with NO only in patients with FS≥20. EAT may be used to identify vascular damage stage, possibly influenced by MDA and type 2 diabetes mellitus.

How cells reach different fates despite using the same DNA template, is a basic question linked to differential patterns of gene expression. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here we asked whether 5hmC is involved in hepatocyte differentiation. We found a genome-wide increase of 5hmC as well as a reduction of 5-methylcytosine at early hepatocyte differentiation, a time when the liver transcript program is already established. Furthermore, we suggest that modifying sadenosylmethionine (SAM) levels through an adenosine derivative could decrease 5hmC enrichment, triggering an impaired acquisition of hepatic identity markers. These results suggest that 5hmC is a regulator of differentiation as well as an imprint related with cell identity.Furthermore, 5hmC modulation could be a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies. Abstract It has been suggested that 5hydroxymethylcytosine (5hmC) is an imprint of cell identity. Here we show that commitment to a hepatocyte transcriptional program is characterized by a demethylation process and emergence of 5hmC at multiple genomic locations. Cells exposed to an adenosine derivative during differentiation did not reach such 5hmC levels, and this was associated with a lower expression of hepatocytemarkers. These results suggest that 5hmC enrichment is an important step on the road to hepatocyte cell fate.