Chronic ethanol feeding alters miRNA expression dynamics during liver regeneration.

BACKGROUND: Adaptation to chronic ethanol (EtOH) treatment of rats results in a changed functional state of the liver and greatly inhibits its regenerative ability, which may contribute to the progression of alcoholic liver disease. METHODS: In this study, we investigated the effect of chronic EtOH intake on hepatic microRNA (miRNA) expression in male Sprague-Dawley rats during the initial 24 hours of liver regeneration following 70% partial hepatectomy (PHx) using miRNA microarrays. miRNA expression during adaptation to EtOH was investigated using RT-qPCR. Nuclear factor kappa B (NFκB) binding at target miRNA promoters was investigated with chromatin immunoprecipitation. RESULTS: Unsupervised clustering of miRNA expression profiles suggested that miRNA expression was more affected by chronic EtOH feeding than by the acute challenge of liver regeneration after PHx. Several miRNAs that were significantly altered by chronic EtOH feeding, including miR-34a, miR-103, miR-107, and miR-122 have been reported to play a role in regulating hepatic metabolism and the onset of these miRNA changes occurred gradually during the time course of EtOH feeding. Chronic EtOH feeding also altered the dynamic miRNA profile during liver regeneration. Promoter analysis predicted a role for NFκB in the immediate-early miRNA response to PHx. NFκB binding at target miRNA promoters in the chronic EtOH-fed group was significantly altered and these changes directly correlated with the observed expression dynamics of the target miRNA. CONCLUSIONS: Chronic EtOH consumption alters the hepatic miRNA expression profile such that the response of the metabolism-associated miRNAs occurs during long-term adaptation to EtOH rather than as an acute transient response to EtOH metabolism. Additionally, the dynamic miRNA program during liver regeneration in response to PHx is altered in the chronically EtOH-fed liver and these differences reflect, in part, differences in miRNA expression between the EtOH-adapted and control livers at the baseline state prior to PHx

Temporal and functional profile of the transcriptional regulatory network in the early regenerative response to partial hepatectomy in the rat

<p>Abstract</p> <p>Background</p> <p>The goal of these studies was to characterize the transcriptional network regulating changes in gene expression in the remnant liver of the rat after 70% partial hepatectomy (PHx) during the early phase response including the transition of hepatocytes from the quiescent (G₀) state and the onset of the G₁phase of the cell cycle.</p> <p>Results</p> <p>The transcriptome of remnant livers was monitored at 1, 2, 4, and 6 hours after PHx using cDNA microarrays. Differentially regulated genes were grouped into six clusters according their temporal expression profiles. Promoter regions of genes in these clusters were examined for shared transcription factor binding sites (TFBS) by comparing enrichment of each TFBS relative to a reference set using the Promoter Analysis and Interaction Network Toolset (PAINT).</p> <p>Analysis of the gene expression time series data using ANOVA resulted in a total of 309 genes significantly up- or down-regulated at <it>any </it>of the four time points at a 20% FDR threshold. Sham-operated animals showed no significant differential expression. A subset of the differentially expressed genes was validated using quantitative RT-PCR. Distinct sets of TFBS could be identified that were significantly enriched in each one of the different temporal gene expression clusters. These included binding sites for transcription factors that had previously been recognized as contributing to the onset of regeneration, including NF-κB, C/EBP, HNF-1, CREB, as well as factors, such as ATF, AP-2, LEF-1, GATA and PAX-6, that had not yet been recognized to be involved in this process. A subset of these candidate TFBS was validated by measuring activation of corresponding transcription factors (HNF-1, NK-κB, CREB, C/EBP-α and C/EBP-β, GATA-1, AP-2, PAX-6) in nuclear extracts from the remnant livers.</p> <p>Conclusion</p> <p>This analysis revealed multiple candidate transcription factors activated in the remnant livers, some known to be involved in the early phase of liver regeneration, and several not previously identified. The study describes the predominant temporal and functional elements to which these factors contribute and demonstrates the potential of this novel approach to define the functional correlates of the transcriptional regulatory network driving the early response to partial hepatectomy.</p

A novel comparative pattern analysis approach identifies chronic alcohol mediated dysregulation of transcriptomic dynamics during liver regeneration.

BACKGROUND: Liver regeneration is inhibited by chronic ethanol consumption and this impaired repair response may contribute to the risk for alcoholic liver disease. We developed and applied a novel data analysis approach to assess the effect of chronic ethanol intake in the mechanisms responsible for liver regeneration. We performed a time series transcriptomic profiling study of the regeneration response after 2/3(rd) partial hepatectomy (PHx) in ethanol-fed and isocaloric control rats. RESULTS: We developed a novel data analysis approach focusing on comparative pattern counts (COMPACT) to exhaustively identify the dominant and subtle differential expression patterns. Approximately 6500 genes were differentially regulated in Ethanol or Control groups within 24 h after PHx. Adaptation to chronic ethanol intake significantly altered the immediate early gene expression patterns and nearly completely abrogated the cell cycle induction in hepatocytes post PHx. The patterns highlighted by COMPACT analysis contained several non-parenchymal cell specific markers indicating their aberrant transcriptional response as a novel mechanism through which chronic ethanol intake deregulates the integrated liver tissue response. CONCLUSIONS: Our novel comparative pattern analysis revealed new insights into ethanol-mediated molecular changes in non-parenchymal liver cells as a possible contribution to the defective liver regeneration phenotype. The results revealed for the first time an ethanol-induced shift of hepatic stellate cells from a pro-regenerative phenotype to that of an anti-regenerative state after PHx. Our results can form the basis for novel interventions targeting the non-parenchymal cells in normalizing the dysfunctional repair response process in alcoholic liver disease. Our approach is illustrated online at http://compact.jefferson.edu

Single-Cell Gene Expression Analysis Identifies Chronic Alcohol-Mediated Shift in Hepatocyte Molecular States After Partial Hepatectomy.

The analysis of molecular states of individual cells, as defined by their mRNA expression profiles and protein composition, has gained widespread interest in studying biological phenomena ranging from embryonic development to homeostatic tissue function and genesis and evolution of cancers. Although the molecular content of individual cells in a tissue can vary widely, their molecular states tend to be constrained within a transcriptional landscape partly described by the canonical archetypes of a population of cells. In this study, we sought to characterize the effects of an acute (partial hepatectomy) and chronic (alcohol consumption) perturbation on the molecular states of individual hepatocytes during the onset and progression of liver regeneration. We analyzed the expression of 84 genes across 233 individual hepatocytes acquired using laser capture microdissection. Analysis of the single-cell data revealed that hepatocyte molecular states can be considered as distributed across a set of four states irrespective of perturbation, with the proportions of hepatocytes in these states being dependent on the perturbation. In addition to the quiescent, primed, and replicating hepatocytes, we identified a fourth molecular state lying between the primed and replicating subpopulations. Comparison of the proportions of hepatocytes from each experimental condition in these four molecular states suggested that, in addition to aberrant priming, a slower transition from primed to replication state could contribute toward ethanol-mediated suppression of liver regenerative response to partial hepatectomy

Transcriptional regulatory network analysis during epithelial-mesenchymal transformation of retinal pigment epithelium

PURPOSE: Phenotypic transformation of retinal pigment epithelial (RPE) cells contributes to the onset and progression of ocular proliferative disorders such as proliferative vitreoretinopathy (PVR). The formation of epiretinal membranes in PVR may involve an epithelial-mesenchymal transformation (EMT) of RPE cells as part of an aberrant wound healing response. While the underlying mechanism remains unclear, this likely involves changes in RPE cell gene expression under the control of specific transcription factors (TFs). Thus, the purpose of the present study was to identify TFs that may play a role in this process. METHODS: Regulatory regions of genes that are differentially regulated during phenotypic transformation of ARPE-19 cells, a human RPE cell line, were subjected to computational analysis using the promoter analysis and interaction network toolset (PAINT). The PAINT analysis was used to identify transcription response elements (TREs) statistically overrepresented in the promoter and first intron regions of two reciprocally regulated RPE gene clusters, across four species including the human genome. These TREs were then used to construct transcriptional regulatory network models of the two RPE gene clusters. The validity of these models was then tested using RT-PCR to detect differential expression of the corresponding TF mRNAs during RPE differentiation in both undifferentiated and differentiated ARPE-19 and primary chicken RPE cell cultures. RESULTS: The computational analysis resulted in the successful identification of specific transcription response elements (TREs) and their cognate TFs that are candidates for serving as nodes in a transcriptional regulatory network regulating EMT in RPE cells. The models predicted TFs whose differential expression during RPE EMT was successfully verified by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, including Oct-1, hepatocyte nuclear factor 1 (HNF-1), similar to mothers against decapentaplegic 3 (SMAD3), transcription factor E (TFE), core binding factor, erythroid transcription factor-1 (GATA-1), interferon regulatory factor-1 (IRF), natural killer homeobox 3A (NKX3A), Sterol regulatory element binding protein-1 (SREBP-1), and lymphocyte enhancer factor-1 (LEF-1). CONCLUSIONS: These studies successfully applied computational modeling and biochemical verification to identify biologically relevant transcription factors that are likely to regulate RPE cell phenotype and pathological changes in RPE in response to diseases or trauma. These TFs may provide potential therapeutic targets for the prevention and treatment of ocular proliferative disorders such as PVR

Effect of postural changes on normal and stenosed common carotid artery using FSI

Gravity associated with postural changes has a strong bearing on haemodynamics of blood flow in arteries. Its effect on stenosed cases has not been widely investigated. In the present study, variation observed in blood flow during postural changes is investigated for different conditions like standing, sleeping and head-down position. A fluid structure interaction study is carried out for idealized normal and 75 % eccentric and concentric stenosed common carotid normal artery. The results clearly indicate the effects of altered gravity on flow conditions. It was found to be very significant during head-down position and demonstrated very high arterial blood pressure in stenosed common carotid when compared with normal carotid

Patient-Specific Genome-Scale Metabolic Models for Individualized Predictions of Liver Disease

The prevalence of liver disease is steadily increasing, coupled with the limited availability of therapeutic treatments. Recent literature points to metabolic reprogramming as a key feature of liver failure. Hence, we sought to uncover the metabolic pathways and mechanisms associated with liver disease and acute liver failure. We generated patient-specific genome scale metabolic models by integrating RNA-seq data from patient liver samples with a generalized human metabolic model. Flux balance analysis simulations showed a distinct separation of non-alcohol associated and alcohol-associated disease states. Our analysis suggests that the alcohol associated liver has an increased flux through nucleotide and glycerophospholipid metabolic pathways. By contrast, non-alcohol associated liver has an increased flux through fatty acid oxidation, the carnitine shuttle, and bile acid recycling pathways. Importantly, there was significant variation of metabolic fluxes between patients within the same clinical category of disease stage, pointing to the necessity and opportunity for personalized medicine in treating liver disease. We conclude that the metabolic reprogramming occurring in alcohol-associated liver disease is likely distinct from the adaptations in non-alcohol associated liver disease, potentially requiring alternative therapeutic approaches

Genome-Scale Metabolic Modeling Reveals Sequential Dysregulation of Glutathione Metabolism in Livers from Patients with Alcoholic Hepatitis

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there is no efficacious treatment aiding most patients. AH manifests differently in individuals, with some patients showing debilitating symptoms more so than others. Previous studies showed significant metabolic dysregulation associated with AH. Therefore, we sought to analyze how the activity of metabolic pathways differed in the liver of patients with varying degrees of AH severity. We utilized a genome-scale metabolic modeling approach that allowed for integration of a generic human cellular metabolic model with specific RNA-seq data corresponding to healthy and multiple liver disease states to predict the metabolic fluxes within each disease state. Additionally, we performed a systems-level analysis of the transcriptomic data and predicted metabolic flux data to identify the regulatory and functional differences in liver metabolism with increasing severity of AH. Our results provide unique insights into the sequential dysregulation of the solute transport mechanisms underlying the glutathione metabolic pathway with increasing AH disease severity. We propose targeting of the solute transporters in the glutathione pathway to mimic the flux activity of the healthy liver state as a potential therapeutic intervention for AH