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

A Single Cell Transcriptomics Map of Paracrine Networks in the Intrinsic Cardiac Nervous System

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease

Integrated Computational Model of Intracellular Signaling and microRNA Regulation Predicts the Network Balances and Timing Constraints Critical to the Hepatic Stellate Cell Activation Process

Activation and deactivation of hepatic stellate cells (HSCs) is an important mechanism contributing to both healthy liver function and development of liver diseases, which relies on the interplay between numerous signaling pathways. There is accumulating evidence for the regulatory role of microRNAs that are downstream from these pathways in HSC activation. However, the relative contribution of these pathways and interacting microRNA regulators to the activation process is unknown. We pursued a computational modeling approach to explore the timing and regulatory balances that are critical to HSC activation and quiescence. We developed an integrated model incorporating three signaling pathways with crosstalk (NF-κB, STAT3 and TGF-β) and two microRNAs (miR-146a, miR-21) that are differentially regulated by these pathways. Simulations demonstrated that TGF-β-mediated regulation of microRNAs is critical to drive the HSC phenotypic switch from quiescence (miR-146ahigh miR-21low) to an activated state (miR-146alow miR-21high). We found that the relative timing between peak NF-κB and STAT3 activation plays a key role driving the initial dynamics of miR-146a. We observed re-quiescence from the activated HSC state upon termination of cytokine stimuli. Our integrated model of signaling and microRNA regulation provides a new computational platform for investigating the mechanisms driving HSC molecular state phenotypes in normal and pathological liver physiology

In Vivo Zonal Variation and Liver Cell-Type Specific NF-κB Localization after Chronic Adaptation to Ethanol and following Partial Hepatectomy.

NF-κB is a major inflammatory response mediator in the liver, playing a key role in the pathogenesis of alcoholic liver injury. We investigated zonal as well as liver cell type-specific distribution of NF-κB activation across the liver acinus following adaptation to chronic ethanol intake and 70% partial hepatectomy (PHx). We employed immunofluorescence staining, digital image analysis and statistical distributional analysis to quantify subcellular localization of NF-κB in hepatocytes and hepatic stellate cells (HSCs). We detected significant spatial heterogeneity of NF-κB expression and cellular localization between cytoplasm and nucleus across liver tissue. Our main aims involved investigating the zonal bias in NF-κB localization and determining to what extent chronic ethanol intake affects this zonal bias with in hepatocytes at baseline and post-PHx. Hepatocytes in the periportal area showed higher NF-κB expression than in the pericentral region in the carbohydrate-fed controls, but not in the ethanol group. However, the distribution of NF-κB nuclear localization in hepatocytes was shifted towards higher levels in pericentral region than in periportal area, across all treatment conditions. Chronic ethanol intake shifted the NF-κB distribution towards higher nuclear fraction in hepatocytes as compared to the pair-fed control group. Ethanol also stimulated higher NF-κB expression in a subpopulation of HSCs. In the control group, PHx elicited a shift towards higher NF-κB nuclear fraction in hepatocytes. However, this distribution remained unchanged in the ethanol group post-PHx. HSCs showed a lower NF-κB expression following PHx in both ethanol and control groups. We conclude that adaptation to chronic ethanol intake attenuates the liver zonal variation in NF-κB expression and limits the PHx-induced NF-κB activation in hepatocytes, but does not alter the NF-κB expression changes in HSCs in response to PHx. Our findings provide new insights as to how ethanol treatment may affect cell-type specific processes regulated by NF-κB activation in liver cells

Active Remodeling of Chromatin and Implications for In Vivo Folding

Building on the observation that chromatin compaction can be locally modulated by activity, we propose a model of in vivo chromatin as an active polymer and study its large scale conformations. In particular, we study an active mechanochemical model of chromosomal folding based on the interplay among polymer elasticity, confinement, topological constraints, and fluctuating active stresses arising from the ATP-dependent action of a variety of chromatin-associated protein machines and chromatin-remodeling proteins and their stochastic turnover. We find that activity drives the chromatin to a nonequilibrium steady state; the statistics of conformations in this nonequilibrium steady state are consistent with recent measurements on intrachromosomal contact probabilities and chromosomal compaction. The contact exponents at steady state show a systematic variation with changes in the nature of activity and the rates of turnover. The steady state configuration of the active chromatin in two dimensions resembles a space-filling Peano curve, which might have implications for the optimization of genome information storage

Validation of expression changes of putative NF-κB target genes in whole-tissue lysates by qPCR analysis.

<p>Results are presented as -ΔΔC_t values, where higher values correspond to increased expression, and lower values to decreased expression. Data were normalized by median-centering raw C_t values within each sample, followed by median-centering within each gene across all samples.</p

Whole-cell levels of NF-κB in the HSCs in the ethanol-adapted condition (N = 2 rats/condition, N = 284 cells total).

<p><b>(A and B)</b> Merged images of tissue stained with Alexa Fluor-488-labeled Phalloidin (green) and Alexa Fluor-647-labeled antibody for NF-κB (red), nuclei which were stained with DAPI (blue) and the HSCs, which were stained with Cy3 labeled-antibody for GFAP (HSC marker). The highlighted regions in the blue and orange rectangles show typical HSCs, magnified further. Scale bar: 40 μm. <b>(C)</b> Distributions of whole-cell intensities measured from the HSCs of both baseline groups. The ethanol group shows two populations, with the second one showing a higher level of whole-cell NF-κB than the carbohydrate group. <b>(D)</b> Cumulative distributions of the whole-cell intensities of the HSCs from both groups. Close to 80% of the HSCs from the ethanol group show higher whole-cell levels of NF-κB than carbohydrate group. (Total no of cells: Baseline Ethanol: 132, Baseline Carbohydrate: 152).</p