Targeting Epigenetic Mechanisms to Alleviate Alcoholic Steatosis

Alcohol-related liver disease (ALD) is a major health concern and recent studies have reported nearly 1 million alcohol-related deaths from 1999 to 2017 in the United States.1 ALD is a spectrum of conditions that ranges from early steatosis or fatty liver to inflammation or alcoholic steatohepatitis progressing to fibrosis and cirrhosis. Approximately 8%–20% of alcoholic steatohepatitis patients develop cirrhosis and, in some, alcoholic steatohepatitis can present in the form of acute-on-chronic liver failure, termed alcoholic hepatitis, owing to excessive drinking episodes. Corticosteroids are the first line of therapy for ALD, however, only marginal short-term survival benefit in patients with severe alcoholic hepatitis has been reported.2 Studies from the National Institute on Alcohol Abuse and Alcoholism consortia have focused on preclinical or early clinical testing of drugs classified on the basis of pathogenic mechanisms such as targeting the gut–liver axis, anti-inflammatory agents, antioxidants, and drugs that promotes liver regeneration.3 Despite several efforts, the treatment for alcoholic hepatitis remains suboptimal and there is an urgent need to develop new, safe, and effective therapies. Uncovering new targets directly involved in regulatory processes that influence gene expression and cellular phenotype could be an attractive strategy

Cellular Signaling Pathways in Alcoholic Liver Disease

Introduction: The pathogenesis of acute and chronic alcohol consumption is complex with diverse consequences in different tissues. Alcohol abuse is associated with a continuum of liver abnormalities ranging from steatosis or fat deposition, steatohepatitis or fat plus inflammation to cirrhosis and hepatocellular carcinoma. The progression of alcohol-induced liver damage involves both parenchymal and non-parenchymal cells of the liver. The signaling pathways affected by direct or indirect alcohol exposure range from oxidative stress mechanims, metabolism related effects, inflammation, and apoptosis. Understanding the interactions of inter- and intra-cellular signaling pathways in the liver during alcohol exposure will aid in identification of new integrative approaches as it relates to alcoholic liver disease and provide potential new directions to develop therapeutic target intervention. The goal of this chapter is to review signaling pathways related to oxidative stress and inflammatory responses modulated by alcohol in parenchymal and nonparenchymal cells of the liver important to ALD

Alcohol and Cancer: Mechanisms and Therapies

Several scientific and clinical studies have shown an association between chronic alcohol consumption and the occurrence of cancer in humans. The mechanism for alcohol-induced carcinogenesis has not been fully understood, although plausible events include genotoxic effects of acetaldehyde, cytochrome P450 2E1 (CYP2E1)-mediated generation of reactive oxygen species, aberrant metabolism of folate and retinoids, increased estrogen, and genetic polymorphisms. Here, we summarize the impact of alcohol drinking on the risk of cancer development and potential underlying molecular mechanisms. The interactions between alcohol abuse, anti-tumor immune response, tumor growth, and metastasis are complex. However, multiple studies have linked the immunosuppressive effects of alcohol with tumor progression and metastasis. The influence of alcohol on the host immune system and the development of possible effective immunotherapy for cancer in alcoholics are also discussed here. The conclusive biological effects of alcohol on tumor progression and malignancy have not been investigated extensively using an animal model that mimics the human disease. This review provides insights into cancer pathogenesis in alcoholics, alcohol and immune interactions in different cancers, and scope and future of targeted immunotherapeutic modalities in patients with alcohol abuse

Periostin and mesothelin: Potential predictors of malignant progression in intrahepatic cholangiocarcinoma

Cholangiocarcinoma (CCA), a heterogeneous group of malignancy arising at any level of the biliary tree based on their anatomical location, is classified into intrahepatic (iCCA), perihilar, and distal subtypes. iCCA, which arises distally to the second‐order bile ducts, is a highly heterogeneous and aggressive malignancy with overall poor prognosis. The rate of iCCA is increasing rapidly, particularly in Western countries; however, its precise etiology and pathogenesis remain elusive.1 While surgical resection is the first line of treatment, liver transplantation is the potential curative treatment for unresectable tumors in patients with iCCA, and posttransplantation 5‐year survival rates are 51% in these patients. Currently, there are no curative medical therapies or targeted molecular therapies approved for use in iCCA. The complex plethora of cell types, extracellular matrix, and soluble factors that influence tumor progression should be considered to understand its pathogenesis and to devise effective strategies for its clinical management.1 Furthermore, identification of biomarker signatures relevant to disease progression and aggressiveness may not only aid in diagnosis but also have prognostic value that will help build a precision approach for the treatment of iCCA...

Targeting Heat Shock Protein 90 Alters Epigenetic Genes in Alcoholic Liver Disease

Chronic alcohol induces acetylation and methylation of chromatin-associated histones that influence transcriptional activity of genes. Molecular chaperone heat shock protein 90 (hsp90) is being recognized as mediator of chromatin remodeling and is upregulated in alcoholic liver disease. We hypothesize that hsp90 plays a pivotal role in altered expression of chromatin modifying enzymes during alcoholic liver injury. To test our hypotheses, C57Bl/6 mice were fed Lieber-deCarli diet with 5% v/v ethanol for 10 days followed by a binge containing 20%v/v alcohol. A single injection of hsp90 inhibitor, 17-DMAG [17-Dimethylamino-ethylamino-17-demethoxygeldanamycin] was administered (30-50 mg/kg BW) i.p. before the binge. Epigenetic PCR array analyzing chromatin modifying enzyme expression was employed to determine effect of chronic alcohol and 17-DMAG treatment. Elevated ALT, triglycerides and steatosis confirmed alcoholic liver injury. Results show significant up-regulation of 5 genes including, ATF2 (p=0.0003), a transcription factor with histone acetyltransferase activity, PRMT6 (p=0.0001) and SETD7 (p=0.002), protein methyltransferases, RPS6KA3 (p=4.4 e-6) kinase and HDAC3 (p=0.0001) whereas HDAC9 (p=0.002) was decreased in alcoholic whole livers. Further, ATF2 was exclusively up-regulated in Kupffer cells (KCs) while PRMT6 increased in alcoholic hepatocytes. HDAC3, SETD7 and RPS6KA3 were increased in both, alcoholic KCs and hepatocytes. HDAC9 was exclusively down regulated in alcohol exposed hepatocytes but not in KCs. Inhibition of hsp90 by 17-DMAG after chronic alcohol exposure alleviated liver injury as noted by significantly lowered serum ALT, TBARS and liver triglycerides. Interestingly, 17-DMAG treatment prevented upregulation of ATF2 (p=3.13 e-5), PRMT6 (p=0.001) and HDAC3 (p=0.001) and inhibited down regulation of HDAC9 (p=2.6 e-8) without any effect on expression of SETD7 and RPS6KA3 genes in liver. In summary, our data indicate that chronic alcohol exposure regulates chromatin modifying epigenetic genes in a cell-specific manner likely via molecular chaperone, hsp90. Inhibition of hsp90 leads to modulation of chromatin modifying enzymes with resolution of liver injury. (Supported by the NIH/NIAAA # AA179086

Toll-Like Receptors in the Pathogenesis of Alcoholic Liver Disease

In the multifactorial pathophysiology of alcoholic liver disease (ALD), inflammatory cascade activation plays a central role. Recent studies demonstrated that Toll-like Receptors, the sensors of microbial and endogenous danger signals, are expressed and activated in innate immune cells as well as in parenchymal cells in the liver and thereby contribute to ALD. In this paper, we discuss the importance of gut-derived endotoxin and its recognition by TLR4. The significance of TLR-induced intracellular signaling pathways and cytokine production as well as the contribution of reactive oxygen radicals is evaluated. The contribution of TLR signaling to induction of liver fibrosis and hepatocellular cancer is reviewed in the context of alcohol-induced liver disease

Editorial: Macrophages in Liver Disease

Macrophages constitute a key component of our immune system and play an important role in immune surveillance. Hepatic macrophages are a heterogeneous population of immune cells that mainly comprises of embryonically-derived resident Kupffer cells (KCs), and circulating monocyte-derived macrophages (MoMFs). They play a critical role in disease initiation and progression as well as contribute to disease resolution. Traditionally, macrophages were defined by two broad subsets: classically-activated pro-inflammatory M1 or alternatively-activated anti-inflammatory M2 macrophages. However, it has been recognized that macrophages can differentiate into multiple phenotypes with distinct functions based on the tissue microenvironment

Alcohol‐Associated Liver Disease Before and After COVID‐19 – An Overview and Call for Ongoing Investigation

The Coronavirus Disease-2019 (COVID-19) pandemic has exacted a heavy toll on patients with alcohol-associated liver disease (ALD) and alcohol use disorder (AUD). The collective burden of ALD and AUD was large and growing prior to the COVID-19 pandemic. There is accumulating evidence that this pandemic has had a large direct effect on these patients and is likely to produce indirect effects via delays in care, psychological strain, and increased alcohol use. Now a year into the pandemic, it is important that clinicians fully understand the effects of the COVID-19 pandemic on patients with ALD and AUD. To fill existing gaps in knowledge, the scientific community must set research priorities for patients with ALD regarding their risk of COVID-19, prevention/treatment of COVID-19, changes in alcohol use during the pandemic, best use of AUD treatments in the COVID-19 era, and downstream effects of this pandemic on ALD. Conclusion: The COVID-19 pandemic has already inflicted disproportionate harms on patients with ALD and ongoing, focused research efforts will be critical to better understand the direct and collateral effects of this pandemic on ALD

Measuring Changes in Brain Metabolite Levels Using Live-animal Magnetic Resonance Spectroscopy and Offline LC-MS Metabolomics in a Binge-ethanol Murine Model

Alcoholism and acute alcohol binge are significant public health concerns. Liquid chromatography-mass spectrometry (LC-MS) based metabolomics is a robust and sensitive technique for determining and quantifying transient or permanent biochemical changes within the central nervous system (CNS). However, access to human tissue and CNS biofluid for such analyses is limited in a clinical context. In-vivo magnetic resonance spectroscopy (MRS) is an attractive alternative for clinical measurement but currently the technique is limited to a small to a number of well-characterized, highly abundant analytes. We therefore seek to correlate LC-MS and MRS measurements to better understand and leverage the strengths of each. Following live animal MRS measurement, metabolites in hippocampal brain punch homogenates were quantified by LC-MS, and a Spearman’s correlation coefficient was calculated. We found that the measurements for glutamine and glutamate,, were significantly correlated. Other established neurochemicals, including NAA and aspartate, showed non-significant correlations. NAAG showed little correlation between the two measurements. Additional experiments are ongoing to resolve these discrepancies, and determine how to achieve better agreement between the two methods. In addition,, we used Elements (Proteome Software) to determine differentially expressed metabolites between ethanol exposed and control mice.. An initial pass shows more than 1000 peak-picked features identified in the two conditions, with approximately 200 analytes identified in the metabolite database (human) based on accurate mass. Differentially expressed candidates can be validated further using tandem mass spectrometry and, where possible, the use of authentic standards. Metabolites that change after binge ethanol exposure are reported along with an overview of comparing MRS with LC-MS datasets

A Comparative Study of Single and Dual Perfusion During End-ischemic Subnormothermic Liver Machine Preservation

Background: It remains controversial if arterial perfusion in addition to portal vein perfusion during machine preservation improves liver graft quality. Comparative studies using both techniques are lacking. We studied the impact of using single or dual machine perfusion of donation after circulatory death rat livers. In addition, we analyzed the effect of pulsatile versus continuous arterial flow. Methods: Donation after circulatory death rat livers (n = 18) were preserved by 6 hours cold storage, followed by 1 hour subnormothermic machine perfusion (20 degrees C, pressure of 40/5 mm Hg) and 2 hours ex vivo warm reperfusion (37 degrees C, pressure of 80/11 mm Hg, 9% whole blood). Machine preservation was either through single portal vein perfusion (SP), dual pulsatile (DPP), or dual continuous perfusion (DCP) of the portal vein and hepatic artery. Hydrodynamics, liver function tests, histopathology, and expression of endothelial specific genes were assessed during 2 hours warm reperfusion. Results: At the end of reperfusion, arterial flow in DPP livers tended to be higher compared to DCP and SP grafts. However, this difference was not significant nor was better flow associated with better outcome. No differences in bile production or alanine aminotransferase levels were observed. SP livers had significantly lower lactate compared to DCP, but not DPP livers. Levels of Caspase-3 and tumor necrosis factor-alpha were similar between the groups. Expression of endothelial genes Kruppel-like-factor 2 and endothelial nitric oxide synthase tended to be higher in dual perfused livers, but no histological evidence of better preservation of the biliary endothelium or vasculature of the hepatic artery was observed. Conclusions: This study shows comparable outcomes after using a dual or single perfusion approach during end-ischemic subnormothermic liver machine preservation