Gavin Edward Arteel

Background: Little is known about the long-term risk of developing cirrhosis in patients with alcohol use disorder (AUD). Methods: This was a retrospective cohort of patients with AUD presenting to healthcare centers between January 2006 and June 2017. Individuals ≥18 years old with a diagnosis of AUD based on ICD-9 and ICD-10 codes were included. Patients with concurrent cirrhosis were excluded. Then, patients were followed until August 2021 for the development of complications or mortality. Results: A total of 32,808 patients with AUD were included in the final analysis. During an average follow-up of 5.7 years, 12.3% of patients developed incident cirrhosis, and the overall mortality was 20.2%. The risk of cirrhosis increased in patients with diabetes mellitus (DM) [hazard ratio (HR), 1.52; 95% CI, 1.41–1.63; p p p p <0.001). The presence of DM, concomitant hepatitis C infection, and non-AA race collectively conferred a greater risk of cirrhosis and mortality in individuals with AUD. Interpretation: Patients presenting with AUD are at high risk for developing incident cirrhosis and mortality. Concurrent DM and hepatitis C increase the risk of cirrhosis and mortality. Early intervention in patients presenting with AUD is urgently warranted.

Background: Ethanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most alcohol‐related liver disease (ALD) patients. Recent studies have highlighted the role of calpain, a calcium‐dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1 (calpain‐4), which stabilizes and modulates the activity of its catalytic isoforms, calpain‐1 and calpain‐2. This study investigated calpain's impact on lipid metabolism in ALD. Methods: Six‐week‐old C57Bl6/J mice were injected with rAAV8 vectors encoding Capns1 shRNA or control vectors. After 4 weeks, mice underwent a 10‐day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. Results: Capns1 knockdown attenuated ethanol‐induced microvesicular steatosis. Hepatic triglyceride and free fatty acid levels were not significantly altered, whereas cholesterol levels were significantly reduced in the ethanol group with Capns1 knockdown. Cpt1a expression increased significantly in the ethanol group with Capns1 knockdown. Western blot analysis revealed increased Cleaved‐3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase (HMGCR) to Pro‐HMGCR ratio in Capns1‐knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXRα expression was mainly increased in the cytoplasm in the ethanol group, and following Capns1 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis indicated that Capns1 knockdown contributes to the reprogramming of ethanol‐induced disruptions in metabolic pathways, primarily those involving cholesterol metabolism. Conclusion: Further investigation into the relationship between Capns1 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol‐related liver disease.

Next-generation sequencing can identify previously uncharacterized gene expression patterns in disease. Beyond differentially expressed gene (DEG) analysis, we investigated the ability of within-population diversity (α-diversity) of the transcriptome to reveal additional biological information in alcohol-associated liver disease (ALD), comparing differential Shannon diversity (DSD) to transcriptome heterogeneity changes. RNA sequencing data from normal livers and patients with early ALD and severe AH were analyzed. α-Diversity indices and percentage Shannon diversity of a gene, which refers to this gene’s contribution to total Shannon entropy, were calculated. Ingenuity pathway analysis identified canonical pathways determined by DEG and DSD approaches. ALD significantly decreased hepatic transcriptome α-diversity, correlating with increased relative contribution of select genes. These changes were driven by lower-abundance gene expression loss. DEG and DSD analyses showed overlapping genes and canonical pathways, but DSD also identified additional genes and pathways not highlighted by DEGs, including fatty acid oxidation, extracellular matrix degradation, and cholesterol metabolism pathways that may represent additional therapeutic targets. Importantly, DSD more effectively identified differences between ASH and AH. Overall, α-diversity analysis revealed that ALD progressively reduces transcriptome heterogeneity, and that DSD provides complementary insights into disease mechanisms missed by standard approaches.

Background & Aims: Ethanol affects lipid metabolism through multiple pathways, leading to fatty liver development in most ALD patients. Recent studies have highlighted the role of calpain, a calcium-dependent protease, in liver inflammation and fibrosis. Calpain activity is regulated by its essential subunit, Capns1, (calpain-4, Capn4), which stabilizes and modulates the activity of its catalytic isoforms, calpain-1 and calpain-2. This study investigated calpain’s impact on lipid metabolism in ALD. Approach & Results: Six-week-old C57Bl6/J mice were injected with rAAV8 vectors encoding Capn4 shRNA or control vectors. After four weeks, mice underwent a 10-day period of ad libitum ethanol consumption, followed by a single gavaged ethanol administration on day 11. Following Capn4 knockdown, microvesicular steatosis was attenuated. While triglycerides and free fatty acids levels showed no significant changes, cholesterol levels were significantly reduced in the ethanol (EtOH) group with Capn4 knockdown. Cpt1a expression increased significantly in the EtOH group with Capn4 knockdown. Western blot analysis revealed increased Cleaved-HMGCR to Pro-HMGCR ratio in Capn4 knockdown mice, suggesting reduced HMGCR activity and suppressed cholesterol biosynthesis. LXRα expression was mainly increased in the cytoplasm in the EtOH group, and following Capn4 knockdown, it was relocalized to the nucleus via its activation. In addition, RNA sequencing analysis suggests that Capn4 knockdown contributes to the reprogramming of ethanol-induced disruptions in metabolic and homeostatic pathways, primarily those involving cholesterol metabolism. Conclusions: Further investigation into the relationship between Capn4 and cholesterol biosynthesis proteins may provide insights into using calpain inhibitors as a therapeutic approach for alcohol-related hepatitis.

Background and aims Alcohol-associated hepatitis (AH) is an acute form of alcohol-related liver disease (ALD) with high mortality rate. AH is histologically characterised by cellular processes, including steatosis, inflammation and cell death. Apoptosis is the most studied form of cell death in AH; however, the role of cellular senescence, another response to cellular injury, in AH is unknown. Here, we explore the mechanisms of ALD pathophysiology and describe the role of senescence in AH. Methods We performed RNA sequencing and bioinformatics analysis of 0- and 28-day transjugular liver biopsies (n=65) from patients with AH participating in the IL-1 Signal Inhibition In Alcoholic Hepatitis (ISAIAH) clinical trial. Additional bioinformatics reanalysis of existing AH transcriptomic datasets was conducted to confirm our findings. We also performed multiomic analysis of an in vitro model of AH with ethanol-treated hepatocytes overexpressing ethanol-metabolising enzymes. Results Our longitudinal analysis revealed that senescence and inflammation were reduced at transcriptomic level following AH resolution; the expression of hepatocyte markers was increased. We identified two senescence-associated protein complexes, cytochrome c oxidase and the proteasome, which may act as senescence-induction mechanisms. We confirmed that senescence markers and pathways were increasingly expressed in hepatocytes as ALD progressed towards AH; this was partially reversed following AH resolution. Our in vitro model revealed that ethanol directly induces senescence and was dependent on ethanol metabolism. Conclusions Our results suggest a possible pathogenic role for senescence in AH and indicate cellular senescence as a potential therapeutic target in early ALD to limit AH severity.

The hepatic extracellular matrix (ECM) is most accurately depicted as a dynamic compartment that comprises a diverse range of players that work bidirectionally with hepatic cells to regulate overall homeostasis. Although the classic meaning of the ECM referred to only proteins directly involved in generating the ECM structure, such as collagens, proteoglycans, and glycoproteins, the definition of the ECM is now broader and includes all components associated with this compartment. The ECM is critical in mediating phenotype at the cellular, organ, and even organismal levels. The purpose of this review is to summarize the prevailing mechanisms by which ECM mediates hepatic phenotype and discuss the potential or established role of this compartment in the response to hepatic injury in the context of steatotic liver disease.

Background: Alcohol-associated hepatitis (AH) is plagued with high mortality and difficulty in identifying at-risk patients. The extracellular matrix undergoes significant remodeling during inflammatory liver injury and could potentially be used for mortality prediction. Methods: EDTA plasma samples were collected from patients with AH (n = 62); Model for End-Stage Liver Disease score defined AH severity as moderate (12–20; n = 28) and severe (>20; n = 34). The peptidome data were collected by high resolution, high mass accuracy UPLC-MS. Univariate and multivariate analyses identified differentially abundant peptides, which were used for Gene Ontology, parent protein matrisomal composition, and protease involvement. Machine-learning methods were used to develop mortality predictors. Results: Analysis of plasma peptides from patients with AH and healthy controls identified over 1600 significant peptide features corresponding to 130 proteins. These were enriched for extracellular matrix fragments in AH samples, likely related to the turnover of hepatic-derived proteins. Analysis of moderate versus severe AH peptidomes was dominated by changes in peptides from collagen 1A1 and fibrinogen A proteins. The dominant proteases for the AH peptidome spectrum appear to be CAPN1 and MMP12. Causal graphical modeling identified 3 peptides directly linked to 90-day mortality in >90% of the learned graphs. These peptides improved the accuracy of mortality prediction over the Model for End-Stage Liver Disease score and were used to create a clinically applicable mortality prediction assay. Conclusions: A signature based on plasma peptidome is a novel, noninvasive method for prognosis stratification in patients with AH. Our results could also lead to new mechanistic and/or surrogate biomarkers to identify new AH mechanisms. Export

Since Karl von Kupffer first described the Sternzellen of the liver in 1876,¹ understanding the origin and function of the cells named after him (i.e. Kupffer cells) has been long and complicated, including debate over which cell types von Kupffer actually identified in his original work.² This conversation continues to this day, with deeper understanding provided by fate mapping and cell-specific genotyping/phenotyping.³ The current understanding is that the identity of a Kupffer cell represents actually a spectrum of hepatic phagocytic cells,⁴ whose functional roles alter in response to injury or stress.³ This updated generalist identity of Kupffer cells is used hereafter in this article. The strategic location of the liver between the intestinal tract and the rest of the body makes it a critical organ for clearance of xenobiotics and toxins that enter the portal blood. The concept that Kupffer cells play a key role in the function of the liver as a physical (and biochemical) barrier has remained rather consistent for the past decades. This phagocytic function of Kupffer cells also serves as a critical bridge between the innate and adaptive immune response, presenting antigens for tolerance and/or immunity. This phagocytic function of Kupffer cells is actually pinocytotic and is more rapid and efficient than true phagocytosis mediated by other macrophages.⁵ In normal liver, the presence of Kupffer cells within the sinusoidal space is ideally suited for this process ( Fig. 1A). However, this function is impaired when the liver is injured, as the normally fenestrated endothelium closes (i.e. capillarization), which limits contact of the Kupffer cells with hepatocytes and other sinusoidal cells ( Fig. 1A).⁶ Moreover, severe fibrotic liver injury impairs normal sinusoidal blood flow, shunting it to intrahepatic (and extrahepatic) collateral vessels ( Fig. 1B),⁷ which lessens even more the ability of Kupffer cells to surveil the portal blood. Although this concept is well understood, how the liver adapts to attempt to maintain this barrier function was largely unknown. The recent work by Peiseler et al.⁸ elegantly and clearly sheds new light on this issue.

Alcohol-related hepatitis (AH) is plagued with high mortality and difficulty in identifying at-risk patients. The extracellular matrix undergoes significant remodeling during inflammatory liver injury that can be detected in biological fluids and potentially used for mortality prediction. EDTA plasma samples were collected from AH patients (n= 62); Model for End-Stage Liver Disease (MELD) score defined AH severity as moderate (12-20; n=28) and severe (>20; n=34). The peptidome data was collected by high resolution, high mass accuracy UPLC-MS. Univariate and multivariate analyses identified differentially abundant peptides, which were used for Gene Ontology, parent protein matrisomal composition and protease involvement. Machine learning methods were used on patient-specific peptidome and clinical data to develop mortality predictors. Analysis of plasma peptides from AH patients and healthy controls identified over 1,600 significant peptide features corresponding to 130 proteins. These were enriched for ECM fragments in AH samples, likely related to turnover of hepatic-derived proteins. Analysis of moderate versus severe AH peptidomes showed a shift in abundance of peptides from collagen 1A1 and fibrinogen A proteins. The dominant proteases for the AH peptidome spectrum appear to be CAPN1 and MMP12. Increase in hepatic expression of these proteases was orthogonally-validated in RNA-seq data of livers from AH patients. Causal graphical modeling identified four peptides directly linked to 90-day mortality in >90% of the learned graphs. These peptides improved the accuracy of mortality prediction over MELD score and were used to create a clinically applicable mortality prediction assay. A signature based on plasma peptidome is a novel, non-invasive method for prognosis stratification in AH patients. Our results could also lead to new mechanistic and/or surrogate biomarkers to identify new AH mechanisms. Lay summary: We used degraded proteins found the blood of alcohol-related hepatitis patients to identify new potential mechanisms of injury and to predict 90 day mortality.