Insights into ALD and AUD diagnosis and prognosis: Exploring AI and multimodal data streams.

The rapid evolution of artificial intelligence and the widespread embrace of digital technologies have ushered in a new era of clinical research and practice in hepatology. Although its potential is far from realization, these significant strides have generated new opportunities to address existing gaps in the delivery of care for patients with liver disease. In this review, we discuss how artificial intelligence and opportunities for multimodal data integration can improve the diagnosis, prognosis, and management of alcohol-associated liver disease. An emphasis is made on how these approaches will also benefit the detection and management of alcohol use disorder. Our discussion encompasses challenges and limitations, concluding with a glimpse into the promising future of these advancements.

Digital pathology and spatial omics in steatohepatitis: Clinical applications and discovery potentials.

Steatohepatitis with diverse etiologies is the most common histological manifestation in patients with liver disease. However, there are currently no specific histopathological features pathognomonic for metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, or metabolic dysfunction-associated steatotic liver disease with increased alcohol intake. Digitizing traditional pathology slides has created an emerging field of digital pathology, allowing for easier access, storage, sharing, and analysis of whole-slide images. Artificial intelligence (AI) algorithms have been developed for whole-slide images to enhance the accuracy and speed of the histological interpretation of steatohepatitis and are currently employed in biomarker development. Spatial biology is a novel field that enables investigators to map gene and protein expression within a specific region of interest on liver histological sections, examine disease heterogeneity within tissues, and understand the relationship between molecular changes and distinct tissue morphology. Here, we review the utility of digital pathology (using linear and nonlinear microscopy) augmented with AI analysis to improve the accuracy of histological interpretation. We will also discuss the spatial omics landscape with special emphasis on the strengths and limitations of established spatial transcriptomics and proteomics technologies and their application in steatohepatitis. We then highlight the power of multimodal integration of digital pathology augmented by machine learning (ML)algorithms with spatial biology. The review concludes with a discussion of the current gaps in knowledge, the limitations and premises of these tools and technologies, and the areas of future research.

Targeting Src SH3 domain-mediated glycolysis of HSC suppresses transcriptome, myofibroblastic activation, and colorectal liver metastasis.

BACKGROUND AND AIMS: Transforming growth factor-beta 1 (TGFß1) induces HSC activation into metastasis-promoting cancer-associated fibroblasts (CAFs), but how the process is fueled remains incompletely understood. We studied metabolic reprogramming induced by TGFß1 in HSCs. APPROACHES AND RESULTS: Activation of cultured primary human HSCs was assessed by the expression of myofibroblast markers. Glucose transporter 1 (Glut1) of murine HSC was disrupted by Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination (Cre/LoxP). Plasma membrane (PM) Glut1 and glycolysis were studied by biotinylation assay and the Angilent Seahorse XFe96 Analyzer. S.c. HSC/tumor co-implantation and portal vein injection of MC38 colorectal cancer cells into HSC-specific Glut1 knockout mice were performed to determine in vivo relevance. Transcriptome was obtained by RNA sequencing of HSCs and spatialomics with MC38 liver metastases. TGFß1-induced CAF activation of HSCs was accompanied by elevation of PM Glut1, glucose uptake, and glycolysis. Targeting Glut1 or Src by short hairpin RNA, pharmacologic inhibition, or a Src SH3 domain deletion mutant abrogated TGFß1-stimulated PM accumulation of Glut1, glycolysis, and CAF activation. Mechanistically, binding of the Src SH3 domain to SH3 domain-binding protein 5 led to a Src/SH3 domain-binding protein 5/Rab11/Glut1 complex that activated Rab11-dependent Glut1 PM transport under TGFß1 stimulation. Deleting the Src SH3 domain or targeting Glut1 of HSCs by short hairpin RNA or Cre recombinase/LoxP sequence derived from bacteriophage P1 recombination suppressed CAF activation in mice and MC38 colorectal liver metastasis. Multi-omics revealed that Glut1 deficiency in HSCs/CAFs suppressed HSC expression of tumor-promoting factors and altered MC38 transcriptome, contributing to reduced MC38 liver metastases. CONCLUSION: The Src SH3 domain-facilitated metabolic reprogramming induced by TGFß1 represents a target to inhibit CAF activation and the pro-metastatic liver microenvironment.

Angiocrine signaling in sinusoidal homeostasis and liver diseases.

The hepatic sinusoids are composed of liver sinusoidal endothelial cells (LSECs), which are surrounded by hepatic stellate cells (HSCs) and contain liver-resident macrophages called Kupffer cells, and other patrolling immune cells. All these cells communicate with each other and with hepatocytes to maintain sinusoidal homeostasis and a spectrum of hepatic functions under healthy conditions. Sinusoidal homeostasis is disrupted by metabolites, toxins, viruses, and other pathological factors, leading to liver injury, chronic liver diseases, and cirrhosis. Alterations in hepatic sinusoids are linked to fibrosis progression and portal hypertension. LSECs are crucial regulators of cellular crosstalk within their microenvironment via angiocrine signaling. This review discusses the mechanisms by which angiocrine signaling orchestrates sinusoidal homeostasis, as well as the development of liver diseases. Here, we summarise the crosstalk between LSECs, HSCs, hepatocytes, cholangiocytes, and immune cells in health and disease and comment on potential novel therapeutic methods for treating liver diseases.

Randomized trial of anakinra plus zinc vs. prednisone for severe alcohol-associated hepatitis.

BACKGROUND & AIMS: Severe alcohol-associated hepatitis (SAH) is associated with high 90-day mortality. Glucocorticoid therapy for 28 days improves 30- but not 90-day survival. We assessed the efficacy and safety of a combination of anakinra, an IL-1 antagonist, plus zinc (A+Z) compared to prednisone using the Day-7 Lille score as a stopping rule in patients with SAH. METHODS: In this phase IIb double-blind randomized trial in adults with SAH and MELD scores of 20-35, participants were randomized to receive either daily anakinra 100 mg subcutaneously for 14 days plus daily zinc sulfate 220 mg orally for 90 days, or daily prednisone 40 mg orally for 30 days. Prednisone or prednisone placebo was stopped if Day-7 Lille score was >0.45. All study drugs were stopped for uncontrolled infection or ≥5 point increase in MELD score. The primary endpoint was overall survival at 90 days. RESULTS: Seventy-three participants were randomized to prednisone and 74 to A+Z. The trial was stopped early after a prespecified interim analysis showed prednisone was associated with higher 90-day overall survival (90% vs. 70%; hazard ratio for death = 0.34, 95% CI 0.14-0.83, p = 0.018) and transplant-free survival (88% vs. 64%; hazard ratio for transplant or death = 0.30, 95% CI 0.13-0.69, p = 0.004) than A+Z. Acute kidney injury was more frequent with A+Z (45%) than prednisone (22%) (p = 0.001), but rates of infection were similar (31% in A+Z vs. 27% in prednisone, p = 0.389). CONCLUSIONS: Participants with SAH treated with prednisone using the Day-7 Lille score as a stopping rule had significantly higher overall and transplant-free 90-day survival and lower incidence of acute kidney injury than those treated with A+Z. IMPACT AND IMPLICATIONS: There is no approved treatment for severe alcohol-associated hepatitis (SAH). In this double-blind randomized trial, patients with SAH treated with prednisone using the Lille stopping rule on Day 7 had higher 90-day overall and transplant-free survival and lower rates of acute kidney injury compared to patients treated with a combination of anakinra and zinc. The data support continued use of glucocorticoids for patients with SAH, with treatment discontinuation for those with a Lille score >0.45 on Day 7. TRIAL REGISTRATION: NCT04072822.

Association between public health policies on alcohol and worldwide cancer, liver disease and cardiovascular disease outcomes.

BACKGROUND & AIMS: The long-term impact of alcohol-related public health policies (PHPs) on disease burden is unclear. We aimed to assess the association between alcohol-related PHPs and alcohol-related health consequences. METHODS: We conducted an ecological multi-national study including 169 countries. We collected data on alcohol-related PHPs from the WHO Global Information System of Alcohol and Health 2010. Data on alcohol-related health consequences between 2010-2019 were obtained from the Global Burden of Disease database. We classified PHPs into five items, including criteria for low, moderate, and strong PHP establishment. We estimated an alcohol preparedness index (API) using multiple correspondence analysis (0 lowest and 100 highest establishment). We estimated an incidence rate ratio (IRR) for outcomes according to API using adjusted multilevel generalized linear models with a Poisson family distribution. RESULTS: The median API in the 169 countries was 54 [IQR 34.9-76.8]. The API was inversely associated with alcohol use disorder (AUD) prevalence (IRR 0.13; 95% CI 0.03-0.60; p = 0.010), alcohol-associated liver disease (ALD) mortality (IRR 0.14; 95% CI 0.03-0.79; p = 0.025), mortality due to neoplasms (IRR 0.09; 95% CI 0.02-0.40; p = 0.002), alcohol-attributable hepatocellular carcinoma (HCC) (IRR 0.13; 95% CI 0.02-0.65; p = 0.014), and cardiovascular diseases (IRR 0.09; 95% CI 0.02-0.41; p = 0.002). The highest associations were observed in the Americas, Africa, and Europe. These associations became stronger over time, and AUD prevalence was significantly lower after 2 years, while ALD mortality and alcohol-attributable HCC incidence decreased after 4 and 8 years from baseline API assessment, respectively (p <0.05). CONCLUSIONS: The API is a valuable instrument to quantify the robustness of alcohol-related PHP establishment. Lower AUD prevalence and lower mortality related to ALD, neoplasms, alcohol-attributable HCC, and cardiovascular diseases were observed in countries with a higher API. Our results encourage the development and strengthening of alcohol-related policies worldwide. IMPACT AND IMPLICATIONS: We first developed an alcohol preparedness index, an instrument to assess the existence of alcohol-related public policies for each country. We then evaluated the long-term association of the country's alcohol preparedness index in 2010 with the burden of chronic liver disease, hepatocellular carcinoma, other neoplasms, and cardiovascular disease. The strengthening of alcohol-related public health policies could impact long-term mortality rates from cardiovascular disease, neoplasms, and liver disease. These conditions are the main contributors to the global burden of disease related to alcohol use. Over time, this association has not only persisted but also grown stronger. Our results expand the preliminary evidence regarding the importance of public health policies in controlling alcohol-related health consequences.

ACG Clinical Guideline: Alcohol-Associated Liver Disease.

ABSTRACT: Alcohol-associated liver disease (ALD) is the most common cause of advanced hepatic disease and frequent indication for liver transplantation worldwide. With harmful alcohol use as the primary risk factor, increasing alcohol use over the past decade has resulted in rapid growth of the ALD-related healthcare burden. The spectrum of ALD ranges from early asymptomatic liver injury to advanced disease with decompensation and portal hypertension. Compared with those with other etiologies of liver disease, patients with ALD progress faster and more often present at an advanced stage. A unique phenotype of advanced disease is alcohol-associated hepatitis (AH) presenting with rapid onset or worsening of jaundice, and acute on chronic liver failure in severe forms conveying a 1-month mortality risk of 20%-50%. The model for end stage disease score is the most accurate score to stratify AH severity (>20 defined as severe disease). Corticosteroids are currently the only available therapeutic with proven efficacy for patients with severe AH, providing survival benefit at 1 month in 50%-60% of patients. Abstinence of alcohol use, a crucial determinant of long-term outcomes, is challenging to achieve in ALD patients with concurrent alcohol use disorder (AUD). As patients with ALD are rarely treated for AUD, strategies are needed to overcome barriers to AUD treatment in patients with ALD and to promote a multidisciplinary integrated care model with hepatology, addiction medicine providers, and social workers to comprehensively manage the dual pathologies of liver disease and of AUD. Liver transplantation, a definitive treatment option in patients with advanced cirrhosis, should be considered in selected patients with AH, who are unresponsive to medical therapy and have a low risk of relapse to posttransplant alcohol use. Level of evidence and strength of recommendations were evaluated using the Grading of Recommendations, Assessment, Development, and Evaluations system. This guideline was developed under the American College of Gastroenterology Practice Parameters Committee.

Artificial Intelligence-Enabled Stool Analysis for Lactulose Titration Assistance in Hepatic Encephalopathy Through a Smartphone Application.

INTRODUCTION: Management of hepatic encephalopathy relies on self-titration of lactulose. In this feasibility trial, we assess an artificial intelligence-enabled tool to guide lactulose use through a smartphone application. METHODS: Subjects with hepatic encephalopathy on lactulose captured bowel movement pictures during lead-in and intervention phases. During the intervention phase, daily feedback on lactulose titration was delivered through the application. Goals were determined according to number of bowel movement and Bristol Stool Scale reports. RESULTS: Subjects completed the study with more than 80% satisfaction. In the lead-in phase, less compliant subjects achieved Bristol Stool Scale goal on 62/111 (56%) of days compared with 107/136 (79%) in the intervention phase ( P = 0.041), while the most compliant subjects showed no difference. Severe/recurrent hepatic encephalopathy group achieved Bristol Stool Scale goal on 80/104 (77%) days in the lead-in phase and 90/110 (82%) days in the intervention phase ( P = NS), compared with 89/143 (62%) days and 86/127 (68%) days in the stable group. DISCUSSION: Dieta application is a promising tool for objective Bowel Movement/Bristol Stool Scale tracking for hepatic encephalopathy and may potentially be used to assist with lactulose titration.

The Liver Cirrhosis Network Cohort Study: Cirrhosis Definition, Study Population, and Endpoints.

BACKGROUND: One of the primary goals of the Liver Cirrhosis Network (LCN) is to develop a cohort study to better understand and predict the risk of hepatic decompensation and other clinical and patient-reported outcomes among patients with Child A cirrhosis. METHODS: The LCN consists of a Scientific Data Coordinating Center (SDCC) and 10 clinical centers whose investigators populate multiple committees. The LCN Definitions and Measurements Committee developed preliminary definitions of cirrhosis and its complications by literature review, expert opinion, and reviewing definition documents developed by other organizations. The Cohort Committee developed the study protocol with the input of the steering committee. RESULTS: The LCN developed a prospective cohort study to describe and predict the rates of incident clinical events pertaining to first decompensation and patient reported outcomes. The LCN developed a pragmatic definition of compensated cirrhosis incorporating clinical, laboratory, imaging, and histological criteria. Definitions of incident and recompensated ascites, overt hepatic encephalopathy, variceal hemorrhage, bleeding due to portal gastropathy, and hepatocellular carcinoma were also codified. CONCLUSION: The LCN Cohort Study design will inform the natural history of cirrhosis in contemporary patients with compensated cirrhosis. The LCN Definitions and Measures Committee developed criteria for the definition of cirrhosis to standardize entry into this multi-center cohort study and standardized criteria for liver-related outcome measures. This effort has produced definitions intended to be both sensitive and specific as well as easily operationalized by study staff such that outcomes critical to the LCN cohort are identified and reported in an accurate and generalizable fashion.

Emerging targets for therapy in ALD: Lessons from NASH.

Alcohol-associated liver disease due to harmful alcohol use and NAFLD associated with metabolic syndrome are the 2 most common liver diseases worldwide. Control of respective risk factors is the cornerstone in the long-term management of these diseases. Furthermore, there are no effective therapies. Both diseases are characterized by metabolic derangements; thus, the focus of this review was to broaden our understanding of metabolic targets investigated in NAFLD, and how these can be applied to alcohol-associated liver disease. Conserved pathogenic pathways such as dysregulated lipid metabolism, cell death pathways including apoptosis and activation of innate immune cells, and stellate cells mediate both alcohol and NAFLDs, resulting in histological abnormalities of steatosis, inflammation, fibrosis, and cirrhosis. However, pathways such as gut microbiome changes, glucose metabolism and insulin resistance, inflammatory signaling, and microRNA abnormalities are distinct in these 2 diseases. In this review article, we describe conserved and distinct pathogenic pathways highlighting therapeutic targets that may be of potential in both diseases and those that are unique to each disease.

Head-to-head comparison of magnetic resonance elastography-based liver stiffness, fat fraction, and T1 relaxation time in identifying at-risk NASH.

BACKGROUND AND AIMS: The presence of at-risk NASH is associated with an increased risk of cirrhosis and complications. Therefore, noninvasive identification of at-risk NASH with an accurate biomarker is a critical need for pharmacologic therapy. We aim to explore the performance of several magnetic resonance (MR)-based imaging parameters in diagnosing at-risk NASH. APPROACH AND RESULTS: This prospective clinical trial (NCT02565446) includes 104 paired MR examinations and liver biopsies performed in patients with suspected or diagnosed NAFLD. Magnetic resonance elastography-assessed liver stiffness (LS), 6-point Dixon-derived proton density fat fraction (PDFF), and single-point saturation-recovery acquisition-calculated T1 relaxation time were explored. Among all predictors, LS showed the significantly highest accuracy in diagnosing at-risk NASH [AUC LS : 0.89 (0.82, 0.95), AUC PDFF : 0.70 (0.58, 0.81), AUC T1 : 0.72 (0.61, 0.82), z -score test z >1.96 for LS vs any of others]. The optimal cutoff value of LS to identify at-risk NASH patients was 3.3 kPa (sensitivity: 79%, specificity: 82%, negative predictive value: 91%), whereas the optimal cutoff value of T1 was 850 ms (sensitivity: 75%, specificity: 63%, and negative predictive value: 87%). PDFF had the highest performance in diagnosing NASH with any fibrosis stage [AUC PDFF : 0.82 (0.72, 0.91), AUC LS : 0.73 (0.63, 0.84), AUC T1 : 0.72 (0.61, 0.83), |z| <1.96 for all]. CONCLUSION: Magnetic resonance elastography-assessed LS alone outperformed PDFF, and T1 in identifying patients with at-risk NASH for therapeutic trials.

Imaging of Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) continues to be a global health concern, responsible for a significant number of deaths worldwide. Although most individuals who consume alcohol do not develop ALD, heavy drinkers and binge drinkers are at increased risk. Unfortunately, ALD is often undetected until it reaches advanced stages, frequently associated with portal hypertension and hepatocellular carcinoma (HCC). ALD is now the leading indication for liver transplant. The incidence of alcohol-associated hepatitis (AH) surged during the COVID-19 pandemic. Early diagnosis of ALD is therefore important in patient management and determination of prognosis, as abstinence can halt disease progression. The spectrum of ALD includes steatosis, steatohepatitis, and cirrhosis, with steatosis the most common manifestation. Diagnostic techniques including ultrasound, CT, and MRI provide useful information for identifying ALD and excluding other causes of liver dysfunction. Heterogeneous steatosis and transient perfusion changes on CT and MRI in the clinical setting of alcohol-use disorder are diagnostic of severe AH. Elastography techniques are useful for assessing fibrosis and monitoring treatment response. These various imaging modalities are also useful in HCC surveillance and diagnosis. This review discusses the imaging modalities currently used in the evaluation of ALD, highlighting their strengths, limitations, and clinical applications.

Angiocrine Signaling in Sinusoidal Health and Disease.

Liver sinusoidal endothelial cells (LSECs) are key players in maintaining hepatic homeostasis. They also play crucial roles during liver injury by communicating with liver cell types as well as immune cells and promoting portal hypertension, fibrosis, and inflammation. Cutting-edge technology, such as single cell and spatial transcriptomics, have revealed the existence of distinct LSEC subpopulations with a clear zonation in the liver. The signals released by LSECs are commonly called "angiocrine signaling." In this review, we summarize the role of angiocrine signaling in health and disease, including zonation in healthy liver, regeneration, fibrosis, portal hypertension, nonalcoholic fatty liver disease, alcohol-associated liver disease, aging, drug-induced liver injury, and ischemia/reperfusion, as well as potential therapeutic advances. In conclusion, sinusoidal endotheliopathy is recognized in liver disease and promising preclinical studies are paving the path toward LSEC-specific pharmacotherapies.

Longitudinal Changes in MR Elastography-based Biomarkers in Obese Patients Treated with Bariatric Surgery.

Obesity-related chronic inflammation contributes to nonalcoholic fatty liver disease (NAFLD) progression in obese patients (body mass index [BMI] >30 kg/m2).1 The early detection of inflammation with noninvasive imaging technology may help identify individuals with a high risk of developing NAFLD.

Adding Inflammatory Markers and Refining National Institute on Alcohol Abuse and Alcoholism Criteria Improve Diagnostic Accuracy for Alcohol-associated Hepatitis.

BACKGROUND & AIMS: Although histology is considered the gold standard for diagnosis of alcohol-associated hepatitis (AH), it is not required for entry into therapeutic studies if patients meet National Institute on Alcohol Abuse and Alcoholism (NIAAA) consensus criteria for probable AH. Our aim was to assess the diagnostic accuracy of NIAAA criteria against liver biopsy and to explore new criteria to enhance diagnostic accuracy for AH. METHODS: A total of 268 consecutive patients with alcohol-related liver disease with liver biopsy were prospectively included: 210 and 58 in the derivation and validation cohorts, respectively. NIAAA criteria and histological diagnosis of alcoholic steatohepatitis (ASH) were independently reviewed by clinical investigators and pathologists from Hospital Clínic and Mayo Clinic. Using biopsy-proven ASH as gold standard we determined diagnostic capability of NIAAA criteria and proposed the new improved criteria. RESULTS: In the derivation cohort, diagnostic accuracy of NIAAA for AH was modest (72%) due to low sensitivity (63%). Subjects who did not meet NIAAA with ASH at liver biopsy had lower 1-year survival compared with subjects without ASH (70% vs 90%; P < .001). NIAAAm-CRP criteria, created by adding C-reactive protein and modifying the variables of the original NIAAA, had higher sensitivity (70%), accuracy (78%), and specificity (83%). Accuracy was also higher in a sensitivity analysis in severe AH (74% vs 65%). In the validation cohort, NIAAAm-CRP and NIAAA criteria had a sensitivity of 56% vs 52% and an accuracy of 76% vs 69%, respectively. CONCLUSION: NIAAA criteria are suboptimal for the diagnosis of AH. The proposed NIAAAm-CRP criteria may improve accuracy for noninvasive diagnosis of AH in patients with alcohol-related liver disease.

The digital transformation of hepatology: The patient is logged in.

The rise in innovative digital health technologies has led a paradigm shift in health care toward personalized, patient-centric medicine that is reaching beyond traditional brick-and-mortar facilities into patients' homes and everyday lives. Digital solutions can monitor and detect early changes in physiological data, predict disease progression and health-related outcomes based on individual risk factors, and manage disease intervention with a range of accessible telemedicine and mobile health options. In this review, we discuss the unique transformation underway in the care of patients with liver disease, specifically examining the digital transformation of diagnostics, prediction and clinical decision-making, and management. Additionally, we discuss the general considerations needed to confirm validity and oversight of new technologies, usability and acceptability of digital solutions, and equity and inclusivity of vulnerable populations.

Research methodologies to address clinical unmet needs and challenges in alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is emerging worldwide as the leading cause of liver-related morbidity, mortality, and indication for liver transplantation. The ALD Special Interest Group and the Clinical Research Committee at the digital American Association for the Study of Liver Diseases meeting in November 2020 held the scientific sessions to identify clinical unmet needs in ALD, and addressing these needs using clinical research methodologies. Of several research methodologies, the sessions were focused on (a) studying disease burden of ALD using large administrative databases, (b) developing biomarkers for noninvasive diagnosis of alcohol-associated hepatitis (AH) and estimation of disease prognosis, (c) identifying therapeutic targets for ALD and AH, (d) deriving accurate models to predict prognosis or posttransplant alcohol relapse as a basis for developing treatment algorithm and a uniform protocol on patient-selection criteria for liver transplantation, and (e) examining qualitative research methodologies in studying the barriers to implementation of multidisciplinary integrated care model by hepatology and addiction teams for the management of dual pathology of liver disease and of alcohol use disorder. Prospective multicenter studies are required to address many of these clinical unmet needs. Further, multidisciplinary care models are needed to improve long-term outcomes in patients with ALD.

Therapeutic advances in alcohol-associated hepatitis.

In recent years, there have been important advances in our understanding of alcohol-associated hepatitis (AH), which have occurred in parallel with a surge in clinical trial activity. Meanwhile, the broader medical field has seen a transformation in care paradigms based on emerging digital technologies. This review focuses on breakthroughs in our understanding of AH and how these breakthroughs are leading to new paradigms for biomarker discovery, clinical trial activity, and care models for patients. It portends a future in which multimodal data from genetic, radiomic, histologic, and environmental sources can be integrated and synthesised to generate personalised biomarkers and therapies for patients with AH.

Mechanotransduction-induced glycolysis epigenetically regulates a CXCL1-dominant angiocrine signaling program in liver sinusoidal endothelial cells in vitro and in vivo.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling in vitro and in vivo. METHODS: Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. RESULTS: Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin ß1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. CONCLUSION: Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. LAY SUMMARY: Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex.

BACKGROUND & AIMS: Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. METHODS: We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. RESULTS: BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. CONCLUSION: Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. LAY SUMMARY: We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.