Malhi, Harmeet

BET bromodomain inhibitors are efficacious as anti-inflammatory compounds, but clinical translation is hampered by dose-limiting toxicities from non-selective inhibition. We herein report compounds 16 and 26, based on previously reported 4-methyl-1,2,3-triazole-based inhibitor, DW34. 16 has a BRD4 BD1 Ki of 10 nM, and up to 13–38-fold selectivity for BRD4 BD1 over other BD2 domains, and an improved hERG affinity of 4.24 µM over DW34. 26, A biscyclopropyl analog of 16 showed further enhancement of BD1 selectivity over BD2 (26–203-fold), but exhibited more potent hERG inhibition. Compound 16 had improved liver and plasma clearance over DW34, with good biodistribution to the liver, and had an intracellular target-engagement affinity Ki of 92 nM, with reduction of Ccl2, Cxcl1, AST, and ALT in in vitro and in vivo mouse models of liver inflammation. 16 represents a useful lead for treating inflammatory liver diseases, with improved specificity, drug-like properties, and reduced hERG affinity.

Abstract Accurate, noninvasive biomarkers for diagnosis and longitudinal monitoring of metabolic dysfunction–associated steatohepatitis (MASH) remain an urgent unmet clinical need. Here, we describe an electrochemical immunoassay that involves capturing hepatic extracellular vesicles (EVs) on antibody-modified electrodes followed by detecting EV-borne biomarkers using redox active gold nanoparticles (AuNPs). The intensity of electrochemical redox signal represents a quantitative measure of biomarker abundance. Three MASH-associated biomarkers—S100A11, TNF-α, and TGF-β1—were analyzed on hepatic EVs to assess steatotic, inflammatory, and fibrotic states of the diseased liver. The electrochemical signals for individual biomarkers were added into a composite EV MASH score. Plasma samples from 28 patients with MASH, 22 healthy controls, and 27 patients with non-MASH liver disease were scored in this manner, revealing 100% accuracy in the training cohort and >90% accuracy in an independent validation cohort. Our electrochemical immunosensor represents a minimally invasive liquid liver biopsy, offering a promising alternative to solid liver biopsies for screening and monitoring of MASH. One Sentence Summary Circulating hepatic extracellular vesicles carry biomarkers of liver injury enabling accurate, minimally invasive MASH diagnosis in blood.

This study presents a refined murine MetALD model designed to distinguish ethanol-modified features from FFC-driven metabolic steatohepatitis. Chronic ethanol did not uniformly worsen liver injury. Instead, it selectively modified disease trajectory through greater early collagen-positive fibrotic remodeling, altering lipid-droplet architecture, and context-dependent inflammatory chemokine signaling. By 24 wk, FFC and FFC/ethanol injury programs converged, supporting MetALD as a timing- and pathway-dependent modification of metabolic steatohepatitis.

Background and Aims: Cholangiocarcinoma (CCA) is a rare but aggressive malignancy with poorly understood genetic susceptibility. To date, genome-wide association studies (GWAS) investigating germline variants associated with CCA risk remain limited. We aimed to identify genetic risk loci for CCA and its clinical subtypes through comprehensive GWAS and post-GWAS analyses. Approach and Results: We conducted a GWAS of 2366 CCA cases and 11,750 controls of European ancestry. Genome-wide significant loci ( p <5×10 −8 ) were identified and further examined through fine-mapping, functional annotation, and HLA imputation. Subgroup analyses were conducted by CCA subtypes and primary sclerosing cholangitis (PSC) status. Cross-trait linkage disequilibrium score regression and Mendelian randomization were employed to investigate the shared genetic architecture and potential causal relationships with a diverse range of traits. We identified 1 new genome-wide significant variant, rs535777 (OR=1.44), near HLA-DRB1/DQA1 associated with CCA, and 2 variants associated with extrahepatic CCA: rs116224263 (OR=0.17) in LINC02506 at 4p15.1 and rs6914950 (OR=1.63) near HLA-DRB1/DQB1 . Stratified analyses revealed rs2395184 (OR=3.51) near HLA-DRA/DRB5 associated with PSC-related CCA, and rs142674434 (OR=2.98) in THSD7A at 7p21.3 associated with non-PSC-related CCA. HLA imputation uncovered new amino acid residues associated with disease risk. Cross-trait analyses identified shared genetic signals between CCA and anthropometric, lipidemic, lifestyle, and medical traits. Mendelian randomization supported putative causal associations for 12 traits with CCA or its subtypes. Conclusions: Our large-scale GWAS highlights new genetic variants and HLA-linked mechanisms underlying CCA susceptibility. Integrating multi-step post-GWAS approaches enhances understanding of CCA pathogenesis and may facilitate the development of risk biomarkers for early detection and precision prevention strategies.

Metabolic dysfunction-associated steatohepatitis (MASH) is a global health care burden. Appropriate large animal models mimicking the main MASH characteristics of steatosis, inflammation, and hepatocyte damage alongside progression of fibrosis are imperative for robust preclinical studies, especially in the field of hepatobiliary surgery. The present study aimed to characterize a novel model of steatohepatitis in fumarylacetoacetate hydroxylase-deficient ( FAH -/- ) pigs. FAH -/- pigs were generated using a CRISPR/Cas9 system. The animals received a choline-deficient, L-amino acid-defined high-fat diet and subtherapeutic doses of nitisinone and were followed for 3 months. Liver biopsies were obtained at baseline and every month during treatment. Steatosis, inflammation, and fibrosis were assessed by conventional histological scoring systems and by an artificial intelligence (AI) model. Serum liver parameters were analyzed every 2 weeks. Steatosis increased significantly throughout the study, with severe steatosis observed as early as 2 months into treatment. The inflammation score was increased in all animals after 3 months of treatment, whereas the AI-based CD45+ cell count showed region-specific trends in the portal and lobular areas. Collagen content and the corresponding fibrosis stage showed an increase over the 3-month period; however, the difference was not significant. Serum liver parameters did not show any relevant elevations during the study. In summary, we successfully developed and characterized a novel model of steatohepatitis in the FAH -/- pig within 3 months. Further studies with prolonged observation time and/or cycling of nitisinone administration are needed to evaluate whether progressive fibrosis and cirrhosis could be achieved with this model.

Metabolic dysfunction–associated steatohepatitis (MASH) is a progressive liver disease characterized by complex interactions between lipotoxicity, ER stress responses, and immune-mediated inflammation. We identified enrichment of the proinflammatory alarmin S100 calcium-binding protein A11 (S100A11) on extracellular vesicles stimulated by palmitate-induced lipotoxic ER stress with concomitant upregulation of hepatocellular S100A11 abundance in an IRE1A-XBP1s–dependent manner. We next investigated the epigenetic mechanisms that regulate this stress response. Publicly available human liver ChIP-Seq GEO datasets demonstrated a region of histone H3 lysine 27 (H3K27) acetylation upstream of the S100A11 promoter. H3K27 acetylation ChIP-qPCR demonstrated a positive correlation between lipotoxic ER stress and H3K27 acetylation of the region, which we termed the lipotoxicity-influenced enhancer (LIE) domain. CRISPR-mediated repression of the LIE domain reduced palmitate-induced H3K27 acetylation and corresponding S100A11 upregulation in Huh7 cells and immortalized mouse hepatocytes. Silencing of the murine LIE in 2 independent steatohepatitis models demonstrated reduced S100a11 upregulation and attenuated liver injury. We confirmed H3K27 acetylation and XBP1s occupancy at the LIE domain in human MASH liver samples and an increase in hepatocyte-derived S100A11-enriched extracellular vesicles in MASH patient plasma. Our studies demonstrate a LIE domain that mediates hepatic S100A11 upregulation. This pathway may be a potential therapeutic target in MASH.

Abstract Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor‐mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor‐induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. © 2013 American Physiological Society. Compr Physiol 3:977‐1010, 2013.

Abstract Extracellular vesicles (EVs) are membrane‐bound nanoparticles released by cells and are an important means of intercellular communication in physiological and pathological states. We provide an overview of recent advances in the understanding of EV biogenesis, cargo selection, recipient cell effects, and key considerations in isolation and characterization techniques. Studies on the physiological role of EVs have relied on cell‐based model systems due to technical limitations of studying endogenous nanoparticles in vivo . Several recent studies have elucidated the mechanistic role of EVs in liver diseases, including nonalcoholic fatty liver disease, viral hepatitis, cholestatic liver disease, alcohol‐associated liver disease, acute liver injury, and liver cancers. Employing disease models and human samples, the biogenesis of lipotoxic EVs downstream of endoplasmic reticulum stress and microvesicles via intracellular activation stress signaling are discussed in detail. The diverse cargoes of EVs including proteins, lipids, and nucleic acids can be enriched in a disease‐specific manner. By carrying diverse cargo, EVs can directly confer pathogenic potential, for example, recruitment and activation of monocyte‐derived macrophages in NASH and tumorigenicity and chemoresistance in hepatocellular carcinoma. We discuss the pathogenic role of EVs cargoes and the signaling pathways activated by EVs in recipient cells. We review the literature that EVs can serve as biomarkers in hepatobiliary diseases. Further, we describe novel approaches to engineer EVs to deliver regulatory signals to specific cell types, and thus use them as therapeutic shuttles in liver diseases. Lastly, we identify key lacunae and future directions in this promising field of discovery and development. © 2023 American Physiological Society. Compr Physiol 13:4631‐4658, 2023.