BACKGROUND & AIMS:Single-cell fixed RNA profiling (FLEX) is a novel technique that captures RNA expression in frozen tissues at a single-cell resolution. We applied FLEX to mouse model of liver fibrosis progression and regression to identify novel antifibrotic targets. METHODS:Mice were administered intraperitoneal thioacetamide for 10 weeks to induce fibrosis, regression was assessed 2 weeks after cessation. The livers were fixed, dissociated, and analysed using FLEX. Molecular validation included immunoblotting, immunohistochemistry, gene silencing/overexpression, cytokine/phosphokinase arrays, and human liver samples. RESULTS:Approximately 40,000 liver cells were profiled, integrated, and annotated into 10 major cell types using lineage-specific markers. Pericentral signature restoration in hepatocytes, scar-resolving genes (Mmp14 and Ctsl), fenestrae restoration in liver sinusoidal endothelial cells, anti-inflammatory Kupffer cells, reduced fibrogenic cholangiocytes, and recovery-associated immune cell subsets were observed. During fibrosis, monocyte-derived macrophages secrete semaphorin-4D (SEMA4D), which binds to Plexin B2 on hepatic stellate cells (HSCs). SEMA4D+ cells were upregulated in mouse fibrotic livers (n = 6, p <0.05). Recombinant SEMA4D induces type I collagen in HSCs, whereas humanised monoclonal IgG4 SEMA4D blockade (VX15/2503) attenuates fibrosis in vivo (n = 5, p <0.05). LIM and cysteine-rich domains 1 (LMCD1) was enriched in fibrotic HSCs and suppressed during regression. LMCD1 knockdown reduced the expression of fibrotic protein, while LMCD1 overexpression promoted the expression of fibrotic protein via AKT/mTOR signalling. LMCD1 and SEMA4D are localised in the fibrotic septa and are correlated with the fibrotic stage of MASLD (n = 34, p <0.05) and HCV (n = 76, p <0.05) in humans. CONCLUSIONS:This FLEX-based single-cell atlas revealed critical transcriptional programs and cell-cell interactions, identifying SEMA4D and LMCD1 as promising therapeutic targets for liver fibrosis. IMPACT AND IMPLICATIONS:In this study, we applied a novel technique, single-cell fixed RNA profiling, to profile 38,136 cells obtained from control, thioacetamide-induced liver fibrosis, and regression-phase mouse livers, generating a high-resolution atlas of liver fibrosis progression and regression. We identified the transcriptomic patterns of regressed cell subpopulations and uncovered two key therapeutic targets: the macrophage-derived factor semaphorin-4D (SEMA4D) and the hepatic stellate cell-specific transcription factor LIM and cysteine-rich domains 1 (LMCD1). Treatment with a humanised monoclonal IgG4 antibody against SEMA4D significantly alleviated liver fibrosis in the thioacetamide-induced mouse model. SEMA4D and LMCD1 expression correlated with the metabolic dysfunction-associated steatotic liver disease-related fibrosis stage, and the attenuation of SEMA4D and LMCD1 after HCV-sustained virologic response reduced the risk of progression to hepatocellular carcinoma.

Metabolic syndrome and excessive alcohol consumption (MetALD) result in liver injury and fibrosis, which are driven by increased collagen production by activated hepatic stellate cells (HSCs). Our previous studies demonstrated that LARP6, an RNA-binding protein, may facilitate collagen production. However, the expression and function of LARP6 as a regulator of fibrosis development in a disease-relevant model remain poorly understood. We demonstrated that LARP6 was upregulated in human activated HSCs in metabolic dysfunction-associated steatohepatitis (MASH) and MetALD. By using single-nucleus RNA-seq and assay for transposase-accessible chromatin sequencing, we showed that JUNB upregulated LARP6 expression in activated HSCs. Moreover, LARP6 knockdown in human HSCs suppressed fibrogenic gene expression. By integrating enhanced crosslinking and IP analysis and ribosome profiling in HSCs, we showed that LARP6 interacted with mature mRNAs comprising more than 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. IP-mass spectrometry analysis demonstrated LARP6 protein-protein interactions with mRNA translation components and the actin cytoskeleton. Furthermore, Dicer substrate siRNA-based HSC-specific gene knockdown or pharmacological inhibition of LARP6 attenuated fibrosis development in human MASH and MetALD liver spheroids. Our results suggest LARP6 plays a key role in fibrogenic gene regulation and that targeting LARP6 in human HSCs may represent a therapeutic approach for liver fibrosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), are strongly linked to heart failure with preserved ejection fraction (HFpEF), yet the mechanisms underlying this association remain unclear because robust integrative preclinical models are lacking and the liver and heart are rarely studied as a coordinated system. Here we show that Alms1 -/- (Foz/Foz) mice fed a Western diet develop MASH with advanced liver fibrosis accompanied by a HFpEF phenotype characterized by left ventricular hypertrophy, impaired cardiomyocyte contractility, reduced β-adrenergic reserve, elevated BNP, and increased mortality despite ejection fraction >50. Liver fibrosis emerged as a strong predictor of cardiac dysfunction. Remarkably, dietary reversal restored hepatic architecture, normalized cardiac function, and improved survival, revealing marked plasticity of the liver-heart axis. Mechanistic analyses revealed coordinated mitochondrial dysfunction, altered substrate utilization, and extracellular matrix remodeling in the left ventricle, with strong concordance to human HFpEF transcriptomic signatures. Ultrastructural studies confirmed mitochondrial injury and sarcomeric disorganization, linking metabolic failure to impaired cardiomyocyte performance. Together, these findings identify mitochondrial dysfunction as a central mediator of MASLD-associated HFpEF and establish the Foz/Foz model as a powerful platform for dissecting liver-to-heart signaling pathways and testing mechanism-based therapeutic strategies.

BACKGROUND & AIMS:The contribution of activated hepatic stellate cells (aHSCs) to cholestatic fibrosis and cancer is well-documented, but the role of portal fibroblasts (PFs), and especially mesothelin (Msln)-mucin 16 (Muc16)- Thy-1 cell surface antigen (Thy-1) signaling in activated portal fibroblasts (aPFs), is unknown. METHODS:The role of aPFs/mesenchymal cells in the pathogenesis of cholestatic fibrosis and hepatocellular carcinoma (HCC) was studied in aged (16 months old) multidrug resistance protein 2 knockout (Mdr2-/-) mice, which mimic primary biliary cholangitis with biliary fibrosis. RESULTS:Aged female Mdr2-/- mice were more susceptible to cholestatic fibrosis and inflammation and developed 4-fold more adenomas and GPC3+SOX9+AFP+ HCC than age-matched male littermates. Deletion of Msln or Muc16 ameliorated cholestatic fibrosis, inflammation and HCC in Mdr2-/-Msln-/- and Mdr2-/-Muc16-/- mice, whereas Mdr2-/- and Mdr2-/-Thy-1-/- mice exhibited similar phenotypes and developed severe fibrosis and HCC. Aged Mdr2-/-Msln-/- and Mdr2-/-Muc16-/- mice developed fewer HCCs and of smaller sizes. Ductular proliferation and hepatocyte and cholangiocyte senescence were suppressed in Mdr2-/-Msln-/- and Mdr2-/-Muc16-/- mice, whereas hepatocyte regeneration was markedly improved. Msln- and Muc16-deficient aPFs exhibited a less fibrogenic and inflammatory phenotype, and downregulated expression of Col1a2, Col3a1, Tgfβ1, MMP3, Cxcl9, Clcl7, Lgals1, and MMP2/3. The lack of MMP3 in Msln-/- aPFs was linked to increased hepatocyte proliferation. Based on in vitro studies, MMP3-mediated shedding of hepatic HGFR (c-Met) was identified as one of the mechanisms by which aPFs suppress HGF-c-Met-induced phosphorylation of AKT, ERK, p38, resulting in proliferation of primary human hepatocytes. In turn, proliferation of MMP3-stimulated human hepatocytes was restored in the presence of MMP3 inhibitor. CONCLUSIONS:These findings demonstrate that aPFs mediate the crosstalk between cholangiocytes and hepatocytes, regulate hepatocyte functions, and that Msln-Muc16 signaling in aPFs is pathogenic for cholestatic fibrosis and HCC. Msln and Muc16 may become novel targets for anti-fibrotic therapy and patients with HCC and sclerosis cholangitis.

Steatotic liver disease (SLD) comprises metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction and alcohol-associated liver disease (MetALD), and alcohol-associated liver disease (ALD), which represent subclasses of liver disorders with overlapping etiologies. MASLD is defined as SLD with cardiometabolic dysfunction, whereas MetALD refers to MASLD with moderate alcohol consumption (140-350 g/week in females and 210-420 g/week in males). Despite being classified as distinct entities, MASLD and MetALD exhibit substantial phenotypic overlap, underscoring the need to delineate their pathological and molecular features and to develop models that capture the synergistic effects of alcohol and metabolic stress. Recent advances in multi-omic technologies have enabled integrated single-cell profiling of genetic, epigenetic, spatial, and proteomic features, providing high-resolution insights into cellular heterogeneity and disease mechanisms. In this review, we examine the pathophysiological landscape of SLD, highlight key distinctions between MASLD and MetALD, and discuss experimental models, including human liver spheroids. These approaches provide deeper insights into disease classification and accelerate the development of targeted therapies.

In the past decades, the pathogenic role of hepatic stellate cells (HSCs) in the development of liver fibrosis and its complications has been deeply characterized, rendering HSCs a primary target for antifibrotic therapies. By contrast, the beneficial roles of HSCs in liver homeostasis and liver disease are only beginning to emerge, revealing critical regulatory and fibrosis-independent functions in hepatic zonation, metabolism, injury, regeneration and non-parenchymal cell identity. Here, we review how HSC mediators, such as R-spondin 3, hepatocyte growth factor and bone morphogenetic proteins, regulate critical and homeostatic liver functions in health and disease via cognate receptors in hepatocytes, Kupffer cells and endothelial cells. We highlight how the balance shifts from protective towards fibropathogenic HSC mediators during the progression of chronic liver disease (CLD) and the impact of this shifted balance on patient outcomes. Notably, the protective roles of HSCs are not accounted for in current therapeutic concepts for CLD. We discuss the concept that reverting the HSC balance from fibrogenesis towards hepatoprotection might represent a novel holistic treatment approach to inhibit fibrogenesis and restore epithelial health in CLD simultaneously. Hepatic stellate cells (HSCs) are best known as the primary fibrogenic liver cell type. This Review provides a comprehensive overview of protective and pathogenic HSC functions in health and disease and discusses novel therapeutic concepts derived from this dual role.

Approximately half of all deaths from liver cirrhosis, the tenth leading cause of mortality in the United States, are related to alcohol use. Chronic alcohol consumption is accompanied by intestinal dysbiosis and bacterial overgrowth, yet little is known about the factors that alter the microbial composition or their contribution to liver disease. We previously associated chronic alcohol consumption with lower intestinal levels of the antimicrobial-regenerating islet-derived (REG)-3 lectins. Here, we demonstrate that intestinal deficiency in REG3B or REG3G increases numbers of mucosa-associated bacteria and enhances bacterial translocation to the mesenteric lymph nodes and liver, promoting the progression of ethanol-induced fatty liver disease toward steatohepatitis. Overexpression of Reg3g in intestinal epithelial cells restricts bacterial colonization of mucosal surfaces, reduces bacterial translocation, and protects mice from alcohol-induced steatohepatitis. Thus, alcohol appears to impair control of the mucosa-associated microbiota, and subsequent breach of the mucosal barrier facilitates progression of alcoholic liver disease.

ABSTRACT Background There are no FDA‐approved therapies for alcohol‐associated liver disease (ALD). Preclinical studies indicate that blocking IL‐23/IL‐17 signalling may reverse liver injury. Guselkumab, an IL‐23‐specific antibody approved for psoriasis, may be beneficial for ALD. Aims We aimed to assess the safety and tolerability of guselkumab in patients with ALD. Methods This phase‐1 dose‐escalation study included patients with ≥ 2 DSM‐5 criteria for alcohol use disorder, significant steatosis (MRI‐PDFF ≥ 8%) and MRE < 3.63 kPa (to exclude advanced disease). Guselkumab was given subcutaneously on Days 1 and 29 in 30, 70 or 100 mg dose cohorts. Primary endpoints were adverse events (AEs) and dose‐limiting toxicity. Results We enrolled 13 patients (three 30 mg, three 70 mg, and seven 100 mg). Eleven completed the study and two early discontinued in the 100 mg group. Of them, 77% were men, and the median age was 53 [IQR 49–61] years. The median MRI‐PDFF and MRE were 18.4% [IQR 8.4%–34.0%] and 2.5 [2.2–2.6] kPa, respectively. The most frequent AEs were hyperuricemia (13%, mild only) and elevated lipase (11%, mild and moderate). There were no serious adverse events or significant variations in liver enzymes. There was a suppression of peripheral interleukin (IL)‐17, IL‐23, IL‐1b and TNF‐α in the 70 and 100 mg groups, and a significant decrease in alcohol consumption over time (AUDIT‐C: 6 [3–7] vs. 5 [1–6], p = 0.023). Conclusions Guselkumab is safe in doses up to 100 mg and may reduce inflammation markers in ALD. These findings support further phase 2 studies to evaluate the efficacy of guselkumab in ALD, particularly in patients with severe phenotypes.

Metabolic dysfunction-associated steatohepatitis (MASH), an advanced stage of metabolic dysfunction-associated steatotic liver disease, is characterized by significant hepatic fibrosis and inflammation. The pan-peroxisome proliferator-activated receptor (pan-PPAR) agonist IVA337 (lanifibranor) has shown potential as an anti-MASH therapeutic, although its mechanisms of action remain incompletely understood. This study explores the effects and mechanisms of IVA337 using two distinct MASH models: two-dimensional (2D) primary human hepatic stellate cells (HSCs) stimulated with transforming growth factor β1 (TGF-β1), and three-dimensional (3D) liver spheroids comprising primary hepatocytes, HSCs, and non-parenchymal cells. In TGF-β1-stimulated HSCs, IVA337 effectively suppressed the expression of fibrosis-related genes, including PAI1, COL1A1, and ACAT2, as well as the inflammatory gene IL6. 3D mouse and human liver spheroid models of MASH, characterized by elevated fibrotic gene expression, were established. IVA337 treatment not only attenuated fibrotic gene expression but also restored lipid content in the MASH spheroids, as evidenced by BODIPY staining. Immunostaining further confirmed a reduction in α-smooth muscle actin and collagen 1 levels after IVA337 treatment. Bulk RNA sequencing and Gene Ontology analysis revealed several lipid metabolism-related genes as key effectors downstream of IVA337. In addition, IVA337 modulated multiple signaling pathways, including IL-17, tumor necrosis factor, NF-κB, phosphatidylinositol 3 kinase/protein kinase B, and mitogen-activated protein kinase. Collectively, these findings show that IVA337 effectively mitigates fibrosis development in both 2D and 3D MASH models by restoring lipid homeostasis and regulating crucial fibrotic and inflammatory pathways.

Metabolic dysfunction-associated steatotic liver disease (MASLD) has limited treatments, and cell type-specific regulatory networks driving MASLD represent therapeutic avenues. We assayed five transcriptomic and epigenomic modalities in 2.4M cells from 86 livers across MASLD stages. Integrating modalities increased annotation of the genome in liver cell types several-fold over previous catalogs. We identified cell type regulatory networks of MASLD progression, including distinct hepatocyte networks driving MASL and mild and severe fibrosis MASH. Our single cell atlas annotated 88% of MASH-associated loci, including a third affecting hepatocyte regulation which we linked to distal target genes. Finally, we characterized hepatocyte heterogeneity, including MASH-enriched populations with altered repression, localization, and signaling. Overall, our results provide high-resolution maps of liver cell types and revealed novel targets for anti-MASH therapy.

Aging is characterized by the progressive deterioration of cell and tissue functions. The liver, which regulates metabolic homeostasis, detoxification, and immune responses, undergoes structural and functional changes with age. These include increasing genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient-sensing and intracellular communication, mitochondrial dysfunction, cell senescence, stem cell exhaustion, chronic inflammation, disabled macroautophagy, and dysbiosis. These alterations contribute to hepatocyte dysfunction, impaired regenerative responses, and fibrosis risk, which all exacerbate existing liver diseases. Senescence involves irreversible cell cycle arrest resulting in an inflammatory, senescence-associated secretory cell phenotype. Senescent hepatocytes, liver sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells accumulate in the aged liver, creating an inflammatory and fibrotic microenvironment that promotes tumorigenesis. As the burden of aging-related liver disease increases, therapeutic strategies targeting hepatic senescence have gained attention. We review these, along with the mechanisms and pathogenic effects of liver aging.

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as nonalcoholic fatty liver disease (NAFLD), is the most prevalent chronic liver disease worldwide, with an estimated global prevalence of approximately 30%; however, effective pharmacotherapies are still limited due to its complex pathogenesis and etiology. Therefore, a more thorough understanding of disease pathogenesis is urgently needed. An increasing number of studies suggest that MASLD and its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), are driven by chronic overnutrition, multiple genetic susceptibility factors, and pathogenic consequences, including hepatocyte damage and liver inflammation. Hepatic inflammation is the key event fueling the conversion from simple steatosis to steatohepatitis and fibrosis. Current therapies for MASH, including the recently approved thyroid hormone receptor-beta agonist resmetirom or the available incretin mimetics, mainly target metabolic injury to the liver but not inflammation directly. In this review, we provide an in-depth discussion of current data related to the immunological mechanisms of MASLD and summarize the effects of current and experimental therapies on immunoregulation in MASLD.

Background & Aims: Metabolic dysfunction and alcohol-associated liver disease (MetALD) results in the development of liver steatosis, inflammation, fibrosis, and hepatocellular carcinoma (HCC). De novo lipogenesis and cholesterol synthesis play an important role in the pathogenesis of MetALD. DHCR7 (7-dehydrocholesterol reductase) regulates the last stages of cholesterol production. Methods: We investigated whether targeting DHCR7 can ameliorate the development of MetALD and HCC using experimental models and 3D human liver spheroids. Results: Here, we demonstrate that partial genetic ablation of the Dhcr7 gene and pharmacological blockade of DHCR7 activity with the AY9944 inhibitor suppresses hepatic steatosis (↓ lipid area, n = 15; p <0.001), inflammation (↓ F4/80, n = 6; p <0.01), fibrosis (↓ Sirius red, n = 6; p <0.01), and HCC (↓ AFP/YAP, n = 6; p <0.01) in diethylnitrosamine (DEN)-challenged high-fat diet (HFD) + ethanol (EtOH)-fed mice treated with AY9944 compared with control mice. To translate our findings, the effect of DHCR7 was tested using 3D human liver spheroids, which mimicked MetALD and MetALD-HCC. MetALD liver spheroids were composed of primary human hepatocytes, non-parenchymal cells, and hepatic stellate cells. In contrast, in MetALD-HCC spheroids, the HCC cell line HepG2 was used instead of hepatocytes. Therapeutic administration of AY9944 inhibited inflammation (↓ TNF, p <0.05) and fibrosis in MetALD spheroids (↓ ACTA2, p <0.001; COL1A1, p <0.05; TIMP1, p <0.01; SERPINE1, p <0.05). In turn, dsiRNA-based knockdown of DHCR7 reduced HepG2 proliferation (↓ PCNA, p <0.05; CCNE, p <0.05) and expression of MetALD-HCC markers (↓ AFP, p <0.05; GPC3, p <0.05; YAP, p <0.01). Conclusions: Our data demonstrate that targeting DHCR7 can become a strategy for the treatment of MetALD and HCC. Impact and implications: This study demonstrates the critical role of de novo lipogenesis and cholesterol synthesis in the pathogenesis of metabolic dysfunction and alcohol-associated liver disease (MetALD) and its progression to hepatocellular carcinoma (HCC). Our findings identified that the upregulation of DHCR7 contributes to the pathogenesis of MetALD and its inhibition suppresses hepatic steatosis, inflammation, fibrosis, and tumor proliferation. These findings are significant for researchers and clinicians, as they establish that genetic and pharmacological inhibition of DHCR7 is effective in both experimental models and translational 3D human liver spheroids. The results uncover the translational potential of DHCR7-targeted therapies for MetALD and HCC, offering practical implications for the development of novel treatment strategies. Further studies are necessary to optimize these approaches and address potential methodological limitations.

Hepatic stellate cells (HSCs) play a crucial role in the pathogenesis of liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), a condition characterized by excessive fat accumulation in the hepatocytes, unrelated to alcohol consumption. In a healthy liver, HSCs are quiescent, store vitamin A, and function as pericytes. However, in response to liver injury and inflammation, HSCs become activated. In MASH, HSC activation is driven by metabolic stress, lipotoxicity, and chronic inflammation. Injured hepatocytes, recruited macrophage, capillarized sinusoidal endothelial cells, and permeable intestinal epithelium may each contribute to activating HSCS. This leads to a unique inflammatory environment that promotes fibrosis. MASH HSCs change their metabolism to favor glycolysis, glutaminolysis, and lactate generation. Activated HSCs transform into myofibroblast-like cells, producing excessive extracellular matrix components that result in fibrosis. In addition, HSCs in MASH have inflammatory and intermediate activated phenotypes. This fibrotic process is a key feature of MASH, which can lead to cirrhosis and liver cancer. Understanding the mechanisms of HSC activation and their role in MASH progression is essential for developing targeted therapies to treat and prevent liver fibrosis in affected individuals.

Alcohol-use disorder and alcohol-associated liver disease (ALD) are major causes of death and liver transplantation1. The gut-liver axis has a crucial yet poorly understood role in ALD pathogenesis, which depends on microbial translocation. Intestinal goblet cells (GCs) educate the immune system by forming GC-associated antigen passages (GAPs) on activation of muscarinic acetylcholine receptor M4 (mAChR4, also known as M4), enabling sampling of luminal antigens by lamina propria antigen-presenting cells. Here we show that chronic alcohol use in humans and mice downregulates small intestinal mAChR4 and reduces GAP formation, disrupting antimicrobial immunity. This is reversed on activation of intestinal IL-6 signal transducer (IL6ST, also known as glycoprotein 130; gp130), which restores mAChR4 expression and GAP formation, enabling induction of downstream type-3 innate lymphoid cell-derived IL-22 and antimicrobial REG3 proteins. This blunts translocation of enteric bacteria to the liver, thereby conferring ALD resistance. GAP induction by GC-specific mAChR4 activation was essential and sufficient for prevention of ethanol-induced steatohepatitis. These results lay the foundation for a therapeutic approach using mAChR4 or IL6ST agonists to promote GAP formation and prevent ALD by inhibiting microbial translocation.

Chronic liver injury results in activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing activated HSCs that make the liver fibrotic. We identified ETS1 and ETS2 (ETS1/2) as lineage-specific transcription factors regulating HSC phenotypes. Here, we investigated the role of ETS1/2 in HSCs in liver fibrosis using toxic liver injury models and 3D human liver spheroids. Liver fibrosis was induced in WT and HSC-specific Ets1-KO (Ets1ΔHSC) and Ets2-KO (Ets2ΔHSC) mice by administration of CCl4 for 6 weeks, followed by cessation of liver injury for 2 weeks. Liver fibrosis was more severe in Ets1ΔHSC and to a lesser extent Ets2ΔHSC mice compared with WT mice. Regression of liver fibrosis was suppressed only in Ets1ΔHSC mice, indicating Ets1 is the predominant isoform maintaining a quiescent-like phenotype in HSCs. Similar results were obtained in a metabolic dysfunction-associated steatohepatitis (MASH) model using 3D human liver spheroids. Knockdown of ETS1 in human HSCs caused upregulation of fibrogenic genes in MASH human liver spheroids and prevented fibrosis regression. ETS1 regulated the quiescent HSC phenotype via the CREB-regulated transcription coactivator 2 (CRTC2)/PGC1α/PPARγ pathway. Knockdown of CRTC2 abrogated PPARγ responses and facilitated HSC activation. These findings suggest that ETS1 may represent a therapeutic target for antifibrotic therapy.

Metabolic syndrome and excessive alcohol consumption result in liver injury and fibrosis, which is characterized by increased collagen production by activated Hepatic Stellate Cells (HSCs). LARP6, an RNA-binding protein, was shown to facilitate collagen production. However, LARP6 expression and functionality as a regulator of fibrosis development in a disease relevant model remains elusive. By using snRNA-sequencing, we show that LARP6 is upregulated mainly in HSCs of liver fibrosis patients. Moreover, LARP6 knockdown in human HSCs suppresses fibrogenic gene expression. By integrating eCLIP analysis and ribosome profiling in HSCs, we show that LARP6 interacts with mature mRNAs comprising over 300 genes, including RNA structural elements within COL1A1 , COL1A2 , and COL3A1 to regulate mRNA expression and translation. Furthermore, LARP6 knockdown in HSC attenuates fibrosis development in human liver spheroids. Altogether, our results suggest that targeting LARP6 in human HSCs may provide new strategies for anti-fibrotic therapy. Highlights:LARP6 is upregulated in liver fibrosis, mainly in HSCs.LARP6 knockdown in human HSCs reduces liver fibrosis development.Of the hundreds of gene targets, LARP6 interacts most with collagen mRNAs.LARP6 regulates mRNA translation via interaction with 5'UTRs.

BACKGROUND:Liver fibrosis is caused by chronic toxic or cholestatic liver injury. Fibrosis results from the recruitment of myeloid cells into the injured liver, the release of inflammatory and fibrogenic cytokines, and the activation of myofibroblasts, which secrete extracellular matrix, mostly collagen type I. Hepatic myofibroblasts originate from liver-resident mesenchymal cells, including HSCs and bone marrow-derived CD45+ collagen type I+ expressing fibrocytes. Recombinant human serum amyloid P (hSAP), a natural inhibitor of fibrocyte activation into myofibroblasts, was shown to ameliorate experimental renal, lung, skin, and cardiac fibrosis. We investigated if hSAP can ameliorate the development of liver fibrosis of different etiologies. METHODS:Reporter Collagen-α(1)I-GFP mice were subjected to cholestatic liver injury (by ligation of the common bile duct) or toxic liver injury (by carbon tetrachloride administration) and treated prophylactically or therapeutically with hSAP (12.5 μg/g). Primary cultures of mouse fibrocytes and HSCs were stimulated to activate with or without incubation with hSAP. RESULTS:We demonstrate that treatment with hSAP suppressed hepatic fibrosis by ≈50% through dual mechanisms. hSAP prevented the recruitment of fibrocytes into the injured liver and their differentiation into myofibroblasts. Remarkably, hSAP also inhibited the activation of HSCs into myofibroblasts. CONCLUSIONS:Since HSCs serve as a major source of collagen type I-producing myofibroblasts and fibrocytes stimulate fibrosis, hSAP may become part of the therapy of liver fibrosis of different etiologies.