System analysis based on weighted gene co-expression analysis identifies SOX7 as a novel regulator of hepatic stellate cell activation and liver fibrosis.

Hepatic stellate cell (HSC) activation is the essential pathological process of liver fibrosis (LF). The molecular mechanisms regulating HSC activation and LF are incompletely understood. Here, we explored the effect of transcription factor SRY-related high mobility group box 7 (SOX7) on HSC activation and LF, and the underlying molecular mechanism. We found the expression levels of SOX7 were decreased in human and mouse fibrotic livers, particularly at the fibrotic foci. SOX7 was also downregulated in primary activated HSCs and TGF-ß1 stimulated LX-2 cells. SOX7 knockdown promoted activation and proliferation of LX-2 cells while inhibiting their apoptosis. On the other hand, overexpression of SOX7 suppressed the activation and proliferation of HSCs. Mechanistically, SOX7 attenuates HSC activation and LF by decreasing the expression of ß-catenin and phosphorylation of Smad2 and Smad3 induced by TGF-ß1. Furthermore, overexpression of SOX7 using AAV8-SOX7 mouse models ameliorated the extent of LF in response to CCl4 treatment in vivo. Collectively, SOX7 suppressed HSC activation and LF. Targeting SOX7, therefore, could be a potential novel strategy to protect against LF.

GSK805 inhibits alpha-smooth muscle expression and modulates liver inflammation without impairing the well-being of mice.

Cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), lead to inflammation and severe hepatic damage with limited therapeutic options. This study assessed the efficacy of the inverse RORγt agonist, GSK805, both in vitro using the hepatic stellate cell-line LX-2 and in vivo using male bile duct-ligated BALB/c mice. In vitro, 0.3 µM GSK805 reduced alpha-smooth muscle actin expression in LX-2 cells. In vivo, GSK805 significantly decreased IL-23R, TNF-α, and IFN-γ expression in cholestatic liver. Despite high concentrations of GSK805 in the liver, no significant reduction in fibrosis was noticed. GSK805 significantly increased aspartate aminotransferase and alanine aminotransferase activity in the blood, while levels of glutamate dehydrogenase, alkaline phosphatase, and bilirubin were not substantially increased. Importantly, GSK805 did neither increase an animal distress score nor substantially reduce body weight, burrowing activity, or nesting behavior. These results suggest that a high liver concentration of GSK805 is achieved by daily oral administration and that this drug modulates inflammation in cholestatic mice without impairing animal well-being.

Senescence of Hepatic Stellate Cells by Specific Delivery of Manganese for Limiting Liver Fibrosis.

Senescence of activated hepatic stellate cells (HSCs) is crucial for the regression of liver fibrosis. However, impaired immune clearance can result in the accumulation of senescent HSCs, exacerbating liver fibrosis. The activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is essential for both senescence and the innate immune response. Additionally, the specific delivery to activated HSCs is hindered by their inaccessible anatomical location, capillarization of liver sinusoidal endothelial cells (LSECs), and loss of substance exchange. Herein, we propose an antifibrotic strategy that combines prosenescence with enhanced immune clearance through targeted delivery of manganese (a cGAS-STING stimulator) via albumin-mediated transcytosis, specifically aimed at inducing senescence and eliminating activated HSCs in liver fibrosis. Our findings demonstrate that only albumin efficiently transfers manganese to activated HSCs from LSECs via transcytosis compared to liposomes, resulting in significant antifibrotic effects in vivo while exhibiting negligible toxicity.

Hepatocyte-derived exosomes deliver the lncRNA CYTOR to hepatic stellate cells and promote liver fibrosis.

Liver fibrosis is characterized by the activation and transformation of hepatic stellate cells (HSCs) induced by various injury factors. The degree of liver fibrosis can be significantly improved, but persistent injury factors present a significant therapeutic challenge. Hepatocytes are the most important parenchymal cell type in the liver. In this study, we explored the molecular mechanisms by which damaged liver cells activate HSCs through extracellular vesicles. We established a coculture model of LO2 and LX2 and validated its exosomal transmission activity. Subsequently, differentially expressed long noncoding RNAs (lncRNAs) were screened through RNA sequencing and their mechanisms of action as competing endogenous RNAs (ceRNAs) further confirmed using biological methods, such as FISH and luciferase assays. Damaged liver cells induced activation of LX2 and upregulation of liver fibrosis-related markers. Exosomes extracted and identified from the supernatant fraction contained differentially expressed lncRNA cytoskeleton regulator RNA (CYTOR) that competed with microRNA-125 (miR-125) for binding to glial cell line-derived neurotrophic factor (GDNF) in HSCs, in turn, promoting LX2 activation. MiR-125 could target and regulate both CYTOR and GDNF and vice versa, as verified using the luciferase assay. In an in vivo model, damaged liver extracellular vesicles induced the formation of liver fibrosis. Notably, downregulation of CYTOR within extracellular vesicles effectively inhibited liver fibrosis. The lncRNA CYTOR in exosomes of damaged liver cells is upregulated and modulates the expression of downstream GDNF through activity as a ceRNA, providing an effective mechanism for activation of HSCs.

ZNF469 is a profibrotic regulator of extracellular matrix in hepatic stellate cells.

Activation of quiescent hepatic stellate cells (HSCs) into proliferative myofibroblasts drives extracellular cellular matrix (ECM) accumulation and liver fibrosis; nevertheless, the transcriptional network that promotes such a process is not completely understood. ZNF469 is a putative C2H2 zinc finger protein that may bind to specific genome sequences. It is found to be upregulated upon HSC activation; however, the molecular function of ZNF469 is completely unknown. Here, we show that knockdown of ZNF469 in primary human HSCs impaired proliferation, migration, and collagen production. Conversely, overexpression of ZNF469 in HSCs yielded the opposite results. Transforming growth factor-ß 1 promoted expression of ZNF469 in a Smad3-dependent manner, where the binding of Smad3 was confirmed at the ZNF469 promoter. RNA sequencing data of ZNF469-knockdown HSCs revealed the ECM-receptor interaction, which provides structural and signaling support to cells, was the most affected pathway, and significant downregulation of various collagen and proteoglycan genes was observed. To investigate the function of ZNF469, we cloned a full-length open reading frame of ZNF469 with an epitope tag and identified a nuclear localization of the protein. Luciferase reporter and chromatin immunoprecipitation assays revealed the presence of ZNF469 at the promoter of ECM genes, supporting its function as a transcription factor. Analysis of human fibrotic and cirrhotic tissues showed increased expression of ZNF469 and a positive correlation between expression levels of ZNF469 and ECM genes. Moreover, this observation was similar in other fibrotic organs, including the heart, lung, and skin, suggesting that myofibroblasts from various origins generally require ZNF469 to promote ECM production. Together, this study is the first to reveal the role of ZNF469 as a profibrotic factor in HSCs and suggests ZNF469 as a novel target for antifibrotic therapy.

Chimeric antigen receptor-modified macrophages ameliorate liver fibrosis in preclinical models.

BACKGROUND & AIMS: Treatments directly targeting fibrosis remain limited. Given the unique intrinsic features of macrophages and their capacity to engraft in the liver, we genetically engineered bone marrow-derived macrophages with a chimeric antigen receptor (CAR) to direct their phagocytic activity against hepatic stellate cells (HSCs) in multiple mouse models. This study aimed to demonstrate the therapeutic efficacy of CAR macrophages (CAR-Ms) in mouse models of fibrosis and cirrhosis and to elucidate the underlying mechanisms. METHODS: uPAR expression was studied in patients with fibrosis/cirrhosis and in murine models of liver fibrosis, including mice treated with carbon tetrachloride, a 5-diethoxycarbonyl-1, 4-dihydrocollidine diet, or a high-fat/cholesterol/fructose diet. The safety and efficacy of CAR-Ms were evaluated in vitro and in vivo. RESULTS: Adoptive transfer of CAR-Ms resulted in a significant reduction in liver fibrosis and the restoration of function in murine models of liver fibrosis. CAR-Ms modulated the hepatic immune microenvironment to recruit and modify the activation of endogenous immune cells to drive fibrosis regression. These CAR-Ms were able to recruit and present antigens to T cells and mount specific antifibrotic T-cell responses to reduce fibroblasts and liver fibrosis in mice. CONCLUSION: Collectively, our findings demonstrate the potential of using macrophages as a platform for CAR technology to provide an effective treatment option for liver fibrosis. CAR-Ms might be developed for treatment of patients with liver fibrosis. IMPACT AND IMPLICATIONS: Liver fibrosis is an incurable condition that afflicts millions of people globally. Despite the clear clinical need, therapies for liver fibrosis are limited. Our findings provide the first preclinical evidence that chimeric antigen receptor (CAR)-macrophages (CAR-Ms) targeting uPAR can attenuate liver fibrosis and cirrhosis. We show that macrophages expressing this uPAR CAR exert a direct antifibrotic effect and elicit a specific T-cell response that augments the immune response against liver fibrosis. These findings demonstrate the potential of using CAR-Ms as an effective cell-based therapy for the treatment of liver fibrosis.

Targeting the liver clock improves fibrosis by restoring TGF-ß signaling.

BACKGROUND & AIMS: Liver fibrosis is the major driver for hepatocellular carcinoma and liver disease related death. Approved anti-fibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSC)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC-phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and validation in cell-based models. Using mouse models for MASH fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC-oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß-activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models uncover the CC as a therapeutic candidate target for liver fibrosis - a rising global unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day being controlled by the circadian clock (CC). Here we demonstrate that the regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncovers a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

Phenotypes and ontogeny of senescent hepatic stellate cells in metabolic dysfunction-associated steatohepatitis.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are the key drivers of fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), the fastest growing cause of hepatocellular carcinoma (HCC) worldwide. HSCs are heterogenous, and a senescent subset of HSCs is implicated in hepatic fibrosis and HCC. Administration of anti-uPAR (urokinase-type plasminogen activator receptor) CAR T cells has been shown to deplete senescent HSCs and attenuate fibrosis in murine models. However, the comprehensive features of senescent HSCs in MASH, as well as their cellular ontogeny have not been characterized; hence, we aimed to comprehensively characterize and define the origin of HSCs in human and murine MASH. METHODS: To comprehensively characterize the phenotype and ontogeny of senescent HSCs in human and murine MASH, we integrated senescence-associated beta galactosidase activity with immunostaining, flow cytometry and single-nucleus RNA sequencing (snRNAseq). We integrated the immunohistochemical profile with a senescence score applied to snRNAseq data to characterize senescent HSCs and mapped the evolution of uPAR expression in MASH. RESULTS: Using pseudotime trajectory analysis, we establish that senescent HSCs arise from activated HSCs. While uPAR is expressed in MASH, the magnitude and cell-specificity of its expression evolve with disease stage. In early disease, uPAR is more specific to activated and senescent HSCs, while it is also expressed by myeloid-lineage cells, including Trem2+ macrophages and myeloid-derived suppressor cells, in late disease. Furthermore, we identify novel surface proteins expressed on senescent HSCs in human and murine MASH that could be exploited as therapeutic targets. CONCLUSIONS: These data define features of HSC senescence in human and murine MASH, establishing an important blueprint to target these cells as part of future antifibrotic therapies. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) are the primary drivers of scarring in chronic liver diseases. As injury develops, a subset of HSCs become senescent; these cells are non-proliferative and pro-inflammatory, thereby contributing to worsening liver injury. Here we show that senescent HSCs are expanded in MASH (metabolic dysfunction-associated steatohepatitis) in humans and mice, and we trace their cellular origin from the activated HSC subset. We further characterize expression of uPAR (urokinase plasminogen activated receptor), a protein that marks senescent HSCs, and report that uPAR is also expressed by activated HSCs in early injury, and in immune cells as liver injury advances. We have integrated high-resolution single-nucleus RNA sequencing with immunostaining and flow cytometry to identify five other novel proteins expressed by senescent HSCs, including mannose receptor CD206, which will facilitate future therapeutic development.

Exploring the impact of the PNPLA3 I148M variant on primary human hepatic stellate cells using 3D extracellular matrix models.

BACKGROUND & AIMS: The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSC biology using transcriptomic data and validated findings in 3D-culture models. METHODS: RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of individuals with obesity, genotyped for the PNPLA3 I148M variant. Data were validated in wild-type (WT) or PNPLA3 I148M variant-carrying HSCs cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic livers, with/without TGFB1 or cytosporone B (Csn-B) treatment. RESULTS: Transcriptomic analyses of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in WT/PNPLA3-I148M HSCs cultured in 3D liver scaffolds. Mitochondrial dysfunction in PNPLA3-I148M cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in PNPLA3-I148M HSCs, while reactive oxygen species secretion was increased in PNPLA3-I148M HSCs and higher in bioengineered cirrhotic vs. healthy scaffolds. TGFB1 signalling followed the same trend. In PNPLA3-I148M cells, expression and activation of the endogenous TGFB1 inhibitor NR4A1 were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in PNPLA3-WT HSCs and to Erk signalling in PNPLA3-I148M HSCs. CONCLUSION: HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant responses, and increased TGFB1 signalling, which dampens antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs requires further investigation. Here, by using comparative transcriptomics and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs' behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.

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.

PFKFB3-mediated glycolysis in hepatic stellate cells promotes liver regeneration.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.

Loss of CEACAM1 in hepatocytes causes hepatic fibrosis.

BACKGROUND: The role of insulin resistance in hepatic fibrosis in Metabolic dysfunction-Associated SteatoHepatitis (MASH) remains unclear. Carcinoembryonic Antigen-related Cell Adhesion Molecule1 protein (CEACAM1) promotes insulin clearance to maintain insulin sensitivity and repress de novo lipogenesis, as bolstered by the development of insulin resistance and steatohepatitis in AlbuminCre + Cc1fl/fl mice with liver-specific mouse gene encoding CEACAM1 protein (Ceacam1) deletion. We herein investigated whether these mice also developed hepatic fibrosis and whether hepatic CEACAM1 is reduced in patients with MASH at different fibrosis stages. METHODS: AlbuminCre + Cc1fl/fl mice were fed a regular or a high-fat diet before their insulin metabolism and action were assessed during IPGTT, and their livers excised for histochemical, immunohistochemical and Western blot analysis. Sirius red staining was used to assess fibrosis, and media transfer was employed to examine whether mutant hepatocytes activated hepatic stellate cells (HSCs). Hepatic CEACAM1 protein levels in patients with varying disease stages were assessed by ELISA. RESULTS: Hepatocytic deletion of Ceacam1 caused hyperinsulinemia-driven insulin resistance emanating from reduced hepatic insulin clearance. AlbuminCre + Cc1fl/fl livers showed inflammation, fibrosis and hepatic injury, with more advanced bridging and chicken-wire hepatic fibrosis under high-fat conditions. Media transferred from hepatocytes isolated from mutant mice activated control HSCs, likely owing to their elevated endothelin1 content. Interestingly, hepatic CEACAM1 levels were lower in the livers of patients with MASH and declined gradually with advanced fibrosis stage. CONCLUSIONS: Hepatic CEACAM1 levels declined with progression of MASH in humans. The phenotype of AlbuminCre + Cc1fl/fl mice assigned a key role to CEACAM1 loss from hepatocytes in hepatic fibrosis independently of other liver cells.

Novel Oridonin Analog CYD0682 Inhibits Hepatic Stellate Cell Activation via the Heat Shock Protein 90-Dependent STAT3 Pathway.

INTRODUCTION: Activated hepatic stellate cells (HSCs) are the primary effector cells in hepatic fibrosis, over depositing extracellular matrix (ECM) proteins. Our previous work found oridonin analog CYD0682 attenuates proliferation, Transforming Growth Factor ß (TGFß)-induced signaling, and ECM production in immortalized HSCs. The underlying mechanism behind these reductions is unclear. The Signal Transduction and Activator of Transcription 3 (STAT3) pathway plays a central role in HSC activation and has been found to be overexpressed in models of hepatic injury. In this study, we will examine the effect of CYD0682 on STAT3 signaling. METHODS: Immortalized human (LX-2) and rat (HSC-T6) HSC lines were treated with CYD0682 or Tanespimycin (17-AAG) with or without TGF-ß. Nuclear and cytosolic proteins were extracted. Protein expression was analyzed with Western blot. DNA binding activity was assessed with STAT3 DNA Binding ELISA. Cell viability was assessed with Alamar blue assay. RESULTS: CYD0682 treatment inhibited STAT3 phosphorylation at tyrosine 705 in a dose-dependent manner in LX-2 and HSC-T6 cells. STAT3 DNA binding activity and STAT3 regulated protein c-myc were significantly decreased by CYD0682. Notably, TGFß-induced STAT3 phosphorylation and ECM protein expression were inhibited by CYD0682. STAT3 is reported to be a Heat Shock Protein 90 (HSP90) client protein. Notably, CYD0682 attenuated the expression of endogenous STAT3 and other HSP90 client proteins FAK, IKKα, AKT and CDK9. HSP90 specific inhibitor 17-AAG suppressed endogenous and TGFß-induced STAT3 phosphorylation and ECM protein production. CONCLUSIONS: CYD0682 attenuates endogenous and TGFß-induced STAT3 activation and ECM production via an HSP90 dependent pathway in HSCs. Further study of this pathway may present new targets for therapeutic intervention in hepatic fibrosis.

Immune signature and therapeutic approach of natural killer cell in chronic liver disease and hepatocellular carcinoma.

Natural killer (NK) cells are one of the key members of innate immunity that predominantly reside in the liver, potentiating immune responses against viral infections or malignant tumors. It has been reported that changes in cell numbers and function of NK cells are associated with the development and progression of chronic liver diseases (CLDs) including non-alcoholic fatty liver disease, alcoholic liver disease, and chronic viral hepatitis. Also, it is known that the crosstalk between NK cells and hepatic stellate cells plays an important role in liver fibrosis and cirrhosis. In particular, the impaired functions of NK cells observed in CLDs consequently contribute to occurrence and progression of hepatocellular carcinoma (HCC). Chronic infections by hepatitis B or C viruses counteract the anti-tumor immunity of the host by producing the sheddases. Soluble major histocompatibility complex class I polypeptide-related sequence A (sMICA), released from the cell surfaces by sheddases, disrupts the interaction and affects the function of NK cells. Recently, the MICA/B-NK stimulatory receptor NK group 2 member D (NKG2D) axis has been extensively studied in HCC. HCC patients with low membrane-bound MICA or high sMICA concentration have been associated with poor prognosis. Therefore, reversing the sMICA-mediated downregulation of NKG2D has been proposed as an attractive strategy to enhance both innate and adaptive immune responses against HCC. This review aims to summarize recent studies on NK cell immune signatures and its roles in CLD and hepatocellular carcinogenesis and discusses the therapeutic approaches of MICA/B-NKG2D-based or NK cell-based immunotherapy for HCC.

Thymoquinone protects thioacetamide-induced chronic liver injury by inhibiting TGF-ß1/Smad3 axis in rats.

Chronic liver injury due to various etiological factors results in excess secretion and accumulation of extracellular matrix proteins, leading to scarring of liver tissue and ultimately to hepatic fibrosis. If left untreated, fibrosis might progress to cirrhosis and even hepatocellular carcinoma. Thymoquinone (TQ), an active compound of Nigella sativa, has been reported to exhibit antioxidant, anti-inflammatory and anticancer activities. Therefore, the effect of TQ against thioacetamide (TAA)-induced liver fibrosis was assessed in rats. Fibrosis was induced with intraperitoneal administration of TAA (250 mg/kg b.w.) twice a week for 5 weeks. TQ (20 mg/kg b.w.) and silymarin (50 mg/kg b.w.) were orally administered daily for 5 weeks separately in TAA administered groups. Liver dysfunction was reported by elevated liver enzymes, increased oxidative stress, inflammation and fibrosis upon TAA administration. Our study demonstrated that TQ inhibited the elevation of liver marker enzymes in serum. TQ administration significantly increased antioxidant markers, such as superoxide dismutase, catalase, glutathione, glutathione peroxidase and glutathione reductase in the liver tissue of rats. Further, TQ significantly attenuated liver fibrosis, as illustrated by the downregulation of TAA-induced interleukin-ß, tumour necrosis factor-α, inducible nitric oxide synthase and fibrosis markers like transforming growth factor-ß (TGF-ß), α-smooth muscle actin, collagen-1, Smad3 and 7. Therefore, these findings suggest that TQ has a promising hepatoprotective property, as indicated by its potential to effectively suppress TAA-induced liver fibrosis in rats by inhibiting oxidative stress and inflammation via TGF-ß/Smad signaling.

Identification of (E)-1-((1H-indol-3-yl)methylene)-4-substitute-thiosemicarbazones as potential anti-hepatic fibrosis agents.

Liver fibrosis remains a global health challenge due to its rapidly rising prevalence and limited treatment options. The orphan nuclear receptor Nur77 has been implicated in regulation of autophagy and liver fibrosis. Targeting Nur77-mediated autophagic flux may thus be a new promising strategy against hepatic fibrosis. In this study, we synthesized four types of Nur77-based thiourea derivatives to determine their anti-hepatic fibrosis activity. Among the synthesized thiourea derivatives, 9e was the most potent inhibitor of hepatic stellate cells (HSCs) proliferation and activation. This compound could directly bind to Nur77 and inhibit TGF-ß1-induced α-SMA and COLA1 expression in a Nur77-dependent manner. In vivo, 9e significantly reduced CCl4-mediated hepatic inflammation response and extracellular matrix (ECM) production, revealing that 9e is capable of blocking the progression of hepatic fibrosis. Mechanistically, 9e induced Nur77 expression and enhanced autophagic flux by inhibiting the mTORC1 signaling pathway in vitro and in vivo. Thus, the Nur77-targeted lead 9e may serve as a promising candidate for treatment of chronic liver fibrosis.

Extracellular vesicles originating from steatotic hepatocytes promote hepatic stellate cell senescence via AKT/mTOR signaling.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid-treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA-EVs) inhibit collagen type 1 and α-smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL-6, IL-1ß, p21 and senescence-associated ß-galactosidase activity) in early activating HSCs via the AKT-mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA-treated hepatocytes inhibit HSC activation by inducing senescence via the AKT-mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.

Cells in the liver microenvironment regulate the process of liver metastasis.

The research of liver metastasis is a developing field. The ability of tumor cells to invade the liver depends on the complicated interactions between metastatic cells and local subpopulations in the liver (including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, and immune-related cells). These interactions are mainly mediated by intercellular adhesion and the release of cytokines. Cell populations in the liver microenvironment can play a dual role in the progression of liver metastasis through different mechanisms. At the same time, we can see the participation of liver parenchymal cells and nonparenchymal cells in the process of liver metastasis of different tumors. Therefore, the purpose of this article is to summarize the relationship between cellular components of liver microenvironment and metastasis and emphasize the importance of different cells in the occurrence or potential regression of liver metastasis.

Lipotoxic hepatocyte derived LIMA1 enriched small extracellular vesicles promote hepatic stellate cells activation via inhibiting mitophagy.

BACKGROUND: Hepatic stellate cells (HSCs) play a crucial role in the development of fibrosis in non-alcoholic fatty liver disease (NAFLD). Small extracellular vesicles (sEV) act as mediators for intercellular information transfer, delivering various fibrotic factors that impact the function of HSCs in liver fibrosis. In this study, we investigated the role of lipotoxic hepatocyte derived sEV (LTH-sEV) in HSCs activation and its intrinsic mechanisms. METHODS: High-fat diet (HFD) mice model was constructed to confirm the expression of LIMA1. The relationship between LIMA1-enriched LTH-sEV and LX2 activation was evaluated by measurement of fibrotic markers and related genes. Levels of mitophagy were detected using mt-keima lentivirus. The interaction between LIMA1 and PINK1 was discovered through database prediction and molecular docking. Finally, sEV was injected to investigate whether LIMA1 can accelerate HFD induced liver fibrosis in mice. RESULTS: LIMA1 expression was upregulated in lipotoxic hepatocytes and was found to be positively associated with the expression of the HSCs activation marker α-SMA. Lipotoxicity induced by OPA led to an increase in both the level of LIMA1 protein in LTH-sEV and the release of LTH-sEV. When HSCs were treated with LTH-sEV, LIMA1 was observed to hinder LX2 mitophagy while facilitating LX2 activation. Further investigation revealed that LIMA1 derived from LTH-sEV may inhibit PINK1-Parkin-mediated mitophagy, consequently promoting HSCs activation. Knocking down LIMA1 significantly attenuates the inhibitory effects of LTH-sEV on mitophagy and the promotion of HSCs activation. CONCLUSIONS: Lipotoxic hepatocyte-derived LIMA1-enriched sEVs play a crucial role in promoting HSCs activation in NAFLD-related liver fibrosis by negatively regulating PINK1 mediated mitophagy. These findings provide new insights into the pathological mechanisms involved in the development of fibrosis in NAFLD.

Hepatitis C virus NS5A and core protein induce fibrosis-related genes regulation on Huh7 cells through activation of LX2 cells.

INTRODUCTION AND OBJECTIVES: Liver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. MATERIALS AND METHODS: We evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. RESULTS: We observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFß1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. CONCLUSIONS: We demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A and Core-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.