Primary Human Hepatic Stellate Cells Research Articles

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-β 1promoted expression of ZNF469 in a Smad3-dependent manner, where the binding of Smad3 was confirmed at the ZNF469 promoter.RNA sequencingdata 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 immunoprecipitationassays 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.

EphB2 deficiency in hepatic stellate cells mitigates fibrogenesis in metabolic dysfunction-associated steatohepatitis by attenuating the TGFβ/SMAD signaling

Background & Aims: The receptor tyrosine kinase EphB2 is now emerging as a critical regulator of inflammation and fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH) though the underlying mechanism remain unclear. We aimed to investigate the role or mechanism by which EphB2 contributes in the progression of MASH fibrogenesis. Hypothesis: We hypothesized that EphB2 in hepatic stellate cells (HSCs) promotes MASH-liver fibrosis Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for EphB2 expression. Human liver biopsies obtained from patients with MASH were used to assess EphB2 expression by immunohistochemistry. Primary human HSCs were stimulated with TGFβ with or without TGFβR/SMAD inhibitors (SB525334, CS208, and SIS3 at 1μM) and EphB2 expression assessed by qPCR, western blot and immunofluorescence staining. EphB2 was silenced in primary human HSCs using siRNA and the resulting HSCs assess for TGBβ-induced HSC-myofibroblasts transition. MASH models were developed in hepatocyte-specific EphB2 knockout and HSCs EphB2 knockout and wild-type mice fed a high fat diet for 26 weeks or a choline-deficient amino-acid defined (CDAA) high fat diet for 10 weeks. Statistical analysis was performed using GraphPad Prism and a p-value < 0.05 was considered significant. Results: The expression of EphB2 was 3-fold significantly upregulated in liver specimens from patients with advanced MASH and correlated with disease severity. EphB2 was modestly expressed in hepatocyte, but strongly induced in HSCs in human and mouse MASH. Hepatocyte-specific EphB2 deficiency does not inhibit the development of steatohepatitis and fibrosis in mice fed the CDAA-diet. TGFβ/SMAD signaling promotes an 8-fold increase of EphB2 expression in human HSCs while siRNA silencing of EphB2 in human HSCs was suffcient to abolished TGFβ-mediated HSCs activation. Interestingly, HSCs-specific EphB2 knockout mice developed 2.5 to 6-fold less fibrosis than wild-type controls in response to high fat diets feeding suggesting that EphB2 expression on HSCs is required for the development of fibrogenesis in MASH. Deletion of EphB2 in activated HSCs after fibrosis was established in mice was suffcient to mitigate MASH fibrosis. Conclusions: EphB2 is upregulated in human and mouse MASH. Upregulation of EphB2 in human HSCs is mediated via the TGFβ/SMAD signaling. HSCs expressing EphB2 regulate the development of MASH-fibrosis. Specific EphB2 inhibitors could potentially mitigates fibrogenesis in MASH, opening up an avenue for the treatment of MASH. Funding sources: NIDDK, NIAMS, APS postdoctoral fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TM7SF3 controls TEAD1 splicing to prevent MASH-induced liver fibrosis

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and invivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs invitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Combinatorial Microgels for 3D ECM Screening and Heterogeneous Microenvironmental Culture of Primary Human Hepatic Stellate Cells.

Nonalcoholic fatty liver disease affects 30% of the United States population and its progression can lead to nonalcoholic steatohepatitis (NASH), and increased risks for cirrhosis and hepatocellular carcinoma. NASH is characterized by a highly heterogeneous liver microenvironment created by the fibrotic activity of hepatic stellate cells (HSCs). While HSCs have been widely studied in 2D, further advancements in physiologically relevant 3D culture platforms for the in vitro modeling of these heterogeneous environments are needed. In this study, the use of stiffness-variable, extracellular matrix (ECM) protein-conjugated polyethylene glycol microgels as 3D cell culture scaffolds to modulate HSC activation is demonstrated. These microgels as a high throughput ECM screening system to identify HSC matrix remodeling and metabolic activities in distinct heterogeneous microenvironmental conditions are further employed. The 6kPa fibronectin microgels are shown to significantly increase HSC matrix remodeling and metabolic activities in single or multiple-component microenvironments. Overall, heterogeneous microenvironments consisting of multiple distinct ECM microgels promoted a decrease in HSC matrix remodeling and metabolic activities compared to homogeneous microenvironments. The study envisions this ECM screening platform being adapted to a broad number of cell types to aid the identification of ECM microenvironments that best recapitulate the desired phenotype, differentiation, or drug efficacy.

Orphan receptor GPR176 in hepatic stellate cells exerts a profibrotic role in chronic liver disease

Background & AimsChronic liver disease (CLD) remains a global health issue associated with a significant disease burden. Liver fibrosis, a hallmark of CLD, is characterised by the activation of hepatic stellate cells (HSCs) that gain profibrotic characteristics including increased production of extracellular matrix protein. Currently, no antifibrotic therapies are available clinically, in part because of the lack of HSC-specific drug targets. Here, we aimed to identify HSC-specific membrane proteins that can serve as targets for antifibrotic drug development. MethodsSmall interfering RNA-mediated knockdown of GPR176 was used to assess the in vitro function of GPR176 in HSCs and in precision cut liver slices (PCLS). The in vivo role of GPR176 was assessed using the carbon tetrachloride (CCl4) and common bile duct ligation (BDL) models in wild-type and GPR176 knockout mice. GPR176 in human CLD was assessed by immunohistochemistry of diseased human livers and RNA expression analysis in human primary HSCs and transcriptomic data sets. ResultsWe identified Gpr176, an orphan G-protein coupled receptor, as an HSC-enriched activation associated gene. In vitro, Gpr176 is strongly induced upon culture-induced and hepatocyte-damage-induced activation of primary HSCs. Knockdown of GPR176 in primary mouse HSCs or PCLS cultures resulted in reduced fibrogenic characteristics. Absence of GPR176 did not influence liver homeostasis, but Gpr176-/- mice developed less severe fibrosis in CCl4 and BDL fibrosis models. In humans, GPR176 expression was correlated with in vitro HSC activation and with fibrosis stage in patients with CLD. ConclusionsGPR176 is a functional protein during liver fibrosis and reducing its activity attenuates fibrogenesis. These results highlight the potential of GPR176 as an HSC-specific antifibrotic candidate to treat CLD. Impact and implicationsThe lack of effective antifibrotic drugs is partly attributed to the insufficient knowledge about the mechanisms involved in the development of liver fibrosis. We demonstrate that the G-protein coupled receptor GPR176 contributes to fibrosis development. Since GPR176 is specifically expressed on the membrane of activated hepatic stellate cells and is linked with fibrosis progression in humans, it opens new avenues for the development of targeted interventions.

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Aucubin inhibits hepatic stellate cell activation through stimulating Nrf2/Smad7 axis

AimLiver fibrosis may develop into end-stage liver disease if left unprevented. The study is attempting to identify a compound to ameliorate liver fibrosis progression with high efficiency and low toxicity, as well as to analyze its potential molecular mechanism. MethodsThe drug screening was performed using human hepatic stellate cell line LX-2 for identifying the compound as collagen I inhibitor. Primary Human hepatic stellate cells and LX-2 cell line were used to detect the antifibrotic function activity and molecular mechanism analysis in vitro. The CCl4-induced mouse experimental model was used to measure the amelioration in liver fibrosis. ResultsThis study identified Aucubin, a natural compound, as a candidate for anti-liver fibrosis. Besides, Aucubin could inhibit the collagen I and α-SMA expressions in LX-2 cells and primary human hepatic stellate cells, as well as the cell proliferation. In terms of mechanism, Aucubin could upregulate Smad7 in hepatic stellate cells in a dose-dependent manner and block TGF-β signaling. We also found that Nrf2 might be a direct target for the action of Aucubin, whose activation was necessary for Smad7 upregulation. In an in-vivo mouse model, Aucubin efficiency ameliorated the progression of CCl4-induced liver fibrosis, and reduced the hepatic levels of collagen deposition, transaminase and inflammatory cytokines. ConclusionCapable of inhibiting the activation of hepatic stellate cells in vitro and in vivo, Aucubin may be a potential therapeutic candidate for liver fibrosis, which is dependent on the suppression of TGF-β signaling through stimulating Nrf2/Smad7 axis.

Optimization of physical microenvironment to maintain the quiescence of human induced pluripotent stem cell-derived hepatic stellate cells.

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis by producing excessive extracellular matrix (ECM) following chronic inflammation. However, studying HSC function has been challenging due to the limited availability of primary human quiescent HSCs (qHSCs) in vitro, and the fact that primary qHSCs quickly activate when cultured on plastic plates. Advances in stem cell technology have allowed for the generation of qHSCs from human induced pluripotent stem cells (hiPSCs) with the potential to provide an unlimited source of cells. However, differentiated quiescent-like HSCs (iqHSCs) also activate spontaneously on conventional plastic plates. In this study, we generated iqHSCs from hiPSCs and developed a culture method to maintain such iqHSCs in a lowly activated state for up to 5 days by optimizing their physical culture microenvironment. We observed that three-dimensional (3D) culture of iqHSCs in soft type 1 collagen hydrogels significantly inhibited their spontaneous activation in vitro while maintaining their ability to convert to activated state. Activation of iqHSC was successfully modeled by stimulating them with the fibrotic cytokine TGFβ1. Hence, our culture method can be used to generate HSCs with functions comparable to those in a healthy liver, facilitating the development of accurate in vitro liver models for identifying novel therapeutic agents.

CCL24 regulates biliary inflammation and fibrosis in primary sclerosing cholangitis.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

GARP on hepatic stellate cells is essential for the development of liver fibrosis

Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis. The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-β. Moreover, cell contraction of HSCs in the context of TGF-β activation was tested in a GARP-dependent fashion. Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-β and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage. GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-β. Considering that systemic blockade of TGF-β has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-β activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis. Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease.

Effect of distinct ECM microenvironments on the genome-wide chromatin accessibility and gene expression responses of hepatic stellate cells

Hepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Cell microarrays demonstrated the cooperative effects of stiffness and ECM composition on H3K4 and H3K9 methylation/acetylation. ATAC sequencing revealed higher chromatin accessibility in HSCs on 1kPa compared to 25kPa substrates for all ECM conditions. Gene set enrichment analysis using RNA sequencing data of HSCs in defined ECM microenvironments demonstrated higher enrichment of NAFLD and fibrosis-related genes in pre-activated HSCs on 1kPa relative to 25kPa. Overall, these findings are indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs. Statement of SignificanceHepatic stellate cells (HSCs) are one of the primary drivers of liver fibrosis in non-alcoholic fatty liver disease. Although HSC activation in liver disease is associated with changes in extracellular matrix (ECM) deposition and remodeling, it remains unclear how ECM regulates the phenotypic state transitions of HSCs. Using high-throughput cellular microarrays, coupled with genome-wide ATAC and RNA sequencing within engineered ECM microenvironments, we investigated the effect of ECM and substrate stiffness on chromatin accessibility and resulting gene expression in activated primary human HSCs. Overall, these findings were indicative of a microenvironmental adaptation response in HSCs, and the acquisition of a persistent activation state. Combined ATAC/RNA sequencing analyses enabled identification of candidate regulatory factors, including HSD11B1 and CEBPb. siRNA-mediated knockdown of HSD11b1 and CEBPb demonstrated microenvironmental controlled reduction in fibrogenic markers in HSCs.

Expression of Bone Morphogenetic Protein 14 in Liver Disease and Cancer

The activation of hepatic stellate cells (HSCs) is the key event of hepatic fibrosis. Furthermore, activated HSCs also play an important role in the progression of hepatocellular cancer (HCC). Bone morphogenetic protein 14 (BMP14) is a member of the TGF-β/BMP superfamily. So far, most studies have analyzed BMP14 in the context of bone and cartilage formation and homeostasis. The aim of this study was to assess the expression and function of BMP14 in liver fibrosis and HCC. The BMP14 expression increased during the in vitro activation of primary human HSCs and also in mouse models of liver fibrosis. In human HCC, as well as non-tumorous liver tissues, there was a significant correlation between the expression of BMP14 and alpha-smooth-muscle actin (α-SMA), an established marker for HSC activation. RNAi-mediated BMP14 suppression in activated HSCs resulted in the reduced expression of the transcription factors inhibitor of differentiation 1 (ID1) and ID2, known targets of BMP signaling. Interestingly, α-SMA and collagen expression was also reduced in BMP14-depleted cells, while treatment with recombinant BMP14 induced ID1, ID2, α-SMA and collagen expression. In human HCC cell lines, treatment with recombinant BMP14 induced proliferation, migratory activity and colony formation. In summary, our data indicate activated HSCs as a major cellular source of enhanced BMP14 expression in fibrotic liver disease and HCC, and show that BMP14 exhibits pro-fibrogenic as well as pro-tumorigenic effects. Future analyses will reveal the potential of this soluble growth factor as a therapeutic target or prognostic marker for the progression of fibrosis and HCC in patients with chronic liver disease.

Multi-omics profiling of primary hepatic stellate cells from advanced liver fibrosis patients reveals distinctive molecular signatures.

Hepatic fibrosis is a common pathogenic outcome of almost all chronic liver diseases and a growing public health problem globally. However, the key genes or proteins driving liver fibrosis and cirrhosis are not well understood. We aimed to identify novel hepatic fibrosis genes of human primary hepatic stellate cells (HSCs). Human primary HSCs were isolated from surgically resected advanced fibrosis liver tissues (n=6) and surgical resection of normal liver tissue around hemangioma (n=5). Differences in the expression levels of mRNA and proteins from HSCs in advanced fibrosis group and the control group were analyzed using RNA sequencing and mass spectrometry as transcriptomic and proteomic approaches. The obtained biomarkers were further validated through real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and Western blot. A total of 2156 transcripts and 711 proteins were found to be differently expressed between the advanced fibrosis group and the control group patients. The Venn diagram shows that a total of 96 upregulated molecules are overlapped in both the transcriptomic and proteomic datasets. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes analysis indicated that those overlapped genes were mainly involved in wound healing, cell adhesion regulation, and actin binding, which reflects the major biological conversions in liver cirrhosis process. Pyruvate kinase M2 and EH domain-containing 2 were identified as potential new markers for advanced liver cirrhosis, which have been validated in primary human HSCs and in vitro cellular hepatic fibrosis model Lieming Xu-2 (LX-2) cells. Our results revealed the major transcriptomic and proteomic changes during liver cirrhosis process and identified new biomarkers and potential therapeutic targets for advanced liver fibrosis.

The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells.

The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.

Identification of Liver Fibrosis-Related MicroRNAs in Human Primary Hepatic Stellate Cells Using High-Throughput Sequencing

MicroRNAs (miRNAs) participate in hepatic stellate cell (HSC) activation, which drives liver fibrosis initiation and progression. We aimed to identify novel hepatic fibrosis targets using miRNA sequencing (miRNA-seq) of human primary HSCs. Surgically resected liver tissues were used to extract HSCs. Based on next-generation sequencing, miRNA-seq was performed on four pairs of HSCs before and after in vitro culture. Additionally, we compared our data with open access miRNA-seq data derived from fourteen cirrhotic and nine healthy liver tissues. Selected miRNAs associated with fibrosis were verified by quantitative real-time PCR. Target mRNAs of differentially expressed (DE) miRNAs were predicted to construct co-expression networks. We identified 230 DEmiRNAs (118 upregulated and 112 downregulated) upon HSC activation. Of the 17 miRNAs with the most significant differences in expression, liver disease-related miRNAs included miR-758-3p, miR-493-5p, miR-409-3p, miR-31-5p, miR-1268a, and miR-381-3p, which might play roles in hepatic fibrosis. Moreover, let-7g-5p, miR-107, miR-122-5p, miR-127-3p, miR-139-5p, miR-148a-3p, miR-194-5p, miR-215-5p, miR-26a-5p, miR-340-5p, miR-451a, and miR-99a-5p were common between our data and the publicly available sequencing data. A co-expression network comprising 1891 matched miRNA-mRNA pairs representing 138 DEmiRNAs and 1414 DEmRNAs was constructed. MiR-1268a and miR-665, possessing the richest target DEmRNAs, may be vital in HSC activation. The targeted genes were involved in collagen metabolism, extracellular matrix structural constituent, cytoskeletal protein binding, and cell adhesion. The miRNAs we identified may provide a basis and reference for the selection of diagnostic and therapeutic targets for hepatic fibrosis.

Pro-fibrogenic role of alarmin high mobility group box 1 in HIV-hepatitis B virus coinfection.

Liver disease is accelerated in people with HIV (PWH) with hepatitis B virus (HBV) coinfection. We hypothesized that liver fibrosis in HIV-HBV is triggered by increased hepatocyte apoptosis, microbial translocation and/or HIV/HBV viral products. Sera from PWH with HBV coinfection versus from those with HBV only or putative mediators were used to examine the pathogenesis of liver disease in HIV-HBV. We applied sera from PWH and HBV coinfection versus HBV alone, or putative mediators (including HMGB1), to primary human hepatic stellate cells (hHSC) and examined pro-fibrogenic changes at the single cell level using flow cytometry. High mobility group box 1 (HMGB1) levels in the applied sera were assessed according to donor fibrosis stage. Quantitative flow cytometric assessment of pro-fibrogenic and inflammatory changes at the single cell level revealed an enhanced capacity for sera from PWH with HBV coinfection to activate hHSC. This effect was recapitulated by lipopolysaccharide, HIV-gp120, hepatocyte conditioned-media and the alarmin HMGB1. Induction of hepatocyte cell death increased their pro-fibrogenic potential, an effect blocked by HMGB1 antagonist glycyrrhizic acid. Consistent with a role for this alarmin, HMGB1 levels were elevated in sera from PWH and hepatitis B coinfection compared to HBV alone and higher in those with HIV-HBV with liver fibrosis compared to those without. Sera from PWH and HBV coinfection have an enhanced capacity to activate primary hHSC. We identified an increase in circulating HMGB1 which, in addition to HIV-gp120 and translocated microbial products, drove pro-fibrogenic changes in hHSC, as mechanisms contributing to accelerated liver disease in HIV-HBV.

MicroRNA-223 Suppresses Human Hepatic Stellate Cell Activation Partly via Regulating the Actin Cytoskeleton and Alleviates Fibrosis in Organoid Models of Liver Injury.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

Role of Hepatocyte Senescence in the Activation of Hepatic Stellate Cells and Liver Fibrosis Progression

Hepatocyte senescence is associated with liver fibrosis. However, the possibility of a direct, causal relation between hepatocyte senescence and hepatic stellate cell (HSC) activation was the subject of this study. Liver biopsy specimens obtained from 50 patients with non-alcoholic fatty liver disease and a spectrum of liver fibrosis stages were stained for p16, αSMA, and picrosirius red (PSR). Primary human HSCs were cultured in conditioned media derived from senescent or control HepG2 cells. Expression of inflammatory and fibrogenic genes in HSCs cultured in conditioned media were studied using RT-PCR. ELISAs were undertaken to measure factors known to activate HSCs in the conditioned media from senescent and control HepG2 cells and serum samples from healthy volunteers or patients with biopsy-proven cirrhosis. There was a strong association between proportion of senescent hepatocytes and hepatic stellate cell activation. Both proportion of hepatocyte senescence and hepatic stellate cell activation were closely associated with fibrosis stage. Inflammatory and fibrogenic genes were up-regulated significantly in HSCs cultured in conditioned media from senescent HepG2 cells compared with control HepG2 cells. PDGF levels were significantly higher in the conditioned media from senescent hepatocytes than control HepG2-conditioned media, and in serum samples from patients with cirrhosis than healthy volunteers. In conclusion, this ‘proof of concept’ study revealed activation of human HSCs by media from senescent HepG2 cells, indicating direct involvement of factors secreted by senescent hepatocytes in liver fibrosis.

Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more β-actin was localized in the cortical layer under SMG.

Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.