Differentiation Of Hepatic Stellate Cells Research Articles

Spatial transcriptomics reveals tumor-derived SPP1 induces fibroblast chemotaxis and activation in the hepatocellular carcinoma microenvironment

BackgroundThe tumor microenvironment (TME) exerts profound effects on tumor progression and therapeutic efficacy. In hepatocellular carcinoma (HCC), the TME is enriched with cancer-associated fibroblasts (CAFs), which secrete a plethora of cytokines, chemokines, and growth factors that facilitate tumor cell proliferation and invasion. However, the intricate architecture of the TME in HCC, as well as the mechanisms driving interactions between tumor cells and CAFs, remains largely enigmatic.MethodsWe analyzed 10 spatial transcriptomics and 12 single-cell transcriptomics samples sourced from public databases, complemented by 20 tumor tissue samples from liver cancer patients obtained in a clinical setting.ResultsOur findings reveal that tumor cells exhibiting high levels of SPP1 are preferentially localized adjacent to hepatic stellate cells (HSCs). The SPP1 secreted by these tumor cells interacts with the CD44 receptor on HSCs, thereby activating the PI3K/AKT signaling pathway, which promotes the differentiation of HSCs into CAFs. Notably, blockade of the CD44 receptor effectively abrogates this interaction. Furthermore, in vivo studies demonstrate that silencing SPP1 expression in tumor cells significantly impairs HSC differentiation into CAFs, leading to a reduction in tumor volume and collagen deposition within the tumor stroma.ConclusionsThis study delineates the SPP1-CD44 signaling axis as a pivotal mechanism underpinning the interaction between tumor cells and CAFs. Targeting this pathway holds potential to mitigate liver fibrosis and offers novel therapeutic perspectives for liver cancer management.

Therapeutic efficacy of mitochondria-targeted esculetin in the improvement of NAFLD-NASH via modulating AMPK-SIRT1 axis

Mitochondrial dysfunction due to deregulated production of mitochondria-derived ROS is implicated in the development and progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH). Recently, we synthesized a novel mitochondria-targeted esculetin (Mito-Esc) and investigated its dose–response therapeutic efficacy in mitigating high-fat diet (HFD)-induced NAFLD and NASH in Apoe-/- mice. Mito-Esc administration, compared to simvastatin and pioglitazone, dose-dependently caused a significant reduction in body weight, improved lipid profile, glucose homeostasis, and pro-inflammatory cytokines level. Mito-Esc administration reduced adipose tissue hypertrophy and lipid accumulation presumably by regulating the levels of CD36, PPAR-γ, EBP-α, and their target genes. Mechanistically, Mito-Esc-induced activation of the AMPK1α-SIRT1 axis inhibited pre-adipocyte differentiation. Conversely, Mito-Esc failed to regulate pre-adipocyte differentiation under AMPK/SIRT1 depleted conditions. In parallel, Mito-Esc administration ameliorated HFD-induced steatosis, fibrosis of the liver, and NAFLD-associated atheromatous plaque formation in the aorta. Importantly, Mito-Esc administration inhibited HFD-induced infiltration of macrophages, a marker of steatohepatitis, in the adipose and liver tissues. The results of the in vitro studies showed that Mito-Esc treatment significantly inhibits TGF-β-induced hepatic stellate cell differentiation as well as the fibrotic markers. Consistent with the above observations, Mito-Esc treatment by activating the AMPK-SIRT1 pathway markedly reversed palmitate-induced mitochondrial superoxide production, depolarization of mitochondrial membrane potential, and lipid accumulation in HepG2 cells. Together, the therapeutic efficacy of Mito-Esc in the mitigation of HFD-induced lipotoxicity, and the associated NASH is in part, mediated by potentiating the AMPK-SIRT1 axis.

Unraveling the activation process and core driver genes of HSCs during cirrhosis by single-cell transcriptome.

Worldwide, cirrhosis is a common cause of death, manifesting itself as fibrosis of the liver tissue. When the liver is damaged, the liver produces fibrotic, proliferative myofibroblasts, which are formed by the differentiation of activated hepatic stellate cells. There are no effective antifibrotic treatment options. To deeply explore the activation process of hepatic stellate cells (HSCs) and to discover better therapeutic target genes, single-cell RNA sequencing data on 13 non-cirrhotic liver tissue samples and 10 cirrhotic liver tissue samples were analyzed. We identified activated HSCs from the mesenchymal cell population with high expression of ACTA2. By pseudo-time analysis, we found that the key genes for the differentiation of HSCs into myofibroblasts were C3, CCDC80, COL1A1, COL3A1, DCN, FBLN1, IGFBP3, MXRA5, SERPINE1, and MYH11. Then, we found that the main regulators of HSCs from inactive to activated state were NTF3, NTRK3, NTRK2, JAG1, NOTCH3, ESAM, and CD46 by cell-cell communication analysis. In addition, we found that the top2 hub genes of activated HSCs were CRIP1 and ACTA2. The experimental results show that the top2 hub genes were significantly overexpressed in cirrhotic samples. Our work dissected key intercellular regulators and core driver genes during hepatic stellate cell activation during cirrhosis through single-cell transcriptome data analysis, providing a research strategy to discover rational therapeutic targets for cirrhosis and some important information for gene targeting therapy.

A pH‑responsive hyaluronic acid nano‑vehicle co‑encapsulating doxorubicin and all‑trans retinoic acid for the inhibition of hepatic stellate cell‑induced tumor growth and metastasis.

All‑trans retinoic acid (ATRA) has been implicated in the differentiation of hepatic stellate cells (HSCs). In the present study, the liver‑targeting hyaluronic acid micelles (ADHG) were prepared for co‑delivery of ATRA and doxorubicin (DOX) to block the HSC‑hepatoma interrelation. To simulate the tumor microenvironment, an invitro dual‑cell model and an invivo co‑implantation mouse model were established for anticancer studies. The experimental methods involved the MTT assay, wound‑healing assay, cellular uptake, flow cytometry and and invivo antitumor study. The results revealed that the HSCs in the research models notably promoted tumor proliferation and migration. Furthermore, ADHG were readily internalized by cancer cells and HSCs simultaneously, and widely distributed in cancer regions. The invivo antitumor studies demonstrated that ADHG could notably decrease HSC activation and extracellular matrix deposition, as well as constrain tumor growth and metastasis. Therefore, ATRA could facilitate DOX‑induced anti‑proliferation and anti‑metastasis effects, and ADHG are a promising nano‑sized formulation for the combination therapy of hepatocellular carcinoma.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways.

Hepatic fibrosis is a serious condition, and the development of hepatic fibrosis can lead to a series of complications. However, the pathogenesis of hepatic fibrosis remains unclear, and effective therapy options are still lacking. Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1 (NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis, but its role in diseases including hepatic fibrosis remains undefined. Therefore, additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment. To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1. Twenty-four male C57BL/6 mice were randomized and separated into three groups, comprising the normal, fibrosis, and calcitriol treatment groups, and liver fibrosis was modeled by carbon tetrachloride (CCl4). To evaluate the level of hepatic fibrosis in every group, serological and pathological examinations of the liver were conducted. TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells. NS3TP1, α-smooth muscle actin (α-SMA), collagen I, and collagen III in every group were examined using a Western blot and real-time quantitative polymerase chain reaction. The activity of the transforming growth factor beta 1 (TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected. The statistical analysis of the data was performed using the Student's t test. NS3TP1 promoted the activation, proliferation, and differentiation of hepatic stellate cells (HSCs) and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways, as evidenced by the presence of α-SMA, collagen I, collagen III, p-smad3, and p-p65 in LX-2 cells, which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference. The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression, as shown by the luciferase assay. NS3TP1 inhibited the apoptosis of HSCs. Moreover, both Smad3 and p65 could bind to NS3TP1, and p65 increased the promoter activity of NS3TP1, while NS3TP1 increased the promoter activity of TGFβ1 receptor I, as indicated by coimmunoprecipitation and luciferase assay results. Both in vivo and in vitro, treatment with calcitriol dramatically reduced the expression of NS3TP1. Calcitriol therapy-controlled HSCs activation, proliferation, and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice. Furthermore, calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1. Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique, prospective therapeutic target in hepatic fibrosis.

Differentiation of Hepatic Stellate Cells from Pluripotent Stem Cells.

Hepatic stellate cells (HSCs) are non-parenchymal cells with a mesenchymal origin involved in vitamin A storage and extracellular matrix (ECM) homeostasis. In response to injury, HSCs activate and acquire myofibroblastic features, participating in the wound healing response. Upon chronic liver injury, HSCs become the main contributors to ECM deposition and to the progression of fibrosis. Due to their relevant roles in liver function and pathophysiology, it is of utmost importance to develop means to obtain HSCs for liver disease modeling and drug development. Here, we describe a directed differentiation protocol from human pluripotent stem cells (hPSCs) to obtain functional HSCs (PSC-HSCs). The procedure is based on the subsequent addition of growth factors during 12days of differentiation. PSC-HSCs can be used for liver modeling and drug screening assays, hence emerging as a promising and reliable source of HSCs.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

OS039 - The proteomic analysis of hepatic stellate cell differentiation from iPSCs identifies RORalpha as an antifibrogenic target

OS039 - The proteomic analysis of hepatic stellate cell differentiation from iPSCs identifies RORalpha as an antifibrogenic target

Extracellular Vesicular Transmission of miR-423-5p from HepG2 Cells Inhibits the Differentiation of Hepatic Stellate Cells.

Liver fibrosis (LF) is a major cause of morbidity and mortality worldwide. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix in the liver and their activation is a central event in LF development. Extracellular vesicles (EVs) are intercellular communication agents, which play important roles in physiological processes in chronic liver diseases. The aim of this study was to examine the crosstalk between hepatocytes and HSCs mediated by hepatocyte-secreted EVs. EVs were purified from primary mouse hepatocytes, HepG2 cell lines, under normal or stressed conditions. The effect of EVs on primary HSCs (pHSCs) differentiation was evaluated by measuring of differentiation markers. In addition, their impact on the carbon tetrachloride (CCl4)-induced fibrosis mouse model was evaluated. The results demonstrated that HepG2-EVs regulate HSC differentiation and that under stress conditions, promoted pHSCs differentiation into the myofibroblast phenotype. The evaluation of miRNA sequences in the HepG2 secreted EVs demonstrated high levels of miR-423-5p. The examination of EV cargo following stress conditions identified a significant reduction of miR-423-5p in HepG2-EVs relative to HepG2-EVs under normal conditions. In addition, pHSCs transfected with miR-423-5p mimic and exhibit lower mRNA levels of alpha smooth muscle actin and Collagen type 1 alpha, and the mRNA expression level of genes targeted the family with sequence-similarity-3 (FAM3) and Monoacylglycerol lipase (Mgll). This study strengthened the hypothesis that EVs are involved in LF and that their cargo changes in stress conditions. In addition, miR-423-5p was shown to be involved in HSCs differentiation and hence, fibrosis development.

Control of Tissue Fibrosis by 5-Methoxytryptophan, an Innate Anti-Inflammatory Metabolite.

Tissue fibrosis causes debilitating human diseases such as liver cirrhosis, heart failure, chronic kidney disease and pulmonary insufficiency. It is a dynamic process orchestrated by specific subsets of monocyte-macrophages, fibroblasts, pericytes and hepatic stellate cells. Fibrosis is linked to tissue inflammation. Pro-inflammatory macrophages promote fibrosis by driving myofibroblast differentiation and macrophage myofibroblast transition. Myofibroblasts express α-smooth muscle cell actin (α-SMA) and secrete extracellular matrix (ECM) proteins notably collagen I and III. Deposition of ECM proteins at injury sites and interstitial tissues distorts normal structure and impairs vital functions. Despite advances in the mechanisms of fibrosis at cellular, molecular and genetic levels, prevention and treatment of fibrotic diseases remain poorly developed. Recent reports suggest that 5-methoxytryptophan (5-MTP) is effective in attenuating injury-induced liver, kidney, cardiac and pulmonary fibrosis. It inhibits macrophage activation and blocks fibroblast differentiation to myofibroblasts. Furthermore, it inhibits hepatic stellate cell differentiation into myofibroblasts. As 5-MTP is an endogenous molecule derived from tryptophan catabolism via tryptophan hydroxylase pathway, it is well-suited as a lead compound for developing new anti-fibrotic drugs. This article provides an overview of 5-MTP synthesis, and a critical review of its anti-fibrotic activities. Its mechanisms of actions and potential therapeutic value will be discussed.

Exogenous Plant gma-miR-159a, Identified by miRNA Library Functional Screening, Ameliorated Hepatic Stellate Cell Activation and Inflammation via Inhibiting GSK-3β-Mediated Pathways.

PurposePlant-derived exogenous microRNAs (miRNAs) regulate human physiological functions by blocking the translation of target mRNAs. Although several computational approaches have been developed to elucidate the interactions of cross-species miRNAs and their targets in mammals, the number of verified plant miRNAs is still limited, and the biological roles of most exogenous plant miRNAs remain unknown.MethodsA miRNA mimic library-based phenotypic screening, which contained 8394 plant mature miRNAs published in the official database miRbase, was performed to identify more novel bioactive plant miRNAs for the prevention of hepatic fibrosis. Inhibition of candidates for the activation of hepatic stellate cells (HSCs) and the underlying mechanisms were evaluated in TGF-β1- and PDGF-exposed HSC models. The protective effects of the candidates against CCl4-induced liver fibrosis were evaluated in a mouse model.ResultsAmong the 8394 plant mature miRNAs reported in the official database miRBase, five candidates were found to effectively inhibit the differentiation of HSCs. gma-miR-159a (miR159a) exerted the strongest inhibitory activities on both TGF-β1- and PDGF-induced HSC activation and proliferation by inhibiting the GSK-3β-mediated NF-κB and TGF-β1 pathways. Moreover, miR159a was mainly accumulated in the liver after intravenous injection, and it reduced CCl4-induced hepatic fibrosis and inflammation in mice.ConclusionResults indicated that miR159a has the therapeutic potential for preventing hepatic fibrosis. This study provides a novel strategy for achieving natural nucleic acid drugs.

Sulfatase 2-Induced Cancer-Associated Fibroblasts Promote Hepatocellular Carcinoma Progression via Inhibition of Apoptosis and Induction of Epithelial-to-Mesenchymal Transition

BackgroundSulfatase 2 (SULF2) removes the 6-O-sulfate groups from heparan sulfate proteoglycans (HSPG) and consequently alters the binding sites for various signaling molecules. Here, we elucidated the role of SULF2 in the differentiation of hepatic stellate cells (HSCs) into carcinoma-associated fibroblasts (CAFs) in the hepatocellular carcinoma (HCC) microenvironment and the mechanism underlying CAF-mediated HCC growth.MethodsThe clinical relevance of SULF2 and CAFs was examined using in silico and immunohistochemical (IHC) analyses. Functional studies were performed to evaluate the role of SULF2 in the differentiation of HSCs into CAFs and elucidate the mechanism underlying CAF-mediated HCC growth. Mechanistic studies were performed using the chromatin immunoprecipitation, luciferase reporter, and RNA immunoprecipitation assays. The in vitro findings were verified using the nude HCC xenograft mouse model.ResultsThe Cancer Genome Atlas (TCGA) database and IHC analyses revealed that the expression of CAF markers, which was positively correlated with that of SULF2 in the HCC tissues, predicted unfavorable postsurgical outcomes. Co-culturing HSCs with HCC cells expressing SULF2 promoted CAF differentiation. Additionally, CAFs repressed HCC cell apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway. Meanwhile, SULF2-induced CAFs promoted epithelial-to-mesenchymal transition (EMT) of HCC cells by modulating the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. Studies using HCC xenograft mouse models demonstrated that OIP5-AS1 induced EMT by upregulating SNAI1 and promoted HCC growth in vivo.ConclusionThese data indicated that SULF2 secreted by the HCC cells induced the differentiation of HSCs into CAFs through the TGFβ1/SMAD3 signaling pathway. SULF2-induced CAFs attenuated HCC apoptosis by activating the SDF-1/CXCR4/PI3K/AKT signaling pathway and induced EMT through the SDF-1/CXCR4/OIP5-AS1/miR-153-3p/SNAI1 axis. This study revealed a novel mechanism involved in the crosstalk between HCC cells and CAFs in the tumor microenvironment, which can aid in the development of novel and efficient therapeutic strategies for primary liver cancer.

Strategies Targeting the Innate Immune Response for the Treatment of Hepatitis C Virus-Associated Liver Fibrosis

Direct-acting antivirals eliminate hepatitis C virus (HCV) in more than 95% of treated individuals and may abolish liver injury, arrest fibrogenesis, and reverse fibrosis and cirrhosis. However, liver regeneration is usually a slow process that is less effective in the late stages of fibrosis. What is more, fibrogenesis may prevail in patients with advanced cirrhosis, where it can progress to liver failure and hepatocellular carcinoma. Therefore, the development of antifibrotic drugs that halt and reverse fibrosis progression is urgently needed. Fibrosis occurs due to the repair process of damaged hepatic tissue, which eventually leads to scarring. The innate immune response against HCV is essential in the initiation and progression of liver fibrosis. HCV-infected hepatocytes and liver macrophages secrete proinflammatory cytokines and chemokines that promote the activation and differentiation of hepatic stellate cells (HSCs) to myofibroblasts that produce extracellular matrix (ECM) components. Prolonged ECM production by myofibroblasts due to chronic inflammation is essential to the development of fibrosis. While no antifibrotic therapy is approved to date, several drugs are being tested in phase 2 and phase 3 trials with promising results. This review discusses current state-of-the-art knowledge on treatments targeting the innate immune system to revert chronic hepatitis C-associated liver fibrosis. Agents that cause liver damage may vary (alcohol, virus infection, etc.), but fibrosis progression shows common patterns among them, including chronic inflammation and immune dysregulation, hepatocyte injury, HSC activation, and excessive ECM deposition. Therefore, mechanisms underlying these processes are promising targets for general antifibrotic therapies.

Intercellular Communication in the Progression from Fibrosis to HCC by Ectosomal HK1-Induced TGF-β Secretion

Extracellular vesicles (EVs) play a crucial role in intercellular communication in the tumor microenvironment (TME). Although EVs released by hepatocellular carcinoma (HCC) cells promote the differentiation of hepatic stellate cells (HSCs) into cancer-associated fibroblasts to remodel the TME, whether HSC-derived EVs directly affect HCC progression remains elusive. Here, we demonstrated that in hepatic fibrosis, transforming growth factor-β (TGF-β) stimulates the palmitoylation and subsequent plasma membrane localization of hexokinase 1 (HK1) in HSCs, leading to ectosomal HK1 secretion. This ectosomal HK1 is hijacked by HCC cells, which not only promotes HCC proliferation by accelerating glycolysis but also enhances the N-glycosylation of pro-TGF-β to stimulate TGF-β secretion from HCC cells, thereby creating a feedforward loop to promote HCC progression. The nuclear receptor Nur77 transcriptionally activates expression of the depalmitoylase ABHD17B to attenuate ectosomal HK1 release. However, TGF-β-activated Akt represses the function of Nur77 by inducing Nur77 phosphorylation and degradation. We further identified a small-molecule compound, PDNPA, that binds Nur77 to disrupt Akt-mediated Nur77 degradation, thereby inhibiting HCC development. This study demonstrates an overlooked function of HK1 in promoting HCC development upon its release from HSCs and highlights PDNPA as a candidate for repressing HCC through the interception of ectosomal HK1 release.

Tyrosine kinase inhibitor neratinib attenuates liver fibrosis by targeting activated hepatic stellate cells

Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl4-induced liver fibrosis. HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-β)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl4. Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.

Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFβ receptor endocytosis.

TGFβ induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGFβ receptor II (TβRII), in response to TGFβ stimulation, is a prerequisite for TGF signaling, we investigated the role of protein diaphanous homolog 1 (known as Diaph1 or mDia1) for the myofibroblastic activation of HSCs. Using shRNA to knockdown Diaph1 or SMIFH2 to target Diaph1 activity of HSCs, we found that the inactivation of Diaph1 blocked internalization and intracellular trafficking of TβRII and reduced SMAD3 phosphorylation induced by TGFβ1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both TβRII and Rab5a directly and that Rab5a activity of HSCs was increased by Diaph1 overexpression and decreased by Diaph1 knockdown. Additionally, expression of Rab5aQ79L (active Rab5a mutant) increased whereas the expression of Rab5aS34N (inactive mutant) reduced the endosomal localization of TβRII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGFβ stimulation promoted HSCs to express tumor-promoting factors, and α-smooth muscle actin, fibronection, and CTGF, markers of myofibroblastic activation of HSCs. Targeting Diaph1 or Rab5a suppressed HSC activation and limited tumor growth in a tumor implantation mouse model. Thus, Dipah1 and Rab5a represent targets for inhibiting HSC activation and the hepatic tumor microenvironment.

A Deactivation Factor of Fibrogenic Hepatic Stellate Cells Induces Regression of Liver Fibrosis in Mice

Hepatic stellate cells (HSCs), a key player in the progression of liver fibrosis, are activated by various inflammatory stimuli and converted to myofibroblast-like cells with excessive collagen production. Despite many attempts to suppress activation of HSCs or inhibit collagen production in activated HSCs, their clinical applications have not been established yet. Recently, the deactivation of HSCs has been reported as a mechanism underlying the reversibility of experimental liver fibrosis. In the present study, we sought for deactivation factors of HSCs that induce regression of established liver fibrosis. We identified transcription factor 21 (Tcf21) as one of the transcription factors whose expression was up-regulated in parallel to the differentiation of fetal HSCs. Expression of Tcf21 in HSCs remarkably decreased during culture-induced activation in vitro and in murine and human fibrotic liver tissue in vivo. This reduced Tcf21 expression was recovered during the spontaneous regression of murine liver fibrosis. Tcf21 was also examined for its effects by adeno-associated virus serotype 6-mediated Tcf21 gene transfer into cultured activated HSCs and mice with carbon tetrachloride- or methionine-choline deficient diet-induced liver fibrosis. Overexpression of Tcf21 in activated HSCs not only suppressed fibrogenic gene expression but also restored cells, at least in part, to a quiescent phenotype both in vitro and in vivo. These phenotypic changes of HSCs were accompanied by the regression of steatohepatitis and fibrosis and improved hepatic architecture and function. Tcf21 has been identified as a deactivation factor of fibrogenic HSCs, providing insight into a treatment strategy for the otherwise intractable liver fibrosis.

Focal Adhesion Kinase Promotes Hepatic Stellate Cell Activation by Regulating Plasma Membrane Localization of TGFβ Receptor 2.

Transforming growth factor β (TGFβ) induces hepatic stellate cell (HSC) differentiation into tumor‐promoting myofibroblast, although underlying mechanism remains incompletely understood. Focal adhesion kinase (FAK) is activated in response to TGFβ stimulation, so it transmits TGFβ stimulus to extracellular signal‐regulated kinase and P38 mitogen‐activated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGFβ receptors. In this study, we tested whether FAK phosphorylated TGFβ receptor 2 (TGFβR2) and regulated TGFβR2 intracellular trafficking in HSCs. The FAKY397F mutant and PF‐573,228 were used to inhibit the kinase activity of FAK, the TGFβR2 protein level was quantitated by immunoblotting, and HSC differentiation into myofibroblast was assessed by expression of HSC activation markers, alpha‐smooth muscle actin, fibronectin, or connective tissue growth factor. We found that targeting FAK kinase activity suppressed the TGFβR2 protein level, TGFβ1‐induced mothers against decapentaplegic homolog phosphorylation, and myofibroblastic activation of HSCs. At the molecular and cellular level, active FAK (phosphorylated FAK at tyrosine 397) bound to TGFβR2 and kept TGFβR2 at the peripheral plasma membrane of HSCs, and it induced TGFβR2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGFβR2 led to lysosomal sorting and degradation of TGFβR2. Using RNA sequencing, we identified that the transcripts of 764 TGFβ target genes were influenced by FAK inhibition, and that through FAK, TGFβ1 stimulated HSCs to produce a panel of tumor‐promoting factors, including extracellular matrix remodeling proteins, growth factors and cytokines, and immune checkpoint molecule PD‐L1. Functionally, targeting FAK inhibited tumor‐promoting effects of HSCs in vitro and in a tumor implantation mouse model. Conclusion: FAK targets TGFβR2 to the plasma membrane and protects TGFβR2 from lysosome‐mediated degradation, thereby promoting TGFβ‐mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment.

CXCR4/TGF-β1 mediated hepatic stellate cells differentiation into carcinoma-associated fibroblasts and promoted liver metastasis of colon cancer

ABSTRACTBackground: Liver metastasis of colon cancer is strongly affected by the tumor microenvironment (TME), with interactions between tumor cells and cancer-associated fibroblasts (CAFs) in particular. TGF-β is well known for its ability to mediate the CAF phenotype, and CXCR4 expression is closely correlated to poor prognosis in CRC. The relationship between these two signaling pathways remains to be delineated in liver metastasis of colon cancer.Methods: Immunohistochemistry was employed to investigate CXCR4 expression in 45 human specimens of primary colorectal cancer (CRC) and liver metastasis. The functions of SDF-1 released by hepatic stellate cells (HSCs) on CXCR4 and TGF-β1 in CRC cells were investigated in vitro. The effects of CRC on HSCs differentiation into CAFs were confirmed using co-culture technology and expression analysis of CAFs markers by qPCR, western blot and immunofluorescence. The involvement of CXCR4 and TGF-β1 was verified with addition of CXCR4 inhibitor AMD3100 and TGF-β1 inhibitor cyclophosphamide (Cy) both in vitro and in vivo.Results: There were more CXCR4-positive cells at the liver metastatic tissues compared to the primary sites. CRC cells activated and transformed HSCs to CAFs after co-cultivating with HSCs. Activated HSCs stimulated TGF-β1 secretion from CRC cells after co-culture with CRC cells in vitro. Moreover, the expression of CAFs markers was increasing in the activated HSCs. In a mouse hepatic metastasis model, treated with AMD3100 or Cy blocked the metastatic potential of HCT116 cells and the hepatic CAFs differentiation.Conclusions: These results indicated that CXCR4/TGF-β1 axis plays an important role in CRC liver metastasis through mediating HSCs differentiation into CAFs, providing preclinical evidences that blockade of the axis might be beneficial for anti-metastasis therapy in CRC.

TAF and TDF attenuate liver fibrosis through NS5ATP9, TGFβ1/Smad3, and NF-κB/NLRP3 inflammasome signaling pathways.

This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis. The effects of TAF/TDF on carbon tetrachloride (CCl4)-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-κB/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout. TAF/TDF significantly inhibited CCl4-induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGFβ1/Smad3 and NF-κB signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro. TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.