Fibrotic Mouse Livers Research Articles

Infusion of Human Mesenchymal Stem Cells Improves Regenerative Niche in Thioacetamide-Injured Mouse Liver.

This study investigated whether xenotransplantation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-β1-stimulation in Smad2 phosphorylation and RhoA upregulation. These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

Hydrogen selenide, a vital metabolite of sodium selenite, uncouples the sulfilimine bond and promotes the reversal of liver fibrosis.

Sodium selenite has alleviating effects on liver fibrosis; however, its therapeutic molecular mechanism remains unclear. Herein, hydrogen selenide, a major metabolite of Na2SeO3, was tested to uncouple the sulfilimine bond in collagen IV, the biomarker of liver fibrosis. A mouse model of liver fibrosis was constructed via a CCl4-induced method, followed by the administration of 0.2 mg kg-1 Na2SeO3 via gavage three times per week for 4 weeks. Changes in H2Se, NADPH, and H2O2 levels were monitored in real time by using NIR-H2Se, DCI-MQ-NADPH, and H2O2 probes in vivo, respectively. H2Se continuously accumulated in the liver throughout the Na2SeO3 treatment period, but the levels of NADPH and H2O2 decreased. The expression of collagen IV was analyzed through Western blot and liquid chromatography-mass spectrometry. Results confirmed that the sulfilimine bond of collagen IV in the fibrotic mouse livers could be broken by H2Se with the Na2SeO3 treatment. Therefore, the therapeutic effect of Na2SeO3 on liver fibrosis could be mainly attributed to H2Se that uncoupled the sulfilimine bond to induce collagen IV degradation. This study provided a reasonable explanation for the molecular mechanism of the in vivo function of Na2SeO3 and the prevention of liver fibrosis by administering inorganic selenium.

Pyruvate Kinase M2 Tetramerization Protects against Hepatic Stellate Cell Activation and Liver Fibrosis

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The invitro and invivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs invitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis invivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.

Ph-responsive polymersomes for matrix metalloproteinase-1 delivery as a promising therapeutic strategy for the treatment of liver fibrosis

Background & Aims: Liver fibrosis is a growing health problem affecting millions of people worldwide. Chronic liver injury leads to the formation of scar tissue due to the excessive accumulation of extracellular matrix, mainly collagen-I and -III produced by activated hepatic stellate cells (HSCs). Currently, there are no therapies available for liver fibrosis. Matrix metalloproteinase-1 (MMP-1) is an enzyme that degrades the scar tissue by degrading collagen-I and -III favoring fibrolysis. We hypothesized liver-specific delivery of MMP-1 to degrade collagen as a promising approach for the treatment of liver fibrosis. Using state-of-the-art technologies, we synthesized innovative pH-responsive smart MMPsomes with the MMP1 decorated on the polymersome surface. Finally, we investigated the therapeutic efficacy of MMP1 and MMPsomes on human HSCs in vitro and on liver fibrosis mouse models in vivo, and ex vivo on fibrotic mouse livers. Methods: Polymersomes (Psomes) were fabricated using the pH switch method and MMP-1 post-loading at pH 5-6 was performed to increase the interaction between MMP-1 and the Psome membrane. Physiochemical analysis and enzymatic assays were performed to characterize the attachment and functionalization of enzyme on the Psomes. In vitro studies were performed on TGF-β activated human HSCs to evaluate the effects of MMP1 and MMPsomes on the cell viability, functionality and the gene and protein expression of collagen and HSCs activation marker α-SMA. Finally, the efficacy of MMP-1 and MMPsomes was tested in vivo and ex vivo on CCl4-induced liver fibrosis mouse models Results: Decoration of MMP-1 on the surface of the polymersome was successfully established with an affinity of 30%, without inhibition of function. Synthesized MMPsomes showed favorable size (~180nm at pH 6 and ~145 nm at pH 8) and charge (positive at pH 6 and negative at pH 8). MMP-1 and more significantly MMPsomes showed dose-dependent inhibition of collagen-I, -III and α-SMA gene expression, and collagen-I protein expression in TGF-β activated human HSCs with no significant effects on cell viability. The studies on CCl4-induced liver fibrosis mouse models have been performed, showed promising preliminary results with no in vivo toxicity however still under investigation. Conclusions: In conclusion, we present an innovative approach of MMP-1 delivery for the treatment of liver fibrosis.

Circular RNA cMTO1 Promotes PTEN Expression Through Sponging miR-181b-5p in Liver Fibrosis.

BackgroundCircular RNAs (circRNAs) are considered as key regulators of cancer biology. Recently, cMTO1 (a circRNA derived from MTO1 gene, hsa_circ_0007874) has been demonstrated to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the roles of cMTO1 in liver fibrosis are largely unknown.MethodsExpressions and roles of cMTO1 were examined in vivo and in vitro during liver fibrosis. The interaction between microRNA-181b-5p (miR-181b-5p) and cMTO1 was analyzed by luciferase activity assays and pull down assays.ResultscMTO1 was shown to be reduced in the liver from patients with cirrhosis. In addition, cMTO1 was down-regulated in the mouse fibrotic livers as well as activated hepatic stellate cells (HSCs). Restoring of cMTO1 led to a reduction in HSC proliferation. Results of immunofluorescence analysis showed that cMTO1 suppressed the expressions of α-SMA and type I collagen. cMTO1 was found to be expressed in the cytoplasm of HSCs. Further studies confirmed that cMTO1 and miR-181b-5p were co-located in the cytoplasm. Interestingly, there was an interaction between cMTO1 and miR-181b-5p. Results of luciferase reporter assays and pull down assays confirmed that miR-181b-5p could bind to cMTO1. cMTO1-inhibited HSC activation was blocked down by miR-181b-5p or PTEN. Meanwhile, PTEN was a target of miR-181b-5p.ConclusioncMTO1 inhibits HSC activation, at least in part, through miR-181b-5p-mediated PTEN expression. Our results also suggest that cMTO1 may be a novel therapeutic target in liver fibrosis.

Osteoprotegerin Is more than a Possible Serum Marker in Liver Fibrosis: A Study into Its Function in Human and Murine Liver

Osteoprotegerin (OPG) serum levels are associated with liver fibrogenesis and have been proposed as a biomarker for diagnosis. However, the source and role of OPG in liver fibrosis are unknown, as is the question of whether OPG expression responds to treatment. Therefore, we aimed to elucidate the fibrotic regulation of OPG production and its possible function in human and mouse livers. OPG levels were significantly higher in lysates of human and mouse fibrotic livers compared to healthy livers. Hepatic OPG expression localized in cirrhotic collagenous bands in and around myofibroblasts. Single cell sequencing of murine liver cells showed hepatic stellate cells (HSC) to be the main producers of OPG in healthy livers. Using mouse precision-cut liver slices, we found OPG production induced by transforming growth factor β1 (TGFβ1) stimulation. Moreover, OPG itself stimulated expression of genes associated with fibrogenesis in liver slices through TGFβ1, suggesting profibrotic activity of OPG. Resolution of fibrosis in mice was associated with decreased production of OPG compared to ongoing fibrosis. OPG may stimulate fibrogenesis through TGFβ1 and is associated with the degree of fibrogenesis. It should therefore be investigated further as a possible drug target for liver fibrosis or biomarker for treatment success of novel antifibrotics.

[18F]-Alfatide PET imaging of integrin αvβ3 for the non-invasive quantification of liver fibrosis

The vitronectin receptor integrin αvβ3 drives fibrogenic activation of hepatic stellate cells (HSCs). Molecular imaging targeting the integrin αvβ3 could provide a non-invasive method for evaluating the expression and the function of the integrin αvβ3 on activated HSCs (aHSCs) in the injured liver. In this study, we sought to compare differences in the uptake of [18F]-Alfatide between normal and injured liver to evaluate its utility for assessment of hepatic fibrogenesis. PET with [18F]-Alfatide, non-enhanced CT, histopathology, immunofluorescence staining, immunoblotting and gene analysis were performed to evaluate and quantify hepatic integrin αvβ3 levels and liver fibrosis progression in mouse models of fibrosis (carbon tetrachloride [CCl4] and bile duct ligation [BDL]). The liver AUC divided by the blood AUC over 30 min was used as an integrin αvβ3-PET index to quantify fibrosis progression. Exvivo analysis of frozen liver tissue from patients with fibrosis and cirrhosis verified the animal findings. Fibrotic mouse livers showed enhanced [18F]-Alfatide uptake and retention compared to control livers. The radiotracer was demonstrated to bind specifically with integrin αvβ3, which is mainly expressed on aHSCs. Autoradiography and histopathology confirmed the PET imaging results. Further, the mRNA and protein level of integrin αvβ3 and its signaling complex were higher in CCl4 and BDL models than controls. The results obtained from analyses on human fibrotic liver sections supported the animal findings. Imaging hepatic integrin αvβ3 with PET and [18F]-Alfatide offers a potential non-invasive method for monitoring the progression of liver fibrosis. Integrin αvβ3 expression on activated hepatic stellate cells (aHSCs) is associated with HSC proliferation during hepatic fibrogenesis. Herein, we show that a radioactive tracer, [18F]-Alfatide, binds to integrin αvβ3 with high affinity and specificity. [18F]-Alfatide could thus be used as a non-invasive imaging biomarker to track hepatic fibrosis progression.

Hepatocarcinoma–intestine–pancreas/pancreatitis-associated protein (HIP/PAP) confers protection against hepatic fibrosis through downregulation of transforming growth factor β receptor II

Hepatocarcinoma–intestine–pancreas/pancreatitis-associated protein (HIP/PAP) has antimicrobial, antioxidant, anti-inflammatory, mitogenic, and antiapoptotic effects and thus exerts important functions in the maintenance of integrity and homeostasis of several organs, such as the gastrointestinal tract, pancreas, and liver. Although the potent hepatoprotective effect of HIP/PAP has been validated, its impact on liver fibrosis has not been reported. In this study, we evaluated the role of HIP/PAP on hepatic fibrosis and explored the possible underlying mechanisms. We found that the expression of HIP/PAP and its mouse counterpart, Reg3B, was markedly upregulated in fibrotic human or mouse livers. Intraperitoneal (i.p.) interleukin (IL)-10, IL-6, and TNF-α but not TGF-β1 significantly induced hepatic overexpression of Reg3B in mice. In both CCl4 and BDL liver fibrosis models, adenovirus-mediated ectopic expression of HIP/PAP markedly alleviated liver injury, inflammation, collagen deposition, hepatic stellate cell activation, and the overexpression of profibrotic cytokines, including transforming growth factor β1 (TGF-β1), platelet-derived growth factor (PDGF)-A, B, connective tissue growth factor (CTGF), and plasminogen activator inhibitor-1 (PAI-1), in mice. In vitro experiments demonstrated that, in addition to suppressing hepatic stellate cell proliferation and accelerating hepatocyte proliferation, HIP/PAP mitigated TGF-β1-induced hepatic stellate cell activation, hepatocyte epithelial-mesenchymal transition (EMT) and upregulated expression of profibrotic cytokines in both hepatic stellate cells and hepatocytes. Moreover, HIP/PAP attenuated the overexpression of TGF-β receptor II (TGF-βRII) in fibrotic mouse livers and decreased the basal expression of TGF-βRII in nonfibrotic mouse livers as well as in cultured hepatocytes and hepatic stellate cells, which is at least partly attributable to the TGF-β1-antagonizing function of HIP/PAP. This study indicates that increased expression of hepatic HIP/PAP serves as a countermeasure against liver injury and fibrosis. Exogenous supplementation of HIP/PAP might be a promising therapeutic agent for hepatic fibrosis as well as liver injury.

Src Inhibition Attenuates Liver Fibrosis by Preventing Hepatic Stellate Cell Activation and Decreasing Connetive Tissue Growth Factor.

The SRC kinase family comprises non-receptor tyrosine kinases that are ubiquitously expressed in all cell types. Although Src is reportedly activated in pulmonary and renal fibrosis, little is known regarding its role in liver fibrosis. This study investigated whether the inhibition of Src protects against liver fibrosis. The expression of Src was upregulated in thioacetamide (TAA)-induced fibrotic mouse liver and cirrhosis of patients, and phospho-Src was upregulated during activation of hepatic stellate cells (HSC). In addition, Src inhibition reduced the expression of α-smooth muscle actin (αSMA) in primary HSCs and suppressed transforming growth factor β (TGF-β)-induced expression of connective tissue growth factor (CTGF) in hepatocytes. Src inhibitor Saracatinib also attenuated TAA-induced expression of type I collagen, αSMA, and CTGF in mouse liver tissues. The antifibrotic effect of Src inhibitors was associated with the downregulation of Smad3, but not of signal transducer and activator of transcription 3 (STAT3). In addition, Src inhibition increased autophagy flux and protected against liver fibrosis. These results suggest that Src plays an important role in liver fibrosis and that Src inhibitors could be treat liver fibrosis.

Toll-Like Receptor 5 Signaling Ameliorates Liver Fibrosis by Inducing Interferon β–Modulated IL-1 Receptor Antagonist in Mice

Bacterial flagellin, recognized by cell surface of Toll-like receptor (TLR) 5, is a potent activator of many types of cells, leading to the activation of innate or adaptive immunity, which are pivotal in regulating fibrotic process. However, the exact role of TLR5 signaling in hepatic fibrogenesis remains unclear, and this study aims to elucidate its underlying mechanisms. Flagellin was injected to hepatotoxin- and cholestasis-induced liver fibrosis murine models. Flagellin-induced TLR5 activation significantly decreased the severity of liver fibrosis. Interestingly, the expression levels of IL-1 receptor antagonist (IL1RN) and interferon (IFN)β markedly increased in fibrotic livers on flagellin treatment. Consistently, invivo activation of TLR5 signaling markedly increased IFNβ and IL1RN expression in the livers. Notably, flagellin injection significantly exacerbated the severity of liver fibrosis in IFN-α/β receptor 1 (IFNAR1) knockout mice. Furthermore, hepatic expression of IL1RN in the fibrotic livers of IFNAR1 knockout mice was significantly lower than those of wild-type mice. In support of these findings, flagellin-mediated IL1RN production is not sufficient to alleviate the severity of hepatic fibroinflammatory responses in IFNAR1-deficient milieu. Finally, hepatic stellate cells treated with IL1RN had significantly decreased cellular activation and its associated fibrogenic responses. Collectively, manipulation of TLR5 signaling may be a promising therapeutic strategy for the treatment of liver fibrosis.

TGF-β- and lipopolysaccharide-induced upregulation of circular RNA PWWP2A promotes hepatic fibrosis via sponging miR-203 and miR-223.

Both transforming growth factor-beta (TGF-β) and lipopolysaccharide (LPS) can activate hepatic stellate cells (HSCs), thus increasing expressions of alpha smooth muscle actin (α-SMA) and type I collagen alpha 1 (Col1α1) and promoting liver fibrosis. However, whether TGF-β and LPS have a common downstream reactor remains unclear. Recently, a strong relationship of circular RNAs (circRNAs) and fibrogenesis has been elucidated. In this study, we compared the expressions of several circRNAs in TGF-β- and LPS-activated HSCs, and found that circ-PWWP2A was upregulated in both TGF-β- and LPS-activated HSCs and in mouse fibrotic liver tissues. Meanwhile, circ-PWWP2A was positively correlated with HSC activation and proliferation. Two microRNAs, miR-203 and miR-223, were identified to be the downstream targets of circ-PWWP2A using luciferase reporter assay and pull-down interaction assay. Circ-PWWP2A was suggested to promote HSC activation and proliferation via sponging miR-203 and miR-223, and subsequently increasing Fstl1 and TLR4, respectively. Furthermore, downregulating circ-PWWP2A was indicated to alleviate hepatic fibrosis in vivo. In conclusion, our findings indicated that circ-PWWP2A is the common downstream reactor of TGF-β and LPS in HSC activation, and that circ-PWWP2A plays a critical role in hepatic fibrogenesis via sponging miR-203 and miR-223.

Single-Cell Transcriptomics Uncovers Zonation of Function in the Mesenchyme during Liver Fibrosis

SummaryIterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.

A cAbl-MRTF-A Feedback Loop Contributes to Hepatic Stellate Cell Activation.

Trans-differentiation of quiescent hepatic stellate cells (HSC) to myofibroblasts is a hallmark event in liver fibrosis. Previous studies have led to the discovery that myocardin-related transcription factor A (MRTF-A) is a key regulator of HSC trans-differentiation or, activation. In the present study we investigated the interplay between MRTF-A and c-Abl (encoded by Abl1), a tyrosine kinase, in this process. We report that hepatic expression levels of c-Abl were down-regulated in MRTF-A knockout (KO) mice compared to wild type (WT) littermates in several different models of liver fibrosis. MRTF-A deficiency also resulted in c-Abl down-regulation in freshly isolated HSCs from the fibrotic livers of mice. MRTF-A knockdown or inhibition repressed c-Abl in cultured HSCs in vitro. Further analyses revealed that MRTF-A directly bound to the Abl1 promoter to activate transcription by interacting with Sp1. Reciprocally, pharmaceutical inhibition of c-Abl suppressed MRTF-A activity. Mechanistically, c-Abl activated extracellular signal-regulated kinase (ERK), which in turn phosphorylated MRTF-A and promoted MRTF-A nuclear trans-localization. In conclusion, our data suggest that a c-Abl-MRTF-A positive feedback loop contributes to HSC activation and liver fibrosis.

Chitinase 3-like 1 deficiency ameliorates liver fibrosis by promoting hepatic macrophage apoptosis.

AimChitinase 3‐like 1 (CHI3L1), an 18‐glycosyl hydrolase‐related molecule, is a member of the enzymatically inactive chitinase‐like protein family. Serum levels of CHI3L1 are strongly correlated with hepatic fibrosis progression during many liver diseases. Therefore, this protein could be involved in the development of hepatic fibrosis pathology; however, its role has not been elucidated. We aimed to elucidate its role in the pathophysiology of liver fibrosis.MethodsChitinase 3‐like 1‐deficient (Chi3l1 −/−) mice were given carbon tetrachloride twice per week for 4 weeks or fed a methionine choline‐deficient diet for 12 weeks to generate mouse liver fibrosis models. Human fibrotic liver tissues were also examined immunohistochemically.ResultsIn human and mouse fibrotic livers, CHI3L1 expression was mainly localized to hepatic macrophages, and the intrahepatic accumulation of CHI3L1+ macrophages was significantly enhanced compared to that in control livers. In the two mouse models, hepatic fibrosis was significantly ameliorated in Chi3l1 −/− mice compared to that in wild‐type mice, which was dependent on hepatic macrophages. The accumulation and activation of hepatic macrophages was also significantly suppressed in Chi3l1 −/− mice compared to that in wild‐type mice. Furthermore, apoptotic hepatic macrophages were significantly increased in Chi3l1 −/− mice. Chitinase 3‐like 1 was found to inhibit hepatic macrophage apoptosis by suppressing Fas expression and activating Akt signaling in an autocrine manner, which resulted in hepatic macrophage accumulation and activation, exaggerating liver fibrosis.ConclusionsChitinase 3‐like 1 exacerbates liver fibrosis progression by suppressing apoptosis in hepatic macrophages. Therefore, this might be a potential therapeutic target for the treatment of liver fibrosis.

The Role of Phospho-c-Jun N-Terminal Kinase Expression on hepatocyte Necrosis and Autophagy in the Cholestatic Liver

BackgroundClinically, liver fibrosis and cholestasis are two major disease entities, ultimately leading to hepatic failure. Although autophagy plays a substantial role in the pathogenesis of these diseases, its precise mechanism has not been determined yet. Materials and methodsMouse models of liver fibrosis or cholestasis were obtained after the serial administration of thioacetamide (TAA) or surgical bile duct ligation (BDL), respectively. Then, after obtaining liver specimens at specific time points, we compared the expression of makers related to apoptosis (cleaved caspases), inflammation (CD68), necrosis (high-mobility group box 1), phospho-c-Jun N-terminal kinase (p-JNK), and autophagy (microtubule-associated protein light chain 3B and p62) in the fibrotic or cholestatic mouse livers, by polymerase chain reaction, Western blot analysis, immunohistochemistry, and immunofluorescence. ResultsAlthough cholestatic livers exhibited the tendency of progressively increasing the expression of most apoptosis-related markers (cleaved caspases), it was not prominent when it was compared with the tendency found in the livers of TAA-treated mice. Contrastingly, the necrosis-related factor (high-mobility group box 1) was significantly increased in the livers of BDL mice over time, reaching their peak values on day 7 after BDL. In addition, the inflammation-related factor (CD68) was highly expressed in BDL mice compared with TAA-treated mice over time. Autophagy marker studies indicated that autophagy was upregulated in fibrotic livers, whereas it was downregulated in cholestatic livers. We also observed mild to moderate activation of p-JNK in the livers of TAA-treated mice, whereas significantly higher p-JNK activation was detected in the livers of BDL mice. ConclusionsUnlike TAA-treated mice, BDL mice exhibited higher expression of the markers related with inflammation and necrosis, especially including p-JNK, while maintaining low levels of autophagic process. Therefore, obstructive cholestasis is characterized by higher p-JNK activation, which could be related with marked necrotic cell death resulting from extensive inflammation and little chance of compensatory autophagy.

SAT-035 Small Molecule Allosteric Agonist of Relaxin Receptor Ml290 Demonstrates Antifibrotic Properties in Liver Fibrosis

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological therapeutic options. The beneficial effects of relaxin peptide treatment have been demonstrated previously in clinically relevant animal models of liver disease. However, the low stability of the recombinant relaxin peptide in vivo requires continuous intravenous delivery for chronic therapeutic application. We have recently identified a first series of small molecule allosteric biased agonists of the human relaxin receptor RXFP1 which showed efficacy similar to relaxin in several functional assays in vitro. Here we investigated the therapeutic effects of small molecule RXFP1 agonist on activated hepatic stellate cells, the main source of excessive collagen production in liver fibrosis. We have demonstrated that RXFP1 expression is increased in fibrotic mouse liver, specifically in activated hepatic stellate cells. The lead compound, ML290, was selected based on its effects on the expression of the genes involved in fibrosis in primary human stellate cells. RNA-Seq analysis of TGFβ1-activated LX-2 hepatic stellate cells showed that about 500 genes were misregulated by ML290. Gene Ontology analysis demonstrated that ML290 treatment primarily affects extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment of human liver organoids with lipopolysaccharide-induced fibrotic phenotype resulted in dramatic reduction of type I collagen. The pharmacokinetics of ML290 in mice after multiple injections demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. The ML290 treatment of mice expressing human RXFP1 gene with carbon tetrachloride-induced liver fibrosis resulted in significantly reduced collagen content, alpha-smooth muscle actin expression and cell proliferation around portal ducts, and the decrease of pro-fibrotic genes’ expression. In summary, ML290, the small molecule agonist of relaxin receptor, has anti-fibrotic effects in liver fibrosis. Funding source: NIH/NIDDK

PI3-kinase/Akt pathway-regulated membrane transportation of acid-sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF-induced HSC Activation.

Acid‐sensing ion channel 1a (ASIC1a) allows Na+ and Ca2+ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non‐neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS‐related proteins were up‐regulated in carbon tetrachloride (CCl4)‐induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS‐related biomarkers GRP78, Caspase12 and IREI‐XBP1. And, ERS inhibition by 4‐PBA down‐regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF‐induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF‐activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.

MicroRNA‐26b‐5p Regulates Bone Marrow Mesenchymal Stem Cell Differentiation to Pericytes via Targeting Platelet‐Derived Growth Factor Receptor‐β and Interacting with Long non‐Coding RNA Maternally Expressed Gene 3 in Liver Fibrosis and Angiogenesis

Background/AimsPericytes are important regulators of vascular morphogenesis and function in multiple biological processes, including liver fibrosis, which is closely associated with angiogenesis. However, the origin and functional role of pericytes during liver fibrosis have not been deciphered yet.MethodsMice received irradiation and transplantation of EGFP‐labeled bone marrow (BM) mesenchymal stem cells (BMSCs), which obtained from EGFP transgenic mice and had been sorted and purified by immunomagnetic cell sorting with anti‐CD146 microbeads. Four weeks later, the BM was reconstructed and the chimera mice with EGFP‐labeled BMSCs were fed with methionine‐choline‐deficient and high fat (MCDHF) diet to build liver injury model. Expression of pericyte markers or fibrosis markers was characterized by qRT‐PCR, Western blot, immunofluerescent staining and microarray analysis. Biotin‐avidin pull‐down and lucifer reporter assay were performed to validate the binding of microRNA (miR)‐26b‐5p and its target platelet‐derived growth factor receptor beta (PDGFR‐β). GO, KEGG and Reactome enrichment analysis was conducted to clarify the function of differentially expressed genes.ResultsIncreased expression of pericyte markers (e.g. PDGFR‐β, neural glial antigen 2, regulator of G protein signaling 5) was detected in MCDHF mice with a positive correlation with fibrosis markers (e.g. TGF‐β1, α‐smooth muscle actin, procollagen α1(I) and (III)). Numerous PDGFR‐β+ pericytes was found in the fibrotic liver not only around mature vessels but also around nascent vessels, and a significant proportion (72.5%) of pericytes in mouse fibrotic liver derived from BMSCs. BMSCs differentiated into pericytes under TGF‐β1 treatment in vitro. Long non‐coding RNA maternally expressed gene 3 (LncMEG3), which had been focused on its effects on angiogenesis recently, positively regulated PDGFR‐β expression during BMSC differentiation to pericytes. Bioinformatics analysis from three databases (miRanda, TargetScan, and RNA22v2) suggested that miR‐26b‐5p was predicted to target PDGFR‐β. MiR‐26b‐5p negatively regulated BMSC differentiation to pericytes via directly targeting PDGFR‐β in cultured BMSCs. GO, KEGG and Reactome enrichment analysis revealed that miR‐26b‐5p overexpression affected a list of genes associated with cell differentiation, angiogenesis and extracellular matrix. LncMEG3 acted as a molecular sponge of miR‐26b‐5p thus contributed to the de‐repression of miRNA target PDGFR‐β. MiR‐26b‐5p agomir blocked BMSC differentiation to pericytes and attenuates liver fibrosis and angiogenesis in vivo.ConclusionmiR‐26b‐5p regulates BMSC differentiation to pericytes via targeting PDGFR‐β and interacting with lncMEG3, which may represent an effective therapeutic strategy for liver fibrosis.Support or Funding InformationThis work was supported by grants from the National Natural and Science Foundation of China (81430013, 81500465) and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality(IDHT20150502)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TLR5 Signaling Ameliorates Liver Fibrosis by Inducing Interferon β-Interleukin-1 Receptor Antagonist in Mice

Background: Bacterial flagellin, recognized by cell surface of toll-like receptor (TLR) 5, is a potent activator of many types of cells, leading to the activation of innate or adaptive immunity, which are pivotal in regulating fibrotic process. However, the exact role of TLR5 signaling in liver fibrosis is still unclear. Methods: Hepatotoxin- and cholestasis-induced liver fibrosis models were used in this study. Real-time PCR, Western blotting, and histopathologic analysis were performed to elucidate the underlying mechanism of TLR5 signaling in the pathogenesis of liver fibrosis. Findings: Flagellin-induced TLR5 activation significantly decreased the severity of liver fibrosis. Notably, hepatic expression of interleukin-1 receptor antagonist (IL-1ra) and interferon (IFN) β was markedly increased in fibrotic livers upon flagellin treatment. Consistently, in vivo activation of TLR5 signaling significantly increased IFNβ and IL-1ra expression in the livers. Interestingly, flagellin injection significantly exacerbated the severity of liver fibrosis in IFN-α/β receptor-1 (IFNAR1)-deficient mice. Furthermore, hepatic expression of IL-1ra in the fibrotic livers of IFNAR1-deficient mice was significantly lower than those of WT mice. In support of these findings, flagellin-mediated IL-1ra production is not sufficient to alleviate the severity of hepatic fibro-inflammatory responses in IFNAR1-deficient milieu. Finally, hepatic stellate cells treated with IL-1ra showed significantly decreased cellular activation and its associated fibrogenic responses. Interpretation: Collectively, manipulation of TLR5 signaling may be a promising therapeutic strategy for the treatment of liver fibrosis. Funding Statement: This research was supported by grant (NRF-2017R1D1A3B03030521, 2017R1A6A3A11032024) of the Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Republic of Korea Declaration of Interests: The authors declare that there are no conflicts of interests. Ethics Approval Statement: Experimental procedures and animal management procedures were undertaken in accordance with the requirements of the Animal Care and Ethics Committees of Chonbuk National University. The animal facility of Chonbuk National University is fully accredited by the National Association of Laboratory Animal Care.

Autophagy promotes hepatic differentiation of hepatic progenitor cells by regulating the Wnt/\u03b2-catenin signaling pathway

Hepatic progenitor cells (HPCs) can be activated when the liver suffers persistent and severe damage and can differentiate into hepatocytes to maintain liver regeneration and homeostasis. However, the molecular mechanism underlying the hepatic differentiation of HPCs is unclear. Therefore, in this study, we aimed to investigate the roles of autophagy and the Wnt/β-catenin signaling pathway during hepatic differentiation of HPCs in vivo and in vitro. First, immunohistochemistry, immunofluorescence and electron microscopy showed that Atg5 and β-catenin were highly expressed in human fibrotic liver and mouse liver injury induced by feeding a 50% choline-deficient diet plus 0.15% ethionine solution in drinking water (CDE diet) for 21 days; in addition, these factors were expressed in CK19-positive HPCs. Second, Western blotting and immunofluorescence confirmed that CK19-positive HPCs incubated in differentiation medium for 7 days can differentiate into hepatocytes and that differentiated HPCs were able to take up ICG and secrete albumin and urea. Further investigation via Western blotting, immunofluorescence and electron microscopy revealed autophagy and the Wnt/β-catenin pathway to be activated during hepatic differentiation of HPCs. Next, we found that inhibiting autophagy by downregulating Atg5 gene expression impaired hepatic differentiation of HPCs and inhibited activation of the Wnt/β-catenin pathway, which was rescued by overexpression of the β-catenin gene. Moreover, downregulating β-catenin gene expression without inhibiting autophagy still impeded the differentiation of HPCs. Finally, coimmunoprecipitation demonstrated that P62 forms a complex with phosphorylated glycogen synthase kinase 3 beta (pGSK3β). Third, in mouse CDE-induced liver injury, immunohistochemistry and immunofluorescence confirmed that downregulating Atg5 gene expression inhibited autophagy, thus impeding hepatic differentiation of HPCs and inhibiting activation of the Wnt/β-catenin pathway. As observed in vitro, overexpression of β-catenin rescued this phenomenon caused by autophagy inhibition, though decreasing β-catenin levels without autophagy inhibition still impeded HPC differentiation. We also found that HPCs differentiated into hepatocytes in human fibrotic liver tissue. Collectively, these results demonstrate that autophagy promotes HPC differentiation by regulating Wnt/β-catenin signaling. Our results are the first to identify a role for autophagy in promoting the hepatic differentiation of HPCs.