Culture-activated Hepatic Stellate Cells Research Articles

MiR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway.

In hepatic fibrosis (HF), hepatic stellate cells (HSCs) form the extracellular matrix (ECM), and the pathological accumulation of ECM in the liver leads to inflammation. Our previous research found that miR-324-3p was down-regulated in culture-activated human HSCs. However, the precise effect of miR-324-3p on HF has not been elucidated. In this study, the HF mouse models were induced through directly injecting carbon tetrachloride (CCl4) into mice; the HF cell models were constructed using TGF-β1-treated LX-2 cells. Next, real-time-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and immunohistochemistry (IHC) were applied to assess the expression levels of miR-324-3p, α-smooth muscle actin (α-SMA), Vimentin or SMAD4; hematoxylin and eosin (H&E), Masson' s trichrome and Sirius red staining to evaluate the liver injury; luciferase reporter assay to verify the targeting relationship between miR-324-3p and SMAD4; enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and cell counting kit-8 (CCK-8) and flow cytometry to evaluate the effects of miR-324-3p on cell proliferation and cycle/apoptosis, respectively. The experimental results showed a reduction in miR-324-3p level in CCl4-induced HF mice as well as transforming growth factor (TGF)-β1-activated HSCs. Interestingly, the miR-324-3p level was rescued following the HF recovery process. In HF mice induced by CCl4, miR-324-3p overexpression inhibited liver tissue damage, decreased serum ALT and AST levels, and inhibited fibrosis-related biomarkers (α-SMA, Vimentin) expression, thereby inhibiting HF. Similarly, miR-324-3p overexpression up-regulated α-SMA and Vimentin levels in HF cells, while knockdown of miR-324-3p had the opposite effect. Besides, miR-324-3p played an antifibrotic role through inhibiting the proliferation of hepatocytes. Further experiments confirmed that miR-324-3p targeted and down-regulated SMAD4 expression. SMAD4 was highly expressed in HF cells, and silencing SMAD4 significantly decreased the α-SMA and Vimentin levels in HF cells. Collectively, the miR-324-3p may suppress the activation of HSCs and HF by targeting SMAD4. Therefore, miR-324-3p is identified as a potential and novel therapeutic target for HF.

An unusual signal transducer GIV/Girdin engages in the roles of adipocyte-derived hormone leptin in liver fibrosis

Obese patients usually have hyperleptinemia and are prone to develop liver fibrosis. Leptin is intimately linked to liver fibrogenesis, a multi-receptor-driven disease. Gα-Interacting Vesicle-associated protein (GIV) functions as a multimodular signal transducer and a guanine nucleotide exchange factor for Gi controling key signalings downstream of diverse receptors. This study aimed to examine the roles of GIV in leptin-caused liver fibrosis and employed the culture-activated hepatic stellate cells (HSCs) and leptin-deficient mice, respectively. Results indicated that leptin upregulated GIV expression in HSCs. GIV was involved in leptin-induced HSC activation and liver fibrosis. GIV mediated leptin regulation of TIMP1, MMP9, PDGFβ receptor and TGFβ receptor and was required for leptin stimulating the pathways of Erk1/2, Akt1, and Smad3. GIV was also a mediator for leptin-regulation of Cyclin D1 and Caspase-3 activity but GIV reduced Caspase-3 level independently of leptin in vivo. Erk1/2 signaling, Egr1 and c-Jun were associated with the effect of leptin on GIV expression in HSCs. Leptin-induced Erk1/2 signaling increased Egr1 and p-c-Jun levels and promoted their binding to GIV promoter at the sites between −190 bp and −180 bp and between −382 bp and − 376 bp, respectively. Egr1 knockdown lessened leptin-upregulation of GIV in vitro and in vivo. In human cirrhotic livers, the increase in GIV protein level parallelled with the elevated p-Erk1/2 and Egr1 levels in HSCs. In summary, the unusual signal transducer GIV was identified as an important mediator in leptin-induced liver fibrosis. GIV may have significant implications in liver fibrosis progression of obese patients with hyperleptinaemia.

Single-cell transcriptomic analysis reveals a novel cell state\xa0and switching genes during hepatic stellate cell activation in vitro

BackgroundThe transformation of hepatic stellate cell (HSC) to myofibroblast is a key event during liver fibrogenesis. However, the differentiation trajectory of HSC-to-myofibroblast transition and the switching genes during this process remains not well understood.MethodsWe applied single-cell sequencing data to reconstruct a single-lineage pseudotime trajectory of HSC transdifferentiation in vitro and analyzed the gene expression patterns along the trajectory. GeneSwitches was used to identify the order of critical gene expression and functional events during HSC activation.ResultsA novel cell state during HSC activation was revealed and the HSCs belonging to this state may be an important origin of cancer-associated fibroblasts (CAFs). Combining single-cell transcriptomics with GeneSwitches analyses, we identified some distinct switching genes and the order at which these switches take place for the new state of HSC and the classic culture-activated HSC, respectively. Based on the top switching genes, we established a four-gene combination which exhibited highly diagnostic accuracy in predicting advanced liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD) or hepatitis B (HBV).ConclusionOur study revealed a novel cell state during HSC activation which may be relevant to CAFs, and identified switching genes that may play key roles in HSC transdifferentiation and serve as predictive markers of advanced fibrosis in patients with chronic liver diseases.

A magnetic resonance imaging modality for non-invasively distinguishing progression of liver fibrosis by visualizing hepatic platelet-derived growth factor receptor-beta expression in mice.

Activated hepatic stellate cells (HSCs) are the most critical cells responsible for liver fibrosis, and platelet-derived growth factor (PDGF) is the most prominent mitogen for HSCs in fibrogenesis. This study aimed to explore the potential of gadolinium (Gd)-labeled cyclic peptides (pPB) targeting PDGF receptor-β (PDGFR-β) as a magnetic resonance imaging (MRI) radiotracer to identify the progression of liver fibrosis by imaging hepatic PDGFR-β expression. Mice treated with carbon tetrachloride (CCl4 ) were used to mimic hepatic fibrosis in vivo. The binding activity of FITC-labeled pPB to PDGFR-β was assessed in cultured human HSCs (HSC-LX2). MRI was performed to visualize hepatic PDGFR-β expression in mice with different degrees of liver fibrosis after Gd-labeled pPB was injected. Hepatic PDGFR-β expression level was correlated with the severity of liver fibrosis, and the majority of cells expressing PDGFR-β were found to be activated HSCs in fibrotic livers. Culture-activated human HSCs expressed abundant PDGFR-β, and FITC-labeled pPB could bind to these cells in a concentration-dependent and time-dependent manner. With Gd-labeled pPB as a tracer, an MRI modality demonstrated that the relative hepatic T1-weighted MRI signal value progressively increased with the severity of hepatic fibrosis and reduced with remission. Hepatic PDGFR-β expression reflects the progression of hepatic fibrosis, and MRI using Gd-labeled pPB as a tracer exhibits potential for distinguishing liver fibrosis staging in mice.

Non-Invasively Distinguishing Progress of Liver Fibrosis by Visualizing Hepatic Platelet Derived Growth Factor Receptor-Beta Expression with an MRI Modality in Mice

Background: Activated hepatic stellate cells (HSCs) are the most critical cell responsible for liver fibrosis. In liver fibrogenesis, platelet-derived growth factor (PDGF) is the most prominent mitogen for HSCs. This study aims to explore the potential of gadolinium (Gd)-labeled cyclic peptides (pPB) targeted to PDGF receptor-β (PDGFR-β) as an MRI radiotracer to identify the progress of liver fibrosis by imaging hepatic PDGFR-β expression. Methods: Mouse models of liver fibrosis caused by bile duct ligation (BDL) and CCl 4 treatment were both used to determine the association of hepatic PDGFR-β expression and the progress of hepatic fibrosis. The binding activity of FITC-labeled pPB to PDGFR-β was assessed in culture-activated human HSCs (HSC-LX2). MR imaging was performed to visualize hepatic PDGFR-β expression in mice with different degrees of liver fibrosis after Gd-labeled pPB were injected. Findings: Hepatic PDGFR-β expression level was found to be paralleled with the severity of liver fibrosis, which was increased with the progression of fibrosis and reduced with the regression. Majority of cells expressing PDGFR-β was determined to be activated HSCs in fibrotic livers. Culture-activated HSC-LX2 expressed abundant PDGFR-β, and FITC-labeled pPB could bind to HSC-LX2 in a concentration and time dependent manner. Interpretation: With Gd-labeled pPB as a tracer, the relative hepatic T1-weighed MR signal value was increased progressively along with severity of hepatic fibrosis and reduced with the remission, suggesting that hepatic PDGFR-β expression reflects the progress of hepatic fibrosis. MR imaging using Gd-labeled pPB as a tracer may distinguish different stages of liver fibrosis in mice. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant NO. 81270007 & 81670513 to Li F), Shanghai Talent Development Funds (Grant NO. 201304 to Li F), Shanghai Rising-Star Program (Grant NO. 13QA1400700 to Li F), And Shanghai Outstanding Young Talent Training Plan in Health System (Grant NO. 13YO56 to Li F). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: The study was approved by Institutional Ethical Committee of Animal Experimentation, all experiments and postoperative animal care procedures were performed according to the governmental and international guidelines on animal experimentation.

Hepatic stellate cell activation: A source for bioactive lipids

Hepatic stellate cells (HSCs) are non-parenchymal liver cells that characteristically contain multiple retinoid (vitamin A)-containing lipid droplets. In this study, we addressed the metabolic fate of non-retinoid lipids originating from lipid droplet loss during HSCs activation. UPLC/MS/MS and qRT-PCR were used to monitor the lipid composition and mRNA expression of selected genes regulating lipid metabolism in freshly isolated, overnight-, 3- and 7-day cultures or primary mouse HSCs. A preferential accumulation of specific C20-C24 fatty acid species, especially arachidonic (C20:4) and docosahexaenoic acids (C22:6), was revealed in culture-activated HSCs along with an upregulation of transcription of fatty acid desaturases (Scd1, Scd2) and elongases (Elovl5, Elovl6). This was accompanied with an enrichment of activated HSCs with 36:4 and 38:4 phosphatidylcholine species containing polyunsaturated fatty acids and associated accumulation of selective lipid mediators, including endocannabinoids and related N-acylethanolamides, as well as ceramides. An increase in 2-arachidonoylglycerol and N-arachydonoylethanolamide concentrations was observed along with an upregulation of Daglα mRNA expression in HSCs during culture activation. N-palmitoylethanolamide was identified as the most abundant endocannabinoid-like species in activated HSCs. An increase in total ceramide levels and enrichment with N-palmitoyl (C16:0), N-tetracosenoyl (C24:1), N-tetracosanoyl (C24:0) and N-docosanoyl (C22:0) ceramides was detected in activated HSC cultures and was preceded by increased mRNA expression of ceramide synthesizing enzymes (CerS2, CerS5 and Smpd1). Our data suggest an active redistribution of non-retinoid lipids in HSCs underlying the formation of low abundance, highly bioactive lipid species that may affect signaling during HSC activation, as well as extracellularly within the liver.

MicroRNA-125b Promotes Hepatic Stellate Cell Activation and Liver Fibrosis by Activating RhoA Signaling

miR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs, but not in hepatocytes, during hepatic fibrogenesis in vivo and upon culture activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor β (TGF-β)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSCs enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA-specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSCs and highlight its importance in hepatic fibrosis.

RNA Sequencing and Bioinformatics Analysis Implicate the Regulatory Role of a Long Noncoding RNA-mRNA Network in Hepatic Stellate Cell Activation

Background/Aims: To analyze the long noncoding (lncRNA)-mRNA expression network and potential roles in rat hepatic stellate cells (HSCs) during activation. Methods: LncRNA expression was analyzed in quiescent and culture-activated HSCs by RNA sequencing, and differentially expressed lncRNAs verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were subjected to bioinformatics analysis. In vivo analyses of differential lncRNA-mRNA expression were performed on a rat model of liver fibrosis. Results: We identified upregulation of 12 lncRNAs and 155 mRNAs and downregulation of 12 lncRNAs and 374 mRNAs in activated HSCs. Additionally, we identified the differential expression of upregulated lncRNAs (NONRATT012636.2, NONRATT016788.2, and NONRATT021402.2) and downregulated lncRNAs (NONRATT007863.2, NONRATT019720.2, and NONRATT024061.2) in activated HSCs relative to levels observed in quiescent HSCs, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that changes in lncRNAs associated with HSC activation revealed 11 significantly enriched pathways according to their predicted targets. Moreover, based on the predicted co-expression network, the relative dynamic levels of NONRATT013819.2 and lysyl oxidase (Lox) were compared during HSC activation both in vitro and in vivo. Our results confirmed the upregulation of lncRNA NONRATT013819.2 and Lox mRNA associated with the extracellular matrix (ECM)-related signaling pathway in HSCs and fibrotic livers. Conclusion: Our results detailing a dysregulated lncRNA-mRNA network might provide new treatment strategies for hepatic fibrosis based on findings indicating potentially critical roles for NONRATT013819.2 and Lox in ECM remodeling during HSC activation.

Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice

Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.

Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase.

Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.

Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells.

Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.

Epigenetic Changes during Hepatic Stellate Cell Activation.

Background and AimsHepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.Methods and ResultsThe analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.ConclusionsIn summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.

Role of R-Spondin1 in the activation of hepatic stellate cells

To investigate the role of R-Spondinl in the activation of hepatic stellate cells (HSCs). Twenty-four healthy male Kunming mice were randomly divided into the following two groups:fibrosis model group (n=16) and control group (n=8). Hepatic fibrosis was induced by subcutaneous injections of CC14 (20% in olive oil) at a dose of 5 ml/kg twice per week. After 10 weeks, the animals were sacrificed by CO(2) over-exposure and liver tissues were harvested.The protein and mRNA levels of R-Spondin1, alphat-SMA,and collagen I were examined by Western blot assay and real-time PCR respectively. Additionally,HSCs were isolated from the mice liver tissues to examine the time-series expression changes of R-Spondinl, alpha-SMA, and nuclear beta-catenin.TCF activity was analyzed by luciferase reporter assay.Moreover,HSCs were cocultured with recombinant R-Spondin1 and DKK1 to evaluate dose-response. R-Spondinl expression was significantly higher in the fibrosis model group than in the control group (protein level:3.16 ± 0.18 vs. 0.99 ± 0.16, t =13.31, P < 0.01; mRNA level:4.36 ± 0.26 vs. 0.98 ± 0.12, t =21.46, P < 0.01).The culture-activated mouse HSCs showed up-regulated TCF activity (5.33 ± 0.34 vs. non-activated: 1.03 ± 0.09, t =20.93, P < 0.01), nuclear beta-catenin expression (4.47 ± 0.21 vs. 0.97 ± 0.14, t =25.25, P < 0.01), and R-Spondin1 expression (protein level: 4.54 ± 0.18 vs. 1.04 ± 0.12, t =31.17, P < 0.01; mRNA level:5.13 ± 0.15 vs. 1.01 ± 0.16, t=38.06, P < 0.01). Exogenous stimulation of freshly isolated mouse HSCs with recombinant R-Spondin1 induced a dose-dependent increase in both TCF activity and the expression of nuclear beta-catenin and alphat-SMA. DKK1 down-regulated activities of factors in the WNT signaling pathway and repressed activation of HSCs. Conclusion R-Spondin1 may promote HSC activation by enhancing the canonical WNT signaling pathway.

Morin, a dietary flavonoid, exhibits anti-fibrotic effect and induces apoptosis of activated hepatic stellate cells by suppressing canonical NF-κB signaling.

In experimental liver fibrosis, activated hepatic stellate cells (HSCs) play a central role and thus, induction of apoptosis of activated HSCs is a promising therapeutic strategy for liver fibrosis. The present study was designed to elucidate the molecular mechanisms of the pro-apoptotic effects of morin, a dietary flavonoid, invitro and invivo. Culture-activated human HSCs (LX-2 cells) were treated with morin (50μM) for 24 and 48h, and the mechanism of cell death induced by morin was evaluated. Also, the anti-fibrotic and pro-apoptotic effect of morin in diethylnitrosamine (DEN)-induced fibrotic rats were determined. Morin induced apoptosis in cultured LX-2 cells by preventing the nuclear translocation of nuclear factor-κBp65 (NF-κBp65) by inhibiting NF-κB activation via inhibition of IκBα degradation and thereby suppressing anti-apoptotic proteins and activating caspases. In fibrotic rats, morin treatment resulted in inhibition of canonical NF-κB signaling and induction of apoptosis, mainly by downregulating Bcl-2, upregulating Bax and cyt c and by activation of caspase-9 and caspase-3. Translocation of phosphatidylserine to the outer membrane, altered nuclear morphology and DNA fragmentation confirmed the induction of apoptosis by morin. Overall, morin treatment ameliorated experimental liver fibrosis, most likely through induction of apoptosis by inhibiting canonical NF-κB signaling in activated HSCs. It is therefore postulated that morin is a potential therapeutic candidate for liver fibrosis.

R-spondin1 arguments hepatic fibrogenesis in vivo and in vitro

BackgroundThe development of liver fibrosis is the key stage toward a number of mortal complications of liver diseases, including cirrhosis and hepatocellular carcinoma. Canonical Wnt pathway is crucial in diverse biological processes and mediates the progression and regression of liver fibrosis. As a potent Wnt pathway agonist, roof plate-specific spondin-1 (R-spondin1) protein's role in the hepatic fibrosis has not been well elucidated. The purpose of this study was to investigate whether R-spondin1 contributed to hepatic stellate cells (HSC) activation, the key event in liver fibrogenesis. Materials and methodsTissue microarrays of human fibrotic liver samples, hepatocellular carcinoma samples, and normal hepatic tissue samples were constructed and immunostained for R-spondin1. Protein expression and transcriptional level of freshly isolated mice HSC were analyzed by Western blot assay and real-time polymerase chain reaction, respectively. Exogenous stimulation with recombinant R-spondin1 and Dickkopf-1 was performed to investigate the functionality. Nuclear β-catenin level and T-cell specific transcription factor activity were analyzed, and HSC proliferation was tested by Methyl-Thiazol-Tetrazolium bromide assay. ResultsOverexpression of R-spondin1 was observed in both fibrotic liver tissues and culture-activated HSC. Coculture with recombinant R-spondin1 induced a dose-dependent increase in both the transcription factor activity and the protein level of α-smooth muscle actin, collagen I, and nuclear β-catenin. Additionally, Dickkopf-1 repressed R-spondin1's effect on HSC. ConclusionsThese findings suggested that R-spondin1 might argument liver fibrogenesis by enhancing the canonical Wnt pathway.

Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.

Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells

Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC, is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150 nm in diameter, negatively charged (-26 mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR-214-transfected activated HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild-type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled by way of exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs.

Gene Expression Profiling of Early Hepatic Stellate Cell Activation Reveals a Role for Igfbp3 in Cell Migration

BackgroundScarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells. Aim and methodsTo identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. ResultsGene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp) 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. ConclusionIgfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.

Alteration of the ERK5 pathway by hydroxysafflor yellow A blocks expression of MEF2C in activated hepatic stellate cells in vitro: Potential treatment for hepatic fibrogenesis

Context: Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. Hepatic stellate cells (HSC) are a main mediator of hepatic fibrosis through the accumulation of extracellular matrix proteins. Suppression activation of passaged HSC has been proposed as therapeutic strategies for the treatment and prevention of hepatic fibrosis.Objective: To evaluate the effect of hydroxysafflor yellow A (HSYA), an active chemical compound derived from the flowers of Carthamus tinctorius L. (Compositae), on HSC inhibition, and to begin elucidating underlying mechanisms.Materials and methods: Primary HSCs were isolated from rats by in situ pronase/collagenase perfusion. Culture-activated HSCs were treated with or without HSYA at 30 μM in the presence or absence of PD98059 for 48 h, and then cell proliferation was measured by MTS assays. Messenger RNA (mRNA) expression was quantified by polymerase chain reaction, and protein was quantified by Western blots or enzyme-linked immunosorbent assays.Results: HSYA significantly inhibits culture-activated HSC proliferation in a dose-dependent and time-dependent manner with an IC50 value of 112.79 μM. HSYA (30 μM) induce the suppression of HSC activation, as indicated by decreases in contents of type I alpha collagen in HSC-cultured media and expression of α-smooth muscle actin protein in culture-activated HSC by 55 and 71%, respectively. HSYA (30 μM) also caused significant decreases in mRNA expression of type III alpha collagen in HSC by 28%. HSYA (30 μM) suppresses myocyte enhancer factor 2 C (MEF2C) expression both at its mRNA and protein levels by 60 and 61%, respectively. Further study demonstrated that HSYA (30 μM) caused significant decreases in p-ERK5 by 49%. Blocking extracellular signal-regulated protein kinase 5 (ERK5) activity by XMD 8--92, an ERK5 inhibitor, markedly abrogated the inhibitive effects of HSYA on HSC activation, and blocked the HSYA-mediated MEF2C down-regulation.Conclusions: HSYA suppress HSC activation by ERK5-mediated MEF2C down-regulation and makes it a potential candidate for prevention and treatment of hepatic fibrogenesis.

Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway

Oxidative stress is involved in hepatic fibrogenesis. Activation of hepatic stellate cells (HSCs), the key effectors in hepatic fibrogenesis, is characterized by overproduction of extracellular matrix. Astragaloside IV, the active component of Radix Astragali, has antioxidant properties and antifibrotic potential in renal fibrosis. Little is known about the role of astragaloside IV in liver and its involvement in hepatic fibrosis. This study aims at evaluating the antifibrotic potential of astragaloside IV and characterizing involved signal transduction pathways in culture-activated HSCs. Our results show that astragaloside IV attenuates oxidative stress in culture-activated HSCs, as demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation, and elevates the level of cellular glutathione by stimulating Nrf2gene expression. Depletion of cellular glutathione by buthionine sulfoximine or abrogation of p38 MAPK by SB-203580 evidently eliminates the inhibitory effects of astragaloside IV on genes relevant to HSC activation. These results demonstrate that astragaloside IV inhibits HSC activation by inhibiting generation of oxidative stress and associated p38 MAPK activation and provide novel insights into the mechanisms of astragaloside IV as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.