Phenotype Of Hepatic Stellate Cells Research Articles

Inhibition of endogenous hydrogen sulfide production reduces activation of hepatic stellate cells via the induction of cellular senescence

ABSTRACT In chronic liver injury, quiescent hepatic stellate cells (HSCs) transdifferentiate into activated myofibroblast-like cells and produce large amounts of extracellular matrix components, e.g. collagen type 1. Cellular senescence is characterized by irreversible cell-cycle arrest, arrested cell proliferation and the acquisition of the senescence-associated secretory phenotype (SASP) and reversal of HSCs activation. Previous studies reported that H2S prevents induction of senescence via its antioxidant activity. We hypothesized that inhibition of endogenous H2S production induces cellular senescence and reduces activation of HSCs. Rat HSCs were isolated and culture-activated for 7 days. After activation, HSCs treated with H2S slow-releasing donor GYY4137 and/or DL-propargylglycine (DL-PAG), an inhibitor of the H2S-producing enzyme cystathionine γ-lyase (CTH), as well as the PI3K inhibitor LY294002. In our result, CTH expression was significantly increased in fully activated HSCs compared to quiescent HSCs and was also observed in activated stellate cells in a in vivo model of cirrhosis. Inhibition of CTH reduced proliferation and expression of fibrotic markers Col1a1 and Acta2 in HSCs. Concomitantly, DL-PAG increased the cell-cycle arrest markers Cdkn1a (p21), p53 and the SASP marker Il6. Additionally, the number of β-galactosidase positive senescent HSCs was increased. GYY4137 partially restored the proliferation of senescent HSCs and attenuated the DL-PAG-induced senescent phenotype. Inhibition of PI3K partially reversed the senescence phenotype of HSCs induced by DL-PAG. Inhibition of endogenous H2S production reduces HSCs activation via induction of cellular senescence in a PI3K-Akt dependent manner. Our results show that cell-specific inhibition of H2S could be a novel target for anti-fibrotic therapy via induced cell senescence.

Neutrophil extracellular traps activate hepatic stellate cells and monocytes via NLRP3 sensing in alcohol-induced acceleration of MASH fibrosis

ObjectiveAlcohol use in metabolic dysfunction-associated steatohepatitis (MASH) is associated with an increased risk of fibrosis and liver-related death. Here, we aimed to identify a mechanism through which repeated alcohol binges...

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.

The m6A reader IGF2BP2 regulates glycolytic metabolism and mediates histone lactylation to enhance hepatic stellate cell activation and liver fibrosis

Evidence for the involvement of N6-Methyladenosine (m6A) modification in the etiology and progression of liver fibrosis has emerged and holds promise as a therapeutic target. Insulin-like growth factor 2 (IGF2) mRNA-binding protein 2 (IGF2BP2) is a newly identified m6A-binding protein that functions to enhance mRNA stability and translation. However, its role as an m6A-binding protein in liver fibrosis remains elusive. Here, we observed that IGF2BP2 is highly expressed in liver fibrosis and activated hepatic stellate cells (HSCs), and inhibition of IGF2BP2 protects against HSCs activation and liver fibrogenesis. Mechanistically, as an m6A-binding protein, IGF2BP2 regulates the expression of Aldolase A (ALDOA), a key target in the glycolytic metabolic pathway, which in turn regulates HSCs activation. Furthermore, we observed that active glycolytic metabolism in activated HSCs generates large amounts of lactate as a substrate for histone lactylation. Importantly, histone lactylation transforms the activation phenotype of HSCs. In conclusion, our findings reveal the essential role of IGF2BP2 in liver fibrosis by regulating glycolytic metabolism and highlight the potential of targeting IGF2BP2 as a therapeutic for liver fibrosis.

Transcriptomic and proteomic investigation of the ameliorative effect of total polyphenolic glycoside extract on hepatic fibrosis in Lamiophlomis rotata Kudo via the AGE/RAGE pathway

Ethnopharmacological relevanceDuring the regression of liver fibrosis, a decrease in hepatic stellate cells (HSCs) can occur through apoptosis or inactivation of activated HSCs (aHSCs). A new approach for antifibrotic therapy involves transforming hepatic myofibroblasts into a quiescent-like state. Lamiophlomis rotata (Benth.) Kudo (L. rotata), an orally available Tibetan herb, has traditionally been used to treat skin disease, jaundice, and rheumatism. In our previous study, we found that the total polyphenolic glycoside extract of L. rotata (TPLR) promotes apoptosis in aHSCs for the treatment of hepatic fibrosis. However, whether TPLR induces aHSCs to become inactivated HSCs (iHSCs) is unclear, and the underlying mechanism remains largely unknown. PurposeThis study aimed to examine the impact of TPLR on the phenotypes of hepatic stellate cells (HSCs) during the regression of liver fibrosis and explore the potential mechanism of action. MethodsThe effect of TPLR on the phenotypes of hepatic stellate cells (HSCs) was assessed using immunofluorescence (IF) staining, reverse transcription-polymerase chain reaction (RT–PCR), and Western blotting. Transcriptomic and proteomic methods were employed to identify the main signaling pathways involved. Based on the omics results, the likely mechanism of TPLR on the phenotypes of aHSCs was confirmed through overexpression and knockdown experiments in TGF-β1-activated LX-2 cells. Using a CCl4-induced liver fibrosis mouse model, we evaluated the anti-hepatic fibrosis effect of TPLR and explored its potential mechanism based on omics findings. ResultsTPLR was found to induce the differentiation of aHSCs into iHSCs by significantly decreasing the protein expression of α-SMA and Desmin. Transcriptomic and proteomic analyses revealed that the AGE/RAGE signaling pathway plays a crucial role in the morphological transformation of HSCs following TPLR treatment. In vitro experiments using RAGE overexpression and knockdown demonstrated that the mechanism by which TPLR affects the phenotype of HSCs is closely associated with the RAGE/RAS/MAPK/NF-κB axis. In a model of liver fibrosis, TPLR obviously inhibited the generation of AGEs and alleviated liver tissue damage and fibrosis by downregulating RAGE and its downstream targets. ConclusionThe AGE/RAGE axis plays a pivotal role in the transformation of activated hepatic stellate cells (aHSCs) into inactivated hepatic stellate cells (iHSCs) following TPLR treatment, indicating the potential of TPLR as a therapeutic agent for the management of liver fibrosis.

Baccharis anomala DC. extract reduces inflammation and attenuates hepatic fibrosis in vivo by decreasing NF-kB and extracellular matrix compounds

Baccharis anomala DC. (BA) is a plant species found in the tropical regions of South America and is widely used for its hepatoprotective effects, as well as for the treatment of gastrointestinal diseases. Studies have recently reported its antioxidant and anti-inflammatory potential. BA extract can reverse the activated phenotype of hepatic stellate cells (HSC), which plays a central role in extracellular matrix (ECM) deposition in the development of liver fibrosis. Thus, this study aimed to evaluate the effects of the treatment with BA extract on liver fibrosis in a CCl4-induced liver fibrosis model in BALB/c mice. Methanolic extract was obtained from BA leaves, a gas chromatography/mass spectrometry (GC/MS) to detect the compounds present was performed, and then administered by intraperitoneal injection in Balb/C mice at a concentration of 50 and 100 mg/kg together with the administration of CCl4 for inducing liver fibrosis. After 10 weeks, blood analysis, histopathology, oxidative stress, as well as protein and gene expression in the hepatic tissue were performed. Treatment with BA extract was able to reduce profibrotic markers by reducing the expression of α-SMA and Col-1 proteins, as well as reducing the formation of free radicals and lipid peroxidation. (BA extract showed anti-inflammatory effects in the liver by suppressing NF-kB activation and reducing gene expression of signaling targets (IL-6 and iNOS). The data obtained showed that BA extract has antifibrotic and anti-inflammatory effects.

Thyroid hormone receptor alpha modulates fibrogenesis in hepatic stellate cells.

Progressive hepatic fibrosis can be considered the final stage of chronic liver disease. Hepatic stellate cells(HSC) play a central role in liver fibrogenesis. Thyroid hormones (TH, e.g. thyroxine; T4 and triiodothyronine; T3) significantly affect development, growth, cell differentiation and metabolism through activation of TH receptor α and/or β (TRα/β). Here, we evaluated the influence of TH in hepatic fibrogenesis. Human liver tissue was obtained from explanted livers following transplantation. TRα-deficient (TRα-KO) and wild-type (WT) mice were fed a control or a profibrogenic methionine-choline deficient (MCD) diet. Liver tissue was assessed by qRT-PCR for fibrogenic gene expression. In vitro, HSC were treated with TGFβ in the presence or absence of T3. HSC with stable TRα knockdown and TRα deficient mouse embryonic fibroblasts (MEF) were used to determine receptor-specific function. Activation of HSC and MEF was assessed using the wound healing assay, Western blotting, and qRT-PCR. TRα and TRβ expression is downregulated in the liver during hepatic fibrogenesis in humans and mice. TRα represents the dominant isoform in HSC. In vitro, T3 blunted TGFβ-induced expression of fibrogenic genes in HSC and abrogated wound healing by modulating TGFβ signalling, which depended on TRα presence. In vivo, TRα-KO enhanced MCD diet-induced liver fibrogenesis. These observations indicate that TH action in non-parenchymal cells is highly relevant. The interaction of TRα with TH regulates the phenotype of HSC via the TGFβ signalling pathway. Thus, the TH-TR axis may be a valuable target for future therapy of liver fibrosis.

Long non-coding RNA HOX transcript antisense intergenic RNA depletion protects against alcoholic hepatitis through the microRNA-148a-3p/sphingosine 1-phosphate receptor 1 axis.

The aggravating role of long noncoding RNA (lncRNA) HOTAIR has been indicated in liver injury caused by hepatic ischemia/reperfusion. However, under the condition of alcoholic hepatitis (AH), its effects remain unclear. The present study aimed to examine the effect of lncRNA HOTAIR on hepatic stellate cell viability and apoptosis during liver injury caused by AH. In the liver tissues of AH rats, HOTAIR and S1PR1 were overexpressed, and microRNA (miR)-148a-3p was poorly expressed. Loss-of-function assays revealed that silencing of HOTAIR alleviated liver injury in AH by inhibiting the activated phenotype of hepatic stellate cells, inflammation, and fibrosis. Using the bioinformatics databases, dual-luciferase, RIP, and FISH assays, we observed that HOTAIR was mainly localized in the cytoplasm of hepatic stellate cells, and HOTAIR could bind specifically to miR-148a-3p. In addition, miR-148a-3p could target S1PR1 expression. Rescue experiments showed that silencing of miR-148a-3p or overexpression of S1PR1 reversed the alleviating effects of HOTAIR silencing on liver injury. Taken together, our findings revealed that HOTAIR regulates hepatic stellate cell proliferation via the miR-148a-3p/S1PR1 axis in liver injury, which may serve as the basis for developing novel therapeutic strategies to treat AH.

WED-524 - GTX-011 improves fibrosis and hepatic stellate cells phenotype in human precision cut liver slices

WED-524 - GTX-011 improves fibrosis and hepatic stellate cells phenotype in human precision cut liver slices

Circular RNA circRSF1 binds to HuR to promote radiation-induced inflammatory phenotype in hepatic stellate cells

To investigate whether circular RNA circRSF1 regulates radiation-induced inflammatory phenotype of hepatic stellate cells (HSCs) by binding to HuR protein and repressing its function. Human HSC cell line LX2 with HuR overexpression or knockdown was exposed to 8 Gy X-ray irradiation, and the changes in the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) were detected by qRT-PCR. The expressions of IκBα and phosphorylation of NF-κB were detected with Western blotting. The binding of circRSF1 to HuR was verified by RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP). The expressions of inflammatory factors, IκBα and the phosphorylation of NF-κB were detected after modifying the interaction between circRSF1 and HuR. Knockdown of HuR significantly up- regulated the expressions of IL-1β, IL-6 and TNF-α, decreased IκBα expression and promoted NF-κB phosphorylation in irradiated LX2 cells, whereas overexpression of HuR produced the opposite changes (P < 0.05). Overexpression or knockdown of circRSF1 did not significantly affect the expression of HuR. RNA pull-down and RIP experiments confirmed the binding between circRSF1 and HuR. Overexpression of circRSF1 significantly reduced the binding of HuR to IκBα and down-regulated the expression of IκBα (P < 0.05). Overexpression of circRSF1 combined with HuR overexpression partially reversed the up-regulation of the inflammatory factors, down-regulated IκBα expression and increased phosphorylation of NFκB in LX2 cells, while the opposite effects were observed in cells with knockdown of both circRSF1 and HuR (P < 0.05). circRSF1 reduces IκBα expression by binding to HuR to promote the activation of NF-κB pathway, thereby enhancing radiation- induced inflammatory phenotype of HSCs.

Bioactive coumarin-derivative esculetin decreases hepatic stellate cell activation via induction of cellular senescence via the PI3K-Akt-GSK3β pathway

BackgroundActivation of hepatic stellate cells (HSC) leads to initiation and progression of hepatic fibrosis. HSC senescence is inversely correlated with HSC proliferation and activation. Therefore, induction of HSC senescence may be a strategy to treat hepatic fibrosis. Coumarin-derivatives like esculetin have been suggested to inhibit proliferation of fibrogenic cells. Therefore, in this study we aimed to investigate the effect of esculetin on HSC activation and senescence. MethodsPrimary rat HSC were used in all experiments. Real-time cell analyzer and BrdU incorporation assay were used to determine HSC proliferation. Gene expression of the senescence-associated genes Cdkn1a (p21), P53, activation markers Acta2, Col1a1 and quiescence markers Pparg and Lrat were measured by RT-qPCR. Senescence associated β-Galactosidase (SA-β-Gal) staining was used to identify senescent HSC. ResultsOur results demonstrate that esculetin increased markers of senescence and decreased proliferation and activation markers in HSC. Protein expression of P21Cip1, accompanied by phosphorylation of Ser473 Akt and Ser9 GSK3β was increased by esculetin. The effect of esculetin was dependent on PI3K-Akt signaling. ConclusionWe conclude that esculetin induces HSC senescence and reverses the activated phenotype of HSC. The induction of senescence depends on PI3K-Akt-GSK3β signaling. Esculetin could be a potential therapeutic drug for the treatment of hepatic fibrosis by inducing stellate cell senescence.

CK2 blockade alleviates liver fibrosis by suppressing activation of hepatic stellate cells via the Hedgehog pathway.

Liver fibrosis is a serious cause of morbidity and mortality worldwide characterized by accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). The protein kinase CK2 is a pro-survival kinase overexpressed in human tumours. However, the biological role of CK2 in liver fibrosis is largely unknown. We aimed to investigate the mechanism by which CK2 promotes liver fibrosis. In vitro, LX-2 cells were stimulated with transforming growth factor-β (TGF-β). HSCs were also isolated for research. In vivo, the adeno-associated virus AAV-sh-csnk2a1 was used to knockdown CK2α specifically in HSCs, and CX-4945 was used to pharmacologically inhibit the enzymatic activity of CK2 in murine models of fibrosis induced by carbon tetrachloride (CCl4 ) and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. Histological and biochemical analyses were performed to study the involvement of CK2 in regulation of fibrogenic and fibrolytic factors as well as activation properties of HSCs. HSC-specific genetic invalidation of CK2α or pharmacological inhibition of CK2 protected mice treated with CCl4 or fed a DDC diet against liver fibrosis and HSC accumulation. Mechanistically, CK2α, which bound to Smoothened (SMO), was a positive regulator of the Hedgehog signal transduction pathway. CK2 prevented ubiquitination and proteasomal degradation of SMO, which was abolished by knockdown of CK2α or pharmacological inhibition of CK2. CK2 activation is critical to sustain the activated and fibrogenic phenotype of HSCs via SMO stabilization. Therefore, inactivation of CK2 by CX-4945 may be of therapeutic interest for liver fibrotic diseases.

Liver sinusoidal endothelial cell expressed vascular cell adhesion molecule 1 promotes liver fibrosis.

BackgroundDuring liver injury, liver sinusoidal endothelial cells (LSECs) dysfunction and capillarization promote liver fibrosis. We have previously reported that the LSEC vascular cell adhesion molecule 1 (VCAM1) plays a key role in liver inflammation in nonalcoholic steatohepatitis (NASH) and we now aim to uncover its role in LSEC capillarization and liver fibrosis.MethodsWild-type C57BL/6J mice were fed either chow or high fat, fructose and cholesterol diet to induce NASH and treated with either anti-VCAM1 neutralizing antibody or control isotype antibody. Inducible endothelial cell-specific Vcam1 deleted mice (Vcam1Δend ) and control mice (Vcam1fl/fl ) were fed choline-deficient high-fat diet (CD-HFD) to induce NASH or injected with carbon tetrachloride to induce liver fibrosis. LSECs isolated from Vcam1fl/fl or Vcam1Δend and hepatic stellate cells (HSCs) isolated from wild-type mice were cocultured in a 3-D system or a μ-Slide 2 well co-culture system.ResultsImmunostaining for Lyve1 (marker of differentiated LSECs) was reduced in Vcam1fl/fl mice and restored in Vcam1Δend mice in both NASH and liver fibrosis models. Co-immunostaining showed increased α-smooth muscle actin in the livers of Vcam1fl/fl mice in areas lacking Lyve1. Furthermore, scanning electron microscopy showed reduced LSEC fenestrae in the Vcam1fl/fl mice but not Vcam1Δend mice in both injury models, suggesting that VCAM1 promotes LSEC capillarization during liver injury. HSCs profibrogenic markers were reduced when cocultured with LSECs from CD-HFD fed Vcam1Δend mice compared to Vcam1fl/fl mice. Furthermore, recombinant VCAM1 activated the Yes-associated protein 1 pathway and induced a fibrogenic phenotype in HSCs in vitro, supporting the profibrogenic role of LSEC VCAM1.ConclusionVCAM1 is not just a scaffold for leukocyte adhesion during liver injury, but also a modulator of LSEC capillarization and liver fibrosis.

Modulation of miR‐21 can reprogram the TGF‐β signaling pathway to alter HSC phenotype in vitro

microRNAs are small non‐coding RNAs capable of modulating gene expression through post‐transcriptional modification affecting fundamental regulatory mechanisms to control cell function and play a significant role in the transition between cell states. Previous studies have demonstrated the differential expression of specific microRNAs in the context of disease states including alcoholic liver disease. Research from our group and others have identified several miRNAs as potential regulators of liver regeneration, such as miR‐21, whose role in liver regeneration and liver disease is well‐characterized, and several other miRNAs that show differential expression in response to altered miR‐21 expression. Dysregulation of miR‐21 expression has been implicated in altered hepatic stellate cell (HSC) activation. A potential mechanism for ethanol‐mediated impairment of liver regeneration is due to TGF‐β/SMAD/SMURF signaling in activated HSCs. In this study, we have characterized the HSCs response to miR‐21 dysregulation followed by TGF‐β stimulation using human hepatic stellate cell line LX2. We manipulated miR‐21 activity using locked nucleic acid oligos to either inhibit or mimic miR‐21. Our findings show that inhibition of miR‐21 activity followed by TGF‐β stimulation leads to the altered expression of components of the TGF‐β signaling pathway that in effect attenuated the downstream response to TGF‐β. By contrast, enhancing miR‐21 activity using a mimic amplified response to TGF‐β signal. This reprogramming of the TGF‐β pathway by exogenous alterations in miR‐21 activity was reflected in the modulation of HSC activation signature and migration phenotype. Taken together, our results show that changes in miR‐21 activity rewires the TGF‐β signaling network with consequences on HSC phenotype in vitro.

Empagliflozin Reduces the Progression of Hepatic Fibrosis in a Mouse Model and Inhibits the Activation of Hepatic Stellate Cells via the Hippo Signalling Pathway.

Hepatic fibrosis is the excessive production and deposition of the extracellular matrix, resulting in the activation of the fibrogenic phenotype of hepatic stellate cells (HSCs). The Hippo/Yes-associated protein (YAP) signalling pathway is a highly conserved kinase cascade that is critical in regulating cell proliferation, differentiation, and survival, and controls stellate cell activation. Empagliflozin, a sodium-glucose cotransporter type-2 inhibitor, is an antidiabetic drug that may prevent fibrotic progression by reducing hepatic steatosis and inflammation. However, little is known about its mechanism of action in liver fibrosis. In this study, we used male C57 BL/6 J mice fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) as a model for hepatic fibrosis. For 5 weeks, the mice received either a vehicle or empagliflozin based on their assigned group. Empagliflozin attenuated CDAHFD-induced liver fibrosis. Thereafter, we identified the Hippo pathway, along with its effector, YAP, as a key pathway in the mouse liver. Hippo signalling is inactivated in the fibrotic liver, but empagliflozin treatment activated Hippo signalling and decreased YAP activity. In addition, empagliflozin downregulated the expression of pro-fibrogenic genes and activated Hippo signalling in HSCs. We identified a mechanism by which empagliflozin ameliorates liver fibrosis.

Macrophage MerTK promotes a profibrogenic cross-talk with hepatic stellate cells via soluble mediators

Background and aims: Activation of Kupffer cells and recruitment of monocytes are key events in fibrogenesis. These cells release soluble mediators which induce the activation of hepatic stellate cells (HSC), the main fibrogenic cell type within the liver. Mer tyrosine kinase (MerTK) signaling regulates multiple processes in macrophages and has been implicated in the pathogenesis of NASH-related fibrosis. In this study, we explored if MerTK activation in macrophages influences the profibrogenic phenotype of HSCs.

Hepatic stellate cell activation and senescence induced by intrahepatic microbiota disturbances drive progression of liver cirrhosis toward hepatocellular carcinoma.

BackgroundThe significance of the relationship between the microbiota and diseases is increasingly being recognized. However, the characterization of tumor microbiome and their precise molecular mechanisms through which microbiota promotes hepatocellular carcinoma (HCC) development are still unclear.MethodsThe intrahepatic microbiota was investigated from tumor, normal adjacent tissues in 46 patients with HCC and normal hepatic tissues in 33 patients with hemangioma by 16S rRNA gene sequencing. Taxonomic composition differences in patients were evaluated using Linear discriminant analysis Effect Size (LefSe) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to predict microbial functional pathways. Associations between the most relevant taxa and clinical characteristics of HCC patients were analyzed by Spearman rank correlations. The effects of microbe on hepatic stellate cells (HSCs) activation and HCC progression were examined.ResultsWe observed intrahepatic microbiota disturbances by reduced microbial diversity in HCC. The tumor microbiota of the HCC patients with cirrhosis showed higher abundance of Stenotrophomonas maltophilia (S. maltophilia). S. maltophilia provoked senescence-associated secretory phenotype (SASP) in HSCs by activating TLR-4-mediated NF-κB signaling pathway, which in turn induced NLRP3 inflammasome complex formation and secreted various inflammatory factors in the liver, thus facilitating HCC progression in mice. Moreover, signs of SASP were also observed in the HSCs in the area of HCC with higher S. maltophilia enrichment arising in patients with cirrhosis.ConclusionsOur analysis of the hepatic microbiota revealed for the first time that patients with HCC exhibited a dysbiotic microbial community with higher S. maltophilia abundance, which induced the expression SASP factors of HSCs and cirrhosis in the liver, concurring in the process of hepatocarcinogenesis.

High throughput interrogation of human liver stellate cells reveals microenvironmental regulation of phenotype

Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. Statement of significanceIn this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics.

Human Liver Stem Cell-Derived Extracellular Vesicles Target Hepatic Stellate Cells and Attenuate Their Pro-fibrotic Phenotype.

Liver fibrosis occurs in response to chronic liver injury and is characterized by an excessive deposition of extracellular matrix. Activated hepatic stellate cells are primarily responsible for this process. A possible strategy to counteract the development of hepatic fibrosis could be the reversion of the activated phenotype of hepatic stellate cells. Extracellular vesicles (EVs) are nanosized membrane vesicles involved in intercellular communication. Our previous studies have demonstrated that EVs derived from human liver stem cells (HLSCs), a multipotent population of adult stem cells of the liver with mesenchymal-like phenotype, exert in vivo anti-fibrotic activity in the liver. However, the mechanism of action of these EVs remains to be determined. We set up an in vitro model of hepatic fibrosis using a human hepatic stellate cell line (LX-2) activated by transforming growth factor-beta 1 (TGF-β1). Then, we investigated the effect of EVs obtained from HLSCs and from human bone marrow-derived mesenchymal stromal cells (MSCs) on activated LX-2. The incubation of activated LX-2 with HLSC-EVs reduced the expression level of alpha-smooth muscle actin (α-SMA). Conversely, MSC-derived EVs induced an increase in the expression of pro-fibrotic markers in activated LX-2. The analysis of the RNA cargo of HLSC-EVs revealed the presence of several miRNAs involved in the regulation of fibrosis and inflammation. Predictive target analysis indicated that several microRNAs (miRNAs) contained into HLSC-EVs could possibly target pro-fibrotic transcripts. In particular, we demonstrated that HLSC-EVs shuttled miR-146a-5p and that treatment with HLSC-EVs increased miR-146a-5p expression in LX-2. In conclusion, this study demonstrates that HLSC-EVs can attenuate the activated phenotype of hepatic stellate cells and that their biological effect may be mediated by the delivery of anti-fibrotic miRNAs, such as miR-146a-5p.

HBeAg mediates inflammatory functions of macrophages by TLR2 contributing to hepatic fibrosis

BackgroundWe and others have confirmed activation of macrophages plays a critical role in liver injury and fibrogenesis during HBV infection. And we have also proved HBeAg can obviously induce the production of macrophage inflammatory cytokines compared with HBsAg and HBcAg. However, the receptor and functional domain of HBeAg in macrophage activation and its effects and mechanisms on hepatic fibrosis remain elusive.MethodsThe potentially direct binding receptors of HBeAg were screened and verified by Co-IP assay. Meanwhile, the function domain and accessible peptides of HBeAg for macrophage activation were analyzed by prediction of surface accessible peptide, construction, and synthesis of truncated fragments. Furthermore, effects and mechanisms of the activation of hepatic stellate cells induced by HBeAg-treated macrophages were investigated by Transwell, CCK-8, Gel contraction assay, Phospho Explorer antibody microarray, and Luminex assay. Finally, the effect of HBeAg in hepatic inflammation and fibrosis was evaluated in both human and murine tissues by immunohistochemistry, immunofluorescence, ELISA, and detection of liver enzymes.ResultsHerein, we verified TLR-2 was the direct binding receptor of HBeAg. Meanwhile, C-terminal peptide (122-143 aa.) of core domain in HBeAg was critical for macrophage activation. But arginine-rich domain of HBcAg hided this function, although HBcAg and HBeAg shared the same core domain. Furthermore, HBeAg promoted the proliferation, motility, and contraction of hepatic stellate cells (HSCs) in a macrophage-dependent manner, but not alone. PI3K-AKT-mTOR and p38 MAPK signaling pathway were responsible for motility phenotype of HSCs, while the Smad-dependent TGF-β signaling pathway for proliferation and contraction of them. Additionally, multiple chemokines and cytokines, such as CCL2, CCL5, CXCL10, and TNF-α, might be key mediators of HSC activation. Consistently, HBeAg induced transient inflammation response and promoted early fibrogenesis via TLR-2 in mice. Finally, clinical investigations suggested that the level of HBeAg is associated with inflammation and fibrosis degrees in patients infected with HBV.ConclusionsHBeAg activated macrophages via the TLR-2/NF-κB signal pathway and further exacerbated hepatic fibrosis by facilitating motility, proliferation, and contraction of HSCs with the help of macrophages.