Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation.

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR, Western blot and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1-treated Schistosoma-infected mice for RNA-sequencing analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in the transdifferentiation of HSCs to myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg-induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum.

Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway.

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.

AICAR and compound C negatively modulate HCC-induced primary human hepatic stellate cell activation in vitro.

Tumor stroma and microenvironment have been shown to affect hepatocellular carcinoma (HCC) growth, with activated hepatic stellate cells (HSC) as a major contributor in this process. Recent evidence suggests that the energy sensor adenosine monophosphate-activated kinase (AMPK) may mediate a series of essential processes during carcinogenesis and HCC progression. Here, we investigated the effect of different HCC cell lines with known TP53 or CTNBB1 mutations on primary human HSC activation, proliferation, and AMPK activation. We show that conditioned media obtained from multiple HCC cell lines differently modulate human hepatic stellate cell (hHSC) proliferation and hHSC AMPK activity in a paracrine manner. Pharmacological treatment of hHSC with AICAR and Compound C inhibited the HCC-induced proliferation/activation of hHSC through AMPK-dependent and AMPK-independent mechanisms, which was further confirmed using mouse embryonic fibroblasts (MEFs) deficient of both catalytic AMPKα isoforms (AMPKα1/α2-/-) and wild type (wt) MEF. Both compounds induced S-phase cell-cycle arrest and, in addition, AICAR inhibited the mTORC1 pathway by inhibiting phosphorylation of 4E-BP1 and S6 in hHSC and wt MEF. Data mining of the Cancer Genome Atlas (TCGA) and the Liver Cancer (LICA-FR) showed that AMPKα1 (PRKAA1) and AMPKα2 (PRKAA2) expression differed depending on the mutation (TP53 or CTNNB1), tumor grading, and G1-G6 classification, reflecting the heterogeneity in human HCC. Overall, we provide evidence that AMPK modulating pharmacological agents negatively modulate HCC-induced hHSC activation and may therefore provide a novel approach to target the mutual, tumor-promoting interactions between hHSC and HCC.NEW & NOTEWORTHY HCC is marked by genetic heterogeneity and activated hepatic stellate cells (HSC) are considered key players during HCC development. The paracrine effect of different HCC cell lines on the activation of primary hHSC was accompanied by differential AMPK activation depending on the HCC line used. Pharmacological treatment inhibited the HCC-induced hHSC activation through AMPK-dependent and AMPK-independent mechanisms. This heterogenic effect on HCC-induced AMPK activation was confirmed by data mining TCGA and LICA-FR databases.

Therapeutic effect of uridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

SOD3 deficiency induces liver fibrosis by promoting hepatic stellate cell activation and epithelial-mesenchymal transition.

Hepatic stellate cell (HSC) activation plays an important role in the pathogenesis of liver fibrosis, and epithelial-mesenchymal transition (EMT) is suggested to potentially promote HSC activation. Superoxide dismutase 3 (SOD3) is an extracellular antioxidant defense against oxidative damage. Here, we found downregulation of SOD3 in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl4 ). SOD3 deficiency induced spontaneous liver injury and fibrosis with increased collagen deposition, and further aggravated CCl4 -induced liver injury in mice. Depletion of SOD3 enhanced HSC activation marked by increased α-smooth muscle actin and subsequent collagen synthesis primarily collagen type I in vivo, and promoted transforming growth factor-ß1 (TGF-ß1)-induced HSC activation in vitro. SOD3 deficiency accelerated EMT process in the liver and TGF-ß1-induced EMT of AML12 hepatocytes, as evidenced by loss of E-cadherin and gain of N-cadherin and vimentin. Notably, SOD3 expression and its pro-fibrogenic effect were positively associated with sirtuin 1 (SIRT1) expression. SOD3 deficiency inhibited adenosine monophosphate-activated protein kinase (AMPK) signaling to downregulate SIRT1 expression and thus involving in liver fibrosis. Enforced expression of SIRT1 inhibited SOD3 deficiency-induced HSC activation and EMT, whereas depletion of SIRT1 counteracted the inhibitory effect of SOD3 in vitro. These findings demonstrate that SOD3 deficiency contributes to liver fibrogenesis by promoting HSC activation and EMT process, and suggest a possibility that SOD3 may function through modulating SIRT1 via the AMPK pathway in liver fibrosis.

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 in vitro and in vivo 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 in vitro. 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 in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.

Curcumin reduces methionine adenosyltransferase 2B expression by interrupting phosphorylation of p38 MAPK in hepatic stellate cells.

The active polyphenol curcumin demonstrates therapeutic effects against various different diseases. Researches revealed the inhibitory roles of curcumin in hepatic stellate cell (HSC) activation and fibrogenesis. HSC activation, a key step in liver fibrogenesis, requires the remodeling of DNA methylation, which is associated with methionine adenosyltransferase II (MATII) composed of catalytic subunit MAT2A and regulatory subunit MAT2B. MATII is essential for HSC activation in vitro. The present researches aimed to investigate the effect of curcumin on MAT2B expression in HSCs in vivo and in vitro. Results demonstrated that curcumin could reduce MAT2B expression in HSCs at multiple levels. The activation of p38 MAPK pathway promoted MAT2B expression in HSCs. The effect of curcumin on MAT2B was through its interruption of p38 MAPK signaling pathway. Knockdown of MAT2B inhibited HSC activation and reduced collagen level in the model of liver fibrosis. Curcumin down-regulation of MAT2B contributed to the inhibitory role of curcumin on HSC activation and collagen expression in mouse livers. This study provided evidences for the effect of curcumin on the expression of MAT2B, an enzyme for the biosynthesis of methyl donor S-adenosylmethionine, in HSCs and demonstrated the function significance of curcumin-induced downregulation of MAT2B in curcumin inhibition of liver fibrosis.

Arachidonate 12S-lipoxygenase of platelet-type in hepatic stellate cells of methionine and choline-deficient diet-fed mice.

A role of 12-lipoxygenase in the progression of non-alcoholic steatohepatitis (NASH) is suggested, although the underlying mechanism is not entirely understood. The catalytic activity of 12S-lipoxygenase that was hardly observed in liver cytosol of normal chow-fed mice was clearly detectable in that of NASH model mice prepared by feeding a methionine and choline-deficient (MCD) diet. The product profile, substrate specificity and immunogenicity indicated that the enzyme was the platelet-type isoform. The expression levels of mRNA and protein of platelet-type 12S-lipoxygenase in the liver of MCD diet-fed mice were significantly increased compared with those of normal chow-fed mice. Immunohistochemical analysis showed that platelet-type 12S-lipoxygenase colocalized with α-smooth muscle actin as well as vitamin A in the cells distributing along liver sinusoids. These results indicate that the expression level of platelet-type 12S-lipoxygenase in hepatic stellate cells was increased during the cell activation in MCD diet-fed mice, suggesting a possible role of the enzyme in pathophysiology of liver fibrosis.

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/ß-catenin signalling pathway.

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl4 -induced liver fibrosis mice and LX-2 cells stimulated with TGF-ß1. Knockdown of PLK1 inhibited α-SMA and Col1α1 expression and reduced the activation of HSCs in CCl4 -induced liver fibrosis mice and LX-2 cells stimulated with TGF-ß1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/ß-catenin signalling pathway may be essential for PLK1-mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.

Alamandine attenuates hepatic fibrosis by regulating autophagy induced by NOX4-dependent ROS.

Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin-angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.

Role of spleen tyrosine kinase in liver diseases.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase expressed in most hematopoietic cells and non-hematopoietic cells and play a crucial role in both immune and non-immune biological responses. SYK mediate diverse cellular responses via an immune-receptor tyrosine-based activation motifs (ITAMs)-dependent signalling pathways, ITAMs-independent and ITAMs-semi-dependent signalling pathways. In liver, SYK expression has been observed in parenchymal (hepatocytes) and non-parenchymal cells (hepatic stellate cells and Kupffer cells), and found to be positively correlated with the disease severity. The implication of SYK pathway has been reported in different liver diseases including liver fibrosis, viral hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis and hepatocellular carcinoma. Antagonism of SYK pathway using kinase inhibitors have shown to attenuate the progression of liver diseases thereby suggesting SYK as a highly promising therapeutic target. This review summarizes the current understanding of SYK and its therapeutic implication in liver diseases.

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 ß (TGF-ß)-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.

Cellular and molecular effects of silymarin on the transdifferentiation processes of LX-2 cells and its connection with lipid metabolism.

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.

AGAP2: Modulating TGFß1-Signaling in the Regulation of Liver Fibrosis.

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-ß1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-ß1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFß1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis.

Targeting Src family kinase member Fyn by Saracatinib attenuated liver fibrosis in vitro and in vivo.

Recent studies suggest that Src family kinase (SFK) plays important roles in systemic sclerosis and pulmonary fibrosis. However, how SFKs contributed to the pathogenesis of liver fibrosis remains largely unknown. Here, we investigated the role of Fyn, a member of SFK, in hepatic stellate cell (HSC) activation and liver fibrosis, and evaluated the anti-fibrotic effects of Saracatinib, a clinically proven safe Fyn inhibitor. Fyn activation was examined in human normal and fibrotic liver tissues. The roles of Fyn in HSC activation and liver fibrosis were evaluated in HSC cell lines by using Fyn siRNA and in Fyn knockout mice. The effects of Saracatinib on HSC activation and liver fibrosis were determined in primary HSCs and CCl4 induced liver fibrosis model. We showed that the Fyn was activated in the liver of human fibrosis patients. TGF-ß induced the activation of Fyn in HSC cell lines. Knockdown of Fyn significantly blocked HSC activation, proliferation, and migration. Fyn deficient mice were resistant to CCl4 induced liver fibrosis. Saracatinib treatment abolished the activation of Fyn, downregulated the Fyn/FAK/N-WASP signaling in HSCs, and subsequently prevented the activation of HSCs. Saracatinib treatment significantly reduced the severity liver fibrosis induced by CCl4 in mice. In conclusions, our findings supported the critical role of Fyn in HSC activation and development of liver fibrosis. Fyn could serve as a promising drug target for liver fibrosis treatment. Fyn inhibitor Saracatinib significantly inhibited HSC activation and attenuated liver fibrosis in mouse model.

Dynamic Changes in Function and Proteomic Composition of Extracellular Vesicles from Hepatic Stellate Cells during Cellular Activation.

: During chronic liver injury, hepatic stellate cells (HSC) undergo activation and are the principal cellular source of collagenous scar. In this study, we found that activation of mouse HSC (mHSC) was associated with a 4.5-fold increase in extracellular vesicle (EV) production and that fibrogenic gene expression (CCN2, Col1a1) was suppressed in Passage 1 (P1; activated) mHSC exposed to EVs from Day 4 (D4; relatively quiescent) mHSC but not to EVs from P1 mHSC. Conversely, gene expression (CCN2, Col1a1, αSMA) in D4 mHSC was stimulated by EVs from P1 mHSC but not by EVs from D4 mHSC. EVs from Day 4 mHSC contained only 46 proteins in which histones and keratins predominated, while EVs from P1 mHSC contained 337 proteins and these were principally associated with extracellular spaces or matrix, proteasome, collagens, vesicular transport, metabolic enzymes, ribosomes and chaperones. EVs from the activated LX-2 human HSC (hHSC) line also promoted fibrogenic gene expression in D4 mHSC in vitro and contained 524 proteins, many of which shared identity or had functional overlap with those in P1 mHSC EVs. The activation-associated changes in production, function and protein content of EVs from HSC likely contribute to the regulation of HSC function in vivo and to the fine-tuning of fibrogenic pathways in the liver.

Carvedilol Inhibits Angiotensin II-Induced Proliferation and Contraction in Hepatic Stellate Cells through the RhoA/Rho-Kinase Pathway.

AIM: Carvedilol is a nonselective beta-blocker used to reduce portal hypertension. This study investigated the effects and potential mechanisms of carvedilol in angiotensin II- (Ang II-) induced hepatic stellate cell (HSC) proliferation and contraction. METHODS: The effect of carvedilol on HSC proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell cycle progression and apoptosis in HSCs were determined by flow cytometry. A collagen gel assay was used to confirm HSC contraction. The extent of liver fibrosis in mice was evaluated by hematoxylin-eosin (H&E) and Sirius Red staining. Western blot analyses were performed to detect the expression of collagen I, collagen III, α-smooth muscle actin (α-SMA), Ang II type I receptor (AT1R), RhoA, Rho-kinase 2 (ROCK2), and others. RESULTS: The results showed that carvedilol inhibited HSC proliferation and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Carvedilol also modulated Bcl-2 family proteins and increased apoptosis in Ang II-treated HSCs. Furthermore, carvedilol inhibited HSC contraction induced by Ang II, an effect that was associated with AT1R-mediated RhoA/ROCK2 pathway interference. In addition, carvedilol reduced α-SMA expression and collagen deposition and attenuated liver fibrosis in carbon tetrachloride (CCl4)-treated mice. The in vivo data further confirmed that carvedilol inhibited the expression of angiotensin-converting enzyme (ACE), AT1R, RhoA, and ROCK2. CONCLUSIONS: The results indicated that carvedilol dose-dependently inhibited Ang II-induced HSC proliferation by impeding cell cycle progression, thus alleviating hepatic fibrosis. Furthermore, carvedilol could inhibit Ang II-induced HSC contraction by interfering with the AT1R-mediated RhoA/ROCK2 pathway.

Regulation of Fibrotic Processes in the Liver by ADAM Proteases.

Fibrosis in the liver is mainly associated with the activation of hepatic stellate cells (HSCs). Both activation and clearance of HSCs can be mediated by ligand-receptor interactions. Members of the a disintegrin and metalloprotease (ADAM) family are involved in the proteolytic release of membrane-bound ligands and receptor ectodomains and the remodelling of the extracellular matrix. ADAM proteases are therefore major regulators of intercellular signalling pathways. In the present review we discuss how ADAM proteases modulate pro- and anti-fibrotic processes and how ADAM proteases might be harnessed therapeutically in the future.

Soluble egg antigen of Schistosoma japonicum induces pyroptosis in hepatic stellate cells by modulating ROS production.

BACKGROUND: Inflammation-induced dysfunction of hepatic stellate cells (HSCs) is involved in schistosomiasis-associated liver fibrosis, and soluble egg antigen (SEA) is a crucial pathogen-associated molecular pattern associated with liver injury in schistosomiasis. In addition, numerous studies have shown that caspase-1-mediated pyroptosis participates in the development of multiple inflammation-related diseases. However, whether pyroptotic cell death of HSCs is involved in SEA-mediated liver damage is not well understood. METHODS: Primary cultured HSCs and Schistosoma japonicum-infected mouse liver tissue were analysed for histological changes and caspase-1 activation, and the role of pyroptosis in the mechanisms underlying SEA-induced HSC death was investigated. Accumulation of reactive oxygen species (ROS) in infected livers and SEA-stimulated HSCs was measured by flow cytometry and immunofluorescence. RESULTS: Caspase-1 activity was elevated in both liver tissues and HSCs of S. japonicum-infected mice. Furthermore, SEA stimulation increased the proportion of pyroptotic HSCs, as shown by lactate dehydrogenase (LDH) release assays and by flow cytometric analysis of propidium iodide (PI) and caspase-1 double staining in cells. In addition, ROS generation was elevated in infected liver tissues and SEA-stimulated HSCs, and ROS inhibition downregulated SEA-induced caspase-1 activation and pyroptosis in HSCs. CONCLUSIONS: Our present study demonstrates that pyroptotic cell death in HSCs induced by SEA via ROS-mediated caspase-1 activation may serve as a significant mechanism to initiate the inflammatory response and thereby exacerbate liver injury during S. japonicum infection.