Human Hepatic Stellate Cells Research Articles

The quantitative proteomic analysis reveals schisantherin a prevents liver fibrosis through regulating extracellular matrix organization

The activation of hepatic stellate cell (HSC) induced by transforming growth factor-β1 (TGF-β1) is the key event in the pathogenesis of liver fibrosis. Schisantherin A (SCA), a main active ingredient of Schisandra chinensis, has anti-tumor and anti-inflammatory activities. Here, we reported that SCA inhibited the activation, proliferation, and cell cycle of human HSC cell line LX2. Then, by performing the tandem mass tag (TMT)-based quantitative proteomic analysis on LX2, we identified a total of 6045 proteins across the control, TGF-β1-activated and SCA treated LX2 groups, of which 544 proteins were significantly changed among the three groups. All the differentially expressed proteins (DEPs) were assigned to 4 clusters by fuzzy c-means (FCM) clustering analysis. The changed expression of DEPs in cluster 3 and 4 was reversed by SCA treatment. Bioinformatic analysis revealed that SCA regulated the expression of several DEPs involved in extracellular matrix organization, such as thrombospondin 1 (THBS1), transgelin (TAGLN) and tissue inhibitor of metalloproteinase 3 (TIMP3). The Western blot and RT-qPCR analysis confirmed these proteins increased in TGF-β1 group and decreased by the SCA treatment. In summary, we found that SCA may exert anti-fibrotic effect on HSCs by regulating the process of extracellular matrix organization in HSCs. Keywords: liver fibrosis, schisantherin A, proteomics, extracellular matrix organization.

Three new feruloyl glucuronopyranosyl glycerols from Eriocaulon buergerianum with their anti-fibrotic effects on hepatic stellate cells

Three new feruloyl glucuronopyranosyl glycerols, 1-O-α-D-(2′-feruloyl) glucuronopyranosyl glycerol (1), 1-O-α-D-(3′-feruloyl) glucuronopyranosyl glycerol (2), and 1-O-α-D-(4′-feruloyl) glucuronopyranosyl glycerol (3) were isolated and purified from the capitulum of Eriocaulon buergerianum Koern. Their structures were identified by spectroscopic and chemical methods. Molecular docking study showed that 1 is a potential inhibitor of TGF-β1 receptor. Bioassay results revealed that 1 exhibited strong inhibitory activity against the TGF-β-induced expression of α-smooth muscle actin (α-SMA) and fibronectin in human hepatic stellate LX-2 cells. The results in this study indicated that the new feruloyl glucuronopyranosyl glycerol 1 has preventive and therapeutic potentials against hepatic fibrosis.

Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more β-actin was localized in the cortical layer under SMG.

SAT025 - Butyrate protects against diet-induced NASH and liver fibrosis and suppresses specific non-canonical TGFB signaling pathways in human hepatic stellate cells

SAT025 - Butyrate protects against diet-induced NASH and liver fibrosis and suppresses specific non-canonical TGFB signaling pathways in human hepatic stellate cells

SAT008 - Transcriptomic analysis confirms that PNPLA3 I148M variant is associated with impaired mitochondrial function and antioxidant response in 3D cultured human hepatic stellate cells and liver tissue of patients with NAFLD

SAT008 - Transcriptomic analysis confirms that PNPLA3 I148M variant is associated with impaired mitochondrial function and antioxidant response in 3D cultured human hepatic stellate cells and liver tissue of patients with NAFLD

Antiproliferative and Antimicrobial Effects of Rosmarinus officinalis L. Loaded Liposomes.

Rosmarinus officinalis L. is a species that is widely known for its culinary and medicinal uses. The purpose of the present study consisted of the evaluation of the antiproliferative and antimicrobial effects of R. officinalis-loaded liposomes (L-R). Characterization of the liposomes was performed by establishing specific parameters. The load of the obtained liposomes was analyzed using an LC-MS method, and antiproliferative assays evaluated the cell viability on a liver adenocarcinoma cell line and on a human hepatic stellate cell line. Antimicrobial assays were performed by agar–well diffusion and by broth microdilution assays. The obtained liposomes showed high encapsulation efficiency, suitable particle size, and good stability. High amounts of caffeic (81.07 ± 0.76), chlorogenic (14.10 ± 0.12), carnosic (20.03 ± 0.16), rosmarinic (39.81 ± 0.35), and ellagic (880.02 ± 0.14) acids were found in their composition, together with other polyphenols. Viability and apoptosis assays showed an intense effect on the cancerous cell line and a totally different pattern on the normal cells, indicating a selective toxicity towards the cancerous ones and an anti-proliferative mechanism. Antimicrobial potential was noticed against all tested bacteria, with a better efficacy towards Gram-positive species. These results further confirm the biological activities of R. officinalis leaf extract, and proposes and characterizes novel delivery systems for their encapsulation, enhancing the biological activities of polyphenols, and overcoming their limitations.

Dehydromevalonolactone ameliorates liver fibrosis and inflammation by repressing activation of NLRP3 inflammasome

Liver fibrosis is an important process in chronic liver disease and is strongly related to poor prognosis. Dehydromevalonolactone (C8) is a natural product isolated from a fungus of Fusarium sp. CPCC 401218, and its pharmacological activity has never been reported before. In this study, the potential of C8 as an anti-hepatic fibrosis agent was investigated. In human hepatic stellate cell (HSC) line LX-2, C8 suppressed the increased expression of COL1A1 and α-SMA induced by TGFβ1, which indicated that C8 could repress the activation of HSCs. In bile duct ligated rats, C8 administration (100 mg/kg, i.p.) markedly attenuated liver injury, fibrosis, and inflammation, and suppressed the expression of the macrophage surface marker F4/80. In terms of mechanism, C8 treatment blocked the activation of the NLRP3 inflammasome, which was stimulated by LPS and nigericin in bone marrow-derived macrophages (BMDMs) and companied by the release of active IL-1β. In addition, the activation of LX-2 cells induced by IL-1β released from BMDMs was also inhibited after C8 administration, which indicated that C8 repressed HSCs activation by inhibiting the activation of NLRP3 inflammasome in macrophages. Furthermore, C8 exhibited the effects of anti-fibrosis and inhibiting the expression of NLRP3 inflammasome in non-alcoholic steatohepatitis (NASH) mice. Finally, C8 can be commendably absorbed in vivo and was safe for mice at the concentration of 1000 mg/kg (p.o.). In summary, our study reveals that C8 ameliorates HSCs activation and liver fibrosis in cholestasis rats and NASH mice by inhibiting NLRP3 inflammasome in macrophages, and C8 might be a safe and effective candidate for the treatment of liver fibrosis.

Improved anti-fibrotic effects by combined treatments of simvastatin and NS-398 in experimental liver fibrosis models.

Background/AimsEfficient anti-fibrotic therapies are required for the treatment of liver cirrhosis. Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) and cyclooxygenase-2 (COX-2) inhibitors have been reported to have anti-fibrotic effects. Here, we investigated whether combined treatment with a statin and a COX-2 inhibitor has synergistic anti-fibrotic effects.MethodsThe effects of treatment strategies incorporating both simvastatin and a COX-2 inhibitor, NS-398, were investigated using an immortalized human hepatic stellate cell line (LX-2) and a hepatic fibrosis mouse model developed using thioacetamide (TAA) in drinking water. Cellular proliferation was investigated via 5-bromo-2-deoxyuridine uptake. Pro- and anti-apoptotic factors were investigated through Western blotting and real-time polymerase chain reaction analysis.ResultsThe evaluation of the anti-proliferative effects on LX-2 cells showed that the observed effects were more pronounced with combination therapy than with single-drug therapy. Moreover, hepatic fibrosis and collagen deposition decreased significantly in TAA-treated mice in response to the combined treatment strategy. The mechanisms underlying the anti-fibrotic effects of the combination therapy were investigated. The effects of the combination therapy were correlated with increased expression levels of extracellular signal-regulated kinase 1/2 signaling molecules, upregulation of the Bax/Bcl-2 signaling pathway, inhibition of the transforming growth factor-β signaling pathway, and inhibition of tissue inhibitor of matrix metalloproteinases 1 and 2.ConclusionsThe combination of simvastatin and NS-398 resulted in a synergistic anti-fibrotic effect through multiple pathways. These findings offer a theoretical insight into the possible clinical application of this strategy for the treatment of advanced liver diseases with hepatic fibrosis.

Upregulation of GLT25D1 in Hepatic Stellate Cells Promotes Liver Fibrosis via the TGF-β1/SMAD3 Pathway In Vivo and In vitro.

Collagen β(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-β1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLC-MS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-β1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

Nanchangmycin regulates FYN, PTK2, and MAPK1/3 to control the fibrotic activity of human hepatic stellate cells.

Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.

Hepatocyte-derived VEGFA accelerates the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma via activating hepatic stellate cells.

Non-alcoholic fatty liver disease (NAFLD) is emerging as an epidemic risk factor for hepatocellular carcinoma (HCC). The progression of NAFLD to HCC is closely associated with paracrine communication among hepatic cells. Vascular endothelial growth factor A (VEGFA) plays a key role in NAFLD and HCC; however, the cellular communication of VEGFA in the pathological transition from NAFLD to HCC remains unclear. Here, we found that VEGFA elevation was considerably distributed in hepatocytes of clinical and murine NAFLD-HCC specimens. Notably, progression from NAFLD to HCC was attenuated in hepatocyte-specific deletion of Vegfa (VegfaΔhep) mice. Mechanistically, VEGFA activated human hepatic stellate cell (HSC) LX2 into a fibrogenic phenotype via VEGF-VEGFR signaling in fatty acid medium, and HSC activation was largely attenuated in VegfaΔhep mice during NAFLD-HCC progression. Additionally, a positive correlation between VEGFA and hepatic fibrosis was observed in the NAFLD-HCC cohort, but not in the HBV-HCC cohort. Moreover, LX2 cells could be activated by conditioned medium from NAFLD-derived organoids, but not from HBV livers, whereas this activation was blocked by a VEGFA antibody. In summary, our findings reveal that hepatocyte-derived VEGFA contributes to NAFLD-HCC development by activating HSCs and highlight the potential of precisely targeting hepatocytic VEGFA as a promising therapeutic strategy for NAFLD-HCC.

MicroRNA‐34a modulates the senescence of activated hepatic stellate cells in alcohol‐associated liver injury

BackgroundReversion of activated hepatic stellate cells (HSCs) to quiescence is likely to be a promising pathway in liver fibrosis regression. The development of senescence in HSCs during alcoholic liver injury may lead to liver compensation against fibrosis. The current study aims to characterize the functional role of miR‐34a regulated cellular senescence during alcohol‐associated hepatitis.MethodsSenescence related gene and miR‐34a expression was assessed using a PCR Array and/or real‐time PCR analysis. Cellular senescence in cultured HSCs were measured by SA‐β‐gal staining. Senescence mediators were defined in LPS treated hepatic stellate cells in vitro, and in TLR‐4 knockout mice or morpholino antisense oligomer against miR‐34a (miR‐34a Morpho/AS) treated mice with chronic ethanol feeding in vivo.ResultsWe identified that 5 weeks of ethanol feeding significantly increased the total liver histopathology score and miR‐34a expression, along with the activation of hepatic stellate cells marked by enhanced α‐SMA staining. Treatment of HSCs with LPS (20 ng/ml) for 24 hr significantly increased miR‐34a expression, along with the enhanced SA‐β‐gal activity, up‐regulated α‐SMA/Collagen A1 expression and increased cellular viability. Silencing of miR‐34a decreased LPS‐induced activation in HSCs by downregulation of fibrosis markers α‐SMA, Col1a1 and TIMP‐1, and increased SA‐β‐gal activity as well as the cellular senescence marker CCl2, whereas silencing of the LPS receptor, TLR4, decreased this marker in the same group of HSCs, suggesting anti‐miR‐34a reversed LPS‐mediated HSC activation through inducing cellular senescence. Furthermore, the expression of miR‐34a and verified miR‐34a related senescence marker CCl2 were significantly altered in hepatic stellate cells from ethanol‐fed mouse liver specimens compared to controls. TLR4 knockout mice and miR‐34a Morpho/AS treated mice displayed less sensitivity to alcoholic injury, along with enhanced SA‐β‐gal activity and CCl2 level in HSCs, and recovered expressions of α‐SMA, Col1a1 and TIMP‐1. Summary andConclusionOur results show that miR‐34a mediated cellular senescence is essential for the potential reversion from activated to quiescent hepatic stellate cells during alcohol‐associated hepatitis. These findings provide new insight into the function of microRNA regulatedcellular senescence in human HSCs and increase opportunities for the development of novel treatment paradigms for the management of alcohol‐associated liver diseases.

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.

FoxO1 Promotes Liver Fibrosis through TGF‐β1 mediated Hepatic Stellate Cell Activation

The O‐class of the forkhead transcription factor FoxO1 is a crucial factor mediating the action of insulin→PI3K→Akt and governs diverse cellular processes. To investigate the role of FoxO1 in control of liver fibrosis, we generated liver‐specific deletion of FoxO1 gene in mice (L‐F1KO). Using the mouse model, we injected intraperitoneally (i.p.) corn oil or 20% solution of carbon tetrachloride (CCL4) twice per week for 4 weeks (with 2.5 ul/g body weight) in WT and L‐F1KO mice. H&E staining and Sirius Red staining revealed that CCl4‐induced liver injury and fibrosis were significantly appeared, but the effects were largely attenuated in L‐F1KO mice. Moreover, we found that both mRNA and protein levels of TGF‐β1, a cytokine that activates hepatic stellate cell (HSC) for fibrogenesis, significantly decreased in the liver L‐F1KO mice compared to WT mice in response to CCL4 treatment. In primary hepatocytes, with FoxO1‐loss of function and gain‐of function, we confirmed the regulation of TGF‐β1 expression by FoxO1. Moreover, conditional medium (CM) collected from WT hepatocytes treated with CCL4 activated human HSC cell line (LX‐2), such effect was attenuated by FoxO1 deletion in primary hepatocytes or neutralization of TGF‐β1 in the CM using TGF‐β1 antibody. These results indicate a crosstalk between hepatocytes and HSC via FoxO1‐regualted TGF‐β1. To further confirm this concept in vivo, we overexpressed FoxO1 in the liver of WT and liver‐specific TGF‐β1 deficient (L‐TGF‐β1KO) mice with adenovirus expressing FoxO1 (adFoxO1), and injected (i.p.) these mice with CCL4. We found that FoxO1 overexpression in WT mice promoted CCL4‐induced liver fibrosis and HSC activation, and such effect was blocked in L‐TGF‐β1KO mice. Taken together, our data indicate that hepatic FoxO1 promotes CCL4‐inducled liver fibrosis via upregulating TGF‐β1 expression in hepatocytes and stimulating TGF‐β1‐mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.

Ca2+ Signaling and Proliferation via Ca2+-Sensing Receptors in Human Hepatic Stellate LX-2 Cells.

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt). However, the regulatory mechanisms underlying Ca2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca2+-sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca2+ restoration (from 0 to 2.2 mM) increased [Ca2+]cyt in these cells. The extracellular Ca2+-induced increase in [Ca2+]cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC50 = 6.0 and 9.5 μM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.

A Novel miRNA From Egg-Derived Exosomes of Schistosoma japonicum Promotes Liver Fibrosis in Murine Schistosomiasis.

Schistosomiasis caused by Schistosoma japonicum is a serious public health problem in China. Granuloma and hepatic fibrosis are the main pathological features of schistosomiasis japonica. The role and mechanism of egg-derived exosomes of S. japonicum in liver fibrosis remain unclear. In this study, we found that egg-derived exosomes of S. japonicum carry a new type of microRNA (miRNA-33). In vitro, this novel miRNA upregulated the expression of smooth muscle actin (α-SMA) and collagen 1 α1 (Col 1 α1) in the human hepatic stellate cell (LX-2) line at both mRNA and protein levels. In vivo, this novel miRNA was upregulated in the serum of infected mice, and when injected into mice through the tail vein using miRNA agomir, α-SMA, Col 1 α1, and Col 3 α1 were upregulated in liver tissue at both mRNA and protein levels. In addition, this novel miRNA downregulated the expression of α-SMA and Col 1 α1 in liver tissue at mRNA and protein levels in mice infected with S. japonicum and treated with miRNA antagomir. The novel miRNA-33 upregulated TGF-β Receptor I (TGF-β RI) at both mRNA and protein levels in LX-2 cells. Our results suggest that this novel miRNA from egg-derived exosomes of S. japonicum can promote liver fibrosis in the host in a cross-species manner, and the degree of fibrosis can be decreased by inhibiting the expression of this miRNA.

One-Step Fabrication of Porous Membrane-Based Scaffolds by Air-Water Interfacial Phase Separation: Opportunities for Engineered Tissues.

Common methods for fabricating membrane-based scaffolds for tissue engineering with (hydrophobic) polymers include thermal or liquid-phase inversion, sintering, particle leaching, electrospinning and stereolithography. However, these methods have limitations, such as low resolution and pore interconnectivity and may often require the application of high temperatures and/or toxic porogens, additives or solvents. In this work, we aim to overcome some of these limitations and propose a one-step method to produce large porous membrane-based scaffolds formed by air-water interfacial phase separation using water as a pore-forming agent and casting substrate. Here, we provide proof of concept using poly (trimethylene carbonate), a flexible and biocompatible hydrophobic polymer. Membrane-based scaffolds were prepared by dropwise addition of the polymer solution to water. Upon contact, rapid solvent–non-solvent phase separation took place on the air-water interface, after which the scaffold was cured by UV irradiation. We can tune and control the morphology of these scaffolds, including pore size and porosity, by changing various parameters, including polymer concentration, solvent type and temperature. Importantly, human hepatic stellate cells cultured on these membrane-based scaffolds remained viable and showed no signs of pro-inflammatory stress. These results indicate that the proposed air-water interfacial phase separation represents a versatile method for creating porous membrane-based scaffolds for tissue engineering applications.

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine–choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.

Bone morphogenetic protein 13 in hepatic stellate cells and hepatic fibrosis.

Hepatic fibrosis can be considered as a deregulated wound healing process in response to chronic liver injury. Bone morphogenetic protein 13 (BMP13) has been described to promote bone and tendon repair. In this study, we aimed to analyze the expression and function of BMP13 in hepatic fibrosis. We found increased BMP13 expression during the activation of hepatic stellate cells (HSCs), which is known as the key event of hepatic fibrosis. Fitting to this, BMP13 was elevated in murine models of hepatic fibrosis, and immunofluorescence staining showed colocalization of BMP13 and α-smooth muscle actin (α-SMA), a marker for activated HSC, in cirrhotic human liver tissue. BMP13 depletion in activated human HSC reduced the phosphorylation of smad1/5/9 and the expression of the transcription factor inhibitor of differentiation 1 (ID1), a known BMP target gene and profibrogenic factor. Furthermore, BMP13-depletion led to reduced proliferation and downregulation of collagen I α1 (COL1A1) and α-SMA, and, interestingly, also reduced phosphorylation of extracellular signal-regulated kinases (ERK). Conversely, stimulation with recombinant BMP13 induced the phosphorylation of smad1/5/9 and ERK, as well as the proliferation and the expression of ID1, COL1A1,and α-SMA in HSCs. These stimulatory effects were inhibited by dorsomorphin 1, a small-molecule inhibitor of the BMP-type I receptors activin receptor-like kinase-2 and -3, which are both expressed by HSC. In summary, these data indicate increased BMP13 expression in hepatic fibrosis as a profibrogenic factor. Thus, this soluble growth factor might have the potential as a new fibrosis marker and antifibrogenic therapeutic target in patients with chronic liver disease.

Hydrostatic pressure induces profibrotic properties in hepatic stellate cells via the RhoA/ROCK signaling pathway.

Elevated interstitial fluid hydrostatic pressure is commonly observed in diseased livers. We herein examined the hypothesis that hydrostatic pressure induces hepatic stellate cells to acquire profibrotic properties under pathological conditions. Human hepatic stellate cells were exposed to 50 mmHg pressure for 24 h. Although we observed few changes of cell growth and morphology, PCR array data on the expression of fibrosis‐associated genes suggested the acquisition of profibrotic properties. The exposure of hepatic stellate cells to 50 mmHg pressure for 24 h also significantly enhanced the expression of RhoA, ROCK1, α‐SMA, TGF‐β1, p‐MLC, and p‐Smad2, and this was effectively attenuated by the ROCK inhibitor Y‐27632. Our ex vivo experimental data suggest that elevated interstitial fluid hydrostatic pressure under pathological conditions may promote liver fibrosis by inducing acquisition of profibrotic properties of hepatic stellate cells through the RhoA/ROCK signaling pathway.