Proliferation Of Human Hepatic Stellate Cells Research Articles

Pioneering PGC-1α-boosted secretome: a novel approach to combating liver fibrosis.

Liver fibrosis is a critical health issue with limited treatment options. This study investigates the potential of PGC-Sec, a secretome derived from peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-overexpressing adipose-derived stem cells (ASCs), as a novel therapeutic strategy for liver fibrosis. Upon achieving a cellular confluence of 70%-80%, ASCs were transfected with pcDNA-PGC-1α. PGC-Sec, obtained through concentration of conditioned media using ultrafiltration units with a 3-kDa cutoff, was assessed through in vitro assays and in vitro mouse models. In vitro, PGC-Sec significantly reduced LX2 human hepatic stellate cell proliferation and mitigated mitochondrial oxidative stress compared to the control-secretome. In an in vivo mouse model, PGC-Sec treatment led to notable reductions in hepatic enzyme activity, serum proinflammatory cytokine concentrations, and fibrosis-related marker expression. Histological analysis demonstrated improved liver histology and reduced fibrosis severity in PGC-Sec-treated mice. Immunohistochemical staining confirmed enhanced expression of PGC-1α, optic atrophy 1 (a mitochondrial function marker), and peroxisome proliferator-activated receptor alpha (an antifibrogenic marker) in the PGC-Sec-treated group, along with reduced collagen type 1A expression (a profibrogenic marker). These findings highlight the therapeutic potential of PGC-Sec in combating liver fibrosis by enhancing mitochondrial biogenesis and function, and promoting antifibrotic processes. PGC-Sec holds promise as a novel treatment strategy for liver fibrosis.

Fibroblast-specific adipocyte enhancer binding protein 1 is a potential pathological trigger and prognostic marker for liver fibrosis independent of etiology.

Aortic carboxypeptidase-like protein (ACLP) is an extracellular protein involved in adipogenesis, epithelial-mesenchymal transition, epithelial cell hyperplasia, and collagen fibrogenesis. This study mainly aimed to analyze the potential role of adipocyte enhancer binding protein 1 (AEBP1), the ACLP-encoding gene, as a pathological target or prognostic marker for liver fibrosis regardless of etiology. Dysregulation pattern, clinical relevance, and biological significance of AEBP1 gene in liver fibrosis were analyzed using publicly available transcriptomic profiles, different liver fibrosis mouse models, biological databases, and AEBP1 gene silencing followed by RNA sequencing in human hepatic stellate cells (HSCs). AEBP1 gene expression was upregulated and positively correlated with liver fibrogenesis independent of etiology, the protein of which was further verified in liver fibrosis mouse models induced by different pathogenic factors. A higher expression of liver AEBP1 gene had the potential to predict poor prognosis in liver fibrosis. Systematic bioinformatic analyses revealed that AEBP1 expression was HSCs-specific and associated with extracellular matrix (ECM) remodeling and its downstream mechanical-chemical signaling transition. AEBP1 knockdown by specific small interfering RNAs (siRNAs) in HSCs inhibited ECM-receptor interaction and immune-related pathways as well as HSC proliferation or activation. A high expression of AEBP1 was specifically associated with liver fibrosis and was related to a poor prognosis and predicted the role of AEBP1 in HSCs, providing a new insight for understanding AEBP1 in liver fibrosis.

Hastatoside attenuatescarbon tetrachloride-induced liver fibrosis by targeting glycogen synthase kinase-3β

BackgroundHastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients. Hypothesis/purposeThis study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms. MethodsC57BL/6 J mice with carbon tetrachloride (CCl4)-induced hepatic fibrosis were used as the in vivo models. Histological features of the liver were observed using Masson's trichrome and hematoxylin–eosin staining. Alanine aminotransferase and aspartate aminotransferase levels and the hepatic fibrosis indices (type 3 procollagen, laminin, and hyaluronic acid) were measured using corresponding assay kits. LX-2 human hepatic stellate cells (HSCs) stimulated with the transforming growth factor β1 were used as the vitro models. Transfection of the glycogen synthase kinase (GSK)-3β small interfering RNA (siRNA) and β-catenin plasmids was also performed in vitro. Protein levels of GSK-3β, phospho-GSK-3β (Ser 9), α-smooth muscle actin, collagen type I alpha 1, c-Myc, cyclin D1, and β-catenin were determined via western blotting. Moreover, the p-GSK-3β:GSK-3β ratio was calculated to determine the GSK-3β activity. ResultsHastatoside prevented CCl4-induced liver injury and histological damage. It inhibited the upregulation of α-SMA and Col1α1 levels in a CCl4-induced mouse hepatic fibrosis model. In vitro, hastatoside inhibited the proliferation and activation of HSCs by decreasing the expression levels of cyclin D1 and c-Myc and the proportion of LX-2 cells activated in the G0/G1 phase. Molecular docking results showed that hastatoside bound to GSK-3β. Hastatoside significantly increased the GSK-3β activity and inhibited the downstream effector expression of β-catenin. ConclusionThese findings suggest that hastatoside can bind to GSK-3β and promote its activity, while inhibiting the GSK-3β downstream effector expression of β-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.

Targeting ER protein TXNDC5 in hepatic stellate cell mitigates liver fibrosis by repressing non-canonical TGFβ signalling

Background and objectivesLiver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide...

Retraction.

Retraction: "Role of RKIP in human hepatic stellate cell proliferation, invasion, and metastasis", by Quanfang Huang, Jinbin Wei, Ling Wei, Xiaolin Zhang, Facheng Bai, Shujuan Wen, Yuanyuan Wei, Shimei Tan, Zhongpeng Lu, and Xing Lin, J Cell Biochem. 2019; 6168-6177: The above article, published online on 10 October 2018 in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/jcb.27904) has been retracted by agreement between the authors, the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. The investigation was conducted by several experts into the accuracy of the data acquisition and concluded that inappropriate manipulation had taken place during Western blotting. Specifically, inconsistencies and duplicated image elements were found in figures 1A, 1B, 1D, 2C, 5D, and 6A. The authors disagree with this decision.

Carvedilol improves liver cirrhosis in rats by inhibiting hepatic stellate cell activation, proliferation, invasion and collagen synthesis.

Portal hypertension (PHT) is one of the most severe consequences of liver cirrhosis. Carvedilol is a first-line pharmacological treatment of PHT. However, the antifibrogenic effects of carvedilol on liver cirrhosis and the intrinsic mechanisms underlying these effects have not been thoroughly investigated. The present study aimed to investigate the antifibrogenic effects of carvedilol on liver cirrhosis in vivo and in vitro. Liver cirrhosis was induced in rats by carbon tetrachloride (CCl4) administration for 9 weeks; carvedilol was administered simultaneously in the experimental group. Blood samples were collected for serum biochemistry. Liver tissues were used for fibrosis evaluation, histological examination, immunohistochemistry and western blot analysis. The human hepatic stellate cell (HSC) line LX-2 was used for in vitro studies. The effects of carvedilol on LX-2 cell proliferation and invasion were evaluated by Cell Counting Kit-8 assay and Transwell invasion assays, respectively. The effect of carvedilol on transforming growth factor β1 (TGFβ1)-induced collagen synthesis in LX-2 cells and the molecular mechanisms were examined by western blot analysis. The results demonstrated that carvedilol improved CCl4-induced structural distortion and fibrosis in the liver. Carvedilol inhibited HSC activation, proliferation and invasion. Carvedilol inhibited HSC collagen synthesis through the TGFβ1/SMAD pathway. In conclusion, carvedilol may alleviate liver cirrhosis in rats by inhibiting HSC activation, proliferation, invasion and collagen synthesis. Carvedilol may be a potential treatment of early-stage liver cirrhosis.

The STAT3 inhibitor S3I-201 suppresses fibrogenesis and angiogenesis in liver fibrosis

Liver fibrosis is a common pathological response to chronic hepatic injury. STAT3 is actively involved in the fibrogenesis and angiogenesis seen in liver fibrosis. S3I-201 (NSC 74859) is a chemical inhibitor of STAT3 activity, which blocks the dimerization of STAT3, STAT3-DNA binding and transcription activity. This study evaluated the effects of S3I-201 against liver fibrosis. S3I-201 inhibited the proliferation, migration, and actin filament formation in primary human hepatic stellate cells (HSCs), as well as the expression of α-SMA, collagen I and TIMP1 in both primary HSC and in a CCl4-induced fibrosis mouse model. S3I-201 induced both apoptosis and cell cycle arrest in the HSC cell line (LX-2). S3I-201 inhibited the expression of fibrogenesis factors TGFβ1 and TGFβRII, as well as the downstream phosphorylation of Smad2, Smad3, Akt and ERK induced by TGFβ1. In addition to fibrogenesis, both in vitro and in vivo assays showed that S3I-201 inhibited angiogenesis through expression suppression of VEGF and VEGFR2. Moreover, S3I-201 also had a synergistic effect with sorafenib, an FDA approved liver cancer drug, in the proliferation, apoptosis, angiogenesis and fibrogenesis of HSC. S3I-201 suppressed liver fibrosis through multiple mechanisms, and combined with sorafenib, S3I-201 could be a potentially effective antifibrotic agent.S3I-201, a chemical inhibitor of the transcription factor STAT3, suppresses liver fibrogenesis and angiogenesis through multiple mechanisms both in vitro and in vivo. Moreover, S3I-201 and sorafenib, an FDA-approved multikinase inhibitor, function synergistically in suppressing fibrogenesis and angiogenesis of human hepatic stellate cells, indicating high potential for liver fibrosis treatment.

Role of RKIP in human hepatic stellate cell proliferation, invasion, and metastasis.

The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, apoptosis, invasion, and metastasis of human hepatic stellate cell line (LX-2). A recombinant plasmid (pcDNA3.1-RKIP) or RKIP-targeting small interfering RNA (siRNA) vector (siRNA-RKIP) was transfected into LX-2 cells to interfere with the RKIP expression. The results demonstrated that increased RKIP expression significantly reduced cell viability, clonogenic growth, and invasion. Further, it promoted cell apoptosis and induced cell cycle arrest in the G1 phase. Overexpression of RKIP led to inactivation of LX-2 cells, as evidenced by the decrease in the expression levels of collagen I and α-smooth muscle actin (α-SMA). In addition, increased RKIP expression significantly reduced the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), the transcriptional activity of nuclear factor-κB (NF-κB), and the levels of matrix metalloproteinases-1 and -2. In conclusion, these findings clearly demonstrate that RKIP inhibits LX-2 cell growth, metastasis, and activation, primarily by downregulating the ERK/MAPK and NF-κB signaling pathways.

PBI-4050 Reduces Stellate Cell Activation and Liver Fibrosis through Modulation of Intracellular ATP Levels and the Liver Kinase B1/AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We previously showed that 2-(3-pentylphenyl)acetic acid (PBI-4050) is a dual G protein-coupled receptor GPR40 agonist/GPR84 antagonist that exerts antifibrotic, anti-inflammatory, and antiproliferative action. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The antifibrotic effect of PBI-4050 was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation-induced liver fibrosis rodent models. Treatment with PBI-4050 suppressed CCl4-induced serum aspartate aminotransferase levels, inflammatory marker nitric oxide synthase, epithelial to mesenchymal transition transcription factor Snail, and multiple profibrotic factors. PBI-4050 also decreased GPR84 mRNA expression in CCl4-induced injury, while restoring peroxisome proliferator-activated receptor γ (PPARγ) to the control level. Collagen deposition and α-smooth muscle actin (α-SMA) protein levels were also attenuated by PBI-4050 treatment in the bile duct ligation rat model. Transforming growth factor-β-activated primary HSCs were used to examine the effect of PBI-4050 and its mechanism of action in vitro. PBI-4050 inhibited HSC proliferation by arresting cells in the G0/G1 cycle phase. Subsequent analysis demonstrated that PBI-4050 signals through a reduction of intracellular ATP levels, activation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), and blockade of mammalian target of rapamycin (mTOR), resulting in reduced protein and mRNA levels of α-SMA and connective tissue growth factor and restored PPARγ mRNA expression. Our findings suggest that PBI-4050 may exert antifibrotic activity in the liver through a novel mechanism of action involving modulation of intracellular ATP levels and the LKB1/AMPK/mTOR pathway in stellate cells, and PBI-4050 may be a promising agent for treating liver fibrosis.

Pathological Roles of Interleukin-22 in the Development of Recurrent Hepatitis C after Liver Transplantation.

ObjectiveThe aim of this study was to longitudinally evaluate and analyze the role of interleukin-22-producing CD4 positive cells (IL-22) in the pathogenesis of Hepatitis C Virus recurrence after Orthotopic Liver Transplantation (HCV-OLT).Methods15 HCV-OLT, 15 age- and gender- matched non-HCV post-OLT (OLT) and 15 hepatitis C virus infected (HCV) patients were enrolled into our study from the liver transplantation and research center at Beijing 302 Hospital. We determined the frequencies of IL-22 using flow cytometry and expression of IL-22 mRNA using PCR in peripheral blood and liver tissue. We also divided HCV-OLT patients into rapid fibrosis progression (RFP) and slow fibrosis progression (SFP), examined IL-22 cells and analyzed the correlations between IL-22 frequencies and liver injury, fibrosis and clinical parameters. Moreover, we investigated the role of IL-22 in Human Hepatic Stellate Cells (HSCs).ResultsThe levels of serum IL-22, frequencies of IL-22 producing cells in peripheral blood mononuclear cells, and expression of IL-22 mRNA and protein in the liver in the HCV-OLT group were significantly higher than that in the HCV and OLT groups. Furthermore, eight (53.3%) patients developed RFP after two years; another three patients were diagnosed liver cirrhosis. The frequencies of IL-22 were much higher in RFP compared with SFP, while no significant difference existed between OLT and SFP. Intrahepatic IL-22 positive cells were located in fibrotic areas and significantly correlated with α-smooth muscle actin (α-SMA) and fibrosis staging scores, not with grading scores and HCRVNA. In vitro, IL-22 administration prevented HSCs apoptosis, promoted HSCs proliferation and activation, up-regulated the expression of HSC-sourced growth factors including α-SMA, TGF-β and TIMP-1, and increased the production of liver fibrosis markers including laminin, hyaluronic acid and collagen type IV.ConclusionPeripheral and intrahepatic IL-22 is up-regulated and plays a pathological role in exacerbating liver fibrosis by activating HSCs in HCV-OLT patients, which may predict RFP and serve as an attractive target for anti-fibrotic therapy.

Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation.

Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor‐inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro‐fibrogenic and pro‐inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl4) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co‐localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFβ in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up‐regulated in CCl4 models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl4 injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF, VEGF and PDGF Signaling

Background and AimsBrivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.Methods In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1.

We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.

Depletion of Thymosin β4 Promotes the Proliferation, Migration, and Activation of Human Hepatic Stellate Cells

Background & Aims: It has recently been reported that thymosin beta-4 (Tβ4) has anti-fibrogenic effects in human hepatic stellate cells (HSCs) in vitro, but the mechanisms underlying these effects remain unclear. The aim of this study was to investigate the roles of Tβ4 in the proliferation, migration, and activation of HSCs. Methods: Enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and western blot assays were utilized to determine the expression levels of Tβ4 in serum, liver tissues, and LX-2 cells. Tβ4 was depleted in LX-2 cells using small interfering RNAs (siRNAs). Cell proliferation was analyzed using cell counting kit-8 (CCK-8) viability assays, and cell migration was investigated using wound-healing and transwell migration assays. Results: The expression of Tβ4 was significantly reduced during the progression of liver fibrosis. The depletion of Tβ4 significantly promoted the proliferation and migration of LX-2 cells via the activation of the PI3K/Akt signaling pathway. The pro-migratory and pro-proliferative effects of Tβ4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tβ4 depletion was also associated with the activation of HSCs via the enhanced expression of α-smooth muscle actin (α-SMA) and vimentin. Conclusions: Our results suggest that Tβ4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tβ4 may be an effective target in the treatment of liver fibrosis.

Activation of TGF-β1 Promoter by Hepatitis C Virus-Induced AP-1 and Sp1: Role of TGF-β1 in Hepatic Stellate Cell Activation and Invasion

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.

Low density lipoprotein receptor‐related protein 1 modulates the proliferation and migration of human hepatic stellate cells

Human hepatic stellate cells (HHSCs) proliferation and migration play a key role in the pathogenesis of liver inflammation and fibrogenesis. Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor involved in intracellular signal transduction. The aim of this work was to analyse the role of low density lipoprotein receptor-related protein (LRP1) in HHSCs proliferation and migration and the mechanisms involved. Human LRP1 deficient-HHSCs were generated by nucleofecting the line HHSCs with siRNA anti-LRP1. HHSCs DNA synthesis was measured by [(3) H]-thymidine incorporation and cell cycle progression by flow cytometry after annexin V and iodure propidium staining. Cell migration was assessed using a wound repair model system. LRP1 expression and extracellular matrix-regulated kinase (ERK1,2) phosphorylation were analysed by Western blot analysis. Transforming growth factor-β (TGF-β) extracellular levels were analysed by ELISA. siRNA-antiLRP1 treatment almost completely inhibited LRP1 mRNA and protein expression. LRP1 deficient HHSCs showed higher proliferative response (172 ± 19 vs. 93 ± 8 [(3) H]-thymidine incorporation; 78.68% vs. 82.69% in G0/G1, 21.32% vs. 17.30% in G2/S) and higher migration rates than control HHSCs. LRP1 deficient cells showed higher levels of phosphorylated ERK1,2. TGF-β extracellular levels were threefold higher in LRP1-deficient than in control HHSCs cells. These results demonstrate that LRP1 regulates HHSCs proliferation and migration through modulation of ERK1,2 phosphorylation and TGF-β extracellular levels. These results suggest that HHSCs-LRP1 may play a key role in the modulation of factors determining hepatic fibrosis.

Significance of the Balance between Regulatory T (Treg) and T Helper 17 (Th17) Cells during Hepatitis B Virus Related Liver Fibrosis

BackgroundHepatitis B virus-related liver fibrosis (HBV-LF) always progresses from inflammation to fibrosis. However, the relationship between these two pathological conditions is not fully understood. Here, it is postulated that the balance between regulatory T (Treg) cells and T helper 17 (Th17) cells as an indicator of inflammation may predict fibrosis progression of HBV-LF.Methodology/Principal FindingsThe frequencies and phenotypes of peripheral Treg and Th17 cells of seventy-seven HBeAg-positive chronic hepatitis B (CHB) patients who underwent liver biopsies and thirty healthy controls were determined by flow cytometry. In the periphery of CHB patients, both Treg and Th17 frequencies were significantly increased and correlated, and a lower Treg/Th17 ratio always indicated more liver injury and fibrosis progression. To investigate exact effects of Treg and Th17 cells during HBV-LF, a series of in vitro experiments were performed using purified CD4+, CD4+CD25+, or CD4+CD25− cells from the periphery, primary human hepatic stellate cells (HSCs) isolated from healthy liver specimens, human recombinant interleukin (IL)-17 cytokine, anti-IL-17 antibody and HBcAg. In response to HBcAg, CD4+CD25+ cells significantly inhibited cell proliferation and cytokine production (especially IL-17 and IL-22) by CD4+CD25− cells in cell-contact and dose-dependent manners. In addition, CD4+ cells from CHB patients, compared to those from HC subjects, dramatically promoted proliferation and activation of human HSCs. Moreover, in a dramatically dose-dependent manner, CD4+CD25+ cells from CHB patients inhibited, whereas recombinant IL-17 response promoted the proliferation and activation of HSCs. Finally, in vivo evidence about effects of Treg/Th17 balance during liver fibrosis was obtained in concanavalin A-induced mouse fibrosis models via depletion of CD25+ or IL-17+ cells, and it’s observed that CD25 depletion promoted, whereas IL-17 depletion, alleviated liver injury and fibrosis progression.Conclusions/SignificanceThe Treg/Th17 balance might influence fibrosis progression in HBV-LF via increase of liver injury and promotion of HSCs activation.

Fabrication and characterization of an inorganic gold and silica nanoparticle mediated drugdelivery system for nitric oxide

Nitric oxide (NO) plays an important role in inhibiting the development of hepatic fibrosisand its ensuing complication of portal hypertension by inhibiting human hepatic stellatecell (HSC) activation. Here we have developed a gold nanoparticle and silicananoparticle mediated drug delivery system containing NO donors, which couldbe used for potential therapeutic application in chronic liver disease. The goldnanoconjugates were characterized using several physico-chemical techniques such asUV–visible spectroscopy and transmission electron microscopy. Silica nanoconjugateswere synthesized and characterized as reported previously. NO released fromgold and silica nanoconjugates was quantified under physiological conditions (pH = 7.4 at37 °C) for a substantial period of time. HSC proliferation and the vascular tube formationability, manifestations of their activation, were significantly attenuated by the NO releasedfrom these nanoconjugates. This study indicates that gold and silica nanoparticle mediateddrug delivery systems for introducing NO could be used as a strategy for thetreatment of hepatic fibrosis or chronic liver diseases, by limiting HSC activation.

Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics

BackgroundPregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16α carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) – which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) – in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN.ResultsQuiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17β-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1.ConclusionLAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.

Role of the receptor for advanced glycation end products inhepatic fbrosis

To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGE-BSA) and N(epsilon)-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-alpha (TNF-alpha). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-alpha. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen alpha1(I) mRNA by AGE-BSA. Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.