Experimental Liver Fibrosis In Mice Research Articles

The antiprotozoal drug nitazoxanide improves experimental liver fibrosis in mice

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg−1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-β1 (TGF-β1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-β1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.

Oxidative stress promotes liver fibrosis by modulating the microRNA-144 and SIN3A-p38 pathways in hepatic stellate cells.

Background & Aims: Reactive oxygen species (ROS) act as modulators triggering cellular dysfunctions and organ damage including liver fibrosis in which hepatic stellate cell (HSC) activation plays a key role. Previous studies suggest that microRNA-144 (miR-144) acts as a pro-oxidant molecule; however, whether and how miR-144 affects HSC activation and liver fibrosis remain unknown. Methods: Carbon tetrachloride (CCl4) and bile duct ligation (BDL)-induced experimental liver fibrosis models were used. Hepatic miR-144 expression was analyzed by miRNA in situ hybridization with RNAscope probe. The in vivo effects of silencing or overexpressing miR-144 were examined with an adeno-associated virus 6 (AAV6) carrying miR-144 inhibitor or mimics in fibrotic mouse experimental models. Results: In this study, we demonstrated that ROS treatment significantly upregulated miR-144 in HSCs, which further promoted HSC activation in vitro. Interestingly, miR-144 was preferentially elevated in HSCs of experimental liver fibrosis in mice and in human liver fibrotic tissues. Furthermore, in vivo loss or gain-of-function experiments via AAV6 carrying miR-144 antagomir or agomir revealed that blockade of miR-144 in HSCs mitigated, while overexpression of miR-144 in HSCs accelerated the development of experimental liver fibrosis. Mechanistically, SIN3 transcription regulator family member A (SIN3A), a transcriptional repressor, was identified to be the target of miR-144 in HSCs. MiR-144 downregulated Sin3A, and in line with this result, specific knockdown of Sin3a in HSCs remarkedly activated p38 MAPK signaling pathway to promote HSC activation, eventually exacerbating liver fibrosis. Conclusions: Oxidative stress-driven miR-144 fuels HSC activation and liver fibrogenesis by limiting the SIN3A-p38 axis. Thus, a specific inhibition of miR-144 in HSCs could be a novel therapeutic strategy for the treatment of liver fibrosis.

MicroRNA-101 suppresses liver fibrosis by downregulating PI3K/Akt/mTOR signaling pathway

MicroRNA-101 (miR-101) is markedly downregulated in both hepatitis B virus-related liver cirrhosis and hepatocellular carcinoma (HCC). In this study, we aimed to investigate the effect and mechanism of miR-101 on hepatic stellate cell (HSC) activation and liver fibrosis. HSC LX-2 was treated with TGF-β1 and with or without miR-101 mimics. LX-2 vitality and proliferation, the expression of F-actin and mRNAs for α-SMA, collagen 1α1 (Col 1α1), and connective tissue growth factor 2 (CCN2) were measured. A 6-week intraperitoneal injection of carbon tetrachloride (CCl4) was used to induce experimental liver fibrosis in mice, which were treated using a miR-101 negative control or miR-101 agomir from the fourth week until the end of the experiment. Liver function, hepatic hydroxyproline, liver histopathology, collagen deposition, α-SMA, type I collagen (Col I) and the protein-expressions of p-PI3K, p-Akt and p-mTOR were measured. MiR-101 significantly suppressed the increased LX-2 vitality and high accumulation of extracellular matrix (ECM) induced by TGF-β1. Exposure to CCl4 led to the impairment of liver function and disruption of normal hepatic parenchyma in mice, as well as obvious liver fibrosis indicated by elevated levels of hydroxyproline, α-SMA, and Col 1α1 in liver tissues. MiR-101 administration significantly improved liver function, relieved hepatic parenchyma damage, and reversed liver fibrosis by decreasing the accumulation of ECM components. Furthermore, miR-101 substantially downregulated the CCl4-increased p-PI3K, p-Akt, and p-mTOR in mouse liver. MiR-101 has antifibrotic effects in experimental liver fibrosis, and downregulating the PI3K/Akt/mTOR signaling pathway may be one of its antifibrotic mechanisms.

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed.Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis.Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-β1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-β1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner.Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

Mogroside IVE attenuates experimental liver fibrosis in mice and inhibits HSC activation through downregulating TLR4-mediated pathways

Liver fibrosis has been emphasized as a serious threat to human health. There is currently no effective clinical drug treatment. Although mogrosides (MGs) have extensive pharmacological effects with minimal toxicity, their effects on liver function, inflammation, matrix metalloproteinases and hepatic stellate cell (HSC) activation remain to be researched. In the current study, we investigated whether mogroside IVE (MGIVE), a main compound isolated from MGs, provided protection against liver fibrosis in mice. MGIVE (25mg/kg) significantly reduced carbon tetrachloride (CCl4)-induced inflammatory infiltration, pro-inflammatory cytokine release, and myeloperioxide (MPO) activity, as well as improved liver function in CCl4-treated mice. Additionally, MGIVE also significantly impaired CCl4-induced increases in liver fibrotic marker expression, such as collagen type I and hypoxia inducible factor-1α (HIF-1α). Further investigation indicated that the possible molecular target of MGIVE is the toll-like receptor 4 (TLR4)-mediated pathway, and MGIVE treatment significantly prevented CCl4-induced transforming growth factor-β1 (TGF-β1) overexpression and the phosphorylation of mitogen activated protein kinase (MAPK) in vivo. In vitro tests of HSCs or RAW 264.7 cells challenged with TGF-β1 or lipopolysaccharide (LPS) demonstrated that TLR4 expression partly mediated the anti-fibrotic effects of MGIVE. In conclusion, supplementation with MGIVE may attenuate liver fibrosis through inhibiting the TLR4 signaling pathway, including MyD88 and MAPKs, as well as HIF-1α. MGIVE may act as a therapeutic potential drug for the treatment of liver fibrosis via the TLR4/HIF-1α cohort signaling pathway.

Correction to: Krithika, Jyothi, Prasantha, and Ramtej, Mechanism of protective effect of phyllanthin against carbon tetrachloride-induced hepatotoxicity and experimental liver fibrosis in mice

"Correction to: Krithika, Jyothi, Prasantha, and Ramtej, Mechanism of protective effect of phyllanthin against carbon tetrachloride-induced hepatotoxicity and experimental liver fibrosis in mice." Toxicology Mechanisms and Methods, 27(6), p. 476

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high-affinity ligand for the aryl hydrocarbon receptor (AhR). Increasing evidence indicates that AhR signaling contributes to wound healing, which involves the coordinated deposition and remodeling of the extracellular matrix. In the liver, wound healing is attributed to the activation of hepatic stellate cells (HSCs), which mediate fibrogenesis through the production of soluble mediators and collagen type I. We recently reported that TCDD treatment increases the activation of human HSCs in vitro. The goal of this study was to determine how TCDD impacts HSC activation in vivo using a mouse model of experimental liver fibrosis. To elicit fibrosis, C57BL6/male mice were treated twice weekly for 8weeks with 0.5ml/kg carbon tetrachloride (CCl4). TCDD (20μg/kg) or peanut oil (vehicle) was administered once a week during the last 2weeks. Results indicate that TCDD increased liver-body-weight ratios, serum alanine aminotransferase activity, and hepatic necroinflammation in CCl4-treated mice. Likewise, TCDD treatment increased mRNA expression of HSC activation and fibrogenesis genes, namely α-smooth muscle actin, desmin, delta-like homolog-1, TGF-β1, and collagen type I. However, TCDD treatment did not exacerbate fibrosis, nor did it increase the collagen content of the liver. Instead, TCDD increased hepatic collagenase activity and increased expression of matrix metalloproteinase (MMP)-13 and the matrix regulatory proteins, TIMP-1 and PAI-1. These results support the conclusion that TCDD increases CCl4-induced liver damage and exacerbates HSC activation, yet collagen deposition and the development of fibrosis may be limited by TCDD-mediated changes in extracellular matrix remodeling.

Mechanism of protective effect of phyllanthin against carbon tetrachloride-induced hepatotoxicity and experimental liver fibrosis in mice

Chronic injury to liver triggers synthesis of extracellular matrix components resulting in progressive fibrosis and eventually cirrhosis. Transforming growth factor-β1 (TGF-β1) transduces its signal by binding to TGF-β type 1 receptor kinase or activin like kinase (ALK5) receptor and mediates hepatic fibrosis by increasing the transcription of downstream entities such as collagen via Smad2 and Smad3. The present study was carried out to investigate the mechanism by which phyllanthin, a hepatoprotective lignin isolated from the plant Phyllanthus amarus (P. amarus) exerts its anti-fibrotic effect. The inhibitory role of phyllanthin on ALK5 was first analyzed using molecular docking experiments. Phyllanthin was found to effectively bind to serine (Ser) 280 at the active site of ALK5 by forming hydrogen bonds. The in vivo protective effect of phyllanthin against carbon tetrachloride (CCl4)-induced hepatic fibrosis was established by studying the protein expressions of TGF-β1, ALK5 and Smad2 and 3 and by determining various biochemical and histopathological parameters. Phyllanthin was found to exert its anti-fibrotic effect by down-regulating TGF signaling pathway via ALK5 and Smad2 and 3 inhibition.

Loss of Gab1 adaptor protein in hepatocytes aggravates experimental liver fibrosis in mice.

Grb2-associated binder 1 (Gab1) adaptor protein amplifies signals downstream of a broad range of growth factors/receptor tyrosine kinases. Although these signals are implicated in liver fibrogenesis, the role of Gab1 remains unclear. To elucidate the role of Gab1, liver fibrosis was examined in hepatocyte-specific Gab1-conditional knockout (Gab1CKO) mice upon bile duct ligation (BDL). Gab1CKO mice developed exacerbated liver fibrosis with activation of hepatic myofibroblasts after BDL compared with control mice. The antifibrotic role of hepatocyte Gab1 was further confirmed by another well-established mouse model of liver fibrosis using chronic injections of carbon tetrachloride. After BDL, Gab1CKO mice also displayed exacerbated liver injury, decreased hepatocyte proliferation, and enhanced liver inflammation. Furthermore, cDNA microarray analysis was used to investigate the potential molecular mechanisms of the Gab1-mediated signal in liver fibrosis, and the fibrosis-promoting factor chemokine (C-C motif) ligand 5 (Ccl5) was identified as upregulated in the livers of Gab1CKO mice following BDL. Interestingly, in vitro studies using primary hepatocytes isolated from control and Gab1CKO mice revealed that the loss of Gab1 resulted in increased hepatocyte CCL5 synthesis upon lipopolysaccharide stimulation. Finally, pharmacological antagonism of CCL5 reduced BDL-induced liver fibrosis in Gab1CKO mice. In conclusion, our results demonstrate that hepatocyte Gab1 is required for liver fibrosis and that hepatocyte CCL5 could be an important contributor to this process. Thus, we present a novel antifibrotic function of hepatocyte Gab1 in liver fibrogenesis.

The chemokine CCL3 promotes experimental liver fibrosis in mice.

Liver fibrosis is associated with infiltrating immune cells and activation of hepatic stellate cells. We here aimed to investigate the effects of the CC chemokine CCL3, also known as macrophage inflammatory protein-1α, in two different fibrosis models. To this end, we treated mice either with carbon tetrachloride or with a methionine- and choline-deficient diet to induce fibrosis in CCL3 deficient and wild-type mice. The results show that the protein expression of CCL3 is increased in wild-type mice after chronic liver injury. Deletion of CCL3 exhibited reduced liver fibrosis compared to their wild-type counterparts. We could validate these results by treating the two mouse groups with either carbon tetrachloride or by feeding a methionine- and choline-deficient diet. In these models, lack of CCL3 is functionally associated with reduced stellate cell activation and liver immune cell infiltration. In vitro, we show that CCL3 leads to increased proliferation and migration of hepatic stellate cells. In conclusion, our results define the chemokine CCL3 as a mediator of experimental liver fibrosis. Thus, therapeutic modulation of CCL3 might be a promising target for chronic liver diseases.

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF+/+ or HB-EGF-null (HB-EGF−/−) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF+/+ or HB-EGF−/− mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF+/+ and HB-EGF−/− mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF+/+ mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF−/− mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF+/+ mice, TAA-stimulated hepatic mRNA levels in HB-EGF−/− mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF+/+ mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.

Protease-activated receptor 2 promotes experimental liver fibrosis in mice and activates human hepatic stellate cells

Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated after proteolytic cleavage by serine proteases, including mast cell tryptase and activated coagulation factors. PAR-2 activation augments inflammatory and profibrotic pathways through the induction of genes encoding proinflammatory cytokines and extracellular matrix proteins. Thus, PAR-2 represents an important interface linking coagulation and inflammation. PAR-2 is widely expressed in cells of the gastrointestinal tract, including hepatic stellate cells (HSCs), endothelial cells, and hepatic macrophages; however, its role in liver fibrosis has not been previously examined. We studied the development of CCl(4) -induced liver fibrosis in PAR-2 knockout mice, and showed that PAR-2 deficiency reduced the progression of liver fibrosis, hepatic collagen gene expression, and hydroxyproline content. Reduced fibrosis was associated with decreased transforming growth factor beta (TGFβ) gene and protein expression and decreased matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1 gene expression. In addition, PAR-2 stimulated activation, proliferation, collagen production, and TGFβ protein production by human stellate cells, indicating that hepatic PAR-2 activation increases profibrogenic cytokines and collagen production both in vivo and in vitro. Our findings demonstrate the capacity of PAR-2 activation to augment TGFβ production and promote hepatic fibrosis in mice and to induce a profibrogenic phenotype in human HSCs. PAR-2 antagonists have recently been developed and may represent a novel therapeutic approach in preventing fibrosis in patients with chronic liver disease.

Role of nitric oxide in the regulation of fibrogenic factors in experimental liver fibrosis in mice.

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl₄-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl₄ with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl₄-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl₄-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl₄-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.

Bone Marrow-Derived Mensenchymal Stem Cells Modified by Mouse Interferon-γ Gene Reduce Fibrosis and Improve Function In a Mouse Model of Liver Fibrosis.

AbstractAbstract 3768 Objective:To investigate the effect of bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ gene on experimental liver fibrosis in mice. Methods:(1) Mesenchymal stem cells (MSCs) derived from BM in BALB/c mice were cultured and identified by the flow cytometry. (2) The Ad-mIFN-γ, a recombinant adenoviral vector encoding mouse IFN-γ gene obtained by PCR from the vector pORF5-mIFN-γ, was created with the AdEasy Adenoviral Vector System. (3) MSCs were transfected with Ad-mIFN-γ. The transfection efficiency was detected by the flow cytometry at 24 hours after infection to determine the best multiplicity of infection (MOI). (4) The liver fibrosis of BALB/c mice were induced with concanavalin A (Con A) challenge weekly for six times. Then MSCs modified by the recombinant adenovirus encoding mouse IFN-γ gene were infused to BALB/c mice induced with Con A at the 24 hours after the first dose. The behavioral changes of mice were monitored closely. One week after the last dose of Con A, all mice were sacrificed and the fibrosis indexes were assessed by several histopathology methods systemicly. Moreover, hepatic hydroxyproline and serum parameters for liver fibrosis and liver function were evaluated and serum mIFN-γ concentrations were measured, respectively. Results:(1) The flow cytometry confirmed that the cells we obtained were MSCs. (2) The target gene mIFN-γ amplified by PCR from the vector pORF5-mIFN-γ, was identified by sequencing, which proved that the mIFN-γ gene was consisted of 468 nucleotides and was completely the same as the sequence published on GenBank. The target gene mIFN-γ was cut out by double endonucleases and then connected to the shuttle vector pAdTrack-CMV. Then the newly constructed vector was linearized by Pme I following transformed to the E.coli. BJ5183, which has the backbone vector of pAdEasy-1. The correct recombinant pAd-mIFN-γ was selected by endonucleases and by Kanamycin resistance and was transfected into AD-293 cells to obtain the adenoviral vector Ad-mIFN-γ. The Ad-mIFN-γ can be propagated in AD-293 cell line, the titre of which was 3.2×109 pfu/ml. (3) MSCs modified by recombinant adenovirus encoding mIFN-γ (IFN-γ/GFP-MSCs) at different multiplicity of infection (MOI) were detected by the flow cytometry. The optimal MOI for Ad-mIFN-γ was 400 with the transfection efficiency of 80.4%, and the optimal MOI for Ad-control was 200 with the transfection efficiency of 72.3%. (4) The changes of liver tissue and serum parameters for liver fibrosis showed liver fibrosis were reducd more obviously in the group treated with IFN-γ/GFP-MSCs (p<0.05), and liver functions were also improved more obviously in this group (p<0.05). The histopathology study using HE staining, masson trichrome staining and silver staining demonstrated that the structure of liver became much better in mice treated with IFN-γ/GFP-MSCs, with liver fibrosis improved significantly. Conclusion:Bone marrow-derived mensenchymal stem cells modified by mouse interferon-γ can reduce liver fibrosis and improve liver function on immuo-mediated liver fibrosis in mice induced by Con A. Disclosures:No relevant conflicts of interest to declare.

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

Resolution of experimental liver fibrosis in mice by targeted delivery of connective tissue growth factor siRNA

Resolution of experimental liver fibrosis in mice by targeted delivery of connective tissue growth factor siRNAConnective tissue growth factor (CTGF) promotes liver fibrosis by stimulating collagen production in activated hepatic stellate cells (HSC). Here, we investigated the curative properties of CTGF siRNA delivered to activated HSC in acute or chronic models of CCl4‐induced liver fibrosis. Modified liposomes containing CTGF siRNA were coated with a collagen Type VI receptor‐binding peptide. Balb/c mice received daily injections of CCl4 or oil control for either 3 or 5 weeks. During the last 2 weeks of these regimens, some mice also received concurrent daily injections of CTGF siRNA in targeted liposomes ("CTGF siRNA‐TL") or scrambled CTGF siRNA in targeted liposomes ("CTGF SsiRNA‐TL"). Administration of CCl4 for 3 or 5 weeks resulted in high levels of α‐SMA‐positive cells and deposition of collagen. Whereas this response was unaffected by administration of CTGF SsiRNA‐TL, neither α‐SMA‐positive cells nor collagen deposits were present in CTGF siRNA‐TL‐treated livers. Real‐time PCR showed that the CCl4‐induced mRNA expression of α‐SMA, collagen Type I α1, TGF‐β, or CTGF was restored to baseline values by CTGF siRNA‐TL (p&lt; 0.01) but not by CTGF SsiRNA‐TL. Thus, this targeted liposomal delivery system designed to deliver CTGF siRNA to activated HSC was effective in either preventing or reversing fibrosis even in the continued presence of the fibrotic insult.

Angiostatin inhibits experimental liver fibrosis in mice

Liver fibrosis is a response to chronic hepatic damage, which ultimately leads to liver failure and necessitates liver transplantation. A characteristic of fibrosis is pathological vessel growth. This type of angiogenesis may contribute to the disturbance of hepatocyte perfusion dynamics and lead to aggravation of disease. We hypothesized that angiostatin can inhibit pathological vessel growth and, consequently, the development of hepatic fibrosis. Hepatic fibrosis was induced by injection of carbon tetrachloride for 5 weeks. Angiostatin mice received carbon tetrachloride for 5 weeks and angiostatin during weeks 4 and 5. After 5 weeks, immunohistochemistry for endothelial cell marker von Willebrand factor and for cell proliferation was performed. Angiogenesis was quantified by counting the number of immunopositive microvessels. Also, the relative fibrotic surface was determined using Sirius Red histostaining and computer image analysis. Immunohistochemistry revealed increased expression for von Willebrand factor in fibrotic livers. Immunopositive microvessels were localized in fibrotic areas surrounding larger vessels and in emerging fibrotic septa. Angiostatin reduced the number of immunopositive microvessels by 69% (p<0.001). In addition, angiostatin reduced the relative fibrotic area in the liver by 63+/-0.1% (p<0.001). Finally, angiostatin treatment was not associated with differences in cell proliferation. Angiostatin inhibits the development of pathological angiogenesis and liver fibrosis in mice. These results warrant further evaluation of angiostatin as an antifibrotic agent, potentially contributing to the deferment of liver transplantation and reduced recurrence of fibrotic disease in the transplanted liver.

Oesophageal chest pain

<h3>Objective</h3> Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. <h3>Methods</h3> Experimental liver fibrosis in mice induced by bile duct ligation or CCl₄ application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. <h3>Results</h3> In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with <i>Listeria</i> that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. <h3>Conclusions</h3> In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis.