Profibrotic effect of miR-33a with Akt activation in hepatic stellate cells

MicroRNAs (miRNAs) attract more attention in the pathophysiology of liver fibrosis and miR-33a has been previously demonstrated as involved in the regulation of cholesterol and lipid metabolism. Transforming growth factor-beta1 (TGF-β1) is generally accepted to be the main stimulating factor in the hepatic stellate cells (HSCs) activation, which plays an important role in hepatic fibrosis. However, the involvement and underlying mechanism of miR-33a and its role in TGF-β1-induced hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-33a in the activation of immortalized human HSCs, Lx-2 cells. Our findings have shown that the expression of miR-33a with its host gene sterol regulatory element-binding protein 2 (SREBP2) was more highly expressed in activation of Lx-2 cells than in quiescent cells. The expression of miR-33a on TGF-β1-induced HSCs activation may be modulated via the activation of PI3K/Akt pathway. In addition, miR-33a significantly correlated with TGF-β1-induced expression of α1 (I) collagen (Col1A1) and α-SMA in HSCs. Bioinformatics analyses predict that peroxisome proliferator activated receptor-alpha (PPAR-α) is the potential target of miR-33a. We further found that anti-miR-33a significantly increases target gene PPAR-α mRNA and protein level, suggesting that miR-33a involved in HSCs function might be modulated by targeting PPAR-α. Finally, our results indicate that the expression of miR-33a increased with the progression of liver fibrosis. These results suggested that anti-miR-33a inhibit activation and extracellular matrix production, at least in part, via the activation of PI3K/Akt pathway and PPAR-α and anti sense of miR-33a may be a novel potential therapeutic approach for treating hepatic fibrosis in the future.     

MyD88 in hepatic stellate cells enhances liver fibrosis via promoting macrophage M1 polarization

During liver fibrosis, quiescent HSCs (qHSCs) are activated to become activated HSCs (aHSCs)/myofibroblasts. The signal adapter MyD88, an essential component of TLR signaling, plays an important role in liver fibrosis. However, far less is known about the specific effects of MyD88 signaling in both qHSCs and aHSCs in the progress of liver fibrosis. Here, we used a CCl₄-induced mouse fibrosis model in which MyD88 was selectively depleted in qHSCs (GFAP^MyD88−/− mice) or aHSCs (α-SMA^MyD88−/− mice). MyD88 deficiency in qHSCs or aHSCs attenuated liver fibrosis in mice and inhibited α-SMA-positive cell activation. Inhibition of MyD88 in HSCs decreased α-SMA and collagen I levels, inflammatory cell infiltration, and pro-inflammatory gene expression. Furthermore, MyD88 signaling in HSCs increased the secretion of CXCL10, which promoted macrophage M1 polarization through CXCR3, leading to activation of the JAK/STAT1 pathway. Inhibition of CXCL10 attenuated macrophage M1 polarization and reduced liver fibrosis. Thus, MyD88 signaling in HSCs crucially contributes to liver fibrosis and provides a promising therapeutic target for the prevention and treatment of liver fibrosis.Subject terms: Mechanisms of disease, Kupffer cells     

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.     

Lymphangiogenesis in renal fibrosis arises from macrophages via VEGF-C/VEGFR3-dependent autophagy and polarization

Inflammation plays a crucial role in the occurrence and development of renal fibrosis, which ultimately results in end-stage renal disease (ESRD). There is new focus on lymphangiogenesis in the field of inflammation. Recent studies have revealed the association between lymphangiogenesis and renal fibrosis, but the source of lymphatic endothelial cells (LECs) is not clear. It has also been reported that macrophages are involved in lymphangiogenesis through direct and indirect mechanisms in other tissues. We hypothesized that there was a close relationship between macrophages and lymphatic endothelial progenitor cells in renal fibrosis. In this study, we demonstrated that lymphangiogenesis occurred in a renal fibrosis model and was positively correlated with the degree of fibrosis and macrophage infiltration. Compared to resting (M0) macrophages and alternatively activated (M2) macrophages, classically activated (M1) macrophages predominantly transdifferentiated into LECs in vivo and in vitro. VEGF-C further increased M1 macrophage polarization and transdifferentiation into LECs by activating VEGFR3. It was suggested that VEGF-C/VEGFR3 pathway activation downregulated macrophage autophagy and subsequently regulated macrophage phenotype. The induction of autophagy in macrophages by rapamycin decreased M1 macrophage polarization and differentiation into LECs. These results suggested that M1 macrophages promoted lymphangiogenesis and contributed to newly formed lymphatic vessels in the renal fibrosis microenvironment, and VEGF-C/VEGFR3 signaling promoted macrophage M1 polarization by suppressing macrophage autophagy and then increased the transdifferentiation of M1 macrophages into LECs.Subject terms: Lymphangiogenesis, End-stage renal disease     

Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12

Quiescent hepatic stellate cells (HSCs), in response to liver injury, undergo characteristic morphological transformation into proliferative, contractile and ECM-producing myofibroblasts. In this study, we investigated the implication of canonical Wnt signaling pathway in HSCs and liver fibrogenesis. Canonical Wnt signaling pathway activation and inhibition using β-catenin/CBP inhibitor ICG001 was examined in-vitro in TGFβ-activated 3T3, LX2, primary human HSCs, and in-vivo in CCl₄-induced acute liver injury mouse model. Fibroblasts-conditioned medium studies were performed to assess the Wnt-regulated paracrine factors involved in crosstalk between HSCs-macrophages and HSCs-endothelial cells. Canonical Wnt signaling pathway components were significantly up-regulated in-vitro and in-vivo. In-vitro, ICG-001 significantly inhibited fibrotic parameters, 3D-collagen contractility and wound healing. Conditioned medium induced fibroblasts-mediated macrophage and endothelial cells activation was significantly inhibited by ICG-001. In-vivo, ICG-001 significantly attenuated collagen accumulation and HSC activation. Interestingly, ICG-001 drastically inhibited macrophage infiltration, intrahepatic inflammation and angiogenesis. We further analyzed the paracrine factors involved in Wnt-mediated effects and found CXCL12 was significantly suppressed both in-vitro and in-vivo following Wnt inhibition. Wnt-regulated CXCL12 secretion from activated HSCs potentiated macrophage infiltration and activation, and angiogenesis. Pharmacological inhibition of canonical Wnt signaling pathway via suppression of stromal CXCL12 suggests a potential therapeutic approach targeting activated HSCs in liver fibrosis.     

Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4)) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6)) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4) treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4) administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4) demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4) treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4) alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4) treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.     

MicroRNA-145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2

Activation of quiescent hepatic stellate cells (HSCs) is the major event in liver fibrosis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. The p53, a guardian of the genome is associated with liver fibrosis, has been shown to regulate HSCs senescence. In this study, we report that microRNA-145 (miR-145) and p53 were downregulated in vivo and in vitro, concomitant with the enhanced expression of zinc finger E-box binding homeobox 2 (ZEB2). In addition, overexpression of miR-145 and p53 led to upregulation of the number of senescence-associated β-galactosidase-positive HSCs and the expression of senescence markers p16 and p21, along with the reduced abundance of HSC activation markers α-smooth muscle actin and type I collagen in activated HSCs. Furthermore, silencing of ZEB2 promoted senescence of activated HSCs. Moreover, we also demonstrated that miR-145 specifically targeted the 3′-untranslated regions of ZEB2. In vitro promoter regulation studies show that ZEB2 could bind to the E-box of the p53 promoter as well as inhibit its promoter activity and thus suppress the expression of p53, which in turn repressed activated HSCs senescence. Taken together, our results describe a novel miR-145-ZEB2-p53 regulatory line might participate in the senescence of activated HSCs and might carry potential therapeutic targets for restraining liver fibrosis.     

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl₄) for 8 weeks, and mice were treated with PlGF siRNA or non‐targeting control siRNA starting two weeks after initiating CCl₄ injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA‐treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia‐inducible factor‐1α (HIF‐1α), VEGF and VEGF receptor‐1. Moreover, down‐regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF‐1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis‐associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.     

CDH11 promotes liver fibrosis via activation of hepatic stellate cells

Liver fibrosis, an important health condition associated with chronic liver injury that provides a permissive environment for cancer development, is characterized by the persistent deposition of extracellular matrix components that are mainly derived from activated hepatic stellate cells (HSCs). CDH11 belongs to a group of transmembrane proteins that are principally located in adherens junctions. CDH11 mediates homophilic cell-to-cell adhesion, which may promote the development of cirrhosis. The goal of this study was to determine whether CDH11 regulates liver fibrosis and to examine its mechanism by focusing on HSC activation. Here we demonstrate that CDH11 expression is elevated in human and mouse fibrotic liver tissues and that CDH11 mediates the profibrotic response in activated HSCs. Our data indicate that CDH11 regulates the TGFβ-induced activation of HSCs. Moreover, cells from CDH11 deficient mice displayed decreased HSC activation in vitro, and CDH11 deficient mice developed liver fibrogenesis in response to chronic damage induced by CCl₄ administration. In addition, CDH11 expression was positively correlated with liver fibrosis in patients with cirrhosis, and could therefore be a prognostic factor in patients with liver fibrosis. Collectively, our findings demonstrate that CDH11 promotes liver fibrosis by activating HSCs and may represent a potential target for anti-fibrotic therapies.     

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.     

Induction of M2 Polarization in Primary Culture Liver Macrophages from Rats with Acute Pancreatitis

Background and Aims

Systemic inflammatory response syndrome (SIRS), a major process of severe acute pancreatitis (SAP), usually occurs after various activated proinflammatory cytokines, which are produced by macrophages such as liver macrophages. Macrophages can secrete not only proinflammatory mediators but also inhibitory inflammatory cytokines such as IL-10, leading to two different functional states defined as “polarization”. The main purpose of this study was to demonstrate the polarization of liver macrophages during severe acute pancreatitis and to explore whether the polarization of these activated Liver macrophages could be reversed in vitro.

Methods

Liver macrophages were isolated from rats with acute pancreatitis. These primary culture macrophages were treated with IL-4 or regulatory T cells in vitro to reverse their polarization and was evaluated by measuring M1/M2 marker expression using real time PCR and immunofluorescence staining.

Results

Acute pancreatitis was induced successfully by intra-pancreatic ductal injection of 5% sodium taurocholate. The liver macrophages demonstrated M1 polarization from 4 h to 16 h after the onset of acute pancreatitis. However, after IL-4 or Treg treatment, the polarization of the liver macrophages was reversed as indicated by increased expression of M2 markers and reduced expression of M1 markers. Furthermore, the effect of Treg on modulating macrophage polarization was slightly better than that of IL-4 in vitro.

Conclusion

Liver macrophages, a pivotal cell type in the pathogenesis of SAP, become M1 polarized during pancreatic inflammation. Treatment of these cells with IL-4 and Treg can reverse this activation in vitro. This method of altering macrophage polarization could be a prospective therapy for SAP.     

Covalent Inhibition of Pyruvate Kinase M2 Reprograms Metabolic and Inflammatory Pathways in Hepatic Macrophages against Non-alcoholic Fatty Liver Disease

Warburg effect of aerobic glycolysis in hepatic M1 macrophages is a major cause for metabolic dysfunction and inflammatory stress in non-alcoholic fatty liver disease (NAFLD). Plant-derived triterpene celastrol markedly inhibited macrophage M1 polarization and adipocyte hypertrophy in obesity. The present study was designed to identify the celastrol-bound proteins which reprogrammed metabolic and inflammatory pathways in M1 macrophages. Pyruvate kinase M2 (PKM2) was determined to be a major celastrol-bound protein. Peptide mapping revealed that celastrol bound to the residue Cys³¹ while covalent conjugation altered the spatial conformation and inhibited the enzyme activity of PKM2. Mechanistic studies showed that celastrol reduced the expression of glycolytic enzymes (e.g., GLUT1, HK2, LDHA, PKM2) and related signaling proteins (e.g., Akt, HIF-1α, mTOR), shifted aerobic glycolysis to mitochondrial oxidative phosphorylation and skewed macrophage polarization from inflammatory M1 type to anti-inflammatory M2 type. Animal experiments indicated that celastrol promoted weight loss, reduced serum cholesterol level, lipid accumulation and hepatic fibrosis in the mouse model of NAFLD. Collectively, the present study demonstrated that celastrol might alleviate lipid accumulation, inflammation and fibrosis in the liver via covalent modification of PKM2.     

Regulation of peroxisome proliferator-activated receptor-gamma activity affects the hepatic stellate cell activation and the progression of NASH via TGF-β1/Smad signaling pathway

Journal of Physiology and Biochemistry - Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into myofibroblasts (activated HSCs), which produce...     

肝纤维化发生发展及逆转过程中肝巨噬细胞亚群分类

肝纤维化是由持续性损伤修复反应引起的,导致肝组织内细胞外基质异常沉积,进一步引发肝脏结构和肝功能异常改变的一种病理过程.已有大量研究表明,肝纤维化在去除损伤因素后是可以逆转的.肝星状细胞作为主要的效应细胞,合成和分泌各种胶原和细胞外基质,一直被认为是肝纤维化发生发展的中心环节.最近的研究发现,巨噬细胞作为主要的调节细胞,能同时调节肝星状细胞的功能和基质胶原的降解,促进肝纤维化的形成.而在肝纤维化逆转过程中,促使活化的肝星状细胞凋亡和纤维胶原的降解,促进肝纤维化的逆转.目前已有研究表明,巨噬细胞亚群在肝纤维化发生发展及逆转中具有双向调控作用,但是对于动物模型体内还没有系统的研究巨噬细胞亚群的分类.本文对巨噬细胞亚群的分类研究做一个全面的综述,对肝脏巨噬细胞在肝纤维化中分子机制的进一步研究具有一定的参考价值和借鉴意义.     

Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.