Autocrine release of TGF-beta by portal fibroblasts regulates cell growth

Portal fibroblasts (PF) are a newly isolated population of fibrogenic cells in the liver postulated to play a significant role in early biliary fibrosis. Because transforming growth factor-beta (TGF)-beta is a key growth factor in fibrosis, we characterized the response of PF to TGF-beta. We demonstrate that PF produce significant amounts of TGF-beta2 and, unlike activated hepatic stellate cells (HSC), express all three TGF-beta receptors and are growth inhibited by TGF-beta1 and TGF-beta2. Fibroblast growth factor (FGF)-2, but not platelet derived growth factor (PDGF), causes PF proliferation. These data suggest a mechanism whereby HSC eclipse PF as the dominant myofibroblast population in biliary fibrosis.     

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.     

Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.     

Scotin: A new p63 target gene expressed during epidermal differentiation

Accumulating evidence indicates that bacteria and bacterial products promote hepatic fibrogenesis. The activation of hepatic stellate cells (HSC) plays a central role in hepatic fibrosis. Here, we demonstrate that HSC express toll-like receptor 9 (TLR9), a pattern recognition receptor that is activated by CpG motifs present specifically in bacterial DNA. Upon CpG stimulation human as well as murine HSC isolated from wild-type (TLR9+/+) mice express increased levels of the profibrogenic chemokine monocyte chemotactic protein 1 (MCP-1). In contrast, HSC isolated from TLR9 deficient (TLR9−/−) mice lacked CpG motif induced MCP-1 expression indicating the functionality of TLR9 in HSC. Bile duct ligation revealed significantly lower hepatic MCP-1 and collagen expression and less hepatic fibrosis in TLR9−/− compared to TLR9+/+ mice. In addition, the expression of hepatic α-smooth-muscle actin, a known marker for HSC activation, was reduced in TLR9−/− mice indicating that bacterial DNA induces the activation of HSC and therefore promotes hepatic fibrosis.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.     

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover, these findings, together with our previous reports and the emerging data from in vivo studies of TIMP inhibition, provide strong evidence that TIMP-1 is mechanistically central to liver fibrosis and an important potential therapeutic target.     

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through alpha1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-alpha1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-kappaB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed alpha(1A)-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed alpha(1A)-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (alpha1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific beta-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-alpha1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-kappaB activation. BAY 11-7082, a specific NF-kappaB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-kappaB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.     

Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line,LI90, by a selective cyclooxygenase 2 inhibitor,NS-398

Cyclooxygenase 2 (COX-2) has been thought to be associated with liver fibrosis whereas it is well known that hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis. There is little evidence of how COX-2 regulates the activation of human HSC or the mechanism involved. In this study, we investigated the effect of a COX-2 inhibitor, NS-398, on a line of human HSC, LI90. Our findings demonstrated that alpha-smooth muscle actin (alpha-SMA) protein expression was inhibited in a dose-dependent manner by treatment with NS-398. Proliferation cell nuclear antigen (PCNA) expression and cell growth were partially down-regulated. The generation of PGE2, IL-8, IL-6, and hyaluronan in the cultured medium was also inhibited. In conclusion, our findings imply that a selective COX-2 inhibitor might be a potential drug for the chemoprevention and treatment of liver fibrosis by inhibiting the activation of HSC.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

MCP-1 in pleural injury: CCR2 mediates haptotaxis of pleural mesothelial cells

Pleural injury results in the death of mesothelial cells and denudation of the mesothelial basement membrane. Repair of the mesothelium without fibrosis requires proliferation and migration of mesothelial cells into the injured area. We hypothesized that monocyte chemoattractant protein-1 (MCP-1) induces proliferative and haptotactic responses in pleural mesothelial cells (PMCs) and that the MCP-1 binding receptor CCR2 mediates the pleural repair process. We demonstrate that PMCs exhibited MCP-1-specific immunostaining on injury. MCP-1 induced proliferative and haptotactic responses in PMCs. PMCs express CCR2 in a time-dependent manner. Fluorescence-activated cell sorting analysis demonstrated that interleukin (IL)-2 upregulated CCR2 protein expression in PMCs, whereas lipopolysaccharide (LPS) downregulated the response at the initial period compared with that in resting PMCs. However, the inhibitory potential of LPS was lost after 12 h and showed a similar response at 24 and 48 h. Haptotactic migration was upregulated in PMCs that were cultured in the presence of IL-2. The increased haptotactic capacity of mesothelial cells in the presence of IL-2 correlated with increased CCR2 mRNA expression. PMCs cultured in the presence of LPS showed decreased haptotactic activity to MCP-1. Blocking the CCR2 with neutralizing antibodies decreased the haptotactic response of PMCs to MCP-1. These results suggest that the haptotactic migration of mesothelial cells in response to MCP-1 are mediated through CCR2, which may play a crucial role in reepithelialization of the denuded basement membrane at the site of pleural injury and may thus contribute to the regeneration of the mesothelium during the process of pleural repair.     

Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl₄)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl₄ induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl₄-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.     

Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression

Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca²⁺ signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca²⁺ signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca²⁺ chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca²⁺ signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca²⁺ signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca²⁺ signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca²⁺ signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca²⁺ are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis.     

Comparative atomic force and scanning electron microscopy: An investigation of structural differentiation of hepatic stellate cells

The molecular mechanism leading to the transdifferentiation of hepatic stellate cells (HSC) into myofibroblast-like cells following liver injury is not well understood. The state of cultured rat HSCs was determined using primarily fluorescence microscopy (UV), immunofluorescence (IF) (Glial fibrillary acidic protein (GFAP), Desmin, alpha-smooth muscle actin (alpha-SMA), F-actin) and immunocytochemistry (ICC) (GFAP, Desmin, alpha-SMA, Fibulin-2). Additionally, tapping-mode atomic force microscopy (TM-AFM) and field-emission scanning electron microscopy (FE-SEM) with low-resistivity indium-tin-oxide (ITO) thin-film were performed to observe the micro-morphological character of cells during HSC differentiation. Quiescent HSCs changed to the activated state were identified via UV, IF, and ICC observations. Normal rat HSCs (NHSCs) and thioacetamide-induced rat HSCs (THSCs) were demonstrated to be UV⁻, GFAP⁺, Desmin⁺, alpha-SMA⁺ and Fibulin-2⁻. After F-actin staining, lamellipodia and filopodia were found in both NHSCs and THSCs, but membrane ruffles were only seen in THSCs. The micro-structures of lamellipodia and filopodia in both NHSCs and THSCs were confirmed using FE-SEM and TM-AFM with ITO; in contrast, the micro-projection was not found. Moreover, “aerial root” structures were observed for the first time in the filopodia of THSCs using TM-AFM. These results reveal that HSC transdifferentiation to a myofibroblastic-like cell (activated HSC) from thioacetamide-induced rat HSC induces extensive changes in the cytoskeleton.     

miR-627/HMGB1/NF-κB regulatory loop modulates TGF-β1-induced pulmonary fibrosis

Pulmonary fibrosis (PF) is a fibroproliferative disease that can eventually lead to fatal lung failure. It is characterized by abnormal proliferation of fibroblasts, dysregulated fibroblast differentiation to myofibroblast, and disorganized collagen and extracellular matrix production, deposition and degradation. There is still a lack of effective treatment strategies for PF. Extracellular high-mobility group box protein 1 (HMGB1) induces PF through NF-κB-mediated TGF-β1 release. Herein, we first validate the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-smooth muscle actin (α-SMA) and collagen I protein expression. In PF, miRNAs exert different effects through targeting various downstream target messenger RNAs. We searched an online database for dysregulated miRNAs in PF tissues; among them, miR-627 was predicted by online tools to target HMGB1 to inhibit its expression. miR-627 overexpression could partially reverse TGF-β1-induced normal human lung fibroblast proliferation, as well as α-SMA and collagen I protein expression. miR-627 inhibition could partially reverse the suppressive effect of HMGB1 knockdown on TGF-β1-induced α-SMA and collagen I protein expression through direct binding to the 3′-untranslated region of HMGB1. Moreover, miR-627/HMGB1 affected TGF-β1 release through RAGE/NF-κB signaling; miR-627/HMGB1 and RAGE/NF-κB signaling formed a regulatory loop to modulate TGF-β1-induced PF in vitro. In conclusion, miR-627 may be a potential agent that targets HMGB1 to inhibit its expression, thereby improving TGF-β1-induced PF in vitro.     

Expression and induction of anaphylatoxin C5a receptors in the rat liver

The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and in-vivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-beta1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.