Activation of matrix metalloproteinase-2 from hepatic stellate cells requires interactions with hepatocytes.

Activation of matrix metalloproteinase (MMP)-2, the 72-kd collagenase IV/gelatinase A, is involved in extracellular matrix remodeling. It has been suggested that a membrane-type MMP (MT-MMP-1) and the tissue inhibitor of metalloproteinase (TIMP)-2 are involved in MMP-2 processing, but the exact mechanism(s) of its activation remains unclear. We have investigated the role of cell-cell cooperation in the activation of pro-MMP-2 in the liver, using pure cultures and co-cultures of hepatocytes and hepatic stellate cells (HSCs). Northern blot analysis and in situ hybridization showed that, in both pure and co-cultures, HSCs, but not hepatocytes, expressed MMP-2, TIMP-2, and MT-MMP-1 mRNA. Zymography analyses revealed the latent form of MMP-2 in medium from 2-day-old pure HSC cultures with higher amounts in medium from hepatocyte/HSC co-cultures. When hepatocytes were added to 10-day-old HSC cultures, the activated form of MMP-2 was detected, concomitantly with the deposition of an abundant extracellular matrix. Incubation of plasma membrane-enriched fractions from hepatocytes with conditioned medium from pure HSC cultures generated the activated species of MMP-2 (62 and 59 kd). Activation of pro-MMP-2 by hepatocyte membranes was inhibited by EDTA, heat, and trypsin but not by serine proteinase inhibitors. These data show that the co-expression of TIMP-2, MMP-2, and MT-MMP-1 by HSCs does not lead to secretion of the activated form of MMP-2. Hepatocytes, which do not express MMP-2, TIMP-2, or MT-MMP-1, induce MMP-2 activation through a plasma membrane-dependent mechanism(s), thus suggesting that cell-cell interactions are involved in this process in vivo.     

CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable in parallel with HSC activation, which suggests that apoptosis might represent an important mechanism terminating proliferation of activated HSC.     

Mechanism of interleukin‐1β‐induced proliferation in rat hepatic stellate cells from different levels of signal transduction

Hepatic stellate cells (HSCs) are the major producers of collagen in the liver. Their conversion from resting cells to proliferative, contractile, and activated cells is a critical step leading to liver fibrosis that is characterized by the deposition of excessive extracellular matrix. Interleukin‐1 (IL‐1) may play a role in maintaining HSC in a proliferative state that is responsible for hepatic fibrogenesis. The aim of this study was to study the roles of the IL‐1 type I receptor (IL‐1R1), c‐Jun N‐terminal kinase (JNK), and activation protein‐1 (AP‐1) in IL‐1β–mediated proliferation in rat HSCs. We showed that IL‐1β can upregulate proliferation in rat HSCs; however, inhibition of the JNK pathway could inhibit HSCs proliferation. Furthermore, IL‐1β activated IL‐1R1 expression, the JNK signaling pathway, and AP‐1 activity in a time‐dependent manner in rat HSCs. These data demonstrate that IL‐1β could promote the proliferation of rat HSCs and that the IL‐1R1, JNK, and AP‐1 pathways were involved in this process. In summary, IL‐1β‐induced proliferation is possibly mediated by the IL‐1R1, JNK, and AP‐1 pathways in rat HSCs. Therefore, drugs that block these pathways may inhibit the proliferation of HSCs and suppress liver fibrosis.     

Oxidative stress effect on the activation of hepatic stellate cells

Collagen is the most excessive extracellular matrix protein in hepatic fibrosis. Activated, but not quiescent, hepatic stellate cells (HSCs) have a high level of collagen and a smooth muscle actin (alpha SMA) expression. HSCs play a key role in the pathogenesis of hepatic fibrosis. We analyzed a mechanism leading to HSC activation by evaluating the role of oxidative stress and the expression of NFkB. In vitro study HSCs were proliferated (PCNA:2% vs 68%) and activated (alpha SMA: 5% vs 78%) by ascorbate/FeSO4, and HSCs activated by type I collagen were blocked (PCNA: 97% vs 4%, a SMA: 86% vs 9%) by a-tocopherol. In vivo study means of a SMA positive cells in liver at 400 x HPF were 48.3+/-5.2 and 15.2+/-1.8 and [3H]thymidine uptake of HSC was 529.2+/-284.8 cpm and 223.0+/-86.3 cpm in control and a-tocopherol treated group respectively at 32 hours after CCl4 injection. Nuclear extracts from activated, but not from quiescent, HSCs formed a complex with the NFkB cognate oligonucleotidesand alpha-tocopherol inhibited this bindings. This study indicates that oxidative stress plays an essential role through the induction of NFkB on HSC activation.     

黏着斑激酶反义寡核苷酸对肝星状细胞增殖及活化的影响

黏着斑激酶(Focal adhesion kinase,FAK)在肝星状细胞(Hepatic stellate cells,HSC)的激活过程中起重要作用.本文旨在研究FAK反义寡核苷酸(FAK antisense oligonucleotides,FAK-ASON)能否抑制体外培养的大鼠肝星状细胞的增殖及活化.用链霉蛋白酶-胶原酶原位灌注、Nycodenz密度梯度离心法分离大鼠HSC,并在体外培养使其激活后,将FAK-ASON转染至活化的HSC.应用MTT法观察HSC细胞增殖的变化,并用RT-PCR、Western blot观察HSC活化的标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达的变化.MTT结果显示FAK-ASON可明显抑制活化HSC的增殖;FAK-ASON处理48 h后,HSC α-SMA mRNA及蛋白的表达亦明显下降.结果进一步提示FAK在HSC激活过程中的作用,而FAK-ASON有可能成为潜在的抗肝纤维化治疗药物.     

Increased SSeCKS Expression in Rat Hepatic Stellate Cells Upon Activation In Vitro and In Vivo

Recent reports suggest that src suppressed c kinase substrates (SSeCKS) are early inflammatory response protein. However, there is only scarce knowledge on the functional role of SSeCKS in liver under conditions of acute inflammation. In the present study, we investigated SSeCKS expression in liver after administration of carbon tetrachloride (CCl₄) in rats and in isolated primary hepatic stellate cells (HSCs) upon activation on a plastic dish. We found that SSeCKS mRNA was hardly detectable in healthy liver tissue and further increased in carbon tetrachloride-mediated acute liver failure. SSeCKS protein expression was mainly found in hepatic stellate cells. In vitro, SSeCKS expression in activated rat HSCs was dramatically increased. The upregulation of SSeCKS protein expression in rat HSCs during activation in vitro and in vivo suggested the possibility of SSeCKS, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries.     

Neutralization of CXCL10 accelerates liver regeneration in carbon tetrachloride-induced acute liver injury

Remodeling of hepatic tissue structure following injury requires the coordinated action of hepatocytes, hepatic stellate cells (HSCs), and endothelial cells. However, their in vivo properties are not fully understood. We report here that the chemokine CXCL10 regulates hepatic tissue remodeling in a carbon tetrachloride (CCl(4))-induced acute liver injury in mice. The production of CXCL10 was enhanced by hepatocytes after CCl(4) exposure. Neutralization of CXCL10 protected mice from acute liver dysfunction and diminished hepatocellular loss. The hepatoprotective effect was associated with increased numbers of 5'-bromo-2' deoxyuridine (BrdU)+ hepatocytes from day 1 and with accumulation of HSCs and endothelial cells within the injured zones from day 3. In vitro, recombinant CXCL10 directly inhibited the proliferation of hepatocytic cells, establishing a novel role of CXCL10 in modulating hepatocyte proliferation, in addition to a previously reported angiostatic role. In summary, neutralization of CXCL10 initially stimulates hepatocyte proliferation and, subsequently, HSC migration and angiogenesis to facilitate remodeling of hepatic cords. Thus, CXCL10 can be a novel therapeutic target for acute hepatocellular damage by regulating liver tissue remodeling.     

Mannan binding lectin‐associated serine protease 1 is induced by hepatitis C virus infection and activates human hepatic stellate cells

Mannan binding lectin (MBL)‐associated serine protease type 1 (MASP‐1) has a central role in the lectin pathway of complement activation and is required for the formation of C3 convertase. The activity of MASP‐1 in the peripheral blood has been identified previously as a highly significant predictor of the severity of liver fibrosis in hepatitis C virus (HCV) infection, but not in liver disease of other aetiologies. In this study we tested the hypotheses that expression of MASP‐1 may promote disease progression in HCV disease by direct activation of hepatic stellate cells (HSCs) and may additionally be up‐regulated by HCV. In order to do so, we utilized a model for the maintenance of primary human HSC in the quiescent state by culture on basement membrane substrate prior to stimulation. In comparison to controls, recombinant MASP‐1 stimulated quiescent human HSCs to differentiate to the activated state as assessed by both morphology and up‐regulation of HSC activation markers α‐smooth muscle actin and tissue inhibitor of metalloproteinase 1. Further, the expression of MASP‐1 was up‐regulated significantly by HCV infection in hepatocyte cell lines. These observations suggest a new role for MASP‐1 and provide a possible mechanistic link between high levels of MASP‐1 and the severity of disease in HCV infection. Taken together with previous clinical observations, our new findings suggest that the balance of MASP‐1 activity may be proinflammatory and act to accelerate fibrosis progression in HCV liver disease.     

Identification of growth differentiation factor 15 as a profibrotic factor in mouse liver fibrosis progression

The aim of this study was elucidate the inhibitory role of growth differentiation factor 15 (GDF15) in liver fibrosis and its possible activation mechanism in hepatic stellate cells (HSCs) of mice. We generated a GDF15neutralizing antibody that can inhibit TGF1induced activation of the TGF/Smad2/3 pathway in LX2 cells. All the mice in this study were induced by carbon tetrachloride and thioacetamide. In addition, primary HSCs from mice were isolated from fresh livers using Nycodenz density gradient separation. The severity and extent of liver fibrosis were evaluated by Sirius Red and Masson staining. The effect of GDF15 on the activation of the TGF pathway was detected using dualluciferase reporter and Western blotting assays. The expression of GDF15 in cirrhotic liver tissue was higher than that in normal liver tissue. Blocking GDF15 with a neutralizing antibody resulted in a delay in primary hepatic stellate cell activation and remission of liver fibrosis induced by carbon tetrachloride or thioacetamide. Meanwhile, TGF pathway activation was partly inhibited by a GDF15neutralizing antibody in primary HSCs. These results indicated that GDF15 plays an important role in regulating HSC activation and liver fibrosis progression. The inhibition of GDF15 attenuates chemicalinducible liver fibrosis and delays hepatic stellate cell activation, and this effect is probably mainly attributed to its regulatory role in TGF signalling.     

Morphologic features in the regenerating liver—a comparative intravital,lightmicroscopical and ultrastructural analysis with focus on hepatic stellate cells

Different cell types play a role in the liver regeneration. The present study reveals morphological key steps of liver regeneration by correlating intravital, light, and electron microscopic with immunohistochemistry results focusing on hepatic stellate cells (HSCs). In Lewis rats, liver regeneration was induced by a 2/3-hepatectomy. Animals (n = 7 each) were killed after 0, 1, 2, 3, 4, 7, and 14 days. Morphological features were investigated by light microscopy, immunohistochemistry [α-smooth muscle actin (α-SMA), Desmin, vascular endothelial growth factor (VEGF)/VEGF receptor, Ki-67, ssDNA], intravital microscopy (sinusoid density, number of hepatocytes, and HSC), and electron microscopy focussed on cell-to-cell interactions. During liver regeneration, HSC were activated at day 3 showing a loss of autofluorescence and simultaneously an increased α-SMA expression and direct cell contact to hepatocytes. HSC activation was followed by increasing VEGF expression and sinusoid density. After 14 days, liver architecture and ultrastructure was restored and HSCs were deactivated showing decreased α-SMA expression as well as increased apoptosis and no more direct cell contact to hepatocytes. HSCs play a central role in the regenerating liver by governing angiogenesis and extracellular matrix remodeling. A direct cell contact to hepatocytes seems to be essential for HSC activation, whereas deactivation is accompanied by loosening of hepatocyte contact and increased apoptosis.     

Inhibitory effects of dimethyl α-ketoglutarate in hepatic stellate cell activation

Aim: The activation of Hepatic stellate cell (HSC) is a pivotal event in the initiation and progression of hepatic fibrosis and a major source of collagen deposition. A recent study found that autophagy fuels the HSC activation. α-ketoglutarate (AKG), an intermediate in the Kerbs CYCLE, has been shown to regulate the level of autophagy. In this study, we aim to investigate the potential effect of dimethyl α-ketoglutarate (DMKG), a membrane-permeable esters of AKG, on the activation of HSC. Methods: HSC and hepatocyte cell lines were treated with DMKG at gradient concentrations, MTT assay was used to assess the cell viability. Concentrations of DMKG that did not affect the cell survival were added to the culture media of HSC cells. Real-time PCR and western blot analysis was carried out to evaluate the expression of fibrogenic genes in HSC after culture for 24 hours. Results: Low dose of DMKG had little cytotoxicity to both HSCs and hepatocytes, while HSCs were more vulnerable to high dose of DMKG than hepatocytes. More importantly, DMKG inhibited the expression of α-SMA and collagen I significantly in HSCs detected by real-time PCR and western blot analysis at the concentrations that didn’t decrease cell viability. Conclusions: DMKG has a significant role of inhibiting the activation of HSC and may attenuate hepatic fibrosis safely.     

辣椒素抑制大鼠肝纤维化的实验研究

 目的：探讨辣椒素（capsaicin）对大鼠肝星状细胞（hepatic stellate cells, HSCs）活性的影响及其对实验性肝纤维化的治疗作用。方法：DCFH-DA法检测辣椒素对HSCs中活性氧的影响;CCK-8法检测辣椒素对HSCs增殖的影响;Western blotting检测HSCs α-平滑肌肌动蛋白的表达;RT-PCR检测辣椒素对HSCs纤维化相关基因表达的影响;流式细胞术检测辣椒素对HSCs凋亡的影响;通过腹腔注射四氯化碳建立鼠肝纤维化模型,经腹腔输注辣椒素,检测肝组织病理切片,观察肝纤维化指标的变化。结果：与对照组比较,辣椒素抑制了HSCs中活性氧的产生,明显抑制了HSCs的活化与增殖(P<0.05),促进了HSCs的凋亡(P<0.05),降低了金属蛋白酶组织抑制物1及转化生长因子β 1的表达(P<0.05),降低了肝羟脯氨酸含量及血清Ⅲ型胶原和透明质酸水平(P<005)。结论：辣椒素抑制HSCs增殖、活化并诱导其凋亡。辣椒素对实验性肝纤维化具有一定的抑制作用。     

Identification and functional characterization of the hepatic stellate cell CD38 cell surface molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.     

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.     

Spatial and temporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis.

In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial relationships between oxidant stress and activation of HSCs in an in vivo model of oxidant-stress-associated fibrogenesis. To this purpose, rats were treated with carbon tetrachloride (CCl4) and livers subjected to in situ perfusion with nitroblue tetrazolium, which, in the presence of superoxide ions, is reduced to an insoluble blue-colored formazan derivative and is readily detectable in the tissue by light microscopy. Moreover, various combinations of in situ hybridization and immunocytochemical analyses were performed. An acute dose of CCl4 caused a transient production of superoxide radicals at 24 hours into pericentral necrotic areas, whereas HSC appearance and expression of collagen mRNA were detectable only at 48 and 72 hours. After chronic CCl4 intoxication, higher levels of oxygen radical production in necrotic areas were detectable along with dramatic and sustained activation of HSCs. However, maximal HSC activation was still delayed as compared with superoxide production. Expression of heme oxygenase, a gene responsive to a variety of oxidant stress mediators, was strongly enhanced by chronic CCl4 administration but remained unchanged in HSCs, both in situ and after isolation of pure HSC fractions from control and CCl4-treated animals. In conclusion, during postnecrotic fibrogenesis, oxidant stress anticipates HSC activation. HSCs do not directly face an oxidant stress while engaged in active fibrogenesis.     

Differential Increases in the Expression of Intermediate Filament Proteins and Concomitant Morphological Changes of Transdifferentiating Rat Hepatic Stellate Cells Observed In Vitro

The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.