Protective mechanism of Lygodium flexuosum extract in treating and preventing carbon tetrachloride induced hepatic fibrosis in rats

The protective effect of Lygodium flexuosum extract in preventive and curative treatments of CCl(4) induced fibrosis was quantified. Hepatic fibrosis was induced in male Wistar rats by CCl(4) administration (150 microL/100 gm rat weight, oral) twice a week for 10 weeks. In preventive treatment daily doses of L. flexuosum n-hexane extract (200 mg/kg, p.o) were administered for 10 weeks. In curative treatment L. flexuosum extract (200 mg/kg, p.o) was given for 2 weeks after the establishment of fibrosis for 10 weeks. Treatment with the n-hexane extract (200 mg/kg) reduced the mRNA levels of proinflammatory cytokines, growth factors and other signaling molecules, which are involved in hepatic fibrosis. The expression levels of tumor necrosis factor-alpha, interleukin-1beta, transforming growth factor-beta1, procollagen-I, procollagen-III and tissue inhibitor of metalloproteinase-1 were elevated during carbon tetrachloride administration and reduced the levels to normal by the treatment with the extract treatment. The increased levels of matrix metalloproteinase-13 in extract treated rats were indicative of the protective action of L. flexuosum n-hexane extract. In conclusion, L. flexuosum n-hexane extract functions as a potent fibrosuppresant, effectively reverses carbon tetrachloride-induced hepatic fibrosis in curative treatment and reduces the effects of ongoing toxic liver injury in preventive treatment by promoting extracellular matrix degradation in the fibrotic liver.     

Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats

BACKGROUND/AIMS: Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS: Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS: Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS: Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.     

Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis

Hepatic fibrosis results from an imbalance between fibrogenesis and fibrolysis in the liver. It remains uninvestigated whether Kupffer cells produce matrix metalloproteinase-13 (MMP-13), which mainly hydrolyzes extracellular matrix (ECM). We sought to determine the role of Kupffer cells in fibrogenesis/fibrolysis. In vivo, we used the rat model of pig serum-induced liver fibrosis. A subset was treated with gadolinium chloride (GdCl(3)), which specifically acts on Kupffer cells. Administration of GdCl(3) remarkably decreased the hydroxyproline content of the liver and increased the expression of MMP-13 mRNA in the liver without a difference in procollagen type I and tissue inhibitors of metalloproteinase-1 (TIMP-1) mRNA expression on Northern blot analysis with the elimination of ED2-positive cells. In vitro, addition of GdCl(3) to isolated Kupffer cells showed increased type I collagen-degrading activity in a dose-dependent manner as well as MMP-13 mRNA expression on Northern blot analysis. It is concluded that Kupffer cells are a major source of MMP-13 and modulation of Kupffer cells by GdCl(3) prevents liver fibrosis with increased expression of MMP-13 mRNA and protein, whereas procollagen type I and TIMP-1 mRNA, which encode two major effectors of fibrogenesis, were unchanged. This is the first report showing that Kupffer cells produce interstitial collagenase (MMP-13) resulting in the reduction of ECM. This discovery may provide new insights into therapy for hepatic 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.     

Changes in the activity of matrix metalloproteinases in regenerating rat liver after CCl4-induced injury

Molecular mechanisms involved in mediating alteration in cell matrix interaction have been examined by studying the changes in the activity of matrix metalloproteinases (MMPs) in CCl4-induced regenerating liver, using zymography and ELISA. Activity of MMPs (72 kD, 92 kD and 130 kD gelatinases) in the rat liver increased progressively during acute injury till the 4th day and then decreased to near normal level after CCl4 administration (0.5 ml/100 g body wt.) on the 6th day. Hepatocyte lysate of injured liver on the 4th day showed significantly higher levels of MMP2 and MMP9 compared to the control. In the culture medium of hepatocytes, the levels of MMP2 and MMP9 increased progressively with the duration of culture, indicating that hepatocytes are the major source of these MMPs in regenerating liver. These results suggest an involvement of MMPs in matrix degradation and remodeling during regeneration after acute liver injury induced by CCl4.     

Aqueous extract of Anoectochilus formosanus attenuate hepatic fibrosis induced by carbon tetrachloride in rats.

The aim of this study was to investigate the effects of aqueous extract of Anoectochilus formosanus (AFE) on liver fibrogenesis in carbon tetrachloride (CCl4)-induced cirrhosis. Fibrosis was induced in rats by oral administration of CCl4 (20%, 0.5 ml/rat, p.o.) twice a week for 8 weeks. AFE (0.5 and 2.0 g/kg, p.o., daily for 8 weeks) was administered to rats simultaneously. AFE showed reducing actions on the elevated levels of GOT and GPT caused by CCl4. Liver fibrosis in rats induced by CCl4 led to the drop of serum albumin concentration; the AFE increased the albumin concentration. The CCl4-induced liver fibrosis markedly caused liver atrophy and splenomegalia, while AFE increased the liver weight, and decreased the spleen weight. The CCl4-induced liver fibrosis decreased the protein content, and increased collagen contents in rat's liver. AFE significantly increased the contents of protein and reduced the amount of collagen in the liver. In CCl4-treated rats, glutathione concentrations of liver were not affected. AFE significantly increased liver glutathione concentrations. All these results clearly demonstrate that AFE can reduce the liver fibrogensis in rats induced by CCl4.     

Effects of Shugan-Huayu powder, a traditional Chinese medicine, on hepatic fibrosis in rat model

Shugan-Huayu powder (SHP) has been administered to outpatients with chronic liver disease without clear anti-fibrosis mechanism. To investigate the anti-fibrotic effects of SHP on liver fibrosis in a rat model and in hepatic stellate cells (HSCs) in vitro, rats were gavaged with CCl4 at 1.0 g/kg body weight twice a week for 8 weeks to induce liver fibrosis and the rats were randomly assigned to one of the three groups: -CCl4 alone, low-dose SHP and high-dose SHP. SHP was given by gavages 5 times a week for 8 weeks. Serum, livers and HSCs were assayed for serology, pathology, western blot, zymography and quantitative RT-PCR. Hepatic function improved as decreased serum aspartate aminotransferase and alanine aminotransferase, and collagen deposition and active HSCs were significantly reduced in CCl4-induced liver by SHP treatment. The expression of matrix metalloproteinase-2 (MMP-2) and transforming growth factor-beta1 (TGF-beta1) mRNA in fibrotic liver showed significant downregulation after SHP treatment. In vitro, inhibition of alpha-smooth muscle actin (alpha-SMA) expression and MMP-2 secretion of active HSCs were also noticed by SHP treatment. SHP has an antifibrotic effect on CCl4-induced liver fibrosis in rats. Anti-fibrotic mechanisms were probably inhibiting activation of HSCs and decreased expression of MMP-2 and TGF-beta1.     

Rapid induction of liver cirrhosis in rats by treatment with ethanol, carbon tetrachloride and progesterone

The administration of progesterone increases the degree of liver cirrhosis in rats treated with CCl4 and ethanol. Pseudolobulation with large amount of interstitial fibrosis are obtained after only 6 weeks of treatment. The ability of progesterone to suppress collagenase activity is supposed to be responsible of the strong increase of cirrhosis.     

Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis.

The activated hepatic stellate cell (HSC) is central to liver fibrosis as the major source of collagens I and III and the tissue inhibitors of metalloproteinase-1 (TIMP-1). During spontaneous recovery from liver fibrosis, there is a decrease of TIMP expression, an increase in collagenase activity, and increased apoptosis of HSC, highlighting a potential role for TIMP-1 in HSC survival. In this report, we use tissue culture and in vivo models to demonstrate that TIMP-1 directly inhibits HSC apoptosis. TIMP-1 demonstrated a consistent, significant, and dose-dependent antiapoptotic effect for HSC activated in tissue culture and stimulated to undergo apoptosis by serum deprivation, cycloheximide exposure, and nerve growth factor stimulation. A nonfunctional mutated TIMP-1 (T2G mutant) in which all other domains are conserved did not inhibit apoptosis, indicating that inhibition of apoptosis was mediated through MMP inhibition. Synthetic MMP inhibitors also inhibited HSC apoptosis. Studies of experimental liver cirrhosis demonstrated that persistent expression of TIMP-1 mRNA determined by PCR correlated with persistence of activated HSC quantified by alpha smooth muscle actin staining, while in fibrosis, loss of activated HSC correlated with a reduction in TIMP-1 mRNA. We conclude that TIMP-1 inhibits apoptosis of activated HSC via MMP inhibition.     

Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells

Hepatic stellate cells are the major source of the extracellular matrix that accumulates in fibrotic liver. During progressive liver fibrosis, hepatic stellate cells proliferate, but during resolution of fibrosis there is extensive stellate cell apoptosis that coincides with degradation of the liver scar. We have examined the possibility that the fate of stellate cells is influenced by the extracellular matrix through the intermediary of alpha(v)beta(3) integrin. alpha(v)beta(3) integrin was expressed by activated, myofibroblastic rat and human stellate cells in culture. Antagonism of this integrin using neutralizing antibodies, echistatin, or small inhibitory RNA to silence alpha(v) subunit expression inhibited stellate cell proliferation and their expression of proliferating cell nuclear antigen and activated forms of p44 and p42 MAPK. These alpha(v)beta(3) antagonists also increased apoptosis of cultured stellate cells, and this was associated with an increase in the BAX/BCL-2 protein ratio, induction of nuclear DNA fragmentation, and activation of intracellular caspase-3. Expression of tissue inhibitor of metalloproteinases-1 by activated stellate cells was reduced by the alpha(v)beta(3) antagonists, while matrix metalloproteinase-9 synthesis was enhanced. Stellate cells incubated with active recombinant matrix metalloproteinase-9 showed enhanced apoptosis, while cells treated with a synthetic inhibitor of this protease showed increased survival. Our studies suggest that alpha(v)beta(3) integrin regulates the fate of hepatic stellate cells. Degradation of alpha(v)beta(3) ligands surrounding activated stellate cells during resolution of liver fibrosis might decrease alpha(v)beta(3) integrin ligation, suppressing stellate cell proliferation and inducing a fibrolytic, matrix metalloproteinase-secreting phenotype that may prime stellate cells for apoptosis.     

Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation

Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murine collagenase-like A and B). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-B genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.     

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.     

Plasminogen activator activity increases during reversal of hepatic fibrosis in murine schistosomiasis

After specific chemotherapy, granulomatous fibrosis undergoes a marked reversal in liver of Schistosoma mansoni-infected mice. We have previously shown that this fibrosis reversal was related to a high proportion of the active form of the interstitial collagenase. In vitro, plasmin has been described as a physiological activator of interstitial procollagenase. Moreover, plasmin itself degrades directly matrix components such as proteoglycans and fibronectin. We have thus followed the course of the plasminogen activator, which converts plasminogen zymogen to plasmin, in liver of S. mansoni-infected mice treated with praziquantel, as schistosomicidal drug. It was found that plasminogen activator activity in the liver increases rapidly until 5 days after treatment as compared to nontreated infected mice and then diminishes gradually. Increased plasminogen activator activity appears to be one of initial events leading to this fibrosis reversal.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.