Maki Niioka

Bone marrow–derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice

Liver fibrosis is usually progressive, but it can occasionally be reversible if the causative agents are adequately removed or if patients are treated effectively. However, molecular mechanisms responsible for this reversibility of liver fibrosis have been poorly understood. To reveal the contribution of bone marrow (BM)‐derived cells to the spontaneous regression of liver fibrosis, mice were treated with repeated carbon tetrachloride injections after hematopoietic reconstitution with enhanced green fluorescent protein (EGFP)‐expressing BM cells. The distribution and characteristics of EGFP‐positive (EGFP+) cells present in fibrotic liver tissue were examined at different time points after cessation of carbon tetrachloride intoxication. A large number of EGFP+ cells were observed in liver tissue at peak fibrosis, which decreased during the recovery from liver fibrosis. Some of them, as well as EGFP‐negative (EGFP−) liver resident cells, expressed matrix metalloproteinase (MMP)‐13 and MMP‐9. Whereas MMP‐13 was transiently expressed mainly in the cells clustering in the periportal areas, MMP‐9 expression and enzymatic activity were detected over the resolution process in several different kinds of cells located in the portal areas and along the fibrous septa. Therapeutic recruitment of BM cells by granulocyte colony‐stimulating factor (G‐CSF) treatment significantly enhanced migration of BM‐derived cells into fibrotic liver and accelerated the regression of liver fibrosis. Experiments using transgenic mice overexpressing hepatocyte growth factor (HGF) indicated that G‐CSF and HGF synergistically increased MMP‐9 expression along the fibrous septa. Conclusion: Autologous BM cells contribute to the spontaneous regression of liver fibrosis, and their therapeutic derivation could be a new treatment strategy for intractable liver fibrosis. (HEPATOLOGY; 2007:213–222.)

Butyrate-producing probiotics reduce nonalcoholic fatty liver disease progression in rats: new insight into the probiotics for the gut-liver axis.

Nonalcoholic fatty liver disease (NAFLD) includes simple steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The gut-derived endotoxin plays an essential role in the pathophysiological development and progression of NAFLD. By using rat models of choline-deficient/L-amino acid-defined (CDAA)-diet-induced NAFLD, we examined whether MIYAIRI 588–a butyrate-producing probiotic – prevents the progression of pathophysiological changes from steatosis to hepatocarcinogenesis. In vivo experiments showed that treatment with MIYAIRI 588 reduced CDAA-diet-induced hepatic lipid deposition and significantly improved the triglyceride content, insulin resistance, serum endotoxin levels, and hepatic inflammatory indexes. We also found that MIYAIRI 588 substantially increased the activation of hepatic adenosine 5′-monophosphate-activated protein kinase (AMPK) and AKT and the expression of lipogenesis- or lipolysis-related proteins. MIYAIRI 588 also improved CDAA-diet-induced delocalization and substantially decreased the expression of the tight-junction proteins intestinal zonula occluden-1 and occludin in CDAA-diet-fed rats. Further, the MIYAIRI 588-treated rats also showed remarkable induction of nuclear factor erythoid 2-related factor 2 (Nrf2) and its targeted antioxidative enzymes, which suppressed hepatic oxidative stress. In vitro studies revealed that treatment with sodium butyrate (NaB) also activated AMPK and AKT and enhanced Nrf2 expression by precluding ubiquitination, thereby increasing the half-life of the Nrf2 protein. Pharmacological studies and siRNA knockdown experiments showed that NaB-mediated AMPK activation induced the phosphorylation and nuclear translocation of Sirtuin 1, leading to the increased assembly of mammalian TOR complex 2 and phosphorylation of AKT at Ser473 and subsequent induction of Nrf2 expression and activation. These favorable changes caused an obvious decrease in hepatic fibrous deposition, GST-P-positive foci development, and hepatocarcinogenesis. Our data clearly established that the probiotic MIYAIRI 588 has beneficial effects in the prevention of NAFLD progression.

Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride

BACKGROUND/AIMS Liver fibrosis is a dynamic state between matrix production and degradation. Since our report in 1974, many studies have examined collagenase and liver fibrosis, but not the identification of cells responsible for collagenase production in vivo. The aim of this study was to investigate the gene expression of interstitial collagenase in the progressive and recovery phases of experimental rat liver fibrosis by in situ hybridization. METHODS We examined the gene expression of interstitial collagenase (MMP-13) in the progressive and recovery phase of experimental rat liver fibrosis induced by chronic CCl4 intoxication by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. In order to identify the cells expressing MMP-13 mRNA by in situ hybridization, immunohistochemistry was performed using serial sections. RESULTS In normal rat liver, a faint band for MMP-13 mRNA was observed by RT-PCR, but not by in situ hybridization. The livers of rats treated with CCl4 for 4 weeks showed fatty metamorphosis but no definite fibrosis. Positive signals for MMP-13 mRNA were observed in scattered mesenchymal cells, within lobules which seem to be stellate cells from immunohistochemical staining. Once the fibrosis became prominent, the faint band for MMP-13 mRNA was detected only by RT-PCR and very few signals, if any, by in situ hybridization. On the other hand, in the recovery phase of liver fibrosis, gene expression of MMP-13 was markedly enhanced. Strong positive cells by in situ hybridization were observed mainly at the interface between the resolving fibrous septa and the parenchyma. Overlapping both images of in situ hybridization and immunohistochemical staining with the help of a computer revealed that some positive cells, but not all cells, were stellate cells stained with a-smooth muscle actin antibody. CONCLUSIONS MMP-13 participates in the degradation of newly-formed matrix in the recovery from rat liver fibrosis more than in the remodeling of extracellular matrix for the formation of fibrosis. Hepatic stellate cells play a crucial role in MMP-13 production in the recovery from fibrosis, though not all stellate cells were positive for MMP-13 mRNA. Further investigation into gene expression of MMP-13 in recovery will lead to new strategies for the treatment of liver cirrhosis.

Dynamic change of cells expressing MMP-2 mRNA and MT1-MMP mRNA in the recovery from liver fibrosis in the rat

BACKGROUND/AIMS The aim of this study was to investigate whether both matrix metalloproteinase-2 (MMP-2) and membrane type 1 MMP (MT1-MMP) participate in the spontaneous resolution of liver fibrosis. METHODS Transcription of both genes was examined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Gelatinase activity was investigated by zymography. RESULTS Gene expression by RT-PCR showed that both genes increased in the process of liver fibrosis, then decreased gradually in the recovery phase. ISH revealed that distribution of positive cells changed quickly in the recovery phase. Positive cells were widely seen in the liver, mainly around fibrous septa, in the aggressive phase, but were exclusively observed at the interface between the resolving fibrous band and the parenchyma, then were diffusely located in the lobules in the recovery phase. Main cells expressing both mRNAs seemed to be stellate cells for their morphology, though they did not express characteristic cell markers. Some hepatocytes and Kupffer cells expressed both mRNAs in the recovery phase. Gelatinase activity of MMP-2 increased in the recovery phase of 8-week-treated rat liver by gelatin zymography. CONCLUSIONS The results of present study suggest that both enzymes participate in the destruction of extracellular matrix in coordination with MMP-13.

Matrix metalloproteinase-13 promotes recovery from experimental liver cirrhosis in rats.

Objective: To evaluate the role of matrix metalloproteinase (MMP)-13 gene expression in the early phase of recovery from liver fibrosis/cirrhosis. Methods: Liver fibrosis was induced in male Wistar rats by administration of carbon tetrachloride (CCl4) for 10 weeks. Recombinant adenovirus-mediated human MMP-13 gene transfer (RAdMMP-13) was performed via the femoral vein on day 3 after the last CCl4 injection. The role of MMP-13 in stably expressing cell lines was also analyzed. Results: Fibrous deposition in the liver was decreased in RAdMMP-13-injected rats by day 3 after gene transfer compared with empty vector RAd66-injected rats. Furthermore, MMP-2 and MMP-9 enzymatic activity was markedly enhanced in the liver of RAdMMP-13 injected rats. Hepatocyte growth factor (HGF) induction was also increased in RAdMMP-13 injected rats. In established stable HT-1080 cells transfected with MMP-13, HGF-α expression and MMP-2 and MMP-9 enzymatic activity were increased. The conversion of precursor HGF into mature HGF was also increased in the MMP-13 expressing cell lines. Conclusion: Forced MMP-13 expression effectively accelerated recovery from liver cirrhosis via the effects of MMP-13-mediated HGF, MMP-2, and MMP-9 expression, which induced the degradation of collagen fibers and promoted hepatic regeneration.

c-jun NH2-terminal kinase pathway is involved in constitutive matrix metalloproteinase-1 expression in a hepatocellular carcinoma-derived cell line

Transcription factor c‐Jun serves for cellular proliferation, survival, differentiation and transformation and is recognized as an important factor in cancer development, including hepatocellular carcinoma (HCC). The purpose of present study is to determine the involvement of c‐Jun in matrix metalloproteinase‐1 (MMP‐1) expression, which is previously reported by us to be expressed only in the early stage of human HCC showing stromal invasion. Of 5 human HCC cell lines examined, only HLE cells revealed mRNA and protein expression as well as enzymatic activity of MMP‐1. Transient transfection of an MMP‐1 promoter/luciferase construct (including 4.4 kb full promoter region) into HLE and HCC‐T cells (MMP‐1 nonproducer) showed that high promoter activity was observed only in HLE cells without inducers, and that this promoter activity was still observed when a shorter 0.6 kb proximal promoter construct was transfected. The 0.6 kb promoter region contained 3 AP‐1 sites, and c‐jun mRNA was constitutively expressed in HLE cells without inducers. Furthermore, phosphorylated c‐Jun and c‐Jun NH2‐terminal kinase (JNK) were detected in HLE cells. Promoter activity of the 0.6 kb construct was suppressed with SP600125, a potent inhibitor of JNK, but not with PD98059 and SB203580, potent inhibitors of MEK1/2 and p38, respectively. The inhibitory effect of SP600125 was also observed at protein expression level and in enzymatic activity of MMP‐1. Taken together, this study suggests that the JNK pathway is involved in the expression of MMP‐1 in HCC cells and may represent a new functional role of c‐Jun for HCC development.

Changes in serum levels of metalloproteinases and their inhibitors by treatment of chronic hepatitis C with interferon.

We treated 18 patients with chronic hepatitis C by recombinant interferon-α (6 MIU for 24 weeks). In seven patients, serum aminotransferase levels declined to normal (responders). To evaluate the effect of interferon on matrix metalloproteinases (MMPs) and their inhibitors, namely tissue inhibitors of metalloproteinases (TIMPs), the serum levels of these enzymes were determined by enzyme immunoassay (EIA) using a specific monoclonal antibody. In responders, there was a tendency, but not a significant one, towards either an increase in serum MMP 1 levels or a decrease in serum TIMP 1 levels. In contrast, in nonresponders, both a significant decrease in MMP 1 and MMP 3 and a significant increase in TIMP 1 were observed. The number of cases of either increase in serum MMP levels or decrease in serum TIMP levels was significantly larger in responders than in nonresponders. Furthermore, the ratio of MMP 1 to TIMP 1 significantly increased in responders, suggesting that the balance between matrix formation and degradation in hepatic fibrosis tended to move toward degradation. These data indicate that interferon may exert a beneficial effect on hepatic fibrosis in parallel with improvement of aminotransferase activity.

Hepatic Fibrolysis and Hepatic Sinusoidal Cells

The authors succeeded in inducing MMP-1 production by coculture of fibroblasts and hepatocytes. This abundant MMP-1 production by fibroblasts contributes to massive necrosis or tissue breakdown in vivo. Bhatnagar et al. [26] revealed that rat Kupffer cell stimulation by lipopolysaccharide releases collagenase. Bacterial antigen and endotoxins stimulate Kupffer cells to secrete collagenase. This finding leads to the possibility that the sinusoidal environment may prevent pericellular collagen deposition. The authors also observed the gene expression of interstitial collagense in CCl4-treated rat liver by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (unpublished data). In normal rats, no signals for interstitial collagenase mRNA were observed in the liver by in situ hybridization. In rats with fatty change induced by treatment with CC14 for 4 weeks, positive signals for interstitial collagenase mRNA were observed in scattered Ito cells which were identified as α-smooth muscle actin-positive cells. In rats treated for 8 weeks, an intense signal for interstitial collagenase mRNA was observed in several kinds of cells within lobules. In contrast, cirrhotic liver in rats treated for 12 weeks revealed weak expression of interstitial collagenase mRNA in Ito cells. RT-PCR analysis also revealed gene expression of interstitial collagenase in rats treated for 8 weeks. No hepatocytes in the liver displayed interstitial collagenase mRNA transcripts, despite CC14 treatment. Moreover the authors demonstrated evidence of participation of interstitial collagenase in the destruction of matrix in the recovery stage of hepatic fibrosis.

Stem Cells Expressing Matrix Metalloproteinase-13 mRNA Appear during Regression Reversal of Hepatic Cirrhosis

Publisher Summary This chapter describes a mechanism that underlies the degradation of newly formed fibrous bands in CCl4-induced liver fibrosis and cirrhosis and demonstrates that neural progenitor cells derived from hematopoietic stem cells express interstitial collagenase in the recovery phase of experimental cirrhosis. It also discusses the availability of hematopoietic stem cells for the treatment of human liver fibrosis or cirrhosis. The reversibility of liver fibrosis in patients with alcoholic cirrhosis and alcoholic fatty liver with fibrosis has been observed after alcohol abstinence. Histologically documented reversibility of liver fibrosis in alcoholic liver diseases was closely correlated with a decrease in serum fibrosis markers of type IV collagen. It is now generally accepted that reversibility of liver fibrosis or cirrhosis occurs under some conditions if the cause of liver damage is removed or adequately treated.

Reversibility of Liver Fibrosis

Reversibility of liver fibrosis has been reported both experimentally and clinically, if the cause of liver damage is removed or adequately treated. We first reported the collagenase activity in the process of experimental liver fibrosis in 1974. The present review discusses the participation of matrix metalloproteinases (MMPs), especially MMP-1/MMP-13, in the spontaneous resolution of liver fibrosis, in association with tissue inhibitor of MMPs (TIMPs). A recent advance in molecular biology has revealed the important role of MMPs and TIMPs in the recovery from liver fibrosis and cirrhosis. In situ hybridization study showed that some stem/progenitor cells expressing MMP-13 mRNA may play an important role in the recovery from liver cirrhosis, and these cells were determined to be neural cells by us to be neural origin. Transfusion of stem cells derived from bone marrow will be applied to patients with liver cirrhosis in near future. Suitable conditions to make stem cells proliferate and differentiate for the expression of MMP-13 should be developed.