Yutaka Yata

Increased expression of plasminogen activator and plasminogen activator inhibitor during liver fibrogenesis of rats: role of stellate cells

BACKGROUND/AIMS Plasminogen activators and plasminogen activator inhibitors are important regulators of the balance between the proteolytic and antiproteolytic activities that determine extracellular matrix turnover. We examined the expression of plasminogen activator-plasmin system components in experimental liver fibrosis of rats. METHODS Liver fibrosis was produced in rats by injecting carbon tetrachloride for 6 to 12 weeks. Gene expression for plasminogen activator inhibitor-1 (PAI-1), urokinase and tissue plasminogen activators (uPA and tPA), urokinase plasminogen activator receptor (uPAR), and transforming growth factor-beta1 (TGF-beta1) was examined by Northern analysis. Western analysis was performed to detect protein expression of PAI-1, uPA and uPAR. An immunohistochemical study was performed to detect the localization of PAI-1. Additionally, primary cultured liver cells were examined by Northern and Western analyses for this protein with or without prior incubation with TGF-beta1. RESULTS At 6 weeks, when fibrosis had occurred, uPA and uPAR mRNAs had increased 2.8-fold and 1.8-fold, respectively; PAI-1 and tPA mRNA levels were unchanged. At the cirrhotic stage (9 to 12 weeks), mRNA levels for PAI-1, uPA, uPAR and tPA were all increased. Western analysis also showed increased uPA and uPAR expressions in fibrotic liver, and increased PAI-1, uPA and uPAR expressions in cirrhotic liver. PAI-1 protein was also demonstrated immunohistochemically along sinusoids, vessels, and bile duct cells of normal and fibrotic liver. In liver cell cultures, Kupffer cells, hepatocytes, and especially stellate cells, expressed PAI-1. Expression was enhanced in stellate cells cultured from fibrotic or cirrhotic liver or stimulated in vitro with TGF-beta1. CONCLUSION Though increased uPA and uPAR may act on matrix degradation in fibrotic liver, increased PAI-1 together with uPA, uPAR and tPA are associated with overall inhibition of matrix degradation in cirrhotic liver. Hepatic stellate cells are an important source of PAI-1 during liver fibrosis.

Evaluation of neuropsychological function in patients with liver cirrhosis with special reference to their driving ability

Ability to drive an automobile was evaluated in 16 patients with well compensated liver cirrhosis. Four tests were performed, namely the emergency reaction test, the continuous emergency reaction test, the signal confirmation test and the accelerator reaction test. Test scores were compared to those of a group of age-matched healthy volunteers. 31% of patients were found to be unfit to drive. Alcoholic cirrhotics fared as poorly as non-alcoholic cirrhotics. In patients with subclinical hepatic encephalopathy (defined by neuropsychologic testing), 44% were unfit to drive. Routine testing of cirrhotic patients for ability to drive could have a major impact on motor vehicle accident rates.

The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-β, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect α1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle α-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis ∼2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.

Expression of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 in acute liver injury.

BACKGROUND/AIMS Matrix metalloproteinase-13, one of the principal neutral proteinases capable of cleaving native fibrillar collagens, is important in the degradation and remodeling of extracellular matrix. However, its precise expression in liver injury has not been characterized. We examined the kinetics of the expression of matrix metalloproteinase-13 and one of its specific inhibitors, tissue inhibitor of metalloproteinase-1, in acute liver injury in rats. METHODS Acute liver injury was induced by administration of carbon tetrachloride or two different doses of D-galactosamine hydrochloride in Wistar rats. Hepatic matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 mRNA levels were then examined by Northern blotting. RESULTS All rats survived after liver injury induced by carbon tetrachloride or low doses of D-galactosamine hydrochloride. However, rats died 5 days after induction of liver injury by high doses of D-galactosamine hydrochloride. In carbon tetrachloride-induced liver injury, matrix metalloproteinase-13 mRNA was transiently increased between 6 h and 1 day after injury. Tissue inhibitor of metalloproteinase-1 mRNA expression was increased between 6 h and 3 days after the peak of matrix metalloproteinase-13 expression. Similar patterns of matrix metalloproteinase-13 and tissue inhibitor of metalloproteinase-1 expression were observed in low-dose D-galactosamine hydrochloride-induced liver injury. In contrast, in high-dose D-galactosamine hydrochloride-induced liver injury, tissue inhibitor of metalloproteinase-1 expression peaked before matrix metalloproteinase-13 expression, which was increased 2 days after injury. Both mRNA levels continued to increase until death. CONCLUSIONS Transient expression of matrix metalloproteinase-13, followed by that of tissue inhibitor of metalloproteinase-1, was observed during recovery from acute liver injury induced by carbon tetrachloride and low-dose D-galactosamine hydrochloride. In contrast, disordered expression of matrix metalloproteinase-13 was observed in fatal liver injury caused by high-dose D-galactosamine hydrochloride. These results indicate that matrix metalloproteinase13 plays an important role in the early phase of recovery from liver injury.

Optimal follow-up time to determine the sustained virological response in patients with chronic hepatitis C receiving pegylated-interferon and ribavirin

Background and Aim:  This study evaluated whether the assessment of hepatitis C virus (HCV)‐RNA at 12 weeks (FW+12) post‐treatment follow‐up was as applicable as FW+24 to evaluate sustained virological response (SVR) using the highly sensitive real‐time polymerase chain reaction (PCR) HCV assay.

Development of osteomalacia in a post-liver transplant patient receiving adefovir dipivoxil

We report the case of a patient treated with living donor-related liver transplantation who suffered from osteomalacia during adefovir dipivoxil (ADV)-containing antiviral therapy for lamivudine-resistant hepatitis B virus infection. The patient had generalized bone pain, with severe hypophosphatemia after 20 mo of ADV therapy. Radiographic studies demonstrated the presence of osteomalacia. The peak plasma ADV level was 38 ng/mL after administration of ADV at 10 mg/d. It was also found that ADV affected the metabolism of tacrolimus, a calcineurin-inhibitor, and caused an increase in the plasma levels of tacrolimus. The disability was reversed with the withdrawal of ADV and with mineral supplementation. ADV can cause an elevation of plasma tacrolimus levels, which may be associated with renal dysfunction. High levels of ADV and tacrolimus can cause nephrotoxicity and osteomalacia. This case highlights the importance of considering a diagnosis of osteomalacia in liver transplantation recipients treated with both ADV and tacrolimus.

Spatial distribution of tissue inhibitor of metalloproteinase-1 mRNA in chronic liver disease

BACKGROUND/AIMS Tissue inhibitor of metalloproteinase-1, a specific inhibitor of matrix metalloproteinases, plays an important role in the pathogenesis of fibrosis and tumor progression. However, the precise expression of tissue inhibitor of metalloproteinase-1 messenger RNA in human hepatic fibrosis has not yet been defined. We investigated the spatial distribution of tissue inhibitor of metalloproteinase-1 messenger RNA in chronic human liver disease. METHODS Northern and in situ hybridization of probes to tissue inhibitor of metalloproteinase-1 messenger RNA were performed in specimens from 16 surgically resected human livers. Immunohistochemical staining of sections for tissue inhibitor of metalloproteinase-1 and immunoelectron microscopy were also performed. RESULTS Northern hybridization demonstrated that expression of tissue inhibitor of metalloproteinase-1 messenger RNA was increased 3.9-fold in mild chronic hepatitis, 6.8-fold in moderate chronic hepatitis, and 6.4-fold in cirrhosis, compared with control liver. In situ hybridization showed the expression of tissue inhibitor of metalloproteinase-1 messenger RNA in spindle-shaped cells in the fibrous septa and lobules in chronic hepatitis and cirrhosis; these cells were immunohistochemically positive for a-smooth muscle actin. Immunoelectron microscopy revealed localization of tissue inhibitor of metalloproteinase-1 in between fibers, to the rough endoplasmic reticula of stellate cells located in the lobules and periportal areas, and to fibroblasts in the fibrous septa. These results indicate that tissue inhibitor of metalloproteinase-1 was produced mainly by stellate cells in the specimens of chronic liver diseases. CONCLUSIONS Expression of tissue inhibitor of metalloproteinase-1 messenger RNA is increased in hepatic fibrosis and stellate cells are involved primarily in its expression.

Modulation of matrix metalloproteinase-9 in hepatic stellate cells by three-dimensional type I collagen: its activation and signaling pathway

Background/AIMS: Hepatic stellate cells (HSCs) play a key role in the production and degradation of extracellular matrix (ECM) in the liver. In the present study, we investigated the interaction between ECM and HSCs in vitro with emphasis on the modulation of matrix metalloproteinases (MMPs) by ECM. METHODS: Freshly isolated rat HSCs were cultured in several conditions on type I collagen- or matrigel-coated dishes, on thick matrigel or in three-dimensional type I collagen (3D-gel), and MMPs expression in HSCs was examined. In addition, activation and signaling pathway of MMP-9 expression modulated by 3D-gel in HSCs were examined. RESULTS: Increased expression of MMP-3, -9, -13 and -14 was markedly detected only in the 3D-gel-treated HSCs. Zymography demonstrated that only 3D-gel-treated cells showed active gelatinase activity of MMP-9 at 82 kDa. MMP-9 expression was inhibited by neutralizing antibody against integrin alpha2beta1, tyrosine kinase inhibitors, or MEK1,2 inhibitor PD 98059, but not by p38 inhibitor SB 203580. Western blotting also showed phosphorylated p38, ERK1,2, and JUN/SAPK was quickly induced in HSCs by 3D-gel. CONCLUSIONS: MMP-9 expression and activation is induced in HSCs by 3D-gel and this observed collagen-dependent induction of MMP-9 requires ERK1,2 activity.

Matrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in the injured liver.

Effective mobilization of hematopoietic stem cells (HSCs) in injured organs has not been established. Matrix metalloproteinase-9 (MMP-9) is known to release HSCs from bone marrow (BM) into the peripheral blood, but its role in the recruitment of HSCs to injured organs is unclear. In this study we tried to clarify the role of the host MMP-9 in trafficking of HSCs toward the injured liver, especially the relation of MMP-9 with the chemokine receptor 4 (CXCR4)–chemokine ligand 12 (CXCL12) axis, and to examine whether MMP-9 deficiency affects BM cell trafficking to the injured liver in mice. In vitro, we investigated the effect of MMP-9 on migration activity and CXCR4 expression on lineage-negative (Lin-) BM cells. In vivo, we induced acute and chronic liver injury in MMP-9 knockout (KO) and control mice by inoculation of carbon tetrachloride, followed by transplantation of Lin- BM cells obtained from enhanced green fluorescent protein (EGFP)-transgenic mice, and counted the BM cells mobilized in the injured liver. In a migration assay, active MMP-9, but not proMMP-9, increased the number of migrated Lin- BM cells, which was inhibited by tissue inhibitor of metalloproteinase-1 or a MMP inhibitor. This chemoattractant function by MMP-9 was synergistic when cotreated with CXCL12. CXCR4 expression on Lin- BM cells was dose- and time-dependently increased by active MMP-9. At the same time, treatment with MMP-9 enhanced CXCL12 expression, and CXCL12 reciprocally increased MMP-9 expression in BM cells. In in vivo studies, many EGFP+ cells were seen in control recipient mice. In contrast, few EGFP+ cells were observed in MMP-9 KO mice. BM cells tended to differentiate into desmin+ cells. In conclusion, MMP-9 contributes to the mobilization of BM cells in the injured liver by upregulating the expression of CXCR4 on Lin- BM cells and attracting BM cells along its gradient of CXCL12. Therefore, host MMP-9 plays an important role in BM cell migration in the injured liver.

An improved method for the purification of stellate cells from rat liver with dichloromethylene diphosphate (CL2MDP).

Hepatic perisinusoidal cell population consists of hepatic stellate cells, Kupffer cells, endothelial cells, and Pit cells. These cells are isolated by enzymic digestion and purified by density gradient centrifugation. With isolation of stellate cells, conventional method is unable to eliminate the contamination of Kupffer cells because the densities of these two cells are similar. We report here an improved method for isolation of highly purified hepatic stellate cells, using dichloromethylene diphosphate (CL2MDP), which has selective cytotoxicity of Kupffer cells. Three days after the single intravenous administration of liposome-encapsulated CL2MDP, the Kupffer cells disappeared almost completely from the liver. Following Percoll density gradient centrifugation, the purity of the hepatic stellate cells exceeded 98% without any contamination of the Kupffer cells. Kupffer cells are reported to affect the physiological functions of stellate cells. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.