Koji Hironaka

Quantitative analysis of liver fibrosis and stellate cell changes in patients with chronic hepatitis C after interferon therapy

Objective: The proliferation and differentiation of stellate (Ito, or fat-storing) cells into myofibroblast-like cells is responsible for the development of liver fibrosis. Using computer image analysis, we evaluated the changes of α smooth muscle actin–positive stellate cells and liver fibrosis after interferon-α or -β (IFN-α, β) therapy in patients with chronic hepatitis C. Methods: Patients with chronic hepatitis C were treated with IFN-α or -β and were divided into three groups on the basis of clinical criteria; a complete responder group (CR, 18 of 51), a partial responder group (PR, 17 to 51), and a nonresponder group (NR, 16 of 51). Liver fibrosis was assessed from specimens stained with Sirius red and was quantitated by computer image analysis. We also evaluated α-smooth muscle actin expression in the liver before and after IFN therapy by a semiquantitative scoring method (the α-smooth muscle actin index). Results: Before IFN therapy, a large number of stellate cells expressing α-smooth muscle actin were present in the liver biopsy specimens. There was a significant correlation (r = 0.699, p < 0.05) between the change in the percent area of fibrosis and the α-smooth muscle actin index before and after IFN therapy in all groups. The complete responder group also showed a significant reduction of α-smooth muscle actin-expressing cells that was correlated with the reduction of serum ALT (r = 0.686, p < 0.05). Conclusion: These results suggest α-smooth muscle actin-expressing cells are responsible for liver fibrosis, and the elimination of factors stimulating matrix synthesis (e.g., hepatitis virus) may decrease liver fibrosis.

Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells

The aim of this study was to investigate whether matrix metalloproteinases (MMP-13, 9) of Kupffer cells induced by gadolinium chloride (GdCl(3)) treatment can reverse dimethylnitrosamine (DMN)-induced liver fibrosis (in vivo) and the effect of GdCl(3) on MAP kinase activity (in vitro). Male Wistar rats 6 weeks of age received DMN (10 mg/kg) three successive days a week for 4 weeks. Then two groups of rats (n = 6 each) were treated twice weekly with either GdCl(3) (7 mg/kg) or saline solution intravenously for the next 4 weeks. Animals were sacrificed to estimate the degree of liver fibrosis. Isolated Kuppfer cells were treated with GdCl(3) and the expressions of MMPs, MAP kinase activity (ERK, SAPK/JNK, P38) as well as apoptosis were also examined. Rats that received DMN for 4 weeks followed by GdCl(3) injection for 4 weeks showed an reduced liver hydroxyproline content compared to rats treated with DEN followed by saline (277 +/- 22 VS 348 +/- 34 microg/g, n = 6 each, P < 0.01). There were significantly increased MMP-13 mRNA levels in GdCl(3)-treated rats. However, no significant change was observed in procollagen type I mRNA levels. Isolated Kuppfer cells treated with GdCl(3) showed increased MAP kinase activity, especially P38 pathway as well as MMP-13, 9 mRNA and type I collagen-degrading activity leading to apoptosis. SB203580, inhibitor of P38 pathway diminished these activation and prevented apoptosis. These results suggest that Kuppfer cells can reverse liver fibrosis via the expression of MMPs mainly through P38 pathway.

Prolyl 4-hydroxylase inhibitor (HOE 077) prevents TIMP-1 gene expression in rat liver fibrosis

Abstract: The effects of a prolyl 4-hydroxylase inhibitor (HOE 077) on tissue inhibitor of metalloproteinase-1 (TIMP-1) and procollagen type I were examined in rat liver fibrosis. HOE 077 (200 ppm) prevented fibrosis by inhibiting the expression of liver type I procollagen and TIMP-1 mRNAs, with liver hydroxyroline content correlated with the reduction in activated stellate cells. HOE 077 prevents fibrosis by inhibiting proline hydroxylation and stellate cell activation, resulting in reduced expression of procollagen and TIMP-1 mRNAs.

Iron chelator deferoxamine reduces preneoplastic lesions in liver induced by choline-deficient L-amino acid-defined diet in rats

The aim of this study was to investigate wheThe ran iron chelator, deferoxamine (DFO) can prevent lipidperoxidation, resulting in reduced liver injury as wellas reducing preneoplastic lesions induced by a choline-deficient L-amino acid-defined(CDAA) diet. CDAA diet administration resulted in anincreased serum ALT level (367 ± 58) after twoweeks, but simultaneous DFO treatment for two weeksreduced this elevation of ALT as well asmalondialdehyde (MDA) production in the liver. Feedingrats a CDAA diet for 12 weeks led to the development ofsevere liver fibrosis and preneoplastic lesions detectedas enzymealtered lesions. DFO treatment preventedthe expression of activated stellate cells, resulting inThe reduction of liver fibrosis as well as reducing thedevelopment of preneoplastic lesions. These results indicate that iron chelation can reducethe development of preneoplastic lesions in a CDAA dietmodel.

Correlation Between Stellate Cell Activation and Serum Fibrosis Markers in Choline-Deficient l-Amino Acid-Defined Diet-Induced Rat Liver Fibrosis

The aim of this study was to investigate the association of stellate cell activation with serum fibrosis markers in a rat model of hepatic fibrosis prepared using a choline-deficient l-amino acid (CDAA) defined diet. CDAA diet administration resulted in increased liver hydroxyproline contents in a time-dependent manner with activated stellate cells, expressing α-smooth muscle actin (α-SMA) as well as increased serum concentrations of amino-terminal procollagen type III peptide (PIIIP) and the 7S fragment of type IV collagen. Hydroxyproline content of the liver showed a closer correlation with the serum 7S (r = 0.75, P < 0.01) concentration than with the serum PIIIP (r = 0.51, P < 0.01) concentration. The percent area of α-SMA-positive cells showed stronger correlation with the serum PIIIP concentration (r = 0.85, P < 0.01) than with the 7S concentration (r = 0.50, P < 0.01). These results indicate that the serum PIIIP concentration reflects the activity of fibrogenesis, while the serum 7S concentration reflects the accumulation of collagen fibers in the liver.

Interferon γ- but not α-treatment prevents fibrosis by inhibiting procollagen and tissue inhibitor of metalloproteinase-1 gene expression in pig serum-induced rat liver fibrosis in vivo

Abstract The aim of this study was to investigate the effect of interferons- γ (IFN- γ ) and - α (IFN- α ) on the synthesis of matrix proteins such as collagens as well as the gene expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in vivo. We investigated the effects of IFN- γ and - α in a model of liver fibrosis induced by pig serum in male Wistar rats, which develop fibrosis without an increase in serum ALT (i.e. without hepatocyte injury). Rats were injected with 0.5 ml of pig serum twice a week for 8 weeks with or without 50 000 U of IFN- γ or 100 000 U of IFN- α . IFN- γ at the dose of 50 000 U day −1 prevented fibrosis, as indicated by reduced hydroxyproline content in the liver. IFN- γ at 50 000 U day −1 also reduced expression of type I procollagen as well as TIMP-1 in the liver. However, fibrosis was not reduced by IFN- α . Histologically, IFN- γ at 50 000 U day −1 also reduced the number of myofibroblast-like cells (activated stellate cells). These results indicate that IFN- γ , but not IFN- α , can prevent fibrosis by inhibiting the activation and proliferation of stellate cells, resulting in reduced expression of procollagen and TIMP-1 mRNA in pig serum-induced rat liver fibrosis in vivo.

Preventive effect of FK 506 (tacrolimus hydrate) on experimentally induced acute liver injury in rats.

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 μg/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) -α mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF-α production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 ± 401 in the saline group versus 119 ± 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF-α concentration was lower in the FK 506-treated group (1419 ± 957 pg/ml) than in the saline group (9205 ± 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF-α production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF-α production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.

HBV-related fulminant hepatic failure: successful intensive medical therapy in a candidate for liver transplantation

Fulminant hepatic failure (FHF) usually has a fatal prognosis without liver transplantation. We describe the case of a woman who developed FHF, and was evaluated as a candidate for liver transplantation, but who was cured without transplantation through intensive medical care that included glucagon-insulin therapy, methylprednisolone pulse therapy, interferon beta and lamivudine administration, cyclosporine administration, and high-volume hemodiafiltration and plasma exchange. In a patient with FHF who is a candidate for liver transplantation but for whom the transplantation cannot be performed for some reason, intensive medical therapy, including regeneration-promoting therapy, immunosuppressive therapy, antiviral therapy, and vigorous hepatic support, should be carried out.

Loss of inhibitory growth regulation by TGF-β1 in preneoplastic lesions in rat liver

Injection of pig serum into rats twice a week for eight weeks induced transforming growth factor-β1 (TGF-β1) mRNA expression and protein production resulting in liver fibrosis without parenchymal cell injury. Eight-week treatment with pig serum reduced bromodeoxyuridine (BrdU) -positive hepatocytes 24 hr after 70% partial hepatectomy compared to that in the livers of rats treated with saline for eight weeks. Administration of a choline-deficient l-amino acid-defined (CDAA) diet for six weeks with pig serum coadministration, after pretreatment with pig serum for eight weeks, led to the development of preneoplastic lesions that were positive for the placental form of glutathione S-transferase (GSTP). Eight-week pretreatment with pig serum induced more GSTP-positive lesions and TGF-β1 mRNA expression and protein concentration in the livers of rats subsequently fed a CDAA diet for six weeks than in rats fed the CDAA diet with saline treatment. These results indicate that TGF-β1 induced by pig serum treatment inhibited hepatocyte proliferation but failed to prevent the development of preneoplastic lesions in a CDAA diet model.

Prevention of Hepatocarcinogenesis by Fibrosuppression

A choline-deficient L-amino acid-defined (CDAA) diet led to the development of liver cirrhosis in 100% of male Wistar rats after 15 weeks and liver neoplasms in 90% of rats after one year. Concurrent administration of a prolyl 4- hydroxylase inhibitor (HOE 077) [2,4-pyridine dicarboxylic acid bis (2-methoxyethy- lamide)] at a dose of 200 ppm as an antifibrotic agent to rats fed a CDAA diet reduced the increase in liver hydroxyproline content without reduction of serum ALT. HOE 077 prevented the activation of stellate cells, as determined histologically, as well as the expression of procollagen type I mRNA, resulting in reduced hydroxyproline levels in the liver. Also, the administration of a CDAA diet for 15 weeks led to a substantial induction of GSTP-positive lesions and the production of 8-hydroxy- deoxyguanosine (8OHdG) in the liver. The concurrent administration of HOE 077 reduced the area of GSTP-positive lesions, in parallel with the reduction in hydroxyproline content. Administration of HOE 077 for 1 year reduced the development of liver neoplasms to 50% of rats fed a CDAA diet with reduced hydroxyproline and 8OHdG content. These data suggest that inhibition of fibrosis may prevent the development of neoplasms.