Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis

Interferon gamma (IFN-gamma) inhibits in vitro the activation of hepatic stellate cells (HSC), the primary extracellular matrix-producing cells in liver fibrosis. This study was undertaken to determine in vivo the effect of IFN-gamma in the rat model of liver fibrosis induced by dimethylnitrosamine (DMN), where HSC activation represents an early response to cell injury. Rats were killed after 1 or 3 weeks of treatment with DMN, IFN-gamma, DMN + IFN-gamma, or saline. Immunohistochemistry was used to identify proliferating (desmin-positive/bromodeoxyuridine (BrdU)-positive cells) and activated (alpha-smooth-muscle actin [alpha-SMA]-positive cells) HSCs. Collagen deposition was determined colorimetrically and by morphometry. The parenchymal extension of desmin- and actin-positive cells and of fibrotic tissue was measured by point-counting technique and expressed as a percentage of area. Western blot was used to determine laminin and fibronectin accumulation. The levels of messenger RNA (mRNA) for procollagen type I, fibronectin, and laminin were evaluated by Northern blot. No differences were observed in rats treated with either saline or IFN-gamma alone. IFN-gamma reduced HSC activation induced by liver injury, as shown by the decreased number of proliferating HSC and the reduction of parenchymal area occupied by alpha-SMA-positive cells observed in DMN + IFN-gamma-treated animals compared with the DMN group. This was associated with reduced collagen, laminin, and fibronectin accumulation and lower levels of mRNA for procollagen type I, fibronectin, and laminin in the DMN + IFN-gamma group. Thus, this study indicates that IFN-gamma reduces extracellular matrix deposition in vivo by inhibition of HSC activation.     

Expression of fibronectin splicing variants in organ transplantation: a differential pattern between rat cardiac allografts and isografts

Allograft rejection is associated with infiltration of inflammatory cells and deposition of extracellular matrix proteins. The extent to which diversity in the extracellular matrix regulates inflammatory cell function in transplants remains unclear. One group of extracellular matrix proteins, termed fibronectins (FNs), exhibits inherent diversity as a consequence of alternative splicing in three segments: EIIIA, EIIIB, or V. Although the EIIIA segment has documented functions in mesenchymal cell differentiation, neither this segment nor the EIIIB segment have been tested for effects specific to leukocyte functions. By contrast, the V region can include the CS-1 segment to which leukocytes may adhere through alpha 4 beta 1 integrins. In this study, we demonstrate that EIIIA+, EIIIB+, and V+ FN variants are synthesized, primarily by macrophages in distinct temporal and spatial patterns in two rat cardiac transplant models: either with antigenic challenge, allografts, or without challenge, isografts. The ratio of EIIIA inclusion into FN increases by day 1 in allografts and isografts and remains high until allografts are rejected (approximately 7 days) but falls to normal levels in tolerated isografts (day 6). EIIIB+ FN ratios in allografts peak later than do EIIIA+ FNs (day 4). EIIIB+ FN ratios remain relatively low in isografts. Interestingly, EIIIA+ and EIIIB+ FNs are deposited prominently in the myocardium of rejecting allografts in close association with infiltrating leukocytes, and FN expression and deposition are prominent at sites of infarction. By contrast, these FNs are largely restricted to the epicardium and to a lesser degree in the immediately adjacent myocardium in isografts. CS-1+ FNs increase in allografts and isografts at 3 hours after transplantation but are particularly prominent in allografts 1 to 3 days before rejection. Our data suggest that FN splicing variants have a differential role in the effector functions of leukocytes in allografts and isografts and provide a foundation for testing their function on leukocytes and a rationale for FN-based therapeutics to modulate allograft rejection in transplant recipients.     

Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates

Hepatocyte growth factor (HGF) decreases transforming growth factor beta1 (TGFbeta1) levels in the liver and attenuates hepatic fibrosis caused by dimethylnitrosamine in rats. In the liver, HGF is presumed to act predominantly on parenchymal cells, and TGFbeta1 is produced mainly by mesenchymal cells. In hepatic fibrosis, stellate cells play a central role with undergoing activation, which also occurs when the cells are cultured on plastic. Thus, we wondered if HGF could act directly on stellate cells. c-Met was detected in rat stellate cells activated by culture for 10 days, but not in the cells cultured for 3 days. Specific binding of HGF to the activated cells was determined, and Scatchard analysis indicated an apparent Kd of 1.5 nM. c-Met mRNA was detected in freshly isolated stellate cells from rats treated with carbon tetrachloride for 8 weeks, but not in those cells from normal rats. These results indicate that stellate cells express c-met when activated in vitro and in vivo. HGF enhanced TGFbeta1 production and DNA synthesis in the activated cells.     

Effect of interferon gamma on intrahepatic haemodynamics of the cirrhotic rat liver

Sublethal injury of the liver with carbon tetrachloride (CCl4) induces the modulation of hepatic stellate cells to their myofibroblast (MFB) phenotype. Pretreatment or concomitant treatment with interferon gamma (IFNgamma) has been shown to inhibit this phenomenon. The aim of this study was to investigate the influence of IFNgamma treatment (50000 IU s.c. each day for 5 days) in rats with an established cirrhosis. Cirrhosis was induced with nine doses of CCl4. Comparison of biopsies before and after treatment with IFNgamma showed that the number of MFB present, identified by their alpha-smooth muscle actin immunoreactivity, was markedly reduced. Pressure-flow curves were constructed in isolated perfused liver preparations from IFNgamma-treated and saline-treated cirrhotic rats and analysed to obtain the extrapolated zero-flow intercept (Po, an index of hepatic vascular distensibility) and the vasodilator-induced change in resistance at a flow rate of 1 mL/min per g (deltaR1, an indication of the level of intrinsic vascular tone). In IFNgamma-treated rats, portal venous pressure measured in vivo was significantly reduced compared with controls (11.9+/-1.2 vs 16.0+/-0.5 mmHg, P< 0.05), Po was lower (2.03+/-0.18 vs 2.87+/-0.32 mmHg, P<0.05) and deltaR1 was decreased (0.39+/-0.15 vs 1.02+/-0.19 mmHg/mL per min per g, P< 0.05). The findings indicate that treatment with IFNgamma is effective in reducing MFB density in established CCl4-cirrhosis in the rat and results in a marked improvement in intrahepatic haemodynamics.     

Dissociation between anxiolytic and hypomnestic effects for combined extracts of zingiber officinale and ginkgo biloba, as opposed to diazepam

Extrahepatic biliary atresia is a severe neonatal liver disease resulting from a sclerosing cholangiopathy of unknown etiology. Although biliary obstruction may be surgically corrected by a "Kasai" hepatoportoenterostomy, most patients still develop progressive hepatic fibrosis, although the source of increased collagen deposition is unclear. This study examined the role of hepatic stellate cells (HSCs) and assessed the source of transforming growth factor-beta (TGF-beta) production in hepatic fibrogenesis in patients with biliary atresia. Liver biopsies from 18 biliary atresia patients (including 5 pre- and post-Kasai) were subjected to immunohistochemistry for alpha-smooth muscle actin and in situ hybridization for either procollagen alpha1 (I) mRNA or TGF-beta1 mRNA. Sections were also subjected to immunohistochemistry for active TGF-beta1 protein. The role of Kupffer cells in TGF-beta1 production was assessed by immunohistochemistry for CD68. Procollagen alpha1 (I) mRNA was colocalized to alpha-smooth muscle actin-positive HSCs within the region of increased collagen protein deposition in fibrotic septa and surrounding hyperplastic bile ducts. The number of activated HSCs was decreased in only one post-Kasai biopsy. TGF-beta1 mRNA expression was demonstrated in bile duct epithelial cells and activated HSCs and in hepatocytes in close proximity to fibrotic septa. Active TGF-beta1 protein was demonstrated in bile duct epithelial cells and activated HSCs. This study provides evidence that activated HSCs are responsible for increased collagen production in patients with biliary atresia and therefore play a definitive role in the fibrogenic process. We have also shown that bile duct epithelial cells, HSCs, and hepatocytes are all involved in the production of the profibrogenic cytokine, TGF-beta1.     

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.     

Morphology of liver stellate cells and liver vitamin A content in 3,4,3',4'-tetrachlorobiphenyl-treated rats

BACKGROUND/AIMS: Because xenobiotics decrease the vitamin A stores localized in the liver stellate cells, we investigated morphological alterations in the liver of rats exposed to 3,4,3',4'-tetrachlorobiphenyl. Special attention was given to the morphology of the liver stellate cells and to their relationship to the liver vitamin A content. METHODS: Six rats received an intraperitoneal injection of 3,4,3',4'-tetrachlorobiphenyl (300 mumol/kg) in soyabean oil. A further six rats received the vehicle alone. After 7 days, all rats were killed and their livers assayed for vitamin A. Liver stellate cells were examined and counted on liver sections, stained with toluidine blue or immunocytochemically for desmin and, for some animals, for alpha-smooth muscle actin. RESULTS: In the livers of 3,4,3',4'-tetrachlorobiphenyl-treated rats, we found spotty and bridging necrosis, with inflammation and accumulation of desmin-positive liver stellate cells. Steatosis and mild portal inflammation were also observed. 3,4,3',4'-Tetrachlorobiphenyl decreased the liver vitamin A content by 38%, whereas morphometric analyses showed a 40% decrease of the number of toluidine blue-detected liver stellate cells and an 11% increase of desmin-detected liver stellate cells, indicating a likely differentiation of liver stellate cells into myofibroblast-like cells. 3,4,3',4'-Tetrachlorobiphenyl treatment did not modify the expression of alpha-smooth muscle actin. Morphological alterations were more pronounced in periportal than in pericentral areas. The liver vitamin A content was positively correlated (r = 0.56, p < 0.005) with the number of toluidine-blue detected liver stellate cells. CONCLUSIONS: 3,4,3',4'-tetrachlorobiphenyl administration results in an accumulation of liver stellate cells that start differentiating into myofibroblast-like cells. The 3,4,3',4'-tetrachlorobiphenyl-induced decrease in liver vitamin A probably results from this differentiation, although other mechanisms cannot be excluded.     

One hundred years of sinusoidal cells in the liver

Kupffer (1898) reported that the stellate cells were phagocytic endothelial cells, retracting his earlier view that these cells were perivascular cells. His new concept stimulated studies on the vital staining and the RES theory, while it ensued several controversies in liver histology. The original stellate cells were rediscovered in 1971, and were proved to be identical with several perisinusoidal cells reported previously. For this cell the term hepatic stellate cell has recently been adopted as the standardized name. The space of Disse is newly defined as the space between the endothelial cell-stellate cell complex and the parenchymal cells. The stellate cells display vitamin A-storage, collagen synthesis and may contract to regulate the sinusoidal blood flow and the fluid-exchange between the sinusoidal lumen and the space of Disse. The sinusoidal endothelial cells uptake injected lithium carmine and macromolecules by coated vesicles. Kupffer cells clear endotoxin, bacteria and apoptotic neutrophils and release various cytokines. The pit cells are the liver-associated NK cells and are activated by the administration of biological response modifiers, preventing tumor metastasis. Extrathymic pathways of T cell differentiation exist in the hepatic sinusoid. The dendritic cells differentiate in the sinusoid and translocate to the Glisson's sheath. Intralobular heterogeneity of sinusoidal cells have been observed. The sinusoidal cells communicate each other with autocrine and paracrine mechanisms to regulate various functions of the liver.     

Extracellular matrix deposition, lysyl oxidase expression, and myofibroblastic differentiation during the initial stages of cholestatic fibrosis in the rat

Early studies showed that during hepatic fibrosis induced by bile duct ligation, fibroblasts within the portal tracts proliferate and express alpha-smooth muscle (SM) actin, suggesting that they may be involved in the deposition of extracellular matrix components in cholestatic fibrosis. Thus, we investigated the deposition of extracellular matrix components (laminin, fibronectin EIIIA, collagen I and IV, procollagen III, elastin, tenascin) as well as the expression of lysyl oxidase and of alpha-SM actin in the portal zone at 24, 48, and 72 hours and 7 days after ligation of the common bile duct. Rat liver tissues were processed for immunofluorescence, in situ hybridization, immunohistochemistry, and for electron and immunoelectron microscopy. At all times examined after bile duct ligation, laminin was observed essentially in the basal membrane of vessels and portal ductules. In sham-operated animals, the fibronectin EIIIA was present exclusively in vessels; at 24 hours postinjury, fibronectin EIIIA expression appeared in both the portal zone and along sinusoids. Two days after ligation, increased expressions of collagen I and IV, procollagen III, and elastin were observed within the portal zone, compared with sham-operated animals. The deposition of these components increased thereafter. Tenascin expression increased soon after bile duct ligation in stroma surrounding proliferating ductules, reaching a maximum at 48 hours; thereafter, expression was restricted to the periphery of proliferating ductules. By in situ hybridization, procollagen I and tissue inhibitor of metalloproteinase-1 mRNA expression was greatly increased in periductular areas at 24 hours postligation and remained elevated throughout the experiment. At 24 hours, a strong reactivity for lysyl oxidase appeared in the portal zone, and, as in controls, alpha-SM actin expression was restricted to vascular SM cells. In the stroma adjacent to proliferating ductules, alpha-SM actin appeared at 48 hours, and the number of alpha-SM actin-positive cells increased until the 7th day. Lysyl oxidase staining increased until 72 hours after bile duct ligation, when it was located in areas surrounding the myofibroblastic cells. At 7 days, lysyl oxidase expression was restricted around myofibroblastic cells present at the periphery of the reactive tissue and appeared to extend into the surrounding parenchyma. These results show that after bile duct ligation, extracellular matrix deposition, and lysyl oxidase expression occur very early in portal connective tissue surrounding proliferating ductules, and precede myofibroblastic differentiation, ie, alpha-SM actin expression. In addition, the data are compatible with the suggestion that in the bile duct ligation model, myofibroblastic differentiation represents an adaptive response to modification of the extracellular matrix environment.     

Sarcomeric gene expression and contractility in myofibroblasts

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.     

Assembly of an active enzyme by the linkage of two protein modules

BACKGROUND & AIMS: Oxidative stress mediates activation and stimulates collagen production of cultured hepatic stellate (Ito) cells. The aim of this study was to assess whether oxidative stress contributes to hepatic fibrogenesis in chronic hepatitis C. METHODS: In liver biopsy specimens of patients with chronic hepatitis C, the following fibrogenesis cascade was analyzed: (1) oxidative stress, determined by the presence of malondialdehyde protein adducts; (2) activation of stellate cells as indicated by their expression of alpha-smooth muscle actin; (3) stimulation of c-myb expression in stellate cells, a critical step in the activation of these cells; and (4) induction of collagen gene expression as detected by in situ hybridization. RESULTS: Treatment with d-alpha-tocopherol (1200 IU/day for 8 weeks) in 6 of these patients, who were refractory to interferon therapy, prevented the fibrogenesis cascade observed before antioxidant treatment. In addition, d-alpha-tocopherol treatment significantly decreased the carbonyl modifications of plasma proteins, a sensitive index of oxidative stress. However, 8 weeks of d-alpha-tocopherol treatment did not significantly affect serum alanine aminotransferase levels, hepatitis C virus titers, or histological degree of hepatocellular inflammation or fibrosis. CONCLUSIONS: These data suggest that enhanced oxidative stress initiates a fibrogenesis cascade in the liver of patients with chronic hepatitis C.     

Mutational analysis of cis-acting sequences in the 3'- and 5'-untranslated regions of RNA2 of red clover necrotic mosaic virus

BACKGROUND/AIMS: Hepatic stellate cells appear to be the main producers of hepatocyte growth factor of the normal liver. Insulin-like growth factors in doses over 20 ng/ml have been reported to stimulate hepatocyte growth factor production in cultured hepatic stellate cells. The aim of the present study was to investigate whether parenchymal cell conditioned medium had insulin-like growth factor-independent effects on hepatic stellate cells. METHODS: Primary rat hepatic stellate cells were cultured for 1-7 days. DNA synthesis was measured by 3H-thymidine incorporation. Hepatocyte growth factor and transforming growth factor beta1 immunoreactivity was quantified by ELISA. Hepatocyte growth factor mRNA levels were determined with gel RNase protection assay. Parenchymal cell conditioned medium was obtained from hepatocytes cultured for 2 days in medium without added serum or hormones. RESULTS: Incubation of 1-7-day-old hepatic stellate cells for 2 days with parenchymal cell conditioned medium enhanced the medium content of hepatocyte growth factor. Parenchymal cell conditioned medium contained less than 5.0 ng/ml immunoreactive insulin-like growth factor-1 as measured by radio immunoassay. Parenchymal cell conditioned medium did not contain any insulin-like growth factor bioactivity measured as phosphorylation of type 1 insulin-like growth factor receptor beta subunit and a protein with a size consistent with that of insulin receptor substrate-1. The stimulatory effect of parenchymal cell conditioned medium on hepatocyte growth factor was time- and dose-dependent. The effects of a high dose of parenchymal cell conditioned medium (dilution 1:2 containing less than 2.5 ng/ml insulin-like growth factor-1) were additive to that of high doses (100 ng/ml) of insulin-like growth factor-1 or des (1-3) insulin-like growth factor-1, an analogue with low affinity to insulin-like growth factor binding proteins. Neither parenchymal cell conditioned medium nor insulin-like growth factor-1 enhanced transforming growth factor beta1 immunoreactivity in the medium. Both parenchymal cell conditioned medium and insulin-like growth factor-1 stimulated DNA synthesis in hepatic stellate cells, confirming previous reports. CONCLUSIONS: The present results indicate that both insulin-like growth factor-1 and insulin-like growth factor-1-independent factors from hepatocytes can stimulate hepatocyte growth factor production by hepatic stellate cells. Therefore, insulin-like growth factor-1 and other hepatocyte-derived factors may indirectly affect hepatocytes via a paracrine loop.     

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.     

Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor

Lysophosphatidic acid (LPA) is a growth factor-like mediator for fibroblasts or smooth muscle cells produced and released by activated platelets. Platelet activation occurs with hepatic necrosis and subsequent liver regeneration and fibrosis. In the fibrosis, hepatic stellate cells proliferate with phenotypic transformation to myofibroblasts. Thus, effects of LPA on proliferation of hepatocytes and stellate cells were investigated. In cultured rat stellate cells, LPA increased DNA synthesis with enhanced MAP kinase activity. Pertussis toxin (PTX) attenuated this mitogenic action. In contrast, LPA decreased DNA synthesis by cultured rat hepatocytes induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting protein synthesis. Enhanced MAP kinase activity by HGF or EGF was not changed by LPA. This anti-mitogenic action was attenuated by PTX. TGFbeta level in the medium was less than the level effective for inhibiting the DNA synthesis in the presence of LPA. Our results suggest that LPA might affect proliferation of hepatocytes and stellate cells in liver diseases complicating platelet activation.