Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.     

Apoptosis of hepatic stellate cells in carbon tetrachloride induced acute liver injury of the rat: analysis of isolated hepatic stellate cells

BACKGROUND/AIMS: Analysis of isolated hepatic stellate cells (HSCs) from the injured liver may provide direct information on HSC apoptosis. However, it has not been established whether apoptotic HSCs would be isolated using the usual density gradient centrifugation method. The aim of this study was to observe the serial pattern of proliferation and apoptosis in isolated HSCs in comparison with that of liver tissue sections in CCl4 induced acute liver injury. METHODS: Male Sprague-Dawley rats were treated with a single intraperitoneal injection of carbon tetrachloride (CCl4) and were killed at various time points after the treatment. RESULTS: HSC proliferation showed a maximal increase at 32 h after CCl4 injection. Apoptosis of HSC, examined by quantitative analysis of annexin-V-fluorescein isothiocyanate (FITC)staining, showed the maximal increase at 64 h. Apoptosis of HSC in liver tissue sections examined by counting desmin and Tdt-mediated-dUTP biotin nick end labeling (TUNEL) double staining cells, peaked at 64 h. The number of TUNEL positive HSCs in liver tissue sections correlated significantly with annexin-V-FITC binding of isolated HSC. CONCLUSIONS: Studying apoptosis using apoptotic HSCs isolated by a usual density gradient centrifugation method from injured tissue sections would be feasible since it correlated with in vivo apoptosis of HSC.     

Activation of hepatic stellate cells after phagocytosis of lymphocytes: A novel pathway of fibrogenesis

Increased CD8-T lymphocytes and reduced natural killer (NK) cells contribute to hepatic fibrosis. We have characterized pathways regulating the interactions of human hepatic stellate cells (HSCs) with specific lymphocyte subsets in vivo and in vitro. Fluorescence-activated cell sorting (FACS) was used to characterize human peripheral blood lymphocytes (PBLs) and intrahepatic lymphocytes (IHLs) obtained from healthy controls and from patients with either hepatitis B virus (HBV) or hepatitis C virus (HCV) with advanced fibrosis. Liver sections were analyzed by immunohistochemistry and confocal microscopy. To investigate in vitro interactions, PBLs from healthy controls or patients with HCV cirrhosis were co-cultured with an immortalized human HSC line (LX2 cells) or with primary HSCs. Significant alterations in lymphocyte distribution were identified in IHLs but not PBLs. The hepatic CD4/CD8 ratio and NK cells were significantly reduced in HBV/HCV patients. Expression of alpha-smooth muscle actin and infiltration of CD4, CD8, and NK cells were readily apparent in liver sections from patients with cirrhosis but not in healthy controls. Lymphocytes from each subset were in proximity to HSCs primarily within the periportal regions, and some were directly attached or engulfed. In culture, HSC activation was stimulated by HCV-derived CD8-subsets but attenuated by NK cells. Confocal microscopy identified lymphocyte phagocytosis within HSCs that was completely prevented by blocking intracellular adhesion molecule 1 (ICAM-1) and integrin molecules, or by irradiation of HSCs. LX2 knockdown of either Cdc42 or Rac1 [members of the Rho-guanosine triphosphatase (GTPase) family] prevented both phagocytosis and the activation of HSC by HCV-derived lymphocytes. CONCLUSION: The CD4/CD8 ratio and NK cells are significantly decreased in livers with advanced human fibrosis. Moreover, disease-associated but not healthy lymphocytes are engulfed by cultured HSCs, which is mediated by the Rac1 and Cdc42 pathways. Ingestion of lymphocytes by HSCs in hepatic fibrosis is a novel and potentially important pathway regulating the impact of lymphocytes on the course of hepatic fibrosis.     

Taurine inhibits oxidative damage and prevents fibrosis in carbon tetrachloride-induced hepatic fibrosis

BACKGROUND/AIMS: The aim of the study was to examine the effects of taurine on hepatic fibrogenesis and in isolated hepatic stellate cells (HSC). METHODS: The rats of the hepatic damage (HD) group were administered carbon tetracholoride (CCl4) for 5 weeks and a subgroup received, in addition, a 2% taurine containing diet for 6 weeks (HDT). The HSC were isolated from normal rats and cultured for 4 days. RESULTS: The hepatic taurine concentration was decreased in the HD group. This loss and the hepatic histological damage and fibrosis (particularly in the pericentral region), were reduced following taurine treatment. Furthermore, the hepatic alpha-SMA, lipid hydroperoxide and 8-OHdG levels in serum and liver, as well as hepatic TGF-beta1 mRNA and hydroxyproline levels were significantly increased in the HD group, and most of these parameters were significantly reduced following taurine treatment. In contrast to the MAP-kinase and Akt expressions, which remained unchanged, the lipid hydroperoxide and hydroxyproline concentrations, as well as TGF-beta1 mRNA levels were significantly reduced by taurine in activated HSC. CONCLUSIONS: Oral taurine administration enhances hepatic taurine accumulation, reduces oxidative stress and prevents progression of hepatic fibrosis in CCl4-induced HD rats, as well as inhibits transformation of the HSC.     

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM:To study the effects of interleukin-10(IL-10)on the expression of α-smooth muscle actin(α-SMA),nuclear factor-κB(NF-κB)and Fas/Fas ligand(FasL)inhepatic stellate cells of experimental rats with hepaticfibrosis.METHODS:Sixty clean SD rats were randomly dividedinto control group(group N),liver fibrotic group(groupC)and IL-10 treatment group(group I).Control groupreceived intraperitoneal injection of saline(2ml·kg~(-1)),twicea week.Fibrotic group was injected intraperitoneallywith 50% carbon tetrachloride(CCl_4)(2 ml·kg~(-1)),twicea week.IL-10 treatment group was given IL-10 at adose of 4 μg·kg~(-1)20 minutes before CCl_4 administrationfrom the third week.Hepatic stellate cells(HSCs)wereisolated from these rats at the seventh and eleventhweeks during the course of liver fibrosis,respectively.The expression of α-SMA and NF-κB in HSCs wasmeasured by S-P immunohistochemistry.The expressionof Fas and FasL mRNA was measured by RT-PCR.Furthermore,liver tissues were harvested from threegroups at the same time.RESULTS:The CCl_4- induced experimental rat hepaticfibrosis model was established successfully.The purityof extracted hepatic stellate cells was about 95% andthe yield of hepatic stellate cells was 1.2-2.3×10~6/g livertissue averagely.The positive expression of α-SMA andNF-κB was 36.5% and 28.5% respectively in group N.The positive levels of α-SMA and NF-κB were increasedsignificantly in group C compared to group N(P<0.01).The positive signals decreased significantly(P<0.05)ingroup I.In the 11~(th)week,the HSCs of group I becameround with visible pyknotic nuclei.The expression ofNF-μB in group C was significantly increased in a time-dependentmanner(P<0.01),but there was no difference in the α-SMA expression(P>0.05).The mRNA of Fasand FasL in group C was significantly increased in a time-dependent manner compared to that in control group.After treated with IL-10,the expression level of Fas andFasL was higher in group I than in group C.CONCLUSION:The positive expression of α-SMA andNF-κB in hepatic stellate cells is decreased by ectogenicIL-10 in liver fibrosis induced by CCl_4.The expression ofFas and FasL is increased in the course of liver fibrosis,and is further increased by IL-10.IL-10 could inhibitthe activation of HSCs and cause apoptosis of activatedHSCs.     

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.     

Molecular imaging of hepatic stellate cell activity by visualization of hepatic integrin αvβ3 expression with SPECT in rat

The key factors in the pathogenesis of liver fibrosis are the activation and proliferation of hepatic stellate cells (HSCs), which express integrin αvβ3 after activation. This study aimed to explore the potential of (99m)Tc-labeled cyclic arginine-glycine-aspartic acid pentapeptide (cRGD) as a single photon emission computed tomography (SPECT) radiotracer to image hepatic integrin αvβ3 expression to reflect HSC activity in fibrotic livers. Rat models of liver fibrosis caused by thioacetamide or carbon tetrachloride (CCl(4)) treatment were employed to examine the expression and distribution of integrin αvβ3 during fibrotic progression or regression. The binding activity of radiolabeled cRGD to integrin αvβ3 was assessed in liver sections. SPECT was performed to determine hepatic integrin αvβ3 expression in rats with different stages of liver fibrosis. Protein and messenger RNA (mRNA) levels of integrin αv and β3 subunits were increased with the progression of liver fibrosis and reduced with its regression. The cell type that expressed the majority of integrin αvβ3 in fibrotic livers was found to be activated HSCs. The cRGD binding to activated HSCs displayed a high receptor-coupling affinity and an abundant receptor capacity. Iodine-125 ((125)I)-labeled cRGD bound to fibrotic liver sections and the binding activity was the highest in advanced fibrosis. Intravenously administered carboxyfluorescein-labeled cRGD was accumulated in fibrotic liver, and the accumulation amount was increased with the progression and reduced with the regression of fibrosis. A SPECT imaging study with (99m)Tc-labeled cRGD as a tracer demonstrated that the radioactivity ratio of liver to heart increased progressively along with severity of hepatic fibrosis. Conclusion: Hepatic integrin αvβ3 expression in fibrotic liver reflects HSC activity and its imaging using (99m)Tc-labeled cRGD as a SPECT radiotracer may distinguish different stages of liver fibrosis in rats.     

Differential expression genes analyzed by cDNA array in the regulation of rat hepatic fibrogenesis.

PURPOSE: To analyze the gene expression pattern in rat hepatic fibrogenesis and further assess the role of some key genes during the pathological process. METHODS: Hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine or carbon tetrachloride (CCl(4)) injection subcutaneously in rats, and identification of the hepatic fibrosis related genes with cDNA microarray was performed. After some key genes up-regulated during the development of hepatic fibrosis were screened and confirmed, their effects on the function of the activated rat hepatic stellate cells (HSC) were assessed using the small interfering RNA (siRNA) technique. RESULTS: Using an Atlas rat cDNA array, a number of differentially expressed genes in fibrotic liver tissues were identified compared with non-diseased control. A total of 15 genes predominantly associated with the mitogen-activated protein kinase (MAPK) signal transduction pathway were upregulated in the fibrotic liver. Immunohistochemical study revealed that the expressions of both extracellular signal-regulated kinases (ERK) and ribosomal protein S6 kinase (RSK), two of the key genes in the MAPK pathway, were remarkably induced, which was closely correlated to that of collagen types I and III during the development of hepatic fibrosis. Transfection of siRNA targeting ERK1 mRNA (siERK1) into HSC led to a 66% and 72% reduction of ERK1 mRNA and protein expression, respectively. Furthermore, siERK1 exerted the inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen types I and III. In addition, siERK1 abolished the effect of platelet-derived growth factor-BB on the proliferation of HSC. CONCLUSIONS: The present study provided strong evidence for the participation of the MAPK pathway in the pathogenesis of hepatic fibrosis. Selective targeting of ERK1 inhibitors to HSC might present as a novel strategy for the treatment of hepatic fibrosis.     

Inverse association between hepatic stellate cell apoptosis and fibrosis in chronic hepatitis C virus infection

Summary.  Perisinusoidal hepatic stellate cells (HSC) are the principal fibrogenic cells in the liver. In animal models, HSC apoptosis is the predominant clearance mechanism of activated HSC, although data evaluating whether the same processes occur in humans are limited. We conducted a cross-sectional study to evaluate the association between HSC apoptosis and fibrosis stage in subjects with chronic hepatitis C virus (HCV) infection ( n  = 44) and HCV-negative controls with normal liver histology ( n  = 9). We used immunohistochemical techniques to identify activated (α-smooth muscle actin⁺), proliferative (Ki-67⁺) and apoptotic (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labelling⁺) HSC in liver biopsy specimens from all subjects. The same pathologist enumerated positive cells per high-power field (HPF, ×200) in 20 periportal/lobular areas. HSC apoptosis was decreased in HCV-positive subjects compared with controls (median 0.4, range 0.0–3.1 vs 1.1, 0.2–3.5 cells/HPF, P  =   0.02). Among HCV-positive subjects, HSC apoptosis was decreased in those with moderate to advanced fibrosis ( P  = 0.04) compared with those with mild fibrosis. By multivariate analysis, HSC apoptosis decreased by an average of 0.14 cells/HPF (95% confidence interval 0.01–0.28 cells/HPF) per increase in fibrosis stage ( P  = 0.04). While the number of activated and proliferative HSC was significantly increased in HCV-infected subjects compared with that in uninfected controls, the numbers of these cells did not differ between HCV-infected subjects with mild vs moderate/advanced fibrosis. In conclusion, the number of apoptotic HSC was significantly decreased in HCV-infected subjects with advanced fibrosis. In chronic HCV infection, inhibition of HSC apoptosis may be one mechanism by which fibrosis progresses.     

Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice

Activation of innate immunity (natural killer [NK] cell/interferon-γ [IFN-γ]) has been shown to play an important role in antiviral and antitumor defenses as well as antifibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2-week or a 10-week carbon tetrachloride (CCl(4) ) challenge, respectively. Injection of polyinosinic-polycytidylic acid (poly I:C) or IFN-γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2-week CCl(4) model. Such activation was diminished in the 10-week CCl(4) model. Consistent with these findings, the inhibitory effect of poly I:C and IFN-γ on liver fibrosis was markedly reduced in the 10-week versus the 2-week CCl(4) model. In vitro coculture experiments demonstrated that 4-day cultured (early activated) HSCs induce NK cell activation via an NK group 2 member D/retinoic acid-induced early gene 1-dependent mechanism. Such activation was reduced when cocultured with 8-day cultured (intermediately activated) HSCs due to the production of transforming growth factor-β (TGF-β) by HSCs. Moreover, early activated HSCs were sensitive, whereas intermediately activated HSCs were resistant to IFN-γ-mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN-γ inhibition of cell proliferation in intermediately activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN-γ signaling and functioning, whereas production of TGF-β by HSCs inhibited NK cell function and cytotoxicity against HSCs. CONCLUSION: The antifibrogenic effects of NK cell/IFN-γ are suppressed during advanced liver injury, which is likely due to increased production of TGF-β and expression of SOCS1 in intermediately activated HSCs.     

Hepatic fibrogenesis requires sympathetic neurotransmitters

BACKGROUND AND AIMS: Hepatic stellate cells (HSC) are activated by liver injury to become proliferative fibrogenic myofibroblasts. This process may be regulated by the sympathetic nervous system (SNS) but the mechanisms involved are unclear. METHODS: We studied cultured HSC and intact mice with liver injury to test the hypothesis that HSC respond to and produce SNS neurotransmitters to promote fibrogenesis. RESULTS: HSC expressed adrenoceptors, catecholamine biosynthetic enzymes, released norepinephrine (NE), and were growth inhibited by alpha- and beta-adrenoceptor antagonists. HSC from dopamine beta-hydroxylase deficient (Dbh(-/-)) mice, which cannot make NE, grew poorly in culture and were rescued by NE. Inhibitor studies demonstrated that this effect was mediated via G protein coupled adrenoceptors, mitogen activated kinases, and phosphatidylinositol 3-kinase. Injury related fibrogenic responses were inhibited in Dbh(-/-) mice, as evidenced by reduced hepatic accumulation of alpha-smooth muscle actin(+ve) HSC and decreased induction of transforming growth factor beta1 (TGF-beta1) and collagen. Treatment with isoprenaline rescued HSC activation. HSC were also reduced in leptin deficient ob/ob mice which have reduced NE levels and are resistant to hepatic fibrosis. Treating ob/ob mice with NE induced HSC proliferation, upregulated hepatic TGF-beta1 and collagen, and increased liver fibrosis. CONCLUSIONS: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitters to promote hepatic fibrosis.     

Inhibition of hepatic stellate cell proliferation and activation by the semisynthetic analogue of fumagillin TNP-470 in rats

Proliferation and activation of hepatic stellate cells (HSCs) are critical steps for the development of postnecrotic fibrosis in the liver. The present study aimed to reveal the inhibitory effect of the semisynthetic analogue of fumagillin TNP-470 on these events for its possible use as an antifibrogenic agent. Rat models of carbon tetrachloride (CCl(4))- and dimethylnitrosamine-induced hepatic fibrosis were used for an in vivo study. In both models, the fibrotic area was considerably decreased by concurrent repetitive subcutaneous injections of 30 mg/kg body weight of TNP-470. In CCl(4)-induced fibrosis, factor VIII-related antigen-positive blood vessels, desmin-, or alpha-smooth muscle actin (alphaSMA)-positive mesenchymal cells, bromodeoxyuridine (BrdU)-positive mesenchymal cells also decreased in number by treatment with TNP-470. In in vitro experiments, a supplement of 1,000 ng/mL TNP-470 suppressed BrdU incorporation and cyclins D1, D2, and E expression by cultured HSCs in the absence and/or presence of platelet-derived growth factor (PDGF). Expression of HSC activation markers, i.e., alphaSMA and PDGF receptor beta, was also suppressed. The present results indicate that TNP-470 inhibits HSC proliferation by blocking the cell-cycle transition from G1 to S and HSC activation, and, as the consequence, prevents the progression of hepatic fibrosis, probably being coupled with its antiangiogenic effect.     

Hepatic fibrogenesis]

In acute injury, liver recovers completely without any scarring change or complication. However, large portion of liver is changed into fibrotic state by excessive production of extracellular matrix (ECM) under chronic injury. Excessive production of ECM results in hepatic fibrosis and repeated process of hepatic fibrosis progress into liver cirrhosis. Liver cirrhosis is an irreversible and terminal state of chronic liver disease and one of the major causes of death in Korea. To block the progression to liver cirrhosis, various studies in the field of virology and immunology have been proceeded. Recently, studies on the hepatic fibrogenesis have progressed with the development of molecular biology. Hepatic stellate cells (HSC) play a key role in the pathogenesis of hepatic fibrosis by producing ECM. The degree of hepatic fibrosis depends on the proliferation and activation of HSC and increased net production of collagen. Therefore, inhibition of HSC activation is one of the main ways to block the progression of hepatic fibrosis. Many kinds of factors such as oxidative stress, acetaldehyde, ascorbic acid, transforming growth factor-beta (TGF-beta) and carbon tetrachloride (CCl4) have been reported to activate HSC and stimulate collagen gene expression. Although there are no definite and effective antifibrogenic agents, possible candidates are antioxidants, interferon, retinoids such as beta-carotene, flavonoids, renin-angiotensin system inhibitors and peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists. We tried to evaluate the charateristics of HSC in order to develop agents that inhibit hepatic fibrogenesis.     

Effect of Losartan, an Angiotensin II Antagonist, on Hepatic Fibrosis Induced by CCl4 in Rats

In addition to regulating blood pressure and body fluid homeostasis, the renin-angiotensin system (RAS) is also involved in hepatic fibrogenesis. We aimed to investigate the effect of losartan, an angiotensin II (Ang II) antagonist, on CCl4-induced hepatic fibrosis in rats. Hepatic fibrosis was induced by a subcutaneous injection with 50% CCl4 in Sprague-Dawley rats. The amount of CCl4 administered was 1 mg/kg. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma and hydroxyproline (Hyp) contents in liver tissue were assayed by spectrophotometry. Hyaluronic acid (HA) and procollagen III (PC III) were assessed by radioimmunoassay. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) levels in culture supernatants of Kupffer cells (KCs) stimulated with Ang II was determined by ELISA. Liver samples collected after 12 weeks of CCl4 treatment were stained with hematoxylin and eosin, then scored. Losartan (2.5, 5, and 10 mg x kg(-1), ig) and captopril (100 mg x kg(-1), ig) significantly decreased liver and spleen indexes, serum transaminase (AST, ALT) activities, HA and PC III levels, and Hyp contents in liver tissue in rats of hepatic fibrosis. Histopathological scores showed that losartan had an inhibitory effect on the progression of hepatic fibrosis. In in vitro experiments, losartan (1 x 10(-9) - 1 x 10(-5) M) significantly reduced TNF-alpha and TGF-beta1 levels in culture supernatants of KCs, but captopril (1 x 10(-5) M) did not. The results showed that losartan significantly inhibited the progression of hepatic fibrosis induced by CCl4, and the inhibitory effect of losartan on hepatic fibrosis might be associated with its ability to inhibit the production of TNF-alpha and TGF-beta1 by activated KCs.     

Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice

Rac, a small, GTP-binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the alpha-smooth muscle actin (alpha-sma) promoter, because alpha-sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac-transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl(4)) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis-as well as higher mortality-than CCl(4)-treated wild-type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin-induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis.     

Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/ enhancer. A model of CCl(4)-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(alpha1)-collagen-I, (alpha2)-collagen-IV, and alpha-smooth muscle actin (alpha-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl(4), however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl(4)-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl(4)-treated TIMP-Tg-mice with a pattern similar to that of alpha-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development.     

Low molecular weight heparin prevents hepatic fibrogenesis caused by carbon tetrachloride in the rat

BACKGROUND/AIMS: In this study, we investigated the effect of dalteparin sodium, a low molecular weight (LMW)-heparin, on hepatic fibrogenesis caused by chronic carbon tetrachloride (CCl4) administration in the rat. METHODS: Female Wistar rats were given a single, or repeated intraperitoneal injections of CCl4 (1ml/kg, twice per week) and dalteparin (50IU/kg, daily) for 7 weeks. RESULTS: Dalteparin did not prevent acute CCl4-induced hepatic necrosis and elevation in serum aminotransferases levels; however, proliferating cell nuclear antigen (PCNA)-positive hepatocytes were dramatically increased 24h after simultaneous administration of CCl4 and dalteparin. Interestingly, serum hepatocyte growth factor (HGF) levels 12h after injection of CCl4 were almost doubled when dalteparin was given simultaneously. Hepatic fibrosis following 7-week CCl4 treatment was markedly ameliorated by daily co-administration of dalteparin. Indeed, dalteparin largely inhibited CCl4-induction of smooth muscle alpha-actin expression, alpha1(I)procollagen and transforming growth factor (TGF)-beta1 mRNA levels in the liver. Further, dalteparin blunted platelet-derived growth factor (PDGF)-induced increases in 5-bromo-2'deoxyuridine (BrdU) uptake in 3-day cultured hepatic stellate cells (HSCs) in a dose-dependent manner. CONCLUSIONS: Dalteparin enhances hepatic regeneration and minimizes hepatic fibrogenesis caused by chronic CCl4 treatment. The mechanism underlying these effects most likely involves both up-regulation of HGF and inhibition of HSC proliferation.