Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats

BACKGROUND & AIMS: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. METHODS: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor beta receptor (AdTbeta-TR), which works as a dominant-negative receptor, bacterial beta-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1-21 days. RESULTS: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTbeta-TR-, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTbeta-TR-treated rats. CONCLUSIONS: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTbeta-TR-treated livers indicate that transforming growth factor beta is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor beta is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells.     

Acetaldehyde increases endogenous adiponectin and fibrogenesis in hepatic stellate cells but exogenous adiponectin inhibits fibrogenesis

Background: Adiponectin has antifibrogenic properties. Acetaldehyde, the principal metabolite of ethanol, is known to stimulate the expression of type I collagen genes and the production of type I collagen by wild‐type (wt) but not by obese gene (ob/ob) stellate cells. The aim of this study was to determine the expression of adiponectin in activated stellate cells obtained from wt and ob/ob mice and to determine the effects of acetaldehyde on adiponectin in relation to the expression of type I collagen. Methods: Stellate cells were isolated from wt and ob/ob mice by perfusion of the portal vein and cultured. Cell adiponectin was visualized by immunohistochemistry and confocal microscopy and determined by radioimmunoassay and by western blot. Adiponectin mRNA and α₁(I) collagen mRNA were determined by quantitative real time polymerase chain reaction. Results: Adiponectin levels were similar in wt and ob/ob stellate cells. Adiponectin receptor 2 mRNA (AdipoR2 mRNA) and AdipoR2 immunoprotein were higher in ob/ob than in wt stellate cells (p < 0.01). Acetaldehyde (200 μM) increased adiponectin both in wt and in ob/ob stellate cells (p < 0.05), but increased AdipoR2 immunoprotein only in ob/ob stellate cells (p < 0.01). However, in the presence of leptin, acetaldehyde decreased adiponectin in ob/ob stellate cells (p < 0.01). Acetaldehyde enhanced α₁(I) collagen mRNA in wt (p < 0.05), but decreased it in ob/ob stellate cells (p < 0.01). Leptin abrogated the effect of acetaldehyde in decreasing α₁(I) collagen mRNA in ob/ob stellate cells (p < 0.01). Adiponectin inhibited α₁(I) collagen mRNA in the basal state in wt stellate cells or when enhanced by acetaldehyde. Conclusions: Adiponectin and adiponectin receptor are present in activated stellate cells. Adiponectin has a negative regulatory role on the enhancing effect of acetaldehyde on fibrogenesis in alcoholic liver disease.     

细胞外信号调节激酶1与肝星状细胞增殖关系的体内实验研究

目的　探讨在纤维化发生中 ,细胞外信号调节激酶1(ERK1)与肝星状细胞 (HSCs)增殖的关系。方法　采用胆总管结扎 (BDL)方法建立大鼠肝纤维化模型 ,应用免疫组织化学及逆转录聚合酶链式反应 (RT PCR)技术研究ERK1及其mRNA在肝纤维化不同时期肝组织中的分布及含量的动态变化 ;采用免疫组织化学方法测定α SMA。结果　正常肝组织有少量α SMA、ERK1分布 ,随着肝纤维化发展 ,α SMA、ERK1阳性细胞明显增多 ;正常大鼠肝组织中有ERK1mRNA表达 ,分别于造模 2d开始上调 ,造模 4w表达最多。ERK1与α SMA呈显著正相关 (r =0 958,P <0 0 5)。结论　肝纤维化形成过程中ERK1及其mRNA表达明显增加 ,ERK1在HSCs增殖及肝纤维化形成过程中发挥重要作用     

Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis.

BACKGROUND & AIMS: The accelerated course of hepatic fibrosis that occurs in some patients after liver transplantation is a major clinical problem. This response may be caused by the antirejection therapeutics, and in an earlier report we showed that FK-506 enhanced the fibrogenic process in in vivo and in vitro models of liver fibrosis. In the present study, the aim was to determine whether a new immunosuppressive agent, rapamycin, enhances or inhibits liver fibrosis. METHODS: Effects of rapamycin were investigated in a carbon tetrachloride model of hepatic fibrosis in rats and on hepatic stellate proliferation in vitro. RESULTS: Rapamycin inhibited extracellular matrix deposition in the rat model of fibrogenesis as determined by histological analysis, collagen content, messenger RNA levels of procollagen and transforming growth factor beta1, and tissue transglutaminase activity. Moreover, rapamycin decreased platelet growth factor-induced proliferation of hepatic stellate cells. CONCLUSIONS: These findings indicate that the new antirejection agent rapamycin inhibits hepatic fibrosis and thus may become a valuable addition to the immunosuppression armamentarium.     

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.     

氧化苦参碱对大鼠肝星状细胞增殖的影响

目的探讨氧化苦参碱对大鼠肝星状细胞增殖的影响及其抗肝纤维化的机理.方法用链酶蛋白酶和胶原酶原位灌流消化正常大鼠肝脏,Nycodenz密度梯度离心分离肝星状细胞,以MTT比色法观察氧化苦参碱对肝星状细胞毒性作用,3H-TdR法观察氧化苦参碱对肝星状细胞增殖的效应.结果氧化苦参碱浓度＞10-5mol/1时对肝星状细胞增殖有抑制作用(P＜0.05).结论氧化苦参碱可抑制肝星状细胞的增殖,有抗肝纤维化的作用.     

Characteristics of the cell proliferation profile of activated rat hepatic stellate cells in vitro in contrast to their fibrogenesis activity

Purpose. Alterations in the kinetics of hepatic stellate cells (HSCs) after the cells are activated once have not been well documented. We investigated the characteristic profiles of cell proliferation of once-activated HSCs in contrast to the in fibrogenesis activity. Methods. HSCs from male Wistar rats were submitted to primary culture for 14 days and to secondary culture for 7 days. The potential for cell proliferation was evaluated by the number of the cells in G2/M phase, based on flow cytometric analysis of the cell cycle. The fibrogenesis activity was assessed by Northern blot analysis of the expression of type I and type III procollagen mRNA. Results. The number of HSCs in G2/M phase was maintained at a low level in primary culture after 6 days, while a significantly (P < 0.05) elevated number of HSCs in G2/M phase was observed on days 3 to 4. In secondary culture, the number of HSCs in G2/M phase was also consecutively maintained at a decreased level. By contrast, HSCs showed progressively increased type I and type III procollagen mRNA expression during the experimental periods of primary culture. Conclusions. These results clearly demonstrated consecutively decreased proliferative activity, evaluated by the potential for cell mitogenesis, in once-activated HSCs, in contrast to their progressively increased fibrogenesis activity. Received: April 12, 2000 / Accepted: December 22, 2000     

Rosmarinic acid attenuates hepatic fibrogenesis via suppression of hepatic stellate cell activation/proliferation and induction of apoptosis

Objective:To investigate the antifibrotic role of rosmarinic acid(RA),a natural polyphenolic compound,on HSCs activation/proliferation and apoptosis in vitro and in vivo. Methods:The impact of RA on stellate cell line(HSC-T6) proliferation,activation and apoptosis was assessed along with its safety on primary hepatocytes. In vivo,rats were divided into:(i) normal;(ii) thioacetamide(TAA)-intoxicated rats for 12 weeks;(iii) TAA+silymarin or(iv) TAA+RA. At the end of experiment,liver functions,oxidative stress,inflammatory and profibrogenic markers,tissue inhibitor metalloproteinases type-1(TIMP-1) and hydroxyproline(HP) levels were evaluated. Additionally,liver histopathology and immunohistochemical examinations of alpha-smooth muscle actin(α-SMA),caspase-3 and proliferation cellular nuclear antigen(PCNA) were determined. Results:RA exhibited anti-proliferative effects on cultured HSCs in a time and concentration dependent manner showing an IC50 of 276 μg/mL and 171 μg/mL for 24 h and 48 h,respectively,with morphological reversion of activated stellate cell morphology to quiescent form. It significantly improved ALT,AST,oxidative stress markers and reduced TIMP-1,HP levels,inflammatory markers and fibrosis score(S1 vs S4). Furthermore,reduction in α-SMA plus elevation in caspase-3 expressions of HSCs in vitro and in vivo associated with an inhibition in proliferation of damaged hepatocytes were recorded. Conclusions:RA impeded the progression of liver fibrosis through inhibition of HSCs activation/proliferation and induction of apoptosis with preservation of hepatic architecture.     

Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury

BACKGROUND/AIM: Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats. METHODS: The authors used transgenic mice (SMP8-IGF-I) expressing IGF-I under control of alphaSMA promoter to study the influence of IGF-I synthesised by activated HSCs on the recovery from liver injury. RESULTS: The transgene was expressed by HSCs from SMP8-IGF-I mice upon activation in culture and in the livers of these animals after CCl4 challenge. Twenty four hours after administration of CCl4 both transgenic and wild type mice showed similar extensive necrosis and increased levels of serum transaminases. However at 72 hours SMP8-IGF-I mice exhibited lower serum transaminases, reduced hepatic expression of alphaSMA, and improved liver morphology compared with wild type littermates. Remarkably, at this time all eight CCl4 treated wild type mice manifested histological signs of liver necrosis that was severe in six of them, while six out of eight transgenic animals had virtually no necrosis. In SMP8-IGF-I mice robust DNA synthesis occurred earlier than in wild type animals and this was associated with enhanced production of HGF and lower TGFbeta1 mRNA expression in the SMP8-IGF-I group. Moreover, Colalpha1(I) mRNA abundance at 72 hours was reduced in SMP8-IGF-I mice compared with wild type controls. CONCLUSIONS: Targeted overexpression of IGF-I by activated HSCs restricts their activation, attenuates fibrogenesis, and accelerates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFbeta1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis.     

Liver fibrogenesis and the role of hepatic stellate cells: New insights and prospects for therapy

Hepatic fibrosis is a wound-healing response to chronic liver injury, which if persistent leads to cirrhosis and liver failure. Exciting progress has been made in understanding the mechanisms of hepatic fibrosis. Major advances include: (i) characterization of the components of extracellular matrix (ECM) in normal and fibrotic liver; (ii) identification of hepatic stellate cells as the primary source of ECM in liver fibrosis; (iii) elucidation of key cytokines, their cellular sources, modes of regulation, and signalling pathways involved in liver fibrogenesis; (iv) characterization of key matrix proteases and their inhibitors; (v) identification of apoptotic mediators in stellate cells and exploration of their roles during the resolution of liver injury. These advances have helped delineate a more comprehensive picture of liver fibrosis in which the central event is the activation of stellate cells, a transformation from quiescent vitamin A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The progress in understanding fibrogenic mechanisms brings the development of effective therapies closer to reality. In the future, targeting of stellate cells and fibrogenic mediators will be a mainstay of antifibrotic therapy. Points of therapeutic intervention may include: (i) removing the injurious stimuli; (ii) suppressing hepatic inflammation; (iii) down-regulating stellate cell activation; and (iv) promoting matrix degradation. The future prospects for effective antifibrotic treatment are more promising than ever for the millions of patients with chronic liver disease worldwide.