The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver

Monospecific antibodies directed against fibronectin, type I collagen, and two basement membrane components, laminin and type IV collagen, were localized in normal rat liver by light and electron microscopy immunohistochemistry. Type I collagen was found in the liver capsule, protal stroma, and in Disse's space where it was often in direct contact with the hepatocyte plasmalemma; along the sinusoidal wall, type I collagen was more abundant at points of branching or inflexion. Collagen type IV was found in all basement membranes: ductal, neural, and vascular. In addition, small, discrete, discontinuous, deposits of type IV collagen were found along the entire length of the sinusoid. Laminin codistributed with type IV collagen in all basement membranes but was not found in the sinusoidal wall. The structural glycoprotein, fibronectin, was found in the liver capsule and portal stroma but not in basement membranes. However, fibronectin was found in direct contact with the hepatocytes microvilli forming an almost continuous structure; it was the most prominent component of the extracellular matrix in Disse's space. These findings provide a new image of the Disse's space. Rather than being an empty space, as suggested by classic electron microscopy, it was found to contain an extracellular matrix with several unique features: type I collagen, in direct contact with hepatocytes and endothelial cells, formed the scaffold of the hepatic lobule. Type IV collagen was found "free," not associated with laminin and not forming part of a basement membrane. Hepatocytes and endothelial cells lacked a basement membrane but were separated by an extracellular matrix containing predominantly fibronectin, some type I collagen, and occasional spotty deposits of type IV collagen. Future studies of the physiology and pathology of the hepatic sinusoid will have to take into account this unique extracellular matrix.     

The role of food restriction on CCl4-induced cirrhosis model in rats.

Effects of food restriction on susceptibility to the toxic effect of some chemicals are controversial. In order to identify an exposure model that could maximize cirrhosis and minimize mortality rate, this study aimed to evaluate the effect of food restriction on tetrachloride carbon (CCl(4))-induced cirrhosis model in rats. Fifty-three male Wistar rats received CCl(4) 0.25 ml/kg weekly intragastrically once a week. Thirty-three had 44% food restriction (group 1); 10 rats had 25% food restriction (group 2); and 10 rats received ad libitum food (group 3). After 10 weeks, the animals were sacrificed and liver sections were collected for histology. Of the 53 animals enrolled for the study, 22 (41.5%) died before completing 10-week CCl(4). Mortality rate was significantly higher in group 1 compared to other groups (p<0.05). Cirrhosis was significantly more prevalent in group 1 than in group 3 (p<0.01), but without significant difference between groups 1 and 2 (p=0.624). We concluded that food restriction is an important issue to be considered when establishing a CCl(4)-induced cirrhosis model in rats. Moreover, there is an ideal range of food intake that predisposes to liver damage without increasing mortality leading to a more effective model.     

卵巢切除对四氯化碳诱导大鼠肝纤维化形成的影响

为探讨卵巢切除对CCl4 诱导大鼠肝纤维化形成的影响 ,采用CCl4 诱导雌性大鼠肝纤维化动物模型 ,观察卵巢切除及雌激素替代治疗 (苯甲酸雌二醇 1mg kg)对肝脏胶原沉积和I、Ⅲ型胶原蛋白表达的影响 ,并分别检测血清学标志及肝脏组织学等变化。结果显示CCl4 模型组大鼠肝脏发生典型的肝纤维化改变 ,卵巢切除组的肝脏胶原沉积更为明显 ,肝脏表达I、Ⅲ型胶原及血清肝纤维化指标也明显高于CCl4 摸型组 (P <0 0 5 ) ,而雌激素干预及替代治疗则可抑制肝纤维化的形成。表明卵巢切除加速CCl4 诱导大鼠肝纤维化的形成 ,其发生可能与卵巢分泌的雌激素对肝纤维化的抑制作用有关。     

The effect of vitamin A on CCl4-induced hepatic injuries in rats: a histochemical, immunohistochemical and ultrastructural study

In this study, we aimed to investigate the effect of vitamin A on the transformation of the Ito cells to fibrogenic form and suppression of the development of fibrosis. Carbon tetrachloride intoxication was performed on rats for 2, 8, 12 or 20 weeks and 5x10(4) IU vitamin A (as retinol palmitate) was injected subcutaneously once every 4 weeks. Ito cells were detected by gold chloride impregnation, as well as desmin and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry. Additionally, all groups were examined ultrastructurally. The number of Ito cells that were labelled positively with gold impregnation decreased in the fibrotic groups; however, alpha-SMA and desmin immunopositive Ito cells increased. The samples from animals that were treated with vitamin A showed an increase in labelling with gold impregnation but a decrease in alpha-SMA immunopositivity. The data showed that vitamin A can prevent hepatic injury, by suppressing the transformation of Ito cells to fibrogenic form. We conclude that vitamin A has potential for the treatment of hepatic fibrotic diseases. Alpha-SMA immunohistochemistry was found to be more informative than desmin immunohistochemistry for monitoring liver fibrosis.     

Rebamipide retards CCl4-induced hepatic fibrosis in rats: Possible role for PGE2

Prostaglandin E₂ (PGE₂) is a potent physiological suppressor of liver fibrosis. Because the anti-ulcer drug rebamipide can induce the formation of endogenous PGE₂, this study investigated the potential effects of rebamipide on development of a hepatic fibrosis that was inducible by carbon tetrachloride (CCl₄). Groups of Wistar rats received intraperitoneal (IP) injections of CCl₄ (0.45 ml/kg [0.72 g CCl₄/kg]) over the course of for 4 weeks. Sub-sets of CCl₄-treated rats were also treated concurrently with rebamipide at 60 or 100?mg/kg. At 24 h after the final treatments, liver function and oxidative stress were indirectly assessed. The extent of hepatic fibrosis was evaluated using two fibrotic markers, hyaluronic acid (HA) and pro-collagen-III (Procol-III); isolated liver tissues underwent histology and were evaluated for interleukin (IL)-10 and PGE₂ content. The results indicated that treatment with rebamipide significantly inhibited CCl₄-induced increases in serum ALT and AST and also reduced oxidative stress induced by CCl₄. Fibrotic marker assays revealed that either dose of rebamipide decreased the host levels of Procol-III and HA that had become elevated due to the CCl₄. At the higher dose tested, rebamipide appeared to be able to permit the hosts to have a normal liver histology and to minimize any CCl₄-induced collagen precipitation in the liver. Lastly, the use of rebamipide was seen to be associated with significant increases in liver levels of both PGE₂ and the anti-inflammatory cytokine IL-10. Based on these findings, it is concluded that rebamipide can retard hepatic fibrosis induced by CCl₄ and that this effect may, in part, be mediated by an induction of PGE₂ and IL-10 in the liver itself.     

The hepatic extracellular matrix

The unique nature of the hepatic extracellular matrix (ECM) is predicted by the special configuration of the space of Disse. Whereas other epithelial organs have two basement membranes (BM) and a substantial ECM interposed between endothelial and epithelial cells, the liver lobule has no BM and only an attenuated ECM, consisting mostly of fibronectin (FN), some collagen type I, and minor quantities of types III, IV, V, and VI. This configuration, together with the abundant fenestrations and gaps of the sinusoidal endothelial cells, seems ideally suited to facilitate the rapid bidirectional exchange of macromolecules normally taking place between plasma and hepatocytes. During organogenesis, the liver anlage is vascularized by continuous capillaries with BM, but by day 13.5 of development (in the rat) the vessels in the immediate proximity of hepatocytes become fenestrated, lacking specialized junctions and BM, suggesting that the hepatocytes produce signals capable of modulating the endothelial phenotype. In regeneration, hepatocyte proliferation precedes vascular proliferation resulting in the formation of hepatocyte clusters that, temporarily, lack sinusoids. Eventually, vascular proliferation follows and the normal hepatocyte-vascular relationships are restored. During this period laminin synthesis by Ito cells is prominent. As soon as hepatocytes become stable, secretion of the sinusoid phenotype-maintaining factors resumes and laminin synthesis and secretion terminates. The interplay between extracellular matrix and liver cells is essential for normal homeostasis and its modification results in derranged hepatic function.Parts of this editorial have been adapted from: A. Martinez-Hernandez, P.S. Amenta. Morphology, localization and origin of the hepatic extracellular matrix. In: Zern MA, Reid L (eds) Extracellular matrix: chemistry, biology, and pathology with emphasis on the liver. Marcel Dekker, New York, (in press)     

The hepatic extracellular matrix

The unique nature of the hepatic extracellular matrix (ECM) is predicated by the special configuration of the space of Disse. Whereas other epithelial organs have two basement membranes (BM) and a substantial ECM interposed between endothelial and epithelial cells, the liver lobule has no BM and only an attenuated ECM, consisting mostly of fibronectin, some collagen type I, and minor quantities of types III, IV, V, and VI. This configuration, together with the abundant fenestrations and gaps of the sinusoidal endothelial cells, seems ideally suited to facilitate the rapid bidirectional exchange of macromolecules normally taking place between plasma and hepatocytes. During organogenesis, the liver anlage is vascularized by continuous capillaries with BM, but by day 13.5 of development (in the rat) the vessels in the immediate proximity of hepatocytes become fenestrated, lacking specialized junctions and BM, suggesting that the hepatocytes produce signals capable of modulating the endothelial phenotype. In regeneration, hepatocyte proliferation precedes vascular proliferation resulting in the formation of hepatocyte clusters that, temporarily, lack sinusoids. Eventually, vascular proliferation follows and the normal hepatocyte-vascular relationships are restored. During this period laminin synthesis by Ito cells is prominent. As soon as hepatocytes become stable, secretion of the sinusoid phenotype-maintaining factors resumes and laminin synthesis and secretion terminates. The interplay between extracellular matrix and liver cells is essential for normal homeostasis and its modification results in derranged hepatic function.Parts of this editorial have been adapted from: A Martinez-Hernandez, PS Amenta. Morphology, localization and origin of the hepatic extracellular matrix. In: Zern MA, Reid L (eds) Extracellular matrix: chemistry, biology, and pathology with emphasis on the liver. Marcel Dekker, New York (in press)     

Functions of bone marrow hemopoiesis-inducing microenvironment during CCl4-induced liver cirrhosis

During late stages of CCl₄-induced liver cirrhosis, production of factors stimulating the growth of erythroid and granulomonocytopoietic precursors by bone marrow adherent and nonadherent fractions of BALB/c mice decreased. Stimulation with the yeast polysaccharide zymosan decreased production of these activities by various bone marrow cells, especially by the adherent fraction. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 283–285, September, 1999     

Growth-inhibitory activity of bone marrow cells during CCl4-induced liver cirrhosis

During CCl₄-induced liver cirrhosis, cells of the hemopoiesis-inducing microenvironment in the bone marrow of BALB/c mice produced activity inhibiting the growth of erythropoiesis and granulomonocytopoiesis precursors. Stimulation with yeast polysaccharide zymosan increased the inhibitory activity (especially in relation to granulomonocytic precursors). The highest growth-inhibitory activity was produced by the bone marrow adherent fraction (residual bone marrow macrophages). Tumor necrosis factor-α is probably responsible for the inhibition of the growth of myeloid precursors in mice with CCl₄-induced liver cirrhosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 645–647, June, 1999     

四氯化碳致大鼠、小鼠肝损伤的对比实验

人们习惯将CCl4 致肝损伤模型作为筛选具有保肝降酶作用药物的常用模型之一[1～ 3] 。常用实验动物为大鼠及小白鼠 ,以血清转氨酶为观察指标来评价被筛选药物的保肝降酶效果。我们在最近筛选保肝药物的实验中发现 ,用CCl4 所致的肝损伤模型动物小白鼠的血清转氨酶量存在很大的个体差异。为此我们对比了CCl4 致大鼠、小鼠肝损伤实验 ,就CCl4 致动物肝损伤的稳定性进行了探讨。1　实验材料1 1　药品和试剂 :四氯化碳 (CCl4 ,分析纯 ,济南化工厂生产 ) ,根据需要用纯花生油配成不同浓度 ;谷丙转氨酶试剂盒(购自北京中国生化药品公司 )。1 2…     

Attenuation of CCl4-induced hepatic oxidative stress in rat by Launaea procumbens

Antioxidant effects of Launaea procumbens methanol extract (LPME) were evaluated against CCl₄-induced oxidative stress in liver of rat. 48 male rats were equally divided in to 8 groups (06 rats each). Group I (control) remained untreated, while Group II was given vehicles (olive oil and DMSO). Animals of Groups III, IV, V, VI and VII were injected intraperitoneally with CCl₄ (3 ml/kg b.w.; i.p., 20% CCl₄/olive oil) twice a week for four weeks. Group III received only CCl₄ while Group IV was given rutin (50 mg/kg b.w.). Group V, VI and VII were administered LPME at a dose of 100, 150 and 200 mg/kg b.w., respectively. Animals of Group VIII received LPME (200 mg/kg b.w.) alone. Oxidative stress induced with CCl₄ in liver was evident by a significant increase in triglycerides, total cholesterol, LDL-cholesterol, and enzymatic activities of AST, ALT, ALP, LDH, γ-GT activities in serum. Level of lipid peroxidation, nitrite, and hydrogen peroxide concentration, DNA injuries in liver samples was also increased with CCl₄. GSH concentration in liver was significantly decreased, as were the activities of antioxidant enzymes; CAT, POD, SOD, GSH-Px, GST, GSR, QR. Co-treatment of rats with LPME and rutin prevented all the changes observed with CCl₄. Hepatic lesions and telomerase activity induced with CCl₄ was also suppressed with LPME and rutin. It is suggested that LPME effectively prevented the CCl₄-induced oxidative injuries in liver, possibly through antioxidant and/or free radical scavenging effects of flavonoids and phenolic compounds in the extract.     

Hepatoprotective effects of prostacyclins on CCl4-induced liver injury in rats

Certain biochemical parameters of acute liver injury induced by carbon tetrachloride were investigated in rats treated with prostacyclin (PGI2) and two of its derivatives. Serum glutamate oxalacetate transaminase elevation and both triglyceride accumulation and reduction of glycogen content in liver were significantly suppressed by PGI2, 7-oxo-PGI2, and 20-methyl-13,14-didehydro-2,4-m-interphenylene-PGI2 48 hr after the injury. Prostacyclins partially restored some of the parameters of injury even in doses of 10 micrograms/kg ip. When the compounds were given 24 hr after CCl4 intoxication, much more pronounced protection was observed than in the case of treatments 1 hr before administration of the hepatotoxin. Thus, all tested prostacyclins exerted significant protective effects on acute liver damage which is obtained mainly in the second phase of the injury.     

Hepatoprotection with a chloroform extract of Launaea procumbens against CCl4-induced injuries in rats

ABSTRACT: BACKGROUND: Launaea procumbens (Asteraceae) is used as a folk medicine to treat hepatic disorders in Pakistan. The effect of a chloroform extract of Launaea procumbens (LPCE) was evaluated against carbon-tetrachloride (CCl4)-induced liver damage in rats. METHODS: To evaluate the hepatoprotective effects of LPCE, 36 male Sprague-Dawley rats were equally divided into six groups. Animals of group 1 (control) had free access to food and water. Group II received 3 ml/kg of CCl4 (30% in olive oil v/v) via the intraperitoneal route twice a week for 4 weeks. Group III received 1 ml of silymarin via gavage (100 mg/kg b.w.) after 48 h of CCl4 treatment whereas groups IV and V were given 1 ml of LPCE (100 and 200 mg/kg b.w., respectively) after 48 h of CCl4 treatment. Group VI received 1 ml of LPCE (200 mg/kg b.w.) twice a week for 4 weeks. The activities of the antioxidant enzymes catalase, peroxidase (POD), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), glutathione reductase (GSR), glutathione (GSH) and lipid peroxidation (thiobarbituric acid reactive substances (TBARS)) were measured in liver homogenates. DNA damage, argyrophilic nucleolar organizer regions (AgNORs) counts and histopathology were studied in liver samples. Serum was analyzed for various biochemical parameters. Phytochemical composition in LPCE was determined through high-performance liquid chromatography (HPLC). RESULTS: LPCE inhibited lipid peroxidation, and reduced the activities of aspartate transaminase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase in serum induced by CCl4. GSH contents were increased as were the activities of antioxidant enzymes (catalase, SOD, GST, GSR, GSH-Px) when altered due to CCl4 hepatotoxicity. Similarly, absolute liver weight, relative liver weight and the number of hepatic lesions were reduced with co-administration of LPCE. Phyochemical analyses of LPCE indicated that it contained catechin, kaempferol, rutin, hyperoside and myricetin. CONCLUSION: These results indicated that Launaea procumbens efficiently protected against the hepatotoxicity induced by CCl4 in rats, possibly through the antioxidant effects of flavonoids present in LPCE. KEYWORDS: Launaea procumbens, hepatic injuries, flavonoids, antioxidant enzymes, carbon tetrachloride.     

Serological and immunohistochemical studies on porcine-serum-induced hepatic fibrosis in rats

We previously reported that the strain difference in the development of porcine-serum (PS)-induced rat hepatic fibrosis was closely related to the difference in the mode of MHC class-II-related genes expression. This study was carried out to clarify the serological and immunohistochemical changes in this hepatic fibrosis model. Six-week-old male Brown Norway (BN) and Wistar rats were injected with 0.5 ml of sterile PS twice a week for up to 8 weeks. The serum levels of PS-specific IgG1, IgG2a, and IgM were elevated more prominently in BN rats than Wistar rats. In the liver, significant increases in the numbers of PS-, OX-6 (RT1.B)-, CD4-, CD8, ED1-, and ED2-positive cells occurred earlier in BN rats than Wistar rats. At 8 weeks, deposition of PS and immunoglobulins was observed in hepatic fibrous septa and renal glomerular mesangium, and IgG1- and IgG2a-positive cells were found in the white pulp of the spleen. The present results suggest that humoral immunity probably regulated by MHC class II molecules and inflammatory cells may be involved in PS-induced hepatic fibrosis in rats.     

Prevention of CCl4-induced liver cirrhosis by ribbon antisense to transforming growth factor-beta1

Transforming growth factor-beta1 (TGF-beta1) is an important mediator of tissue fibrosis, including liver cirrhosis. Ribbon-type antisense oligonucleotide to TGF-beta1 (TGF-beta1 RiAS) was designed and combined with cationic peptide derived from the nuclear localization signal of human immunodeficiency virus-1 Tat protein for enhanced cellular uptake. When Hepa1c1c7 cells were transfected with TGF-beta1 RiAS, the level of TGF-beta1 mRNA was reduced by >70%. TGF-beta1 RiAS, mismatched RiAS, and normal saline were each injected into mice via the tail vein, beginning the week after intraperitoneal CCl4 injection and continuing for 7 weeks, in order to determine whether TGF-beta1 RiAS prevents the fibrotic changes induced by the CCl4 injection. After 8 weeks of the experiment, all of the mice treated with TGF-beta1 RiAS survived, compared to 50% of the control group and 65% of the mismatched RiAS-treated group. Upon examining the biochemical effects on the liver, TGF-beta1 mRNA levels were reduced significantly only in the TGF-beta1 RiAS-treated group. Immunohistochemical studies showed a reduced accumulation of collagen and alpha-smooth muscle actin. Our experimental results suggest that ribbon antisense to TGF-beta1, with efficient uptake, effectively blocks the expression of TGF-beta1 and prevents fibrosis of the liver.     

Suppression of hemopoiesis during CCl4-induced hepatic fibrosis: Role of systemic endotoxemia

CCl₄-induced hepatic fibrosis in mice was accompanied by insufficiency of bone marrow myelo- and erythropoiesis. Polymyxin B completely abolished these changes. This phenomenon can be explained by the development of macrophage endotoxin tolerance during hepatic fibrosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 172–175, August, 2000     

Suppression of hemopoiesis during CCl4-induced hepatic fibrosis: Role of systemic endotoxemia

CCl₄-induced hepatic fibrosis in mice was accompanied by insufficiency of bone marrow myelo- and erythropoiesis. Polymyxin B completely abolished these changes. This phenomenon can be explained by the development of macrophage endotoxin tolerance during hepatic fibrosis. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 172–175, August, 2000     

Experimental studies on hepatic cirrhosis and hepatocarcinogenesis. II. Influence of cirrhotic liver on 2-FAA hepatocarcinogenesis in rats

The effects of hepatic cirrhosis on chemical hepatocarcinogenesis were studied in rats using an experimental model for hepatic cirrhosis established by chronic furfural administration (described in a previous report) and N-2-fluorenylacetamide (2-FAA). Sixteen male Wistar rats were fed with 2-FAA feeding 3 cycles, which consisted of 3 weeks consuming 0.03% 2-FAA diet and 1 week basal diet, after 120 days of furfural feeding. Grossly and histologically all livers of experimental animals showed formation of multiple hyperplastic nodules with atypical features in parts. These hyperplastic nodules were positively immunostained with alpha fetoprotein. On the other hand, none of the livers of 16 rats fed with furfural alone showed formation of hyperplastic changes and only 2 livers of 16 rats fed 2-FAA without preadministration of furfural showed one small hyperplastic nodule each, respectively. These results indicate that the cirrhotic liver induced by chronic furfural feeding has an enhancing effect on 2-FAA chemical hepatocarcinogenesis, and suggests that the presence of hepatic cirrhosis induces increased susceptibility to hepatocarcinogenic stimuli.     

Hematological and blood pressure studies in the CCl4 treated rats

Hematological parameters were studied in normotensive and spontaneous hypertensive rats along with those treated with chronic subcutaneous injections of CCl4. The normotensive animals treated with CCl4 demonstrated an erythrocytosis with an increase in the hematocrit levels. A lymphocytosis was seen with neutropenia in both CCl4 treated and untreated normotensive groups. The SHR animals compared to their normotensive counterparts had a lesser degree of lymphocytosis with an increase in the number of neutrophils. There was no blood pressure change in the normotensive CCl4 treated group, however a significant blood pressure difference was observed in the SHR group after CCl4 treatment.     

The extracellular matrix in pigmented skin lesions: an immunohistochemical study

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.