Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins

The sketches in Figures 11 and 12, necessarily simplified, summarize some of the points discussed in the previous paragraphs. It can be concluded that the hepatic ECM and the ECM in general is a complicated supramolecular assembly of numerous defined macromolecules, each of which is endowed with a specific potential for interactions with one another and with various cell types. This matrix not only adapts rapidly to slight metabolic changes of the cells producing it, but also itself modulates the biochemical and morphologic phenotype of the cells anchored to it. Since mesenchymal cells appear to be the major source of hepatic ECM proteins in health and disease, it follows that the mesenchyme determines to a significant degree the phenotype of liver epithelial cells. Based on the growing knowledge of the hepatic ECM, immunoassays for ECM proteins or peptides in serum may provide information about the dynamics of fibrogenesis or fibrolysis in individual patients on a day to day basis, or permit detection of derangements of the hepatic ECM before they become clinically apparent. The clinical value of such assays is already being studied. Finally, knowing the molecules and the cells involved in ECM pathologic states offers us the tools to develop a specific targeted antifibrogenic therapy.     

Expression of the novel extracellular matrix component tenascin in normal and diseased human liver. An immunohistochemical study.

The novel extracellular matrix glycoprotein tenascin was studied immunohistochemically in normal and fibrotic human liver. Its localization was compared to that of laminin, fibronectin and collagen type IV. In the normal liver, a weak staining for tenascin was detected along sinusoids, while portal tracts were negative. In both alcoholic and cholestatic liver disease and acute and chronic hepatitis, sinusoidal immunoreactivity for tenascin was variably increased as compared to the normal liver. Most striking, however, was the preferential accumulation of tenascin at connective tissue-parenchymal interfaces between proliferating ductules and in areas of piecemeal necrosis. As compared to laminin, fibronectin and collagen type IV, tenascin has the most restricted distribution. Our findings indicate that tenascin is a component of the extracellular matrix of the human liver. Its preferential expression at connective tissue-parenchymal interfaces in fibrosing areas in contrast to its absence from mature fibrous septa suggest a transient role in early matrix organization.     

Formation of extracellular matrix in normal rat liver: lipocytes as a major source of proteoglycan

Proteoglycans are a major component of the normal hepatic extracellular matrix and undergo quantitative and qualitative changes in hepatic fibrosis. The cellular sources of proteoglycans are as yet incompletely defined. We examined this question using primary cultures of hepatocytes and lipocytes isolated from normal rat liver. Proteoglycan synthesis was assessed by measuring production of sulfated glycosaminoglycan, the polysaccharide moiety of proteoglycans. The findings indicate that lipocytes produce sixfold more glycosaminoglycan, per cell, than do hepatocytes. Two-thirds of the newly synthesized material is cell- or matrix-associated. Of the individual glycosaminoglycan species produced by lipocytes, dermatan sulfate represents 60% of the total; heparan sulfate and chondroitin sulfate are measurable but relatively minor. In hepatocyte cultures, heparan sulfate accounted for essentially all of the glycosaminoglycan detected. We conclude that lipocytes are an important source of proteoglycan in normal liver and may be the principal source of dermatan sulfate associated with hepatic fibrosis.     

Localization of cellular retinol-binding protein in several rat tissues.

The distribution of cellular retinol-binding protein (CRBP) in rat liver, ileum, and epididymis was examined by the peroxidase-antiperoxidase immunolocalization technique. Positive cytoplasmic staining was seen in the liver when antiserum prepared against purified CRBP was used but not when antiserum absorbed with purified CRBP was used. Ileal mucosa, a tissue that contains no detectable CRBP, showed no positive staining. The epididymis showed strong positive staining in the caput but not in the cauda. Staining was present in principal and basal cells but not in peritubular or interstitial cells. Radioimmunoassay revealed that the CRBP within the caput epididymidis was localized in the initial segment and proximal region, areas known to be involved in the synthesis and secretion of factors necessary for sperm maturation. The results demonstrate that the expression of CRBP may vary within the same cell type, as well as between different cell types within the same tissue.     

Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.     

胞外基质Matrilin-2在肝再生中与大鼠卵圆细胞的关系

目的:探讨肝脏胞外基质Matrilin-2在肝再生中与卵圆细胞的关系及作用.方法:采用改良的Soft-Farber建立大鼠肝脏卵圆细胞增殖模型,对照组灌喂生理盐水.分别取术后2、4、6、9、12、15 d大鼠肝组织,采用免疫组织化学以及Western blot的方法动态观察大鼠卵圆细胞增殖模型中肝脏胞外基质成分Matrilin-2的变化与卵圆细胞的关系.结果:肝脏部分切除术(partial hepatectomy,PH)后第2天,卵圆细胞开始向门静脉周围区域增殖,Matrilin-2主要出现在门静脉周围的肝窦状隙内;术后第9天,卵圆细胞进一步向肝实质内增殖,Matrilin-2表达增加;术后第12天,随着卵圆细胞分化为小肝细胞结节,大多数Matrilin-2位于结节周边,少数出现在结节内.Matrilin-2的含量自肝切除后第2天开始升高,第9天达到高峰,第12天后逐步恢复生理水平.结论:肝脏胞外基质成分Matrilin-2与卵圆细胞介导的肝脏再生存在紧密联系并发挥重要的调控作用.     

Polymicrobial sepsis disrupts normal neutrophil extracellular matrix protein interactions.

The purpose of this study was to examine how intra-abdominal sepsis and extracellular matrix proteins (fibronectin, laminin) affect adherent polymorphonuclear leukocyte (PMN) function. Two groups of swine were studied: Group I (n = 5) underwent sham laparotomy; Group II (n = 8) underwent cecal ligation and incision. PMN adherent to either fibronectin (F) or laminin (L) had increased candicidal activity over buffer (B) by Group I but not by post-operative day 8 Group II PMN. (Percent specific release 51Cr-Group I--35.00, 68.25, 64.75% for B, F, and L; P less than 0.001 comparing B vs. F or L; Group II--14.25, 12.50, 12.75% for B, F, and L; P = NS comparing B vs. F or L.) To determine the mechanism for this finding, PMN priming was then assessed by evaluating both PMN adherence to extracellular matrix proteins and the cell surface expression of CR1/CR3 by using sheep RBC opsonized with C3b or C3bi. PMN activation was assayed by using MTT-Formazan, myeloperoxidase, and hypochlorous acid (HOCl) production. Fibronectin and laminin increased PMN adherence and CR1/CR3 expression over buffer by Group I and Group II animals. Fibronectin and laminin increased MTT-Formazan, myeloperoxidase, and HOCl production over buffer by Group I PMN but not POD 8 Group II PMN. These results suggest that untreated intra-abdominal sepsis partially abrogates the effect of extracellular matrix proteins on PMN function; in particular, the activation but not priming of adherent PMN by extracellular matrix proteins is reduced in this clinical situation.     

Extracellular matrix in normal and fibrotic human lungs

Polyclonal affinity-purified antibodies to human collagen types I, III, and IV, and laminin were used to compare the extracellular matrix (ECM) in 10 normal and 32 abnormal lungs by indirect immunofluorescence. In normal lungs, type IV collagen and laminin codistributed in a uniform linear pattern along the epithelial and endothelial basement membranes. Type III collagen was found within the alveolar septa and interstitium in an interrupted ribbonlike pattern and was aggregated at the entrance rings of the alveoli. Type I collagen was distributed irregularly within the alveolar wall and was less prominent than type III collagen. In patients with pulmonary disease not characterized by interstitial fibrosis (n = 15), the distribution of ECM components studied was essentially normal. In pulmonary disease in which interstitial fibrosis was the characteristic feature, such as idiopathic pulmonary fibrosis (IPF) and adult respiratory distress syndrome (ARDS) (n = 17), collagen types I and III accumulated in the expanded interstitium. Type III collagen was initially predominant in the thickened alveolar septa and interstitium, whereas type I collagen appeared to be the principal collagen at later stages in the disease course. The basement membrane was disrupted early in the disease course with invasion of the alveolar spaces by interstitial collagens similar in type to those present in the adjacent interstitium.     

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice

BACKGROUND/AIMS: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied. METHODS: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis. RESULTS: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice. CONCLUSIONS: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.     

Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury.

Tenascin is an extracellular matrix molecule synthesized and released by young astrocytes during embryonic and early postnatal development of the nervous system, and it is concentrated in boundaries around emerging functional neuronal units. In the adult nervous system, tenascin can be detected only in very low levels. Distinct spatial and temporal distributions of tenascin during developmental events suggest a role in the guidance and/or segregation of neurons and their processes within incipient functional patterns. We show here, using in situ hybridization and immunocytochemistry, that stab wounds of the adult mouse cerebellar and cerebral cortices result in an enhanced expression of tenascin in a discrete region around the lesion site that is associated with a subset of glial fibrillary acidic protein-positive astrocytes. Tenascin up-regulation in the lesioned adult brain may be directly involved in failed regeneration or indirectly involved through its interactions with other glycoconjugates that either inhibit or facilitate neurite growth.     

IL-10基因修饰骨髓源性肝干细胞移植对肝纤维化大鼠细胞外基质积聚的影响

目的: 评价IL-10基因修饰骨髓源性肝干细胞(BDLSCs)移植对肝纤维化大鼠细胞外基质(ECM)积聚的影响.方法: 利用免疫磁珠细胞分选(magnetic beadcell sorting, MACS)方法分选大鼠β2m-/Thy-1++BDLSCs. 将腺病毒介导的IL-10基因转导至BDLSCs, 采用ELISA法检测IL-10蛋白分泌水平. 将大鼠随机分为4组: 正常组、模型组、BDLSCs组及BDLSCs/IL-10组. 采用PCR法检测Y染色体性别决定基因Sry; 采用VG染色法观测肝组织胶原沉积面积; Western blot法检测肝组织α-平滑肌肌动蛋白(α-smooth muscleactin, α-SMA)表达; 采用碱水解法检测肝组织羟脯氨酸(hydroxyproline, Hyp)浓度; ELISA法检测血清透明质酸(HA)、层粘连蛋白(LN)和Ⅲ型前胶原(PCⅢ)等细胞外基质含量.结果: MASC法能成功分选纯化BDLSCs.IL-10修饰BDLSCs持续高浓度分泌IL-10蛋白至胞外, 移植后能顺利种植于肝内, 减轻胶原沉积, 减少α-SMA表达从而抑制肝星状细胞活化, 与BDLSCs组比较, BDLSCs/IL-10组大鼠肝组织Hyp水平显著降低(255.0±50.5 μg/gvs 373.0±26.7 μg/g, P<0.01), 血清HA, LN及PCⅢ水平也有所降低, 差异具有统计学意义(40.5±7.7 μg/L vs 79.4±10.3 μg/L, 61.5±16.4 μg/L vs 77.7±12.6 μg/L, 14.3±0.8 μg/Lvs 14.9±1.5 μg/L, P<0.01或0.05).结论: IL-10基因修饰BDLSCs移植能有效减少肝纤维化大鼠ECM的积聚, 为治疗肝纤维化提供了新思路.     

Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human.

Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.     

Integrin and extracellular matrix interactions regulate engraftment of transplanted hepatocytes in the rat liver

BACKGROUND & AIMS: Recognition and circumvention of the hepatic endothelial barrier is critical in the engraftment of transplanted cells. We examined whether interactions between integrin and extracellular matrix component receptors could be manipulated for improving transplanted cell engraftment and liver repopulation. METHODS: Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV-deficient rats. Coating of cells or of liver sinusoids with natural collagen, natural laminin, or an engineered fibronectin-like polymer was studied with analysis of cell engraftment and liver repopulation using histologic and molecular assays. Focal adhesion complexes were identified by vinculin immunostaining. The role of integrin receptors in cell engraftment was analyzed with RGD peptide inhibition assays. RESULTS: Coating of cells with extracellular matrix components before transplantation did not enhance cell engraftment. In contrast, intraportal infusion of collagen or fibronectin-like polymer in recipients prior to cell transplantation increased cell engraftment. Adherence of transplanted cells to the hepatic endothelium resulted in rapid activation of vinculin-containing focal adhesion complexes. Superior cell engraftment in animals treated with fibronectin-like polymer was RGD sensitive, verifying the integrin-dependent nature of this process. Moreover, studies in the retrorsine-partial hepatectomy rat model showed that intraportal infusion of the fibronectin-like polymer before cell transplantation significantly accelerated liver repopulation. CONCLUSIONS: Integrin-extracellular matrix component interactions can be manipulated for enhancing cell engraftment in the liver. Such mechanisms will be relevant for engraftment of other cell types and for strategies concerning liver-directed cell therapy.     

Collagen in the cellular and fibrotic stages of scleroderma.

The collagen in localized and systemic scleroderma skin was studied by light microscopy with silver impregnation (50 patients), electron microscopy (14 patients), and immunofluorescence microscopy using specific antibodies against Type I and Type III collagens (12 patients). In the cellular stage, the dermis and adipose tissue revealed perivascular or diffuse cellular infiltrates (mostly lymphocytes, plasma cells, and macrophages), accompanied by deposition of Type III collagen. The lower dermis also showed an increase in Type III collagen. In the fibrotic stage, the papillary layer showed a reduction and/or clumping of Type III collagen as compared to normal skin. The lower dermis and the adipose tissue revealed compact collagen consisting exclusively of Type I collagen or a mixture of Type I and Type III collagen. The pattern of Type III collagen distribution was similar to that of reticulin, thus suggesting that at least some reticulin fibrils may represent Type III collagen.     

Tyrosyl protein kinases in normal rat liver: identification and partial characterization.

Rat livers were fractionated and subcellular components were assayed for tyrosyl protein kinase activity. About 60% of the kinase activity in the cytoplasm sedimented with the microsomal fraction, whereas 40% remained in the supernatant. Purification of cytosolic and microsomal kinases by ion-exchange and gel filtration chromatography resolved a major species whose molecular mass was 75 kilodaltons (referred to as TPK 75) and a minor one whose molecular mass was greater than 160 kilodaltons. Partially purified TPK 75 phosphorylated a protein of the same molecular mass on tyrosine residues. The activity associated with TPK 75 was not stimulated by growth factors and was sensitive to thiol re-agents.