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.     

Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection. Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.     

Immunolocalization of laminin in normal rat liver and biosynthesis of laminin by hepatic lipocytes in primary culture

Laminin, a glycoprotein with a molecular weight of approximately 850,000 daltons, is a major constituent of most epithelial basement membranes. Its presence in the extracellular matrix of normal liver, however, is debated. Using two affinity-purified antibodies directed against laminin, we have localized the glycoprotein within normal rat liver and identified its cellular source. Immunofluorescent staining of rat liver sections revealed laminin in a continuous distribution around hepatic sinusoids, adjacent to hepatocytes and sinusoidal lining cells. To determine the cellular origin of laminin, three perisinusoidal cell populations (hepatocytes, sinusoidal endothelial cells, and lipocytes) were purified from enzymatically dispersed rat liver and were established in primary culture. By immunofluorescence, laminin was associated almost exclusively with lipocytes. Synthesis of laminin was demonstrated by immunoprecipitation of the protein from lipocyte culture medium pulse-labeled with radioactive methionine. These results show that in adult liver, laminin is present in the perisinusoidal matrix and is produced by hepatic lipocytes. Lipocytes, which have the capacity to produce collagen as well as laminin, may be the principal source of extracellular matrix proteins in the perisinusoidal space, and may contribute to subendothelial fibrosis resulting from liver injury.     

Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1.

The endothelins (ETs) form a group of three vasoactive peptides (ET-1, ET-2, and ET-3) for which two types of cellular receptors have been identified, types A and B ET receptors (ETA and ETB receptors, respectively). To address possible targets for ETs within the liver, we isolated the four principal liver cell populations and placed them in short-term primary culture. By ligand-binding assay and mRNA levels, expression of ET receptors was greatest on hepatic lipocytes (Ito cells or fat-storing cells), which are perisinusoidal cells exhibiting features of smooth muscle cells. Moreover, lipocytes expressed both ETA and ETB receptors. The mRNA for ETB receptor, but not for ETA receptor, was detectable in sinusoidal endothelial cells and Kupffer cells; neither mRNA was detectable in hepatocytes. Both ET-1 and ET-3 elicited contraction of activated lipocytes cultured on collagen lattices; the EC50 value for ET-1 was 3 +/- 1 nM and for ET-3 was 17 +/- 12 nM. In cell isolates from injured liver (after administration of carbon tetrachloride), expression of ET receptors was unchanged. However, mRNA for ET-1 was significantly increased in activated lipocytes, suggesting an autocrine loop for the initiation of lipocyte contraction. The findings imply that ET-1 may play a role in regulating sinusoidal perfusion through its effect on lipocytes, particularly in injury states.     

Alcoholic liver injury in baboons: transformation of lipocytes to transitional cells

Ultrastructure of the lipocytes (the main perisinusoidal cells) and their alterations in the progression of hepatic fibrosis were studied in liver biopsy specimens of baboons pair-fed with diets containing alcohol, or isocaloric carbohydrate, for up to 112 mo. In control baboons, 97% of the cells in the Disse space were lipocytes characterized by a volume density of lipid droplets occupying greater than 20% of the cell volume and by the presence of microfilament bundles with associated dense bodies and pinocytic vesicles. Intercellular junctions of the adherens type were present between lipocytes and hepatocytes. After alcohol consumption, the number of lipocytes (as assessed by light microscopy) was significantly decreased in fatty livers and at various stages of hepatic fibrosis; this was associated with a decreased hepatic vitamin A content. In baboons fed alcohol, only 48% of cells were lipocytes, whereas 52% were transitional cells defined by a volume of lipid droplets less than 20% of the cell. Like the lipocytes, transitional cells exhibited microfilament bundles, dense bodies, and pinocytic vesicles, and were attached to the hepatocytes by cell junctions. The rough endoplasmic reticulum in transitional cells was conspicuous and had an area significantly greater than that in lipocytes of controls and alcohol-fed animals (69% and 37%, respectively). There was a significant correlation between the percentage of transitional cells as well as the area of their rough endoplasmic reticulum and the degree of hepatic fibrosis. Thus, in baboons fed alcohol, the progression of hepatic fibrosis is associated with transformation of lipocytes to transitional cells characterized by a depletion of lipid droplets and a hypertrophy of the rough endoplasmic reticulum; these transitional cells may play a role in promoting hepatic fibrosis in alcoholic liver injury.     

Hepatic blood flow regulation by stellate cells in normal and injured liver

Hepatic stellate cells have received considerable attention as key components of the fibrogenic response to injury. Beyond this feature, they also have been implicated as regulators of sinusoidal vascular tone, and in disease states, in the pathogenesis of intrahepatic portal hypertension. The basis for this latter concept is derived from the following: (a) stellate cells are situated in a perisinusoidal orientation within the sinusoid, optimized for sinusoidal constriction; (b) a series of studies performed over the past decade have demonstrated that perisinusoidal stellate cells exhibit a remarkable capacity for cellular contraction, a characteristic that is most prominent after liver injury and stellate cell activation; and (c) in vivo microscopy studies have revealed that stellate cells can mediate sinusoidal constriction. Available evidence indicates that liver injury leads to a vascular disorder in which endothelin-1 is overproduced by stellate cells and endothelial cell-derived nitric oxide production is reduced. These abnormalities, in the context of exaggerated stellate cell contractility after liver injury, set up a paradigm in which stellate cells contribute to the increased intrahepatic resistance typical of portal hypertension. Furthermore, because stellate cell contractility and the mediators that control this function are dynamic processes, strategies that target exaggerated contractility provide an opportunity for novel therapeutics in intrahepatic portal hypertension.     

Effects of ethanol and hepatic vitamin A on the proliferation of lipocytes in regenerating rat liver

The effects of ethanol and hepatic vitamin A on lipocyte proliferation were studied in regenerating rat livers. Positive identification of proliferating lipocytes was achieved by a double immunostaining sequence for cytoplasmic desmin and bromodeoxyuridine-labelled nuclei. Ethanol administration caused a 76 and 36% reduction in labelling indices of lipocytes 2 and 3 days after hepatectomy, respectively, thereby delaying the onset of the proliferative peak of lipocytes by 1 day compared to the control. Hepatic vitamin A did not decrease until 4 days after hepatectomy. The relationship between hepatic vitamin A and lipocyte proliferation was further evaluated in rats fed diets containing four different amounts of vitamin A. Despite the striking differences in liver vitamin A levels, the proliferative activity of lipocytes was unaffected, as measured at the peak of lipocyte proliferation 2 days after hepatectomy. These data indicate that ethanol ingestion suppresses the proliferative response of lipocytes to partial hepatectomy but that this ethanol effect is independent of changes of vitamin A levels in the liver.     

Hepatic stellate cells and alcoholic liver disease.

Liver fibrosis represents a significant health problem worldwide for which no effective therapy exists. A great deal of research has been carried out to understand the molecular mechanisms responsible for the development of liver fibrosis. Activated stellate cells are the primary cell type responsible for the production of collagen I, the key protein involved in the development of liver fibrosis. Excessive deposition of collagen I occurs along with impaired extracellular matrix remodeling. Following a fibrogenic stimulus stellate cells transform into an activated collagen type I-producing cell. Numerous changes in gene expression are associated with stellate cell activation, including the induction of several intracellular signaling cascades, which help maintain the activated phenotype and control the fibrogenic and proliferative state of the cell. Activation of stellate cells is mediated by factors released from hepatocytes and Kupffer cells as they produce reactive oxygen species, nitric oxide, cytokines, growth factors, and cyclooxygenase and lipoxygenase metabolites, which provide pivotal paracrine effects in the liver milieu. Inhibition of stellate cell activation, proliferation, and the increased production of extracellular matrix (i.e. collagen type I) are therefore crucial steps for intervention in hepatic fibrogenesis.     

Hepatic venography of normal and regenerating dog liver

Total and selective hepatic vein angiographies were performed preoperatively and 6 weeks after 70 per cent liver resection in 6 dogs. The alterations in the hepatic vein system, increased diameter and elongation of the remaining lobar veins and their major tributaries plus increased angle between the radicles, are consistent with the findings earlier described by angiography of the hepatic arterial and portal venous system after major hepatic resection in dogs.     

Isolation and characterization of biliary epithelial cells from normal rat liver

A cell fraction enriched in biliary epithelial cells (BEC) has been isolated from the liver of normal rats. The procedure involved proteolytic digestion by trypsin and mild mechanical disruption of biliary ductular and connective tissue that remained undigested after collagenase-hyaluronidase perfusion. An adherence procedure removed the large majority of contaminating Kupffer cells. The majority (87.4 +/- 3.5%) of the cells were positive to an indirect immunofluorescence staining that used an antiserum against bovine hoof prekeratin that specifically recognizes intermediate filaments of biliary epithelium. Similar results were obtained by histochemical staining for gamma-glutamyltranspeptidase activity. The contamination of the BEC fraction with Kupffer cells and hepatocytes was approximately 7% and 2%, respectively. The viability of the BEC population was always more than 90%. The BEC and hepatocytes were analysed for their lipid composition; the BEC were found to have a cholesterol content approximately 6-times higher than hepatic parenchymal cells, with a cholesterol/phospholipid molar ratio of 0.53 in comparison to a value of 0.11 for hepatocytes. No detectable evidence of cytochrome P-450 or cytochrome P-450-related enzymatic activities was found in the BEC.     

Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts. Increased proportion of high collagen-producing cells in systemic sclerosis fibroblasts

Objective. The goal of this study was to quantitatively analyze the distribution of collagen synthesis in normal and systemic sclerosis (SSc) fibroblast populations in order to determine the extent of activation in SSc populations. Methods. We used quantitative in situ hybridization to assess the population distribution of type I collagen synthesis. Fibroblast cultures were derived from both clinically involved and uninvolved skin regions of patients with SSc, and from healthy adults, and assessed for levels of α1(I) procollagen messenger RNA (mRNA). Results. Dermal fibroblasts from both patients with SSc and normal adults were heterogeneous for distribution of α1(I) procollagen mRNA when assessed by in situ hybridization, with a wide range of grains per cell. In contrast, clones of neonatal fibroblasts showed a relatively homogeneous distribution of grain counts. Involved SSc skin fibroblasts had a larger proportion of cells in the high collagen-producing mRNA subpopulation (mean ± SEM 28.4 ± 6.85%), compared with normal fibroblasts (1.75 ± 1.44%) and uninvolved fibroblasts (9.6 ± 6.73%). Conversely, within the low collagen-producing mRNA subpopulation, involved fibroblasts had a smaller proportion of cells (mean ± SEM 14.0 ± 5.63%) than did uninvolved fibroblasts (37.8 ± 13.69%), while normal fibroblasts had a majority of the cells in this subpopulation (53.5 ± 8.68%). Conclusion. These results suggest that only a specific subset of fibroblasts are activated in SSc, as evidenced by an increased proportion of cells with high levels of α1(I) procollagen mRNA. Differences between the SSc and normal fibroblast populations appeared to be quantitative rather than qualitative. This may be a result of either clonal selection or selective activation in the pathogenesis of SSc.     

Hepatic stem cells and liver repopulation

Research on hepatic stem cells has entered a new era of controversy, excitement, and great expectations. Although adult liver stem cells have not yet been isolated, an enormous repopulating capacity of transplanted mature hepatocytes under conditions of continuous liver injury has been discovered. Stem/progenitor cells from fetal liver have been successfully isolated and transplanted, repopulating up to 10% of normal liver. However, progenitor cell lines from adult and embryonic liver have not shown significant repopulating activity. Intensive research on embryonic stem cells has revealed the first promising attempts to use these cells as a source of hepatic progenitors. Conditions for their differentiation in vitro, isolation of purified hepatic progenitor cells, and liver repopulation are currently being evaluated. Multilineage adult progenitor cells of mesenchymal origin from bone marrow, muscle, and brain may turn out to be the long-sought primitive potential stem cells remaining in adult tissues.     

肝干细胞与肝病

在较长时间内,有关肝干细胞的研究主要集中在基础方面,但近年研究表明它与肝脏多种病理生理过程密切相关,并展示了良好的应用前景。现将有关研究进展综述安等。     

Vitamin A-containing lipocytes and formation of type III collagen in liver injury.

Hepatocellular necrosis in carbon tetracholride-induced injury of rats is associated with an accumulation of lipocytes (perisinusoidal cells or Ito cells) containing fat droplets and giving vitamin A fluorescence. In the subsequent formation of connective tissue septa, transitional cells having morphologic characteristics of lipocytes and fibroblasts are abundant and are associated with the appearance of type III collage-. The features suggest that the lipocyte is the precursor of the fibroblasts responsible for parenchymal fibrillogenesis and under these conditions forms type III collagen. The process is a postulated link between hepatocellular necrosis and fibrosis.     

Hepatic stellate cells: a target for the treatment of liver fibrosis

Hepatic fibrosis is a wound-healing process that occurs when the liver is injured chronically. Hepatic stellate cells (HSC) are responsible for the excess production of extracellular matrix (ECM) components. The activation of HSC, a key issue in the pathogenesis of hepatic fibrosis, is mediated by various cytokines and reactive oxygen species released from the damaged hepatocytes and activated Kupffer cells. Therefore, inhibition of HSC activation and its related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. This is especially true when the etiology is unknown or there is no established therapy for the cause of the chronic injury. This review explores the rationale for choosing HSC as a target for the pharmacological, molecular, and other novel therapeutics for hepatic fibrosis. One focus of this review is the inhibition of two cytokines, transforming growth factor-β and platelet-derived growth factor, which are important in hepatic fibrogenesis. A number of new agents, such as Chinese herbal recipes and herbal extracts, silymarin, S-adenosyl-l-methionine, polyenylphosphatidylcholine, and pentoxifylline are also discussed. Received: April 3, 2000 / Accepted: April 28, 2000     

Hepatic clearance of rat liver aspartate aminotransferase isozymes: evidence for endocytotic uptake via different binding sites on sinusoidal liver cells

Rat liver aspartate aminotransferase (AAT) (EC 2.6.1.1) exists in two isozymic forms, cytosolic (c-AAT) and mitochondrial (m-AAT). The previous study (Kamimoto, Y. et al., Hepatology an accompanying paper in this issue) demonstrated that these isozymes were cleared from blood at different half-lives via adsorptive endocytosis by sinusoidal liver cells. To understand the cellular mechanism for the differential uptake of the isozymes, we have further studied in vivo uptake of 125I-labeled AAT isozymes by sinusoidal cells. The results indicated that the uptake of the isozymes occurred via a typical endocytotic pathway: the initial binding, internalization and subsequent degradation in the lysosomes. Quantitation of the isozymes bound to the cell surface prior to internalization either by binding at 4 degrees C or by a combined use of anti-AAT antibody and 125I-protein A at 37 degrees C revealed that the differential plasma clearance of AAT isozymes could be attributable to the isozymic difference in capacity of surface membranes to bind the isozymes. The uptake of 125I-c-AAT was inhibited by unlabeled c-AAT more significantly than by m-AAT, but not by other ligands known to be taken up by sinusoidal cells via receptor-mediated pathways. Similarly, the uptake of 125I-m-AAT was inhibited predominantly by unlabeled m-AAT. Similar ligand specificity was also demonstrated in the binding study at 4 degrees C. The binding of 125I-m-AAT and c-AAT followed saturation kinetics with an apparent Kd of 1.3 X 10(-6) M and 1.7 X 10(-6) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic stellate cells: it's role in normal and pathological conditions

Hepatic fibrosis is a dynamic and sophisticatedly regulated wound healing response to chronic hepatocellular injury. This fibrotic process results from the accumulation of extracellular matrix (ECM) including collagen, proteoglycan, and adhesive glycoproteins which are principally produced by hepatic stellate cells (HSC), a mesenchymal cell type located between parenchymal cell plates and sinusoidal endothelial cells in the space of Disse. In physiological conditions, quiescent HSCs play important roles in the regulation of retinoid homeostasis and ECM remodeling by producing ECM components as well as metalloproteases and its inhibitor. However during hepatic fibrogenesis, HSCs are known to be activated or "transdifferentiated" to myofibroblast-like cells which play a pivotal role in ECM remodeling and hepatic blood flow regulation. Activation of HSC is now well established as the key process involved in the development of hepatic fibrosis. Both basic morphology and functions of HSCs in normal conditions and its role in pathological fibrosis will be discussed in this review.