Perisinusoidal Stellate Cells Research Articles

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.

THREE-DIMENSIONAL STRUCTURE OF EXTRACELLULAR MATRIX REVERSIBLY REGULATES MORPHOLOGY, PROLIFERATION AND COLLAGEN METABOLISM OF PERISINUSOIDAL STELLATE CELLS (VITAMIN A-STORING CELLS)

The three-dimensional structure of the extracellular substratum was found to regulate reversibly the morphology, proliferation and collagen synthesis of perisinusoidal stellate cells (lipocytes, i.e. fat-storing ‘Ito’ cells). On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended their cellular processes. On the surface of type I collagen gels, the cells gathered and formed a mesh-like structure. However, in type I collagen gel where the cells were surrounded by type I collagen three-dimensionally, the cells extended their fine cellular processes and resembled the star-shaped stellate cells seen in vivo. The cell proliferation was more prominent in culture dishes coated with type I collagen or in polystyrene culture dishes than on or in type I collagen gels. The collagen synthesis was affected in the same manner. These data indicate that the nature and the three-dimensional structure of the extracellular matrix (ECM) can regulate morphology, proliferation and functions of the perisinusoidal stellate cells. In order to examine the reversibility of these regulations, we liberated cultured cells with trypsin or with purified bacterial collagenase and re-seeded them onto or into each substratum. The cells changed their shape, rate of proliferation and collagen synthesis according to each new substratum. These results indicate that the three-dimensional structure of ECM reversibly regulates the morphology, proliferation rate and functions of the perisinusoidal stellate cells.

Glial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast-like phenotypes

Glial fibrillary acidic protein (GFAP) has recently been shown to provide a marker for normal perisinusoidal stellate cells (PSC) in the liver. However, nothing was known so far about the changes in the intercellular abundancy of GFAP during transformation of PSC into myofibroblasts, a process characterized by marked changes in the expression of elements of the cytoskeleton. In order to address this question, we have used double-labelling immunofluorescence techniques for detecting smooth muscle alpha-actin (SMAA) and the intermediate filament proteins, GFAP, desmin and vimentin, taking advantage of the fact that PSC present in primary cultures of rat hepatocytes proliferate and transform. GFAP and vimentin were expressed in PSC throughout cultivation, while desmin which stained only about half of the PSC in early cultures was expressed in all GFAP-positive cells later on. The intensity of staining for GFAP in PSC transiently increased till the second day of cultivation followed by a decrease. At the 6th day of cultivation, staining for GFAP was seen only in the perinuclear region and as a faint rim at the contours of the cells. In contrast, SMAA, an established marker for transformed PSC, started to be expressed at the third day. Thereafter, immunoreactivity for SMAA increased continuously, indicating an inverse expression of GFAP and SMAA during transformation. These results indicate that GFAP, due to the plasticity of its expression, can discriminate between the normal untransformed and transformed phenotypes of PSC. Furthermore, they suggest a role of GFAP not only as a reliable marker, but also in the maintenance and/or function of the normal differentiated phenotype of liver PSC.

Glial fibrillary acidic protein as a marker of perisinusoidal stellate cells that can distinguish between the normal and myofibroblast‐like phenotypes

Glial fibrillary acidic protein (GFAP) has recently been shown to provide a marker for normal perisinusoidal stellate cells (PSC) in the liver. However, nothing was known so far about the changes in the intercellular abundancy of GFAP during transformation of PSC into myofibroblasts, a process characterized by marked changes in the expression of elements of the cytoskeleton. In order to address this question, we have used double-labelling immunofluorescence techniques for detecting smooth muscle alpha-actin (SMAA) and the intermediate filament proteins, GFAP, desmin and vimentin, taking advantage of the fact that PSC present in primary cultures of rat hepatocytes proliferate and transform. GFAP and vimentin were expressed in PSC throughout cultivation, while desmin which stained only about half of the PSC in early cultures was expressed in all GFAP-positive cells later on. The intensity of staining for GFAP in PSC transiently increased till the second day of cultivation followed by a decrease. At the 6th day of cultivation, staining for GFAP was seen only in the perinuclear region and as a faint rim at the contours of the cells. In contrast, SMAA, an established marker for transformed PSC, started to be expressed at the third day. Thereafter, immunoreactivity for SMAA increased continuously, indicating an inverse expression of GFAP and SMAA during transformation. These results indicate that GFAP, due to the plasticity of its expression, can discriminate between the normal untransformed and transformed phenotypes of PSC. Furthermore, they suggest a role of GFAP not only as a reliable marker, but also in the maintenance and/or function of the normal differentiated phenotype of liver PSC.

The Cellular Derivation and the Life Span of the Myofibroblast

The presence of myofibroblasts in granulation tissue and various fibrotic settings is well established. Recent work on this cell has shown that myofibroblasts derive mainly from local fibroblasts, but also from pericytes and smooth muscle cells as well as from specialized cells such as perisinusoidal stellate cells of the liver and mesangial cells of the kidney glomerulus. During the healing of an open wound, myofibroblasts disappear by means of apoptosis when the wound is closed and granulation tissue gradually transforms into scar tissue. The possibility exists that an altered regulation of this process leads to the development of a hypertrophic scar.

Expression of neural cell adhesion molecule (N-CAM) in perisinusoidal stellate cells of the human liver.

Neural cell adhesion molecule (N-CAM) is distributed in most nerve cells and some non-neural tissues. The present immunohistochemical study has revealed, for the first time, the expression of N-CAM in perisinusoidal stellate cells of the human liver. Liver specimens were stained with monoclonal antibody against human Leu19 (N-CAM) by a streptoavidin-biotin-peroxidase-complex method. Light- and electron-microscopic analyses have shown that N-CAM-positive nerve fibers are distributed in the periportal and intermediate zones of the liver lobule. Perisinusoidal stellate cells in these zones are also positive for N-CAM. N-CAM is expressed on the surface of the cell, including cytoplasmic projections. Close contact of N-CAM-positive nerve endings with N-CAM-positive stellate cells has been observed. On the other hand, stellate cells in the centrilobular zone exhibit weak or no reaction for N-CAM. Perivascular smooth muscle cells and fibroblasts in the portal area and myofibroblasts around the central veins are negative for N-CAM. The present results indicate that the perisinusoidal stellate cells in the periportal and intermediate zones of the liver lobule characteristically express N-CAM, unlike other related mesenchymal cells, and suggest that the intralobular heterogeneity of N-CAM expression by stellate cells is related to the different maturational stages of these cells.

Sodium butyrate reversibly blocks mouse hepatocytes early in G1 and inhibits the spontaneous expression of p53 and CDK4: [formula omitted] INSERM U-49, Hôpital Pontchaillou, Rennes 35033, France

Proliferating lipocytes (fat-storing cells or perisinusoidal stellate cells of the liver) were detected by in vivo incorporation of bromodeoxyuridine (BrdU) in an experimental model of cirrhosis in the rat by dimethylnitrosamine. Lipocytes were identified by sequential double immunohistochemical staining on frozen sections using anti-desmin antibodies as a marker of cytoplasmic intermediate filaments followed by anti-BrdU antibodies to identify S-phase nuclei in animals treated for 7, 14 or 21 days. The number of desmin-positive (lipocytes) and desmin-negative (Kupffer and endothelial cells) sinusoidal cells incorporating BrdU was recorded. The labelling index of lipocytes was calculated as the percentage of BrdU-labelled desmin-positive cells with respect to total number of lipocytes. In control animals, when the total number of lipocytes was 153.9 ± 11/mm2 (mean ± 1 S.E.) the number of desmin-posilive S-phase sinusoidal cells never exceeded 6.8 ± 1.2/mm2 with a maximum labelling index of 4.3 ± 0.5%. At 7 days of treatment, the values were respectively 236 ± 26.5/mm2, 53.2 ± 5.9/mm2 and 22.6 ± 0.5% (p < 0.001 vs. controls), while, at 21 days they were 272.5 ± 21.2/mm2, 23.3 ± 4.0/mm2 and 8.5 ± 1.1% respectively (p < 0.01). These results show that hyperplasia of lipocytes represents an early reaction to dimethylnitrosamine-induced liver injury. The local accumulation of lipocytes appear to occur in areas where fibrous septa develop later on.

Kupffer cell depletion shifts the balance of growth-modulatory cytokines to promote hepatocyte proliferation: [formula omitted] Dept. of Medicine, Johns Hopkins University, Baltimore, MD

Proliferating lipocytes (fat-storing cells or perisinusoidal stellate cells of the liver) were detected by in vivo incorporation of bromodeoxyuridine (BrdU) in an experimental model of cirrhosis in the rat by dimethylnitrosamine. Lipocytes were identified by sequential double immunohistochemical staining on frozen sections using anti-desmin antibodies as a marker of cytoplasmic intermediate filaments followed by anti-BrdU antibodies to identify S-phase nuclei in animals treated for 7, 14 or 21 days. The number of desmin-positive (lipocytes) and desmin-negative (Kupffer and endothelial cells) sinusoidal cells incorporating BrdU was recorded. The labelling index of lipocytes was calculated as the percentage of BrdU-labelled desmin-positive cells with respect to total number of lipocytes. In control animals, when the total number of lipocytes was 153.9 ± 11/mm2 (mean ± 1 S.E.) the number of desmin-posilive S-phase sinusoidal cells never exceeded 6.8 ± 1.2/mm2 with a maximum labelling index of 4.3 ± 0.5%. At 7 days of treatment, the values were respectively 236 ± 26.5/mm2, 53.2 ± 5.9/mm2 and 22.6 ± 0.5% (p < 0.001 vs. controls), while, at 21 days they were 272.5 ± 21.2/mm2, 23.3 ± 4.0/mm2 and 8.5 ± 1.1% respectively (p < 0.01). These results show that hyperplasia of lipocytes represents an early reaction to dimethylnitrosamine-induced liver injury. The local accumulation of lipocytes appear to occur in areas where fibrous septa develop later on.

Liver of juvenile Atlantic salmon, Salmo salar L.: a light, transmission, and scanning electron microscopic study, with special reference to the sinusoid.

This report provides a detailed description of sinusoidal and perisinusoidal structures in the normal liver of the juvenile Atlantic salmon (Salmo salar L.), a teleost species. The liver was studied by light, transmission, and scanning electron microscopy, and organ specimens were sampled after retrograde, whole-body perfusion through the dorsal aorta using 3% glutaraldehyde. Detailed characterization of perisinusoidal stellate cells also included immunohistochemical staining for desmin and evaluation of autofluorescence of the same cells upon excitation in ultraviolet (UV) light. The sinusoid is lined by one cell type only: the endothelial cell. No intraluminal pit cells or Kupffer cells are present. The space of Disse contains reticulin fibres, visualized by Gomori's silver stain, and perisinusoidal stellate cells (PSC). PSC exhibited autofluorescence in UV light, indicating that these cells store vitamin A in cytoplasmic lipid droplets. Immunohistochemically, PSC were found negative for desmin. The space of Disse, extending deep down between adjacent hepatocytes, receives long, slender microvilli from parenchymal cells. In addition to scattered macrophages, interhepatocytic cells (IHC) are found perisinusoidally. Hepatocytes of Atlantic salmon form branching and anastomosing tubules. The sinusoids of Atlantic salmon liver are lined by a fenestrated endothelium, with PSC located in the space of Disse, with macrophages and IHC as inhabitants of the interhepatocytic space. IHC show ultrastructural similarities to mammalian pit cells and teleostean large granular lymphocytes, as well as to piscine monocytes. PSC might be storage cells for vitamin A in Atlantic salmon as shown by autofluorescence in these cells, while immunohistochemical studies indicate that desmin does not seem to be an adequate immunohistochemical marker for PSC in the juvenile Atlantic salmon. Methodologically, fixation for electron microscopy was performed by a new and convenient perfusion method: arterial retrograde whole body perfusion. Liver specimens intended for scanning electron microscopy were fractured at room temperature after prolonged osmium postfixation, leaving hepatocytes intact and producing images well suited to document the three-dimensional structure of cells and tissue.

Extracellular Matrix Regulates and L-Ascorbic Acid 2-Phosphate Further Modulates Morphology, Proliferation, and Collagen Synthesis of Perisinusoidal Stellate Cells

Morphology, proliferation, and collagen synthesis of perisinusoidal stellate cells (fat-storing cells, lipocytes, Ito cells) varied according to the nature of the substratum. On a basement membrane gel, the stellate cells formed a mesh-like structure and proliferated slowly. On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended cellular processes. The cell proliferation and collagen synthesis were more prominent on culture dishes coated with type I collagen than on polystyrene dishes. On each extracellular substrate, proliferation and collagen synthesis were stimulated by a long-acting vitamin C derivative, L-ascorbic acid 2-phosphate. These results indicate that morphology, proliferation, and collagen metabolism of the stellate cells are regulated by extracellular matrix and modulated further by L-ascorbic acid 2-phosphate.

Capillarization and venularization of hepatic sinusoids in porcine serum-induced rat liver fibrosis: A mechanism to maintain liver blood flow

The processes of capillarization and venularization of sinusoids after porcine serum-induced rat liver fibrosis were studied by light and electron microscopy. Accompanying the development of fibrosis in the walls of central veins, most of the sinusoidal outlets collapsed, resulting in the formation of hepatic limiting plates around central veins. A few remaining sinusoids underwent capillarization (the development of a basal lamina and the defenestration of the sinusoidal endothelial cell), followed by venularization (the transformation into venules of sinusoids, characterized by the enlargement of the diameters with the lumina being lined with several endothelial cells, which lose fenestrae and develop a basal lamina). These newly formed venules served to maintain blood flow from sinusoids into central veins and thus have been designated the “outlet venules.” Diameters of these venules could reach about 25 μm. They were classified into two types: (a) the septal outlet venules, which developed inside the septa; and (b) the angular outlet venules, which drained blood directly from the parenchyma into the fibrotic central veins at the angles between two septa. Associated with venularization, perisinusoidal stellate cells (fat-storing cells or Ito cells) differentiated to myofibroblasts. (HEPATOLOGY 1993;18:1450–1458.)

Capillarization and venularization of hepatic sinusoids in porcine serum-induced rat liver fibrosis: A mechanism to maintain liver blood flow

The processes of capillarization and venularization of sinusoids after porcine serum-induced rat liver fibrosis were studied by light and electron microscopy. Accompanying the development of fibrosis in the walls of central veins, most of the sinusoidal outlets collapsed, resulting in the formation of hepatic limiting plates around central veins. A few remaining sinusoids underwent capillarization (the development of a basal lamina and the defenestration of the sinusoidal endothelial cell), followed by venularization (the transformation into venules of sinusoids, characterized by the enlargement of the diameters with the lumina being lined with several endothelial cells, which lose fenestrae and develop a basal lamina). These newly formed venules served to maintain blood flow from sinusoids into central veins and thus have been designated the "outlet venules." Diameters of these venules could reach about 25 microns. They were classified into two types: (a) the septal outlet venules, which developed inside the septa; and (b) the angular outlet venules, which drained blood directly from the parenchyma into the fibrotic central veins at the angles between two septa. Associated with venularization, perisinusoidal stellate cells (fat-storing cells or Ito cells) differentiated to myofibroblasts.

Granuloma formation in the liver of Balb/c mice intoxicated with carbon tetrachloride.

Hepatic granulomas induced by a single or several subcutaneous injections of carbon tetrachloride (CCl4) in Balb/c mice were examined electronmicroscopically and immunocytochemically. Stellate cells (fat-storing cells; lipocytes; Ito cells) were identified by the detection of cytoplasmic desmin, while T-lymphocytes and monocytes/macrophages were identified with monoclonal antibodies Thy 1.2 and MOMA-2, respectively. Following pericentral necrosis induced with CCl4, clear foci containing lymphocytes, monocytes, macrophages and perisinusoidal stellate cells occurred in the surrounding hepatic parenchyma on day 5. These clear foci developed to granulomas with increasing numbers of macrophages and stellate cells. Mitotic and apoptotic figures in randomly distributed macrophages, and direct contacts between macrophages and stellate cells were frequently seen within the granulomas. The stellate cells were characterized by a well-developed granular endoplasmic reticulum and Golgi complex. Collagen fibrils were closely applied to the stellate cells and connective tissue septa extended between neighboring granulomas and/or the pericentral necrotic areas after several injections of CCl4. CCl4-induced hepatic granulomas provide a model for investigating paracrine and/or autocrine modulation within a well-organized microenvironment during progressive hepatic inflammation and fibrosis.

Intralobular heterogeneity of perisinusoidal stellate cells in porcine liver.

The aim of the present investigation was to elucidate the intralobular heterogeneity of the perisinusoidal stellate cells (fat-storing cells, lipocytes) in the porcine liver. Their three-dimensional structure, desmin immunoreactivity and vitamin-A storage were studied by use of the Golgi silver, immunocytochemical and gold chloride methods. In order to locate the stellate cells, the hepatic lobules were divided into 10 zones. The stellate cells were readily identified in Golgi preparations by their striking dendritic appearance with branching processes encompassing the sinusoids. The stellate cells in the centrolobular zones were conspicuously dendritic with longer processes in comparison to those emitted by periportal elements. Such arborizations were studded with numerous thorn-like microprojections. Desmin immunoreaction in the periportal zones was stronger than that in the centrolobular zones. Vitamin-A storage in the stellate cells was well developed in zones 2-4, but reduced gradually toward the central region. The perisinusoidal stellate cells display marked heterogeneity in morphology and function based on their zonal location in the hepatic lobule.

Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells.

Rat liver stellate cells were cocultured with HepG2 human hepatoma cells, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was studied by cryoimmunoelectron microscopy. In stellate cells, human RBP was found on the cell surface and within endosomes. The transfer of human RBP from HepG2 cells to stellate cells was blocked by addition of RBP antibodies to the culture medium. Very little uptake of RBP was observed when fibroblasts were cocultured with HepG2 cells. In a series of experiments, RBP was bound to its putative cell surface receptor at 4 degrees C, and the stellate cells were washed and then incubated at 37 degrees C in order to allow them to internalize a pulse of RBP. About 50% of the RBP was internalized after 6 min of incubation. The RBP-positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membranes. After 2 hr, however, most RBP was localized in intracellular vesicles at a distance from the vesicular membranes, suggesting that RBP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with 125I-RBP at 4 degrees C and cell-associated radioactivity was determined. The calculated dissociation constant for the specific binding was 12.7 +/- 3.2 nM. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cells bound about 30 times more 125I-labeled RBP than did parenchymal cells (based on mass of cell protein). These data suggest that RBP mediates the paracrine transfer of retinol from hepatocytes to perisinusoidal stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis.

Vitamin A metabolism in rat liver: a kinetic model

Vitamin A metabolism in the liver involves both hepatocytes and the nonparenchymal perisinusoidal stellate cells. To describe and quantitate the dynamic relationships between retinol in these cells and in plasma, we administered either chylomicrons labeled with [3H]retinyl esters or plasma containing [3H]retinol-retinol-binding protein-transthyretin to rats. Radioactivity and retinol masses were measured in plasma, liver, and isolated hepatocytes for 15 days; data were analyzed by model-based compartmental analysis. The resulting model predicts that: 1) approximately 20% of the total plasma turnover of retinol goes to the liver (vs. nonhepatic tissues) and approximately 20% of plasma retinol input is from liver (vs. nonhepatic tissues), 2) about one-half of the retinol recycling from plasma to liver is taken up by hepatocytes and about one-half by nonparenchymal cells, 3) retinyl esters in both cell types are derived preferentially from newly taken up retinol rather than from the main intracellular retinol pools, and 4) at least one-half of the retinol secreted by hepatocytes of rats consuming low levels of vitamin A is directly transferred to nonparenchymal cells. In addition, the data are compatible with the hypothesis that retinol-binding protein is the vehicle for transfer of retinol from hepatocytes to nonparenchymal stellate cells and between plasma and liver cells.

Role of mesenchymal cell populations in porcine serum-induced rat liver fibrosis.

The role of liver mesenchymal cell populations in porcine serum-induced rat liver fibrosis were studied morphologically and immunohistochemically. Five-week-old rats were intraperitoneally injected with porcine serum twice a week and examined at various intervals between 3 and 24 wk after the initial injection. At an early phase, numbers of fibroblasts and extracellular matrix increased in the walls of central veins and in portal and capsular connective tissues. In the walls of central veins, the number of "second-layer cells" (i.e., the fibroblasts located at the second layer of the wall) increased. Connective tissue septa, accompanying some fibroblasts, extended from these interstitial tissues into the hepatic parenchyma, and their foremost edges came into direct contact with the perisinusoidal stellate cells. The sinusoids adjacent to the newly formed septa collapsed and later disappeared; this process resulted in the formation of hepatic limiting plates along the septa. At a more advanced stage, the interstitial fibroblasts and septal cells-which were derived from interstitial fibroblasts and the stellate cells-increased and became multilayered, constructing three-dimensional cell networks. These networks, together with increased collagen fibrils and elastic fibers, constitute the fibrotic dense connective tissue. In the control rat, smooth muscle cells were positive on vimentin, desmin and smooth muscle-alpha-actin staining. The stellate cells, second-layer cells, capsular and portal fibroblasts were shown to be vimentin and desmin positive and smooth muscle-alpha-actin negative. In the fibrotic liver, septal(fibroblastic) cells were vimentin and desmin positive and smooth muscle-alpha-actin negative. We conclude that not only the perisinusoidal stellate cells but also the interstitial fibroblasts, including the second-layer cells, play substantial role in the development of porcine serum-induced septal fibrosis in rat liver.

Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-induced cirrhosis: An immunohistochemical study

Proliferating lipocytes (fat-storing cells or perisinusoidal stellate cells of the liver) were detected by in vivo incorporation of bromodeoxyuridine (BrdU) in an experimental model of cirrhosis in the rat by dimethylnitrosamine. Lipocytes were identified by sequential double immunohistochemical staining on frozen sections using anti-desmin antibodies as a marker of cytoplasmic intermediate filaments followed by anti-BrdU antibodies to identify S-phase nuclei in animals treated for 7, 14 or 21 days. The number of desmin-positive (lipocytes) and desmin-negative (Kupffer and endothelial cells) sinusoidal cells incorporating BrdU was recorded. The labelling index of lipocytes was calculated as the percentage of BrdU-labelled desmin-positive cells with respect to total number of lipocytes. In control animals, when the total number of lipocytes was 153.9 ± 11/mm 2 (mean ± 1 S.E.) the number of desmin-posilive S-phase sinusoidal cells never exceeded 6.8 ± 1.2/mm 2 with a maximum labelling index of 4.3 ± 0.5%. At 7 days of treatment, the values were respectively 236 ± 26.5/mm 2, 53.2 ± 5.9/mm 2 and 22.6 ± 0.5% ( p < 0.001 vs. controls), while, at 21 days they were 272.5 ± 21.2/mm 2, 23.3 ± 4.0/mm 2 and 8.5 ± 1.1% respectively ( p < 0.01). These results show that hyperplasia of lipocytes represents an early reaction to dimethylnitrosamine-induced liver injury. The local accumulation of lipocytes appear to occur in areas where fibrous septa develop later on.

網内系学説と現在の肝類洞壁細胞像

The wall of hepatic sinusoids is a morphological and functional unit made up of four different cell populations, i.e., endothelial cells, Kupffer cells, perisinusoidal stellate cells (fat-storing cells), and lymphocytes including pit cells (liver-associated LGLs).The core element of this unit is the endothelial lining which provides a bed for other sinusoidal cells. Endothelial cells are highly fenestrated and sieve the fluids exchanged between the sinusoidal lumen and the space of Disse. Presence of numerous receptors on endothelial cells supports the receptor-mediated endocytosis of various substances. Adhesion molecules are expressed on the surface of these cells. Kupffer cells are resident macrophages, and clear endotoxin from the blood stream. When activated, Kupffer cells secrete metabolites of arachidonic acid, TNF, IL-1, IFN, and reactive oxygen products. Stellate cells are located perisinusoidally surrounding the sinusoidal tubes and, on the other hand, send thorn-like microprojections to parenchymal cells. Stellate cells store vitamin A and produce Type I, III and IV collagens and other extracellular matrix. Pit cells are characterized by the presence of dense granules and rod-cored vesicles. After administration of BRMs, number of pit cells increases adhering to the endothelial lining and liver-associated NK activity is augmented. Paracrine and autocrine secretions of mediators and cytokines from sinusoidal cells modulate functions of the sinusoidal wall and parenchymal cells.Currently the RES is restricted only to the system of macrophages. From the modern concept of sinusoidal cells in the liver, however, phagocytosis is only the trigger for starting the cascade of broad RES functions beyond the single cell line. The liver RES is not only Kupffer cells but the sinusoidal wall itself which provides a microenvironment between the blood stream and parenchymal cells. The RES is specifically organized tissue in the body, as suggested by early investigators.