Biomedical subjects

E Wisse

Publications and source records attributed to E Wisse.

At least 19 recordsLinked to original sources

PubMed1999

Pit cells (Hepatic natural killer cells) of the rat induce apoptosis in colon carcinoma cells by the perforin/granzyme pathway.

The high mortality of colon cancer is to a large extent caused by the frequent occurrence of liver metastasis. This is remarkable, because the liver harbors two distinct cell populations that can eliminate invading cancer cells, namely hepatic natural killer (NK) cells and Kupffer cells. These hepatic NK cells, known as pit cells, are the most cytotoxic cells of the naturally occurring NK cells. However, the mechanism by which pit cells eliminate tumor cells is largely unknown. Because we recently found an indication that apoptosis is involved, we tried to assess the role of this mode of cell death using an in vitro system with isolated pure pit cells (>90%) and CC531s cells, a rat colon carcinoma (CC) cell line. Pit cells induced apoptosis in CC531s cells as shown by quantitative DNA fragmentation, agarose gel electrophoresis, and different modes of microscopy. When extracellular Ca2+ was chelated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,-tetraacetic acid (EGTA) during coincubation or when the pit cells were preincubated with the granzyme inhibitor 3,4-dichloroisocoumarin (DCI), the induction of apoptosis was abolished. These results show that pit cells use the Ca2+-dependent perforin/granzyme pathway to induce apoptosis in the CC531s cells, and not the alternative Ca2+-independent Fas pathway. To further exclude the possibility of the involvement of the Fas pathway, we treated CC531s cells with recombinant Fas ligand. This treatment did not result in the induction of apoptosis, indicating that CC531s cells are resistant to Fas-mediated apoptosis. We conclude therefore that pit cells induce apoptosis in CC cells in vitro by the perforin/granzyme pathway.

Animals↗

PubMed1998

A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide.

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.

Actins↗

PubMed1998

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages.

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented. AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip-sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.

Adenocarcinoma↗

PubMed1997

AFM imaging and elasticity measurements on living rat liver macrophages.

The authors investigated the morphology and the elastic properties of living cultured rat liver macrophages (Kupffer cells) with an atomic force microscope (AFM). Continuous imaging and elasticity mapping of individual cells in physiological buffer was carried out for several hours without damaging the cells as judged by their persistent undisturbed morphology. Dynamic events such as protrusive activity were observed in time course. The importance of the cytoskeleton for the mechanical properties of the cell has been investigated by measuring the cell's elasticity as a function of position. Chemical disassembly of the actin network by applying 10 microgram/ml cytochalasin B decreased the cell's average elastic modulus seven-fold within less than 40 minutes. Treating the cells with 0.1 micrograms/ml latrunculin A resulted in a two-fold decrease in the elastic modulus merely in the perinuclear region after 40 minutes, whereas other parts of the cell were not affected.

Animals↗

PubMed1997

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.

Critical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.

Animals↗

PubMed1997

On the function of pit cells, the liver-specific natural killer cells.

Pit cells are liver-specific natural killer (NK) cells and belong to the group of sinusoidal cells, together with Kupffer, endothelial, and fat-storing cells. Pit cells are lymphoid cells containing specific granules, classifying them also as large granular lymphocytes (LGL). They probably originate from the bone marrow, circulate in the blood, and marginate in the liver, where they develop into pit cells by lowering their density and increasing the number of granules, which decrease in size. Pit cells remain in the liver about 2 weeks and are dependent on Kupffer cells. Pit cells also proliferate locally, when stimulated with interleukin-2, biological response modifiers, or other agents. Pit cells adhere to tumor target cells during killing. They possess a high level of natural cytotoxicity against a variety of tumor cell lines, which is comparable to the cytotoxicity level of lymphokine-activated killer (LAK) cells. Tumor cell killing is synergistically enhanced when pit cells attack tumor cells together with Kupffer cells. Further investigations are needed to clarify the mechanisms of pit cell cytotoxicity and the role of these cells in killing virus-infected cells, such as during viral hepatitis.

Animals↗

PubMed1996

Insulin-like growth factor II/mannose 6-phosphate-receptor expression in liver and serum during acute CCl4 intoxication in the rat.

The liver is reported to be the main source of soluble insulin-like growth factor-II/mannose 6-phosphate (IGF-II/M6P) receptor in adults. In view of the role of this receptor in the activation of transforming growth factor beta (TGF-beta) during hepatic fibrogenesis, we have investigated the correlation between serum levels and tissue expression of the receptor during acute CCl4 intoxication of the rat. Sixteen hours after CCl4, injection, the level of the soluble receptor in serum, as measured by radioimmunoassay (RIA), increased threefold. At 24 hours, values almost returned to normal, but increased again by twofold at 48 hours. By 96 hours, nearly normal values were obtained. Northern blot analysis showed peaks in tissue IGF-II/M6P receptor messenger RNA (mRNA) levels at 24 hours and at 48 hours. In normal liver, immunostaining for IGF-II/M6P receptor showed weak positivity in parenchymal cells. CCl4-induced hydropic changes appeared in centrilobular parenchymal cells (PCs) at 8 hours. These changes extended to the midzonal region at 16 hours. Hydropic cells were devoid of receptor staining. The hydropic wave became extinct at 32 hours. At 48 hours, we observed a collapse of PCs in the centrilobular zone, coinciding with strongly positive staining for IGF-II/M6P receptor in fat-storing cells (FSCs), identified by dual IGF-II/M6P receptor and desmin immunostaining. Between 48 and 72 hours, the liver gradually regained its normal appearance. As shown by Western blotting, in vitro differentiated FSCs released soluble receptor in the medium. Northern blot analysis showed this release to be preceded by an increased receptor-mRNA expression, whereas immunostaining showed an increase of intracellular receptor. In conclusion, we have shown that acute CCl4 intoxication induces two peaks in serum levels of soluble receptor. While the first peak at 16 hours coincides with the loss of receptor-staining in hydropically damaged PCs, the second peak at 48 hours is paralleled by an increase in positive staining in FSCs and tissue mRNA level. Differentiated FSCs shed soluble receptor in vitro. As a consequence, these cells might contribute to the serum levels of soluble receptor in vivo. These results indicate that measuring serum soluble IGF-II/M6P receptor might useful in the diagnosis of early acute liver damage.

Animals↗

PubMed1996

Comparison of glial fibrillary acidic protein and desmin staining in normal and CCl4-induced fibrotic rat livers.

Fat-storing cells are the major producers of extracellular matrix in the liver. A good immunocytochemical marker is, however, still lacking for this cell type. Desmin, frequently used by most investigators, fails to stain many pericentral fat-storing cells in normal rat liver. The aim of the present study is to evaluate glial fibrillary acidic protein (GFAP) as an alternative marker of fat-storing cells. In normal rat liver, immunostaining of GFAP revealed numerous fat-storing cells with characteristic cytoplasmic extensions. Unlike desmin, which was preferentially expressed in periportal fat-storing cells, GFAP-positive fat-storing cells were distributed more evenly in the lobules. In a narrow periportal zone, however, GFAP-positive cells were occasionally absent. Dual GFAP/desmin staining revealed colocalization of these markers, but fat-storing cells positive only for GFAP or desmin were also present. Chronic carbon tetrachloride exposure induced a spatial change in the expression of GFAP and desmin. At 3 weeks, accumulation of GFAP/desmin double-positive cells was observed in developing fibrotic septa. At 8 weeks, the GFAP positivity in the septa persisted but became weak, while desmin expression became stronger. In contrast, the expression of GFAP within the lobule was gradually decreased as fibrosis progressed. We conclude that GFAP is expressed by a subpopulation of fat-storing cells, which differs partially from the population that expresses desmin. Because in normal rat liver desmin-negative fat-storing cells can be identified by GFAP staining and vice versa, dual GFAP/desmin staining allows more complete identification of fat-storing cells. In chronically injured liver, GFAP may not be as useful as in normal rat liver. The coexpression of GFAP/desmin in developing septa and the subsequent downregulation of GFAP in an advanced stage of fibrosis may reflect different stages of fat-storing cell activation. Further investigation is required to determine the functional significance of alteration of GFAP expression in fat-storing cells.

Animals↗

PubMed1996

Dexamethasone alters messenger RNA levels but not synthesis of collagens, fibronectin, or laminin by cultured rat fat-storing cells.

Glucocorticoids have been shown to suppress collagen synthesis and gene expression by fibroblasts. However, little is known about their effects on fat-storing cells, the major matrix-producing cells in liver fibrosis. In this study we investigated the effect of dexamethasone on the extracellular matrix expression by cultured rat fat-storing cells. Fat-storing cells were isolated from male Wistar rats by collagenase/pronase digestion and purified by density gradient centrifugation. Fat-storing cells in early primary culture (3-day-old, representing a relatively quiescent phenotype) and in subculture (one passage, about 2-week-old, representing an activated phenotype) were treated with 10(-6) mol/L dexamethasone for messenger RNA (mRNA) study or with 10(-8) to 10(-6) mol/L dexamethasone for protein study. Expression of collagen type I, III, IV, fibronectin, and laminin was analyzed at the mRNA level by Northern hybridization, and at the protein level by metabolic labeling and immunoprecipitation. Dexamethasone had a variable effect on the expression of collagen alpha1(I) mRNA level. While a tendency for modest suppression was observed (5%-50%) in primary cells, the difference was not statistically significant. Variable response was observed in subcultured cells. Collagen alpha1(III) mRNA level showed a tendency for stimulation. Dexamethasone stimulated the expression of collagen alpha1 (IV), fibronectin, and laminin B1 mRNA levels by 1.4-, 2.4-, and 1.6-fold respectively, in primary fat-storing cells. Subcultured cells showed a similar response, but the magnitude of stimulation was more variable than that of primary cells. Unexpectedly, at the protein level dexamethasone had no effect on the expression of these proteins. Our results indicate that glucocorticoids do not possess a net suppressive effect on extracellular matrix synthesis by fat-storing cells. Beneficial effects of glucocorticoids may be attributable to other mechanisms of action, such as their anti-inflammatory effect.

Animals↗

PubMed1996

Centrolobular liver fibrosis in the hypercholesterolemic rabbit.

During a study on the development of atheromatous lesions in rabbits fed a diet with a low or high cholesterol supplement, we found a moderate to pronounced centrolobular liver fibrosis. This fibrosis developed in three stages. Early after supplementation of cholesterol, we observed increased immunoreactivity of collagen types I, III, and IV, and fibronectin, around central veins and in adjacent sinusoids. In the second stage, we observed further increase of collagen and fibronectin immunoreactivity, together with the appearance of alpha-smooth muscle actin (alpha-SM actin)-positive cells and anti-rabbit macrophage monoclonal antibody (RAM 11)-positive cells. In the third stage, we observed large numbers of alpha-SM actin-positive cells, together with heavy deposition of connective tissue proteins in pericentral sinusoids, in addition to focal atrophy of parenchymal cells. By transmission electron microscopy (TEM), fat-storing cells in the pericentral regions were shown to be enlarged, to lose their lipid-droplets, and to acquire dilated rough endoplasmic reticulum corresponding to an activated phenotype. Parenchymal cells were either normal or contained numerous small lipid-droplets. They sometimes were smaller and distorted. Northern hybridization performed on total RNA of whole liver showed an increased level of collagen alpha1(I), alpha1(III), and alpha1(IV) messenger RNA (mRNA) after 24 weeks of low dietary cholesterol supplementation. These data show enhanced expression of extracellular matrix proteins. We conclude that cholesterol overload induces pericentral liver fibrosis in rabbits. The diet clearly activates fat-storing cells to become fibrogenic effector cells. At present, we have no explanation why hypercholesterolemia induces phenotypic transition of fat-storing cells.

Actins↗

PubMed1996

The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver.

Previous studies showed that blood large granular lymphocytes (LGL), which possess natural killer (NK) activity, develop within rat liver sinusoids into high-density (HD) and subsequently into low-density (LD) pit cells which show an increasing level and spectrum of tumor cytotoxicity. In this study, we investigated the role of adhesion molecules, such as CD2, CD11a, CD18, and CD54 in the recruitment of pit cells to the liver. Immunostaining for electron microscopy, and two color flow cytometry showed that most pit cells expressed CD2, CD11a, CD18, and CD54. After intravenous injections into rats with anti-CD2, anti-CD11a, and anti-CD18 antibodies, the number of pit cells per square millimeter in frozen sections of liver tissue decreased. Treatment of rats with zymosan increased the number of pit cells fivefold, whereas subsequent treatment with anti-adhesion-molecule antibodies resulted in approximately 60% lower number of pit cells. Anti-CD54, supposed to block CD54 expression on sinusoidal endothelial cells, also decreased the number of pit cells. The number of blood LGL was, however, not affected by these antibodies. These results indicate that blocking of CD2, CD11a, CD18, and CD54 antigens on blood LGL and/or liver endothelium decreased the number of pit cells in the liver. These adhesion molecules therefore play an important role in the recruitment of pit cells in the liver.

Animals↗

PubMed1996

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Breast Neoplasms↗

PubMed1996

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro.

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparations techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (-15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

Animals↗

PubMed1996

Microfilament-disrupting agent latrunculin A induces and increased number of fenestrae in rat liver sinusoidal endothelial cells: comparison with cytochalasin B.

This report describes the effect of the actin-disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of liver endothelial cells (LECs). Fluorescence microscopy and whole mount-transmission electron microscopic preparations of isolated, purified, and cultured LECs showed that latrunculin A, which sequesters actin monomers and depolymerizes actin filaments, caused profound changes in microfilament organization in LECs. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 minutes, whereas the diameter is only slightly reduced. All new fenestrae possess the earlier described fenestrae-associated cytoskeleton ring. Cytochalasin B, which disrupts the network of actin filaments, principally by capping the fast growing end of actin filaments, produced comparable effects with regard to actin organization and the number and size of fenestrae. After 1 hour of treatment, an equal maximum number of fenestrae was observed for both agents. The effect of latrunculin A was obtained at concentrations about 100 times lower than cytochalasin B. Thus, two agents that alter the state of actin organization in LECs, albeit by different mechanisms, cause the doubling of the number of fenestrae within 10 to 30 minutes. This indicates that the state of assembly of the actin cytoskeleton is important in the numerical dynamics of LEC fenestrae and that the actin cytoskeleton of LECs is probably the main mechanical regulator for sieving between the sinusoidal blood and the parenchymal cells. Latrunculin A represents a new agent in the study of the de novo formation of fenestrae.

Actin Cytoskeleton↗

PubMed1996

Comparative scanning, transmission and atomic force microscopy of the microtubular cytoskeleton in fenestrated liver endothelial cells.

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.

Animals↗

PubMed1995

Insulinlike growth factor-II/mannose 6-phosphate receptor is expressed on CCl4-exposed rat fat-storing cells and facilitates activation of latent transforming growth factor-beta in cocultures with sinusoidal endothelial cells.

Transforming growth factor beta (TGF-beta), a potent fibrogenic cytokine, is secreted in latent form. We examined which cell type in both normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver bears surface type II IGF/mannose 6-phosphate (IGF-II/M6P) receptor, known to facilitate activation of TGF-beta. In addition, the role of the IGF-II/M6P receptor in activation of latent TGF-beta was investigated in a coculture system with sinusoidal endothelial cells. Northern hybridization analysis for IGF-II/M6P receptor messenger RNA (mRNA) was performed on total RNA of different isolated and purified liver cell types. In normal liver, cells expressed little IGF-II/M6P receptor mRNA. In fibrotic liver, we found significant expression only in fat-storing cells. The presence of IGF-II/M6P receptors was established by [125I]IGF-II binding assays on freshly isolated fat-storing cells from normal and CCl4-exposed rat livers. We found specific binding of [125I]IGF-II only on CCl4 exposed fat-storing cells. As determined by polyacrylamide gel electrophoresis after affinity labeling, the specific binding involved 220 kD type II IGF receptors. Scatchard analysis revealed the presence of 3,043 +/- 1,378 IGF-II/M6P high-affinity receptors/fat-storing cell, with a Kd of 387 = 165 pmol/L. With a mink lung epithelial cell (Mv1Lu) proliferation inhibition assay, inhibition of proliferation (a measure of active TGF-beta function) was determined using conditioned media of activated fat-storing cells, cocultures of fat-storing cells, and endothelial cells and pure endothelial cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Experimental biliary fibrosis correlates with increased numbers of fat-storing and Kupffer cells, and portal endotoxemia.

In the present study, we have investigated the correlation between hepatic fibrosis in rats subjected to bile duct ligation, the numbers of Kupffer and fat-storing cells, and the level of endotoxin in both the portal and systemic circulation. The extent of hepatic fibrosis was measured by morphometry. Kupffer cells were identified by indirect immunoperoxidase staining using ED-2 anti-macrophage antibody. Fat-storing cells were stained with DE-B-5 anti-desmin antibody. Endotoxin levels were determined by the Limulus Lysate test. Following bile duct ligation, connective tissue septa rapidly developed in periportal areas. After 1 week, the volume density of connective tissue had increased from 0.6 +/- 0.1% in control animals to 3.8 +/- 1.1%. After 2 weeks, this volume increased to 19.9 +/- 1.3%, and after 3 weeks to 34.3% +/- 2.7%. The number of periportal fat-storing cells increased 2.8-fold during the first 2 weeks, whereas pericentral fat-storing cells increased only 1.7-fold. After 2 weeks, no further increase was observed. During the first week of bile duct ligation, the number of Kupffer cells increased nearly two-fold. Thereafter, no further increase was detected. In control rats, only two of ten rats showed low amounts of endotoxin in the portal blood. Portal endotoxemia increased with time after bile duct ligation. After 3 weeks, all rats were positive. The measured endotoxin levels were approximately 7 times higher than in control rats. We conclude that the development of fibrosis secondary to experimental bile duct ligation is accompanied by protal endotoxemia, and increases in the numbers of Kupffer and periportal fat-storing cells. We found a significant correlation between portal endotoxemia, the number of Kupffer and fat-storing cells, and the extent of fibrous septa, supporting the view that high endotoxemia levels coincide with Kupffer cell activation and fibrogenesis.

Adipocytes↗

PubMed1995

New observations on cytoskeleton and fenestrae in isolated rat liver sinusoidal endothelial cells.

Fenestrae control the exchange of fluids, dissolved compounds and small particles between the blood and the space of Disse, and are primarily limited at one side by parenchymal cells. We recently described a simple and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells. With regard to the purity and morphology of liver endothelial cells, a detailed microscopic study was performed. Purity and viability after selective adherence was 74 and 95%, respectively. Liver endothelial cell purity was further enhanced to about 95% during adherence and spreading on collagen after 8 h of culture. Liver endothelial cells isolated by this method provide a viable cell population, enabling the study of structure and function of these cells in vitro. We investigated the cytoskeleton associated with fenestrae and sieve plates of liver endothelial cells. Cultured cells were slightly fixed and treated with cytoskeleton extraction buffer containing 0.1% Triton. Whole mounts of extracted liver endothelial cells were prepared for scanning and transmission electron microscopy. Extracted liver endothelial cells show an integral, intricate cytoskeleton. Sieve plates and fenestrae are clearly delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton ring with an average filament thickness of 16 nm. Additionally, sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. Microtubuli are sometimes found delineating linear arrangements of fenestrae. In conclusion, liver endothelial cells possess a cytoskeleton, that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton rings are involved in determining the size of fenestrae. The fenestrae-associated cytoskeleton therefore probably controls the important hepatic function of endothelial filtration.

Animals↗