Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

1988 ◽  
Vol 253 (1) ◽  
Author(s):  
B�rd Smedsr�d ◽  
Marie Malmgren ◽  
Jan Ericsson ◽  
TorvardC. Laurent
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Chondroitin Sulphate ◽  
Sulphate Proteoglycan ◽  
Morphological Studies ◽  
Chondroitin Sulphate Proteoglycan ◽  
Liver Endothelial Cells

scholarly journals Endocytosis and degradation of chondroitin sulphate by liver endothelial cells

1985 ◽  
Vol 229 (1) ◽  
pp. 63-71 ◽  
Author(s):  
B Smedsrød ◽  
L Kjellén ◽  
H Pertoft
Keyword(s):  
Endothelial Cells ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Binding Studies ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Primary Monolayer ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells ◽  
Primary Monolayer Cultures

Intravenously administered chondroitin sulphate, chemically labelled by [3H]acetylation of partially deacetylated polysaccharide, was taken up and degraded by the non-parenchymal cells of the liver. Studies using primary monolayer cultures of pure Kupffer cells, liver endothelial cells and parenchymal cells revealed that [3H]chondroitin sulphate was taken up and degraded by the liver endothelial cells only. Binding studies at 4 degrees C with [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan indicated that the glycosaminoglycan and the proteoglycan are recognized by the same binding sites on the liver endothelial cells. The ability of hyaluronic acid to compete with the labelled ligands for binding suggested that the binding site is identical with the recently described hyaluronate receptor on the liver endothelial cells [Smedsrød, Pertoft, Eriksson, Fraser & Laurent (1984) Biochem. J. 223, 617-626]. Fluorescein-labelled chondroitin sulphate proteoglycan accumulated in perinuclear vesicles of the liver endothelial cells, indicating that the proteoglycan is internalized and transported to the lysosomes. The finding that [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan were degraded by the liver endothelial cells to low-molecular-mass radioactive products suggested that both the polysaccharide chain and the core protein were catabolized by the cells.

scholarly journals Endocytosis of hyaluronic acid by rat liver endothelial cells. Evidence for receptor recycling

1989 ◽  
Vol 257 (3) ◽  
pp. 875-884 ◽  
Author(s):  
C T McGary ◽  
R H Raja ◽  
P H Weigel
Keyword(s):  
Endothelial Cells ◽  
Hyaluronic Acid ◽  
Rat Liver ◽  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Receptor Recycling ◽  
Density Lipoprotein Receptor ◽  
Liver Endothelial Cells

Hyaluronic acid (HA) is cleared from the blood by liver endothelial cells through receptor-mediated endocytosis [Eriksson, Fraser, Laurent, Pertoft & Smedsrod (1983) Exp. Cell Res. 144, 223-238]. We have measured the capacity of cultured rat liver endothelial cells to endocytose and degrade 125I-HA (Mr approximately 44,000) at 37 degrees C. Endocytosis was linear for 3 h and then reached a plateau. The rate of endocytosis was concentration-dependent and reached a maximum of 250 molecules/s per cell. Endocytosis of 125I-HA was inhibited more than 92% by a 150-fold excess of non-radiolabelled HA. HA, chondroitin sulphate and heparin effectively competed for endocytosis of 125I-HA, whereas glucuronic acid, N-acetylglucosamine, DNA, RNA, polygalacturonic acid and dextran did not compete. In the absence of cycloheximide, endothelial cells processed 13 times more 125I-HA in 6 h than their total (cell-surface and intracellular) specific HA-binding capacity. This result was not due to degradation and rapid replacement of receptors, because, even in the presence of cycloheximide, these cells processed 6 times more HA than their total receptor content in 6 h. Also, in the presence of cycloheximide, no decrease in 125I-HA-binding capacity was seen in cells processing or not processing HA for 6 h, indicating that receptors are not degraded after the endocytosis of HA. During endocytosis of HA at 37 degrees C, at least 65% of the intracellular HA receptors became occupied with HA within 30 min. This indicates that the intracellular HA receptors (75% of the total) function during continuous endocytosis. Hyperosmolarity inhibits endocytosis and receptor recycling in the asialoglycoprotein and low-density-lipoprotein receptor systems by disrupting the coated-pit pathway [Heuser & Anderson (1987) J. Cell Biol. 105, 230a; Oka & Weigel (1988) J. Cell. Biochem. 36, 169-183]. Hyperosmolarity inhibited 125I-HA endocytosis in liver endothelial cells by more than 90%, suggesting use of a coated-pit pathway by this HA receptor. We conclude that liver endothelial cell HA receptors are recycled during the continuous endocytosis and processing of HA.

scholarly journals Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate

1990 ◽  
Vol 265 (1) ◽  
pp. 61-68 ◽  
Author(s):  
H Morita ◽  
T Takeuchi ◽  
S Suzuki ◽  
K Maeda ◽  
K Yamada ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans ◽  
Cell Layers ◽  
Aortic Endothelial

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.

scholarly journals Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1538 ◽  
Author(s):  
Marta Mellai ◽  
Laura Annovazzi ◽  
Ilaria Bisogno ◽  
Cristiano Corona ◽  
Paola Crociara ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Chondroitin Sulphate ◽  
Molecular Genetic ◽  
Malignant Gliomas ◽  
Malignant Cell ◽  
Wild Type ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Proteoglycan 4

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

HDL from type III dysbetalipoproteinaemic patients show decreased capacity to inhibit VLDL uptake in rat liver endothelial cells

1986 ◽  
Vol 62 (2) ◽  
pp. 145-149
Author(s):  
S GUSTAFSON ◽  
B VESSBY ◽  
A OSTLUNDLINDGVIST ◽  
C EHNHOLM
Keyword(s):  
Endothelial Cells ◽  
Rat Liver ◽  
Type Iii ◽  
Liver Endothelial Cells

Tissue Plasminogen Activator is Endocytosed by Mannose and Galactose Receptors of Rat Liver Cells

1988 ◽  
Vol 59 (03) ◽  
pp. 480-484 ◽  
Author(s):  
Bård Smedsrød ◽  
Monica Einarsson ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Side Chains ◽  
Diisopropyl Fluorophosphate ◽  
Tissue Plasminogen ◽  
Rat Liver Cells ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryExperiments were carried out to charact erize the specificity of uptake of tPA in rat liver cells. Endocytosis in liver endothelial cells of the native carbohydrate variants of tissue plasminogen activator (tPA), and tPA inactivated by diisopropyl fluorophosphate was found to be competitive, suggesting that the determinant being recognized by these cells is different from the active site. Fibronectin and urokinase, which show partial homology with tPA, did not compete with tPA for uptake in liver endothelial cells. Hyaluronic acid, collagen, or IgG, which are endocytosed by specific receptors in liver endothelial cells, did not interfere with the uptake.Reduced endocytosis by liver endothelial cells was observed with tPA modified in the carbohydrate side chains, suggesting that these structures are important for uptake. Ovalbumin, mannan, mannose, fructose, and EDTA, but not galactose, effectively inhibited uptake in liver endothelial cells of both native and diisopropyl fluorophosphate-inhibited tPA, but had very little effect on the uptake of tPA modified in the carbohydrate side chains.Endocytosis of native tPA by parenchymal cells could be inhibited by galactose, ovalbumin, and EDTA, but not by mannose.These results suggest that endocytosis of tPA by liver endothelial cells and parenchymal cells is mediated by the mannose and galactose receptors, respectively.

Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

scholarly journals Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns

1986 ◽  
Vol 235 (2) ◽  
pp. 469-479 ◽  
Author(s):  
B C Wightman ◽  
E A Weltman ◽  
L A Culp
Keyword(s):  
Extracellular Matrix ◽  
Affinity Chromatography ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Heparan Sulphate Proteoglycan ◽  
3T3 Cells ◽  
Platelet Factor ◽  
Sulphate Proteoglycan ◽  
Adhesion Sites ◽  
Chondroitin Sulphate Proteoglycan

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.

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