scholarly journals Binding of hyaluronate and chondroitin sulphate to liver endothelial cells

1986 ◽  
Vol 234 (3) ◽  
pp. 653-658 ◽  
Author(s):  
T C Laurent ◽  
J R E Fraser ◽  
H Pertoft ◽  
B Smedsrød
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Chondroitin Sulphate ◽  
Mutual Exclusion ◽  
Degree Of Polymerization ◽  
Multiple Site ◽  
Large Molecules ◽  
Liver Endothelial Cell ◽  
Liver Endothelial Cells ◽  
Inhibition Studies

Hyaluronate is taken up and metabolized in liver endothelial cells by means of a receptor. To characterize the interaction with the receptor, two preparations of 3H-labelled hyaluronate, of Mr 4 × 10(5) and 6.4 × 10(6), and a series of hyaluronate oligosaccharides were bound to cultured liver endothelial cells at 7 degrees C. The dissociation constant varied between 4.6 × 10(-6) M for an octasaccharide and 9 × 10(-12) M for the largest polymer. The Mr-dependence for the series of oligosaccharides was explained by the increased probability of binding due to the repetitive sequence along the chain. The high affinity of high-Mr hyaluronate for the receptor could also be mainly ascribed to this effect, which rules out any major contribution of co-operative multiple-site attachment to the cell surface. Each liver endothelial cell can bind 10(5) oligosaccharides, about 10(4) molecules with Mr 4 × 10(5) and about 10(3) molecules with Mr 6.4 × 10(6). This is explained by mutual exclusion of large molecules from the cell surface. Chondroitin sulphate is also bound to liver endothelial cells. Inhibition studies showed that it binds to the same receptor as hyaluronate and with an affinity that is about 3-fold higher than that of hyaluronate of the same degree of polymerization.

scholarly journals Studies in vivo and in vitro on the uptake and degradation of soluble collagen α1(I) chains in rat liver endothelial and Kupffer cells

1985 ◽  
Vol 228 (2) ◽  
pp. 415-424 ◽  
Author(s):  
B Smedsrød ◽  
S Johansson ◽  
H Pertoft
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Kupffer Cell ◽  
Kupffer Cells ◽  
Cultured Cells ◽  
Chondroitin Sulphate ◽  
Liver Cells ◽  
Type I ◽  
Liver Endothelial Cell ◽  
Liver Endothelial Cells

Intravenously administered 125I-labelled monomeric alpha 1 chains (125I-alpha 1) of collagen type I were rapidly cleared and degraded by the liver of rats. Isolation of the liver cells after injection of the label revealed that the uptake per liver endothelial cell equalled the uptake per Kupffer cell, whereas the amount taken up per hepatocyte was negligible. The uptake of 125I-alpha 1 in cultured cells was 10 times higher per liver endothelial cell than per Kupffer cell. The ligand was efficiently degraded by cultures of both cell types. However, spent medium from cultures of Kupffer cells, unlike that from cultures of other cells, contained gelatinolytic activity which degraded 125I-alpha 1. The presence of hyaluronic acid, chondroitin sulphate or mannose/N-acetylglucosamine-terminal glycoproteins, which are endocytosed by the liver endothelial cells via specific receptors, did not interfere with binding, uptake or degradation of 125I-alpha 1 by these cells. Unlabelled alpha 1 and heat-denatured collagen inhibited the binding to a much greater extent than did native collagen. The presence of fibronectin or F(ab')2 fragments of anti-fibronectin antibodies did not affect the interaction of the liver endothelial cells, or of other types of liver cells, with 125I-alpha 1. The accumulation of fluorescein-labelled heat-denatured collagen in vesicles of cultured liver endothelial cells is evidence that the protein is internalized. Moreover, chloroquine, 5-dimethylaminonaphthalene-1-sulphonylcadaverine (dansylcadaverine), monensin and cytochalasin B, which impede one or more steps of the endocytic process, inhibited the uptake of 125I-alpha 1 by the liver endothelial cells. Leupeptin, an inhibitor of cathepsin B and ‘collagenolytic cathepsins’, inhibited the intralysosomal degradation of 125I-alpha 1, but had no effect on the rate of uptake of the ligand. The current data are interpreted as follows. (1) The ability of the liver endothelial cells and the Kupffer cells to sequester circulating 125I-alpha 1 efficiently may indicate a physiological pathway for the breakdown of connective-tissue collagen. (2) The liver endothelial cells express receptors that specifically recognize and mediate the endocytosis of collagen alpha 1(I) monomers. (3) The receptors also recognize denatured collagen (gelatin). (4) Fibronectin is not involved in the binding of alpha 1 to the receptors. (5) Degradation occurs intralysosomally by leupeptin-inhibitable cathepsins.

scholarly journals Degradation of Sulphated Glycosaminoglycans at the Surface of Cultured Human Endothelial Cells by a Platelet Heparitinase

1977 ◽  
Author(s):  
C. Busch ◽  
B. Glimelius ◽  
Å Wastesson ◽  
B. Westermark
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Coagulation Factors ◽  
Platelet Lysate ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Sulphated Glycosaminoglycans

The non-thrombogenic property of the endothelial cell surface is a prerequisite for maintainance of blood circulation. The nature of this property is poorly understood. Recent advances in culturing techniques of endothelial cells in vitro may facilitate studies of the surface biochemistry. Human endothelial cells (EC) isolated from umbilical veins were shown to synthesize and secrete sulphated glycosaminoglycans (GAG). The recent finding of a platelet enzyme capable of degrading heparin sulphate (HS) raised the question:Can platelet lysate or a purified heparitinase detach and degrade endothelial HS? EC cultured in the presence of 35S-sulphate, produce 35S-labelled GAG which was isolated from the incubation medium from a cell associated trypsin labile pool and from a cellular pool not liberated by trypsin. After 48 hours of incorporation about 95% of 35S-GAG was found in the medium fraction, 5% in the trypsin fraction and negligible amounts in the cell fraction. In the trypsin pool (“surface fraction”) heparin sulphate comprised about 85%, while the remaining 15% consisted of chondroitin sulphate and/or dermatan sulphate. Incubation of 35S-labelled EC with platelet lysate or a partially purified preparation of the enzyme from the same source caused a marked release of cell-surface associated HS to the incubation medium as oligosaccharides. These effects could be ascribed to heparitinase activity and may alter the properties of the EC-surface and influence the interaction between these cells on one hand and blood cells or plasma components, e.g., coagulation factors on the other.

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 Intracellular transport of formaldehyde-treated serum albumin in liver endothelial cells after uptake via scavenger receptors

1989 ◽  
Vol 258 (2) ◽  
pp. 511-520 ◽  
Author(s):  
W Eskild ◽  
G M Kindberg ◽  
B Smedsrød ◽  
R Blomhoff ◽  
K R Norum ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Serum Albumin ◽  
Intravenous Injection ◽  
Intracellular Transport ◽  
Liver Homogenate ◽  
Scavenger Receptors ◽  
Coated Pits ◽  
Mitochondrial Fractions ◽  
Liver Endothelial Cells

Endocytosis of formaldehyde-treated serum albumin (FSA) mediated by the scavenger receptor was studied in rat liver endothelial cells. Suspended cells had about 8000 receptors/cell, whereas cultured cells had about 19,000 receptors/cell. Kd was 10(-8) M in both systems. Cell-surface scavenger receptors were found exclusively in coated pits by electron microscopy, by using ligand labelled with colloidal gold. Cell-surface-bound FSA could be released by decreasing the pH to 6.0; it was therefore possible to assess the rate of internalization of surface-bound ligand. This rate was very high: t1/2 for internalization of ligand prebound at 4 degrees C was 24 s. The endocytic rate constant at 37 degrees C, Ke, measured as described by Wiley & Cunningham [(1982) J. Biol. Chem. 257, 4222-4229], was 2.44 min-1, corresponding to t1/2 = 12 s. Uptake of FSA at 37 degrees C after destruction of one cell-surface pool of receptors by Pronase was decreased to 60%. This finding is compatible with a relatively large intracellular pool of receptors. The intracellular handling of 125I-tyramine-cellobiose-labelled FSA (125I-TC-FSA) was studied by subcellular fractionation in sucrose gradients, Nycodenz gradients or by differential centrifugation. The density distributions of degraded and undegraded 125I-TC-FSA after fractionation of isolated non-parenchymal cells and whole liver were similar, when studied in Nycodenz and sucrose gradients, suggesting that the subcellular distribution of the ligand was not influenced by the huge excess of non-endothelial material in a whole liver homogenate. Fractionation in sucrose gradients showed that the ligand was sequentially associated with organelles banding at 1.14, 1.17 and 1.21 g/ml. At 9-12 min after intravenous injection the ligand was in a degradative compartment, as indicated by the accumulation of acid-soluble radioactivity at 1.21 g/ml. A rapid transfer of ligand to the lysosomes was also indicated by the finding that a substantial proportion of the ligand could be degraded by incubating mitochondrial fractions prepared 12 min after intravenous injection of the ligand. The results indicate that FSA is very rapidly internalized and transferred through an endosomal compartment to the lysosomes. The endosomes are gradually converted into lysosomes between 9 and 12 min after injection of FSA. The rate-limiting step in the intracellular handling of 125I-TC-FSA is the degradation in the lysosomes.

scholarly journals Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells

1990 ◽  
Vol 271 (2) ◽  
pp. 345-350 ◽  
Author(s):  
B Smedsrød ◽  
J Melkko ◽  
L Risteli ◽  
J Risteli
Keyword(s):  
Endothelial Cells ◽  
Parenchymal Cell ◽  
Mannose Receptor ◽  
Type I ◽  
Main Site ◽  
Type I Procollagen ◽  
Liver Endothelial Cell ◽  
Liver Endothelial Cells ◽  
Terminal Mannose

The fate of the circulating C-terminal propeptide of type I procollagen (PICP) was studied. Trace amounts of 125I-PICP administered intravenously to rats disappeared from the blood with an initial t1/2 of 6.1 min. After 45 min the radioactivity was distributed as follows: liver, 36%; blood, 23%; kidneys, 18%; urine, 20%; spleen, 1%; lungs, 2%; heart, 0.4%. To prevent escape of label from the site of uptake, PICP was labelled with 125I-tyramine cellobiose (125I-TC), which is trapped intralysosomally. With this ligand a serum t1/2 of 8.7 min was recorded, and 70% and 20% was traced in the liver and kidneys respectively. The uptake per liver endothelial cell (LEC) was 1000 times that per parenchymal cell and twice that per Kupffer cell. At 1 h and 6 h after addition of 125I-PICP to cultured LEC, 15% and 45% respectively, had been endocytosed. Only ligands for the mannose receptor could compete with PICP for endocytosis. To study whether the same specificity was operative in vivo, 125I-PICP was injected along with an excess of ovalbumin, which is known to be endocytosed by the mannose receptor of LEC. The serum t1/2 was prolonged from 6 to 16 min, signifying that terminal mannose residues are an important signal for clearance of PICP. In conclusion, these studies show that LEC constitute the main site of uptake of circulating PICP. The uptake is mediated by endocytic receptors which recognize terminal mannose residues.

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.

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