Studies in vivo and in vitro on the uptake and degradation of soluble collagen alpha 1(I) chains in rat liver endothelial and Kupffer 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.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

Binding of hyaluronate and chondroitin sulphate to liver endothelial cells.

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 X 10(5) and 6.4 X 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 X 10(-6) M for an octasaccharide and 9 X 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 X 10(5) and about 10(3) molecules with Mr 6.4 X 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.     

几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响

目的：比较几丁聚糖和透明质酸钠对血管内皮细胞增殖的影响。方法：用含不同浓度的几丁聚糖和透明质酸钠的培养液对血管内皮细胞（EV304）进行培养,以四唑盐比色法测细胞增殖,并用流式细胞仪测定细胞周期。结果：几丁聚糖在≥0.1mg/ml时促进血管内皮细胞的增殖,透明质酸钠对血管内皮细胞的增殖有抑制作用,几丁聚糖可使细胞周期中G1期比例下降,而透明质酸钠使细胞周期中G1期比例上升。结论：几丁聚糖促进血管内皮细胞的增殖,而透明质酸钠对血管内皮细胞的增殖有抑制作用。     

Intracellular degradation by liver endothelial cells

Investigations were carried out on the intracellular fate of formaldehyde treated bovine serum albumin (F-BSA), in liver non-parenchymal cells. This paper reports the observations and results obtained by us. The first part of our work involved the injecting of the compound into either a) normal rats, b) rats injected with Triton WR 1339 or c) rats treated with mannan. Fractions obtained after differential and isopycnic centrifugation in sucrose gradients, were analysed by SDS-gel electrophoresis and fluorography. The degradation takes place in a two step process. The molecule is first split into radiolabeled compounds that are still acid precipitable. This is followed by the appearance of acid soluble radioactive molecules. In a sucrose gradient the first kind of degradation products exhibit a distribution totally different from that of acid soluble degradation compounds. In the second part of our experiments, fairly pure fractions of the organelles, known to be involved in the endocytic pathway i.e. endosomes, transfer lysosomes and accumulation lysosomes (marked by the presence of either Triton WR 1339 or mannan) were isolated and incubated with [¹²⁵I]-F-BSA. These experiments revealed that endosomes, isolated by us, are incapable of degradation. Accumulation lysosomes arising exclusively from liver non-parenchymal cells (in which mannan had accumulated) though rich in certain hydrolases eg. arylsulfatase did not have an efficient proteolytic machinery. Our results, both fromin vivo andin vitro studies, suggest that the first degradation step occurs in one type of structure (probably not endosomes), a sort of hybrid endosome-lysosome (as they are not affected by glycyl-l-phenyl-2-napthylamide [1]) and the second step in a different type of lysosomes, what we have designated transfer lysosomes.     

Uptake and degradation of human very-low-density lipoproteins by rat liver endothelial cells in culture

Rat liver endothelial cells in primary cultures take up and degrade 125I-labelled human very-low-density lipoproteins (VLDL) in a saturable fashion at physiological triacylglycerol concentrations. The iodinated VLDL are readily taken up by the freshly isolated endothelial cells and degradation products appear in the medium about 30 min after the addition of VLDL to the cultures. Uptake and degradation at 37 degrees C are effectively inhibited by unlabelled human VLDL, low-density lipoproteins (LDL), high-density lipoproteins and lymph chylomicrons, but only modestly by acetylated LDL. Purified apolipoproteins E and C-III:1 also compete with the uptake of iodinated VLDL, but when degradation was studied for longer periods of time, such a competition could not be demonstrated. This may be due to the fact that the added apolipoproteins become associated with the lipoproteins. In binding experiments at 7 degrees C, iodinated apolipoprotein C III:1 bound to the liver endothelial cells in a manner characteristic of receptor binding with a dissociation constant of 0.5 microM. This binding could not only be inhibited by unlabelled apolipoprotein C-III:1 but also by unlabelled apolipoprotein E. The results indicate that rat liver endothelial cells carry receptors for VLDL and that these recognize the apolipoproteins E, C-III and B on the lipoprotein surface. Considering the large endothelial surface and high blood flow through the liver, significant quantities of lipoproteins can be taken up and degraded, thus influencing the levels of circulating lipoproteins in the in vivo situation.     

Uptake and degradation in vivo and in vitro of laminin and nidogen by rat liver cells.

Laminin antigens are known to be present in the blood of normal individuals. In the present study we have investigated the fate of laminin-related molecules in the circulation. After intravenous injection in rats, the native laminin-nidogen complex, as well as the separated proteins, were rapidly eliminated from the blood (half-lives 2-10 min) by the liver. The large laminin fragments E1 and E8 (Mr 400,000 and 280,000 respectively), which contain the major cell-adhesion-promoting activities of laminin, were also cleared from the blood mainly by the liver, but the rate of uptake was an order of magnitude lower for these fragments than for laminin. This indicates that the uptake of laminin did not occur via cell-adhesion receptors. The endothelial cells of liver were the most important cell type in the uptake of laminin-nidogen complex, nidogen, laminin and fragment E1, whereas the parenchymal cells were responsible for more than 50% of the uptake of E8 in the liver. Studies in vitro with cultured liver endothelial cells and parenchymal cells demonstrated that the ligands were endocytosed and degraded independently of plasma factors. The results reveal that the level of laminin antigens in blood is a very complex parameter. It is not only dependent on the turnover of basement membranes, but also on the degree of degradation of the material released into the blood and on the functional state of the liver, particularly the liver endothelial cells.     

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

Endocytosis and degradation of chondroitin sulphate by liver endothelial cells.

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.     

The uptake and metabolism of chylomicron-remnant lipids by rat liver parenchymal and non-parenchymal cells in vitro.

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.     

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (øynebråten et al.(2000) J. Leukocyte Biol. 67; 183–188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [³H] in the acetyl groups of N-acetyl galactosamine residues, [¹⁴C] in the pyranose rings, or [³⁵S] in the sulfate groups were identified as[³H]-acetate, [¹⁴C]-lactate and [³⁵S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with ¹²⁵I in the tyrosine residues, or biosynthetically with [³⁵S] or [³H] in the sulfate or acetyl groups, respectively, showed that ¹²⁵I appeared more rapidly in the medium than [³⁵S]-sulfate and [³H]-acetate. Judging from the speed of appearance of free ¹²⁵I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains.Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.     

Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro.

1. The uptake of 125I-labelled high density lipoproteins (HDL) in various organs of the rat was determined after an intravenous injection. The uptake of 125I-labelled polyvinylpyrrolidone in the same organs was determined in order to assess uptake by fluid endocytosis. The uptake/organ was highest for the liver. The adrenals showed the highest uptake/unit weight of the organs studied. The liver, the kidneys and the spleen showed comparable values for uptake/g of tissue. The uptake of 125I-labelled HDL exceeded by far that of 125I-labelled polyvinylpyrrolidone in the liver, the kidneys, the spleen and the adrenals, indicating that the uptake of 125I-labelled HDL was mediated by adsorptive endocytosis. 2. The in vivo uptake of 125I-labelled HDL was determined in purified hepatocytes and non-parenchymal cells prepared by collagenase perfusion of livers from animals after intravenous injections of 125I-labelled HDL. When expressed per cell, the hepatocytes and the non-parenchymal liver cells took up about the same amount of 125I-labelled HDL. 3. The in vitro uptake and degradation of 125I-labelled HDL in isolated rat hepatocytes was studied. The uptake at increasing concentrations of 125I-labelled HDL was saturable indicating uptake mediated through binding sites. 125I-labelled HDL were easily degraded by contaminating proteases from the perfusate. 4. Subcellular fractionation by isopycnic centrifugation indicated that the accumulation of 125I-labelled HDL did not take place in the lysosomes, but rather on the plasma membrane and possibly in the endosomes (phagosomes). 5. 125I-labelled HDL were internalized into the cells and degraded in the lysosomes. Leupetin and chloroquine, inhibitors of the lysosomal function effectively inhibited the formation of 125I-labelled acid-soluble radioactivity by the cells. Chloroquine, but not the protease inhibitor leupeptin, reduced the hydrolysis of the cholesteryl ester moiety of HDL.     

Zinc uptake by isolated rat liver parenchymal cells.

Primary cultures of rat liver parenchymal cells maintained as a monolayer in serum-free culture medium were used to investigate the characteristics of zinc accumulation in vitro. Liver parenchymal cells accumulated zinc by a temperature-dependent, saturable process that was inhibited by cyanide, azide, oligomycin, N-ethylmaleimide and iodoacetamide. Cadmium reversibly inhibited zinc accumulation in both serum-free and serum-containing media. Gel filtration chromatographic studies showed that recently accumulated intracellular zinc was present as a low molecular weight complex smaller than metallothionein, the zinc storage protein, but larger than individual amino acids. The quantity of zinc accumulated was affected by preincubation of the cells with various hor?ONES. Dexamethasone, prednisone and prednisolone each increased zinc uptake by 40--50% when either insulin or glucagon was also present. Hydrocortisone, cortisone and sex steroids did not influence zinc accumulation. Removal of the polypeptide hormones from the medium abolished the stimulatory effect of the synthetic glucocorticoid steroid hormones on zinc accumulation.     

Junctions between sinusoidal endothelial cells in fetal rat liver.

During a freeze-fracture study of tight junctions in fetal rat liver (Montesano et al., '75) unusual patterns of intramembranous particles were observed in regions of contact between sinusoidal endothelial cells. These patterns were mainly represented by arrays of particles, often associated with linear elevations or crests in the membrane A-face; they may represent abortive forms of tight junctions.     

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.     

Intracellular transport of endocytosed proteins in rat liver endothelial cells.

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.