Search

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Rat liver Kupffer cell membranes contain a protein that recognizes specifically oxidized low-density lipoproteins (oxLDL). Visualization after blotting under reducing conditions indicates that the receptor is a monomeric protein, with an estimated molecular mass of 115-120 kDa. N-Glycosidase F and endoglycosidase F treatment resulted in a fall in estimated molecular mass of 24 and 11 kDa respectively, whereas O-glycosidase was ineffective. No effect on the extent of interaction with oxLDL was noticed, suggesting that glycans are not essential for ligand recognition. Using a polyclonal antibody to mouse macrosialin, we visualized macrosialin on blot, and compared this glycoprotein with the oxLDL-binding protein. It appears that the two glycoproteins have a similar molecular mass and are comparably affected by treatment with the different glycosidases. Incubation with trypsin resulted in a reduction in the estimated molecular mass of about 25 kDa for both the oxLDL-binding protein and macrosialin. These results indicate that the oxLDL-binding protein and macrosialin are identical, suggesting a role for macrosialin in modified LDL catabolism.

Background: Clinical application of tissue plasminogen activator (TPA) as a fibrinolytic agent is complicated by its rapid clearance from the bloodstream, which is caused by TPA liver uptake. The mannose receptor on endothelial liver cells and the LDL receptor-related protein (LRP) on parenchymal liver cells were reported to contribute to liver uptake., Methods and Results: In this study, we addressed whether TPA clearance can be delayed by inhibiting receptor-mediated endocytosis of TPA. A series of cluster mannosides was synthesized, and their affinity for the mannose receptor was determined. A cluster mannoside carrying six mannose groups (M6L5) displayed a subnanomolar affinity for the mannose receptor (Ki = 0.41 +/- 0.09 nmol/L). Preinjection of M6L5 (1.2 mg/kg) reduced the clearance of 125I-TPA in rats by 60% because of specific inhibition of the endothelial cell uptake. The low toxicity of M6L5, combined with its accessible synthesis and high specificity for the mannose receptor, makes it a promising agent to improve the pharmacokinetics of TPA. Blockade of LRP by 39-kD receptor-associated protein (GST-RAP) also inhibited TPA clearance by 60%. Finally, combined preinjection of M6L5 and GST-RAP almost completely abolished reduced liver uptake of TPA and delayed its clearance by a factor of 10., Conclusions: It can be concluded that (1) the mannose receptor and LRP appear to be the sole major receptors responsible for TPA clearance and (2) therapeutic levels of TPA can be maintained for a prolonged time span by coadministration of the aforementioned receptor antagonists.

Both type I and type II MSRs are integral membrane proteins containing a collagenous domain and elicit an extraordinarily wide range of ligand binding capability. They were found during the search for the molecule(s) responsible for the accumulation of modified LDL during atherogenesis. However, all prior the evidence relating to their physiological and pathophysiological roles in vivo had been indirect. Targeted disruption of the MSR gene results in a reduction in the size of atherosclerotic lesions in an apo E deficient animal. Macrophages from MSR deficient mice exhibit a marked decrease in modified LDL uptake in vitro, whereas modified LDL clearance from plasma remains normal, suggesting that there are alternative mechanisms for the uptake of modified LDL from the circulation. In addition, MSR knockout mice are more susceptible to L. monocytogenes and HSV-1 infection, indicating a role for MSR in host defense against various pathogens.

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron-remnants and beta-migrating very-low-density lipoprotein (beta-VLDL) is under active dispute. We have investigated the effect of the 39-kDa receptor-associated protein (RAP) on the recognition site for activated alpha 2-macroglobulin and beta-VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiolabelled trypsin-activated alpha 2-macroglobulin (alpha 2M-T) was cleared rapidly by the liver (maximal uptake of 80.8 +/- 1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione-S-transferase-linked RAP (GST-RAP)/kg rat reduced the liver uptake to 62.2 +/- 2.3%, 59.3 +/- 1.1%, or 2.9 +/- 0.1% of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST-RAP/kg rat but this did not affect the serum decay and liver uptake of 125I-beta-VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I-alpha 2M-T was 98% inhibited by GST-RAP with an IC50 of 0.3 microgram/ml (4.2 nM), whereas the binding of 125I-beta-VLDL and 125I-beta-VLDL + recombinant apolipoprotein E (rec-apoE) was unaffected by GST-RAP up to 50 micrograms/ml (700 nM). Also, the cell association and degradation of alpha 2M-T was blocked by RAP, while the association and degradation of beta-VLDL and beta-VLDL + rec-apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of alpha 2M-T lasted for 1-2 h of incubation at 37 degrees C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I-labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (the alpha 2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of beta-VLDL by rat liver parenchymal cells is not mediated by the alpha 2Mr/LRP. The properties of binding of beta-VLDL to rat liver parenchymal cells points to an apoE-specific recognition site for lipoprotein remnants which differs from the alpha 2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.

We have previously developed triantennary galactosides [TG(4A)C and TG(20A)C] that lower cholesterol levels by inducing liver uptake of lipoproteins via galactose-recognizing hepatic receptors. In this study, we have investigated whether this strategy could also be applied to reduce elevated serum levels of the atherogenic lipoprotein(a) [Lp(a)]. Both TG(4A)C and TG(20A)C could be incorporated into Lp(a). Incorporation of these glycolipids induced a rapid clearance of Lp(a). Concomitantly, the hepatic uptake of 125I-Lp(a) was enhanced from 4 +/- 1% to 80 +/- 4% of the injected dose for TG(4A)C (P < .0001) and to 17 +/- 4% of the injected dose for TG(20A)C (P < .006). TG(4A)C was apparently more effective in accelerating the serum decay of 125I-Lp(a), which may be caused by the higher hydrophobicity of this glycolipid relative to TG(20A)C. The TG(4A)C- and TG(20A)C-induced stimulation of the serum decay and liver uptake of 125I-Lp(a) could be significantly inhibited (> 85%) by preinjection of N-acetyl-galactosamine (150 mg), indicating that galactose-recognizing receptors are involved in the liver uptake of the glycolipid/Lp(a) complexes. The TG(4A)C-induced liver uptake of 125I-Lp(a) could be ascribed mainly to Kupffer cells (76 +/- 7%), whereas the parenchymal liver cell was the major site for liver uptake of TG(20A)C-laden 125I-Lp(a) (55 +/- 12%). In conclusion, both TG(4A)C and TG(20A)C stimulate the catabolism of 125I-Lp(a) by enhancing hepatic uptake. Because endocytosis of the substrate via galactose-recognizing receptors on Kupffer and parenchymal liver cells is followed by lysosomal degradation, we anticipate that both approaches for Lp(a) targeting may prove valuable as therapeutic modalities for lowering atherogenic levels of Lp(a).

In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal alpha-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 A). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18-23 microM to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional alpha-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.

The hepatitis B virus (HBV) is the world's most important chronic virus infection. The immunomodulator interferon-alpha (IFN alpha) is the only clinically applied drug available, despite its low response rate (approximately 30%) even in highly selected chronic carriers. Antiviral nucleoside analogues have proven to be potent inhibitors of viral replication in vitro, but their significant adverse effects which are, at least partially, due to their nonspecific body distribution, have forced the cessation of their clinical development in the past. For example, vidarabine causes severe neuromuscular toxicity, and fialuridine has caused fatal cases of liver and kidney failure in a recent clinical trial. Furthermore, the potential clinical application of (modified) antisense oligodeoxynucleotides, which are very specific inhibitors of viral replication, is hampered by their nonspecific body distribution, instability in serum and poor cell penetration. As infection and replication of HBV mainly occur in liver parenchymal cells, selective targeting of antiviral nucleoside analogues as well as antisense oligodeoxynucleotides to the liver would theoretically improve therapeutic efficacy. At present, conjugates of vidarabine and neoglycoproteins have entered clinical trials, and initial data suggest that therapeutic concentrations are achieved at lower dosages with minor adverse effects. These data have stimulated preclinical research on other liver-specific drug carriers for the selective delivery of HBV-active drugs such as glycosylated polymers and neolipoproteins: these approaches are outlined in this paper.

Selective delivery of cytotoxic anti-neoplastic drugs can diminish the severe side-effects associated with these drugs. Many malignant tumours express high levels of low-density lipoprotein (LDL) receptors on their membranes. Therefore, LDL may be used as a carrier to obtain selective delivery of anti-neoplastic drugs to tumours. The present study was performed to investigate the feasibility of the murine B16 tumour/mouse model for the evaluation of LDL-mediated tumour therapy. LDL binds with high affinity to LDL receptors on cultured B16 cells (Kd, 5.9 +/- 2.3 micrograms ml-1; Bmax 206 +/- 23 ng LDL mg-1 cell protein). After binding and internalisation, LDL was very efficiently degraded: 724 +/- 19 ng LDL mg-1 cell protein h-1. Chloroquine and ammonium chloride completely inhibited the degradation of LDL by the B16 cells, indicating involvement of lysosomes. LDL receptors were down-regulated by 70% after preincubation of B16 cells with 300 micrograms ml-1 LDL, indicating that their expression is regulated by intracellular cholesterol. To evaluate the uptake of LDL by the B16 tumour in vivo, tissue distribution studies were performed in C57/B1 mice inoculated with B16 tumours. For these experiments, LDL was radiolabelled with tyramine cellobiose, a non-degradable label, which is retained in cells after uptake. At 24 h after injection of LDL, the liver, adrenals and the spleen were found to be the major organs involved in LDL uptake, with tissue-serum (T/S) ratios of 0.82 +/- 0.08, 1.17 +/- 0.20 and 0.69 +/- 0.08 respectively. Of all the other tissues, the tumour showed the highest uptake of LDL (T/S ratio of 0.40 +/- 0.07). A large part of the LDL uptake was receptor mediated, as the uptake of methylated LDL was much lower. Although the LDL uptake by the liver, spleen and adrenals is higher than that by the tumour, the LDL receptor-mediated uptake by these organs may be selectively down-regulated by methods that do not affect the expression of LDL receptors on tumour cells. It is concluded that the B16 tumour-bearing mouse constitutes a good model to evaluate the effectiveness of LDL-mediated delivery of cytotoxic (pro)drugs to tumours in vivo.

We recently reported the conversion of the water-soluble antiviral drug iododeoxyuridine (IDU) into the lipophilic prodrug dioleoyl-iododeoxyuridine (IDU-Ol2). The prodrug was incorporated into reconstituted high-density lipoprotein (NeoHDL) particles with physical and biological properties similar to those of native HDL. We also found, in initial experiments, that lactosylation of the prodrug-loaded NeoHDL increases its liver uptake. Because this offers the attractive perspective of using these particles for the delivery of drugs to the liver, we now analyze the characteristics and biological fate of lactosylated IDU-Ol2-loaded NeoHDL. The particles (containing approximately 25 prodrug molecules) have the same size and charge as native HDL, indicating that lactosylation does not cause aggregation or oxidative modification. At 10 min after intravenous injection of lactosylated [3H]IDU-Ol2-loaded NeòHDL into rats, only 13.5 +/- 2.8% of the dose was left in plasma and 75.9 +/- 2.4% of the dose was recovered in the liver. The relative specific uptake by the liver was 1-2 orders of magnitude higher than that of any other tissue. The hepatic uptake of lactosylated [3H]IDU-Ol2-loaded NeoHDL was much higher than that of free [3H]IDU ( < 20% of the dose). Both parenchymal liver cells and Kupffer cells express galactose-specific receptors. By isolating liver cells after injection of the prodrug-loaded particles, it was established that hepatic uptake occurred mainly (for 84.4 +/- 3.8%) in parenchymal liver cells. Preinjection with asialofetuin substantially reduced the liver uptake of lactosylated [3H]IDU-Ol2-loaded NeoHDL, which points to uptake by the asialoglycoprotein receptor. Subcellular fractionation of the liver indicated that lactosylated [3H]IDU-Ol2-loaded NeoHDL does not merely associate to cells, but is internalized and delivered to the lysosomes. In conclusion, we show that IDU can be specifically targeted to the parenchymal liver cell. Conversion of the water-soluble parent drug into a lipophilic prodrug that is incorporated into a lactosylated reconstituted HDL particle, is an approach that may also be used to deliver other water-soluble drugs to the parenchymal liver cells. This may lead to more effective therapy for liver diseases such as hepatitis B.

Apolipoprotein E (apoE) is an important determinant for the liver uptake of triglyceride-rich lipoproteins and emulsions by the remnant receptor. In the current study, we assessed an additional role of apoE as modulator of the metabolism of triglyceride-rich lipoproteins in vitro and in vivo. Glycerol tri[3H]oleate [14C]cholesteryl oleate double-labeled triglyceride-rich emulsions were injected into fasted rats. The serum half-life of glycerol tri[3H]oleate was 3-fold faster (5.4 min) than that of [14C]cholesteryl oleate (16.7 min), confirming lipoprotein lipase (LPL)-mediated processing. To establish a specific effect of apoE on emulsion lipolysis rather than liver uptake, rats were functionally hepatectomized, and hypo(apo)lipoproteinemia was induced by 17alpha-ethinyl estradiol treatment. An apoE concentration-dependent inhibition of emulsion-triglyceride hydrolysis was observed, reaching a 14.8-fold increased half-life of glycerol tri[3H]oleate as compared with that in the absence of exogenous apoE. The mechanism and specificity of the effect of apoE on emulsion lipolysis by purified LPL was assessed in vitro. Addition of apoE to glycerol tri[3H]oleate-labeled emulsions led to a concentration-dependent inhibition of [3H]oleate release (9.5% residual LPL activity at 60 microg/ml apoE), while apoA-I was ineffective. The inhibitory effect of apoE was not abolished by reductive methylation of lysine residues, whereas selective modification of arginine residues by 1,2-cyclohexadione completely cancelled the inhibitory effect of apoE. It is concluded that apoE can specifically inhibit the LPL-mediated hydrolysis of emulsion triglycerides both in vitro and in vivo, and that arginine residues in apoE are essential for this effect. We suggest that in addition to its role in receptor recognition, apoE also modulates the LPL-mediated processing of triglyceride-rich lipoproteins.

The effect of lipopolysaccharide (LPS) on cultured cerebral endothelial cells was investigated to assess the changes in the trans endothelial electrical resistance (TEER) across the blood-brain barrier that may occur during inflammatory diseases of the central nervous system. Primary cultures of bovine cerebral endothelial cells were cultured to tight monolayers with a TEER of 250 to 300 omega.cm2 on polycarbonate Transwell filters. LPS induced a time- and dose-dependent decline in TEER. Transport of the hydrophilic model compounds sodium fluorescein and fluorescein dextran (MR, 4 kDa) across monolayers of bovine cerebral endothelial cells increased more than 3-fold after treatment of the cells with LPS (50 ng/ml). Treatment of the monolayers with various concentrations of LPS caused a 3-to 4-fold increase in the permeability of bovine cerebral endothelial cells for [125I]bovine serum albumin, which was also preceded by a decrease in TEER. The reduction of TEER by LPS could be inhibited completely by indomethacin (10(-6)M for 30 min), a cyclooxygenase inhibitor, but not by dexamethasone, a glucocorticoid (10(-7) M for 16 hr). In conclusion, LPS administration to blood-brain barrier endothelial cells causes a decrease in TEER which leads to enhanced transport of low and high molecular weight molecules. During this process the production of eicosanoids by the endothelial cells seem to play a key role.

Gaucher's disease is an inherited lysosomal storage disorder that is caused by a deficiency of glucocerebrosidase. The resulting accumulation of the substrate glucosylceramide in macrophages of liver, spleen, and bone marrow causes severe clinical symptoms. Gaucher's disease is treated by intravenous administration of a modified glucocerebrosidase (Alglucerase), which has exposed mannose residues to promote uptake by target macrophages. To evaluate the effectiveness of the targeting of Alglucerase, we studied the fate of the enzyme in the rat. Intravenously injected Alglucerase was rapidly cleared from the circulation (half-life 2.0 +/- 0.5 min). The liver was the main site of uptake, with 65.6 +/- 1.2% of the dose present at 10 min after injection. Smaller amounts ( < 3% of the dose) were taken up by spleen and bone marrow. Previous injection with mannan substantially increased the plasma half-life of the enzyme (14.8 +/- 3.2 min versus 1.7 +/- 0.3 min in solvent-preinjected controls) and uptake of the enzyme by liver, spleen and bone marrow was reduced by > 90%. These findings indicate that the enzyme is taken up by these organs via mannose-specific receptors. Subcellular fractionation of the liver indicated that the enzyme is internalized and transported to the lysosomes. By isolating various liver cell types after injection of the Alglucerase, it was found that endothelial cells are the main site of uptake of the enzyme: 60.8 +/- 3.4% of the total liver uptake. Parenchymal and Kupffer cells were responsible for 31.0 +/- 3.1% and 8.2 +/- 0.7% of the hepatic uptake, respectively. We conclude that Alglucerase is rapidly cleared from the circulation by mannose-specific receptors in liver, spleen, and bone marrow. However, less than 10% of the enzyme taken up by the liver is accounted for by Kupffer cells, the hepatic target cells for therapeutic intervention. It is suggested that alterations of the formulation of the therapeutic enzyme may lead to a higher uptake by Kupffer cells and other macrophages, and thus to a more (cost)effective therapy of Gaucher's disease.

The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells.

1. Lactoferrin and aminopeptidase M-modified lactoferrin (APM-lactoferrin; which lacks its 14 N-terminal amino acids) inhibit the liver uptake of lipoprotein remnant. In the present study, the role of proteoglycans in the initial interaction of beta-migrating very-low-density lipoprotein (beta-VLDL), native and APM-lactoferrin with isolated rat parenchymal liver cells was investigated. Treatment of the cells with chondroitinase lowered the Kd of lactoferrin binding (from 10 to 2.4 microM), and the number of sites/cell (from 20 x 10(6) to 7 x 10(6)), while heparinase treatment did not affect the binding. The binding characteristics of APM-lactoferrin and beta-VLDL were not altered by treatment of the cells with chondroitinase or heparinase. It is concluded that proteoglycans are not involved in the initial binding of APM-lactoferrin and beta-VLDL to parenchymal cells, while chondroitin sulphate proteoglycans are mainly responsible for the massive, low-affinity binding of native lactoferrin..2. The binding of lactoferrin, APM-lactoferrin and beta-VLDL to parenchymal liver cells was not influenced by the glutathione S-transferase-receptor-associated protein (GST-RAP) (97.2% +/- 4.0%, 95.5 +/- 3.7% and 98.5% of the control binding), while the binding of alpha 2-macroglobulin was fully blocked at 10 micrograms/ml GST-RAP (1.8 +/- 0.5% of the control binding). Since GST-RAP blocks the binding of all the known ligands to the low-density lipoprotein (LDL)-receptor-related protein (LRP), it is concluded that LRP is not the initial primary recognition site for lactoferrin, APM-lactoferrin and beta-VLDL on parenchymal liver cells. 3. We showed earlier that.APM-lactoferrin, as compared with lactoferrin, is a more effective inhibitor of the liver uptake of lipoprotein remnants (49.4 +/- 4.0% versus 80.8 +/- 4.8% of the control at 500 micrograms/ml respectively). We found in the present study that beta-VLDL is able to inhibit the binding of APM-lactoferrin to parenchymal liver cells significantly (74.9 +/- 3.3% of the control; P < 0.002), while the lactoferrin binding was unaffected. It is concluded that a still unidentified specific recognition site (the putative remnant receptor) is responsible for the initial binding of remnants to parenchymal cells and it is suggested that the partial cross-competition between APM-lactoferrin and beta-VLDL may be of further help in the elucidation of the molecular nature of this recognition site.

To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL; one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF-treated macrophages. Treatment of human monocyte-derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated approximately 50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte-derived macrophages, which might have implications for foam cell formation.

The effects of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on the permeability of monolayers of rat cerebral endothelial cells (RCEC) were investigated to assess potential changes in the integrity of the blood-brain barrier (BBB). RCEC were cultured to tight monolayers with a trans endothelial electrical resistance (TEER) of 100-150 ohm . cm2 on polycarbonate filters. Exposure of the RCEC to TNF-alpha, IL-1 beta and IL-6 induced a decline in the TEER, which could be completely abolished by 1 muM of indomethacin, a cyclooxygenase inhibitor. In addition, the effect of IL-1 beta on TEER across monolayers of RCEC could be completely inhibited by IL-1 receptor antagonist. In conclusion, cytokines induce a disruption of the BBB in vitro. In this process, cyclooxygenase activation within the endothelial cells seems to play a key role.

Oxidation of low-density lipoprotein (LDL) may play a causal role in atherosclerosis. In this study we analyzed whether the severity of progression of coronary atherosclerosis is related to the susceptibility of LDL to oxidative modification. On the basis of repeated coronary angiography, 28 coronary bypass patients were divided into two groups: group A, 12 patients with, and group B, 16 patients without progression of coronary atherosclerosis. The lag time, reflecting the resistance of LDL to oxidative modification, was significantly smaller in group A as compared with group B (81 +/- 10 and 93 +/- 15 min, respectively). Besides differences in cholesterol and apolipoprotein B concentrations, the difference in susceptibility of LDL to oxidation significantly contributes to the differences between the progression and the non-progression group (P = 0.02). In the combined groups of patients, the lag phase of LDL for oxidation was positively correlated with LDL cholesterol ester to protein ratio (r = 0.53, P = 0.01). It is concluded that LDL samples obtained from coronary bypass patients differ with respect to their oxidizability depending on progression of atherosclerosis following coronary bypass surgery.

The catabolism of the novel plasminogen activator reteplase (BM 06.022) was described. For this purpose BM 06.022 was radiolabelled with 125I or with the accumulating label 125I-tyramine cellobiose (125I-TC). BM 06.022 was injected at a pharmacological dose of 380 micrograms/kg b.w. and it was cleared from the plasma in a biphasic manner with a half-life of about 1 min in the alpha-phase and t1/2 of 20-28 min in the beta-phase. 28% and 72% of the injected dose was cleared in the alpha-phase and beta-phase, respectively. Initially liver, kidneys, skin, bones, lungs, spleen, and muscles contributed mainly to the plasma clearance. Only liver and the kidneys, however, were responsible for the uptake and subsequent degradation of BM 06.022 and contributed for 75% to the catabolism of BM 06.022. BM 06.022 was degraded in the lysosomal compartment of both organs. Parenchymal liver cells were responsible for 70% of the liver uptake of BM 06.022. BM 06.022 associated rapidly to isolated rat parenchymal liver cells and was subsequently degraded in the lysosomal compartment of these cells. BM 06.022 bound with low-affinity to the parenchymal liver cells (550 nM) and the binding of BM 06.022 could be displaced by t-PA (IC50 5.6 nM), indicating that the low-density lipoprotein receptor-related protein (LRP) could be involved in the binding of BM 06.22. GST-RAP, which is an inhibitor of LRP, could in vivo significantly inhibit the uptake of BM 06.022 in the liver. It is concluded that BM 06.22 is metabolized primarily in the liver and the kidneys. These organs take up and degrade BM 06.022 in the lysosomes. The uptake mechanism of BM 06.022 in the kidneys is unknown, while LRP is responsible for a low affinity binding and uptake of BM 06.022 in parenchymal liver cells.

The liver plays a decisive role in the regulation of the plasma levels of atherogenic lipoproteins. The primary liver interaction site for chylomicron-remnants and VLDL remnants (beta-VLDL) is still unidentified, while the subsequent cellular uptake is likely to be mediated in concert by the LDL receptor related protein (LRP) and the LDL receptor. The nature of the primary interaction site of remnants (remnant-receptor) might be a liver-specific proteoglycan or a liver-specific protein. Atherogenic modified LDL can be recognized by a family of scavenger-receptors. A newly identified 95-kDa protein forms the most likely candidate for mediating the in vivo uptake of oxidized LDL from the circulation and might thus protect the body against the presence of oxidized LDL in the blood compartment.

Purpose: The transport of the hydrophilic model compound sodium fluorescein into the cerebrospinal fluid (CSF) of rats was studied during experimental allergic encephalomyelitis (EAE), as a model for local central nervous system (CNS) inflammatory disease, and after a single injection of a pyrogenic dose of lipopolysaccharide (LPS), as a model for a general inflammation., Methods: Transport of sodium fluorescein was measured by means of serial CSF and plasma sampling. Transport of this hydrophilic model compound was studied in Lewis rats suffering from EAA and three hours after LPS administration in male Wistar rats., Results: During acute EAE, sodium fluorescein concentrations in the CSF increased twofold compared to control animals, whereas plasma kinetics were comparable within both groups. After i.v. LPS administration, however, plasma as well as CSF kinetic parameters of sodium fluorescein concentration were significantly changed from those seen in control animals. Transport of sodium fluorescein from plasma into the CSF was calculated as the ratio Area Under the Curve (AUC)CSF/AUCPLASMA. During acute EAE this ratio increased 2-fold compared to control animals, whereas after i.v. LPS administration it was not significantly different from the one obtained in control animals., Conclusions: These results suggest an opening of the blood-brain barrier (BBB) during a cerebral inflammatory response, like acute EAE, but not after LPS administration.

The present study was undertaken in order to determine the recognition site for tissue-type plasminogen activator-plasminogen activator inhibitor type 1 [t-PA-PAI-1] complexes in rat liver in vivo and in vitro. After intravenous injection into rats t-PA-PAI-1 complexes were rapidly removed from the plasma and the liver took up 80% of the injected dose. Within the liver parenchymal and endothelial liver cells contributed mainly to the uptake of t-PA-PAI-1, and were responsible for 62% and 24% of the liver uptake, respectively. The interaction of t-PA-PAI-1 with isolated rat parenchymal liver cells was of high affinity (Kd 17 nM). A well-known antagonist of the alpha 2-macroglobulin receptor (alpha 2MR/low-density lipoprotein receptor-related protein (LRP), GST-39kDa protein (GST-39kDaP) efficiently inhibited the binding (IC50 0.7 nM) of t-PA-PAI-1 to rat parenchymal liver cells. The interaction of t-PA-PAI-1 with LRP on rat parenchymal liver cells was not Ca2(+)-dependent and is most probably mediated by a specific determinant on PAI-1, since an anti-PAI-1 monoclonal antibody inhibited the binding of t-PA-PAI-1, where as free t-PA did not. The binding of t-PA-PAI-1 to rat hepatocytes could not be inhibited by a complex of plasmin and alpha 2-antiplasmin nor by various other ligands of LRP like beta-VLDL and lactoferrin. Binding of t-PA-PAI-1 to rat parenchymal liver cells was followed by internalization and subsequent degradation in the lysosomal compartment. It is concluded that parenchymal and endothelial liver cells mediate the removal of t-PA-PAI-1 complexes from the circulation. LRP on rat parenchymal liver cells is responsible for the uptake and degradation of t-PA-PAI-1 and may therefore be important for the regulation of the t-PA levels in the circulation.

During sepsis the infiltration of leukocytes plays a pivotal role in tissue damage. Induction of septic shock results in an early accumulation of polymorphonuclear leukocytes in the liver (after 3 hours), which is followed by an infiltration of mononuclear phagocytes (after 30 hours). Expression of adhesion molecules may contribute to the migration of leukocytes to the site of inflammation. Therefore, in the present study we determined the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on hepatocytes, liver endothelial cells, and Kupffer cells after lipopolysaccharide (LPS) treatment of rats in vivo. Parenchymal cells showed no constitutive expression of VCAM-1 and the expression could not be upregulated by LPS treatment in vivo, whereas Kupffer and endothelial cells had a low basal expression of VCAM-1 and this expression was increased 40-fold by LPS treatment in vivo. All three cell types showed a basal expression of ICAM-1 and the expression on endothelial liver cells of untreated rats was two times higher than the expression on parenchymal and Kupffer cells. Stimulation with LPS increased the expression of ICAM-1 2.5 times per parenchymal cells and approximately 4 times for endothelial and Kupffer cells. It is concluded that the expression of adhesion molecules may contribute to the influx of leukocytes during septic shock and, therefore, play a role in tissue damage during septic shock.

Periportal and perivenous rat liver parenchymal cells were isolated according to the digitonin-collagenase perfusion method. Affinities and maximal specific binding of a conjugate of glutathione S-transferase and the alpha 2-macroglobulin receptor-associated protein (GST-39kDaP), of lactoferrin and of transferrin to freshly isolated periportal parenchymal cells in vitro were not significantly different from values obtained with perivenous cells. It is concluded that the receptors for these three ligands show a zonally homogeneous expression in rat liver. The zonal homogeneity in binding observed for GST-39kDaP is at variance with the 1.5-fold higher periportal over perivenous binding of trypsin-activated alpha 2-macroglobulin. Since GST-39kDaP as well as trypsin-activated alpha 2-macroglobulin are ligands for the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein, it is suggested that GST-39kDaP can bind to (an) additional receptor(s) with a higher perivenous expression. The zonal homogeneity observed with lactoferrin, an inhibitor of ligand binding to the lipoprotein remnant receptor, may indicate zonal homogeneity of the lipoprotein remnant receptor. The observed zonal homogeneity of the transferrin receptor suggests an equal and essential need for iron by parenchymal cells across the rat liver acinus in vivo.

The recognition systems mediating the clearance of glycosylated high molecular weight single-chain urokinase-type plasminogen activator (HMW-scu-PA, produced in human embryonic kidney cells) and recombinant non-glycosylated scu-PA (rscu-PA, produced in E. coli) were analyzed by studying their binding characteristics to freshly isolated rat parenchymal liver cells. The binding of 125I-HMW-scu-PA at 4 degrees C was calcium-dependent and of high affinity (Kd = 37.6 nM) and could be inhibited by low molecular weight two-chain u-PA (LMW-tcu-PA) and lactose, but not by the low density lipoprotein receptor-related protein (LRP)-associated 39-kDa protein (RAP), rscu-PA or a mutant form lacking amino acids 11-135 (Delta 125-rscu-PA). Removal of the carbohydrate side chain of HMW-scu-PA by treatment with N-glycosidase F, completely reduced the specific binding to the parenchymal cells and strongly reduced its competition with 125I-HMW-scu-PA in cell binding. Recombinant scu-PA also bound with high affinity (Kd = 38.7 nM) to the parenchymal liver cells. The binding of 125I-rscu-PA could be competed for by unlabeled rscu-PA while Delta 125-rscu-PA, LMW-tcu-PA or lactose were ineffective. In contrast to HMW-scu-PA, binding of 125I-rscu-PA could be effectively inhibited by RAP (Ki = 1.1 nM), while also its association and degradation, as determined at 37 degrees C, were inhibited by RAP. Pretreatment of the parenchymal cells with proteinase K supplied further evidence for the involvement of two different receptor systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures of bovine cerebral endothelial cells were used as an in vitro model for the blood-brain barrier to study the transport and interactions of high-density lipoprotein (HDL) across monolayers of these cells. Transport of 125I-apoE free HDL across a monolayer of bovine cerebral endothelial cells occurred in a linear fashion up to a concentration of 70 micrograms/mL, suggesting paracellular transport of HDL. Bovine cerebral endothelial cells possess a high affinity binding site for HDL with a mean dissociation constant (KD) of 10.8 +/- 2.6 micrograms/mL (N = 4). Maximal binding of apoE free HDL to cerebral endothelial cells proved to be temperature-dependent: at 4 degrees a Bmax value of 42 +/- 9.3 ng/mg cell protein was found, while at 37 degrees this value was 177 +/- 70.4 ng/mg cell protein. Cell association of 125I-HDL could be effectively displaced by HDL, not by low-density lipoprotein or acetylated low-density lipoprotein, and association was not coupled to degradation. The in vitro blood-brain barrier cell system possesses high affinity binding sites for HDL, which are probably not involved in the transport of HDL across cerebral endothelial cells.

The capacity of rat cerebral endothelial cells (RCEC) to form eicosanoids was determined after incubation with 14C-labelled arachidonic acid. Prostaglandin E2 (PGE2) was the main metabolite formed by RCEC and was responsible for 54% of the total amount of eicosanoids produced. In contrast, in primary cultures of rat aorta endothelial cells, 32% of the amount of prostaglandins was 6-keto-PGF1 alpha). RCEC treated with 50 ng/ml LPS for 24 h responded with an augmented PGE2 synthesis and 6-keto-PGF1 alpha of 3.4-fold and 2.2-fold, respectively. Cultures treated with IL-1 beta (50 ng/ml) for 3 h showed a stimulation of the release of PGE2 and 6-keto-PGF1 alpha of 2.5- and 4.5-fold, respectively, and 2.0-fold and 2.3-fold, respectively, after IL-6 (50 ng/ml) incubation for 3 h. PGE2 is the main eicosanoid formed by RCEC in response to inflammatory agents, suggesting an important role of the cerebral endothelial cells in the transduction of an inflammatory response in the central nervous system.

Cholesterol-derivatized galactosides have been devised in order to induce liver uptake of lipoproteins via the galactose-recognizing asialoglycoprotein receptor in the liver. In this study we describe the derivatization of a newly developed triantennary cluster galactoside having high affinity for the asialoglycoprotein receptor, N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D-galactopyranosyl)metho xym ethyl] -N alpha-[1-(6-methyladipyl)]glycinamide (TG(20A)) with cholesterol. Hereto, TG(20A) was coupled to glycine-(5-cholesten-3 beta-yl ester) in the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, affording N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D- galactopyranosyl)methoxymethyl]methyl]-N alpha-[1-(6-(5-cholesten-3 beta-yloxy)glycyl)adipyl]glycinamide (TG(20A)C) in 46% yield. This compound is an amphiphilic, water-soluble compound. In aqueous solution it readily formed small micelles (4.9 +/- 1.2 nm) consisting of approximately 20 molecules. Upon incubation with human serum, TG(20A)C spontaneously incorporated into the most prominent serum lipoproteins, i.e., low-density lipoprotein (LDL) and high-density lipoprotein (HDL), thereby inducing an increase in buoyant density of these lipoproteins. The integrity of HDL and LDL, as judged from particle size analysis of both lipoproteins, was not altered by incubation with up to 0.33% of TG(20A)C (w/v). Following intravenous bolus injection into rats, TG(20A)C induced a dose-dependent decrease in the serum cholesterol content of maximally 44%, at a dose of 1.9 mg kg-1. This makes TG(20A)C at least 30-fold more effective than the previously developed N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[4-(5- cholesten-3 beta-yloxy)succinyl]glycinamide (TG(4A)C), provided with a cluster galactoside that displayed a 2000-fold lower affinity for the asialoglycoprotein receptor than TG(20A). In conclusion, the hypocholesterolemic activity of a cholesterylated galactoside can be strongly enhanced by using a cluster galactoside with higher affinity for the asialoglycoprotein receptor.

High-affinity ligands for the asialoglycoprotein receptor, which is uniquely localized on the parenchymal liver cell and recognizes oligoantennary galactosides, might be utilized as homing device to specifically target drugs or genes to parenchymal liver cells. In the present study, the synthesis of galactose-terminated triantennary glycosides, provided with various spacers between the beta-galactopyranosyl moieties and the branching point of the dendrite, is described. N-[Tris[[(methylthio)methoxy]methyl]methyl]-N alpha-[1-(6- methyladipy)]glycinamide (3b) was glycosylated with monogalactosyl derivatives, containing propanediol or ethylene glycol units as hydrophilic spacer moieties, to yield the corresponding cluster galactosides. To determine the affinity of the cluster galactosides for the asialoglycoprotein receptor, we have performed competition studies of [125I]ASOR binding, a specific ligand for the asialoglycoprotein receptor, to isolated parenchymal cells. The affinity for the asialoglycoprotein receptor significantly increased with increasing spacer length. N-[[[Tris-O-(beta-D-galactopyranosyl)-3,6,9-trioxaunde- canoxy]methoxy]methyl]-N-alpha-[1-(6-methyladipyl)]glycinami de (4e), a cluster galactoside provided with a 20 A spacer, possessed an at least 2000-fold higher affinity for the receptor than N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[1-(6- methyladipyl)]glycinamide (4a), a cluster galactoside lacking the spacer. It is concluded that vicinal galactosyl moieties within a cluster galactoside are more optimal recognized by the galactose binding sites of the asialoglycoprotein receptor upon proper spacing. The most potent galactoside, TG(20A), may constitute an attractive targeting device for the specific delivery of drugs and/or genes to the parenchymal liver cell.

Background: Previous studies have demonstrated that cholesterol-derivatized galactosides exert a hypocholesterolemic effect by inducing hepatic uptake of atherogenic lipoproteins by means of galactose-recognizing receptors in the liver. However, a prolonged infusion of high concentrations of these compounds was required for this effect, possibly because of low affinity for the galactose-recognizing asialoglycoprotein receptor on the parenchymal liver cell., Methods and Results: We have designed a new series of triantennary galactosides to optimize the affinity and specificity for this receptor. The affinity of a triantennary galactoside for the asialoglycoprotein receptor appeared to be dramatically enhanced by proper spacing of the three terminal galactose groups. In rats, a single injection of N-[tris-O-(3,6,9-trioxaundecanyl-beta-D-galacto- pyranosyl)methoxymethyl]methyl-N alpha-[1-(6-(5-cholesten-3 beta- yloxy)glycyl)adipyl]glycinamide [TG(20A)C], the cholesterol derivative of the most selective galactoside, causes a dose-dependent decrease of < or = 45% in the serum cholesterol concentration (P < .001). This decrease is mainly attributed to a decrease in the level of serum HDL (P = .0066) and, to a lesser extent, serum LDL (P = .036). In addition, TG(20A)C strongly enhances the bile-acid secretion in rats during the first 2 hours after administration, which indicates that TG(20A)C-induced clearance of cholesterol from the bloodstream is efficiently coupled to hepatic bile-acid secretion., Conclusions: We conclude that TG(20A)C efficiently directs lipoproteins that contain cholesterol to the liver at a 30-fold-lower concentration than previously developed cholesterol-derived cluster galactosides. This newly developed approach to lower cholesterol levels may prove valuable for familial hypercholesterolemic patients or those with familial defective apolipoprotein B-100 who do not respond or who respond insufficiently, respectively, to conventional therapies.

Hepatitis B virus (HBV) infection is the world's most important chronic virus infection. No safe and effective treatment is available at present, and clinical exploration of promising antiviral agents, such as nucleoside analogues is hampered because of significant side-effects due to their aspecific body distribution. We are exploring the possibility of the selective delivery of antiviral active drugs to liver parenchymal cells, the main site of infection and replication of HBV. Chylomicrons, which transport dietary lipids into the liver via apolipoprotein E-specific receptors, could serve as drug carriers. However, their endogenous nature hampers their application as pharmaceutical drug carriers. We report here that incorporation of a derivative of the nucleoside analogue iododeoxyuridine into recombinant chylomicrons leads to selective targeting to liver parenchymal cells. Potentially effective intracellular drug concentrations of 700 nM can be achieved, and we therefore anticipate that these drug carrier complexes represent a conceptual advance in the development of an effective and safe therapy for hepatitis B.

We investigated the potential use of reconstituted HDL particles (NeoHDL) as a carrier for lipophilic (pro)drugs. The antiviral drug iododeoxyuridine (IDU) was used as model compound. [3H]-IDU was derivatized with two oleoyl residues to dioleoyl[3H]iododeoxyuridine ([3H]IDU-Ol2), and the lipophilic prodrug was incorporated into NeoHDL by cosonication of [3H]IDU-Ol2 with lipids and HDL apoproteins. NeoHDL particles with the same density, size, and electrophoretic mobility as native HDL were obtained, which contained 7.3 +/- 0.8% (w/w) [3H]IDU-Ol2 (about 30 molecules of prodrug per particle). NeoHDL-associated [3H]IDU-Ol2 was stable during 2 h of incubation with human plasma; the prodrug was not appreciably hydrolyzed, nor exchanged with LDL. After intravenous injection of [3H]-IDU-Ol2-loaded 125I-NeoHDL into rats, [3H]IDU-Ol2 disappeared more rapidly from the circulation than the 125I-apoproteins (78.0 +/- 8.0% vs 30.1 +/- 4.5% of the dose cleared from plasma in 60 min, respectively). The hepatic association of the prodrug was higher than that of the apoproteins (21.6 +/- 0.5 vs 5.2 +/- 1.0% of the dose at 10 min after injection, respectively). As selective clearance and uptake of lipid esters is also observed with native HDL, this suggests that, in vivo, prodrug-loaded NeoHDL may be subject to physiological HDL-specific processing. Lactosylated [3H]IDU-Ol2-loaded 125I-NeoHDL, which contains galactose residues that can be recognized by galactose receptors on parenchymal liver cells, was rapidly cleared from plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies showed that both endothelial and Kupffer cells contain specific recognition sites of oxidized low-density lipoprotein (OxLDL), in addition to recognition sites which recognize OxLDL and acetylated LDL (AcLDL). We have determined the binding characteristics of the recognition sites for OxLDL on Kupffer cells and endothelial cells (OxLDL-specific binding-site) in comparison to the recognition site for AcLDL on endothelial cells, which recognizes both AcLDL and OxLDL (Ac/OxLDL binding site). The capacity of Kupffer cells to bind OxLDL (Bmax. = 779 ng of 125I-OxLDL/mg of cell protein; Kd = 6 micrograms/ml) was comparable to the binding-capacity of endothelial cells (Bmax. = 803 ng of 125I-OxLDL/mg of cell protein; Kd = 5 micrograms/ml). The effect of net charge of modified LDL on its affinity for the recognition sites on Kupffer and endothelial cells was evaluated using competition studies. The affinity of AcLDL for the Ac/OxLDL binding site was greatly increased from 460 micrograms/ml to 4 micrograms/ml with increasing extent of modification and thus net charge. The Ac/OxLDL binding-site on endothelial cells also displayed an increased affinity towards LDL with an increasing degree of oxidation. The affinity of OxLDL for the Ac/OxLDL binding-site appeared to be about 4-fold higher than that of AcLDL with a similar extent of modification. At higher degrees of oxidation of LDL, the affinity for the OxLDL-specific site on endothelial and Kupffer cells was also strongly enhanced; the OxLDL-specific binding-site possesses a higher affinity for mildly oxidized LDL as compared with the Ac/OxLDL binding-site. It is concluded that recognition of OxLDL by both the OxLDL-specific binding-site and the Ac/OxLDL binding-site on liver endothelial and Kupffer cells depends on the net negative charge of modified LDL. The similarity in binding pattern of these binding sites makes it likely that the newly described 95 kD OxLDL binding protein on Kupffer cells [Y. B. De Rijke and Th. J. C. van Berkel, J. Biol. Chem. (1994), 269, 824-827] contains a recognition site with similar structural elements as described earlier for scavenger receptors.

Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

In this study, we describe the development and characterization of lactosylated poly-L-lysine as a potential carrier for targeting anti-viral drugs to the parenchymal liver cell. Poly-L-lysine (M(r) 38,000) was modified with 2 to 130 lactose residues per molecule poly-L-lysine. In vitro competition studies for the asialoglycoprotein receptor on parenchymal liver cells using 125I-asialoorosomucoid as radioligand revealed that mild modification of poly-L-lysine with only five lactose residues was sufficient for high affinity competition. In vivo studies showed that, after injection of poly-L-lysine modified with at least five lactose residues, about 70-80% of the injected dose was taken up by the liver. Preinjection of N-acetyl galactosamine almost completely blocked the hepatic uptake of lactosylated poly-L-lysine, indicating that galactose-recognizing receptors are involved. At 10 min following injection, the contribution of the various liver cell types to the hepatic uptake of lactosylated poly-L-lysine was determined; the parenchymal cell appeared to be responsible for more than 98% of the total liver uptake. To assess the applicability of lactosylated poly-L-lysine as an anti-viral drug carrier, it was derivatized with 4 to 15 residues of the antiviral drug 5-iodo 2'-deoxyuridine, 5'-monophosphate per molecule poly-L-lysine (4-16% by weight) via an acid-labile phosphamide bond. Maximally 0.7% of the conjugated 5-iodo 2'-deoxyuridine 5'-monophosphate was released after 1 h incubation of the drug/carrier conjugate with serum at 37 degrees C, thus establishing the stability of the conjugate in serum. The drug-carrier conjugate was rapidly cleared from the bloodstream within 1 min. Approximately 90% of the injected dose could be recovered in the liver. The parenchymal liver cell was responsible for 97% of the hepatic uptake. In vitro studies on the kinetics of endocytosis of lactosylated poly-L-lysine, derivatized with 5-iodo 2'-deoxyuridine 5'-monophosphate, by parenchymal liver cells revealed that the ligand was immediately internalized and, after a 10-min lag phase, deacetylated. Internalization and degradation did not occur in the presence of 100 mM N-acetyl galactosamine. In conclusion, the bioavailability of 5-iodo 2'-deoxyuridine 5'-monophosphate to the parenchymal liver cell is dramatically enhanced as a result of the conjugation of the anti-viral drugs to lactosylated poly-L-lysine. Accordingly, lactosylated poly-L-lysine constitutes a suitable carrier for targeting anti-viral drugs to the parenchymal liver cell.(ABSTRACT TRUNCATED AT 400 WORDS)

The liver plays a decisive role in the regulation of the plasma levels of atherogenic lipoproteins. The primary liver interaction site of chylomicron remnants and VLDL remnants (beta-VLDL) is still unidentified, whereas the subsequent cellular uptake is likely to be mediated in concert by the LDL receptor-related protein and the LDL receptor. The nature of the primary interaction site of remnants (remnant receptor) might be a liver-specific proteoglycan or a liver-specific protein. Atherogenic modified LDL can be recognized by a family of scavenger receptors. A newly identified 95 kDa protein forms the most likely candidate for mediating the in-vivo uptake of oxidized LDL from the circulation and may, therefore, protect the body against the presence of oxidized LDL in the blood compartment.

Serum clearance of alpha 2M-Me or alpha 2M-Tr is rapid and identical. Alpha 2M-Tr is almost exclusively taken up in the liver by the parenchymal cells; the uptake of alpha 2M-Me is equally shared between endothelial and parenchymal cells. Blocking the scavenger receptor on endothelial cells by polyinosinic acid reduces the uptake of alpha 2M-Me to 40% of the control value; under these conditions, alpha 2M-Me is only associated with the parenchymal cells. These results show the following: (1) activation of alpha 2M by methylamine or trypsin is different; (2) the scavenger receptor on endothelial cells functions as a system for the uptake of alpha 2M-Me in addition to the specific alpha 2M receptor on parenchymal cells.

We have developed a new triantennary galactoside, in which the terminal galactose moieties are connected to the branching point of the cluster galactoside via a 20 A (2 nm) spacer [TG(20A)]. In vitro binding studies have demonstrated that introduction of a 20 A spacer resulted in avid and specific binding of the triantennary galactoside to the asialoglycoprotein receptor on the parenchymal liver cell. Derivatization of this galactoside with a cholesterol moiety afforded a compound [TG(20A)C] that lowered the serum cholesterol concentration when injected into rats. In the present study we have evaluated the direct effect of TG(20A)C on the in vivo fate of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). A direct association of TG(20A)C with HDL and LDL was observed on mixing these components. Incorporation of TG(20A)C into 125I-HDL and 125I-LDL significantly accelerated the serum decay and concomitantly stimulated the hepatic uptake of these lipoproteins in rats. The liver uptake of TG(20A)C-loaded 125I-HDL or 125I-LDL could be inhibited by 81% and 82% respectively by preinjection of 150 mg of N-acetylgalactosamine, indicating that the enhanced liver uptake proceeded via galactose-specific receptors. More than 96% of the hepatic uptake of TG(20A)C-loaded 125I-HDL could be attributed to the parenchymal cell. Surprisingly, the parenchymal cell also accounted for 93% of the liver association of TG(20A)C-loaded 125I-LDL, suggesting that TG(20A)C stimulates the uptake and processing of both lipoproteins by the asialoglycoprotein receptor on the parenchymal liver cell. This contrasts with earlier data indicating that a triantennary cluster galactoside provided with a 4 A spacer between the terminal galactose moieties and the branching point of the dendrite stimulated hepatic uptake of LDL via the Kupffer cells. The parenchymal cell is the only liver cell type that is capable of irreversibly removing cholesterol from the body in the form of bile acids. The above results imply that administration of TG(20A)C not only facilitates the hepatic uptake of lipoprotein-derived cholesterol (esters) but also their elimination from the body. In addition, it might be possible to utilize TG(20A)C as a targeting device to selectively deliver large drug carriers and possibly genes to the parenchymal liver cell.

The presence and upregulation of adhesion molecules on bovine brain endothelial cells (BBEC) were investigated. Monolayers of BBEC were incubated with lipopolysaccharide (LPS), interleukin-1 beta (rhIL-1 beta), and interleukin-6 (rhIL-6) to simulate in vitro an inflammatory site in the cerebral capillaries. Adhesion of lymphocytes to BBEC increased 4.1-fold after stimulation of the endothelial cells for 4 h with 5 or 10 ng/ml LPS. Lymphocyte adhesion increased after incubation of the BBEC for 4 h with IL-1 and was increased 3.7-fold using 100 ng/ml IL-1. BBEC pre-incubated with IL-6 for 4 h also showed an increase in adhesion of lymphocytes, and cells pretreated with 100 ng/ml IL-6 showed a 3-fold increase in lymphocyte adherence. Specific monoclonal antibodies directed against CD11a, CD18, and VLA-4 were able to block adherence of lymphocytes to stimulated BBEC. These results indicate that the in vitro activation of BBEC may serve as a model for the study of inflammation of the blood-brain barrier.

Human low-density lipoprotein was labeled in its cholesteryl ester moiety with [3H]cholesteryl oleate or [3H]cholesteryl oleoyl ether and oxidized by exposure to 10 mumol/L of cupric sulfate. The in vivo metabolism of cholesteryl esters of oxidized low-density lipoprotein was determined after injection into rats. When oxidized low-density lipoprotein was labeled with [3H]cholesteryl oleoyl ether, a nonhydrolyzable analog of cholesteryl oleate, Kupffer cells contributed to 55.1% +/- 4.1% of the total liver uptake 10 min after injection. When [3H]cholesteryl oleate-labeled oxidized low-density lipoprotein was injected, the radiolabeled cholesterol esters were nearly completely hydrolyzed within 1 hr of injection. Within this time, the Kupffer cell-associated radioactivity declined to 32% of the maximal uptake value. In serum, the highest specific resecreted [3H]cholesteryl (esters) were associated with the serum high-density lipoprotein fraction, suggesting a role for high-density lipoprotein as an in vivo cholesterol acceptor. The kinetics of biliary secretion were studied in rats equipped with catheters in the bile duct, duodenum and heart. One hour after injection of [3H]cholesteryl oleate-labeled oxidized low-density lipoprotein, 4.15% +/- 0.67% of the injected dose was secreted in the bile, mainly as bile acids. Six hours after injection, this value was 19.2% +/- 1.2%. These values are three times higher than those for injected [3H]cholesteryl oleate-labeled acetylated low-density lipoprotein, which is initially mainly taken up by liver endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactosylated low density lipoprotein (lac-LDL) is a potential carrier for the site-specific delivery of lipophilic drugs to liver macrophages (Kupffer cells). In the present study we evaluated the application of lac-LDL as a carrier to target the immunomodulator muramyl tripeptide-phosphatidylethanolamine (MTP-PE) to rat Kupffer cells, to specifically activate these cells to tumor-killing cells. The drug carrier 125I-labeled lac-LDL interacted with a galactose-specific recognition system on isolated rat Kupffer cells. The in vitro association of 125I-lac-LDL at 37 degrees was maximal after 20 min, whereas degradation of 125I-lac-LDL was observed after a lag period of 10 min. Cultured rat Kupffer cells were activated after incubation with MTP-PE incorporated into lac-LDL. Lac-LDL-MTP-PE induced a 2-fold increase in the amount of newly synthesized proteins secreted by Kupffer cells. Lac-LDL-MTP-PE induced a concentration-dependent increase in the cytostatic and cytolytic activities of Kupffer cells towards tumor cells (B16F10 melanoma cells) in vitro. Treatment of rats with lac-LDL-MTP-PE also resulted in dose-dependent activation of Kupffer cells to tumoricidal cells, whereas the drug carrier alone had only a minor effect on this activity of Kupffer cells. The present data show that lac-LDL is an effective carrier for the delivery of the lipophilic immunomodulator MTP-PE to rat Kupffer cells. The specific activation of Kupffer cells to tumoricidal cells by lac-LDL-MTP-PE may be beneficial for the treatment of liver metastases.

Previous in vivo studies have demonstrated that small galactose-exposing particles are preferentially internalized by the asialoglycoprotein receptor on the parenchymal liver cell and large particles by the galactose-particle receptor on the Kupffer cell. In this study, we have investigated using in vitro binding studies whether the affinity for either receptor is affected by the ligand size. The asialoglycoprotein receptor appeared to bind and process lactosylated proteins irrespective of their size. In contrast, recognition of galactose-exposing proteins by the galactose-particle receptor on the Kupffer cell was strongly dependent on size. The affinity increased 3000-fold with protein sizes increasing from 5 to 15 nm, reaching its maximum at approx. 1 nM for ligands larger than 15 nm. Apparently, the preferential in vivo uptake of large galactose-exposing ligands by Kupffer cells does not result from an inability of the parenchymal liver cells to internalize these ligands, but from the high affinity of large ligands for the galactose-particle receptor and the strategic anatomical localization of the Kupffer cells in the liver. In the preceding paper [Kuiper, Bakkeren, Biessen and Van Berkel (1994) Biochem. J. 299, 285-290] the galactose-particle receptor on the Kupffer cell was suggested to be identical with the fucose receptor. 125I-Lac-LDL-binding studies clearly showed that the galactose-particle receptor exhibited high-affinity binding of fucose-exposing proteins also. The affinity of fucosylated proteins for the galactose-particle receptor was greatly affected by ligand size. The above data strongly support the hypothesis that the galactose-particle receptor is identical with the fucose receptor. The size of neoglycoproteins can be appreciated as a new major determinant of affinity for the fucose receptor.

The characteristics of the recognition system involved in the binding of galactose-exposing particles to freshly isolated rat Kupffer cells were determined. For this purpose we used iodinated lactosylated low-density lipoprotein (125I-Lac-LDL) as a ligand for the galactose receptor on Kupffer cells. The affinity of the binding of 125I-Lac-LDL to Kupffer cells was saturable (23,500 galactose-specific binding sites per cell) and of high affinity (2.4 +/- 0.3 nM). The order of potency of various carbohydrates in inhibiting the association of 125I-Lac-LDL with Kupffer cells was as follows: N-acetylgalactosamine > L-fucose >> N-acetylglucosamine/mannan. Association of 125I-Lac-LDL with Kupffer cells in the absence of Ca2+ was at the same level as in the presence of 50 mM N-acetylgalactosamine. A polyclonal antibody raised against the rat asialoglycoprotein receptor inhibited the binding of 125I-Lac-LDL to Kupffer cells and reacted in a Western blot with two proteins (molecular mass 88 and 77 kDa), which correspond to the molecular mass of the fucose receptor [Lehrman, Haltiwanger and Hill (1986) J. Biol. Chem. 261, 7426-7432]. Furthermore, the ability of fucosylated neoglycoproteins to displace 125I-Lac-LDL from Kupffer cells was equally dependent on the extent of fucosylation as previously reported for the fucose receptor. We conclude that the fucose receptor and not the C-reactive protein, as recently proposed [Kempka, Roos and Kolb-Bachofen (1990) J. Immunol. 144, 1004-1009], functions as the galactose-particle receptor on the Kupffer cell. The binding of galactose-exposing particles to the fucose receptor is a previously unknown property of this receptor.

The liver is the major organ responsible for the uptake of oxidized low density lipoproteins (Ox-LDL) from the blood circulation with Kupffer cells as major cellular uptake site. Candidate binding proteins for Ox-LDL on membranes from Kupffer and endothelial liver cells were identified with ligand blots. Under nonreducing conditions, a major binding protein with an estimated molecular mass of 95 kDa and a minor stained protein of 220 kDa were detected on Kupffer cell membranes, while endothelial cell membranes expressed only a 220-kDa binding protein. Both the 95-kDa protein of Kupffer cell membranes and the 220-kDa protein of endothelial membranes displayed saturable binding of 125I-Ox-LDL with a Kd of 15 and 5 micrograms/ml, respectively. LDL was a weak competitor for the binding of 125I-Ox-LDL to the 95-kDa protein, while the degree of competition appeared to be dependent on the oxidation grade of LDL with a complete competition with LDL oxidized for 20 h with 10 microM Cu2+. We conclude that the 95-kDa binding protein, highly concentrated on rat Kupffer cells, forms the most likely candidate for mediating the in vivo uptake of Ox-LDL from the blood circulation.