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T J van Berkel

Publications and source records attributed to T J van Berkel.

At least 19 recordsLinked to original sources
PubMed2001

Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein.

The efficacy of antisense oligonucleotides depends on the ability to reach in vivo their target cells. We aim to develop strategies to enhance uptake of phosphorothioate oligodeoxynucleotides by Kupffer cells. To this end, we conjugated cholesterol to ISIS-3082, a phosphorothioate oligodeoxynucleotide specific for intercellular adhesion molecule-1. The cholesterol-conjugated oligonucleotide, denoted ISIS-9388, associated readily with lactosylated low-density lipoprotein (LacLDL), a lipidic carrier that is taken up by galactose receptors on Kupffer cells. Association of up to 10 molecules of ISIS-9388 per LacLDL particle did not induce aggregation. LacLDL-associated [3H]ISIS-9388 was rapidly taken up by the liver after injection into rats (52.9+/-1.8% of the dose within 2 min versus 18.6+/-2.8% for ISIS-3082). N-acetylgalactosamine inhibited hepatic uptake, indicating involvement of galactose-specific receptors. Liver cells were isolated at 60 min after injection of LacLDL-associated [3H]ISIS-9388. Kupffer cells displayed the highest uptake: 88.1+/-24.7 ng of oligonucleotide/mg of cell protein, which is 6-14 times higher than after injection of free ISIS-9388 or ISIS-3082 (15.0+/-3.8 ng and 6.3+/-1.4 ng, respectively). It can be calculated that Kupffer cells contribute 43.9+/-5.4% to the liver uptake (free ISIS-9388 and ISIS-3083 14.5+/-3.1% and 8.3+/-3.2%, respectively). In conclusion, conjugation of a phosphorothioate oligodeoxynucleotide with cholesterol and its subsequent association with LacLDL results in a substantially increased Kupffer cell uptake of the oligonucleotide. As Kupffer cells play a key role in inflammation, our approach may be utilized to improve antisense-based therapeutic intervention during inflammation.

Animals↗
PubMed2001

Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.

The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) >> galactose) and the effects of ligand valency (tetraantennary > triantennary >> diantennary >> monoantennary) and sugar spacing (20 A 10 A 4 A) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Here too, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc(3)-terminated cluster glycoside with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [(3)H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 +/- 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.

Animals↗
PubMed2001

Macrophage p53 deficiency leads to enhanced atherosclerosis in APOE*3-Leiden transgenic mice.

Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (p53(-/-)) or control (p53(+/+)) donor mice. Reconstitution of mice with p53(-/-) bone marrow did not result in any hemopoietic abnormalities as compared with p53(+/+) transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with p53(-/-) bone marrow showed a significant (P=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with p53(+/+) bone marrow. Although likely a secondary effect of the increased lesion area, p53(-/-) transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1+/-1.3 versus 0.2+/-0.7; P=0.04) and lesion macrophages (macrophage area, 79.9+/-40.0 versus 39.7+/-27.3x10(3) micrometer(2) per section; P=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei going from 0.42+/-0.39 to 0.14+/-0.15%, P=0.071), whereas the number of proliferating cells (5'-bromo-2'-deoxyuridine-positive nuclei) was not affected (3.75+/-0.98 versus 4.77+/-2.30%; P=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.

Animals↗
PubMed2001

Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting.

Lipoproteins are endogenous particles that transport lipids through the blood to various cell types, where they are recognised and taken up via specific receptors. These particles are, therefore, excellent candidates for the targeted delivery of drugs to various tissues. For example, the remnant receptor and the asialoglycoprotein receptor (ASGPr), which are uniquely localised on hepatocytes, recognise chylomicrons and lactosylated high density lipopoteins (HDL), respectively. In addition, tumour cells of various origins overexpress the low density lipoprotein (LDL) receptor that recognises apolipoprotein E (apoE) on small triglyceride-rich particles and apoB-100 on LDL. Being endogenous, lipoproteins are biodegradable, do not trigger immune reactions, and are not recognised by the reticuloendothelial system (RES). However, their endogenous nature also hampers large-scale pharmaceutical application. In the past two decades, various research groups have successfully synthesised recombinant lipoproteins from commercially available natural and synthetic lipids and serum-derived or recombinant apolipoproteins, which closely mimic the metabolic behaviour of their native counterparts in animal models as well as humans. In this paper, we will summarise the studies that led to the development of these recombinant lipoproteins, and we will address the possibility of using these lipidic particles to selectively deliver a wide range of lipophilic, amphiphilic, and polyanionic compounds to hepatocytes and tumour cells. In addition, the intrinsic therapeutic activities of recombinant chylomicrons and HDL in sepsis and atherosclerosis will be discussed.

Animals↗
PubMed2001

Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.

BACKGROUND: Perivascular collar placement has been used as a means for localized atherosclerosis induction in a variety of experimental animal species. In mice, however, atherosclerosis-like lesions have thus far not been obtained by this method. The aim of this study was the development of a mouse model of rapid, site-controlled atherogenesis. METHODS AND RESULTS: Silastic collars were placed around the carotid arteries of apolipoprotein E-deficient (apoE-/-) and LDL receptor-deficient (LDLr-/-) mice. The development of collar-induced lesions was found to occur predominantly in the area proximal to the collar and to be dependent on a high-cholesterol diet. Lesions were evident in apoE-/- mice after 3 weeks and in LDLr-/- mice after 6 weeks and were overtly atherosclerotic in appearance. Lumen stenosis reached 85% in apoE-/- mice and 61% in LDLr-/- mice 6 weeks after collar insertion. Expression levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were increased both proximal and distal to the collar, whereas endothelial nitric oxide synthase expression was downregulated at the proximal site. CONCLUSIONS: We propose that this model of collar-induced acceleration of carotid atherogenesis is of hemodynamic cause. It may serve as a substrate for sequential mechanistic studies concerned with the underlying cause and pathogenesis of atherosclerosis. The rapidity of lesion development will also aid the efficient screening of new potentially antiatherogenic chemical entities and the evaluation of therapies with limited duration of effectiveness, such as adenoviral gene therapy.

Animals↗
PubMed2001

A structure-function study of ligand recognition by CD22beta.

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.

Animals↗
PubMed2001

Effect of dietary elaidic versus vaccenic acid on blood and liver lipids in the hamster.

Male hamsters (30 per group) were fed five different semi-purified diets ad libitum. The diets, containing 30% of energy (en%) as fat, differed in their dietary fat composition (specified fatty acids exchanged at 10 en%) and were fed for 4 weeks. The five fatty acids compared in mixed triglycerides were elaidic acid (C18:1 9t), vaccenic acid (C18:1 11t), their cis-counterpart oleic acid (C18:1 9c), medium-chain fatty acids (MCFA; C8:0 and C10:0), and palmitic acid (C16:0). Compared with oleic acid, dietary MCFA and palmitic acid tended to increase blood cholesterol levels in the hamsters. The effect of elaidic and vaccenic acid on blood cholesterol did not differ from that of oleic acid. When elaidic acid and vaccenic acids were compared directly, the ratio of LDL/HDL-cholesterol in plasma was significantly higher in hamsters fed vaccenic acid than in those fed elaidic acid, and elaidic acid was incorporated at low levels, but more efficiently than vaccenic acid at the sn-2 position of platelet phospholipids. Biological consequences of this low incorporation are considered unlikely as levels of arachidonic acid (C20:4 n-6) and docosohexaenoic acid (C22:6 n-3) in the platelet phospholipids of all dietary groups did not differ. With respect to the effect on the LDL/HDL-cholesterol ratio, elaidic acid may be preferable to vaccenic acid. We conclude that this animal study does not provide evidence for the suggestion, based on epidemiological observations, that elaidic acid would be more detrimental to cardiovascular risk than vaccenic acid.

Animals↗
PubMed2001

Design and synthesis of a multivalent homing device for targeting to murine CD22.

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail. and lead to an undesired immune response, for example in cases of allergy, rheumatoid arthritis and Crohn's disease. In this situation, synthetic high-affinity ligands for CD22 may be of therapeutic value as inhibitors of B-cell activation. Recent studies have revealed that natural ligands for CD22 contain the trisaccharide NeuAc alpha-2,6-Lac as the basic binding motif. In addition, it has been demonstrated that binding to CD22 is strongly enhanced by multivalent presentation of the basic binding motif (cluster effect). In this paper. the stepwise development of a novel multivalent high-affinity ligand for CD22 is described. In the first stage, a series of monovalent NeuAc alpha-2,6-Glc(Y)X type binding motifs was prepared, and their affinity for murine CD22 was monitored, to obtain more insight into the effect of separate structure elements on ligand recognition. In the second stage, we prepared a trivalent cluster, based on the monovalent motif that displayed the highest affinity for CD22, NeuAc alpha-2,6-GlcNBzNO2OMe (7). This cluster, TRIS(NeuAc alpha-2,6-GlcNBzNO2)3 (52), displayed a more than 58-fold higher affinity for CD22 than the reference structure NeuAc alpha-2,6-LacOMe (10). To our knowledge, the cluster 52 is one of the most potent antagonists for CD22 yet synthesised.

Animals↗
PubMed2001

Carrier-mediated delivery improves the efficacy of 9-(2-phosphonylmethoxyethyl)adenine against hepatitis B virus.

We recently synthesized a lipophilic prodrug of 9-(2-phosphonyl-methoxyethyl)adenine (PMEA), designated PMEA-LO, and incorporated it into reconstituted lactosylated high-density lipoprotein (LacNeoHDL). In a rat model, LacNeoHDL-associated PMEA-LO was internalized by the asialoglycoprotein receptor on parenchymal liver cells and converted into its active diphosphorylated metabolite. To further evaluate the therapeutic potential of the carrier-associated prodrug, we examined in this study the processing of (125)I-labeled PMEA-LO--loaded LacNeoHDL by HepG2 cells. Upon incubation with HepG2 cells, PMEA-LO--loaded LacNeoHDL became rapidly cell-associated. The association was saturable and of high-affinity (k(d) = 3.8 +/- 0.4 nM). Asialofetuin, an established ligand for the asialoglycoprotein receptor, inhibited the association by >75%, which confirms the role of the asialoglycoprotein receptor. Association of the prodrug-loaded particles to HepG2 cells was coupled to degradation. Radiolabeled degradation products appeared in the culture medium with a lag phase of 2 h. Asialofetuin and chloroquine inhibited secretion of degradation products by 75 to 80%, indicating that PMEA-LO--loaded LacNeoHDL is internalized via the asialoglycoprotein receptor and lysosomally processed. The therapeutic potential of LacNeoHDL-associated PMEA-LO was studied by measuring its effects on hepatitis B virus (HBV) replication in Hep AD38 cells (HBV-transfected HepG2 cells). LacNeoHDL-associated PMEA-LO effectively inhibited HBV DNA synthesis. The EC(50) value of carrier-associated PMEA-LO was 35 times lower than that of free PMEA (3.4 +/- 0.4 and 120 +/- 18 ng of PMEA/ml, respectively). We conclude that the present results, combined with our earlier in vivo disposition data, underscore the therapeutic potential and utility of PMEA-LO--loaded LacNeoHDL for treatment of chronic hepatitis B.

Adenine↗
PubMed2001

Tissue distribution of factor VIII gene expression in vivo--a closer look.

Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.

Animals↗
PubMed2001

Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice.

Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3(-/-)) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3(-/-) mice with apoE(-/-) mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE(+/+) apoC3(-/-) mice, apoE(-/-)apoC3(-/-) mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3(-/-) mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3(-/-) mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3(-/-) mice. Thus the mechanisms underlying the hypolipidemia in apoC3(-/-) mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma. In summary, our studies of apoC3(-/-) mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters.

Animals↗
PubMed2000

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Animals↗
PubMed2000

Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein.

Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid-ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/- 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins.

Animals↗
PubMed2000

Apolipoprotein E is resistant to intracellular degradation in vitro and in vivo. Evidence for retroendocytosis.

Apolipoprotein E (apoE) is an important determinant for the uptake of triglyceride-rich lipoproteins and emulsions by the liver, but the intracellular pathway of apoE following particle internalization is poorly defined. In the present study, we investigated whether retroendocytosis is a unique feature of apoE as compared with apoB by studying the intracellular fate of very low density lipoprotein-sized apoE-containing triglyceride-rich emulsion particles and LDL after LDLr-mediated uptake. Incubation of HepG2 cells with [(3)H]cholesteryl oleate-labeled particles at 37 degrees C led to a rapid release of [(3)H]cholesterol within 30 min for both LDL and emulsion particles. In contrast, emulsion-derived (125)I-apoE was more resistant to degradation (>/=120 min) than LDL-derived (125)I-apoB (30 min). Incubation at 18 degrees C, which allows endosomal uptake but prevents lysosomal degradation, with subsequent incubation at 37 degrees C resulted in a time-dependent release of intact apoE from the cells (up to 14% of the endocytosed apoE at 4 h). The release of apoE was accelerated by the presence of protein-free emulsion (20%) or high density lipoprotein (26%). Retroendocytosis of intact particles could be excluded since little intact [(3)H]cholesteryl oleate was released (<3%). In contrast, the degradation of LDL was complete with virtually no secretion of intact apoB into the medium. The intracellular stability of apoE was also demonstrated after hepatic uptake in C57Bl/6 mice. Intravenous injection of (125)I-apoE and [(3)H]cholesteryl oleate-labeled emulsions resulted in efficient LDLr-mediated uptake of both components by the liver (45-50% of the injected dose after 20 min). At 1 h after injection, only 15-20% of the hepatic (125)I-apoE was degraded, whereas 75% of the [(3)H]cholesteryl oleate was hydrolyzed. From these data we conclude that following LDLr-mediated internalization by liver cells, apoE can escape degradation and can be resecreted. This sequence of events may allow apoE to participate in its hypothesized intracellular functions such as mediator of the post-lysosomal trafficking of lipids and very low density lipoprotein assembly.

Animals↗
PubMed2000

Scavenger receptors on liver Kupffer cells mediate the in vivo uptake of oxidatively damaged red blood cells in mice.

In vitro studies have shown that damaged red cells and apoptotic cells are efficiently phagocytosed by scavenger receptors from macrophages, even under non-opsonizing conditions. Damaged red blood cells are in vivo effectively removed from the blood circulation, but the responsible receptor systems are largely unknown. We used a murine model in which (51)Cr-labeled oxidized red blood cells were injected intravenously, and the cellular uptake sites and the potential involvement of scavenger receptors were analyzed. The decay of damaged red cells was rapid (more than 50% removed within 10 minutes after injection), whereas native red cells were not cleared. The main site of uptake of damaged red cells was the liver Kupffer cells, which contained 24% of the injected dose at 10 minutes after injection. The blood decay and liver uptake were inhibited by typical ligands for scavenger receptors, such as polyinosinic acid, liposomes containing phosphatidylserine, oxidized low-density lipoprotein, and fucoidan, but not by polyadenosinic acid or liposomes without phosphatidylserine. Mice lacking scavenger receptors class A type I and II showed no significant decrease in the ability to take up damaged red cells from the circulation. We conclude that Kupffer cells are mainly responsible for the removal of damaged red cells from the blood circulation, a process mediated by polyinosinic acid- and phosphatidylserine-sensitive scavenger receptors, different from scavenger receptor class A type I and II. Our data indicate that scavenger receptors, as pattern-recognizing receptors, play an important role in vivo in the removal of apoptotic, damaged, or other unwanted cells from the blood circulation. (Blood. 2000;95:2157-2163)

Animals↗
PubMed2000

Carrier-mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to parenchymal liver cells: a novel therapeutic approach for hepatitis B.

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3alpha-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [(3)H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% +/- 7.7% of the dose (free [(3)H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [(3)H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% +/- 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

Adenine↗