Selective liver targeting of antivirals by recombinant chylomicrons--a new therapeutic approach to hepatitis B.

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.

Analytical study of microsomes and isolated subcellular membranes from rat liver. IX. Nicotinamide adenine dinucleotide glycohydrolase: a plasma membrane enzyme prominently found in Kupffer cells.

The distribution of nicotinamide adenine dinucleotide (NAD) glycohydrolase in rat liver was investigated by subcellular fractionation and by isolation of hepatocytes and sinusoidal cells. The behavior of NAD glycohydrolase in subcellular fractionation was peculiar because, although the enzyme was mainly microsomal, plasma membrane preparations contained distinctly more NAD glycohydrolase than could be accounted for by their content in elements derived from the endoplasmic reticulum or the Golgi complex identified by glucose-6-phosphatase and galactosyltransferase, respectively. When microsomal and plasmalemmal preparations were brought to equilibrium in a linear-density gradient, NAD glycohydrolase differed from these enzymes and behaved like 5'-nucleotidase and alkaline phosphodiesterase I. NAD glycohydrolase was markedly displaced towards higher densities after treatment with digitonin. This behavior in density-gradient centrifugation strongly suggests that NAD glycohydrolase is an exclusive enzyme of the plasma membrane. NAD glycohydrolase differed clearly from other plasmalemmal enzymes when the liver was fractionated into hepatocytes and sinusoidal cells; its specific activity was considerably greater in sinusoidal cell than in hepatocyte preparations. Further subfractionation of sinusoidal cell preparations into endothelial and Kupffer cells by counterflow elutriation showed that NAD glycohydrolase is more active in Kupffer cells. We estimate that the specific activity of NAD glycohydrolase activity is at least 65-fold higher at the periphery of Kupffer cells than at the periphery of hepatocytes. As the enzyme shows not structure-linked latency and is an exclusive constituent of the plasma membranes, we conclude that it is an ectoenzyme that cannot lead to a rapid turnover of the cytosolic pyridine nucleotides.

CC chemokine ligand-5 (CCL5/RANTES) and CC chemokine ligand-18 (CCL18/PARC) are specific markers of refractory unstable angina pectoris and are transiently raised during severe ischemic symptoms.

BACKGROUND: Chemokines play an important role in atherogenesis and in ischemic injury and repair; however, prospective data on individual chemokines in unstable angina pectoris (UAP) are scarce. Therefore, we assessed chemokine patterns in a prospective cohort of patients with UAP. METHODS AND RESULTS: Plasma samples of 54 patients with Braunwald class IIIB UAP were examined at baseline for 11 chemokines and 5 inflammatory mediators via multiplex analysis. Levels of CC chemokine ligand (CCL)-5 (also known as RANTES [regulated on activation, normally T-cell expressed, and secreted]; 32.7 versus 23.1 ng/mL, P=0.018) and CCL18 (also known as PARC [pulmonary and activation-regulated chemokine]; 104.4 versus 53.7 ng/mL, P=0.011) were significantly elevated in patients with refractory ischemic symptoms versus stabilized patients. Temporal monitoring by ELISA of CCL5, CCL18, and soluble CD40 ligand (sCD40) levels revealed a drop in CCL5 and sCD40L levels in all UAP patients from day 2 onward (CCL5 12.1 ng/mL, P<0.001; sCD40L 1.35 ng/mL, P<0.05), whereas elevated CCL18 levels were sustained for at least 2 days, then were decreased at 180 days after inclusion (34.5 ng/mL, P<0.001). Peripheral blood mononuclear cells showed increased protein expression of chemokine receptors CCR3 and CCR5 in CD3+ and CD14+ cells at baseline compared with 180 days after inclusion, whereas mRNA levels were downregulated, which was attributable in part to a postischemic release of human neutrophil peptide-3-positive neutrophils and in part to negative feedback. Finally, elevated CCL5 and CCL18 levels predicted future cardiovascular adverse events, whereas C-reactive protein and sCD40L levels did not. CONCLUSIONS: We are the first to report that CCL18 and CCL5 are transiently raised during episodes of UAP, and peak levels of both chemokines are indicative of refractory symptoms. Because levels of both chemokines, as well as of cognate receptor expression by circulating peripheral blood mononuclear cells, are increased during cardiac ischemia, this may point to an involvement of CCL5/CCL18 in the pathophysiology of UAP and/or post-UAP responses.

Reduced leucocyte cholesteryl ester transfer protein expression in acute coronary syndromes.

OBJECTIVE: Cholesterol ester transfer protein (CETP) plays an important role in HDL cholesterol metabolism. Leucocytes, including monocyte-derived macrophages in the arterial wall synthesize and secrete CETP, but its role in atherosclerosis is unclear. The aim of the current study was to investigate the effect of acute coronary syndromes (ACS) on leucocyte CETP expression. RESEARCH DESIGN: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from hospitalized ACS patients displaying Braunwald class IIIB unstable angina pectoris (UAP) on admission (t = 0) and at 180 days post inclusion (t = 180) for analysis of CETP expression. In addition, to prove the potential correlation between leucocyte CETP and ACS the effect of acute myocardial infarction on leucocyte CETP expression was studied in CETP transgenic mice. RESULTS: Upon admission, UAP patients displayed approximately 3-6 fold (P < 0.01) lower CETP mRNA and nearly absent CETP protein expression in PBMCs, as compared to healthy age-/sex-matched controls. Interestingly, CETP mRNA and protein levels were significantly elevated in PBMCs isolated from UAP patients (both stabilized and refractory) at t = 180 as compared to t = 0 (P < 0.01), which was correlated with a reduced inflammatory status after medical treatment. In agreement with the data obtained in UAP patients, markedly down-regulated leucocyte CETP mRNA expression was observed after coronary artery ligation in CETP transgenic mice, which also correlated with increased serum amyloid A levels. CONCLUSIONS: We are the first to report that episodes of UAP in humans and myocardial infarction in CETP transgenic mice are associated with reduced leucocyte CETP expression. We propose that the impairment in leucocyte CETP production is associated with an enhanced inflammatory status, which could be clinically relevant for the pathogenesis of ACS.

Induction of oral tolerance to oxidized low-density lipoprotein ameliorates atherosclerosis.

BACKGROUND: Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis. METHODS AND RESULTS: Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor-/- mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+ CD25+ Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-beta production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis. CONCLUSIONS: OxLDL-specific T cells, present in LDL receptor-/- mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+ CD25+ Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Human recombinant apolipoprotein E redirects lipopolysaccharide from Kupffer cells to liver parenchymal cells in rats In vivo.

Chylomicrons have been shown to protect mice and rats against a lethal dose of lipopolysaccharide and may serve as a therapeutic means to protect against endotoxemia. However, the requisite of isolation from human lymph hampers pharmaceutical application. Recently, we developed recombinant chylomicrons from commercially available lipids and human recombinant apolipoprotein E. The current study explored the effectiveness of these apoE-enriched emulsions in redirecting LPS from Kupffer cells to liver parenchymal cells. Upon injection into rats, 125I-LPS rapidly and specifically associated with the liver (64.3+/-3.1% of the injected dose) and spleen (4.1+/-0.7%). The uptake of LPS by the spleen was four- to fivefold reduced upon incubation with the apoE-enriched emulsion or free apoE (P < 0.0001), but not with emulsion alone or Lipofundin. Within the liver, 125I-LPS mainly associated with Kupffer cells. The uptake by Kupffer cells was eight- to ninefold reduced by the apoE-enriched emulsion or apoE alone (P < 0.01), and a 19.6-fold increased uptake ratio by liver parenchymal cells over Kupffer cells was observed. The emulsion without apoE had no effect on the in vivo kinetics of LPS. LPS interacted selectively with the apoE moiety of the recombinant chylomicron. Emulsion-associated and free apoE bound approximately two molecules of LPS, possibly by its exposed hydrophilic domain involving arginine residues. We anticipate that the protecting effect of endogenous chylomicrons against LPS-induced endotoxemia may result from the apoE moiety and that human recombinant apoE may serve as a therapeuticum to protect against endotoxemia.

Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E- deficient mouse hepatocytes.

To explore mechanisms underlying triglyceride (TG) accumulation in livers of chow-fed apo E-deficient mice (Kuipers, F., J.M. van Ree, M.H. Hofker, H. Wolters, G. In't Veld, R.J. Vonk, H.M.G. Princen, and L.M. Havekes. 1996. Hepatology. 24:241-247), we investigated the effects of apo E deficiency on secretion of VLDL-associated TG (a) in vivo in mice, (b) in isolated perfused mouse livers, and (c) in cultured mouse hepatocytes. (a) Hepatic VLDL-TG production rate in vivo, determined after Triton WR1339 injection, was reduced by 46% in apo E-deficient mice compared with controls. To eliminate the possibility that impaired VLDL secretion is caused by aspecific changes in hepatic function due to hypercholesterolemia, VLDL-TG production rates were also measured in apo E-deficient mice after transplantation of wild-type mouse bone marrow. Bone marrow- transplanted apo E-deficient mice, which do not express apo E in hepatocytes, showed normalized plasma cholesterol levels, but VLDL-TG production was reduced by 59%. (b) VLDL-TG production by isolated perfused livers from apo E-deficient mice was 50% lower than production by livers from control mice. Lipid composition of nascent VLDL particles isolated from the perfusate was similar for both groups. (c) Mass VLDL-TG secretion by cultured apo E-deficient hepatocytes was reduced by 23% compared with control values in serum-free medium, and by 61% in the presence of oleate in medium (0. 75 mM) to stimulate lipogenesis. Electron microscopic evaluation revealed a smaller average size for VLDL particles produced by apo E-deficient cells compared with control cells in the presence of oleate (38 and 49 nm, respectively). In short-term labeling studies, apo E-deficient and control cells showed a similar time-dependent accumulation of [3H]TG formed from [3H]glycerol, yet secretion of newly synthesized VLDL-associated [3H]TG by apo E-deficient cells was reduced by 60 and 73% in the absence and presence of oleate, respectively. We conclude that apo E, in addition to its role in lipoprotein clearance, has a physiological function in the VLDL assembly-secretion cascade.

Blockade of interleukin-12 function by protein vaccination attenuates atherosclerosis.

BACKGROUND: Interleukin-12 (IL-12) has been identified as a key inducer of a type 1 T-helper cell cytokine pattern, which is thought to contribute to the development of atherosclerosis. We sought to study the role of IL-12 in atherosclerosis by inhibition of IL-12 using a newly developed vaccination technique that fully blocks the action of IL-12. METHODS AND RESULTS: LDL receptor-deficient (LDLr(-/-)) mice were vaccinated against IL-12 by 5 intramuscular injections of IL-12-PADRE complex in combination with adjuvant oil-in-water emulsion (low dose)/MPL/QS21 every 2 weeks. Two weeks thereafter, atherogenesis was initiated in the carotid artery by perivascular placement of silicone elastomer collars. IL-12 vaccination resulted in the induction of anti-IL-12 antibodies that functionally blocked the action of IL-12 as determined in an IL-12 bioassay. Blockade of IL-12 by vaccination of LDLr(-/-) mice resulted in significantly reduced (68.5%; P<0.01) atherogenesis compared with control mice without a change in serum cholesterol levels. IL-12 vaccination also resulted in a significant decrease in intima/media ratios (66.7%; P<0.01) and in the degree of stenosis (57.8%; P<0.01). On IL-12 vaccination, smooth muscle cell and collagen content in the neointima increased 2.8-fold (P<0.01) and 4.2-fold (P<0.01), respectively. CONCLUSIONS: Functional blockade of endogenous IL-12 by vaccination resulted in a significant 68.5% reduction in atherogenesis in LDLr(-/-) mice. Vaccination against IL-12 also improved plaque stability, from which we conclude that the blockade of IL-12 by vaccination may be considered a promising new strategy in the treatment of atherosclerosis.

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.

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.

Prolonged serum half-life of antineoplastic drugs by incorporation into the low density lipoprotein.

In vitro and in vivo data have indicated that tumor cells actively internalize the low density lipoprotein (LDL) from the circulation. In order to achieve a selective delivery of drugs to tumor cells via the LDL pathway, we have incorporated oleyl derivatives of methotrexate and floxuridine (FdUrd) into LDL particles. Three different incorporation procedures were studied: Method A, the dry film method; Method B, the transfer protein method; and Method C, the delipidation-reconstitution method. In all cases, 3H-labeled drug was incorporated into 125I-labeled LDL to yield double-labeled particles so that the behavior of both drug and carrier could be followed simultaneously. The last method led to the highest drug loading and it was possible to incorporate 50-70 molecules of dioleoyl-FdUrd per LDL particle as compared with about 18 molecules of drug when utilizing the transfer protein procedure. In vitro studies on the interaction of dioleoyl-FdUrd-LDL particles, obtained by the delipidation-reconstitution method, with the hepatocellular carcinoma cell line Hep G2, indicated that these reconstituted particles were equally effective in competing for LDL binding as native LDL. Moreover, drug delivery to Hep G2 cells occurred at the same rate as cellular association of the apolipoprotein B. In vivo studies on the fate of dioleoyl-FdUrd-LDL complexes in rats indicated that the serum decay was increased as compared with native LDL. The half-life of 6-9 min is, however, considerably prolonged as compared to the free drug (t1/2 less than 1 min). It is suggested that the 6-fold increased serum half-life of the drug-LDL complex accompanied by the possibly more specific tumor delivery may lead to an increased therapeutic effect.

In vivo characteristics of a specific recognition site for LDL on non-parenchymal rat liver cells which differs from the 17 alpha-ethinyl estradiol-induced LDL receptor on parenchymal liver cells.

Chemical modification of lysine or arginine residues of apolipoprotein B-100 in human low-density lipoprotein (LDL) with respectively reductive methylation (Me-LDL) or cyclohexanedione treatment (CHD-LDL) was applied to determine the role of these amino acids in LDL recognition by the various liver cell types. The cell association of native human LDL, Me-LDL and CHD-LDL to parenchymal and non-parenchymal cells was determined in vivo by isolating the various cell types 30 min after intravenous injection of the lipoproteins. In order to prevent degradation or release of cell-bound apolipoproteins during cell dissociation and purification, a low-temperature (8 degrees C) liver perfusion and cell isolation procedure was performed. It was found that reductive methylation of LDL inhibits the association of LDL to both parenchymal and non-parenchymal cells, indicating that lysine residues are important for recognition of LDL by both these cell types. In contrast, cyclohexanedione treatment of LDL did not influence the cell association of LDL to non-parenchymal cells. 17 alpha-Ethinyl estradiol treatment selectively increases the cell association of LDL by parenchymal cells (16-fold), leaving the non-parenchymal cell association uninfluenced. The increased cell-association of LDL to parenchymal cells is almost completely blocked by cyclohexanedione treatment of LDL (by 81%) or by methylation of LDL (by 97%). These data indicate that the arginine residues in LDL are not important for the recognition of LDL by non-parenchymal cells, whereas for the cell association of LDL to the estrogen-stimulated binding site on parenchymal cells both arginine and lysine residues are essential. The in vivo cell association of CHD-LDL or native LDL to non-parenchymal cells was lowered to the level of Me-LDL by ethyl oleate treatment of the rats, while no effect of ethyl oleate on parenchymal cells was noticed. These data suggest that the specific site for LDL on non-parenchymal cells, which need lysine residues on LDL for recognition, can be down-regulated by ethyl oleate treatment. The LDL, internalized by non-parenchymal cells, is effectively degraded. This degradation occurs at least partly in the lysosomes. It is suggested that the unique recognition site for LDL on non-parenchymal cells may be quantitatively important for serum LDL catabolism.

A saturable, high-affinity binding site for human low density lipoprotein on freshly isolated rat hepatocytes.

Freshly isolated rat hepatocytes bind the solely apolipoprotein B-containing human low density lipoprotein (LDL) with a high-affinity component. After 1 h of incubation less than 30% of the cell-associated human LDL is internalized and no evidence for any subsequent high-affinity degradation was obtained. Scatchard analysis of the binding data for human 125I-labeled LDL indicates that the high-affinity receptor for human LDL on rat hepatocytes possesses a Kd of 2.6 x 10(-8)M, while the binding is dependent on the extracellular Ca2+ concentration. Competition experiments indicate that both the apolipoprotein B-containing lipoproteins (human LDL and rat LDL) as well as the apolipoprotein E-containing lipoproteins (human HDL and rat HDL) do compete for the same surface receptor. It is concluded that hepatocytes freshly isolated from untreated rats do contain, in addition to the earlier described rat lipoprotein receptor which does not interact with human apolipoprotein B-containing LDL, a high-affinity receptor which interacts both with solely apolipoprotein B-containing human LDL and apolipoprotein E-containing lipoproteins.

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Nonparenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.

Uptake and degradation of rat and human very low density (remnant) apolipoprotein by parenchymal and non-parenchymal rat liver cells.

1. The relative contribution of parenchymal and non-parenchymal cells to the in vivo hepatic uptake of serum apolipoproteins was measured 30 min after intravenous injection of radioiodinated rat very low density lipoprotein (VLDL) remnants, rat and human VLDL, low density lipoprotein (LDL) and high density lipoprotein (HDL). Using rat VLDL, VLDL-remnants, LDL and HDL, respectively, the non-parenchymal cells contain 4.7, 4.9, 6.1 and 5.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For human VLDL, LDL and HDL these values are 5.1, 12.0 and 5.9 respectively. 2. The abilities of homogenates of human liver, rat liver parenchymal cells and rat liver non-parenchymal cells to hydrolyze human and rat iodinated VLDL apoprotein were determined by measuring the amount of trichloroacetic acid-soluble (non-iodide) radioactivity liberated upon incubation at the optimal pH of 4.2. Non-parenchymal cells possess a 8--21-fold higher maximal capacity to degrade VLDL apoprotein per mg of cell protein than parenchymal cells. This factor is 5--6 for VLDL-remnant apoprotein degradation measured at low (suboptimal) apolipoprotein concentrations. 3. It is concluded that, in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake and possibly degradation of VLDL-(remnant) apoprotein.

Rapid transport of fatty acids from rat liver endothelial to parenchymal cells after uptake of cholesteryl ester-labeled acetylated LDL.

Acetylated low-density lipoprotein (acetyl-LDL) radiolabeled in the oleate moiety of cholesteryloleate was injected into rats. Isolation of the various liver cell types at different times after acetyl-LDL injection by a low-temperature procedure allowed the intrahepatic metabolism of the oleate moiety to be followed in vivo. The cholesteryloleate radioactivity is rapidly cleared from the circulation and at 5 min after injection recovered into parenchymal and endothelial liver cells, mainly as cholesteryloleate ester. At longer time intervals after injection, the amount of cholesteryl esters associated with the endothelial cells was sharply decreased and the [14C]oleate was redistributed within the liver and mainly recovered in the parenchymal cells. The cholesteryl ester initially directly taken up by the parenchymal cells was also rapidly hydrolysed but, in contrast to the endothelial cells, the [14C]oleate remained inside the cells and was incorporated into triacylglycerols and phospholipids. The 14C radioactivity in parenchymal cells taken up between 5 and 30 min after injection of the cholesteryl [14C]oleate-labeled acetyl-LDL (transported as oleate from endothelial cells), followed a similar metabolic route as the amount which was directly associated to parenchymal cells. The data indicate that the liver and, in particular, the liver endothelial cell has the full capacity to rapidly catabolize modified lipoproteins. In this catabolism, the liver functions as an integrated organ in which fatty acids, formed from cholesteryl esters in endothelial cells, are rapidly transported to parenchymal cells, indicating the concept of metabolic cooperation between the various liver cell types.

Identification of prostaglandin D2 as the major eicosanoid from liver endothelial and Kupffer cells.

The capacity of freshly isolated endothelial, Kupffer and parenchymal rat liver cells to produce eicosanoids from [1-14C]arachidonic acid was investigated in order to determine the relative importance of these cells to total liver eicosanoid production. Based upon the total formation of [1-14C]arachidonate metabolites in the liver, it can be calculated that Kupffer and endothelial cells are responsible for 65 and 23%, respectively, of the total amount of eicosanoids produced by the liver. Consequently, parenchymal liver cells, representing 92.5% of the total liver mass, contribute only 12% to the total liver production of eicosanoids. The main product of Kupffer cells was prostaglandin D2 (PGD2), representing 55% of the total amount of eicosanoids produced. Liver endothelial cells produced about 4-times less eicosanoids (per mg cell protein) than Kupffer cells, and PGD2 was also the main product of these cells (44%). The production of eicosanoids by parenchymal cells was lower by a factor of 180 (per mg cell protein) than that in Kupffer cells. Besides the ability to form eicosanoids from added 14C-labeled arachidonic acid, Kupffer and endothelial liver cells were also able to produce significant amounts of PGD2 (the main liver prostaglandin) from endogenous arachidonic acid, as determined by a radioimmunoassay. It is concluded that inside the liver, Kupffer cells together with endothelial cells are of major importance in the production of eicosanoids, while the parenchymal cells may be considered metabolic target cells for these products, as indicated by the finding that the major liver prostaglandin, PGD2, could stimulate the glucose output in isolated parenchymal cells.

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.

On the lipid peroxidation of rat liver hepatocytes, the formation of fluorescent chromolipids and high molecular weight protein.

1. The formation of malondialdehyde by intact hepatocytes, induced by ADP/Fe3+ or cumene hydroperoxide, can be inhibited by the addition of thiourea. This may indicate that hydroxyl radicals are involved in this process. 2. Lipid peroxidation of intact hepatocytes leads to the formation of fluorescent chromolipids. When similar amounts of malondialdehyde are formed by either ADP/Fe3+ or cumene hydroperoxide, the lipid peroxidation induced by cumene hydroperoxide generates more fluorescent chromolipids than does the lipid peroxidation induced by ADP/Fe3+. 3. The formation of chromolipids is accompanied by the genesis of high molecular weight protein. With cumene hydroperoxide more high molecular weight protein is formed than with ADP/Fe3+. 4. It can be concluded that the defense system against lipid peroxidation of intact hepatocytes does not prevent the formation of lipofuscin-like chromolipids and high molecular weight protein as found earlier in microsomes. Cumene hydroperoxide, at least in this system, can be considered as an effective inducer of chromolipids.

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells.

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.