Targeting of tumor cells by low density lipoproteins: principle and use of ellipticin derivatives]

Cells acquire cholesterol via de novo synthesis and high affinity receptor-mediated uptake of low-density lipoprotein (LDL). Some tumor tissues display increased receptor-mediated uptake of LDL as compared with the corresponding normal tissues. This increased LDL receptor activity is unexplained: a high cholesterol demand for cell growth or a mechanism directly linked to cell transformation. LDL has therefore been proposed as a potential carrier for chemotherapeutic agents. Various methods have been used to incorporate antineoplastic lipophilic drugs into LDL. The resultant drug-LDL complexes have been shown to be cytotoxic towards tumor cells in vitro, via the LDL receptor dependent pathway. However little is now on the in vivo fate of this complex. We described the incorporation of lipophilic derivatives of ellipticine into LDL by a fusion or facilitated transfer technique between drug containing microemulsions and LDL. The drug-LDL complex expressed similar metabolic activity, in vitro and in vivo, than native LDL. Initial experiments with melanoma B16 tumor-bearing mice suggest that LDL may be a potential drug carrier in the treatment of malignant diseases. The knowledge of the molecular mechanism of the expression of the LDL receptor in tumor cells and the ability to downregulate the LDL receptor in the normal tissues, will define the application field of this targeting approach.     

Role of a calf thymus preparation in the degradation of native and reductively methylated low density lipoprotein.

1. The clearance of low density lipoprotein (LDL) is mediated by a specific LDL receptor pathway and by an alternative metabolic pathway that is responsible for the receptor-independent LDL catabolism. 2. This alternative catabolism can be studied in vivo using a preparation of chemically modified LDL that are reductively methylated. 3. Recently we showed that a calf thymus protein extract affects the cholesterol metabolism via activation of LDL catabolism. 4. The aim of this study was to investigate whether in vivo the specific LDL receptor pathway and the independent LDL receptor pathway are affected by thymus treatment. 5. The results obtained injecting in rats native and chemically modified 125I-LDL to probe the receptor independent pathway, show that the thymus gland decreases serum cholesterol by activation of the specific LDL receptor pathway. 6. This effect is mainly evident in liver and kidney that represent organs in which the specific LDL receptors are widely present.     

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.     

LDL transcytosis by protein membrane diffusion

Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.     

A synthetic low density lipoprotein particle capable of supporting U937 proliferation in vitro.

A synthetic LDL (sLDL) has been prepared by combining a lipid microemulsion with amphipathic peptides containing the apoprotein B receptor domain. The biological properties of sLDL have been investigated using the U937 in vitro cell proliferation assay. sLDL exhibits a concentration dependent and saturable stimulation of U937 proliferation. By utilizing different amphipathic peptides, variable proliferation is achieved, indicating a specific interaction between sLDL and the U937 LDL receptor are possible. U937 proliferation is reduced by the addition of an anti-LDL receptor antibody, indicating that sLDL is assimilated via the LDL receptor pathway.The behavior of sLDL mimics that of native LDL, and this approach represents a viable technique for the production of an sLDL particle on a large scale for research and general application.     

Mathematical Model for Low Density Lipoprotein (LDL) Endocytosis by Hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL–LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.     

Faster lactate transport across red blood cell membrane in sickle cell trait carriers.

The physical and physiological behavior of sickle cell trait carriers (AS) is somewhat equivocal under strenuous conditions, although this genetic abnormality is generally considered to be a benign disorder. The occurrence of incidents and severe injuries in AS during exercise might be explained, in part, by the lactic acidosis due to a greater lactate influx into AS red blood cells (RBCs). In the present study, the RBC lactate transport activity via the different pathways was compared between AS and individuals with normal hemoglobin (AA). Sixteen Caribbean students, nine AS and seven AA, performed a progressive and maximal exercise test to determine maximal oxygen consumption. Blood samples were obtained at rest to assess haematological parameters and RBC lactate transport activity. Lactate influxes [total lactate influx and monocarboxylate transporter (MCT-1)-mediated lactate influx] into erythrocytes were measured at four external [14C]-labeled lactate concentrations (1.6, 8.1, 41, and 81.1 mM). The two groups had similar maximal oxygen consumption. Total lactate influx and lactate influx via the MCT-1 pathway were significantly higher in AS compared with AA at 1.6, 41, and 81.1 mM. The maximal lactate transport capacity for MCT-1 was higher in AS than in AA. Although AS and AA had the same maximal aerobic physical fitness, the RBCs from the sickle cell trait carriers took up more lactate at low and high concentrations than the RBCs from AA individuals. The higher MCT-1 maximal lactate transport capacity found in AS suggests greater content or greater activity of MCT-1 in AS RBC membranes.     

Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.     

Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma

In vitro, metabolism of modified forms of low density lipoprotein (LDL) by macrophages via the acetyl-LDL receptor pathway promotes the massive cellular accumulation of lipid. It has been postulated that in vivo this contributes to foam cell formation in the atherosclerotic lesion. Recent studies have shown that arterial wall cells in vitro can secrete a number of cytokines, several of which have been reported to modulate macrophage cell function. Thus, cytokines have the potential to modulate the acetyl-LDL receptor pathway and to influence the rate of foam cell generation. To study the regulation of this pathway by cytokines, the effect of cytokines on the degradation of acetyl-LDL protein by mouse peritoneal macrophages was examined. Initially, supernatant from stimulated lymphocytes was used as a source of cytokines. Macrophages preincubated with supernatants obtained after the stimulation of T-cell helper type 1 (Th1) clone HDK-1 or BALB/c spleen cells degraded acetyl-LDL at a slower rate, whereas supernatant from stimulated T-cell helper type 2 (Th2) clone D-10 had no effect. Comparison of the lymphokine profiles showed that spleen and HDK-1 cells secreted several lymphokines in common including significant levels of interferon-gamma. Interferon-gamma was then directly shown to be inhibitory; an anti-interferon-gamma monoclonal antibody blocked the HDK-1-mediated inhibition by 70% and the addition of recombinant interferon-gamma (IFN-gamma) to macrophages inhibited the specific degradation of acetyl-LDL in a dose- and time-dependent manner with a maximum suppression to approximately 40% of control. The inhibition was not accompanied by an increase in the amount of cell-associated acetyl-LDL and was not due to cell death nor could it be accounted for by the presence of endotoxin. To study the mechanism of the inhibition, the effects of IFN-gamma on the itinerary of acetyl-LDL and its receptor were examined. IFN-gamma decreased specific acetyl-LDL binding only to a small degree, and the rate of lysosome-mediated degradation was not affected. The principal alteration was in the rate of transport to the lysosome which was markedly slowed. Since the receptors eventually returned to the surface to maintain a steady state, and there was not an increase in cell-associated lipoprotein, there must be other changes in the itinerary that were not identified with the techniques used. Thus, the receptor cycle is being regulated at a discrete point. IFN-gamma also suppressed the LDL receptor pathway in macrophages, but this pathway was not affected by IFN-gamma in mouse fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

Newly absorbed retinol is transported in association with chylomicrons and their remnants. In addition, after intake of high doses of retinol, significant amounts are also found in low-density lipoprotein (LDL). As both chylomicron remnants and LDL may be taken up by cells via the LDL receptor, and retinoids inhibit proliferation of some leukaemic cells, we have studied the uptake of retinol in leukaemic cells via the LDL-receptor pathway. HL-60 cells contain saturable binding sites for LDL. The binding of LDL to its receptor has a dissociation constant of about 3.2 x 10(-9) M, and the number of receptors per cell was calculated to be about 2700. Uptake of 125I-LDL by HL-60 cells was increased 2-fold by preincubating the cells with mevinolin. The presence of specific receptors for LDL on HL-60 cells was further confirmed by the finding that exogenous LDL cholesterol was able to up-regulate the ACAT (acyl-CoA: cholesterol acyltransferase) activity of HL-60 cells. We then tested the uptake of retinyl ester in leukaemic cells via the LDL-receptor pathway. HL-60 cells were incubated with LDL or chylomicron remnants labelled with [3H]retinyl palmitate. Uptake of retinyl ester associated with both LDL and chylomicron remnants was observed. Furthermore, the presence of excess LDL decreased the uptake by 75-100%, supporting the hypothesis that the uptake of retinyl ester occurred via the LDL receptor in HL-60 cells.     

Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells.

The uptake and transport of cholesterol-carrying low density lipoprotein (LDL) by the arterial wall is a continuous dynamic process, contributing to the cholesterol homeostasis in the plasma and in the cellular components of the vessel wall. Upon exposure to endothelial cells (EC), LDL interacts in part, with specific surface receptors (LDL-R). In this study we questioned: (i) the distribution of LDL receptors on the apical and basal cell membranes in endothelial cells; (ii) the role of LDL receptors in the control of cholesterol homeostasis and (iii) the translocation of LDL receptor across the EC. To this purpose bovine aortic EC were cultured on filters in a double-chamber system, in Dulbecco's medium supplemented either with 10% fetal calf serum (FCS) or with 10% lipoprotein-deficient serum (LPDS). The cells were exposed for 3h to 13H]acetate (40 microCi) added to both compartments of the cell culture inserts. The newly synthesized [3H]cholesterol was detected by thin layer chromatography and quantified by liquid scintillation counting. The LDL-R were detected in EC protein homogenates by immunoblotting using a monoclonal antibody against LDL-R (IgG-C7); the intracellular pathway of LDL-R was examined by electron microscopy using a complex made of protein A 5 nm or 20 nm colloidal gold particles and an anti-LDL receptor antibody (Au-PA-C7). To evaluate the distribution and the transport of LDL-R from one cell surface to the other, EC grown in LPDS were radioiodinated either on the apical or on the basolateral surface, incubated on the same surface with LDL, and subsequently biotinylated on the opposite non-radiolabeled surface. The EC were further solubilized and the protein extract immunoprecipitated with anti-LDL-R antibody or with mouse IgG (as control). The eluted antigen-antibody complexes were precipitated with streptavidin-agarose beads, solubilized, and subjected to SDS-PAGE. The results showed that: (a) the LDL-R were present on both endothelial cell fronts; (b) using the complex Au-PA-C7, the LDL-R were localized in endothelial plasmalemmal vesicles as well as coated pits and coated vesicles in multivesicular bodies and lysosomes, irrespective of the cell surface exposed to the complex; (c) biochemical assays indicated that upon ligand binding, the LDL-R were translocated preferentially from the apical to the basal plasma membrane.     

Ligand size as a determinant for catabolism by the low density lipoprotein (LDL) receptor pathway. A lattice model for LDL binding

Low density lipoproteins (LDL) are large (Mr = 2.5 x 10(6)) in comparison to LDL receptors (Mr = 115,000). Since most LDL receptors are clustered in coated pits, we tested the hypothesis that crowding of receptor-bound LDL particles would cause steric effects. The apparent affinity of LDL for receptors on cultured fibroblasts decreased near saturation causing concave-upward Scatchard plots. Both the higher and lower affinity components of binding were up-regulated by the cholesterol synthesis inhibitor, lovastatin, indicating that the entire binding curve was sterol-responsive. In contrast, neither component of LDL binding was present on lovastatin-treated or untreated null fibroblasts which are incapable of expressing LDL receptors. Therefore, the concave-upward Scatchard plots were entirely due to binding to LDL receptors. These results are consistent with a lattice model in which receptor-bound LDL are large enough to decrease binding to adjacent receptors. A lattice model implies that large LDL should produce steric effects at a lower receptor occupancy than should small LDL. This was tested using seven LDL fractions that differed in diameter from 20 to 27 nm. Fewer large than small LDL were bound to the cell surface at 4 degrees C and 37 degrees C, and fewer were internalized and degraded at 37 degrees C. Since large LDL bound via both apolipoprotein (apo) E and apoB100, receptor cross-linking could have caused fewer large LDL to be bound at saturation. However, when the potential for cross-linking was prevented by an apo-E-specific monoclonal antibody (1D7), the difference in binding by large versus small LDL was not eliminated; instead, it was exaggerated. Taken together, these results support a lattice model for LDL binding and indicate that steric hindrance associated with crowding of LDL particles on receptor lattices is a major determinant for catabolism by the LDL receptor pathway in vitro.     

A Chinese hamster ovarian cell line imports cholesterol by high density lipoprotein degradation

Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway, HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein-deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density lipoprotein (LDL) receptor pathway but also degrades LDL. 25-Hydroxycholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells, and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycans and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line.     

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.     

Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.     

A New Function for the LDL Receptor: Transcytosis of LDL across the Blood–Brain Barrier

Lipoprotein transport across the blood–brain barrier (BBB) is of critical importance for the delivery of essential lipids to the brain cells. The occurrence of a low density lipoprotein (LDL) receptor on the BBB has recently been demonstrated. To examine further the function of this receptor, we have shown using an in vitro model of the BBB, that in contrast to acetylated LDL, which does not cross the BBB, LDL is specifically transcytosed across the monolayer. The C7 monoclonal antibody, known to interact with the LDL receptor-binding domain, totally blocked the transcytosis of LDL, suggesting that the transcytosis is mediated by the receptor. Furthermore, we have shown that cholesterol-depleted astrocytes upregulate the expression of the LDL receptor at the BBB. Under these conditions, we observed that the LDL transcytosis parallels the increase in the LDL receptor, indicating once more that the LDL is transcytosed by a receptor-mediated mechanism. The nondegradation of the LDL during the transcytosis indicates that the transcytotic pathway in brain capillary endothelial cells is different from the LDL receptor classical pathway. The switch between a recycling receptor to a transcytotic receptor cannot be explained by a modification of the internalization signals of the cytoplasmic domain of the receptor, since we have shown that LDL receptor messengers in growing brain capillary ECs (recycling LDL receptor) or differentiated cells (transcytotic receptor) are 100% identical, but we cannot exclude posttranslational modifications of the cytoplasmic domain, as demonstrated for the polymeric immunoglobulin receptor. Preliminary studies suggest that caveolae are likely to be involved in the potential transport of LDL from the blood to the brain.The maintenance of the homeostasis of brain interstitial fluid, which constitutes the special microenvironment for neurons, is established by the presence of the blood–brain barrier (BBB)¹ at the transition area from endothelial cells (ECs) to brain tissue. Of primary importance in the formation of a permeability barrier by these cells is the presence of continuous tight junctions that seal together the margins of the ECs and restrict the passage of substances from the blood to the brain. Furthermore, in contrast to ECs in many other organs, the brain capillary ECs contain no direct transendothelial passageways such as fenestrations or channels. But obviously, the BBB cannot be absolute. The brain is dependent upon the blood to deliver metabolic substrates and remove metabolic waste, and the BBB therefore facilitates the exchange of selected solutes. Carrier-mediated transport systems that facilitate the uptake of hexoses, amino acids, purine compounds, and mono-carboxylic acids have been revealed in the cerebral endothelium (Betz and Goldstein, 1978), but until now little information has come to light regarding the cerebral uptake of lipids.There is growing evidence that the brain is equipped with a relatively self-sufficient transport system for maintaining cholesterol and lipid homeostasis. The presence of a low density lipoprotein (LDL) receptor has been demonstrated by immunocytochemistry in rat and monkey brains; and apolipoprotein (apo) E and apo AI-containing particles have been detected in human cerebrospinal fluid (Pitas et al., 1987). Furthermore, enzymes involved in lipid metabolism have been located within the brain: LCAT mRNA has been shown to be expressed in rat brains and cholesteryl ester transfer protein, which plays a key role in cholesterol homeostasis, has been detected in human cerebrospinal fluid and seems to be synthesized in the brain (Albers et al., 1992). The distribution of the LDL receptor-related protein, a multifunctional receptor that binds apoE, is highly restricted and limited to the gray matter, primarily associated with neuronal cell population (Wolf et al., 1992). The difference in cellular expression of ligand (apoE) and receptor (LDL receptor-related protein) may provide a pathway for intracellular transport of apoE-containing lipoproteins in the central nervous system. All these data leave little doubt that the brain is equipped with a relatively self-sufficient transport system for cholesterol.Cholesterol could be derived from de novo synthesis within the brain and from plasma via the BBB. Malavolti et al. (1991) indicate the presence of unexpectedly close communications between extracerebral and brain cholesterol. Changes in the extracerebral cholesterol levels are readily sensed by the LDL receptor in the brain and promptly provoke appropriate modifications in its activity. Méresse et al. (1989a) provided direct evidence for the occurrence in vivo of an LDL receptor on the endothelium of brain capillaries. Furthermore, the fact that enzymes involved in the lipoprotein metabolism are present in the brain microvasculature (Brecher and Kuan, 1979) and that the entire fraction of the drug bound to lipoproteins is available for entry into the brain strongly suggest that this cerebral endothelial receptor plays a role in the interaction of plasma lipoproteins with brain capillaries. These results pinpoint the critical importance of the interactions between brain capillary ECs and lipoproteins. Owing to the fact that the neurological abnormalities that result from the inadequate absorption of dietary vitamin E can be improved by the oral administration of pharmacological doses of vitamin E, Traber and Kayden (1984) have suggested that LDL functions as a transport system for tocopherol to the brain. Furthermore, the trace amounts of apolipoprotein B that were detected by Salem et al. (1987) in cerebrospinal fluid from healthy patients using a very sensitive immunoblot technique confirm that, at most, small amounts of apolipoprotein B normally pass through the BBB. However, whether LDL is involved in the exchange is not known.Using an in vitro model of the BBB that imitates an in vivo situation by culturing capillary ECs and astrocytes on opposite sides of a filter (Dehouck et al., 1990a , 1992), we have demonstrated that in culture, like in vivo, in contrast to peripheral endothelium and in spite of the tight apposition of ECs and their contact with physiological concentrations of lipoproteins, brain capillary ECs express an LDL receptor (Méresse et al., 1991; Dehouck et al., 1994). The capacity of ECs to bind LDLs is greater when cocultured with astrocytes than in their absence. Futhermore, we have shown that the lipid requirement of astrocytes increases the expression of the LDL receptor on brain capillary ECs. Taken together, the presence of LDL receptors on brain capillary ECs and the modulation of the expression of these receptors by the lipid composition of astrocytes suggest that cholesterol used by cells in the central nervous system may be derived, at least in part, from the periphery via transport across the BBB.In the present study, we provide direct evidence that after binding to brain capillary ECs, there is a specific mechanism for the transport of LDL across the endothelial monolayer from the apical to the abluminal surface. This mechanism might be best explained by a process of receptor-mediated transcytosis. Preliminary results pinpoint the role of caveolae in the transcellular transport of LDL across the brain endothelium.     

Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes

The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO(4) (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80-90%), LDL receptor (-30-40%), CD36 (-40%) and phagocytosis (-35-40%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.