A simple binding assay for the determination of low-density lipoprotein receptors in cell homogenates

A convenient binding assay has been developed for the determination of low-density lipoprotein (LDL) receptors in homogenates of cultured and freshly-isolated normal and malignant human cells. Cell homogenates were incubated with 125I-labeled LDL and the ligand bound to the homogenate particulates was separated from the unbound ligand by filtration. When the particulates of the homogenates were subsequently incubated with heparin, a fraction of the bound 125I-LDL was released. Previous studies on intact cells have shown that heparin exclusively releases LDL bound to its cell surface receptor. The heparin-sensitive binding of 125I-LDL to cell homogenate particulates represents LDL bound to its cell surface receptor as judged from the following criteria: (a) it was quantitatively similar to the heparin-sensitive binding of 125I-LDL to intact cells, (b) it showed a direct correlation to the receptor-mediated degradation of 125I-LDL by intact cells, (c) no heparin-sensitive binding could be detected in homogenates prepared from normal erythrocytes or from cultured fibroblasts from a patient with homozygous familial hypercholesterolemia (two types of cell lacking LDL receptors), (d) it was dependent on calcium and inhibited by EDTA, (e) it was susceptible to treatment with pronase, and (f) it was heat-labile. The assay developed should be of value in determining the number of LDL receptors in tissues, since it is far less time-consuming and requires less material than currently available methods.     

Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages

The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.     

Carboxymethylated beta-1,3-glucan inhibits the binding and degradation of acetylated low density lipoproteins in macrophages In Vitro and modulates their plasma clearance In Vivo

In atherosclerotic lesions, macrophages are transformed into foam cells accumulating modified low density lipoproteins (LDL) via the scavenger receptor pathway. We have investigated the effects of carboxymethylated beta-1,3-glucan (CMG) on acetylated LDL (AcLDL) metabolism in murine peritoneal macrophages in vitro and upon the clearance of AcLDL by rat liver in vivo. In cultured murine peritoneal macrophages, CMG reduced substantially the AcLDL-induced synthesis of cholesteryl esters, decreased the binding and degradation of [¹²⁵I]-AcLDL in a dose-dependent manner with complete inhibition at 20–30 nM , but had no effect on the binding and degradation of native [¹²⁵I]–LDL. In contrast, other polysaccharides studied, namely zymosan, lipopolysaccharide, non-modified glucan and mannan Rhodexman, had a slight effect at concentrations significantly exceeding the concentrations of CMG. [¹²⁵I]-AcLDL injected intravenously into rats was cleared from the blood with a half-life of 3.7 min. About 56 per cent of the label of injected [¹²⁵I]-AcLDL was recovered in the liver 15 min after administration. Co-injection of the labelled AcLDL with CMG (25 mg kg^?1 b.w.) decreased the rate of AcLDL clearance so that the half-life increased to 6.0 min. Injections of CMG (25 mg kg^?1 b.w.) 48 and 24 h before the determination increased the rate of [¹²⁵I]-AcLDL clearance (with a half-life of about 2.3 min) and increased the uptake of AcLDL by the liver. We suggest that CMG competed with AcLDL for scavenger receptors in vitro and in vivo and repeated CMG injections before the measurements of AcLDL resulted in the induction of scavenger receptor function.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.     

Influence of the cytocidal macrophage phenotype on the degradation of acetylated low density lipoproteins: Dual regulation of scavenger receptor activity and of intracellular degradation of endocytosed ligand

The appearance of lipid-laden macrophages is a characteristic feature in the development of the atherosclerotic plaque. The functional status of macrophages located within the intima of atherosclerotic lesions is as yet unknown; nevertheless, macrophages are known to be exceedingly responsive to their environment and can differentiate to different functional states. The objective of this study was to determine the influence of two definable macrophage functional states, namely the IFN-primed state and the cytocidal state, on the capacity of macrophages to bind and degrade lipoproteins. We report that priming of macrophages with IFN-beta or IFN-gamma failed to influence the ability of macrophages to degrade native low density lipoprotein or acetylated low density lipoprotein (AcLDL). However, challenge with stimuli that induce expression of the cytocidal state (poly[I:C] and LPS) resulted in a marked inhibition of the capacity of the cells to degrade both lipoproteins. The poly[I:C]-induced inhibition of 125I-AcLDL degradation was accompanied by a proportional decrease in the binding of the ligand to its receptor which Scatchard analysis revealed was due to a decrease in receptor number rather than a change in receptor affinity for 125I-AcLDL. However, in addition to the down-regulation of receptor activity, the degradation of endocytosed 125I-AcLDL was also suppressed in macrophages that had been exposed to poly[I:C]. This latter observation suggests that the degradation of endocytosed lipid is also regulated at a second, previously unidentified level, independent of the availability of cell surface ligand receptors. We speculate that this down-regulation in the intracellular hydrolysis of endocytosed lipid may account for the observed accumulation of 125I-AcLDL in these cells.     

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Thematic review series: the immune system and atherogenesis. Recent insights into the biology of macrophage scavenger receptors

Scavenger receptors were originally defined by their ability to bind and internalize modified lipoproteins. Macrophages express at least six structurally different cell surface receptors for modified forms of LDL that contribute to foam cell formation in atherosclerosis. In addition to their role in the pathology of atherosclerosis, macrophage scavenger receptors, especially SR-A, play critical roles in innate immunity, apoptotic cell clearance, and tissue homeostasis. In this review, we highlight recent advances in understanding the biology of macrophage scavenger receptors as pattern recognition receptors for both infectious nonself (pathogens) and modified self (apoptotic cells and modified LDL). We critically evaluate the potential of scavenger receptors and their ligands as targets for therapeutic intervention in human disease.     

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.     

Interaction of a high-affinity heparin subfraction with low-density lipoprotein stimulates cholesteryl ester accumulation in mouse macrophages

A high-affinity heparin subfraction accounting for 8% of whole heparin from bovine lung was isolated by low-density lipoprotein (LDL)-affinity chromatography. When compared to whole heparin, the high-affinity subfraction was relatively higher in molecular weight (11,000 vs. 17,000) and contained more iduronyl sulfate as hexuronic acid (76% vs. 86%), N-sulfate ester (0.75 vs. 0.96 mol/mol hexosamine), and O-sulfate ester (1.51 vs. 1.68 mol/mol hexosamine). Although both heparin preparations formed insoluble complexes with LDL quantitatively in the presence of 30 mM Ca2+, the concentrations of NaCl required for 50% reduction in maximal insoluble complex formation was markedly higher with high-affinity subfraction (0.55 M vs. 0.04 M). When compared to complex of 125I-LDL and whole heparin (H-125I-LDL), complex of 125I-LDL and high-affinity heparin subfraction (HAH-125I-LDL) produced marked increase in the degradation of lipoproteins by macrophages (7-fold vs. 1.4-fold over native LDL, after 5 h incubation) as well as cellular cholesteryl ester synthesis (16.7-fold vs. 2.2-fold over native LDL, after 18 h incubation) and content (36-fold vs. 2.7-fold over native LDL, after 48 h incubation). After a 5 h incubation, macrophages accumulated 2.3-fold more cell-associated radioactivity from HAH-125I-LDL complex than from [125I]acetyl-LDL. While unlabeled HAH-LDL complex produced a dose-dependent inhibition of the degradation of labeled complex, native unlabeled LDL did not elicit any effect even at a 20-fold excess concentration. Unlabeled particulate LDL aggregate competed for 33% of degradation of labeled complex; however, cytochalasin D, known inhibitor of phagocytosis, did not effectively inhibit the degradation of labeled complex. Unlabeled acetyl-LDL produced a partial (33%) inhibition of the degradation of labeled complex. These results indicate that (1) the interaction of high-affinity heparin subfraction with LDL leads to scavenger receptor mediated endocytosis of the lipoprotein, and stimulation of cholesteryl ester synthesis and accumulation in the macrophages; and (2) with respect to macrophage recognition and uptake, HAH-LDL complex was similar but not identical to acetyl-LDL. These observations may have implications for atherogenesis, because both mast cells and endothelial cells can synthesize heparin in the arterial wall.     

Increased uptake of alpha-hydroxy aldehyde-modified low density lipoprotein by macrophage scavenger receptors

Reactive aldehydes can be formed during the oxidation of lipids, glucose, and amino acids and during the nonenzymatic glycation of proteins. Low density lipoprotein (LDL) modified with malondialdehyde are taken up by scavenger receptors on macrophages. In the current studies we determined whether alpha-hydroxy aldehydes also modify LDL to a form recognized by macrophage scavenger receptors. LDL modified by incubation with glycolaldehyde, glyceraldehyde, erythrose, arabinose, or glucose (alpha-hydroxy aldehydes that possess two, three, four, five, and six carbon atoms, respectively) exhibited decreased free amino groups and increased mobility on agarose gel electrophoresis. The lower the molecular weight of the aldehyde used for LDL modification, the more rapid and extensive was the derivatization of free amino groups. Approximately 50-75% of free lysine groups in LDL were modified after incubation with glyceraldehyde, glycolaldehyde, or erythrose for 24-48 h. Less extensive reductions in free amino groups were observed when LDL was incubated with arabinose or glucose, even at high concentration for up to 5 days. LDL modified with glycolaldehyde and glyceraldehyde labeled with (125)I was degraded more extensively by human monocyte-derived macrophages than was (125)I-labeled native LDL. Conversely, LDL modified with (125)I-labeled erythrose, arabinose, or glucose was degraded less rapidly than (125)I-labeled native LDL. Competition for the degradation of LDL modified with (125)I-labeled glyceraldehyde was nearly complete with acetyl-, glycolaldehyde-, and glyceraldehyde-modified LDL, fucoidin, and advanced glycation end product-modified bovine serum albumin, and absent with unlabeled native LDL.These results suggest that short-chain alpha-hydroxy aldehydes react with amino groups on LDL to yield moieties that are important determinants of recognition by macrophage scavenger receptors.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.     

Regulation of low density lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo

Membranes prepared from the adrenal gland of mice and rats possess high affinity binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These binding sites resemble the functional LDL receptors that mediate the uptake of LDL by cultured mouse and bovine adrenal cells. The number of LDL binding sites per mg of membrane protein increased 2- to 5-fold over 24 h when mice or rats were treated with adrenocorticotropin (ACTH). In rats, this increase was accompanied by a similar ACTH-induced increase in the adrenal uptake of intravenously administered 125I-LDL, suggesting that the LDL binding sites mediate the uptake of LDL by the adrenal in the intact animal. The number of LDL binding sites on adrenal membranes rose by 5-fold when animals were rendered lipoprotein-deficient, either by treatment of mice with 4-aminopyrazolopyrimidine or by treatment of rats with 17 alpha-ethinyl estradiol. This increase was prevented when endogenous ACTH secretion was blocked by administration of dexamethasone, suggesting that ACTH was required. The current experiments suggest that LDL receptors provide one source of cholesterol for the mouse and rat adrenal in vivo and that the number of LDL receptors of this organ is regulated by ACTH.     

Cellular localization of the receptor-dependent and receptor-independent uptake of human LDL in the liver of normal and 17 alpha-ethinyl estradiol-treated rats

The cellular localization in the liver of the receptor-dependent and -independent uptake of human low density lipoprotein (LDL) in normal and 17 alpha-ethinyl estradiol-treated rats was investigated by the simultaneous in vivo injection of human 131I-LDL and human reductive methylated 125I-LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor-dependent liver-association of human LDL occurs in non-parenchymal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor-dependent cell-association of human LDL with hepatocytes (17-fold), while the receptor-dependent cell-association with non-parenchymal cells is not affected.     

A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.     

Downstream effects on human low density lipoprotein of homocysteine exported from endothelial cells in an in vitro system

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposed to HUVECs exporting Hcy undergo time-related lipid oxidation, a process inhibited by the thiol trap dithionitrobenzoate. This is likely to be related to the generation of hydroxyl radicals, which we show are associated with Hcy export. Although Hcy is the major oxidant, cysteine also contributes, as shown by the effect of glutamate. Finally, the LDL oxidized in this system showed a time-dependent increase in uptake by human macrophages, implying an upregulation of the scavenger receptor. These results suggest that continuous export of Hcy from endothelial cells contributes to the generation of extracellular hydroxyl radicals, with associated oxidative modification of LDL and incorporation into macrophages, a key step in atherosclerosis. Factors that regulate intracellular Hcy metabolism modulate these effects.