Scavenger receptor BI mediates the selective uptake of oxidized cholesterol esters by rat liver

High density lipoprotein (HDL) can protect low density lipoprotein (LDL) against oxidation. Oxidized cholesterol esters from LDL can be transferred to HDL and efficiently and selectively removed from the blood circulation by the liver and adrenal in vivo. In the present study, we investigated whether scavenger receptor BI (SR-BI) is responsible for this process. At 30 min after injection, the selective uptake of oxidized cholesterol esters from HDL for liver and adrenal was 2.3- and 2.6-fold higher, respectively, than for native cholesterol esters, whereas other tissues showed no significant difference. The selective uptake of oxidized cholesterol esters from HDL by isolated liver parenchymal cells could be blocked for 75% by oxidized LDL and for 50% by phosphatidylserine liposomes, both of which are known substrates of SR-BI. In vivo uptake of oxidized cholesterol esters from HDL by parenchymal cells decreased by 64 and 81% when rats were treated with estradiol and a high cholesterol diet, respectively, whereas Kupffer cells showed 660 and 475% increases, respectively. These contrasting changes in oxidized cholesterol ester uptake were accompanied by similar contrasting changes in SR-BI expression of parenchymal and Kupffer cells. The rates of SR-BI-mediated selective uptake of oxidized and native cholesterol esters were analyzed in SR-BI-transfected Chinese hamster ovary cells. SR-BI-mediated selective uptake was 3.4-fold higher for oxidized than for native cholesterol esters (30 min of incubation). It is concluded that in addition to the selective uptake of native cholesterol esters, SR-BI is responsible for the highly efficient selective uptake of oxidized cholesterol esters from HDL and thus forms an essential mediator in the HDL-associated protection system for atherogenic oxidized cholesterol esters.     

Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus.

Oxidized low-density lipoprotein (Ox-LDL) is known to be involved in the generation and progression of atherosclerosis. Ox-LDL has a number of potentially atherogenic effects on vascular cells, including the uncontrolled uptake by scavenger receptors. We have previously shown that Asp-hemolysin binds to Ox-LDL in a concentration-dependent manner. The present study was undertaken to clarify the binding specificity of Asp-hemolysin to Ox-LDL. We examined the binding specificity of Asp-hemolysin to Ox-LDL using several modified lipoproteins and scavenger receptor ligands. Asp-hemolysin bound to Ox-LDL with shorter LDL oxidation times. However, Asp-hemolysin did not bind to the acetylated LDL. The native high-density lipoprotein (n-HDL) and modified HDL (e.g., acetylated HDL, oxidized HDL) also had no Asp-hemolysin binding. Furthermore, inhibitors of the scavenger receptor binding, including maleylated BSA, polyinosinic acid, dextran sulfate and fucoidin, had no effect on the binding of Ox-LDL to Asp-hemolysin. Surface plasmon resonance studies revealed that Ox-LDL binds with high affinity (K(D)=0.63 microg/ml) to Asp-hemolysin. We concluded that Asp-hemolysin is a specific binding protein with a high affinity for Ox-LDL, and its binding specificity is distinct from any receptor for Ox-LDL. The present studies suggest that Asp-hemolysin may bind to Ox-LDL using a mechanism different from the scavenger receptors.     

Enhanced macrophage uptake of elastase-modified high-density lipoproteins

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.     

Modification of LDL by platelet secretory products induces enhanced uptake and cholesterol accumulation in macrophages

LDL modified by incubation with platelet secretory products caused cholesterol accumulation and stimulation of cholesterol esterification in mouse peritoneal macrophages. Its uptake by the macrophages was a receptor-mediated process, not susceptible to competition by acetyl-LDL or polyanions suggesting independence of the scavenger receptor. Stimulation of the esterification process in macrophages by this modified LDL was inhibited by the lysosomal inhibitor chloroquine, indicating requirement for cellular uptake and lysosomal hydrolysis of the lipoprotein. Within the cell, the modified LDL inhibited cellular biosynthesis of triglycerides in a manner similar to the action of acetyl-LDL but different to the effect of native LDL. In the presence of HDL, acting in the medium as an acceptor for cholesterol, a low rate of cholesterol efflux from cells incubated with this modified LDL as well as with acetyl-LDL was demonstrated. A small reduction in cholesteryl ester synthesis was found in these cells, compared to a 60% reduction in cells incubated with native LDL. Thus it was demonstrated that LDL modified by platelet secretory products could induce macrophage cholesterol accumulation even though it was recognized and taken up via the regulatory LDL receptor.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

In vivo and in vitro catabolism of native and biologically modified LDL

Incubation of human low density lipoprotein (LDL) at 37 degrees C in the presence of human umbilical-vein endothelial cells (EC) causes a time-dependent shift in the charge and density of LDL. After intravenous injection into rats, native LDL is merely cleared from the circulation by Kupffer cells while EC-modified LDL is rapidly cleared by endothelial liver cells. The uptake of native LDL by Kupffer cells and EC-modified LDL by endothelial cells in vivo can be explained by the presence of two different specific receptors on these cell types. It is concluded that the liver endothelial cells form an important protection against a possible atherogenic action of EC-modified LDL.     

Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus

Oxidized low-density lipoprotein (Ox-LDL) is known to be involved in the generation and progression of atherosclerosis. Ox-LDL has a number of potentially atherogenic effects on vascular cells, including the uncontrolled uptake by scavenger receptors. We have previously shown that Asp-hemolysin binds to Ox-LDL in a concentration-dependent manner. The present study was undertaken to clarify the binding specificity of Asp-hemolysin to Ox-LDL. We examined the binding specificity of Asp-hemolysin to Ox-LDL using several modified lipoproteins and scavenger receptor ligands. Asp-hemolysin bound to Ox-LDL with shorter LDL oxidation times. However, Asp-hemolysin did not bind to the acetylated LDL. The native high-density lipoprotein (n-HDL) and modified HDL (e.g., acetylated HDL, oxidized HDL) also had no Asp-hemolysin binding. Furthermore, inhibitors of the scavenger receptor binding, including maleylated BSA, polyinosinic acid, dextran sulfate and fucoidin, had no effect on the binding of Ox-LDL to Asp-hemolysin. Surface plasmon resonance studies revealed that Ox-LDL binds with high affinity (K_D=0.63 μg/ml) to Asp-hemolysin. We concluded that Asp-hemolysin is a specific binding protein with a high affinity for Ox-LDL, and its binding specificity is distinct from any receptor for Ox-LDL. The present studies suggest that Asp-hemolysin may bind to Ox-LDL using a mechanism different from the scavenger receptors.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells

Human low density lipoprotein was oxidized (Ox-LDL) by exposure to 5 microM Cu2+ and its fate in vivo was compared to acetylated low density lipoprotein (Ac-LDL). Ox-LDL, when injected into rats, is rapidly removed from the blood circulation by the liver, similarly as Ac-LDL. A separation of rat liver cells into parenchymal, endothelial, and Kupffer cells at 10 min after injection of Ox-LDL or Ac-LDL indicated that the Kupffer cell uptake of Ox-LDL is 6.8-fold higher than for Ac-LDL, leading to Kupffer cells as the main liver site for Ox-LDL uptake. In vitro studies with isolated liver cells indicated that saturable high affinity sites for Ox-LDL were present on both endothelial and Kupffer cells, whereby the capacity of Kupffer cells to degrade Ox-LDL is 6-fold higher than for endothelial cells. Competition studies showed that unlabeled Ox-LDL competed as efficiently (90%) as unlabeled Ac-LDL with the cell association and degradation of 125I-labeled Ac-LDL by endothelial and Kupffer cells. However, unlabeled Ac-LDL competed only partially (20-30%) with the cell association and degradation of 125I-labeled Ox-LDL by Kupffer cells, while unlabeled Ox-LDL or polyinosinic acid competed for 70-80%. It is concluded that the liver contains, in addition to the scavenger (Ac-LDL) receptor which interacts efficiently with both Ac-LDL and Ox-LDL and which is concentrated on endothelial cells, an additional specific Ox-LDL receptor which is highly concentrated on Kupffer cells. In vivo the specific Ox-LDL recognition site on Kupffer cells will form the major protection system against the occurrence of the atherogenic Ox-LDL particles in the blood.     

Uptake of lipoprotein-associated alpha-tocopherol by primary porcine brain capillary endothelial cells

From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with alpha-tocopherol (alphaTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of alphaTocH in cells constituting the blood-brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of alphaTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein-associated alphaTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4 degrees C, pBCECs bound LDL with high affinity (K(D) = 6 nM) and apolipoprotein E-free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. alphaTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]alphaTocH-labeled lipoprotein preparations were used. The majority of HDL3-associated alphaTocH was taken up in a lipoprotein particle-independent manner, exceeding HDL3 holoparticle uptake 8- to 20-fold. This uptake route is less important for LDL-associated alphaTocH (alphaTocH uptake approximately 1.5-fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that alphaTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic alphaTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with alphaTocH in vivo.     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification

The uptake of native and modified low density lipoprotein (LDL) in foam cells in atherosclerotic tissue was studied in an in vitro perfusion system for rabbit aorta. Experimental atherosclerosis was induced in rabbits by a combination of cholesterol feeding and mechanical injury. The aorta was perfused in an incubation chamber. A trace-label, radioiodinated tyramine-cellobiose, was used to study cellular uptake of lipoproteins. After perfusion, the tissue was digested and cells were isolated by centrifugation in a density gradient. About 40 times more LDL per cell was accumulated in the foam cell fraction than in the smooth muscle cell fraction. When the cellular uptake of LDL and acetylated LDL (AcLDL) was compared, about 4 times more AcLDL than LDL was taken up by the foam cells, suggesting that the scavenger receptor is expressed in these cells. In a competition experiment, the uptake of LDL into foam cells was reduced by 70% when a tenfold excess of AcLDL was added. This experiment suggests that native LDL is taken up by the same mechanism as AcLDL. The accumulation of radiolabeled LDL in plaques and in foam cells was reduced by 30-55% by adding vitamin E (0.1 mg/ml) to the system. These studies show an uptake of LDL by foam cells in the atherosclerotic tissue. Furthermore, these cells seem to express the scavenger receptor. The competition experiment would suggest that native LDL is taken up by the scavenger receptor. The observation that an antioxidant, vitamin E, may decrease this uptake suggests that oxidative modification of LDL is of importance for this process.     

Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates.

Human endothelial cells (EA.hy 926 line) were loaded with cationized low density lipoprotein (LDL) and subsequently incubated with fatty acid/bovine serum albumin complexes. The fatty acids were palmitic, oleic, linoleic, arachidonic, and eicosapentaenoic acids. The preincubations resulted in extensively modified fatty acid profiles in cell membrane phospholipids and in cellular cholesteryl esters. The cholesterol efflux from these fatty acid-modified cells was measured using 0.2 mg high density lipoprotein3 (HDL3)/ml medium. The efflux was significantly higher for the palmitic acid-treated cells, compared to all other fatty acid treatments. These differences in efflux rates were not caused by changes in the binding of HDL3 to high affinity receptors on the EA.hy 926 cells. Efflux mediated by dimethyl suberimidate-treated HDL3, which does not interact with high affinity HDL receptors, was similar to efflux induced by native HDL3 after all fatty acid treatments. Our results indicate that high affinity HDL receptors are not important for HDL-mediated efflux of cell cholesterol. The fatty acid composition of the cell membrane phospholipids may be an important determinant.     

CD36 does not play a direct role in HDL or LDL metabolism

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.     

Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.

The scavenger receptor-BI (SR-BI) delivers sterols from circulating lipoproteins to tissues, but the relative potency of individual lipoproteins and the transported cholesterol has not been studied in detail. In this study, we used Chinese hamster ovary cells that express recombinant mouse SR-BI but have no functional low density lipoprotein (LDL) receptors (ldlA7-SRBI cells) to compare the fate of lipids transferred from high or low density lipoproteins to cells by SR-BI. HDL and LDL were equally effective in mediating the transfer of [(3)H]cholesterol to cells. Only 5% of the free cholesterol transferred to cells was esterified, in direct contrast to the findings in the cells that express LDL receptors in which 50% of the transported cholesterol was esterified. Almost all the free cholesterol transferred from lipoproteins to cells was rapidly excreted when the ldlA7-SRBI cells were switched to media containing unlabeled lipoproteins. SR-BI expression was associated with an increase in selective cholesteryl ester uptake from both lipoproteins, but HDL was a more effective donor. HDL and LDL were equally effective in delivering cholesterol to the intracellular regulatory pool via SR-BI. These data indicate that SR-BI is able to exchange cholesterol rapidly between lipoproteins and cell membranes and can mediate the uptake of cholesteryl esters from both classes of lipoproteins.     

Uptake of LDL in parenchymal and non-parenchymal rabbit liver cells in vivo. LDL uptake is increased in endothelial cells in cholesterol-fed rabbits.

1. Hepatic uptake of low-density lipoprotein (LDL) in parenchymal cells and non-parenchymal cells was studied in control-fed and cholesterol-fed rabbits after intravenous injection of radioiodinated native LDL (125I-TC-LDL) and methylated LDL (131I-TC-MetLDL). 2. LDL was taken up by rabbit liver parenchymal cells, as well as by endothelial and Kupffer cells. Parenchymal cells, however, were responsible for 92% of the hepatic LDL uptake. 3. Of LDL in the hepatocytes, 89% was taken up via the B,E receptor, whereas 16% and 32% of the uptake of LDL in liver endothelial cells and Kupffer cells, respectively, was B,E receptor-dependent. 4. Cholesterol feeding markedly reduced B,E receptor-mediated uptake of LDL in parenchymal liver cells and in Kupffer cells, to 19% and 29% of controls, respectively. Total uptake of LDL in liver endothelial cells was increased about 2-fold. This increased uptake is probably mediated via the scavenger receptor. The B,E receptor-independent association of LDL with parenchymal cells was not affected by the cholesterol feeding. 5. It is concluded that the B,E receptor is located in parenchymal as well as in the non-parenchymal rabbit liver cells, and that this receptor is down-regulated by cholesterol feeding. Parenchymal cells are the main site of hepatic uptake of LDL, both under normal conditions and when the number of B,E receptors is down-regulated by cholesterol feeding. In addition, LDL is taken up by B,E receptor-independent mechanism(s) in rabbit liver parenchymal, endothelial and Kupffer cells. The non-parenchymal liver cells may play a quantitatively important role when the concentration of circulating LDL is maintained at a high level in plasma, being responsible for 26% of hepatic uptake of LDL in cholesterol-fed rabbits as compared with 8% in control-fed rabbits. The proportion of hepatic LDL uptake in endothelial cells was greater than 5-fold higher in the diet-induced hypercholesterolaemic rabbits than in controls.     

Interaction in vivo and in vitro of apolipoprotein E-free high-density lipoprotein with parenchymal, endothelial and Kupffer cells from rat liver.

The interaction of apolipoprotein (apo) E-free high-density lipoprotein (HDL) with parenchymal, endothelial and Kupffer cells from liver was characterized. At 10 min after injection of radiolabelled HDL into rats, 1.0 +/- 0.1% of the radioactivity was associated with the liver. Subfractionation of the liver into parenchymal, endothelial and Kupffer cells, by a low-temperature cell-isolation procedure, indicated that 77.8 +/- 2.4% of the total liver-associated radioactivity was recovered with parenchymal cells, 10.8 +/- 0.8% with endothelial cells and 11.3 +/- 1.7% with Kupffer cells. It can be concluded that inside the liver a substantial part of HDL becomes associated with endothelial and Kupffer cells in addition to parenchymal cells. With freshly isolated parenchymal, endothelial and Kupffer cells the binding properties for apo E-free HDL were determined. For parenchymal, endothelial and Kupffer cells, evidence was obtained for a saturable, specific, high-affinity binding site with Kd and Bmax. values respectively in the ranges 10-20 micrograms of HDL/ml and 25-50 ng of HDL/mg of cell protein. In all three cell types nitrosylated HDL and low-density lipoproteins did not compete for the binding of native HDL, indicating that lipids and apo B are not involved in specific apo E-free HDL binding. Very-low-density lipoproteins (VLDL), however, did compete for HDL binding. The competition of VLDL with apo E-free HDL could not be explained by label exchange or by transfer of radioactive lipids or apolipoproteins between HDL and VLDL, and it is therefore suggested that competition is exerted by the presence of apo Cs in VLDL. The results presented here provide evidence for a high-affinity recognition site for HDL on parenchymal, liver endothelial and Kupffer cells, with identical recognition properties on the three cell types. HDL is expected to deliver cholesterol from peripheral cells, including endothelial and Kupffer cells, to the liver hepatocytes, where cholesterol can be converted into bile acids and thereby irreversibly removed from the circulation. The observed identical recognition properties of the HDL high-affinity site on liver parenchymal, endothelial and Kupffer cells suggest that one receptor may mediate both cholesterol efflux and cholesterol influx, and that the regulation of this bidirectional cholesterol (ester) flux lies beyond the initial binding of HDL to the receptor.     

Sialic acid content of human low density lipoproteins affects their interaction with cell receptors and intracellular lipid accumulation.

Low density lipoproteins (LDL) isolated from the plasma of patients with angiographically demonstrable coronary heart disease (CHD) induced accumulation of triglycerides, free cholesterol, and cholesteryl esters in cultured macrophages, smooth muscle cells, and endothelial cells derived from uninvolved intima of human aorta, but not in skin fibroblasts or hepatoma cells. The sialic acid content of LDL from CHD patients was 40-75% lower than that from healthy donors. There was a negative correlation between LDL sialic acid content and the LDL-induced accumulation of total intracellular cholesterol. Neuraminidase treatment of LDL from normal healthy donors produced sialic acid-depleted LDL (Ds-LDL) which was able to stimulate intracellular lipid accumulation. Neuraminidase treatment of LDL from CHD patients further increased its capacity to induce intracellular lipid accumulation. Sialic acid-poor LDL isolated by affinity chromatography of LDL from CHD patients induced a 2- to 4-fold increase of free and esterified cholesterol in human intimal smooth muscle cells. Binding, uptake, and degradation of 125I-labeled Ds-LDL by macrophages and endothelial cells were 1.5- to 2-fold higher than for native LDL. Binding and uptake of Ds-LDL was inhibited 64-93% by the addition of 20-fold excess acetylated LDL (Ac-LDL); in the inverse experiment, the level of inhibition was 35-54%. These data indicate that a sialic acid-poor form of LDL isolated from CHD patients can interact with both native and scavenger LDL receptors. A sialic acid-poor form of LDL may be a naturally occurring ligand that interacts with the scavenger receptor(s) on macrophages and endothelial cells.     

Enzyme immunoassay of the receptors for modified low density lipoprotein

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 micrograms/ml. This effect was completely blocked by polyinosinic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.