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.     

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.     

Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein

The mass efflux of free and esterified cholesterol was studied in skin fibroblasts loaded with cholesterol by incubation with low density lipoproteins (LDL) isolated from normal or hypercholesterolemic cynomolgus monkeys. Cells incubated with hypercholesterolemic LDL accumulated 2-3 times more cholesteryl ester than did cells incubated with the same amount of normal LDL. Cholesteryl oleate was the principal cholesteryl ester species to accumulate in cells incubated with both normal and hypercholesterolemic LDL. Efflux of this accumulated cholesterol was absolutely dependent on the presence of a cholesterol acceptor in the culture medium. Lipoprotein-deficient serum (LPDS) was the most potent promoter of cholesterol efflux tested, with maximum efflux occurring at LPDS concentrations greater than 1.5 mg protein/ml. Upon addition of efflux medium containing LPDS, there was a reduction in both the free and esterified cholesterol concentration of the cells. Greater than 90% of the cholesteryl esters that were lost from the cells appeared in the culture medium as free cholesterol, indicating that hydrolysis of cholesteryl esters preceded efflux. Efflux was not inhibited by chloroquine, however, suggesting a mechanism independent of lysosomes. Loss of cellular free cholesterol was maximum by 6 hr and changed very little thereafter up to 72 hr. Cholesteryl ester loss from cells decreased in a log linear fashion for efflux periods of 6-72 hr, with an average half-life for cholesteryl ester efflux of 30 hr, but with a range of 20-50 hr, depending upon the specific cell line. The rate of efflux of cellular cholesteryl esters was similar for cells loaded with normal or hypercholesterolemic LDL. In cells loaded with cholesteryl esters, cholesterol synthesis was suppressed and cholesterol esterification and fatty acid synthesis were enhanced. During efflux, cholesterol synthesis remained maximally suppressed while cholesterol esterification decreased for the first 24 hr of efflux, then plateaued at a level approximately 5-fold higher than control levels, while fatty acid synthesis was slightly stimulated. There was little difference in the rate of efflux of individual cholesteryl ester species. There was, however, the suggestion that reesterification of cholesterol principally to palmitic acid occurred during efflux. Since the rate of cellular cholesteryl ester efflux was similar regardless of whether the cells had been loaded with cholesterol by incubation with normal LDL or hypercholesterolemic LDL, the greater accumulation of cholesterol in cells incubated with hypercholesterolemic LDL cannot be explained by differences in rates of efflux.-St. Clair, R. W., and M. A. Leight. Cholesterol efflux from cells enriched with cholesteryl esters by incubation with hypercholesterolemic monkey low density lipoprotein.     

Reduced uptake of cholesterol esterase-modified low density lipoprotein by macrophages.

Changes in low density lipoprotein (LDL) lipid composition were shown to alter its interaction with the LDL receptor, thus affecting its cellular uptake. Upon incubation of LDL with 5 units/ml cholesterol esterase (CEase) for 1 h at 37 degrees C, there was a 33% reduction in lipoprotein cholesteryl ester content, paralleled by an increment in its unesterified cholesterol. CEase-LDL, in comparison to native LDL, was smaller in size, possessed fewer free lysine amino groups (by 14%), and demonstrated reduced binding to heparin (by 83%) and reduced immunoreactivity against monoclonal antibodies directed toward epitopes along the LDL apoB-100. Incubation of CEase-LDL with the J-774 macrophage-like cell line resulted in about a 30% reduction in lipoprotein binding and degradation in comparison to native LDL, and this was associated with a 20% reduction in macrophage cholesterol mass. Similarly, CEase-LDL degradation by mouse peritoneal macrophages, human monocyte-derived macrophages, and human skin fibroblasts was reduced by 20-44% in comparison to native LDL. CEase-LDL uptake by macrophages was mediated via the LDL receptor and not the scavenger receptor. CEase activity toward LDL was demonstrated in plasma and in cells of the arterial wall such as macrophages and endothelial cells. Thus, CEase modification of LDL may take place in vivo, and this phenomenon may have a role in atherosclerosis.     

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.     

Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous fibroblasts

The esterification of cholesterol derived from human low density lipoprotein (LDL) or fetal bovine serum (FBS) was deficient in cultured fibroblasts from subjects with heterozygous and homozygous type C Niemann-Pick (NPC) disease. Failure to significantly esterify LDL-derived cholesterol resulted in abnormal accumulation of predominantly unesterified cholesterol in homozygous NPC fibroblasts. Compared with normal and homozygous fibroblasts, heterozygous NPC fibroblasts synthesized intermediate levels of cholesteryl ester during the initial 6 h of incubation with LDL. The rate of cholesterol esterification in heterozygous cells was normal when measured over a 24-h period of incubation with LDL. In addition to demonstrating a defect in cholesterol esterification, homozygous NPC fibroblasts accumulated more total cholesterol when incubated with LDL or FBS than normal fibroblasts accumulated. When heterozygous NPC fibroblasts were incubated with LDL or FBS, cellular accumulation of cholesterol reached levels that were high-normal or intermediary between levels observed in normal and homozygous NPC fibroblasts. The partial expression of these metabolic errors in the heterozygous genotype relevantly links these errors to the primary mutation of this disorder.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.     

Low density lipoprotein modification by cholesterol oxidase induces enhanced uptake and cholesterol accumulation in cells.

Oxidation of low density lipoprotein (LDL) by cells of the arterial wall or in the presence of copper ions was shown to result in the peroxidation of its fatty acids as well as its cholesterol moiety. LDL incubation with cholesterol oxidase (CO) resulted in the conversion of up to 85% of the lipoprotein unesterified cholesterol (cholest-5-en-3-ol) to cholestenone (cholest-4-en-3-one) in a dose- and time-dependent pattern. Plasma very low density lipoprotein (VLDL) and high density lipoprotein (HDL) could be similarly modified by CO. In cholesterol oxidase-modified LDL (CO-LDL), unlike copper ion-induced oxidized LDL (Cu-Ox-LDL), there was no fatty acids peroxidation, and lipoprotein size or charge as well as LDL cholesteryl ester, phospholipids, and triglycerides content were not affected. CO-LDL, however, demonstrated enhanced susceptibility to oxidation by copper ions in comparison to native LDL. Upon incubation of CO-LDL with J-774 A.1 macrophage-like cell line, cellular uptake and degradation of the lipoprotein was increased by up to 62% in comparison to native LDL but was 15% lower than that of Cu-Ox-LDL. Similarly, the binding of CO-LDL to macrophages increased by up to 80%, and cellular cholesterol mass was increased 51% more than the mass obtained with native LDL. Several lines of evidence indicate that CO-LDL was taken up via the LDL receptor: 1) Excess amounts of unlabeled LDL, but not acetyl-LDL (Ac-LDL), effectively competed with 125I-CO-LDL for the uptake by cells. 2) The degradation of CO-LDL by various types of macrophages and by fibroblasts could be dissociated from that of Ac-LDL and was always higher than that of native LDL. 3) A monoclonal antibody to the LDL receptor (IgG-C7) and a monoclonal antibody to the LDL receptor binding domains on apoB-100 (B1B6) inhibited macrophage degradation of CO-LDL. The receptor for Cu-Ox-LDL, which is not shared with Ac-LDL, was also partially involved in macrophage uptake of CO-LDL, since Cu-Ox-LDL demonstrated some competition capability with CO-125I-LDL for its cellular degradation. CO-LDL cellular degradation was inhibited by chloroquine, thus implying lysosomal involvement in the cellular processing of the lipoprotein. Incubation of macrophages with LDL in the presence of increasing concentrations of cholestenone resulted in up to 52% enhanced lipoprotein cellular degradation suggesting that the cholestenone in CO-LDL might be involved in the enhanced cellular uptake of the modified lipoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein

Summary The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized. Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS). The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties. Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls. Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL. Cellular cholesterol content saturated at 4 hours with 50 g/ml LDL; this effect may be associated with receptor saturation. Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content. However, deposition of CE was not observed below Ac-LDL concentrations of 100 g/ml. This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM. In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS. Cholesterol esterification in response to LDL and Ac-LDL was examined by ¹⁴C-oleic acid incorporation into CE. These results confirmed the mass cellular cholesterol and CE measurements. Kinetic studies conducted with RAW264 cells treated with 50 or 100 g/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6–12 hours of incubation. Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.Abbreviations LDL Low Density Lipoprotein - Ac-LDL Acetylated Low Density Lipoprotein - FBS Fetal Bovine Serum - DM Defined Medium - PBS Phosphate Buffered Saline - CE Cholesteryl Ester     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism

A lipoprotein particle designated A-LDL, which contains apolipoprotein B (apoB) and which is the size of plasma low density lipoproteins (LDL), was isolated from homogenates of human aortic athersclerotic plaques by a combination of affinity chromatography and gel-filtration. Compared to plasma LDL, A-LDL was more electronegative, its hydrated density was lower and more heterogeneous, and its protein-to-lipid ratio was lower. In addition, apoB in A-LDL was highly degraded, and A-LDL was recognized by mouse peritoneal macrophages (MPM) as indicated by its ability to stimulate cholesterol esterification. Cholesterol esterification was saturable with an apparent Km of 100 micrograms of A-LDL cholesterol/ml. Stimulation of cholesterol esterification was linear with time, leading to extensive accumulation of cholesteryl ester in MPM over a 48-hr time interval. The uptake or degradation of acetyl-LDL (radiolabeled either in the protein with 125I or hydrophobic core with [3H]cholesteryl ether) was markedly decreased by excess unlabeled acetyl-LDL but not by A-LDL, and excess acetyl-LDL did not inhibit the uptake or degradation of labeled A-LDL. However, a 10-fold excess of A-LDL also failed to inhibit the uptake of labeled A-LDL. This finding was consistent with the observation that, unlike the saturable stimulation of cholesterol esterification in MPM induced by A-LDL, the uptake of cholesteryl ether-labeled A-LDL was almost linear over a 0-400 micrograms cholesterol/ml range. This discrepancy between dose response curves for A-LDL, which did not occur for acetyl-LDL, could be eliminated by a 24-hr postincubation period in the absence of lipoprotein, suggesting that A-LDL is catabolized less efficiently than acetyl-LDL following internalization. In summary, we conclude that A-LDL uptake by MPM occurs via a low affinity-high capacity process. Although the uptake of A-LDL is not readily saturated, it is of sufficient affinity to lead to lipid loading of macrophages even when A-LDL is present at relatively low concentrations. If these mechanisms are operative in vivo, they could explain how foam cells in human fatty streak lesions develop.     

Modified low density lipoprotein isolated from atherosclerotic lesions does not cause lipid accumulation in aortic smooth muscle cells

Foam cells in atherosclerotic lesions are derived not only from blood monocytes but also from smooth muscle cells (SMC). To better understand the mechanisms by which SMC may become lipid-laden, we have studied the catabolism by cultured rabbit aortic SMC of LDL derived from atherosclerotic lesions (A-LDL) previously shown to be chemically modified. A-LDL was isolated either from homogenates of atherosclerotic plaques in human aortas by affinity chromatography and gel filtration, or from nonhomogenized extracts of plaque minces by ultracentrifugation and gel filtration. Internalization of A-LDL by SMC or fibroblasts appeared to be mediated primarily via the LDL receptor since: 1) either unlabeled LDL or A-LDL could inhibit the degradation of 125I-labeled A-LDL or of 125I-labeled LDL, 2) the uptake of both A-LDL and LDL, as estimated by their abilities to stimulate cholesterol esterification, was reduced in cells in which LDL receptor expression was down-regulated; and 3) the uptake of both [3H]cholesteryl ether-labeled A-LDL and LDL by normal fibroblasts was significant and could be inhibited by excess LDL, but was negligible in receptor-negative fibroblasts. At saturating concentrations of lipoproteins, maximum cholesterol esterification in SMC was greater for LDL than for A-LDL. Over a 48-h incubation, A-LDL, like LDL, was unable to induce cellular cholesteryl ester accumulation. Cross-competition studies suggested that either the affinity of A-LDL for the LDL receptor was less than that of LDL, or that some particles in A-LDL are not internalized by SMC. The latter alternative was supported by the observations that some A-LDL particles had undergone aggregation, especially at high concentrations, and that aggregated forms of A-LDL or plasma LDL failed to be internalized and degraded by SMC. Collectively, these results are consistent with recognition of some of the A-LDL particles by the LDL receptor, but also suggest that, at least under in vitro conditions, A-LDL is unlikely to induce lipid accumulation in SMC resulting in SMC-derived foam cells.     

Preferential enrichment of large-sized very low density lipoprotein populations with transferred cholesteryl esters

The effect of lipid transfer proteins on the exchange and transfer of cholesteryl esters from rat plasma HDL2 to human very low (VLDL) and low density (LDL) lipoprotein populations was studied. The use of a combination of radiochemical and chemical methods allowed separate assessment of [3H]cholesteryl ester exchange and of cholesteryl ester transfer. VLDL-I was the preferred acceptor for transferred cholesteryl esters, followed by VLDL-II and VLDL-III. LDL did not acquire cholesteryl esters. The contribution of exchange of [3H]cholesteryl esters to total transfer was highest for LDL and decreased in reverse order along the VLDL density range. Inactivation of lecithin: cholesterol acyltransferase (LCAT) and heating the HDL2 for 60 min at 56 degrees C accelerated transfer and exchange of [3H]cholesteryl esters. Addition of lipid transfer proteins increased cholesterol esterification in all systems. The data demonstrate that large-sized, triglyceride-rich VLDL particles are preferred acceptors for transferred cholesteryl esters. It is suggested that enrichment of very low density lipoproteins with cholesteryl esters reflects the triglyceride content of the particles.     

氧化修饰低密度脂蛋白与动脉粥样硬化

动脉粥样硬化的发生发展与低密度脂蛋白受到氧化修饰有关。本文从以下四个方面对本室的工作进行了综述：（１）动脉粥样硬化机体受到脂质过氧化损伤;（２）Ｏｘ－ＬＤＬ对内皮细胞、平滑肌细胞和巨噬细胞的毒性效应;（３）Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ的比较及与Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ结合的清道夫受体的特征;（４）不同方法对ＬＤＬ氧化修饰的比较和以ＬＤＬ氧化修饰为模型对某些物质的抗氧化修饰研究。研究结果为动脉粥     

Type C Niemann-Pick disease. A parallel loss of regulatory responses in both the uptake and esterification of low density lipoprotein-derived cholesterol in cultured fibroblasts

Low density lipoprotein (LDL) internalization by mutant type C Niemann-Pick (NPC) fibroblasts results in uptake of excess total cholesterol. Uptake of excess lipoprotein cholesterol appears to be mediated by the specific LDL receptor pathway. Associated with excessive LDL-cholesterol uptake is a lesion in early intracellular cholesteryl ester synthesis. In vitro acylCoA:cholesterol acyltransferase activity is normal in cell-free extracts of mutant cells. The ability of exogenous sterols to enhance intracellular esterification of [3H]mevalonate-derived [3H]cholesterol was severely limited in mutant cell cultures suggesting that in vivo activation and/or expression of activated acylCoA:cholesterol acyltransferase may be compromised by the primary mutation of type C Niemann-Pick disease. After 2 days of LDL uptake, rates of intracellular cholesteryl ester synthesis in mutant cells paralleled the rates of esterification in normal cells suggesting that specific early in vivo expression of the acyltransferase may be affected in this disorder.     

Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein

This paper describes a procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of the unique structure of low density lipoprotein, a plasma cholesterol transport protein that enters cells by receptor-mediated endocytosis. LDL contains a core of cholesteryl ester that can be extracted and reconstituted with hydrophobic molecules that convert the LDL into a toxic or fluorescent particle. Mutagenized Chinese hamster ovary cells were incubated with reconstituted LDL containing toxic 25-hydroxycholesteryl oleate. Wild-type cells take up this lipoprotein via the LDL receptor, liberate the 25-hydroxycholesterol in lysosomes, and die. To identify colonies of receptor-deficient cells from among the few survivors of the first selection step, we incubated the cells with LDL reconstituted with a fluorescent cholesteryl ester and picked colonies that failed to accumulate fluorescence. The two-step isolation procedure yielded receptor-deficient cells at a frequency of 1 in 10⁵. The mutant cells grew in the presence of LDL reconstituted with 25-hydroxycholesteryl oleate at concentrations 100-fold higher than those that killed parental cells. The altered phenotypes have remained stable for more than 200 population doublings under non-selective conditions. Inasmuch as LDL can be coupled to ligands that bind to receptors other than the LDL receptor, the above method may have general utility in isolating cells with mutations affecting other receptor systems.     

Lipoproteins of the extravascular space: enhanced macrophage degradation of low density lipoproteins from interstitial inflammatory fluid

Current evidence has demonstrated that cholesteryl ester-loaded macrophages are important components of the atherosclerotic lesion. Additional studies have implicated low density lipoproteins (LDL) and circulating monocytes as central to the origin of lipid-laden foam cells found in the arterial wall. This is a result of the finding of accelerated macrophage uptake of LDL chemically modified by reaction with malondialdehyde (MDA-LDL), acetic anhydride (Ac-LDL), or incubation with arterial cells in vitro. In concert with these chemical modifications, we have previously demonstrated selective in vivo modification of LDL isolated from interstitial inflammatory fluid (IF) of the rabbit. Utilizing the polyvinyl sponge implant model, we reported that IF-LDL had an altered chemical composition, electrophoretic mobility, and particle size distribution when compared to LDL isolated from homologous plasma (WP-LDL). In this study reported herein, we examined the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM) in culture. IF-LDL was degraded substantially faster by MPM, and resulted in a substantial increase in cellular cholesteryl ester when compared to cells incubated with WP-LDL. IF-LDL binding to MPM was inhibited by Ac-LDL derived from WP-LDL, but only minimally by unmodified WP-LDL. Transmission electron microscopy of MPM revealed extensive lipid deposition in cells incubated with Ac-LDL and IF-LDL. These results implicate LDL from interstitial inflammatory fluid as an in vivo modified lipoprotein that can enhance uptake via the acetyl LDL receptor pathway in resident macrophages.     

Inhibition of acyl coenzyme A:cholesterol acyl transferase in J774 macrophages enhances down-regulation of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and prevents low density lipoprotein-induced cholesterol accumulation

Cholesteryl ester accumulation in arterial wall macrophages (foam cells) is a prominent feature of atherosclerotic lesions. We have previously shown that J774 macrophages accumulate large amounts of cholesteryl ester when incubated with unmodified low density lipoprotein (LDL) and that this is related to sluggish down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. To further explore intracellular cholesterol metabolism and regulatory events in J774 macrophages, we studied the effect of inhibitors of acyl-CoA:cholesterol acyl transferase (ACAT) on the cells' ability to accumulate cholesterol and to down-regulate receptor and reductase. Treatment of J774 cells with LDL in the presence of ACAT inhibitor 58-035 (Sandoz) prevented both cholesteryl ester and total cholesterol accumulation. Furthermore, 58-035 markedly enhanced down-regulation of the J774 LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase in the presence of LDL. In dose-response studies, down-regulation of the receptor by 58-035 paralleled its inhibition of ACAT activity. Compound 58-035 also increased the down-regulation of the J774 LDL receptor in the presence of 25-hydroxycholesterol and acetyl-LDL but not in the presence of cholesteryl hemisuccinate, which is not an ACAT substrate. The ability of 58-035 to enhance LDL receptor down-regulation was negated when cells were simultaneously incubated with recombinant high density lipoprotein3 discs, which promote cellular cholesterol efflux. In contrast to the findings with J774 macrophages, down-regulation of the human fibroblast LDL receptor was not enhanced by 58-035. These data suggest that in J774 macrophages, but not in fibroblasts, ACAT competes for a regulatory pool of intracellular cholesterol, contributing to diminished receptor and reductase down-regulation, LDL-cholesterol accumulation, and foam cell formation.