Low-density lipoprotein and scavenger receptor activities are modulated by secretory products derived from cells of the arterial wall

The atherosclerotic lesion consists of cholesterol-loaded macrophages, smooth muscle cells, and other cells of the arterial wall. Conditioned medium from human monocyte-derived macrophages (HMDM) stimulated both acetylated and native low-density lipoprotein (Ac-LDL and LDL, respectively) degradation in autologous cells by 25% and 90%, respectively, and this was due to an increase in the number of both LDL and Ac-LDL receptors. Macrophage conditioned medium also resulted in an approximate doubling of LDL degradation by human arterial smooth muscle cells (HASMC), endothelial cells (HEC), and skin fibroblasts (HSF). Macrophage degradation of both Ac-LDL and LDL was enhanced 15% to 45% by conditioned medium derived from HASMC and HSF, respectively, but not by HEC-conditioned medium. Conditioned medium from HASMC, like that from macrophages, could also enhance LDL degradation by smooth muscle cells, fibroblasts, endothelial cells, and macrophages. Thus, the current study demonstrated that arterial wall cells secretory products can affect cellular lipoprotein receptor activities. This phenomenon could lead to increased cellular cholesterol accumulation and foam cell formation.     

Effect of probucol on macrophages, leading to regression of xanthomas and atheromatous vascular lesions

To explain the strong effect of probucol on xanthomas, the drug's effect on lipid storage in macrophages in the presence of denatured low-density lipoprotein (LDL) was studied. Two macrophage cell lines, UE-12 and THP-1, were used. Those cells stored lipids and became foam cells when they were incubated with acetylated LDL (acetyl-LDL). When probucol was added into the medium either in ethanolic solution or in the form bound to LDL, the storage of cholesterol and other lipids and the development of macrophages into foam cells were greatly suppressed. Two functions of probucol should be considered: (1) It inhibited the uptake of acetyl-LDL by macrophages; and (2) it enhanced the release of cholesterol from these cells. Cells were first incubated with probucol. After the cells were washed with fresh medium, the radiolabeled acetyl-LDL was added to the medium and the degradation of acetyl-LDL was measured. Increasing the concentration of probucol led to a decrease in degradation of acetyl-LDL by macrophages. Probucol also suppressed the uptake of albumin. Macrophages were incubated with acetyl-LDL, washed once, then incubated with or without probucol and high-density lipoprotein (HDL). Addition of HDL caused a rapid decrease in cholesterol content in the cells, and this phenomenon was enhanced by probucol for both kinds of cells. The secretion of apolipoprotein E was also stimulated by the addition of probucol. These 2 sets of experimental results suggest that probucol prevents lipid storage in macrophages by both suppressing the uptake and stimulating the release of cholesterol and other lipids into or from the macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arterial blood derived low density lipoprotein increases platelet aggregation and macrophage cholesterol content in comparison to lipoprotein derived from veinous blood

We studied the biochemical and biological properties of plasma lipoproteins taken from blood derived from either the aorta or femoral vein of patients with normal coronary arteriography. There were no significant differences in concentrations of cholesterol, triglycerides, apoprotein A-1 and apoprotein B derived from either source. The cholesterol content of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) was similar in both aortic and venous blood. The low density lipoprotein (LDL) concentration, however, was significantly higher in the aortic blood sample. Arterial LDL significantly enhanced in vitro platelet aggregation when compared to venous LDL. (p less than 0.02). When incubated with mouse peritoneal macrophages (MPM) arterial LDL and VLDL caused an increased cholesterol accumulation and enhanced cholesterol esterification within these macrophages. The venous lipoproteins had little effect. The differences noted in the arterial lipoproteins in composition and biological function when compared to venous lipoproteins might be related to the much higher incidence of atherosclerosis found in the arterial tree.     

含载脂蛋白B的脂蛋白对小鼠腹腔巨噬细胞内脂质的影响

将小鼠腹腔巨噬细胞和含载脂蛋白Ｂ的两种脂蛋白、这两种脂蛋白与硫酸葡聚糖或相应抗体形成的复合物一起培养２４ｈ后，用薄层层析法测定巨噬细胞内胆固醇酯的含量。结果发现，天然脂蛋白（ａ）对巨噬细胞内胆固醇酯含量影响不大（增加８％，Ｐ〉０．０５）；天然低密度脂蛋白具有促进巨噬细胞内脂质蓄积作用（增加３５．４％，Ｐ〈０．０５）；上述两种脂蛋白与硫酸葡聚糖一起共同作用，促进巨噬细胞内胆醇酯蓄积作用明显加强（分别     

Glycation of low-density lipoproteins by methylglyoxal and glycolaldehyde gives rise to the in vitro formation of lipid-laden cells

AIMS/HYPOTHESIS: Previous studies have implicated the glycoxidative modification of low-density lipoprotein (LDL) by glucose and aldehydes (apparently comprising both glycation and oxidation), as a causative factor in the elevated levels of atherosclerosis observed in diabetic patients. Such LDL modification can result in unregulated cellular accumulation of lipids. In previous studies we have characterized the formation of glycated, but nonoxidized, LDL by glucose and aldehydes; in this study we examine whether glycation of LDL, in the absence of oxidation, gives rise to lipid accumulation in arterial wall cell types. METHODS: Glycated LDLs were incubated with macrophage, smooth muscle, or endothelial cells. Lipid loading was assessed by HPLC analysis of cholesterol and individual esters. Oxidation was assessed by cholesterol ester loss and 7-ketocholesterol formation. Cell viability was assessed by lactate dehydrogenase release and cell protein levels. RESULTS: Glycation of LDL by glycolaldehyde and methylglyoxal, but not glucose (in either the presence or absence of copper ions), resulted in cholesterol and cholesterol ester accumulation in macrophage cells, but not smooth muscle or endothelial cells. The extent of lipid accumulation depends on the degree of glycation, with increasing aldehyde concentration or incubation time, giving rise to greater extents of particle modification and lipid accumulation. Modification of lysine residues appears to be a key determinant of cellular uptake. CONCLUSIONS/INTERPRETATION: These results are consistent with LDL glycation, in the absence of oxidation, being sufficient for rapid lipid accumulation by macrophage cells. Aldehyde-mediated "carbonyl-stress" may therefore facilitate the formation of lipid-laden (foam) cells in the artery wall.     

Inhibition of cholesterol synthesis by mevinolin stimulates low density lipoprotein receptor activity in human monocyte-derived macrophages

Mevinolin inhibited the incorporation of [14C]acetate into cholesterol by human monocyte-derived macrophages (HMD macrophages) when both mevinolin and [14C]acetate were added simultaneously to the culture medium. Longer incubation with mevinolin, 24 h, led to a marked increase in the degradation of [125I]low density lipoprotein (LDL) via the high affinity receptor for LDL by HMD macrophages. Furthermore, this increased LDL receptor activity in cells incubated for 24 h with mevinolin and lipoprotein-depleted serum (LPDS) led to a nearly 3-fold increase in the formation of cholesteryl esters when these cells were then incubated with LDL for 24 h. Continuous exposure to mevinolin did not result in a constant increase in LDL receptor activity. Cells exposed to mevinolin for 24 h on day 3 had increased LDL receptor activity, but continuous exposure to mevinolin with 5% human serum (HS) from day 3-9 resulted in degradation of [125I]LDL at the same rate as observed in cells incubated with 5% HS alone. Cholesterol synthesis from [14C]acetate was decreased in cells incubated for 24 h in 5% HS + mevinolin compared with 5% HS alone, and in 10% LPDS + mevinolin compared with 10% LPDS alone; however, cells incubated with LPDS + mevinolin synthesized cholesterol at higher rates than did cells cultured in 5% HS alone. Continuous culture with 5% HS + mevinolin for 5 days resulted in cholesterol synthesis at control levels. These data show that following inhibition of cholesterol synthesis by mevinolin, HMD macrophages maintain cholesterol homeostasis by increasing LDL receptor activity to obtain cholesterol from LDL.     

Platelet secretory products enhance LDL receptor activity and inhibit scavenger receptor activity in human monocyte derived macrophages

Macrophage cholesterol accumulation is an early event in atherogenesis. Platelet secretory products have the potential to affect macrophage cholesterol accumulation through their effect on cellular lipoprotein uptake via the low density lipoprotein (LDL) or the scavenger receptor pathways. Preincubation of human monocyte-derived macrophages (HMDM) for 16 hours at 37 degrees C with serotonin, ADP, fibrinogen, fibronectin and platelet-derived growth factor (PDGF), followed by washout of these substances, significantly enhanced LDL uptake by 25% to 75%, whereas acetyl LDL (AcLDL) degradation (AcLDL is taken up by the scavenger receptor), was substantially reduced by 40% to 60% (except for ADP). The effect of serotonin (0 to 75 mumol/L) on macrophage interaction with lipoproteins was further analyzed and revealed a dose-dependent effect on both stimulation of macrophage LDL degradation and cholesterol esterification by up to 2.5 times, as well as an inhibition of the cellular uptake of AcLDL by up to 1.5 times. Analysis of the regulatory effect of serotonin on macrophage lipoprotein uptake revealed that the main effect of serotonin on the uptake of both lipoproteins was to change the affinity of the lipoproteins toward their specific receptor without a significant effect on the number of binding sites. The next questions addressed are whether substances that are known to be secreted by activated platelets can also modify LDL, and whether this modification can alter the interaction between LDL and macrophages. LDL treated with all of the studied substances demonstrated significantly enhanced cellular degradation compared with untreated LDL. The data thus demonstrate that substances such as those released from activated platelets can selectively affect macrophage LDL and scavenger receptor activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sterol regulatory element binding proteins downregulate LDL receptor-related protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by human macrophages

OBJECTIVE: In the extracellular intima, extracellular matrix proteoglycans favor LDL retention and aggregation (agLDL). In contrast to native LDL (nLDL), agLDL induces high intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that LDL receptor-related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding proteins (SREBPs) modulate LRP1 expression and LRP1-mediated agLDL uptake by human monocyte-derived macrophages (HMDM). METHODS AND RESULTS: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66+/-5 microg CE/mg protein) was reduced by 95.76+/-5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1 even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. CONCLUSIONS: These results suggest that high levels of active SREBPs downregulate LRP1 expression and intracellular CE accumulation in HMDM.     

Transfer of cholesterol in vitro between normal arterial smooth muscle tissue and serum lipoproteins of normo-lipidemic rabbits.

During incubation of normal arterial tissue with serum lipoproteins, net transfer of cholesterol was observed in the direction from the lipoproteins into the arterial tissue. Such transfer was only observed during incubations with single, isolated lipoprotein fractions. It was independent of the type of lipoprotein, VLDL, LDL or HDL, which was incubated with the arterial tissue. On the other hand, no net transfer of cholesterol was observed during incubations of arterial tissue with a combination of serum lipoproteins equivalent to that in native serum. Studies on the transfer of radioactive cholesterol suggested that cholesterol elimination from arterial tissue in vitro was not affected by the composition of the incubation medium. Therefore, it is suggested that cholesterol accumulation more easily is influenced by the serum lipoprotein composition, and that serum lipoprotein dysbalance may promote cholesterol accumulation in the tissue. This effect may be present even when the dysbalance involves a decrease of specific serum lipoprotein fractions.     

Macrophages exposed to hypoxia secrete proteoglycans for which LDL has higher affinity

Macrophages are prominent in hypoxic areas of atherosclerotic lesions. Their secreted proteoglycans (PG) can modulate the retention of lipoproteins as well as the activity of enzymes, cytokines, and growth factors involved in atherogenesis. Versican appears to be one of the main extracellular matrix components binding LDL in the arterial intima. We have recently shown that hypoxia increases versican and perlecan expression in macrophages, and that this increase was regulated by the hypoxia inducible factor (HIF). Here we report effects of hypoxia on human monocyte-derived macrophage (HMDM) secreted glycosaminoglycans (GAG), and its interaction with LDL. After 24 h exposure to 0.5% O2 (hypoxia), metabolically labeled GAG of secreted PG had higher affinity for LDL compared to 21% O2 (control cells). GAG secreted by HMDM in hypoxia were found to be more sulfated and longer which might be responsible for the increased affinity of LDL for these GAG chains. These results indicate that hypoxia induced changes in macrophage GAG biosynthesis have important consequences for the interaction with LDL. If present in vivo, an augmented association of GAG with LDL might contribute to the development of atherosclerosis in hypoxic intima.     

Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein.

The uptake of low density lipoprotein (LDL) by cultured mouse macrophages was markedly promoted by isolated rat mast cell granules present in the culture medium. The granule-mediated uptake of LDL enhanced the rate of cholesteryl ester synthesis in the macrophages, the result being accumulation of cholesteryl esters in these cells. Binding of LDL to the granules was essential for the granule-mediated uptake of LDL by macrophages, for the uptake process was prevented by treating the granules with avidin or protamine chloride or by treating LDL with 1,2-cyclohexanedione, all of which inhibit the binding of LDL to the granules. Inhibition of granule phagocytosis by the macrophages with cytochalasin B also abolished the granule-mediated uptake of LDL. Finally, mouse macrophage monolayers and LDL were incubated in the presence of isolated rat serosal mast cells. Stimulation of the mast cells with compound 48/80, a degranulating agent, resulted in dose-dependent release of secretory granules from the mast cells and a parallel increase in cholesteryl ester synthesis in the macrophages. The results show that, in this in vitro model, the sequence of events leading to accumulation of cholesteryl esters in macrophages involves initial stimulation of mast cells, subsequent release of their secretory granules, binding of LDL to the exocytosed granules, and, finally, phagocytosis of the LDL-containing granules by macrophages.     

Influence of probucol on early experimental atherogenesis in hypercholesterolemic rats

A possible cellular action for probucol in early atherogenesis was investigated. In diet-induced hypercholesterolemic rats, probucol reduced aortic accumulation of cholesterol without ameliorating monocyte attachment to the arterial endothelium. Under the imposed conditions, circulating cholesterol levels were not significantly altered by probucol. 125I-labeled acetyl LDL uptake and degradation studies with mouse peritoneal macrophages revealed that probucol had an inhibitory effect on the scavenger receptor pathway. The data suggested that the observed beneficial effects of probucol were not related to an early cholesterol-induced injury phase which might involve calcium. Instead, probucol probably through its free radical scavenging property, intervened at a subsequent cellular level to restrict lipid accumulation.     

Lipid-protein particles secreted from activated platelets reduce macrophage uptake of low density lipoprotein.

Cellular uptake of low density lipoprotein (LDL) was reduced by 30-40% in macrophages that were preincubated with platelet conditioned medium (PCM) obtained from activated platelets. LDL mediated cholesterol accumulation and cholesterol esterification in macrophages were substantially inhibited by macrophages preincubation with PCM. This inhibitory effect was found to be dose dependent, and resulted from a reduction in the number of LDL receptors (decrement of 35% in "apparent Vmax"). The active component in PCM was present only in medium obtained from activated platelets and was found to be of a molecular weight higher than 25,000 dalton. It comprised of both protein and cholesterol but upon PCM delipidation only the lipid fraction demonstrated the inhibitory effect on macrophage uptake of LDL. Specific uptake of the PCM lipoprotein-like particle via the scavenger receptor on macrophages was found to be essential for the expression of LDL receptor reduced activity. Furthermore, LDL mediated cholesterol esterification was not inhibited by PCM in U937 macrophages, a cell line that lacks the scavenger receptors. It is concluded that activated platelets secrete a lipoprotein-like particle which is recognized by the macrophage scavenger receptor. Subsequent to PCM-macrophage interaction, cellular LDL uptake was reduced. This effect could be attributed to the PCM lipid constituents.     

Non-saturable degradation of LDL by monocyte-derived macrophages leads to a reduction in HMG-CoA reductase activity with little synthesis of cholesteryl esters

In monocyte-derived macrophages from both normal and familial hypercholesterolaemic (FH) subjects, degradation of low density lipoprotein (LDL) through non-saturable pathways produced the same fall in 3-hydroxy-3-methylglutaryl-CoA reductase activity as receptor-mediated degradation of acetylated LDL, yet did not lead to as great an increase in incorporation of [14C]oleate into cholesteryl esters. Studies using FH cells showed that the simultaneous addition of LDL did not reduce oleate incorporation resulting from degradation of acetylated LDL, and that there was a similar relationship for both lipoproteins between the increase in net oleate incorporation and the increase in the cholesteryl ester content of the cells. FH cells maintained in serum-free medium accumulated more free cholesterol than cells in lipoprotein-deficient serum when incubated with LDL but not when incubated with acetylated LDL. The results suggest that the cholesterol released from non-saturable degradation of LDL is more easily removed from the cells by acceptors in the medium than cholesterol released from receptor-mediated uptake of acetylated LDL, and is not readily available for esterification.     

Cholesteryl ester accumulation in macrophages incubated with low density lipoprotein pretreated with cigarette smoke extract.

Although cigarette smoking is one of the major risk factors for atherosclerosis and coronary heart disease, the precise mechanisms of its adverse effects have not been fully elucidated. We incubated low density lipoprotein (LDL) with cigarette smoke (CS) extract and examined the incorporation of the lipoprotein by macrophages in vitro. When incubated with macrophages, LDL pretreated with CS extract (100 micrograms/ml) stimulated cholesteryl [14C]oleate synthesis approximately equal to 12.5-fold that with unmodified LDL and transformed macrophages to cells rich in lipid droplets positively stained with oil red O. Enhancement in cholesteryl ester synthesis was dependent on the concentration of CS-modified LDL and exhibited saturation kinetics. When subjected to electrophoreses, CS-modified LDL migrated to a more anionic position than did unmodified LDL and showed extensive fragmentation of apolipoprotein B. This LDL modification depended upon the incubation time and concentration of the CS extract. Superoxide dismutase inhibited modification of LDL by 52%, suggesting that superoxide anion is, at least in part, involved. These results suggest that CS extract alters LDL into a form recognized and incorporated by macrophages. Such modification if it occurs in vivo, could explain the increased incidence of atherosclerosis and coronary heart disease in smokers.     

Probucol prevents lipid storage in macrophages

Effects of probucol on lipid storage in macrophages in vitro in the presence of acetylated low density lipoprotein (acetyl-LDL) were observed using macrophage-like cells (UE-12) established from a human histiocytic lymphoma cell line (U-937). Under ordinary light microscopy as well as under electron microscopy we found that probucol added to the medium, either in ethanolic solution or bound to LDL, markedly prevented the development of macrophages into foam cells. Microscale enzymatic assay of cholesterol also showed that the intracellular accumulation of esterified cholesterol caused by acetyl-LDL was markedly decreased by the addition of probucol. The concentration of probucol added to the medium was almost comparable to the plasma concentration of the drug usually obtained in patients under treatment with probucol. The possibility that probucol interferes with the binding of acetyl-LDL to the receptors on the cell surface was suggested. The results of the present investigation coincide with the clinical findings that probucol causes a more marked regression of xanthomas than would be expected from the extent of lowering of LDL cholesterol.     

Accumulation and loss of cholesterol esters in monkey arterial smooth muscle cells exposed to normal and hyperlipemic serum lipoproteins.

The effects of high low and very low density lipoprotein fractions from normal or hyperlipemic rhesus monkey serum on the accumulation or removal of cholesterol esters from rhesus monkey smooth muscle cells in tissue culture were determined. Serum or serum lipoproteins were labeled with [14C] free cholesterol and adjusted to the same free cholesterol level in the incubation medium. Of the two normal lipoproteins examined, the LDL fraction caused more esterification than the HDL. Cells incubated in hyperlipemic serum showed a 2-fold stimulation in esterification as compared to cells in normal serum. This was contributed by hyperlipemic VLDL and LDL and led to a concomitant increase in cellular cholesterol ester content. Both hyperlipemic LDL and HDL stimulated esterification when compared to their normal counterparts. Cholesterol ester removal was examined by incubating the serum or lipoprotein fractions with cells enriched in cholesterol ester through a prior exposure to hyperlipemic serum. The cells incubated in normal or hyperlipemic HDL or lipoprotein-deficient serum had the lowest cholesterol ester content. Thus, the lipoprotein fractions which caused the lowest levels of cholesterol esterification were also the most efficient in the removal of cellular cholesterol esters.     

Effect of stanol ester on postabsorptive squalene and retinyl palmitate

Stanol ester dissolved in margarine inhibits cholesterol absorption in general and, despite increasing cholesterol synthesis, decreases serum total and low-density lipoprotein (LDL) cholesterol levels, but its effects on postprandial lipid metabolism are unknown. We performed fat tolerance tests in 11 men at baseline and during short-term stanol ester consumption without and with stanol esters added to the test meal also containing retinol and squalene. Cholesterol, triglycerides, retinyl palmitate, and squalene were analyzed in plasma, chylomicrons, and very-low-density lipoprotein (VLDL) at baseline and 3, 4, 6, 9, 12, and 24 hours after the test meal. Serum total and LDL cholesterol only tended to diminish after the 2-week stanol ester consumption. However, the proportion of plasma plant sterol and cholesterol-precursor sterol to cholesterol was significantly altered, suggesting that cholesterol absorption was diminished and cholesterol synthesis was increased. Postprandial peak times of squalene and retinyl palmitate in plasma, chylomicrons, and VLDL were significantly reduced by stanol esters, but their concentrations in chylomicrons were unchanged. Stanol esters reduced the VLDL squalene peak concentration by 23% (P < .05) and the incremental area under the curve (AUIC) in plasma and VLDL by 22% and 32% (P < .01 for both). Chylomicron remnant metabolism measured with triglycerides only tended to diminish. The effects of stanol esters in the diet only and both in the diet and with supplementation did not differ significantly. We conclude that dietary stanol esters reduce postprandial lipoproteins measured with dietary retinyl palmitate and especially squalene, and the reduction is observed even though serum total and LDL cholesterol are only inconsistently decreased after short-term stanol ester consumption.     

Modified plasma-derived lipoproteins in human atherosclerotic plaques

Low density lipoproteins extracted from surgical specimens of human atherosclerotic plaques (A-LDL) showed altered electrophoretic mobility indicating a greater negative charge than that of plasma LDL (P-LDL). A-LDL but not P-LDL showed high affinity binding/degradation by human monocyte-derived macrophages; this was inhibited by acetylated LDL but not by native P-LDL. Following injection of 125I-labelled autologous P-LDL prior to reconstructive arterial surgery, polyacrylamide and agarose gel electrophoresis of A-LDL extracted from arterial intima showed that the A-LDL and its apolipoprotein B moiety were derived from P-LDL; the electrophoretic mobility of the product A-LDL was greater than that of native P-LDL. The compositions of arterial intermediate density lipoprotein (A-IDL) and A-LDL differed from those obtained from human plasma intermediate density lipoprotein (P-IDL) and P-LDL. A-IDL showed a reduced triglyceride content and increased esterified and unesterified cholesterol. Although the total cholesterol content of A-LDL was similar to that of P-LDL, there was an increase in unesterified cholesterol and a decrease of cholesteryl ester. These studies indicate that LDL extracted from human atherosclerotic plaque is derived from and modified from P-LDL in vivo. Compared with native P-LDL, A-LDL showed differences in charge and composition, associated with its high affinity binding by the acetyl LDL receptor of human macrophages.