Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein

Oxidation of low density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification with 12/15-lipoxygenase (LO). We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells expressing 12/15LO. This mmLDL activates macrophages inducing membrane ruffling and cell spreading, activation of ERK1/2 and Akt signaling, and secretion of proinflammatory cytokines. In this study, we found that many of the biological activities of mmLDL were associated with cholesteryl ester (CE) hydroperoxides and were diminished by ebselen, a reducing agent. Liquid chromatography coupled with mass spectroscopy demonstrated the presence of many mono- and polyoxygenated CE species in mmLDL but not in native LDL. Nonpolar lipid extracts of mmLDL activated macrophages, although to a lesser degree than intact mmLDL. The macrophage responses were also induced by LDL directly modified with immobilized 12/15LO, and the nonpolar lipids extracted from 12/15LO-modified LDL contained a similar set of oxidized CE. Cholesteryl arachidonate modified with 12/15LO also activated macrophages and contained a similar collection of oxidized CE molecules. Remarkably, many of these oxidized CE were found in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE(-/-) mice. These results suggest that CE hydroperoxides constitute a class of biologically active components of mmLDL that may be relevant to proinflammatory activation of macrophages in atherosclerotic lesions.     

Postprandial oxidative stress

Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified particles could account for a relevant link between nutrition and early biological processes that foster the development of atherosclerosis.     

Low-density lipoproteins modified by lipid transfer protein have altered biological activity

Low-density lipoproteins (LDL) were modified by incubation with very-low-density lipoproteins (VLDL) and lipid transfer protein(s) to yield LDL particles that were enriched in triacylglycerol, depleted in cholesteryl esters, and contained apolipoprotein C. The uptake and degradation of these 125I-labeled modified LDL particles by cultured skin fibroblasts was reduced by approx. 30% when compared with LDL that had not been exposed to lipid transfer protein. Incubation of fibroblasts for 24 h in the presence of modified LDL resulted in less inhibition of LDL receptor activity and sterol synthesis than did incubation with control LDL. Both the degradation of 125I-labeled modified LDL and the effect of unlabeled modified LDL on the regulation of LDL binding and sterol synthesis were progressively decreased as the extent of modification of the LDL was increased. Even when identical amounts of modified LDL or control LDL protein were degraded, less inhibition of LDL receptor activity and sterol synthesis was observed with modified LDL than with control LDL, suggesting that the effects of modified LDL on these regulatory events are related to both the reduced degradation of the modified lipoprotein particles and to the alteration in its chemical composition. Uptake and degradation of modified LDL by human monocyte-derived macrophages in culture was reduced in a manner similar to that observed in the cultured fibroblasts, and was considerably less than that observed with acetylated LDL. No differences were observed between modified LDL prepared by exposure to lipid transfer activity in the lipoprotein deficient fraction of serum or when partially purified lipid transfer was used. Modified LDL, with similar composition to that used in the experiments, has been observed in certain diabetic and non-diabetic hypertriglyceridemic states. Thus, it is possible that the cellular metabolism of LDL in vivo might be altered in the presence of hypertriglyceridemia.     

Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.     

Protein Hydroperoxides are a Major Product of Low Density Lipoprotein Oxidation During Copper,Peroxyl Radical and Macrophage-mediated Oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

Chondroitin sulfate-modified LDL induces increased cholesteryl ester synthesis and down-regulation of LDL receptors in smooth muscle cells and macrophages

Hypercholesterolemia induces increased transcytosis and accumulation of plasma lipoproteins in the arterial intima, where they interact with matrix proteins and become modified and reassembled lipoproteins. Chondroitin 6-sulfate-modified LDL (CS-mLDL) induces migration, proliferation, and lipid accumulation in human aortic smooth muscle cells (SMCs). To search for the mechanism(s) responsible for lipid accumulation, cultured SMC and macrophages were exposed to CS-mLDL, minimally modified LDL (mmLDL), and native LDL (as a control). Then the cellular uptake, degradation and expression of the LDL receptor (LDL-R) was determined using radioiodinated ligands, ACAT activity assay, fluorescence microscopy and RT-PCR. The uptake of CS-mLDL was 2-fold higher in SMC and 3-to 4-fold higher in macrophages as compared to LDL and mmLDL; the lysosomal degradation of CS-mLDL was slower in SMCs and considerably diminished in macrophages. Compared with LDL, CS-mLDL induced increased synthesis and accumulation of esterified cholesterol in SMCs (∼2-fold) and macrophages (∼10-fold) within an expanded acidic compartment. CS-mLDL and mmLDL down-regulate the gene expression of the LDL-R in the both cell types. Mechanisms of CS-mLDL-induced lipid accumulation in SMC and macrophages involve increased cellular uptake, and diminished cellular degradation that stimulates cholesterol ester synthesis and accumulation in cytoplasmic inclusions and in the lysosomal compartment in an undegraded form; modified lipoproteins induce down-regulation of LDL-R.     

Luminescence in the study of lipid metabolism

Using bioluminescence assays for glycerol, free fatty acids, β-hydroxybutyrate and lactate, we were able to perform complex studies of human energy and lipid metabolism both in serum samples in vivo and in isolated fat cells in vitro. These studies would have been impossible without reliable, specific and highly sensitive luminescence methods. Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. Adaptation of a chemiluminescence assay for lipid hydroperoxides to LDL isolated by specific precipitation from serum makes it possible to measure LDL oxidation in vivo. Cell dependent chemiluminescence was used to investigate whether receptor mediated endocytosis of LDL by macrophages leads to oxygen radical production in these cells. No activation of the membrane NAD(P)H oxidase was observed.     

Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants

The high cardiovascular mortality in patients receiving hemodialysis (HD) has been attributed, in part, to oxidative stress. Here we examined the effectiveness of antioxidants introduced by means of a novel hemolipodialysis (HLD) procedure in terms of reducing oxidative stress during ex vivo blood circulation. Oxidative stress was studied in a model HD system resembling the extracorporeal circulation of blood during clinical HD. Blood circulation produced an increase of up to 280% in free hemoglobin levels and an increase of 320% in electronegative LDL (LDL(-)) subfraction. A significant correlation between LDL(-) and free hemoglobin levels confirmed previous findings that LDL(-) formation during ex vivo circulation of blood can be mediated by the oxidative activity of free hemoglobin. These effects were significantly attenuated during HLD using a dialysis circuit containing vitamin E with or without vitamin C. By contrast, HLD with vitamin C alone had a marked pro-oxidant effect. TBARS, lipid hydroperoxides, vitamin E and beta-carotene content in LDL were not significantly altered by the HD procedure. These findings demonstrate the occurrence of oxidative stress in human plasma where lipoproteins are a target and indicate antioxidant-HLD treatment as a specific new approach to decreasing the adverse oxidative stress frequently associated with cardiovascular complications in high-risk populations of uremic patients.     

Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH(3))(3) resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.     

Esterified lipid hydroperoxide-derived modification of protein: formation of a carboxyalkylamide-type lysine adduct in human atherosclerotic lesions

We have recently identified Nepsilon-azelayllysine (AZL) as a carboxyalkylamide-type novel lysine adduct in the reaction of linoleic acid hydroperoxides with the lysine derivative. To examine the formation of AZL in vivo, a novel monoclonal antibody (mAb19D5) specific to AZL moiety was prepared. The mAb19D5 scarcely recognized oxidized low-density lipoprotein (oxLDL), whereas the treatment of oxLDL with alkali or phospholipase A2 significantly increased the immunoreactivity. Similarly, the immunopositive materials were detected in alkali- or phospholipase A2-treated sections from human atherosclerotic aorta but not in untreated sections. These results suggest that esterified lipid hydroperoxide-derived modification of protein may serve as one mechanism for the oxidative modification of LDL and subsequent formation of atherosclerotic lesions in vivo.     

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.     

巨噬细胞对氧化和丙二醛修饰的低密度脂蛋白结合与降解特性的比较研究

用Cu~(2+)(引发氧化修饰)和脂质过氧化降解产物丙二醛对低密度脂蛋白(LDL)进行修饰,分别测定了巨噬细胞系P~(300)D_1和小鼠腹腔巨噬细胞对两种被修饰LDL的结合量(包括内移量)和降解量。结果显示:LDL经氧化修饰和丙二醛修饰后被两类巨噬细胞的结合量与降解量均高于正常LDL,在修饰程度相近(琼脂糖电泳迁移率相近)时,两类巨噬细胞对氧化修饰LDL的结合量与降解量高于丙二醛修饰的LDL。竞争性抑制结果显示,丙二醛修饰的LDL和乙酰化修饰的LDL均可部分抑制巨噬细胞对氧化修饰LDL的结合与降解。     

巨噬细胞对氧化和丙二醛修饰的低密度脂蛋白结合与降解特性的比较研究

用Cu~(2+)(引发氧化修饰)和脂质过氧化降解产物丙二醛对低密度脂蛋白(LDL)进行修饰,分别测定了巨噬细胞系P~(300)D_1和小鼠腹腔巨噬细胞对两种被修饰LDL的结合量(包括内移量)和降解量。结果显示:LDL经氧化修饰和丙二醛修饰后被两类巨噬细胞的结合量与降解量均高于正常LDL,在修饰程度相近(琼脂糖电泳迁移率相近)时,两类巨噬细胞对氧化修饰LDL的结合量与降解量高于丙二醛修饰的LDL。竞争性抑制结果显示,丙二醛修饰的LDL和乙酰化修饰的LDL均可部分抑制巨噬细胞对氧化修饰LDL的结合与降解。     

Cholesteryl ester enrichment of plasma low-density lipoproteins in hamsters fed cereal-based diets containing cholesterol

Male Syrian hamsters were fed 0.02, 0.03, or 0.05% cholesterol to test the hypothesis that moderate cholesterol intake increases the cholesteryl ester content of the plasma low-density lipoproteins (LDL). Dietary cholesterol levels of 0.02%-0.05% were chosen to reflect typical human intakes of cholesterol. Hamsters were fed ad libitum a cereal-based diet (modified NIH-07 open formula) for 15 weeks. Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased plasma LDL and high-density lipoprotein cholesterol concentration, increased liver cholesterol concentration, and increased total aorta cholesterol content. The cholesteryl ester content of plasma LDL was determined as the molar ratio of cholesteryl ester to apolipoprotein B and to surface lipid (i.e., phospholipid + free cholesterol). Increasing dietary cholesterol from 0.02% to 0.05% resulted in significantly increased cholesteryl ester content of LDL particles. Furthermore, cholesteryl ester content of LDL was directly associated with increased total aorta cholesterol, whereas a linear relationship between plasma LDL cholesterol concentration and aorta cholesterol was not observed. Thus, the data suggest that LDL cholesteryl ester content may be an important atherogenic feature of plasma LDL.     

Measurement of Lipid Peroxidation

Lipid peroxidation results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. The approach to the quantitative assessment of lipid peroxidation depends on whether the samples involve complex biological material obtained in vivo, or whether the samples involve relatively simple mixtures obtained in vituo. Samples obtained in vivo contain a large number of products which themselves may undergo metabolism. The measurement of conjugated diene formation is generally applied as a dynamic quantitation e.g. during the oxidation of LDL, and is not generally applied to samples obtained in vivo. Lipid hydroperoxides readily decompose, but can be measured directly and indirectly by a variety of techniques. The measurement of MDA by the TBAR assay is non-specific, and is generally poor when applied to biological samples. More recent assays based on the measurement of MDA or HNE-lysine adducts are likely to be more applicable to biological samples, since adducts of these reactive aldehydes are relatively stable. The discovery of the isoprostanes as lipid peroxidation products which can be measured by gas chromatography mass spectrometry or immunoassay has opened a new avenue by which to quantify lipid peroxidation in vivo, and will be discussed in detail.     

Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS

A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated.     

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.     

Concentrations of iron correlate with the extent of protein, but not lipid, oxidation in advanced human atherosclerotic lesions

Previous studies have provided compelling evidence for the presence of oxidized proteins and lipids in advanced human atherosclerotic lesions. The catalyst responsible for such oxidation is unknown and controversial. We have previously provided evidence for elevated levels of iron in lesions. In this study we hypothesized that if iron ions catalyzed protein and lipid oxidation in the artery wall, then there should be a positive correlation between these parameters. Iron concentrations in ex vivo healthy human arteries and advanced carotid lesions were quantified by electron paramagnetic resonance spectroscopy. Four specific side-chain oxidation products of proteins, and the lipid oxidation products 7-ketocholesterol and cholesterol ester alcohols and hydroperoxides, were quantified by HPLC in the same samples used for the iron measurements. Parent amino acids, cholesterol, and cholesterol esters were also quantified. Statistically elevated levels of iron, cholesterol, cholesterol esters, 7-ketocholesterol, and cholesterol ester alcohols and hydroperoxides were detected in advanced lesions compared with healthy control tissue. Iron levels correlated positively and strongly with all four markers of protein oxidation, but not with either marker of lipid oxidation. These data support the hypothesis that elevated levels of iron contribute to the extent of protein, but not lipid, oxidation in advanced human lesions.     

The effect of felodipine on the uptake and degradation of acetylated LDL in mouse peritoneal cells and on the distribution of acetylated LDL in macrophage-rich organs of the rat

The effect of felodipine on lipoprotein metabolism ex vivo and in vivo was investigated. In the ex vivo studies mice were given felodipine (40–125 μ mol/kg body weight) or vehicle for one week. Peritoneal macrophages from these animals and controls were isolated and used in binding and degradation studies with human iodinated acetylated LDL (Ac-LDL). Macrophages from felodipine-treated mice showed a significant decrease of binding and degradation of Ac-LDL compared to macrophages from control animals (P<0.05). The in vivo studies were performed in rats pretreated with felodipine or vehicle. To determine the distribution and plasma turnover of LDL and Ac-LDL, ¹²⁵I-tyramine cellobiose labelled LDL or Ac-LDL were given i.v. No differences in the removal rate of Ac-LDL or LDL were observed between felodipine-treated or untreated rats. However, an increased uptake of Ac-LDL could be seen in the liver of the felodipine-treated rats. This increased uptake could be ascribed to the parenchymal cells because no differences in uptake could be seen in the liver endothelial cells. However, a significant decreased uptake was seen in the Kuppfer cells and in the spleen, a macrophage-rich organ, of the felodipine-treated rats. The present study suggests a possible mechanism behind the antiatherogenic effects of calcium antagonists, a decreased uptake of atherogenic modified lipoproteins by peripheral macrophages and an increased uptake by the liver.