Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts.(ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation-for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration-LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).The lipid-laden foam cells in atherosclerotic lesions are derived largely or in part from monocyte/macrophages (1, 2). These cells have only low levels of the classical low density lipoprotein (LDL) receptor and take up native LDL in vitro at relatively low rates, insufficient to cause lipid accumulation to the extent found in vivo (3). It has been suggested that this paradox may be explained if the LDL particle is in some way altered in vivo to a form taken up more readily than native LDL. Certain chemically modified forms of LDL, including acetylated LDL, are indeed taken up much more rapidly than native LDL by macrophages, and this uptake involves a different receptor, designated the acetyl-LDL receptor (3-5). Incubation of LDL with cultured endothelial cells generates a modified form (or forms) of LDL (endothelial cell-modified LDL; EC-modified LDL) that is taken up 3-to 10-fold more rapidly by macrophages and at least in part by way of the acetyl-LDL receptor (6-8). The modification in biological properties is accompanied by a marked increase in electrophoretic mobility and hydrated density but the mechanisms involved are still poorly understood.LDL is highly sensitive to metal-catalyzed oxidation (9-11) and oxidized LDL has been shown to be toxic to some cultured cells (12,13). Endothelial cells in culture have been shown to be capable of oxidizing LDL (14). Since the modification of LDL by endothelial cells involves long incubation under aerobic conditions, we examined the possible role of oxidative changes in the process. In the present paper, we report that generation of EC-modified LDL is associated with lipid peroxidation and with extensive hydrolysis of LDL phosphatidylcholine (PtdCho) to lysophosphatidylcholine (lyso-PtdCho).MATERIALS...

Abstract-CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO-1, and Prx I protein levels. Because PPAR-␥ can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

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A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

1. The kinetics of the depletion of alpha-tocopherol in human low-density lipoprotein (LDL) were measured during macrophage-mediated and cell-free oxidation. The formation of oxidatively modified, high-uptake species of LDL in these systems was not detectable until all of the endogenous alpha-tocopherol had been consumed. 2. Supplementation of the alpha-tocopherol content of LDL by loading in vivo extended the duration of the lag period during which no detectable oxidative modification occurred. 3. The addition of a flavonoid (morin) prevented both alpha-tocopherol consumption and oxidative modification of LDL. 4. The alpha-tocopherol contents of LDLs from a range of individual donors could not be used to predict their relative resistance to oxidation, indicating that other endogenous antioxidants may also be present, and quantitatively significant, in human LDL.

Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18:1 do not appear to have a significant effect on the blood lipid profile.

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Abstract-The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. Key Words: atherosclerosis Ⅲ ceroid Ⅲ lysosome Ⅲ iron Ⅲ oxidized low density lipoprotein T he local oxidation of low density lipoprotein (LDL) within atherosclerotic lesions is widely believed to be of importance in the pathogenesis of atherosclerosis. 1 LDL is thought to be oxidized within the extracellular space of atherosclerotic lesions and then to be bound by scavenger receptors and taken up by macrophages, which become cholesterol-laden foam cells, a major feature of atherosclerotic lesions. 2 Among many other effects, oxidized LDL increases the expression of cellular adhesion molecules and chemokines, 3,4 increases the production of metalloproteinases, 5 which probably destabilize the fibrous caps over advanced lesions, and induces apoptosis in cells. 6 The mechanisms by which LDL is oxidized in atherosclerotic lesions remain uncertain, despite a great deal of work. 7 The oxidation hypothesis of atherosclerosis needs to address the high antioxidant capacity of extracellular fluids. Even a few percent of serum or interstitial fluid can inhibit greatly the oxidation of LDL by cells. 8,9 We postulated that LDL oxidation might occur not within the interstitial fluid of atherosclerotic lesions but within lysosomes in macrophages in atherosclerotic lesions. Materials and Methods LDL Isolation and ModificationBlood was taken from healthy volunteers with EDTA as the anticoagulant (final concentration 3 mmol/L). LDL (1.019 to 1.063 g/mL) was isolated from the plasma by sequential density ultracentrifugation at 4°C, as described previously. 10 LDL was stored in the dark under argon at 4°C and used within 1 month. Aggregation of LDL was achieved by vortexing 11 or acetylation. 12 Acetylation of LDL was confirmed by agarose gel electrophoresis (Paragon gels; Beckman), as seen by an increase of about 4.5 in electrophoretic mobility relative to native LDL. Cell CultureCell culture media (DMEM, RPMI 1640, and Ham's F-10) and phosphate buffered saline (PBS) (without calcium or magnesium) were obtained from Gibco Life Technologies. The media used in this study were supplemented with 20% (v/v) fetal calf serum, Glutamax (2 mmol/L), penicillin (50 IU/mL), streptomycin (50 g/mL), and amphotericin B (0.95 g/mL), unless otherwise stated. Humidified 95% air/5% carbon dioxide at 37°C was used for cell culture. J774 cells were regularly cultured in supplement...