Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, referred to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7alpha-, 7beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O2*-) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O2*- overproduction quantified with hydroethidine was identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, with cholesterol-5alpha, 6alpha-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7beta-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.

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Background: Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C‐reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7β‐hydroxycholesterol, 7‐ketocholesterol, and 25‐hydroxycholesterol), and various cytokines. Methods: Different flow cytometric bead‐based assays were used to quantify some cytokines (IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, IL‐12, IL‐13, IL‐17, G‐CSF, GM‐CSF, IFN‐γ, MCP‐1, MIP‐1β, or TNF‐α) in the culture media of oxysterol‐treated U937 and THP‐1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL‐8 mRNA levels, intracellular IL‐8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal‐regulated kinase1/2 (MEK/ERK1/2) signaling pathway. Results: All oxysterols investigated are potent in vitro inducers of MCP‐1, MIP‐1β, TNF‐α, and/or IL‐8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL‐8, IL‐1β, IL‐6, IL‐10, TNF‐α, IL‐12, and MCP‐1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF‐α, and IL‐10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered. Conclusions: Flow cytometric bead‐based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol‐treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages. © 2006 International Society for Analytical Cytology

Vitamin E is composed of closely related compounds, including tocopherols and tocotrienols. Studies of the last decade provide strong support for a specific role of alpha-tocopherol in cell signalling and the regulation of gene expression. It produces significant effects on inflammation, cell proliferation and apoptosis that are not shared by other vitamin E isomers with similar antioxidant properties. The different behaviours of vitamin E isomers might relate, at least in part, to the specific effects they exert at the plasma membrane. alpha-Tocopherol is not randomly distributed throughout the phospholipid bilayer of biological membranes, and as compared with other isomers, it shows a propensity to associate with lipid rafts. Distinct aspects of vitamin E transport and metabolism is discussed with emphasis on the interaction between alpha-tocopherol and lipid rafts and the consequences of these interactions on cell metabolism.

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike ␥-tocopherol, the ␣-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, ␣-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre-and co-treatment of the cells with ␣-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of ␣-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by ␣-tocopherol, but not ␥-tocopherol pretreatment.

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On treatment with 7-ketocholesterol (7-keto) or 7beta-hydroxycholesterol (7beta-OH), which are major oxysterols in atherosclerotic plaques, the simultaneous identification of oncotic and apoptotic cells suggests that these compounds activate different metabolic pathways leading to various modes of cell death. With U937, MCF-7 (caspase-3 deficient), MCF-7/c3 cells (stably transfected with caspase-3), we demonstrate that caspase-3 is essential for caspase-9, -7, -8 activation, for Bid degradation mediating mitochondrial cytochrome c release, for cleavage of poly(ADP-ribose) polymerase and inhibitor of the caspase-activated deoxyribonuclease, and, at least in part, for internucleosomal DNA fragmentation. The crucial role of caspase-3 was supported by the use of z-VAD-fmk and z-DEVD-fmk, which abolished apoptosis and the associated events. However, inactivation or lack of caspase-3 did not inhibit 7-keto- and 7beta-OH-induced cell death characterized by staining with propidium iodide, loss of mitochondrial potential. The mitochondrial release of apoptosis-inducing factor and endonuclease G was independent of the caspase-3 status, which conversely played major roles in the morphological aspects of dead cells. We conclude that caspase-3 is essential to trigger 7-keto- and 7beta-OH-induced apoptosis, that these oxysterols simultaneously activate caspase-3-dependent and/or -independent modes of cell death.