Death-signaling pathways in human myeloid cells by oxLDL and its cytotoxic components 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide

Oxidized low-density lipoprotein contains many potentially proatherogenic molecules, including oxysterols, which have been shown to induce apoptosis in various cell lines. The aim of this study was to investigate the pathway of apoptosis induced by oxidized low-density lipoprotein and the oxysterols, 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide, in two human monocytic cell lines. The HL-60 cells appeared to be more sensitive to oxidized low-density lipoprotein than U937 cells, whereas the isolated oxysterols were more potent inducers of apoptosis in the U937 cells. Caspase-2 inhibition decreased the number of viable cells in oxidized low-density lipoprotein-treated samples; however, it protected against cholesterol-5beta,6beta-epoxide-induced cell death. Western blot analysis was utilized to examine the effect of caspase-2 inhibition on the expression of the antiapoptotic protein Bcl-2. Pretreatment with the inhibitor protected against the decrease in Bcl-2 expression in oxidized low-density lipoprotein- and 7beta-hydroxycholesterol-treated U937 cells. In HL-60 cells, Bcl-2 was overexpressed in oxidized low-density lipoprotein-treated cells, but in the presence of the inhibitor Bcl-2 expression was returned to control levels. Depleted ATP concentrations in the cells suggest that both apoptosis and necrosis may have occurred simultaneously. Our results highlight differences in the signaling pathways induced by oxidized low-density lipoprotein, 7beta-hydroxycholesterol, and cholesterol-5beta,6beta-epoxide in U937 and HL-60 cells.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.     

Activation of caspase-3-dependent and -independent pathways during 7-ketocholesterol- and 7beta-hydroxycholesterol-induced cell death: a morphological and biochemical study

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.     

In vivo interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol, potential surrogate markers for oxidative stress

The oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7beta-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11beta-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7beta-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7beta-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7beta-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in humans.     

Different patterns of IL-1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α-, 7β-hydroxycholesterol, or 7-ketocholesterol

Among oxysterols oxidized at C7 (7α-, 7β-hydroxycholesterol, and 7-ketocholesterol), 7β-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-1β and/or tumor necrosis factor (TNF)-α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1β secretion. TNF-α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α- or β-hydroxyl radical position plays a key role in apoptosis and IL-1β secretion.     

7-Ketocholesterol and staurosporine induce opposite changes in intracellular pH,associated with distinct types of cell death in ECV304 cells

Incubation of ECV304 cells with 7-ketocholesterol, a lipid component of oxidized low-density lipoproteins, caused a concentration- and time-dependent decrease in the number of viable cells. Other cholesterol oxides, 7 beta-hydroxycholesterol and 25-hydroxycholesterol, but not cholesterol, were only weakly cytotoxic. No evidence for activation of caspase-3 and -8, DNA laddering, or release of cytochrome c from mitochondria into the cytoplasm was obtained in 7-ketocholesterol-treated cells, indicating that cell death was not due to apoptosis. As a positive control for apoptosis, ECV304 cells were treated with staurosporine, which indeed caused significant activation of caspase-3 activity, DNA laddering, and cytochrome c release. Cellular morphology and actin cytoskeletal organization were distinctly different after exposure to the two drugs. Furthermore, staurosporine caused intracellular acidification, whereas 7-ketocholesterol induced a significant alkalinization, which was abolished by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid. In conclusion, in ECV304 cells 7-ketocholesterol induces some typical hallmarks of necrotic cell death but not of apoptosis.     

Glycyrrhizin Prevents 7-Ketocholesterol Toxicity Against Differentiated PC12 Cells by Suppressing Mitochondrial Membrane Permeability Change

Defects in mitochondrial function participate in the induction of neuronal cell injury. In neurodegenerative conditions, oxidative products of cholesterol are elevated and oxysterols seem to be implicated in neuronal cell death. The present work was designed to study the inhibitory effect of licorice compounds glycyrrhizin and 18β-glycyrrhetinic acid against the toxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, loss of the mitochondrial transmembrane potential, increase in the cytosolic Bax and cytochrome c levels, caspase-3 activation and cell death in differentiated PC12 cells. Glycyrrhizin and 18β-glycyrrhetinic acid prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death. The results obtained show that glycyrrhizin and 18β-glycyrrhetinic acid may prevent the 7-ketocholesterol-induced neuronal cell damage by suppressing changes in the mitochondrial membrane permeability.     

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 μg/mL corresponding to 50 μM, 24–48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3β), and with degradation of Mcl-1. With α-tocopherol (400 μM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3β dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3β/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.     

Cholesterol is superior to 7-ketocholesterol or 7 alpha-hydroxycholesterol as an allosteric activator for acyl-coenzyme A:cholesterol acyltransferase 1

We compared the abilities of cholesterol versus various oxysterols as substrate and/or as activator for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by monitoring the activity of purified human ACAT1 in response to sterols solubilized in mixed micelles or in reconstituted vesicles. The results showed that 5 alpha,6 alpha-epoxycholesterol and 7 alpha-hydroxycholesterol are comparable with cholesterol as the favored substrates, whereas 7-ketocholesterol, 7 beta-hydroxycholesterol, 5 beta,6 beta-epoxycholesterol, and 24(S),25-epoxycholesterol are very poor substrates for the enzyme. We then tested the ability of 7-ketocholesterol as an activator when cholesterol was measured as the substrate, and vice versa. When cholesterol was measured as the substrate, the addition of 7-ketocholesterol could not activate the enzyme. In contrast, when 7-ketocholesterol was measured as the substrate, the addition of cholesterol significantly activated the enzyme and changed the shape of the substrate saturation curve from sigmoidal to essentially hyperbolic. Additional results show that, as an activator, cholesterol is much better than all the oxysterols tested. These results suggest that ACAT1 contains two types of sterol binding sites; the structural requirement for the ACAT activator site is more stringent than it is for the ACAT substrate site. Upon activation by cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.     

alpha-Tocopherol, but not gamma-tocopherol inhibits 7 beta-hydroxycholesterol-induced apoptosis in human U937 cells.

Oxysterols, particularly those oxidised at position 7, are toxic to cells in culture and have been shown to induce apoptosis in cell types such as vascular endothelial cells, smooth muscle cells and monocytes. The precise mechanism by which oxysterols induce apoptosis is unknown but may involve the generation of oxidative stress. In the present study we examined the ability of alpha-TOC, alpha-TOC acetate (alpha-TOCA) and gamma-TOC to protect against 7 beta-hydroxycholesterol (7 beta-OHC)-induced apoptosis of human monocytic U937 cells. 7 beta-OHC is one of the most commonly detected oxysterols in foods and its level in plasma has been positively associated with an increased risk of atherosclerosis. The present study demonstrates a significant decrease in cell membrane integrity and cellular glutathione levels when U937 cells were treated with 30 microM 7 beta-OHC. DNA fragmentation also occurred, as measured by agarose gel electrophoresis, and the number of apoptotic cells increased as assessed by nuclear morphology. Analysis by HPLC showed that there was a greater incorporation of gamma-TOC into U937 cells after a 48 h incubation, than either alpha-TOC or alpha-TOCA. However, despite the increased uptake of gamma-TOC, only alpha-TOC, and not gamma-TOC or alpha-TOCA was effective at inhibiting 7 beta-OHC-induced apoptosis in U937 cells.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occuring during 7-ketocholesterol-induced apoptosis

Previous investigations of our laboratory have shown that 7-ketocholesterol was a potent inducer of apoptosis involving a release of cytochrome c into the cytosol, and a lipid peroxidation process that could be the consequence of a production of radical oxygen species. According to these considerations, we asked whether some antioxidants were able to counteract 7-ketocholesterol-induced apoptosis, and whether prevention of cell death was associated with the impairment of mitochondrial events implied in the commitment to apoptosis, i.e., opening of the mitochondrial megachannels leading to the loss of the mitochondrial transmembrane potential (DeltaPsim), and release of cytochrome c from mitochondria into the cytosol. To this end, we studied the effects of glutathione (15 mM), N-acetylcysteine (15 mM), vitamin E (100 microM), vitamin C (50 microM) and melatonin (1 mM) on U937 cells treated with 7-ketocholesterol (40 microg/ml). Only glutathione, N-acetylcysteine, and vitamin E prevented apoptosis measured by the occurrence of cells with condensed and/or fragmented nuclei, as well as the loss of DeltaPsim, and the release of cytochrome c. However, all the antioxidants used were potent inhibitors of the production of O(2)(*) occuring under treatment with 7-ketocholesterol. Collectively, our data demonstrate that impairment of apoptosis by glutathione, N-acetylcysteine, and vitamin E correlates with the prevention of mitochondrial dysfunctions, and they underline that the ability of antioxidants to counteract 7-ketocholesterol-induced apoptosis does not only depend on their capability to inhibit the production of O(2)(*).     

7-Ketocholesterol-induced apoptosis. Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways

Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the 'BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK-->ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway.     

Tyrosine Kinase Inhibitor AG126 Reduces 7-Ketocholesterol-Induced Cell Death by Suppressing Mitochondria-Mediated Apoptotic Process

The preventive effect of tyrosine kinase inhibitor AG126 against the 7-ketocholesterol toxicity was investigated in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, the mitochondrial membrane permeability changes, the formation of reactive oxygen species and the depletion of GSH, which leads to cell death in differentiated PC12 cells. Tyrphostin AG126 significantly attenuated the 7-ketocholesterol-induced decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic pro-apoptotic Bax levels, mitochondrial membrane potential loss, cytochrome c release and subsequent caspase-3 activation. The inhibitory effect of tyrphostin AG126 may be supported by the inhibitory effect on another oxysterol 25-hydroxycholesterol-induced cell death. The results show that tyrphostin AG126 may prevent the 7-ketocholesterol toxicity by suppressing the mitochondrial membrane permeability change that leads to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and the depletion of GSH.     

Generation of an oxidative stress precedes caspase activation during 7beta-hydroxycholesterol-induced apoptosis in U937 cells

The oxysterol 7beta-hydroxycholesterol (7beta-OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf-1 causes activation of the initiator caspase, caspase-9, which in turn activates caspase-3 ultimately leading to the degradation of poly(ADP-ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7beta-OH-induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7beta-OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase-3, -8, and -9 and cleavage of the caspase-3 substrate PARP. The sequence proceeded with activation of caspase-9 after 9 h, caspase-3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7beta-OH. Caspase-8 did not appear to play a major role in this particular apoptotic pathway.     

Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis

7-Ketocholesterol is a component of oxidized LDL, which plays a central role in atherosclerosis. It is a potent inducer of cell death towards a wide number of cells involved in atherosclerosis. In this study, it is reported that 7-ketocholesterol treatment induces an increase of cytosolic-free Ca(2+) in THP-1 monocytic cells. This increase is correlated with the induction of cytotoxicity as suggested from experiments using the Ca(2+) channel blockers verapamil and nifedipine. This 7-ketocholesterol-induced apoptosis appears to be associated with the dephosphorylation of serine 75 and serine 99 of the proapoptotic protein Bcl-2 antagonist of cell death (BAD). We demonstrated that this dephosphorylation results mainly from the activation of calcium-dependent phosphatase calcineurin by the oxysterol-induced increase in Ca(2+). Moreover, this Ca(2+) increase appears related to the incorporation of 7-ketocholesterol into lipid raft domains of the plasma membrane, followed by the translocation of transient receptor potential calcium channel 1, a component of the store operated Ca(2+) entry channel, to rafts.     

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7 beta-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7 beta-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.     

Membrane properties of oxysterols. Interfacial orientation, influence on membrane permeability and redistribution between membranes

The membrane properties of cholesterol auto-oxidation products, 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol and 25-hydroxycholesterol were examined. Monolayer studies show that these oxysterols are perpendicularly orientated at the interphase. Only 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol are tilted at low surface pressures. In mixed monolayers with dioleoylphosphatidylcholine, 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol show a condensing effect in this order, although to a lesser extent that that observed for cholesterol. In liposomes these oxysterols also reduce glucose permeability and in the same order as their condensing effect. On the other hand 25-hydroxycholesterol shows no condensing effect in monomolecular layers whereas glucose permeability in liposomes is enormously increased. The permeability increase is already maximal at 2.5 mol% 25-hydroxycholesterol. Differential scanning calorimetry experiments reveal that all four oxysterols tested reduce the heat content of the gel----liquid-crystalline phase transition. It is concluded that 7-ketocholesterol, 7 beta-hydroxycholesterol and 7 alpha-hydroxycholesterol have a cholesterol like effect, although less efficient than cholesterol, whereas 25-hydroxycholesterol showing no condensing effect acts as a spacer molecule. Packing defects in the hydrophobic core of the bilayer due to the presence of the C-25 hydroxyl group are believed to cause the permeability increase. The transfer of radiolabelled (oxy)sterols from the monolayer to lipoproteins or vesicles in the subphase was studied. The transfer rate increases in the following order 7-ketocholesterol, 7 beta-hydroxycholesterol, 7 alpha-hydroxycholesterol, 25-hydroxycholesterol. The difference in rate between 7-ketocholesterol and 25-hydroxycholesterol is 20-fold. A higher rate of transfer is observed in the presence of high density lipoproteins and small unilamellar vesicles. A transfer rate for cholesterol is hardly measurable under these conditions. The transfer measured is consistent with the involvement of a water-soluble intermediate.     

Oxysterol mixtures, in atheroma-relevant proportions, display synergistic and proapoptotic effects

Apoptotic cells in atheroma lesions may contribute to plaque development and instability. Oxysterols constitute the major toxic component in oxLDL and are present in mixed forms in human atheroma lesions. However, the cellular effects of oxysterols have been mostly studied individually. In the present study, we investigated the cytotoxic effects of 7beta-hydroxycholesterol (7betaOH), 7-ketocholesterol (7keto), 25-hydroxycholesterol (25OH), and 27-hydroxycholesterol (27OH) on U937 monocytic cells, both individually and in atheroma-relevant mixtures mimicking the oxysterol composition reported in human atheroma lesions. Apoptosis and necrosis were studied by examining cell morphology, phosphatidylserine exposure, caspase activation, and the terminal dUTP nick end-labeling technique. Cellular reactive oxygen species and total amount of reduced thiols were measured by using fluorescence probes and 5,5'-dithiobis-(2-nitrobenzoic acid), respectively. We found that 7betaOH and 7keto induced caspase activation, ROS production, cellular thiol depletion, permeabilization of lysosomal and mitochondrial membranes, and cell death. 25OH and 27OH did not cause any of the above alterations, whereas 7betaOH and 7keto exerted synergistic toxic effects. Although single 25OH or 27OH exhibited quenching effects on both 7betaOH- and 7keto-induced cell death, the combination of all four oxysterols in atheroma-relevant proportions was proapoptotic. Our findings indicate that the major oxysterols accumulated in human atheroma are proapoptotic and may contribute to atherosclerotic lesion development.