Knock-down of the oxysterol receptor LXRα impairs cholesterol efflux in human primary macrophages: lack of compensation by LXRß activation.

Liver X Receptors (LXRs) α and ß are oxysterol-activated nuclear receptors involved in the control of lipid metabolism and inflammation. Pharmacological activation of LXR is promising in the treatment of atherosclerosis since it can promote cholesterol efflux from macrophages and prevent foam cell formation. However, the development of LXR agonists has been limited by undesirable side-effects such as hepatic steatosis mediated by LXRα activation. Therefore, it has been proposed that targeting LXRα activators to extrahepatic tissues or using LXRß-specific activators could be used as alternative strategies. It is not clear whether these molecules will retain the full atheroprotective potential of non-selective agonists. Our aim was therefore to determine the contribution of LXRα and LXRß to the control of cholesterol efflux in human macrophages. LXRα and/or LXRß expression was suppressed by small interfering RNAs in human primary macrophages treated or not with synthetic LXRα/ß dual agonists T0901317 and GW3965. We observed that LXRß silencing had no detectable impact on the expression of LXR-target genes such as ABCA1 and ABCG1. Moreover it did not affect cholesterol efflux. In contrast, LXRα silencing reduced the response of these LXR-target genes to LXR agonist and inhibited cholesterol efflux to ApoA-I, HDL2 or to endogenous ApoE. Importantly, no differences were observed between LXRα and LXRα/ß knockdown conditions. Altogether, our data demonstrate that LXRß activation is unable to maintain maximal cholesterol efflux capacities in human primary macrophages when LXRα expression is impaired. In contrast to earlier mouse studies, LXRα levels appear as a limiting factor for macrophage cholesterol efflux in humans.

Induction of transglutaminase 2 by a liver X receptor/retinoic acid receptor alpha pathway increases the clearance of apoptotic cells by human macrophages.

RATIONALE: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that are involved in the control of cholesterol homeostasis and inflammatory response. Human monocytes and macrophages express high levels of these receptors and are appropriate cells to study the response to LXR agonists. OBJECTIVE: The purpose of this study was to identify new LXR targets in human primary monocytes and macrophages and the consequences of their activation. METHODS AND RESULTS: We show that LXR agonists significantly increase the mRNA and protein levels of the retinoic acid receptor (RAR)alpha in primary monocytes and macrophages. LXR agonists promote RARalpha gene transcription through binding to a specific LXR response element on RARalpha gene promoter. Preincubation of monocytes or macrophages with LXR agonists before RARalpha agonist treatment enhances synergistically the expression of several RARalpha target genes. One of these genes encodes transglutaminase (TGM)2, a key factor required for macrophage phagocytosis. Accordingly, the combination of LXR and RARalpha agonists at concentrations found in human atherosclerotic plaques markedly enhances the capabilities of macrophages to engulf apoptotic cells in a TGM2-dependent manner. CONCLUSIONS: These results indicate an important role for LXRs in the control of phagocytosis through an RARalpha-TGM2-dependent mechanism. A combination of LXR/RARalpha agonists that may operate in atherosclerosis could also constitute a promising strategy to improve the clearance of apoptotic cells by macrophages in other pathological situations.

Cholesterol accumulation is increased in macrophages of phospholipid transfer protein-deficient mice: normalization by dietary alpha-tocopherol supplementation.

OBJECTIVE: Phospholipid transfer protein (PLTP) is a multifunctional, extracellular lipid transport protein that plays a major role in lipoprotein metabolism and atherosclerosis. Recent in vivo studies suggested that unlike systemic PLTP, macrophage-derived PLTP would be antiatherogenic. The present study aimed at characterizing the atheroprotective properties of macrophage-derived PLTP. METHODS AND RESULTS: Peritoneal macrophages were isolated from PLTP-deficient and wild-type mice and their biochemical characteristics were compared. It is shown that macrophages isolated from PLTP-deficient mice have increased basal cholesterol content and accumulate more cholesterol in the presence of LDL compared with wild-type cells. Cholesterol parameters in macrophages of PLTP-deficient mice were normalized by dietary alpha-tocopherol supplementation. CONCLUSIONS: The antiatherogenic properties of macrophage-derived PLTP are related at least in part to its ability to reduce cholesterol accumulation in macrophages through changes in the alpha-tocopherol content and oxidative status of the cells.

7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.

BACKGROUND: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis. METHODS: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. RESULTS: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures. CONCLUSIONS: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.

Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death.

OBJECTIVE: To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images. RESULTS: By UV-CLSM, 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures. CONCLUSION: The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.

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.

Chronology of cellular alterations during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells: analysis by time lapse-video microscopy and conventional fluorescence microscopy.

BACKGROUND: Time-lapse video microscopy was used to determine whether mitochondrial and nuclear changes (decrease in mitochondrial transmembrane potential, condensation, and/or fragmentation of the nuclei, morphologic features typical of apoptosis) occurring during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells took place before or after the loss of cell adhesion. In addition, changes in actin organization were followed by conventional fluorescence microscopy. METHODS: Morphologic, functional, and spatial changes at the mitochondrial level were investigated with 3,3'-dihexyloxacarbocyanine iodide and/or MitoTracker Red, and nuclear morphology was characterized by staining with Hoechst 33342. Actin fibers, which are major components of the filament network of the cytoskeleton, were visualized with phalloidin linked to fluorescein. The numbers of adherent and nonadherent cells were determined by cell counting. RESULTS: 7-Ketocholesterol-induced cell death was associated with a rapid alteration of actin fibers, a loss of intercellular junctions, and cell shape modifications. Analysis of mitochondrial transmembrane potential showed successively a hyperpolarization and a more or less pronounced progressive decrease followed by a dramatic drop associated with an increase in Hoechst 33342 staining, reflecting chromatin condensation and morphologic changes in the nuclei. CONCLUSIONS: During cell death induced by 7-ketocholesterol in A7R5 rat smooth muscle cells, the different methods of microscopy allowed us to establish that alterations of actin fibers and mitochondrial dysfunctions occurred before condensation and/or fragmentation of the nuclei, which preceded the loss of cell adhesion.

Analysis of oxidative processes and of myelin figures formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis: comparison with various pro-apoptotic chemicals.

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol) 7beta-hydroxycholesterol and 7-ketocholesterol are potent inducers of cell death and probably play central roles in atherosclerosis. As suggested by our previous investigations, 7-ketocholesterol might be a causative agent of vascular damage by inducing apoptosis and enhancing superoxide anion (O2*-) production. To determine the precise relationships between cytotoxicity and oxidative stress, the ability of oxysterols oxidized at C7 to induce apoptosis, to stimulate O2*- production and to promote lipid peroxidation was compared with different pro-apoptotic chemicals: antitumoral drugs (VB, Ara-C, CHX, and VP-16) and STS. All compounds, except 7alpha-hydroxycholesterol, induced apoptosis characterized by the occurrence of cells with fragmented and/or condensed nuclei, loss of mitochondrial potential, caspase-3 activation, PARP degradation, and internucleosomal DNA fragmentation. The highest proportion of apoptotic cells was found with antitumoral drugs and STS, whereas the highest overproduction of O2*- detected before and after the loss of mitochondrial potential was obtained with 7beta-hydroxycholesterol and 7-ketocholesterol. Overproduction of O2*- was always correlated with enhanced lipid peroxidation. Vit E was only capable to significantly counteract apoptosis and oxidative stress induced by 7beta-hydroxycholesterol, 7-ketocholesterol, VB and STS. By electron and fluorescence microscopy, myelin figures evocating autophagic vacuoles were barely observed under treatment with 7beta-hydroxycholesterol and 7-ketocholesterol, and their formation occurring before the loss of mitochondrial potential was reduced by Vit E. In the presence of 7alpha-hydroxycholesterol, no enhancement of O2*- production, no lipid peroxidation, and no formation of myelin figures were observed. Collectively, our data demonstrate, that there can be a more or less important stimulation of oxidative stress during apoptosis. They also suggest that enhancement of O2*- production associated with lipid peroxidation during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis could contribute to in vivo vascular injury, and that myelin figures could constitute suitable markers of oxysterol-induced cell death.

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death.

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.