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

Abstract

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.

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

“…Moreover, it has been reported that 7-ketocholesterol can favor in vitro monocyte differentiation and foam cell formation (16). Interestingly, in the atherosclerotic process, the part played by unesterified cholesterol in intracellular lipid accumulation is well documented, and the associated cellular modifications, such as the presence of multilamellar cytoplasmic structures, recall the modifications observed under treatment with cytotoxic oxysterols (7-ketocholesterol, 7ß-hydroxycholesterol, cholesterol-5ß, 6ß epoxide) in various cell types (24,31,32,46): presence of multilamellar cytoplasmic structures (also called myelin figures) of various sizes and shapes (27) and intracellular accumulation of polar lipids revealed by Nile Red staining and subcellular fractionation associated with gaseous chromatography coupled with mass spectrometry (30,38). When the cells were treated with 7-ketocholesterol, these biochemical analyses also revealed an accumulation of cholesterol and 7-ketocholesterol in myelin figures.…”

“…Whereas the involvement of an autophagic process is supported by the identification of LC3-II in 7-ketocholesterol-treated human aortic smooth muscle cells, the presence of cytoplasmic structures stained by the lysosome cationic lipophilic dye, monodansylcadaverine, which is detected before the loss of mitochondrial potential in 7-ketocholesterol-treated U937 cells, suggests instead that 7-ketocholesterol is a potent inducer of phospholipidosis (29). Indeed, under treatment with cytotoxic oxysterols, these structures, which are identified both in cells with condensed and/or fragmented nuclei characteristic of apoptotic cells and in cells with swollen nuclei considered as oncotic cells, can also be stained with Nile Red, which colors neutral and polar lipids yellow and orange/red, respectively (30). In addition, their biochemical characterization revealed that they contain a high concentration of phosphatidylcholine and sphingomyelin.…”

“…In addition, their biochemical characterization revealed that they contain a high concentration of phosphatidylcholine and sphingomyelin. Moreover, at cytoplasmic levels, since oxysterolinduced cell death is associated with important modifications (presence of multilamellar cytoplasmic structures) revealed by transmission electron microscopy (27,30,31), we asked whether 7-ketocholesterol-induced apoptosis was associated with an activation of reticulum, stress inducing a coordinated activated program called the unfolded protein response (UPR). In 7-ketocholesterol-treated human aortic smooth muscle cells, an activation of the UPR pathway characterized by the following events was observed (32): early transient calcium oscillations and induction of the expression of the cell death effector CHOP and GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the UPR.…”

“…Moreover, with 7-ketocholesterol, it has been reported that myelin figures were localized in acidic compartments revealed by staining with monodansylcadaverine, which is a lysosomotropic cationic fluorescent probe (27). Moreover, when 7-ketocholesterol-treated cells were cultured in the presence of 7-ketocholesterol for periods of time preceding the loss of transmembrane mitochondrial potential considered as a point of no return and reintroduced in 7-ketocholesterol-free medium, the percentage of cells with depolarized mitochondria was substantially reduced and lower levels of polar lipids per cell (evaluated by Nile Red staining and characterized by large red cytoplasmic structures) were found (30).…”

“…It has also been suggested, with cytotoxic oxysterols (15) and cationic amphiphilic drugs (29), that the rupture of the acidic compartments containing multilamellar structures might favor the release of some proteolytic enzymes into the cytoplasm, which could subsequently contribute to activating certain apoptotic pathways. Moreover, since vitamin E and caspase inhibitors (z-VAD-fmk: broad-spectrum caspase inhibitor; z-VDVADfmk: caspase-2 inhibitor) can counteract phospholipidosis, as well as certain apoptotic-associated events (caspase activation, lysosomal degradation) (30,41), it is tempting to speculate that phospholipidosis and cell death may have common signaling pathways. Therefore, the ability of cytotoxic oxysterols to activate intracellular lipid accumulation (mainly polar lipids) favors the hypothesis that the balance between these oxysterols and vitamin E might play key roles in the lipid profile of atherosclerotic lesions (Figure 3), Figure 3.…”