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

Abstract

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, 3,3Ј-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 orangered 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.

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…In previous investigations, we demonstrated that the maximum proportions of U937 cells simultaneously characterized by a typical morphology of apoptosis (condensed and/ or fragmented nuclei) and by an important accumulation of polar lipids measured by staining with NR, were identified at 18 h when they are cultured in the presence of 7KC used at 20-40 lg/ml (7,10). Therefore in the present study, the relationships between polar lipid accumulation and caspase activity were analyzed in the same experimental conditions.…”

“…Similarly, preliminary experiments were also performed to determine the most convenient band pass filter settings able to differentiate emissions. As a result, yellow, orange, and red fluorescence emissions could not be differentiated properly and advanced spectral analysis was therefore processed (7,10).…”

“…Among these cholesterol oxide derivatives, 7-ketocholesterol (7KC) is one of the most prominent oxysterols present in atheromatous plaques (5). We previously reported that 7KC-induced cytotoxic effects include numerous features of apoptosis such as activation of caspases-3, -7, -8, and -9, which are associated to the formation of multilamellar cytoplasmic structures named myelin figures (6,7). These ultrastructural features 1 which can be stained with Nile Red (NR) (a metachromatic fluorescent probe which puts yellow and orange/red colors on neutral and polar lipids, respectively) (8)(9)(10) are rich in 7KC, accumulate polar lipids (phosphatidylcholine and sphingomyelin) and contain cholesterol (7).…”

“…We previously reported that 7KC-induced cytotoxic effects include numerous features of apoptosis such as activation of caspases-3, -7, -8, and -9, which are associated to the formation of multilamellar cytoplasmic structures named myelin figures (6,7). These ultrastructural features 1 which can be stained with Nile Red (NR) (a metachromatic fluorescent probe which puts yellow and orange/red colors on neutral and polar lipids, respectively) (8)(9)(10) are rich in 7KC, accumulate polar lipids (phosphatidylcholine and sphingomyelin) and contain cholesterol (7). These data, which provide experimental evidence that 7KC-induced cell death is associated with altered lipid metabolism, are in agreement with previous investigations performed on J774 A.1 murine macrophage like cells cultured in the presence of oxidatively modified LDL (11).…”

“…Hence, the reconstructed 3D image resulted in a distorted sphere, itself containing small spheres. As a result each yellow spot (neutral lipids) inside a flattened cellular background can be visualized (7,10). As a consequence also, unmixing is provided when the distance between two yellow spots makes it possible to differentiate them visually or according to resolution criteria (7,10).…”