A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy

Abstract

SUMMARYWe introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 m in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vessicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions ( Ͼ 0.5 mm diameter) had a "nested" distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center.

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

“…This oxysterol, frequently detected at enhanced levels in atherosclerotic plaques (8) and in the plasma of patients with high cardiovascular risks (42), is a potent inducer of apoptosis characterized by various cellular dysfunctions: phosphatidylserine externalization (43), loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADPribose) polymerase (36), morphologic nuclear changes (cells with swollen, condensed, and/or fragmented nuclei) (44), and formation of multilamellar cytoplasmic structures (22,23). Because multilamellar cytoplasmic structures (also termed myelin figures), considered as lipid storage organelles (21), were observed in aortic cells from cholesterol-fed rabbits (25,26), the effects of 7KC (20 g/ ml) on the cellular lipid content were studied on human promonocytic U937 cells after staining with filipin (27,28) and NR (29,34). Moreover, because an increased cellular content of 7KC can disturb numerous signaling pathways (12), the interaction between 7KC and cellular lipids was investigated by FRET.…”

“…This dye stains neutral lipids yellow (570 -590 nm) and polar lipids orange-red (Ն590 nm) when excited at 488 nm (29,34). In the present investigation, NR was prepared at 100 g/ml in dimethylsulfoxide.…”

“…Briefly, after 24 h of culture in the absence or presence of 7KC (20 g/ml), U937 cells were incubated at 37°C with NR (0.1 g/ml, 15 min). NR stains neutral lipids yellow (570 -590 nm) and polar lipids orange-red (Ն590 nm) when excited at 488 nm (29,34). The medium was removed.…”

“…2C,D). To determine whether the formation of myelin figures occurring during 7KC-induced cell death is associated with modifications of cellular lipid content, untreated and 7KC-treated U937 cells were stained at different times of culture (0, 6, 14, 18, 24, and 30 h) with filipin, which permits quantification of free cholesterol (unesterified cholesterol) (27,28), and with NR, which emits a yellow or orange-red fluorescence in the presence of neutral lipid or polar lipid, respectively, when excited with blue light (29). Under these conditions, incubation of U937 cells with 7KC induced a time-dependent cellular accumulation of free cholesterol.…”

“…In the present study the effects of 7KC on cellular lipid content were investigated by flow cytometry after staining with filipin and Nile Red (NR). Filipin permits quantification of free cholesterol (27,28) and NR emits a yellow or orange-red fluorescence in the presence of neutral lipids (esterified cholesterol) and polar lipids (phospholipids), respectively, when excited with blue light (29). Because intracellular accumulation of 7KC can disturb numerous metabolic pathways (12), interactions between 7KC and NR-stained cellular lipids were determined by FRET on spectral images obtained in the biphoton excitation mode of confocal microscopy (30).…”