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Oxysterol mixtures prevent proapoptotic effects of 7-ketocholesterol in macrophages: implications for proatherogenic gene modulation ¹

FIORELLA BIASI^*,, GABRIELLA LEONARDUZZI^*, BARBARA VIZIO^*, DANIELLA ZANETTI^*, ALEX SEVANIAN^#, BARBARA SOTTERO^*, VERONICA VERDE^*, BARBARA ZINGARO^*, ELENA CHIARPOTTO^* and GIUSEPPE POLI^*,2

^* Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, Torino, Italy;
Italian National Research Council, Torino, Italy; and
^# Department of Molecular Pharmacology and Toxicology, University of Southern California, School of Pharmacy, Los Angeles, California, USA

² Correspondence: Department of Clinical and Biological Sciences, University of Torino, S. Luigi Gonzaga Hospital, 10043 Orbassano, Torino, Italy. E-mail: giuseppe.poli{at}unito.it

SPECIFIC AIMS

Oxysterols are commonly encountered in oxidized LDL (oxLDL) and in the core region of fibrotic plaque, and exist as a mixture of several major components that may have a variety of effects and molecular interactions. We compared the proapoptotic effect of 7-ketocholesterol plus or minus 7ß-hydroxycholesterol, unoxidized cholesterol, and a mixture of oxysterols that are representative of plasma samples analyzed in our laboratory and similar to proportions in oxLDL as reported by others.

PRINCIPAL FINDINGS

1. The effects of 7-ketocholesterol, unoxidized cholesterol, and a biologically representative oxysterol mixture are different in terms of caspase-3 activation in J774A.1 macrophages
In a concentration range detectable in hypercholesterolemic patients (20–30 µM), a biologically representative oxysterol mixture did not trigger programmed cell death in cultivated J774A.1 murine macrophages, as demonstrated by the lack of effect on caspase-3 activity. The latter activity was monitored by 7-amino-4-methylcumarin (AMC) fluorogenic enzyme product release. Incubation of cells with unoxidized cholesterol (20 µM) had no effect on caspase-3 activity. By contrast, identical amounts of 7-ketocholesterol significantly enhanced caspase-3, starting from 12 h treatment. Consistent findings were obtained when a 30 µM concentration of cholesterol and the oxysterol mixture were compared. Conclusive evidence of a strong quenching by oxysterols in mixture on the stimulating effect of 7-ketocholesterol on caspase-3 activity was obtained by comparing 20 µM 7-ketocholesterol with 60 µM oxysterol mixture, in which the toxic oxysterol represented 30% of total amount. Indeed, when mixed to other oxysterols, 7-ketocholesterol exerted much less stimulation on caspase-3 activity.

2. Activation of the mitochondrial pathway of apoptosis by 7-ketocholesterol only
After 9 h incubation, a significant release of cytochrome c was consistently demonstrated in cells challenged with 20 µM 7-ketocholesterol but not for treatments with either 60 µM oxysterol mixture or 60 µM unoxidized cholesterol (Fig. 1 ). This corresponded with a significant rise in caspase-9 activity observed in cultures treated with 7-ketocholesterol. After 24 h incubation, caspase-9 activity was 287 ± 25 (mean of 3 experiments ±SD) pmol/mg protein/min in 7-ketocholesterol samples, and 151 ± 35, 113 ± 28, and 110 ± 27 in the oxysterol mixture, cholesterol, and control samples, respectively.

View larger version (74K): [in this window] [in a new window]   Figure 1. Cytochrome c release in J774A.1 macrophages incubated 9 and 18 h with cholesterol, oxysterol mixture, or 7-ketocholesterol. A) 12.5 µM ethanol; B) 60 µM cholesterol; C) 20 µM oxysterol mixture; D) 60 µM oxysterol mixture; E) 20 µM 7-ketocholesterol. Fluorescence was detected by laser scanning confocal microscopy: the laser band of excitation was 488 nm with a 515 nm long pass filter emission; the lens was an oil immersion plan neofluar 100x/1.3 (image dimension 326 µm2). The image was elaborated using LSCM 510 Image Examiner Program (Zeiss). Exciting light intensity, black level, and photomultiplier gain were adjusted on control specimens; the same settings were used to scan the experimental material.

3. Increased p21 expression in J774.A1 macrophages treated with 7-ketocholesterol only: protective effect of 7ß-hydroxycholesterol cotreatment
Gene expression involved in triggering and executing the apoptotic program was investigated by profiling gene expression in J774.A1 cells incubated for 6 h with 20 µM 7-ketocholesterol or 20 µM oxysterol mixture or solvent vehicle. In cells treated with 7-ketocholesterol, two of the genes tested showed modulation above the arbitrary cutoff: p21 and p53. No modulation of any studied genes was found after treatments with the oxysterol mixture. The marked difference in p21 expression between the two experimental groups was also confirmed by Western blot. When macrophages were challenged with the same amounts of 7-ketocholesterol and 7ß-hydroxycholesterol (7ßOH-cholesterol), the up-regulation of p21 synthesis induced by 7-ketocholesterol disappeared. Consistent with this, p21 up-regulation by 20 µM 7-ketocholesterol was no more evident when an identical amount of the toxic sterol was given to cells mixed with other oxysterols, such as in the 60 µM oxysterol mixture.

4. Quenching of 7-ketocholesterol-induced caspase-3 activation and apoptosis by simultaneous treatment with 7ßOH-cholesterol
The pronounced toxicity of 7-ketocholesterol when administered singly was further analyzed by simultaneously challenging cells with equimolar amounts of 7ßOH-cholesterol (20 µM). The strong proapoptotic effect of 7-ketocholesterol was reduced by the combined treatment, as shown by a significant reduction in caspase-3 activity. 7ßOH-Cholesterol (20 µM) alone showed no effect on caspase-3. Further support was provided by Hoechst staining of challenged macrophages after 24 h incubation. Several apoptotic bodies were evident in 7-ketocholesterol-treated cells but were rare or absent in macrophages incubated with the solvent vehicle, oxysterol mixture, 7ßOH-cholesterol, or unoxidized cholesterol. Thus, the proapoptotic effect of 7-ketocholesterol was markedly reduced by cotreatment with 7ßOH-cholesterol.

5. Early increase in steady-state levels of reactive oxygen species (ROS) in 7-ketocholesterol-treated macrophages: quenching effect by the simultaneous addition of 7ßOH-cholesterol
The protective mechanism exerted by 7ßOH-cholesterol against the proapoptotic effect of 7-ketocholesterol was investigated for ROS production by confocal microscopic measurements of dichlorofluorescein (DCF) fluorescence (Fig. 2 ). A significant increase of fluorescence was detected only in cells incubated with 7-ketocholesterol alone. Treatments with the oxysterol mixture, unoxidized cholesterol, or 7ßOH-cholesterol produced no increase in DCF fluorescence. Stimulated ROS generation by 7-ketocholesterol through up-regulation of NADPH oxidase was competitively inhibited by 7ßOH-cholesterol. When cell uptake of the individual sterol oxides was measured by gas chromatography, no difference was observed in intracellular recovery of the two oxysterols administered individually or in combination.

View larger version (110K): [in this window] [in a new window]   Figure 2. Generation of reactive oxygen species (ROS) in J774A.1 macrophages under different experimental conditions. Cells were incubated for 30, 60, or 180 min with 12.5 µM ethanol, 20 µM cholesterol, 20 µM oxysterol mixture, 20 µM 7ßOH-cholesterol (7ßOH), 20 µM 7-ketocholesterol (7K), 20 µM 7-ketocholesterol + 20 µM 7ßOH-cholesterol. ROS production was visualized in macrophages as DCF fluorescence by laser scanning confocal microscopy. Histograms report the fluorescence in arbitrary units/pixel as means ± SD of 3 different experiments. *Significantly different vs. the other experimental groups (P<0.001). Pictures are related to a whole representative experiment.

CONCLUSIONS AND SIGNIFICANCE

A composite mixture of oxysterols rather than single cholesterol oxides is usually found in oxidized LDL; some of these oxysterols are detectable in the fully developed atherosclerotic lesion, 7-ketocholesterol being the most prominent. Oxidized LDL and its component lipids appear to assimilate into cells in a "stealthy" manner without damage until large amounts of oxidized lipid have been incorporated. Various lipid oxidation products, including oxysterols, contribute to the evolution of inflammatory and fibrogenic events. In a second phase, promotion and progression of the lesion are favored through cycles of cell toxicity, repair, proliferation, and death. Our findings provide a compelling argument that: 1) oxysterols found in circulating or phagocytized oxLDL do not exert overt toxicity due to counteracting effects on cells, 2) 7-ketocholesterol, but not oxysterols in a mixture, triggers an oxidative burst in macrophages that may initiate the apoptotic program involving p21 up-regulation and caspase-3 activation; 3) cell cotreatment with 7ßOH-cholesterol counteracts the proapoptotic effect of 7-ketocholesterol (see Fig. 3 ); 4) the simultaneous addition of 7-ketocholesterol and 7ßOH-cholesterol does not interfere with the uptake of the two oxysterols.

View larger version (81K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized Trojan horse-like behavior of an oxysterol mixture on macrophages. Oxysterols in the complex chemical composition found in oxLDL quench the apoptotic effect of some oxysterols, like 7-ketocholesterol: competition among oxysterols, apparently at the level of NADPH oxidase, diminishes ROS overproduction and p21 up-regulation. Because of less oxysterol mixture toxicity, cell viability is preserved and a more subtle gene modulation becomes facilitated in the vascular cell.

In terms of the quenching effect among oxysterols in a mixture, only one other report has indicated that the simultaneous treatment of U-937 human promonocytic cells by 7ßOH-cholesterol and 25OH-cholesterol leads to a considerable decrease in cytotoxicity induced by these oxysterols when added singly. The lack of effect by unoxidized cholesterol in the present model indirectly suggests that the sterol must be oxidized, most likely by macrophages of the fibrotic plaque, to become more proatherogenic.

ROS production is implicated in the oxysterol effect and is the result of macrophage NADPH oxidase activation. Concomitant addition of 7ßOH-cholesterol inhibited the 7-ketocholesterol-induced ROS production. In conclusion, we propose that challenging macrophages with oxysterols points to oxidation as an important biochemical reaction that must occur for cholesterol to exert proatherogenic effects. Oxysterols in the complex chemical composition found in oxLDL may quench the inherent toxic effects of some oxysterols, enabling accumulation of several proatherogenic factors that promote lesion formation before eventual macrophage death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0401fje; doi: 10.1096/fj.03-0401fje

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