Atherosclerosis is a complex and chronic arterial process which is characterized by a remodeling of the vascular wall, associated with inflammatory reactions, proliferation and cell death process, and with accumulation of oxidized lipids among which oxysterols (cholesterol oxidation products) which might play key roles in the initiation and development of atheromatous lesions.

Our work performed on U937 and THP1 promonocytic cells, rat aorta embryonic A7R5 cells, and breast carcinoma MCF7 cells (caspase-3 deficient). Different oxysterols, present in large quantity in atheromatous lesions, were used: 7-cétocholestérol (7KC), 7Β-hydroxycholestérol, 25-hydroxycholestérol, cholestérol-5Α, 6Α-epoxide, cholestérol-5Β, 6Β-epoxide).

The first part of work shown that 7KC-induced cell death was a complex phenomenon presenting of apoptotic characteristics accompanied by a synthesis of cytoplasmic multilamellar structures called myelin body highlighted by transmission electron microscopy. The synthesis of these structures is an early phenomenon preceding the cytotoxic effects: lost of the mitochondrial membrane potential, increase in the permeability to propidium iodide and modifications of nuclear morphology (condensation, fragmentation, swelling of the nuclei). The isolation of these structures by differential ultracentrifugation after staining with monodansylcadaverine or with Nile Red (emitting a yellow fluorescence in the presence of neutral lipids and red in the presence of polar lipids) allowed to determine that they are rich in polar lipids (sphingomyelin, phosphatidylcholine) and in cholesterol and that they constitute sites of accumulation of the 7KC. Within these myelin structures, a co-localization of 7KC with polar lipids was shown by the technique of FRET carried out by mono and bi-photon confocal microscopy. The morphological and biochemical characteristics of myelin figures allowed to establish that 7-ketocholesterol is a powerful inducer of phospholipidosis.

Taking into account the space-time, quantitative and qualitative lipid modifications, induced by the 7KC and revealed by Nile Red, the second part of work was to specify the relationships between cell death, the synthesis of myelin structures, accumulation of polar lipids and caspase activity. With different oxysterols studied, the myelin structures are observed only with cytotoxic oxysterols (7KC, 7Β-hydroxycholestérol, cholesterol-5Β,6Β-epoxide) and their synthesis are independent of caspase activity. On the other hand, the polar lipid accumulation induced by 7KC is inhibited in the presence of z-VAD-fmk (caspase broad spectrum inhibitor) and of z-VDVAD-fmk (caspase-2 inhibitor). These results suggest that some caspases and in particular the caspase-2 would contribute to the polar lipid accumulation.

The third part of work resulted in studying the effects of the Vitamin E (VitE) on 7KC-induced cell death 7KC according to its anti-apoptotic and antioxidant properties. VitE protect from 7KC-induced-cell death. These protective effects could partly be with the capacity of VitE to maintain functional the PI3-K/c-Akt pathway while being opposed to the dephosphorylations of PDK-1 and c-Akt, and by preserving PI3-K activity. In addition, VitE is opposed to the lipid modifications on the cytoplasmic membrane level and to the lipid polar accumulation. VitE is also opposed to the degradation of the pro-form of caspase-2L and increases the rates of ARNm corresponding.

These work shown relationships between oxysterol-induced cell death and lipid metabolism. They revealed that VitE protects from 7KC-induced-cell death, probably while acting on the level of the PI3-K/c-Akt pathway. However, VitE is opposed to the lipid membranous and cytoplasmic modifications associated with 7KC-induced-cell death.

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   |texte=   Caracterization of myelin figures associated with polar lipid accumulation induced by different cytotoxic oxysterols identified in atheromatous lesions: study of relationships between apoptosis and lipid metabolism.
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