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.
Identifieur interne : 000023 ( Hal/Curation ); précédent : 000022; suivant : 000024Caracterization 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.
Auteurs : Anne Vejux [France]Source :
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English descriptors
Abstract
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.
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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<front><div type="abstract" xml:lang="en">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.</div>
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<title xml:lang="fr">Caractérisation des figures myéliniques associées à l'accumulation de lipides polaires induites par différents oxystérols cytotoxiques identifiés dans les lésions athéromateuses: étude des relations entre apoptose et métabolisme des lipides</title>
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<abstract xml:lang="en">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.</abstract>
<abstract xml:lang="fr">L'athérosclérose est une pathologie artérielle complexe à évolution lente définie par un remodelage de la paroi vasculaire, associée à des réactions inflammatoires, à des processus de prolifération, de mort cellulaire et à une accumulation de lipides oxydés tels que les oxystérols (dérivés oxydés du cholestérol) dont le rôle est fortement suspecté dans le développement de l'athérome.
Le travail réalisé a été effectué sur des cellules monocytaires humaines U937 et THP-1, aortiques de rat A7R5 et carcinomateuses humaines MCF-7 (déficientes en caspase-3). Différents oxystérols, présents en quantité importante dans les lésions athéromateuses, ont été utilisés (7-cétocholestérol (7KC), 7Β-hydroxycholestérol, 25-hydroxycholestérol, cholestérol-5Α, 6Α-epoxide, cholestérol-5Β, 6Β-epoxide) ainsi que du cholestérol.
La première partie du travail a montré que la mort cellulaire induite par le 7KC est un phénomène complexe présentant des caractéristiques apoptotiques accompagnées d'une synthèse de structures multilamellaires cytoplasmiques appelées corps myéliniques visualisés par microscopie électronique à transmission. La synthèse rapide de ces structures précède les effets cytotoxiques : chute du potentiel membranaire mitochondrial, augmentation de la perméabilité à l'iodure de propidium et modifications de la morphologie nucléaire (condensation, fragmentation, gonflement des noyaux). L'isolement de ces structures par ultracentrifugation après coloration à la monodansylcadavérine (fluorochrome lysosomotropique acide) ou au Nile Red (fluorescence jaune en présence de lipides neutres et rouge en présence de lipides polaires) a permis de montrer qu'elles sont riches en lipides polaires (sphingomyéline, phosphatidylcholine) et en cholestérol et qu'elles accumulent le 7KC. Au sein de ces corps myéliniques, une co-localisation du 7KC avec les lipides polaires a été démontrée par la technique de FRET réalisée par microscopie confocale mono- et bi-photonique. Les caractéristiques morphologiques et biochimiques des figures myéliniques ont permis d'établir que le 7-cétocholestérol est un puissant inducteur de phospholipidose.
Compte tenu des modifications lipidiques spatiotemporelles, quantitatives et qualitatives, induites par le 7KC et révélé par le Nile Red, la seconde partie du travail a précisé les relations entre la mort cellulaire, la synthèse de corps myéliniques, l'accumulation de lipides polaires et l'activité caspase. Avec les différents oxystérols étudiés, les corps myéliniques ne sont observés qu'avec des composés cytotoxiques (7KC, 7Β-hydroxycholestérol, cholestérol-5Β, 6Β-epoxide) et leur synthèse est indépendante d'activité caspase. En revanche, l'accumulation de lipides polaires induite par le 7KC est inhibée en présence de z-VAD-fmk (inhibiteurs de caspases large spectre) et de z-VDVAD-fmk (inhibiteur de caspase-2). Certaines caspases et en particulier la caspase-2 pourraient contribuer à l'accumulation de lipides polaires.
La troisième partie du travail a conduit à étudier les effets de la Vitamine-E (VitE) sur la mort cellulaire induite par le 7KC en raison de ses propriétés anti-apoptotiques et anti-oxydantes. La VitE protège de la mort cellulaire induite par le 7KC. Les effets protecteurs pourraient en partie être dus à la capacité de la VitE à maintenir fonctionnelle la voie PI3-K/c-Akt en s'opposant aux déphosphorylations de PDK-1 et de c-Akt, et en préservant l'activité PI3-K. Par ailleurs, la VitE s'oppose aux modifications lipidiques au niveau de la membrane cytoplasmique et à l'accumulation de lipides polaires. La VitE réduit aussi la dégradation de la pro-forme de la caspase-2L et augmente les taux d'ARNm correspondants.
Ces travaux montrent des relations entre la mort cellulaire induite par des oxystérols et le métabolisme des lipides. Ils révèlent aussi que la VitE protège de la mort induite par le 7KC en agissant au niveau de la voie PI3-K/c-Akt. Par ailleurs, la VitE s'oppose aux modifications lipidiques membranaires et cytoplasmiques associées à la mort cellulaire induite par le 7KC.</abstract>
<particDesc> <org type="consortium">Dr Edmond Kahn</org>
<org type="consortium">INSERM U678</org>
<org type="consortium">CHU Pitié-Salpêtrière</org>
<org type="consortium">75634 Paris Cedex 13</org>
<org type="consortium">France.</org>
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