Abstract
Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment.
Highlights
It has been suggested that cholesterol oxide-induced apoptosis is a key event in the initiation and progression of atherosclerosis lesions [1, 2]
Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane
In more advanced stages and as the atherosclerotic lesion progresses, cholesterol oxides could contribute to the destruction of foam cells and vascular smooth muscle cells, to the formation of the lipid core, to the reduction of cell proliferation, and eventually to plaque destabilization [1, 2]
Summary
The red fluorescence was immediately measured on a Galaxy flow cytometer (Partec, Munster, Germany) at excitation and emission wavelengths of 488 and 585/42 nm, FIGURE 1. A, cell permeability to PI was measured by flow cytometry. Fluorescence associated with DIOC6(3) was measured by flow cytometry, and 10,000 cells were analyzed for each assay. 7-Ketocholesterol, cycloheximide, staurospotial (⌬⌿m) were measured with DIOC6(3) (Exmax, 484 nm; Emmax, 501 nm) used at a final concentration of 40 nmol/liter [17] after 24 h of treatment. PKB polyclonal antibody, the anti-Akt/PKB phospho-Thr-308 Hoechst 33342—Nuclear morphology of control and treated polyclonal antibody, and the anti-Akt/PKB phospho-Ser-473 cells was studied by fluorescence microscopy after staining with monoclonal antibody were purchased from Cell Signaling Hoechst 33342 (Exmax, 346 nm; Emmax, 420 nm) used at 10 Technology (Hitchin, UK). The formation of dichlorofluorescein was monitored at excitation/emission wavelengths of 485/538 nm over 30 min using a VICTOR 1420 multilabel counter
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
