Choice of cyclodextrin for cellular cholesterol depletion for vascular endothelial cell lipid raft studies: cell membrane alterations, cytoskeletal reorganization and cytotoxicity.
Choice of cyclodextrin for cellular cholesterol depletion for vascular endothelial cell lipid raft studies: cell membrane alterations, cytoskeletal reorganization and cytotoxicity.
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发表时间：
2012-10
影响因子：
1.4
通讯作者：
A. Hinzey;M. Kline;S. Kotha;S. Sliman;E. O. Butler;A. B. Shelton;T. Gurney;N. Parinandi
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文献类型：
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作者：
A. Hinzey;M. Kline;S. Kotha;S. Sliman;E. O. Butler;A. B. Shelton;T. Gurney;N. Parinandi
使用环糊精作为工具来确定胆固醇筏在细胞功能中的作用已成为一种被广泛接受的方法。然而，环糊精作为胆固醇消耗剂对细胞结构和功能的不利影响尚未详细报道。因此，在本研究中，我们研究了两种广泛使用的细胞胆固醇消耗环糊精甲基-β-环糊精（MbetaCD）和羟丙基-β-环糊精（HPCD）在我们完善的牛肺动脉内皮细胞（BPAEC）体外模型系统中的膜扰动作用和细胞毒性。用不同浓度的MbetaCD和HPCD（2%和5%，wt/vol.）15-180 min显示膜胆固醇、细胞毒性、细胞形态学改变、肌动蛋白细胞骨架重组、细胞蛋白和膜脂肪酸组成的改变以及跨内皮电阻（TER）的降低。与相同条件下HPCD引起的细胞蛋白质损失相比，MbetaCD诱导了细胞蛋白质的显著损失。更值得注意的是，MbetaCD导致BPAEC中膜脂脂肪酸的急剧损失，与未能引起这种改变的HPCD相比。通过环糊精（特别是MbetaCD）处理去除胆固醇显然引起细胞膜流动性的损失和包括蛋白质和脂肪酸的重要细胞分子的泄漏，从而引起BPAEC中的细胞毒性和细胞形态的损失。通过MbetaCD处理耗尽胆固醇后用胆固醇补充细胞显著减弱了BPAEC中细胞胆固醇的耗尽、细胞毒性和形态学改变，表明膜胆固醇在血管EC完整性中的重要性。此外，目前的研究提供了一种更安全的方法，通过HPCD从膜和脂筏中去除胆固醇，这表明其可用于研究脂筏相关胆固醇在细胞功能中的作用。
The use of cyclodextrins as tools to establish the role of cholesterol rafts in cellular functions has become a widely accepted procedure. However, the adverse effects of cyclodextrins as the cholesterol-depleting agents on cellular structure and functions are not reported in detail. Therefore, in the current study, we investigated the membrane-perturbing actions and cytotoxicity of the two widely used cellular cholesterol-depleting cyclodextrins methyl-beta-cyclodextrin (MbetaCD) and hydroxypropyl-beta-cyclodextrin (HPCD) in our well-established bovine pulmonary artery endothelial cell (BPAEC) in vitro model system. BPAECs treated with different concentrations of MbetaCD and HPCD (2% and 5%, wt/vol.) for 15-180 min showed significant loss of membrane cholesterol, cytotoxicity, cell morphology alterations, actin cytoskeletal reorganization, alterations in cellular proteins and membrane fatty acid composition, and decrease in trans-endothelial electrical resistance (TER). MbetaCD induced a marked loss of cellular proteins, as compared to that caused by HPCD under identical conditions. More noticeably, MbetaCD caused a drastic loss of membrane lipid fatty acids in BPAECs, as compared to HPCD which failed to cause such alteration. Removal of cholesterol by cyclodextrin (especially MbetaCD) treatment apparently caused loss of fluidity of the cell membrane and leakage of vital cellular molecules including proteins and fatty acids, and thus caused cytotoxicity and loss of cell morphology in BPAECs. Replenishment of cells with cholesterol following its depletion by MbetaCD treatment significantly attenuated the depletion of cellular cholesterol, cytotoxicity and morphological alterations in BPAECs, indicating the importance of membrane cholesterol in vascular EC integrity. Also, the current study offered a safer method of cholesterol removal from membranes and lipid rafts by HPCD, suggesting its use in studies to investigate the role of lipid raft-associated cholesterol in cellular functions.