6 research outputs found
Choice of cyclodextrin for cellular cholesterol depletion for vascular endothelial cell lipid raft studies: Cell membrane alterations, cytoskeletal reorganization and cytotoxicity
329-341The 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-b-cyclodextrin (MbCD) and
hydroxypropyl-b-cyclodextrin (HPCD) in our well-established bovine pulmonary artery
endothelial cell (BPAEC) in vitro
model system. BPAECs treated with different concentrations of MβCD 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). MbCD induced a
marked loss of cellular proteins, as compared to that caused by HPCD under
identical conditions. More noticeably, MbCD 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 MβCD)
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 MbCD 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