A Simple Method for Effective and Safe Removal of Membrane Cholesterol from Lipid Rafts in Vascular Endothelial Cells: Implications in Oxidant-Mediated Lipid Signaling

Abstract

Lipid raft-associated cholesterol has been identified as a pivotal player among membrane lipids in regulating cellular functions. Cholesterol of the vascular endothelial cell (EC) membranes is also being recognized as an important element in the vascular EC signaling. However, methods utilized in studying the important role of lipid raft-associated cholesterol in cell signaling involve removal of the raft cholesterol with the aid of chemical agents called cyclodextrins. Caution should be exercised in using cyclodextrins to remove the cellular lipid raft-associated cholesterol as the cyclodextrins cause adverse effects on cells such as loss of cell viability or induction of cytotoxicity. Therefore, the choice of a cyclodextrin to remove the cellular lipid raft-associated cholesterol is extremely important in order to ensure effective and safe removal of cholesterol from the cellular lipid rafts. In order to achieve this, here, we have selected the bovine pulmonary artery endothelial cells (BPAECs) and subjected them to the removal of cholesterol using two different beta-cyclodextrin compounds, methyl-beta-cyclodextrin (MbetaCD) and hydroxypropyl-beta-cyclodextrin (HPCD). Phospholipase D (PLD), which generates one of the most potent bioactive lipid signal mediators (phosphatidic acid), is activated by oxidants. Therefore, we examined the effects of cholesterol removal by utilizing our current methods on the hydrogen peroxide (H(2)O(2))-activated PLD in BPAECs. Differences in the loss of cholesterol and the resulting effects on the cell membrane, cell viability, morphology, and the extent of oxidant-induced PLD activation were determined. The results revealed that both MbetaCD and HPCD caused loss of cholesterol, loss of cell viability, and altered cell morphology in the chosen EC system. It was also determined that the HPCD compound caused far less extensive damage to the cells than the MbetaCD, therefore making the HPCD compound a safer tool for EC cholesterol removal.

Hydroxypropyl‑β‑cyclodextrin attenuates the epithelial‑to‑mesenchymal transition via endoplasmic reticulum stress in MDA‑MB‑231 breast cancer cells

“…Thus, the present study focused on the effects of another important lipid raft component, cholesterol, in the context of breast cancer. HP-β-CD can deplete cholesterol in lipid rafts and this may affect the construction of lipid rafts and then cancer cell viability by regulating the transmembrane signaling (15,27). For example, Alawin et al (28) found that antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells and Badana et al (29).…”

“…Therefore, the present study sought to explore the effects of the change in cholesterol in lipid rafts in the development of EMT regulated by the TGF-β/Smad signaling pathway. Toward accomplish this, hydroxypropyl-β-cyclodextrin (HP-β-CD) was used for in vitro treatment of MDA-MB-231 cells to deplete cholesterol in lipid rafts (15).…”

Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts

“…C-Laurdan imaging was performed with and without treatment with sterol-specific drugs MBCD and Amphotericin B (Amp B), which respectively target cholesterol 50 – 52 and ergosterol 53 . Both MBCD and Amp B have been used extensively in previous investigations of lipid rafts 51 , 52 , 54 – 57 .…”

Highlighting the Role of Polymer Length, Carbohydrate Size, and Nucleic Acid Type in Potency of Glycopolycation Agents for pDNA and siRNA Delivery

“…These results could indicate that free polymer aids in endosomal escape of siRNA formulations, which intern, aids gene knockdown and was particularly noticed with the cyclodextrin analogs. We speculate that the known affinity for cyclodextrin to extract cholesterol from cell membranes 36-39 could play a role in aiding endosomal disruption and polyplex release into the cytoplasm with the CD4 polymer series.…”