Direct regulation of prokaryotic Kir channel by cholesterol.

Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by (86)Rb(+) uptake. Our results show that (86)Rb(+) flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses (86)Rb(+) flux by >50%, and activity is completely inhibited at 12-15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated (86)Rb(+) uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 microg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.

Influence of membrane cholesterol and substrate elasticity on endothelial cell spreading behavior.

Interactions between implanted materials and the surrounding host cells critically affect the fate of bioengineered materials. In this study, the biomechanical response of bovine aortic endothelial cells (BAECs) with different membrane cholesterol levels to polyacrylamide (PA) gels was investigated by measuring cell adhesion and spreading behaviors at varying PA elasticity. The elasticity of gel substrates was manipulated by cross-linker content. Type I collagen (COL1) was coated on PA gel to provide a biologically functional environment for cell spreading. Precise quantitative characterization of changes in cell area and perimeter of cells across two treatments and three bioengineered substrates were determined using a customized software developed for computational image analysis. We found that the initial response of endothelial cells to changes in substrate elasticity was determined by membrane cholesterol levels, and that the extent of endothelial cell spreading increases with membrane cholesterol content. All of the BAECs with different cholesterol levels showed little growth on substrates with elasticity below 20 kPa, but increased spreading at higher substrate elasticity. Cholesterol-depleted cells were consistently smaller than control and cholesterol-enriched cells regardless of substrate elasticity. These observations indicate that membrane cholesterol plays an important role in cell spreading on soft biomimetic materials constructed with appropriate elasticity.

OxLDL and substrate stiffness promote neutrophil transmigration by enhanced endothelial cell contractility and ICAM-1.

Elevated levels of oxLDL in the bloodstream and increased vasculature stiffness are both associated with cardiovascular disease in patients. However, it is not known how oxLDL and subendothelial matrix stiffness together regulate an immune response. Here, we used an in vitro model of the vascular endothelium to explore the combined effects of oxLDL and subendothelial matrix stiffening on neutrophil transmigration. We prepared fibronectin-coated polyacrylamide gels of varying stiffness and plated human umbilical vein endothelial cells (ECs) onto the gels. We observed that oxLDL treatment of the endothelium promoted neutrophil transmigration (from <1% to 26% on soft 0.87kPa substrates), with stiffer substrates further promoting transmigration (54% on 5kPa and 41% on 280kPa). OxLDL exposure enhanced intercellular adhesion molecule-1 (ICAM-1) expression on the endothelium, which was likely responsible for the oxLDL-induced transmigration. Importantly, inhibition of MLCK-mediated EC contraction reduced transmigration to ∼9% on all substrates and eliminated the effects of subendothelial matrix stiffness. In addition, large holes, thousands of square microns in size, formed in monolayers on stiff substrates following transmigration, indicating that oxLDL treatment and subsequent neutrophil transmigration caused serious damage to the endothelium. Our results reveal that an interplay between ICAM-1 and MLCK-dependent contractile forces mediates neutrophil transmigration through oxLDL-treated endothelium. Thus, microvasculature stiffness, which likely varies depending on tissue location and health, is an important regulator of the transmigration step of the immune response in the presence of oxLDL.