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Under hypercholesterolemic conditions, exposure of cells to lipoproteins results in a subtle membrane increase in the levels of cholesterol and 7-ketocholesterol, as compared to normal conditions. The effect of these physiologically relevant concentration increases on multicomponent bilayer membranes was investigated using coarse-grained molecular dynamics simulations. Significant changes in the structural and dynamic properties of the bilayer membranes resulted from these subtle increases in sterol levels, with both sterol species inducing decreases in the lateral area and inhibiting lateral diffusion to varying extents. Cholesterol and 7-ketocholesterol, however, exhibited opposite effects on lipid packing and orientation. The results from this study indicate that the subtle increases in membrane sterol levels induced by exposure to lipoproteins result in molecular-scale biophysical perturbation of membrane structure.

Bioactive omega-3 polyunsaturated fatty acids have been shown to reduce the risk of death in patients with cardiovascular disease and alleviate the symptoms of other inflammatory diseases. However, the mechanisms of action of these effects remain unclear. It has been postulated that omega-3 polyunsaturated fatty acids modify cell membranes by incorporation into the membrane and altering the signaling properties of cellular receptors. In this chapter, we explore the effects of omega-3 polyunsaturated fatty acids on cell membrane structure and function. We present a review of the current evidence for the health benefits of these compounds and explore the molecular mechanisms through which omega-3 polyunsaturated fatty acids interact with membrane lipids and modulate bilayer structure. Using computational models of multicomponent phospholipid bilayers, we assess the consequences of incorporation of these fatty acids on membrane lipid packing, water permeation, and membrane structure.

Cholesterol is a major regulator of multiple types of ion channels, but the specific mechanisms and the dynamics of its interactions with the channels are not well understood. Kir2 channels were shown to be sensitive to cholesterol through direct interactions with “cholesterol-sensitive” regions on the channel protein. In this work, we used Martini coarse-grained simulations to analyze the long (μs) timescale dynamics of cholesterol with Kir2.2 channels embedded into a model membrane containing POPC phospholipid with 30 mol% cholesterol. This approach allows us to simulate the dynamic, unbiased migration of cholesterol molecules from the lipid membrane environment to the protein surface of Kir2.2 and explore the favorability of cholesterol interactions at both surface sites and recessed pockets of the channel. We found that the cholesterol environment surrounding Kir channels forms a complex milieu of different short- and long-term interactions, with multiple cholesterol molecules concurrently interacting with the channel. Furthermore, utilizing principles from network theory, we identified four discrete cholesterol-binding sites within the previously identified cholesterol-sensitive region that exist depending on the conformational state of the channel—open or closed. We also discovered that a twofold decrease in the cholesterol level of the membrane, which we found earlier to increase Kir2 activity, results in a site-specific decrease of cholesterol occupancy at these sites in both the open and closed states: cholesterol molecules at the deepest of these discrete sites shows no change in occupancy at different cholesterol levels, whereas the remaining sites showed a marked decrease in occupancy.

Regulation of cell volume is a fundamental property of all mammalian cells. Multiple signaling pathways are known to be activated by cell swelling and to contribute to cell volume homeostasis. Although cell mechanics and membrane tension have been proposed to couple cell swelling to signaling pathways, the impact of swelling on cellular biomechanics and membrane tension have yet to be fully elucidated. In this study, we use atomic force microscopy under isotonic and hypotonic conditions to measure mechanical properties of endothelial membranes including membrane stiffness, which reflects the stiffness of the submembrane cytoskeleton complex, and the force required for membrane tether formation, reflecting membrane tension and membrane-cytoskeleton attachment. We find that hypotonic swelling results in significant stiffening of the endothelial membrane without a change in membrane tension/membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin, which, as expected, results in a dramatic decrease in the cellular elastic modulus of both the membrane and the deeper cytoskeleton, indicating a collapse of the cytoskeleton scaffold, does not abrogate swelling-induced stiffening of the membrane. Instead, this swelling-induced stiffening of the membrane is enhanced. We propose that the membrane stiffening should be attributed to an increase in hydrostatic pressure that results from an influx of solutes and water into the cells. Most importantly, our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for activating volume-sensitive mechanisms in hypotonically swollen cells.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs’ proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1. Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1-2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K-dependent and -independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

The effects of osmotic swelling on key cellular biomechanical properties are explored in this chapter. We present the governing equations and theoretical backgrounds of the models employed to estimate cell membrane tension and elastic moduli from experimental methods, and provide a summary of the prevailing experimental approaches used to obtain these biomechanical parameters. A detailed analysis of the current evidence of the effects of osmotic swelling on membrane tension and elastic moduli is provided. Briefly, due to the buffering effect of unfolding membrane reservoirs, mild hypotonic swelling does not change membrane tension or the adhesion of the membrane to the underlying cytoskeleton. Conversely, osmotic swelling causes the cell membrane envelope to stiffen, measured as an increase in the membrane elastic modulus.

Numerous ion channels have been shown to be regulated by the level of membrane cholesterol, but the mechanisms responsible for these effects are still not well understood. The key question in the field is how to discriminate between the contributions of the two central mechanisms that might be responsible for the sensitivity of ion channels to cholesterol: specific sterol-protein interactions or regulation of channels by the bilayer physical properties. Comparative analysis of cholesterol and its isomers on the function of an ion channel is a powerful tool to achieve this goal. An increasing number of studies show that cholesterol regulates several types of ion channels in a stereospecific manner, suggesting an involvement of specific sterol-protein interactions. However in this chapter, we present evidence that the stereospecificity of cholesterol-ion channel interactions may be mediated, not by a lack of binding, as has been generally assumed, but by the specificity of the interaction, which results in a functional effect, in the case of native cholesterol, and a lack of functional effect, in the case of a cholesterol isomer. In other words, accumulating evidence suggests that the structural requirements of ion channel cholesterol-binding sites are lax, allowing chiral isomers of cholesterol to bind to the same site in a nonstereospecific way, but the ability of a sterol to confer a functional effect on the channel activity can still be stereospecific. This is an important distinction both conceptually and methodologically. Indeed, our analysis shows that the orientations of cholesterol and its chiral isomer ent-cholesterol within a hydrophobic binding pocket of Kir2.2 are significantly different, and we propose that this difference may underlie distinct functional outcomes.

Cell stiffness or deformability is a fundamental property that is expected to play a major role in multiple cellular functions. It is well known that cell stiffness is dominated by the intracellular cytoskeleton that, together with the plasma membrane, forms a membrane-cytoskeleton envelope. However, our understanding of how lipid composition of plasma membrane regulates physical properties of the underlying cytoskeleton is only starting to emerge. In this review, we first briefly describe the impact of cholesterol on the physical properties of lipid bilayers in model membranes and in living cells, with the dominant effect of increasing the order of membrane lipids and decreasing membrane fluidity. Then, we discuss accumulating evidence that removal of cholesterol, paradoxically, decreases the mobility of membrane proteins and increases cellular stiffness, with both effects being dependent on the integrity of the cytoskeleton. Finally, we discuss emerging evidence that oxidized modifications of low-density lipoproteins (oxLDL) have the same effects on endothelial biomechanical properties as cholesterol depletion, an effect that is mediated by the incorporation of oxysterols into the membrane.

Oxidatively modified low-density lipoprotein (OxLDL) has been implicated in a range of disorders, including certain cardiovascular, metabolic, and immunological diseases. The bioactive oxidation products of this lipoprotein include a vast array of oxidized lipids, sterols, and triglycerides. Some of these species may play a significant role in the pathophysiological effects observed at elevated OxLDL concentrations. However, the particular biological effects of specific OxLDL components, as well as the mechanisms by which they elicit these responses, are poorly understood. To elucidate some of the effects of OxLDL, we undertake a molecular-scale investigation of changes induced in endothelial membranes in the presence of various oxidation products. Specifically, in order to disaggregate the individual effects of particular oxidized species, we utilize coarse-grained molecular dynamics simulations. With this technique, we probe alterations to the biomechanical properties (structure and dynamics) of multi-component model membranes by compounds that have been previously shown, through experimental means, to induce endothelial stiffening. This study may begin to clarify some of the molecular mechanisms underlying endothelial cell dysfunction resulting from exposure to OxLDL.

Compounds with nominally similar biological activity may exhibit differential toxicity due to differences in their interactions with cell membranes. Many pharmaceutical compounds are amphiphilic and can be taken up by phospholipid bilayers, interacting strongly with the lipid-aqueous interface whether or not subsequent permeation through the bilayer is possible. Bolaamphiphilic compounds, which possess two hydrophilic ends and a hydrophobic linker, can likewise undergo spontaneous uptake by bilayers. While membrane-spanning bolaamphiphiles can stabilize membranes, small molecules with this characteristic have the potential to create membrane defects via disruption of bilayer structure and dynamics. When compared to the amphiphilic therapeutic anticoagulant, warfarin, the bolaamphiphilic analogue, brodifacoum, exhibits heightened toxicity that goes beyond superior inhibition of the pharmacological target enzyme. We explore, herein, the consequences of anticoagulant accumulation in a dipalmitoylphosphatidylcholine (DPPC) bilayer. Coarse-grained molecular dynamics simulations reveal that permeation of phospholipid bilayers by brodifacoum causes a disruption of membrane barrier function that is driven by the bolaamphiphilic nature and size of this molecule. We find that brodifacoum partitioning into bilayers causes membrane thinning and permeabilization and promotes lipid flip-flop - phenomena that are suspected to play a role in triggering cell death. These phenomena are either absent or less pronounced in the case of the less toxic, amphiphilic compound, warfarin.

Inwardly rectifying potassium (Kir) channels form a major family of potassium channels and have been shown to play a critical role in setting resting membrane potential, regulating potassium homeostasis, and modulating membrane excitability. Cholesterol, which forms a major component of mammalian cell membranes, has been shown in the past to suppress Kir family channel function in high concentrations and is linked to a number of pathological conditions. Recently, it has been proposed that cholesterol binds to specific non-annular sites between the transmembrane domains of the Kir protein, and that this bonding helps to stabilize a closed conformation for the channel. In order to better understand this interaction, we have taken a computational approach which utilizes coarse-grained molecular dynamics simulations - employing the MARTINI force field - to further investigate the spatial and temporal mechanics of cholesterol binding. In addition to the mechanics of binding, this approach has allowed us to more closely examine the physiological effects of cholesterol binding on Kir channel function in a more physiologically relevant membrane environment. Through this we have been able to examine a greater range of binding effects, such as binding time, binding instances, changes in channel conformation, and ion flux through the channel.