Cellular Membrane Cholesterol Research Articles

MiRNA Regulation of Cerebrovascular Inwardly Rectifying K+ Channels During Onset of Alzheimer Disease: Role of Cholesterol and Cannabidiol Treatment

Currently, more than 6 million Americans live with Alzheimer disease (AD). Recent studies demonstrate that impaired blood flow in the brain is integral to the development of AD. Vascular inwardly rectifying K+ channels, such as KIR2.x and KIR6.x, are important for regulating vasoconstriction and vasodilation to tune cerebral perfusion in response to metabolic demand. We’ve previously identified that disruption in membrane cholesterol during advanced AD pathology restores function of KIR2.1 channels to that of young, healthy conditions or better. Additionally, we’ve identified specific microRNAs (miRNAs) in cerebral vessels that can be used to track early development of AD. In this study, we seek to understand molecular pathways stemming from miRNAs that directly or indirectly modulate activity of KIR2.x ( Kcnj2, Kcnj12) and KIR6.x ( Kcnj8, Kcnj11) channels. These ion channels are abundant in cerebrovascular endothelium and smooth muscle cells while particularly sensitive to dyslipidemia. We hypothesize that cerebrovascular miRNAs marking AD pathology are involved in dysregulation of inwardly rectifying K+ channels via cholesterol signaling. With employing QIAGEN’s Ingenuity Pathway Analysis (IPA), we first identified miRNAs that generally regulate each of the target genes for select KIR channels. With applying acquired knowledge of cerebrovascular miRNAs implicated in AD, we found which of these miRNAs also regulate cellular membrane cholesterol levels. Finally, we pinpointed miRNAs sensitive to cannabidiol (CBD) treatment during the development of AD as a potential therapeutic alternative to statins and cyclodextrins. In total, there are 233 miRNAs that regulate Kcnj2, 43 for Kcnj8, 203 for Kcnj11, and 177 for Kcnj12. When factoring in miRNAs that are AD-related, remaining miRNAs include nine that regulate Kcnj2, and one each for Kcnj8, Kcnj11, and Kcnj12. Remaining miRNAs that are cholesterol-related include one for Kcnj2, 43 for Kcnj8, 191 for Kcnj11, and one for Kcnj12. In general, at AD onset, CBD upregulates KIR2.2 and downregulates KIR2.1, KIR6.1, and KIR6.2 channels. CBD reversed expression of miRNAs involved in KIR2.x channel regulation in endothelial and smooth muscle/pericytes (e.g., miR-133 and miR-145) during AD onset in 3xTg-AD animals relative to wild-type controls. CBD also reverses expression of miR-129 and miR-151 to effectively upregulate genes modulating KIR6.x channels from onset of AD relative to wild-type. Altogether, we evidence specific pathways underpinning regulation of KIR2.x and KIR6.x channels in the transition from a healthy to diseased brain. This work was supported by the National Institutes of Health (R01AG073230). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A basic model for the association of ligands with membrane cholesterol: application to cytolysin binding

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol.

Effect of the water-soluble cholesterol-methyl-β-cyclodextrin complex on the development of pulmonary edema with an analysis of the osmotic resistance of erythrocytes in rats

Introduction. Methyl-β-cyclodextrin is a potent acceptor of cellular membrane cholesterol and, at the same time, used as a solubilizer, which makes it a potential target for targeted delivery of hydrophobic compounds.Aim. To assess the effect of administration of methyl-β-cyclodextrin containing cholesterol on the degree of lung hydration and osmotic resistance of erythrocytes against the background of pulmonary edema in adult rats caused by the administration of mezaton.Materials and methods. We investigated the degree of hydration of the lungs by the value of the pulmonary coefficient and their dry residue. Osmotic resistance of erythrocytes were assessed by the intensity of their hemolysis in a series of hypotonic sodium chloride solutions in adult male Wistar rats (220±40 g) of the control group (intact, n=10) and two experimental groups with pulmonary edema caused by the administration of mezaton (n=10) and with the subsequent introduction of a water-soluble complex cholesterol-methyl-β-cyclodextrin against the background of the development of pulmonary edema (n=10). We used additional criteria for assessing the osmotic resistance of erythrocytes: minimum resistance – the concentration of sodium chloride solution, at which the first “weak” erythrocytes begin to hemolyze; maximum resistance is the concentration of sodium chloride solution at which all or almost all erythrocytes hemolyzed.Results. The introduction of mezaton led to the development of pronounced hemolysis and hydration of the lungs with the development of their edema and a narrowing of the range of minimum and maximum osmotic resistance of erythrocytes. The introduction of a complex of cholesterol with methyl-β-cyclodextrin after edemogenic exposure led to a decrease in the severity of pulmonary edema, determined by a decrease in the pulmonary coefficient and an increase in the dry residue, and to an increase in the osmotic resistance of erythrocytes with an expansion of the range of their minimum and maximum osmotic resistance.Conclusion. The introduction of a water-soluble complex of cholesterol with methyl-β-cyclodextrin against the background of the development of hemodynamic pulmonary edema significantly reduced the degree of hydration of the lungs, which combined with an increase in the osmotic resistance of erythrocytes.

Cholesterol-Rich Lipid Rafts in the Cellular Membrane Play an Essential Role in Avian Reovirus Replication.

Cholesterol is an essential component of lipid rafts in cellular plasma membranes. Although lipid rafts have been reported to have several functions in multiple stages of the life cycles of many different enveloped viruses, the mechanisms by which non-enveloped viruses, which lack outer lipid membranes, infect host cells remain unclear. In this study, to investigate the dependence of non-enveloped avian reovirus (ARV) infection on the integrity of cholesterol-rich membrane rafts, methyl-β-cyclodextrin (MβCD) was used to deplete cellular membrane cholesterol at the ARV attachment, entry, and post-entry stages. Treatment with MβCD significantly inhibited ARV replication at both the entry and post-entry stages in a dose-dependent manner, but MβCD had a statistically insignificant effect when it was added at the attachment stage. Moreover, MβCD treatment markedly reduced syncytium formation, which occurs at a relatively late stage of the ARV life cycle and is involved in cell-cell transmission and release. Furthermore, the addition of exogenous cholesterol reversed the effects mentioned above. Colocalization data also showed that the ARV proteins σC, μNS, and p10 prefer to localize to cholesterol-rich lipid raft regions during ARV infection. Altogether, these results suggest that cellular cholesterol in lipid rafts plays a critical role in ARV replication.

Identification of the caveolae/raft-mediated endocytosis as the primary entry pathway for aquareovirus

Grass carp reovirus (GCRV), a member of the Aquareovirus genus in the Reoviridae family, is considered the most pathogenic aquareovirus. However, its productive viral entry pathways remain largely unclear. Using a combination of quantum dot (QD)-based live-virus tracking and biochemical assays, we found that extraction of cellular membrane cholesterol with methyl-β-cyclodextrin (MβCD) and nystatin strongly inhibited the internalization of GCRVs, and supplementation with cholesterol restored viral infection. In addition, the entry of the virus was restrained by genistein, an inhibitor known to block caveolar endocytosis. Subsequent real-time tracking experiments revealed that the QD-labeled GCRV particles were colocalized with caveolin-1, and transfection of cells with dominant-negative mutant (caveolin-1 Y14F) significantly reduced GCRV infection. In contrast, no effects on virus infection were detected when the clathrin-mediated endocytosis or the macropinocytosis inhibitors were used. Our results collectively suggest that aquareoviruses can use caveolae/raft-mediated endocytosis as the primary entry pathway to initiate productive infection.

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes.

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

Magnification of Cholesterol-Induced Membrane Resistance on the Tissue Level: Implications for Hypoxia.

High cellular membrane cholesterol is known to generate membrane resistance and reduce oxygen (O2) permeability. As such, cholesterol may contribute to the Warburg effect in tumor cells by stimulating intracellular hypoxia that cannot be detected from extracellular oxygen measurements. We probe the tissue-level impact of the phenomenon, asking whether layering of cells can magnify the influence of cholesterol, to modulate hypoxia in relation to capillary proximity. Using molecular dynamics simulations, we affirm that minimally hydrated, adjacent lipid bilayers have independent physical behavior. Combining this insight with published experimental data, we predict linearly increasing impact of membrane cholesterol on oxygen flux across cells layered in tissue.

Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion.

ABSTRACTHantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and a highly fatal hantavirus cardiopulmonary syndrome (HCPS) in the New World. No vaccines or antiviral therapies are currently available to prevent or treat hantavirus disease, and gaps in our understanding of how hantaviruses enter cells challenge the search for therapeutics. We performed a haploid genetic screen in human cells to identify host factors required for entry by Andes virus, a highly virulent New World hantavirus. We found that multiple genes involved in cholesterol sensing, regulation, and biosynthesis, including key components of the sterol response element-binding protein (SREBP) pathway, are critical for Andes virus entry. Genetic or pharmacological disruption of the membrane-bound transcription factor peptidase/site-1 protease (MBTPS1/S1P), an SREBP control element, dramatically reduced infection by virulent hantaviruses of both the Old World and New World clades but not by rhabdoviruses or alphaviruses, indicating that this pathway is broadly, but selectively, required by hantaviruses. These results could be fully explained as arising from the modest depletion of cellular membrane cholesterol that accompanied S1P disruption. Mechanistic studies of cells and with protein-free liposomes suggested that high levels of cholesterol are specifically needed for hantavirus membrane fusion. Taken together, our results indicate that the profound dependence on target membrane cholesterol is a fundamental, and unusual, biophysical property of hantavirus glycoprotein-membrane interactions during entry.

African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis

The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication.

Myeloid cell-specific ABCA1 deletion does not worsen insulin resistance in HF diet-induced or genetically obese mouse models

Obesity-associated low-grade chronic inflammation plays an important role in the development of insulin resistance. The membrane lipid transporter ATP-binding cassette transporter A1 (ABCA1) promotes formation of nascent HDL particles. ABCA1 also dampens macrophage inflammation by reducing cellular membrane cholesterol and lipid raft content. We tested the hypothesis that myeloid-specific ABCA1 deletion may exacerbate insulin resistance by increasing the obesity-associated chronic low-grade inflammation. Myeloid cell-specific ABCA1 knockout (MSKO) and wild-type (WT) mice developed obesity, insulin resistance, mild hypercholesterolemia, and hepatic steatosis to a similar extent with a 45% high-fat (HF) diet feeding or after crossing into the ob/ob background. Resident peritoneal macrophages and stromal vascular cells from obese MSKO mice accumulated significantly more cholesterol. Relative to chow, HF diet markedly induced macrophage infiltration and inflammatory cytokine expression to a similar extent in adipose tissue of WT and MSKO mice. Among pro-inflammatory cytokines examined, only IL-6 was highly upregulated in MSKO-ob/ob versus ob/ob mouse peritoneal macrophages, indicating a nonsignificant effect of myeloid ABCA1 deficiency on obesity-associated chronic inflammation. In conclusion, myeloid-specific ABCA1 deficiency does not exacerbate obesity-associated low-grade chronic inflammation and has minimal impact on the pathogenesis of insulin resistance in both HF diet-induced and genetically obese mouse models.

Luminol Electrochemiluminescence for the Analysis of Active Cholesterol at the Plasma Membrane in Single Mammalian Cells

A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.

HDL and ApoA-I inhibit antigen presentation-mediated T cell activation by disrupting lipid rafts in antigen presenting cells

ObjectiveDepletion of cholesterol by methyl-β-cyclodextrin (MCD) on peptide-loaded antigen presenting cells (APCs) inhibits antigen presentation and T cell activation. However, whether membrane cholesterol efflux induced by high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) also results in inhibition of antigen presentation and T cell activation is still unknown. Methods and resultsVarious types of APCs, including B cells, macrophages and dendritic cells (DCs), were first loaded with antigen, then incubated with HDL and apoA-I to decrease cellular membrane cholesterol content. After being treated with HDL and apoA-I, APCs demonstrated decreased potential to activate T cells, and this decrease correlated with an increase in cholesterol efflux from APCs. Cholesterol repletion reversed the inhibitory effects of HDL and apoA-I, demonstrating that the observed reduction in T cell proliferation is mediated through cholesterol. Furthermore, lipid raft analysis showed that HDL and apoA-I reduced cholesterol and major histocompatibility (MHC) class II protein content in lipid rafts, suggesting that cholesterol efflux from APCs to HDL and apoA-I inhibits antigen presentation and T cell activation by reducing lipid rafts assembly in APCs. ConclusionHDL and apoA-I inhibit the capacity of APCs to stimulate T cell activation, and this inhibition can be attributed to cholesterol efflux and the ensuing disruption of plasma membrane lipid rafts in APCs. Overall, these findings suggest that cholesterol efflux mediated by HDL and apoA-I may serve to link immunity and cardioprotection.

Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Cholesterol represents one of the key constituents of small, dynamic, sterol- and sphingolipid-enriched domains on the plasma membrane. It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection. In this study, the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus (PRRSV) infection of MARC-145 cells was investigated. Pretreatment of MARC-145 cells with methyl-β-cyclodextrin (MβCD), a drug used to deplete cholesterol from cellular membrane, significantly reduced PRRSV infection in a dose-dependent manner. This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment, suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol. Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry, especially virus attachment and release. These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.

The Influence of Myrj 59 on the Solubility, Toxicity and Activity of Amphotericin B

The effect of Myrj 59 (a polyoxyethyleneglycol derivative of stearic acid) on amphotericin B (Am B) solubility, toxicity and activity has been investigated. We showed that Myrj 59 could solubilize the antibiotic. Moreover, it also decreased and abolished the haemolytic activity of the drug by increasing the resistance of the red blood cells and impairing the interaction of Am B with the cellular membrane cholesterol, but it did not modify the in-vitro antifungal activity of the drug. On the other hand, Myrj 59 did not decrease the acute in-vivo toxicity of the drug (LD50 and nephrotoxicity). In a previous study we have shown that a polyoxyethleneglycol derivative of cholesterol could solubilize Am B and was able to decrease the in-vitro and in-vivo toxicity of the antibiotic without altering its in-vitro antifungal activity. The results of the present study suggest that the cholesterol moiety of the surfactant is not necessary to decrease the in-vitro lytic activity of the drug but could play a role in the reduction of the in-vivo toxicity.

Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus

Cellular membrane cholesterol has been shown to support various membrane proteins. However, the role and function of membrane cholesterol in viral production are still unclear. Here, we investigated the effects of cholesterol depletion from the cell membrane on the production of hemagglutinating virus of Japan (HVJ; Sendai virus). Cholesterol depletion from LLC-MK2 cells by methyl-beta cyclodextrin treatment resulted in a marked increase in the production of both HVJ from the infected cells and virus-like particles from M-gene-transfected cells. The HVJ produced from cholesterol-depleted cells possessed a reduced amount of envelope cholesterol and showed a rather wide range of particle sizes and amount of envelope protein compared to HVJ produced from untreated cells. Direct depletion of envelope cholesterol from HVJ significantly impaired its infectivity, even without a change in envelope protein composition. These results suggest that membrane cholesterol plays important roles in regulating the production of infectious HVJ.

Force Generation in Lamellipodia is a Discontinuous Process with Nanometer Discrete Forward and Backward Jumps

The progressive addition of actin monomers to the existing network of actin filaments underlies force generation in lamellipodia. By using optical tweezers, we have characterized the dynamics by which lamellipodia of Dorsal Root Ganglia neurons exerted force on encountered obstacles such as silica beads. Because of the presence of adhesion forces, beads in close contact with a lamellipodium could seal on its membrane so that the standard deviation of Brownian fluctuations could be reduced by 10 times. In several experiments, the bead remained within 300 nm from the center of the optical trap where voltage sensitivity of the detector and trap stiffness is constant. Under these conditions, if the lamellipodium pushed the bead, we could detect discrete jumps possibly constituting the elementary events underlying force generation. Jumps were detected by using algorithms based on nonlinear diffusion methods and on numerical differentiation. These jumps occurred within 1 ms and had an amplitude varying from 5 to 20 nm. When the lamellipodium retracted, pulling the beads with it, no discrete events were observed. These discrete events were not observed in the presence of Latrunculin A, a blocker of actin polymerization or when neurons were fixed with paraformaldehyde. The amplitude of these jumps increased by 20-40% when cholesterol in cellular membrane was depleted by treatment with cyclodextrin. These jumps show that force generation in lamellipodia is not a smooth process but is a discontinuous process in which bursts of actin polymerization and depolymerization alternate continuously.

Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na(+)/H(+) ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.

Molecular View of Cholesterol Flip-Flop and Chemical Potential in Different Membrane Environments

The relative stability of cholesterol in cellular membranes and the thermodynamics of fluctuations from equilibrium have important consequences for sterol trafficking and lateral domain formation. We used molecular dynamics computer simulations to investigate the partitioning of cholesterol in a systematic set of lipid bilayers. In addition to atomistic simulations, we undertook a large set of coarse grained simulations, which allowed longer time and length scales to be sampled. Our results agree with recent experiments (Steck, T. L.; et al. Biophys. J. 2002, 83, 2118-2125) that the rate of cholesterol flip-flop can be fast on physiological time scales, while extending our understanding of this process to a range of lipids. We predicted that the rate of flip-flop is strongly dependent on the composition of the bilayer. In polyunsaturated bilayers, cholesterol undergoes flip-flop on a submicrosecond time scale, while flip-flop occurs in the second range in saturated bilayers with high cholesterol content. We also calculated the free energy of cholesterol desorption, which can be equated to the excess chemical potential of cholesterol in the bilayer compared to water. The free energy of cholesterol desorption from a DPPC bilayer is 80 kJ/mol, compared to 67 kJ/mol for a DAPC bilayer. In general, cholesterol prefers more ordered and rigid bilayers and has the lowest affinity for bilayers with two polyunsaturated chains. Overall, the simulations provide a detailed molecular level thermodynamic description of cholesterol interactions with lipid bilayers, of fundamental importance to eukaryotic life.

Abstract: 1062 PLTP, MACROPHAGES AND ATHEROSCLEROSIS

The two main functions of phospholipid transfer protein (PLTP) are the transfer of phospholipids between plasma lipoproteins and the conversion of high density lipoprotein (HDL), where preβ-HDL particles are generated. HDL is considered an anti-atherogenic lipoprotein due to its function in the reverse cholesterol transport, where preβ-HDL accepts cellular membrane cholesterol from peripheral tissues. However, the anti-atherogenic properties of native HDL may be abolished by oxidation/modification. Hypochlorous acid/hypochlorite (HOCl/OCl−)—a potent oxidant generated in vivo only by the myeloperoxidase–H2O2–chloride system of activated phagocytes—alters the physiological properties of HDL by generating a pro-atherogenic lipoprotein particle. Therefore, we have studied the effect of HOCl on the function of HDL subclass 3 (HDL3) and triglyceride-enriched HDL3 (TG-HDL3) in PLTP-mediated processes in vitro. Modification of HDL3 and TG-HDL3 with increasing HOCl concentrations (oxidant:lipoprotein molar ratio between 25:1 and 200:1) decreased the capacity of the corresponding lipoprotein particles to accept phospholipids. Although binding of PLTP to unmodified and HOCl-modified lipoprotein particles was similar, the degree of PLTP-mediated HDL conversion was decreased upon HOCl oxidation. PLTP released apolipoprotein A-I (apoA-I) from HOCl-modified HDL3, but the particles formed displayed no preβ-mobility. Based on these findings, we conclude that the substrate properties of HOCl-modified HDL3 and TG-HDL3 in PLTP-mediated processes are impaired, which indicates that the anti-atherogenic properties of HDL are impaired.