Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids.

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.

Cholesterol binding to ion channels.

Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

The role of oxysterols in control of endothelial stiffness.

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE⁻/⁻ mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein.

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390µM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3ß-17 ß-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Hypoxia modulates the expression of leucine zipper-positive MYPT1 and its interaction with protein kinase G and Rho kinases in pulmonary arterial smooth muscle cells.

We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ(+)MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ(+) MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ(+) MYPT1 and PKG, resulting in increased MLC(20) phosphorylation, a higher pMLC(20)/MLC(20) ratio and SMC contraction. In normoxic SMC PKG overexpression, LZ(+) MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ(+) MYPT1 overexpression enhanced the interaction between PKG and LZ(+) MYPT1. Overexpression of a mutant LZ(-) MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC(20)/MLC(20) ratio. Collectively, our data suggest that hypoxia downregulates LZ(+) MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ(+) MYPT1 with PKG and promotes LZ(+) MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction.

oxLDL-induced decrease in lipid order of membrane domains is inversely correlated with endothelial stiffness and network formation.

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

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.

Green and black tea extracts inhibit HMG-CoA reductase and activate AMP kinase to decrease cholesterol synthesis in hepatoma cells.

Recent studies have demonstrated that green and black tea consumption can lower serum cholesterol in animals and in man, and suppression of hepatic cholesterol synthesis is suggested to contribute to this effect. To evaluate this hypothesis, we measured cholesterol synthesis in cultured rat hepatoma cells in the presence of green and black tea extracts and selected components. Green and black tea decreased cholesterol synthesis by up to 55% and 78%, respectively, as measured by a 3-h incorporation of radiolabeled acetate. Inhibition was much less evident when radiolabeled mevalonate was used, suggesting that the inhibition was mediated largely at or above the level of HMG-CoA reductase. Both extracts directly inhibited HMG-CoA reductase when added to microsomal preparations, although the extent of inhibition was considerably less than the decrease in cholesterol synthesis observed in whole cells. As HMG-CoA reductase activity also can be decreased by enzyme phosphorylation by AMP kinase, the phosphorylation state of HMG-CoA reductase and AMP kinase, which is activated by phosphorylation, was determined in lysates from cells treated with tea extracts. Both extracts increased AMP-kinase phosphorylation and HMG-CoA reductase phosphorylation by 2.5- to 4-fold, but with different time courses: maximal phosphorylation with green tea was evident within 30 min of treatment, whereas with black tea phosphorylation was slower to develop, with maximal phosphorylation occurring > or =3 hours after treatment. These results suggest that both green and black tea decrease cholesterol synthesis in whole cells by directly inhibiting HMG-CoA reductase and by promoting its inactivation by AMP kinase.

Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity.

We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60-80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids.

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2.

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.

Policosanol inhibits cholesterol synthesis in hepatoma cells by activation of AMP-kinase.

Policosanol is a mixture of long-chain primary alcohols that has been shown to decrease serum cholesterol in animals and in humans. The hypocholesterolemic effect results from a decrease in cholesterol synthesis by suppression of HMG-CoA reductase activity, but the mechanism of this suppression and the active components of policosanol have not been established. In the present study, we investigated the ability of policosanol and its principal components to inhibit cholesterol synthesis in cultured rat hepatoma cells. Maximal inhibition by policosanol yielded a 30% decrease in [(14)C]acetate incorporation without evidence of cellular toxicity. Octacosanol (C28, the major constituent of policosanol), heptacosanol (C27), and hexacosanol (C26) yielded smaller and statistically insignificant decreases in cholesterol synthesis, whereas triacontanol (1-hydroxytriacontane; C30) replicated the inhibition obtained with policosanol. At pharmacological concentrations (<5 microg/ml), policosanol and triacontanol decreased [(14)C]acetate incorporation into cholesterol without affecting the incorporation of [(14)C]mevalonate, indicating that these compounds act at or above HMG-CoA reductase. Policosanol and triacontanol did not directly inhibit HMG-CoA reductase, and incubation of these compounds with hepatoma cells did not affect reductase enzyme levels. However, reductase activity was decreased by up to 55% in lysates prepared from these cells, suggesting that HMG-CoA reductase activity was down-regulated by policosanol treatment. Consistent with this hypothesis, a 3-fold increase in AMP-kinase phosphorylation was noted in policosanol-treated cells. Because AMP-kinase is activated by phosphorylation and is well established to suppress HMG-CoA reductase activity, these results suggest that policosanol or a metabolite decreases HMG-CoA reductase activity by activating AMP-kinase.

Inhibition of sterol 4alpha-methyl oxidase is the principal mechanism by which garlic decreases cholesterol synthesis.

Clinical and experimental evidence indicates that garlic ingestion lowers blood cholesterol levels, and treatment of cells in culture with garlic and garlic-derived compounds inhibits cholesterol synthesis. To identify the principal site of inhibition in the cholesterolgenic pathway and the active components of garlic, cultured hepatoma cells were treated with aqueous garlic extract or its chemical derivatives, and radiolabeled cholesterol and intermediates were identified and quantified. Garlic extract reduced cholesterol synthesis by up to 75% without evidence of cellular toxicity. Levels of squalene and 2,3-oxidosqualene were not altered by garlic, indicating that the site of inhibition was downstream of lanosterol synthesis, and identical results were obtained with 14C-acetate and 14C-mevalonate, confirming that 3-hydroxy-3-methylglutaryl-CoA reductase activity was not affected in these short-term studies. Several methylsterols that accumulated in the presence of garlic were identified by coupled gas chromatography-mass spectrometry as 4,4'-dimethylzymosterol and a possible metabolite of 4-methylzymosterol; both are substrates for sterol 4alpha-methyl oxidase, pointing to this enzyme as the principal site of inhibition in the cholesterolgenic pathway by garlic. Of 9 garlic-derived compounds tested for their ability to inhibit cholesterol synthesis, only diallyl disulfide, diallyl trisulfide, and allyl mercaptan proved inhibitory, each yielding a pattern of sterol accumulation identical with that obtained with garlic extract. These results indicate that compounds containing an allyl-disulfide or allyl-sulfhydryl group are most likely responsible for the inhibition of cholesterol synthesis by garlic and that this inhibition is likely mediated at sterol 4alpha-methyl oxidase.

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro.

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

Rat supernatant protein factor-like protein stimulates squalene monooxygenase and is activated by protein kinase A.

Rat supernatant protein factor-like protein (SPF2) shares 90% sequence identity with rat SPF and 77% identity with human SPF, both of which have been shown to stimulate squalene monooxygenase in the cholesterol biosynthetic pathway. SPF2 appears to be predominantly expressed in respiratory and epithelial tissues, whereas SPF is expressed in liver. To determine if SPF2 was also able to stimulate squalene monooxygenase activity, we have cloned, expressed, and purified the protein following heterologous expression in Escherichia coli. SPF2 was only half as effective as SPF in stimulating squalene epoxidation and was more strongly inhibited by GTP and GDP. The inhibition by guanine nucleotides was fully prevented by alpha-tocopherol, a reported ligand for these proteins. Incubation of SPF2 with protein kinase A and ATP increased its activity by about twofold, has been found for SPF. These results indicate that SPF2 activity is modulated by guanine nucleotides and alpha-tocopherol, as well as by phosphorylation.

Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase.

Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold. Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.