Cholesterol-enriched Liposomes Research Articles

Influence of lipid vesicle properties on the function of conjugation dependent membrane active peptides.

Membrane active peptides (MAPs) can provide novel means to trigger the release of liposome encapsulated drugs to improve the efficacy of liposomal drug delivery systems. Design of MAP-based release strategies requires possibilities to carefully tailor the interactions between the peptides and the lipid bilayer. Here we explore the influence of lipid vesicle properties on the function of conjugation-dependent MAPs, specifically focusing on two de novo designed peptides, JR2KC and CKV4. Utilizing liposomes with differences in size, lipid composition, and surface charge, we investigated the mechanisms and abilities of the peptides to induce controlled release of encapsulated cargo. Our findings indicate that liposome size modestly affects the structural changes and function of the peptides, with larger vesicles facilitating a minor increase in drug release efficiency due to higher peptide-to-liposome ratios. Notably, the introduction of negatively charged lipids significantly enhanced the release efficiency, predominantly through electrostatic interactions that favor peptide accumulation at the lipid bilayer interface and subsequent membrane disruption. The incorporation of cholesterol and a mix of saturated and unsaturated lipids was shown to alter the vesicle's phase behavior, thus modulating the membrane activity of the peptides. This was particularly evident in the cholesterol-enriched liposomes, where JR2KC induced lipid phase separation, markedly enhancing cargo release. Our results underscore the critical role of lipid vesicle composition in the design of MAP-based drug delivery systems, suggesting that precise tuning of lipid characteristics can significantly influence their performance.

A CRAC-like motif in BAX sequence: Relationship with protein insertion and pore activity in liposomes

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, –L/V-(X)(1–5)-Y-(X)(1–5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.

Herpes Simplex Virus Glycoprotein B Associates with Target Membranes via Its Fusion Loops

Herpes simplex virus (HSV) glycoproteins gB, gD, and gH/gL are necessary and sufficient for virus entry into cells. Structural features of gB are similar to those of vesicular stomatitis virus G and baculovirus gp64, and together they define the new class III group of fusion proteins. Previously, we used mutagenesis to show that three hydrophobic residues (W174, Y179, and A261) within the putative gB fusion loops are integral to gB function. Here we expanded our analysis, using site-directed mutagenesis of each residue in both gB fusion loops. Mutation of most of the nonpolar or hydrophobic amino acids (W174, F175, G176, Y179, and A261) had severe effects on gB function in cell-cell fusion and null virus complementation assays. Of the six charged amino acids, mutation of H263 or R264 also negatively affected gB function. To further analyze the mutants, we cloned the ectodomains of the W174R, Y179S, H263A, and R264A mutants into a baculovirus expression system and compared them with the wild-type (WT) form, gB730t. As shown previously, gB730t blocks virus entry into cells, suggesting that gB730t competes with virion gB for a cell receptor. All four mutant proteins retained this function, implying that fusion loop activity is separate from gB-receptor binding. However, unlike WT gB730t, the mutant proteins displayed reduced binding to cells and were either impaired or unable to bind naked, cholesterol-enriched liposomes, suggesting that it may be gB-lipid binding that is disrupted by the mutations. Furthermore, monoclonal antibodies with epitopes proximal to the fusion loops abrogated gB-liposome binding. Taken together, our data suggest that gB associates with lipid membranes via a fusion domain of key hydrophobic and hydrophilic residues and that this domain associates with lipid membranes during fusion.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3beta-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

Cholesterol enrichment upregulates intercellular adhesion molecule-1 in human vascular endothelial cells

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-κB (NF-κB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-κB-dependent expression was not affected. The AP-1/Ets (−891 to −908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.

Cholesterol and ω-3 fatty acids inhibit Na, K-ATPase activity in human endothelial cells

We have investigated the effects of cholesterol and ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) on Na, K-ATPase activity in human endothelial cells (HUVEC). Cultured HUVEC were incubated for 18 h with pure egg phosphatidylcholine (PC), or cholesterol-enriched liposomes (4 mg PC/ml). EPA and DHA α-tocopherol-acetate were emulsified with PC and incubated with HUVEC (10 mM). Na, K-ATPase and 5′-nucleotidase activities were determined using the coupled assay method on microsomal fractions obtained from cultured cells using non treated cells as control. Cholesterol enrichment significantly reduced both Na, K-ATPase and 5′-nucleotidase activities by a similar level (−40%), whereas pure phospholipid liposomes inhibited this activity only by 22%. The dose–response curves of Na, K-ATPase activity were all biphasic assuming the presence of two independent sites exhibiting different affinities for ouabain of nM and μM respectively. The cholesterol induced inhibitory effect was greater for low affinity sites (−54%) as compared to that of the high affinity sites (−24%) whereas ω-3 fatty acids reduced the activity of both sites by 22%. Short term effects of EPA and DHA on Na, K-ATPase activity were determined by incubating microsomal fractions from untreated cells with various concentrations of free fatty acids (from 1 to 200 μM) for 20 min. Both EPA and DHA significantly reduced Na, K-ATPase activity but inhibition by EPA seems to be more effective than DHA. These results suggest that cholesterol and ω-3 fatty acids reduce Na, K-ATPase activity in HUVEC.

Nuclear membrane cholesterol can modulate nuclear nucleoside triphosphatase activity.

Previous work has suggested that changes in nuclear membrane cholesterol may induce a stimulation in nuclear nucleoside triphosphatase (NTPase) activity. The purpose of the present study was to directly investigate if nuclear membrane cholesterol can stimulate nuclear NTPase activity. The cholesterol content of nuclei was altered with a liposomal methodology. The cholesterol content of nuclei isolated from hepatic tissue was relatively low in comparison to that typically exhibited by other membrane fractions. Because of this, it was difficult to further deplete the nuclear membrane of cholesterol, but we could successfully increase the cholesterol content after exposure to cholesterol-enriched liposomes. Nuclear NTPase activity was potently stimulated (approximately 150-200% of control) by an increase in the nuclear membrane cholesterol content. The Vmax of the NTPase activity in the presence of ATP or GTP was significantly increased after cholesterol enrichment without altering the affinity of the enzyme for these moieties. Mg2+ dependency of NTPase activity was also altered by cholesterol incorporation into the nuclear membrane. Cholesterol enrichment of the nuclear membrane also left the nuclei more susceptible to damage by salt-induced lysis than control nuclei. Our results clearly demonstrate that the cholesterol content of the nuclear membrane will have significant, direct effects on nuclear integrity and NTPase activity.

Cholesterol enrichment inhibits Na +/K + pump in endothelial cells

We have previously shown that cholesterol enrichment reduces 3H-ouabain binding in cultured vascular endothelial cells. The present study aimed to determine the effect of cholesterol enrichment on ouabain-sensitive 86Rb (as a substitute for K +) influx, i.e. K+ transport via the pump, and to examine whether cellular K+ content was affected in human umbilical vein endothelial cells. 86Rb influx was inhibited by both ouabain and bumetanide in a dose-dependent manner. Consistent with an earlier report [1], inhibition achieved was greater for ouabain (∼ 70% at mM range) than for bumetanide (∼ 55% at 0.1 mM), indicating that K + influx via Na +/K + pump was greater than that via Na +K +Cl − cotransport in these cells. After incubation of 18 h or more with cholesterol-enriched liposomes (2:1 cholesterol to phospholipid ratio), a significant reduction (>20%) of the ouabain-sensitive K + influx and an increase in cellular cholesterol content were observed. The inhibitory effect was observed only at liposome concentrations above 2 mg/ml. Following 18 h incubation with 2 mg/ml cholesterol-enriched liposomes, cellular K + content was significantly decreased. The phospholipid liposome treatment did not alter K + content, suggesting that the inhibitory effect on Na +/K + pump and the cellular K + content reduction was not due to liposome fusion alone or to the phospholipid present. These findings indicate that cholesterol enrichment inhibits Na +/K + pump and thus reduces cellular K + content in endothelial cells, and may play a role in the widely observed abnormal endothelium-dependent vascular response induced by hypercholesterolemia.

Sphingomyelin and cholesterol modulate sodium coupled uptakes in proximal tubular cells

Sphingomyelin (SM) and cholesterol are major lipid species of apical membranes in renal proximal tubular cells and confer to these membranes a low fluidity. Changes in membrane fluidity and/or lipidic composition were shown to affect the activity of cotransport systems of renal apical membranes. We evaluated the effect of decreasing membrane SM content on lipidic composition, membrane fluidity and sodium (Na)coupled uptakes in rabbit proximal tubular cells in primary culture. Sphingomyelinase (SMase) (30 to 250 mU/ml) decreased [3H]choline-labeled SM content, decreased cholesterol content, and increased cholesterol esterification. SMase did not modify membrane fluidity on isolated brush border membranes. SMase decreased Vmax of Na-dependent uptake of phosphate and alpha-methyl-D-glucoside, but not of alanine. SMase did not influence protein kinase C-induced inhibition of phosphate and glucose uptake. Increasing membrane cholesterol content with cholesterol-enriched liposomes subsequently to SMase action restored in part glucose uptake, but not phosphate uptake. In conclusion, SM degradation affected Na-phosphate and Na-glucose cotransports through changes in both SM and cholesterol contents of apical proximal membranes; these changes seemed to occur independently from changes in bulk membrane fluidity. These results suggest that SM and cholesterol have distinct and intricated roles in accessibility and/or activity of apical cotransport systems.

Modulation of Cellular Cholesterol and Its Effect on Cornified Envelope Formation in Cultured Human Epidermal Keratinocytes

When cultured human epidermal keratinocytes (NHK) reach confluence they start to differentiate and an increase in the total cellular cholesterol content is observed. This increase parallels the appearance of a characteristic feature of terminal keratinocyte differentiation, the spontaneous formation of cornified envelopes (CE). Synthesis of CE is catalyzed by the plasma membrane-associated transglutaminase (TGm). Supplementation of the medium with inhibitors of cholesterologenesis suppressed increase in cholesterol levels and CE formation but did not interfere with TGm expression or TGm activity. Modulation of the plasma membrane cholesterol-phospholipid ratio of confluent NHK cultures using either pure phospholipid liposomes or liposomes enriched in cholesterol strongly affected spontaneous CE formation. Pure phospholipid liposomes completely inhibited CE formation, whereas cholesterol-enriched liposomes ensured envelope formation, even in the presence of inhibitors of cholesterol synthesis. From these results we conclude that in differentiating NHK an increase in the cellular cholesterol level is part of the differentiation program and is essential for the spontaneous CE formation.

Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.

We examined the role of cholesterol in altering the activity of the microsomal cytochrome P-450 enzyme, cholesterol-NADPH:oxygen oxidoreductase (cholesterol 7 alpha-hydroxylase). Liposomes were used to deliver cholesterol to hepatic microsomes. Formation of 7 alpha-hydroxycholesterol was quantitated by isotope dilution/gas chromatography-mass spectrometry. As the liposomal cholesterol/phospholipid molar ratio increased, 7 alpha-hydroxylase activity increased, whereas the activity of another microsomal cytochrome P-450 enzyme, ethylmorphine N-demethylase, decreased. To determine if the degree of stimulation was affected by the endogenous activity (without liposomes), microsomes, from rats fed chow alone or chow containing cholestyramine, taurocholate, or cholesterol were challenged with cholesterol-enriched liposomes. The degree of stimulation was dependent upon the endogenous activity: cholestyramine-fed much greater than cholesterol = chow control greater than taurocholate-fed. To determine if cholesterol stimulates 7 alpha-hydroxylase by increasing membrane viscosity, microsomes were incubated with liposomes having the same cholesterol/phospholipid molar ratio as microsomes, but different viscosities. Dipalmitoylphosphatidylcholine (high viscosity) liposomes increased microsomal viscosity and decreased 7 alpha-hydroxylase activity. In contrast, dioleoylphosphatidylcholine (low viscosity) liposomes decreased microsomal viscosity and increased enzyme activity. Since greater viscosity inhibits 7 alpha-hydroxylase, cholesterol cannot stimulate the enzyme by increasing membrane viscosity. The data suggest that cholesterol stimulates production of 7 alpha-hydroxycholesterol by providing substrate.

Cholesterol-induced changes in rabbit arterial smooth muscle sensitivity to adrenergic stimulation.

We evaluated the effects of exposure to high cholesterol levels for 3 h on arterial smooth muscle responses to adrenergic stimulation. Femoral arteries from Dutch belted rabbits were perfused in vitro with a constant-flow variable-pressure perfusion apparentus. After equilibration the vessels were perfused for 180 min more with media supplemented with cholesterol-phospholipid (C/PL) liposomes of molar ratios of 2:1 or 0.5:1. Although resting vascular resistance was unchanged, norepinephrine (NE) concentration-response analyses revealed a fivefold increase in NE sensitivity (P less than 0.001) in the arteries perfused with the cholesterol-enriched liposomes (2:1) compared with control arteries perfused with the 0.5:1 liposome medium. The arteries perfused with the cholesterol-enriched liposomes demonstrated a 60% increase in cholesterol content and a marked (90%) reduction in Na+-K+-ATPase activity. The increased sensitivity of the cholesterol-enriched arteries was not mediated by acute reductions in Na-pump activity, altered endothelial function, adrenergic nerve function, or prostaglandin production. Cholesterol-induced sensitization to NE did demonstrate an absolute dependence on extra-cellular calcium. These findings suggest that an increase in the free cholesterol content of the arterial smooth muscle cell plasma membrane alters membrane permeability to extracellular calcium during adrenergic stimulation.

5′-Nucleotidase is activated upon cholesterol-depletion of liver plasma membranes

The cholesterol content of rat liver plasma membranes was manipulated using either cholesterol-free or cholesterol-enriched liposomes. Removal of cholesterol from the membranes led to a marked increase in 5′-nucleotidase activity. However, increase in cholesterol content failed to exert any significant effect on 5′-nucleotidase activity. Arrhenius plots of the activity of the native enzyme exhibited a break at around 28°C with the activation energy of the reaction less above this temperature than below. In cholesterol-depleted membranes a single break at around 26°C was observed with activation energies greater above this temperature than below it. In cholesterol-enriched membranes Arrhenius plots were linear over the range examined. It is suggested that the lipid environment of the external half of the bilayer only influences 5′-nucleotidase activity in these membranes and that cholesterol exerts controlling effects on both the activity and conformation of the enzyme in native membranes.

Modifications of membrane cholesterol content affects electrical properties and prolactin release of cultured pituitary cells.

Treatment of cloned pituitary cells (GH3/B6) with cholesterol-enriched liposomes, which presumably increases membrane cholesterol content, affects the passive and active electrophysiological properties and stimulates the release of prolactin (PRL).

Cholesterol modulates activity of calcium-dependent ATPase of the sarcoplasmic reticulum

BIOMEMBRANES consist of an asymmetric lipid bilayer matrix into which and around which the various proteins are situated. The proteins may be attached to the outside of the lipid bilayer (extrinsic proteins), but in many cases the proteins (intrinsic proteins) are embedded within, and can span, the bilayer. Associated with this is the idea that in many cases the lipid matrix is in a fluid condition in which the lipids are essentially above their transition temperature (Tc) and able to diffuse along the bilayer length. The perturbation introduced into the lipid bilayer by the presence of an intrinsic protein has recently been discussed2,3. Some workers4,5 have suggested that intrinsic proteins, for example the Ca2+-ATPase of the sarcoplasmic reticulum, carry with them, even when excess bulk fluid lipid occurs, a shell of immobilised lipid, referred to as an annulus, which controls the enzyme activity. The shell is said to exclude cholesterol so that cholesterol molecules do not influence the enzyme activity. We report here the use of cholesterol-enriched liposomes to reversibly vary the content of cholesterol in the sarcoplasmic membranes. We show in contrast to the previous work that as the cholesterol content of the membrane varies so does the activity of the Ca2+-ATPase.