betadex and 25-hydroxycholesterol

In the present work, the mechanisms involved in the recently reported antiviral activity of zebrafish C-reactive protein-like protein (CRP1-7) against the spring viraemia of carp rhabdovirus (SVCV) in fish are explored. The results neither indicate blocking of the attachment or the binding step of the viral replication cycle nor suggest the direct inhibition of G protein fusion activity or the stimulation of the host's interferon system. However, an antiviral state in the host is induced. Further results showed that the antiviral protection conferred by CRP1-7 was mainly due to the inhibition of autophagic processes. Thus, given the high affinity of CRPs for cholesterol and the recently described influence of the cholesterol balance in lipid rafts on autophagy, both methyl-β-cyclodextrin (a cholesterol-complexing agent) and 25-hydroxycholesterol (a cholesterol molecule with antiviral properties) were used to further describe CRP activity. All the tested compounds exerted antiviral activity by affecting autophagy in a similar manner. Further assays indicate that CRP reduces autophagy activity by initially disturbing the cholesterol ratios in the host cellular membranes, which in turn negatively affects the intracellular regulation of reactive oxygen species (ROS) and increases lysosomal pH as a consequence. Ultimately, here we propose that such pH changes exert an inhibitory direct effect on SVCV replication by disrupting the pH-dependent membrane-fusogenic ability of the viral glycoprotein G, which allows the release of the virus from endosomes into cytoplasm during its entry phase.

Topics: Animals; Autophagy; beta-Cyclodextrins; C-Reactive Protein; Cell Line; Cell Membrane; Cholesterol; Hydrogen-Ion Concentration; Hydroxycholesterols; Protein Isoforms; Reactive Oxygen Species; Rhabdoviridae; Rhabdoviridae Infections; Virus Replication; Zebrafish; Zebrafish Proteins

Classical swine fever virus (CSFV), the etiological agent of classical swine fever in pigs, is a member of the Pestivirus genus within the Flaviviridae family. It has been proposed that CSFV infection is significantly inhibited by methyl-β-cyclodextrin (MβCD) treatment. However, the exact engagement of cellular cholesterol in the life cycle of CSFV remains unclear. Here, we demonstrated that pretreatment of PK-15 cells with MβCD significantly decreased the cellular cholesterol level and resulted in the inhibition of CSFV infection, while replenishment of exogenous cholesterol in MβCD-treated cells recovered the cellular cholesterol level and restored the viral infection. Moreover, we found that depletion of cholesterol acted on the early stage of CSFV infection and blocked its internalization into the host cells. Furthermore, we showed that 25-hydroxycholesterol, a regulator of cellular cholesterol biosynthesis, exhibited a potent anti-CSFV activity by reducing cellular cholesterol level. Taken together, our findings highlight the engagement of cholesterol in the life cycle of CSFV and its potential use as an antiviral target.

Topics: Animals; Antiviral Agents; beta-Cyclodextrins; Cell Line; Cholesterol; Classical Swine Fever Virus; Epithelial Cells; Hydroxycholesterols; Swine; Virus Internalization

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Topics: Androstenes; beta-Cyclodextrins; Blotting, Western; Carrier Proteins; Cell Death; Cholesterol; Fibroblasts; Flow Cytometry; Humans; Hydroxycholesterols; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lysosomal Membrane Proteins; Lysosomes; Membrane Glycoproteins; Microscopy, Phase-Contrast; Mutation; Neurons; Niemann-Pick C1 Protein; Quinacrine; Statistics, Nonparametric

Previously we showed that insulin-like growth factor-I (IGF-I) promotes sustained phosphorylation of Akt in oligodendrocyte progenitor cells (OPCs) and that Akt phosphorylation is required for survival of these cells. The direct mechanisms, however, by which IGF-I promotes Akt phosphorylation are currently undefined. Recently, cholesterol-enriched membranes (CEMs) have been implicated in regulation of growth factor-mediated activation of the PI3K/Akt pathway and survival of mature oligodendrocytes; however, less is know about their role in OPC survival. In the present study, we investigate the role of CEMs in IGF-I-mediated Akt phosphorylation and OPC survival. We report that acute disruption of membrane cholesterol with methyl-beta-cyclodextrin results in altered OPC morphology and inhibition of IGF-I-mediated Akt phosphorylation. We also report that long-term inhibition of cholesterol biosynthesis with 25-hydroxycholesterol blocks IGF-I stimulated Akt phosphorylation and cell survival. Moreover, we show that the PI3K regulatory subunit, p85, Akt, and the IGF-IR are sequestered within cholesterol-enriched fractions in steady-state stimulation of the IGF-IR and that phosphorylated Akt and IGF-IR are present in cholesterol-enriched fractions with IGF-I stimulation. Together, the results of these studies support a role for CEMs or "lipid rafts" in IGF-I-mediated Akt phosphorylation and provide a better understanding of the mechanisms by which IGF-I promotes OPC survival.

Topics: Animals; Animals, Newborn; beta-Cyclodextrins; Cell Survival; Cells, Cultured; Cholesterol; Hydroxycholesterols; Insulin-Like Growth Factor I; Membrane Microdomains; Oligodendroglia; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 1; Signal Transduction; Stem Cells

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.

Topics: Anticholesteremic Agents; beta-Cyclodextrins; Carcinoma, Hepatocellular; Cells, Cultured; Cellular Structures; Cholesterol; Genome, Viral; Hepacivirus; Host-Parasite Interactions; Humans; Hydroxycholesterols; Lipid Metabolism; Lipids; Replicon; RNA, Viral; Virus Replication

Oxidized phospholipids, including oxidation products of palmitoyl-arachidonyl-phosphatidyl choline (PAPC), are mediators of inflammation in endothelial cells (ECs) and known to induce several chemokines, including interleukin-8 (IL-8). In this study, we show that oxidized PAPC (OxPAPC), which accumulates in atherosclerotic lesions, paradoxically depletes endothelial cholesterol, causing caveolin-1 internalization from the plasma membrane to the endoplasmic reticulum and Golgi, and activates sterol regulatory element-binding protein (SREBP). Cholesterol loading reversed these effects. SREBP activation resulted in increased transcription of the low-density lipoprotein receptor, a target gene of SREBP. We also provide evidence that cholesterol depletion and SREBP activation are signals for OxPAPC induction of IL-8. Cholesterol depletion by methyl-beta-cyclodextrin induced IL-8 synthesis in a dose-dependent manner. Furthermore, cholesterol loading of ECs by either the cholesterol-cyclodextrin complex or caveolin-1 overexpression inhibited OxPAPC induction of IL-8. These observations suggest that changes in cholesterol level can modulate IL-8 synthesis in ECs. The OxPAPC induction of IL-8 was mediated through the increased binding of SREBP to the IL-8 promoter region, as revealed by mobility shift assays. Overexpression of either dominant-negative SREBP cleavage-activating protein or 25-hydroxycholesterol significantly suppressed the effect of OxPAPC on IL-8 transcription. A role for SREBP activation in atherosclerosis is suggested by the observation that EC nuclei showed strong SREBP staining in human atherosclerotic lesions. The current studies suggest a novel role for endothelial cholesterol depletion and subsequent SREBP activation in inflammatory processes in which phospholipid oxidation products accumulate.

Topics: Animals; Aorta; Arteriosclerosis; beta-Cyclodextrins; Cattle; Caveolin 1; Caveolins; CCAAT-Enhancer-Binding Proteins; Cell Compartmentation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cholesterol; DNA-Binding Proteins; Endoplasmic Reticulum; Endothelial Cells; Endothelium, Vascular; Golgi Apparatus; HeLa Cells; Humans; Hydroxycholesterols; Inflammation; Interleukin-8; Intracellular Signaling Peptides and Proteins; Membrane Lipids; Membrane Proteins; Phosphatidylcholines; Phospholipid Ethers; Recombinant Fusion Proteins; STAT3 Transcription Factor; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection

The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised approximately 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by approximately 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; beta-Cyclodextrins; Cholesterol; Cyclodextrins; Endoplasmic Reticulum; Fibroblasts; Humans; Hydroxycholesterols; Membrane Lipids; Rats; Surface-Active Agents; Tumor Cells, Cultured

PC12 cells induced to differentiate with nerve growth factor were used to study the neurotoxicity of 25-OH-cholesterol. This agent induced a dose- and time-dependent cell death in neuronal PC12 cells. Cells treated with this agent showed condensed nuclei, a morphology similar to that of cells dying of programmed cell death. However, agents known to prevent neuronal programmed cell death (cyclic AMP, KCl, aurintricarboxylic acid, and cycloheximide) failed to prevent the 25-OH-cholesterol-mediated cytotoxicity. On the other hand, cell death induced by 25-OH-cholesterol was prevented by treatment with vitamin E and methyl-beta-cyclodextrin. In contrast to observations made in other cell types, whole-cell patch clamp recording of neuronal PC12 cells revealed that treatment with 25-OH-cholesterol did not significantly alter calcium influx through voltage-dependent channels. These results provide the first characterization of the toxicity of cholesterol oxides toward neuronal PC12 cells, which should be useful in future studies on the interactions between cholesterol oxides and cells from the nervous system.

Topics: Animals; Apoptosis; Ascorbic Acid; Aurintricarboxylic Acid; beta-Cyclodextrins; Blood; Calcium Channels; Cell Differentiation; Cyclic AMP; Cyclodextrins; Hydroxycholesterols; Nerve Growth Factors; PC12 Cells; Potassium Chloride; Rats; Vitamin E

Cultured rat sympathetic neurons derived from postnatal rat superior cervical ganglia (SCG) were used to compare the neurotoxicity of several cholesterol oxides. The cholesterol oxides tested included: 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH-, and 25-OH-cholesterol. These agents caused an acute as well as a delayed toxicity in sympathetic neurons with 25-OH-cholesterol appearing to be the most toxic. A time-dependent experiment indicated that 25-OH-cholesterol at 4 microg/ml (10 microM) was able to kill 50% of the cells in 36 h. Morphological studies indicate that most of the cells do not exhibit a structural change similar to that observed in neuronal programmed cell death. Whole-cell patch clamp recording of untreated controls and 25-OH-cholesterol (2 microg/ml)-treated cells indicated that this toxicity was not accompanied by significant changes in voltage-dependent calcium channel activity. A number of pharmacological agents including ethylene glycolbis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), cycloheximide, KCl, vitamin E, and methyl-beta-cyclodextrin were able to prevent the 25-OH-cholesterol-induced cell death to various degrees. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may induce neurotoxicity in sympathetic neurons.

Topics: Animals; Animals, Newborn; beta-Cyclodextrins; Calcium Channels; Cells, Cultured; Cyclodextrins; Cycloheximide; Egtazic Acid; Hydroxycholesterols; Kinetics; Membrane Potentials; Neurons; Neurotoxins; Patch-Clamp Techniques; Potassium Chloride; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion; Time Factors; Vitamin E

Cultured rat cerebellar granule cells were used to determine the potential neurotoxicity of cholesterol oxides. The cholesterol oxides tested included: 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH- and 25-OH- cholesterol. Among them, 7-beta-OH- and 7-keto-cholesterol were the most efficacious in causing neuronal death such that 20 microg/ml (50 microM) of these agents killed more than 80% of cells in 2 days. 7-beta-OH-cholesterol at this concentration killed 50% of cells in approximately 7 h. A number of pharmacological agents were tested for their abilities to prevent neuronal death induced by cholesterol oxides. Among them, aurintricarboxylic acid, vitamin E and methyl-beta-cyclodextrin were able to prevent cholesterol oxide-induced neurotoxicity in a dose-dependent manner. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may induce toxicity in neurons of the central nervous system.

Topics: Animals; Ascorbic Acid; Aurintricarboxylic Acid; beta-Cyclodextrins; Cell Death; Cells, Cultured; Cerebellum; Cyclic AMP; Cyclodextrins; Cycloheximide; Drug Interactions; Hydroxycholesterols; Neurons; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Vitamin E

N9 microglial cells were used as a model to examine the effect of cholesterol oxides on central nervous system microglia. Results indicated that 25-OH-cholesterol was the most cytotoxic agent among the cholesterol oxides tested. During the process of cell death, this agent caused prominent nuclei condensation and significant DNA fragmentation, a phenomenon association with programmed cell death. Cholesterol oxides were able to potentiate the bacterial lipopolysaccharide (LPS)-induced nitric oxide production to various degrees. Consistent with this finding, Northern blot analysis indicated that 25-OH-cholesterol potentiated the LPS-induced nitric oxide synthase RNA levels. The cytotoxicity of 25-OH-cholesterol could be prevented by methyl-beta-cyclodextrin, a glucose polymer known to cause cholesterol oxide efflux from cells. While much attention has been focused on the cytotoxicity of cholesterol oxides on immune cells within the blood, including lymphocytes and macrophages, the results from this study indicated for the first time that these agents are toxic to microglial cells derived from the central nervous system.

Topics: Animals; Apoptosis; beta-Cyclodextrins; Cell Line; Cyclodextrins; DNA Fragmentation; Drug Synergism; Hydroxycholesterols; Lipopolysaccharides; Mice; Microglia; Nitric Oxide; Nitric Oxide Synthase; RNA, Messenger

The utility of 2-hydroxypropyl-beta-cyclodextrin for increasing the sensitivity of assays for the microsomal acylCoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-beta-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-beta-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-beta-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-beta-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Amides; Animals; beta-Cyclodextrins; Cell Fractionation; Cholesterol; Cyclodextrins; Cytoplasm; Enzyme Activation; Enzyme Inhibitors; Hydroxycholesterols; Kinetics; Lysosomes; Microsomes, Liver; Mitochondria, Liver; Organosilicon Compounds; Rats; Sodium Fluoride; Solubility; Sterol Esterase; Sterol O-Acyltransferase