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BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11βHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11βHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1⁺/⁺/apoE⁻/⁻ and 11βHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.

This work shows that 6-ketocholestanol (kCh) inhibits the effect of carbonyl cyanide-m-chlorophenyl hydrazone (CCP) on mitochondrial Ca2+ efflux. Such an effect proved to be caused by diminution of membrane fluidity, therefore, it is affected by the incubation temperature. Furthermore, kCh reversed CCP-induced Ca2+ efflux depending on the accumulation of phosphate. It is also shown that kCh enhances the effect of carboxyatractyloside on membrane permeability transition.

In a dairy spread (800 g lipid/kg, 10 g salt/kg) based on 750 g milk fat/kg and 250 g rapeseed oil/kg fat in 15 g extruded catering packaging, there was a more significant accumulation of cholesterol oxidation products than in butter (minimum 800 g lipid/kg, 12 g salt/kg) in 10 g extruded catering packaging when stored at 4 or at 20 degrees C. There was a lag phase of 7 weeks in cholesterol oxidation in dairy spread stored at 4 degrees C, while no lag phase was observed for storage at 20 degrees C. Total concentrations of oxysterols were, however, very similar for dairy spread stored at 4 and 20 degrees C after 13 weeks storage (approximately 12 micrograms/g milk lipid); storage at -18 degrees C almost prevented cholesterol oxidation (approximately 4 micrograms/g milk lipid). For butter, cholesterol oxidation was less pronounced at 4 degrees C (

Various cholesterol oxides generated during the oxidation of low-density lipoproteins have been reported to exert cytotoxic effects on cultured endothelial cells and to decrease their barrier function. The cytoskeleton, and in particular the actin microfilament meshwork, is one of the preferential targets in oxidative stress-and thiol-depleting agent-induced cell injury. The alterations occurring in the microfilament network were investigated using the endothelial cell line 73/73 treated with increasing concentrations (0.5-10 micrograms/ml) of cholestane-3 beta, 5 alpha, 6 beta-triol, CH, 5-cholesten-3beta-ol-7one, KC, and 25-OH-cholesterol, COH, for up to 6 h. The distribution of microfilaments was visualized using immunofluorescence and laser scanner confocal microscopy. All cholesterol oxides caused a progressive disruption of actin microfilaments that was characterized by the disappearance of the stress fibers within the cell body and, in selected cells, by a complete marginalization and clustering of the filaments to one edge of the cell. In addition, COH promoted F-actin fragmentation, as revealed by the presence of scattered fragments of F-actin in various cell regions. The redistribution of actin microfilaments was associated with a similar redistribution of alpha-actinin, an actin-binding protein involved in bundle formation and in the anchorage of actin filaments to the adhesion plaques. Concomitantly, cholesterol oxides promoted a loss of vinculin, another actin-binding protein, from the focal adhesion plaques located under the cell body and their marginalization and thinning. These alterations preceded cell detachment and cell death by apoptosis as revealed by the subsequent leakage of cytosolic enzymes and nuclear fragmentation. These results suggest that cytoskeletal (microfilament) alterations caused by cholesterol oxides may be one of the cytopathological events involved in the detachment of endothelial cells from the inner vascular surface promoted by cholesterol oxides.

The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 microM 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of .NO and O2- in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged .NO, or with L-NMMA, an inhibitor of .NO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging O2-.

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains.

7-Ketocholesterol (7-KC)-induced apoptosis of macrophages is considered a key event in the development of human atheromas. In the present study, the effect of indicaxanthin (Ind), a bioactive pigment from cactus pear fruit, on 7-KC-induced apoptosis of human monocyte/macrophage THP-1 cells was investigated. A pathophysiological condition was simulated by using amounts of 7-KC that can be reached in human atheromatous plaque. Ind was assayed within a micromolar concentration range, consistent with its plasma level after dietary supplementation with cactus pear fruit. Pro-apoptotic effects of 7-KC were assessed by cell cycle arrest, exposure of phosphatidylserine at the plasma membrane, variation of nuclear morphology, decrease of mitochondrial trans-membrane potential, activation of Bcl-2 antagonist of cell death and poly(ADP-ribose) polymerase-1 cleavage. Kinetic measurements within 24 h showed early formation of intracellular reactive oxygen species over basal levels, preceding NADPH oxidase-4 (NOX-4) over-expression and elevation of cytosolic Ca2+, with progressive depletion of total thiols. 7-KC-dependent activation of the redox-sensitive NF-κB was observed. Co-incubation of 2·5 μmof Ind completely prevented 7-KC-induced pro-apoptotic events. The effects of Ind may be ascribed to inhibition of NOX-4 basal activity and over-expression, inhibition of NF-κB activation, maintaining cell redox balance and Ca homeostasis, with prevention of mitochondrial damage and consequently apoptosis. The findings suggest that Ind, a highly bioavailable dietary phytochemical, may exert protective effects against atherogenetic toxicity of 7-KC at a concentration of nutritional interest.

Abstract We have shown that oxidized low-density lipoprotein (Ox-LDL) modulates various endothelial cell (EC) functions. C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family to be discovered, is secreted from peripheral vascular ECs and regulates body fluid homeostasis, vascular tone, and vascular growth. This study was designed to investigate the effects of lipoproteins on CNP secretion from cultured ECs. Treatment of bovine carotid ECs with Ox-LDL and its extracted lipids resulted in a concentration-dependent suppression of the spontaneous and transforming growth factor-β1–stimulated secretion of CNP. Native LDL, its extracted lipids, and acetylated LDL were inactive. Ox-LDL depleted of its amphiphilic lipids, which was prepared by incubation with defatted albumin, lost its suppressive effect on CNP secretion. 7-Ketocholesterol, one of the amphiphilic lipids in Ox-LDL that is transferable from Ox-LDL to defatted albumin, suppressed CNP secretion by ECs, thus mimicking the effect of Ox-LDL. Coincubation with high-density lipoprotein (HDL), which alone had no effect on CNP release, significantly prevented Ox-LDL–induced inhibition of CNP secretion by ECs. Analysis by thin-layer chromatography demonstrated that oxysterols, including 7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL to HDL during coincubation of these two lipoproteins. These results indicate that Ox-LDL suppresses CNP secretion from ECs by 7-ketocholesterol or other transferable hydrophilic lipids in Ox-LDL, and the suppressive effect of Ox-LDL is reversed by HDL. Lipoproteins thus may regulate CNP secretion from the endothelium of atherosclerotic arteries.

Oxygenated derivatives of cholesterol and related compounds (oxysterols) have long been known to be cytotoxic to many different cell types. The mechanism of this cytotoxic effect is not fully understood. Our laboratory has earlier reported that oxysterol cytotoxicity resembles that of nonsteroidal antiestrogens in some aspects: (i) the cytotoxic action of both types of compounds is blocked by inhibitors of protein or RNA synthesis, and (ii) both classes of compounds bind with high affinity to the microsomal antiestrogen binding site, a protein which may mediate the cytotoxicity of its ligands. We have now extended these studies by developing cell lines which are resistant to the cytotoxic action of oxysterols. Oxysterol-resistant cells were isolated by exposing two murine lymphoma cell lines, K36 and EL4, to incremental concentrations of 7-ketocholestanol. Intriguingly, the resistant cells thus obtained also exhibited considerable resistance to the cytotoxic effects of nonsteroidal antiestrogens such as tamoxifen and clomiphene, having LD 50 values which were 10–100-times higher than that of the parental cells. The resistance appeared to be selective for oxysterols and antiestrogens and did not extend to non-specific toxic agents such as azide, ethanol, Triton-X100, or heat. The biochemical basis of the resistance is not clear but is not due to diminished cellular uptake or increased metabolism of the cytotoxic agents or to changes in the antiestrogen binding protein. The availability of the resistant cell lines should facilitate further studies on the mechanism of oxysterol- and antiestrogen-induced cell death.

Abstract Macrophage uptake of oxidatively modified LDL (Ox-LDL), unlike the uptake of acetylated LDL (Ac-LDL), resulted in lysosomal accumulation of unesterified cholesterol (UC). As sphingomyelin (SM) binds UC with high affinity, we considered whether lysosomes also accumulate Ox-LDL–derived SM, and if such a phenomenon could be involved in the lysosomal trapping of Ox-LDL–derived UC. Incubation of J-774 A.1 macrophages with Ox-LDL increased the lysosomal accumulations of UC by 75% and SM by 63% compared with the effect of Ac-LDL. The addition of chlorpromazine, an inhibitor of lysosomal sphingomyelinase (SMase), to macrophages that were incubated with [ 3 H]cholesteryl ester–labeled Ac-LDL also led to lysosomal accumulation of both SM and UC. 7-Ketocholesterol (7-KC), the major oxysterol in Ox-LDL, inhibited lysosomal SMase in a cell-free system. The addition of 7-KC to cells in the presence of [ 3 H]choline- or [ 3 H]cholesteryl ester–labeled Ac-LDL led to macrophage accumulation of SM or UC, respectively. Niemann-Pick type C disease (NP-C) is an inherited cholesterol-storage disease in which lysosomal SMase activity is attenuated after uptake of LDL. Incubation of monocyte-derived macrophages from two NP-C patients with Ac-LDL or Ox-LDL resulted in an accumulation of UC in the lysosomes, whereas normal monocyte-derived macrophages accumulate UC in their lysosomes after incubation with Ox-LDL but not Ac-LDL. These results suggest that inhibition of lysosomal SMase in NP-C cells or by 7-KC is required for lysosomal accumulation of UC. Analysis of the macrophage lysosomal extract (following cell incubation with Ox-LDL) by density-gradient ultracentrifugation and gel-filtration chromatography revealed the presence of a particle consisting of UC, SM, 7-KC, and apoB-100. We conclude that 7-KC in Ox-LDL can inhibit lysosomal SMase, thus leading to the accumulation of SM, which binds UC avidly and inhibits its further cellular processing out of the lysosome. As UC-SM particles of lysosomal origin exist in the atherosclerotic lesion, the formation of such particles may result from an impaired processing of Ox-LDL by arterial wall macrophages during early atherogenesis.

Abstract Although oxidized low-density lipoprotein (OxLDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I–containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells. Furthermore, the subcellular distributions of both cholesterol and 7-ketocholesterol in individual subcellular organelle fractions were identical to that of free cholesterol in nonloaded cells, indicating that these sterols in OxLDL-loaded cells are not selectively sequestered in lysosomes. 7-Ketocholesterol is released much less efficiently than cholesterol from OxLDL-loaded cells. In addition, OxLDL-loaded cells release cholesterol less efficiently than do cells derived from AcLDL. It is possible that this impairment of efflux from OxLDL-loaded cells influences the generation and persistence of the foam cell phenotype in vivo and may therefore contribute to the atherogenicity of OxLDL.

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 microM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of performed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.

7-Ketocholesterol (a major cholesterol oxidation product) and phytosterols are important indicators of lipoprotein oxidation and lipoprotein metabolism respectively. We describe a simple, sensitive and reproducible method for the simultaneous measurement of these sterols in human lipoprotein samples by capillary column gas liquid chromatography. The method is suitable for clinical studies as small quantities of lipoprotein are required. Sterols are analysed after extraction from lipoprotein samples obtained by sequential flotation ultracentrifugation. The method involves briefly: extraction from lipoprotein samples using chloroform-methanol, saponification of sterol esters using cold potassium hydroxide, purification and derivatisation to trimethylsilyl ethers using BSTFA and 1% TMCS. Oxidation is prevented by drying under nitrogen and the use of powerful antioxidants. Separation is achieved using a DB-1 capillary column and a two-stage temperature ramp from 180-250 degrees C and detection using FID. The identity of sterols can be confirmed by GC-MS. Phytosterols and 7-ketocholesterol are present at low concentration in all the major lipoproteins. Using [3,4-13C]cholesterol and GC-MS we present evidence that cholesterol oxidation does not occur during the processing of lipoproteins using this technique.

Free radical products including reactive oxygen species are potent to oxidize lipids and reliable measurements have been established mostly in human and rodent. To date, robust biomarkers were not used to assess the peroxidation in marine fish. The changes of oxidized lipid products from polyunsaturated fatty acids and cholesterol were assessed after exposure of H(2)O(2) to fish (medaka). Oxidized lipid products released by free radical reaction (F(2)-isoprostanes and metabolites, F(3)-isoprostanes, neuroprostanes, 7-ketocholesterol, 7β-hydroxycholesterol), by lipoxygenase enzymes (5(S)-, 8(S)-, 12(S)- and 15(S)-HETE, and resolvin D1) and by cytochrome P450 (9(S)-, 11(S)- and 20-HETE, and 27-hydroxycholestrol) were measured in fish muscle using LC/MS/MS. Arachidonate, docosahexaenoate, eicosapentaenoate and cholesterol levels, and antioxidant enzymes activity (catalase, SOD and gluthathione reductase) measurement were also determined. Activity of antioxidant enzymes especially catalase were elevated in presence of H(2)O(2) however longer exposure time suppressed the antioxidant activities. Arachidonate, docosahexaenoate, eicosapentaenoate and cholesterol levels were reduced in presence of H(2)O(2) and oxidized lipid products (isoprostanes, neuroprostanes 5(S)-HETE, 20-HETE, 7-ketocholesterol, 27-hydroxycholesterol and resolvin D1) were rapidly released in the fish muscle. This study validates oxidized lipid products, noticeably isoprostanes are measurable in marine fish muscle and should be considered when assessing oxidative stress especially due to exogenous factors.

Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. Na(V)1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. Na(V)1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for Na(V)1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated Na(V)1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that Na(V)1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that Na(V)1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-β-cyclodextrin (MβCD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and Na(V)1.8. By calcium imaging we demonstrated that the lack of association between rafts and Na(V)1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of Na(V)1.8 in nociceptors and highlight the importance of the association between Na(V)1.8 and lipid rafts in the control of nociceptor excitability.

Phenylketonuria (PKU) possibly leads to hypocholesterolemia and lowered vitamin D (VD) status. Metabolism of oxysterols linking with those of cholesterol and VD has never been examined in PKU.Blood oxysterols along with blood phenylalanine, lipids and VD were examined for 33 PKU adults aged 21-38 years and 20 age-matched healthy controls.Total- and low-density cholesterols, and 25-hydroxy VD(3) were decreased significantly in the PKU group (cholesterols, 10% decrease; 25-hydroxy VD(3) 35% decrease vs. the control group). 24S-hydroxycholesterol (24S-OHC) eliminating brain cholesterol, and 27-OHC and 7α-hydroxycholesterol (7α-OHC) representing peripheral and hepatic cholesterol elimination, respectively, were significantly decreased in PKU group: 24S-OHC, 25% decrease, p.01; 27-OHC and 7α-OHC, 35-40% decrease, p.001. 7β-Hydroxycholesterol (7β-OHC) reflecting oxidative stress was increased significantly in PKU group (p.05). 7α-OHC and 27-OHC levels in PKU group always showed similar values, regardless of other parameters while the 24S-OHC and 7β-OHC levels decreased and increased, respectively, showing significant correlations with phenylalanine level (p.005). 27-OHC level showed a significant positive correlation with the 25-hydroxy VD(3) level in this group (p.001).Blood oxysterol changes predominate over blood cholesterol changes and influence on VD status in adult PKU patients.

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC-induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE(-/-)) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC-induced cell death in human aortic smooth muscle cells.

Membrane structural organization is an intrinsic property of a cell membrane. Any changes in lipid composition, and/or any stimuli that affect molecular packing induce structural re-organization. It membrane dynamics provide a means by which changes in structure organization can be determined, upon a change in the membrane internal or external environment. Here, we report on the effect of thermo-stress on membranes containing cholesterol liquid crystal (LC) compounds cholesterol benzoate (BENZO) and oxidized cholesterols. We have (1) revealed that lipid vesicles containing this artificial cholesterol derivative (BENZO) is thermo-responsive, and that this thermo-sensitivity is significantly similar to naturally oxy-cholesterols (2) elucidated the mechanism behind the membrane perturbation. Using Langmuir monolayer experiments, we have demonstrated that membrane perturbation was due to an increase in the molecular surface area, (3) discussed the similarities between cholesterol benzoate in the cholesterol LC state and in lipid bilayer membranes. Last, (4) drawing from previously reported findings, our new data on membrane dynamics, and the discussion above, we propose that artificial cholesterol derivatives such as BENZO, open new possibilities for controlled and tailored design using model membrane systems. Examples could include the development of membrane technology and provide a trigger for progress in thermo-tropical liquid crystal engineering.

Oxysterols are structurally similar to cholesterol, but are characterized by one or more additional oxygen-containing functional groups. These compounds are implicated in inflammation given their ability to cause irreversible damage to vascular cells. The aim of this study was to study the alteration of some inflammatory biomarkers in Wistar rats in response to dietary oxysterols. Eighteen rats were randomly divided into three groups of six rats each. A standard diet supplemented with 1% (w/w) pure cholesterol (Chol group) or 1% (w/w) of an oxidized cholesterol mixture (COPs group) was fed for 8 weeks. Blood serum was separated; abdominal, pericardial, and epididymal adipose tissue was removed carefully. The COPs subjects exhibited significant increase in blood pressure and serum triacylgycerols as well as increased body fat index and pericardic, abdominal, and epididymal adipose tissue. These effects were accompanied by elevated circulating levels of plasma high-sensitivity C-reactive protein, tumor necrosis factor alpha, and resistin. We suggest that dietary oxysterols have an important pro-inflammatory effect.

Objectives Oxidized sterols are toxic to endothelial cells and play a central role in promoting atherogenesis. In this study, we evaluated the impact of anthocyanin, a class of flavonoid compounds, on oxysterol efflux from endothelial cells and the underlying mechanism. Methods and results The human aortic ECs (HAECs) were incubated with anthocyanin cyanidin-3- O -β-glucoside (C3G) for different times. C3G treatment upregulates ABCG1 and ABCA1 expression in a dose-dependent manner in HAECs. Moreover, C3G promotes the efflux of cholesterol mainly 7-ketocholesterol (7-KC) from HAECs in an ABCG1-dependent manner. As a result, C3G abrogated the 7-KC-mediated increase of reactive oxygen species (ROS) and apoptosis in HAECs. Furthermore, C3G treatment reverses the inhibition of endothelial nitric oxide synthase (eNOS) activity by 7-KC, leading to the preservation of nitric oxide (NO) bioavailability. The induction of ABCG1 and its mediated 7-KC efflux from HAECs by C3G resulted from liver X receptor α (LXRα) activation, which was confirmed by its blockage of ABCG1 expression after pharmacological or small interfering RNA inhibition of LXRα. Conclusions These data uncover a novel mechanism by which C3G ameliorates oxysterol-induced oxidative damage on endothelial cells.

Purpose: This study aimed at elucidating the molecular mechanisms involved in the regulation of IL-8 production by several oxysterols in retinal pigment epithelium (RPE) cells. Methods: A human cell line from RPE (ARPE-19) was used to test the role of cholesterol and several oxysterols (25-OH, 7-KC and 7b-OH) in the expression and secretion of IL-8. Expression of IL-8 was assessed by real-time PCR, while IL-8 secretion was evaluated by ELISA. PI3K-, MEK1 ⁄ 2-, ERK1 ⁄ 2- and NF-jB-specific inhibitors were used to assess the specific role of the several players on the regulation of IL-8 production by oxysterols. A gene-reporter assay for AP-1 activity was also conducted to evaluate the putative role of this transcription factor on IL-8 expression induced by oxysterols. Results: Here, we demonstrate that 25-OH specifically increases transcription and secretion of the cytokine IL-8 in ARPE-19 cells. Indeed, treatment of ARPE-19 with 25-OH, but not with 7-KC, 7b-OH or cholesterol, induced the secretion of IL8 from cells. 25-OH also induced the activation ⁄ phosphorylation of ERK1 ⁄ 2 through a mechanism dependent on MEK, ERK1 ⁄ 2 and PI3K kinase activity. Real-time PCR and ELISA experiments demonstrated that 25-OH increased transcription and secretion of IL-8 through a mechanism that is dependent on ERK1 ⁄ 2 and PI3K activity. Furthermore, 25-OH triggered the activation ⁄ phosphorylation of the AP-1 component c-Jun and, consistently, increased the transcriptional activity of AP-1. Additionally, we also found that 25-OH decreases the levels of IjB and increases the nuclear levels of NF-jB p65 subunit and that inhibition of NF-jB activity partially prevents the increased secretion of IL-8 induced by 25-OH. Conclusions: The results presented in this study suggest a role for 25-OH in inducing IL-8 production through pathways that are likely to involve AP-1 and NF-j Bi n ARPE-19 cells. Our data may also provide new molecular targets for the treatment of AMD.

Background: Hepatitis C virus infection is characterised by enhanced oxidative stress, which can be measured quantitatively by plasma oxysterol concentration. These molecules may affect lipid metabolism through the activation of Liver X Receptors. Hepatitis C virus exploits host lipid metabolism to facilitate its replication and diffusion. In our study we aimed to evaluate and highlight the potential pathogenetic role of oxysterols, 7-ketocholesterol and 7-β-hydroxycholesterol, in hepatitis C virus-related lipid dysmetabolism. Methods: The study was performed in 42 patients with chronic hepatitis C (93% genotype 1b) and 38 non-alcoholic fatty liver disease patients. Plasma oxysterols 7-ketocholesterol and 7-β-hydroxycholesterol were determined by isotope dilution gas chromatography/mass spectrometry. Results: Gas chromatography/mass spectrometry revealed higher 7-ketocholesterol (71.2 ± 77.3 vs 30.4 ± 14.5; p

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Only acetylcholinesterase inhibitors and the NMDA antagonist memantine are approved for AD treatment. Recent preclinical and epidemiological studies proposed statins as novel therapeutics for AD, but the mechanisms of action are still unknown. Here, we demonstrate that atorvastatin (80 mg/d for 14.5 months) treatment resulted in an up-regulation of the inducible isoform of haem oxygenase (HO-1), an enzyme with significant neuroprotective activity. Atorvastatin selectively increased HO-1 in the parietal cortex but not cerebellum. In contrast, HO-2 was increased in cerebellum but not parietal cortex. No changes were observed in HO-1 or HO-2 in the liver. Significant negative correlations between HO-1 and oxidative stress indices and positive correlations with glutathione levels in parietal cortex were found. HO-1 up-regulation significantly correlated with lower discrimination learning error scores in aged beagles. Reference to therapeutic applications of atorvastatin in AD is discussed.

The effect of a keto-derivative of cholesterol, namely, 6-ketocholestanol (5 alpha-cholestan-3 beta-ol-6-one; kCh) on the uncoupling of oxidation and phosphorylation by various uncouplers was studied in rat heart mitochondria. kCh was found to completely abolish the uncoupling effect (the increase in the respiration rate under the respiratory control conditions and the decrease in the membrane potential) caused of FCCP, CCCP and SF6847 and partially by TTFB at low concentrations of uncouplers. It was without effect on the uncoupling by PCP, DNP and palmitate. Carboxyatractylate, a specific inhibitor of the ADP/ATP-antiporter, was shown to almost completely abolish the uncoupling induced by palmitate and partially by low concentration of TTFB, PCP and DNP. Effects of high concentrations of all these uncouplers as well as of any concentrations of gramicidin proved to be kCh- and carboxyatractilate-insensitive. The data are discussed in terms of the hypothesis on the protein-mediated mechanism of the protonophorous uncoupling.

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

A group of oxygenated sterols has been identified as physiological regulators of hepatic cholesterol biosynthesis. However, the regulatory effects of these oxysterols on cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, is not clearly elucidated. We administered 0.1% 7-ketocholesterol (15 mg/day), a strong inhibitor of sterol synthesis, to rats orally for 6 days. Then, the levels of accumulated oxysterols in liver microsomes and microsomal 7 alpha-hydroxylase activity were determined. The results were compared to those in the groups of rats treated with either control diet or diets containing 0.1 or 1% cholesterol, 0.1% butylated hydroxytoluene, 3% cholestyramine or 1% taurocholate. 7-Ketocholesterol feeding resulted in significant increase of both 7-ketocholesterol and 7 beta-hydroxycholesterol in microsomal fraction (449.4 +/- 36.8 and 438.2 +/- 46.8 ng/mg protein, respectively; mean +/- S.E.). Hepatic microsomal 7 alpha-hydroxylase activity in the rats fed 7-ketocholesterol was significantly elevated as compared with those of control rats; 44.70 +/- 5.97 vs. 16.57 +/- 2.46 pmol/min per mg protein. Addition of BHT to 7-ketocholesterol reduced the accumulation of 7 beta-hydroxycholesterol, and the stimulatory effect of 7-ketocholesterol on 7 alpha-hydroxylase activity was suppressed. Our results demonstrate that oxysterols do not inhibit but rather stimulate hepatic microsomal 7 alpha-hydroxylase.

The cytotoxicity of oxidized low-density lipoprotein (Ox-LDL) to arterial smooth muscle cells (SMCs) may contribute to atherogenesis by causing cell death in core regions of plaques. The aim of the present study was to identify the components of copper-oxidized LDL responsible for its toxicity to porcine aortic SMCs. Toxicity to SMCs was assessed as the decrease in viable cell counts after 3-day cell incubation. Extracts of LDL were tested for toxicity at concentrations equivalent to that derived from 100 micrograms LDL protein per milliliter. Lipid extracts of Ox-LDL but not native-LDL were toxic to SMCs. When separated into neutral and polar lipid classes, only the neutral lipids were toxic (89.7 +/- 0.7% cell loss). The neutral lipids were fractionated further by use of solid-phase extraction and high-performance liquid chromatography (HPLC). Two toxic fractions, causing 93.3% and 60.3% cell loss, were isolated from HPLC and analyzed by gas chromatography/mass spectrometry. The most toxic of these fractions contained 7-ketocholesterol, and the other contained 7 alpha- and 7 beta-hydroxycholesterol. Quantification of these unesterified oxysterols in LDL extracts and comparison with the toxicity of authentic standards indicate that 7-ketocholesterol and 7-hydroxycholesterol are present in Ox-LDL at levels sufficient to account for its toxicity to SMCs.

Ketocholesterol (7KC) is a cytotoxic oxy- sterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 mM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cy- totoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications. Biotechnol. Bioeng. 2012;109: 2409-2415.

Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-α and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis.

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Background Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Methods Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. Results In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. Conclusion In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.

7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (k(cat)/K(m) = 3 × 10(4) m(-1) s(-1)). P450 7A1 converted lathosterol (Δ(5)-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (~1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a Fe(III)-O-C-C(+) intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482-5489). Accordingly, reaction of P450 7A1 with 7-[(2)H(1)]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F(2)-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F(4)-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F(2)-isoprostanes, HETEs, and 8-hydroxy-2'-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F(2)-isoprostanes, allantoin, and 7β-hydroxycholesterol and urinary F(2)-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F(2)-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2'-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress.

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′- dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H2O2 induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H 2O2 treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited. © 2011 Elsevier Ireland Ltd. All rights reserved.

It is now well accepted that oxysterols play important roles in the formation of atherosclerotic plaque, involving cytotoxic, pro-oxidant and proinflammatory processes. It has been recently suggested that tomato lycopene may act as a preventive agent in atherosclerosis, although the exact mechanism of such a protection is not clarified. The main aim of this study was to investigate whether lycopene is able to counteract oxysterol-induced proinflammatory cytokines cascade in human macrophages, limiting the formation of atherosclerotic plaque. Therefore, THP-1 macrophages were exposed to two different oxysterols, such as 7-keto-cholesterol (4-16 μM) and 25-hydroxycholesterol (2-4 μM), alone and in combination with lycopene (0.5-2 μM). Both oxysterols enhanced pro-inflammatory cytokine [interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor α) secretion and mRNA levels in a dose-dependent manner, although at different extent. These effects were associated with an increased reactive oxygen species (ROS) production through an enhanced expression of NAD(P)H oxidase. Moreover, a net increment of phosphorylation of extracellular regulated kinase 1/2, p-38 and Jun N-terminal kinase and of nuclear factor kB (NF-κB) nuclear binding was observed. Lycopene prevented oxysterol-induced increase in pro-inflammatory cytokine secretion and expression. Such an effect was accompanied by an inhibition of oxysterol-induced ROS production, mitogen-activated protein kinase phosphorylation and NF-κB activation. The inhibition of oxysterol-induced cytokine stimulation was also mimicked by the specific NF-κB inhibitor pyrrolidine dithiocarbamate. Moreover, the carotenoid increased peroxisome proliferator-activated receptor γ levels in THP-1 macrophages. Taken all together, these data bring new information on the anti-atherogenic properties of lycopene, and on its mechanisms of action in atherosclerosis prevention.

International audience; In the context of multiple sclerosis and X-linked adrenoleukodystrophy, 7-ketocholesterol (7KC) and very long chain fatty acids (C24:0, C26:0) are supposed to induce side effects respectively on oligodendrocytes which are myelin (which is a lipoproteic complex) synthesizing cells. The effects of 7KC (25, 50 mu M), C24:0 and C26:0 (10, 20 mu M) on cell viability and lipid membrane organization were investigated on 158N murine oligodendrocytes. Concerning 7KC and fatty acids (at 20 mu M only):1) cell growth was strongly inhibited; 2) marked induction of cell death was revealed with propidium iodide (PI); 3) no apoptotic cells were found with C24:0 and C26:0 (absence of cells with condensed and/or fragmented nuclei, of FLICA positive cells and of PI negative/SYTO16 negative cells); 4) some apoptotic cells were detected with 7KC. Fatty acids (at 20 mu M only) and 7KC also induced a disorganization of lipid membranes revealed with Merocyanine 540. So, to point out the effects of 7KC (25 mu M), C24:0 and C26:0 (20 mu M) on the lateral organization of lipid membranes, we used LAURDAN, which gives simultaneous information about morphology and phase state of lipid domains:its emission is blue in the ordered lipid phase, green in the disordered lipid phase. To overcome the qualitative filtering settings of blue and green emission colors, data obtained by mono- and bi-photon confocal microscopy were analyzed by spectral analysis. Sequences of emission images were obtained on both mono-and bi-photon confocal microscopes and processed by means of Factor Analysis of Medical Image Sequences (FAMIS), which is a relevant tool to unmix emission spectra and provide pure color images. Only 7KC was capable to induce a green emission with LAURDAN. Thus, at concentrations inducing oligodendrocyte cell death, 7KC (25 mu M) is more efficient than C24:0 and C26:0 (20 mu M), to trigger lateral lipid membrane disorganization. (C) 2011 International Society for Advancement of Cytometry

Rats of the Sprague-Dawley strain were infused intravenously with a fat emulsion (Intralipid, trademark of Kabi Pharmacia, Uppsala, Sweden) containing 7-oxocholesterol. This resulted in an increased cholesterol 7 alpha-hydroxylase activity in liver microsomes as compared to controls and was accompanied by increased levels of cholesterol 7 alpha-hydroxylase mRNA and microsomal cholesterol 7 alpha-hydroxylase protein. Rats were also fed a cholestyramine-supplemented diet and infused with 7-oxocholesterol. These animals excreted about half as much bile acids in faeces as cholestyramine-fed controls. Addition of 7-oxocholesterol to liver microsomes from normal rats in amounts corresponding to those present in microsomes from 7-oxocholesterol-treated rats inhibited the cholesterol 7 alpha-hydroxylase activity by about 75%. Cholesterol induced a type-I binding spectrum when added to a purified bacterial-expressed cholesterol 7 alpha-hydroxylase (P-450c7 delta 2-24). 7-Oxocholesterol competitively inhibited the cholesterol binding spectrum, while 7 beta-hydroxycholesterol did not interfere with binding of cholesterol to the enzyme. It is concluded that treatment with the competitive inhibitor 7-oxocholesterol leads to a reduced bile acid biosynthesis and, as a consequence of reduced bile acid inhibition, a compensatory increase in cholesterol 7 alpha-hydroxylase synthesis. The high enzyme activity measured in microsomal preparations from 7-oxocholesterol-treated rats may be due to a continuous conversion of 7-oxocholesterol into less inhibitory metabolites, e.g. 7 beta-hydroxycholesterol. The latter compound was found in high concentrations in liver microsomes from rats treated with 7-oxocholesterol. The physiological importance of these results is discussed in relation to the previous findings that 7-oxocholesterol is accumulated in liver after cholesterol feeding and that 7-oxocholesterol is formed from cholesterol during lipid peroxidation.

Several oxysterols were examined for their effect on gap junctional communication between rat hepatocytes in primary culture. 25-Hydroxycholesterol, 22(S)-hydroxycholesterol and 7 beta-hydroxycholesterol, in decreasing order of potency, markedly inhibited gap junctional communication. In contrast, 7-ketocholesterol showed no inhibitory effect. The inhibition of gap junctional communication by oxysterols was not a consequence of changes in cell viability, as measured by lactate dehydrogenase leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity. The addition of exogenous cholesterol to the culture medium did not abolish the effect of 25-hydroxycholesterol, suggesting that the capacity of oxysterols to inhibit gap junctional communication is independent of their inhibitory effect on cholesterol synthesis. We suppose that inhibition of gap junctional communication may be an early sign of oxysterols-induced toxicity on hepatocytes.

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.

Cholesterol oxidation products formed under the enhanced oxidative stress in the brain are suggested to induce neuronal cell death. However, it is still unknown whether oxysterol-induced apoptosis in neuronal cells is mediated by Akt and NF-κB pathways. We assessed the apoptotic effect of 7-ketocholesterol against differentiated PC12 cells in relation to activation of the reactive oxygen species-dependent nuclear factor (NF)-κB, which is mediated by the Akt pathway. 7-Ketocholesterol induced a decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic Bax levels, cytochrome c release, caspase-3 activation and upregulation of p53. 7-Ketocholesterol induced an increase in phosphorylated inhibitory κB-α, NF-κB p65 and NF-κB p50 levels, binding of NF-κB p65 to DNA, and activation of Akt. Treatment with Bay 11-7085 (an inhibitor of NF-κB activation) and oxidant scavengers, including N-acetylcysteine, prevented the 7-ketocholesterol-induced formation of reactive oxygen species, activation of NF-κB, Akt and apoptosis-related proteins, and cell death. Results from this study suggest that 7-ketocholesterol may exert an apoptotic effect against PC12 cells by inducing activation of the caspase-8-dependent pathway as well as activation of the mitochondria-mediated cell death pathway, leading to activation of caspases, via the reactive oxygen species-dependent activation of NF-κB, which is mediated by the Akt pathway.

To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties.Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion. The overproduction of reactive oxygen species (ROS) was determined by FCM with hydroethidine.Whereas the nanoparticles had no cytotoxic or inflammatory effects, they could stimulate ROS production. QDs were not incorporated. When 7KC was used, LDH release was enhanced and QDs potentialized IL-8 secretion. The incorporation and exit dynamics of nanoparticles were visualized to differentiate the emission spectra of SYTOX Green and nanoparticles and to precisely determine the cellular localization of nanoparticles.The selected nanoparticles, which accumulate at the inner or outer cytoplasmic membrane level, can induce biologic activities and are able to interfere with those of chemically defined molecules such as 7KC.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC(50)=2.9+/-0.38 microM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC(50)=1.5+/-1.2 microM and 2.2+/-1.1 microM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT.

As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

Suzuki K, Sakiyama Y, Usui M, Obama T, Kato R, Itabe H, Yamamoto M. Oxidized low-density lipoprotein increases interleukin-8 production in human gingival epithelial cell line Ca9-22. J Periodont Res 2010; 45: 488–495. © 2010 John Wiley & Sons A/S Background and Objective: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low-density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. Material and Methods: Cells of the human gingival epithelial cell line Ca9-22 were cultured in media containing OxLDL, and the amounts of interleukin-8 (IL-8) and prostaglandin E2 (PGE2) produced were measured using ELISAs. Results: Production of IL-8 by Ca9-22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE2 by Ca9-22 cells was enhanced by co-incubation with OxLDL and interleukin-1β (IL-1β). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL-induced IL-8 and PGE2 production in the presence of IL-1β. The p38 MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL-induced IL-8 production. Among oxidized lipids and chemically modified LDL, 7-ketocholesterol enhanced IL-8 production. Conclusion: This is the first report to show that OxLDL enhances IL-8 production in epithelial cells.

Objective— Adenosine monophosphate activated protein kinase (AMPK) has been identified as a regulator of vascular function via the preservation of endothelial cell (EC) function. In this study, we examined whether the beneficial effects of AMPK on ECs are dependent on its involvement in cholesterol efflux and its impact on hypercholesterolemia-induced endothelial dysfunction. Methods and Results— Using human aortic ECs and bovine aortic ECs, we show that AMPK activation upregulates ATP binding cassette G1 (ABCG1) expression independently of liver X receptor alpha (LXRα) transcriptional activity but through a posttranscriptional mechanism that increases mRNA stability. Using a heterologous system and a luciferase reporter, we further identify that the 3′-untranslated region of the ABCG1 mRNA is responsible for the regulatory effects of AMPK activation. 5-Aminoimidazole-4-carboxamide-1-β- d -riboside treatment promotes endothelial 7-ketocholesterol efflux and prevents 7-ketocholesterol (7-KC)–induced reactive oxygen species production in an ABCG1-dependent manner, thus preserving endothelial nitric oxide synthase activity and nitric oxide bioavailability. Notably, in vivo studies using C57BL/6J mice receiving a high-cholesterol diet revealed that the infusion of 5-aminoimidazole-4-carboxamide-1-β- d -riboside increases vascular ABCG1 expression and improves vascular reactivity. These effects are abrogated by the AMPK antagonist compound C and by the vascular gene transfer of ABCG1 small interfering RNA. Conclusion— Our current findings uncover a novel mechanism by which AMPK protects against hypercholesterolemia-mediated endothelial dysfunction.

We studied the effects of cholesterol, its oxidized derivatives mevalonate, and nuclear receptor agonists LXR, RXR, and FXR on the production of transforming growth factor-beta1 (TGF- beta1) by macrophages. After recruiting of macrophage monocytes into the focus of inflammation, the production of TGF-beta1 increased by 3.5 times in comparison with control macrophages. Cholesterol diet stimulated the production of TGF-beta1 by 2.5 times. Cholesterol directly stimulated macrophage production of TGF-beta1 in vitro, while addition of mevalonate to the incubation medium effectively reduced this induced production. Agonists of nuclear receptor sharply reduced the production of TGF-beta1 in recruited macrophages. Under conditions of inflammation, hypercholesterolemia can be a factor of fibrogenesis due to TGF-beta1 induction in macrophages, which depends on the products of mevalonate biochemical chain.