Release Of Cholesterol Research Articles

Reduced Plasma Levels of 25-Hydroxycholesterol and Increased Cerebrospinal Fluid Levels of Bile Acid Precursors in Multiple Sclerosis Patients

Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography–mass spectrometry (LC–MS) exploiting multistage fragmentation (MSn). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol (25-HC, P < 0.05) in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid (7αH,3O-CA, P < 0.05) were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophages.

Caveolin-1 facilitates internalization and degradation of ABCA1 and probucol oxidative products interfere with this reaction to increase HDL biogenesis

Background and aimsExpression of ATP binding cassette transporter (ABC) A1, a key membrane protein for biogenesis of high-density lipoprotein (HDL), is regulated not only by its gene transcription but also by its intracellular degradation to modulate plasma HDL concentration. We previously showed that inhibition of ABCA1 degradation by probucol oxidative products, spiroquinone (SQ) and diphenoquinone (DQ), increased HDL biogenesis and reverse cholesterol transport, and achieved reduction of atherosclerosis in animal models. The background mechanism has thus been investigated. MethodsInvolvement of caveolin-1, a protein of multiple functions in cell biology, particularly in cholesterol trafficking, has been examined for its roles in ABCA1 degradation as well as the effects of SQ and DQ on the reaction. ResultsABCA1 protein was increased in caveolin-1-deficient mouse embryonic fibroblasts, not by increase of transcription but by decrease in its internalization and degradation. Transfection and expression of caveolin-1 normalized the protein level and the rate of degradation of ABCA1. Immunoprecipitation experiments demonstrated association between ABCA1 and caveolin-1 and SQ and DQ disrupted this interaction. The effects of SQ and DQ to increase ABCA1 and cell cholesterol release induced by apolipoprotein A-I were dependent on expression of caveolin-1. Fluorescence imaging of ABCA1 and caveolin-1 in cultured cells demonstrated their co-localization as well as its disruption by SQ and DQ, being consistent with the biochemical findings. ConclusionsCaveolin-1 enhances internalization and degradation of ABCA1 by its association with ABCA1. Interference of this interaction by probucol oxidative products suppresses ABCA1 degradation and increase HDL biogenesis.

Abstract 378: Cholecystokinin Upregulates Cholesterol Absorption in Human Intestinal Epithelial Cells

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. We previously reported that cholecystokinin (CCK) increases intestinal cholesterol absorption and plasma cholesterol level in mouse models. The goal of this study was to investigate the effect of CCK on cholesterol absorption and Niemann-Pick C1 Like 1 (NPC1L1) expression in human primary intestinal epithelial cells (HPIECs). Normal HPIECs were isolated from small bowl resection specimens, and purchased from the Lonza Group (Walkersville, MD) . Cholesterol absorption was determined by measuring transcellular cholesterol transport in adherent cell culture and cholesterol association and release in suspension cell culture. Surface NPC1L1 was isolated using a biotinylation kit and detected by western blotting. Our data demonstrate that HPIECs express both CCK receptor-1 and -2 (CCK1R; CCK2R). Treatment of HPIECs with 3 nM [Thr28, Nle31]-CCK for 60 min increased transcellular cholesterol transport, cholesterol association and release by ~38, 32 and 44%. Selective inhibition of CCK1R and CCK2R with antagonists (1 μM lorglumide or L365260) or selective knockdown of CCK1R and CCK2R with siRNAs attenuated CCK-induced cholesterol absorption. In the cells cultured on transwell membranes, CCK increased the level of NPC1L1 in the apical membrane by ~35% but did not alter the total NPC1L1 protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. These data imply that activation of CCK1R/2R enhances cholesterol absorption by induction of NPC1L membrane translocation. [This study was supported by NIH grants U54MD0007593, UL1TR000445, and SC1HL101431]

β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells.

Atherosclerosis is an inflammatory disease that leads to an aberrant accumulation of cholesterol in vessel walls forming atherosclerotic plaques. During this process, the mechanism regulating complex cellular cholesterol pools defined as the reverse cholesterol transport (RCT) is altered as well as expression and functionality of transporters involved in this process, namely ABCA1, ABCG1, and SR-BI. Macrophages, arterial endothelial and smooth muscle cells (SMCs) have been involved in the atherosclerotic plaque formation. As macrophages are widely described as the major cell type forming the foam cells by accumulating intracellular cholesterol, RCT alterations have been poorly studied at the arterial endothelial cell and SMC levels. Amongst the therapeutics tested to actively counteract cellular cholesterol accumulation, the methylated β-cyclodextrin, KLEPTOSE® CRYSMEβ, has recently shown promising effects on decreasing the atherosclerotic plaque size in atherosclerotic mouse models. Therefore we investigated in vitro the RCT process occurring in SMCs and in arterial endothelial cells (ABAE) as well as the ability of some modified β-CDs with different methylation degree to modify RCT in these cells. To this aim, cells were incubated in the presence of different methylated β-CDs, including KLEPTOSE® CRYSMEβ. Both cell types were shown to express basal levels of ABCA1 and SR-BI whereas ABCG1 was solely found in ABAE. Upon CD treatments, the percentage of membrane-extracted cholesterol correlated to the methylation degree of the CDs independently of the lipid composition of the cell membranes. Decreasing the cellular cholesterol content with CDs led to reduce the expression levels of ABCA1 and ABCG1. In addition, the cholesterol efflux to ApoA-I and HDL particles was significantly decreased suggesting that cells forming the blood vessel wall are able to counteract the CD-induced loss of cholesterol. Taken together, our observations suggest that methylated β-CDs can significantly reduce the cellular cholesterol content of cells forming atherosclerotic lesions and can subsequently modulate the expression of ABC transporters involved in RCT. The use of methylated β-CDs would represent a valuable and efficient tool to interfere with atherosclerosis pathogenesis in patients, nonetheless their mode of action still needs further investigations to be fully understood and finely controlled at the cellular level.

Abstract 539: Soluble Oxidized Apolipoprotein A-I, a Precursor of Amyloid Fibrils, Activates Secretion of Inflammatory Cytokines in Macrophages

Atherosclerosis is often described as an inflammatory disease of the arteries. One mechanism whereby apolipoprotein A-I (apoA-I) exerts its anti-atherosclerotic effect is by mitigating the inflammatory response of cells involved in atherosclerosis progression. However, oxidation transforms apoA-I from an anti-inflammatory to a pro-inflammatory protein. We previously reported that oxidation can also promote apoA-I aggregation and formation of amyloid fibrils. In this study, we investigated the mechanistic interplay between oxidation, amyloid formation and the inflammatory response of macrophages to apoA-I. We hypothesized that amyloid fibrils constituted of oxidized apoA-I activate production of inflammatory cytokines in macrophages. To test this hypothesis, amyloidogenic apoA-I was generated by oxidation with an excess of H 2 O 2 (H 2 O 2 -ApoA-I). Intracellular and secreted levels of IL-1β were determined upon incubation of mouse bone marrow derived macrophages (BMDM) with intact-apoA-I, soluble H 2 O 2 -ApoA-I and pre-formed H 2 O 2 -ApoA-I amyloid fibrils. Cellular cholesterol release from RAW264.7 cells was also measured. Soluble H 2 O 2 -ApoA-I (amyloid precursor) retained the cellular cholesterol release capacity of intact-ApoA-I. In BMDM incubated with soluble H 2 O 2 -ApoA-I however, levels of IL-1β synthesis and secretion were at least 2-fold higher than those induced by intact-ApoA-I. In contrast, incubation with H 2 O 2 -ApoA-I amyloid fibrils did not increase the levels of IL-1β synthesis and secretion, compared to intact-ApoA-I. Thus, soluble and functional oxidized apoA-I activates inflammatory cytokine synthesis and secretion in macrophages. Notably, this pro-inflammatory potential was completely neutralized when oxidized apoA-I was aggregated in amyloids. Therefore in atherosclerotic lesions, amyloid formation could reduce, rather than exacerbate, the inflammatory burden produced by pro-inflammatory soluble oxidized apoA-I species.

The effects of apolipoprotein B depletion on HDL subspecies composition and function

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Probucol-Oxidized Products, Spiroquinone and Diphenoquinone, Promote Reverse Cholesterol Transport in Mice.

Oxidized products of probucol, spiroquinone and diphenoquinone, were shown to increase cell cholesterol release and plasma high-density lipoprotein (HDL) by inhibiting degradation of ATP-binding cassette transporter A1. We investigated whether these compounds enhance reverse cholesterol transport in mice. Spiroquinone and diphenoquinone increased ATP-binding cassette transporter A1 protein (2.8- and 2.6-fold, respectively, P<0.01) and apolipoprotein A-I-mediated cholesterol release (1.4- and 1.4-fold, P<0.01 and P<0.05, respectively) in RAW264.7 cells. However, diphenoquinone, but not spiroquinone, enhanced cholesterol efflux to HDL (+12%, P<0.05), whereas both increased ATP-binding cassette transporter G1 protein, by 1.8- and 1.6-fold, respectively. When given orally to mice, both compounds significantly increased plasma HDL-cholesterol, by 19% and 20%, respectively (P<0.05), accompanied by an increase in hepatic and macrophage ATP-binding cassette transporter A1 but not ATP-binding cassette transporter G1. We next evaluated in vivo reverse cholesterol transport by injecting RAW264.7 cells labeled with (3)H-cholesterol intraperitoneally into mice. Both spiroquinone and diphenoquinone increased fecal excretion of the macrophage-derived (3)H-tracer, by 25% and 28% (P<0.01 and P<0.05), respectively. spiroquinone/diphenoquinone did not affect fecal excretion of HDL-derived (3)H-cholesterol, implying that macrophage-to-plasma was the most important step in spiroquinone/diphenoquinone-mediated promotion of in vivo reverse cholesterol transport. Finally, spiroquinone significantly reduced aortic atherosclerosis in apolipoprotein E null mice when compared with the vehicle. Spiroquinone and diphenoquinone increase functional ATP-binding cassette transporter A1 in both the macrophages and the liver, elevate plasma HDL-cholesterol, and promote overall reverse cholesterol transport in vivo. These compounds are promising as therapeutic reagents against atherosclerosis.

Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism.

Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL) plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL) and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression.

Apolipoprotein A-I induces tubulin phosphorylation in association with cholesterol release in fetal rat astrocytes

We previously identified cytosolic lipid–protein particles (CLPP) having size and density of HDL in rat astrocytes, to which apoA-I induces translocation of cholesterol, caveolin-1 and protein kinase Cα (PKCα) following its association with microtubules prior to cholesterol release/biogenesis of HDL (JBC 277: 7929, 2002; JLR 45: 2269, 2004). To further understand the physiological relevance of these findings, we investigated the CLPP/microtubule association and its role in intracellular cholesterol trafficking by using a technique of reconstituted microtubule-like filaments (rMT) in rat astrocyte cytosol. When the cells were pretreated with apoA-I, α-tubulin as a 52-kDa protein in rMT was found phosphorylated while α-tubulin in a soluble monomeric form was little phosphorylated. The phosphorylation took place coincidentally to apoA-I-induced association with rMT of CLPP, a complex containing PKCα, and was suppressed by a PKC inhibitor, Bis indolylmaleimide 1 (BIM). α-Tubulin dissociated from CLPP when phosphorylated, and it poorly bound to CLPP once dissociated. BIM did not influence association of PKCα with rMT but suppressed apoA-I-induced cholesterol translocation to the cytosol from the ER/Golgi apparatus and apoA-I-mediated cholesterol release. We thereby concluded that α-tubulin phosphorylation by PKCα on CLPP is involved in reversible CLPP association with the microtubules and intracellular cholesterol trafficking for apoA-I-dependent HDL biogenesis/cholesterol release in rat astrocytes.

Abstract 447: Acrolein-Modified HDL Impairs Cholesterol Transport and HDL Function

The interaction between scavenger receptor BI (SR-BI) and HDL reduces the risk for cardiovascular disease (CVD) by playing key roles in the reverse cholesterol transport pathway (RCT). In the beginning of RCT, SR-BI stimulates release of free cholesterol (FC) from peripheral macrophages to HDL, whereas in the final step of RCT, HDL binds to hepatic SR-BI for delivery of excess cholesteryl esters (CE) into the liver for excretion via bile. While “functioning” HDL is crucial in protection against CVD, environmental factors such as acrolein, a highly reactive aldehyde found in tobacco smoke, can induce oxidative stress that promotes HDL modifications and renders the particle “dysfunctional”. Although there is a strong correlation between cigarette smoking and CVD risk, much less is known about the effects of smoking on RCT. We hypothesized that acrolein modifications to HDL (acro-HDL) generate a dysfunctional particle by: (i) impairing SR-BI-mediated selective uptake of HDL-CE and efflux of FC and (ii) upregulating inflammatory pathways in macrophages that promote atherogenesis. To test this hypothesis, human HDL was modified with 250μM acrolein and acrolein adducts on HDL were confirmed by immunoblot analysis and mass spectrometry. To evaluate acro-HDL’s functionality at the beginning of RCT, SR-BI-mediated FC release to native- or acro-HDL was measured in SR-BI-transfected COS-7 cells. Our data revealed that there was 30% less efflux of FC in the presence of acro-HDL as compared to native HDL. To assess acro-HDL functionality at the end of RCT, HDL binding and HDL-CE uptake assays were performed and acro-HDL (0-50μg/mL) was 10-25% less efficient at delivering CE to cells via SR-BI. Finally, to determine whether acro-HDL was rendered atherogenic, we used qRT-PCR to demonstrate that acro-HDL, but not native HDL, paralleled the effects of oxidized LDL by upregulating mRNA scavenger receptor expression (CD36 and LOX-1) and inflammatory marker expression (MCP-1, IL-6, ICAM-1) in THP-1 macrophages. Altogether, our data reveal that acrolein modification produces a dysfunctional HDL particle that upregulates inflammatory pathways. More detailed investigations into the impact of acrolein adducts on HDL function may help prevent smoking-related CVD deaths.

Abstract 425: Development of a Vascular Cell Adhesion Molecule-1 Targeted Esterase Nanofiber that Binds Activated Endothelial Cells

Introduction: Novel therapies capable of directly targeting and regressing atherosclerotic plaques would provide tremendous benefit to patients with atherosclerotic cardiovascular disease. Molecular imaging studies have shown the feasibility of targeting vascular adhesion molecule (VCAM-1) for non-invasive atherosclerosis imaging. Histidine contains an imidazole ring with esterase activity that can convert cholesterol esters to free cholesterol. Thus, we hypothesize that a peptide amphiphile (PA) functionalized with a VCAM-1 binding peptide and histidine will spontaneously form a targeted nanofiber that binds activated endothelium and liberates free cholesterol from cholesterol esters. Methods: PAs were synthesized by solid phase methods and purified by reversed phase HPLC. Three different VCAM-1 binding peptide sequences were conjugated to the PAs (free N-terminus, free C-terminus, and cyclic). To assess 3-dimensional structure of the co-assembled PAs, cryogenic transmission electron microscopy (TEM) was used. To assess targeting of the VCAM-PAs (50μM), human umbilical endothelial cells (HUVECs) were stimulated with TNFα (10ng/ml). To assess the ability of the histidine-PA to liberate free cholesterol, a modified Amplex Red Cholesterol Assay was used. Results: Using TEM, we demonstrated that the co-assembled VCAM- and histidine-PAs formed characteristic nanofibers with diameters of 5-8nm and lengths ranging from 300nm to 1μm. TNFα-activated HUVECs were confirmed to express VCAM-1 protein by Western blot analysis. Confocal imaging revealed that the VCAM-1 binding peptide with a free N-terminus conjugated to the PA had the greatest binding affinity to the TNFα-activated HUVECs. Co-incubation of the histidine-PA with cholesterol esters demonstrated that free cholesterol was liberated from cholesterol esters in a concentration-dependent manner with a five-fold increase in free cholesterol release with 25μM of PA versus 1μM. Conclusion: We have designed and characterized a VCAM-1 targeted nanofiber that successfully binds activated endothelium and generates free cholesterol from cholesterol esters. This novel therapeutic nanotechnology has the capacity to target and regress atherosclerotic plaques.

Involvement of cdc42/Rho kinase in ApoA‐I‐mediated cholesterol efflux through interaction between cytosolic lipid‐protein particles and microtubules in rat astrocytes

Activation of cdc42 reportedly enhances apoA-I-mediated cholesterol release through ATP-binding cassette transporter A1 (ABCA1). We examined the involvement of cdc42 and Rho kinase in intracellular cholesterol transport for release of cholesterol after the interaction between apoA-I and ABCA1 in astrocytes. Exogenously added apoA-I increased the GTP-bound form of cdc42 and enhanced Rho kinase activity in rat astrocytes. Suppression of ABCA1 expression by siRNA substantially repressed both cellular level of GTP-bound cdc42 and Rho kinase activity, indicating that these reactions require ABCA1. ApoA-I-mediated lipid release and Rho kinase activation were inhibited by not only Rho kinase inhibitor but also cdc42 siRNA. These findings suggest that cdc42 is activated by the interaction between apoA-I and ABCA1 and enhances cholesterol release through the activation of Rho kinase. ApoA-I increased the binding of Rock1, one of the Rho kinases, to reconstituted microtubule-like filaments (rMT). Y-27632 suppressed not only the association of rMT with the cytosolic lipid-protein particles (CLPP)-related proteins and lipids but also the intracellular transport of newly synthesized cholesterol to the plasma membrane in rat astrocytes treated with apoA-I without inhibiting cholesterol synthesis. Finally, cdc42 siRNA reduced apoA-I-induced interaction between rMT and major players in intracellular cholesterol trafficking, such as caveolin-1 and Rock1, suggesting a regulatory role of Rho family proteins in the apoA-I-mediated intracellular cholesterol transport. We conclude that ABCA1/cdc42/Rho kinase signaling is involved in apoA-I-induced intracellular cholesterol transport and apoA-I-mediated cholesterol release in rat astrocytes.

Detailed characterization of the endocannabinoid system in human macrophages and foam cells, and anti-inflammatory role of type-2 cannabinoid receptor

ObjectiveCannabinoid receptors are activated in murine macrophages upon exposure to oxidized low-density lipoproteins (oxLDL), and type-1 cannabinoid receptor (CB1R) is considered as a risk factor in atherosclerosis, because it promotes cholesterol accumulation and release of inflammatory mediators. Conversely, accumulated evidence suggests a protective role for type-2 cannabinoid receptor (CB2R). Here, we sought to ascertain whether different elements of the endocannabinoid system (ECS) were activated in human lipid-laden macrophages, and whether CB2R played any role in atherogenesis and inflammation of these cells. Methods and resultsHuman macrophages were exposed to oxLDL in order to obtain lipid-laden foam cells. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the production of the endocannabinoids in both macrophages and foam cells, and radiometric assays were performed to measure cannabinoid receptor binding and activity of endocannabinoid metabolizing enzymes. OxLDL accumulation was investigated by confocal imaging, and cytokine production and release were measured by means of flow cytometry and ELISA. The results showed that human macrophages possess a fully functional ECS, which was modulated by oxLDL. Selective CB2R activation reduced cellular oxLDL accumulation, which was associated with decreased expression of CD36 scavenger receptor, and decreased production of TNFα, IL-12 and IL-10. These anti-atherogenic and anti-inflammatory effects were reverted by the selective CB2R antagonist SR144528. ConclusionsA fully active ECS is present in human macrophages and macrophage-derived foam cells. Selective activation of CB2R reduces CD36-dependent oxLDL accumulation and modulates production of inflammatory cytokines, thus representing a potential therapeutic strategy to combat atherosclerosis.

Platelet-activating factor antagonists enhance intracellular degradation of amyloid-β42 in neurons via regulation of cholesterol ester hydrolases.

IntroductionThe progressive dementia that is characteristic of Alzheimer’s disease is associated with the accumulation of amyloid-beta (Aβ) peptides in extracellular plaques and within neurons. Aβ peptides are targeted to cholesterol-rich membrane micro-domains called lipid rafts. Observations that many raft proteins undertake recycling pathways that avoid the lysosomes suggest that the accumulation of Aβ in neurons may be related to Aβ targeting lipid rafts. Here we tested the hypothesis that the degradation of Aβ by neurons could be increased by drugs affecting raft formation.MethodsPrimary neurons were incubated with soluble Aβ preparations. The amounts of Aβ42 in neurons or specific cellular compartments were measured by enzyme-linked immunosorbent assay. The effects of drugs on the degradation of Aβ42 were studied.ResultsAβ42 was targeted to detergent-resistant, low-density membranes (lipid rafts), trafficked via a pathway that avoided the lysosomes, and was slowly degraded by neurons (half-life was greater than 5 days). The metabolism of Aβ42 was sensitive to pharmacological manipulation. In neurons treated with the cholesterol synthesis inhibitor squalestatin, less Aβ42 was found within rafts, greater amounts of Aβ42 were found in lysosomes, and the half-life of Aβ42 was reduced to less than 24 hours. Treatment with phospholipase A2 inhibitors or platelet-activating factor (PAF) antagonists had the same effects on Aβ42 metabolism in neurons as squalestatin. PAF receptors were concentrated in the endoplasmic reticulum (ER) along with enzymes that constitute the cholesterol ester cycle. The addition of PAF to ER membranes triggered activation of cholesterol ester hydrolases and the release of cholesterol from stores of cholesterol esters. An inhibitor of cholesterol ester hydrolases (diethylumbelliferyl phosphate) also increased the degradation of Aβ42 in neurons.ConclusionsWe conclude that the targeting of Aβ42 to rafts in normal cells is a factor that affects its degradation. Critically, pharmacological manipulation of neurons can significantly increase Aβ42 degradation. These results are consistent with the hypothesis that the Aβ-induced production of PAF controls a cholesterol-sensitive pathway that affects the cellular localization and hence the fate of Aβ42 in neurons.

Oxidative Stress and FGF-1 Release from Astrocytes

The brain is an organ to consume highly oxygen and to produce Reactive Oxygen Species (ROS) at high level. ROS facilitate to produce many kinds of peroxide lipids in the lipid-rich brain. Accordingly, it is considered that the brain is an organ to undergo easily oxidative stress. Severe diseases of brain such as Parkinsonism and Alzheimer’s disease are seemingly accompanied with oxidative stress. The construction of protection system against oxidative stress is very important to maintain normal neuron function in the brain. We are studying the production and function of Fibroblast Growth Factor 1 (FGF-1) in astrocytes undergoing oxidative stress. Astrocytes increase FGF-1 release under stressful condition such as oxidative stress and long-term cultured stress. In vitro treatment of rat astrocytes with 100 μM Hydrogen Peroxide (H2O2) for 10 min enhances the release of FGF-1 along with cytosolic proteins such as HSP90, HSP70, PK-Cδ without inducing apoptosis. The treatment with H2O2 enhanced the flow into the cells of exogenous compounds such as trypsin and Rhodamin-phalloidin also, suggesting increase in permeability of plasma membrane in astrocytes treated with H2O2. Oxidative stress suppressed transiently cholesterol synthesis and enhanced cholesterol release from the cell surface, resulted in impairment of the cholesterol metabolism. The change of cholesterol metabolism may participate in the increase in membrane permeability in astrocytes undergone oxidative stress. FGF-1 released from rat astrocytes enhances apoE-containing HDL-like lipoproteins (apoE/HDL) generation of astrocytes in the manner of autocrine action. In this review, we describe physiological significance of FGF-1 released from astrocytes stimulated by oxidative stress to relate with generation of apoE/HDL of astrocytes in the brain.

Curcumin promotes exosomes/microvesicles secretion that attenuates lysosomal cholesterol traffic impairment

Exosomes/microvesicles are originated from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of curcumin, a polyphenol, on exosomes/microvesicles secretion in different cells lines, using U18666A as a model of intracellular cholesterol trafficking impairment. In both HepG2 hepatocarcinoma cells and THP-1 differentiated macrophages, treatment with curcumin affected the size and the localization of endosome/lysosomes accumulated by U18666A, and reduced the cholesterol cell content. To ascertain the mechanism, we analyzed the incubation medium. Curcumin stimulated the release of cholesterol and the lysosomal β-hexosaminidase enzyme, as well as the exosome markers, flotillin-2 and CD63. Electron microscopy studies demonstrated the presence of small vesicles similar to exosomes/microvesicles in the secretion fluid. These vesicles harbored CD63 on their surface, indicative of their endolysosomal origin. These effects of curcumin were particularly intense in cells treated with U18666A. These findings indicate that curcumin ameliorates the U18666A-induced endolysosomal cholesterol accumulation by shuttling cholesterol and presumably other lipids out of the cell via exosomes/microvesicles secretion. This action may contribute to the potential of curcumin in the treatment of lysosomal storage diseases.

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of 'toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

Abstract 404: Altered Interaction of Modified High Density Lipoprotein with Scavenger Receptor BI

High density lipoprotein (HDL) combats atherosclerosis, largely through its role in the reverse cholesterol transport (RCT) pathway where excess cholesterol from peripheral tissues is transported by HDL to the liver for excretion. High HDL-cholesterol (HDL-C) levels have been traditionally linked to a lower risk for cardiovascular disease (CVD). However, recent evidence suggests that HDL “function”, rather than HDL levels, is a better indicator of CVD risk as modifications to HDL under oxidative stress can render the particles “dysfunctional”. Scavenger receptor BI (SR-BI), the HDL receptor, mediates the selective uptake of HDL-cholesteryl ester (CE) into the liver during RCT. We hypothesized that SR-BI would be unable to mediate its cholesterol transport functions in the presence of oxidized or modified HDL due to an inability to engage in productive binding interactions with modified ligands. To test this hypothesis, we assessed HDL binding and selective uptake of HDL-CE in COS7 cells transiently expressing SR-BI using native HDL or HDL modified with: 1) copper (Cu2+), 2) 4-hydroxynonenal (HNE), or 3) acrolein. Our data revealed that, compared to native HDL, SR-BI bound 20-50% less Cu2+-HDL and acrolein-HDL, and mediated 40%-60% less selective uptake of CE from these modified particles, respectively. On the other hand, while SR-BI was able to bind HNE-HDL, it could not efficiently mediate cholesterol uptake (20% less compared to native HDL). Interestingly, our data also revealed that the ability of SR-BI to mediate the release of free cholesterol from COS7 cells did not differ when modified HDL served as acceptor particles, as compared to native HDL. Taken together, only the HDL binding and HDL-CE selective uptake functions of SR-BI are influenced by the type of modification on the HDL particle. These data have significant implications as they suggest that higher levels of plasma HDL-C may, in part, be the result of the inability of SR-BI to recognize and mediate cholesterol removal from HDL particles that have been exposed to oxidative stress. More detailed investigations of the interactions between SR-BI and various populations of oxidized HDL will improve our understanding of the mechanisms that render HDL dysfunctional, and ultimately, atherogenic.

P73: The possible role of nitrogen dioxide produced in the field of bifurcation of vessels, in the processes of their damage in hemorrhagic strokes, and the formation of atherosclerotic plaques

According to modern views the formation of atherosclerotic plaques is associated with accumulation of cholesterol in the vascular wall. This is due to an imbalance between the intake of cholesterol in the intima of vessels, together with the low-density lipoproteins (LDL) and its output with high-density lipoprotein (HDL). Change of LDL (glycosylation, lipid peroxidation, hydrolysis of phospholipids) and the effective release of cholesterol from the endothelium of the vascular wall are the factors that cause an imbalance in cholesterol metabolism. In this paper we propose a new concept of the mechanism of initial formation of atherosclerotic plaques, which can complement the existing concepts. According to this concept an important role in the early stages of atherosclerosis are highly reactive molecules of nitrogen dioxide (NO2), resulting from the violation of the cycles of nitric oxide and superoxide anion radical [1] , [2] , [3] , [4] . Hypothesized that the mechanism of antiradical protection of cells and the organism as a whole, above all, laid out in most of the cyclic organization of metabolic processes that involve the formation of free radicals. Violation of this cyclic mechanism may be one of the causes of many diseases associated with hypoxia/ischemia and inflammation. The review considers the hypothesis of the possibility of participation of NO2 and OH-radicals formed in violation of the cycles of NO and superoxide, in the mechanisms of vascular damage with hemorrhagic stroke and in the formation of atherosclerotic plaques. Disclosure Supported by a research grant from RFFI.

Cholesterol pericarditis associated with rheumatoid arthritis: A rare cause of pericardial effusion

Cholesterol pericarditis is an uncommon form of pericardial disease, of unknown pathophysiology, that is characterized by chronic relapsing, usually large, pericardial effusions that are distinctive due to high levels of cholesterol in the pericardial fluid, with or without the formation of crystals [ 1 Alexander S.J. A pericardial effusion of “gold paint” appearance due to presence of cholesterin. Br Med J. 1919; 2: 463 Crossref PubMed Scopus (27) Google Scholar , 2 Brawley R.K. Vasko J.S. Monrow A.G. Cholesterol pericarditis: consideration of its pathogenesis and treatment. Am J Cardiol. 1966; 41: 235-248 Scopus (33) Google Scholar ]. It is usually idiopathic, but can be associated with various systemic diseases such as hypothyroidism, rheumatoid arthritis and tuberculosis. In the case of rheumatoid arthritis (RA), release of cholesterol from pericardial cell membranes or from necrotic rheumatoid nodules, lysis of red blood cells, lymphatic obstruction and reduced pericardial absorption capacity have all been suggested as possible mechanisms [ 3 Knobel B. Rosman P. Cholesterol pericarditis associated with rheumatoid arthritis. Harefuah. 2001 Jan; 140 (87): 10-12 PubMed Google Scholar , 4 Fernandes F. Vieira G.S. Arteaga E. Ianni B.M. Pego-Fernandes P. Mady C. Cholesterol pericarditis. A specific but rare cause of pericardial disease. Arq Bras Cardiol. May 2001; 76: 391-394 Crossref PubMed Scopus (15) Google Scholar , 5 Stanley R.J. Subramanian R. Lie J.T. Cholesterol pericarditis terminating as constrictive calcific pericarditis. Am J Cardiol. 1980; 46: 511-514 Abstract Full Text PDF PubMed Scopus (14) Google Scholar , 6 Ford E.J. Bear P.A. Adams R.W. Cholesterol pericarditis causing cardiac tamponade. Am Heart J. 1991; 122: 877-879 Abstract Full Text PDF PubMed Scopus (8) Google Scholar ]. Although its clinical course is usually indolent and complications such as cardiac tamponade and chronic constrictive pericarditis are relatively rare, the need for surgery for complete treatment has been reported in at least 10% of cases [ [7] Raposo Luís et al. Cholesterol pericarditis — relapsing pericardial effusion in a patient with rheumatoid arthritis. Rev Port Cardiol. 2005; 24: 737-745 PubMed Google Scholar ].