Nigella and Milk Thistle Seed Oils: Potential Cytoprotective Effects against 7ß-Hydroxycholesterol-Induced Toxicity on SH-SY5Y Cells.

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7ß-hydroxycholesterol (7ß-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7ß-OHC-induced side effects. The impact of 7ß-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7ß-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7ß-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7ß-OHC.

27-Hydroxycholesterol contributes to cognitive deficits in APP/PS1 transgenic mice through microbiota dysbiosis and intestinal barrier dysfunction.

BACKGROUND: Research on the brain-gut-microbiota axis has led to accumulating interest in gut microbiota dysbiosis and intestinal barrier dysfunction in Alzheimer's disease (AD). Our previous studies have demonstrated neurotoxic effects of 27-hydroxycholesterol (27-OHC) in in vitro and in vivo models. Here, alterations in the gut microbiota and intestinal barrier functions were investigated as the possible causes of cognitive deficits induced by 27-OHC treatment. METHODS: Male APP/PS1 transgenic and C57BL/6J mice were treated for 3 weeks with 27-OHC (5.5 mg/kg/day, subcutaneous injection) and either a 27-OHC synthetase inhibitor (anastrozole, ANS) or saline. The Morris water maze and passive avoidance test were used to assess cognitive impairment. Injuries of the intestine were evaluated by histopathological examination. Intestinal barrier function was assessed by plasma diamine oxidase (DAO) activity and D-lactate. Systemic and intestinal inflammation were evaluated by IL-1ß, TNF-α, IL-10, and IL-17 concentrations as determined by ELISA. The fecal microbiome and short-chain fatty acids (SCFAs) were analyzed using 16S rDNA sequencing and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Tight junction proteins were evaluated in the ileum and colon by qRT-PCR and Western blots. Tight junction ultrastructure was examined by transmission electron microscopy. RESULTS: Treatment with 27-OHC resulted in severe pathologies in the ileum and colon. There was impaired intestinal barrier integrity as indicated by dilated tight junctions and downregulation of tight junction proteins, including occludin, claudin 1, claudin 5, and ZO-1, and signs of inflammation (increased IL-1ß, TNF-α, and IL-17). Fecal 16S rDNA sequencing and taxonomic analysis further revealed a decreased abundance of Roseburia and reduced fecal levels of several SCFAs in 27-OHC-treated mice. Meanwhile, co-treatment with ANS reduced intestinal inflammation and partially preserved intestinal barrier integrity in the presence of 27-OHC. CONCLUSIONS: The current study demonstrates for the first time that 27-OHC treatment aggravates AD-associated pathophysiological alterations, specifically gut microbiota dysbiosis and intestinal barrier dysfunction, which suggests that the gut microbiome and intestinal barrier function warrant further investigation as potential targets to mitigate the neurotoxic impact of 27-OHC on cognitive function and the development of AD.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

25-hydroxycholesterol impairs neuronal and muscular development in zebrafish.

Oxysterols have essential effects on brain homeostasis and their levels are often altered in neurodegenerative and neuroinflammatory diseases. Several studies have demonstrated the cytotoxic effects of 25-HC on different cell lines, however, not much is known about its effects on neurons in vivo. In this study, we examined the effects of 25-HC exposure on the nervous system development in the zebrafish. We showed that survival rate of zebrafish embryos/larvae is significantly decreased at doses of 25-HC above 40 µM. 25-HC was found to affect the motility of zebrafish larvae, primary motor axon and muscle morphology. Furthermore, larvae treated with 25-HC showed a reduced neuronal network and number of HuC-positive cells in the brain. An increased cell death was also observed in both the brain and spinal cord of zebrafish treated with 25-HC. Interestingly, administration of 25-HC at later stages of development (24 and 48 h post fertilization) had no detrimental effects on motor axons. Altogether, our findings show that elevated levels of 25-HC may have important consequences on neuronal development and cell survival.

An in vitro developmental neurotoxicity screening assay for retinoic acid-induced neuronal differentiation using the human NT2/D1 cell line.

Traditional approaches (e.g., neurobehavior, neuropathology) can detect alterations in apical endpoints indicative of developmental neurotoxicity (DNT). However, there is an increasing desire to understand mode-of-action (MOA) for DNT effects; thus, this short communication describes initial work on a neuronal differentiation assay. Basically, our laboratory used the human NT2/D1 cell line to develop an assay to evaluate toxicants for effects on all-trans retinoic acid (RA)-induced neuronal differentiation. Based on literature reports, we selected a neuronal protein, neuronal class III ß-tubulin (ß3-tubulin), as a marker of differentiation. For this assay, cultured RA-treated NT2 cells were trypsinized to individual cells, methanol fixed, and labeled with a ß3-tubulin specific monoclonal antibody (TUJ1). Characterization studies using 100,000 cells/sample showed that NT2 cells had appreciable expression of ß3-tubulin starting around day 7 of the differentiation process with a peak expression noted around day 12. Methylmercury, 22(R)-hydroxycholesterol, N-(4-hydroxyphenol)retinamide (4HPR), and 9-cis retinoic acid were selected as initial test compounds. Of these, only 9-cis RA, which is known to affect the RA pathway, was positive for specific impacts on differentiation. These results demonstrate the feasibility of using a flow cytometry method targeting specific cellular biomarkers for evaluating effects on neuronal differentiation. Additional assays are needed to detect compounds targeting other (non-RA) neuronal differentiation pathways. Ultimately, a battery of in vitro assays would be needed to evaluate the potential MOAs involved in altered neuronal differentiation.

The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-hydroxycholesterol-induced cellular senescence in nerve cells.

The oxysterol 27-hydroxycholesterol (27HC) is a selective estrogen receptor modulator (SERMs), which like endogenous estrogen 17ß-estradiol (E2) induces the proliferation of ER-positive breast cancer cells in vitro. Interestingly, the observation that 27HC induces adverse effects in neural system, distinguishing it from E2. It has been suggested that high levels of circulating cholesterol increase the entry of 27HC into the brain, which may induce learning and memory impairment. Based on this evidence, 27HC may be associated with neurodegenerative processes and interrupted cholesterol homeostasis in the brain. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that 27HC induced apparent cellular senescence in nerve cells. Senescence-associated ß-galactosidase (SA-ß-Gal) assay revealed that 27HC induced senescence in both BV2 cells and PC12 cells. Furthermore, we demonstrated that 27HC promoted the accumulation of cellular reactive oxygen species (ROS) in nerve cells and subsequently activation of IL-6/STAT3 signaling pathway. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly blocked 27HC-induced ROS production and activation of IL-6/STAT3 signaling pathway. Either blocking the generation of ROS or inhibition of IL-6/STAT3 both attenuated 27HC-induced cellular senescence. In sum, these findings not only suggested a mechanism whereby 27HC induced cellular senescence in nerve cells, but also helped to recognize the 27HC as a novel harmful factor in neurodegenerative diseases.

27-Hydroxycholesterol induces invasion and migration of breast cancer cells by increasing MMP9 and generating EMT through activation of STAT-3.

Breast carcinoma plays a vital role in the reasons of global women's death. ER-related invasion and migration play an important part in the development and prognosis of breast cancer. Here, we found that 27-Hydroxycholesterol (27HC) could induce epithelial-mesenchymal transition (EMT) and increase the expression of the matrix metalloproteinase 9 (MMP9) at mRNA level and the active form. Meanwhile, interestingly, we found 27HC activated signal transducer and activator of transcription 3 (STAT-3) in ER positive cells except activation of ER signaling. Furthermore, inhibition of STAT-3 by siRNA attenuated the 27HC-induced improvement of MMP9 and decreased the invasion and migration ability in MCF7 and T47D cells. In addition, 27HC could also promote MMP9, vimentin and active STAT-3 in the ER negative cells MDA-MB-231. All these results not only raise a mechanism whereby 27HC enhances the invasion and metastasis, but also is helpful to realize 27HC as a potential endogenous detrimental factor in breast tumor patients.

α-Asarone blocks 7ß-hydroxycholesterol-exposed macrophage injury through blocking elF2α phosphorylation and prompting beclin-1-dependent autophagy.

Macrophage apoptosis is salient in advanced atherosclerotic lesions and is induced by several stimuli including endoplasmic reticulum (ER) stress. This study examined that α-asarone present in purple perilla abrogated macrophage injury caused by oxysterols via ER stress- and autophagy-mediated mechanisms. Nontoxic α-asarone at 1-20 µM attenuated 7ß-hydroxycholesterol-induced activation of eukaryotic initiation factor 2α in macrophages leading to C/EBP homologous protein (CHOP) expression and apoptosis due to sustained ER stress. The α-asarone treatment increased the formation of autophagolysosomes localizing in perinuclear regions of 7ß-hydroxycholesterol-exposed macrophages. Consistently, this compound promoted the induction of the key autophagic proteins of beclin-1, vacuolar protein sorting 34 and p150 responsible for vesicle nucleation, and prompted the conversion of microtubule-associated protein 1A/1B-light chain 3 and the induction of p62, neighbor of BRCA1 and autophagy-related (Atg) 12-Atg5-Atg16L conjugate involved in phagophore expansion and autophagosome formation. Additionally, α-asarone increased ER phosphorylation of bcl-2 facilitating beclin-1 entry to autophagic process. Furthermore, the deletion of Atg5 or beclin-1 gene enhanced apoptotic CHOP induction. Collectively, α-asarone-stimulated autophagy may be potential multi-targeted therapeutic avenues in treating ER stress-associated macrophage apoptosis.

The ROS-mediated activation of STAT-3/VEGF signaling is involved in the 27-hydroxycholesterol-induced angiogenesis in human breast cancer cells.

Breast cancer (BC) is the leading cause of cancer-related mortality among females worldwide, and angiogenesis plays a crucial role in BC progression. 27-Hydroxycholesterol (27HC) is an endogenous selective estrogen receptor modulator, which promotes the growth and metastasis of BC. Here, we further found that, 27HC improved the angiogenic ability of BC in a VEGF-dependent manner. For the molecular mechanisms, on one hand, as an estrogen-like factor, 27HC enhanced the expression of VEGF by the classical ERα/VEGF signaling in ER-positive BC cells; on the other hand, in both ER-positive and ER-negative BC cells, 27HC enhanced the generation of ROS, which in turn activated the STAT-3/VEGF signaling in an ER independent manner. Either blocking the generation of ROS or knockdown of STAT-3 attenuated the 27HC-induced autocrine of VEGF and angiogenesis. These findings not only suggested a mechanism whereby 27HC enhanced the angiogenesis, but also helped to recognize the 27HC as a novel potential harmful factor in BC, especially in the menopause patients.

Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol.

Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.

Diclofenac inhibits 27-hydroxycholesterol-induced inflammation.

27-Hydroxycholesterol (27OHChol) is a cholesterol oxidation product that induces inflammation. In the current study we investigated the effects of diclofenac on inflammatory responses caused by 27OHChol using human monocyte/macrophage (THP-1) cells. Transcription and secretion of CCL2, CCL3, and CCL4 chemokines enhanced by 27OHChol were significantly attenuated by diclofenac in a concentration dependent manner. Migrations of monocytic cells and CCR5-positive Jurkat T cells were reduced proportionally to the concentrations of diclofenac. Superproduction of CCL2 and monocytic cell migration induced by 27OHChol plus LPS were significantly attenuated by diclofenac. Diclofenac also attenuated transcription of MMP-9 and release of its active gene product. These results indicate that diclofenac inhibits 27OHChol-induced inflammatory responses, thereby suppressing inflammation in a milieu rich in cholesterol oxidation products.

Dietary compound α-asarone alleviates ER stress-mediated apoptosis in 7ß-hydroxycholesterol-challenged macrophages.

SCOPE: Prolonged endoplasmic reticulum (ER) stress has lost the function of protein folding capacity and the ER accumulation of unfolded proteins that eventually triggers apoptosis. Oxysterols are emerging as contributing factors in atherogenesis known to involve macrophage apoptosis. This study determined the inhibitory effect of α-asarone present in purple perilla, on 7ß-hydroxycholesterol-induced macrophage apoptosis, targeting against ER stress signaling pathway. METHODS AND RESULTS: J774A1 murine macrophages were exposed to 28 µM 7ß-hydroxycholesterol and treated with 1-10 µM α-asarone. Macrophage apoptosis and ER stress were examined by and α-Asarone blocked 7ß-hydroxycholesterol-induced DNA fragmentation and apoptosome formation. Immunoblotting showed that the oxysterol activated the ER transmembrane resident kinases of IRE1α, PERK and ATF4 and triggered caspase-12 signaling cascades, which was reversed by α-asarone. Additionally, 7ß-hydroxycholesterol activated TRAF2-ASK1-JNK1/2 complex following the IRE1α activation, and α-asarone blunted such IRE1α-mediated pathway. Real-time PCR and dual-luciferase reporter analyses revealed that α-asarone reduced transcriptional activation of ER stress-responsive genes including XBP1 and CHOP by 7ß-hydroxycholesterol. Finally, α-asarone disturbed oxysterol-elicited signaling of PERK and ATF4 responsible for CHOP induction. CONCLUSION: α-Asarone blocked 7ß-hydroxycholesterol-induced macrophage apoptosis through allaying ER stress-specific signaling involving caspase activation and CHOP induction. α-Asarone was an anti-atherosclerotic agent antagonizing ER stress-mediated macrophage apoptosis by 7ß-hydroxycholesterol.

27-Hydroxycholesterol contributes to disruptive effects on learning and memory by modulating cholesterol metabolism in the rat brain.

Cholesterol metabolism is important for neuronal function in the central nervous system (CNS). The oxysterol 27-hydroxycholesterol (27-OHC) is a cholesterol metabolite that crosses the blood-brain barrier (BBB) and may be a useful substitutive marker for neurodegenerative diseases. However, the effects of 27-OHC on learning and memory and the underlying mechanisms are unclear. To determine this mechanism, we investigated learning and memory and cholesterol metabolism in rat brain following the injection of various doses of 27-OHC into the caudal vein. We found that 27-OHC increased cholesterol levels and upregulated the expression of liver X receptor-α (LXR-α) and adenosine triphosphate (ATP)-binding cassette transporter protein family member A1 (ABCA1). In addition, 27-OHC decreased the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR) and low-density lipoprotein receptor (LDLR) in rat brain tissues. These findings suggest that 27-OHC may negatively modulate cognitive effects and cholesterol metabolism in the brain.

Quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside attenuates cholesterol oxidation product-induced apoptosis by suppressing NF-κB-mediated cell death process in differentiated PC12 cells.

Cholesterol oxidation products are suggested to be involved in neuronal cell degeneration. We examined the preventive effect of quercetin-3-O-(2″-galloyl)-α-L-rhamnopyranoside (QGR), a quercetin derivative, on the cholesterol oxidation product-induced neuronal cell death using differentiated PC12 cells in relation to nuclear factor (NF)-κB-mediated apoptotic process. 7-Ketocholesterol and 25-hydroxycholesterol induced a decrease in the levels of BH3 interacting-domain death agonist (Bid) and B cell lymphoma 2 (Bcl-2), increase in the levels of Bcl-2-associated X protein (Bax) and p53, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases, and cleavage of poly(ADP-ribose) polymerase 1 (PARP-1). 7-Ketocholesterol induced increase in cytosolic and nuclear NF-κB p65, nuclear phospho-NF-κB p65, cytosolic NF-κB p50, and cytosolic phospho-IκB-α levels. The addition of QGR, N-acetyl cysteine, or Bay 11-7085 attenuated the cholesterol oxidation product-induced changes in the apoptosis-related protein levels, activation of NF-κB, formation of reactive oxygen species, depletion of glutathione (GSH), nuclear damage, and cell death. The results show that QGR may attenuate the cholesterol oxidation product-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways that is mediated by NF-κB activation. The preventive effect appears to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

The enzyme lecithin-cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH-SY5Y cells.

Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture.

Differential effects of the estrogen receptor agonist estradiol on toxicity induced by enzymatically-derived or autoxidation-derived oxysterols in human ARPE-19 cells.

PURPOSE/AIM OF THE STUDY: Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER. MATERIALS AND METHODS: ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels. RESULTS: Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC. CONCLUSIONS: Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.

Expression of Egr1 and p53 in human carotid plaques and apoptosis induced by 7-oxysterol or p53.

Egr-1 and p53 are involved in pathology of both atherosclerosis and cancer. However, it is unknown whether p53 and Egr1 are interactively involved in apoptosis in atherosclerosis. We found that in human carotid plaques, the expression of p53 was inversely correlated with Egr1. In U937 cells, 7ß-hydroxycholesterol and 7-ketocholesterol induced production of reactive oxygen species (ROS), transient up-regulation of Egr1 followed by late induction of p53 and apoptosis. Cells with nuclear fragmentation induced by 7-oxysterol or p53 showed increased levels of p53, but decreased levels of Egr1. In conclusion, ROS induced by 7-oxysterols may function as an early initiator of Egr1 expression. The late induced p53 by 7-oxysterols contributes to apoptotic cell death and is linked to the reduction of Egr1 levels, which resembles the differential expression of p53 and Egr1 in human atheroma progression.

Biological activities of the LXRα and ß agonist, 4ß-hydroxycholesterol, and of its isomer, 4α-hydroxycholesterol, on oligodendrocytes: effects on cell growth and viability, oxidative and inflammatory status.

The biochemical and biological properties of 4ß-hydroxycholesterol and of its isomer, 4α-hydroxycholesterol, are not well known. So, we determined the ability of 4α- and 4ß-hydroxycholesterol to react with LXRα and LXRß, and we characterized the activities of these oxysterols on oligodendrocytes which are myelin synthesizing cells. The effects of 4α- and 4ß-hydroxycholesterol were studied on 158N murine oligodendrocytes to assess their activities on cell growth and viability, oxidative and inflammatory status. To this end different parameters were used: cell counting with trypan blue; identification of dead cells and cell cycle analysis with propidium iodide; evaluation of mitochondrial depolarization, lysosomal membrane integrity, actin depolimerization, nuclear morphology, and superoxide anion production after staining with JC-1, acridine orange, rhodamine-phalloidin, Hoechst 33342, and dihydroethidium, respectively; evaluation of ultrastructural changes by transmission electron microscopy, and cytokine quantification with a cytometric bead array. Only 4ß-hydroxycholesterol is a LXRα and ß agonist. No cytotoxic effects were found with 4α-hydroxycholesterol except a slight inhibition of cell growth at elevated concentrations. At high concentrations, 4ß-hydroxycholesterol was not only able to inhibit cell growth, but also to induce cell death associated with a loss of mitochondrial transmembrane potential, dysfunctions of lysosomal membrane integrity, and superoxide anion overproduction. These side effects were lower than those observed with 7-ketocholesterol and 25-hydroxycholesterol used as positive controls. On oligodendrocyte murine primary cultures, only lysosomal membrane integrity was slightly affected under treatment with 4α- and 4ß-hydroxycholesterol. So, 4α- and 4ß-hydroxycholesterol have different biological activities. Their ability to induce cytotoxic effects on oligodendrocytes can be considered as weak comparatively to 7-ketocholesterol and 25-hydroxycholesterol.

7ß-Hydroxycholesterol-induced energy stress leads to sequential opposing signaling responses and to death of C6 glioblastoma cells.

7ß-Hydroxycholesterol cytotoxicity has been shown in vivo and in vitro to be dependent on the accumulation of its esters. We show in our study, using a detergent-free raft preparation and LC/MS lipid content analysis, that membrane microdomains isolated from 7ß-hydroxycholesterol-treated C6 cells have a reduced cholesterol: cholesterol ester ratio and accumulate 7keto-hydroxycholesterol, 7ß-hydroxycholesterol and 7ß-hydroxycholesterol esters. These modifications in lipid content are accompanied by a redistribution of flotillin-1 in the lipid rafts. Transient increases of AMPK phosphorylation and mitochondrial activity during the first 12 h of 7ß-hydroxycholesterol treatment indicate that C6 cells undergo energy stress and increase oxidative phosphorylation. Even so, ATP levels are maintained during 15 h until glucose uptake decreases. The cell's answers to raft modifications and energy stress are sequential activations of different signaling pathways such as ERK, AMPK and PI3K/Akt. These pathways, known to be activated under energy stress conditions, are transiently activated at 6 h (ERK, AMPK) and 12 h (Akt) of treatment respectively suggesting a shift from cell survival to cell proliferation. The persistence of 7ß-hydroxycholesterol-induced stress led after 24 h to P38 activation, loss of GSK3ß activation and to cell death. Finally we demonstrate that the observed signaling responses depend on 7ß-hydroxycholesterol esterification, confirming that esterification of 7ß-hydroxycholesterol is essential for cytotoxicity.

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols.

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7ß-hydroxycholesterol (7ßOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24h, which was preceded by early lipid accumulation (6h) and lysosomal membrane permeabilization (6-12h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7ßOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7ßOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.