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Auto-oxidized oxysterols are implicated in the pathogenesis of various chronic diseases. Their concentrations are indicators of oxidative stress in vivo and associated with atherosclerosis. Subclinical hypothyroidism is related with cardiac diseases and oxidative stress, but the exact mechanisms underlying these associations are not clear yet.To investigate the auto-oxidized oxysterols, 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (chol-triol), in patients with subclinical hypothyroidism, as well as to evaluate the impact of restoring euthyroidism on oxysterol concentrations.In this prospective observational study, 64 patients with newly diagnosed autoimmune thyroiditis (41 with subclinical hypothyroidism and 23 euthyroidism), and 45 healthy controls were enrolled. Age, gender, and body mass index were matched among patient groups and healthy controls. Anthropometric measurements were obtained and fasting plasma 7-ketocholesterol and cholestane-3β,5α,6β-triol concentrations were measured by using liquid chromatography coupled with tandem mass spectrometry. Levothyroxine was then administered to all patients with subclinical-hypothyroidism. After three months, measurements of the oxysterols and serum cholesterols from the patients who have become euthyroid were repeated.Concentrations of 7-ketocholesterol and cholestane-3β,5α,6β-triol were significantly higher in patients with subclinical-hypothyroidism when compared to both euthyroid patients and healthy controls (p 0.001 for both oxysterols). After restoration of euthyroidism, concentrations of 7-ketocholesterol and cholestane-3β,5α,6β-triol decreased significantly and reached similar concentrations observed in healthy controls (p 0.001 for both oxysterols).Auto-oxidized oxysterol species are higher in patients with mild thyroid dysfunction, and supported the rationale for treating subclinical-hypothyroidism.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1β IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Background: Abnormal cholesterol metabolism changes the neuronal membrane and may promote amyloidogenesis. Oxysterols in cerebrospinal fluid (CSF) are related to Alzheimer's disease (AD) biomarkers in mild cognitive impairment and dementia. Cholesterol turnover is important for axonal and white matter (WM) microstructure maintenance. Objective: We aim to demonstrate that the association of oxysterols, AD biomarkers, and WM microstructure occurs early in asymptomatic individuals. Methods: We studied the association of inter-individual variability of CSF 24-hydroxycholesterol (24-OHC), 27-hydroxycholesterol (27-OHC), 7-ketocholesterol (7-KC), 7 beta-hydroxycholesterol (7 beta-OHC), amyloid-beta(42) (A beta(42)), total-tau (t-tau), phosphorylated-tau (p-tau), neurofilament (NfL), and WM microstructure using diffusion tensor imaging, generalized linear models and moderation/mediation analyses in 153 healthy adults. Results: Higher 7-KC levels were related to lower A beta(42), indicative of greater AD pathology (p = 0.041). Higher 7-KC levels were related to lower fractional anisotropy (FA) and higher mean (MD), axial (AxD), and radial (RD) diffusivity. 7-KC modulated the association between AxD and NfL in the corpus callosum splenium (B = 39.39, p = 0.017), genu (B = 68.64, p = 0.000), and fornix (B = 10.97, p = 0.000). Lower A beta(42) levels were associated to lower FA and higher MD, AxD, and RD in the fornix, corpus callosum, inferior longitudinal fasciculus, and hippocampus. The association between AxD and A beta(42) was moderated by 7K-C (p = 0.048). Conclusion: This study adds clinical evidence to support the role of 7K-C on axonal integrity and the involvement of cholesterol metabolism in the A beta(42) generation process.

Cholestane-3β,5α,6β-triol (3β,5α,6β-triol) is formed from cholestan-5,6-epoxide (5,6-EC) in a reaction catalysed by cholesterol epoxide hydrolase, following formation of 5,6-EC through free radical oxidation of cholesterol. 7-Oxocholesterol (7-OC) and 7β-hydroxycholesterol (7β-HC) can also be formed by free radical oxidation of cholesterol. Here we investigate how 3β,5α,6β-triol, 7-OC and 7β-HC are metabolised to bile acids. We show, by monitoring oxysterol metabolites in plasma samples rich in 3β,5α,6β-triol, 7-OC and 7β-HC, that these three oxysterols fall into novel branches of the acidic pathway of bile acid biosynthesis becoming (25R)26-hydroxylated then carboxylated, 24-hydroxylated and side-chain shortened to give the final products 3β,5α,6β-trihydroxycholanoic, 3β-hydroxy-7-oxochol-5-enoic and 3β,7β-dihydroxychol-5-enoic acids, respectively. The intermediates in these pathways may be causative of some phenotypical features of, and/or have diagnostic value for, the lysosomal storage diseases, Niemann Pick types C and B and lysosomal acid lipase deficiency. Free radical derived oxysterols are metabolised in human to unusual bile acids via novel branches of the acidic pathway, intermediates in these pathways are observed in plasma., Graphical abstract Image 1, Highlights • Free radical derived oxysterols metabolised to unusual bile acids. • Metabolism proceeds via novel branches of the acidic pathway. • Bile acids with:- 3β,5α,6β-triol; 3β,7β-diol-5-ene or 3β-ol-5-en-7-one in A/B-ring. • Unusual bile acids found in plasma from patients with NPC, NPB and LALD. • Intermediates in the biosynthesis of unusual bile acids found in plasma.

Growth differentiation factor (GDF)11 has been reported to reverse age-related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The present study attempted to test a hypothesis that GDF11 counteracts the pathologic dedifferentiation of mouse carotid arterial smooth muscle cells (CASMCs) due to deficient autophagy. By real-time RT-PCR and Western blot analysis, exogenously administrated GDF11 was found to promote CASMC differentiation with increased expression of various differentiation markers (α-smooth muscle actin, myogenin, myogenic differentiation, and myosin heavy chain) as well as decreased expression of dedifferentiation markers (vimentin and proliferating cell nuclear antigen). Upregulation of the GDF11 gene by trichostatin A (TSA) or CRISPR-cas9 activating plasmids also stimulated the differentiation of CASMCs. Either GDF11 or TSA treatment blocked 7-ketocholesterol-induced CASMC dedifferentiation and autophagosome accumulation as well as lysosome inhibitor bafilomycin-induced dedifferentiation and autophagosome accumulation. Moreover, in CASMCs from mice lacking the CD38 gene, an autophagy deficiency model in CASMCs, GDF11 also inhibited its phenotypic transition to dedifferentiation status. Correspondingly, TSA treatment was shown to decrease GDF11 expression and reverse CASMC dedifferentiation in the partial ligated carotid artery of mice. The inhibitory effects of TSA on dedifferentiation of CASMCs were accompanied by reduced autophagosome accumulation in the arterial wall, which was accompanied by attenuated neointima formation in partial ligated carotid arteries. We concluded that GDF11 promotes CASMC differentiation and prevents the phenotypic transition of these cells induced by autophagosome accumulation during different pathological stimulations, such as Western diet, lysosome function deficiency, and inflammation.NEW & NOTEWORTHY The present study demonstrates that growth differentiation factor (GDF)11 promotes autophagy and subsequent differentiation in carotid arterial smooth muscle cells. Upregulation of GDF11 counteracts dedifferentiation under different pathological conditions. These findings provide novel insights into the regulatory role of GDF11 in the counteracting of sclerotic arterial diseases and also suggest that activation or induction of GDF11 may be a new therapeutic strategy for the treatment or prevention of these diseases.

Age-related diseases for which there are no effective treatments include cardiovascular diseases; neurodegenerative diseases such as Alzheimer's disease; eye disorders such as cataract and age-related macular degeneration; and, more recently, Severe Acute Respiratory Syndrome (SARS-CoV-2). These diseases are associated with plasma and/or tissue increases in cholesterol derivatives mainly formed by auto-oxidation: 7-ketocholesterol, also known as 7-oxo-cholesterol, and 7β-hydroxycholesterol. The formation of these oxysterols can be considered as a consequence of mitochondrial and peroxisomal dysfunction, leading to increased in oxidative stress, which is accentuated with age. 7-ketocholesterol and 7β-hydroxycholesterol cause a specific form of cytotoxic activity defined as oxiapoptophagy, including oxidative stress and induction of death by apoptosis associated with autophagic criteria. Oxiaptophagy is associated with organelle dysfunction and in particular with mitochondrial and peroxisomal alterations involved in the induction of cell death and in the rupture of redox balance. As the criteria characterizing 7-ketocholesterol- and 7β-hydroxycholesterol-induced cytotoxicity are often simultaneously observed in major age-related diseases (cardiovascular diseases, age-related macular degeneration, Alzheimer's disease) the involvement of these oxysterols in the pathophysiology of the latter seems increasingly likely. It is therefore important to better understand the signalling pathways associated with the toxicity of 7-ketocholesterol and 7β-hydroxycholesterol in order to identify pharmacological targets, nutrients and synthetic molecules attenuating or inhibiting the cytotoxic activities of these oxysterols. Numerous natural cytoprotective compounds have been identified: vitamins, fatty acids, polyphenols, terpenes, vegetal pigments, antioxidants, mixtures of compounds (oils, plant extracts) and bacterial enzymes. However, few synthetic molecules are able to prevent 7-ketocholesterol- and/or 7β-hydroxycholesterol-induced cytotoxicity: dimethyl fumarate, monomethyl fumarate, the tyrosine kinase inhibitor AG126, memantine, simvastatine, Trolox, dimethylsufoxide, mangafodipir and mitochondrial permeability transition pore (MPTP) inhibitors. The effectiveness of these compounds, several of which are already in use in humans, makes it possible to consider using them for the treatment of certain age-related diseases associated with increased plasma and/or tissue levels of 7-ketocholesterol and/or 7β-hydroxycholesterol.

Oxidized cholesterols and lipids accumulate in Bruch’s membrane in age-related macular degeneration (AMD). It remains unknown what causal relationship exists between these substances and AMD pathophysiology. We addressed the hypothesis that a prevalent form, 7-ketocholesterol (7KC), promotes choroidal endothelial cell (CEC) migration and macular neovascularization in AMD. Compared to control, 7KC injection caused 40% larger lectin-stained lesions, but 70% larger lesions measured by optical coherence tomography one week after laser-injury. At two weeks, 7KC-injected eyes had 86% larger alpha smooth muscle actin (αSMA)-labeled lesions and more collagen-labeling than control. There was no difference in cell death. 7KC-treated RPE/choroids had increased αSMA but decreased VE-cadherin. Compared to control-treated CECs, 7KC unexpectedly reduced endothelial VE-cadherin, CD31 and VEGFR2 and increased αSMA, fibroblast activation protein (FAP) and transforming growth factor beta (TGFβ). Inhibition of TGFβ receptor-mediated signaling by SB431542 abrogated 7KC-induced loss of endothelial and increase in mesenchymal proteins in association with decreased transcription factor, SMAD3. Knockdown of SMAD3 partially inhibited 7KC-mediated loss of endothelial proteins and increase in αSMA and FAP. Compared to control, 7KC-treatment of CECs increased Rac1GTP and migration, and both were inhibited by the Rac1 inhibitor; however, CECs treated with 7KC for 72 hours had reduced tube formation. These findings suggest that 7KC, which increases in AMD and with age, induces mesenchymal transition in CECs making them invasive and migratory, and causing fibrosis in macular neovascularization. Further studies to interfere with this process may reduce fibrosis and improve responses to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

Approximately two-thirds of US infants receive infant formula (IF) as a primary or sole nutritional source during the first six months of life. IF is available in a variety of commercial presentations; from a manufacturing standpoint, they can be categorized as powder- (PIF) or liquid- (LIF) based formulations. Thirty commercial IFs were analyzed in their oxidative and non-oxidative lipid profiles. We identified 7-ketocholesterol - a major end-product of cholesterol oxidation - as a potential biomarker of IF manufacturing. The statistical analysis allowed a re-classification of IF based on their metabolomic fingerprint, resulting in three groups assigned with low-to-high oxidative status. Finally, we modeled the dietary intake of cholesterol, sterols, and 7-ketocholesterol in the first year of life. The database provided in this study will be instrumental for scientists interested in infant nutrition, to establish bases for epidemiological studies aimed to find connections between nutrition and diet-associated diseases, such as sitosterolemia.

Background Serum neurofilament light chain (sNfL) is an established marker of neuroaxonal injury in multiple sclerosis (MS). Objectives To investigate if oxysterols produced from non-enzymatic and enzymatic cholesterol oxidation are differentially associated with sNfL measurements in MS. Methods This longitudinal study included 62 relapsing-remitting (RR-MS) and 36 progressive MS (PMS) patients with baseline and 5-year follow-up measures of serum levels of 6 oxysterols, sNfL and lipids. The oxysterols, 24-hydroxycholesterol (24HC), 25HC, 27HC, 7αHC, 7βHC and 7-ketocholesterol (7KC), were measured using liquid chromatography-mass spectrometry. sNfL was measured using single molecular array assay. Serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were obtained from a lipid profile. Results The enzymatically produced oxysterols 24HC, 25HC, 27HC and 7αHC were not associated with sNfL. However, baseline levels of reactive oxygen species (ROS) produced oxysterols, 7KC (p = 0.032) and 7βHC (p = 0.0025), were positively associated with sNfL levels at follow-up. Follow-up 7KC (p = 0.038) levels were also associated with follow-up sNfL levels. The associations of 7KC or 7βHC with sNfL remained significant after adjusting for LDL-C or HDL-C. Conclusions 7KC and 7βHC, produced by ROS-mediated cholesterol oxidation are associated with neuroaxonal injury as assessed by sNfL in MS.

Non-alcoholic fatty liver disease is strongly associated with obese and type 2 diabetes. It has been reported that an oxidized cholesterol, 7-ketocholesterol (7KC), might cause inflammatory response in macrophages and plasma 7KC concentration were higher in patients with cardiovascular diseases or diabetes. Therefore, we have decided to test whether small amount of 7KC in diet might induce hepatic steatosis and inflammation in two types of obese models. We found that addition of 0.01% 7KC either in chow diet (CD, regular chow diet with 1% cholesterol) or western type diet (WD, high fat diet with 1% cholesterol) accelerated hepatic neutral lipid accumulation by Oil Red O staining. Importantly, by lipid extraction analysis, it has been recognized that triglyceride rather than cholesterol species was significantly accumulated in CD+7KC compared to CD as well as in WD+7KC compared to WD. Immunostaining revealed that macrophages infiltration was increased in CD+7KC compared to CD, and also in WD+7KC compared to WD. These phenotypes were accompanied by inducing inflammatory response and downregulating fatty acid oxidation. Furthermore, RNA sequence analysis demonstrated that 7KC reduced expression of genes which related to autophagy process. Levels of LC3-II protein were decreased in WD+7KC compared to WD. Similarly, we have confirmed the effect of 7KC on acceleration of steatohepatitis in db/db mice model. Collectively, our study has demonstrated that small amount of dietary 7KC contributed to accelerate hepatic steatosis and inflammation in obese mice models.

Oxysterols play a critical role in human health and diseases associated with high cholesterol and oxidative stress. Given that a positive correlation was observed between cholesterol and collagen type 1 fragment (CTX-1) or serum reactive oxygen species (ROS) in humans, we hypothesized that oxidized cholesterol metabolites may participate in cholesterol-induced bone loss. Therefore, this study aimed to identify the metabolite responsible for cholesterol-associated bone loss and evaluate its effect on osteoclasts (OCs) leading to bone loss. An atherogenic diet in mice increased the levels of the oxysterol, 7-ketocholesterol (7-KC) in bone, as well as serum ROS. 7-KC increased the number and activity of OCs by enhancing autophagy via the ROS-transcription factor EB signaling pathway. These findings suggest that 7-KC acts as a cholesterol metabolite and is at least partially responsible for cholesterol-induced bone loss by inducing autophagy in OCs.

BackgroundNiemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by mutations of NPC1 or NPC2, which encode the proteins that are responsible for intracellular cholesterol trafficking. Loss of this function results in the accumulation of cholesterol-related products, such as oxysterols, sphingolipids, and NPC-related bile acids, which were recently used as biochemical biomarkers for the diagnosis of NPC. Bile acid-408 is a new significant compound we found in Japanese NPC patients, and it likely belongs to the category of bile acids. However, the diagnosis of NPC using a single biomarker is not satisfactory for clinical application because of the high instance of false negatives or positives. Therefore, we proposed an application of NPC diagnosis using a combination of 7-ketocholesterol (7-KC), lysosphingomyelin (lysoSM), bile acid-408 and/or glucosylsphingosine (lysoGL-1).Methods and findings7-KC, lysoSM and lysoGL-1 in sera and bile acid-408 in dried blood spots (DBS) were quantified within 17 minutes using tandem mass spectrometry and high-resolution mass spectrometry, respectively. We measured these biomarkers in NPC patients (n = 19), X-linked adrenoleukodystrophy (X-ALD) patients (n = 5), patients with other lysosomal diseases (n = 300), newborns (n = 124) and healthy people (n = 74). Our results showed a promising accuracy (97%) for NPC diagnosis using the combination of 7-KC, lysoSM and bile acid-408. However, contrary to our expectations, lysoGL-1 levels did not present at a significantly greater amount in NPC patients than other patients and negative controls.ConclusionsThe combination of 7-KC, lysoSM and bile acid-408 improves the accuracy of NPC diagnosis and is feasible for mass screening due to its simple sample preparation and measurement. Future research should investigate the chemical structure of bile acid-408 to further facilitate its advantage in diagnosis.

The Mediterranean diet is associated with health benefits due to bioactive compounds such as polyphenols. The biological activities of three polyphenols (quercetin (QCT), resveratrol (RSV), apigenin (API)) were evaluated in mouse neuronal N2a cells in the presence of 7-ketocholesterol (7KC), a major cholesterol oxidation product increased in patients with age-related diseases, including neurodegenerative disorders. In N2a cells, 7KC (50 &micro, M, 48 h) induces cytotoxic effects characterized by an induction of cell death. When associated with RSV, QCT and API (3.125, 6.25 &micro, M), 7KC-induced toxicity was reduced. The ability of QCT, RSV and API to prevent 7KC-induced oxidative stress was characterized by a decrease in reactive oxygen species (ROS) production in whole cells and at the mitochondrial level, by an attenuation of the increase in the level and activity of catalase, by attenuating the decrease in the expression, level and activity of glutathione peroxidase 1 (GPx1), by normalizing the expression, level and activity of superoxide dismutases 1 and 2 (SOD1, SOD2), and by reducing the decrease in the expression of nuclear erythroid 2-like factor 2 (Nrf2) which regulates antioxidant genes. QCT, RSV and API also prevented mitochondrial dysfunction in 7KC-treated cells by counteracting the loss of mitochondrial membrane potential (&Psi, &Delta, m) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase &alpha, (AMPK&alpha, ), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor &gamma, coactivator-1&alpha, (PGC-1&alpha, ) implicated in mitochondrial biogenesis. At the peroxisomal level, QCT, RSV and API prevented the impact of 7KC by counteracting the decrease in ATP binding cassette subfamily D member (ABCD)3 (a peroxisomal mass marker) at the protein and mRNA levels, as well as the decreased expresssion of genes associated with peroxisomal biogenesis (Pex13, Pex14) and peroxisomal &beta, oxidation (Abcd1, Acox1, Mfp2, Thiolase A). The 7KC-induced decrease in ABCD1 and multifunctional enzyme type 2 (MFP2), two proteins involved in peroxisomal &beta, oxidation, was also attenuated by RSV, QCT and API. 7KC-induced cell death, which has characteristics of apoptosis (cells with fragmented and/or condensed nuclei, cleaved caspase-3, Poly(ADP-ribose) polymerase (PARP) fragmentation) and autophagy (cells with monodansyl cadaverine positive vacuoles, activation of microtubule associated protein 1 light chain 3&ndash, I (LC3-I) to LC3-II, was also strongly attenuated by RSV, QCT and API. Thus, in N2a cells, 7KC induces a mode of cell death by oxiapoptophagy, including criteria of OXIdative stress, APOPTOsis and autoPHAGY, associated with mitochondrial and peroxisomal dysfunction, which is counteracted by RSV, QCT, and API reinforcing the interest for these polyphenols in prevention of diseases associated with increased 7KC levels.

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 μM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

International audience; 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 7beta-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 7beta-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 7beta-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.

Objective To investigate the link between Human Herpesvirus 8 (HHV8) infection and plasma oxidative stress in patients with diabetes mellitus type 2 (DM2). Results Blood samples collected from DM2 and control subjects were screened for the presence of antibodies against HHV8 and for biomarkers of oxidative stress. We determined the products of radical damage on the plasma lipid fraction, such as malondialdehyde (MDA), fatty acid hydroperoxides (HP) and 7-ketocholesterol (7-keto), the oxidation products of unsaturated fatty acids (UFA) and cholesterol, respectively. The level of plasma antioxidant α-tocopherol (α-toc) was also assessed. Relevant differences were observed in the redox status in DM2 and either HHV8-positive or -negative control subjects. The level of α-toc significantly decreased in both DM2 and HHV8-positive subjects. Levels of MDA, HP and 7-keto were much higher in HHV8-positive and DM2 subjects, indicating that plasma oxidative stress is a common feature in both DM2 and HHV8-infection. In addition, 7-keto was further increased in HHV8-positive DM2 patients. We hypothesized that the HHV8-infection may contribute to the production of ROS, and hence to the oxidative stress closely related to the pathogenesis and development of DM2.

Cholesterol oxidation products, also named oxysterols, can be formed either by cholesterol auto-oxidation, enzymatically or both. Among these oxysterols, 7-ketocholesterol (7KC) is mainly formed during radical attacks that take place on the carbon 7 of cholesterol. As increased levels of 7KC have been found in the tissues, plasma and/or cerebrospinal fluid of patients with major diseases, especially age-related diseases (cardiovascular diseases, eye diseases, neurodegenerative diseases), some cancers, and chronic inflammatory diseases, it is suspected that 7KC, could contribute to their development. Since 7KC, provided by the diet or endogenously formed, is not or little efficiently metabolized, except in hepatic cells, its cellular accumulation can trigger numerous side effects including oxidative stress, inflammation and cell death. To counteract 7KC-induced side effects, it is necessary to characterize the metabolic pathways activated by this oxysterol to identify potential targets for cytoprotection and geroprotection. Currently, several natural compounds (tocopherols, fatty acids, polyphenols, etc) or mixtures of compounds (oils) used in traditional medicine are able to inhibit the deleterious effects of 7KC. The different molecules identified could be valued in different ways (functional foods, recombinant molecules, theranostic) to prevent or treat diseases associated with 7KC.

Mammalian cell entry protein (Mce4A) is a member of MCE-family, and is being considered as a potential drug target of Mycobacterium tuberculosis infection because it is required for invasion and latent survival of pathogen by utilizing host's cholesterol. In the present study, we performed molecular docking followed by 100 ns MD simulation studies to understand the mechanism of interaction of Mce4A to the cholesterol derivatives and probucol. The selected ligands, cholesterol, 25-hydroxycholesterol, 5-cholesten-3β-ol-7-one and probucol bind to the predicted active site cavity of Mce4A, and complexes remain stable during entire simulation of 100 ns. In silico studies were further validated by fluorescence-binding studies to calculate actual binding affinity and number of binding site(s). The non-toxicity of all ligands was confirmed on human monocytic cell (THP1) by MTT assay. This work provides a deeper insight into the mechanism of interaction of Mce4A to cholesterol derivatives, which may be further exploited to design potential and specific inhibitors to ameliorate the Mycobacterium pathogenesis.

To investigate the effects of 7-oxygenated cholesterol molecules on the expression of tight junction proteins, we examined the outcomes effects of 7-ketocholesterol (7K), 7α-hydroxycholesterol (7αOHChol) and 7β-hydroxycholesterol (7βOHChol) on the expression of the tight-junction protein zonula occludens-1 (ZO-1) using vascular cells. Vascular smooth muscle cells (VSMCs) constitutively express ZO-1, and this expression remained unaffected in the presence of cholesterol. However, the level of ZO-1 protein decreased after exposure to 7K and, to a lesser extent, 7αOHChol and 7βOHChol. ZO-1 was translocated to the nucleus following treatment with 7K; this translocation was inhibited by z-VAD-fmk, a pan-caspase inhibitor. ZO-1 protein was found to disintegrate in the aorta of ApoE knockout mice fed a high cholesterol diet, whereas it remained intact in the wild-type control. THP-1 monocyte/macrophage cells, which show no expression of ZO-1, were not influenced by treatment with cholesterol, 7K, and 7βOHChol. However, the treatment of THP-1 cells with 7αOHChol resulted in ZO-1 expression, which largely remained localized on the cytoplasmic membrane. These results indicate the varying effects of 7-oxygenated cholesterol molecules on the expression and localization of ZO-1 depending on cell types, and suggest the contribution of 7-oxygeneted cholesterol molecules to the structural alteration of tight junctions.

Purpose of review To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. Recent findings Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3β,5α,6β-triol (3β,5α,6β-triol), has recently been shown to be metabolized to the corresponding bile acid, 3β,5α,6β-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3β,5α,6β-triol is likely to be 3β-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. Summary Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5β,6β-epoxycholesterol, 7α- and 7β-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.

Graphical abstract, 7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

7-ketocholesterol, a toxic oxidative product of oxysterol is a causative agent of several diseases and disabilities concomitant to aging including cardiovascular diseases like atherosclerosis. Auto-oxidation of cholesterol esters present in low-density lipoprotein (LDL) deposits lead to the formation of oxidized LDL (Ox-LDL) along with its byproducts, namely 7KCh. It is predominantly found in atherosclerotic plaque and also found to be more atherogenic than cholesterol by being cytotoxic, interfering with cellular homeostasis. This makes it a serious threat by being the foremost cause of morbidity and mortality worldwide and is likely to become more serious during forth coming years. It involves in mediating inflammatory mechanisms characterized by the advancement of fibroatheroma plaques. The atherosclerotic lesion is composed of Ox-LDL along with fibrotic mass consisting of immune cells and molecules. Macrophages being the specialized phagocytic cells, contribute to removal of detrimental contents of the lesion along with accumulated lipids leading to alteration of its biology and functionality due to its plasticity. Here, we have explored the known as well as proposed mechanisms involved with 7KCh associated atherogenesis along with potential therapeutic strategies for targeting 7KCh as a diagnostic and target in medicine.

In this research we investigate the connection between the cytoplasmic machinery of apoptosis and the plasma membrane organization by studying the coupling of caspase-3 activation and inhibition with PS exposure and the change of lipid order in plasma membrane sensed by a fluorescent membrane probe NR12S. First, we performed in silico molecular dynamics simulations, which suggest that the mechanism of response of NR12S to lipid order may combine both sensitivity to membrane polarity/hydration and change in the fluorophore orientation. Second, cellular studies revealed that upon triggering apoptosis with IPA-3 and camptothecin the NR12S response is similar to that observed after decrease of lipid order induced by cholesterol depletion, 7-ketocholesterol enrichment or sphingomyelin hydrolysis. NR12S response can be influenced by a caspase-3 inhibitor Z-DEVD-FMK. Flow cytometry data further indicate that the NR12S response correlates with the response of FITC-labeled DEVD-FMK peptide and GFP-labeled Annexin V on the whole time scale (0–24 h) of apoptosis induction by camptothecin. We conclude that fine changes in lipid order observed by NR12S are coupled with early steps of cellular events in apoptosis.

Rationale: 7-Ketocholesterol (7-KC), a form of cholesterol oxidation product, plays an essential role in the atherogenesis in animal models. Objective: We sought to determine the association of circulating 7-KC with clinical cardiovascular outcomes and total mortality in patients with stable coronary artery disease. Methods and Results: We measured the plasma 7-KC levels by high-performance liquid chromatography in a prospective cohort study of 1016 patients (mean age, 63.2 years; male 61.1%) with stable coronary artery disease who were recruited from December 2008 to December 2011 and followed up for a median of 4.6 years. We adjudicated myocardial infarction, hospitalization of heart failure, cardiovascular death, all-cause death, and composite end points of myocardial infarction/heart failure/death by review of medical records and death certificates. We used multivariable Cox proportional hazards analysis to compare the incidence rate of cardiovascular events and all-cause death according to the quartile of the plasma 7-KC. During the median 4.6 years follow-up, totally 221 participants (21.8%) experienced a cardiovascular event or death. The adjusted risk of the composite end points was higher in the highest 7-KC quartile than in the lowest quartile (hazard ratio, 1.76; 95% confidence interval, 1.42–2.21; P P =0.007). Plasma 7-KC clearly improved various model performance measures, including C statistics, integrated discrimination, and category-free net reclassification. Conclusions: High 7-KC levels are associated with increased risk of cardiovascular events, total death, and composite outcomes in patients with stable coronary artery disease.

In multiple sclerosis (MS) a process of white matter degradation leading to demyelination is observed. Oxidative stress, inflammation, apoptosis, necrosis and/or autophagy result together into a progressive loss of oligodendrocytes. 7-ketocholesterol (7KC), found increased in the cerebrospinal fluid of MS patients, triggers a rupture of RedOx homeostasis associated with mitochondrial dysfunctions, aptoptosis and autophagy (oxiapoptophagy) in cultured murine oligodendrocytes (158N). α-tocopherol is able to mild the alterations induced by 7KC partially restoring the cellular homeostasis. In presence of 7KC, the amount of adherent 158N cells was decreased and oxidative stress was enhanced. An increase of caspase-3 and PARP degradation (evidences of apoptosis), and an increased LC3-II/LC3-I ratio (criterion of autophagy), were detected. These events were associated with a decrease of the mitochondrial membrane potential (ΔΨm) and by a decrease of oxidative phosphorylation revealed by reduced NAD+ and ATP. The cellular lactate was higher while pyruvate, citrate, fumarate, succinate (tricarboxylic acid (TCA) cycle intermediates) were significantly reduced in exposed cells, suggesting that an impairment of mitochondrial respiratory functions could lead to an increase of lactate production and to a reduced amount of ATP and acetyl-CoA available for the anabolic pathways. The concentration of sterol precursors lathosterol, lanosterol and desmosterol were significantly reduced together with satured and unsatured long chain fatty acids (C16:0 − C18:0, structural elements of membrane phospholipids). Such reductions were milder with α-tocopherol. It is likely that the cell death induced by 7KC is associated with mitochondrial dysfunctions, including alterations of oxidative phosphorylation, which could result from lipid anabolism dysfunctions, especially on TCA cycle intermediates. A better knowledge of mitochondrial associated dysfunctions triggered by 7KC will contribute to bring new information on the demyelination processes which are linked with oxidative stress and lipid peroxidation, especially in MS.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

Background: Dyslipidemia causes renal damage; however, the detailed molecular mechanism has not been clarified. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. In the present study, we investigated whether the accumulation of lipids induced by 7-ketocholesterol (7-KC) in tubular epithelial cells produce a fibrotic and inflammatory response through CPT1a. Methods: Using an epithelial cell line, NRK-52E, we determine if intracellular accumulation of 7-KC modulates profibrotic and inflammatory events through CPT1a gene expression. In addition, the direct effects of CPT1a genetic modification has been studied. Results: Our results revealed that high levels of 7-KC induce increased expression of CPT1a, TGF-β1, α- SMA and NLRP3 that was correlated with lipid content. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC laden cells and decreased the expression of CPT1a, TGF-β1, α- SMA and NLRP3. Furthermore, CPT1a Knockdown and C75 pre-treatment increased lipid content but decreased TGF-β1, α- SMA and NLRP3. Conclusions: Our findings reveal that the profibrotic effect of 7-KC on renal epithelial cells are mediated by CPT1a overexpression, which acts on TGF-β1, α-SMA and NLRP3. Thus, CPT1a downregulation protects against 7-KC-induced fibrosis in tubular epithelial cells by downregulating TGF-β1, α- SMA and NLRP3., This study was supported by grants from Ministerio de Economía y Competitividad, awarded to G.H reference SAF2015-67770-R.P.C is performing the studies under the PhD program in Medicine ofthe Universitat de Barcelona, funded by the mentioned grant

Silicosis is one of the prolonged and irreversible occupational diseases. Crystalline silica dust, which has been linked with silicosis, occurs in different industrial areas such as constructions, ceramic, quarry, and pottery. There are significant numbers of newly diagnosed cases every year in Turkey. Patients with silicosis suffer from inflammatory respiratory disorders and silicosis-related complications such as rheumatoid arthritis, systemic sclerosis, and vasculitis. Oxysterols are defined as 27-carbon intermediates or end products of cholesterol. They are also implicated in the etiology of disease states such as atherosclerosis, neurodegenerative, and inflammatory diseases. The aim of the study is to evaluate cholesterol oxidation products in the patients with silicosis and determination of sphingosine-1-phosphate (S1P) levels which is a sphingolipid metabolite. In addition to these parameters, it is aimed to determine the possible lipid peroxidation by different parameters. For this purpose, blood samples and urine were collected from 47 patients and 30 healthy individual with their consents. In order to evaluate oxysterols, 7-ketocholesterol and cholestan 3β,5α,6β-triol levels were measured by LC–MS/MS method. The measured levels of 7-KC were 0.101 ± 0.005 µmol/l in patient and 0.050 ± 0.003 µmol/l in control plasma samples. Triol levels were measured as 0.038 ± 0.005 µmol/l in patient group and 0.033 ± 0.004 µmol/l in control group (p

7-Ketocholesterol (7KC) is a toxic oxysterol that is associated with many diseases and disabilities of aging, as well as several orphan diseases. 7KC is the most common product of a reaction between cholesterol and oxygen radicals and is the most concentrated oxysterol found in the blood and arterial plaques of coronary artery disease patients as well as various other disease tissues and cell types. Unlike cholesterol, 7KC consistently shows cytotoxicity to cells and its physiological function in humans or other complex organisms is unknown. Oxysterols, particularly 7KC, have also been shown to diffuse through membranes where they affect receptor and enzymatic function. Here, we will explore the known and proposed mechanisms of pathologies that are associated with 7KC, as well speculate about the future of 7KC as a diagnostic and therapeutic target in medicine., Graphical abstract Image 1, Highlights • 7KC is mainly a nonenzymatically produced oxysterol known at the moment for its cytotoxicity. • 7KC is implicated in many age-related diseases. • 7KC appears to be a good target for disease, age related and otherwise. • Removal of 7KC from the body could stall and/or reverse disease progression.

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSs+) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSs+ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients' 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD.

7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.

Abdominal aortic aneurysm (AAA) is a degenerative vascular disease with a complex aetiology that remains to be fully elucidated. Clinical management of AAA is limited to surgical repair, while an effective pharmacotherapy is still awaited. Endoplasmic reticulum (ER) stress and mitochondrial dysfunction have been involved in the pathogenesis of cardiovascular diseases (CVDs), although their contribution to AAA development is uncertain. Therefore, we aimed to determine their implication in AAA and investigated the profile of oxysterols in plasma, specifically 7-ketocholesterol (7-KC), as an ER stress inducer.In the present study, we determined aortic ER stress activation in a large cohort of AAA patients compared with healthy donors. Higher gene expression of activating transcription factor (ATF) 6 (ATF6), IRE-1, X-binding protein 1 (XBP-1), C/EBP-homologous protein (CHOP), CRELD2 and suppressor/enhancer of Lin-12-like (SEL1L) and greater protein levels of active ATF6, active XBP1 and of the pro-apoptotic protein CHOP were detected in human aneurysmatic samples. This was accompanied by an exacerbated apoptosis, higher reactive oxygen species (ROS) production and by a reduction in mitochondrial biogenesis in the vascular wall of AAA. The quantification of oxysterols, performed by liquid chromatography-(atmospheric pressure chemical ionization (APCI))-mass spectrometry, showed that levels of 7-KC were significantly higher while those of 7α-hydroxycholesterol (HC), 24-HC and 27-HC were lower in AAA patients compared with healthy donors. Interestingly, the levels of 7-KC correlate with the expression of ER stress markers.Our results evidence an induction of ER stress in the vascular wall of AAA patients associated with an increase in circulating 7-KC levels and a reduction in mitochondrial biogenesis suggesting their implication in the pathophysiology of this disease., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO)-Instituto de Salud Carlos III (ISCIII) [grant numbers CP15/00126, PI17/08137 (to M.G.), PI18/0919 (to C.R.) and RTI2018-094727-B-100 (to J.M.G.)]; the CIBERCV [grant number CB16/11/00257]; the ISCIII, Miguel Servet I program (grant number CP15/00126 (to M.N.M. and M.G.)]; and by Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR; program of support to Research Groups. Ref. 2017-SGR-00333). The study was co-founded by Fondo Europeo de Desarrollo Regional (FEDER)-The way to build Europe

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE−/− mice. Application of ApoE−/− mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.

Atherogenesis is associated with chronic gut infections; however, the mechanisms are not clear. The aim of the study was to determine whether lipopolysaccharide of E. coli (E. coli LPS) may affect endothelial barrier and modify IL-10 expression in dendritic cells (DCs). Human umbilical vein endothelial cells (HUVECs) and monocyte-derived DCs were treated with E. coli LPS, apolipoprotein B100 (ApoB100) and 7-ketocholesterol (7-kCH) - harmful oxidized form of cholesterol. The effect of E. coli LPS, 7-kCH and ApoB100 on the barrier functions of HUVECs in real-time cell electric impedance sensing system (RTCA-DP) was assessed. Furthermore, the effect of 7-kCH and ApoB100 on barrier functions of HUVECs co-cultured with DCs previously treated with LPS was analyzed. Both E. coli LPS and 7-kCH decreased barrier functions of HUVECs and reduced tight junction protein mRNA expression, whereas ApoB100 increased endothelial barrier. In DCs, ApoB100 and E. coli LPS decreased IL-10 mRNA expression. In HUVECs co-cultured with DCs treated with LPS and subsequently pulsed with ApoB100 or 7-kCH, IL-10 mRNA expression was lower. E. coli LPS-exposed DCs diminished the protective effect of ApoB100 on endothelial integrity and led to the decrease in occludin mRNA expression. LPS potentially derived from gut microflora may destabilize endothelial barrier together with oxidized cholesterol and intensify the immunogenicity of ApoB100.

Objective. Cholesterol oxidation products have an established proatherogenic and cytotoxic effect. An increased exposure to these substances may be associated with the development of atherosclerosis and cancers. Relatively little, though, is known about the effect of phytosterol oxidation products, although phytosterols are present in commonly available and industrial food products. Thus, the aim of the research was to assess the effect of 5α,6α-epoxyphytosterols, which are important phytosterol oxidation products, on redox state in rats.Material and Methods. The animals were divided into 3 groups and exposed to nutritional sterols by receiving feed containing 5α,6α-epoxyphytosterols (ES group) and 5α,6α-epoxycholesterol (Ech group) or sterol-free feed (C group). The levels of malondialdehyde (MDA), conjugated dienes (CD), and ferric reducing antioxidant potential (FRAP) were assayed in the plasma; anti-7-ketocholesterol antibodies and activity of paraoxonase-1 (PON1) were determined in serum, whereas the activity of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), S-glutathione transferase (GST), and superoxide dismutase (SOD) were assayed in RBCs.Results. During the experiment, the levels of lipid peroxidation products increased, such as CD and anti-7-ketocholesterol antibodies. At the same time, the plasma levels of FRAP and serum activity of PON1 decreased alongside the reduced activity of GPx, GR, and SOD in RBCs. There was no effect of the studied compounds on the plasma MDA levels or on the activity of CAT and GST in RBCs.Conclusions. Both 5α,6α-epoxyphytosterols and 5α,6α-epoxycholesterols similarly dysregulate theredoxstate in experimental animal model and may significantly impact atherogenesis.

[Background] Macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated., [Methods] Using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied., [Results] Our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles., [Conclusions] Altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype., This study was supported by grants from Ministerio de Economía y Competitividad, reference SAF2015–67770-R, awarded to G.H and from Fondo de Investigación Sanitaria (FIS), reference PI17/01411, awarded to A.S.

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1–2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

// Mei-Chi Chang 1,2 , Yi-Jane Chen 3,* , Eric Jein-Wein Liou 2 , Wan-Yu Tseng 3 , Chiu-Po Chan 2 , Hseuh-Jen Lin 4 , Wan-Chuen Liao 3 , Ya-Ching Chang 5 , Po-Yuan Jeng 3 and Jiiang-Huei Jeng 3 1 Biomedical Science Team, Chang Gung University of Science and Technology, Kwei-Shan, Taoyuan, Taiwan 2 Department of Dentistry, Chang Gung Memorial Hospital, Taipei, Taiwan 3 School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital 4 Department of Dentistry, Show Chwan Memorial Hospital, Changhua, Taiwan 5 Department of Dentistry, Mackey Memorial Hospital, Taipei, Taiwan * This author have contributes equally to the first author Correspondence to: Jiiang-Huei Jeng, email: // Chiu-Po Chan, email: // Keywords : apoptosis, atherosclerosis, cytotoxicity, endothelial cells, inflammation,Gerotarget Received : September 13, 2016 Accepted : October 06, 2016 Published : October 11, 2016 Abstract Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer’s disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 µg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV, quercetin, QCT, and apigenin, API) as well as &omega, 3 and &omega, 9 unsaturated fatty acids (&alpha, linolenic acid, ALA, eicosapentaenoic acid, EPA, docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 &micro, M)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: &le, 6.25 &micro, M, fatty acids: &le, 25 &micro, M) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of &alpha, tocopherol (400 &micro, M) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (&Delta, &Psi, m) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as &omega, 9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.

This review will focus on the inflammatory and toxic mechanism of action of 7-ketocholesterol (7KCh) and the potential implications of its accumulation, especially in the retina. 7KCh is a pro-inflammatory oxysterol usually associated with oxidized lipoprotein deposits present in aged retinas. High amounts of 7KCh can be generated in situ in these lipoprotein deposits possibly through a free radical-mediated mechanism catalyzed by iron. 7KCh seems to activate several kinase signaling pathways that work via multiple transcription factors to induce cytokines and intracellular effectors causing cell death. There seems to be a controversy in the literature in relation to the mechanisms of death induced by 7KCh. Some of the discrepancies arise from the way the oxysterol is delivered because different signaling pathways are activated in different experimental setups. The elucidation of the inflammatory and toxic mechanisms is crucial for the discovery and design of new therapies. Importantly, there is little evidence of 7KCh detoxifying mechanisms in the retina, although some potential enzymes have been described. Thus, continuous formation throughout life and potential toxicity of 7KCh points it out as an "age-related" risk factor in pathologies such as age-related macular degeneration.

The purpose of this work was to investigate whether changes in oxysterol and apolipoprotein levels over 5 years are associated with disease course and disability progression in multiple sclerosis (MS). This study included 139 subjects [39 healthy controls (HCs), 61 relapsing-remitting MS (RR-MS) patients, and 39 progressive MS (P-MS) patients]. Oxysterols [24-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC), 27-hydroxycholesterol (27HC), 7α-hydroxycholesterol (7αHC), and 7-ketocholesterol (7KC)] were measured at baseline and 5 years using a novel mass spectrometric method, and apolipoproteins were measured using immunoturbidometric diagnostic kits. Levels of 24HC (P = 0.004), 25HC (P = 0.029), and 27HC (P = 0.026) increased in P-MS patients. 7KC (P = 0.047) and 7αHC (P = 0.001) levels decreased in RR-MS patients, and there were no changes in any oxysterols in HCs. In MS patients, ApoC-II (all P ≤ 0.01) and ApoE (all P ≤ 0.01) changes were positively associated with all oxysterol levels. Increases in 24HC (P = 0.038) and ApoB (P = 0.038) and decreases in 7KC (P = 0.020) were observed in RR-MS patients who converted to secondary P-MS (SP-MS) at follow-up and in SP-MS patients compared with RR-MS patients. Oxysterols and their associations with apolipoproteins differed between MS patients and HCs over 5 years. Oxysterol and apolipoprotein changes were associated with conversion to SP-MS.

PubMed ID: 30008986, Involvement of high cholesterol and oxidative stress in cardiovascular diseases is well studied, as it can be hypothesized that various products originated from lipid peroxidation, such as oxysterols, or affected protein expression might lead to cardiomyocyte damage followed by the pathological modifications. Although oxidation of excessive cholesterol to oxysterols in elevated stress conditions is identified by a number of studies, the role of a high cholesterol diet in regulating fatty acid and oxysterol accumulation, together with scavenger receptor mRNA levels, in the heart remains little investigated. Our study provides a detailed analysis of the changes in fatty acid, oxysterol, and scavenger receptor profiles and its relation with histological alterations in the heart tissue. We evaluated alterations of fatty acid composition, by the GC-MS method, while 4ß-, 25-, and 27-hydroxycholesterol and 7-ketocholesterol levels by means of LC-MS/MS in high cholesterol diet-fed rabbits. Additionally, a number of proteins related to lipid metabolism and scavenger receptor mRNA expressions were evaluated by Western blotting and RT-PCR. According to our in vivo results, a high cholesterol diet enhances a number of unsaturated fatty acids, oxysterols, and LXR?, in addition to CD36, CD68, CD204, and SR-F1 expressions while ?-tocopherol supplementation decreases LXR? and SR expressions together with an increase in 27-hydroxycholesterol and ABCA1 levels. Our results indicated that the high cholesterol diet modulates proteins related to lipid metabolism, which might result in the malfunction of the heart and ?-tocopherol shows its beneficial effects. We believe that this work will lead the generation of different theories in the development of heart diseases.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-019-00060-y) contains supplementary material, which is available to authorized users.

It is now established that cholesterol oxidation products (oxysterols) are involved in several events underlying Alzheimer's disease (AD) pathogenesis. Of note, certain oxysterols cause neuron dysfunction and degeneration but, recently, some of them have been shown also to have neuroprotective effects. The present study, which aimed to understand the potential effects of 24-hydroxycholesterol (24-OH) against the intraneuronal accumulation of hyperphosphorylated tau protein, stressed these latter effects. A beneficial effect of 24-OH was demonstrated in SK-N-BE neuroblastoma cells, and is due to its ability to modulate the deacetylase sirtuin 1 (SIRT1), which contributes to preventing the neurotoxic accumulation of the hyperphosphorylated tau protein. Unlike 24-OH, 7-ketocholesterol (7-K) did not modulate the SIRT1-dependent neuroprotective pathway. To confirm the neuroprotective role of 24-OH, in vivo experiments were run on mice that express human tau without spontaneously developing tau pathology (hTau mice), by means of the intracerebroventricular injection of 24-OH. 24-OH, unlike 7-K, was found to completely prevent the hyperphosphorylation of tau induced by amyloid β monomers. These data highlight the importance of preventing the loss of 24-OH in the brain, and of maintaining high levels of the enzyme SIRT1, in order to counteract neurodegeneration. Graphical abstract: A hypothetical scheme of the molecular mechanisms underlying the effects of 24-OH on hyperphosphorylated tau accumulation.fx1 Keywords: 24-hydroxycholesterol, Sirtuin 1, Tau, Oxysterols, Alzheimer's disease

International audience; Little is known about K+ regulation playing major roles in the propagation of nerve impulses, as well as in apoptosis and inflammasome activation involved in neurodegeneration. As increased levels of 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC) and tetracosanoic acid (C24:0) have been observed in patients with neurodegenerative diseases, we studied the effect of 24 and/or 48 h of treatment with 7KC, 24S-OHC and C24:0 on Kv3.1b potassium channel level, intracellular K+ concentration, oxidative stress, mitochondrial dysfunction, and plasma membrane permeability in 158N oligodendrocytes and BV-2 microglial cells. In 158N cells, whereas increased level of Kv3.1b was only observed with 7KC and 24S-OHC but not with C24:0 at 24 h, an intracellular accumulation of K+ was always detected. In BV-2 cells treated with 7KC, 24S-OHC and C24:0, Kv3.1b level was only increased at 48 h; intracellular K+ accumulation was found at 24 h with 7KC, 24S-OHC and C24:0, and only with C24:0 at 48 h. Positive correlations between Kv3.1b level and intracellular K+ concentration were observed in 158N cells in the presence of 7KC and 24S-OHC, and in 7KC-treated BV-2 cells at 48 h. Positive correlations were also found between Kv3.1b or the intracellular K+ concentration, overproduction of reactive oxygen species, loss of transmembrane mitochondrial potential and increased plasma membrane permeability in 158N and BV-2 cells. Our data support that the lipid environment affects Kv3.1b channel expression and/or functionality, and that the subsequent rupture of K+ homeostasis is relied with oligodendrocytes and microglial cells damages.

This work was presented as an oral presentation at the 7th ENOR (European Network for Oxysterol Research) Symposium ‘Oxysterols and Sterol Derivatives in Health and Disease’, September 21–22, 2017, Université catholique de Louvain, Brussels, Belgium (https://www.oxysterols.net/).; International audience; The involvement of organelles in cell death is well established especially for endoplasmic reticulum, lysosomes and mitochondria. However, the role of the peroxisome is not well known, though peroxisomal dysfunction favors a rupture of redox equilibrium. To study the role of peroxisomes in cell death, 158 N murine oligodendrocytes were treated with 7-ketocholesterol (7 KC: 25-50 mu M, 24 h). The highest concentration is known to induce oxiapoptophagy (OXIdative stress + APOPTOsis + autoPHAGY), whereas the lowest concentration does not induce cell death. In those conditions (with 7 KC: 50 mu M) morphological, topographical and functional peroxisome alterations associated with modifications of the cytoplasmic distribution of mitochondria, with mitochondrial dysfunction (loss of transmembrane mitochondrial potential, decreased level of cardiolipins) and oxidative stress were observed: presence of peroxisomes with abnormal sizes and shapes similar to those observed in Zellweger fibroblasts, lower cellular level of ABCD3, used as a marker of peroxisomal mass, measured by flow cytometry, lower mRNA and protein levels (measured by RT-qPCR and western blotting) of ABCD1 and ABCD3 (two ATP-dependent peroxisomal transporters), and of ACOX1 and MFP2 enzymes, and lower mRNA level of DHAPAT, involved in peroxisomal beta-oxidation and plasmalogen synthesis, respectively, and increased levels of very long chain fatty acids (VLCFA: C24:0, C24:1, C26:0 and C26:1, quantified by gas chromatography coupled with mass spectrometry) metabolized by peroxisomal beta-oxidation. In the presence of 7 KC (25 mu M), slight mitochondrial dysfunction and oxidative stress were found, and no induction of apoptosis was detected; however, modifications of the cytoplasmic distribution of mitochondria and clusters of mitochondria were detected. The peroxisomal alterations observed with 7 KC (25 mu M) were similar to those with 7 KC (50 mu M). In addition, data obtained by transmission electron microcopy and immunofluorescence microscopy by dual staining with antibodies raised against p62, involved in autophagy, and ABCD3, support that 7 KC (25-50 mu M) induces pexophagy. 7 KC (25-50 mu M)-induced side effects were attenuated by alpha-tocopherol but not by alpha-tocotrienol, whereas the anti-oxidant properties of these molecules determined with the FRAP assay were in the same range. These data provide evidences that 7 KC, at concentrations inducing or not cell death, triggers morphological, topographical and functional peroxisomal alterations associated with minor or major mitochondrial changes.

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H2DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography–mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [3H]-cholesterol labeled HPEC and [14C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7β-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL3 (78 ± 17%, 40 ± 9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.

Recent studies have expanded the appreciation of the roles of oxysterols triggering inflammatory, immune cytotoxic and apoptotic processes, but have not been considered for proteome analysis. A comparative proteomic study in intestinal epithelial cell cultures incubated (60 μM/24 h) with 7keto-cholesterol or 7keto-stigmasterol was performed. The influence of both compounds was studied following the nLC-TripleTOF analysis. Findings were compared to results for control cultures. In the principal component analysis (PCA) of proteome patterns, two components were extracted accounting for 99.8% of the variance in the protein expression. PCA analysis clearly discriminated between the perturbations in the proteome of cell cultures incubated with 7keto-cholesterol and 7keto-stigmasterol. These proteins participate in mitochondrial function, lipid homeostasis, inflammation and immunity and cell proliferation. Remarkable differences between proteome patterns in cell cultures exposed to 7keto-cholesterol and 7keto-stigmasterol affect macrophage migration inhibitory factor, apolipoprotein E, Bcl-2-associated transcription factor and cellular retinoic acid-binding protein. Besides, exposure to 7keto-stigmasterol increased the concentration of ubiquitin-conjugating enzyme E2 and the mitochondrial superoxide dismutase protein. Such findings raise new questions about safety studies and the regulatory potential of oxysterols in the differentiation and function of intestinal and associated immune cells, their response to environmental stimuli and impairment of absorption processes.

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3β,5α,6β-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.