Investigating the effects of 7-ketocholesterol on retinal pigment epithelium bioenergetics.

Age-related macular degeneration (AMD) is associated with formation of drusen, clusters of lipids, and oxidized lipid products under the retinal pigment epithelium (RPE). 7-Ketocholesterol (7KC) is a form of oxidized cholesterol present in drusen and is hypothesized to play a role in AMD pathogenesis. The association of 7KC with cellular toxicity and inflammation, key elements of AMD pathology, has been demonstrated. However, the effects of 7KC on altering RPE bioenergetics, a potentially important pathologic process in AMD, are unclear. Herein, we describe the effects of non-lethal doses of 7KC on the bioenergetics and phenotype of RPE cells in culture. Metabolic analysis demonstrated a significant dose-dependent increase in total ATP production rates that was driven primarily by an increase in glycolysis. The increase in glycolysis was accompanied by an increase in glucose uptake and increased expression of hexokinase 1. Increased levels of Translocase of Outer Mitochondrial Membrane 20 and NADH:Ubiquinone Oxidoreductase Core Subunit S1, Succinate dehydrogenase, Ubiquinol-Cytochrome C Reductase Core Protein 2, Cytochrome C Oxidase II, and ATP synthase subunit beta, proteins involved in oxidative phosphorylation (OXPHOS), were also seen. However, specific electron transport chain activity remained unchanged. 7KC-treated cells also demonstrated a change in cellular morphology with decreased expression of epithelial markers. In summary, 7KC has significant effects on the bioenergetics and morphology of RPE cells reflective of findings seen in clinical AMD.

Development and validation of a new genotype-phenotype correlation for Niemann-Pick disease type C1.

Hundreds of NPC1 variants cause highly heterogeneous phenotypes. This study aims to explore the genotype-phenotype correlation of NPC1, especially for missense variants. In a well-characterized cohort, phenotypes are graded into three clinical forms: mild, intermediate, and severe. Missense residue structural location was stratified into three categories: surface, partially, and fully buried. The association of phenotypes with the topography of the amino acid substitution in the protein structure was investigated in our cohort and validated in two reported cohorts. One hundred six unrelated NPC1 patients were enrolled. A significant correlation of genotype-phenotype was found in 81 classified individuals with two or one (the second was null variant) missense variant (p < 0.001): of 25 patients with at least one missense variant of surface (group A), 19 (76%) mild, six (24%) intermediate, and none severe; of 31 cases with at least one missense variant of partially buried without surface variants (group B), 11 (35%) mild, 16 (52%) intermediate, and four (13%) severe; of the remaining 25 patients with two or one buried missense variants (group C), eight (32%) mild, nine (36%) intermediate, and eight (32%) severe. Additionally, 7-ketocholesterol, the biomarker, was lower in group A than in group B (p = 0.024) and group C (p = 0.029). A model was proposed that accurately predicted phenotypes of 72 of 90 (80%), 73 of85 (86%), and 64 of 69 (93%) patients in our cohort, Italian, and UK cohort, respectively. This study proposed a novel genotype-phenotype correlation in NPC1, linking the underlying molecular pathophysiology with clinical phenotype and aiding genetic counseling and evaluation in clinical practice.

A Novel Method for the Determination of Squalene, Cholesterol and Their Oxidation Products in Food of Animal Origin by GC-TOF/MS.

Cholesterol present in food of animal origin is a precursor of oxysterols (COPs), whose high intake through diet can be associated with health implications. Evaluation of the content of these contaminants in food is associated with many analytical problems. This work presents a GC-TOF/MS method for the simultaneous determination of squalene, cholesterol and seven COPs (7-ketocholesterol, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, 25-hydroxycholesterol, 5,6α-epoxycholesterol, 5,6ß-epoxycholesterol, cholestanetriol). The sample preparation procedure includes such steps as saponification, extraction and silylation. The method is characterized by high sensitivity (limit of quantification, 0.02-0.25 ng mL-1 for instrument, 30-375 µg kg of sample), repeatability (RSD 2.3-6.2%) and a wide linearity range for each tested compound. The method has been tested on eight different animal-origin products. The COP to cholesterol content ratio in most products is about 1%, but the profile of cholesterol derivatives differs widely (α = 0.01). In all the samples, 7-ketocholesterol is the dominant oxysterol, accounting for 31-67% of the total COPs level. The levels of the other COPs range between 0% and 21%. In none of the examined products are cholestanetriol and 25-hydroxycholesterol present. The amount of squalene, which potentially may inhibit the formation of COPs in food, ranges from 2 to 57 mg kg-1.

Accumulation of oxysterols in the erythrocytes of COVID-19 patients as a biomarker for case severity.

BACKGROUND: Due to the high risk of COVID-19 patients developing thrombosis in the circulating blood, atherosclerosis, and myocardial infarction, it is necessary to study the lipidome of erythrocytes. Specifically, we examined the pathogenic oxysterols and acylcarnitines in the erythrocyte homogenate of COVID-19 patients. These molecules can damage cells and contribute to the development of these diseases. METHODS: This study included 30 patients and 30 healthy volunteers. The erythrocyte homogenate extract was analyzed using linear ion trap mass spectrometry combined with high-performance liquid chromatography. The concentrations of oxysterols and acylcarnitines in erythrocyte homogenates of healthy individuals and COVID-19 patients were measured. Elevated levels of toxic biomarkers in red blood cells could initiate oxidative stress, leading to a process known as Eryptosis. RESULTS: In COVID-19 patients, the levels of five oxysterols and six acylcarnitines in erythrocyte homogenates were significantly higher than those in healthy individuals, with a p-value of less than 0.05. The mean total concentration of oxysterols in the red blood cells of COVID-19 patients was 23.36 ± 13.47 µg/mL, while in healthy volunteers, the mean total concentration was 4.92 ± 1.61 µg/mL. The 7-ketocholesterol and 4-cholestenone levels were five and ten times higher, respectively, in COVID-19 patients than in healthy individuals. The concentration of acylcarnitines in the red blood cell homogenate of COVID-19 patients was 2 to 4 times higher than that of healthy volunteers on average. This finding suggests that these toxic biomarkers may cause the red blood cell death seen in COVID-19 patients. CONCLUSIONS: The abnormally high levels of oxysterols and acylcarnitines found in the erythrocytes of COVID-19 patients were associated with the severity of the cases, complications, and the substantial risk of thrombosis. The concentration of oxysterols in the erythrocyte homogenate could serve as a diagnostic biomarker for COVID-19 case severity.

Malonyl-CoA Accumulation as a Compensatory Cytoprotective Mechanism in Cardiac Cells in Response to 7-Ketocholesterol-Induced Growth Retardation.

The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-13C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of ß-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene (Mlycd-/-) alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells.

Protective Effect of Ergothioneine against 7-Ketocholesterol-Induced Mitochondrial Damage in hCMEC/D3 Human Brain Endothelial Cells.

Recent findings have suggested that the natural compound ergothioneine (ET), which is synthesised by certain fungi and bacteria, has considerable cytoprotective potential. We previously demonstrated the anti-inflammatory effects of ET on 7-ketocholesterol (7KC)-induced endothelial injury in human blood-brain barrier endothelial cells (hCMEC/D3). 7KC is an oxidised form of cholesterol present in atheromatous plaques and the sera of patients with hypercholesterolaemia and diabetes mellitus. The aim of this study was to elucidate the protective effect of ET on 7KC-induced mitochondrial damage. Exposure of human brain endothelial cells to 7KC led to a loss of cell viability, together with an increase in intracellular free calcium levels, increased cellular and mitochondrial reactive oxygen species, a decrease in mitochondrial membrane potential, reductions in ATP levels, and increases in mRNA expression of TFAM, Nrf2, IL-1ß, IL-6 and IL-8. These effects were significantly decreased by ET. Protective effects of ET were diminished when endothelial cells were coincubated with verapamil hydrochloride (VHCL), a nonspecific inhibitor of the ET transporter OCTN1 (SLC22A4). This outcome demonstrates that ET-mediated protection against 7KC-induced mitochondrial damage occurred intracellularly and not through direct interaction with 7KC. OCTN1 mRNA expression itself was significantly increased in endothelial cells after 7KC treatment, consistent with the notion that stress and injury may increase ET uptake. Our results indicate that ET can protect against 7KC-induced mitochondrial injury in brain endothelial cells.

Identification of 7-Ketocholesterol-Modulated Pathways and Sterculic Acid Protective Effect in Retinal Pigmented Epithelium Cells by Using Genome-Wide Transcriptomic Analysis.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. AMD is characterized by the formation of lipidic deposits between the retinal pigment epithelium (RPE) and the choroid called drusen. 7-Ketocholesterol (7KCh), an oxidized-cholesterol derivative, is closely related to AMD as it is one of the main molecules accumulated in drusen. 7KCh induces inflammatory and cytotoxic responses in different cell types, and a better knowledge of the signaling pathways involved in its response would provide a new perspective on the molecular mechanisms that lead to the development of AMD. Furthermore, currently used therapies for AMD are not efficient enough. Sterculic acid (SA) attenuates the 7KCh response in RPE cells and is presented as an alternative to improve these therapies. By using genome-wide transcriptomic analysis in monkey RPE cells, we have provided new insight into 7KCh-induced signaling in RPE cells, as well as the protective capacity of SA. 7KCh modulates the expression of several genes associated with lipid metabolism, endoplasmic reticulum stress, inflammation and cell death and induces a complex response in RPE cells. The addition of SA successfully attenuates the deleterious effect of 7KCh and highlights its potential for the treatment of AMD.

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia.

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1trg/ECcre) and their control littermates (Smpd1trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1trg/ECcre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1trg/ECcre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1trg/ECcre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1trg/ECcre mice than in their control littermates. Using ECs from Smpd1trg/ECcre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1ß levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

A Dietary Oxysterol, 7-Ketocholesterol, Exacerbates Imiquimod-Induced Psoriasis-like Dermatitis in Steatohepatitic Mice.

Patients with psoriasis are at a higher risk of developing nonalcoholic fatty liver disease. We previously identified an oxidized derivative of cholesterol, 7-ketocholesterol (7KC), in diet-induced steatohepatitic mice. Here, we investigated whether 7KC exacerbates psoriasis-like dermatitis by accelerating steatohepatitis in mice. A high-fat/high-cholesterol/high-sucrose/bile salt diet (nonalcoholic steatohepatitis (NASH) diet) with or without 0.0125% 7KC was fed to C57BL/6 mice (7KC or control group) for three weeks to induce steatohepatitis. A 5% imiquimod cream was then applied to the ears and dorsal skin for four days to induce psoriasis-like dermatitis. Hepatic lipid accumulation and inflammatory cell infiltration were exacerbated in the 7KC group compared with the control group after three weeks. Serum tumor necrosis factor-α (TNF-α) levels were also elevated in the 7KC group (108.5 ± 9.8 vs. 83.1 ± 13.1 pg/mL, p < 0.005). Imiquimod cream increased the psoriasis area severity index (PASI) score in mice in the 7KC group (9.14 ± 0.75 vs. 5.17 ± 1.17, p < 0.0001). Additionally, Tnfa, Il23a, Il17a, and Il22 mRNA levels in the dorsal lesion were significantly upregulated. Finally, Th17 cell differentiation and the TNF signaling pathway were enhanced in the dorsal lesions and liver of mice in the 7KC group. These data suggest that steatohepatitis and psoriasis are linked by a potent, diet-related factor.

Accumulation of Deleterious Effects in Gastric Epithelial Cells and Vascular Endothelial Cells In Vitro in the Milieu of Helicobacter pylori Components, 7-Ketocholesterol and Acetylsalicylic Acid.

The Gastric pathogen Helicobacter pylori (HP) may influence the development of coronary heart disease (CHD). H. pylori induce reactive oxygen species (ROS), which transform cholesterol to 7-ketocholesterol (7-kCh), a CHD risk factor. Acetylsalicylic acid (ASA)-an Anti-aggregation drug used in CHD patients-may increase gastric bleeding and inflammation. We examined whether H. pylori driven ROS effects in the cell cultures of gastric epithelial cells (AGS) and vascular endothelial cells (HUVEC) progress in the milieu of 7-kCh and ASA. Cell cultures, exposed to 7-kCh or ASA alone or pulsed with the H. pylori antigenic complex-Glycine acid extract (GE), urease (UreA), cytotoxin associated gene A (CagA) protein or lipopolysaccharide (LPS), alone or with 7-kCh and ASA-were examined for ROS, apoptosis, cell integrity, interleukin (IL)-8, the activation of signal transducer, the activator of transcription 3 (STAT3), and wound healing. ASA and 7-kCh alone, and particularly in conjunction with H. pylori components, increased the ROS level and the rate of apoptosis, which was followed by cell disintegration, the activation of STAT3, and IL-8 elevation. AGS cells were unable to undergo wound healing. The cell ROS response to H. pylori components may be elevated by 7-kCh and ASA.

7-Ketocholesterol-Induced Micro-RNA-107-5p Increases Number and Activity of Osteoclasts by Targeting MKP1.

Osteoclasts (OCs), which are responsible for bone resorption, play a critical role in cholesterol-induced bone loss and recent studies have suggested that various micro-RNAs (miRs) contribute to modulating OCs. We hypothesized that 7-ketocholesterol (7-KC), a metabolite responsible for cholesterol-induced bone loss, induces miR-107-5p, which affects OCs. Overexpression and knock-down of miR-107-5p were performed using miR-107-5p mimic and anti-miR-107-5p, respectively. The effects of miR-107-5p on OCs were analyzed by tartrate-resistant alkaline phosphatase staining, qPCR, and Western blot. MiR-107-5p was upregulated after 7-KC exposure in receptor activator of nuclear factor kappa-Β ligand-stimulated OCs. Furthermore, miR-107-5p upregulation was also observed in tibiae from an atherogenic diet-fed mice compared with mice fed with a normal diet. MiR-107-5p overexpression enhanced the area and number of OCs, whereas inhibiting the endogenous expression of miR-107-5p generated by 7-KC had the opposite effect. Among the possible candidates, mitogen-activated protein kinase phosphatase-1, a stress-responsive dual-specificity phosphatase that inactivates mitogen-activated protein kinase (MKP1), has been proven to be a target gene of miR-107-5p, as demonstrated by the direct interaction between miR-107-5p and the 3'-untranslated region of MKP1. Collectively, our findings demonstrate that 7-KC-induced miR-107-5p promotes differentiation and function of OCs by downregulating MKP1.

7-ketocholesterol induces endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment.

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 (TGFß). Inhibition of TGFß 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 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 responsiveness to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

Apoptosis, autophagy and atherosclerosis: Relationships and the role of Hsp27.

Atherosclerosis is a multifactorial chronic inflammatory disease of the arterial wall, and an important pathological basis of coronary heart disease. Endothelial cells, vascular smooth muscle cells, and macrophages play important roles in the development of atherosclerosis. Of note, apoptosis and autophagy, two types of programmed cell death, influence the development and progression of atherosclerosis via the modulation of such cells. The small heat shock protein Hsp27 is a multifunctional protein induced by various stress factors and has a protective effect on cells. A large number of studies have demonstrated that Hsp27 plays an important role in regulating apoptosis. Recently, some studies have suggested that Hsp27 also participates in the autophagic process. Moreover, Hsp27 is closely related to the occurrence and development of atherosclerosis. Here, we summarize the molecular mechanisms of apoptosis and autophagy and discuss their effects on endothelial cells, vascular smooth muscle cells, and macrophages in the context of atherosclerotic procession. We further explore the involvement of Hsp27 in apoptosis, autophagy, and atherosclerosis. We speculate that Hsp27 may exert its anti-atherosclerotic role via the regulation of apoptosis and autophagy; this may provide the basis for the development of new approaches for the prevention and treatment of atherosclerosis.

Influential role of 7-Ketocholesterol in the progression of Alzheimer's disease.

Millions of people are affected by neurodegenerative diseases worldwide. They occur due to the loss of brain functions or peripheral nervous system dysfunction. If untreated, prolonged condition ultimately leads to death. Mostly they are associated with stress, altered cholesterol metabolism, inflammation and organelle dysfunction. Endogenous cholesterol and phospholipids in brain undergo auto-oxidation by enzymatic as well as non-enzymatic modes leading to the formation of by-products such as 4-hydroxynonenal and oxysterols. Among various oxysterols, 7-ketocholesterol (7KCh) is one of the major toxic components involved in altering neuronal lipid metabolism, contributing to inflammation and nerve cell damage. More evidently 7KCh is proven to induce oxidative stress and affects membrane permeability. Loss in mitochondrial membrane potential affects metabolism of cell organelles such as lysosomes and peroxisomes which are involved in lipid and protein homeostasis. This in turn could affect amyloidogenesis, tau protein phosphorylation and accumulation in pathological conditions of neurodegenerative diseases. Lipid alterations and the consequent pathogenic protein accumulation, results in the damage of cell organelles and microglial cells. This could be a reason behind disease progression and predominantly reported characteristics of neurodegenerative disorders such as Alzheimer's disease. This review focuses on the role of 7KCh mediated neurodegenerative Alzheimer's disease with emphasis on alterations in the lipid raft microdomain. In addition, current trends in the significant therapies related to 7KCh inhibition are highlighted.

7-Ketocholesterol- and 7ß-Hydroxycholesterol-Induced Peroxisomal Disorders in Glial, Microglial and Neuronal Cells: Potential Role in Neurodegeneration : 7-ketocholesterol and 7ß-hydroxycholesterol-Induced Peroxisomal Disorders and Neurodegeneration.

Peroxisomopathies are qualitative or quantitative deficiencies in peroxisomes which lead to increases in the level of very-long-chain fatty acids (VLCFA) and can be associated with more or less pronounced dysfunction of central nervous system cells: glial and microglial cells. Currently, in frequent neurodegenerative diseases, Alzheimer's disease (AD) and multiple sclerosis (MS), peroxisomal dysfunction is also suspected due to an increase in VLCFA, which can be associated with a decrease of plasmalogens, in these patients. Moreover, in patients suffering from peroxisomopathies, such as X-linked adrenoleukodystrophy (X-ALD), AD, or MS, the increase in oxidative stress observed leads to the formation of cytotoxic oxysterols: 7-ketocholesterol (7KC) and 7ß-hydroxycholesterol (7ß-OHC). These observations led to the demonstration that 7KC and 7ß-OHC alter the biogenesis and activity of peroxisomes in glial and microglial cells. In X-ALD, AD, and MS, it is suggested that 7KC and 7ß-OHC affecting the peroxisome, and which also induce mitochondrial dysfunctions, oxidative stress, and inflammation, could promote neurodegeneration. Consequently, the study of oxisome in peroxisomopathies, AD and MS, could help to better understand the pathophysiology of these diseases to identify therapeutic targets for effective treatments.

Prevention of 7-Ketocholesterol-Induced Overproduction of Reactive Oxygen Species, Mitochondrial Dysfunction and Cell Death with Major Nutrients (Polyphenols, ω3 and ω9 Unsaturated Fatty Acids) of the Mediterranean Diet on N2a Neuronal Cells.

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 ω3 and ω9 unsaturated fatty acids (α-linolenic acid, ALA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 µM)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: ≤6.25 µM; fatty acids: ≤25 µM) 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 α-tocopherol (400 µM) 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 (ΔΨm) 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 ω3 and ω9 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.

CPT1a downregulation protects against cholesterol-induced fibrosis in tubular epithelial cells by downregulating TGFß-1 and inflammasome.

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-ß1, 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-ß1, α- SMA and NLRP3. Furthermore, CPT1a Knockdown and C75 pre-treatment increased lipid content but decreased TGF-ß1, 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-ß1, α-SMA and NLRP3. Thus, CPT1a downregulation protects against 7-KC-induced fibrosis in tubular epithelial cells by downregulating TGF-ß1, α- SMA and NLRP3.

7-Ketocholesterol disturbs RPE cells phagocytosis of the outer segment of photoreceptor and induces inflammation through ERK signaling pathway.

7-Ketocholesterol (7KCh), an oxidized form of cholesterol, is present at a high level in drusen and has been believed to play a role in the pathogenesis of age-related macular degeneration (AMD). Therefore, we developed a rat model to study the direct impact of 7KCh on retina. We delivered 7KCh to the rat retina by intravitreal injection using hydroxypropyl-ß-cyclodextrin as a vehicle. We observed that 7KCh mainly deposited in the retinal pigment epithelial (RPE) cells and induced marked photoreceptor apoptosis. Transmission electron microscope examination demonstrated cytoplasmic vacuoles in RPE cells and the microvilli detached from the outer segment after 7KCh treatment. In vitro experiments also revealed that RPE cells could take up 7KCh in culture. Moreover, 7KCh up-regulated IL-1ßmRNA, TNF-αmRNA, IL-6 mRNA, and IL-1ß secretion of RPE. U0126, a MEK1/2 inhibitor, down regulated the expression of these inflammation factors. Our findings may help elucidate the potential role of 7KCh in the pathogenesis of AMD.

Inflammatory and cell death mechanisms induced by 7-ketocholesterol in the retina. Implications for age-related macular degeneration.

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.

Prevention of 7-ketocholesterol-induced side effects by natural compounds.

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.