Cholesterol and Its Oxidation Derivatives Content in Market Dairy Products.

Cholesterol oxidation products (COPs) are contaminants of food of animal origin. Increased levels of these compounds in the human body are associated with an increased risk of many non-communicable diseases. Dairy products are mentioned among the main sources of these compounds in the diet. The objective of this study was to evaluate the contents of cholesterol and its oxidized derivatives in eleven groups of dairy products, willingly consumed in European countries. The levels of COPs were determined by applying the GC-TOF/MS method. In the tested products, cholesterol and its oxidation derivatives, such as 7-ketocholesterol, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, 5,6ß-epoxycholesterol and 5,6α-epoxycholesterol, were determined. The studied dairy products differed in their contents and profiles of oxysterols. The highest contents of COPs were found in cheese with internal mold (13.8 ± 2.5 mg kg-1) and Cheddar (11.7 ± 3.5 mg kg-1), while the lowest levels were detected in yoghurt (0.94 ± 0.30 mg kg-1) and kefir (0.57 ± 0.11 mg kg-1). 7-ketocholesterol and 5,6ß-epoxycholesterol were the dominant oxysterols. The ratio of oxidized derivatives to total cholesterol was on average 1.7%. Our results confirmed that dairy products are an important dietary source of COPs. Their levels should be monitored in dairy products to provide the best health quality.

Effect of 7-ketocholesterol incorporation on substrate binding affinity and turnover rate of the organic cation transporter 2 (OCT2).

The organic cation transporter 2 (OCT2) is pivotal in the renal elimination of several positively charged molecules. OCT2 mode of transport is profoundly influenced by the level of membrane cholesterol. The aim of this study was to investigate the effect of oxidized cholesterol on OCT2 transport activity in human embryonic kidney 293 cells stably transfected with OCT2 (OCT2-HEK293) and in primary renal proximal tubular epithelial cells (RPTEC). Cholesterol was exchanged with 7-ketocholesterol, the main product of cholesterol auto-oxidation, by exposing cells to sterol-saturated methyl-ß-cyclodextrin (mßcd). After a 30 min-exposure, approximately 50% of the endogenous cholesterol was replaced by 7-ketocholesterol without significant changes in total sterol level. In the presence of 7-ketocholesterol, [3H]1-methyl-4-phenylpyridinium (MPP+) uptake was significantly reduced in both cell lines. 7-ketocholesterol incorporation did not affect lipid raft integrity, nor OCT2 surface expression and spatial organization. The inhibitory effect of 7-ketocholesterol on MPP+ uptake was abolished by the presence of MPP+ in the trans-compartment. In the presence of 7-ketocholesterol, both Kt and Vmax of MPP+ influx decreased. Molecular docking using OCT2 structure in outward occluded conformation showed overlapping poses and similar binding energies between cholesterol and 7-ketocholesterol. The thermal stability of OCT2 was not changed when cholesterol was replaced with 7-ketocholesterol. We conclude that 7-ketocholesterol confers a higher rigidity to the carrier by reducing its conformational entropy, arguably as a result of changes in plasma membrane physical properties, thereby facilitating the achievement of a higher affinity state at the expense of the mobility and overall cycling rate of the transporter.

Effect of PCSK9 inhibition on plasma levels of small dense low density lipoprotein-cholesterol and 7-ketocholesterol.

BACKGROUND: Oxidized forms of cholesterol (oxysterols) are implicated in atherogenesis and can accumulate in the body via direct absorption from food or through oxidative reactions of endogenous cholesterol, inducing the formation of LDL particles loaded with oxidized cholesterol. It remains unknown whether drastic reductions in LDL-cholesterol (LDL-C) are associated with changes in circulating oxysterols and whether small dense LDL (sdLDL) are more likely to carry these oxysterols and susceptible to the effects of PCSK9 inhibition (PCSK9i). OBJECTIVE: We investigate the effect of LDL-C reduction accomplished via PCSK9i on changes in plasma levels of sdLDL-cholesterol (sdLDL-C) and a common, stable oxysterol, 7-ketocholesterol (7-KC), among 134 patients referred to our Preventive Cardiology clinic. METHODS: Plasma lipid panel, sdLDL-C, and 7-KC measurements were obtained from patients before and after initiation of PCSK9i. RESULTS: The intervention caused a significant lowering of LDL-C (-55.4 %). The changes in sdLDL-C levels (mean reduction 51.4 %) were highly correlated with the reductions in LDL-C levels (R = 0.829, p < 0.001). Interestingly, whereas changes in plasma free 7-KC levels with PCSK9i treatment were much smaller than (-6.6 %) and did not parallel those of LDL-C and sdLDL-C levels, they did significantly correlate with changes in triglycerides and very low-density lipoprotein-cholesterol (VLDL-C) levels (R = 0.219, p = 0.025). CONCLUSION: Our findings suggest a non-preferential clearance of LDL subparticles as a consequence of LDL receptor upregulation caused by PCSK9 inhibition. Moreover, the lack of significant reduction in 7-KC with PCSK9i suggests that 7-KC may be in part carried by VLDL and lost during lipoprotein processing leading to LDL formation.

7-Ketocholesterol plays a key role in cholesterol-induced hepatitis via macrophage and neutrophil infiltration.

This study sought to explore the role of 7-ketocholesterol (7-KC) in liver damage caused by high cholesterol intake and its potential pathological mechanism in mice. Our in vivo findings indicated that mice fed a high-cholesterol diet had elevated serum levels of 7-KC, accompanied by liver injury and inflammation, similar to human nonalcoholic steatohepatitis. Furthermore, the high-cholesterol diet induced neutrophil infiltration, which played a critical role in liver damage through myeloperoxidase (MPO) activity. Upon stimulation with 7-KC, macrophages exhibited increased expression of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2, as well as ATP-binding cassette transporter A1 (ABCA1) and ABCG1. Hepatocytes, on the other hand, exhibited increased expression of CXCL2 and ABCG1. The infiltration of neutrophils in the liver was primarily caused by CXCL1 and CXCL2, resulting in hepatocyte cell death due to elevated MPO activity. Our data also revealed that the activation of macrophages by 7-KC via ABCA1 or ABCG1 was not associated with lipid accumulation. Collectively, these findings suggest that high cholesterol-induced hepatitis in mice involves, at least partially, the recruitment of neutrophils to the liver by 7-KC-activated macrophages. This is mediated by increased expression of CXCL1 and CXCL2 through ABCA1 or ABCG1, which act as 7-KC efflux transporters. Additionally, hepatocytes contribute to this process by increased expression of CXCL2 through ABCG1. Therefore, our findings suggest that 7-KC may play a role in high cholesterol-induced hepatitis in mice by activating macrophages and hepatocytes, ultimately leading to neutrophil infiltration.

7ß-Hydroxycholesterol and 7-ketocholesterol: New oxidative stress biomarkers of sarcopenia inducing cytotoxic effects on myoblasts and myotubes.

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7ß-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7ß-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7ß-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7ß-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7ß-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7ß-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.

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.

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.

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.

Oxiapoptophagy in Age-Related Diseases. Comment on Ouyang et al. 7-Ketocholesterol Induces Oxiapoptophagy and Inhibits Osteogenic Differentiation in MC3T3-E1 Cells. Cells 2022, 11, 2882.

Due to the increase in life span and life expectancy, which can, however, be more or less pronounced depending on the economic, social and cultural context [...].

Dietary 7-ketocholesterol exacerbates myocardial ischemia-reperfusion injury in mice through monocyte/macrophage-mediated inflammation.

Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most abundant dietary oxysterols, causes inflammation and cardiovascular diseases. Here we show the deteriorating effects of dietary 7-KC on myocardial ischemia-reperfusion (IR) injury and detailed the molecular mechanisms. A high-fat high-cholesterol diet containing 7-KC (7KWD) for 3 weeks increased the plasma 7-KC level compared with high-fat high-cholesterol diet in mice. In wild-type mice but not in CCR2-/- mice, dietary 7-KC increased the myocardial infarct size after IR. Flow cytometry revealed that the ratio of Ly-6Chigh inflammatory monocytes to total monocytes was increased in the 7KWD group. Unbiased RNA sequencing using murine primary macrophages revealed that 7-KC regulated the expression of transcripts related to inflammation and cholesterol biosynthesis. We further validated that in vitro, 7-KC induced endoplasmic reticulum stress, mitochondrial reactive oxygen species production, and nuclear factor-kappa B activation, which are associated with increased mRNA levels of proinflammatory cytokines. Administration of N-acetyl-L-cysteine or siRNA-mediated knockdown of PKR-like endoplasmic reticulum kinase or endoplasmic reticulum oxidase 1α suppressed the levels of 7-KC-induced inflammation. Dietary 7-KC exacerbates myocardial IR injury through monocyte/macrophage-mediated inflammation. Endoplasmic reticulum stress and oxidative stress are involved in the 7-KC-induced proinflammatory response in macrophages.

7-Ketocholesterol Induces Oxiapoptophagy and Inhibits Osteogenic Differentiation in MC3T3-E1 Cells.

7-Ketocholesterol (7KC) is one of the oxysterols produced by the auto-oxidation of cholesterol during the dysregulation of cholesterol metabolism which has been implicated in the pathological development of osteoporosis (OP). Oxiapoptophagy involving oxidative stress, autophagy, and apoptosis can be induced by 7KC. However, whether 7KC produces negative effects on MC3T3-E1 cells by stimulating oxiapoptophagy is still unclear. In the current study, 7KC was found to significantly decrease the cell viability of MC3T3-E1 cells in a concentration-dependent manner. In addition, 7KC decreased ALP staining and mineralization and down-regulated the protein expression of OPN and RUNX2, inhibiting osteogenic differentiation. 7KC significantly stimulated oxidation and induced autophagy and apoptosis in the cultured MC3T3-E1 cells. Pretreatment with the anti-oxidant acetylcysteine (NAC) could effectively decrease NOX4 and MDA production, enhance SOD activity, ameliorate the expression of autophagy-related factors, decrease apoptotic protein expression, and increase ALP, OPN, and RUNX2 expression, compromising 7KC-induced oxiapoptophagy and osteogenic differentiation inhibition in MC3T3-E1 cells. In summary, 7KC may induce oxiapoptophagy and inhibit osteogenic differentiation in the pathological development of OP.

Cytoprotective activities of representative nutrients from the Mediterranean diet and of Mediterranean oils against 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cytotoxicity: Application to age-related diseases and civilization diseases.

7-ketocholesterol and 7ß-hydroxycholesterol are two oxysterols mainly formed by the autoxidation of cholesterol. These two molecules are interconvertible via specific enzymes. These two oxysterols are often observed at increased amounts in biological fluids as well as tissues and organs affected during age-related diseases and in diseases of civilization such as cardiovascular, neurodegenerative, and ocular diseases as well as type 2 diabetes and metabolic syndrome. Noteworthy, 7-ketocholesterol and 7ß-hydroxycholesterol induce oxidative stress and inflammation, which are frequently observed in patients with age-related and civilization diseases. For this reason, the involvement of these two oxysterols in the pathophysiology of these diseases is widely suspected. In addition, the toxicity of these oxysterols can lead to death by oxiapoptophagy characterized by oxidative stress, apoptosis induction and autophagy criteria. To prevent, or even treat, certain age-related or civilization diseases associated with increased levels of 7-ketocholesterol and 7ß-hydroxycholesterol, the identification of molecules or mixtures of molecules attenuating or inhibiting the toxic effects of these oxysterols allows to consider new treatments. In this context, many nutrients present in significant amounts in the Mediterranean diet, especially tocopherols, fatty acids, and polyphenols, have shown cytoprotective activities as well as several Mediterranean oils (argan and olive oils, milk thistle seed oil, and pistacia lentiscus seed oil). Consequently, a nutraceutical approach, rich in nutrients present in the Mediterranean diet, could thus make it possible to counteract certain age-related and civilization diseases associated with increased levels of 7-ketocholesterol and 7ß-hydroxycholesterol.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria.

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

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.

Effect of Withanolide A on 7-Ketocholesterol Induced Cytotoxicity in hCMEC/D3 Brain Endothelial Cells.

Withanolide A is a naturally occurring phytochemical that is found in Ashwagandha (Withania somnifera, fam. Solanaceae) or Indian Ginseng. In the current study, we elucidated the effect of withanolide A on 7-ketocholesterol (7KC) induced injury in hCMEC/D3 human brain endothelial cells. 7KC is a cholesterol oxidation product or oxysterol that is present in atherosclerotic plaques and is elevated in the plasma of patients with hypercholesterolemia and/or diabetes mellitus. Results showed that withanolide A significantly reduced the effects of 7KC, which include loss of endothelial cell viability, increase in expression of pro-inflammatory genes-IL-1ß, IL-6, IL-8, TNF-α, cyclooxygenase-2 (COX-2), increased COX-2 enzyme activity, increased ROS formation, increased expression of inducible nitric oxide synthase and genes associated with blood clotting, including Factor 2/thrombin, Factor 8, von Willebrand factor, and thromboxane A synthase, and increased human thrombin enzyme activity. Some of the above effects of withanolide A on 7KC were reduced in the presence of the glucocorticoid receptor antagonist, mifepristone (RU486). These findings suggest that the glucocorticoid receptor could play a role in the cytoprotective, antioxidant, and anti-clotting effects of withanolide A against 7KC. Further studies are necessary to elucidate the detailed mechanisms of action of withanolide A against oxysterol-induced injury.

Mammalian Target of Rapamycin Inhibitor Rapamycin Alleviates 7-Ketocholesterol Induced Inflammatory Responses and Vascular Endothelial Growth Factor Elevation by Regulating MAPK Pathway in Human Retinal Pigment Epithelium Cells.

Purpose: To validate the protective effect of a mammalian target of rapamycin (mTOR) inhibitor on human retinal pigment epithelium (RPE) cells challenged with 7-ketocholesterol (7-KC) and explored the underlying mechanisms. Methods: Human primary RPE (hRPE) cells and ARPE-19 cells were cultured with or without 10 nM of rapamycin for 6 h before being exposed to 10 µM of 7-KC for 24 h. The transcriptome of 7-KC challenged ARPE-19 cells was investigated by RNA sequencing (RNA-seq). The effects of 7-KC and rapamycin on the viability of ARPE-19 cells were measured with CCK-8. Gene expression was verified by real-time PCR, and protein levels were determined by ELISA or Western blotting. Results: The expression of IL-6, IL-8, and vascular endothelial growth factor (VEGF) in RPE cells was markedly increased after stimulation with 7-KC for 12/24 h compared with the controls. RNA-seq showed that a total of 10,243 genes were differentially expressed, with 5,518 genes upregulated and 4,725 genes downregulated between the 7-KC treated and the control group. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that 7-KC stimulation activated mTOR signaling and other pathways, including adherent junction, MAPK, and Wnt signalings. mTOR inhibitor rapamycin significantly suppressed the elevation of IL-6, IL-8, and VEGF stimulated by 7-KC. Rapamycin not only decreased the level of phosphorylated mTOR, P70S6K, 4EBP1 but also inhibited the activation of MAPK pathway. Conclusions: Inhibition of mTOR signaling pathway suppressed the elevation of inflammatory cytokines IL-6, IL-8, and the angiogenic agent VEGF induced by 7-KC. The protective effect of rapamycin was associated with its downregulation on MAPK pathway.

7-Ketocholesterol Induces Lipid Metabolic Reprogramming and Enhances Cholesterol Ester Accumulation in Cardiac Cells.

7-Ketocholesterol (7KCh) is a major oxidized cholesterol product abundant in lipoprotein deposits and atherosclerotic plaques. Our previous study has shown that 7KCh accumulates in erythrocytes of heart failure patients, and further investigation centered on how 7KCh may affect metabolism in cardiomyocytes. We applied metabolomics to study the metabolic changes in cardiac cell line HL-1 after treatment with 7KCh. Mevalonic acid (MVA) pathway-derived metabolites, such as farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, phospholipids, and triacylglycerols levels significantly declined, while the levels of lysophospholipids, such as lysophosphatidylcholines (lysoPCs) and lysophosphatidylethanolamines (lysoPEs), considerably increased in 7KCh-treated cells. Furthermore, the cholesterol content showed no significant change, but the production of cholesteryl esters was enhanced in the treated cells. To explore the possible mechanisms, we applied mRNA-sequencing (mRNA-seq) to study genes differentially expressed in 7KCh-treated cells. The transcriptomic analysis revealed that genes involved in lipid metabolic processes, including MVA biosynthesis and cholesterol transport and esterification, were differentially expressed in treated cells. Integrated analysis of both metabolomic and transcriptomic data suggests that 7KCh induces cholesteryl ester accumulation and reprogramming of lipid metabolism through altered transcription of such genes as sterol O-acyltransferase- and phospholipase A2-encoding genes. The 7KCh-induced reprogramming of lipid metabolism in cardiac cells may be implicated in the pathogenesis of cardiovascular diseases.

Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis.

Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule-cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell-mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid "shields" against self-destruction.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.