Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.

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.

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.

7-Oxygenated cholesterol molecules differentially affect the expression of zonula occludens-1 in vascular smooth muscle cells and monocyte/macrophage cells.

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 7ß-hydroxycholesterol (7ßOHChol) 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 7ßOHChol. 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 7ßOHChol. 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.

Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium.

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 7ß-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 ±â€¯17%, 40 ±â€¯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.

Mitochondrial Oxidative Stress Promotes Atherosclerosis and Neutrophil Extracellular Traps in Aged Mice.

RATIONALE: Mitochondrial oxidative stress (mitoOS) has been shown to be increased in various cell types in human atherosclerosis and with aging. However, the role of cell type-specific mitoOS in atherosclerosis in the setting of advanced age and the molecular mechanisms remains to be determined in vivo. OBJECTIVE: The aim of this study was to examine the role of myeloid cell mitoOS in atherosclerosis in aged mice. APPROACH AND RESULTS: Lethally irradiated low-density lipoprotein receptor-deficient mice (Ldlr-/-) were reconstituted with bone marrow from either wild-type or mitochondrial catalase (mCAT) mice. mCAT transgenic mice contain ectopically expressed human catalase gene in mitochondria, which reduces mitoOS. Starting at the age of 36 weeks, mice were fed the Western-type diet for 16 weeks. We found that mitoOS in lesional myeloid cells was suppressed in aged mCAT→Ldlr-/- chimeric mice compared with aged controls, and this led to a significant reduction in aortic root atherosclerotic lesion area despite higher plasma cholesterol levels. Neutrophil extracellular traps (NETs), a proinflammatory extracellular structure that contributes to atherosclerosis progression, were significantly increased in the lesions of aged mice compared with lesions of younger mice. Aged mCAT→Ldlr-/- mice had less lesional neutrophils and decreased NETs compared with age-matched wild-type→Ldlr-/- mice, whereas young mCAT→ and wild-type→Ldlr-/- mice had comparable numbers of neutrophils and similar low levels of lesional NETs. Using cultured neutrophils, we showed that suppression of mitoOS reduced 7-ketocholesterol-induced NET release from neutrophils of aged but not younger mice. CONCLUSIONS: MitoOS in lesional myeloid cells enhanced atherosclerosis development in aged mice, and this enhancement was associated with increased lesional NETs. Thus, mitoOS-induced NET formation is a potentially new therapeutic target to prevent atherosclerosis progression during aging.

Oxidized LDL-Exposed Human Macrophages Display Increased MMP-9 Expression and Secretion Mediated by Endoplasmic Reticulum Stress.

Oxidatively modified low-density lipoproteins (oxLDL) alter the proper function of the endoplasmic reticulum (ER), inducing ER stress (ERS), which consequently activates inflammatory pathways in macrophages. Matrix metalloproteinase-9 (MMP-9) is the main protease acting on the degradation of the extracellular matrix and the ensuing destabilization of the atherosclerotic plaque. We aimed to investigate whether ERS induced by oxLDL or tunicamycin (TM) in human macrophages is associated with the stimulation of MMP-9 expression and secretion. The results showed that oxLDL induced in THP-1 macrophages: (i) increase of MMP-9 gene expression and its pro-form secretion, (ii) intracellular accumulation of 7-ketocholesterol, (iii) ERS activation (increased eIF2α phosphorylation, XBP1 and CHOP mRNA levels, and Grp78 protein expression), and (iv) oxidative stress (increased levels of reactive oxygen species and NADPH oxidase activity). Incubation of macrophages with ERS inducer, TM determined the secretion of both pro- and active-form of MMP-9 and oxidative stress. Treatment of oxLDL or TM-incubated cells with ERS inhibitor, sodium phenylbutyrate decreased MMP-9 gene expression, secretion, and activity. The inhibitor of NADPH oxidase, apocynin, decreased XBP-1 and CHOP mRNA levels, and MMP-9 gene expression and secretion in oxLDL-exposed cells. In conclusion, oxLDL stimulate MMP-9 expression and secretion in human macrophages by mechanisms involving ERS. J. Cell. Biochem. 118: 661-669, 2017. © 2016 Wiley Periodicals, Inc.

Caspase-3 activation decreases lipid order in the outer plasma membrane leaflet during apoptosis: A fluorescent probe study.

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 (0-24h) 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.

Oxysterols Versus Cholesterol in Model Neuronal Membrane. I. The Case of 7-Ketocholesterol. The Langmuir Monolayer Study.

Oxysterols are products of cholesterol oxidation. They can be formed endogenously (in both enzymatic and non-enzymatic reactions) as well as exogenously (delivered with food). Recent studies clearly demonstrate cytotoxic properties of these compounds, being mainly due to their incorporation into natural lipid bilayers. This process can influence mechanical and physicochemical properties of biomembrane-mainly by modifying the interactions between its components, which may result in the disruption of proper functioning of cell membrane and could lead to its degradation. Therefore, it can be assumed that oxysterols may affect the initiation of neurodegenerative diseases, including Alzheimer's disease. However, the mode of action of these molecules at the molecular level is not fully known. To get a better understanding of the role of oxysterols in neurodegeneration, it is of great importance to examine mutual interactions between oxysterols and neuronal membrane components. One of the most promising techniques that can be used to analyze such interactions is the Langmuir monolayer technique. In this work, we have prepared an artificial neuronal membrane modeled as multicomponent Langmuir monolayer built up with cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and sphingomyelin (SM). To examine whether there are any changes in the membrane properties under oxidative stress, in this paper we have investigated the impact of the representative ring-oxidized oxysterol: 7-ketocholesterol (7-KC). Our results show that replacing cholesterol with 7-KC increases the interaction between molecules in the model membrane.

Biotransformation of cholesterol and 16,17-alpha epoxypregnenolone by novel Cladosporium sp. strain IS547.

Nowadays, there are a few steroid drugs or intermediates that have been obtained via the transformation of microorganisms, and many strains of transformed steroids have not been found yet. Therefore, it is very significant to screen for the strains that have the abilities to transform steroids to produce valuable products. This study has focused on the screen and identification of strains, the structural identification of converted products, and the optimization of transformation conditions, as well as the establishment of transformation systems. A soil microbiota was screened for strain involved in the biotransformation of steroids. A new isolate IS547 is capable of converting a variety of steroids (such as cholesterol, ergosterol, hydrocortisone, progesterone, pregnenolone, and 16,17-alpha-epoxypregnenolone). Based on the 18S rDNA gene sequence comparison, the isolate IS547 has been demonstrated to be very closely related to Cladosporium sp. genus. Present paper is the first report regarding the microbial transformation by Cladosporium sp. to produce active intermediates, which include 7-hydroxy cholesterol, 20-droxyl-16α,17α-epoxypregna-4-dien-3-one, 7-ketocholesterol, and 7-droxyl-16α,17α-epoxypregna-4-dien-3,20-dione. Under the optimum conditions, the yields of product 3 and product 4 were 20.58 and 17.42%, respectively, higher than that prior to the optimization. The transformation rate increased significantly under the optimum fermentation conditions. This study describes an efficient, rapid, and inexpensive biotransformation system for the production of active pharmaceutical intermediates.

Evaluation of plasma cholestane-3ß,5α,6ß-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism.

Oxysterols are intermediates of cholesterol metabolism and are generated from cholesterol via either enzymatic or nonenzymatic pathways under oxidative stress conditions. Cholestan-3ß,5α,6ß-triol (C-triol) and 7-ketocholesterol (7-KC) have been proposed as new biomarkers for the diagnosis of Niemann-Pick type C (NP-C) disease, representing an alternative tool to the invasive and time-consuming method of fibroblast filipin test. To test the efficacy of plasma oxysterol determination for the diagnosis of NP-C, we systematically screened oxysterol levels in patients affected by different inherited disorders related with cholesterol metabolism, which included Niemann-Pick type B (NP-B) disease, lysosomal acid lipase (LAL) deficiency, Smith-Lemli-Opitz syndrome (SLOS), congenital familial hypercholesterolemia (FH), and sitosterolemia (SITO). As expected, NP-C patients showed significant increase of both C-triol and 7-KC. Strong increase of both oxysterols was observed in NP-B and less pronounced in LAL deficiency. In SLOS, only 7-KC was markedly increased, whereas in both FH and in SITO, oxysterol concentrations were normal. Interestingly, in NP-C alone, we observed that plasma oxysterols correlate negatively with patient's age and positively with serum total bilirubin, suggesting the potential relationship between oxysterol levels and hepatic disease status. Our results indicate that oxysterols are reliable and sensitive biomarkers of NP-C.

Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2α and SOAT1 followed by selective efflux to HDL.

Accumulation of 7-ketocholesterol (7KCh) in tissues has been previously associated with various chronic aging diseases. Orally ingested 7KCh is readily metabolized by the liver and does not pose a toxicity threat. However, 7KCh formed in situ, usually associated with lipoprotein deposits, can adversely affect surrounding tissues by causing inflammation and cytotoxicity. In this study we have investigated various mechanisms for extra-hepatic metabolism of 7KCh (e.g. hydroxylation, sulfation) and found only esterification to fatty acids. The esterification of 7KCh to fatty acids involves the combined action of cytosolic phospholipase A2 alpha (cPLA2α) and sterol O-acyltransferase (SOAT1). Inhibition of either one of these enzymes ablates 7KCh-fatty acid ester (7KFAE) formation. The 7KFAEs are not toxic and do not induce inflammatory responses. However, they can be unstable and re-release 7KCh. The higher the degree of unsaturation, the more unstable the 7KFAE (e.g. 18:0>18:1>18:2>18:3≫20:4). Biochemical inhibition and siRNA knockdown of SOAT1 and cPLA2α ablated the 7KFAE synthesis in cultured ARPE19 cells, but had little effect on the 7KCh-induced inflammatory response. Overexpression of SOAT1 reduced the 7KCh-induced inflammatory response and provided some protection from cell death. This effect is likely due to the increased conversion of 7KCh to 7KFAEs, which reduced the intracellular 7KCh levels. Addition of HDL selectively increased the efflux of 7KFAEs and enhanced the effect of SOAT1 overexpression. Our data suggests an additional function for HDL in aiding extra-hepatic tissues to eliminate 7KCh by returning 7KFAEs to the liver for bile acid formation.

7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.

Hypertension is a high-risk symptom in atherosclerotic patients, and vascular rigidity is one of the main factors leading to hypertension. ß1-Subunit of BKCa channel (KCNMB1; MaxiKß1) has been reported as a modulator of vascular flexibility. To determine the relationship between atherosclerosis and KCNMB1, we studied some atherogenic factors affecting vascular tone. Blood of atherosclerotic patients shows increased concentration of 7-ketocholesterol (7K), which has been studied as a harmful lipid to blood vessels. Our data showed that KCNMB1 was significantly down-regulated in the presence of 7K, in a dose-/time-dependent manner in vascular smooth muscle cells (VSMCs). And, the reduction of KCNMB1 was confirmed in cell images of 7K-stimulated VSMCs and in vessel tissue images of ApoE knock-out mice. To determine whether aryl hydrocarbon receptor (AhR) was involved in the reduction of KCNMB1 by 7K-stimulation, protein level of AhR was analyzed by Western blot. Our data showed that the reduction of KCNMB1 was modulated through the AhR pathway. In conclusion, results of our study suggest that 7K induces the reduction of KCNMB1 through the AhR pathway.

Cholesterol hydroperoxides and their degradation mechanism.

Cholesterol is one of the oxidizable lipids constituting biomembranes and plasma lipoproteins. Cholesterol hydroperoxides (Chol-OOH) are the primary products if cholesterol is subjected to attack by reactive oxygen species. In particular, singlet molecular oxygen reacts with cholesterol to yield cholesterol 5α-hydroperoxide as the major hydroperoxide species. Chol-OOH may accumulate in biological systems because of its resistance to glutathione-dependent enzymatic detoxification reactions. Their degradation products (including hydroxycholesterol and 7-ketocholesterol) participate in the pathophysiological functions of oxysterols. Highly reactive cholesterol 5,6-secosterol present in atherosclerotic lesions can be derived from the degradation of cholesterol 5α-hydroperoxide. Chol-OOH themselves may affect the lipid rafts of biomembranes, thereby leading to the modification of signal transduction pathways.

Oxysterol mixture in hypercholesterolemia-relevant proportion causes oxidative stress-dependent eryptosis.

BACKGROUND/AIMS: Oxysterol activity on the erythrocyte (RBC) programmed cell death (eryptosis) had not been studied yet. Effects of an oxysterol mixture in hyper-cholesterolemic-relevant proportion, and of individual compounds, were investigated on RBCs from healthy humans. METHODS: Membrane phosphatidylserine (PS) externalization, calcium entry, ROS production, amino-phospholipid translocase (APLT) activity were evaluated by cytofluorimetric assays, cell volume from forward scatter. Prostaglandin PGE2 was measured by ELISA; GSH-adducts and lipoperoxides by spectrophotometry. Involvement of protein kinase C and caspase was investigated by inhibitors staurosporin, calphostin C, and Z-DEVD-FMK, respectively. RESULTS: Oxysterols caused PS externalization and cell shrinkage, associated with PGE2release, opening of PGE2-dependent calcium channels, ROS production, GSH depletion, membrane lipid oxidation. Addition of antioxidants prevented Ca(2+) influx and eryptosis. Calcium removal prevented cell shrinkage, with small effect (-20%) on the PS exposure, whereas ROS generation was unaltered. Either in the presence or absence of calcium i) oxysterols inhibited APLT, ii) staurosporin, calphostin C, Z-DEVD-FMK blunted and iii) antioxidants fully prevented the oxysterol-induced PS externalization. Only 7-ketocholesterol and cholestan-3ß,5α,6ß-triol were individually active. Eryptosis was observed in RBCs isolated after ex vivo spiking of human whole blood with the oxysterol mixture. CONCLUSIONS: Oxysterols induce an oxidative stress-dependent eryptosis, involving calcium-independent mechanisms. Eryptotic activity of oxysterols may be relevant in vivo.

7-Ketocholesterol as marker of cholesterol oxidation in model and food systems: when and how.

Cholesterol can undergo oxidation through enzymatic or chemical mechanisms, generating a wide range of oxidation products (COPs) with adverse biological effects. COPs are characterized by different functional groups and are produced in different ratios or amounts, depending on the treatment and storage conditions. To follow the cholesterol oxidation process, 7-ketocholesterol (7-KC) has been often used as an oxidation marker in both model and food systems, since it is easily formed and is one of the most representative ring COPs. However, 7-KC does not always rise with increasing time/temperature conditions, especially in complex systems and high-protein or extensively processed foods. The following review provides a critical picture of the utilization of 7-KC as a cholesterol oxidation marker in model and food systems, focusing on the possible causes and effects of the different behaviours and trends, as well as on the advantages and disadvantages of using 7-KC when the extent of cholesterol oxidation is to be assessed.

Oxysterols and symptomatic versus asymptomatic human atherosclerotic plaque.

Atherosclerosis is the most common cause of mortality in the Western world, contributing to about 50% of all deaths. Atherosclerosis is characterized by deposition of lipids onto the coronary or carotid arterial wall and formation of an atherosclerotic plaque. Atherosclerotic plaques are categorized into two groups: symptomatic and asymptomatic. The symptomatic plaques tend to be unstable and prone to rupture, and are associated with an increase in ischemic events. Oxysterols, products of cholesterol oxidation, are cytotoxic materials. Their level and type may be associated with plaque formation, development and stability. Oxysterols stimulate the formation of foam cells, advance atherosclerotic plaque progression, and contribute to plaque vulnerability and instability due to their cytotoxicity and their ability to induce cell apoptosis. Studies indicate that plasma 7ß-OH CH level can be used as a biomarker for detecting carotid and coronary artery disease. Further clinical studies are needed to evaluate the potential of oxysterols for use as biomarkers for plaque vulnerability and instability. The identification of biomarkers in the blood that can distinguish between symptomatic and asymptomatic plaques remains an unresolved issue.

Diverse functions of 24(S)-hydroxycholesterol in the brain.

24(S)-hydroxycholesterol (24S-OHC) which is enzymatically produced in the brain plays important physiological roles in maintaining brain cholesterol homeostasis. We found that 24S-OHC at sub-lethal concentrations down-regulated amyloid precursor protein (APP) trafficking via enhancement of the complex formation of APP with up-regulated glucose-regulated protein 78, an endoplasmic reticulum chaperone. In accordance with this mechanism, 24S-OHC suppressed amyloid-ß production in human neuroblastoma SH-SY5Y cells. Furthermore, 24S-OHC at sub-lethal concentrations induced adaptive responses via transcriptional activation of the liver X receptor signaling pathway, thereby protecting neuronal cells against the forthcoming oxidative stress induced by 7-ketocholesterol. On the other hand, we found that high concentrations of 24S-OHC induced apoptosis in T-lymphoma Jurkat cells which endogenously expressed caspase-8, and induced necroptosis - a form of programmed necrosis - in neuronal SH-SY5Y cells which expressed no caspase-8. In this Article, we show the diverse functions of 24S-OHC and consider the possible importance of controlling 24S-OHC levels in the brain for preventing neurodegenerative diseases.

7-ketocholesterol accumulates in ocular tissues as a consequence of aging and is present in high levels in drusen.

We analyzed by LCMS lipid extracts of lens, retina (MNR) and RPE/Choroid (MPEC) from macaque monkeys 2-25 yr in age to determine their content of 7-ketocholesterol (7KCh) as function of age. In addition we also analyzed drusen capped with retinal pigment epithelium (RPE), RPE, and neural retina from human donors age 72-95 yr. The lowest 7KCh levels were found in monkey lens (<0.5-3.5 pmol 7KCh per nmol Ch), the second highest in MNR (1-15 pmol/nmol), and the highest in MPEC (1 to >60 pmol/nmol). Despite individual variability all three tissues demonstrated a strong age-related increase. In older human donors 7KCh levels were significantly higher. The levels in human neural retina ranged from 8 to 20 pmol/nmol, similar to the oldest monkeys, but 7-KCh levels in RPE ranged from 200 to 17,000 pmol/nmol, and in RPE-capped drusen from 200 to 2000 pmol/nmol, levels that would be lethal in most cultured cell systems. Most of the 7KCh is sequestered and not readily available to the surrounding tissue, based on published histochemical evidence that extracellular cholesterol (Ch) and cholesteryl fatty acid esters (CEs) are highly concentrated in Bruch's membrane and drusen. However, adjacent tissues, especially RPE but also choriocapillaris endothelium, could be chronically inflamed and in peril of receiving a lethal exposure. Implications for initiation and progression of age-related macular degeneration are discussed.

[Screening and optimization of cholesterol conversion strain].

OBJECTIVE: Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized. The transformation products were identified. Fermentation conditions were optimized for conversion. METHODS: Cholesterol was used as sole carbon source to isolate strain SE-1. Morphology, physiological and biochemical characteristics of strain SE-1 were studied. 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fermentation supernatants were extracted with chloroform, the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20. Their structures were identified by 1H-NMR and 13C-NMR. Fermentation medium including carbon and nitrogen, methods of adding substrates and fermentation conditions for Strain SE-1 were optimized. RESULTS: Strain SE-1 was a Gram-negative bacterium, exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis. The sequence analysis of 16S rRNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B. cepacia (Genbank No. U96927). The similarity was 99%. The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products, 7beta-hydroxycholesterol and the minor product was 7-oxocholesterol. The optimum culture conditions were: molasses 5%, (NH4 )2SO4 0.3%, 4% of inoculation, pH 7.5 and 36 degrees C. Under the optimum culture condition, the conversion rate reached 34.4% when concentration of cholesterol-Tween 80 was 1 g/L. Cholesterol 7beta-hydroxylation conversion rate under optimal conditions was improved by 20.8%. CONCLUSION: Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale.

[The effect of 7-ketocholesterol on surface ICAM-1 and PECAM-1 expression in human aortic endothelial cells]. / Ocena wplywu 7-ketocholesterolu na powierzchniowa ekspresje molekul adhezyjnych ICAM-1 i PECAM-1 w ludzkich komórkach sródblonka aortalnego.

UNLABELLED: Proatherogenic factors lead to activation of endothelial cells, which symptom is an increased expression of surface adhesion molecules enabling the initiation of a local inflammatory response. 7-ketocholesterol (7-KCH) is a product of oxidation of cholesterol with proven pro-apoptotic effect on the cells of the vessel wall. So far, however, the impact of 7-KCH on surface expression of adhesion molecules has not been assessed. The aim of this study was to evaluate the influence of 7-KCH on the surface expression of adhesion molecules--intracellular adhesion molecule 1 (ICAM-1, CD 54) and .platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31) on human aortic endothelial cells (HAECs). MATERIAL AND METHODS: After treatment with 7-KCH surface expression of adhesion molecules on HAEC was measured with antihuman CD31 and anti-human CD54 antibodies using flow cytometer. RESULTS: 7-KCH significantly increases percentages of CD 54 on viable HEAC, but does not affect expression of CD 31. CONCLUSION: 7-KCH may enhance the initiation of a local inflammatory response in atherosclerosis by increasing the expression of ICAM-1.