Soliman Khatib

Brain sterol dysregulation in sporadic AD and MCI: relationship to heme oxygenase-1.

The objective of this study was to ascertain the impact of aging and Alzheimer’s disease (AD) on brain cholesterol (CH), CH precursors, and oxysterol homeostasis. Altered CH metabolism and up‐regulation of heme oxygenase‐1 (HO‐1) are characteristic of AD‐affected neural tissues. We recently determined that HO‐1 over‐expression suppresses total CH levels by augmenting liver X receptor‐mediated CH efflux and enhances oxysterol formation in cultured astroglia. Lipids and proteins were extracted from postmortem human frontal cortex derived from subjects with sporadic AD, mild cognitive impairment (MCI), and no cognitive impairment (n = 17 per group) enrolled in the Religious Orders Study, an ongoing clinical‐pathologic study of aging and AD. ELISA was used to quantify human HO‐1 protein expression from brain tissue and gas chromatography–mass spectrometry to quantify total CH, CH precursors, and relevant oxysterols. The relationships of sterol/oxysterol levels to HO‐1 protein expression and clinical/demographic variables were determined by multivariable regression and non‐parametric statistical analyses. Decreased CH, increased oxysterol and increased CH precursors concentrations in the cortex correlated significantly with HO‐1 levels in MCI and AD, but not no cognitive impairment. Specific oxysterols correlated with disease state, increasing neuropathological burden, neuropsychological impairment, and age. A model featuring compensated and de‐compensated states of altered sterol homeostasis in MCI and AD is presented based on the current data set and our earlier in vitro work.

Human carotid atherosclerotic plaque increases oxidative state of macrophages and low-density lipoproteins, whereas paraoxonase 1 (PON1) decreases such atherogenic effects

Human atherosclerotic plaque contains a variety of oxidized lipids, which can facilitate further oxidation. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated esterase (lipolactonase), exhibiting antiatherogenic properties. The aims of the present study were to examine the oxidizing potency of the human carotid plaque lipid extract (LE), and the antiatherogenic role of PON1 on LE oxidation competence. Human carotid plaques were extracted by organic solvent, and the extract was incubated with lipoprotein particles, with macrophages, or with probes sensitive to oxidative stress, with or without preincubation with PON1, followed by oxidative-stress assessment. Our findings imply that the LE oxidized LDL, macrophages, and exogenous probes and decreases HDL-mediated cholesterol efflux from macrophages, in a dose-dependent manner. Incubation of PON1 with LE significantly affects LE composition, reduces LE atherogenic properties, and decreases the extracts total peroxide concentration by 44%, macrophage oxidation by 25%, and probe oxidation by up to 52%. We conclude that these results expand our understanding of how the plaque itself accelerates atherogenesis and provides an important mechanism for attenuation of atherosclerosis development by the antioxidant action of PON1 on the atherosclerotic plaque.

Inhibition of serotonin re-uptake by licorice constituents

Ovarian steroid hormones, estrogen and progestin, affect the function of the serotonin neural system by inhibiting serotonin re-uptake through allosteric interaction with the serotonin transporter (SERT) in a nongenomic mechanism. Blocking or reducing serotonin re-uptake at the synapse alleviates depression. The aim of this study was to test the effect of compounds of the isoflavan and isoflavene groups, subclasses of the flavonoids family, on serotonin re-uptake and to compare the results with the effect of other known phytoestrogens like genistein and daidzein to relate the activity of these compounds to their structure. The effect of these compounds on the re-uptake of radioactive serotonin was assayed in HEK-293 cells stably expressed the recombinant human serotonin transporter (hSERT). The results demonstrated that the isoflavans glabridin and 4′-O-methylglabridin (4′-OMeG) and the isoflavene glabrene inhibited serotonin re-uptake by 60, 53 and 47%, respectively, at 50 µM, whereas resorcinol, the isoflavan 2′-O-methylglabridin (2′-OMeG), and the isoflavones genistein and daidzein were inactive. The inhibition of serotonin re-uptake is dose dependant with glabridin and estradiol. These results emphasize the importance of the lipophilic part of the isoflavans, as well as the hydroxyl at position 2′ on ring B. In conclusion, this study showed that several isoflavans are unique phytoestrogens, which like estradiol, affects the serotonergic system and inhibits serotonin re-uptake and, thus, potentially may be beneficial for mild to moderate depression in pre- and postmenopausal women.

Human carotid lesion linoleic acid hydroperoxide inhibits paraoxonase 1 (PON1) activity via reaction with PON1 free sulfhydryl cysteine 284.

Paraoxonase 1 (PON1) is an HDL-associated lactonase with antiatherogenic properties. These include dampening the oxidation properties of human carotid lesion lipid extract (LLE), which in turn inactivates the enzyme. The aims of this study were to identify the PON1 inhibitor in LLE and explore the mechanism of inhibition. LLE inhibited both recombinant PON1 and HDL-PON1 lactonase activity in a dose- and time-dependent manner. Addition of antioxidants or electrophiles to LLE did not prevent PON1 inhibition. LLE was unable to inhibit a PON1 mutant lacking Cys284, whereas it did inhibit all other PON1 mutants tested. The inhibitor in the LLE was identified as linoleic acid hydroperoxide (LA-OOH) and inhibition was specific to this hydroperoxide. During its inhibition, PON1 acted like a peroxidase enzyme, reducing LA-OOH to LA-hydroxide via its Cys284. A similar reaction occurred with external thiols, such as DDT or cysteine, which also prevented PON1 inhibition and restored enzyme activity after inhibition. Thus, the antiatherogenic properties of HDL could be, at least in part, related to the sulfhydryl-reducing characteristics of its associated PON1, which are further protected and recycled by the sulfhydryl amino acid cysteine.

Characterization of the PON1 active site using modeling simulation, in relation to PON1 lactonase activity

Paraoxonase1 (PON1) is a HDL bound enzyme and many of the anti-atherogenic properties of HDL are attributed to PON1. The enzyme precise mechanism of protective action and its endogenous substrate remain elusive. PON1 hydrolyzes organophosphates, arylesters and lactones, whereas the lactones activity is assumed as the physio/pathological one. This study is aimed to predict the location of the PON1 active site within PON1 crystal structure, and the lactone structure suitability as PON1 ligand, by employing modeling techniques. Based on such calculations the ligands-PON1 interactions were characterized, and relating lactones rate of hydrolysis revealed an inverse correlation with the docking energy of the ligands-PON1 complex, and a direct correlation with the lactone side chain length. In conclusion, this study characterized the PON1 possible active site and proposes a tool which may make it possible to envisage the structure of potential endogenous and exogenous lactones such as the PON1 ligand.

Characterization of oxidative stress in blood from diabetic vs. hypercholesterolaemic patients, using a novel synthesized marker

Abstract In the present study, we extend our novel concept of designing and using exogenous markers for the characterization of oxidative stress (OS) and OS-associated diseases. The aim was to use such a synthetic compound as a tool for studying OS in blood from diabetic and hypercholesterolaemic (Hc) patients. The marker used N-linoleoyl tyrosine (LT) was constructed from tyrosine and linoleic acid (LA); both components are known to be easily oxidized upon exposure to different types of reactive oxygen/nitrogen species (ROS/RNS), and to generate specific oxidized products, depending on the type of oxidants present in vivo. Using the LT probe, we showed that the ratios of oxidized LT to total LT (Ox-LT/LT) is significantly higher in blood samples obtained from diabetic patients, than in Hc patients or healthy control subjects. LC/MS analysis revealed that blood from diabetic patients oxidizes the marker with predominant formation of Ox-LT hydroperoxide (LT-OOH) and epoxide (epoxy-LT), where the LA moiety is oxidized to hydroperoxide and to epoxide, respectively. Analysis of oxysterol levels in these samples (GC/MS) revealed that the blood of both diabetic and Hc patients contained significantly more oxysterols than blood of control subjects. Consumption of pomegranate juice by diabetic patients for 3 months suppressed their blood capacity to oxidize the LT and similarly also reduced their blood oxysterol/total cholesterol ratio by 93%. The use of an exogenous marker to characterize OS in blood samples yields important information on the extent of OS, and can provide a fingerprint for the early identification of different pathological conditions associated with OS.

Paraoxonase 1 protects macrophages from atherogenicity of a specific triglyceride isolated from human carotid lesion

Human atherosclerotic lesions contain oxidized lipids that facilitate further oxidation of macrophages, LDLs, and oxidative stress (OS)-sensitive markers and inhibit the antiatherogenic enzyme paraoxonase 1 (PON1). Our aim was to isolate and identify the oxidizing agent in a human atherosclerotic lesion lipid extract (LLE) and to explore the mechanisms of oxidation and of PON1s effect on the oxidizing agent. Of the five main fractions separated from the LLE, only fraction 2 (F2) promoted macrophage reactive oxygen species (ROS) production via a mechanism requiring mitochondrial involvement, whereas the NADPH oxidase system was not involved. Incubation of F2 with PON1 abridged the formers peroxide value and reduced its capacity to oxidize OS markers. The active agent was a triglyceride composed of palmitic, oleic, and linoleic acids, with 0.3% of its linoleic moiety in oxidized form. Incubation of either F2 or an identical synthetic triglyceride with PON1 reduced their ability to oxidize macrophages, without affecting cellular accumulation of triglycerides. We conclude that macrophage ROS production by LLE occurs in the presence of a specific triglyceride and requires mitochondrial involvement. Lipid peroxide in the triglyceride can also facilitate lipid autoxidation. Both atherogenic pathways are suppressed by PON1, which acts as an antiatherogenic element.

Human atherosclerotic plaque lipid extract promotes expression of proinflammatory factors in human monocytes and macrophage-like cells

OBJECTIVE The potential of the atherogenic human carotid plaque to stimulate the inflammatory process was examined in human monocytes and macrophages, in vitro. METHODS AND RESULTS Exposure of monocytes to human carotid plaque lipid extract (LE) elevated the transcription level of the proinflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α, by 2.9 and 100.2 fold, respectively (as determined by real time PCR), and induced TNF-α secretion (as measured by enzyme-linked immunosorbent assay). Furthermore, LE caused an increase of 1.3-3.1 fold in the mRNA expression of the proinflammatory factors, IL-1β, IL-6, TNF-α, cyclooxygenase-2 and intercellular adhesion molecule-1, in macrophage-like cells. In order to investigate the proinflammatory components in the extract, two fractions, obtained by silica gel separation of LE, were characterized. The cholesterol-oxysterol rich fraction was found to have the most significant proinflammatory effect. It caused an increase in TNF-α expression in monocytes, and upregulated IL-6, TNF-α, intercellular adhesion molecule-1 and cyclooxygenase 2 by 1.5-2.5 fold in macrophages. The triglyceride fraction had almost no effect on the cells. CONCLUSIONS The human carotid plaque lipid extract was demonstrated to promote inflammation, in vitro. These data support the atherogenic character of the plaque and imply that its lipid composition may have ramifications on the progress of atherosclerosis.

Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia

Up‐regulation of heme oxygenase‐1 (HO‐1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer‐diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO‐1 over‐expression and its byproducts iron (Fe2+), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO‐1/LXR interactions were also investigated in the context of CH efflux. hHO‐1 over‐expression for 3 days (∼2–3‐fold increase) resulted in a 30% increase in CH biosynthesis and a two‐fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM‐3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three‐fold. Co‐treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer’s disease and other neuropathological states, glial HO‐1 induction may transduce ambient noxious stimuli (e.g. β‐amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO‐1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.

Glabridin protects paraoxonase 1 from linoleic acid hydroperoxide inhibition via specific interaction: A fluorescence-quenching study

The enzyme paraoxonase 1 (PON1) binds to high-density lipoprotein (HDL) and is responsible for many of HDLs antiatherogenic properties. We previously showed that recombinant PON1 is inhibited by linoleic acid hydroperoxide (LA-OOH) present in the lipid fraction of the human carotid plaque (LLE) via oxidation of the enzymes Cys284 thiol. Here we explore the effect of glabridin, an isoflavan isolated from licorice root, on preventing LA-OOHs inhibitory effect on rePON1 using the tryptophan-fluorescence-quenching technique and modeling calculations. Glabridin significantly prevented rePON1 inhibition by LLE or oxidized linoleic acid (by 22% and 15%, respectively), whereas ascorbic acid and Trolox, strong antioxidants, had no effect. Glabridin quenched the intrinsic fluorescence of rePON1 in a concentration-dependent manner. Binding parameters and modeling calculations demonstrated a major role for hydrophobic forces in the rePON1-glabridin interaction, indicating that it is not the antioxidant capacity of glabridin that protects rePON1 from LA-OOH inhibition, but rather its specific interaction with the enzyme.