Jacob Vaya

Reduced Progression of Atherosclerosis in Apolipoprotein E–Deficient Mice Following Consumption of Red Wine, or Its Polyphenols Quercetin or Catechin, Is Associated With Reduced Susceptibility of LDL to Oxidation and Aggregation

The effect of consuming red wine, or its major polyphenol constituents catechin or quercetin, on the development of atherosclerotic lesions, in relation to the susceptibility of plasma LDL to oxidation and to aggregation, was studied in atherosclerotic apolipoprotein E deficient (E degree) mice. Forty E degree mice at the age of 4 weeks were divided into four groups, 10 mice in each group, and were supplemented for up to 6 weeks in their drinking water with placebo (1.1% alcohol); catechin or quercetin (50 micrograms/d per mouse), or red wine (0.5 mL/d per mouse). Consumption of catechin, quercetin, or red wine had no effect on plasma LDL or HDL cholesterol levels. The atherosclerotic lesion area was smaller in the treated mice by 39%, 46%, and 48%, respectively, in comparison with E degree mice that were treated with placebo. In accordance with these findings, cellular uptake of LDL derived after catechin, quercetin, or red wine consumption was found to be reduced by 31%, 40%, and 52%, respectively. These results were associated with reduced susceptibility to oxidation (induced by different modes such as copper ions, free radical generator, or macrophages) of LDL isolated after red wine or quercetin and, to a lesser extent after catechin consumption, in comparison with LDL isolated from the placebo group. Similar results were obtained when LDL was preincubated in vitro with red wine or with the polyphenols prior to its oxidation. Even in the basal oxidative state (not induced oxidation), LDL isolated from E degree mice that consumed catechin, quercetin, or red wine for 2 weeks was found to be less oxidized in comparison with LDL isolated from E degree mice that received placebo, as evidenced by 39%, 48%, and 49% reduced content of LDL-associated lipid peroxides, respectively. This effect could be related to enhanced serum paraoxonase activity in the polyphenol-treated mice. LDL oxidation was previously shown to lead to its aggregation. The present study demonstrated that the susceptibility of LDL to aggregation was reduced in comparison with placebo-treated mice, by 63%, 48%, or 50% by catechin, quercetin, and red wine consumption, respectively, and this effect could be shown also in vitro. The inhibition of LDL oxidation by polyphenols could be related, at least in part, to a direct effect of the polyphenols on the LDL, since both quercetin and catechin were found to bind to the LDL particle via the formation of an ether bond. We thus conclude that dietary consumption by E degree mice of red wine or its polyphenolic flavonoids quercetin and, to a lesser extent, catechin leads to attenuation in the development of the atherosclerotic lesion, and this effect is associated with reduced susceptibility of their LDL to oxidation and aggregation.

Human Serum Paraoxonases (PON1) Q and R Selectively Decrease Lipid Peroxides in Human Coronary and Carotid Atherosclerotic Lesions PON1 Esterase and Peroxidase-Like Activities

BACKGROUND Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. METHODS AND RESULTS The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions. CONCLUSIONS We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.

Oxysterols, cholesterol homeostasis, and Alzheimer disease

Aberrant cholesterol metabolism has been implicated in Alzheimer disease (AD) and other neurological disorders. Oxysterols and other cholesterol oxidation products are effective ligands of liver X activated receptor (LXR) nuclear receptors, major regulators of genes subserving cholesterol homeostasis. LXR receptors act as molecular sensors of cellular cholesterol concentrations and effectors of tissue cholesterol reduction. Following their interaction with oxysterols, activation of LXRs induces the expression of ATP‐binding cassette, sub‐family A member 1, a pivotal modulator of cholesterol efflux. The relative solubility of oxysterols facilitates lipid flux among brain compartments and egress across the blood‐brain barrier. Oxysterol‐mediated LXR activation induces local apoE biosynthesis (predominantly in astrocytes) further enhancing cholesterol re‐distribution and removal. Activated LXRs invoke additional neuroprotective mechanisms, including induction of genes governing bile acid synthesis (sterol elimination pathway), apolipoprotein elaboration, and amyloid precursor protein processing. The latter translates into attenuated β‐amyloid production that may ameliorate amyloidogenic neurotoxicity in AD brain. Stress‐induced up‐regulation of the heme‐degrading enzyme, heme oxygenase‐1 in AD‐affected astroglia may impact central lipid homeostasis by promoting the oxidation of cholesterol to a host of oxysterol intermediates. Synthetic oxysterol‐mimetic drugs that activate LXR receptors within the CNS may provide novel therapeutics for management of AD and other neurological afflictions characterized by deranged tissue cholesterol homeostasis.

Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins

The presence of oxidized sterols (oxysterols) in human serum and lesions has been linked to the initiation and progression of atherosclerosis. Data concerning the origin, identity and quantity of oxysterols in biological samples are controversial and inconsistent. This inconsistency may arise from different analytical methods or handling conditions used by different investigators. In the present study, oxysterol levels and distribution were analyzed by an optimized GC-MS method, in human atherosclerotic coronary and carotid lesions, in atherosclerotic apolipoprotein E deficient mice (E° mice) and in native and in vitro oxidized human low and high density lipoproteins. Oxysterol levels were analyzed with a limit of detection of 0.06 – 0.24 ng, with 25-hydroxycholesterol (25-OH) being the least sensitive. In human coronary and carotid lesions, obtained from endatherectomic samples, 27-hydroxycholesterol (27-OH) was the major oxysterol, with about 85% as sterols esterified to fatty acids. While total cholesterol and oxysterols levels were similar in both kinds of human lesions, oxysterol distribution was significantly different. In coronary lesions the mean levels of 27-OH and 7β-hydroxycholesterol (7β-OH) were 38% and 20% of total oxysterols, whereas in carotid lesions their mean levels were 66% and 5%, respectively. Unlike in human aortic lesions, 27-OH was entirely absent in E° mice, whereas the level of 7α-hydroxycholesterol (7α-OH) was 28% of the total oxysterols, vs. 5% in human coronary lesions. As 27-OH is an enzymatic product of cholesterol oxidation, this finding may indicate that such an enzymatic process does not take place in E° mice.

The effect of oxidative stress on ERα and ERβ expression

Abstract The formation of intracellular reactive oxygen and nitrogen species (ROS and RNS) has been implicated in the pathogenesis of a variety of diseases. In excess, ROS and their byproducts may cause oxidative damage and be cytotoxic to cells. Recently, it has been established that these oxidants can also act as subcellular messengers in gene regulatory and signal transduction pathways. Estrogen, on the other hand, is known to offer protection from coronary artery diseases in post-menopausal women and to be involved in various ROS-related diseases, such as Alzheimer’s and Parkinson’s diseases, diabetes and aging. The existence of estrogen receptors in these tissues lead us to investigate whether ROS can regulate their expression. We demonstrated here, for the first time, that oxidative stress induced by hydrogen peroxide (H2O2), Fe2+, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) and activated macrophages, affect the expression of estrogen receptors α and β (ERα and ERβ) differently, demonstrating cell-specific response which can be blocked by antioxidants. This data suggest that oxidative stress and the production of ROS/RNS function as physiological regulators of ERα and ERβ expression. This may provide a new insight into the ERβ-dependent protective action of estrogen and phytoestrogens in inflammation involving diseases, and may contribute to the development of novel therapeutic treatment strategies.

The Relation Between the Chemical Structure of Flavonoids and Their Estrogen-Like Activities

Estrogens are steroid hormones exhibiting a broad range of physiological activities, which are important in the homeostatic regulation of many cellular and biochemical events. Estradiol (E(2)) is an estrogen which is active in the development of the mammary glands and the uterus, in the maintenance of pregnancy and bone density, in protection from cardiovascular diseases, and in the relief of menopausal symptoms. However, it can also stimulate malignant growths, and hence contribute to the development of estrogen-dependent tumors, such as cancer of the breast and uterus. Phytoestrogens are plant-derived chemicals with estrogen-like activities, which could have a beneficial role in humans against estrogen deficiency. Several studies relate the lower incidence of estrogen deficiency-related disease among women in the Eastern word to a diet rich in phytoestrogens. This compound comprises a variety of structurally diverse chemicals, with flavonoids as their largest group. In the present review we summarize knowledge gained on the relationship between the structure of flavonoids to their ability to mimic estrogen activity, together with the methods adapted to perform these studies. The methods reviewed are qualitative and quantitative measurements of the estrogen-like activities of phytoestrogens, superposition analysis, docking analysis and quantitative structure-activity relationship (QSAR) models. The potential of various new flavonoid derivatives in hormone replacment therapy is discussed.

Estrogen-like activity of licorice root constituents: glabridin and glabrene, in vascular tissues in vitro and in vivo.

Post-menopausal women have higher incidence of heart diseases compared to pre-menopausal women, suggesting a protective role for estrogen. The recently Womens Health Initiative (WHI) randomized controlled trial concluded that the overall heart risk exceeded benefits from use of combined estrogen and progestin as hormone replacement therapy for an average of five years among healthy postmenopausal US women. Therefore, there is an urgent need for new agents with tissue-selective activity with no deleterious effects. In the present study, we tested the effects on vascular tissues in vitro and in vivo of two natural compounds derived from licorice root: glabridin, the major isoflavan, and glabrene, an isoflavene, both demonstrated estrogen-like activities. Similar to estradiol-17beta (E2), glabridin (gla) stimulated DNA synthesis in human endothelial cells (ECV-304; E304) and had a bi-phasic effect on proliferation of human vascular smooth muscle cells (VSMC). Raloxifene inhibited gla as well as E2 activities. In animal studies, both intact females or after ovariectomy, gla similar to E2 stimulated the specific activity of creatine kinase (CK) in aorta (Ao) and in left ventricle of the heart (Lv). Glabrene (glb), on the other hand, had only the stimulatory effect on DNA synthesis in vascular cells, with no inhibition by raloxifene, suggesting a different mechanism of action. To further elucidate the mechanism of action of glb, cells were pre-incubated with glb and then exposed to either E2 or to gla; the DNA stimulation at low doses was unchanged but there was abolishment of the inhibition of VSMC cell proliferation at high doses as well as inhibition of CK stimulation by both E2 and by gla. We conclude that glb behaved differently than E2 or gla, but similarly to raloxifene, being a partial agonist/antagonist of E2. Glabridin, on the other hand, demonstrated only estrogenic activity. Therefore, we suggest the use of glb with or without E2 as a new agent for modulation of vascular injury and atherogenesis for the prevention of cardiovascular diseases in post-menopausal women.

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.

Paraoxonase 1 activities, regulation, and interactions with atherosclerotic lesion

Purpose of review Improving serum levels of HDL and its subfractions, as well as, oxidative/inflammatory properties has become a fundamental aim in todays atherosclerosis research. Efforts to reach this goal are paralleled by achievements in drug development toward decreasing serum LDL levels and oxidative status. Recent findings Paraoxonase1 (PON1) is an HDL-associated enzyme that is deemed responsible for many of the HDLs antiatherogenic and cardioprotective characteristics. PON1 is highly sensitive to variations in its milieu, and endogenous compounds (fatty acids, phospholipids), nutritional ingredients (flavonoids and other antioxidants), and environmental elements (reactive nitrogen and oxygen species, metals, surfactants), significantly affect the enzymes activities. PON1 was shown to be responsible for some of the HDL antiatherogenic characteristics such as HDL-mediated cholesterol efflux from macrophages, and the inhibition of LDL oxidation. Summary The present review summarizes the recent literature related to various elements in PON1s milieu that regulate its activities, with an emphasis on its interrelation with components of the human carotid atherosclerotic lesion (plaque) which are in constant contact with circulating HDL-associated PON1.