The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation

The antioxidant activity of β-carotene and oxygenated carotenoids lutein, canthaxanthin, and astaxanthin was investigated during spontaneous and peroxyl-radical-induced cholesterol oxidation. Cholesterol oxidation, measured as generation of 7-keto-cholesterol (7-KC), was evaluated in a heterogeneous solution with cholesterol, AAPH, and carotenoids solubilized in tetrahydrofuran and in water, and in a homogeneous solution of chlorobenzene, with AIBN as a prooxidant. The formation of 7-KC was dependent on temperature and on cholesterol and prooxidant concentrations. All the carotenoids tested, exhibited significant antioxidant activity by inhibiting spontaneous, AAPH- and AIBN-induced formation of 7-KC, although the overall order of efficacy of these compounds was astaxanthin > canthaxanthin > lutein = β-carotene. The finding that carotenoids exert protective effects on spontaneous and free radical-induced cholesterol oxidation may have important beneficial effects on human health, by limiting the formation of atheroma and by inhibiting cholesterol oxidation in food processing or storage.     

Cholesterol photosensitized oxidation in food and biological systems

Lipid oxidation is one of the main chemical degradations occurring in biological systems and leads to the formation of compounds that are related to aging and various chronic and degenerative diseases. The extent of oxidation will depend on the presence of antioxidants/pro-oxidants, the unsaturation degree of fatty acids, and environmental conditions. Lipid oxidation can also affect other molecules that have double bonds in their chemical structures, such as cholesterol. Cholesterol oxidation products (COPs) have been studied in depth, because of their negative and controversial biological effects. The formation of COPs can be particularly favored in the presence of light and photosensitizers, since they generate excited singlet oxygen that rapidly reacts with the double bond by a non radical mechanism and without any induction period. The present review intends to provide an overall and critical picture of cholesterol photosensitized oxidation in food and biological systems, and its possible impact on human health and well-being.     

Induction of monocyte differentiation and foam cell formation in vitro by 7-ketocholesterol.

Oxidized LDL (OxLDL) is composed of many potentially proatherogenic molecules, including oxysterols. Of the oxysterols, 7-ketocholesterol (7-KC) is found in relatively large abundance in OxLDL, as well as in atherosclerotic plaque and foam cells in vivo. Although there is evidence that 7-KC activates endothelial cells, its effect on monocytes is unknown. We tested the hypothesis that 7-KC may induce monocyte differentiation and promote foam cell formation. THP-1 cells were used as a monocyte model system and were treated with 7-KC over a range of concentrations from 0.5 to 10 microg/ml. Changes in cell adhesion properties, cell morphology, and expression of antigens characteristic of differentiated macrophages were monitored over a 7-day period. 7-KC promoted cells to firmly adhere and display morphologic features of differentiated macrophages; this effect was time and dose dependent and was markedly more potent than cholesterol treatment (45% of cells became adherent after 7 days of treatment with 7-KC at 10 microg/ml vs. less then 5% for control cells, P < 0.01). Similar effects were obtained when LDL enriched with 7-KC or OxLDL were added to THP-1 cells. 7-KC-differentiated cells expressed CD11b, CD36, and CD68, phagocytized latex beads, and formed lipid-laden foam cells after exposure to acetylated LDL or OxLDL. In contrast to 7-KC, oxysterols with known cell regulatory effects such as 25-hydroxycholesterol, 7beta-hydroxycholesterol, and (22R)-hydroxycholesterol did not effectively promote THP-1 differentiation.In conclusion, these results demonstrate for the first time that 7-KC, a prominent oxysterol formed in OxLDL by peroxidation of cholesterol, may play an important role in promoting monocyte differentiation and foam cell formation. These studies also suggest that 7-KC induces monocyte differentiation through a sterol-mediated regulatory pathway that remains to be characterized.     

Edta Differentially and Incompletely Inhibits Components of Prolonged Cell-Mediated Oxidation of Low-Density Lipoprotein

The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 μM copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of preformed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo.     

Short-term effects of 7-ketocholesterol on human adipose tissue mesenchymal stem cells in vitro

Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Among them, 7-ketocholesterol (7-KC) is one of the most important. It has potent effects in cell death processes, including cytoxicity and apoptosis induction. Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from two young, healthy women. After 24 h incubation with 7-KC, mitochondrial hyperpolarization was observed, followed by a slight increase in the level of apoptosis and changes in actin organization. Finally, the IC₅₀ of 7-KC was higher in these cells than has been observed or described in other normal or cancer cell lines.     

Gas chromatography-mass spectrometry determination of conjugated linoleic acids and cholesterol oxides and their stability in a model system

A gas chromatography-mass spectrometry (GC-MS) method was developed to simultaneously separate cholesterol, eight cholesterol oxidation products (COPs), and two conjugated linoleic acids (9-cis,11-trans-CLA and 10-trans,12-cis-CLA) and to evaluate their stability in a model system during heating. Among four capillary columns tested, an Equity-5 column with low-polar stationary phase provided better resolution within 30 min. A high-performance liquid chromatography method was also developed to determine cholesterol hydroperoxides by using a YMC C30 column with diphenyl-1-pyrenylphosphine as fluorescence reagent. No formation of COPs or degradation of cholesterol and CLAs occurred at 100 °C, but the levels of COPs rose drastically at 150 °C. The first-order rate of cholesterol degradation declined following a rise in CLA concentration. For 0-, 100-, and 500-μg/ml CLA levels, the formation profiles of 7-hydroxycholesterol, 7-ketocholesterol, and 5,6-epoxycholesterol at 150 °C were fitted as multiple first-order curves, whereas a single first-order model could adequately describe 7-hydroperoxycholesterol and cholestane-3β,5α,6β-triol formation. A CLA-to-cholesterol mole ratio of 0.49 was required to prevent cholesterol oxidation at 150 °C.     

Interactions of cholesterol and 7‑ketocholesterol with unsaturated fatty acids of different unsaturation degree – The monolayer study

Unsaturated fatty acids (UFAs) are known to lower the level of sterols in blood, which accounts for their cardioprotective effect. To understand the molecular basis of this effect, Langmuir monolayer studies have been performed. A series of UFAs differing in the length of the fatty acid chain and the number of double bonds (oleic acid, OA; linoleic acid, LA; stearidonic acid, SDA; eicosanoic acid, EA) were mixed with cholesterol and its more toxic oxidized derivative, 7‑ketocholesterol (7-KC), abundantly present in atheroma plaques. Strong attractive UFA-sterol interactions were attributed to the formation of “surface complexes”, in which sterol molecules are bound, thereby reducing the amount of free sterol molecules. It has been found that strength of interactions increases with the degree of unsaturation of the acyl chain in UFA molecule. The most attractive interactions correspond to mixtures with SDA containing 70 mol% of 7-KC and 50 mol% of cholesterol. In both cases, the formation of high stability complexes of, respectively, 2:1 and 1:1 sterol/SDA stoichiometry has been proposed. Other complexes of lower stability and 1:2 stoichiometry were postulated for chol (or 7-KC)/LA systems. The complexes of the lowest stability correspond to chol (or 7-KC) mixtures with OA and EA of 1:1 stoichiometry. In all the cases, the interactions of 7-KC with UFAs are more energetically favorable versus cholesterol. The elongation of the hydrophobic chain of UFAs decreased the interactions with the studied sterols. The obtained results can be related to different conformations of the fatty acids chains.     

Oxysterols are substrates for cholesterol sulfotransferase

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7alpha/7beta-hydroxycholesterol and 5alpha,6alpha/5beta,6beta-epoxycholesterol as well as the 7alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.     

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.     

7-Ketocholesterol and cholestane-triol increase expression of SMO and LXRα signaling pathways in a human breast cancer cell line

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of 7-ketocholesterol (7-KC), cholestane-3β-5α-6β-triol (triol), and a mixture of 5α-cholestane-3β,6β-diol and 5α-cholestane-3β,6α-diol (diol) to promote cell death in a human breast cancer cell line (MDA-MB-231). We determined cell viability, after 24-h incubation with oxysterols. These oxysterols promoted apoptosis. At least part of the observed effects promoted by 7-KC and triol arose from an increase in the expression of the sonic hedgehog pathway mediator, smoothened. However, this increased expression was apparently independent of sonic hedgehog expression, which did not change. Moreover, these oxysterols led to increased expression of LXRα, which is involved in cellular cholesterol efflux, and the ATP-binding cassette transporters, ABCA1 and ABCG1. Diols did not affect these pathways. These results suggested that the sonic hedgehog and LXRα pathways might be involved in the apoptotic process promoted by 7-KC and triol.     

Changes in Brain Cholesterol Metabolome After Excitotoxicity

Excitotoxicity due to excess stimulation of glutamate receptors in neurons is accompanied by increased Ca²⁺ influx, stimulation of Ca²⁺-dependent enzymes, ATP depletion, increase in lipid peroxidation products, and loss of glutathione. These changes resemble neurochemical alterations in acute neuronal injury (stroke, spinal cord injury, and traumatic brain injury) and chronic neurodegenerative diseases such as Alzheimer’s disease. Intracerebroventricular injection of the potent glutamate analog kainate in rats results in increased cholesterol concentration in the hippocampus at short to medium time intervals, i.e., 3 days–1 week post-injection, as detected by gas chromatography–mass spectrometry in the lesioned hippocampus. This is accompanied by an early increase in levels of cholesterol biosynthetic precursors and increases in both enzymatically derived oxysterols such as 24-hydroxycholesterol and cholesterol oxidation products (COPs) generated by reactive oxygen species, including cholesterol epoxides and 7-ketocholesterol. In contrast to COPs, no change in concentration of the neurosteroid pregnenolone was found after KA injury. Cholesterol and COPs significantly increase exocytosis in cultured PC12 cells and neurons, and both oxysterols and COPs are able to induce cytotoxic and apoptotic injuries in different cell types, including neurons. Together, the findings suggest that increased cholesterol and COPs after KA excitotoxicity could themselves lead to disturbed neuronal ion homeostasis, increased neurotransmitter release, and propagation of excitotoxicity.     

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.     

Redox regulation of 7-ketocholesterol-induced apoptosis by beta-carotene in human macrophages

The aim of this study was to verify the hypothesis that beta-carotene may prevent 7-ketocholesterol (7-KC)-induced apoptosis in human macrophages. Therefore, THP-1 macrophages were exposed to 7-KC (5-50 microM) alone and in combination with beta-carotene (0.25-1 microM). 7-KC inhibited the growth of macrophages in a dose- and a time-dependent manner by inducing an arrest of cell cycle progression in the G0/G1 phase and apoptosis. Concomitantly, p53, p21, and Bax expressions were increased by 7-KC, whereas the levels of AKT, Bcl-2, and Bcl-xL were decreased. beta-Carotene prevented the growth-inhibitory effects of 7-KC in a dose- and time-dependent manner as well as the effects of 7-KC on the expression of cell cycle- and apoptosis-related proteins. 7-KC also enhanced reactive oxygen species (ROS) production through an increased expression of NAD(P)H oxidase (NOX-4). The effects of 7-KC were counteracted by the addition of the NAD(P)H oxidase inhibitor DPI or by cotransfection of siNOX-4 mRNA. beta-Carotene prevented 7-KC-induced increase in ROS production and in NOX-4 expression, as well as the phosphorylation of p38, JNK, and ERK1/2 induced by 7-KC. These data suggest a possible antiatherogenic role of beta-carotene through the prevention of 7-KC toxicity in human macrophages.     

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.     

Perilipin 2 (PLIN2)-deficiency does not increase cholesterol-induced toxicity in macrophages

Interventions on macrophages/foam cells to redirect intracellular cholesterol towards efflux pathways could become a very valuable addition to our therapeutic arsenal against atherosclerosis. However, certain manipulations of the cholesteryl ester cycle, such as the inhibition of ACAT1, an ER-resident enzyme that re-esterifies cholesterol, are not well tolerated. Previously we showed that targeting perilipin-2 (PLIN2), a major lipid droplet (LD)-associated protein in macrophages, prevents foam cell formation and protects against atherosclerosis. Here we have assessed the tolerance of PLIN2-deficient bone marrow derived macrophages (BMM) to several lipid loading conditions similar to the found during atherosclerosis development, including exposure to modified low-density lipoprotein (mLDL) and 7-ketocholesterol (7-KC), a free cholesterol (FC) metabolite, in media with or without cholesterol acceptors. BMM isolated from mice that do or do not express PLIN2 were tested for apoptosis (TUNEL and cleaved caspase-3), ER stress (CHOP induction and XBP-1 splicing), and inflammation (TNF-α and IL-6 mRNA levels). Like in other cell types, PLIN2 deficiency impairs LD buildup in BMM. However, while most stress parameters were elevated in macrophages under ACAT inhibition and 7-KC loading, PLIN2 inactivation was well tolerated. The data support the safety of targeting PLIN2 to prevent foam cell formation and atherosclerosis.     

The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-alpha

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

7-Ketocholesterol inhibits isocitrate dehydrogenase 2 expression and impairs endothelial function via microRNA-144

Oxysterol is associated with the induction of endothelial oxidative stress and impaired endothelial function. Mitochondria play a central role in oxidative energy metabolism and the maintenance of proper redox status. The purpose of this study was to determine the effects and mechanisms of 7-ketocholesterol (7-KC) on isocitrate dehydrogenase 2 (IDH2) and its impact on endothelial function in both human aortic endothelial cells (HAECs) and C57BL/6J mice. HAECs treated with 7-KC showed significant reductions of IDH2 mRNA and protein levels and enzyme activity, leading to decreased NADPH concentration and an increased ratio of reduced-to-oxidized glutathione in the mitochondria. 7-KC induced the expression of a specific microRNA, miR-144, which in turn targets and downregulates IDH2. In silico analysis predicted that miR-144 could bind to the 3′-untranslated region of IDH2 mRNA. Overexpression of miR-144 decreased the expression of IDH2 and the levels of NADPH. A complementary finding is that a miR-144 inhibitor increased the mRNA and protein expression levels of IDH2. Furthermore, miR-144 level was elevated in HAECs in response to 7-KC. Anti-Ago1/2 immunoprecipitation coupled with a real-time polymerase chain reaction assay revealed that 7-KC increased the functional targeting of miR-144/IDH2 mRNA in HAECs. Infusion of 7-KC in vivo decreased vascular IDH2 expression and impaired vascular reactivity via miR-144. 7-KC controls miR-144 expression, which in turn decreases IDH2 expression and attenuates NO bioavailability to impair endothelial homeostasis. The newly identified 7-KC–miR-144–IDH2 pathway may contribute to atherosclerosis progression and provides new insight into 7-KC function and microRNA biology in cardiovascular disease.     

7-ketocholesterol is an endogenous modulator for the arylhydrocarbon receptor

We have identified 7-ketocholesterol (7-KC) as an endogenous modulator that inhibits transactivation by the arylhydrocarbon receptor (AhR) through competitive binding against xenobiotic ligands. 7-KC binds AhR and displaces labeled dioxin (2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)). IC(50) is 5 x 10(-7) m in vivo and 7 x 10(-6) m in vitro. These figures are consistent with its concentration in human blood plasma and tissues. Association with 7-KC prevents AhR binding to DNA. 7-KC blocks the TCDD-mediated transactivation of stably expressed reporter gene constructs in T47-D cells as well as the expression of the endogenous CYP 1A1 gene in HepG2 cells and in primary porcine aortic endothelial cells. Injection of 7-KC to rats blocks the induction of CYP 1A1 messenger RNA and protein in endothelial cells from myocardial blood vessels. The differential sensitivity of mammalian species to toxic effects of AhR ligands, especially dioxin (TCDD), correlates with the expression of 7-hydroxycholesterol dehydrogenase, which synthesizes 7-KC from 7-hydroxycholesterol. The documented involvement of AhR ligands in cardiovascular diseases through lipid peroxidation and endothelium dysfunction can now be examined in the context of displacement of this protective modulator.