High Cholesterol Diet-Induced Changes in Oxysterol and Scavenger Receptor Levels in Heart Tissue.

Involvement of high cholesterol and oxidative stress in cardiovascular diseases is well studied, as it can be hypothesized that various products originated from lipid peroxidation, such as oxysterols, or affected protein expression might lead to cardiomyocyte damage followed by the pathological modifications. Although oxidation of excessive cholesterol to oxysterols in elevated stress conditions is identified by a number of studies, the role of a high cholesterol diet in regulating fatty acid and oxysterol accumulation, together with scavenger receptor mRNA levels, in the heart remains little investigated. Our study provides a detailed analysis of the changes in fatty acid, oxysterol, and scavenger receptor profiles and its relation with histological alterations in the heart tissue. We evaluated alterations of fatty acid composition, by the GC-MS method, while 4ß-, 25-, and 27-hydroxycholesterol and 7-ketocholesterol levels by means of LC-MS/MS in high cholesterol diet-fed rabbits. Additionally, a number of proteins related to lipid metabolism and scavenger receptor mRNA expressions were evaluated by Western blotting and RT-PCR. According to our in vivo results, a high cholesterol diet enhances a number of unsaturated fatty acids, oxysterols, and LXRα, in addition to CD36, CD68, CD204, and SR-F1 expressions while α-tocopherol supplementation decreases LXRα and SR expressions together with an increase in 27-hydroxycholesterol and ABCA1 levels. Our results indicated that the high cholesterol diet modulates proteins related to lipid metabolism, which might result in the malfunction of the heart and α-tocopherol shows its beneficial effects. We believe that this work will lead the generation of different theories in the development of heart diseases.

Vitamin D supplementation decreases serum 27-hydroxycholesterol in a pilot breast cancer trial.

PURPOSE: 27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue. METHODS: 27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery. RESULTS: A significant increase (p = 4.3E-5) in 25OHD and a decrease (p = 1.7E-1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E-3). CONCLUSIONS: Vitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.

Effects of age and soybean isoflavones on hepatic cholesterol metabolism and thyroid hormone availability in acyclic female rats.

Soy-food and its isoflavones, genistein (G) and daidzein (D), were reported to exert mild cholesterol-lowering effect, but the underlying mechanism is still unclear. In this research, first we studied age-related alterations in hepatic cholesterol metabolism of acyclic middle-aged (MA) female rats. Then we tested if purified isoflavones may prevent or reverse these changes, and whether putative changes in hepatic thyroid hormone availability may be associated with this effect. Serum and hepatic total cholesterol (TChol), bile acid and cholesterol precursors, as well as serum TSH and T4 concentrations, hepatic deiodinase (Dio) 1 enzyme activity and MCT8 protein expression were determined by comparing data obtained for MA with young adult (YA) intact (IC) females. Effects of subcutaneously administered G or D (35mg/kg) to MA rats were evaluated versus vehicle-treated MA females. MA IC females were characterized by: higher (p<0.05) serum TChol, lower (p<0.05) hepatic TChol and its biosynthetic precursors, lower (p<0.05) hepatic 7α-hydroxycholesterol but elevated (p<0.05) 27- and 24-hydroxycholesterol in comparison to YA IC. Both isoflavone treatments decreased (p<0.05) hepatic 27-hydroxycholesterol, G being more effective than D, without affecting any other parameter of Chol metabolism. Only G elevated hepatic Dio1 activity (p<0.05). In conclusion, age-related hypercholesteremia was associated with lower hepatic Chol synthesis and shift from main neutral (lower 7α-hydroxycholesterol) to alternative acidic pathway (higher 27-hydroxycholesterol) of Chol degradation to bile acid. Both isoflavones lowered hepatic 27-hydroxycholesterol, which may be considered beneficial. Only G treatment increased hepatic Dio1 activity, thus indicating local increase in thyroid hormones, obviously insufficient to induce prominent cholesterol-lowering effect.

Effect of Statin Treatment on Plasma 4ß-Hydroxycholesterol Concentrations.

The endogenous oxysterol 4ß-hydroxycholesterol may be used as a marker for the drug-metabolizing enzymes cytochrome P450 3A (CYP3A). The primary aim of this study was to investigate the effect of statin treatment on plasma 4ß-hydroxycholesterol concentrations. Plasma samples from a previously performed clinical study where gallstone patients had been treated with placebo (n = 6), 20 mg fluvastatin (n = 9) or 80 mg atorvastatin (n = 9) daily for 4 weeks were analysed. Hepatic CYP3A mRNA levels had previously been shown to be unchanged in all three treatment groups. Plasma 4ß-hydroxycholesterol did not change significantly (p = 0.92) in the placebo group, but treatment with low-dose fluvastatin or high-dose atorvastatin resulted in reductions in plasma concentration of 10.7% (p < 0.05) and 36.5% (p < 0.01), respectively. However, the 4ß-hydroxycholesterol/cholesterol ratio did not change significantly for the patients receiving placebo or patients receiving low-dose fluvastatin. The ratio for patients receiving high-dose atorvastatin increased by 12% (p < 0.05). In conclusion, the total plasma cholesterol level is an important determinant for the plasma 4ß-hydroxycholesterol level.

4ß-Hydroxycholesterol as an endogenous biomarker of CYP3A activity in cynomolgus monkeys.

It has been proposed that in humans 4ß-hydroxycholesterol is formed mainly by CYP3A-catalyzed metabolism of cholesterol and thus may serve as an endogenous marker for CYP3A activity. The cynomolgus monkey is widely used as one of the nonrodent preclinical safety species in pharmaceutical research. In the current study, the potential application of 4ß-hydroxycholesterol as an endogenous biomarker of CYP3A in response to drug treatment was evaluated in cynomolgus monkeys. Following multiple oral administration of rifampicin (a known CYP3A inducer) at 15 mg/kg/d in cynomolgus monkeys, the mean serum 4ß-hydroxycholesterol levels increased 4-fold from the baseline of 55.3 ± 21.7 to 221 ± 53.4 ng/ml. The mean concentration ratios of 4ß-hydroxycholesterol to cholesterol increased 5-fold. The data suggest that 4ß-hydroxycholesterol formation from cholesterol metabolism was induced by rifampicin treatment in monkeys. This observation correlated with the metabolism of midazolam (a probe substrate of CYP3A activity) monitored in the same study. The serum exposure (area under the curve) of midazolam was markedly decreased by ∼95%, confirming the induction of CYP3A catalytic activity by rifampicin treatment in monkeys. The formation of 4ß-hydroxycholesterol from cholesterol was specifically mediated by recombinant cynomolgus CYP3A8 and CYP3A5. The Km values of CYP3A8 and CYP3A5 for 4ß-hydroxycholesterol formation from cholesterol were 204 and 104 µM, respectively, and Vmax values were 0.600 and 0.310 pg/pmol/min, respectively. The results suggest that 4ß-hydroxycholesterol can be used as an endogenous biomarker to identify strong CYP3A inducers in cynomolgus monkeys, which may help to evaluate drug-drug interaction potential of drug candidates in preclinical settings.

Serum levels of 25-hydroxyvitamin D and the CYP3A biomarker 4ß-hydroxycholesterol in a high-dose vitamin D supplementation study.

The primary aim was to study the relationship between individual serum levels of 25-hydroxyvitamin D and 4ß-hydroxycholesterol, which is an endogenous biomarker of the drug-metabolizing CYP3A enzymes. In addition, the relationship between this biomarker and inflammation, measured as C-reactive protein (CRP), was investigated. Serum samples were used from a recently performed clinical trial in patients with antibody deficiency or increased susceptibility to respiratory tract infections that were randomized to either placebo or high-dose (4000 IU/day) vitamin D for 12 months. One hundred sixteen patients were included in the final analyses, and serum samples collected 6 months after study start were analyzed. At this time point, 25-hydroxyvitamin D levels were found to range between 10 and 284 nM. Individual levels of 25-hydroxyvitamin D as well as CRP were compared with 4ß-hydroxycholesterol levels. In addition, all participants were genotyped for two polymorphisms (Taq1 and Foq1) in the vitamin D receptor gene. There was no significant correlation between individual serum levels of 25-hydroxyvitamin D and 4ß-hydroxycholesterol. However, a moderate, but statistically significant, negative correlation between CRP and 4ß-hydroxycholesterol levels was observed. This study in patients with highly variable serum levels of 25-hydroxyvitamin D could not reveal any relationship between vitamin D and 4ß-hydroxycholesterol, an endogenous biomarker of CYP3A activity. However, the negative correlation between CRP and 4ß-hydroxycholesterol supports earlier experimental results that inflammation may suppress hepatic CYP3A activity, a finding of potentially high clinical relevance that warrants further exploration.

Association of cholesterol oxidation and abnormalities in fatty acid metabolism in cystic fibrosis.

BACKGROUND: Disarrangement in fatty acids and oxidative stress are features of cystic fibrosis. Cholesterol is very sensitive to oxidative stress. OBJECTIVES: The objectives were to examine whether cholesterol oxidation products are altered in cystic fibrosis and whether they are associated with fatty acids and with characteristics of the disease state. DESIGN: 7-Ketocholesterol and 7beta-hydroxycholesterol (prototype molecules of free radical-mediated cholesterol oxidation) and the fatty acid profile were assessed by mass spectrometry in patients and in sex- and age-matched control subjects. RESULTS: In a comparison with control subjects, mean (+/-SD) cholesterol oxidation was higher (7-ketocholesterol: 11.31 +/- 5.1 compared with 8.33 +/- 5.5 ng/mL, P = 0.03; 7beta-hydroxycholesterol: 14.5 +/- 6.8 compared with 9.7 +/- 4.1 ng/mL, P = 0.004), total saturated fatty acids were higher (31.90 +/- 1.93% compared with 30.31 +/- 0.98%, P < 0.001), monounsaturated fatty acids were higher (29.14 +/- 3.85% compared with 25.88 +/- 2.94%, P = 0.004), omega-6 (n-6) polyunsaturated fatty acids were lower (34.84 +/- 4.77 compared with 39.68 +/- 2.98%, P < 0.0001), and omega-3 (n-3) polyunsaturated fatty acids were comparable in patients with cystic fibrosis. Oxysterols were inversely associated with 24:0 and 18:2 omega-6 fatty acids but did not correlate with the increased oleic acid or with any of the omega-3 fatty acids. CONCLUSIONS: Cystic fibrosis is characterized by relevant cholesterol oxidation that is associated with an abnormal fatty acid profile. The interplay between oxysterols and fatty acids potentially provides insight into the biological mechanisms that underlie this complex disease.

High-dose statin treatment does not alter plasma marker for brain cholesterol metabolism in patients with moderately elevated plasma cholesterol levels.

Statins inhibit endogenous cholesterol synthesis, up-regulate low-density lipoprotein (LDL) receptor expression in mammalian liver cells, and thus decrease circulating LDL-cholesterol concentrations. As cholesterol seems to play a role in the development of neurodegenerative diseases, it is of interest to evaluate the effect of high dosages of statins (eg, atorvastatin or simvastatin) on brain cholesterol metabolism. Plasma samples from 44 participants (aged 30-69 years, 16 men and 18 women) of an earlier randomized, placebo-controlled, double-blind trial, who took 40 mg atorvastatin or 80 mg simvastatin daily for 2 months, were used to analyze total cholesterol, its precursor lathosterol, and its metabolites 24(S)-hydroxycholesterol and 27-hydroxycholesterol. Despite a significant decrease in absolute plasma concentrations of oxysterols, total cholesterol, and its endogenous synthesis rate, indicated by a decreased ratio of lathosterol to cholesterol, the plasma 24(S)-hydroxycholesterol to cholesterol ratio, a surrogate marker of brain cholesterol homeostasis, remained unchanged. Short-term high-dose atorvastatin and simvastatin treatment does not seem to influence brain cholesterol metabolism in patients with moderately elevated plasma cholesterol levels.

Short-term hepatic effects of depleted uranium on xenobiotic and bile acid metabolizing cytochrome P450 enzymes in the rat.

The toxicity of uranium has been demonstrated in different organs, including the kidneys, skeleton, central nervous system, and liver. However, few works have investigated the biological effects of uranium contamination on important metabolic function in the liver. In vivo studies were conducted to evaluate its effects on cytochrome P450 (CYP) enzymes involved in the metabolism of cholesterol and xenobiotics in the rat liver. The effects of depleted uranium (DU) contamination on Sprague-Dawley were measured at 1 and 3 days after exposure. Biochemical indicators characterizing liver and kidney functions were measured in the plasma. The DU affected bile acid CYP activity: 7alpha-hydroxycholesterol plasma level decreased by 52% at day 3 whereas microsomal CYP7A1 activity in the liver did not change significantly and mitochondrial CYP27A1 activity quintupled at day 1. Gene expression of the nuclear receptors related to lipid metabolism (FXR and LXR) also changed, while PPARalpha mRNA levels did not. The increased mRNA levels of the xenobiotic-metabolizing CYP3A enzyme at day 3 may be caused by feedback up-regulation due to the decreased CYP3A activity at day 1. CAR mRNA levels, which tripled on day 1, may be involved in this up-regulation, while mRNA levels of PXR did not change. These results indicate that high levels of depleted uranium, acting through modulation of the CYP enzymes and some of their nuclear receptors, affect the hepatic metabolism of bile acids and xenobiotics.

Effects of dietary fat and oxidized cholesterol on gene expression in rat liver as assessed by cDNA expression array analysis.

BACKGROUND: Specific oxysterols acting as ligands for nuclear transcription factors were shown to affect expression of genes involved in lipid metabolism. However, the various biological effects of oxysterols such as cytotoxicity, atherogenicity or mutagenicity suggest that other genes may be also affected by oxysterols than lipid metabolism. AIM OF THE STUDY: The present study was conducted to investigate the effects of dietary oxidized cholesterol containing significant amounts of oxysterols and its interactions with different dietary fats on gene expression profiles as assessed by DNA array technology in rats. METHODS: 54 male Sprague-Dawley rats were assigned to six groups and were fed six semisynthetic diets, which varied in the type of dietary fat (coconut oil vs. salmon oil) and dietary cholesterol (none cholesterol vs. 5 g unoxidized cholesterol/kg vs. 5 g oxidized cholesterol/kg). RESULTS: Changes in gene expression as observed in response to dietary oxidized cholesterol were strongly dependent on the type of fat. In the rats fed coconut oil, the expression of 7 genes (5 up- and 2 down-regulated) was altered by dietary oxidized cholesterol, while in the rats fed salmon oil, the expression of 50 genes (16 up- and 34 down-regulated) was altered. 29 genes (22 up- and 7 down-regulated) were identified as possible targets for an altered gene expression by dietary salmon oil as compared to dietary coconut oil. CONCLUSION: The present study showed that dietary oxidized cholesterol transcriptionally affects many genes involved in xenobiotic metabolism and stress response--an effect that was amplified by the administration of fish oil as dietary fat.

Synthesis of long-chain polyunsaturated fatty acids is inhibited in vivo in hypercholesterolemic rabbits and in vitro by oxysterols.

Plasma total lipids, total cholesterol (cholesterol esters and free cholesterol) and oxysterol (mainly 7 beta-hydroxycholesterol (7 beta OH)) concentrations were significantly elevated in New Zealand rabbits fed a 2% cholesterol-containing diet with respect to controls fed the same diet without cholesterol. In addition, linoleic (18:2 n-6) and alpha-linolenic acid (18:3 n-3) plasma concentrations were significantly elevated in hypercholesterolemic rabbits, while concentrations of long-chain n-6 and n-3 derivatives were reduced. Studies in monocytic cell line THP-1 revealed that 7 beta OH markedly inhibited the conversion of 18:2 to 20:4 n-6 and of 18:3 to 22:6 n-3, indicating depression of the desaturation steps; in particular the inhibition was greater for the Delta 5 desaturation step. Furthermore, experiments of Real-Time PCR showed that 5-10 microM 7 beta OH decreased the Delta 5 gene expression. In conclusion, atherogenic oxysterols interfere with the production of long-chain polyunsaturated fatty acids from their precursors both in hypercholesterolemic rabbits and in cultured cells.

Vitamin E supplementation in patients with carotid atherosclerosis: reversal of altered oxidative stress status in plasma but not in plaque.

OBJECTIVE: Oxidative stress is believed to play a pivotal role in the initiation and progression of atherosclerosis. We analyzed whether vitamin E supplementation influences oxidative stress in plasma and atherosclerotic plaques of patients with severe atherosclerosis. METHODS AND RESULTS: In 16 patients who were candidates for carotid endarterectomy and in 32 age- and sex-matched controls, plasma levels of 7beta-hydroxycholesterol, 7-ketocholesterol, cholesterol, and vitamin E were measured. Patients were randomly allocated to standard treatment with or without 900 mg/d vitamin E. After 6 weeks of treatment, the reported variables were measured in plasma and plaques. The plasma vitamin E/cholesterol ratio was significantly lower in patients than in controls (3.05+/-0.6 versus 6.3+/-1.7 micromol/mmol cholesterol, P<0.001). Plasma 7beta-hydroxycholesterol was significantly higher in patients than in controls (5.0+/-1.04 versus 4.4+/-0.6 ng/mL, P<0.05). Patients who were given vitamin E supplementation showed a significant increase of plasma vitamin E with concomitant decrease of 7beta-hydroxycholesterol. Conversely, no treatment dependence was observed in oxysterol or vitamin E content of plaques. CONCLUSIONS: An imbalance between oxidative stress and antioxidant status is present in patients with advanced atherosclerosis. Vitamin E supplementation improves this imbalance in plasma but not in plaques.

Oral alpha-tocopherol supplementation inhibits lipid oxidation in established human atherosclerotic lesions.

BACKGROUND: Much experimental evidence suggests that lipid oxidation is important in atherogenesis and in epidemiological studies dietary antioxidants appear protective against cardiovascular events. However, most large clinical trials failed to demonstrate benefit of oral antioxidant vitamin supplementation in high-risk subjects. This paradox questions whether ingestion of antioxidant vitamins significantly affects lipid oxidation within established atherosclerotic lesions. METHODS AND RESULTS: This placebo-controlled, double blind study of 104 carotid endarterectomy patients determined the effects of short-term alpha-tocopherol supplementation (500 IU/day) on lipid oxidation in plasma and advanced atherosclerotic lesions. In the 53 patients who received alpha-tocopherol there was a significant increase in plasma alpha-tocopherol concentrations (from 32.66 +/- 13.11 at baseline to 38.31 +/- 13.87 (mean +/- SD) micromol/l, p < 0.01), a 40% increase (compared with placebo patients) in circulating LDL-associated alpha-tocopherol (p < 0.0001), and their LDL was less susceptible to ex vivo oxidation than that of the placebo group (lag phase 115.3 +/- 28.2 and 104.4 +/- 15.7 min respectively, p < 0.02). Although the mean cholesterol-standardised alpha-tocopherol concentration within lesions did not increase, alpha-tocopherol concentrations in lesions correlated significantly with those in plasma, suggesting that plasma alpha-tocopherol levels can influence lesion levels. There was a significant inverse correlation in lesions between cholesterol-standardised levels of alpha-tocopherol and 7beta-hydroxycholesterol, a free radical oxidation product of cholesterol. CONCLUSIONS: These results suggest that within plasma and lesions alpha-tocopherol can act as an antioxidant. They may also explain why studies using < 500 IU alpha-tocopherol/day failed to demonstrate benefit of antioxidant therapy. Better understanding of the pharmacodynamics of oral antioxidants is required to guide future clinical trials.

Bioavailability of vitamin E as function of food intake in healthy subjects: effects on plasma peroxide-scavenging activity and cholesterol-oxidation products.

Clinical trials with vitamin E have yielded contrasting results. In these trials, the amount of vitamin E given was different, and the compliance was not assessed in all studies. In addition, the modality of intake, ie, in relation to food, was not specified in any trial. Vitamin E is lipophilic, and its absorption is expected to be increased by food. We studied the bioavailability of vitamin E in relation to food intake and the effect on the lipid peroxide-scavenging activity of plasma and on 7beta-hydroxycholesterol and 7-ketocholesterol (oxysterols) as markers of oxidant stress. Twenty healthy Italian subjects were randomly assigned to take vitamin E at 300 mg/d on an empty stomach (group A) or during dinner (group B) for 15 days. Plasma vitamin E markedly increased in group B (84%) compared with group A (29%). The lipid peroxide-scavenging activity of plasma increased significantly in group B (14%, P=0.005) but did not change in group A. All subjects showed very low levels of plasma oxysterols, which were not affected by vitamin E supplementation in either group. This study shows that plasma concentration of vitamin E and plasma antioxidant activity in response to oral supplementation are markedly affected by food intake. Healthy Italian subjects show very low levels of cholesterol oxidation products; these low levels are possibly related to the Mediterranean diet. To obtain maximal absorption, vitamin E must be given at meals. These data should be taken into account in clinical trials with vitamin E.

Characterization of a cholesterol response element (CRE) in the promoter of the cholesteryl ester transfer protein gene: functional role of the transcription factors SREBP-1a, -2, and YY1.

Cholesteryl ester transfer protein (CETP) is expressed in human adipocytes, where it acts to promote selective uptake of HDL-CE (Benoist, F., M. McDonnell, P. Lau, R. Milne, and R. McPherson. 1997. J. Biol. Chem. 272: 23572;-23577). In contrast to other major sterol-responsive genes such as 3-hydroxy-3-methylglutaryl coenzyme A reductase CETP expression is up-regulated rather than down-regulated in response to cholesterol. To define elements involved in cholesterol-mediated up-regulation of CETP gene expression, deletion derivatives of the CETP promoter were cloned into a luciferase reporter construct and transfected into the human liposarcoma cell line SW872, cultured in the presence or absence of lipoproteins. A fragment associated with a positive cholesterol response was identified between nucleotides -361 and -138 (relative to the initiation site of transcription) of the promoter. This region contains a tandem repeat of a sequence known to mediate sterol dependent regulation of the hamster HMG-CoA reductase gene. We have putatively denoted this region, the cholesterol response element (CRE). Using gel mobility shift assays we demonstrate that both YY1 and SREBP-1 interact with the CRE of CETP. Furthermore, in transient co-transfection experiments, both YY1 and SREBP-1a were found to trans-activate, in a dose-dependent manner, the luciferase activity of constructs harboring the CRE. We also demonstrate that SREBP-2, is able to trans-activate a luciferase construct harboring the CRE although much less effectively as compared to SREBP-1. Finally, functional analysis of the CRE confirms its regulatory role in modulating CETP gene expression through its interaction with YY1 and SREBP-1a.