This paper has been retracted at the request of the authors.
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The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids... more
The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs) and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol) combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial...
Advanced prostate cancer (PCa) is a clinical challenge as no curative therapeutic is available. In this context, a better understanding of metastasis and resistance mechanisms in PCa is an important issue. As phosphatase and tensin... more
Advanced prostate cancer (PCa) is a clinical challenge as no curative therapeutic is available. In this context, a better understanding of metastasis and resistance mechanisms in PCa is an important issue. As phosphatase and tensin homolog (PTEN) loss is the most common genetic lesion in such cancer, we investigate human data sets for mechanisms that can constrain cancer evolution in this setting. Here we report a liver X receptor (LXR) signature, which tightly correlates with PTEN loss, in PCa. Accordingly, the LXR pathway is deregulated in prostate carcinomas in Pten-null mice. Genetic ablation of LXRs in Pten-null mice, exacerbates PCa invasiveness and metastatic dissemination, which involves mesenchymal transition and accumulation of matrix metalloproteinases. Mechanistically, PTEN deletion governed LXR transcriptional activity through deregulation of cholesterol de novo synthesis, resulting in accumulation of endogenous LXR ligands. Our study therefore reveals a functional circ...
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Context: Increased oxidative stress in adipose tissue emerges as an inducer of obesity-linked insulin resistance. Here we tested whether free-radical derived oxysterols are formed by, and accumulate in, human adipocytes. Moreover, we... more
Context: Increased oxidative stress in adipose tissue emerges as an inducer of obesity-linked insulin resistance. Here we tested whether free-radical derived oxysterols are formed by, and accumulate in, human adipocytes. Moreover, we asked whether increased accumulation of oxysterols characterizes the adipose cells of obese patients with type 2 diabetes (T2D) (OBT2D) compared with lean, nondiabetic controls (CTRLs). Finally, we studied the effects of the free radical–derived oxysterols on adipogenic differentiation of adipose-derived stem cells (ASCs). Main Outcome Measures: Adipocytes and ASCs were isolated from sc abdominal adipose tissue biopsy in four OBT2D and four CTRL subjects. Oxysterols in adipocytes were detected by gas chromatography/mass spectrometry. The cellular and molecular effects of oxysterols were then evaluated on primary cultures of ASCs focusing on cell viability, adipogenic differentiation, and “canonical” WNT and MAPK signaling pathways. Results: 7-ketocholes...
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The oxysterols 7β-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in... more
The oxysterols 7β-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7β-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7β-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11β-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7β-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7β-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7β-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7β-hydroxycholesterol and 7-ketocholesterol in humans.
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Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product,... more
Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.
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Research Interests: Cell Cycle, Oxidative Stress, Apoptosis, Cellular, Humans, and 12 morePhosphorylation, Cellular Physiology, Medical Physiology, Mitogen Activated Protein Kinase, Caspase, Time Factors, Hepatocytes, Necrosis, Cell Proliferation, Sterols, Protein Kinase Inhibitors, and Mitogen- Activated Protein Kinases
Radiolabelled autologous low density lipoprotein (LDL) has previously been used to study in vivo distribution and metabolism of native-LDL. Non-invasive imaging of atherosclerotic lesions using 99mTc-LDL was shown to be feasible in animal... more
Radiolabelled autologous low density lipoprotein (LDL) has previously been used to study in vivo distribution and metabolism of native-LDL. Non-invasive imaging of atherosclerotic lesions using 99mTc-LDL was shown to be feasible in animal models and patients but the clinical utility remains to be assessed. Since recent reports suggest that oxidized LDL may play a major role in the pathogenesis of
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The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen,... more
The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.
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ABSTRACT Oxysterols are oxygenated derivatives of cholesterol with a very short life-time relative to cholesterol. Oxysterols are present in nanomolar concentrations in biological fluids underscoring their role as important intermediates... more
ABSTRACT Oxysterols are oxygenated derivatives of cholesterol with a very short life-time relative to cholesterol. Oxysterols are present in nanomolar concentrations in biological fluids underscoring their role as important intermediates in a number of biochemical pathways including bile acid synthesis, reverse cholesterol transport, control of cholesterol synthesis in the brain, and oxidative stress. Most oxysterols are produced by enzymes of the cytochrome P450 family while others are produced by free radical reactions. This last group of oxysterols (stress oxysterols, or SOX) , which are mainly oxygenated in the C6 or C7 position, have attracted interest for mechanistic studies in the context of oxidative stress, and for probing oxidative stress in vivo . Sensitive and specific mass spectrometric methods have been prepared to measure SOX in a number of clinical settings, and to follow the changes induced by pharmacological treatments. Additional interest in oxidative stress oxysterols is linked to the increasing number of biological effects, obtained at cellular level and in animal models, implicated in the pathophysiological mechanisms that play a role in several diseases, including atherosclerosis, neurodegeneration, and cancer. Oxysterols have in fact been shown to induce apoptosis, cell differentiation, cytotoxicity, and impairment of endothelial function. This review is an evaluation of the recent literature on oxysterols, in particular on the role of oxysterols as bioactive compounds.
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ABSTRACT Assessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E... more
ABSTRACT Assessing vitamin E status in humans is critical for nutritional evaluation and verification of clinical and biological compliance of supplemented subjects. An accurate analytical method for measuring the two main vitamin E isoforms, i.e. α- and γ-tocopherol (α- and γ-TOH) in small volumes of plasma can facilitate the application of this analysis to clinical trials and in situations where a limited amount of sample is available. We have developed a micro method, which uses only 5 μL plasma, based on isotope dilution, trimethylsilation and GC-MS. The method was validated according to the guidelines of the International Conference on Harmonization of analytical procedures. The method was also applied to 5 μL of whole blood for the potential use in conditions were the availability of specimens is limited. Accurate quantitation of α-TOH and γ-TOH was achieved at levels ≥ 0.417 μM and ≥ 0,007 μM, respectively. Within-day coefficient of variation was 1.31 % and 4.70 % for α-tocopherol and γ-tocopherol, respectively. Between-day coefficient of variation was 1.32 % and 2.88 % for α-tocopherol and γ-tocopherol, respectively. Recovery, assessed at three concentration levels, ranged 98-103 % and 100-102 % for α-tocopherol and γ-tocopherol, respectively. The method allowed the detection of α-tocopherol and γ-tocopherol in 5 μL whole blood and in membranes of red blood cells washed from 5 μL of blood as well. The analytical performance was assessed in plasma from a cohort of Italian healthy subjects (n = 205). The mean plasma concentrations were 28.01 ± 6.31 and 0.68 ± 0.48 μM (mean ± SD) for α-TOH and γ-TOH, respectively. Alpha-TOH correlated with total cholesterol (r = 0.617, p < 0.0001) and triglycerides (r = 0.420, p < 0.0001) while γ-TOH correlated modestly with total cholesterol (r = 0.213, p < 0.0001) but not with triglycerides. γ-TOH, but not α-TOH, was significantly lower in smokers than in non-smokers (0.72 ± 0.50 vs. 0.56 ± 0.37, μM, mean ± SD, p = 0.017). Given the high sensitivity, the method allowed to be applied to 5 μM whole blood without specific modification. This micro-method represents an analytical advancement in α- and γ-TOH assay that is available to accurately verify the nutritional status and compliance after supplementation in large-scale settings, and to measure the two vitamers in conditions where sample availability is limited. Copyright © 2015. Published by Elsevier B.V.
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Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified... more
Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O2-) and hydroxyl radical (OH ) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O2- and OH(0), respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O2- and OH was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O2- and OH(0) formation. To further analyze this relationship, O2- and OH(0) formation was measured during AA-stimulated platelet aggregation (PA); we observed that O2- and OH(0) release were dependent upon AA concentration. Furthermore, we found that the incubation of...
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Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. ... Succeeding onset of hepatic, splenic,... more
Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. ... Succeeding onset of hepatic, splenic, and renal infarction in polyarteritis nodosa.
Research Interests: Humans, Kidney, Liver, Male, Clinical Sciences, and 4 moreAged, The American, Polyarteritis Nodosa, and Infarction
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Correspondence from The New England Journal of Medicine — Implantable Cardioverter–Defibrillator Therapy after Myocardial Infarction.
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... Francesco Violi, MD Andrea Ghiselli, MD Cesare Alessandri, MD Stefano Frattaroli, MD Luigi Iuliano, MD Francesco Balsano, MD University of Rome 'La Sapienza', Rome 00161, Italy. 12 References: 1. ... Lipids... more
... Francesco Violi, MD Andrea Ghiselli, MD Cesare Alessandri, MD Stefano Frattaroli, MD Luigi Iuliano, MD Francesco Balsano, MD University of Rome 'La Sapienza', Rome 00161, Italy. 12 References: 1. ... Lipids (in press). 3. Saniabadi AR, Lowe GDO, Barbenel JC, Forbes CD. ...
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Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is... more
Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/dl). We found lower GSH levels (7.7 +/- 1.4 mumol/g hemoglobin [Hb]), higher GSSG levels (0.35 +/- 0.09 mumol/g Hb), and lower GSH/GSSG ratios (23.9 +/- 7.7) in diabetics compared with controls (n = 15; 9.8 +/- 0.8 mumol/g Hb, P < .001; 0.17 +/- 0.02, P < .001; and 58.3 +/- 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 +/- 0.7 mumol/g Hb, P < .01), a decrease in GSSG (0.25 +/- 0.06 mumol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 +/- 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.
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Atherosclerosis is the commonest lesion of blood vessels and is responsible for life-threatening events such as myocardial infarction and stroke. In the last two decades a series of excellent studies unraveled biochemical mechanisms that... more
Atherosclerosis is the commonest lesion of blood vessels and is responsible for life-threatening events such as myocardial infarction and stroke. In the last two decades a series of excellent studies unraveled biochemical mechanisms that provided the background for a theory of atherogenesis. This theory is centered on foam cells and on free radical-mediated modification of low density lipoprotein (LDL). Foam cells are the main cell type of atherosclerotic lesions and originate from monocytes migrated from blood and from smooth muscle cells of the arterial wall. Foam cells are engulfed of lipids taken from LDL. Paradoxically, accumulation of LDL in developing foam cells does not occur via the classic LDL receptor. Incubation of macrophages with even very high concentrations of LDL does not appreciably increase cholesterol content. Chemically modified LDL easily enter the cells of atherosclerotic plaque via an unregulated receptor, the scavenger receptor. The most studied chemical modification of LDL is that induced by free radicals.