Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occuring during 7-ketocholesterol-induced apoptosis.

Previous investigations of our laboratory have shown that 7-ketocholesterol was a potent inducer of apoptosis involving a release of cytochrome c into the cytosol, and a lipid peroxidation process that could be the consequence of a production of radical oxygen species. According to these considerations, we asked whether some antioxidants were able to counteract 7-ketocholesterol-induced apoptosis, and whether prevention of cell death was associated with the impairment of mitochondrial events implied in the commitment to apoptosis, i.e., opening of the mitochondrial megachannels leading to the loss of the mitochondrial transmembrane potential (DeltaPsim), and release of cytochrome c from mitochondria into the cytosol. To this end, we studied the effects of glutathione (15 mM), N-acetylcysteine (15 mM), vitamin E (100 microM), vitamin C (50 microM) and melatonin (1 mM) on U937 cells treated with 7-ketocholesterol (40 microg/ml). Only glutathione, N-acetylcysteine, and vitamin E prevented apoptosis measured by the occurrence of cells with condensed and/or fragmented nuclei, as well as the loss of DeltaPsim, and the release of cytochrome c. However, all the antioxidants used were potent inhibitors of the production of O(2)(*) occuring under treatment with 7-ketocholesterol. Collectively, our data demonstrate that impairment of apoptosis by glutathione, N-acetylcysteine, and vitamin E correlates with the prevention of mitochondrial dysfunctions, and they underline that the ability of antioxidants to counteract 7-ketocholesterol-induced apoptosis does not only depend on their capability to inhibit the production of O(2)(*).

Characterization and comparison of the mode of cell death, apoptosis versus necrosis, induced by 7beta-hydroxycholesterol and 7-ketocholesterol in the cells of the vascular wall.

Oxidized low density lipoproteins (LDLs) play a central role in atherosclerosis, and their toxicity is due, at least in part, to the formation of oxysterols that have been shown to induce apoptosis in various cell types. As 7beta-hydroxycholesterol and 7-ketocholesterol are the major oxysterols found in oxidized LDLs, we have investigated and compared the mode of cell death, apoptosis versus necrosis, that they induce in the cells of the vascular wall, ie, endothelial cells, smooth muscle cells, and fibroblasts. To this end, human vascular endothelial cells from umbilical cord veins (HUVECs), human artery smooth muscle cells, A7R5 rat smooth muscle cells, MRC5 human fibroblasts, and human fibroblasts isolated from umbilical cord veins were taken at confluence and incubated for 48 hours with 7beta-hydroxycholesterol or 7-ketocholesterol (concentration range, 5 to 80 microg/mL). In all cells, both 7beta-hydroxycholesterol and 7-ketocholesterol exhibited toxic effects characterized by a loss of cell adhesion and an increased permeability to propidium iodide. In oxysterol-treated endothelial and smooth muscle cells, typical features of apoptosis were revealed: condensed and/or fragmented nuclei were detected by fluorescence microscopy after staining with Hoechst 33342, oligonucleosomal DNA fragments were visualized in situ in the cell nuclei by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and internucleosomal DNA fragmentation was found on agarose gel. In contrast, in oxysterol-treated fibroblasts, fragmented and/or condensed nuclei were never revealed, and no DNA fragmentation was observed either by the TUNEL method or by DNA analysis on agarose gel, indicating that these oxysterols induced necrosis in these cells but not apoptosis. In addition, acetylated Asp-Glu-Val-L-aspartic acid aldehyde (an inhibitor of Asp-Glu-Val-L-aspartic acid-sensitive caspases) prevented 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death in HUVECs and smooth muscle cells but not in fibroblasts. Thus, 7beta-hydroxycholesterol and 7-ketocholesterol have dual cytotoxic effects on the cells of the vascular wall by their ability to induce apoptosis in endothelial and smooth muscle cells and necrosis in fibroblasts.

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production.

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Different patterns of IL-1beta secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7alpha-, 7beta-hydroxycholesterol, or 7-ketocholesterol.

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.

Evaluation of the cytotoxic effects of some oxysterols and of cholesterol on endothelial cell growth: methodological aspects.

The effects of various oxysterols (7 beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5 alpha, 6 alpha-epoxide, and 25-hydroxycholesterol) and of cholesterol were investigated on cell growth of bovine aortic endothelial (BAE) cells by cell counting, MTT reduction, and 3H-thymidine incorporation in a 5 to 80 micrograms/ml concentration range. By cell counting, a dose related decrease in the number of adherent cells was observed with oxysterols; MTT reduction also indicated a decreased number of viable cells, and both method give similar IC50. A lower 3H-thymidine incorporation was generally detected with oxysterols but no effect on 3H-thymidine incorporation was found with 25-hydroxycholesterol. With cholesterol, no modification of cell growth was shown by cell counting and 3H-thymidine incorporation, whereas an important decrease in MTT reduction was observed. Noteworthy, with the highest cholesterol concentration no change in cellular morphology occurred, and no modification of mitochondrial activity was found with Rhodamine 123. It is concluded that MTT and 3H-thymidine incorporation are not suitable for the evaluation of a putative toxicity of cholesterol and 25-hydroxycholesterol, respectively. Therefore, cell counting seems the most accurate method to determine the effects of oxysterols and of cholesterol on endothelial cell growth.

[Flow cytometry measurement of DNA fragmentation in the course of cell death via apoptosis. New techniques for evaluation of DNA status for the pathologist]. / Mise en évidence de la fragmentation d'ADN par cytométrie en flux au cours de la mort cellulaire par apoptose. Nouvelles techniques d'évaluation de l'état de l'ADN pour le pathologiste.

Cell death by apoptosis is characterized by DNA fragmentation in 200-250 and/or 30-50 kb followed or not by internucleosomal DNA fragmentation in 180-200 pb. Such characteristics have been used to distinguish between necrotic and apoptotic cells, and also to identify and quantify apoptotic cells by flow cytometry. In the case of internucleosomal DNA fragmentation, the analysis of DNA content constitutes the easiest method to identify apoptotic cells giving an hypoploid cell population commonly called "Sub G1". The identification of the "Sub G1" does not depend on the dyes used; however according to the method of cell fixation and permeabilization, of the divalent cations (Ca2+, Mg2+) present in the staining buffers and of the use of trypsin, the "Sub G1" population may be more or less difficult to identify. To detect apoptotic cells whatever the pattern of DNA fragmentation, the most commonly used methods are either in situ nick-translation or TUNEL (TdT dUTP Nick End Labelling). Thus, flow cytometry offers a wide range of attractive techniques to characterize apoptotic cells but it requires the use of methodological controls for validating results.

Induction of apoptosis and of interleukin-1beta secretion by 7beta-hydroxycholesterol and 7-ketocholesterol: partial inhibition by Bcl-2 overexpression.

The oxysterols, 7beta-hydroxycholesterol and 7-ketocholesterol, are involved in the cytotoxicity of oxidized LDL. To elucidate their molecular mechanisms, the human promonocytic leukemia cells U937 and U4 were used. U4 cells overexpressing Bcl-2 were obtained by transfection of U937 cells. 7Beta-hydroxycholesterol and 7-ketocholesterol induced nuclear condensation and/or fragmentation, internucleosomal DNA fragmentation, and IL-1beta secretion, which were partially inhibited by Bcl-2 overexpression. These findings underline that these oxysterols could constitute major risk factors in atherosclerosis by their cytotoxicity and their ability to induce IL-1beta release which might favor the recruitment of immunocompetent cells in the atherosclerotic plaque.

Effect of lipid transfer activity and lipolysis on low density lipoprotein (LDL) oxidizability: evidence for lipolysis-generated non-esterified fatty acids as inhibitors of LDL oxidation.

Low density lipoproteins (LDL) were modified in vitro in the presence of lipid transfer activity and lipolysis, which induced alterations in the size and lipid composition of LDL particles but not in their antioxidant content. Subsequently, modified LDL were oxidized with copper sulfate and the extent of LDL oxidation was evaluated. Lipid transfer activity alone, or in combination with lipolysis, led to a significant reduction of LDL oxidability as compared with starting homologous LDL. Furthermore, the combined effect of lipid transfers and lipolysis reduced LDL oxidability to a significantly greater extent than did lipid transfers alone. Consistent results were obtained by measuring either the formation of lipid peroxides, the appearance of thiobarbituric acid reactive substances (TBARS), the disappearance of polyunsaturated fatty acids (PUFA), or the generation of cholesterol oxides. Non-esterified fatty acids (NEFA) arose as putative candidates in reducing oxidation susceptibility of LDL: NEFA-containing LDL were less oxidizable; the enrichment of LDL with either oleic acid or linoleic acid reduced significantly their oxidability; the oxidation susceptibility of either in vitro modified LDL or LDL isolated from normal or analbuminemic patients significantly increased after reduction of their NEFA content with fatty acid-poor albumin. After NEFA depletion, small-sized LDL resulting from the combined effects of lipid transfer and triglyceride hydrolysis activities became more oxidizable than large-sized LDL treated with lipid transfer activity alone. In addition, the PUFA to total fatty acid ratio and the oxidability of modified LDL varied accordingly after NEFA depletion, showing that in the present study not only lipoprotein-bound NEFA but also the total fatty acid composition of LDL could account for alterations in their oxidability.

Effect of D- and L-1,3-butanediol isomers on glycolytic and citric acid cycle intermediates in the rat brain.

DL-1,3-butanediol (DL-BD) is an ethanol dimer which affords cerebral protection in various experimental models of hypoxia and ischemia but its mechanism of action is unknown. DL-BD is a ketogenic alcohol and it has been proposed that its protective effect was accomplished through cerebral utilization of ketone bodies. Since DL-BD is a racemic, its metabolic effects could be due to D, L or both isomers. The effects of equimolar doses of DL-, D- and L-BD (25 mmol/Kg) on cerebral metabolism were studied by measuring the cortical levels of the main glycolytic (glycogen, glucose, glucose 6-phosphate, fructose 1,6-diphosphate, pyruvate and lactate) and citric acid cycle (citrate, alpha-ketoglutarate and L-malate) intermediates. The two BD isomers exerted different effects on cerebral metabolism. Unlike L-BD, D- and DL-BD treatments resulted in a slight (+10%) but significant increase in citrate level whereas L-BD treatment led to significant reduction in pyruvate (-12%) and lactate (-24%) levels. These effects were apparently not linked to hyperketonemia, since DL-BHB treatment, which mimicked hyperketonemia induced by DL-BD, had no effect on cerebral metabolites but might be due to intracerebral metabolism of BD.

[Agents inducing cell death of the vascular wall]. / Les inducteurs de mort des cellules de la paroi vasculaire.

Arterial wall is constituted by endothelial cells, smooth muscle cells and fibroblasts. A number of agents are capable of inducing damages in these cells leading either to apoptosis or necrosis involved in various pathologies. Such agents are constituted by: bacterial endotoxins, mediators and modulators of immunitary and/or inflammatory systems, drugs, reactive oxygen metabolites, free radicals, cholesterol oxidation derivatives included or not in lipoproteins. The study of the mechanisms of action implied in cell death would permit a better understanding of vascular diseases and open new therapeutic perspectives.

Effect of 1,3-butanediol on cerebral energy metabolism. Comparison with beta-hydroxybutyrate.

Previous studies have shown that 1,3-butanediol (BD) has beneficial effects in experimental models of hypoxia or ischemia but the mechanism by which it exerts its protective effects remains unknown. BD is converted in the body to beta-hydroxybutyrate (BHB) and it has been proposed that its effects were linked to its ketogenic effect. The effects of BD (25 and 50 mmol/kg) on cerebral energy metabolism of rats were studied by measuring the cerebral level of energy metabolites and by evaluating the cerebral metabolic rate according to the Lowry's method. BD induced an increase in [cortical glucose]/[plasma glucose] ratio which was associated with a decrease in lactate level and an increase in glucose and glycogen stores. In contrast, BHB treatment which mimicked hyperketonemia equivalent to BD did not modify cerebral glycolysis metabolites. Calculation of the energy reserve flux after decapitation showed that BD did not reduce the cerebral metabolic rate excluding a protective effect due to a depressant, barbiturate-like, action. These results suggest that BD induces a reduction of cerebral glycolytic rate. However, the effect is not linked to hyperketonemia but might be due to intracerebral conversion of BD to BHB.

Changes in extracellular and rat brain tissue concentrations of D-beta-hydroxybutyrate after 1,3-butanediol treatment.

1,3-Butanediol (BD) treatment was previously shown to produce a dose-related increase of the plasma levels of D-beta-hydroxybutyrate (BHB) and to protect brain tissue against hypoxia and ischemia. The purpose of this study was to test whether BD-induced hyperketonemia was associated with changes in brain extracellular and tissue concentrations of BHB. Changes in extracellular levels of BHB were continuously monitored in anesthetized rats before and after intraperitoneal injection of BD (25 mmol/kg), using intracerebral microdialysis coupled to online analysis of BHB in the dialysate. Cortical tissue concentrations of BHB were determined in control and BD-treated rats (25 and 50 mmol/kg, i.p.) after freezing of the brain in situ. Butanediol produced a rapid increase in dialysate levels of BHB, with a linear relationship between dialysate and plasma BHB concentrations (r = 0.81, p < 0.001). In contrast, and although brain tissue levels of BHB were markedly increased after BD treatment, they were not related to the plasma concentration of BHB. Our results suggest that BHB produced from BD did not accumulate in brain and that BD protects against hypoxia or ischemia by increasing brain BHB availability.

Beneficial effect of 1,3-butanediol on cerebral energy metabolism and edema following brain embolization in rats.

We assessed the effect of 1,3-butanediol on cerebral energy metabolism and edema after inducing multifocal brain infarcts in 108 rats by the intracarotid injection of 50-microns carbonized microspheres. An ethanol dimer that induces systemic ketosis, 25 mmol/kg i.p. butanediol was injected every 3 hours to produce a sustained increase in the plasma level of beta-hydroxybutyrate. Treatment significantly attenuated ischemia-induced metabolic changes by increasing the concentrations of phosphocreatine, adenosine triphosphate, and glycogen and by reducing the concentrations of pyruvate and lactate. Lactate concentration 2, 6, and 12 hours after embolization decreased by 13%, 44%, and 46%, respectively. Brain water content increased from 78.63% in six unembolized rats to 80.93% in 12 saline-treated and 79.57% in seven butanediol-treated rats 12 hours after embolization. (p less than 0.05). The decrease in water content was associated with significant decreases in the concentrations of sodium and chloride. The antiedema effect of butanediol could not be explained by an osmotic mechanism since equimolar doses of urea or ethanol were ineffective. Our results support the hypothesis that the beneficial effect of butanediol is mediated through cerebral utilization of ketone bodies arising from butanediol metabolism, reducing the rate of glycolysis and the deleterious accumulation of lactic acid during ischemia.

Fasting prior to transient cerebral ischemia reduces delayed neuronal necrosis.

A transient brain ischemia of 30-min duration was induced by the four-vessel occlusion technique in normally fed and in 48-hr-fasted rats. Evaluation of brain damage 72 hr after ischemia showed that fasting reduced neuronal necrosis in the striatum, the neocortex, and the lateral part of the CA1 sector of hippocampus. Signs of status spongiosis in the pars reticulata of the substantia nigra were seen in 75% of fed rats and in only 19% of fasted rats. The protective effect was associated with reduction in mortality and in postischemic seizure incidence. The metabolic changes induced by fasting were evaluated before and during ischemia. After 30 min of four-vessel occlusion, fasted rats showed a marked decrease in brain lactate level (14.7 vs 22.5 mumol/g in fed rats; P less than 0.001). The decrease in brain lactate concentration might explain the beneficial effect of fasting by minimizing the neuropathological consequences of lactic acidosis. Several factors may account for lower lactate production during ischemia in fasted rats: hypoglycemia, reduction in preischemic stores of glucose and glycogen, or increased utilization of ketone bodies aiming at reducing the glycolytic rate.

Comparison of the effects of valproate, ethosuximide, phenytoin, and pentobarbital on cerebral energy metabolism in the rat.

The acute effects of valproate (200 and 400 mg/kg), ethosuximide (200 and 400 mg/kg), phenytoin (25 and 50 mg/kg), and pentobarbital (30 and 60 mg/kg) on cerebral energy metabolism of rats were studied by measuring the cerebral content of energy metabolites and by evaluating the rate of metabolite utilization following decapitation. The treatments did not affect the levels of phosphocreatine (PCr), ATP, ADP, and AMP, but did enhance the glycogen or glucose stores. Pentobarbital induced a decrease in lactate, whereas valproate led to a decrease in pyruvate and an increase in lactate. Calculation of the metabolite fluxes after decapitation showed that all treatments delayed the rate of ATP utilization. The response was dose-dependent except with valproate. In addition, pentobarbital led to reductions in glucose utilization and lactate production, but the other drugs had no significant effect on glycolysis rate. The sparing effect on ATP utilization may be related to a membrane-stabilizing effect and may provide brain protection in case of excessive neuronal activation.