Death-signaling pathways in human myeloid cells by oxLDL and its cytotoxic components 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide

Oxidized low-density lipoprotein contains many potentially proatherogenic molecules, including oxysterols, which have been shown to induce apoptosis in various cell lines. The aim of this study was to investigate the pathway of apoptosis induced by oxidized low-density lipoprotein and the oxysterols, 7beta-hydroxycholesterol and cholesterol-5beta,6beta-epoxide, in two human monocytic cell lines. The HL-60 cells appeared to be more sensitive to oxidized low-density lipoprotein than U937 cells, whereas the isolated oxysterols were more potent inducers of apoptosis in the U937 cells. Caspase-2 inhibition decreased the number of viable cells in oxidized low-density lipoprotein-treated samples; however, it protected against cholesterol-5beta,6beta-epoxide-induced cell death. Western blot analysis was utilized to examine the effect of caspase-2 inhibition on the expression of the antiapoptotic protein Bcl-2. Pretreatment with the inhibitor protected against the decrease in Bcl-2 expression in oxidized low-density lipoprotein- and 7beta-hydroxycholesterol-treated U937 cells. In HL-60 cells, Bcl-2 was overexpressed in oxidized low-density lipoprotein-treated cells, but in the presence of the inhibitor Bcl-2 expression was returned to control levels. Depleted ATP concentrations in the cells suggest that both apoptosis and necrosis may have occurred simultaneously. Our results highlight differences in the signaling pathways induced by oxidized low-density lipoprotein, 7beta-hydroxycholesterol, and cholesterol-5beta,6beta-epoxide in U937 and HL-60 cells.     

Plasma oxysterols and angiographically determined coronary atherosclerosis: a case control study

Several in vitro and in vivo experiments have implicated oxysterols in the aetiology and progression of atherosclerosis. Oxysterols may be formed endogenously by oxidation of cholesterol and thus may form a marker of LDL oxidation. They may also be obtained exogenously through dietary intake. We investigated the association of oxysterols with the degree of coronary stenosis in patients undergoing coronary angiography. Cases with severe coronary atherosclerosis 80 stenosis in one of the major coronary vessels, n =80 were compared with controls with no or minor stenosis 50 stenosis in all three major coronary vessels, n =79 . Cases and controls were prestratified on age, gender and smoking habits. Evaluated were plasma levels of unesterified 7 hydroxycholesterol, 7 hydroxycholesterol, 25 hydroxycholesterol, 7 ketocholesterol, cholestane triol and 5,6 epoxycholestanol. 7 Hydroxycholesterol made up 67 of the total amount of plasma oxysterol concentration and was the only one significantly higher in cases 1.53 mu g per 100 ml vs 1.27 mu g per 100 ml, p 0.05 . Further, cases had somewhat higher LDL cholesterol levels and significantly lower HDL cholesterol levels than controls. After multivariate adjustment to account for this difference in lipid levels and for the prestratification factors the mean difference between cases and controls for 7 hydroxycholesterol 0.14 mu g per 100 ml was no longer significant. Also the other oxysterols showed no significant association with the degree of coronary stenosis. Multiple logistic regression analyses showed an adjusted odds ratio of 1.07 95 CI, 0.45-2.59 in the highest tertile of total plasma oxysterol level. We conclude, that this study does not support the hypothesis that plasma oxysterols form an additional risk factor for coronary atherosclerosis.     

Generation of an oxidative stress precedes caspase activation during 7beta-hydroxycholesterol-induced apoptosis in U937 cells

The oxysterol 7beta-hydroxycholesterol (7beta-OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf-1 causes activation of the initiator caspase, caspase-9, which in turn activates caspase-3 ultimately leading to the degradation of poly(ADP-ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7beta-OH-induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7beta-OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase-3, -8, and -9 and cleavage of the caspase-3 substrate PARP. The sequence proceeded with activation of caspase-9 after 9 h, caspase-3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7beta-OH. Caspase-8 did not appear to play a major role in this particular apoptotic pathway.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

Plasma oxysterols and angiographically determined coronary atherosclerosis: a case control study

Several in vitro and in vivo experiments have implicated oxysterols in the aetiology and progression of atherosclerosis. Oxysterols may be formed endogenously by oxidation of cholesterol and thus may form a marker of LDL oxidation. They may also be obtained exogenously through dietary intake. We investigated the association of oxysterols with the degree of coronary stenosis in patients undergoing coronary angiography. Cases with severe coronary atherosclerosis 80 stenosis in one of the major coronary vessels, n =80 were compared with controls with no or minor stenosis 50 stenosis in all three major coronary vessels, n =79 . Cases and controls were prestratified on age, gender and smoking habits. Evaluated were plasma levels of unesterified 7 hydroxycholesterol, 7 hydroxycholesterol, 25 hydroxycholesterol, 7 ketocholesterol, cholestane triol and 5,6 epoxycholestanol. 7 Hydroxycholesterol made up 67 of the total amount of plasma oxysterol concentration and was the only one significantly higher in cases 1.53 mu g per 100 ml vs 1.27 mu g per 100 ml, p 0.05 . Further, cases had somewhat higher LDL cholesterol levels and significantly lower HDL cholesterol levels than controls. After multivariate adjustment to account for this difference in lipid levels and for the prestratification factors the mean difference between cases and controls for 7 hydroxycholesterol 0.14 mu g per 100 ml was no longer significant. Also the other oxysterols showed no significant association with the degree of coronary stenosis. Multiple logistic regression analyses showed an adjusted odds ratio of 1.07 95 CI, 0.45-2.59 in the highest tertile of total plasma oxysterol level. We conclude, that this study does not support the hypothesis that plasma oxysterols form an additional risk factor for coronary atherosclerosis.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Effects of cholesterol,hydroxycholesterols and calcium in pregnenolone production rates in mitochondrial fractions from rat testes

The in vitro regulationof the mitochondrial conversion of cholesterol to pregnenolone in rat testis tissue has been further investigated. Pregnenolone production rates by isolated mitochondrial fractions could be stimulated by the addition of cholesterol. The stimulation was always highest in mitochondria isolated from lutropin-treated testes relative to control and cycloheximide-treated testes.Additionof 20- ro 25-hydroxycholesterol resulted in a greater stimulation of pregnenolone production rates and these rates were unaffected by prior treatmetn with cycloheximide. When both cholesterol and 20- or 25-hydroxy-cholesterol were present in the incubation medium, prepgnenolone production rates were mainly influenced by the hydroxycholesterol, even in the presence of a ten-fold excess of cholesterol.Ca²⁺ in vitro stimulated pregnenolone production rates from endogenous cholesterol as well as from added choleterol. However, pregnenolone production rates in the presence of hydroxycholesterol were not influenced by the addition of Ca²⁺ in vitro.