Oxidized low-density lipoprotein-induced apoptosis

Cultured cells are able to oxidize low-density lipoproteins (LDL) and oxidized LDL (oxLDL), which are present in atherosclerosis areas, exhibit a variety of biological properties potentially involved in atherogenesis. This review is focused on the toxicity of oxLDL, more precisely on the toxic compounds generated during LDL oxidation, the features and the mechanisms of cell death (apoptosis or necrosis) induced by oxLDL. After internalization, toxic oxidized lipids, namely lipid peroxides, oxysterols and aldehydes, induce modifications of cell proteins, elicit oxidative stress, lipid peroxidation and alter various signaling pathways and gene expression. These events may participate in the toxic effect, and converge to trigger an intense, delayed and sustained calcium peak which elicits either apoptosis or necrosis processes. OxLDL-induced apoptosis involves both mitochondrial and death-receptor (Fas/FasL) apoptotic pathways, thereby activating the classical caspase cascade and subsequent biochemical and morphological apoptotic features. When apoptosis is blocked by overexpression of Bcl-2, oxLDL trigger necrosis through a calcium-dependent pathway. Apoptosis occurring in atherosclerotic areas is potentially involved in endothelial cell lining defects, necrotic core formation and plaque rupture or erosion which may trigger atherothrombotic events. However, the precise role of oxLDL in apoptosis/necrosis occurring in vivo in atherosclerotic plaques remains to be clarified.     

Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.     

Aspirin-induced apoptosis in Jurkat cells is not mediated by peroxisome proliferator-activated receptor delta

Apoptosis-inducing agents have been reported to cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1) in endothelial cells (EC). Oxidized LDL (oxLDL) has also been known to induce apoptosis of EC and to inhibit proliferation of EC. In the present study, we show that oxLDL also causes shedding of TNFR1 in EC and that EC transfected with soluble TNFR1 (sTNFR1 ), which is an extracellular domain of TNFR1, can antagonize the toxicity induced by oxLDL. These results suggest that transfection with the sTNFR1 gene plays a protective role against the injury of EC induced by oxLDL. We speculate therefore that sTNFR1 can be a new strategy for treatment of atherogenesis possibly by preventing shedding of TNFR1.     

The role of oxysterols in control of endothelial stiffness

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE^−/− mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.     

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.     

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.     

Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL

During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr(-/-) mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDL-specific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.     

OxLDL-induced macrophage cytotoxicity is mediated by lysosomal rupture and modified by intralysosomal redox-active iron

Oxidized low density lipoprotein (oxLDL) is believed to play a central role in atherogenesis. LDL is oxidized in the arterial intima by mechanisms that are still only partially understood. OxLDL is then taken up by macrophages through scavenger receptor-mediated endocytosis, which then leads to cellular damage, including apoptosis. The complex mechanisms by which oxLDL induces cell injury are mostly unknown. This study has demonstrated that oxLDL-induced damage of macrophages is associated with iron-mediated intralysosomal oxidative reactions, which cause partial lysosomal rupture and ensuing apoptosis. This series of events can be prevented by pre-exposing cells to the iron-chelator, desferrioxamine (DFO), whereas it is augmented by pretreating the cells with a low molecular weight iron complex. Since both DFO and the iron complex would be taken up by endocytosis, and thus directed to the lysosomal compartment, the results suggest that the normal contents of lysosomal low molecular weight iron may play an important role in oxLDL-induced cell damage, presumably by catalyzing intralysosomal fragmentation of lipid peroxides and the formation of toxic aldehydes and oxygen-centered radicals.     

Isolation of a somatic cell mutant resistant to the induction of apoptosis by oxidized low density lipoprotein

Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 microg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25-hydroxycholesterol.     

The Variability of oxLDL-induced Cytotoxicity on Different Types of Cell Lines

The epidemiologic studies indicated an association of obesity with increased incidence of colorectal, breast and ovarian cancer. Further studies found a positive correlation between increased serum oxLDL and an increased risk of the three cancers. In contrast, our previous studies found a negative correlation between the serum oxLDL levels and the risk of leukemia and esophageal cancer. Identification of the variability of cytotoxicity of oxLDL-induced on different types of cell lines is important for understanding the mechanism of oxLDL involved in the tumorigenesis. In the present study, we investigated the effective impacts of oxLDL on the proliferation and apoptosis for the human umbilical vein endothelial cells (HUVEC) and two cancer cell lines (EC-9706 and K562/AO2 with multi-drug resistance). HUVEC, K562/AO2 and EC-9706 cell lines were cultured in the presence of oxLDL, and cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis and cell cycle by flow cytometer, mRNA expression by RT-PCR and protein expression by Western blot. OxLDL could inhibit proliferation and apoptosis of the three cell lines; however, there were significant differences of effective action on the viability and apoptosis. The dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on the two tumor cells. The antibody of lectin-like oxLDL receptor-1 (LOX-1-ab) can block oxLDL-induced cytotoxicity. Cells apoptosis is mediated by reducing Bcl-2 and increasing Bax and caspase-3 mRNA and protein expression. This study showed the dose of oxLDL-induced cytotoxicity on HUVEC was higher than that on K562/AO2 and EC-9706 tumor cells. The antibody of LOX-1 receptor can block the oxLDL-induced cytotoxicity.     

Oxidized HDL Induces Cytotoxic Effects: Implications for Atherogenic Mechanism

Atherosclerosis can be considered as an inflammatory disease and oxidized low‐density lipoprotein (oxLDL) is a critical factor in atherogenesis. Although high‐density lipoprotein (HDL) is generally an antiatherogenic lipoprotein, this property can be compromised by functional impairment mainly due to oxidative modification. As such, understanding the proatherogenic properties exerted by oxidized‐HDL (oxHDL) becomes more important. This study was focused on examining the role of oxHDL as a proatherogenic agent, using oxLDL as a positive control. The comparative toxicity of oxHDL and oxLDL having same range of malondialdehyde, to monocytes was evaluated. After treatment, markers for oxidative stress, inflammation, and cytotoxicity were quantitated. The results showed that like oxLDL, oxHDL induced significant oxidative stress, cytotoxicity, and release of TNF ‐alpha and MMP‐9 in monocytes/macrophages, but was less potent than oxLDL in promoting these proatherogenic effects. Further, the effects of oxHDL for the enhanced formation of MMP‐9 were found to be mediated by NADPH oxidase/ROS‐JNK/ERK pathway, as one mechanism.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Oxysterols modulate calcium signalling in the A7r5 aortic smooth muscle cell-line

Prolonged exposure to oxidized low density lipoprotein (oxLDL) can alter various aspects of cell biology, including modification of vasomotor responses and downregulation of calcium channel proteins in aortic smooth muscle cells. However, the components of oxLDL responsible for these effects have not been fully elucidated. The study reported here aimed at examining the consequences of extended exposure to oxysterols, cholesterol oxidation products whose levels are elevated in oxLDL as compared to unmodified LDL, on calcium signalling mechanisms in A7r5 cells, a model aortic smooth muscle cell-line. Within 24 h of exposure, all three oxysterol congeners tested caused an elevation in the resting cytoplasmic Ca²⁺ concentration. These oxysterols also inhibited Ca²⁺ transients in response to arginine vasopressin and bradykinin, and some but not all congeners ablated Ca²⁺ signals triggered by platelet activating factor, the ryanodine receptor calcium channel agonist 4-choloro-meta-cresol, or thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ uptake. The effects of long-term exposure to the oxysterol congener 7β-hydroxycholesterol on arginine vasopressin stimulated Ca²⁺ signals were mainly at the level of Ca²⁺ release from intracellular stores rather than on Ca²⁺ influx mechanisms. Of the calcium signalling proteins tested, only the type 1 ryanodine receptor and the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) were significantly downregulated by 24 h exposure to oxysterols. Decreases in IP₃R1 protein triggered by 7β-hydroxycholesterol were both time and concentration dependent, occurring over a concentration range encountered within atherosclerotic lesions. IP₃R1 downregulation by certain oxysterols is mediated by proteasomal proteolysis, since it can be abolished by co-incubation with epoxomicin. Overall, these data demonstrate that major oxysterol components of oxLDL cause long-term alterations in Ca²⁺ signalling in a model aortic smooth muscle cell. Such effects could contribute to the pathology of atherosclerotic disease.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Oxidised LDL up-regulate CD36 expression by the Nrf2 pathway in 3T3-L1 preadipocytes

The effect of oxLDL on CD36 expression has been assessed in preadipocytes induced to differentiate. Novel evidence is provided that oxLDL induce a peroxisome proliferator-activated receptor gamma-independent CD36 overexpression, by up-regulating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). The nuclear translocation of Nrf2 appeared to depend on PKC pathway activation. In adipocytes, the CD36 up-regulation may indicate a compensation mechanism to meet the demand of excess oxLDL and oxidised lipids in blood, reducing the risk of atherogenesis. Besides strengthening the hypothesis that oxLDL can contribute to the onset of insulin-resistance, data herein presented highlight the significance of oxLDL-induced CD36 overexpression within the cellular defence response.     

Caspase-dependent apoptosis in THP-1 cells exposed to oxidized low-density lipoproteins

Oxidized low-density lipoproteins (oxLDL) play a critical role in atherogenesis. We investigated the apoptotic process in human monocytic THP-1 cell line, exposed to oxLDL generated by treatment of native LDL either with hypochlorous acid (HOCl), mainly affecting the protein moiety, or with copper sulfate (CuSO(4)), mainly affecting the lipid moiety. After incubation with both types of oxLDL, we observed: (i) microscopy signs of apoptosis in THP-1 cells, (ii) a significant increase of apoptotic cells proportional to LDL protein concentration, either by annexin V or by cell cycle phase analysis with propodium iodide flow cytometry, (iii) a reduction of THP-1 cell apoptosis in presence of the caspase inhibitor Z-VAD.fmk, (iv) the resistance of THP-1 cells apoptosis after PMA-elicited differentiation. In conclusion, HOCl-oxLDL are as potent as Cu-oxLDL to induce high rates of apoptosis in monocytes through a caspase-dependent pathway. Moreover, the resistance of differentiated THP-1 cells to oxLDL-induced apoptosis is compatible with the hypothesis that mature macrophages have prolonged survival and thereby enhance the atherogenic process.     

Oxidized low density lipoprotein suppresses lipopolysaccharide-induced inflammatory responses in microglia: oxidative stress acts through control of inflammation

Low density lipoprotein (LDL) is readily oxidized under certain conditions, resulting in the formation of oxidized LDL (oxLDL). Despite numerous in vitro reports that reveal the pathogenic role of oxidative stress, anti-oxidative strategies have underperformed in the clinic. In this study, we examine the role of oxLDL in brain inflammatory responses using cultured rat brain microglia. We demonstrate that oxLDL inhibits lipopolysaccharide (LPS)-induced inflammatory responses in these cells. It also decreases LPS-induced expression of inducible nitric oxide synthase and production of nitric oxide, and reduces LPS-induced secretion of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Oxysterols, known components of oxLDL and endogenous agonists of liver X receptor, can simulate the inhibitory effects of oxLDL in LPS-activated microglia. In addition, their inhibitory effects were mimicked by liver X receptor (LXR) agonists and potentiated by a retinoid X receptor agonist, suggesting these molecules heterodimerize to function as oxysterol receptors. Taken together, our results demonstrate that oxLDL inhibits LPS-induced inflammatory responses in brain microglia and that these inhibitory effects are mediated by oxysterols and, at least in part, by the nuclear receptor LXR. Our results suggest an additional mechanism of action for oxidative stress that acts indirectly via modulation of inflammatory responses. Although further studies are needed, these results answer in part the question of why anti-oxidative strategies have not been successful in clinical situations. Moreover, as brain inflammation participates in the initiation and progression of several neurodegenerative disorders, the present data provide information that should prove a useful guide for designing therapeutic strategies to combat oxidative brain diseases.     

Cholestane-3beta,5alpha,6beta-triol inhibits osteoblastic differentiation and promotes apoptosis of rat bone marrow stromal cells

Converging lines of evidence suggest that oxidized lipids, long recognized as a risk factor in atherogenesis, also contribute to osteoporosis, but the underlying mechanism is not understood in detail. The effect of atherogenesis related factors including oxysterols on the differentiation and survival of marrow stromal cells (MSCs) would be very important in understanding the link between atherosclerosis and osteoporosis. In the present study, the effect of oxysterol cholestane-3beta,5alpha,6beta-triol (Triol) on osteoblastic differentiation and apoptosis of primary rat bone MSCs as well as the related mechanisms were studied. Triol inhibited MSCs osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, osteocalcin secretion, and matrix mineralization. In the other aspect, Triol promoted MSCs apoptosis, as characterized by condensed or fragmented nuclei as well as active externalization of phosphatidyl serine to the cell surface. In addition, Triol was found to induce increases of intracellular Ca2+ and Ca2+-dependent reactive oxygen species generation in MSCs. These effects were involved in the action of Triol on apoptosis, but not on osteoblastic differentiation of MSCs. These results suggested that Triol might contribute to the decreased bone formation by inhibition of osteoblastic differentiation and promotion of apoptosis of MSCs, providing insights about common factors underlying the pathogenesis of atherosclerosis and osteoporosis.     

Oxidized low-density lipoprotein (LDL) enhances thromboxane A(2) synthesis by platelets, but lysolecithin as a product of LDL oxidation has an inhibitory effect

Oxidation of low-density lipoprotein (LDL) by copper sulfate led to a significant increase in lysophosphatidylcholine (lyso PC) at the expense of phosphatidylcholine. Incubation of different concentrations of oxidized LDL (oxLDL) (32-650 microg protein/ml) with platelets for 1 h at 37 degrees C increased lyso PC content. The increase was dependent on oxLDL concentration. Incubation of platelets with various concentrations of lyso PC in solution for 5 or 15 min showed that lyso PC percentage was increased in the platelet membrane and the increase was dose dependent. Platelets incubated with various concentrations of lyso PC (2-100 microM) for 5 or 15 min and then triggered with thrombin also showed a significant decrease of thromboxane A(2) (TXA(2)) release as lyso PC concentration reached 10 microM or 6 microM, respectively. The decrease of TXA(2) release was more significant as lyso PC concentration was increased. The present study showed that this inhibition of TXA(2) release by lyso PC was due to 1) inhibition of phospholipase A(2) and the decrease of free arachidonic acid liberation from platelet phospholipid and 2) inhibition of cyclooxygenase. These inhibitory effects of lyso PC were discussed in relation to its effect on membrane fluidity. Lyso PC at concentrations of 30, 50, and 100 microM caused a sudden drop in TXA(2) release and a sudden increase of lactic dehydrogenase loss from the platelets due to their lysis and inhibition of cyclooxygenase enzyme. The present study shows that oxLDL contains high levels of lyso PC that are transferable to the platelets and can weaken their responsiveness to thrombin and decrease TXA(2) release. In our previous study, we found that oxLDL also contained high levels of oxysterols and thiobarbituric acid reactive substances (TBARS), which enhanced platelet reactivity to thrombin and increased TXA(2) release. We conclude that the net effect of oxLDL on platelets will depend on its degree of oxidation and the ratio between oxysterols plus TBARS/lyso PC. Variations in this ratio may explain some of the contradictions cited in the literature concerning the effect of oxLDL on platelet activation.