Thomas Montange

Comparison of the cytotoxic, pro-oxidant and pro-inflammatory characteristics of different oxysterols.

Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, refered to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7α-, 7β-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O2⋅ −) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O2⋅ − overproduction quantified with hydroethidine was identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, with cholesterol-5α, 6α-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7β-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.

Measurement of inflammatory cytokines by multicytokine assay in tears of patients with glaucoma topically treated with chronic drugs

Aim: To investigate the ocular surface inflammatory response to chronic topical treatments in patients with glaucoma by measuring the cytokine level in tears using multiplex bead analysis. Methods: Tear samples were collected from 21 patients with glaucoma and 12 healthy volunteers. Tears were analysed for the presence of 17 cytokines: interleukin (IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, IL17, granulocyte-colony stimulating factor, granulocyte-macrophage stimulating factor, interferon (INF)γ, monocyte chemotactic protein (MCP)1, macrophage inflammatory protein 1β and tumour necrosis factor (TNF)α. The cytokines in each sample of tears were measured using multiplex bead analysis with microspheres as solid support for immunoassays. Results: In the tears of treated patients, proinflammatory cytokines (IL1β, IL6, IL12, TNFα) were significantly increased compared with controls. T helper (Th)1 (INFγ, IL2) and Th2 (IL5, IL10, IL4) type cytokines were also significantly higher (p<0.05); however, the most marked increase was observed with Th1 cytokines. The expression of chemokine IL8 and MCP1 was also increased in the treated group. Conclusion: This study shows that pro-inflammatory cytokine secretion by conjunctival cells is increased in response to topical treatments for glaucoma. The characterisation of cytokines in tears was previously limited by the small volume attainable, a limitation that has been overcome by multiplex analysis.

Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients

Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C‐reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7β‐hydroxycholesterol, 7‐ketocholesterol, and 25‐hydroxycholesterol), and various cytokines.

7-Ketocholesterol-induced apoptosis. Involvement of several pro-apoptotic but also anti-apoptotic calcium-dependent transduction pathways.

Oxysterols, and particularly 7‐ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7‐ketocholesterol‐induced apoptosis is triggered by a sustained increase of cytosolic‐free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium‐dependent phosphatase calcineurin, leading to dephosphorylation of the ‘BH3 only’ protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7‐ketocholesterol‐induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium‐dependent pathways during 7‐ketocholesterol‐induced apoptosis. The activation of the MEK→ERK pathway by the calcium‐dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro‐apoptotic BH3 only protein, Bim. Indeed, 7‐ketocholesterol treatment of human monocytic THP‐1 cells induces the release of Bim‐LC8 from the microtubule‐associated dynein motor complex, and its association with Bcl‐2. Therefore, it appears that 7‐ketocholesterol‐induced apoptosis is a complex phenomenon resulting from calcium‐dependent activation of several pro‐apoptotic pathways and also one survival pathway.

Phospholipidosis and down-regulation of the PI3-K/PDK-1/Akt signalling pathway are vitamin E inhibitable events associated with 7-ketocholesterol-induced apoptosis.

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (alpha-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

Effect of Plasma Phospholipid Transfer Protein Deficiency on Lethal Endotoxemia in Mice

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide-binding protein (LBP), CD14, and MD-2, which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide-binding protein gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction toward circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies, which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of proinflammatory cytokines were found in PLTP-deficient as compared with wild type mice. Similar inflammatory damage occurred in tissues from wild type and PLTP-deficient mice 24 h after one single intraperitoneal injection of LPS but with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild type and PLTP-deficient mice further supported the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.

Cytotoxic oxysterols induce caspase-independent myelin figure formation and caspase-dependent polar lipid accumulation.

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7β-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7β-hydroxycholesterol, cholesterol-5β, 6β-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7β-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.

Analogies entre processus athéromateux et dégénérescence maculaire liée à l’âge : rôles présumés des oxystérols

Les mecanismes physiopathologiques de la degenerescence maculaire liee a l’âge (DMLA) sont encore peu connus. Comme d’autres pathologies du vieillissement telles que la maladie d’Alzheimer ou l’atherosclerose, la DMLA est associee a l’apparition de depots macromoleculaires anormaux appeles drusen. Ces derniers sont localises au niveau de la membrane de Bruch. De recentes investigations sur la nature de ces depots, leurs relations avec les reactions inflammatoires et immunitaires ainsi que sur le role important du stress oxydatif ont permis d’elargir les hypotheses sur les mecanismes physiopathologiques conduisant aux lesions de DMLA. De nombreuses analogies avec les mecanismes impliques dans la genese des lesions d’atherosclerose suggerent des similitudes physiopathologiques entre ces deux maladies. Le role pro-atherogene du cholesterol et de ses derives oxydes, les oxysterols, a ete largement demontre dans les processus atheromateux. Il semblerait que ces composes interviennent egalement dans la genese de la DMLA entrainant des activites cytotoxiques, pro-oxydantes et pro-inflammatoires.

Branched-chain fatty acids, increased in tears of blepharitis patients, are not toxic for conjunctival cells

Aim: The composition of the meibum of blepharitis patients is characterised by increased levels of branched-chain fatty acids (BCFAs) that return to normal values in patients treated with cyclins and lid hygiene. The aim of this study was to determine if BCFAs had toxic effects on conjunctival cells related to the disease. Methods: Chang and IOBA-NHC conjunctival human cells were treated with BCFAs (isoC16 and isoC20) or palmitic acid as a control for 4 h or 24 h at 50 μM or 100 μM. Morphological and functional changes were investigated by measuring mitochondrial dehydrogenase activity, cell permeability, mitochondrial depolarisation, chromatin condensation, IL-1β and reactive oxygen species production. Results: None of the fatty acids modified the parameters of cytotoxicity in conjunctival cells in Chang or IOBA-NHC cell lines. Only the mitochondrial dehydrogenase activity was significantly decreased in relation to the isoC20 concentration increase. Conclusions: The increase in BCFAs in the tears of blepharitis patients does not consistently participate in the conjunctival cell changes throughout the course of the disease. Instead, it is likely an adaptive response of the ocular surface to the lack of tears, possibly increasing meibum fluidity, thus enhancing lacrimal film stability.

Activation of a Caspase-3-Independent Mode of Cell Death Associated with Lysosomal Destabilization in Cultured Human Retinal Pigment Epithelial Cells (ARPE-19) Exposed to 7β-Hydroxycholesterol

Purpose: To characterize the possible cytotoxic effects of oxysterols (7β-hydroxycholesterol (7β-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects. Methods: ARPE-19 cells were treated with 7β-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated. Results: Cell viability of ARPE-19 cells was decreased with 7β-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7β- OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death. Conclusion: 7β-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7β-OH.