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Biomedical subjects

Anne Nègre-Salvayre

Publications and source records attributed to Anne Nègre-Salvayre.

16 recordsLinked to original sources
PubMed2006

Desensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers.

The platelet-derived growth factor receptor-beta (PDGFRbeta) signaling pathway regulates smooth muscle cell (SMC) migration and proliferation and plays a role in the vascular wall response to injury. Oxidized low-density lipoprotein (oxLDL) in atherosclerotic lesions can activate the PDGFRbeta pathway, but the long-term effects of oxLDL on PDGFRbeta function are not well understood. We found that oxLDL induced a dual effect on PDGFRbeta signaling. Initial activation of the PDGFR was followed by desensitization of the receptor. PDGFRbeta desensitization was not attributable to PDGFRbeta degradation or changes in localization to the caveolae but instead resulted from decreased PDGF binding and inhibition of PDGFRbeta tyrosine kinase activity. This inhibition was associated with formation of (4HNE)- and acrolein-PDGFRbeta adducts and was mimicked by preincubation of cells with 4HNE. These PDGFRbeta adducts were also detected in aortae of apolipoprotein-deficient mice and hypercholesterolemic rabbits and in human carotid plaques. The aldehyde scavengers DNPH and Hydralazine prevented both oxLDL- and 4HNE-induced structural modification and PDGFRbeta signaling dysfunction in cells and in vivo. OxLDL inhibition of PDGF signaling may contribute to defective SMC proliferation and decrease the stability of a vulnerable plaque.

Aldehydes↗
PubMed2005

Design, synthesis, and evaluation of pharmacological properties of cinnamic derivatives as antiatherogenic agents.

A series of cinnamic and phosphonocinnamic derivatives have been synthesized and their ability to inhibit cell-mediated LDL oxidation and oxidized LDL-induced cytotoxicity was investigated. Electron-donating substituents surrounding the necessary 4-OH group of the aromatic ring showed the best results. Among the different series tested, amide 1, thioester 5c, phosphonoester 7c, and the fluorophosphonocinnamic analogue 12c exhibited a potent inhibitory effect against LDL oxidation (and subsequent toxicity) mediated by cultured human microvascular endothelial cells (HMEC-1), with an efficacy comparable to that observed with probucol. Beside this indirect protective effect, these compounds exhibited a direct protective effect against the toxicity of previously oxidized LDL in HMEC-1. These data suggest that the newly synthesized cinnamic compounds should protect against early events (cell-mediated LDL oxidation) occurring within the vascular wall in atherosclerosis.

Antioxidants↗
PubMed2005

The grape-derived polyphenol resveratrol differentially affects epidermal and platelet-derived growth factor signaling in human liver myofibroblasts.

The grape-derived polyphenol resveratrol is anti-proliferative for human liver myofibroblasts, which may be beneficial for the treatment of liver fibrosis. However, its mechanism of action is ill understood. Here, we have studied how resveratrol interfered with signaling pathways used by epidermal or platelet-derived growth factors to induce the proliferation of these cells. We found that resveratrol inhibited epidermal growth factor or platelet-derived growth factor-induced DNA synthesis. Resveratrol did not, however, decrease epidermal growth factor receptor autophosphorylation or activation of extracellular regulated kinases, but strongly inhibited the phosphorylation of Akt and of its substrate forkhead related transcription factor. This suggested that resveratrol inhibited epidermal growth factor-induced mitogenic signaling through inhibition of the phosphatidylinositol 3-kinase /Akt pathway. The phosphatidylinositol 3-kinase inhibitor LY 294002, also, inhibited epidermal growth factor-dependent DNA synthesis and Akt phosphorylation but did not decrease extracellular regulated kinases phosphorylation. In contrast, resveratrol inhibited platelet-derived growth factor-stimulated receptor autophosphorylation and every subsequent signaling step. Resveratrol did not directly inhibit phosphatidylinositol 3-kinase activity measured on immunoprecipitates from epidermal growth factor-stimulated myofibroblasts, but it strongly reduced the autophosphorylation of the phosphatidylinositol 3-kinase downstream target phospho-inositide-dependent kinase-1 that phosphorylates Akt. We, thus, show that resveratrol has growth factor-specific effects: it inhibits platelet-derived growth factor signaling via reduced receptor activation, whereas it reduces epidermal growth factor-dependent DNA synthesis via inhibition of the phosphatidylinositol 3-kinase/Akt pathway, possibly through inhibition of phospho-inositide-dependent kinase-1 activity.

Cell Proliferation↗
PubMed2005

Structural modifications of HDL and functional consequences.

High density lipoproteins (HDL) are susceptible to structural modifications mediated by various mechanisms including oxidation, glycation, homocysteinylation or enzymatic degradation. Structural alterations of HDL may affect their functional and atheroprotective properties. Oxidants, such as hypochlorous acid, peroxyl radicals, metal ions, peroxynitrite, lipoxygenases and smoke extracts, can alter both surface and core components of HDL. The formation of lipid peroxidation derivatives, such as thiobarbituric acid reactive substances, conjugated dienes, lipid hydroperoxides and aldehydes, is associated with changes of physical properties (fluidity, molecular order) and of apoprotein conformation. Non-enzymatic glycation, generally associated with lipoxidation, leads to form irreversible complexes called advanced glycation end products. These HDL modifications are accompanied with altered biological activities of HDL and associated enzymes, including paraoxonase, CETP and LCAT. Homocysteine-induced modification of HDL is mediated by homocysteine-thiolactone, and can be prevented by a calcium-dependent thiolactonase/paraoxonase. Tyrosylation of HDL induces the formation of dimers and trimers of apo AI, and alters cholesterol efflux. Phospholipases and proteolytic enzymes can also modify HDL lipid and apoprotein structure. HDL modification induces generally the loss of their anti-inflammatory and cytoprotective properties. This could play a role in the pathogenesis of atherosclerosis and neurodegenerative diseases such as Alzheimer's disease.

Antioxidants↗
PubMed2005

Effect of 4-hydroxynonenal on phosphatidylethanolamine containing condensed monolayer and on its interaction with apolipoprotein A-I.

4-Hydroxynonenal (4HNE), generated during polyunsaturated fatty acid oxidation, is present in atherosclerotic lesions. As 4HNE is able to react with phosphatidylethanolamine (PE), we investigated, using AC polarography, whether it may alter the physico-chemical state of a condensed PE-containing phospholipid monolayer and its interaction with apoA-I. The stability of a phospholipid monolayer relative to potential (around the potential of zero charge) is dependent on lipid composition (PE>PC>PE/PC). ApoA-I insertion into PE/PC monolayer is easier than in PC monolayer. Pre-treatment of PE/PC monolayer by 4HNE does not alter monolayer stability, but decreases apo A-I insertion into the monolayer.

Aldehydes↗
PubMed2005

Activation of the {beta}-catenin/T-cell-specific transcription factor/lymphoid enhancer factor-1 pathway by plasminogen activators in ECV304 carcinoma cells.

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.

Active Transport, Cell Nucleus↗
PubMed2004

Two distinct calcium-dependent mitochondrial pathways are involved in oxidized LDL-induced apoptosis.

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular endothelial cells may contribute to plaque erosion and rupture. We aimed to clarify the relationship between the oxLDL-induced calcium signal and induction of apoptotic pathways. METHODS AND RESULTS: Apoptosis was evaluated by biochemical methods, including studies of enzyme activities, protein processing, release of proapoptotic factors, chromatin cleavage, and especially by morphological methods that evaluate apoptosis/necrosis by SYTO-13/propidium iodide fluorescent labeling. The oxLDL-induced sustained calcium rise activated 2 distinct calcium-dependent mitochondrial apoptotic pathways in human microvascular endothelial cells. OxLDLs induced calpain activation and subsequent Bid cleavage and cytochrome C release, which were blocked by calpeptin. Cyclosporin-A inhibited cytochrome C release, possibly by inhibiting the opening of the mitochondrial permeability transition pore (mPTP). Calcineurin, another cyclosporin-sensitive step, was not implicated, because oxLDLs inhibited calcineurin and FK-506 treatment was ineffective. Cytochrome C release in turn induced caspase-3 activation. In addition, oxLDLs triggered release and nuclear translocation of mitochondrial apoptosis-inducing factor through a mechanism dependent on calcium but independent of calpains, mPTP, and caspases. CONCLUSIONS: OxLDL-induced apoptosis involves 2 distinct calcium-dependent pathways, the first mediated by calpain/mPTP/cytochrome C/caspase-3 and the second mediated by apoptosis-inducing factor, which is cyclosporin-insensitive and caspase-independent.

Apoptosis↗
PubMed2004

Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation.

BACKGROUND: Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. METHODS AND RESULTS: OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2-/- cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2-/- cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. CONCLUSIONS: Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

Animals↗
PubMed2004

The sphingomyelin/ceramide pathway is involved in ERK1/2 phosphorylation, cell proliferation, and uPAR overexpression induced by tissue-type plasminogen activator.

Plasminogen activators (tPA and uPA) are serine proteases that convert the circulating zymogen plasminogen to active plasmin and mediate fibrin degradation. These multifunctional proteins trigger various biological events such as extracellular matrix degradation, cell adhesion, migration, and proliferation, through not yet fully characterized mechanisms. We report that, in smooth muscle cells and ECV-304 carcinoma cells, tPA and ATF (the N-terminal catalytically inactive fragment of tPA) elicited DNA synthesis that requires activation of the sphingomyelin/ceramide/sphingosine-1-phosphate (Spm/Cer/S1P), signaling pathway and was blocked by D-erythro-2-(N-myristoylamino)-1-phenyl-propanol (D-MAPP) and N-N'-dimethyl sphingosine (DMS), two classical inhibitors of sphingosine-1-phosphate biosynthesis. Binding of tPA to its receptor uPAR triggered the coordinated activation of two key enzymes of the Spm/Cer/S1P pathway, the neutral sphingomyelinase and the sphingosine kinase-1 that was mediated by a common pertussis toxin (PTX)-sensitive mechanism. The tPA-induced sphingosine kinase-1 activation was mediated by Src, since it was inhibited by herbimycin A and in SrcK- cells (overexpressing a dominant negative kinase defective form of Src) and by ERK1/2 (early phase peaking at 15 min). Sphingosine kinase-1 activation was followed by a second phase of ERK1/2 phosphorylation (peaking at 120 min) and subsequent DNA synthesis, which were inhibited by D-MAPP and DMS, by anti-EGD-1 antibodies and in SrcK- cells (in which the mitogenic signaling was rescued by sphingosine-1-phosphate). Altogether, these data underline a pivotal role for the Spm/Cer/S1P pathway in the tPA-induced mitogenic signaling.

Benzoquinones↗
PubMed2003

Mitochondria play a central role in apoptosis induced by alpha-tocopheryl succinate, an agent with antineoplastic activity: comparison with receptor-mediated pro-apoptotic signaling.

alpha-Tocopheryl succinate (alpha-TOS) is a semisynthetic vitamin E analogue with high pro-apoptotic and anti-neoplastic activity [Weber, T et al. (2002) Clin. Cancer Res. 8, 863-869]. Previous studies suggested that it acts through destabilization of subcellular organelles, including mitochondria, but compelling evidence is missing. Cells treated with alpha-TOS showed altered mitochondrial structure, generation of free radicals, activation of the sphingomyelin cycle, relocalization of cytochrome c and Smac/Diablo, and activation of multiple caspases. A pan-caspase inhibitor suppressed caspase-3 and -6 activation and phosphatidyl serine externalization, but not decrease of mitochondrial membrane potential or generation of radicals. For alpha-TOS, but not Fas or TRAIL, apoptosis was suppressed by caspase-9 inhibition, while TRAIL- and Fas-resistant cells overexpressing cFLIP or CrmA were susceptible to alpha-TOS. The central role of mitochondria was confirmed by resistance of mtDNA-deficient cells to alpha-TOS, by regulation of alpha-TOS apoptosis by Bcl-2 family members, and by anti-apoptotic activity of mitochondrially targeted radical scavengers. Co-treatment with alpha-TOS and anti-Fas IgM showed their cooperative effect, probably by signaling via different, convergent pathways. These data provide an insight into the molecular mechanism, by which alpha-TOS kills malignant cells, and advocate its testing as a potential anticancer agent or adjuvant.

Antineoplastic Agents↗
PubMed2003

Impairment of the cytotoxic and oxidative activities of 7 beta-hydroxycholesterol and 7-ketocholesterol by esterification with oleate.

Atherosclerosis involves inflammatory processes, as well as cytotoxic and oxidative reactions. In atherosclerotic plaques, these phenomena are revealed by the presence of dead cells, oxidized lipids, and oxidative DNA damage, but the molecules triggering these events are still unknown. As 7 beta-hydroxycholesterol and 7-ketocholesterol, which are present at elevated concentrations in atherosclerotic lesions, are strongly cytotoxic and pro-oxidative, their effects were determined on cell death, superoxide anion and nitric oxide production, lipid peroxidation, and oxidative DNA damage. 7-Ketocholesterol- and 7 beta-hydroxycholesterol-induced cell death leads to a loss of mitochondrial potential, to increased permeability to propidium iodide, and to morphological nuclear changes (swelling, fragmentation, and/or condensation of nuclei). These effects are preceded by the formation of cytoplasmic monodansylcadaverine-positive structures and are associated with a rapid enhancement of cells overproducing superoxide anions, a decrease in cells producing nitric oxide, lipid peroxidation (formation of malondialdehyde and 4-hydroxynonenal adducts, low ratio of [unsaturated fatty acids]/[saturated fatty acids]) as well as oxidative DNA damage (8-oxoguanine formation). Noteworthy, none of the cytotoxic features previously observed with 7 beta-hydroxycholesterol and 7-ketocholesterol were noted with cholesterol, 7 beta-hydroxycholesteryl-3-oleate and 7-ketocholesteryl-3-oleate, with the exception of a slight increase in superoxide anion production with 7 beta-hydroxycholesteryl-3-oleate. This finding supports the theory that 7 beta-hydroxycholesterol and 7-ketocholesterol could induce cytotoxic and oxidative processes observed in atherosclerotic lesions and that esterification of these compounds may contribute to reducing atherosclerosis progression.

Arteriosclerosis↗
PubMed2002

Mildly oxidized LDL particle subspecies are distinct in their capacity to induce apoptosis in endothelial cells: role of lipid hydroperoxides.

The risk of atherosclerosis is intimately related to the heterogeneity of low-density lipoprotein (LDL) particles. The potential relationship between oxidative modification of distinct LDL subspecies and induction of apoptosis in arterial wall cells is indeterminate. The capacity of light LDL3 versus dense LDL5 to induce cytotoxicity in endothelial cells as a function of the degree of copper-mediated oxidation was compared. Mildly oxidized LDL3 (oxLDL3) exerted potent cytotoxicity, which was intimately related to both the degree of oxidation and the oxLDL3 concentration based on either cholesterol content or particle number. In contrast, dense LDL5 particles exerted a minor effect on cell viability. Cells incubated with oxLDL3 exhibited apoptotic features, with cytoplasmic condensation, cell or nuclear fragmentation, and accumulation of DNA fragments. OxLDL3-induced apoptosis involved cytoplasmic release of cytochrome c, with a concomitant increase in caspase-3-like protease activity. OxLDL3 particles were uniquely distinct from oxLDL5 particles in their elevated content of lipid hydroperoxides. Hydroperoxide removal by NaBH4 markedly reduced oxLDL3-induced cytotoxicity, leading to an increase in cell viability. Lipid hydroperoxide content of oxidatively modified LDL subclasses is therefore a major determinant of the induction of apoptosis in endothelial cells. These data are highly relevant to atherogenic hypercholesterolemia, in which the LDL phenotype is dominated by elevated concentrations of light LDL3.

Apoptosis↗
PubMed2002

Mitochondrial oxidative stress is modulated by oleic acid via an epidermal growth factor receptor-dependent activation of glutathione peroxidase.

Mitochondria generate reactive oxygen species (ROS) under various pathophysiological conditions. In isolated mitochondria, fatty acids (FA) exhibit an uncoupling effect of the respiratory activity and modulate ROS generation. The effect of FA on intact cultured cells remains to be elucidated. The present study reports that FA (buffered by BSA) decrease the level of cellular ROS generated by the mitochondrial respiratory chain in cultured cells incubated with antimycin A. Both saturated and unsaturated FA are effective. This fatty acid-induced antioxidant effect does not result from a decrease in ROS production, but is subsequent to cellular glutathione peroxidase (GPx) activation and enhanced ROS degradation. This fatty acid-induced GPx activation is mediated through epidermal growth factor receptor (EGFR) signalling, since this response is (i) abrogated by the EGFR inhibitor AG1478 or by a defect in EGFR (in EGFR-deficient B82L fibroblasts), (ii) restored in B82LK+ cells expressing EGFR and (iii) mimicked by epidermal growth factor. These findings indicate that FA contribute to enhance cellular antioxidant defences against mitochondrial oxidative stress through EGFR-dependent GPx activation.

Cell Line↗
PubMed2002

Increased reactive oxygen species production with antisense oligonucleotides directed against uncoupling protein 2 in murine endothelial cells.

Uncoupling protein 2 (UCP-2) belongs to the mitochondrial anion carrier family. It is ubiquitously expressed but is most abdundant in the reticuloendothelial system. In addition to uncoupling function, UCP-2 modulates the production of reactive oxygen species (ROS) by isolated mitochondria. Using an antisense oligonucleotide strategy, we investigated whether a defect in UCP-2 expression modulates ROS in intact endothelial cells. Murine endothelial cells (CRL 2181) pretreated by antisense oligonucleotides directed against UCP-2 mRNA exhibited a significant and specific increase in membrane potential and intracellular ROS level compared with control scrambled or anti-UCP-1 and -UCP-3 antisense oligonucleotides. These specific changes induced by UCP-2 antisense oligonucleotides were correlated with a rise in extracellular superoxide anion production and oxidative stress assessed by thiobarbituric acid reactive substance values. Taken together, these data suggest a role for UCP-2 in control of ROS production and subsequent oxidation of surrounding compounds mediating oxidative stress of endothelial cells. These data also support the notion that manipulations of UCP-2 at the genetic level could control ROS metabolism at the cellular level.

Animals↗
PubMed2002

Advanced glycation end product precursors impair epidermal growth factor receptor signaling.

Formation of advanced glycation end products (AGEs) is considered a potential link between hyperglycemia and chronic diabetic complications, including disturbances in cell signaling. It was hypothesized that AGEs alter cell signaling by interfering with growth factor receptors. Therefore, we studied the effects of two AGE precursors, glyoxal (GO) and methylglyoxal (MGO), on the epidermal growth factor receptor (EGFR) signaling pathway in cultured cells. Both compounds prevented tyrosine autophosphorylation induced by epidermal growth factor (EGF) in a time- and dose-dependent manner as well as phospholipase Cgamma1 recruitment and subsequent activation of extracellular signal-regulated kinases. AGE precursors inhibit EGF-induced EGFR autophosphorylation and tyrosine kinase activity in cell membranes and in EGFR immunoprecipitates. In addition, AGE precursors strongly inhibited cellular phosphotyrosine phosphatase activities and residual EGFR dephosphorylation. AGE precursors induced the formation of EGFR cross-links, as shown by the cross-reactivity of modified EGFR with an anti-N(epsilon)(carboxymethyl)lysine antibody, suggesting that altered EGFR signaling was related to carbonyl-amine reactions on EGFR. Aminoguanidine, an inhibitor of AGE formation, partially prevented the EGFR dysfunction induced by GO and MGO. These data introduce a novel mechanism for impaired cellular homeostasis in situations that lead to increased production of these reactive aldehydes, such as diabetes.

Cell Line, Transformed↗