Biomedical subjects

C Mazière

Publications and source records attributed to C Mazière.

At least 19 recordsLinked to original sources

PubMed2004

Impact of inflammation and oxidative stress on vascular calcifications in chronic kidney disease.

Vascular and/or valvular calcifications in patients with chronic kidney disease (CKD) appear to indicate a poor prognosis in terms of overall survival and cardiovascular morbidity and mortality. Inflammation and oxidative stress represent new features of the arterial and/or valvular calcification process. However, only limited observational and epidemiological data are available in these areas. Therefore, the link between inflammation, oxidation and vascular and/or valvular calcifications deserves careful consideration in CKD patients, since they may become targets for the development of new therapeutic strategies.

Calcinosis↗

PubMed2002

[Oxidative stress, atherogenesis and cardiovascular risk factors].

Cardiovascular diseases are the main cause of mortality in the western world. It is widely accepted that atherosclerosis, the first etiology, is influenced by free radicals and the oxidizing stress that they cause. In the oxidative theory of atherosclerosis, the atheromatous lesion is initiated by oxidation of two density lipoproteins (LDL), a process still known as lipid peroxidation. Oxidized LDL have many effects on the cells of the vessel wall which, provide an explanation to most of the cellular and tissular changes observed in the plaque. The vascular complications of hypercholesterolaemia, diabetes, hyperhomocysteinemia, hypertension and smoking may, in part, be secondary to oxidizing stress that impairs endothelial function and modify the lipids in the intima of the vessels. The aim of this paper is to review the modes of free radical production, to determine the role of oxidizing stress in the development of atherosclerosis and to show how the different risk factors may initiate atheroma through oxidizing stress.

Arteriosclerosis↗

PubMed2001

Activation of JAK2 by the oxidative stress generated with oxidized low-density lipoprotein.

Atherosclerosis includes a series of cellular and molecular responses characteristic of an inflammatory disease. We provide evidence that cupric-ion-oxidized LDL (CuLDL) or endothelial cell-oxidized LDL (ELDL) induced the activation by Tyr-phosphorylation of JAK2, one of the Janus kinase involved upstream of STATs in the JAK/STAT pathway of cytokine transduction. Oxidized LDL (OxLDL) also initiated STAT1 and STAT3 Tyr-phosphorylation and translocation to the nucleus, with a more marked effect for the extensively modified CuLDL. Genistein, a nonspecific Tyr-kinase inhibitor, and AG490, a specific inhibitor of JAKs, markedly prevented the CuLDL-induced enhancement of STAT1 and STAT3 Tyr-phosphorylation and DNA-binding activity, suggesting that JAKs are the main kinases involved in STATs' activation by oxidized LDL. In addition, the lipid extract of CuLDL increased the intracellular levels of lipid peroxidation products and the Tyr-phosphorylation of JAK2, STAT1, and STAT3, whereas the antioxidant vitamin E prevented all these effects. These results demonstrate that OxLDL induces the activation by Tyr-phosphorylation of JAK2, STAT1, and STAT3 by generation of an intracellular oxidative stress by means of its lipid peroxidation products, and thus include JAK2 within the range of oxidative stress-activated kinases.

Cell Line↗

PubMed2001

Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts.

Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1-0.3 U/ml SMase induced a dose-dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose-dependent Tyr-phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose-dependent manner. Concomitantly, SMase enhanced the Tyr-phosphorylation of Janus kinase (JAK) 2, a Tyr-kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr-kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr-phosphorylation, together with STAT1 and STAT3 Tyr-phosphorylation and binding activity. The SMase-induced increase in STAT1/3 binding activity was prevented by methyl-beta-cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER-activated intracellular kinases.

Cell Line↗

PubMed2001

UVA radiation stimulates ceramide production: relationship to oxidative stress and potential role in ERK, JNK, and p38 activation.

Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4-9 J/cm(2). The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.

Cell Line↗

PubMed2001

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes.

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24 h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitonin-permeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.

Adenosine Triphosphate↗

PubMed2000

Oxidized LDL induces an oxidative stress and activates the tumor suppressor p53 in MRC5 human fibroblasts.

It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.

Binding Sites↗

PubMed2000

Biphasic effect of UVA radiation on STAT1 activity and tyrosine phosphorylation in cultured human keratinocytes.

The effect of ultraviolet A (UVA) radiation on the DNA-binding activity of the transcription factor STAT1 was studied by electromobility shift assay in the human keratinocyte cell line NCTC 2544. The STAT1-binding activity exhibited a biphasic pattern as a function of UVA doses. For UVA doses lower than 0.6 J/cm(2), a dose-dependent increase in STAT1 activity was observed. In a second phase, with higher UVA doses (1.5 to 9 J/cm(2)), the activity decreased and reached control value at 6 J/cm2. The enhancement of STAT1 activity was transient, peaked at 1 h after UV irradiation, and regularly decreased to control value 24 h after UV. Genistein, a tyrosine kinase inhibitor, H7, a serine/threonine kinase inhibitor, and PD 98059, a MEK inhibitor, prevented the UVA-induced enhancement of STAT1-binding activity, suggesting the involvement of Tyr, Ser/Thr kinases, and MEK in the observed effect. Immunoblot analysis directly demonstrated that the amount of Tyr-phosphorylated STAT1 was parallel to its DNA-binding activity. Immunoblot analysis also demonstrated the nuclear transport of STAT1 after UVA irradiation at low doses. At high doses, a decrease in the STAT1 level was observed both in the cytoplasmic and the nuclear compartments, suggesting that the inactivation was due to a degradation process. UVA irradiation initiated a dose-dependent increase in lipid peroxidation products and reactive oxygen species. Furthermore, the involvement of the oxidative stress in the UVA-induced effect on STAT1 activity is suggested by the protective action of the antioxidants alpha-tocopherol and N-acetylcysteine on both the activation phase (UVA doses lower than 1.5 J/cm(2)) and the inhibitory phase. By contrast, the pro-oxidant drug buthionine sulfoximine enhanced the effect of UVA on STAT1-binding activity. Since STATs are known as transducers of cytokine action, the enhancement of STAT1 activity by low doses of UVA might be related to the proinflammatory effect of solar radiations at the skin level.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones.

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.

Anti-Infective Agents↗

PubMed1999

Oxidized LDL activates STAT1 and STAT3 transcription factors: possible involvement of reactive oxygen species.

The effect of cupric ion-oxidized low density lipoprotein (Cu-LDL) or endothelial cell-oxidized LDL (E-LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu-LDL exhibited a more marked effect than E-LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid-reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol-containing compound N-acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu-LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.

DNA-Binding Proteins↗

PubMed1999

Cellular enrichment with polyunsaturated fatty acids induces an oxidative stress and activates the transcription factors AP1 and NFkappaB.

A 48-h incubation of cultured human fibroblasts with 5 x 10(-5) M oleic acid or polyunsaturated fatty acids (PUFA) from the (n-6) (linoleic, gamma-linolenic and arachidonic acids) or (n-3) (alpha-linolenic and eicosapentaenoic acids) series resulted in an enrichment of the cells with the introduced fatty acid. Cell enrichment with PUFA initiated a rise in the intracellular level of reactive oxygen species (ROS) and lipid peroxidation products (thiobarbituric reactive substances TBARS). Simultaneously, cell enrichment with all the studied PUFA induced an increase in AP1 and NFkappaB binding activity measured by electrophoretic mobility shift assay, whereas no significant effect was observed with the monounsaturated oleic acid. Furthermore, the antioxidants vitamin E (alpha-tocopherol) and N-acetyl cysteine prevented both the arachidonic acid-induced increase in intracellular ROS and TBARS, and the activation of AP1 and NFkappaB. These results indicate that the accumulation of PUFA from (n-6) and (n-3) series elicited an intracellular oxidative stress, resulting in the activation of oxidative stress-responsive transcription factors such as AP1 and NFkappaB.

Arachidonic Acid↗

PubMed1998

Polyunsaturated fatty acid enrichment enhances endothelial cell-induced low-density-lipoprotein peroxidation.

Oxidative modification of low-density lipoprotein (LDL) is an important feature in the initiation and progression of atherosclerosis. LDL modification by endothelial cells was studied after supplementation of the cells with oleic acid and polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. In terms of the lipid peroxidation product [thiobarbituric acid reactive substances (TBARS)] content and diene level of the LDL particle, oleic acid had no significant effect, and linoleic acid was poorly effective. Gamma linolenic acid (C18:3,n-6) and arachidonic acid (C20:4,n-6) increased by about 1.6-1.9-fold the cell-mediated LDL modification. PUFA from the n-3 series, alpha linolenic acid (C18:3,n-3), eicosapentaenoic acid (C20:5,n-3) and docosahexaenoic acid (C22:6,n-3), induced a less marked effect (1. 3-1.6-fold increase). The relative electrophoretic mobility of the LDL particle and its degradation by macrophages were enhanced in parallel. Concomitantly, PUFA stimulated superoxide anion secretion by endothelial cells. The intracellular TBARS content was also increased by PUFA. Comparison of PUFA from the two series indicates a good correlation between LDL oxidative modification, superoxide anion secretion and intracellular lipid peroxidation. The lipophilic antioxidant vitamin E decreased the basal as well as the PUFA-stimulated LDL peroxidation. These results indicate that PUFAs with a high degree of unsaturation of the n-6 and n-3 series could accelerate cell-mediated LDL peroxidation and thus aggravate the atherosclerotic process.

Cells, Cultured↗

PubMed1998

8-Methoxypsoralen potentiates the photocytotoxic effect of Photofrin II towards EMT-6 murine tumor cells.

Pretreatment of EMT-6 murine tumor cells for 24 h with 10(-4) M 8-methoxypsoralen (8-MOP) increased the photocytotoxicity of Photofrin II (P2) after cell exposure to low doses (1-1.5 J/cm2) of UVA by two- to three-fold. 8-MOP alone had no cytotoxic action under these experimental conditions and did not significantly change the amount of P2 recovered in cells. 8-MOP enhanced the lipid peroxidation end product formation measured as thiobarbituric acid reactive substances (TBARS) during cell photosensitization by P2. The psoralen alone also slightly increased the TBARS level after UVA exposure. These results suggest that 8-MOP, albeit non-photocytotoxic by itself under our experimental conditions, could enhance the efficiency of P2 by increasing cellular lipid peroxidation following light exposure.

Animals↗

PubMed1997

Copper and cell-oxidized low-density lipoprotein induces activator protein 1 in fibroblasts, endothelial and smooth muscle cells.

The effect of cupric ion- or endothelial cell-oxidized low-density lipoproteins (LDL) on transcription factor AP1 activation was investigated by electrophoretic mobility shift assay. Both oxidized LDL induced AP1 activation in fibroblasts, endothelial and smooth muscle cells. This phenomenon was also observed in the presence of cycloheximide. alpha-Tocopherol, a lipophilic free radical scavenger, and N-acetylcysteine, an hydrophilic antioxidant, partially inhibited the stimulatory effect of Cu2+-oxidized LDL. LDL modified by the mixture of the oxygen radicals OH. and O2.-, which generated lipid peroxidation products, also initiated AP1 activation, whereas LDL modified by OH. alone, which did not lead to marked LDL lipid peroxidation, was ineffective. Thus, lipid peroxidation products seem at least partially involved in the activation mechanism. Since AP1 activity is essential for the regulation of genes involved in cell growth and differentiation, our study suggests that the oxidative stress induced by oxidized LDL might be related to the fibroproliferative response observed in the atherosclerotic plaque.

Animals↗

PubMed1997

Diphenylhexatriene (DPH)-labeled lipids as a potential tool for studies on lipid peroxidation in monolayer films.

Using the fluorescence of diphenylhexatriene (DPH), lipid peroxidation in monomolecular films of phospholipids has been monitored dynamically to elucidate the efficacy of such a probe and to elucidate the effects of molecular organization on such peroxidation processes. Behavior in well-controlled model systems may be used to obtain insight into oxidative processes in complex biological systems. Mixed monolayers of hexadecanoyl-DPH-phosphatidylcholine (HDPH-PC) and diarachidonoyl-PC (DAA-PC) 1/10 (mol/mol) were prepared on a Langmuir trough. With pure water as subphase, and under an atmosphere of N2/O2 (3/1, v/v), DPH fluorescence remained stable over several hours, even under continuous illumination. However, over copper sulfate solution, rapid decay in the fluorescence intensity was observed and correlated with both the copper ion concentration and film pressure. No change in fluorescence was observed in absence of subphase copper ions or under an N2 atmosphere. Substitution of dioleoyl phosphatidylcholine for DAA-PC in systems above gave no decrease in fluorescence intensity. This lipid alone is not susceptible to free radical chain reaction. Absorption spectra from HDPH-PC/DAA-PC monolayers confirm that DPH is actually destroyed during film oxidation. We thus propose this system as a new experimental model for studies on lipid peroxidation in organized systems.

Copper↗

PubMed1996

Ultraviolet-A induces activation of AP-1 in cultured human keratinocytes.

UV-A irradiation induces a time-dependent activation of AP-1 in NCTC 2544 human keratinocytes. 4 h after irradiation, a 2-3-fold increase in AP-1 activity is observed in human keratinocytes and fibroblasts. Activation is still detectable 24 h later. The UV-A induced AP-1 binding complex is shown to contain c-Fos and c-Jun proteins. Lipophilic vitamin E impedes UV-A induced lipid peroxidation but does not prevent AP-1 activation which is inhibited by N-acetylcysteine, a hydrophilic antioxidant. This finding suggests that UV-A-dependent AP-1 activation is sensitive to the cellular redox state but is not related to membrane lipid peroxidation.

Acetylcysteine↗

PubMed1996

Interleukin 1 stimulates cholesterol esterification and cholesterol deposition in J774 monocytes-macrophages.

The effects of interleukin 1beta (IL1) in the range of concentration of 10-30 ng/ml on cholesterol metabolism were investigated in the monocyte-macrophage cell line J774. IL1 enhanced cholesterol esterification by [14C]oleic acid and acyl-coenzyme A cholesterol acyl transferase activity in a dose-dependent manner. Incubation of IL1-treated cells with acetylated low density lipoproteins labelled with [3H]cholesteryllinoleate resulted in accumulation of radioactive cholesterol in free and esterified form. Concomitantly, IL1 increased the free and esterified cholesterol intracellular content measured by the cholesterol oxidase technique. The effect of IL1 on cholesterol esterification by oleic acid was not observed in the presence of cycloheximide or of the ACAT inhibitor Sandoz 58 035. IL1 also stimulated cholesterol esterification in other cell types such as human fibroblasts and murine endothelial and smooth muscle cells. The effect of IL1 is specific, since IL2 or tumor necrosis factor (TNF) exhibited no significant activity, whereas oncostatin M only slightly enhanced cholesterol esterification. Since cholesterol deposition is involved in the initiation and progression of the atherosclerotic lesions, these findings highlight the role of the inflammatory cytokine IL1 on this process.

Amides↗

PubMed1996

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926.

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64 +/- 0.18 (m +/- SE, n = 11) and 3.12 +/- 0.24 (n = 11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14 +/- 3% (n = 34) and 22 +/- 13% (n =10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12 +/- 0.04 (n = 11) and 0.07 +/- 0.01 nmol/min/mg cell protein (n = 11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.

Cell Line↗