Biomedical subjects

R Salvayre

Publications and source records attributed to R Salvayre.

At least 19 recordsLinked to original sources

PubMed2004

Detection and incidence of muscular adverse drug reactions: a prospective analysis from laboratory signals.

AIMS: The awareness of muscular adverse drug reactions (ADRs) increased since the withdrawal of cerivastatin, a HMG-CoA reductase inhibitor, from the market in August 2001. Our objectives were to assess the detection and incidence of muscular ADRs in a University Hospital using biochemical laboratory data and to evaluate the underreporting rate of drug-induced muscular disorders. METHODS: A prospective study was undertaken at Toulouse University Hospital, France, for 1 week per month from November 2001 to October 2002. Patients were selected by means of a computerized process using biochemical laboratory data based on serum creatine phosphokinase (CPK) values (over twofold normal). Medical records of all selected patients were then consulted. RESULTS: During the period of the study, 2017 CPK tests were performed, among which 171 values were over twofold normal corresponding to 129 patients. Because of lack of data, 26 patients were excluded. Among these patients ( n=103), 28 cases of muscular ADRs were suspected, 22 of which were detected in outpatient departments. Four patients were totally asymptomatic and five had an increase of CPK over fivefold normal. Nine cases were classified as "serious". Withdrawal of suspected drugs were done in 16 cases with regression of ADRs in 13 cases. According to hospitalization data, the incidence of muscular ADRs was estimated as 7.2 (2.6-15.7) per 10,000 inpatients and 9.3 (5.8-14.1) per 10,000 outpatients over 12 weeks. The involved drugs were mainly: statins (46.4%), fibrates (14.3%), antiretrovirals (14.3%), angiotensin-II receptor antagonists (10.7%), immunosuppressants (7.1%) or hydroxychloroquine (7.1). Only two cases, judged as "serious", were spontaneously reported by physicians during the same period. CONCLUSION: The results of this survey underline the importance to take into account drug hypothesis in muscular injuries diagnosis.

Adult↗

PubMed2002

Choice of different dyes to label tyrosine and nitrotyrosine.

In this work, we will present some attempts to analyze tyrosine and nitrotyrosine using capillary electrophoresis and either UV-Visible detection or laser-induced fluorescence (LIF) detection. An argon ion (488 nm) laser is used for fluorescein isothiocyanate (FITC) and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F). A near infrared (780 nm) laser is used for NIR 780 derivatives. The UV-Visible limit of detection is 2.5 microM whereas it is in the range of 30 nM for LIF detection.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Assay of total homocysteine and other thiols by capillary electrophoresis and laser-induced fluorescence detection. II. Pre-analytical and analytical conditions.

In recent papers, we presented a new analytical method for thiol quantification in serum. This method was developed with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze thiol-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results for homocysteine, glutathione, cysteinylglycine, and cysteine were presented (Caussé E., et al., Clin. Chem. 45 (1999) 412). An exhaustive comparison of the quantitation of homocysteine in plasma, using high-performance liquid chromatography with either conventional fluorescence detection or fluorescence polarization immunoassay was also reported (Caussé E., et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. Recently we studied protein precipitation in serum with different organic agents (Caussé E., et al., J. Chromatogr. A 895 (2000) 173). In this work, we evaluated the conditions of protein precipitation in function of the amounts of acetonitrile and their influence on quantitation and quality of the electropherograms. Then, we looked at the variation of thiol concentrations in the haemolysis states and studied the thiol stability of blood samples cooled on ice.

Adult↗

PubMed2002

Fenofibrate induces a selective increase of protein-bound homocysteine in rodents: a PPARalpha-mediated effect.

Elevated levels of plasma homocysteine (Hcy) are associated with increased risk of cardiovascular disease though it is uncertain whether increases in Hcy represent a cause or a consequence of the disease process. Plasma Hcy exists in reduced, free oxidized, and protein-bound forms, that together comprise total Hcy (tHcy). Free reduced Hcy is thought to be the atherogenic, though minor, sub-fraction of tHcy. Recent reports have indicated that fenofibrate and other fibrates are capable of moderately increasing plasma tHcy. As many of the effects of fibrates are known to be mediated by the nuclear receptor PPARalpha, we determined the effect of fenofibrate on tHcy in PPARalpha-deficient mice. We further examined the effect of fenofibrate and fenofibrate plus folate supplementation on total as well as protein-bound Hcy in rats. Fenofibrate significantly increased serum tHcy in wild-type mice but not in PPARalpha deficient mice. In rats, fenofibrate increased serum tHcy by 69%, while the co-administration of folate with fenofibrate increased tHcy by only 7%. In spite of the above increase in tHcy in rats, only the protein-bound fraction of Hcy was increased. In a further study, fenofibrate also induced a significant increase in tHcy, while in spite of this, ex vivo peroxidation of VLDL+LDL was beneficially lowered and the lag time prolonged. In summary, fenofibrate increases serum tHcy in rodents in a PPARalpha-dependent manner. The increase in rats is solely due to protein-bound Hcy as atherogenic, reduced Hcy was unchanged. While awaiting corroboration in human, our results suggest that the extent and mechanism of the increase in total Hcy in patients treated with fenofibrate should not a priori be associated with relevant risk.

Animals↗

PubMed2001

Angiotensin II induces phenotype-dependent apoptosis in vascular smooth muscle cells.

Angiotensin II regulates vascular structure through growth and apoptosis, with implications in pathophysiology. Subtypes of vascular smooth muscle cells with specific morphology, growth, or apoptotic features have been isolated. Here, we investigated the effects of angiotensin II on apoptosis of 2 morphologically different rat aortic smooth muscle cell phenotypes. Spindle and epithelioid cell lines cultured under low serum conditions were stimulated by angiotensin II. Responsiveness was evaluated by calcium signaling. In both phenotypes, an angiotensin II type 1 receptor-mediated transient intracellular calcium peak arose from intracellular pools. However, a sustained nifedipine-sensitive calcium entry occurred specifically in epithelioid cells. Angiotensin II did not impair spindle cell survival, whereas a delayed reduction in cell number occurred in epithelioid cells. Cell death through apoptosis was characterized by cellular and nuclear morphology. Consistently, DNA fragmentation, evaluated by biochemical quantification, nuclei staining, and ladders, and caspase 3-like activity were promoted by angiotensin II in epithelioid cells. Kinetics of annexin V binding showed that apoptosis was a delayed process. Angiotensin II-induced apoptosis of epithelioid cells was prevented by angiotensin II type 1 but not type 2 receptor antagonists and was inhibited by a calcium chelator or calcium antagonist. Conversely, epithelioid cell apoptosis could be induced by a calcium ionophore. Thus, the death signaling promoted by angiotensin II in epithelioid cells involves type 1 receptor-mediated calcium entry. These data suggest that angiotensin II can promote angiotensin II type 1 receptor-mediated apoptosis in vascular smooth muscle cells, depending on their phenotype. This process may play a role in vascular remodeling in cardiovascular diseases.

Angiotensin II↗

PubMed2001

Sphingolipid mediators in cardiovascular cell biology and pathology.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Animals↗

PubMed2001

Mildly oxidized LDL induces activation of platelet-derived growth factor beta-receptor pathway.

BACKGROUND: Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor beta (PDGFRbeta), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. METHODS AND RESULTS: In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFRbeta signaling pathway, as shown by PDGFRbeta tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFRbeta activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFRbeta activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFRbeta (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFRbeta adduct formation and subsequent PDGFRbeta activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H(2)O(2) (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFRbeta adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFRbeta activation may occur during atherogenesis. CONCLUSIONS: MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFRbeta acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFRbeta activation in atherosclerotic areas.

Aldehydes↗

PubMed2001

Ceramide in apoptosis signaling: relationship with oxidative stress.

Ceramide is one of the major sphingosine-based lipid second messengers that is generated in response to various extracellular agents. However, while widespread attention has focused on ceramide as a second messenger involved in the induction of apoptosis, important issues with regard to the mechanisms of ceramide formation and mode of action remain to be addressed. Several lines of evidence suggest that ceramide and oxidative stress are intimately related in cell death induction. This review focuses on the putative relationships between oxidative stress and sphingolipid metabolism in the apoptotic process and discusses the potential mechanisms that connect and regulate the two phenomena.

Animals↗

PubMed2001

A neutral sphingomyelinase resides in sphingolipid-enriched microdomains and is inhibited by the caveolin-scaffolding domain: potential implications in tumour necrosis factor signalling.

Sphingomyelinases hydrolyse sphingomyelin to ceramide, a process involved in signal-transduction routes leading to apoptosis and various other cellular responses. In the present study, we investigated the sphingomyelinase content of caveolae, invaginated plasma-membrane microdomains that contain a variety of signalling molecules. These structures are highly enriched in sphingomyelin as well as in ceramide, which suggests that metabolism of these lipids might, to some extent, occur locally. By cell fractionation, we demonstrate that, in addition to a previously reported minute amount of acidic sphingomyelinase activity, a substantial amount of neutral sphingomyelinase activity resides in caveolae of human skin fibroblasts. This caveolar neutral sphingomyelinase activity was also detected in Niemann-Pick disease type A fibroblasts, which are completely devoid of functional acidic sphingomyelinase. Neutral (but not acidic) sphingomyelinase activity was specifically inhibited by a peptide that corresponds to the scaffolding domain of caveolin, which suggests a direct molecular interaction between the two proteins. In addition, this finding implies a cytosolic orientation of the caveolar neutral sphingomyelinase. Interestingly, stimulation of fibroblasts with tumour necrosis factor alpha (TNFalpha) resulted in a partial shift of its p55 receptor to caveolin-enriched membrane fractions and the appearance of caveolin-sensitive neutral sphingomyelinase activity in the non-caveolar fractions. These results suggest that (part of) the presently identified caveolar neutral sphingomyelinase activity is involved in TNFalpha signalling.

Caveolae↗

PubMed2001

Phenolic antioxidants trolox and caffeic acid modulate the oxidized LDL-induced EGF-receptor activation.

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL act in part through alteration of intracellular signalling pathways in cells of the vascular wall. We recently reported that the EGF receptor (EGFR) signalling pathway is activated by lipid peroxidation products (among them 4-hydroxynonenal, 4-HNE) contained in oxLDL. The use of phenolic antioxidants, such as trolox, alpha-tocopherol, caffeic acid and tyrphostins A-25, A-46 or A-1478, showed that the oxLDL-induced EGFR activation is constituted by two separate components, the first (early) one being antioxidant-insensitive, the second (late) being antioxidant-sensitive. 4-HNE derivatization of EGFR and EGFR activation induced by exogenous 4-HNE, suggest that the early (0.5 - 3 h) component of oxLDL-induced EGFR activation is mediated (at least in part) by 4-HNE (and possibly by other oxidized lipids). This early component is antioxidant-insensitive. The second component (4 - 5 h) of the oxLDL-induced EGFR activation is antioxidant-sensitive, since it is blocked by antioxidants such as trolox, caffeic acid or PDTC, which act by blocking the cellular oxidative stress (H(2)O(2) generation) evoked by oxLDL. Conversely, exogenous H(2)O(2) induced EGFR autophosphorylation (thus mimicking the second component) and was also inhibited by antioxidants. This effect is mediated in part through inhibition by oxidative stress of protein tyrosine phosphatases involved in EGFR dephosphorylation.

Aldehydes↗

PubMed2001

Involvement of FAN in TNF-induced apoptosis.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Glutathione peroxidase-1 overexpression prevents ceramide production and partially inhibits apoptosis in doxorubicin-treated human breast carcinoma cells.

Reduced glutathione and N-acetylcysteine can inhibit both apoptosis and necrosis of several cell types, suggesting a critical role for reactive oxygen species (ROS) in cell death. However, how the cellular defense against oxidative stress is connected with other cell death mediators remains unclear. We selectively investigated the interaction of seleno-glutathione peroxidase-1 (GPx-1), the major enzyme responsible for peroxide detoxification in mammalian cells, with the cytotoxic response of T47D human breast cancer cells to doxorubicin, an anticancer drug known to promote production of ROS and apoptotic mediator ceramide. The sensitivity to doxorubicin-mediated cell death was compared in T47D/H3 containing low levels of endogenous GPx and T47D/GPx2 transfectant cells, which overexpress GPx-1. We show that T47D/GPx2 cells were significantly more resistant than T47D/H3 cells to doxorubicin (1 microM). The glutathione precursor, N-acetylcysteine also partially protected T47D/H3 cells from the lethal effect of doxorubicin, whereas L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione biosynthesis, sensitized both GPx-1--deficient and -proficient cells. Interestingly, in addition to a decrease in ROS production, the activation of neutral sphingomyelinase, sphingomyelin hydrolysis, and ceramide generation in response to doxorubicin was impaired in T47D/GPx2 cells compared with control cells. In contrast, GPx overexpression did not protect breast cancer cells from cell death induced by exogenous cell-permeant ceramide. Moreover, the basal activity of neutral sphingomyelinase was considerably lower in T47D/GPx2. Taken together, these results indicate that GPx-1 can regulate doxorubicin-induced cell death signaling at least in part by interfering with the activation of the sphingomyelin-ceramide pathway.

Acetylcysteine↗

PubMed2000

Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.

Apoptosis↗

PubMed2000

Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection. I. Preanalytical condition studies.

In recent papers, we presented a new analytical method for thiol quantification in serum. It is based on the use of capillary electrophoresis and laser-induced fluorescence to analyze thiol 6-iodoacetamidofluoresceine (IAF) derivatives. Quantitative results of homocysteine, glutathione, cysteine-glycin, and cysteine were shown (Clin. Chem. 45 (1999) 412). A comprehensive comparison of the quantitation of homocysteine in serum, using high-performance liquid chromatography/conventional fluorescence detection and fluorescence polarization immunoassay was also used (E. Caussé et al., Electrophoresis 21 (2000) 2074). Sample preparation prior to derivatization with IAF had never been investigated. In this work we present the results of quantitation of thiols in serum and plasma with three different anticoagulants widely used: ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate. We show that serum and EDTA plasma gave the same results. Then serum protein precipitations by acetonitrile, acetone, sulfosalicylic acid, perchloric acid and trichloracetic acid, prior to derivatization by IAF, were also investigated. Their influence on the concentrations of the thiols were determined. Sulfosalicylic acid and acetonitrile precipitations are well adapted, whereas acetone cannot be used.

Adult↗

PubMed2000

Chlamydia pneumoniae induces interleukin-10 production that down-regulates major histocompatibility complex class I expression.

Recently, it was demonstrated that CD8(+) T cells are important for the response against Chlamydia pneumoniae. By use of the human monocytic cell line U937 and human monocytes taken from peripheral blood, we investigated the effect of infection on various molecules critical for CD8(+) T cell function. A strong secretion of interleukin (IL)-10 by infected cells was observed, together with an inhibited expression of major histocompatibility complex (MHC) class I antigens, but without significant alteration of tumor growth factor-beta secretion or MHC class II expression. Recombinant IL-10 added to uninfected U937 cells decreased the expression of MHC class I, whereas blocking antibodies to IL-10 and its receptor abolished the C. pneumoniae-induced inhibition of MHC class I expression. Analysis of our data provides evidence that IL-10 secretion induced by C. pneumoniae infection of monocytic cells down-regulates the expression of MHC class I molecules and thereby might reduce the presentation of bacterial epitopes by MHC. This would decrease the ability of CD8(+) T cells to eliminate infected cells.

CD18 Antigens↗

PubMed2000

Determination of asymmetrical dimethylarginine by capillary electrophoresis-laser-induced fluorescence.

Asymmetric dimethyl-L-arginine (ADMA) is a naturally occurring analogue of L-arginine (L-Arg), the substrate of nitric oxide synthase (NOS). ADMA is a potent endogenous inhibitor of NOS and accumulates in the plasma of patients with renal failure, with peripheral arterial occlusive disease or with clinically asymptomatic hypercholesterolemia. We measured circulating concentrations of L-arginine, symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) in human serum. We developed a new method for the rapid determination of these molecules using capillary electrophoresis and laser-induced fluorescence (CE-LIF). All methylated arginines were labeled with fluorescein isothiocyanate (FITC) prior to analysis. Under the capillary electrophoresis (CE) conditions used, methylated arginine derivatives were well separated, with a migration time of around 10 min. These migration times were smaller than the ones of other amino acids which do not have the same charge at pH 10. Consequently, such basic amino acids were well separated from most of the other amines or amino acids. Moreover, CE allowed one to separate all the analogues of fluorescein thiocarbamyl-arginine. The results indicated that CE-LIF is useful as a selective, rapid, cheap and sensitive tool for the determination of methylated arginine products. This new technology might appreciate the endogenous substrate for NO synthase and facilitate the knowledge of the physiological and pathophysiological regulation of NO synthesis.

Arginine↗

PubMed2000

Plasma total homocysteine and other thiols analyzed by capillary electrophoresis/laser-induced fluorescence detection: comparison with two other methods.

We present a new analytical method for thiol quantification in plasma, based on the use of capillary electrophoresis (CE) and laser-induced fluorescence (LIF) to analyze 6-iodoacetamidofluorescein derivatives. Quantitative results of homocysteine, glutathione, cysteinylglycine, and cystationine are presented. A comparison of the quantitation of homocysteine in plasma, using high performance liquid chromatography/fluorescence detection and fluorescence polarization immunoassay is proposed. The results indicate that these techniques for plasma total homocysteine (tHcy) determination can be used interchangeably. The major advantage of CE-LIF is that it can quantitate the thiols in one run while keeping the price of consumables reasonable.

Chromatography, High Pressure Liquid↗

PubMed2000

Sphingomyelin metabolites in vascular cell signaling and atherogenesis.

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.

Arteriosclerosis↗