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Michel Lagarde

Publications and source records attributed to Michel Lagarde.

At least 37 records · Page 2Linked to original sources
PubMed2004

Involvement of gangliosides in glucosamine-induced proliferation decrease of retinal pericytes.

The hexosamine pathway (HP) is a biochemical hypothesis recently proposed explaining cellular alterations occurring during diabetic microvascular complications. Diabetic retinopathy is a common microvascular complication of diabetes, and it is known that cell proliferation is severely affected during the development of the disease. Particularly, early stages are characterized by death of the retinal microvascular cells, pericytes. Gangliosides have often been described to regulate cell growth; however, very few studies focused on the potential role of gangliosides in diabetic microvascular alterations. The aim of this article was to investigate the effect of the HP activation on pericyte proliferation and determine the potential implication of gangliosides in this process. Results indicate first that HP activation, mimicked by glucosamine treatment, decreased pericyte proliferation. Second, glucosamine treatment induced a modification of gangliosides pattern, particularly GM1 and GD3 were significantly increased. Next, results showed that exogenous addition of a-series gangliosides (GM3, GM2, GM1, GD1a) and b-series ganglioside (GD3) caused a decrease of pericyte proliferation, whereas nonsialylated precursors glucosylceramide and lactosylceramide were without effect. Furthermore, when ganglioside biosynthesis was blocked using PPMP, a glucosylceramide synthase inhibitor, the effects of glucosamine on pericyte proliferation were partially reversed. Our results suggest that in retinal pericytes, gangliosides and particularly GM1 and GD3 that are increased in response to glucosamine, are involved in the antiproliferative effect of glucosamine. These observations also underlie the potential involvement of gangliosides in a pathological context, such as diabetic microvascular complications.

Animals↗
PubMed2004

Phospholipase D is involved in myogenic differentiation through remodeling of actin cytoskeleton.

We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.

1-Butanol↗
PubMed2004

Phorbol ester-induced differentiation of L6 myogenic cells involves phospholipase D activation.

TPA, a potent PKC activator, inhibits myogenic differentiation and activates phospholipase D (PLD). We evaluated the involvement of PLD in the TPA effects on L6 myoblasts differentiation. TPA, at concentrations inhibiting differentiation of L6 cells, induced a strong, though transient, PLD activation. Surprisingly, at nanomolar concentration, TPA induced both myogenic differentiation and sustained activation of PLD. Differential effect of TPA can be ascribed to PKC downregulation induced by highest TPA concentrations. TPA-induced differentiation was inhibited by 1-butanol, confirming the involvement of PLD in this effect. These data suggest that prolonged elevation of PLD activity is required for myogenic differentiation.

1-Butanol↗
PubMed2004

Covalent binding of isoketals to ethanolamine phospholipids.

Free radicals have been strongly implicated in the pathogenesis of many human diseases. We previously identified the formation of highly reactive gamma-ketoaldehydes, isoketals, in vivo as products of free radical-induced peroxidation of arachidonic acid. Isoketals react with lysine residues on proteins at a rate that far exceeds that of 4-hydroxynonenal and demonstrate a unique proclivity to crosslink proteins. Hydroxynonenal has been shown to react with aminophospholipids, particularly phosphatidylethanolamine. We explored whether isoketals also react with phosphatidylethanolamine. Using liquid chromatography/electrospray mass spectrometry, we found that isoketals form pyrrole and Schiff base adducts with phosphatidylethanolamine. In addition, the ability of isoketals to covalently modify phosphatidylethanolamine is greater than that of 4-hydroxynonenal. These studies identify in vitro novel isoketal adducts. This provides the basis to explore the formation of isoketal-aminophospholipid adducts in vivo and the biological consequences of the formation of these adducts.

Aldehydes↗
PubMed2004

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture.

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.

Amino Acid Chloromethyl Ketones↗
PubMed2004

Bimodal effect of advanced glycation end products on mesangial cell proliferation is mediated by neutral ceramidase regulation and endogenous sphingolipids.

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 microm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 microm) markedly reduced it (-24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 microm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.

Amidohydrolases↗
PubMed2004

Diabetic patients without vascular complications display enhanced basal platelet activation and decreased antioxidant status.

Vascular complications are the leading causes of morbidity and mortality in diabetic patients. The contribution of platelets to thromboembolic complications is well documented, but their involvement in the initiation of the atherosclerotic process is of rising interest. Thus, the aim of the present study was to evaluate basal arachidonic acid metabolism in relation to the redox status of platelets in both type 1 and type 2 diabetic patients, in the absence of vascular complications, as compared with respective control subjects. For the first time, we show that basal thromboxane B(2), the stable catabolite of thromboxane A(2), significantly increased in resting platelets from both type 1 and type 2 diabetic patients (58 and 88%, respectively), whereas platelet malondialdehyde level was only higher in platelets from type 2 diabetic subjects (67%). On the other hand, both vitamin E levels and cytosolic glutathione peroxidase activities were significantly lower in platelets from diabetic patients as compared with respective control subjects. We conclude that platelet hyperactivation was detectable in well-controlled diabetic patients without complications. This abnormality was associated with increased oxidative stress and impaired antioxidant defense in particular in type 2 diabetic patients. These alterations contribute to the increased risk for occurrence of vascular diseases in such patients.

Antioxidants↗
PubMed2003

Plasmalogens protect unsaturated lipids against UV-induced oxidation in monolayer.

Oxidative stress results from the attack by free radicals of several cellular targets (proteins, DNA and lipids). The cell equilibrium is a direct consequence of the pro-/antioxidant balance. In order to understand the physiological processes involved in oxidative stress, we followed oxidation of unsaturated lipids using a biomimetic system: Langmuir monolayers. The oxidation mode chosen was UV-irradiation and the lipid model was a polyunsaturated phospholipid: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). The monomolecular film technique was used to measure membrane rheology before and after UV-irradiation. We showed that the UV-irradiation of a DLPC monomolecular film led to a molecular area and surface elasticity modulus decrease that attests to the apparition of new molecular species at the air-water interface. The antioxidant effect of a synthetic plasmalogen (1-O-(1'-(Z)-hexadecenyl)-2-O-oleoyl-sn-glycero-3-phosphocholine or P(PLM)OPE) was tested on the oxidation of DLPC. Indeed, for about 25% mol P(PLM)OPE in mixed DLPC/P(PLM)OPE monolayers, a complete inhibition of the molecular area and the surface elasticity modulus decreases was observed in our experimental conditions. Lower P(PLM)OPE quantities delayed but did not prevent the DLPC oxidation in mixed monolayers.

Antioxidants↗
PubMed2003

Ceramide in the antiapoptotic effect of ischemic preconditioning.

Although the mechanism by which ischemic preconditioning (PC) inhibits myocardial apoptosis during ischemia-reperfusion is unclear, evidence indicates a role for the secondary messenger ceramide. We investigated in vivo whether PC may affect ceramide and sn-1,2-diacylglycerol (DAG) production, and attenuate apoptosis during ischemia. Rabbits underwent 30 min of ischemia, followed by 4 h of reperfusion. Before this, they received either no intervention (control group) or one episode of 5 min of ischemia, followed by 5 min of reperfusion (PC group), or an intravenous administration of the sphingomyelinase inhibitor D609. Myocardial content of ceramide and DAG was measured using the DAG kinase assay at different time points of the experiment. Apoptosis was detected and quantified by a sandwich enzyme immunoassay. Both AR and infarct size were measured using blue dye injection and triphenyltetrazolium chloride staining. Control hearts exhibited a peak of ceramide production at 5 min of the prolonged ischemia, with a mean value averaging 64 +/- 5 ng/mg tissue (P < 0.05 vs. 48 +/- 4 ng/mg at baseline). In contrast, ischemic PC and D609 prevented ceramide increase during the prolonged ischemia. Myocardial DAG content was increased only in PC hearts at 30 min of ischemia. Preconditioned and D609 groups developed less apoptosis, as well as a limited infarct size, compared with the control group. These results suggest that the antiapoptotic effect of PC may be due to a reduced ceramide production during sustained ischemia in the rabbit heart.

Animals↗
PubMed2003

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets.

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.

Acetylcysteine↗
PubMed2003

Fibroblast cells: a sensing bioelement for glucose detection by impedance spectroscopy.

Modifying the electrical properties of fibroblasts against various glucose concentrations can serve as a basis for a new, original sensing device. The aim of the present study is to test a new biosensor based on impedancemetry measurement using eukaryote cells. Fibroblast cells were grown on a small optically transparent indium tin oxide semiconductor electrode. Electrochemical impedance spectroscopy (EIS) was used to measure the effect of D-glucose on the electrical properties of fibroblast cells. Further analyses of the EIS results were performed using equivalent circuits in order to model the electrical flow through the interface. The linear calibration curve was established in the range 0-14 mM. The specification of the biosensors was verified using cytochalasin B as an inhibitor agent of the glucose transporters. The nonreactivity to sugars other than glucose was demonstrated. Such a biosensor could be applied to a more fundamental study of cell metabolism.

3T3 Cells↗
PubMed2003

Covalent coupling of reduced glutathione with ribose: loss of cosubstrate ability to glutathione peroxidase.

Glycation (nonenzymatic glycosylation of proteins) is known to be increased as a result of hyperglycaemia in diabetes. Moreover, cell glutathione concentration has been found to be lower in diabetics and such depletion may impair the cell defence against toxic radical species. Ribose being a potent reducing sugar expected to be increased in cells of diabetics where the pentose phosphate pathway is enhanced, its putative condensation with glutathione was investigated. Reduced glutathione (GSH) was incubated with ribose and the structure of the resultant product was assessed by mass spectrometry, as well as the measurement of its remaining thiol group. A covalent reaction clearly occurred between the reducing sugar and GSH, to give an adduct named N-ribosyl-1-glutathione. This adduct appears to be the Amadori product resulting from the condensation of the primary amine group of GSH with the aldehyde group of ribose. Interestingly, the adduct could not be used as a proper substrate by glutathione peroxidase although it keeps its thiol group. We conclude that the coupling of GSH with a monosaccharide such as ribose might contribute to the decreased cell GSH and glutathione peroxidase activity observed in diabetics.

Chromatography, High Pressure Liquid↗
PubMed2003

Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE, and 4-HNE to ethanolamine phospholipid subclasses.

Lipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl. We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes.

Aldehydes↗
PubMed2003

Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney.

Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.

Alkaline Phosphatase↗
PubMed2003

Mechanisms involved in the stimulation of prostacyclin synthesis by human lymphocytes in human umbilical vein endothelial cells.

1 Endothelial cells play an important role in the modulation of vascular tone because of their ability to produce vasoactive substances such as prostacyclin (PGI(2)). Cell-cell contact between human umbilical vein endothelial cells (HUVEC) and peripheral blood lymphocytes has been shown to stimulate endothelial PGI(2) synthesis by increasing free arachidonic acid availability through endothelial cytosolic phospholipase A2 (cPLA(2)) activation. In this study, we sought to determine whether phospholipase C (PLC) and D (PLD) activation also contributes, besides cPLA(2), to the lymphocyte-induced PGI(2) synthesis in HUVEC, and to delineate further the potential mechanisms of cPLA(2) activation triggered by the interaction of HUVEC with lymphocytes. 2 Pretreatment of endothelial cells with the PI-PLC inhibitor U-73122 before the coincubation with lymphocytes markedly inhibited the PGI(2) output whereas the diacylglycerol (DAG) lipase inhibitor RHC 80267 and ethanol had no effect. These results suggest that PLC may be involved through inositol trisphosphate generation and calcium mobilization, and that neither DAG nor phosphatidic acid (PtdOH) was used as sources of arachidonic acid. 3 The stimulated PGI(2) synthesis was protein kinase C (PKC)-independent but strongly inhibited by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U-0126 and by the Src kinase inhibitor PP1. 4 Immunoblot experiments showed an increased phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) upon lymphocyte addition till 4 h coincubation. Phosphorylation was markedly inhibited by U-0126 and PP1 addition. 5 Collectively, these results suggest that the signaling cascade triggered by lymphocytes in endothelial cells involves an Src kinase/ERK1/2 pathway leading to endothelial cPLA(2) activation.

Butadienes↗
PubMed2003

Platelets may inhibit leucotriene biosynthesis by human neutrophils at the integrin level.

Polymorphonuclear leucocytes and blood platelets co-operate in several pathophysiological processes, and arachidonic acid (AA) metabolites produced in response to the activation of these cells are potent mediators of their functions. We studied the role of platelets in the formation of 5-lipoxygenase products from AA by autologous neutrophils, especially the chemotactic agent leucotriene (LT) B4. The formation of all products, namely 5-hydroxy-eicosatetraenoic acid (5-HETE), LTB4 and the other LTA4-derived metabolites, in response to the calcium ionophore A23187 was evaluated by high-performance liquid chromatography. All the 5-lipoxygenase products were significantly diminished by physiological concentrations of platelets. This inhibitory effect was lost when platelets were previously degranulated by thrombin in non-aggregating conditions. Peptides containing the Arg-Gly-Asp-Ser or His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val sequence, which prevent the adhesion of platelets to neutrophils via the fibrinogen released from platelet granules and the integrin glycoprotein IIb/IIIa, markedly decreased the inhibitory effect of non-degranulated platelets. The production of transcellular metabolites of AA such as LTC4, the dual 5- and 12-lipoxygenase product 5,12-diHETE and lipoxins could not account for the decreased formation of 5-HETE and LTA4-derived metabolites. It is concluded that platelets may inhibit the neutrophil 5-lipoxygenase activity at the integrin level and in turn may play a role in slowing down the production of LTB4 in the course of inflammation.

Arachidonate 5-Lipoxygenase↗
PubMed2003

Influence of very low dietary intake of marine oil on some functional aspects of immune cells in healthy elderly people.

Ageing is a multifactorial process involving decreased antioxidant defences and immune functions. n-3 Polyunsaturated fatty acids have been associated with human health benefits, especially against inflammatory and autoimmune diseases. However, their immunomodulatory effects were usually observed with high dosages (>2 g/d) known to increase lipid peroxidation. In contrast, very low doses, that may prevent lipid peroxidation, might affect the immune system differently. To study the latter hypothesis further, we investigated whether the supplementation of healthy elderly people with very low doses of marine oil (MO), a docosahexaenoate (DHA)- and eicosapentaenoate (EPA)-rich triacylglycerol, was able to affect lymphocyte proliferation and biochemical markers known to be altered with age. In a randomized, double-blind design, twenty healthy elderly subjects were assigned to a placebo group (600 mg sunflower oil/d) or to a group consuming 600 mg MO/d providing 150 mg DHA + 30 mg (EPA) for 6 weeks. At day 42, the proliferative responses of lymphocytes to several mitogens were significantly (P<0.01) decreased in the MO group compared with control values. This was accompanied by a slight lowering of their cytosolic cyclic nucleotide phosphodiesterase (PDE) activity, a marked and significant (P<0.05) increase of their particulate PDE activity (+56-57 %) and a slight but significant (P<0.05) increase in cyclic nucleotide intracellular levels. At the same time, the glutathione peroxidase activity was markedly and significantly (P<0.01) depressed in the MO group. None of these modifications could be seen in the placebo group. Collectively, these results demonstrate that even very low doses of n-3 fatty acids are sufficient to affect the immune responses of elderly subjects.

3',5'-Cyclic-AMP Phosphodiesterases↗