Benjamin Guillet

The neuronal excitatory amino acid transporter EAAC1/EAAT3: does it represent a major actor at the brain excitatory synapse?

EAAC1/EAAT3 is a transporter of glutamate (Glu) present at the post‐synaptic neuronal element, in opposition to the two other main transporters, GLAST/EAAT1 and GLT1/EAAT2, expressed at the excitatory amino acid (EAA) synapse by surrounding astrocytes. Although, in the adult, EAAC1/EAAT3 exhibits a rather low expression level and is considered to make a minor contribution to Glu removal from the synapse, its early expression during brain development, before the astrocytes are functional, suggests that such a neuronal transporter is involved in the developmental effects of EAA and, possibly, in the biosynthesis and trophic role of GABA, which is excitatory in nature in different brain regions during the earlier stages of brain development. This neuronal Glu transporter is considered to have a dual action as it is apparently involved in the neuronal uptake of cysteine, which acts as a key substrate for the synthesis of glutathione, a major anti‐oxidant, because the neurones do not express the Xc– transport system in the mature brain. Interestingly, EAAC1/EAAT3 activity/expression was shown to be highly regulated by neuronal activity as well as by intracellular signalling pathways involving primarily α protein kinase C (αPKC) and phosphatidylinositol‐3‐kinase (PI3K). Such regulatory processes could act either at the post‐traductional level or at the transcriptional level. It is worth noting that EAAC1/EAAT3 exhibits specificity, compared with other EAA transporters, because it is present mainly in the intracellular compartment and only for about 20% at the plasma membrane. Variations in neuronal Glu uptake were shown to be associated with rapid changes in the trafficking of the transporter protein altering the membranar location of the transporter. More recent data show that astrocyte‐secreted factors such as cholesterol could also influence rapid changes in the location of EAAC1/EAAT3 between the plasma membrane and the cytoplasmic compartment. Such a highly regulated process of EAAC1/EAAT3 activity/expression may have implications in the physiopathology of major diseases affecting EAA brain signalling, which is further supported by data obtained in animal models of hypoxia–anoxia, for example.

Neuroprotective Effects of Propofol in a Model of Ischemic Cortical Cell Cultures Role of Glutamate and Its Transporters

Background During cerebral ischemia, excess of glutamate release and dysfunction of its high affinity transport induce an accumulation of extracellular glutamate, which plays an important role in neuronal death. The authors studied the relationship among propofol neuroprotection, glutamate extracellular concentrations, and glutamate transporter activity in a model of ischemic cortical cell cultures. Methods Thirteen-day-old primary cortical neuronal-glial cultures were exposed to a 90-min combined oxygen–glucose deprivation (OGD) in an anaerobic chamber, followed by reoxygenation. Propofol was added only during the OGD period, and its effect was compared to that of the N-methyl-d-aspartate receptor antagonist dizocilpine (MK-801). Twenty-four hours after the injury, cell death was quantified by lactate dehydrogenase release and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Extracellular concentrations of glutamate in culture supernatants and glutamate uptake were performed at the end of OGD period by high-performance liquid chromatography and incorporation of l-[3H]glutamate into cells, respectively. Results At clinically relevant concentrations (0.05–10 &mgr;m), propofol offered protection equivalent to that of MK-801. It significantly reduced lactate dehydrogenase release and increased the reduction of MTT. At the end of the ischemic injury, propofol was able to reverse the OGD-induced increase in glutamate extracellular concentrations and decrease of glutamate uptake. The inhibition of the glial GLT1 transporter by 3-methyl-glutamate did not further modify the effect of propofol on glutamate uptake, suggesting that GLT1 was not the major target of propofol. Conclusion Propofol showed a neuroprotective effect in this in vitro model of OGD, which was apparently mediated by a GLT1-independent restoration of the glutamate uptake impaired during the injury.

Sevoflurane preconditioning against focal cerebral ischemia: inhibition of apoptosis in the face of transient improvement of neurological outcome.

Background:Preconditioning the brain with volatile anesthetics seems to be a viable option for reducing ischemic cerebral injury. However, it is uncertain whether this preconditioning effect extends over a longer period of time. The purpose of this study was to determine if sevoflurane preconditioning offers durable neuroprotection against cerebral ischemia. Methods:Rats (Sprague-Dawley) were randomly allocated to two groups: nonpreconditioned control group (n = 44) and preconditioned group (n = 45) exposed to 2.7 vol% sevoflurane (45 min) 60 min before surgery. Animals in both groups were anesthetized with 3.0vol% sevoflurane and subjected to transient middle cerebral artery occlusion. After 60 min of awake focal ischemia, the filament was removed. Functional neurologic outcome (range 0–18; 0 = no deficit), cerebral infarct size (Nissl staining), and apoptosis (Terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling; cleaved caspase-3 staining) were evaluated at 3, 7, and 14 days after ischemia. Results:Sevoflurane preconditioning significantly improved functional outcome and reduced infarct volume (109 ± 43 vs. 148 ± 56 mm3) 3 days after ischemia compared to the control group. However, after 7- and 14-day recovery periods, no significant differences were observed between groups. The number of apoptotic cells was significantly lower in the preconditioned group than in the control group after 3- and 7-day recovery periods. Fourteen days after ischemia, no differences were observed between groups. Conclusion:In this model of transient focal cerebral ischemia, sevoflurane preconditioning induced effective but transient neuroprotective effects. Sevoflurane preconditioning also decreased ischemia-induced apoptosis in a more sustained way because it was observed up to 7 days after injury.

Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia

CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases.

Accelerated senescence of cord blood endothelial progenitor cells in premature neonates is driven by SIRT1 decreased expression.

Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n = 18), ECFCs isolated from PT (n = 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated β-galactosidase activity. Increased p16(INK4a) expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy.

Sevoflurane Protects Rat Mixed Cerebrocortical Neuronal–glial Cell Cultures against Transient Oxygen–glucose Deprivation: Involvement of Glutamate Uptake and Reactive Oxygen Species

Background:The purpose of this study was to clarify the role of glutamate and reactive oxygen species in sevoflurane-mediated neuroprotection on an in vitro model of ischemia–reoxygenation. Methods:Mature mixed cerebrocortical neuronal–glial cell cultures, treated or not with increasing concentrations of sevoflurane, were exposed to 90 min combined oxygen–glucose deprivation (OGD) in an anaerobic chamber followed by reoxygenation. Cell death was quantified by lactate dehydrogenase release into the media and cell viability by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium by mitochondrial succinate dehydrogenase. Extracellular concentrations of glutamate and glutamate uptake were assessed at the end of the ischemic injury by high-performance liquid chromatography and incorporation of L-[3H]glutamate into cells, respectively. Free radical generation in cells was assessed 6 h after OGD during the reoxygenation period using 2′,7′-dichlorofluorescin diacetate, which reacts with intracellular radicals to be converted to its fluorescent product, 2′,7′-dichlorofluorescin, in cell cytosol. Results:Twenty-four hours after OGD, sevoflurane, in a concentration-dependent manner, significantly reduced lactate dehydrogenase release and increased cell viability. At the end of OGD, sevoflurane was able to reduce the OGD-induced decrease in glutamate uptake. This effect was impaired in the presence of threo-3-methyl glutamate, a specific inhibitor of the glial transporter GLT1. Sevoflurane counteracted the increase in extracellular level of glutamate during OGD and the generation of reactive oxygen species during reoxygenation. Conclusion:Sevoflurane had a neuroprotective effect in this in vitro model of ischemia–reoxygenation. This beneficial effect may be explained, at least in part, by sevoflurane-induced antiexcitotoxic properties during OGD, probably depending on GLT1, and by sevoflurane-induced decrease of reactive oxygen species generation during reoxygenation.

CD146 Short Isoform Increases the Proangiogenic Potential of Endothelial Progenitor Cells In Vitro and In Vivo

Rationale: CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined. Objective: The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential. Methods and Results: Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell–derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow. Conclusions: Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

Priming of late endothelial progenitor cells with erythropoietin before transplantation requires the CD131 receptor subunit and enhances their angiogenic potential

Summary.  Background:  Endothelial colony‐forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the β‐common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects.

Early anesthetic preconditioning in mixed cortical neuronal-glial cell cultures subjected to oxygen-glucose deprivation: the role of adenosine triphosphate dependent potassium channels and reactive oxygen species in sevoflurane-induced neuroprotection.

BACKGROUND: The purpose of the present study, on mixed cortical neuronal-glial cell cultures subjected to transient oxygen-glucose deprivation (OGD) was: i) to compare the neuroprotection afforded by sevoflurane added either before (preconditioning) or during (direct neuroprotection) the OGD and ii) to explore the possible involvement of adenosine triphosphate-sensitive potassium (KATP) channels and intracellular reactive oxygen species (ROS) levels in the mechanism of the early preconditioning effect of sevoflurane. METHODS: Mature mixed cortical neuronal-glial cell cultures were exposed to 90-min OGD in an anaerobic chamber followed by reoxygenation. Sevoflurane (0.03–3.4 mM) was randomly administered for 90 min and discontinued 60 min before OGD (early preconditioning) or during the 90-min OGD (direct neuroprotection). Cell death was quantified 24 h after the OGD by lactate dehydrogenase release into the bathing medium. Intracellular ROS generation was assessed at the end of sevoflurane preconditioning using 2′,7′-dichlorofluorescin diacetate. RESULTS: Sevoflurane preconditioning elicited a potent threshold-dependent neuroprotective effect at concentrations higher than 0.07 mM and sevoflurane added during OGD elicited a dose dependent neuroprotective effect. Blockers of KATP channels (glibenclamide 0.3 &mgr;M and 5 hydroxydecanoic acid 50 &mgr;M), or ROS-scavengers (N-2-mercaptopropionyl glycine 100 &mgr;M and N-acetylcysteine 50 &mgr;M), although they did not affect cell viability, counteracted the neuroprotection produced by early sevoflurane preconditioning. Sevoflurane exposure during preconditioning induced a significant increase in ROS levels which was prevented by both ROS scavengers and blockers of KATP channels. CONCLUSION: Early sevoflurane preconditioning induced a threshold-dependent protection of mixed cortical neuronal-glial cell cultures against OGD by mechanisms that seem to involve opening KATP channels, thereby leading to generation of ROS.

Erythropoietin protects newborn rat against sevoflurane-induced neurotoxicity.

Recent data on newborn animals exposed to anesthetics have raised safety concerns regarding anesthesia practices in young children. Indeed, studies on rodents have demonstrated a widespread increase in brain apoptosis shortly after exposure to sevoflurane, followed by long‐term neurologic impairment. In this context, we aimed to evaluate the protective effect of rh‐EPO, a potent neuroprotective agent, in rat pups exposed to sevoflurane.