Pascale Pisano

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.

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.

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.

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.

Exposure of medical personnel to radiation during radionuclide therapy practices.

ObjectivesRadioisotopes that emit &bgr;− radiation are used for the treatment of hepatocellular carcinoma, of arthritic patients (radiosynovectomy) and treatment of bone metastases with, respectively, 131I-labelled lipiodol, colloidal citrate of 90Y or and 153Sm-labelled EDTMP. Radiation energy of these radioisotopes that emit &bgr;− or &bgr;− and gamma radiation (from 300 to 2000 keV) leads to an increase in radiation dose received by nuclear medicine staff. In this paper we focused on clinical and laboratory staff exposure during these types of metabolic radiation therapies. MethodsCylindrical LiF thermoluminescence dosimeters were used to measure radiation-related whole-body doses (WBDs) and finger doses of the clinical staff. ResultsExposure of the two radiopharmacists and three nurses taking part in 131I-labelled lipiodol, 90Y-colloid and 153Sm-EDTMP therapies, for 12 months in succession, were 146 μSv and 750 μSv, respectively, considering WBD, and 14.6 mSv and 6.5 mSv, respectively, considering finger doses. Extrapolated annual exposures (six radiosynovectomies per year) for the rheumatologists were estimated to be 21 μSv (WBD) and 13.2 mSv (finger dose). Extrapolated annual WBDs and finger doses (25 131I-labelled lipiodol treatments per year) for radiologists were estimated to 165 μSv and 3.8 μSv, respectively. ConclusionFortunately, these doses were always lower than the limits reported in the European Directive EURATOM 96/29 05/13/1996 (WBD <20 mSv·year−1; finger dose: 500 mSv·year−1) but have to be added to those relative to other metabolic radiotherapies such as radioiodine treatments and new metabolic radiotherapies (90Y-conjugated peptides or antibodies). Nevertheless, the global exposure of medical staff involved in all these clinical practices justifies dosimetry studies to validate protocols and radiation protection devices for each institution.

Therapeutic benefit of a combined strategy using erythropoietin and endothelial progenitor cells after transient focal cerebral ischemia in rats

Abstract Objective: Many studies have demonstrated beneficial effects of either erythropoietin (EPO) or endothelial progenitor cell (EPC) treatment in cerebral ischemia. To improve post-ischemic tissue repair, we investigated the effect of systemic administration of endothelial colony-forming cells (ECFCs), considered as relevant endothelial progenitors due to their specific vasculogenic activity, in the presence or absence of EPO, on functional recovery, apoptosis, angiogenesis, and neurogenesis in a transient focal cerebral ischemia model in the adult rat. Design: Experimental study. Intervention: The rats were divided into four groups 24 hours after ischemia,, namely control, ECFCs, EPO, and ECFCs+EPO, and received a single intravenous injection of ECFCs (5×106 cells) and/or intraperitoneal administration of EPO (2500 UI/kg per day for 3 days). Measurement: Infarct volume, functional recovery, apoptosis, angiogenesis, and neurogenesis were assessed at different time points after ischemia. Main results: The combination of EPO and ECFCs was the only treatment that completely restored neurological function. The ECFCs+EPO treatment was also the most effective to decrease apoptosis and to increase angiogenesis and neurogenesis in the ischemic hemisphere compared to controls and to groups receiving ECFCs or EPO alone. Conclusion: These results suggest that EPO could act in a synergistic way with ECFCs to potentiate their therapeutic benefits.

Chocolate Intake Associated with Failed Labeling of 99mTc Red Blood Cells

Red blood cells (RBC) labeled in vivo with 99mTc-pertechnetate are used worldwide in nuclear medicine departments. Methods: Here, we present a case of 99mTc-RBC labeling failure associated with chocolate intake in a 25-y-old woman, resulting in uninterpretable images. Because of this clinical observation, we performed in vitro RBC labeling on blood samples from volunteers after they consumed chocolate. Results: Chocolate intake inhibited the labeling rate, compared with the control condition, and significantly increased the 99mTc free fraction (34.1% ± 11.3% vs. 14.0% ± 1.2%). Conclusion: We cannot explain how this interaction could occur, but cacao components are known to modulate red cell and plasma oxidoreductive status and to modify red cell membrane permeability and plasticity. Therefore, for patients who can be considered likely to consume chocolate, such as young patients, we recommend that they limit their consumption of chocolate for 12 h before RBC labeling.

Single photon emission computed tomography imaging of cerebral blood flow, blood-brain barrier disruption, and apoptosis time course after focal cerebral ischemia in rats.

Background Cerebral ischemia is a leading cause of disability worldwide and no other effective therapy has been validated to date than intravenous thrombolysis. In this context, many preclinical models have been developed and recent advances in preclinical imaging represent promising tools. Thus, we proposed here to characterize in vivo time profiles of cerebral blood flow, blood–brain barrier disruption and apoptosis following a transient middle cerebral artery occlusion in rats using SPECT/CT imaging. Methods Rats underwent a 1-h middle cerebral artery occlusion followed by reperfusion. Cerebral blood flow, blood–brain barrier disruption and apoptosis were evaluated by SPECT/CT imaging using respectively 99mTc-HMPAO, 99mTc-DTPA and the experimental 99mTc-Annexin V-128, up to 14 days after middle cerebral artery occlusion. Histological evaluation of apoptosis has been performed using TUNEL method to validate the 99mTc-Annexin V-128 uptake. Results 99mTc-HMPAO cerebral blood flow evaluation showed hypoperfusion during occlusion, partially restored on days 4 and 7 and sustained up to 14 days after middle cerebral artery occlusion. 99mTc-DTPA SPECT/CT showed a blood–brain barrier disruption starting on day 1 post-middle cerebral artery occlusion, peaking on day 2, with barrier integrity totally restored on day 7. 99mTc-Annexin V-128 SPECT/CT imaging showed significant positive correlation with TUNEL immunohistochemistry and allowed ischemic-induced apoptosis to be detected from day 2 to day 7, peaking on day 3 after middle cerebral artery occlusion. Conclusions Using SPECT/CT imaging, we showed that after transient middle cerebral artery occlusion in rat there was a sustained decrease in cerebral blood flow followed by blood–brain barrier disruption preceding meanwhile apoptosis. Rodent SPECT/CT imaging of cerebral blood flow, blood–brain barrier disruption and apoptosis appears to be an efficient tool for evaluating neuroprotective drugs and regenerative therapies against cerebral ischemia and time-windows for therapeutic intervention.