The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after strok

Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells

Background CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. Methods After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. Results We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. Conclusion Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers

The blood brain barrier and ischemia : in vitro study of microvascular protection and dysfunction

La barrière hémato-encéphalique (BHE) est une interface localisée au niveau des cellules endothéliales des capillaires cérébraux. Elle présente des caractéristiques physiques et métaboliques spécifiques restreignant les échanges entre le sang et le cerveau dans le but de maintenir l’homéostasie du système nerveux central. Dans des conditions pathologiques comme l’ischémie cérébrale, la perte de son intégrité provoque l’apparition d’un oedème vasogénique qui aggrave considérablement le pronostic vital des patients. Malheureusement, les mécanismes impliqués dans l’hyperperméabilité vasculaire demeurent inconnus, ce qui limite l’utilisation de la seule thérapie disponible à 5% des patients. Depuis qu’aucun agent pharmacologique n’a réussit à être neuroprotecteur, notre compréhension des rapports entre le sang et le cerveau est remise en cause. La complexité des interactions entre la BHE et les cellules nerveuses a mené au concept d’une unité fonctionnelle dite neurovasculaire. Ainsi de nouvelles stratégies de protection émergent à partir d’observations au niveau vasculaire. Ainsi la première partie de nos travaux a consisté à étudier l’effet vasculoprotecteur potentiel du fénofibrate, un hypolipémiant agoniste du récepteur nucléaire PPAR-a (Peroxisome Proliferator- Activated Receptor-alpha), dont le bénéfice est observé en clinique depuis quelques années et plus récemment dans une étude expérimentale menée chez la souris. Les mécanismes de cette protection aujourd’hui inconnus, pourraient impliquer la BHE réputée très peu perméable à ce fibrate. Un renforcement de la BHE limiterait la formation de l’oedème cérébral. Pour cela nous avons adapté un modèle in vitro syngénique murin de BHE aux études de perméabilité en condition d’OGD (oxygen and glucose deprivation) mimant les conséquences immédiates de l’occlusion, toute première étape de l’accident vasculaire cérébral (AVC) ischémique. Le modèle consiste en une co-culture de cellules endothéliales primaires de capillaires cérébraux et de cellules gliales primaires. Nos travaux démontrent qu’un traitement préventif au fénofibrate protège l’endothélium en limitant l’hyperperméabilité induite par l’OGD. Cette action protectrice cible exclusivement l’endothélium et dépend de l’activation de PPAR-a démontré par l’absence d’effet protecteur sur les cellules endothéliales dont le gène codant pour PPAR-a a été invalidé. La seconde partie de l’étude s’est intéressée aux dommages vasculaires de la reperfusion, étape plus tardive de l’ischémie cérébrale connue pour aggraver l’oedème vasogénique et mener à des hémorragies fatales. A l’aide de notre modèle in vitro, nous avons étudié l’effet de la réoxygénation sur la perméabilité vasculaire dans le but de se rapprocher des conditions ischémiques in vivo. Après une incubation en condition d’OGD, la co-culture est replacée dans un milieu réoxygéné pendant une période allant de 2h à 24h. La mesure de la perméabilité vasculaire a démontré un profil multiphasique de l’ouverture de la BHE dépendant de la présence des cellules gliales. L’analyse en microscopie électronique des cellules endothéliales a suggéré une modulation fine de la fonctionnalité des jonctions serrées endothéliales. De plus, l’étude en IRM de diffusion chez la souris in vivo a révélé des mouvements d’eau qui suggèrent une perturbation de l’homéostasie hydrique du parenchyme cérébral au voisinage de l’occlusion dans les étapes précoces mais aussi dans les étapes tardives. En conclusion, l’ensemble des travaux met en avant la possibilité d’une préservation pharmacologique de l’intégrité de la BHE au début de l’ischémie cérébrale. Celle-ci montre l’intérêt des approches in vitro utilisant un modèle cellulaire pertinent et caractérisé. La validation de la cible cellulaire et moléculaire du fénofibrate à l’aide de notre modèle ouvre une première voie d’exploration des mécanismes impliqués dans ce phénomène de protection microvasculaire précoce. Cependant, la dysfonction retardée de la BHE est également un élément à prendre en compte pour se rapprocher de la physiopathologie de l’ischémie in vivo et espérer à terme une amélioration de l’approche thérapeutique de cette pathologie.The Blood brain barrier (BBB) is an interface localised at brain capillary endothelial cells. The BBB possesses both physical and metabolic restrictive properties aiming at the maintenance of the central nervous system homeostasis. But under pathological conditions like ischemic stroke, the loss of BBB integrity induces a cerebral vasogenic edema which considerably worsens the vital prognosis of patients. The mechanisms underlying this vascular hyperpermeability are currently unknown thus limiting the use of the only medical intervention available at only 5% of stroke patients. Since no pharmacological molecule succeeded in being neuroprotective, our understanding of the relationships between blood and brain is questioned. The complex interactions between the BBB and nervous cells have lead to the concept of a functional unit, termed the neurovascular unit. Thus, new strategies are recently emerging from observation of vascular events. Thus, the first aim of our study was to test the potential vasculoprotective action of fenofibrate, a hypolipemic drug known as an activator of the nuclear receptor PPAR-a a (Peroxisome Proliferator-Activated Receptor-alpha), as benefit against stroke was observed in clinics since a few years, and recently reported in an experimental study. Yet unknown mechanisms, the protective effect may be exerted on the BBB since reported as impermeable to this compound. An early tightening of the BBB would limit the extent of brain edema. Hence, we have adapted a mouse syngenic BBB in vitro cell model to permeability studies under the stress condition found at the early stage of ischemic stroke defined in vitro as oxygen and glucose deprivation (OGD). This stress simulates the early consequences of occlusion. This model consists of a co-culture of primary brain capillary endothelial cells together with primary glial cells. We have demonstrated that a preventive treatment with fenofibrate has a protective effect on the BBB by limiting the hyperpermeability induced by the OGD condition. This effect targets endothelial cells exclusively and depends on PPAR-a activation, as revealed by the absence of protective action of fenofibrate on PPAR-a deficient endothelial cells. The second part of the study has focused on vascular reperfusion injury, a later stage of ischemia known to worsen vasogenic oedema and to lead to fatal haemorrhage. Using our in vitro BBB model, we have studied the effect of reoxygenation on vascular permeability in order to closely simulate in vivo ischemic condition. Following incubation under OGD condition, the co-cultures were placed into an oxygenated culture medium from 2h to 24h. The BBB permeability demonstrated a multiphasic opening of the BBB which depended on glial cells presence. Electronic microscopy analysis of BBB endothelial cells suggested a fine modulation of tight junction functionality. Moreover, the MRI diffusion analysis in mice has revealed particular water movements suggesting an early disturbance in water homeostasis of brain parenchyma in the vicinity of occlusion. In conclusion, this work put forward the idea of a pharmacological BBB protection at the early stage of ischemic stroke. This demonstrates the relevance of in vitro approaches using a pertinent and well characterised cell model. The validation of cellular and molecular targets of fenofibrate opens a way of first exploration of mechanisms involved in this early microvascular protection phenomenon. But the late BBB dysfunction also needs to be taken into account for a complete fitting with in vivo stroke pathophysiology and an improvement of the therapeutic approaches to this pathology

La barrière hémato-encéphalique et l'ischémie cérébrale (étude in vitro de la dysfonction et de la protection microvasculaire)

La barrière hémato-encéphalique (BHE) est une interface localisée au niveau des cellules endothéliales des capillaires cérébraux. Elle présente des caractéristiques physiques et métaboliques spécifiques restreignant les échanges entre le sang et le cerveau dans le but de maintenir l homéostasie du système nerveux central. Dans des conditions pathologiques comme l ischémie cérébrale, la perte de son intégrité provoque l apparition d un oedème vasogénique qui aggrave considérablement le pronostic vital des patients. Malheureusement, les mécanismes impliqués dans l hyperperméabilité vasculaire demeurent inconnus, ce qui limite l utilisation de la seule thérapie disponible à 5% des patients. Depuis qu aucun agent pharmacologique n a réussit à être neuroprotecteur, notre compréhension des rapports entre le sang et le cerveau est remise en cause. La complexité des interactions entre la BHE et les cellules nerveuses a mené au concept d une unité fonctionnelle dite neurovasculaire. Ainsi de nouvelles stratégies de protection émergent à partir d observations au niveau vasculaire. Ainsi la première partie de nos travaux a consisté à étudier l effet vasculoprotecteur potentiel du fénofibrate, un hypolipémiant agoniste du récepteur nucléaire PPAR-a (Peroxisome Proliferator- Activated Receptor-alpha), dont le bénéfice est observé en clinique depuis quelques années et plus récemment dans une étude expérimentale menée chez la souris. Les mécanismes de cette protection aujourd hui inconnus, pourraient impliquer la BHE réputée très peu perméable à ce fibrate. Un renforcement de la BHE limiterait la formation de l oedème cérébral. Pour cela nous avons adapté un modèle in vitro syngénique murin de BHE aux études de perméabilité en condition d OGD (oxygen and glucose deprivation) mimant les conséquences immédiates de l occlusion, toute première étape de l accident vasculaire cérébral (AVC) ischémique. Le modèle consiste en une co-culture de cellules endothéliales primaires de capillaires cérébraux et de cellules gliales primaires. Nos travaux démontrent qu un traitement préventif au fénofibrate protège l endothélium en limitant l hyperperméabilité induite par l OGD. Cette action protectrice cible exclusivement l endothélium et dépend de l activation de PPAR-a démontré par l absence d effet protecteur sur les cellules endothéliales dont le gène codant pour PPAR-a a été invalidé. La seconde partie de l étude s est intéressée aux dommages vasculaires de la reperfusion, étape plus tardive de l ischémie cérébrale connue pour aggraver l oedème vasogénique et mener à des hémorragies fatales. A l aide de notre modèle in vitro, nous avons étudié l effet de la réoxygénation sur la perméabilité vasculaire dans le but de se rapprocher des conditions ischémiques in vivo. Après une incubation en condition d OGD, la co-culture est replacée dans un milieu réoxygéné pendant une période allant de 2h à 24h. La mesure de la perméabilité vasculaire a démontré un profil multiphasique de l ouverture de la BHE dépendant de la présence des cellules gliales. L analyse en microscopie électronique des cellules endothéliales a suggéré une modulation fine de la fonctionnalité des jonctions serrées endothéliales. De plus, l étude en IRM de diffusion chez la souris in vivo a révélé des mouvements d eau qui suggèrent une perturbation de l homéostasie hydrique du parenchyme cérébral au voisinage de l occlusion dans les étapes précoces mais aussi dans les étapes tardives. En conclusion, l ensemble des travaux met en avant la possibilité d une préservation pharmacologique de l intégrité de la BHE au début de l ischémie cérébrale. Celle-ci montre l intérêt des approches in vitro utilisant un modèle cellulaire pertinent et caractérisé. La validation de la cible cellulaire et moléculaire du fénofibrate à l aide de notre modèle ouvre une première voie d exploration des mécanismes impliqués dans ce phénomène de protection microvasculaire précoce. Cependant, la dysfonction retardée de la BHE est également un élément à prendre en compte pour se rapprocher de la physiopathologie de l ischémie in vivo et espérer à terme une amélioration de l approche thérapeutique de cette pathologie.The Blood brain barrier (BBB) is an interface localised at brain capillary endothelial cells. The BBB possesses both physical and metabolic restrictive properties aiming at the maintenance of the central nervous system homeostasis. But under pathological conditions like ischemic stroke, the loss of BBB integrity induces a cerebral vasogenic edema which considerably worsens the vital prognosis of patients. The mechanisms underlying this vascular hyperpermeability are currently unknown thus limiting the use of the only medical intervention available at only 5% of stroke patients. Since no pharmacological molecule succeeded in being neuroprotective, our understanding of the relationships between blood and brain is questioned. The complex interactions between the BBB and nervous cells have lead to the concept of a functional unit, termed the neurovascular unit. Thus, new strategies are recently emerging from observation of vascular events. Thus, the first aim of our study was to test the potential vasculoprotective action of fenofibrate, a hypolipemic drug known as an activator of the nuclear receptor PPAR-a a (Peroxisome Proliferator-Activated Receptor-alpha), as benefit against stroke was observed in clinics since a few years, and recently reported in an experimental study. Yet unknown mechanisms, the protective effect may be exerted on the BBB since reported as impermeable to this compound. An early tightening of the BBB would limit the extent of brain edema. Hence, we have adapted a mouse syngenic BBB in vitro cell model to permeability studies under the stress condition found at the early stage of ischemic stroke defined in vitro as oxygen and glucose deprivation (OGD). This stress simulates the early consequences of occlusion. This model consists of a co-culture of primary brain capillary endothelial cells together with primary glial cells. We have demonstrated that a preventive treatment with fenofibrate has a protective effect on the BBB by limiting the hyperpermeability induced by the OGD condition. This effect targets endothelial cells exclusively and depends on PPAR-a activation, as revealed by the absence of protective action of fenofibrate on PPAR-a deficient endothelial cells. The second part of the study has focused on vascular reperfusion injury, a later stage of ischemia known to worsen vasogenic oedema and to lead to fatal haemorrhage. Using our in vitro BBB model, we have studied the effect of reoxygenation on vascular permeability in order to closely simulate in vivo ischemic condition. Following incubation under OGD condition, the co-cultures were placed into an oxygenated culture medium from 2h to 24h. The BBB permeability demonstrated a multiphasic opening of the BBB which depended on glial cells presence. Electronic microscopy analysis of BBB endothelial cells suggested a fine modulation of tight junction functionality. Moreover, the MRI diffusion analysis in mice has revealed particular water movements suggesting an early disturbance in water homeostasis of brain parenchyma in the vicinity of occlusion. In conclusion, this work put forward the idea of a pharmacological BBB protection at the early stage of ischemic stroke. This demonstrates the relevance of in vitro approaches using a pertinent and well characterised cell model. The validation of cellular and molecular targets of fenofibrate opens a way of first exploration of mechanisms involved in this early microvascular protection phenomenon. But the late BBB dysfunction also needs to be taken into account for a complete fitting with in vivo stroke pathophysiology and an improvement of the therapeutic approaches to this pathology.ARRAS-Bib.electronique (620419901) / SudocSudocFranceF

La barrière hémato-encéphalique lors de l’ischémie cérébrale : une cible thérapeutique

Depuis la preuve de son existence et de son rôle protecteur cérébral, la barrière hémato-encéphalique (BHE), caractérisée par la perméabilité restreinte des cellules endothéliales des capillaires cérébraux, représente un obstacle pour 95 % des futurs médicaments à visée centrale. À l’heure actuelle, une dysfonction de la BHE est trouvée dans un nombre croissant de pathologies telles que les accidents vasculaires cérébraux ischémiques, dont la seule thérapie, une thrombolyse pharmacologique, est limitée à quelques pour cent des patients admis, à cause des effets toxiques des thrombolytiques. Et depuis l’échec clinique de composés neuroprotecteurs prometteurs, de nombreuses études sur l’ischémie cérébrale ont été menées, avec des approches physiopathologiques ou pharmacologiques recentrées sur la BHE dont la complexité structurale s’est élargie à l’ensemble des cellules périvasculaires qui forment une unité fonctionnelle appelée unité neurovasculaire (UNV). Et pourtant, malgré l’identification de nombreux mécanismes moléculaires, le processus de dysfonction de la BHE au décours de l’ischémie/reperfusion demeure insuffisamment décrypté à l’heure actuelle pour expliquer l’action pléiotrope de nouveaux composés pharmacologiques qui pourraient protéger toute l’UNV et représenter de nouveaux traitements

Monitoring how changes in pedagogical practices have improved student interest and performance for an introductory biochemistry course

International audienceThis study describes feedback on the effects of changes introduced in our teaching practices for an introductory biochemistry course in the Life Sciences curriculum. Students on this course have diverse educational qualifications and are taught in large learning groups, creating challenges for the management of individual learning. We used the constructive alignment principle, refining the learning contract and re‐drafting the teaching program to introduce active learning and an organization of activities that promotes the participation of all the students and helps their understanding. We also created teaching resources available through the university virtual work environment. Our research aimed to measure the effects of those changes on the students’ success. Monitoring of the student performance showed a continuous increase in the percentage of students who passed the course, from 2.13% to 33.5% in 4 years. Analysis of student perceptions highlighted that the teaching methodology was greatly appreciated by the students, whose attendance also improved. The recent introduction of clickers‐questions constituted a complementary leverage. The active involvement of the students and better results for summative assessments are altogether a strong motivation for teaching staff to continue to make improvements

Adapting coculture in vitro models of the blood–brain barrier for use in cancer research: maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration

International audienceAlthough brain metastases are the most common brain tumors in adults, there are few treatment options in this setting. To colonize the brain, circulating tumor cells must cross the blood-brain barrier (BBB), which is situated within specialized, restrictive microvascular endothelium. Understanding how cancer cells manage to transmigrate through the BBB might enable this process to be prevented. In vitro models are dedicated tools for characterizing the cellular and molecular mechanisms that underlie transendothelial migration process, as long as they accurately mimic the brain endothelium's in vivo characteristics. The objective of the present study was to adapt an existing in vitro model of the human BBB for use in studying cancer cell transmigration. The model is based on the coculture of endothelial cells (ECs, derived from cord blood hematopoietic stem cells) and brain pericytes. To allow the migration of cancer cells into the lower compartment, our model had to be transposed onto inserts with a larger pore size. However, we encountered a problem when culturing ECs on large (3-μm)-pore inserts: the cells crossed the membrane and formed a non-physiological second layer on the lower face of the insert. Using 3-μm-pore inserts (in a 12-well plate format), we report here on a method that enables the maintenance of a single monolayer of ECs on the insert's upper face only. Under these chosen conditions, the ECs exhibited typical BBB properties found in the original model (including restricted paracellular permeability and the expression of continuous tight junctions). This modified in vitro model of the human BBB enabled us to investigate the migratory potential of the MDA-MB-231 cell line (derived from highly metastatic human breast cancer cells). Last, the results obtained were compared with the rate of transmigration through endothelia with no BBB features

Transient oxygen–glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation

International audienceThrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective

A polarized localization of amino acid/carnitine transporter B<SUP>0,+</SUP> (ATB<SUP>0,+</SUP>) in the blood-brain barrier

International audienceBrain capillary endothelial cells control the uptake and efflux from the brain of many hydrophilic compounds due to highly specialized transporters often localized in a polarized way. Localization of Na+- and Cl--dependent amino acid and carnitine transporter B0,+ (ATB0,+) was studied in a co-culture of bovine brain capillary endothelial cells (BBCEC) grown on filters above astrocytes (an in vitro blood-brain barrier model). Immunoblotting and three-dimensional immunocytochemistry analysis with anti- B0,+antibodies demonstrated the presence of this transporter and its prevalent co-localization with P-glycoprotein i.e. at the apical side. The sensitivity of leucine uptake through the apical membrane to 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH), D-serine as well as sodium and chloride replacement confirm the functioning of ATB0,+ and suggests an important physiological role of ATB0,+ in controlling the delivery of amino acids and carnitine to the brain