17 research outputs found
Influence of the brain perivascular environment on the blood-brain barrier dysfunction during a transient ischemia
Ces derniĂšres annĂ©es, alors quâaucun agent neuroprotecteur nâa Ă©tĂ© efficace en clinique pour parer les dommages de lâischĂ©mie cĂ©rĂ©brale, le concept dâunitĂ© neurovasculaire (UNV) est apparu comme un nouveau paradigme pour lâinvestigation et le traitement des accidents vasculaires cĂ©rĂ©braux ischĂ©miques. La rupture de la barriĂšre hĂ©mato-encĂ©phalique (BHE) localisĂ©e au niveau des capillaires cĂ©rĂ©braux, et ses corollaires lâĆdĂšme vasogĂ©nique et lâhĂ©morragie intracĂ©rĂ©brale, constituent des Ă©vĂ©nements critiques de la maladie, et restreignent considĂ©rablement lâĂ©ligibilitĂ© des patients Ă la thrombolyse au rtPA, seul traitement de phase aiguĂ« disponible actuellement en clinique. La complexitĂ© des intercommunications qui sâexercent au sein de lâUNV rend difficile lâapprĂ©hension de la dysfonction microvasculaire in vivo, soulignant lâimportance des Ă©tudes in vitro pour complĂ©ter les connaissances dans ce domaine. Câest par cette approche combinĂ©e que les travaux effectuĂ©s au cours de ce doctorat dĂ©montrent lâimpact de la nĂ©crose cĂ©rĂ©brale sur la cinĂ©tique de la perte dâintĂ©gritĂ© de la BHE au dĂ©cours de la reperfusion. Cependant, mĂȘme si lâendothĂ©lium microvasculaire demeure fonctionnel aprĂšs un Ă©pisode ischĂ©mique dans un contexte non lĂ©sionel, il devient vulnĂ©rable Ă certaines molĂ©cules comme le rtPA dans une situation de thrombolyse. Ces rĂ©sultats illustrent le rĂŽle dĂ©terminant de lâenvironnement molĂ©culaire pĂ©rivasculaire sur la dysfonction de la BHE lors de lâischĂ©mie cĂ©rĂ©brale, et orientent les nouvelles stratĂ©gies thĂ©rapeutiques vers des approches ciblant la protection de lâensemble de lâUNV.In the recent years, while no neuroprotective agent was clinically effective in reducing brain ischemic damage, the neurovascular unit (NVU) concept emerged as a new paradigm for stroke investigation and treatment. The breakdown of the blood-brain barrier (BBB), localized in brain capillaries, with ensuing vasogenic edema and intracerebral hemorrhage, appears as a critical event of this disease, and severely restricts the eligibility of patients for rtPA thrombolysis, the only acute-phase treatment currently available. The complex intercommunications occurring within the NVU makes the microvascular dysfunction difficult to study in vivo, highlighting the importance of in vitro approaches to complete the knowledge in this field. In this context, the work done in this PhD demonstrates that brain tissue necrosis influences the kinetics of the loss of BBB integrity during reperfusion. However, even when the BBB remains functional in a non-lesional ischemic context, it becomes vulnerable to certain molecules such as rtPA in a thrombolysis situation. These results illustrate the key role of molecular perivascular environment on the BBB dysfunction during cerebral ischemia, and orientate new therapeutic strategies towards the protection of the entire NVU
Influence of the brain perivascular environment on the blood-brain barrier dysfunction during a transient ischemia
Ces derniĂšres annĂ©es, alors quâaucun agent neuroprotecteur nâa Ă©tĂ© efficace en clinique pour parer les dommages de lâischĂ©mie cĂ©rĂ©brale, le concept dâunitĂ© neurovasculaire (UNV) est apparu comme un nouveau paradigme pour lâinvestigation et le traitement des accidents vasculaires cĂ©rĂ©braux ischĂ©miques. La rupture de la barriĂšre hĂ©mato-encĂ©phalique (BHE) localisĂ©e au niveau des capillaires cĂ©rĂ©braux, et ses corollaires lâĆdĂšme vasogĂ©nique et lâhĂ©morragie intracĂ©rĂ©brale, constituent des Ă©vĂ©nements critiques de la maladie, et restreignent considĂ©rablement lâĂ©ligibilitĂ© des patients Ă la thrombolyse au rtPA, seul traitement de phase aiguĂ« disponible actuellement en clinique. La complexitĂ© des intercommunications qui sâexercent au sein de lâUNV rend difficile lâapprĂ©hension de la dysfonction microvasculaire in vivo, soulignant lâimportance des Ă©tudes in vitro pour complĂ©ter les connaissances dans ce domaine. Câest par cette approche combinĂ©e que les travaux effectuĂ©s au cours de ce doctorat dĂ©montrent lâimpact de la nĂ©crose cĂ©rĂ©brale sur la cinĂ©tique de la perte dâintĂ©gritĂ© de la BHE au dĂ©cours de la reperfusion. Cependant, mĂȘme si lâendothĂ©lium microvasculaire demeure fonctionnel aprĂšs un Ă©pisode ischĂ©mique dans un contexte non lĂ©sionel, il devient vulnĂ©rable Ă certaines molĂ©cules comme le rtPA dans une situation de thrombolyse. Ces rĂ©sultats illustrent le rĂŽle dĂ©terminant de lâenvironnement molĂ©culaire pĂ©rivasculaire sur la dysfonction de la BHE lors de lâischĂ©mie cĂ©rĂ©brale, et orientent les nouvelles stratĂ©gies thĂ©rapeutiques vers des approches ciblant la protection de lâensemble de lâUNV.In the recent years, while no neuroprotective agent was clinically effective in reducing brain ischemic damage, the neurovascular unit (NVU) concept emerged as a new paradigm for stroke investigation and treatment. The breakdown of the blood-brain barrier (BBB), localized in brain capillaries, with ensuing vasogenic edema and intracerebral hemorrhage, appears as a critical event of this disease, and severely restricts the eligibility of patients for rtPA thrombolysis, the only acute-phase treatment currently available. The complex intercommunications occurring within the NVU makes the microvascular dysfunction difficult to study in vivo, highlighting the importance of in vitro approaches to complete the knowledge in this field. In this context, the work done in this PhD demonstrates that brain tissue necrosis influences the kinetics of the loss of BBB integrity during reperfusion. However, even when the BBB remains functional in a non-lesional ischemic context, it becomes vulnerable to certain molecules such as rtPA in a thrombolysis situation. These results illustrate the key role of molecular perivascular environment on the BBB dysfunction during cerebral ischemia, and orientate new therapeutic strategies towards the protection of the entire NVU
La barriĂšre hĂ©mato-encĂ©phalique lors de lâischĂ©mie cĂ©rĂ©brale : une cible thĂ©rapeutique
Depuis la preuve de son existence et de son rÎle protecteur cérébral, la barriÚre
hémato-encéphalique (BHE), caractérisée par la perméabilité restreinte des cellules
endothéliales des capillaires cérébraux, représente un obstacle pour 95 % des futurs
mĂ©dicaments Ă visĂ©e centrale. Ă lâheure actuelle, une dysfonction de la BHE est trouvĂ©e
dans un nombre croissant de pathologies telles que les accidents vasculaires cérébraux
ischĂ©miques, dont la seule thĂ©rapie, une thrombolyse pharmacologique, est limitĂ©e Ă
quelques pour cent des patients admis, Ă cause des effets toxiques des thrombolytiques. Et
depuis lâĂ©chec clinique de composĂ©s neuroprotecteurs prometteurs, de nombreuses Ă©tudes sur
lâischĂ©mie cĂ©rĂ©brale ont Ă©tĂ© menĂ©es, avec des approches physiopathologiques ou
pharmacologiques recentrĂ©es sur la BHE dont la complexitĂ© structurale sâest Ă©largie Ă
lâensemble des cellules pĂ©rivasculaires qui forment une unitĂ© fonctionnelle
appelée unité neurovasculaire (UNV). Et pourtant, malgré
lâidentification de nombreux mĂ©canismes molĂ©culaires, le processus de
dysfonction de la BHE au dĂ©cours de lâischĂ©mie/reperfusion demeure
insuffisamment dĂ©cryptĂ© Ă lâheure actuelle pour expliquer lâaction plĂ©iotrope de nouveaux
composĂ©s pharmacologiques qui pourraient protĂ©ger toute lâUNV et reprĂ©senter de nouveaux
traitements
Accumulation of plastid lipidâassociated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species
International audienc
Transient oxygenâglucose 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
Dynamics of the localization of the plastid terminal oxidase PTOX inside the chloroplast
International audienceThe plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. PTOX activity has been previously shown to depend on its localization at the thylakoid membrane. Here we investigated the dynamics of PTOX localization in dependence on the proton motive force. Infiltrating illuminated leaves with uncouplers led to a partial dissociation of PTOX from the thylakoid membrane. In vitro reconstitution experiments showed that the attachment of purified recombinant MBP-OsPTOX to liposomes and isolated thylakoid membranes was strongest at slightly alkaline pH values in the presence of lower millimolar concentrations of KCl or MgCl2. In A. thaliana overexpressing GFP-PTOX, confocal microscopy images showed that PTOX formed distinct spots in chloroplasts of dark-adapted or uncoupler-treated leaves while the protein was more equally distributed in a network-like structure in the light. We propose a dynamic PTOX association with the thylakoid membrane depending on the presence of a proton motive force
Protein kinase C restricts transport of carnitine by amino acid transporter ATB0,+ apically localized in the bloodâbrain barrier
International audienceCarnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated
In vitro discrimination of the role of LRP1 at the BBB cellular level: Focus on brain capillary endothelial cells and brain pericytes
International audienceSeveral studies have demonstrated that the blood-brain barrier (BBB) (dynamic cellular complex composed by brain capillary endothelial cells (BCECs) and surrounded by astrocytic end feet and pericytes) regulates the exchanges of amyloid ÎČ (AÎČ) peptide between the blood and the brain. Deregulation of these exchanges seems to be a key trigger for the brain accumulation of AÎČ peptide observed in Alzheimer's disease (AD). Whereas the involvement of receptor for advanced glycation end-products in AÎČ peptide transcytosis has been demonstrated in our laboratory, low-density lipoprotein receptor's role at the cellular level needs to be clarified. For this, we used an in vitro BBB model that consists of a co-culture of bovine BCECs and rat glial cells. This model has already been used to characterize low-density lipoprotein receptor-related peptide (LRP)'s involvement in the transcytosis of molecules such as tPA and angiopep-2. Our results suggest that AÎČ peptide efflux across the BCEC monolayer involves a transcellular transport. However, the experiments with RAP discard an involvement of LRP family members at BCECs level. In contrast, our results show a strong transcriptional expression of LRP1 in pericytes and suggest its implication in AÎČ endocytosis. Moreover, the observations of pericytes contraction and local downregulation of LRP1 in response to AÎČ treatment opens up perspectives for studying this cell type with respect to AÎČ peptide metabolism and AD
Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-ÎČ Peptides in an In Vitro Model of the Human Blood-Brain Barrier
International audienceOne of the prime features of Alzheimer's disease (AD) is the excessive accumulation of amyloid-ÎČ (AÎČ) peptides in the brain. Several recent studies suggest that this phenomenon results from the dysregulation of cholesterol homeostasis in the brain and impaired bidirectional AÎČ exchange between blood and brain. These mechanisms appear to be closely related and are controlled by the blood-brain barrier (BBB) at the brain microvessel level. In animal models of AD, the anticancer drug bexarotene (a retinoid X receptor agonist) has been found to restore cognitive functions and decrease the brain amyloid burden by regulating cholesterol homeostasis. However, the drug's therapeutic effect is subject to debate and the exact mechanism of action has not been characterized. Therefore, the objective of this present study was to determine bexarotene's effects on the BBB. Using an in vitro model of the human BBB, we investigated the drug's effects on cholesterol exchange between abluminal and luminal compartments and the apical-to-basolateral transport of AÎČ peptides across the BBB. Our results demonstrated that bexarotene induces the expression of ABCA1 but not ApoE. This upregulation correlates with an increase in ApoE2-, ApoE4-, ApoA-I-, and HDL-mediated cholesterol efflux. Regarding the transport of AÎČ peptides, bexarotene increases the expression of ABCB1, which in turn decreases AÎČ apical-to-basolateral transport. Our results showed that bexarotene not only promotes the cholesterol exchange between the brain and the blood but also decreases the influx of AÎČ peptides across BBB, suggesting that bexarotene is a promising drug candidate for the treatment of AD
Stroke-Induced Brain Parenchymal Injury Drives BloodâBrain Barrier Early Leakage Kinetics: A Combined in Vivo / in Vitro Study
International audienceThe disappointing clinical outcomes of neuroprotectants challenge the relevance of preclinical stroke models and data in defining early cerebrovascular events as potential therapeutic targets. The kinetics of bloodâbrain barrier (BBB) leakage after reperfusion and the link with parenchymal lesion remain debated. By using in vivo and in vitro approaches, we conducted a kinetic analysis of BBB dysfunction during early reperfusion. After 60âminutes of middle cerebral artery occlusion followed by reperfusion times up to 24âhours in mice, a non-invasive magnetic resonance imaging method, through an original sequence of diffusion-weighted imaging, determined brain water mobility in microvascular compartments (D*) apart from parenchymal compartments (apparent diffusion coefficient). An increase in D* found at 4âhours post reperfusion concurred with the onset of both Evans blue/Dextran extravasations and in vitro BBB opening under oxygen-glucose deprivation and reoxygenation (R). The BBB leakage coincided with an emerging cell death in brain tissue as well as in activated glial cells in vitro. The co-culture of BBB endothelial and glial cells evidenced a recovery of endothelium tightness when glial cells were absent or non-injured during R. Preserving the ischemic brain parenchymal cells within 4âhours of reperfusion may improve therapeutic strategies for cerebrovascular protection against stroke