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The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

That promising neuroprotectants failed to demonstrate benefit against stroke highlights the great difficulties to translate preclinical pharmacological effects in clinical outcomes. Part of this hurdle implies the complex response to injury of the neurovascular unit increasing the cerebrovascular permeability at the level of the blood-brain barrier (BBB). Previous studies reported neuroprotection in animal models upon activation of the nuclear receptor PPARalpha(peroxisome proliferator-activated receptor)alpha, but the cellular targets at the BBB level remain largely unexplored. Here, to study whether PPAR-alpha activation acts on BBB permeability, we adapted a mouse BBB cell model to ischaemic conditions at the stage of occlusion defined in vitro as oxygen-glucose deprivation (OGD). This model consists of a co-culture of brain capillary endothelial cells (ECs) on a filter insert placed upon a rat glial cell culture. The EC monolayer permeability increase induced by 4 h of OGD was significantly restricted after treatment with the PPAR-alpha agonist fenofibric acid (FA) 24 h before or at the onset of OGD. Treatments of separated ECs or glial cells showed that this protective effect was conferred by BBB ECs but not glial cells. Furthermore, co-cultures with ECs from PPAR-alpha-deficient mice revealed that FA had no effect on OGD-induced hyperpermeability. No transcriptional modulation of classical PPAR-alpha target genes such as SOD, ICAM-1, VCAM-1, ACO, CPT-1, PDK-4 or ET-1 was observed in wild type mouse ECs. In conclusion, these results suggest that part of the preventive PPAR-alpha-mediated protection may occur via BBB ECs by limiting hyperpermeability.

International audience; Brain 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.

International audience; The toxicity of monosubstituted n-alkyldimethylammonium-β-cyclodextrins (DMA-Cn-CD with n = 2, 4 and 12) towards endothelial cells of an in vitro model of the blood-brain barrier (BBB) was evaluated and compared to that of the native β-CD. DMA-C12-CD was found to be non-toxic below 10 mM due to the self-inclusion of the alkyl chain in the CD cavity. A high percentage of passage (30%) of DMA-C12-CD through the endothelial cells has been measured.

International audience; The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brainconc./Plasmaconc.) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood–brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2 = 0.43) and MDCKwt (r2 = 0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2 = 0.74), MDCKwt (r2 = 0.65) and Caco-2 (r2 = 0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of ‘‘batteries'' of in vitro tests are likely to be necessary in order to improve in vitro–in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

International audience; Les cellules endothéliales de capillaires cérébraux, support anatomique de la barrière hémato-encéphalique, présentent des caractéristiques structurales et métaboliques qui restreignent considérablement les échanges entre le sang et le parenchyme cérébral. Nécessaire au maintien de I'homéostasie du système nerveux central, ce filtre sélectif reste un problème pour le traitement des maladies cérébrales. Afin de recréer les interactions qui existent in vivo et d'étudier les mécanismes cellulaires et moléculaires qui régissent le passage des molécules vers le cerveau, nous avons établi un modèle de barrière hémato-encéphalique in vitro. Ce modèle consiste en une coculture de cellules endothéliales de capillaires cérébraux et de cellules gliales ensemencées de part et d'autre d'un filtre. Dans ces conditions de culture, nous avons démontré que les cellules endothéliales exprimaient toutes les caractéristiques, tant physiques que métaboliques, de l'endothélium invivo. Ces résultats font de notre modèle un outil pertinent et précieux dans le développement de nouvelles molécules à visée cérébrale.

International audience; Association between doxorubicin (DOX) and γ-cyclodextrin (γ-CD) or hydroxypropyl-γ-CD (HP-γ-CD) has been examined to increase the delivery of this antitumoral agent to the brain. The stoichiometry and the stability constant of γ-CD or HP-γ-CD and DOX complexes were determined in physiological medium by UV-visible spectroscopy. By using an in vitro model of the blood-brain barrier (BBB), endothelial permeability and toxicity toward the brain capillary endothelial cells of DOX, γ-CD, and HP-γ-CD were performed. For each CD, endothelial permeability was relatively low and a disruption of the BBB occurred at 20 μM, 20 mM, and 50 mM DOX, γ-CD, and HP-γ-CD, respectively. Increasing amounts of CDs were added to a fixed DOX concentration. Addition of γ-CD or HP-γ-CD, up to 15 and 35 mM, respectively, decreased the DOX delivery, probably due to the low complex penetration across the BBB and the decrease in free DOX concentration. Higher CD concentrations increased the DOX delivery to the brain, but this effect is due to a loss of BBB integrity. In contrast to what was observed on Caco-2 cell model with various drugs, CDs are not able to increase the delivery of DOX across our in vitro model of BBB.

International audience; Primary brain tumors, particularly glioblastomas (GB), remain a challenge for oncology. An element of the malignant brain tumors' aggressive behavior is the fact that GB are among the most densely vascularized tumors. To determine some of the molecular regulations occuring at the brain tumor endothelium level during tumoral progression would be an asset in understanding brain tumor biology. Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling, oncogenesis, and angiogenesis. In this work we investigated regulation of caveolin-1 expression in brain endothelial cells (ECs) under angiogenic conditions. In vitro, brain EC caveolin-1 is down-regulated by angiogenic factors treament and by hypoxia. Coculture of brain ECs with tumoral cells induced a similar downregulation. In addition, activation of the p42/44 MAP kinase is demonstrated. By using an in vivo brain tumor model, we purified ECs from gliomas as well as from normal brain to investigate possible regulation of caveolin-1 expression in tumoral brain vasculature. We show that caveolin-1 expression is strikingly down-regulated in glioma ECs, whereas an increase of phosphorylated caveolin-1 is observed. Whole-brain radiation treatment, a classical way in which GB is currently being treated, resulted in increased caveolin-1 expression in tumor isolated ECs. The level of tumor cells spreading around newly formed blood vessels was also elevated. The regulation of caveolin-1 expression in tumoral ECs may reflect the tumoral vasculature state and correlates with angiogenesis kinetics.

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International audience; The blood–brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.

Several lines of evidence indicate that alterations in iron metabolism and/or immune-mediated pathophysiology might contribute to neuronal loss. Recently, lactoferrin, an iron-binding protein and a modulatory agent of the inflammatory response, has been associated with neurodegenerative disorders. We have shown that the source of lactoferrin in brain tissues is double, either synthezised in situ or originating from the circulating blood, and we investigated the mechanisms by which lactoferrin accumulates in pathological conditions. Lactoferrin is synthezised in situ in the mouse brain. An animal model of Parkinson's disease was used to study the expression of lactoferrin in pathological conditions. Lactoferrin expression was increased after treatment with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) which is selectively toxic to the dopaminergic neurons, suggesting that lactoferrin might be produced in response to the oxidative stress generate by the neurodegenerative process occurring in Parkinson's disease. Lactoferrin crosses the blood-brain barrier (BBB). An in vitro model of the BBB was used to characterize the transport of lactoferrin through the brain capillary endothelial cells. We next showed, that this transport was increased in inflammatory conditions, leading to lactoferrin accumulation in the cerebral compartment. This accumulation may account for the increased level of lactoferrin observed in the vicinity of the inflammatory foci.

International audience; Lactoferrin (Lf) is an iron-binding protein involved in host defense against infection and severe inflammation; it accumulates in the brain during neurodegenerative disorders. Before determining Lf function in brain tissue, we investigated its origin and demonstrate here that it crosses the blood-brain barrier. An in vitro model of the blood-brain barrier was used to examine the mechanism of Lf transport to the brain. We report that differentiated bovine brain capillary endothelial cells exhibited specific high (Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 μM; n = 900,000 sites/cell) affinity binding sites. Only the latter were present on nondifferentiated cells. The surface-bound Lf was internalized only by the differentiated cell population leading to the conclusion that Lf receptors were acquired during cell differentiation. A specific unidirectional transport then occurred via a receptor-mediated process with no apparent intraendothelial degradation. We further report that iron may cross the bovine brain capillary endothelial cells as a complex with Lf. Finally, we show that the low density lipoprotein receptor-related protein might be involved in this process because its specific antagonist, the receptor-associated protein, inhibits 70% of Lf transport.

International audience; The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.

International audience; In contrast to the endothelial cells in large vessels where low density lipoprotein receptors are down-regulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced 3 fold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL receptor expression. We have shown that the lipid requirement of astrocytes increases the expression of endothelial cell LDL receptors. Experiments with dialysis membranes of different pore size showed that this effect is mediated by a soluble factor(s) with a molecular mass somewhere between 3,500 and 14,000. Substituting astrocytes with smooth muscle cells or brain endothelium with endothelium from the aorta or the adrenal cortex did not enhance the luminal LDL receptor expression on endothelial cells, demonstrating the specificity of the interactions. This study demonstrates that a cross-talk takes place between brain capillary endothelial cells and astrocytes.

International audience; The aim of the present studies was to compare the transport of drugs using two in vitro models routinely used in our laboratories: a primary culture of brain microvessel endothelial cells and a coculture of brain capillary endothelial cells and astrocytes. For this purpose we selected a set of compounds corresponding to a wide range of lipid solubility. Additionally the in vitro results were compared to in vivo results obtained with the single carotid injection, and a good correlation between in vivo extraction ratios (Et) and in vitro permeability coefficients (Pe) was shown as indicated by the Spearman's correlation coefficient (r = 0.90 and r = 0.96 for primary culture and coculture, respectively). The studies show that the use of brain capillary endothelial cells together with astrocytes is slightly more predictive and significantly easier than the use of primary cultured brain microvessels.

International audience; Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of acoculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxiareoxygenation at the BBB level . The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. Adecrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury . Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation .

International audience; Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood-brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these patho-Iogical conditions. In our in vitro reconstructed BBB model, 1-hr exposure of recombinant human TNF-α (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelial flux of either sucrose (m.w.342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P < 0.05) in sucrose permeation at 2-4 hr with the highest dose of TNF-α. On the other hand, at 16 hr after the 1-hr challenge with TNF-α(delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F-actin filaments into stress fibers, while the intracellutar distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF- α by a delayed increase of permeability and reorganization of actin filaments.

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