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Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling. [ABSTRACT FROM AUTHOR]

Background: Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology: Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings: Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion: These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. [ABSTRACT FROM AUTHOR]

Septic shock is a severe disease state characterised by the body's life threatening response to infection. Complex interactions between endothelial cells and circulating monocytes are responsible for microvasculature dysfunction contributing to the pathogenesis of this syndrome. Here, we intended to determine whether microparticles derived from activated monocytes contribute towards inflammatory processes and notably vascular permeability. We found that endotoxin stimulation of human monocytes enhances the release of microparticles of varying phenotypes and mRNA contents. Elevated numbers of LPS-induced monocytic microparticles (mMP) expressed CD54 and contained higher levels of transcripts for pro-inflammatory cytokines such as TNF, IL-6 and IL-8. Using a prothrombin time assay, a greater reduction in plasma coagulation time was observed with LPS-induced mMP than with non-stimulated mMP. Co-incubation of mMP with the human brain endothelial cell line hCMEC/D3 triggered their time-dependent uptake and significantly enhanced endothelial microparticle release. Unexpectedly, mMP also modified signalling pathways by diminishing pSrc (tyr416) expression and promoted endothelial monolayer tightness, as demonstrated by endothelial impedance and permeability assays. Altogether, these data strongly suggest that LPS-induced mMP have contrasting effects on the intercellular communication network and display a dual potential: enhanced pro-inflammatory and procoagulant properties, together with protective function of the endothelium. [ABSTRACT FROM AUTHOR]

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality. [ABSTRACT FROM AUTHOR]

Objective: The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of ‘specialized endothelial cells’. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions. Approach and Results: First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers. Conclusions: We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays. [ABSTRACT FROM AUTHOR]

Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation. [ABSTRACT FROM AUTHOR]

30% of epilepsy patients receiving antiepileptic drugs (AEDs) are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3) were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp) of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp) but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine. [ABSTRACT FROM AUTHOR]

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues. [ABSTRACT FROM AUTHOR]

Endothelial cells (EC) form the inner lining of blood vessels and are positioned between circulating lymphocytes and tissues. Hypotheses have formed that EC may act as antigen presenting cells based on the intimate interactions with T cells, which are seen in diseases like multiple sclerosis, cerebral malaria (CM) and viral neuropathologies. Here, we investigated how human brain microvascular EC (HBEC) interact with and support the proliferation of T cells. We found HBEC to express MHC II, CD40 and ICOSL, key molecules for antigen presentation and co-stimulation and to take up fluorescently labeled antigens via macropinocytosis. In co-cultures, we showed that HBEC support and promote the proliferation of CD4+ and CD8+ T cells, which both are key in CM pathogenesis, particularly following T cell receptor activation and co-stimulation. Our findings provide novel evidence that HBEC can trigger T cell activation, thereby providing a novel mechanism for neuroimmunological complications of infectious diseases. [ABSTRACT FROM AUTHOR]

In central nervous system (CNS)-directed gene therapy, efficient targeting of brain parenchyma through the vascular route is prevented by the endothelium and the epithelium of the blood-brain and the blood-cerebrospinal fluid barriers, respectively. In this study, we evaluated the feasibility of the combined genetic and chemical adenovirus capsid modification technology to enable transcellular delivery of targeted adenovirus (Ad) vectors across the blood-brain barrier (BBB) in vitro models. As a proof-of-principle ligand, maleimide-activated full-length human transferrin (hTf) was covalently attached to cysteine-modified Ad serotype 5 vectors either to its fiber or hexon protein. In transcytosis experiments, hTf-coupled vectors were shown to be redirected across the BBB models, the transcytosis activity of the vectors being dependent on the location of the capsid modification and the in vitro model used. The transduction efficiency of hTf-targeted vectors decreased significantly in confluent, polarized cells, indicating that the intracellular route of the vectors differed between unpolarized and polarized cells. After transcellular delivery the majority of the hTf-modified vectors remained intact and partly capable of gene transfer. Altogether, our results demonstrate that i) covalent attachment of a ligand to Ad capsid can mediate transcellular targeting across the cerebral endothelium in vitro, ii) the attachment site of the ligand influences its transcytosis efficiency and iii) combined genetic/chemical modification of Ad vector can be used as a versatile platform for the development of Ad vectors for transcellular targeting. [ABSTRACT FROM AUTHOR]

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane. [ABSTRACT FROM AUTHOR]

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pretreatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors. [ABSTRACT FROM AUTHOR]

Introduction: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. Methods: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. Results: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. Conclusion: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of proangiogenic and survival factors. [ABSTRACT FROM AUTHOR]

Background: An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function. Methodology/Principal Findings: As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time. Conclusion: Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment. [ABSTRACT FROM AUTHOR]

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies. [ABSTRACT FROM AUTHOR]

Reproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation. [ABSTRACT FROM AUTHOR]

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues. [ABSTRACT FROM AUTHOR]

Objective: The vascular system is adapted to specific functions in different tissues and organs. Vascular endothelial cells are important elements of this adaptation, leading to the concept of 'specialized endothelial cells'. The phenotype of these cells is highly dependent on their specific microenvironment and when isolated and cultured, they lose their specific features after few passages, making models using such cells poorly predictive and irreproducible. We propose a new source of specialized endothelial cells based on cord blood circulating endothelial progenitors (EPCs). As prototype examples, we evaluated the capacity of EPCs to acquire properties characteristic of cerebral microvascular endothelial cells (blood-brain barrier (BBB)) or of arterial endothelial cells, in specific inducing culture conditions., Approach and Results: First, we demonstrated that EPC-derived endothelial cells (EPDCs) co-cultured with astrocytes acquired several BBB phenotypic characteristics, such as restricted paracellular diffusion of hydrophilic solutes and the expression of tight junction proteins. Second, we observed that culture of the same EPDCs in a high concentration of VEGF resulted, through activation of Notch signaling, in an increase of expression of most arterial endothelial markers., Conclusions: We have thus demonstrated that in vitro culture of early passage human cord blood EPDCs under specific conditions can induce phenotypic changes towards BBB or arterial phenotypes, indicating that these EPDCs maintain enough plasticity to acquire characteristics of a variety of specialized phenotypes. We propose that this property of EPDCs might be exploited for producing specialized endothelial cells in culture to be used for drug testing and predictive in vitro assays.

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional invivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.