Impaired induction of blood-brain barrier properties in aortic endothelial cells by astrocytes from GFAB-deficient mice (original) (raw)
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Neurochemistry International, 1996
Ahstraet-Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood brain barrier (BBB) models, using the coculture in a double chamber system : rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in L-glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the Lglucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.
A comparison of the induction of immortalized endothelial cell impermeability by astrocytes
Neuroreport, 2001
The suitability of various commercially available endothelial cell lines in studies of astrocytic/endothelial cell interactions was assessed. The endothelial-like cell line ECV304 was compared with T24/83, Eahy929, and b.End5 and rat cerebral endothelial cells in their ability, when co-cultured with rat (C6) glioma cells, to form a transendothelial electrical resistance (TEER), an indicator of tight junction formation which is an important property of the blood±brain barrier. As reported previously, the basal TEER of ECV304 cell monolayers was signi®cantly enhanced upon co-culture, an effect reproduced by human 1321N1 astrocytes and primary rat astrocytes. T24/83 cells formed a patchy, gapped monolayer, which produced a poor basal TEER with little in the way of an increase upon coculture. Similarly, all the other cell monolayers analysed demonstrated poor TEERs that were only moderately increased upon co-culture. These data con®rm that while no endothelial cell line with ideal features is available, ECV304 cells remain an appropriate choice especially for studies of astrocyte/endothelial cell interactions. NeuroReport 12:1329± 1334 & 2001 Lippincott Williams & Wilkins.
Cytotechnology, 1997
Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-relat...
Brain Endothelial Barrier in Vitro
2013
The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/ metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).
Brain Research, 2007
The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC.
Experimental Cell Research, 2007
The blood-brain barrier (BBB) is composed of the cerebral microvascular endothelium, which, together with astrocytes, pericytes, and the extracellular matrix (ECM), contributes to a "neurovascular unit". It was our objective to clarify the impact of endogenous extracellular matrices on the barrier function of BBB microvascular endothelial cells cultured in vitro. The study was performed in two consecutive steps: (i) The ECM-donating cells (astrocytes, pericytes, endothelial cells) were grown to confluence and then removed from the growth substrate by a protocol that leaves the ECM behind. (ii) Suspensions of cerebral endothelial cells were seeded on the endogenous matrices and barrier formation was followed with time. In order to quantify the tightness of the cell junctions, all experiments were performed on planar gold-film electrodes that can be used to read the electrical resistance of the cell layers as a direct measure for endothelial barrier function (electric cell-substrate impedance sensing, ECIS). We observed that endogenously isolated ECM from both, astrocytes and pericytes, improved the tightness of cerebral endothelial cells significantly compared to ECM that was derived from the endothelial cells themselves as a control. Moreover, when cerebral endothelial cells were grown on extracellular matrices produced by non-brain endothelial cells (aorta), the electrical resistances were markedly reduced. Our observations indicate that glia-derived ECMas an essential part of the BBBis required to ensure proper barrier formation of cerebral endothelial cells. ava i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s ev i e r. c o m / l o c a t e / yexc r
Developmental Brain Research, 2004
We sought to establish a blood -neural barrier (BNB) model of astrocyte contact with endothelial cells (EC) to test the hypothesis that transforming growth factor h (TGFh) promotes an EC barrier-phenotype. Astrocyte -EC contact induced BNB properties in EC. Transendothelial resistance was augmented by direct contact between astrocytes -EC, but not by astrocyte-conditioned medium or astrocyte -EC coculture conditioned medium. Coculture of EC and astrocytes led to significant increase in endothelial occludin levels and junctional localization. EC g-glutamyl-transferase (GGT) activity was increased by direct contact with astrocytes, by conditioned medium from cocultures or by TGFh1. Coculture inhibited EC proliferation with no effect on astrocyte proliferation. A neutralizing antibody to TGFh decreased GGT activity in cocultures and increased cell number. Whereas total TGFh was not significantly altered by coculture, activated TGFh increased in astrocyte -EC cocultures. In summary, astrocyte -EC contact induces BNB characteristics in EC and locally activated TGFh is responsible for part of the induction. D 2004 Elsevier B.V. All rights reserved.
Brain Research, 2002
Blood-brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of flow and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.
Annals of Biomedical Engineering, 2010
The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L p ), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L p and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.
Journal of Neuroscience Methods, 2002
The specific structure of the blood Á/brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Vcm 2 showing their ability to limit the passage of ions. A 15-fold increase in g-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function. #