The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two diVerent classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaVolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diVusion. In many diseases, disruption of this regulated barrier occurs. This review will brieXy describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The integrity of the BBB is compromised in many disorders of the human CNS; therapeutical strategies for several of these diseases include treatment with glucocorticoids, but the molecular basis of how glucocorticoids regulate BBB permeability is not understood. Here, we report the generation and characterization of a murine immortalized brain (cerebral) capillary endothelial (cEND) cell line which expresses the BBB marker occludin at intercellular tight junctions (TJ). Hydrocortisone at physiological concentrations induced upregulation of occludin, accompanied by a threefold enhancement of transendothelial electrical resistance to values up to 1000 Ωcm 2 . Insulin enhanced the glucocorticoid response. At the molecular level, hydrocortisone induces increase of occludin at protein and mRNA levels by activation of the glucocorticoid receptor (GR) and its binding to putative glucocorticoid responsive elements in the occludin promoter. At the same time, insulin potentiated the ligand-dependent GR transactivation via induction of the GR in this in vitro system. This study thus provides insights into the molecular processes of barrier genesis, and may help to elucidate mechanisms of brain pathology at the microvascular level.

Homeostasis of the central nervous system (CNS) microenvironment is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and back. Many disorders change the functionality and integrity of the BBB. Glucocorticoids are being used sucessfully in the treatment of some disorders while their effects on others are questionable. In addition, conflicting results between clinical and experimental experience using animal models has arisen, so that the results of molecular studies in animal models need to be revisited in an appropriate in vitro model of the human BBB for more effective treatment strategies. Using the human brain microvascular endothelial cell line hCMEC/D3, the influence of glucocorticoids on the expression of barrier constituting adherens junction and tight junction transmembrane proteins (VE-cadherin, occludin, claudins) was investigated and compared to other established BBB models. In hCMEC/D3 cells the administration of glucocorticoids induced expression of the targets occludin 2.75 ± 0.04-fold and claudin-5 up to 2.32 ± 0.11-fold, which is likely to contribute to the more than threefold enhancement of transendothelial electrical resistance reflecting barrier tightness. Our analyses further provide direct evidence that the GC hydrocortisone prevents endothelial barrier breakdown in response to pro-inflammatory stimuli (TNFα administration), which could be demonstrated to be partly based on maintenance of occludin levels. Our studies strongly suggest stabilization of BBB function as a mode of GC action on a molecular level in the human brain vasculature.

The mammary glands of prepubertal estrogen receptor (ER)␤؊͞؊ mice are morphologically indistinguishable from those of WT littermates. It appears that, although ER␤ is expressed in the mouse mammary gland, it is not involved in ductal growth of the gland. In this study, we examined the possibility that ER␤ has a role in the differentiated function of the mammary gland. Pregnancy is rare in ER␤؊͞؊ mice, but an intensive breeding program produced seven pregnant ER␤؊͞؊ mice, of which five did not eat their offspring and continued to successful lactation. Histomorphological comparison of lactating glands revealed that alveoli were larger and there was less secretory epithelium in ER␤؊͞؊ than in WT mice. Ultrastructural analysis showed abundant milk droplets and normal apical villi in the luminal epithelial cells, but the extracellular matrix and lamina basalis were reduced, and very frequently the interepithelial cell space was increased. Levels of the adhesion molecules, E-cadherin, connexin 32, occludin, and integrin ␣2 were reduced, and no zona occludens was detectable. In addition, there was widespread expression of the proliferation marker, Ki-67, in luminal epithelial cells in ER␤؊͞؊ but not in WT mice. These findings suggest a role for ER␤ in organization and adhesion of epithelial cells and hence for differentiated tissue morphology. We speculate that, because a reduced risk for breast cancer is conferred on women who breast-feed at an early age, ER␤ could contribute to this risk reduction by facilitating terminal differentiation of the mammary gland.lactation ͉ cadherin ͉ integrin ͉ tight junction T argeted disruption of estrogen receptor (ER)␤ in mice has revealed that this is a functional receptor in both males and females and that, in addition to its role in reproductive functions (1), it is also important in the cardiovascular (2) and central nervous systems (3). Female mice homozygous for the mutated ER␤ gene (ER␤Ϫ͞Ϫ mice) have been described as subfertile or infertile (4, 5). The ovarian defect is due to early atresia of antral follicles and failure to ovulate. As a result, corpora lutea are rare in ER␤Ϫ͞Ϫ mouse ovaries (6).Prepubertal ER␤Ϫ͞Ϫ females appear to have a normal mammary histology (5) with unaffected ductal outgrowth of the mammary gland anlage. However, because corpora lutea are rare, little progesterone is produced in the ovaries and, in contrast to their WT littermates, ER␤Ϫ͞Ϫ mammary glands fail to develop ductal side branches and alveoli after puberty. On administration of progesterone, side branching occurs, and mammary glands of ER␤Ϫ͞Ϫ mice appear morphologically indistinguishable from those of their WT littermates (7). As judged by nursing behavior and growth of their pups, ER␤Ϫ͞Ϫ mice have previously been reported to lactate effectively (5). In contrast, in female mice lacking aromatase (8) or ER␣ (9), mammary glands fail to develop beyond the prepubertal stage. From these studies, it has been concluded (5) that estrogen, acting through ER␣ but not through ER␤, is essential for norm...

The blood–brain barrier choline transporter (BBB-ChT) may have utility as a drug delivery vector to the central nervous system (CNS). We therefore initiated molecular docking studies with the AutoDock and AutoDock Vina (ADVina) algorithms to develop predictive models for compound screening and to identify structural features important for binding to this transporter. The binding energy predictions were highly correlated with r2=0.88, F=692.4, standard error of estimate =0.775, and P-value<0.0001 for selected BBB-ChT-active/inactive compounds (n=93). Both programs were able to cluster active (Gibbs free energy of binding <−6.0 kcal*mol−1) and inactive (Gibbs free energy of binding >−6.0 kcal*mol−1) molecules and dock them significantly better than at random with an area under the curve value of 0.86 and 0.84, respectively. In ranking smaller molecules with few torsional bonds, a size-related bias in scoring producing false-negative outcomes was detected. Finally, important blood–brain barrier parameters, such as the logBBpassive and logBBactive values, were assessed to predict compound transport to the CNS accurately. Knowledge gained from this study is useful to better understand the binding requirements in BBB-ChT, and until such time as its crystal structure becomes available, it may have significant utility in developing a highly predictive model for the rational design of drug-like compounds targeted to the brain.

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not in lean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associated macrophage activation a homeostatic role to restore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.

Endothelial cells (ECs) from different vascular beds display certain common qualities, but each subtype is uniquely adapted to meet the demands of the underlying tissues. The structural peculiarities of intercellular junctions are, for instance, considered to account for the differences in permeability displayed by various vascular beds: strong occludin expression is unique to cerebral ECs and considered to account for the high electrical resistance and low paracellular permeability of brain microvessels which constitute the blood-brain barrier (BBB). The integrity of the BBB is compromised in many disorders of the human CNS; therapeutic strategies include treatment with glucocorticoids (GCs), which improve barrier properties of the BBB. In contrast, positive effects of GCs on peripheral vascular permeability could not be demonstrated clearly, while side-effects of prolonged GC treatment are considerable. In an effort to elucidate this difference, we analysed the expression of occludin and the glucocorticoid receptor (GR) in BBB and non-BBB (myocardium) endothelial cells. Our results demonstrate complete GR downregulation by GCs in murine non-BBB endothelial cells in vivo, whereas GC administration led to nuclear concentration of GRs in BBB endothelium. In correlation with these in vivo data, the use of cerebral and myocardial endothelial cell lines proved GR downregulation in non-BBB cells in vitro in response to GC treatment. Divergent transactivating activity of GRs in the BBB and non-BBB endothelial cellular context could be demonstrated after transfection of endothelial cells with a model GC-responsive test promoter plasmid in the presence and absence of dexamethasone. Our results thus suggest differential signalling mechanisms involved in endothelial barrier regulation, arguing for the development of tissue-specific drugs for therapeutic applications.