Ignacio A. Romero

Blood-brain barrier-specific properties of a human adult brain endothelial cell line

Establishment of a human model of the blood‐brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact‐inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up‐regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood‐brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood‐brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well‐differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Transporting therapeutics across the blood-brain barrier

In 1996, we are half-way through the Decade of the Brain, yet we still have few effective treatments for major disorders of the central nervous system. These include affective disorders, epilepsy, neurodegenerative disorders, brain tumours, infections and HIV encephalopathy; sufferers far outnumber the morbidity of cancer or heart disease. Increased understanding of the pharmacology of the brain and its blood supply, and methods for rational drug design, are leading to potential new drug therapies based on highly specific actions on particular target sites, such as neurotransmitter receptors and uptake systems. These methods are capable of reducing the side effects that are common with more general treatments. However, all these treatments and potential treatments meet a formidable obstacle--the blood-brain barrier. In this article, we review the properties of this barrier that complicate drug delivery to the brain, and some of the most hopeful strategies for overcoming or bypassing the barrier in humans.

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling

The blood‐brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskel‐eton rearrangements and redistribution and disappearance of TJ proteins claudin‐5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS‐induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/ Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.—Schreibelt, G., Kooij, G., Reijerkerk, A., van Doorn, R., Gringhuis, S. I., van der Pol, S., Weksler, B. B., Romero, I. A., Couraud, P.‐O., Piontek, J., Blasig, I. E., Dijkstra, C. D., Ronken, E., de Vries, H. E. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase and PKB signaling. FASEB J. 21, 3666–3676 (2007)

The hCMEC/D3 cell line as a model of the human blood brain barrier

Since the first attempts in the 1970s to isolate cerebral microvessel endothelial cells (CECs) in order to model the blood–brain barrier (BBB) in vitro, the need for a human BBB model that closely mimics the in vivo phenotype and is reproducible and easy to grow, has been widely recognized by cerebrovascular researchers in both academia and industry. While primary human CECs would ideally be the model of choice, the paucity of available fresh human cerebral tissue makes wide-scale studies impractical. The brain microvascular endothelial cell line hCMEC/D3 represents one such model of the human BBB that can be easily grown and is amenable to cellular and molecular studies on pathological and drug transport mechanisms with relevance to the central nervous system (CNS). Indeed, since the development of this cell line in 2005 over 100 studies on different aspects of cerebral endothelial biology and pharmacology have been published. Here we review the suitability of this cell line as a human BBB model for pathogenic and drug transport studies and we critically consider its advantages and limitations.

Differential effects of hydrocortisone and TNFα on tight junction proteins in an in vitro model of the human blood-brain barrier

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 human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies

The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood‐brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para‐ or transcellular permeabilities (Pe) of inulin (0.74 × 10−3 cm/min), sucrose (1.60 × 10−3 cm/min), lucifer yellow (1.33 × 10−3 cm/min), morphine (5.36 × 10−3 cm/min), propranolol (4.49 × 10−3 cm/min) and midazolam (5.13 × 10−3 cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1–5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1‐mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p‐glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood‐brain barrier.

Secretion of interleukin‐1β by astrocytes mediates endothelin‐1 and tumour necrosis factor‐α effects on human brain microvascular endothelial cell permeability

Evidence suggests that endothelin‐1 (ET‐1) plays an essential role in brain inflammation. However, whether ET‐1 contributes directly to blood–brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co‐cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET‐1 by BMVECs upon stimulation with tumour necrosis factor (TNF)‐α, which plays an essential role in the induction and synthesis of ET‐1 during systemic inflammatory responses. Increased ET‐1 mRNA was detected in the human BMVECs 24 h after TNF‐α treatment. This was correlated with an increase in ET‐1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF‐α and ET‐1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF‐α was partially prevented by antibody neutralization of ET‐1 and completely by monoclonal antibody against IL‐1β. Concomitantly, TNF‐α induced IL‐1β mRNA expression by astrocytes in co‐culture and this effect was partially prevented by ET‐1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET‐1 for 24 h induced IL‐1β mRNA synthesis and IL‐1β protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET‐1, TNF‐α and IL‐1β between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.

Regulation of chemokine receptor expression in human microglia and astrocytes

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.

Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from the blood to the extracellular fluid environment of the brain. An immortalised rat brain endothelial cell line (GPNT) was used to investigate the mechanisms underlying dexamethasone-induced decrease in paracellular permeability. Following treatment with 1 microM dexamethasone there was a decrease in transmonolayer paracellular permeability mainly to sucrose, fluorescein and dextrans of up to 20 KDa. According to pore theory, these differences in permeability were consistent with a decrease in the number of pores between brain endothelial cells. This effect was accompanied by a concentration of filamentous actin and cortactin to the cell periphery. Concomitantly, the continuity of the tight junctional protein ZO-1 at the cell borders was improved and was associated with an increase in both ZO-1 and occludin expression. By contrast, the expression and distribution of adherens junctional proteins such as beta-catenin and p100/p120 remained unchanged. These observations suggest that glucocorticoids induce a more differentiated BBB phenotype in cultured brain endothelial cells through modification of tight junction structure.