Carola Förster

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different 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 scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.

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.

Occludin as direct target for glucocorticoid-induced improvement of blood–brain barrier properties in a murine in vitro system

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 Ωcm2. 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.

In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

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.

Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific

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.

Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND

In many neuroinflammatory conditions, including multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia, the matrix metalloproteinases (MMPs) play an important role in disrupting the blood–brain barrier (BBB). Normally under tight regulation, increased MMP‐9 cerebrospinal fluid levels and excessive proteolytic activity is detected in the blood and cerebrospinal fluid in patients with acute MS. MMP‐9 is a member of the type IV collagenases, which attack components of the endothelial basal lamina, including type IV collagen. The disruption of the BBB and clinical symptoms can be reduced with different inhibitors to MMPs including activators of tissue inhibitor of metalloproteinases‐1 (TIMP‐1), the cognate tissue inhibitor of MMP‐9. Since intravenous glucocorticoid (GC) treatment reduces the levels of MMP‐9 markedly in patients, we hypothesized that GC effects might be mediated by transcriptional activation of the TIMP‐1 gene in addition to reported repressive effects on MMP‐9 transcription. Our results provide direct evidence that GCs increase TIMP‐1 in the brain endothelial cell line cEND, prevent alterations in microvascular integrin α1 subunit expression and help maintain endothelial barrier function in response to pro‐inflammatory stimuli (TNFα administration). GC‐induced up‐regulation of TIMP‐1 expression by the CNS vascular endo‐thelium may thus play a role in preservation of the endothelial basal lamina and maintain integrin α1 and tight junction protein expression important for vessel wall integrity.

Claudin-5 as a Novel Estrogen Target in Vascular Endothelium

Objective—Estrogens have multiple effects on vascular physiology and function. In the present study, we look for direct estrogen target genes within junctional proteins. Methods and Results—We use murine endothelial cell lines of brain and heart origin, which express both subtypes of estrogen receptor, ER&agr; and ER&bgr;. Treatment of these cells with 17&bgr;-estradiol (E2) led to an increase in transendothelial electric resistance and a most prominent upregulation of the tight junction protein claudin-5 expression. A significant increase of claudin-5 promoter activity, mRNA, and protein levels was detected in cells from both vascular beds. In protein lysates and in immunoreactions on brain sections from ovariectomized E2-treated mice, we noticed an increase in claudin-5 protein and mRNA content. Treatment of cells with a specific ER&bgr; agonist, diarylpropionitrile, revealed the same effect as E2 stimulation. Moreover, we detected significantly lower claudin-5 mRNA and protein content in ER&bgr; knockout mice. Conclusions—We describe claudin-5 as a novel estrogen target in vascular endothelium and show in vivo (brain endothelium) and in vitro (brain and heart endothelium) effects of estrogen on claudin-5 levels. The estrogen-induced increase in junctional protein levels may lead to an improvement in vascular structural integrity and barrier function of vascular endothelium.

Differential susceptibility of cerebral and cerebellar murine brain microvascular endothelial cells to loss of barrier properties in response to inflammatory stimuli.

Multiple sclerosis (MS) is a chronic autoimmune disease whose symptoms are caused by an inflammatory invasion of the central nervous system (CNS). The molecular pathogenesis of MS includes an increased permeability of the blood brain barrier (BBB) along with an inability of the BBB to fulfill its normal function of protecting the CNS. The cerebellar BBB seems to be especially vulnerable, as the development of experimental autoimmune encephalomyelitis (EAE) as an animal model of MS often takes its beginning in the cerebellum. Inflammatory lesion development seems to correlate with increased permeability of the local BBB. Responsible for the BBB are cerebral and cerebellar capillary endothelial cells. We therefore generated an in vitro model of the cerebellar BBB (cerebEND) and compared its response to inflammatory stimuli (TNFalpha administration) with a cerebral BBB in vitro model (cEND) characterised previously [Förster, C., Silwedel, C., Golenhofen, N., Burek, M., Kietz, S., Mankertz, J., Drenckhahn, D., 2005. Occludin as direct target for glucocorticoid-induced improvement of blood brain-barrier properties in a murine in vitro system. J. Physiol. 565(Pt 2), 475-486]. We could demonstrate a faster and more pronounced increase in permeability in the cerebellar BBB manifested by reduced transendothelial electrical resistance and reduced tight junction protein expression. This cell line cerebEND could thus be valuable to identify genes differently expressed within the BBB in the future and therefore be helpful in finding new ways of treatment of MS.

Glucocorticoid Insensitivity at the Hypoxic Blood–Brain Barrier Can Be Reversed by Inhibition of the Proteasome

Background and Purpose— Glucocorticoids potently stabilize the blood–brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffective in patients with acute ischemic stroke. The reasons for this discrepancy are unresolved. Methods— To address the molecular basis for the paradox unresponsiveness of the blood–brain barrier during hypoxia, we used murine brain microvascular endothelial cells exposed to O2/glucose deprivation as an in vitro model. In an in vivo approach, mice were subjected to transient middle cerebral artery occlusion to induce brain infarctions. Blood–brain barrier damage and edema formation were chosen as surrogate markers of glucocorticoid sensitivity in the presence or absence of proteasome inhibitors. Results— O2/glucose deprivation reduced the expression of tight junction proteins and transendothelial resistance in murine brain microvascular endothelial cells in vitro. Dexamethasone treatment failed to reverse these effects during hypoxia. Proteasome-dependent degradation of the glucocorticoid receptor impaired glucocorticoid receptor transactivation thereby preventing physiological glucocorticoid activity. Inhibition of the proteasome, however, fully restored the blood–brain barrier stabilizing properties of glucocorticoid during O2/glucose deprivation. Importantly, mice treated with the proteasome inhibitor Bortezomib in combination with steroids several hours after stroke developed significantly less brain edema and functional deficits, whereas respective monotherapies were ineffective. Conclusions— We for the first time show that inhibition of the proteasome can overcome glucocorticoid resistance at the hypoxic blood–brain barrier. Hence, combined treatment strategies may help to combat stroke-induced brain edema formation in the future and prevent secondary clinical deterioration.

Glucocorticoids increase VE-cadherin expression and cause cytoskeletal rearrangements in murine brain endothelial cEND cells

Recent studies have shown the influence of glucocorticoids on the expression of the tight junction protein occludin in the brain capillary endothelial cell line cEND, contributing to improvement in endothelial barrier functions. In this study, we investigated glucocorticoid effects on the expression of the adherens junction proteins VE- (vascular-endothelial) cadherin, α-catenin and β-catenin as well as that of ZO-1, the plaque protein shared by both adherens and tight junctions on stimulation with dexamethasone. We were able to show a positive influence of dexamethasone administration on VE-cadherin protein levels as well as a rearrangement of VE-cadherin protein to the cytoskeleton after dexamethasone treatment. Investigation of transcriptional activation of the VE-cadherin promoter by dexamethasone, however, did not point to direct glucocorticoid-mediated VE-cadherin gene induction but rather suggested indirect steroid effects leading to increased VE-cadherin protein synthesis. Dexamethasone was further shown to induce cellular differentiation into a cobblestone cellular morphology and reinforcement of adherens junctions concomitant with the increased anchorage of VE-cadherin to the actin cytoskeleton. We thus propose that glucocorticoid effects on VE-cadherin protein synthesis and organization are important for the formation of both adherens and tight junction, and for improved barrier properties in microvascular brain endothelial cells.