Carola Förster

Publications of Carola Förster

• Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome.

  Authors: Christoph Kleinschnitz, Kinga Blecharz, Timo Kahles, Tobias Schwarz, Peter Kraft, Kerstin Göbel, Sven G Meuth, Malgorzata Burek, Thomas Thum, Guido Stoll, Carola Förster

  Stroke; a journal of cerebral circulation. 02/2011; 42(4):1081-9.

  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 ineffectiveGlucocorticoids 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. 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 O(2)/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. O(2)/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 O(2)/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. 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 regulate the human occludin gene through a single imperfect palindromic glucocorticoid response element.

  Authors: Nina Harke, Jörg Leers, Silke Kietz, Detlev Drenckhahn, Carola Förster

  Molecular and cellular endocrinology. 09/2008;

  The 65kDa protein occludin is an essential element of the blood-brain barrier. This integral membrane protein represents an important part of the tight junctions, which seal and protect the bloodThe 65kDa protein occludin is an essential element of the blood-brain barrier. This integral membrane protein represents an important part of the tight junctions, which seal and protect the blood brain barrier against paracellular diffusion of solutes to the brain parenchyme and are therefore responsible for the high resistance and low permeability between cerebral capillary endothelial cells. However, the molecular basis for the regulation of occludin gene expression is only incompletely understood. In former projects we showed that treatment of a brain microvascular cell line, cEND, with glucocorticoids resulted in increased occludin expression in cell-cell-contacts [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]. Induction of occludin expression by glucocorticoids was shown to be dependent on the glucocorticoid receptor. This study aims to identify the underlying molecular mechanism of gene expression and to identify potential glucocorticoid receptor binding sites within the occludin promoter, the glucocorticoid response elements. We identified one candidate glucocorticoid response element within the distal part of the occludin promoter that differs from the consensus glucocorticoid response element by the presence of a 4-basepair instead of a 3-basepair spacer between two highly degenerate halfsites (5'-ACATGTGTTTACAAAT-3'). Chromatin immunoprecipitation assay and site-directed mutagenesis confirmed binding of the glucocorticoid receptor to this site. The need for glucocorticoid receptor dimerization to induce gene expression was further confirmed by transfection studies using wild type and glucocorticoid receptor dimerization-deficient expression vectors, indicating that transactivation of occludin occurs through the glucocorticoid response element (GRE).

• Tight junctions and the modulation of barrier function in disease.

  Authors: Carola Förster

  Histochemistry and cell biology. 08/2008; 130(1):55-70.

  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 theTight 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 TNFalpha on tight junction proteins in an in vitro model of the human blood-brain barrier.

  Authors: Carola Förster, Malgorzata Burek, Ignacio A Romero, Babette Weksler, Pierre-Olivier Couraud, Detlev Drenckhahn

  The Journal of physiology. 05/2008; 586(7):1937-49.

  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. ManyHomeostasis 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 (TNFalpha 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.

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

  Authors: Carola Förster, Timo Kahles, Silke Kietz, Detlev Drenckhahn

  The Journal of physiology. 06/2007; 580(Pt.3):937-49.

  In many neuroinflammatory conditions, including multiple sclerosis (MS), encephalitis, meningitis, brain tumours and cerebral ischaemia, the matrix metalloproteinases (MMPs) play an important role inIn 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 alpha1 subunit expression and help maintain endothelial barrier function in response to pro-inflammatory stimuli (TNFalpha 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 alpha1 and tight junction protein expression important for vessel wall integrity.

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

  Authors: Christine Silwedel, Carola Förster

  Journal of neuroimmunology. 11/2006; 179(1-2):37-45.

  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 anMultiple 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.

• DAX-1 expression is regulated during mammary epithelial cell differentiation.

  Authors: Luisa A Helguero, Malin Hedengran Faulds, Carola Förster, Jan-Ake Gustafsson, Lars-Arne Haldosén

  Endocrinology. 08/2006; 147(7):3249-59.

  In recent studies, we have found that DAX-1 (dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome) is expressed in the mouse mammary epithelial cell lineIn recent studies, we have found that DAX-1 (dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome) is expressed in the mouse mammary epithelial cell line HC11. In this study, we focused on the regulation of DAX-1 expression and subcellular localization throughout mouse mammary epithelial cell differentiation and its hormonal regulation in the mouse mammary gland. Proliferating HC11 cells grown in epidermal growth factor (EGF)-containing medium, expressed very low levels of DAX-1 as detected by Western blotting and quantitative real-time PCR, whereas, upon EGF withdrawal and induction of differentiation, DAX-1 expression increased. Inhibition of MAPK pathway with PD 098059 resulted in increased DAX-1 levels even in the presence of EGF. Using confocal microscopy, we showed that DAX-1 cytoplasmic levels increased as cells differentiated. DAX-1 staining was nuclear in luminal cells of mouse mammary glands from 3-month-old virgin mice. A nucleo-cytoplasmic pattern was observed in pseudopregnant mice and a cytoplasmic pattern was found in mammary glands from 6-d lactating mice. The influence of DAX-1 on transcriptional activity of endogenously expressed estrogen receptors alpha (ERalpha) and beta (ERbeta) in HC11 mammary epithelial cells was evaluated with an estrogen response element-luciferase reporter assay and by quantitative real-time PCR of the ER-regulated gene receptor-interacting protein 140 kDa. Cotransfection of HC11 cells with human DAX-1 inhibited estrogen response element-reporter and receptor-interacting protein 140 kDa expression induced by 17beta-estradiol, the ERalpha-selective agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, or the ERbeta-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. In summary, DAX-1 expression increased upon differentiation induced by EGF withdrawal, and DAX-1 decreased response to estrogens in HC11 cells. Further studies are needed to determine whether DAX-1 is also important in regulation of differentiation of HC11 cells.

• Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific.

  Authors: Carola Förster, Jens Waschke, Malgorzata Burek, Jörg Leers, Detlev Drenckhahn

  The Journal of physiology. 07/2006; 573(Pt 2):413-25.

  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 peculiaritiesEndothelial 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.

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

  Authors: Carola Förster, Christine Silwedel, Nikola Golenhofen, Malgorzata Burek, Silke Kietz, Joachim Mankertz, Detlev Drenckhahn

  The Journal of physiology. 07/2005; 565(Pt 2):475-86.

  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 moleculesHomeostasis 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 Omegacm2. 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.

• Characterization of the ERbeta-/-mouse heart.

  Authors: Carola Förster, Silke Kietz, Kjell Hultenby, Margaret Warner, Jan-Ake Gustafsson

  Proceedings of the National Academy of Sciences of the United States of America. 10/2004; 101(39):14234-9.

  Although the heart responds to estrogen, it is not clear whether estrogen acts directly on heart muscle or indirectly by means of the vascular, immune, or nervous system. No role for estrogenAlthough the heart responds to estrogen, it is not clear whether estrogen acts directly on heart muscle or indirectly by means of the vascular, immune, or nervous system. No role for estrogen receptor (ER) beta in the heart has been established, but ERbeta(-/-) mice are hypertensive, and as they age, their hearts become enlarged. Histological and ultrastructural analysis of the heart revealed a disarray of myocytes, a disruption of intercalated discs, an increase in the number and size of gap junctions, and a profound alteration in nuclear structure, concomitantly with a loss of expression of lamin A/C from the nuclear envelope. In the lungs of ERbeta(-/-) mice, lamin A/C was located in the nuclear membrane, indicating that lamin A/C is not an ERbeta-regulated gene. Immunohistochemical studies with ERbeta antibodies failed to detect ERbeta in the myocardium. We conclude that abnormalities in heart morphology in ERbeta(-/-) mice are likely due to stress on the nuclear envelope as a result of the chronic sustained systolic and diastolic hypertension observed in ERbeta(-/-) mice. Because neither ERalpha nor ERbeta could be detected in heart muscle, the effects of estrogen on the myocardium seem to be indirect.

• Involvement of estrogen receptor beta in terminal differentiation of mammary gland epithelium.

  Authors: Carola Förster, Sari Mäkela, Anni Wärri, Silke Kietz, David Becker, Kjell Hultenby, Margaret Warner, Jan-Ake Gustafsson

  Proceedings of the National Academy of Sciences of the United States of America. 12/2002; 99(24):15578-83.

  The mammary glands of prepubertal estrogen receptor (ER)beta-- mice are morphologically indistinguishable from those of WT littermates. It appears that, although ERbeta is expressed in the mouseThe mammary glands of prepubertal estrogen receptor (ER)beta-- mice are morphologically indistinguishable from those of WT littermates. It appears that, although ERbeta 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 ERbeta has a role in the differentiated function of the mammary gland. Pregnancy is rare in ERbeta-- mice, but an intensive breeding program produced seven pregnant ERbeta-- 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 ERbeta-- 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 alpha2 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 ERbeta-- but not in WT mice. These findings suggest a role for ERbeta 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, ERbeta could contribute to this risk reduction by facilitating terminal differentiation of the mammary gland.