Malgorzata Burek

Publications of Malgorzata Burek

• Addition of NMDA-receptor antagonist MK801 during oxygen/glucose deprivation moderately attenuates the upregulation of glucose uptake after subsequent reoxygenation in brain endothelial cells.

  Authors: Winfried Neuhaus, Malgorzata Burek, Cholpon S Djuzenova, Serge C Thal, Hermann Koepsell, Norbert Roewer, Carola Y Förster

  Neuroscience letters. 01/2012; 506(1):44-9.

  During stroke the blood-brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose toDuring stroke the blood-brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7-fold after 6h OGD, which was significantly reduced by 10μM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after 6h OGD and was still higher (210%) after the 20h reoxygenation phase compared to normoxia. Ten micromolar MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.

• 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.

• Claudin-5 as a Novel Estrogen Target in Vascular Endothelium.

  Authors: Malgorzata Burek, Paula A Arias-Loza, Norbert Roewer, Carola Y Förster

  Arteriosclerosis, thrombosis, and vascular biology. 11/2009;

  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 useOBJECTIVE: 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, ERalpha and ERbeta. Treatment of these cells with 17beta-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 ERbeta agonist, diarylpropionitrile, revealed the same effect as E2 stimulation. Moreover, we detected significantly lower claudin-5 mRNA and protein content in ERbeta 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.

• Cloning and characterization of the murine claudin-5 promoter.

  Authors: Malgorzata Burek, Carola Y Förster

  Molecular and cellular endocrinology. 11/2008;

  Claudin-5, an integral tight junction protein component, plays a critical role in permeability of the endothelial cell barrier. Recently, we have shown that claudin-5 protein is down-regulated by theClaudin-5, an integral tight junction protein component, plays a critical role in permeability of the endothelial cell barrier. Recently, we have shown that claudin-5 protein is down-regulated by the proinflammatory cytokine TNF alpha and its levels restored by dexamethasone treatment. In order to investigate the regulation of claudin-5 at the transcriptional level, we have cloned the murine claudin-5 promoter. The claudin-5 promoter sequence (1131bp) showed no consensus TATA-box. We identified putative transcription factor binding sites, including six full and two half sites degenerated glucocorticoid-response elements (GREs), two NFkappaB, three Sp1, one Sp2, one Ap2, as well as three E-boxes. Serially deleted promoter constructs showed high basal activity. TNF alpha significantly reduced the promoter activity and mRNA levels of claudin-5 in brain cEND and myocardial MyEND endothelial cells. Dexamethasone treatment led to a significant increase of the murine claudin-5 promoter activity and mRNA levels in cEND cells. However, no claudin-5 induction could be observed in MyEND cells in response to dexamethasone. Our studies suggest tissue-specific regulation of the claudin-5 gene via glucocorticoids and a high vulnerability of claudin-5 to TNF alpha. This could be an important mechanism in diseases accompanied by the release of proinflammatory cytokines, for example in patients with chronic heart failure or multiple sclerosis.

• 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.

• 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.