M01 as a novel drug enhancer for specifically targeting the blood-brain barrier

Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain

Nitronyl nitroxides, a novel group of protective agents against oxidative stress in endothelial cells forming the blood-brain barrier

Nitronyl nitroxides (NN) effectively decompose free radicals (Haseloff, Zoellner, Kirilijuk, Grigoriev, Reszka, Bernhardt, Mertsch, Roloff and Blasig, 1997a Free Radical Research 26, 7-17). As brain endothelium, forming the blood-brain barrier (BBB), is both the main source and the target of reactive species during cerebral oxidative stress, we studied the effect of NN on brain endothelial cells injured by the mediator of oxidative stress H2O2 (Kondo, Kinouchi, Kawase and Yoshimoto, 1996. Neuroscience Letters 215, 103-106). H2O2 caused hydroxyl radical generation, lipid peroxidation, membrane dysfunction, membrane leak and cell death, concentration dependently. Due to 0.5 mM H2O2, oxy-radical-induced membrane phospholipid peroxidation (malondialdehyde) increased to 0.61±0.04 nmol/mg protein vs control (0.32±0.03, p<0.05), cells lost cytosolic proteins into the medium and viability decreased to 28±2% of control (p<0.05). Permeability through the endothelial monolayer (measure for the tightness of the BBB) rose to 250±40% after 0.15 mM H2O2 (p<0.001). Addition of 10 μM of the NN 5,5-dimethyl-2,4-diphenyl-4-methoxy-2-imidazoline-3-oxide-1-oxyl (NN-2), 1 mM phenylbutyl nitrone (PBN), or 10 μM of the lazaroid U83836E improved cell viability during incubation with 0.5 mM H2O2 to 57±1%, 49±2%, and 42±3% (p<0.05, vs drug-free H2O2 group). The permeability enhancement by 0.15 mM H2O2 was reduced to 171±21%, 170±25%, and 118±32% (p<0.05 vs drug-free H2O2 group). Generally, the assumption is supported that during cerebral oxidative stress the protection should also be directed to the cells of the BBB, which can be provided by antioxidative approaches. NN represent a new group of antioxdatively acting cytoprotectiva improving the survival and function of the endothelium against oxidative stress

Calcium/Calmodulin-dependent protein kinase IIdelta2 and gamma insoforms regulate potassium currents of rat brain capillary endothelial cells under hypoxic conditions

Endothelial K+ and Ca2+ homeostasis plays an important role in the regulation of tissue supply and metabolism under normal and pathological conditions. However, the exact molecular mechanism of how Ca2+ is involved in the regulation of K+ homeostasis in capillary endothelial cells, especially under oxidative stress, is not clear. To reveal Ca2+-triggered pathways, which modulate K+ homeostasis, Ca2+/calmodulin-dependent protein kinase II and voltage-gated outward K+ currents were studied in rat brain capillary endothelial cells under hypoxia. Whole cell voltage-clamp measurements showed voltage-gated outward K+ current with transient and sustained components. mRNA and protein of Ca2+/calmodulin-dependent protein kinase II {delta}2 and two {gamma} isoenzymes were identified. Activation of the isoforms (autophosphorylation) was typically achieved by the Ca2+ ionophore ionomycin, which was prevented by the Ca2+/calmodulin-dependent protein kinase II-specific inhibitor KN-93. Hypoxia resulted in autophosphorylation of the {delta}2 and {gamma}B isoforms, augmented the current amplitude, increased the inactivation time constant, and decreased the extent of inactivation of the transient current. KN-93 prevented both the activation of the isoforms and the alterations in the K+ current characteristics. It is concluded that the activation of Ca2+/calmodulin-dependent protein kinase II decreases inactivation of the voltage-gated outward K+ current, thereby counteracting depolarization of the hypoxic endothelium

Protein kinase C regulates the phosphorylation and cellular localization of occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions

Entwicklung von in vitro-Methoden als Ersatz von Tierversuchen in der praeklinischen Schlaganfallforschung. Teilprojekt 2: In vitro-Modell der Blut-Hirnschranke Abschlussbericht

The blood-brain barrier (BBB) formed by brain capillary endothelial cells (BEC) under the influence of astrocytes (AC) has been neglected in the treatment of blood flow disturbances of the brain, such as ischemia and related injury. On the other hand, these cells can be reached easily by systemic administration of a drug. Among others (i.e., accumulation of cellular Ca&quot;2&quot;+, energy deficiency), reactive oxygen species play an important pathogenic role under ischemic conditions. Therefore, a test hierarchy was developed based on BEC, AC and cocultures of both, which allows to characterized a potentially protective agent with respect to: radical scavenger activity, cytotoxicity, cytoprotectivity against oxidative (hypoxic) stress, and protectivity on the tightness of a cell culture model of the BBB under hypoxic conditions. Moreover, the permeability of test compounds through the BBB model can be determined to predict the permeation of substances into the brain. This test system was validated using neuroprotective agents of different classes and various radical scavengers as research standards. The research strategy described here is expected to partly replace animal experiments in the development of novel neuroprotective drugs. (orig.)SIGLEAvailable from TIB Hannover: F00B620 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

Involvement of claudins in zebrafish brain ventricle morphogenesis

Zebrafish brain ventricle morphogenesis involves an initial circulation-independent opening followed by a blood flow- and circulation-dependent expansion process. Zebrafish claudin-5a is required for the establishment of a neuroepithelial-ventricular barrier, which maintains the hydrostatic pressure within the ventricular cavity, thereby contributing to brain ventricle opening and expansion. In mammalia, several claudin family members, including claudin-3 and claudin-5, are expressed within microvessel endothelial cells of the blood-brain barrier. Whether zebrafish brain ventricle morphogenesis provides a model for studying these claudins during early embryonic development was unknown. This review focuses on the expression and function of these zebrafish claudins during brain ventricle morphogenesis

Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats

Eighteen month old spontaneously hypertensive rats (SHR-rats) showed myocardial dysfunction and autoantibodies directed against the {beta} 1-adrenoceptor similarly as known in human dilated cardiomyopathy or Chagas' disease. The agonist-like antibodies were able to activate the {beta} 1,-adrenoceptor mediated signal transduction cascade in cultured rat cardiomyocytes and induced a long-lasting stimulatory effect resulting in a harmful adrenergic overdrive. The antibodies recognized an epitope of the second extracellular loop of the {beta} 1-adrenoceptor identical to that epitope identified in Chagas' disease. In conclusion, our assumption is supported that old SHR-rat are an useful animal model for investigating the role of anti-{beta} 1-adrenoceptor antibodies in the induction of human cardiomyopath

Tight junction proteins at the blood-brain barrier: far more than claudin-5

At the blood-brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions