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

Redox regulation of cell contacts by tricellulin and occludin: redox-sensitive cysteine sites in tricellulin regulate both tri- and bicellular junctions in tissue barriers as shown in hypoxia and ischemia

Tight junctions (TJs) seal paracellular clefts in epithelia/endothelia and form tissue barriers for proper organ function. TJ-associated marvel proteins (TAMPs: tricellulin, occludin, marvelD3) are thought to be regulatory relevant. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. Aim: As pathological disturbances have not been analyzed, the structure and function of human tricellulin, including potentially redox-sensitive Cys-sites, were investigated under reducing/oxidizing conditions at 3- and 2-cell contacts. Results: Ischemia, hypoxia and reductants redistributed tricellulin from 3- to 2-cell contacts. The extracellular loop 2 (ECL2; conserved Cys321, Cys335) trans-oligomerized between three opposing cells. Substitutions of these residues caused bicellular localization. Cys362 in transmembrane domain 4 contributed to bicellular heterophilic cis-interactions along cell membrane with claudin-1 and marvelD3, while Cys395 in the cytosolic C-terminal tail promoted homophilic tricellullar cis-interactions. The Cys-sites included in homo-/heterophilic bi-/tricellular cis-/trans-interactions contributed to cell barrier tightness for small/large molecules. Innovation: Tricellulin forms TJs via trans- and cis-association in 3-cell contacts demonstrated electron and quantified fluorescence microscopically; it tightens 3- and 2-cell contacts. Tricellulin's ECL2 specifically seals 3-cell contacts redox-dependently; a structural model is proposed. Conclusions: TAMPs' ECL2 and claudins' ECL1 share functionally and structurally similar features involved in homo-/heterophilic tightening of cell-cell contacts. Tricellulin is a specific redox-sensor and sealing element at 3-cell contacts and may compensate as redox-mediator for occludin loss at 2-cell contacts in vivo and in vitro. Molecular interaction mechanisms were proposed that contribute to tricellulin's function. In conclusion, tricellulin is a junctional redox-regulator for ischemia-related alterations

Protective effects of peroxiredoxin-1 at the injured blood-brain barrier.

Contains fulltext : 71338.pdf (publisher's version ) (Closed access)Reactive oxygen species (ROS) play a pivotal role in the development of neuroinflammatory disorders, such as multiple sclerosis (MS). Here, we studied the effect of ROS on protein expression in brain endothelial cells (BECs) using proteomic techniques and show that long-term exposure to ROS induces adaptive responses in BECs to counteract an oxidative attack. ROS induce differential protein expression in BECs, among which is peroxiredoxin-1 (Prx1). To further study the role of Prx1 we established a BEC line overexpressing Prx1. Our data indicate that Prx-1 overexpression protects BECs from ROS-induced cell death, reduces adhesion and subsequent transendothelial migration of monocytes by decreasing intercellular adhesion molecule-1 expression, and enhances the integrity of the BEC layer. Interestingly, vascular Prx1 immunoreactivity was markedly upregulated in inflammatory lesions of experimental autoimmune encephalomyelitis (EAE) animals and active demyelinating MS lesions. These findings indicate that enhanced vascular Prx1 expression may reflect the occurrence of vascular oxidative stress in EAE and MS. On the other hand, it may function as an endogenous defense mechanism to inhibit leukocyte infiltration and counteract ROS-induced cellular injury

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