The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.

The design and synthesis of a polyphenylene dendrimer (PPD 3) with discrete binding sites for lipophilic guest molecules and characteristic surface patterns is presented. Its semi-rigidity in combination with a precise positioning of hydrophilic and hydrophobic groups at the periphery yields a refined architecture with lipophilic binding pockets that accommodate defined numbers of biologically relevant guest molecules such as fatty acids or the drug doxorubicin. The size, architecture, and surface textures allow to even penetrate brain endothelial cells that are a major component of the extremely tight blood-brain barrier. In addition, low to no toxicity is observed in in vivo studies using zebrafish embryos. The unique PPD scaffold allows the precise placement of functional groups in a given environment and offers a universal platform for designing drug transporters that closely mimic many features of proteins.

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.

Neurological disorders are undoubtedly among the most alarming diseases humans might face. In treatment of neurological disorders, the blood‐brain barrier (BBB) is a challenging obstacle preventing drug penetration into the brain. Advances in dendrimer chemistry for central nervous system (CNS) treatments are presented here. A poly(amido)amine (PAMAM) dendrimer bioconjugate with a streptavidin adapter for the attachment of dendrons or any biotinylated drug is constructed. In vitro studies on porcine or murine models and in vivo mouse studies are performed and reveal the permeation of dendronized streptavidin (DSA) into the CNS. The bioconjugate is taken up mainly by the caveolae pathway and transported across the BBB via transcytosis escaping from lysosomes. After transcytosis DSA are delivered to astrocytes and neurons. Furthermore, DSA offer high biocompatibility in vitro and in vivo. In summary, a new strategy for implementing therapeutic PAMAM function as well as drug delivery in neuropathology is presented here.

Meprin α and β, zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1β, interleukin 18, or tumor growth factor α. Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin α and a K(i) of 1.1 × 10(-6) M meprin β. This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin α and β inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin α activity was 40 times weaker than that of meprin β activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin α (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin β. Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

The synthesis of hybrid hydrogels by pH‐controlled structural transition with exceptional rheological properties as cellular matrix is reported. “Depsi” peptide sequences are grafted onto a polypeptide backbone that undergo a pH‐induced intramolecular O–N–acyl migration at physiological conditions affording peptide nanofibers (PNFs) as supramolecular gelators. The polypeptide–PNF hydrogels are mechanically remarkably robust. They reveal exciting thixotropic behavior with immediate in situ recovery after exposure to various high strains over long periods and self‐repair of defects by instantaneous reassembly. High cytocompatibility, convenient functionalization by coassembly, and controlled enzymatic degradation but stability in 2D and 3D cell culture as demonstrated by the encapsulation of primary human umbilical vein endothelial cells and neuronal cells open many attractive opportunities for 3D tissue engineering and other biomedical applications.

Nanotheranostics, combining diagnostics and therapy, has the potential to revolutionize treatment of neurological disorders. But one of the major obstacles for treating central nervous system diseases is the blood–brain barrier (BBB) preventing systemic delivery of drugs and optical probes into the brain. To overcome these limitations, nanodiamonds (NDs) are investigated in this study as they are a powerful sensing and imaging platform for various biological applications and possess outstanding stable far‐red fluorescence, do not photobleach, and are highly biocompatible. Herein, fluorescent NDs encapsulated by a customized human serum albumin–based biopolymer (polyethylene glycol) coating (dcHSA‐PEG) are taken up by target brain cells. In vitro BBB models reveal transcytosis and an additional direct cell–cell transport via tunneling nanotubes. Systemic application of dcHSA‐NDs confirms their ability to cross the BBB in a mouse model. Tracking of dcHSA‐NDs is possible at the single cell level and reveals their uptake into neurons and astrocytes in vivo. This study shows for the first time systemic NDs brain delivery and suggests transport mechanisms across the BBB and direct cell–cell transport. Fluorescent NDs are envisioned as traceable transporters for in vivo brain imaging, sensing, and drug delivery.