Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Shifting from ependyma to choroid plexus epithelium and the changing expressions of aquaporin-1 and aquaporin-4.

The cells of the choroid plexus (CP) epithelium are specialized ependymal cells (ECs) but have distinct properties. The CP cells and ECs form single-cell sheets contiguous to each other at a transitional zone. The CP is underlined by a basal lamina and has barrier properties, whereas the ECs do not. The basal lamina of the CP is continuous with the glia limitans superficialis and, consequently, the CP stroma is continuous with the meninges along entering blood vessels. The CP has previously been reported to express aquaporin-1 (AQP1) mostly apically, and ECs show mostly basolateral aquaporin-4 (AQP4) expression. Recent evidence in various systems has shown that in changing conditions the expression and distribution of AQP4 can be modified, involving phosphorylation and calmodulin-triggered translocation. Studies on the human CP revealed that AQP4 is also expressed in some CP cells, which is likely to be increased during ageing based on mouse data. Moreover, subependymal astrocytic processes in the ependyma-CP transition, forming a glial plate around blood vessels and facing the CP stroma, were strongly positive for AQP4. We propose that the increased AQP4 expression might be a compensatory mechanism for the observed reduction in CSF production in the ageing human brain. The high AQP4 density in the transition zone might facilitate the transport of water into and out of the CP stroma and serve as a drainage and clearing pathway for metabolites in the CNS.

Aquaporin-4 expression in the human choroid plexus.

The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

Evidence of SARS-CoV2 Entry Protein ACE2 in the Human Nose and Olfactory Bulb.

Usually, pandemic COVID-19 disease, caused by SARS-CoV2, presents with mild respiratory symptoms such as fever, cough, but frequently also with anosmia and neurological symptoms. Virus-cell fusion is mediated by angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) with their organ expression pattern determining viral tropism. Clinical presentation suggests rapid viral dissemination to the central nervous system leading frequently to severe symptoms including viral meningitis. Here, we provide a comprehensive expression landscape of ACE2 and TMPRSS2 proteins across human postmortem nasal and olfactory tissue. Sagittal sections through the human nose complemented with immunolabelling of respective cell types represent different anatomically defined regions including olfactory epithelium, respiratory epithelium of the nasal conchae and the paranasal sinuses along with the hardly accessible human olfactory bulb. ACE2 can be detected in the olfactory epithelium as well as in the respiratory epithelium of the nasal septum, the nasal conchae, and the paranasal sinuses. ACE2 is located in the sustentacular cells and in the glandular cells in the olfactory epithelium as well as in the basal cells, glandular cells, and epithelial cells of the respiratory epithelium. Intriguingly, ACE2 is not expressed in mature or immature olfactory receptor neurons and basal cells in the olfactory epithelium. Similarly, ACE2 is not localized in the olfactory receptor neurons albeit the olfactory bulb is positive. Vice versa, TMPRSS2 can also be detected in the sustentacular cells and the glandular cells of the olfactory epithelium. Our findings provide the basic anatomical evidence for the expression of ACE2 and TMPRSS2 in the human nose, olfactory epithelium, and olfactory bulb. Thus, they are substantial for future studies that aim to elucidate the symptom of SARS-CoV2 induced anosmia via the olfactory pathway.

Bacterial Cellulose Shifts Transcriptome and Proteome of Cultured Endothelial Cells Towards Native Differentiation.

Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K-means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular microenvironment to regulate cellular differentiation, and demonstrates, for the first time, the potential of Xellulin as versatile tool promoting an in vivo-like phenotype in primary and propagated cell culture.

DJ-1/Park7 Sensitive Na+ /H+ Exchanger 1 (NHE1) in CD4+ T Cells.

DJ-1/Park7 is a redox-sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ-1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+ /H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study, we explored whether DJ-1 is expressed in CD4+ T cells, and affects ROS production as well as NHE1 in those cells. To this end, DJ-1 and NHE1 transcript, and protein levels were quantified by qRT-PCR and Western blotting, respectively, intracellular pH (pHi ) utilizing bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ-1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ-1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N-acetyl-cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ-1 deficient CD4+ T cells, and blunted the difference between DJ-1-/- and DJ-1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up-regulation of NHE1 in DJ-1-/- CD4+ T cells. In conclusion, DJ-1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. J. Cell. Physiol. 232: 3050-3059, 2017. © 2016 Wiley Periodicals, Inc.

Micafungin-Induced Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The antifungal drug Micafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. Side effects of Micafungin treatment include microangiopathic hemolytic anemia and thrombocytopenia with microvascular thrombosis. The development of thrombosis may be fostered by stimulation of eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. The present study explored, whether Micafungin induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Micafungin (10 - 25 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Micafungin (25 µg/ml) did not significantly modify Fluo3-fluorescence, DCFDA fluorescence, or ceramide abundance. The effect of Micafungin on annexin-V-binding was not significantly modified by removal of extracellular Ca2+, by PKC inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). CONCLUSIONS: Micafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Aquaporin-4 in Astroglial Cells in the CNS and Supporting Cells of Sensory Organs-A Comparative Perspective.

The main water channel of the brain, aquaporin-4 (AQP4), is one of the classical water-specific aquaporins. It is expressed in many epithelial tissues in the basolateral membrane domain. It is present in the membranes of supporting cells in most sensory organs in a specifically adapted pattern: in the supporting cells of the olfactory mucosa, AQP4 occurs along the basolateral aspects, in mammalian retinal Müller cells it is highly polarized. In the cochlear epithelium of the inner ear, it is expressed basolaterally in some cells but strictly basally in others. Within the central nervous system, aquaporin-4 (AQP4) is expressed by cells of the astroglial family, more specifically, by astrocytes and ependymal cells. In the mammalian brain, AQP4 is located in high density in the membranes of astrocytic endfeet facing the pial surface and surrounding blood vessels. At these locations, AQP4 plays a role in the maintenance of ionic homeostasis and volume regulation. This highly polarized expression has not been observed in the brain of fish where astroglial cells have long processes and occur mostly as radial glial cells. In the brain of the zebrafish, AQP4 immunoreactivity is found along the radial extent of astroglial cells. This suggests that the polarized expression of AQP4 was not present at all stages of evolution. Thus, a polarized expression of AQP4 as part of a control mechanism for a stable ionic environment and water balanced occurred at several locations in supporting and glial cells during evolution. This initially basolateral membrane localization of AQP4 is shifted to highly polarized expression in astrocytic endfeet in the mammalian brain and serves as a part of the neurovascular unit to efficiently maintain homeostasis.

Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells.

Stage-specific embryonic antigen-4 (SSEA-4) is a glycosphingolipid, which is overexpressed in some cancers and has been linked to disease progression. However, little is known about the functions of SSEA-4 and the characteristics of SSEA-4 expressing tumor cells. Our studies identified SSEA-4 expression on a subpopulation of cells in many solid tumor cell lines but not in leukemic cell lines. Fluorescence-activated cell sorting-sorted SSEA-4(+) prostate cancer cells formed fibroblast-like colonies with limited cell-cell contacts, whereas SSEA-4(-) cells formed cobblestone-like epithelial colonies. Only colonies derived from SSEA-4(+) cells were enriched for pluripotent embryonic stem cell markers. Moreover, major epithelial cell-associated markers Claudin-7, E-cadherin, ESRP1 and GRHL2 were down-regulated in the SSEA-4(+) fraction of DU145 and HCT-116 cells. Similar to cell lines, SSEA-4(+) primary prostate tumor cells also showed down-regulation of epithelial cell-associated markers. In addition, they showed up-regulation of epithelial-to-mesenchymal transition as well as mesenchymal markers. Furthermore, SSEA-4(+) cells escape from adhesive colonies spontaneously and form invadopodia-like migratory structures, in which SSEA-4, cortactin as well as active pPI3K, pAkt and pSrc are enriched and colocalized. Finally, SSEA-4(+) cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in decreased cellular adhesion to different extracellular matrices. In conclusion, we introduce SSEA-4 as a novel marker to identify heterogeneous, invasive subpopulations of tumor cells. Moreover, increased cell-surface SSEA-4 expression is associated with the loss of cell-cell interactions and the gain of a migratory phenotype, suggesting an important role of SSEA-4 in cancer invasion by influencing cellular adhesion to the extracellular matrix.

Enhanced suicidal erythrocyte death contributing to anemia in the elderly.

BACKGROUND/AIMS: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+)-concentration ([Ca(2+)]i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. METHODS: Blood was drawn from healthy young (n=11, age 31.3 ± 1.7 years) and elderly (n=16, age 88.6 ± 0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. RESULTS: Elderly individuals suffered from severe anemia (hemoglobin 10.5 ± 0.3 g/100 ml) despite enhanced number of reticulocytes (2.3 ± 0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5 ± 0.2%) than in the young volunteers (1.3 ± 0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca(2+)]i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. CONCLUSIONS: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Expression of stromal cell-derived factor-1 receptors CXCR4 and CXCR7 on circulating platelets of patients with acute coronary syndrome and association with left ventricular functional recovery.

BACKGROUND: Surface expression of stromal cell-derived factor-1 (SDF-1) on platelets is enhanced during ischaemic events and might play an important role in peripheral homing and myocardial repair. As SDF-1 effects are mediated through CXCR4/CXCR7, we investigated platelet expression of SDF-1/CXCR4/CXCR7 in patients with coronary artery disease (CAD). METHODS AND RESULTS: Expression of SDF-1, CXCR4, and CXCR7 in platelets was investigated by western blot analysis, immunofluorescence confocal microscopy, and flow cytometry among healthy subjects and patients with acute coronary syndrome (ACS) and stable CAD. In a cohort study, platelet surface expression of CXCR4, CXCR7, and SDF-1 was measured in 215 patients with symptomatic CAD (stable CAD = 112, ACS = 103) at the time of percutaneous coronary intervention. Course of left ventricular ejection fraction (LVEF) was followed up during intrahospital stay and at 3 months. Both CXCR4 and CXCR7 are surface expressed on human platelets and to a higher degree in CAD patients when compared with healthy controls. Platelet surface expression of CXCR7 but not CXCR4 was enhanced in patients with ACS when compared with patients with stable CAD (mean fluorescence intensity 17.8 vs. 15.3, P = 0.004 and 29.0 vs. 26.3, P = 0.122, respectively). CXCR4 and CXCR7 significantly correlated with their ligand SDF-1 on platelets (ρ = 0.273, P < 0.001 and ρ = 0.454, P < 0.001, respectively). Additionally, high CXCR7 expression above the median correlated with the absolute improvement of LVEF% after 5 days and 3 months (46.2, 49.8, 53.7; P = 0.003). CONCLUSION: These findings indicate that platelet surface expression of CXCR4 and CXCR7 might differentially contribute to SDF-1-mediated effects on regenerative mechanisms following ACS. Studies are warranted to further evaluate the regulatory mechanisms of CXCR4/-7 expression and its prognostic impact on CAD.

Blocking TGF-ß- and Epithelial-to-Mesenchymal Transition (EMT)-mediated activation of vessel-associated mural cells in glioblastoma impacts tumor angiogenesis.

Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. GBM displays excessive and unfunctional vascularization which may, among others, be a reason for its devastating prognosis. Pericytes have been identified as the major component of the irregular vessel structure in GBM. In vitro data suggest an epithelial-to-mesenchymal transition (EMT)-like activation of glioma-associated pericytes, stimulated by GBM-secreted TGF-ß, to be involved in the formation of a chaotic and dysfunctional tumor vasculature. This study investigated whether TGF-ß impacts the function of vessel associated mural cells (VAMCs) in vivo via the induction of the EMT transcription factor SLUG and whether this is associated with the development of GBM-associated vascular abnormalities. Upon preventing the TGF-ß-/SLUG-mediated EMT induction in VAMCs, the number of PDGFRß and αSMA positive cells was significantly reduced, regardless of whether TGF-ß secretion by GBM cells was blocked or whether SLUG was specifically knocked out in VAMCs. The reduced amount of PDGFRß+ or αSMA+ cells observed under those conditions correlated with a lower vessel density and fewer vascular abnormalities. Our data provide evidence that the SLUG-mediated modulation of VAMC activity is induced by GBM-secreted TGF-߬ and that activated VAMCs are key contributors in neo-angiogenic processes. We suggest that a pathologically altered activation of GA-Peris in the tumor microenvironment is responsible for the unstructured tumor vasculature. There is emerging evidence that vessel normalization alleviates tumor hypoxia, reduces tumor-associated edema and improves drug delivery. Therefore, avoiding the generation of an unstructured and non-functional tumor vasculature during tumor recurrence might be a promising treatment approach for GBM and identifies pericytes as a potential novel therapeutic target.

Cyclophilin A is a ligand for RAGE in thrombo-inflammation.

AIMS: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA. METHODS AND RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation. CONCLUSION: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

Aquaporin-1 and Aquaporin-4 Expression in Ependyma, Choroid Plexus and Surrounding Transition Zones in the Human Brain.

The choroid plexus (CP) is a structure in the brain ventricles that produces the main part of the cerebrospinal fluid (CSF). It is covered with specialized cells which show epithelial characteristics and are the site of the blood-CSF barrier. These cells form a contiguous cell sheet with ventricle-lining ependymal cells which are known to express aquaporin-4 (AQP4). In contrast, CP epithelial cells express aquaporin-1 (AQP1) apically. We investigated the expression patterns of aquaporins in the CP-ependyma transition from human body donors using immunofluorescence and electron microscopy. Ependymal cells and subependymal astrocytes at the base of the CP showed a particularly high AQP4 immunoreactivity. Astrocytic processes formed a dense meshwork or glial plate around the blood vessels entering the CP. Interestingly, some of these astrocytic processes were in direct contact with the CP stroma, which contains fenestrated blood vessels, separated only by a basal lamina. Electron microscopy confirmed the continuity of the subastrocytic basal lamina with the CP epithelium. We also probed for components of the AQP4 anchoring dystrophin-dystroglycan complex. Immunolabeling for dystrophin and AQP4 showed an overlapping staining pattern in the glial plate but not in previously reported AQP4-positive CP epithelial cells. In contrast, dystroglycan expression was associated with laminin staining in the glial plate and the CP epithelium. This suggests different mechanisms for AQP4 anchoring in the cell membrane. The high AQP4 density in the connecting glial plate might facilitate the transport of water in and out of the CP stroma and could possibly serve as a drainage and clearing pathway for metabolites.

Distribution of corpora amylacea in the human midbrain: using synchrotron radiation phase-contrast microtomography, high-field magnetic resonance imaging, and histology.

Corpora amylacea (CA) are polyglucosan aggregated granules that accumulate in the human body throughout aging. In the cerebrum, CA have been found in proximity to ventricular walls, pial surfaces, and blood vessels. However, studies showing their three-dimensional spatial distribution are sparse. In this study, volumetric images of four human brain stems were obtained with MRI and phase-contrast X-ray microtomography, followed up by Periodic acid Schiff stain for validation. CA appeared as hyperintense spheroid structures with diameters up to 30 µm. An automatic pipeline was developed to segment the CA, and the spatial distribution of over 200,000 individual corpora amylacea could be investigated. A threefold-or higher-density of CA was detected in the dorsomedial column of the periaqueductal gray (860-4,200 CA count/mm3) than in the superior colliculus (150-340 CA count/mm3). We estimated that about 2% of the CA were located in the immediate vicinity of the vessels or in the peri-vascular space. While CA in the ependymal lining of the cerebral aqueduct was rare, the sub-pial tissue of the anterior and posterior midbrain contained several CA. In the sample with the highest CA density, quantitative maps obtained with MRI revealed high R2* values and a diamagnetic shift in a region which spatially coincided with the CA dense region.

Structural and functional protein network analyses predict novel signaling functions for rhodopsin.

Orchestration of signaling, photoreceptor structural integrity, and maintenance needed for mammalian vision remain enigmatic. By integrating three proteomic data sets, literature mining, computational analyses, and structural information, we have generated a multiscale signal transduction network linked to the visual G protein-coupled receptor (GPCR) rhodopsin, the major protein component of rod outer segments. This network was complemented by domain decomposition of protein-protein interactions and then qualified for mutually exclusive or mutually compatible interactions and ternary complex formation using structural data. The resulting information not only offers a comprehensive view of signal transduction induced by this GPCR but also suggests novel signaling routes to cytoskeleton dynamics and vesicular trafficking, predicting an important level of regulation through small GTPases. Further, it demonstrates a specific disease susceptibility of the core visual pathway due to the uniqueness of its components present mainly in the eye. As a comprehensive multiscale network, it can serve as a basis to elucidate the physiological principles of photoreceptor function, identify potential disease-associated genes and proteins, and guide the development of therapies that target specific branches of the signaling pathway.

Dynamics of expression patterns of AQP4, dystroglycan, agrin and matrix metalloproteinases in human glioblastoma.

In human glioblastoma, the blood-brain barrier (BBB) is disturbed. According to our concept, the glio-vascular relationships and thus the control of the BBB are essentially dependent on the polarity of astroglial cells. This polarity is characterized by the uneven distribution of the water channel protein aquaporin-4 (AQP4), dystroglycan and other molecules. Recently, we were able to show that the extracellular matrix component agrin is important for the construction and localization of the so-called orthogonal arrays of particles (OAPs), which consist in AQP4. Here, combining freeze-fracture electron microscopy, immunohistochemistry and Western blotting, we describe alterations of expression and distribution of AQP4, dystroglycan, agrin and the matrix metalloproteinases (MMP) 2, 3 and 9 in human primary glioblastomas (eight primary tumours, six recurrent tumours). Increase of MMP3- and MMP2/9 immunoreactivities went along with loss of agrin and dystroglycan respectively. On the protein level, AQP4 expression was increased in glioblastoma compared to control tissue. This was not accompanied by an increase of OAPs, suggesting that AQP4 can also occur without forming OAPs. The results underline our concept of the loss of glioma cell polarity as one of the factors responsible for the disturbance of the neurovascular unit and as an explanation for the formation of edemas in the glioblastoma.

Microvascular imaging of the unstained human superior colliculus using synchrotron-radiation phase-contrast microtomography.

Characterizing the microvasculature of the human brain is critical to advance understanding of brain vascular function. Most methods rely on tissue staining and microscopy in two-dimensions, which pose several challenges to visualize the three-dimensional structure of microvessels. In this study, we used an edge-based segmentation method to extract the 3D vasculature from synchrotron radiation phase-contrast microtomography (PC-µCT) of two unstained, paraffin-embedded midbrain region of the human brain stem. Vascular structures identified in PC-µCT were validated with histology of the same specimen. Using the Deriche-Canny edge detector that was sensitive to the boundary between tissue and vascular space, we could segment the vessels independent of signal variations in PC-µCT images. From the segmented volumetric vasculature, we calculated vessel diameter, vessel length and volume fraction of the vasculature in the superior colliculi. From high resolution images, we found the most frequent vessel diameter to be between 8.6-10.2 µm. Our findings are consistent with the known anatomy showing two types of vessels with distinctive morphology: peripheral collicular vessels and central collicular vessels. The proposed method opens up new possibilities for vascular research of the central nervous system using synchrotron radiation PC-µCT of unstained human tissue.

Doublecortin in the Fish Visual System, a Specific Protein of Maturing Neurons.

Doublecortin (DCX) is a microtubule associated protein, essential for correct central nervous system development and lamination in the mammalian cortex. It has been demonstrated to be expressed in developing-but not in mature-neurons. The teleost visual system is an ideal model to study mechanisms of adult neurogenesis due to its continuous life-long growth. Here, we report immunohistochemical, in silico, and western blot analysis to detect the DCX protein in the visual system of teleost fish. We clearly determined the expression of DCX in newly generated cells in the retina of the cichlid fish Astatotilapia burtoni, but not in the cyprinid fish Danio rerio. Here, we show that DCX is not associated with migrating cells but could be related to axonal growth. This work brings to light the high conservation of DCX sequences between different evolutionary groups, which make it an ideal marker for maturing neurons in various species. The results from different techniques corroborate the absence of DCX expression in zebrafish. In A. burtoni, DCX is very useful for identifying new neurons in the transition zone of the retina. In addition, this marker can be applied to follow axons from maturing neurons through the neural fiber layer, optic nerve head, and optic nerve.