Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow.

BACKGROUND: Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts. METHODS: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques. RESULTS: By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. CONCLUSIONS: SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs.

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.

3D-electron microscopic characterization of interstitial cells in the human bladder upper lamina propria.

AIMS: To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. METHODS: Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. RESULTS: 3View-SEM image stacks as large as 59 × 59 × 17 µm3 (xyz) at a resolution of 16 × 16 × 50 nm3 and high resolution (5 × 5 × 10 nm3 ) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. CONCLUSIONS: Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

Propofol Related Infusion Syndrome: Ultrastructural Evidence for a Mitochondrial Disorder.

OBJECTIVE: The objective of this report of a fatal propofol-related infusion syndrome in a young adult was to present-to our knowledge for the first time-direct ultrastructural evidence for the central role of mitochondrial damage in the pathogenesis of this syndrome. DATA SOURCES: Histological and electron microscopical analysis of liver, skeletal, and heart muscle obtained by autopsy and blood obtained from patient. STUDY SELECTION: Case report. DATA EXTRACTION: In addition to conventional macroscopical and histological investigations, electron-microscopical analysis of myocardial- and skeletal muscle and liver tissue obtained at autopsy from a young man was performed in order to search for ultrastructural changes of mitochondria. Acylcarnitine concentrations of his blood were determined by ultra-high performance liquid chromatography mass spectrometry. DATA SYNTHESIS: A 19-year-old male was admitted with acute left-side hemiparesis. The patient was intubated, then propofol infusion started, and a craniotomy was performed to remove an intracerebral hematoma. In the postoperative period, the patient presented with elevated intracranial pressure and brain edema. After repeat surgery, the patient showed impaired systolic left ventricular function, increasing fever, anuria, hyperkalemia, and metabolic acidosis, and he finally expired. Electron microscopy revealed dark, electron dense amorphous structures associated with mitochondria in heart muscle and liver tissue obtained at autopsy. Peripheral blood analysis revealed increased levels of acetyl-, propionyl-, butyryl-, malonyl-, and valeryl-carnitine as an indicator for propofol-related infusion syndrome, as well as for propofol-mediated inhibition of free fatty acid uptake into mitochondria, affecting beta-oxidation. CONCLUSIONS: Electron dense bodies found in association with mitochondria in muscle and liver cells probably correspond to accumulation of free fatty acid provide direct morphological evidence for the mitochondrial damage in propofol-related infusion syndrome.

Loss of Mpdz impairs ependymal cell integrity leading to perinatal-onset hydrocephalus in mice.

Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ (MUPP1) are the only known human gene loci associated with non-syndromic hydrocephalus. To investigate functions of the tight junction-associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin-positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz-deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz-deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma.

Labeling Human Melanoma Cells With SPIO: In Vitro Observations.

OBJECTIVES: To use the superparamagnetic iron oxide (SPIO) contrast agent Resovist (±transfection agent) to label human melanoma cells and determine its effects on cellular viability, microstructure, iron quantity, and magnetic resonance imaging (MRI) detectability. MATERIALS AND METHODS: Human SK-Mel28 melanoma cells were incubated with Resovist (±liposomal transfection agent DOSPER). The cellular iron content was measured, and labeled cells were examined at 1.5 T and 3.0 T. The intracellular and extracellular distributions of the contrast agent were assessed by light and electron microscopy. RESULTS: The incubation of melanoma cells with SPIO does not interfere with cell viability or proliferation. The iron is located both intracellularly and extracellularly as iron clusters associated with the exterior of the cell membrane. Despite thorough washing, the extracellular SPIO remained associated with the cell membrane. The liposomal transfection agent does not change the maximum achievable cellular iron content but promotes a faster iron uptake. The MRI detectability persists for at least 7 days. CONCLUSION: The transfection agent DOSPER facilitates the efficient labeling of human metastatic melanoma cells with Resovist. Our findings raise the possibility that other Resovist-labeled cells may collect associated extracellular nanoparticles. The SPIO may be available to other iron-handling cells and not completely compartmentalized during the labeling procedure.

Brain endothelial TAK1 and NEMO safeguard the neurovascular unit.

Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood-brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB-independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1-NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP.

Mode of action of claudin peptidomimetics in the transient opening of cellular tight junction barriers.

In epithelial/endothelial barriers, claudins form tight junctions, seal the paracellular cleft, and limit the uptake of solutes and drugs. The peptidomimetic C1C2 from the C-terminal half of claudin-1's first extracellular loop increases drug delivery through epithelial claudin-1 barriers. However, its molecular and structural mode of action remains unknown. In the present study, >100 µM C1C2 caused paracellular opening of various barriers with different claudin compositions, ranging from epithelial to endothelial cells, preferentially modulating claudin-1 and claudin-5. After 6 h incubation, C1C2 reversibly increased the permeability to molecules of different sizes; this was accompanied by redistribution of claudins and occludin from junctions to cytosol. Internalization of C1C2 in epithelial cells depended on claudin-1 expression and clathrin pathway, whereby most C1C2 was retained in recyclosomes >2 h. In freeze-fracture electron microscopy, C1C2 changed claudin-1 tight junction strands to a more parallel arrangement and claudin-5 strands from E-face to P-face association - drastic and novel effects. In conclusion, C1C2 is largely recycled in the presence of a claudin, which explains the delayed onset of barrier and junction loss, the high peptide concentration required and the long-lasting effect. Epithelial/endothelial barriers are specifically modulated via claudin-1/claudin-5, which can be targeted to improve drug delivery.

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.

UNLABELLED: 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 relevant to regulation. Under normal conditions, tricellulin tightens tricellular junctions against macromolecules. Traces of tricellulin occur in bicellular junctions. AIMS: 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 the 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, as 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: TAMP 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 a 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.

A new kind of labyrinth-like capillary is responsible for leakage from human choroidal neovascular endothelium, as investigated by high-resolution electron microscopy.

PURPOSE: This study reports the clinicopathologic findings of leaky sites in pathological vessels after submacular removal of choroidal neovascular membranes (CNV). As the site that causes fluid exudation from neovascular vessels is unknown, specific attention was focused on the formation of fenestrations, cellular junctions, and morphologic alteration which can cause endothelial leakage. METHODS: Choroidal neovascular membranes of 15 patients who underwent submacular surgery for CNV were investigated. Five patients received bevacizumab treatment before surgery, and another five received photodynamic therapy before surgery. The remaining five did not receive any other treatment before surgery. All membranes were embedded for transmission electron microscopy. CNVs were analyzed for pathological cell-to-cell connections, fenestrations, or other pathological conditions which can cause leakage of plasma. RESULTS: The morphology of the newly formed blood channels was very variable, and in principle was not different in treated and untreated patients. The sources of leakage in neovascular choroidal vessels were caused by insufficient endothelial cell connections and by capillaries with microvillar projections into the vessel lumen which blocked cellular perfusion but still allowed the flow of plasma. Fenestrations were only infrequently observed. CONCLUSIONS: A newly discovered type of pathological capillary, called a labyrinth capillary, is very likely responsible for the permanent leakage of fluid. Due to the small vessel lumen, thrombocytes cannot enter these capillaries to close the leakages. Fenestrations did not appear to play a significant role in vascular leakiness.

Highly conserved cysteines are involved in the oligomerization of occludin-redox dependency of the second extracellular loop.

UNLABELLED: The tight junction (TJ) marker occludin is a 4-transmembrane domain (TMD) protein with unclear physiological and pathological functions, interacting with other TJ proteins. It oligomerizes and is redox sensitive. However, oligomerization sites and mechanisms are unknown. AIMS: To identify hypoxia-sensitive binding sites, we investigated the consequences of amino-acid substitutions of highly conserved cysteines in human occludin, under normal and hypoxic incubations. RESULTS: (i) The extracellular loop 2 (ECL2) showed homophilic trans- and cis-association between opposing cells and along the cell membrane, respectively, caused by a loop properly folded via an intraloop disulfide bridge between the shielded C216 and C237. Hypoxia and reductants prevented the associations. (ii) C82 in TMD1 directly cis-associated without disulfide formation. (iii) C76 in TMD1 and C148 in TMD2 limited the trans-interaction; C76 also limited occludin-related paracellular tightness and changed the strand morphology of claudin-1. (iv) The diminished binding strength found after substituting C82, C216, or C237 was accompanied by increased occludin mobility in the cell membrane. INNOVATION: The data enable the first experimentally proven structural model of occludin and its homophilic interaction sites, in which the ECL2, via intraloop disulfide formation, has a central role in occludin's hypoxia-sensitive oligomerization and to regulate the structure of TJs. CONCLUSION: Our findings support the new concept that occludin acts as a hypoxiasensor and contributes toward regulating the TJ assembly redox dependently. This is of pathogenic relevance for tissue barrier injury with reducing conditions. The ECL2 disulfide might be a model for four TMD proteins in TJs with two conserved cysteines in an ECL.

Cytotoxic effect and tissue penetration of phenol for adjuvant treatment of giant cell tumours.

Local adjuvant treatment of giant cell tumours (GCTs) of the bone with phenol has led to a significant reduction in recurrence rates. In the current study, the optimal phenol concentration and duration of intralesional exposure were evaluated. Specimens of GCTs were exposed to various concentrations of phenol solution (6, 60 and 80%) for either 1 or 3 min. Following embedding in glutaraldehyde, the tumour cell layers were examined by transmission electron microscopy. Destroyed cell organelles indicated the penetration depth as a sign of denaturation. Incubation of GCT specimens with 6% phenol solution for 3 min resulted in the most tissue damage and the deepest tissue penetration of ∼200 µm. Incubation with 60 and 80% phenol solution reached a penetration depth of only ∼100 µm. Phenol instillation may be used for the treatment of small scattered cellular debris following intralesional curettage; however, it is not suitable for treatment of remaining solid tumour tissue of GCT. The use of high phenol concentrations has no benefit and increases the risk of local or systemic intoxication.

Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae.

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases.

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.

BACKGROUND: Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. METHODS: Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. RESULTS: PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. CONCLUSIONS: Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier.

The Gram-positive bacterium Listeria monocytogenes can enter the human central nervous system and cause life-threatening meningitis. During this process the pathogen has to invade and cross diverse cellular barriers involving the functions of the surface proteins Internalin (InlA) and InlB. Whereas the internalin-dependent crossing of the intestinal epithelium and the fetoplacental barrier have been subject to intensive investigation, limited research elucidating the crossing of the human blood-cerebrospinal fluid barrier (BCSFB) has been reported. We have recently established a functional in vitro model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells. We show polarized expression of receptors involved in listerial invasion (i.e. E-Cadherin, Met) in HIBCPP cells. Infecting HIBCPP cells with the L. monocytogenes strain EGD, we demonstrate polar invasion exclusively from the in vivo relevant basolateral cell side. Intracellular listeria were found in vacuoles and the cytoplasm, where they were often associated with "actin tail"-like structures. Furthermore, the L. monocytogenes wild type strain shows significantly higher internalization rates than isogenic mutants lacking either InlA, InlB or both surface proteins. Deletion of either one or both proteins leads to a similarly decreased invasion, suggesting an interdependent function of InlA and InlB during invasion of choroid plexus epithelial cells.

A novel porcine in vitro model of the blood-cerebrospinal fluid barrier with strong barrier function.

Epithelial cells of the plexus choroideus form the structural basis of the blood-cerebrospinal fluid barrier (BCSFB). In vitro models of the BCSFB presenting characteristics of a functional barrier are of significant scientific interest as tools for examination of BCSFB function. Due to a lack of suitable cell lines as in vitro models, primary porcine plexus epithelial cells were subjected to a series of selective cultivation steps until a stable continuous subcultivatable epithelial cell line (PCP-R) was established. PCP-R cells grow in a regular polygonal pattern with a doubling time of 28-36 h. At a cell number of 1.5×10(5) in a 24-well plate confluence is reached in 56-72 h. Cells are cytokeratin positive and chromosomal analysis revealed 56 chromosomes at peak (84th subculture). Employing reverse transcription PCR mRNA expression of several transporters and components of cell junctions could be detected. The latter includes tight junction components like Claudin-1 and -3, ZO-1, and Occludin, and the adherens junction protein E-cadherin. Cellular localization studies of ZO-1, Occludin and Claudin-1 by immunofluorescence and morphological analysis by electron microscopy demonstrated formation of a dense tight junction structure. Importantly, when grown on cell culture inserts PCP-R developed typical characteristics of a functional BCSFB including high transepithelial electrical resistance above 600 Ω×cm(2) as well as low permeability for macromolecules. In summary, our data suggest the PCP-R cell line as a suitable in vitro model of the porcine BCSFB.

An allograft glioma model reveals the dependence of aquaporin-4 expression on the brain microenvironment.

Aquaporin-4 (AQP4), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. In contrast, most cultivated glioma cell lines don't express AQP4, and primary cell cultures of human glioblastoma lose it during the first passages. Accordingly, in C6 cells and RG2 cells, two glioma cell lines of the rat, and in SMA mouse glioma cell lines, we found no AQP4 expression. We confirmed an AQP4 loss in primary human glioblastoma cell cultures after a few passages. RG-2 glioma cells if grafted into the brain developed AQP4 expression. This led us consider the possibility of AQP4 expression depends on brain microenvironment. In previous studies, we observed that the typical morphological conformation of AQP4 as orthogonal arrays of particles (OAP) depended on the extracellular matrix component agrin. In this study, we showed for the first time implanted AQP4 negative glioma cells in animal brain or flank to express AQP4 specifically in the intracerebral gliomas but neither in the extracranial nor in the flank gliomas. AQP4 expression in intracerebral gliomas went along with an OAP loss, compared to normal brain tissue. AQP4 staining in vivo normally is polarized in the astrocytic endfoot membranes at the glia limitans superficialis and perivascularis, but in C6 and RG2 tumors the AQP4 staining is redistributed over the whole glioma cell as in human glioblastoma. In contrast, primary rat or mouse astrocytes in culture did not lose their ability to express AQP4, and they were able to form few OAPs.

Influence of dichloroacetate (DCA) on lactate production and oxygen consumption in neuroblastoma cells: is DCA a suitable drug for neuroblastoma therapy?

Many cancer cells metabolize glucose preferentially via pyruvate to lactate instead to CO(2) and H(2)O (oxidative phosphorylation) even in the presence of oxygen (Warburg effect). Dichloroacetate (DCA) is a drug which is able to shift pyruvate metabolism from lactate to acetyl-CoA (tricarboxylic acid cycle) by indirect activation of pyruvate dehydrogenase (PDH). This can subsequently lead to an increased flow of oxygen in the respiratory chain, associated with enhanced generation of reactive oxygen species (ROS) which may cause apoptosis. In order to investigate if DCA may be suitable for neuroblastoma therapy, it was investigated on three human neuroblastoma cell lines whether DCA can reduce lactate production and enhance oxygen consumption. The data show, that DCA (in the low millimolar range) is able to reduce lactate production, but there was only a slight shift to increased oxygen consumption and almost no effect on cell vitality, proliferation and apoptosis of the three cell lines investigated. Therefore, DCA at low millimolar concentrations seems to be only of minor efficacy for neuroblastoma treatment.

The disturbed blood-brain barrier in human glioblastoma.

The aim of this article is to describe alterations of the blood-brain barrier (BBB) in gliomas. The main clinical problem of human gliomas is the edematous swelling and the dramatic increase of intracerebral pressure, also compromising healthy areas of the brain. According to our concept, one of the main reasons on the cellular level for these clinical problems is the loss or reduction of astroglial polarity. Astroglial polarity means the specific accumulation of potassium and water channels in the superficial and perivascular astroglial endfeet membranes. The most important water channel in the CNS is the astroglial water channel protein aquaporin-4 (AQP4) which is arranged in a morphologically spectacular way, the so-called orthogonal arrays of particles (OAPs) to be observed in freeze-fracture replicas. In brain tumors, but also under conditions of trauma or inflammation, these OAPs are redistributed to membrane domains apart from endfeet areas. Probably, this dislocation might be due to the degradation of the proteoglycan agrin by the matrix metalloproteinase 3 (MMP3). Agrin binds to the dystrophin-dystroglycan-complex (DDC), which in turn is connected to AQP4. As a consequence, agrin loss may lead to a redistribution of AQP4 and a compromised directionality of water transport out of the cell, finally to cytotoxic edema. This in turn is hypothesized to lead to a breakdown of the BBB characterized by disturbed tight junctions, and thus to the development of vasogenic edema. However, the mechanism how the loss of polarity is related to the disturbance of microvascular tight junctions is completely unknown so far.

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.