The splicing-regulatory lncRNA NTRAS sustains vascular integrity.

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA ) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Role of the soluble epoxide hydrolase in the hair follicle stem cell homeostasis and hair growth.

Polyunsaturated fatty acids (PUFAs) are used as traditional remedies to treat hair loss, but the mechanisms underlying their beneficial effects are not well understood. Here, we explored the role of PUFA metabolites generated by the cytochrome P450/soluble epoxide hydrolase (sEH) pathway in the regulation of the hair follicle cycle. Histological analysis of the skin from wild-type and sEH-/- mice revealed that sEH deletion delayed telogen to anagen transition, and the associated activation of hair follicle stem cells. Interestingly, EdU labeling during the late anagen stage revealed that hair matrix cells from sEH-/- mice proliferated at a greater rate which translated into increased hair growth. Similar effects were observed in in vitro studies using hair follicle explants, where a sEH inhibitor was also able to augment whisker growth in follicles from wild-type mice. sEH activity in the dorsal skin was not constant but altered with the cell cycle, having the most prominent effects on levels of the linoleic acid derivatives 12,13-epoxyoctadecenoic acid (12,13-EpOME), and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Fitting with this, the sEH substrate 12,13-EpOME significantly increased hair shaft growth in isolated anagen stage hair follicles, while its diol; 12,13-DiHOME, had no effect. RNA sequencing of isolated hair matrix cells implicated altered Wnt signaling in the changes associated with sEH deletion. Taken together, our data indicate that the activity of the sEH in hair follicle changes during the hair follicle cycle and impacts on two stem cell populations, i.e., hair follicle stem cells and matrix cells to affect telogen to anagen transition and hair growth.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

Structure and Function of the Blood-Brain Barrier (BBB).

The blood-brain barrier (BBB) protects the vertebrate central nervous system from harmful blood-borne, endogenous and exogenous substances to ensure proper neuronal function. The BBB describes a function that is established by endothelial cells of CNS vessels in conjunction with pericytes, astrocytes, neurons and microglia, together forming the neurovascular unit (NVU). Endothelial barrier function is crucially induced and maintained by the Wnt/ß-catenin pathway and requires intact NVU for proper functionality. The BBB and the NVU are characterized by a specialized assortment of molecular specializations, providing the basis for tightening, transport and immune response functionality.The present chapter introduces state-of-the-art knowledge of BBB structure and function and highlights current research topics, aiming to understanding in more depth the cellular and molecular interactions at the NVU, determining functionality of the BBB in health and disease, and providing novel potential targets for therapeutic BBB modulation. Moreover, we highlight recent advances in understanding BBB and NVU heterogeneity within the CNS as well as their contribution to CNS physiology, such as neurovascular coupling, and pathophysiology, is discussed. Finally, we give an outlook onto new avenues of BBB research.

Norrin restores blood-retinal barrier properties after vascular endothelial growth factor-induced permeability.

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through ß-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/ß (GSK-3α/ß) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a ß-catenin-dependent manner.

Functional morphology of the blood-brain barrier in health and disease.

The adult quiescent blood-brain barrier (BBB), a structure organised by endothelial cells through interactions with pericytes, astrocytes, neurons and microglia in the neurovascular unit, is highly regulated but fragile at the same time. In the past decade, there has been considerable progress in understanding not only the molecular pathways involved in BBB development, but also BBB breakdown in neurological diseases. Specifically, the Wnt/ß-catenin, retinoic acid and sonic hedgehog pathways moved into the focus of BBB research. Moreover, angiopoietin/Tie2 signalling that is linked to angiogenic processes has gained attention in the BBB field. Blood vessels play an essential role in initiation and progression of many diseases, including inflammation outside the central nervous system (CNS). Therefore, the potential influence of CNS blood vessels in neurological diseases associated with BBB alterations or neuroinflammation has become a major focus of current research to understand their contribution to pathogenesis. Moreover, the BBB remains a major obstacle to pharmaceutical intervention in the CNS. The complications may either be expressed by inadequate therapeutic delivery like in brain tumours, or by poor delivery of the drug across the BBB and ineffective bioavailability. In this review, we initially describe the cellular and molecular components that contribute to the steady state of the healthy BBB. We then discuss BBB alterations in ischaemic stroke, primary and metastatic brain tumour, chronic inflammation and Alzheimer's disease. Throughout the review, we highlight common mechanisms of BBB abnormalities among these diseases, in particular the contribution of neuroinflammation to BBB dysfunction and disease progression, and emphasise unique aspects of BBB alteration in certain diseases such as brain tumours. Moreover, this review highlights novel strategies to monitor BBB function by non-invasive imaging techniques focussing on ischaemic stroke, as well as novel ways to modulate BBB permeability and function to promote treatment of brain tumours, inflammation and Alzheimer's disease. In conclusion, a deep understanding of signals that maintain the healthy BBB and promote fluctuations in BBB permeability in disease states will be key to elucidate disease mechanisms and to identify potential targets for diagnostics and therapeutic modulation of the BBB.

ß-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function.

UNLABELLED: The canonical Wnt/ß-catenin signaling pathway is crucial for blood-brain barrier (BBB) formation in brain endothelial cells. Although glucose transporter 1, claudin-3, and plasmalemma vesicular-associated protein have been identified as Wnt/ß-catenin targets in brain endothelial cells, further downstream targets relevant to BBB formation and function are incompletely explored. By Affymetrix expression analysis, we show that the cytochrome P450 enzyme Cyp1b1 was significantly decreased in ß-catenin-deficient mouse endothelial cells, whereas its close homolog Cyp1a1 was upregulated in an aryl hydrocarbon receptor-dependent manner, hence indicating that ß-catenin is indispensable for Cyp1b1 but not for Cyp1a1 expression. Functionally, Cyp1b1 could generate retinoic acid from retinol leading to cell-autonomous induction of the barrier-related ATP-binding cassette transporter P-glycoprotein. Cyp1b1 could also generate 20-hydroxyeicosatetraenoic acid from arachidonic acid, decreasing endothelial barrier function in vitro In mice in vivo pharmacological inhibition of Cyp1b1 increased BBB permeability for small molecular tracers, and Cyp1b1 was downregulated in glioma vessels in which BBB function is lost. Hence, we propose Cyp1b1 as a target of ß-catenin indirectly influencing BBB properties via its metabolic activity, and as a potential target for modulating barrier function in endothelial cells. SIGNIFICANCE STATEMENT: Wnt/ß-catenin signaling is crucial for blood-brain barrier (BBB) development and maintenance; however, its role in regulating metabolic characteristics of endothelial cells is unclear. We provide evidence that ß-catenin influences endothelial metabolism by transcriptionally regulating the cytochrome P450 enzyme Cyp1b1 Furthermore, expression of its close homolog Cyp1a1 was inhibited by ß-catenin. Functionally, Cyp1b1 generated retinoic acid as well as 20-hydroxyeicosatetraenoic acid that regulated P-glycoprotein and junction proteins, respectively, thereby modulating BBB properties. Inhibition of Cyp1b1 in vivo increased BBB permeability being in line with its downregulation in glioma endothelia, potentially implicating Cyp1b1 in other brain pathologies. In conclusion, Wnt/ß-catenin signaling regulates endothelial metabolic barrier function through Cyp1b1 transcription.

Personalized translational epilepsy research - Novel approaches and future perspectives: Part II: Experimental and translational approaches.

Despite the availability of more than 15 new "antiepileptic drugs", the proportion of patients with pharmacoresistant epilepsy has remained constant at about 20-30%. Furthermore, no disease-modifying treatments shown to prevent the development of epilepsy following an initial precipitating brain injury or to reverse established epilepsy have been identified to date. This is likely in part due to the polyetiologic nature of epilepsy, which in turn requires personalized medicine approaches. Recent advances in imaging, pathology, genetics, and epigenetics have led to new pathophysiological concepts and the identification of monogenic causes of epilepsy. In the context of these advances, the First International Symposium on Personalized Translational Epilepsy Research (1st ISymPTER) was held in Frankfurt on September 8, 2016, to discuss novel approaches and future perspectives for personalized translational research. These included new developments and ideas in a range of experimental and clinical areas such as deep phenotyping, quantitative brain imaging, EEG/MEG-based analysis of network dysfunction, tissue-based translational studies, innate immunity mechanisms, microRNA as treatment targets, functional characterization of genetic variants in human cell models and rodent organotypic slice cultures, personalized treatment approaches for monogenic epilepsies, blood-brain barrier dysfunction, therapeutic focal tissue modification, computational modeling for target and biomarker identification, and cost analysis in (monogenic) disease and its treatment. This report on the meeting proceedings is aimed at stimulating much needed investments of time and resources in personalized translational epilepsy research. This Part II includes the experimental and translational approaches and a discussion of the future perspectives, while the diagnostic methods, EEG network analysis, biomarkers, and personalized treatment approaches were addressed in Part I [1].

Personalized translational epilepsy research - Novel approaches and future perspectives: Part I: Clinical and network analysis approaches.

Despite the availability of more than 15 new "antiepileptic drugs", the proportion of patients with pharmacoresistant epilepsy has remained constant at about 20-30%. Furthermore, no disease-modifying treatments shown to prevent the development of epilepsy following an initial precipitating brain injury or to reverse established epilepsy have been identified to date. This is likely in part due to the polyetiologic nature of epilepsy, which in turn requires personalized medicine approaches. Recent advances in imaging, pathology, genetics and epigenetics have led to new pathophysiological concepts and the identification of monogenic causes of epilepsy. In the context of these advances, the First International Symposium on Personalized Translational Epilepsy Research (1st ISymPTER) was held in Frankfurt on September 8, 2016, to discuss novel approaches and future perspectives for personalized translational research. These included new developments and ideas in a range of experimental and clinical areas such as deep phenotyping, quantitative brain imaging, EEG/MEG-based analysis of network dysfunction, tissue-based translational studies, innate immunity mechanisms, microRNA as treatment targets, functional characterization of genetic variants in human cell models and rodent organotypic slice cultures, personalized treatment approaches for monogenic epilepsies, blood-brain barrier dysfunction, therapeutic focal tissue modification, computational modeling for target and biomarker identification, and cost analysis in (monogenic) disease and its treatment. This report on the meeting proceedings is aimed at stimulating much needed investments of time and resources in personalized translational epilepsy research. Part I includes the clinical phenotyping and diagnostic methods, EEG network-analysis, biomarkers, and personalized treatment approaches. In Part II, experimental and translational approaches will be discussed (Bauer et al., 2017) [1].

Sonic hedgehog causes mural cells to jump 'n' run.

In this issue of Blood, Yao and colleagues report that the morphogen sonic hedgehog (Shh) is driven by platelet-derived growth factor B (PDGF-BB) in vascular smooth muscle cells, contributing to vessel maturation in an autocrine manner.

Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

ß-Catenin-Gli1 interaction regulates proliferation and tumor growth in medulloblastoma.

BACKGROUND: The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB). In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known. FINDINGS: Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch(+/-)) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as "tumor take". Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining. CONCLUSION: We propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of "tumor take" in mice.

Paired box gene 8 (PAX8) expression is associated with sonic hedgehog (SHH)/wingless int (WNT) subtypes, desmoplastic histology and patient survival in human medulloblastomas.

AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

Nucleoside diphosphate kinase B regulates angiogenesis through modulation of vascular endothelial growth factor receptor type 2 and endothelial adherens junction proteins.

OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.

Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols.

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF-induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH(-/-) mice. Similarly, sEH(-/-) mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

Novel insights into the development and maintenance of the blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/ß-catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.

Wnt signaling in the vasculature.

The development of the vascular system requires orchestrated activities of various molecular pathways to assure the formation of a hierarchically branched tubular network. Furthermore, endothelial cell (EC) populations are heterogeneous to meet organ-specific requirements in the mature vasculature. This developmental scheme is probably best represented by the acquisition and maintenance of unique barrier properties known as the blood-brain barrier (BBB) in microvessels of the central nervous system (CNS). Only recently, the canonical Wnt/ß-catenin pathway was implicated in many aspects of angiogenesis, vascular remodeling and differentiation in various species and organ systems. Beside its major contribution to brain angiogenesis and barrier formation, the Wnt/ß-catenin pathway influences vascular sprouting, remodeling and arterio-venous specification by modulating the Notch pathway. Furthermore, canonical Wnt signaling has been implicated in heart valve formation by initiating endothelial-mesenchymal transition. Growing evidence also points to a role of the non-canonical Wnt pathway in vascular development by regulating VEGF availability. Several novel findings regarding the role of the Wnt pathway in developmental as well as in pathological angiogenesis prompted us to review its emerging function in the vasculature.

Acute myocardial infarction activates progenitor cells and increases Wnt signalling in the bone marrow.

AIMS: We aimed to characterize the influence of acute myocardial infarction (AMI) on the metabolic activity of the bone marrow (BM) and on the composition and functional activity of BM-derived mononuclear cells (BMC). Acute ischaemia or other stressors induce the mobilization of progenitor cells from the BM stem cell niche. The effect of AMI on the numbers and functional activity of cells within the BM is unknown. METHODS AND RESULTS: In patients of the REPAIR-AMI trial as well as in mice, the number and functionality of BMC was compared with respect to the time interval from AMI. Activation of Wnt signalling was assessed after AMI induction in TOP-GAL transgenic reporter mice, carrying a ß-galactosidase gene driven by an LEF/TCF/ß-catenin responsive promoter. The metabolic activity of the BM, as determined by F-18-fluorodeoxyglucose-positron emission tomography, was significantly higher in patients with AMI compared with patients with chronic post-ischaemic heart failure. Moreover, the number of haematopoietic CD34(+) (P < 0.05) and CD133(+) (P < 0.05) cells in the BM aspirates was significantly increased in patients within 7 days after AMI. In order to confirm these clinical data, we induced AMI in mice, which time-dependently increased the number of c-kit + Sca-1 + lin- cells and colony-forming units in the BM. Activation of the BM by AMI induced a significant increase in Wnt signalling, which is known to induce proliferation of haematopoietic stem cells, and demonstrated increased levels of the Wnt target Axin-2 in BM-derived cells on Day 7 (P < 0.01 vs. control). CONCLUSION: Acute myocardial infarction is associated with an increased metabolic activity and increased levels of progenitor cells within days after AMI. These findings document an activation of the stem cell niche within the BM following AMI, which may have important implications for the optimal timing of cell aspirations used for therapeutic application in patients with AMI.

The neurovasculature as a target in temporal lobe epilepsy.

The blood-brain barrier (BBB) is a physiological barrier maintaining a specialized brain micromilieu that is necessary for proper neuronal function. Endothelial tight junctions and specific transcellular/efflux transport systems provide a protective barrier against toxins, pathogens, and immune cells. The barrier function is critically supported by other cell types of the neurovascular unit, including pericytes, astrocytes, microglia, and interneurons. The dysfunctionality of the BBB is a hallmark of neurological diseases, such as ischemia, brain tumors, neurodegenerative diseases, infections, and autoimmune neuroinflammatory disorders. Moreover, BBB dysfunction is critically involved in epilepsy, a brain disorder characterized by spontaneously occurring seizures because of abnormally synchronized neuronal activity. While resistance to antiseizure drugs that aim to reduce neuronal hyperexcitability remains a clinical challenge, drugs targeting the neurovasculature in epilepsy patients have not been explored. The use of novel imaging techniques permits early detection of BBB leakage in epilepsy; however, the detailed mechanistic understanding of causes and consequences of BBB compromise remains unknown. Here, we discuss the current knowledge of BBB involvement in temporal lobe epilepsy with the emphasis on the neurovasculature as a therapeutic target.