12 research outputs found
Tecnomatix Plant Simulation: modeling and programming by means of examples
No full textThis book systematically introduces the development of simulation models as well as the implementation and evaluation of simulation experiments with Tecnomatix Plant Simulation. It deals with all users of Plant Simulation, who have more complex tasks to handle. It also looks for an easy entry into the program. Particular attention has been paid to introduce the simulation flow language SimTalk and its use in various areas of the simulation. The author demonstrates with over 200 examples how to combine the blocks for simulation models and how to deal with SimTalk for complex control and analy
Wnt/beta-catenin signaling controls development of the blood–brain barrier
No full textThe blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown
The epithelial membrane protein 1 (EMP-1) is a novel tight junction protein of the blood-brain barrier.
No full textNRC publication: Ye
Kinetic parameters and tissue distribution of 5-oxo-L-prolinase determined by a fluorimetric assay
No full textWnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro
No full textReproducing the characteristics and the functional responses of the blood–brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation
Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression
No full textEndothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy
Differentiation of the brain vasculature : the answer came blowing by the Wnt
No full textVascularization of the vertebrate brain takes place during embryonic development from a preformed perineural vascular plexus. As a consequence of the intimate contact with neuroectodermal cells the vessels, which are entering the brain exclusively via sprouting angiogenesis, acquire and maintain unique barrier properties known as the blood-brain barrier (BBB). The endothelial BBB depends upon the close association of endothelial cells with pericytes, astrocytes, neurons and microglia, which are summarized in the term neuro-vascular unit. Although it is known since decades that the CNS tissue provides the cues for BBB induction and differentiation in endothelial cells, the molecular mechanism remained obscure. Only recently, the canonical Wnt/beta-catenin pathway and the Wnt7a/7b growth factors have been implicated in brain angiogenesis on the one hand and in BBB induction on the other. This breakthrough in understanding the differentiation of the brain vasculature prompted us to review these findings embedded in the emerging concepts of Wnt signaling in the vasculature. In particular, interactions with other pathways that are crucial for vascular development such as VEGF, Notch, angiopoietins and Sonic hedgehog are discussed. Finally, we considered the potential role of the Wnt pathway in vascular brain pathologies in which BBB function is hampered, as for example in glioma, stroke and Alzheimer's disease
Targeting tumor vasculature
No full textTargeting tumor vasculature is the common theme for the three separate studies presented in this thesis. The studies comprise the roles of components of three signaling pathways (networks) in several steps of blood vessel formation, and related tumor growth and dissemination. With our first study, we wanted to elucidate the role of selected Wnt signaling members in in vitro and in vivo angiogenesis. The aim of the second study was to reveal the advantages of simultaneous targeting of all of the constituents of the VEGFR family over the sole inhibition of VEGF and VEGFR2. Finally, in our third study we wanted to delineate the role of ErbB2 signaling mediator Memo in tumorigenesis, vasculogenesis and metastatic abilities of 4T1 mouse mammary carcinoma cells. Wnt signaling is highly conserved signaling pathway involved in several developmental processes and regulation of adult tissue homeostasis. Accumulating data from mouse knock out and in vitro angiogenesis studies indicate its role in blood vessel formation. To decipher the role of Wnt signaling components in angiogenesis, we used human umbilical vein endothelial cells (HUVEC), and well vascularized Lewis lung carcinomas grown in mice, as our in vitro and in vivo models, respectively. In our in vitro studies, we checked for the abilities of recombinant Wnt3a, a member of the family considered to activate only canonical Wnt pathway, and Wnt5a, so called non canonical Wnt, to induce HUVEC proliferation, migration and survival. We found that Wnt3a is a novel proangiogenic factor with the ability to induce HUVEC proliferation and migration, but without an effect on their survival. The effects were mediated through the common Wnt downstream effector protein Dishevelled. Proliferation induced by Wnt3a is VEGFR signaling independent. Wnt5a did not show an effect on any of those processes, but it activated signaling, demonstrated by Dishevelled phosphorylation. To test the role of Fz6, a member of Wnt receptor family repeatedly shown to be expressed on vasculature from different tissues, in in vivo angiogenesis, we injected Fz6 knock out mice and their wild type littermates with Lewis lung carcinoma cells. The ablation of Fz6 did not have an effect on the kinetics of tumor growth, quantity nor appearance of tumor vasculature. While the knowledge about the role of Wnt signaling in angiogenesis is only emerging, the importance of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) in vessel formation is well documented. In our work, we wanted to test the effects of simultaneous inhibition of VEGFR1, VEGFR2 and VEGFR3 in comparison to inhibition of VEGFR2 or VEGF solely, on tumor growth and spread. We found that targeting all three VEGFRs is more effective in inhibition of functionality of the lymphatics surrounding the primary tumor and subsequent metastatic spread, than targeting VEGFR2 or VEGF alone. Moreover, we show that B16/BL6 melanoma cancer cells display an in vitro autocrine VEGF/VEGFR signaling loop whose inhibition chemosensitizes them to platinum based chemotherapeutics. ErbB2 receptor tyrosine kinase belongs to the family of epidermal growth factor receptors. It is overexpressed in 20% of human breast cancers, and its expression correlates with highly metastatic disease and poor clinical outcome for patients. In a screen for ErbB2 effectors, Memo, a novel signaling protein that is mediating migration induced through ErbB2 and several other receptor tyrosine kinases, was previoulsy identified in our lab. To test the role of Memo in cancer growth, we made use of 4T1, a highly metastatic mouse mammary carcinoma cell line. Downregulation of Memo via stable transfection of shRNA in these cells decreased in vivo primary tumor growth when compared to control cells. The inspection of tumor vasculature revealed that tumors derived from Memo knock down (KD) clones were less vascularized, which could possibly explain their growth delay. To delineate the mechanisms behind decreased vascularization, we measured the concentration of secreted VEGF in the medium of control and Memo KD clones. Our results show that Memo KD clones secrete less VEGF into the medium than control clones, indicating that this might relate to the impairment in vascularization of tumors derived from these cells. To check the role of Memo downregulation in metastasis formation, we injected control and Memo KD clones into the tail vein of BALB/c mice. We found that Memo downregulation decreases the metastatic behavior of these cells. In conclusion, with this study, we describe the effects of Memo downregulation on several aspects of tumorigenesis in mouse mammary carcinoma model. Further studies are needed to decipher the signaling pathway that Memo is a part of, and that is responsible for the described effects on primary tumor, its vascularization and metastasis formation
