The TNF-receptor subtype 2 mediates prothrombotic effects in vivo [Elektronische Ressource] / Joachim Pircher. Betreuer: Florian Krötz

Joachim Pircher

Description

Aus der Medizinischen Poliklinik, Abteilung Kardiologie, Kilnikum der Universität München Komm. Direktor: Prof. Dr. med. M. Reincke und dem Walter-Brendel-Zentrum für Experimentelle Medizin der Universität München Vorstand: Prof. Dr. med. U. Pohl The TNF-receptor subtype 2 mediates prothrombotic effects in vivo Dissertation Zum Erwerb des Doktorgrades der Medizin An der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Joachim Pircher aus Meran (Italien) 2011 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Priv. Doz. Dr. Florian Krötz Mitberichterstatter: Prof. Dr. Bernd Engelmann Priv. Doz. Dr. Michael Fiegl Dekan der Medizinischen Fakultät: Prof. Dr. Dr. h.c. Maximilan Reiser, FACR, FRC R Tag der mündlichen Prüfung: 13.10.2011 ii TABLE OF CONTENTS 1 INTRODUCTION ......................... ....... 1......1.1 TNFα – a proinflammatory cytokine ....................................... ....................... 11.1.1 What is TNFα? ............................................................................ .1. ...1.1.2 TNF-receptors ............................................................................. .1 ...1.1.3 Signaling pathways ....................................................................... .1. .....1.1.

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 37
• Poids de l'ouvrage: 1 Mo

Extrait

Aus der Medizinischen Poliklinik, Abteilung Kardiologie, Klinikum der Universität München Komm. Direktor: Prof. Dr. med. M. Reincke und dem Walter-Brendel-Zentrum für Experimentelle Medizin der Universität München Vorstand: Prof. Dr. med. U. Pohl The TNF-receptor subtype 2 mediates prothrombotic effectsin vivo Dissertation Zum Erwerb des Doktorgrades der Medizin An der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Joachim Pircher aus Meran (Italien) 2011

Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Priv. Doz. Dr. Florian Krötz Mitberichterstatter: Prof. Dr. Bernd Engelmann  Priv. Doz. Dr. Michael Fiegl Dekan der Medizinischen Fakultät: Prof. Dr. Dr. h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung:13.10.2011

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TABLE OF CONTENTS INTRODUCTION....................................................1 1.1TNFα – a proinflammatory cytokine .............................................................. 1 1.1.1........................................................................................... 1What is TNFα? 1.1.2TNF-receptors............................................................................................ 1 1.1.3Signaling pathways.................................................................................... 1 1.1.4TNFα’s crucial role in inflammatory processes ......................................... 2 1.2Inflammation and cardiovascular diseases..................................................... 3 1.2.1Links between inflammation and atherothrombotic diseases ................. 3 1.2.2Vascular effects of TNFα ........................................................................... 4 1.3Platelet activation and arterial thrombosis in inflammation .......................... 4 1.3.1Activation of platelets and inflammation ................................................. 4 1.3.2Arterial thrombus formation..................................................................... 5 1.3.3Clinical observations ................................................................................. 5 1.3.4TNFα and arterial thrombosisin vivo........................................................ 5

OBJECTIVE OF THE STUDY.....................................7

MATERIALS AND METHODS ..................................8 3.1Chemicals ..................................................................................................... 8 3.2Cell culture.................................................................................................... 9 3.2.1Isolation and cultivation of human umbilical vein endothelial cells....... 10 3.2.2Cultivation of human microvascular endothelial cells............................ 10 3.2.3Passaging of cells..................................................................................... 10 3.3Animals........................................................................................................11 3.4In vitroandex vivoexperiments...................................................................11 3.4.1Flow cytometry ....................................................................................... 11 3.4.2................................................................... 12Immunofluorescent staining 3.4.3Western blotting ..................................................................................... 13 3.4.4Quantitative real-time PCR ..................................................................... 16 3.4.5Cytochrome C assay ................................................................................ 17 3.4.6Platelet aggregation studies.................................................................... 18 3.5In vivoexperiments......................................................................................18 3.5.1Anaesthesia ............................................................................................. 18 3.5.2Dorsal skinfold chamber implantation.................................................... 19 3.5.3Carotid catheterization ........................................................................... 19 3.5.4Intravital microscopy............................................................................... 20 3.5.5In vivoTNFα-stimulation ......................................................................... 21 3.5.6Intravital assessment of arterial thrombosis .......................................... 21 3.5.7Mouse platelet isolation and staining..................................................... 22 3.5.8Intravital analysis of platelet-vessel wall-interaction ............................. 22

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Statistical analysis ........................................................................................23

RESULTS ............................................................. 24 4.1Superoxide formation in endothelial cells.....................................................24 4.2Translocation of the p65-subunit of NF-κB....................................................24 4.3Endothelial adhesion molecule expression ...................................................26 4.4Tissue factor expression on endothelial cells ................................................27 4.5PAI-1 mRNA in endothelial cells ...................................................................27 4.6Platelet aggregation in PRP ..........................................................................28 4.7Arterial thrombus formationin vivo.............................................................30 4.8Platelet-endothelium-interactionin vivo......................................................30 4.9TNF-receptor subtypes on endothelial cells ..................................................33 4.10..............33TNF-receptor subtype expression in WT and TNF-receptor KO mice 4.11Arterial thrombus formationin vivoin TNF-receptor KO mice.......................33 4.12Platelet-vessel wall-interactionin vivoin TNF-R1-/- mice..............................36 4.13Platelet aggregation in TNF-R1-/- mice .........................................................36

DISCUSSION ....................................................... 39 5.1TNFα and atherothrombotic disease ............................................................39 5.2The dorsal skinfold chamber as a mouse model to investigate  arterial thrombosis and platelet-vessel wall-interactionin vivo....................39 5.2.1Assessment of arterial thrombosisin vivo.............................................. 40 5.2.2Analysis of platelet-endothelium-interactionin vivo.............................. 40 5.3TNFα – pro- or antithrombotic?....................................................................41 5.4What are the mechanisms underlying the prothrombotic effects of TNFα? ...42 5.4.1................... 43ROS formation and activation of NF-κB in endothelial cells 5.4.2Upregulation of endothelial adhesion molecules................................... 43 5.4.3Mechanisms other than primary hemostasis ......................................... 43 5.4.4Direct effects of TNFα on platelets ......................................................... 44 5.5What TNF-receptor subtype is responsible for the effects? ...........................45 5.5.1TNF-receptor subtype expression on endothelial cells .......................... 46 5.5.2TNF-receptor expression in TNF-receptor deficient mice....................... 46 5.5.3Arterial thrombosis and platelet-endothelium-interaction in  TNF-receptor deficient mice ................................................................... 46 5.5.4Direct effects of TNFα on TNF-R1 deficient platelets ............................. 47 5.6Pathophysiological and clinical consequences ..............................................48

SUMMARY ......................................................... 50

ZUSAMMENFASSUNG......................................... 52

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APPENDIX .......................................................... 54 8.1Non-standard abbreviations and acronyms ..................................................54 8.2References ...................................................................................................55 8.3Publications .................................................................................................62 8.4Acknowledgements......................................................................................64

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1.1

INTRODUCTION

TNFα – a proinflammatory cytokine

1.1.1 What is TNFα? Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine and the prototypical member of the TNF protein superfamily. The polypeptide of approximately 17 kDa is produced by a variety of immune cells including T-lymphocytes, B-lymphocytes, natural killer cells and macrophages (1). TNFαa key regulatory role in several inflammatory plays processes, in host defense against bacterial infection as well in autoimmune disorders (2,3). TNFαgiven its name as it was first discovered in 1975 with the cytotoxic ability to kill was mouse fibrosarcoma cells (4). 1.1.2 TNF-receptors The cellular response to TNFαmediated through its interaction with two different is membrane receptors (5-7). These receptor subtypes, namely TNF-receptor 1 (TNF-R1) and TNF-receptor 2 (TNF-R2), are of different size and are expressed to different levels in different cells (8). While TNF-R1, with its 55 kDa also referred to as p55-receptor, is widely expressed, expression of the 75-kDa TNF-R2 (p75-receptor) is mainly found on immune and endothelial cells (9). Whereas the extracellular domains of the two receptors are strikingly similar in structure, their intracellular domains appear to be unrelated (10). 1.1.3 Signaling pathways By interacting with its membrane receptors TNFαcan activate distinctive signaling pathways in the cell (1,6). Since TNF-R1 and TNF-R2 are differentially expressed on cells and tissues TNFαlead to a range of cellular responses and initiate both distinct and overlapping can signal transduction pathways resulting in either cell death on one side or survival, differentiation, proliferation, and migration on the other side. Among many intracellular factors involved in TNF-receptor signaling the families of the death domain homologues and of the TNF-receptor associated factors (TRAFs) should be mentioned (11-13). TRAF-signaling is described upon activation of both TNF-receptor subtypes and associated with the activation (TRAF2) or inactivation (TRAF1) of NF-κB and consecutive regulation of proinflammatory effects (14-16). TNF-R1 has an intracellular death domain, which can interact with the death domains of other cytosolic proteins such as TRADD (TNF-receptor associated death domain), FADD (Fas associated death domain) or RIP (receptor interacting protein). So, by activation of caspase-8 and other caspases TNFα similarly to the cell death signaling receptor Fas (CD95) induces apoptosis (17). Other cellular responses described upon activation of TNF-R1 include the activation of kinases such as PKC (proteinkinase C), JNK (c-Jun NH2-terminal kinase) and NIK (NF-κB induced kinase) and the activation of intracellular lipases (18). PKC is well described to activate

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