Identification of MKP-1 as a central mediator of cytoprotective effects in human endothelial cells [Elektronische Ressource] : pathways of induction / Robert Fürst

Fürst Maximilian Lieven, Henry M. Robert

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 34
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





Identification of MKP-1 as a central mediator of
cytoprotective effects in human endothelial cells:
pathways of induction





Robert Fürst
aus Temeschburg
2005


Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar
1998 von Frau PD Dr. Alexandra K. Kiemer und Frau Prof. Dr. Angelika M. Vollmar betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.



München, 03.12.2004

____________________
(Robert Fürst)








Dissertation eingereicht am: 03.12.2004
1. Gutachter: Frau Prof. Dr. Angelika M. Vollmar
2. Gutachter: Herr Prof. Dr. Ernst Wagner
Mündliche Prüfung am: 03.02.2005
















deeply grateful and with love
dedicated to my family Contents I




Contents
II Contents
Contents III
1 INTRODUCTION 1
1.1 The endothelium 3
1.1.1 Endothelial cell characteristics 3
1.1.2 Atherosclerosis, inflammation, and endothelial cell activation 4
1.2 Aim of the study 6
1.3 MAP kinases and MAP kinase phosphatases 7
1.4 Atrial natriuretic peptide 10
1.5 ROS and NAD(P)H oxidase 13
1.6 Glucocorticoids 15

2 MATERIALS AND METHODS 19
2.1 Materials 21
2.2 Cell culture 22
2.2.1 Solutions 22
2.2.2 Culture of HUVEC and HMEC 22
2.2.3 Passaging 23
2.2.4 Long time storage 23
2.2.5 Starvation 23
2.3 Western blot analysis 24
2.3.1 Solutions 24
2.3.2 In vivo samples 25
2.3.3 In vitro samples 25
2.3.4 Stripping, reprobing, and coomassie staining 26
2.4 RT-PCR 26
2.4.1 Isolation and characterization of total RNA 26
2.4.2 Reverse transcription 27
2.4.3 Polymerase chain reaction 27
IV Contents

2.5 ROS measurement in cultured endothelial cells 28
2.6 rement in the endothelium of intact rat lung vessels 31
2.6.1 Animals 31
2.6.2 Isolated-perfused rat lung preparation 31
2.6.3 In situ fluorescence microscopy 32
2.7 Electrophoretic mobility shift assay (EMSA) 33
2.7.1 Extraction of nuclear protein 33
2.7.2 Radioactive labeling of consensus oligonucleotides 33
2.7.3 Binding reaction and electrophoretic separation 34
2.8 Transfection experiments 35
2.8.1 Rac1 mutants 35
2.8.2 Antisense oligodesoxynucleotides 35
2.8.3 AP-1 decoy 36
2.9 Rac pull-down assay 36
2.9.1 Principle of the assay 36
2.9.2 Preparation of the GST-PBD sepharose beads 37
2.9.3 Pull-down assay 38
2.10 Immunocytochemistry and confocal microscopy 39
2.11 Flow cytometry (FACS) 39
2.12 Statistical analysis 40

3 RESULTS 41
3.1 ANP and MKP-1 43
3.1.1 MKP-1 induction by ANP 43
3.1.1.1 Time and concentration course of MKP-1 induction 43
3.1.1.2 Influence of cycloheximide on MKP-1 induction 44
3.1.1.3 MKP-1 induction depends on cGMP/NPR-A 44
3.1.1.4 PKG I is not expressed in HUVEC 45