Signaling through NADPH oxidases [Elektronische Ressource] : role in vascular remodeling processes / Talija Djordjevic

Djordjevic Talija

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

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









Signaling through NADPH oxidases:
Role in vascular remodeling processes







Talija Djordjevic
Aus Belgrad
2005




Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Frau PD Dr. Agnes Görlach betreut und von Frau Prof. Dr. Angelika M.
Vollmar vor der Fakultät für Chemie und Pharmazie vertreten.






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



München, 19.12.2005.



_________________________________
(Talija Djordjevic)












Dissertation eingereicht am ______19.12.2005___________
1. Gutachter PD Dr. Agnes Görlach
2. Gutachter Prof. Dr. Angelika M. Vollmar
Mündliche Prüfung am ________23.02.2006__________
Johann Wolfgang von Goethe

........



Natur und Kunst Sie scheinen sich zu fliehen,
Und haben sich, eh’ man es denkt, gefunden;
Der Widerwille ist auch mir verschwunden,
Und beide scheinen gleich mich anzuziehen.

Es gilt wohl nur ein redliches Bemühen!
Und wenn wir erst in abgemess’nen Stunden
Mit Geist und Fleiss uns an die Kunst gebunden,
Mag frei Natur im Herzen wieder glühen.

So ist’s mit aller Bildung auch beschaffen:
Vergebens werden ungebundne Geister
Nach der Vollendung reiner Höhe streben.

Wer Grosses will muss sich zusammenraffen;
In der Beschränkung zeigt sich erst der Meister,
Und das Gesetz nur kann uns Freiheit geben.

........


Nur allein der Mensch
Vermag das Unmögliche:
Er unterscheidet,
Wählet und richtet;
Er kann den Augenblick
Dauer verleihen.










With all my love dedicated to
my mother Ljiljana and my grandmother Živana



Contents
List of the contents

1. Introduction 1
1.1 Vascular emodeling 1
1.1.1 Humoral factors in the pathogenesis of vascular remodeling 3
1.1.1.1 Vasoactive peptides in vascular remodeling 3 1.1.1.2 Prothrombotic state in vascular remodeling 6
1.2 Vascular signaling cascades 8
1.2.1 Reactive oxygen species in the vasculature 8
1.2.2 Vascular NADPH oxidases 10
1.2.2.1 p22phox 13
1.2.2.2 Rac 13
1.2.3 Protein kinases and reactive oxygen species 15
1.2.4 Redox-sensitive transcription factors 16
1.2.4.1. NF κB transcription factor 16
1.2.4.2. HIF-1 factor 17
1.3 Aim of the study 19

2. Materials and methods 20
2.1 Materials 20
2.1.1 Chemicals 20
2.1.2 Plasmids 3
2.1.2.1 Expression plasmids 23 2.1.2.2 Reporter constructs 24
2.1.2.3 Enhancer plasmids 25 2.1.2.4 Transformation and purification of plasmids 26
2.1.3 Antibodies 27
2.1.4. Solutions 29
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2.1.4.1 Solutions for Northern blot 29 2.1.4.2 Solufor Western blot 31
2.2 Cel cultre 34
2.2.1 Endothelial cells 34
2.2.2 Smooth muscle cells
2.2.3 Other cl types 35
2.2.4 Storage of the cells 35
2.3 Transfection experiments 36
2.3.1 Transfection of endothelial cells 36
2.3.2 Transfection of smooth muscle cells 36
2.3.3 Transfection of other cell types 37
2.4 Luciferase reporter gene assay 38
2.4.1 Principle of the
2.4.2 Luciferase assay 39
2.5 Measurements of ROS production 40
2.5.1 DCF fluorescence in the plate reader 40
2.5.2 Flow cytometric analyses using DCF fluorescence 42
2.5.3 Dihydroethidium staining 43
2.6 Single cell two photon confocal laser microscopy 45
2.7 Northern blot analysis 47
2.7.1 Preparation of total RNA 47
2.7.2 Electrophoresis and detection 47
2.8 Western blot analysis 49
2.8.1 Preparation of protein samples 49
2.8.2 Electrophoresis and immunodetection 49
2.8.3 Stripping and reprobing 50
2.9 Rac-1 pull down assay 51
2.9.1 Principle of the assay 51
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2.9.2 Rac-1 pull-down assay 52
2.10 Procoagulant activity 53
2.11 Proliferation assay 54
2.11.1 Principle of the BrdU incorporation assay 54
2.11.2 Proliferation assay 54
2.12 Statistical analysis 55

3. Results 56
3.1 p22phox regulates ROS production and proliferation of endothelial cells
in response to thrombin 56
3.1.1 Thrombin activates ROS production and increases p22phox expression 56
3.1.2 Expression of p22phox colocalizes with intracellular ROS production 58
3.1.3 Thrombin regulates p22phox expression in a redox-sensitive manner 60
3.1.4 Thrombin-stimulated ROS production and p22phox expression require
kinase activation 63
3.1.5 H O induces ROS production and p22phox expression via kinase 2 2
activaon 65
3.1.6 Thrombin and H O regulate p22phox via transcription and 2 2
de novo synthesis 67
3.1.7 Low levels of H O or thrombin stimulate NADPH oxidase-dependent 2 2
proliferative activity 69
3.2 Rac-1 regulates ROS production and proliferation of pulmonary artery
smooth muscle cells in response to thrombin 71
3.2.1 Rac-1 modulates ROS production in response to thrombin 71
3.2.2 Rac-1 expression and activity are increased in response to thrombin 73
3.2.3 Rac-1 activity and ROS production in response to thrombin
are dependent on calcium 74
3.2.4 Rac-1 modulates cell proliferation in response to thrombin 75
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3.3 Rac-1 regulates redox-sensitive tissue factor expression by thrombin in
pulmonary artery smooth muscle cells: The role of the NF κB pathway 77
3.3.1 Thrombin enhances tissue factor expression and promoter activity 77
3.3.2 Tissue factor expression in response to thrombin is redox-sensitive 79
3.3.3 Rac-1 mutants modulate thrombin-induced tissue factor expression
and procoagulant activity 80
3.3.4 Rac-1 modulates tissue factor promoter activity via the nuclear factor
kappa B pathway 82
3.4 Rac-1 regulates redox-sensitive PAI-1 expression by thrombin in pulmonary
artery smooth muscle cells: Role of hypoxia-inducible factor-1 86
3.4.1 Thrombin enhances PAI-1 mRNA and protein levels 86
3.4.2 Thrombin induces PAI-1 by activation of HIF-1 87
3.4.3 PAI-1 and HIF-1 α expression are redox- and calcium-sensitive 89
3.4.4 Rac-1 modulates thrombin-induced PAI-1 promoter activity and
PAI-1 expression 90
3.4.5 Rac-1 modulates HIF-1 α protein levels and HIF-1 activity 92
3.4.6 Rac-1 activates HIF-1 and induces PAI-1 promoter activity via the
activation of PAK in response to thrombin 94
3.4.7 PAI-1 regulates cell proliferation in response to thrombin 97
3.5 p22phox and NOX4 regulate redox-sensitive PAI-1 expression and
proliferation of pulmonary artery smooth muscle cells in response to the
vasoactive peptide urotensin II 98
3.5.1 Urotensin II elevates ROS 98
3.5.2 p22phox and NOX4 contribute to urotensin-II -induced ROS levels 100
3.5.3 hU-II increases protein kinase activity 103
3.5.4 p22phox and NOX4 are involved in uotensin-II stimulated protein
kinase activity 105
3.5.5 Urotensin-II induces PAI-1 expression 106
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3.5.6 Urotensin-II induces PAI-1 expression by MAP kinases and the
Pl3 kinase/Akt pathway involving NADPH oxidases 107
3.5.7 Urotensin-II stimulates proliferation via protein kinases and
NADPH oxidases 109

4. Discussion 112
4.1 Thrombin induces NADPH oxidase-dependent ROS production in
e