Endothelial barrier protection by natural compounds [Elektronische Ressource] : crataegus extract WS 1442 and atrial natriuretic peptide inhibit endothelial hyperpermeability / Martin Friedrich Bubik

Martin Bubik

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 24
• Poids de l'ouvrage: 4 Mo

Extrait

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





ENDOTHELIAL BARRIER PROTECTION
BY NATURAL COMPOUNDS
-
® Crataegus extract WS 1442 and atrial natriuretic peptide
inhibit endothelial hyperpermeability




Martin Friedrich Bubik
aus Pforzheim
2009
Erklärung:
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Frau Prof. Dr. Angelika M. Vollmar betreut am Lehrstuhl
für Pharmazeutische Biologie.




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





München, den 20.11.2009







Martin Bubik





Dissertation eingereicht am: 20.11. 2009
1. Gutachter: Prof. Dr. Angelika Vollmar
2. Gutachter: Prof. Dr. Christian Wahl-Schott
Mündliche Prüfung am: 18.12. 2009

























meinen Eltern CONTENTS I

CONTENTS

I INTRODUCTION....................................................................................................................1
1 THE ENDOTHELIAL BARRIER AND INFLAMMATION...................................................................1
2 INFLAMMATION ACTIVATED ENDOTHELIUM............1
2.1 Signaling of endothelial activation: ICAM-expression ................................................4
2.2 Barrier-disturbing signaling: endothelial hyperpermeability........5
3 ENDOTHELIAL BARRIER PROTECTIVE CAMP- SIGNALING.......................9
®4 HAWTHORN EXTRACT WS 1442................................................................11
® 4.1 Pharmacology and clinical efficancy of WS 1442...................13
5 AIM OF THE STUDY............................................................................................................15
II MATERIALS AND METHODS ...........................................................................................16
1 MATERIALS .......................................................16
®1.1 Crataegus extract WS 1442...................16
1.2 Biochemicals, Inhibitors and Dyes ...........................................................................17
1.3 Technical equipment ................................18
2 CELL CULTURE .................................................18
2.1 Solutions and reagents.............................................................18
2.2 HMEC-1 – human microvascular endothelial cells...................20
2.3 HUVEC – human umbilical vein endothelial cells.....................20
2.4 Passaging.................................................................................................................21
2.5 Long-time storage....21
3 PROTEIN SAMPLE PREPARATION........................22
3.1 Total cell lysate.........................................................................................................22
3.2 Membrane fractionation............................23
3.3 Extraction of nuclear protein.....................................................................................24
4 PROTEIN QUANTIFICATION.................................25
4.1 Bicinchoninic protein assay (BCA)...........25
4.2 Bradford assay .........................................................................25
5 WESTERN BLOT TRANSFER................................26
5.1 SDS-PAGE...............................................26
5.2 Tank-electroblotting..................................................................27
5.3 Protein detection......28
5.4 Enhanced chemiluminescence.................................................................................28
5.5 Infrared Imaging .......................................29 CONTENTS II

5.6 Stripping and reprobing............................................................................................30
6 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)...........................30
6.1 Radioactive labeling of consensus oligonucleotides ................................................30
6.2 Binding reaction and electrophoretic separation......................31
7 TRANSFECTION OF CELLS ..................................................................................................33
8 RAC, RHO AND RAP PULL-DOWN ASSAY.............33
9 MACROMOLECULAR PERMEABILITY ASSAY..........34
2+10 CA -MEASUREMENT .......................................................................................................34
11 CAMP ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)...............36
12 CONFOCAL MICROSCOPY.36
12.1 Microscopy with fixed cells.....................................................................................36
12.2 Live cell imaging.....................................................................................................37
13 FLOW CYTROMETRY........38
14 F-ACTIN QUANTIFICATION39
15 MEASUREMENT OF VASCULAR PERMEABILITY IN THE MOUSE CREMASTER MUSCLE IN VIVO
...........................................................................................................................................39
16 STATISTICAL ANALYSIS....40
III RESULTS ..........................................................................................................................41
® 1 ANTI-INFLAMMATORY POTENTIAL OF WS 1442 ON THE ENDOTHELIUM................................41
® 1.1 WS 1442 reduces TNFα induced ICAM-1 surface expression ...............................41
® 1.2 WS 1442 does not affect NF-κB activity. ................................42
® 1.3 WS 1442 does not affect p38 MAPK activity..........................43
®1.4 WS 1442 does not affect AP-1 activity....................................44
®2 EFFECTS OF WS 1442 ON ENDOTHELIAL HYPERPERMEABILITY..........45
2.1 Inhibition of inflammation-induced endothelial hyperpermeability in vitro ................45
2.2 Inhibition of endothelial permeability in vivo.............................................................46
® 2.3 WS 1442 modulates key parameters of endothelial permeability ...........................48
® 2.4 NO does not affect the protective effect of WS 1442 on the endothelial barrier
function................................................................................................55
® 2+2.5 WS 1442 inhibits the inflammatory Ca -signaling..................57
® 2.6 Activation of the barrier protective cAMP signaling by WS 1442............................63
® 2.7 Protection of the endothelial barrier function by WS 1442 fractions .......................70
IV DISCUSSION ....................................................................................................................78
® 1 EFFECT OF WS 1442 ON ICAM-1 EXPRESSION.................................80
1.1 Conclusion................81
® 2 EFFECTS OF WS 1442 ON INFLAMMATION-ACTIVATED ENDOTHELIAL HYPER-PERMEABILITY 82 CONTENTS III

® 2.1 Influence of WS 1442 on endothelial hyperpermeability signaling..........................83
2.2 Conclusion................................................................................................................88
3 POSSIBLE ASPECTS OF FUTURE RESEARCH........89
V SUMMARY .........................................................................................................................90
® WS 1442 and endothelial ICAM expression.................................90
® WS 1442 and inflammation-induced endothelial hyperpermeability .............................90
VI ANP ...................................................................................................92
VII REFERENCES ...............................................................................................................119
VIII APPENDIX....................126
1 ABBREVIATIONS..............126
2 PUBLICATIONS ................................................................................................................128
2.1 Original publications...............................................................................................128
2.2 Oral presentations..................................128
2.3 Poster presentations..............................129
3 CURRICULUM VITAE.........................................................................130
4 ACKNOWLEDGEMENTS....................................131 Introduction 1

I INTRODUCTION

1 The endothelial barrier and inflammation

The barrier function is the central nature of the endothelium. The maintenance of a
semi-permeable barrier by the endothelium is particularly important for controlling the
passage of macromolecules and fluid between the blood and interstitial space and for
establishing the formation of a transendothelial protein gradient (the colloid osmotic
gradient) required for tissue fluid homeostasis. The endothelium controls the flux of
fluid and solutes across the vessel wall