Novel mechanisms of flavopiridol [Elektronische Ressource] : protection against inflammation-induced endothelium-leukocyte interactions in vivo and in vitro / Ulrike Schmerwitz

Ulrike Schmerwitz

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 37
• Poids de l'ouvrage: 1 Mo

Extrait

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











Novel mechanisms of flavopiridol:
protection against inflammation-induced endothelium-leukocyte
interactions in vivo and in vitro



Ulrike Karla Ilse Schmerwitz
aus Leipzig
2010 2 Contents

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.




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




München, 20. Mai 2010






____________________________
Ulrike Schmerwitz







Dissertation eingereicht am: 20. Mai 2010
1. Gutachter: Frau Prof. Dr. Angelika M. Vollmar
2. Gutachter: Herr Prof. Dr. Christian Wahl-Schott
Mündliche Prüfung am: 28. Juni 2010















dedicated to my family Contents I

1 Contents
II Contents

1 Contents ........................................................................................ I
2 Introduction .................. 1
2.1 Background and aim of the study ................................................................ 2
2.2 Inflammation ................................... 4
2.2.1 The endothelium ........................................................ 4
2.2.2 Leukocytes ................................. 5
2.2.3 Leukocyte-endothelial cell interactions ....................... 6
2.2.4 The selectin family...................................................................................... 8
2.2.5 Integrins and the immunoglobulin (Ig) superfamily of CAMs 8
2.2.6 Regulation of VCAM-1 and ICAM-1 ........................... 8
2.2.7 The nuclear factor-kappa B (NF- B) transcription factor ............................ 9
2.2.8 The NF- B signaling pathway .................................................................. 10
2.3 Cyclin dependent kinases ........... 12
2.3.1 Cell cycle Cdks ......................................................................................... 12
2.3.2 Transcriptional Cdks ................. 13
2.4 The Cdk9-cyclin T1 complex ....................................................................... 14
2.4.1 Function of the P-TEFb complex .............................. 15
2.5 Cyclin dependent kinase inhibitors ............................................................ 16
2.5.1 Flavopiridol (Alvocidib) ............................................................................. 16
2.5.2 Flavopiridol in clinical trials ....... 18
2.6 Cdk inhibitors as anti-inflammatory approach .......... 18
3 Materials and Methods ............................................................... 20
3.1 Materials ....................................................................... 21
3.1.1 Flavopiridol ............................................................... 21
3.1.2 Biochemicals, inhibitors, dyes, and cell culture reagents ......................... 21
3.1.3 Technical equipment ................................................................................ 23
3.2 Cell culture ................................... 24
3.2.1 Isolation and cultivation of human umbilical vein endothelial cells ........... 24 Contents III

3.2.2 Isolation and cultivation of neutrophils...................................................... 25
3.3 Cytotoxicity assays ................................ 26
3.3.1 CellTiter-Blue™ cell viability assay .......................... 26
3.3.2 Quantification of DNA fragmentation by PI staining (Nicoletti method) .... 26
3.4 Concanavalin A-induced liver injury model ............................................... 28
3.4.1 Procedures ............................................................................................... 28
3.4.2 Hematoxylin and eosin (H&E) staining ..................... 28
3.4.3 Granulocyte staining................................................................................. 28
3.4.4 Myeloperoxidase (MPO) activity ............................... 29
3.5 Cremaster muscle preparation and intravital microscopy ....................... 29
3.5.1 Surgical procedure ................................................................................... 29
3.5.2 Intravital microscopy ................. 30
3.6 Cell adhesion assay ..................... 30
3.7 Flow cytometry ............................................................................................. 31
3.7.1 Determination of cell surface expression of adhesion molecules ............. 31
3.7.2 Determination of cell surface expression of CD11b ................................. 31
3.7.3 Determination of oxidative stress in neutrophils ....... 31
3.8 Quantitative RT-PCR .................................................................................... 32
3.8.1 Isolation of RNA of tissue sections ........................... 32
3.8.2 Isolation of RNA of HUVECs .................................................................... 32
3.8.3 Reverse transcription ............... 33
3.8.4 Quantitative Real-Time PCR .... 33
3.9 Immunocytochemistry ................................................................................. 34
3.10 Western blot analysis .................. 35
3.10.1 Preparation of samples ............................................................................ 35
3.10.2 Protein quantification: bicinchoninic protein assay ... 36
3.10.3 SDS-PAGE ............................................................................................... 36
3.10.4 Electroblotting .......................... 37
3.10.5 Protein detection ...................... 38
3.10.6 Unspecific protein staining of gels and membranes ................................. 39
3.11 Transfer of nucleic acids into HUVECs ...................................................... 40 IV Contents

3.11.1 Transfection of siRNA .............................................................................. 40
3.11.2 Experimental procedure ........... 41
3.11.3 Application of shRNA via adenoviral vectors ............ 41
®3.11.4 Dual Luciferase Reporter assay system ................................................. 41
3.12 Electrophoretic mobility shift assay ........................... 42
3.12.1 Extraction of nuclear proteins ................................................................... 42
3.12.2 Protein quantification: Bradford assay ...................... 43
3.12.3 Electrophoretic Mobility Shift Assay ......................... 43
3.13 In vitro IKKβ kinase activity assay ............................................................. 44
3.14 Kinome array (PepChip) .............................................................................. 45
33 ®3.15 Protein Kinase Assay ( PanQinase Activity Assay) ............................... 45
3.16 Statistical Analysis ...................................................................................... 46
4 Results ........................................................................................ 47
4.1 Anti-inflammatory effects of flavopiridol in vivo ....................................... 48
4.1.1 Concanavalin A-induced liver injury model ............... 48
4.1.1.1 Flavopiridol protects against concanavalin A-induced liver injury ........ 48
4.1.1.2 Liver necrosis is reduced by flavopiridol .............................................. 49
4.1.1.3 Flavopiridol inhibits the infiltration of neutrophils into the liver ............. 50
4.1.1.4 Flavopiridol reduces levels of ICAM-1 and E-selectin in the liver ........ 51
4.1.2 Cremaster muscle model ......................................................................... 52
4.1.2.1 Flavopiridol reduces leukocyte-endothelium interactions in vivo ......... 52
4.1.2.2 Microvascular parameters in cremaster muscle venules ..................... 53
4.1.2.3 Motility of interstitially migrating leukocytes ......................................... 54
4.1.2.4 Flavopiridol inhibits ICAM-1 expression in cremaster muscle tissue ... 55
4.2 Cell viability and apoptosis ......................................... 56
4.2.1 Effects of flavopiridol on endothelial cell viability ...................................... 56
4.2.2 Effects of flavopiridol on apoptosis ........................................................... 57
4.3 Anti-inflammatory actions of flavopiridol in vitro ..... 58
4.3.1 Flavopiridol reduces neutrophil-endothelium interactions ......................... 58
4.3.2 Influence of flavopiridol on leukocyte activation ....................................... 59
4.3.3 Flavopiridol reduces the expression of cell adhesion molecules .............. 60 Contents V

4.4 Effects of flavopiridol on NF- B signaling ................................................. 62
4.4.1 Flavopiridol reduces NF- B promoter activity ........... 62
4.4.2 Flavopiridol has no effect on IKK /  and I B  ...