Resveratrol attenuates vascular smooth muscle cell hypertrophy and hyperplasia [Elektronische Ressource] : elucidation of signalling pathways / von Ursula Haider

Ursula Haider

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 34
• Langue: Deutsch

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Resveratrol Attenuates Vascular Smooth Muscle
Cell Hypertrophy and Hyperplasia:
Elucidation of Signalling Pathways
von
Ursula Haider
aus
Trostberg
2003

Erklärung


Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der
Promotionsordung vom 29. Januar 1998 von Priv. Doz. Dr. V. M. Dirsch betreut.

Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, am 05.02.03



(Ursula Haider)





Dissertation eingereicht am 07.02.03
1. Gutachter Priv. Doz. Dr. V. M. Dirsch
2. Gutachter Prof. Dr. M. Biel
Mündliche Prüfung am 14.03.03
A.Contents
A. CONTENTS.............................................................................................. I
B. INTRODUCTION..................................................................................... 1
1 Background and aim of the work.......................................................................... 1
2 Vascular Smooth Muscle Cells .............................................................................. 2
2.1 VSMCs in neointima formation and atherosclerosis ........................................ 2
2.2 VSMCs in culture ............................................................................................. 2
3 Resveratrol .............................................................................................................. 2
3.1 Occurrence and history.....................................................................................2
3.2 Biosynthesis and biological function................................................................ 2
3.3 Properties..........................................................................................................4
3.3.1 Antioxidant activity..................................................................................4
3.3.2 Oestrogenic 5
3.3.3 Chemopreventive and anti-inflammatory activity ................................... 5
3.3.4 Influence on cell cycle and proliferation .................................................. 6
3.3.5 Cardiovascular effects...............................................................................6
3.3.6 Bioavailability...........................................................................................6
4 Angiotensin II 7
4.1 History..............................................................................................................7
4.2 Structure and biosynthesis ................................................................................ 7
4.3 Receptors..........................................................................................................8
4.4 Role in haemodynamics.................................................................................... 9
4.5 Role in development of cardiovascular disease.............................................. 10
4.5.1 Ang II actions in the vessel wall............................................................. 10
4.5.2 Ang II and VSMCs ................................................................................. 11
5 EGF-Receptor transactivation............................................................................. 12
5.1 EGF-Receptors................................................................................................12
5.2 EGF-R transactivation by the AT -R..............................................................1
6 Phosphoinositide 3-kinases................................................................................... 13
6.1 Phosphoinositides...........................................................................................13
6.2 Phosphoinositide 3-kinases- classification and activation.............................. 14
6.3 Inhibitors of phosphoinositide 3-kinases ........................................................ 15 II Contents
S67 Akt, p70 kinase and MAPK...............................................................................15
7.1 Akt kinase........................................................................................................15
S6k7.2 p70 ...............................................................................................................17
7.3 MAPK.............................................................................................................18
8 c-Src kinase ............................................................................................................19
9 Cell Cycle20
9.1 Regulation of G1-phase...................................................................................20
9.2 Inhibitors of cyclin-dependent kinases............................................................21
9.3 p53...................................................................................................................22
9.4 The cell cycle as a therapeutic target in cardiovascular disease .....................23
C. MATERIALS AND METHODS .............................................................. 24
1 Preparation of major stock solutions ..................................................................24
2 Cell culture.............................................................................................................25
2.1 Solutions..........................................................................................................25
2.2 Cell isolation...................................................................................................26
2.3 Passaging of VSMCs.......................................................................................
2.4 Freezing, storage and thawing of VSMCs ......................................................26
3 Detection of cell death and cell viability..............................................................27
3.1 Apoptosis.........................................................................................................27
3.1.1 Staining of apoptotic nuclei with Hoechst 33342 ...................................
3.1.2 Nucleosome ELISA.................................................................................27
3.2 Necrosis...........................................................................................................28
3.3 Cytotoxicity/ Cell viability..............................................................................29
3.3.1 Propidium iodide exclusion assay...........................................................29
3.3.2 MTT cell viability assay..........................................................................29
4 Western blot analysis ............................................................................................30
4.1 Solutions..........................................................................................................30
4.2 Procedure.........................................................................................................32
5 Immunoprecipitation34
6 Assessment of VSMC hypertrophy......................................................................35
6.1 [³H]leucine incorporation................................................................................35
6.2 Cell counting...................................................................................................
7 Cell cycle analysis..................................................................................................36
7.1 PI staining........................................................................................................36 Contents III
7.2 BrdU/7-amino-actinomycin D co-staining. .................................................... 36
8 Statistical analysis................................................................................................. 37
D. RESULTS.............................................................................................. 38
1 Characterisation of VSMCs................................................................................. 38
2 Influence of resveratrol on Ang II- and EGF-treated VSMCs......................... 38
2.1 VSMC hypertrophy.........................................................................................38
2.1.1 Ang II induces hypertrophy but not hyperplasia in VSMCs ..................
2.1.2 Resveratrol inhibits Ang II-induced VSMC hypertrophy....................... 39
S6k2.2 Phosphorylation of Akt, p38, Erk 1/2 and p70 ........................................... 40
S6k2.2.1 Ang II induces phosphorylation of Akt, p38, Erk 1/2 and p70 ........... 40
S6k2.2.2 Resveratrol inhibits phosphorylation of Akt, Erk 1/2 and p70 41
2.3 Protein phosphatase 2A .................................................................................. 43
2.4 Phosphorylation of PI 3-kinase p85 subunit................................................... 44
2.5 Calf serum-induced Akt activation................................................................. 45
2.6 EGF-R transactivation....................................................................................46
2.6.1 Ang II-induced transactivation of the EGF-R is involved in Akt
activation............