Molecular mechanism of intracellular signal transduction by the angiotensin converting enzyme [Elektronische Ressource] / vorgelegt von Cynthia Gershome

Gershome

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2007
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Fachbereich Medizin
Der Johann Wolfgang Goethe-Universität
Frankfurt am Main



Institut für KARDIOVASKULÄRE PHYSIOLOGIE
Direktor: Prof. Dr. Rudi Busse


Molecular mechanism of intracellular signal transduction
by the angiotensin-converting enzyme






Dissertation

zur Erlangung des Doktorgrades der theoretischen Medizin
des Fachbereichs Medizin der Johann Wolfgang Goethe-Universität
Frankfurt am Main





vorgelegt von

Cynthia Gershome

aus Chennai, Indien




Frankfurt am Main 2007

































Dekan: Prof. Dr. J. Pfeilschifter

Referent: Prof. Dr. Ingrid Fleming

Korreferent: Prof. Dr. Dr. Gerd Geislinger

Tag der mündlichen Prüfung: 20.12.2007





Table of contents
Table of contents

1.Introduction……………………………………………………..3

1.1. The Renin Angiotensin System…………………………………………………….4
1.2. ACE inhibitors……………………………………………………………………...6
1.3. The Angiotensin Converting Enzyme……………………………………………....7
1.3.1. Structure………………………………………………………………………...7
1.3.2. ACE isoforms…………………………………………………………………...9
1.3.3. ACE genetics…………………………………………………………………. 10
1.3.4. ACE polymorphisms…………………………..10
1.4. ACE secretion......................................................................................................... 10
1.5. ACE in other tissues……………………………………………………………… 11
1.5.1 Human monocytes…………….. 11
1.5.2. Heart and vasculature………………………… 12
1.5.3. Adipose tissue……………………………………………………………….... 12
1.5.4. Pancreas………………………………………………………………………. 12
1.6. Homologues of ACE .............................................................................................. 13
1.7. ACE as a signal transduction molecule.................................................................. 13
1.8. Aim......................................................................................................................... 16

2.Materials and Methods ............................................................17

2.1. Materials................................................................................................................. 17
2.2. Cell Culture ............................................................................................................ 18
2.3. Stable transfection of endothelial cells........................................... 19
2.4. Adenoviral infection of human endothelial cells............................ 19
2.5. Protein isolation and Western blot analysis............................................................ 19
2.6. Immunoprecipitation ...................................................................... 20
2.8. JNK activity Assay......................................................................... 21
2.9. RNA isolation......................................................................................................... 21
2.10. Gene array (Affymetrix) for expression analysis ................................................. 22
2.12. Nuclear isolation of proteins ................................................................................ 23
2.13. Electrophoretic Mobility Shift Assay (EMSA) .................................................... 23
2.14. Statistical analysis ........................................................................ 24




Table of contents
3. Results ......................................................................................25

3.1. ACE dimerization in endothelial cells.................................................................... 25
3.3 ACE inhibitors induce dimerization of ACE in endothelial cells ........................... 28
3.4. Effect of Carbohydrates on ACE dimerization ...................................................... 30
3.5. Effect of monoclonal antibodies on ACE dimerization and shedding ................... 31
3.6. Ramiprilat-induced dimerization initiates ACE signalling in CHO cells .............. 33
3.6. Divergent gene expression induced by ramiprilat in primary endothelial cells ..... 35
3.7. Expression patterns of selected genes in human endothelial cells ......................... 38
3.8. Ramiprilat enhances cellular retinol binding protein-1 (CRBP-1) levels in human
plasma) .......................................................................................................................... 39

3.9. CRBP-1 overexpression increases the activity of retinoid X receptor (RXR) and
PPAR response element (PPRE) promoter in endothelial cells………………………41
3.10. ACE signalling in human monocytes................................................................... 42
3.11. Effect of ramiprilat on nuclear peroxisome proliferator-activated receptor gamma
(PPAR γ) levels in human monocytes .................................................... 44
3.12. Effect of ramiprilat on nuclear PPAR γ levels in human endothelial cells ........... 46
3.13. ACE inhibitor regulates adiponectin, a downstream target of PPAR γ................ 47
3.14. NF- κB activation by ramiprilat in human monocytes .......................................... 50

4. Discussion.................................................................................51

4.1. ACE dimerization initiates ACE signalling in endothelial cells ............................ 51
4.2. Downstream effectors of the ACE signalling pathways in endothelial cells ......... 55
4.3. ACE signalling in monocytes................................................................................. 58
4.4 ACE inhibitors regulate adiponectin, a downstream target of PPAR γ.................... 60
5. Summary……………………………………………………...62
6. Zusammenfassung……………………………………………65
7. List of abbreviations………………………………………….66
8. Reference List…………………………………………………69



Introduction 3
1. Introduction

Angiotensin I converting enzyme (ACE) is a zinc peptidyl dipeptidase that catalyses the
conversion of angiotensin I to angiotensin II, which is a potent vasoconstrictor and is also
involved in the inactivation of bradykinin, a potent vasodilator (1). ACE plays an important
role in blood pressure regulation by virtue of generating angiotensin II, which increases blood
pressure by inducing aldosterone secretion, leading to sodium and fluid retention and the
release of norepinephrine from the sympathetic nervous system (2). Apart from regulating
blood pressure, angiotensin II, an important effector of the renin-angiotensin system (RAS), is
a potent proinflammatory agent leading to expression of growth factors, cytokines,
chemokines and adhesion molecules, in pathological conditions wherein RAS is activated
(36). Increased local ACE activity leading to increased de novo production of angiotensin II
coupled with degradation of bradykinin, impairs the balance between vasoconstriction and
vasodilation, thrombogenesis and fibrinolysis, proinflammation and anti-inflammation thus
promoting pathological effec