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Expression and function of thrombin receptors (PAR1, PAR3 and PAR4) in human umbilical vein endothelial cells [Elektronische Ressource] : thrombin-mediated CX3CL1 expression / submitted by Milan Popović

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116 pages
University of Ulm Department of Pharmacology of Natural Products and Clinical Pharmacology Head: Prof. Thomas Simmet, MD. Ph.D Expression and Function of Thrombin Receptors (PAR1, PAR3 and PAR4) in Human Umbilical Vein Endothelial Cells: Thrombin-mediated CX3CL1 Expression Thesis Presented to the Faculty of Medicine, University of Ulm, to obtain the degree of a Doctor of Human Biology Submitted by Milan Popovi from Kragujevac, Serbia December 2006 ć Amtierender Dekan: Prof. Klaus Debatin 1. Berichterstatter: Prof. Thomas Simmet 2. Berichterstatter: Prof. Nikolaus Marx thTag der Promotion: 8 December 2006 In memoriam Mojoj dragoj babi, Danici Table Of Contents Table of Contents TABLE OF CONTENTS I ABBREVATIONS III 1 INTRODUCTION 1 1.1 P RINCIPLES OF THE VASCULAR TRANSMIGRATION OF LEUKOCYTES 1 1.1.1 E NDOTHELIUM AS A SENSOR FOR INFLAMMATION 1 1.1.2 I NTERACTION BETWEEN ENDOTHELIAL CELLS AND LEUKOCYTES 2 1.2 H UMAN CYTOMEGALOVIRUS 11 1.2.
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University of Ulm
Department of Pharmacology of Natural Products and Clinical Pharmacology
Head: Prof. Thomas Simmet, MD. Ph.D




Expression and Function of Thrombin Receptors (PAR1,
PAR3 and PAR4) in Human Umbilical Vein Endothelial Cells:
Thrombin-mediated CX3CL1 Expression



Thesis

Presented to the Faculty of Medicine, University of Ulm, to obtain the degree
of a Doctor of Human Biology


Submitted by

Milan Popovi
from Kragujevac, Serbia

December 2006


ć























Amtierender Dekan: Prof. Klaus Debatin

1. Berichterstatter: Prof. Thomas Simmet

2. Berichterstatter: Prof. Nikolaus Marx

thTag der Promotion: 8 December 2006


































In memoriam

Mojoj dragoj babi, Danici







Table Of Contents

Table of Contents


TABLE OF CONTENTS I
ABBREVATIONS III
1 INTRODUCTION 1
1.1 P RINCIPLES OF THE VASCULAR TRANSMIGRATION OF LEUKOCYTES 1
1.1.1 E NDOTHELIUM AS A SENSOR FOR INFLAMMATION 1
1.1.2 I NTERACTION BETWEEN ENDOTHELIAL CELLS AND LEUKOCYTES 2
1.2 H UMAN CYTOMEGALOVIRUS 11
1.2.1 V IRUS STRUCTURE, GENOME ORGANIZATION AND VIRUS REPLICATION 12
1.3 T HE SERINE PROTEASE THROMBIN 18
1.4 T HROMBIN RECEPTORS 19
1.4.1 G PROTEIN-COUPLED RECEPTORS - GENERAL INFORMATIONS 19
1.4.2 S TRUCTURE OF THE THROMBIN RECEPTORS 19
1.4.3 PAR1, THE PROTOTYPICAL THROMBIN RECEPTOR 21
1.4.4 T HE PAR3 RECEPTOR 22
1.4.5 T HE PAR4 RECEPTOR 23
1.4.6 M OLECULAR MECHANISM OF RECEPTOR ACTIVATION 23
1.4.7 I NTRACELLULAR SIGNALING 25
2 MATERIALS AND METHODS 28
2.1 M ATERIALS 28
2.1.1 I NSTRUMENTS 28
2.1.2 E NZYMES 29
2.1.3 R EAGENTS 29
2.1.4 B UFFERS 31
2.1.5 S OLUTIONS 33
2.1.6 A NTIBODIES 33
2.1.7 P RIMERS 34
ITable Of Contents
2.1.8 C ONSUMABLE SUPPLIES 34
2.1.9 C ELL CULTURE 35
2.1.10 V IRUS STRAINS AND DETERMINATION OF VIRUS TITER 37
2.2 M ETHODS 39
2.2.1 I SOLATION OF TOTAL RNA 39
2.2.2 R EVERSE TRANSCRIPTION OF RNA 39
2.2.3 S EMIQUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE-CHAIN REACTION
(RT-PCR) 40
2.2.4 C ONSTRUCTION OF LUCIFERASE REPORTERS, TRANSIENT TRANSFECTION AND
LUCIFERASE ASSAY 41
2.2.5 A NALYSIS OF PROTEIN EXPRESSION 45
3 RESULTS 49
3.1 R EGULATION OF THROMBIN RECEPTORS EXPRESSION BY HCMV INFECTION IN
HUMAN UMBILICAL ENDOTHELIAL CELLS (HUVEC) 49
3.1.1 D ETERMINATION OF OPTIMAL CONDITIONS FOR HCMV INFECTION OF HUVEC
49
3.2 T HROMBIN-INDUCED EXPRESSION OF CX3CL1/FRACTALKINE STIMULATES
PRODUCTION AND RELEASE OF MCP-1 FROM HUMAN UMBILICAL VEIN ENDOTHELIAL
CELLS AFTER COCULTIVATION WITH MONOMAC6 58
4 DISCUSSION 75
4.1 R EGULATION OF THROMBIN RECEPTORS, PAR1, PAR3 AND PAR4
EXPRESSION IN HCMV-INFECTED HUVEC 75
4.2 T HROMBIN-INDUCED EXPRESSION OF CX3CL1/FRACTALKINE POTENTIATES
PRODUCTION AND RELEASE OF MCP-1 FROM HUMAN UMBILICAL VEIN ENDOTHELIAL
CELLS AFTER COCULTIVATION WITH MONO-MAC-6 78
5 SUMMARY 82
6 REFERENCES 83


IIAbbreviations
Abbrevations

AIDS Acquired immunodeficiency syndrome
Ala Alanine
Arg Arginine
Asp Aspartate
BSA Bovine serum albumine
cAMP Cyclic adenosine monophosphate
cDNA complementary DNA
CMV Cytomegalovirus
CPE Cytopathic effect
CREB cAMP response element-binding protein
DAG Diacylglycerol
DARC Duffy antigen receptor for cytokines
DNA Deoxyribonucleic acid
EC Endothelial cell
EDTA Ethylendiamin-N, N, N’, N’-tetraacetic acid
FKN Fractalkine
g Glycoprotein
Gly Glycine
GPCR G protein-coupled receptors
GRK G protein-coupled receptor kinase
HCMV Human CMV
HF Human fibroblasts
His Histidine
Hpi Hours post infection
HRP Horse radish peroxidase
HUVEC Human umbilical vein endothelial cells
ICAM-1 Intracellular adhesion molecule-1
IE Immediate early genes
IFN Interferon
IKK IkB kinase
Ig Immunoglobulin
IIIAbbreviations
IL Interleukine
IP3 Inositol-1,4,5-triphosphate
IkB Inhibitor of NF-kB
JNK c-Jun amino-terminal protein kinase
kDa Kilodalton
L Late genes
LAD Leukocyte adhesion deficiency
LFA Lymphocyte activation-related antigen
LPS Lipopolysaccharide
Lys Lysine
MAPK Mitogen Activated Protein Kinase
MCP-1 Monocyte chemoattractant protein-1
mFKN Membrane-anchored fractalkine
mRNA messenger RNA
NF-kB k light chain enhancer binding nuclear factor in B cells
ORF Open reading frame
PAR Protease-activated receptor
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PDGF Platelet-derived growth factor
PE Phycoerithrin
PI-3 Phosphatidylinositol-3
PIP Phosphatidylinositol 4,5-bisphosphate 2
PLC PIP phospholipase C 2
Pp Phosphoprotein
RAEC Rat aortic endothelial cells
RASMC Rat aortic smooth muscle cells
RNA Ribonucleic acid
Rpm Rotation per minute
rRNA Ribosomal RNA
RT-PCR Reverse transcription-PCR
Ser Serine
sFKN Soluble fractalkine
IVAbbreviations
Thr Threonine
TNF-a Tumor necrosis factor-a
TRAF TNF receptor activated factor
VCAM-1 Vascular cell adhesion molecule-1
VLA-1 Very late activation-1
VSMC Vascular smooth muscle cells




































VIntroduction


1 Introduction

1.1 Principles of the vascular transmigration of
leukocytes

1.1.1 Endothelium as a sensor for inflammation

The vascular wall comprises three different functional layers: the tunica
intima, the tunica media, and the tunica externa or adventitia. The tunica
intima is formed by the monolayer of endothelial cells located at the luminal
side of the vascular wall, a layer of connective tissue and the basal
membrane. The tunica media is composed of circularly arranged vascular
smooth muscle cells (VSMC) mixed with elastic fibers. The tunica externa
consists of connective tissue containing elastic and collagenous fibers that run
1parallel to the long axis of the vessel . Vascular endothelium represents a
functional monolayer interface between blood and tissue. It influences the
three classical interacting components of hemostasis: the vessel, the blood
platelets and the clotting and fibrinolytic systems of the plasma, but also its
natural sequelae: inflammation and tissue repair. Two principal modes of
2endothelium behavior can be defined: anti- and pro-inflammatory state .
Under physiological conditions the endothelium tends to support and to
3, 4 5, 6maintain an anti-inflammatory state by mediating vascular dilatation , by
7preventing platelet adhesion and activation , and by inhibiting generation of
8thrombin . Additionally, under these conditions, adhesion and consequently
9,10the transmigration of inflammatory leukocytes are attenuated and oxygen
radicals generated under normal cell metabolism are efficiently scavenged to
prevent cell damage. Inversely, in conditions such as disruption of the
endothelium, or functional perturbation of its functions by acute or chronic
inflammation (i.e. atherosclerosis, diabetes, or chronic arterial hypertension),
11, 12pro-inflammatory state is activated . In case of activation of pro-
1Introduction
inflammatory state by disruption of the endothelium, the pathological
environment is referred to as pro-thrombotic since it is associated with
thrombin generation. Pro-inflammatory state is characterized by
5, 13, 14vasoconstriction , by platelet adhesion and activation, by promotion of
thrombin formation and subsequently coagulation and fibrin deposition to the
vascular wall. Thrombotic and inflammatory characters of the vascular wall
15, 16participate to the enhancement of endothelial permeability and to the
production of cytokines, chemokines, and growth factors. It also increased the
expression of adhesion molecules involved in interactions with leukocytes and
platelets.


1.1.2 Interaction between endothelial cells and leukocytes

Located at the interface between blood and tissue, the endothelium has the
ability to quickly respond to changes caused by trauma or inflammation. In the
situation where no inflammatory signals are generated, leukocytes monitor the
integrity of the endothelial surface by circulating as nonadherent cells through
17the blood and the lymph . However, pro-inflammatory signals enhance
expression of adhesion and signaling molecules by the endothelium, driving
and homing circulating leukocytes from blood and lymph to the inflammatory
sites. These two functions, circulation and adhesion, are providing an efficient
immune surveillance of the host by leukocytes.
Migration of leukocytes from the blood stream to the inflamed tissue is
mediated by multicellular interactions established through 4 major steps: 1) an
initial, usually reversible attachment to the endothelium, 2) the activation of
attached cells, 3) the development of a strong, shear-resistant adhesion, and
4) the migration through the endothelium into tissues.




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