Regulation of blood-brain barrier tightness and polarity following ischemic stroke [Elektronische Ressource] / vorgelegt von Ayman El Ali

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2010
• Nombre de lectures: 40
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Regulation of blood-brain barrier tightness and
polarity following ischemic stroke






Inaugural-Dissertation
zur
Erlangung des Doktorgrades

Dr. rer. nat.

der Fakultät
Biologie und Geografie

an der

Universität Duisburg-Essen







vorgelegt von

Ayman EL ALI
aus dem LIBANON

Juni 2010 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Institut für
Zellbiologie, in der Abteilung für Vaskuläre Neurologie, Demenz- und Altersforschung
am Universitätsklinikum Essen der Universität Dusiburg-Essen durchgeführt.

1. Gutachter: Herrn Prof. Dr. Süleyman Ergün

2. Gutachter: Herrn Prof. Dr. Dirk Hermann

Vorsitzender des Prüfungsausschusses: Frau Prof. Dr. Andrea Vortkamp

Tag der mündlichen Prüfung: 03.11.2010


TABLE OF CONTENTS i


LIST OF ABBREVIATIONS vi


FIGURE TITELS xii


ABSTRACT 1


1 INTRODUCTION 3

1.1 Blood-brain barrier (BBB) in central nervous system 3
(CNS) diseases

1.2 Biology of the BBB 6
1.2.1 Neurovascular unit 8
1.2.2 Signal transduction at the neurovascular unit 9
1.2.2.1 Regulation of BBB integrity 11
1.2.2.2 Regulation of BBB polarity 14

1.3 Transport across the BBB 15
1.3.1 ATP binding cassette (ABC) transporters 17
1.3.1.1 Structure of ABC transporters 17
1.3.1.2 Function of ABC transporters 20
1.3.2 Physiological role of ABC transporters 21
1.3.3 Brain distribution of ABC transporters 22
1.3.4 ABC transporter regulation at the BBB 23

1.4 BBB in ischemic stroke 25

1.5 BBB in hypercholesterolemia 30

i 1.6 BBB in brain tumors 31

1.7 BBB in epilepsy 32

1.8 BBB in multiple sclerosis 33

1.9 BBB in Parkinson’s disease 34


2 AIM OF THE STUDY 35

2.1 Part I: Effect of LXR activation on BBB tightness 35

2.2 Part II: Effect of apolipoprotein-E (ApoE) on BBB polarity 36

2.3 Part III: Effect of hypercholesterolemia on the ischemic BBB 37


3 MATERIALS AND METHODS 38

3.1 Animal groups and pharmacological experiments 38

3.2 Induction of focal cerebral ischemia 39

3.3 Infarct and edema size evaluation studies 41

3.4 IgG extravasation studies 41

3.5 Microvessel isolation and protein extraction 42

3.6 Western blot analysis 43

3.7 Rho GTPase affinity binding (=pull down) assays 44

3.8 Co-precipitation assays with GST tagged recombinant proteins 45
ii 3.9 Immunoprecipitation and co-immunoprecipitation assays 47

3.10 Immunofluorescence staining 48

3.11 Caseinase microplate assay for calpain-1/2 48

3.12 Gelatin polyacrylamide gel zymography for MMP-2/9 49

3.13 Gelatinase microplate assay for MMP-2/9 50

3.14 Reverse transcriptase (RT) - polymerase chain reaction 51
(PCR) analysis

3.15 Enzyme-linked immunosorbent assay (ELISA) for FK506 52

3.16 Statistics 53


4 RESULTS 54

4.1 Part I: Effect of LXR activation on BBB tightness 54
4.1.1 Effect of LXR activation on ischemic injury and 54
vascular permeability
4.1.2 Effect of T0901317 on LXRαβ expression 56
4.1.3 Calpain-1/2 and MMP-2/9 activities regulation 56
after LXR activation
4.1.4 T0901317 increases the expression of LXR’s 56
target gene calpastatin
4.1.5 LXR activation stabilizes p120 catenin 58
4.1.6 LXR activation differentially regulates RhoA 59
and Cdc42 activation
4.1.7 p120 catenin controls RhoA and Cdc42 activity in 59
reperfused microvessels
4.1.8 Regulation of occludin and ZO-1 after LXR activation 61

iii 4.1.9 LXR activation deactivates JNK1/2 pathways and 62
caspase-3
4.1.10 T0901317 regulates ABC transporters at the BBB, 63
without influencing their polarity

4.2 Part II: Effect of apolipoprotein-E (ApoE) on BBB polarity 65
4.2.1 Severity of injury does not differ between wildtype 65
-/- and ApoE mice
4.2.2 ABCB1 and ABCC1 are inversely regulated upon 65
ischemia in wildtype and ApoE-/- mice
4.2.3 ApoE and its receptor, ApoE receptor-2 (ApoER2), 67
are present on ischemic brain capillaries
4.2.4 ApoER2 binds Jun kinase-1/2 (JNK1/2), its kinase 68
and phosphatase via JNK1/2-interacting protein-1/2
(JIP1/2)
4.2.5 ApoE-/- abolishes post-ischemic deactivation of 69
JNK1/2, thereby overactivating c-Jun
4.2.6 Delivery of recombinant human ApoEε3 or of JNK1/2 70
inhibitor restores ABC transporter levels
4.2.7 ApoE deficiency enhances FK506 accumulation in 71
ischemic brain, whereas ApoE delivery partly
restores drug elimination

4.3 Part III: Effect of hypercholesterolemia on the ischemic BBB 73
4.3.1 Increased brain swelling and IgG extravasation in 73
animals receiving cholesterol-rich (Western) diet
4.3.2 Overactivation of calpain-1/2 and MMP-2/9 in 75
ischemic microvessels of hypercholesterolemic mice
4.3.3 Cholesterol-rich diet enhances the activation of 77
RhoA and downregulate tight junction protein occludin
4.3.4 Angiotensin II type 1 receptor (AT1R) upregulation 78
in cholesterol-rich diet enhances the activation of
RhoA in ischemic brain microvessels
4.3.5 Hypercholesterolemia does not influence ABC 80
transporter expression
iv 5 DISCUSSION 81

5.1 General aspects 81

5.2 Part I: LXR activation enhances BBB tightness but not 82
polarity in the ischemic brain

5.3 Part II: Apolipoprotein-E controls BBB polarity without 85
influencing BBB tightness

5.4 Part III: Hypercholesterolemia impairs post-ischemic 88
BBB tightness but not polarity


6 CONCLUSION AND OUTLOOKS 90


7 REFERENCES 92


8 PERMISSIONS AND IMAGE SOURCES 113


9 CURRICULUM VITAE 115


10 PUBLICATIONS 116


11 ERKLÄRUNG 117





v LIST OF ABREVIATIONS

Aβ amyloid-beta
ABC transporter ATP binding cassette transporter
ACA anterior cerebral artery
ACAT acyltransferase enzyme
ACE angiotensin converting enzyme
ACcomA anterior communicating artery
AJ adherens junction
AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ANG1 angiopoetin 1
ANOVA analysis of variances
AP-1 activator protein-1
Apaf-1 apoptosis-activating factor-1
ApoE apolipoprotein E
ApoER2 apolipoprotein E receptor 2
ASA American Stroke Association
ASA acetylsalicylic acid
AT1R Angiotensin II type 1 receptor
AT2 angiotensin 2
ATP adenosine triphosphate
BA basal artery
BBB blood-brain barrier
BCRP breast cancer resistance protein (ABCG2; MXR)
BDNF brain-derived neurotrophic factor
BCSFB blood cerebrospinal fluid barrier
bFGF basic fibroblast growth factor
BH1 – BH4 Bcl-2 homology domains 1 – 4
BHT 2,6-di-tert-butyl-4-methylphenol
BMI body mass index
BSA bovine serum albumin
CASK dependent serine protein kinase
CCA common carotid artery
CD31 see also PECAM-1
CDB Cdc42 binding domain of WASP
vi
aCINC cytokine-induced neutrophil chemo-attractant protein
CNTF ciliary neurotrophic factor
CNS central nervous system
Cox-2 cyclooxygenase-2
CSF cerebrospinal fluid
CSLM confocal scanning laser microscopy
DAPI 4,6-diaminido-2-phenylidole
DNA deoxyribonucleic acid
EAAT1–3 excitatory amino acid transporters 1–3
ECA external carotid artery
ECL enhanced chemoluminescence
EDTA ethylenediaminetetraacetic acid
ELAM-1 endothelial leukocyte adhesion molecule-1
ELISA enzyme-linked immunosorbent assay
eNOS endothelial nitric-oxide synthase
ER endoplasmic reticulum
ERK-1/2 extracellular-signal-regulated kinases-1/2
ESAM endothelial selective adhesion molecule
ET1 endothelin 1
17ßE 17ß-estradiol
317ßE-d3 D-labeled 17ß-estradiol
17ßEG 17ß-estradiol-17ß-D-glucuronide
FK506 synonym for tacrolimus (immunosuppressant)
FKBP FK506 binding proteins
FMOCCI 9-fluorenylmethyl chloroformate
GABA gamma aminobutyric acid
GAP GTPase activating protein
GDI GDP dissociation inhibitor
GDNF glial-derived neurotrophic factor
GDP guanine diphosphate
GEF guanine exchange factor
GFAP glial fibrillary acidic protein
GLUT1 glucose transporter 1