Identification of novel chemokine and chemokine like mechanisms in leukocyte adhesion and atherosclerotic lesion formation [Elektronische Ressource] / vorgelegt von Regina Krohn

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Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2008
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Identification of Novel Chemokine and Chemokine-
like Mechanisms in Leukocyte Adhesion and
Atherosclerotic Lesion Formation

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
RWTH Aachen University zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften genehmigte Dissertation
vorgelegt von


Diplom-Biologin
Regina Krohn

aus Xanten, Deutschland

Berichter:
Universitätsprofessor Dr. med. Christian Weber
Privatdozent Dr. rer. nat. Christoph Peterhänsel
Tag der mündlichen Prüfung: 11.06.2008

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
The results of this work were in part published in:

Bernhagen, J., Krohn, R., Lue, H., Gregory, J. L., Zernecke, A., Koenen, R. R., Dewor,
M., Georgiev, I., Schober, A., Leng, L., Kooistra, T., Fingerle-Rowson, G., Ghezzi, P.,
Kleemann, R., McColl, S. R., Bucala, R., Hickey, M. J., Weber, C. (2007). MIF is a
noncognate ligand of CXC receptors in inflammatory and atherogenic cell recruitment. Nat
Med 13, 587-96.

Krohn, R., Raffetseder, U., Bot, I., Zernecke, A., Shagdarsuren, E., Liehn, E. A., von
Sandbrick, J. P., Nelson, P. J., Biessen, E. A., Mertens, P. R., Weber, C. (2007). Y-box
binding protein-1 controls CC chemokine ligand-5 (CCL5) expression in smooth muscle
cells and contributes to neointima formation in atherosclerosis-prone mice. Circulation
116, 1812-20.





Table of Contents

Table of Contents
Tabel of contents…………………………………………………………...…………….i
Abbreviations…………………………………………………………………….............v

I Introduction ........................................................................................................... 1
I.1 Atherosclerosis ...................................................................................................... 1
I.1.1 Therapeutic strategies................................................................................. 2
I.2 Chemokines in atherosclerosis .............................................................................. 3
I.2.1 The chemokine RANTES in atherosclerosis.............................................. 5
RANTES expression .................................................................................. 6
Transcription factor YB-1 .......................................................................... 7
I.2.2 The chemokine-like function chemokine Macrophage Migration
Inhibitory Factor (MIF) .............................................................................. 8
MIF and atherosclerosis ............................................................................. 8
MIF structure and functional features ........................................................ 9
MIF interaction partners ........................................................................... 10
I.3 Aim of this study ................................................................................................. 13
II Material and Methods.......................................................................................... 15
II.1 General equipment............................................................................................... 15
II.2 General solutions ................................................................................................. 16
II.3 Mice..................................................................................................................... 16
II.4 Chemokines, antagonists, and recombinant proteins .......................................... 16
II.5 Antibodies............................................................................................................ 18
II.5.1 Primary Antibodies................................................................................... 18
II.5.2 Directly conjugated antibodies ................................................................. 18
II.5.3 Blocking antibodies .................................................................................. 19
II.5.4 Isotype controls ........................................................................................ 19
II.5.5 Secondary antibodies................................................................................ 20
II.6 Mammalian cell culture....................................................................................... 20
II.6.1 Culturing of adherent cell monolayers ..................................................... 20
II.6.2 Culturing of cells in suspension ............................................................... 21
II.6.3 Freezing and thawing of mammalian cells............................................... 21
II.6.4 Isolation of PBMC.................................................................................... 23
II.6.5 Isolation of T cells .................................................................................... 23
II.6.6 Isolation of monocytes ............................................................................. 23
II.6.7 Isolation of neutrophils............................................................................. 23
II.7 RNA and DNA techniques .................................................................................. 24
II.7.1 Transformation of E. coli.......................................................................... 24
Bacteria growth medium .......................................................................... 24
i Table of Contents

Preparation of heat-shock competent E. coli............................................ 25
Heat-shock transformation of competent E. coli...................................... 25
II.7.2 Plasmids.................................................................................................... 26
II.7.3 Miniprep: Small-scale purification of plasmid DNA ............................... 27
II.7.4 Midiprep and Maxiprep: Large-scale purification of plasmid DNA........ 27
II.7.5 Restriction endonuclease digestion of DNA ............................................ 28
II.7.6 Agarose gel electrophoresis...................................................................... 28
II.7.7 Quantification of DNA and RNA............................................................. 29
II.7.8 Sequencing of DNA ................................................................................. 29
II.7.9 Transfection of eukaryotic cells ............................................................... 29
Stable transfection of L1.2 and HEK-293 cells........................................ 29
Transient transfection of Jurkat T cells, VSMC, HCASMC and L1.2
cells........................................................................................................... 29
II.7.10 Quantitative real time-PCR ...................................................................... 30
II.8 Protein assays ...................................................................................................... 32
II.8.1 Electrophoretic mobility shift assay (EMSA) .......................................... 32
II.8.2 Enzyme-linked immunosorbant assay (ELISA)....................................... 33
II.8.3 Flow cytometry......................................................................................... 34
II.8.4 Coimmunoprecipitation............................................................................ 35
II.8.5 SDS-polyacrylamide gel electrophoresis (PAGE) ................................... 36
II.8.6 Western blot analysis................................................................................ 37
II.8.7 Histochemistry.......................................................................................... 38
II.8.8 Immunofluorescence ................................................................................ 40
II.9 Functional assays................................................................................................. 40
II.9.1 Luciferase reporter assay.......................................................................... 40
II.9.2 Parallel plate flow chamber adhesion assay ............................................. 41
II.9.3 αLβ2 integrin activation assay ................................................................. 41
II.9.4 Calcium mobilization assay...................................................................... 42
II.10 Animal experiments............................................................................................. 43
II.10.1 Mouse model of arterial wire-injury and lentiviral transduction.............. 43
II.10.2 Mouse model of atherosclerotic disease progression ............................... 44
II.10.3 Ex vivo perfusion and intravital microscopy of murine carotid arteries... 44
II.10.4 Bone marrow repopulation ....................................................................... 45
Model of acute inflammation in the peritoneal cavity.............................. 45
II.11 Data illustration and statistical analysis............................................................... 46
III Results ................................................................................................................. 47
III.1 The Role of YB-1 in RANTES-mediated atherosclerosis.....