Monocytes and neutrophils [Elektronische Ressource] : accomplices in initiation and development of atherosclerosis / Maik Drechsler

Maik Röder

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• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Monocytes and Neutrophils – Accomplices in Initiation and
Development of Atherosclerosis

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH
Aachen University zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation

vorgelegt von



Master of Science
Maik Drechsler
aus Pirna (Sächsische Schweiz), Deutschland

Berichter:
Universitätsprofessor Dr. med. Christian Weber
Universitätr. rer. nat. Jürgen Bernhagen

Tag der mündlichen Prüfung: 06.07.2011

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
Monocytes and

Neutrophils-

Accomplices in
Initiation and

Development

of
Atherosclerosis

Der Fakultät für Mathematik, Informatik und
Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule
Aachen vorgelegte Dissertation
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer nat.)

von MSc Maik Drechsler
aus Pirna
The results of this work were partially published in:

Drechsler, M., Megens, R.T., van Zandvoort, M., Weber, C., Soehnlein, O. Hyperlipidemia-
triggered neutrophilia promotes early atherosclerosis. Circulation 122(18) 1837-1845 (2010).

*Oliver Soehnlein , Maik Drechsler*, Yvonne Döring, Santosh Vijayan, Dirk Lievens, Helene
Hartwig, Delia Projahn, Rory R. Koenen, Mihail Hristov, Esther Lutgens, Alma Zernecke,
Christian Weber. Chemokine receptor axes sequentially control the prominent
proatherogenic function of classical monocytes. JACC (submitted) (2011)
Table of Contents
Table of Contents
Table of Contents ......................................................................................................................... i
Abbreviations .............................. v
1 Introduction ........................................................................................................................ 1
1.1 Atherosclerosis ............. 1
1.1.1 Getting there - The leukocyte adhesion cascade ................................................. 2
1.1.2 Trigger units – Chemokines and their receptors in atherosclerosis ..................... 4
1.2 Myeloid cells in atherosclerosis ................................................................................... 6
1.2.1 Monocytes ............................................ 7
1.2.2 Neutrophils ........................................................................ 12
1.2.3 Other myeloid cell subsets ................. 16
1.3 Aim of the study ........................................................................................................ 17
2 Material and Methods ..... 19
2.1 Material ..................................................................................................................... 19
2.1.1 General equipment and consumables ............................... 19
2.1.1.1 General equipment ..................................................................................... 19
2.1.1.2 Consumables .............................. 19
2.1.1.3 Kits .............................................................................................................. 19
2.1.2 Buffers, chemicals, media, solutions . 20
2.1.3 Peptides, oligonucleotides, antagonists and antibodies ................................... 20
2.1.4 Mice ................................................................................................................... 23
2.2 Methods .................... 24
2.2.1 Biomolecular Methods ....................................................................................... 24
2.2.1.1 RNA Isolation .............................. 24
2.2.1.2 RNA Quantification ..................................................................................... 24
i
Table of Contents
2.2.1.3 Reverse Transcription ................................................................................. 24
2.2.1.4 Real-time PCR ............................. 25
2.2.1.5 PCR Array .................................................................................................... 25
2.2.2 Protein Assays .... 26
2.2.2.1 Flow Cytometry .......................................................................................... 26
2.2.2.2 Fluorescence Activated Cell Sorting (FACS) ................ 27
2.2.2.3 Enzyme-linked immunosorbent assay (ELISA) ............................................ 27
2.2.2.4 Immunohistochemistry .............................................. 27
2.2.2.5 Immunfluorescence .................................................... 28
2.2.2.6 Myeloperoxidase (MPO) Assay of lung tissue ............ 28
2.2.3 Animal Experiments ........................................................................................... 28
2.2.3.1 Mouse model of atherosclerosis ................................ 28
2.2.3.2 Bone marrow transplantation .................................... 29
2.2.3.3 Blood drawing, animal dissection and organ preparation ......................... 29
2.2.3.4 Adoptive cell transfer ................................................................................. 30
2.2.3.5 Mobilization assays ..................... 30
2.2.3.6 In vivo depletion of leukocyte subsets ....................................................... 31
2.2.3.7 Intravital microscopy .................................................. 31
2.2.3.8 Model of acute peritonitis .......................................... 32
2.2.3.9 In vivo Two-Photon microscopy ................................. 32
2.2.4 Statistics ............................................................................. 33
3 Results .............................................................................................................................. 35
3.1. Role of neutrophils during the onset of atherosclerosis .......................................... 35
3.1.1 Hypercholesterolemia induces neutrophilia ..................... 35
3.1.2 Hyperlipidemia interferes with neutrophil homeostasis ................................... 36
3.1.3 Neutrophils infiltrate large arteries ................................... 37
ii
Table of Contents
3.1.4 Neutrophils are crucial during onset of atherosclerosis .................................... 39
3.1.5 Neutrophils express a variety of chemokine receptors ..... 40
3.1.6 Arterial neutrophil recruitment depends on CCR1, CCR2, CCR5 and CXCR2 .... 41
3.1.7 Endothelium in micro- and macrocirculation presents different chemokines . 43
3.1.8 Platelet derived CCL5 promotes arterial neutrophil recruitment ..................... 44
3.2 Importance of monocytes in atheroprogression ...................................................... 45
3.2.1 Hyperlipidemia induces a manifest monocytosis .............. 45
3.2.2 The CXCR2-CXCL1 (KC) axis is partially responsible for monocytosis ................ 46
+ -3.2.3 Gr1 monocytes but not Gr1 monocytes dominate atheroprogression ........... 48
3.2.4 Monocyte subsets differentially effect plaque phenotype ............................... 50
3.2.5 Chemokine receptors are key players in atherogenesis and -progression ....... 51
3.2.6 Monocytes employ chemokine receptors for arterial recruitment .................. 52
3.3 Neutrophil/Monocyte interaction in atherogenesis ................................................. 54
3.3.1 Neutrophils pave the way for monocytes in atherogenesis .............................. 54
4 Discussion......................................................................................................................... 57
4.1 Neutrophils initiate early atherosclerosis development .......................................... 57
4.1.1 Hypercholesterolemia alters neutrophil homeostasis ...... 57
4.1.2 Neutrophils fuel atherosclerosis during early stages ........................................ 58
4.1.3 Chemokine receptors trigger arterial recruitment of neutrophils .................... 60
4.1.4 Perspective – Neutrophils .................................................................................. 61
+4.2 Inflammatory Gr1 monocytes dominate atheroprogression .................................. 62
+4.2.1 New insights into HFD-induced Gr1 monocytosis ............ 62
+ 4.2.2 Gr1 monocytes dominate atheroprogression .................................................. 63
+4.2.3 Chemokine receptors differentially affect Gr1 monocytes at various levels ... 66
4.2.4 Perspectives – Monocytes ................................................................................. 68
4.3 Neutrophils and monocytes – ill alliance in atherogenesis ...... 69
iii
Table of Contents
Summary .................................................................................................................................. 71
Zusammenfassung ................................................................................................................... 73
Literature ................................................................................................................................. 75
Danksagung .............................. 85
Curriculum Vita