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Interactions of Bartonella Henselae with Myeloid Angiogenic Cells and Consequences for Pathological Angiogenesis

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Interactions of Bartonella Henselae with Myeloid Angiogenic Cells and Consequences for Pathological Angiogenesis Interactions of Bartonella henselae with Myeloid Angiogenic Cells and Consequences for Pathological Angiogenesis Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Fiona OʼRourke aus Calgary (Kanada) Frankfurt am Main 2015 vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität als Dissertation angenommen. Dekanin: Prof. Dr. Meike Piepenbring 1. Gutachter: Prof. Dr. Volker Müller 2. Gutachter: Prof. Dr. Volkhard A. J. Kempf Datum der Disputation: 02.12.2015 I Table of Contents 1. Introduction .................................................................................................... 1 1.1 Bartonella ................................................................................................................................1 1.2 Bartonella henselae ..................................................................................................................2 1.3 Infection-associated pathological angiogenesis ..........................................................................2 1.4 B. henselae pathogenicity strategy ............................................................................................4 1.4.1 Mitogenic stimulus ....................................................................................................................... 6 1.4.2 Inhibtion of apoptosis .................................................................................................................. 7 1.4.3 HIF/ VEGF ..................................................................................................................................... 7 1.4.4 Myeloid cells and the inflammatory response............................................................................. 8 1.4.5 Immune evasion ........................................................................................................................... 9 1.5 B. henselae pathogenicity factors ............................................................................................ 10 1.5.1 Bartonella Adhesin A (BadA) ...................................................................................................... 10 1.5.2 VirB/D4 Type IV Secretion System (T4SS) .................................................................................. 11 1.5.3 Interaction of BadA and T4SS ..................................................................................................... 12 1.6 Angiogenesis .......................................................................................................................... 13 1.6.1 Development of the vascular network ...................................................................................... 13 1.6.2 The angiogenic process .............................................................................................................. 13 1.6.3 Angiogenesis in health and disease ........................................................................................... 16 1.7 Tumor angiogenesis ................................................................................................................ 17 1.8 Angiogenic progenitor cells ..................................................................................................... 18 1.8.1 Endothelial Progenitor Cells (EPCs) ............................................................................................ 18 1.8.2 Myeloid Angiogenic Cells (MACs) .............................................................................................. 19 1.9 Monocytes and macrophages .................................................................................................. 22 1.9.1 Macrophage functions ............................................................................................................... 22 1.9.2 Macrophage polarization ........................................................................................................... 23 1.10 Macrophages and angiogenesis ............................................................................................. 25 1.10.1 Macrophages and pathological angiogenesis .......................................................................... 27 1.11 Tumor Associated Macrophages (TAM)s ................................................................................ 27 1.12 Interaction of bacteria with stem and progenitor cells ........................................................... 30 1.13 Interaction of B. henselae with MACs .................................................................................... 32 2. Objectives ..................................................................................................... 33 3. Materials and Methods ................................................................................. 34 3.1 Laboratory equipment and analytical instruments ................................................................... 34 3.2 Laboratory consumables ......................................................................................................... 35 3.3 Chemicals and reagents .......................................................................................................... 36 3.4 Kits ......................................................................................................................................... 37 3.5 Bacteria .................................................................................................................................. 38 3.5.1 Bacterial strains and growth conditions .................................................................................... 38 3.5.2 Preparation of Bartonella henselae bacterial stocks ................................................................. 38 II 3.6 Cell culture ............................................................................................................................. 39 3.6.1 Ethics statement ........................................................................................................................ 39 3.6.2 Human umbilical vein endothelial cells (HUVECs) ..................................................................... 40 3.6.3 Isolation and cultivation of Myeloid Angiogenic Cells (MACs) .................................................. 40 3.7 Infection experiments ............................................................................................................. 42 3.8 Electron microscopy ................................................................................................................ 42 3.9 Nicoletti apoptosis assay ......................................................................................................... 43 3.10 HIF-1 Western Blot ................................................................................................................ 43 3.10.1 Cell stimulation and preparation of protein extracts .............................................................. 44 3.10.2 Sodium dodecyl sulfate- polyacrylamice gel electrophoresis (SDS-PAGE) .............................. 44 3.10.3 Western Blot ............................................................................................................................ 44 3.11 Spheroid assay of sprouting angiogenesis .............................................................................. 47 3.12 Matrigel capillary formation assay ......................................................................................... 48 3.13 16S rDNA PCR ....................................................................................................................... 49 3.14 Flow cytometry ..................................................................................................................... 51 3.15 Immunohistochemistry ......................................................................................................... 53 3.16 Quantitative real-time PCR (qRT-PCR) .................................................................................... 56 3.16.1 Isolation of total RNA ............................................................................................................... 56 3.16.2 Reverse Transcription PCR (RT-PCR) ........................................................................................ 56 3.16.3 Quantitative RT-PCR ................................................................................................................. 57 3.17 Microarray gene expression profiling ..................................................................................... 58 3.18 Secretome analysis ............................................................................................................... 60 3.19 Enzyme linked immunosorbent assays (ELISAs) ...................................................................... 60 3.20 Statistical analysis ................................................................................................................. 61 4. Results .......................................................................................................... 62 4.1 B. henselae invades MACs and resides in intracellular vacuoles .......................................... 62 4.2 B. henselae infected MACs maintain high viability ............................................................. 62 4.3 B. henselae infection of MACs induces BadA dependent HIF-1 activation ..........................
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