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 ..........................