EFFECTS OF DIABETES AND HOXA3 UPON MACROPHAGE FUNCTION A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy Developmental Biology in the Faculty Life Sciences 2015 MATTHEW BURGESS FACULTY OF LIFE SCIENCES List of contents List of contents .............................................................................................................................. 2 List of figures ................................................................................................................................. 7 List of tables .................................................................................................................................. 9 Declaration .................................................................................................................................. 12 Copyright statement ................................................................................................................... 12 Acknowledgement ...................................................................................................................... 13 The author ................................................................................................................................... 14 1 Introduction ........................................................................................................................ 15 1.1 Cutaneous wound healing .......................................................................................... 15 1.1.1 Inflammatory phase ........................................................................................... 17 1.1.2 Inflammation and the chronic wound ................................................................ 19 1.1.3 Proliferation phase .............................................................................................. 19 1.1.4 The proliferation phase and chronic wounds ..................................................... 20 1.1.5 Neovascularisation .............................................................................................. 21 1.1.6 Neovascularisation and chronic wounds ............................................................ 23 1.1.7 Remodelling phase .............................................................................................. 25 1.2 Macrophages .............................................................................................................. 25 1.2.1 Origins of macrophages ...................................................................................... 25 1.2.2 Maturation and activation .................................................................................. 26 1.2.3 Myeloid cells and neovascularisation ................................................................. 28 1.2.4 Macrophages and diabetes ................................................................................. 29 1.3 Endothelial progenitor cells .............................................................................................. 30 1.3.1 Origins of endothelial progenitor cells....................................................................... 30 1.3.2 Isolation of endothelial progenitor cells ............................................................. 31 1.3.3 Roles of endothelial progenitor cells in neovascuarisation ................................ 35 1.4 Transcriptional basis of lineage plasticity ................................................................... 37 1.4.1 Reprogramming in haematopoiesis ........................................................................... 38 1.4.2 The epigenetic landscape – erythroid/monocyte fate........................................ 40 1.5 Hypothesis and Experimental Aims ............................................................................ 41 1.5.1 Investigation of the phenotypic differences between non-diabetic and diabetic macrophages ....................................................................................................................... 41 1) Differentiation of macrophages in models of diabetes .......................................... 41 2) Activation potential of diabetic macrophages ........................................................ 42 3) Interaction of diabetic macrophages with neovascularisation ............................... 42 2 1.5.2 Identification of transcription factors with monocytic to endothelial reprogramming potential.................................................................................................... 43 1) Identification of transcription factors to be tested for endothelial reprogramming potential .......................................................................................................................... 43 2) Development of an assay to screen transcription factors for monocytic to endothelial reprogramming ............................................................................................ 43 3) Assess the effects of the validated transcription factors upon THP-1 cell phenotype in the transdifferentiation assay ..................................................................................... 44 1.5.3 Investigation of the effects of Hoxa3 transcriptional activity upon macrophages 44 1) Effects of Hoxa3 upon macrophage development ................................................. 44 2) Effects of Hoxa3 upon macrophage activation ....................................................... 45 3) Effects of Hoxa3 upon macrophage interactions with neovascularisation ............ 45 2 Materials and methods ....................................................................................................... 46 2.1 Cell culture .................................................................................................................. 46 2.1.1 Cell lines .............................................................................................................. 46 2.1.2 Cell Culture .......................................................................................................... 46 RAW 264.7 cells............................................................................................................... 46 bEnd.5 ............................................................................................................................. 47 L929 cells ......................................................................................................................... 47 THP-1 cells ....................................................................................................................... 47 HUVECs ............................................................................................................................ 48 2.1.3 Primary cell lines ................................................................................................. 48 Murine primary bone marrow cells ................................................................................ 48 Macrophage activation ................................................................................................... 49 2.1.4 Cryostorage of cells ............................................................................................. 50 2.1.5 Nucleofection ...................................................................................................... 50 2.1.6 Conditioned medium culture .............................................................................. 54 Calcium phosphate transfection ..................................................................................... 54 Conditioned medium collection ...................................................................................... 55 Treatment of cells with SP.Hoxa3.mCherry conditioned medium ................................. 55 Pleiotrophin and macrophage colony stimulating factor supplemented medium......... 56 2.1.7 Neovascularisation assays ................................................................................... 56 2.2 PCR .............................................................................................................................. 58 2.2.1 RNA extraction .................................................................................................... 58 2.2.2 DNase treatment ................................................................................................. 58 2.2.3 cDNA generation ................................................................................................. 59 3 2.2.4 Quantitative PCRs ............................................................................................... 59 TaqMan real time PCRs ................................................................................................... 59 SYBR real-time PCRs ........................................................................................................ 59 Relative expression analysis ............................................................................................ 60 2.2.5 Generation of pCRII-TOPO pleiotrophin ............................................................. 64 2.2.6 Sub-cloning of pcDNA3.1/myc-His pleiotrophin ................................................. 65 2.2.7 Colony PCR .......................................................................................................... 66 2.3 Western blotting ......................................................................................................... 66 2.3.1 Reagents .............................................................................................................