Investigating Biomarkers of Keloid Scarring Zoe Drymoussi 2015 A thesis presented for the degree of Doctor of Philosophy Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London 1 Declaration I, Zoe Drymoussi, declare that the work presented in this thesis is my own and has not been submitted in any form for another degree or diploma at any university or other institute of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. Zoe Drymoussi, PhD Student 1st August 2015 2 Abstract Keloids are fibroproliferative scars that form in response to abnormal healing processes. The extracellular matrix (ECM) remodelling of the dermis in the maturation phase of normal wound healing is insufficient in keloids, leading to excessive ECM proteins being deposited in the granulation tissue. Keloid scars are unique to humans, and show increased prevalence in darker skin types. Current treatments rarely lead to permanent regression, and despite decades of study, the key molecular processes responsible for keloid scarring are still largely elusive. The research presented in this thesis aims to investigate markers of keloid scars, and to examine the impact of both the dermis and epidermis in keloid pathogenesis. Histological examination of the keloid scars showed a thickened epidermis and densely collagenous dermis, both of which demonstrated a higher level of cell proliferation and myofibroblast expression, as compared to normal skin. Differences between the central and marginal regions of the scars were also noted. Protein expression patterns of Matrix Metalloproteinases (MMPs) 1, 2, 13 and 14 were examined in formalin fixed paraffin embedded biopsies from the same keloids (n=10) and healthy skin (n=9). MMPs 2 and 14 showed a distinct pattern of protein expression across the central, marginal, and adjacent non-keloid regions of the scar. In the dermis, MMP2 and 14 were most evident at the leading edge, whereas the epidermis revealed a strong but variable pattern along all regions. This was in contrast to healthy skin, where expression in both epidermis and dermis was much lower overall, and showed a more uniform pattern across the tissue. Gene expression levels of key ECM and adhesion molecules, as well as select target genes across 10 signal transduction pathways, were analysed through Real-Time qPCR in keloid scars and healthy control skin from unaffected individuals (n=5). MMPs 1, 13 and 14 gene expression levels were significantly increased by 52, 24, and 3-fold respectively in keloid, compared to normal tissue. SPP1 and SPARC were also upregulated by 29.6 and 9.2-fold respectively, in keloid tissue. Hedgehog and Wnt pathway target genes were significantly altered in keloid tissue, with WISP1 and VEGFA being overexpressed by 10 and 2-fold respectively, and BMP2 and BMP4 underexpressed by 5 and 3.7-fold respectively. Eighteen of the most altered of these 168 genes were examined further in primary cultured fibroblasts (n=6) and keratinocytes (n=4), from both keloid and healthy control skin. Gene expression profiles appeared complementary between fibroblast and keratinocyte cell-types, either of which did not necessarily match the gene expression profiles demonstrated earlier in whole tissue. This might be due to the epidermal and dermal cells being in isolation from each other, and therefore 3D culture containing both cell-types would be more representative of the original tissue environment. SPP1 was the only gene that consistently showed significant over- 3 expression in whole tissue and primary keloid cells. Protein expression levels of SPP1 appeared increased in keloid tissue compared to normal skin, particularly in the basal epidermis and edge dermis of the keloid scars. In summary, a high level of inter-patient variability was observed throughout all biomarker investigations, though the leading edge of all keloids consistently appeared to be the most active region. The consistent detection of changes in expression SPP1 indicates a potential role for osteogenic-linked signalling in the fibrotic nature of keloid scarring. 4 Table of Contents Investigating Biomarkers of Keloid Scarring ............................................................... 1 Declaration ............................................................................................................... 2 Abstract .................................................................................................................... 3 Table of Contents ..................................................................................................... 5 List of Figures ........................................................................................................... 9 List of Tables ........................................................................................................... 12 Abbreviations ......................................................................................................... 14 Acknowledgements ................................................................................................ 17 1 Introduction ...................................................................................................... 18 1.1 Human Skin ............................................................................................................................... 19 1.1.1 Epidermis .................................................................................................................................. 19 1.1.2 Dermis ........................................................................................................................................ 22 1.2 Normal cutaneous wound healing ................................................................................... 26 1.2.1 Haemostasis and inflammation ...................................................................................... 29 1.2.2 Proliferation and re-epithelialisation .......................................................................... 30 1.2.3 Tissue remodelling ................................................................................................................ 31 1.3 Aberrant scarring ................................................................................................................... 31 1.3.1 Hypertrophic scarring......................................................................................................... 32 1.3.2 Other dermal fibroses .......................................................................................................... 34 1.4 Keloid Scarring ........................................................................................................................ 37 1.4.1 Clinical aspects ....................................................................................................................... 37 1.4.2 Clinical management of keloids ...................................................................................... 38 1.4.3 Genetics of keloid scarring ................................................................................................ 41 1.4.4 ECM remodelling in keloids .............................................................................................. 43 1.4.5 Fibrogenic Signalling Pathways ..................................................................................... 46 1.5 Research aims and objectives ............................................................................................ 54 5 1.5.1 Research aim ........................................................................................................................... 54 1.5.2 Objectives .................................................................................................................................. 54 2 Materials and Methods ..................................................................................... 55 2.1 Patient samples ....................................................................................................................... 56 2.1.1 Tissue fixation and wax embedding .............................................................................. 59 2.1.2 Snap-freezing fresh tissue ................................................................................................. 61 2.2 Protein assays .......................................................................................................................... 61 2.1.1 Haematoxylin and Eosin (H&E) .......................................................................................... 61 2.1.2 Herovici’s special stain ........................................................................................................... 61 2.2.1 3,3’-Diaminobenzidine (DAB) Immunohistochemistry (IHC) ............................ 63 2.2.2 Immunofluorescence ........................................................................................................... 65 2.2.3 Protein sample preparation ............................................................................................. 65 2.2.4 Western Blotting ................................................................................................................... 69 2.3 Nucleic acid assays ................................................................................................................. 75 2.3.1 RNA extraction from tissue