Investigating Biomarkers of Keloid Scarring.

PhD (Med Res)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- 4 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

Effectiveness and acceptability of myo-inositol nutritional supplement in the prevention of gestational diabetes (EMmY): a protocol for a randomised, placebo-controlled, double-blind pilot trial.

INTRODUCTION: Gestational diabetes increases maternal and offspring complications in pregnancy and cardiovascular complications in the long term. The nutritional supplement myo-inositol may prevent gestational diabetes; however, further evaluation is required, especially in multiethnic high-risk mothers. Our pilot trial on myo-inositol to prevent gestational diabetes will evaluate trial processes, assess acceptability to mothers and obtain preliminary estimates of effect and cost data prior to a large full-scale trial. METHODS AND ANALYSIS: EMmY is a multicentre, placebo-controlled, double-blind, pilot, randomised trial, with qualitative evaluation. We will recruit pregnant women at 12-15+6 weeks' gestation, with gestational diabetes risk factors, from five maternity units in England between 2018 and 2019. We will randomise 200 women to take either 2 g of myo-inositol powder (intervention) or placebo, twice daily until delivery. We will assess rates of recruitment, randomisation, adherence to intervention and follow-up. Gestational diabetes will be diagnosed at 24-28 weeks as per the National Institute for Health and Care Excellence (NICE) criteria (fasting plasma glucose: ≥5.6 mmol/L and 2-hour plasma glucose: ≥7.8 mmol/L). We will assess the effects of myo-inositol on glycaemic indices at 28 weeks and on other maternal, fetal and neonatal outcomes at postnatal discharge. Qualitative evaluation will explore the acceptability of the trial and the intervention among women and healthcare professionals. Cost data and health-related quality of life measures will be captured. We will summarise feasibility outcomes using standard methods for proportions and other descriptive statistics, and where appropriate, report point estimates of effect sizes (eg, mean differences and relative risks) and associated 95% CIs. ETHICS AND DISSEMINATION: Ethical approval was obtained through the London Queen Square Research Ethics Committee (17/LO/1741). Study findings will be submitted for publication in peer-reviewed journals. Newsletters will be made available to participants, healthcare professionals and members of Katie's Team (a patient and public advisory group) to disseminate. TRIAL REGISTRATION NUMBER: ISRCTN48872100. PROTOCOL VERSION AND DATE: Version 4.0, 15 January 2018

Effectiveness and acceptability of myo-inositol nutritional supplement in the prevention of gestational diabetes (EMmY): a protocol for a randomised, placebo-controlled, double-blind pilot trial.

INTRODUCTION: Gestational diabetes increases maternal and offspring complications in pregnancy and cardiovascular complications in the long term. The nutritional supplement myo-inositol may prevent gestational diabetes; however, further evaluation is required, especially in multiethnic high-risk mothers. Our pilot trial on myo-inositol to prevent gestational diabetes will evaluate trial processes, assess acceptability to mothers and obtain preliminary estimates of effect and cost data prior to a large full-scale trial. METHODS AND ANALYSIS: EMmY is a multicentre, placebo-controlled, double-blind, pilot, randomised trial, with qualitative evaluation. We will recruit pregnant women at 12-15+6 weeks' gestation, with gestational diabetes risk factors, from five maternity units in England between 2018 and 2019. We will randomise 200 women to take either 2 g of myo-inositol powder (intervention) or placebo, twice daily until delivery. We will assess rates of recruitment, randomisation, adherence to intervention and follow-up. Gestational diabetes will be diagnosed at 24-28 weeks as per the National Institute for Health and Care Excellence (NICE) criteria (fasting plasma glucose: ≥5.6 mmol/L and 2-hour plasma glucose: ≥7.8 mmol/L). We will assess the effects of myo-inositol on glycaemic indices at 28 weeks and on other maternal, fetal and neonatal outcomes at postnatal discharge. Qualitative evaluation will explore the acceptability of the trial and the intervention among women and healthcare professionals. Cost data and health-related quality of life measures will be captured. We will summarise feasibility outcomes using standard methods for proportions and other descriptive statistics, and where appropriate, report point estimates of effect sizes (eg, mean differences and relative risks) and associated 95% CIs. ETHICS AND DISSEMINATION: Ethical approval was obtained through the London Queen Square Research Ethics Committee (17/LO/1741). Study findings will be submitted for publication in peer-reviewed journals. Newsletters will be made available to participants, healthcare professionals and members of Katie's Team (a patient and public advisory group) to disseminate. TRIAL REGISTRATION NUMBER: ISRCTN48872100. PROTOCOL VERSION AND DATE: Version 4.0, 15 January 2018

Metformin in the prevention of type 2 diabetes after gestational diabetes in postnatal women (OMAhA): a UK multicentre randomised, placebo-controlled, double-blind feasibility trial with nested qualitative study

Objective: To determine the feasibility of a definitive trial of metformin to prevent type 2 diabetes in the postnatal period in women with gestational diabetes. Design: A multicentre, placebo-controlled, double-blind randomised feasibility trial with qualitative evaluation. Setting: Three inner-city UK National Health Service hospitals in London. Participants: Pregnant women with gestational diabetes treated with medication. Interventions: 2 g of metformin (intervention) or placebo (control) from delivery until 1 year postnatally. Primary outcome measures: Rates of recruitment, randomisation, follow-up, attrition and adherence to the intervention. Secondary outcome measures: Preliminary estimates of glycaemic effects, qualitative exploration, acceptability of the intervention and costs. Results: Out of 302 eligible women, 57.9% (175/302) were recruited. We randomised 82.3% (144/175) of those recruited, with 71 women in the metformin group and 73 women in the placebo group. Of the participants remaining in the study and providing any adherence information, 54.1% (59/109) took at least 75% of the target intervention dose; the overall mean adherence was 64% (SD 33.6). Study procedures were found to be acceptable to women and healthcare professionals. An increased perceived risk of developing type 2 diabetes, or a positive experience of taking metformin during pregnancy, encouraged participation and adherence to the intervention. Barriers to adherence included disruption to the medication schedule caused by the washout periods ahead of each study visit or having insufficient daily reminders. Conclusions: It is feasible to run a full-scale definitive trial on the effectiveness of metformin to prevent type 2 diabetes in women with gestational diabetes, during the early postnatal period. Adherence and engagement with the study could be improved with more regular reminders and potentially the addition of ongoing educational or peer support to reinforce messages around type 2 diabetes prevention

Type VI Collagen Regulates Dermal Matrix Assembly and Fibroblast Motility

This work was funded by a PhD studentship from the Queen Mary EPSRC Doctoral Training Account and the BBSRC project grant BB/J000914/