Peritoneal adhesions are fibrous bands of tissue joining normally separated organs and occur frequently as a side effect of abdominal surgery. Despite being a major cause of infertility, chronic pelvic pain and bowel obstruction, there are currently no adequate methods of therapy or prevention. Post-operative adhesions are known to develop from fibrin clots deposited between damaged serosa in combination with insufficient fibrinolytic capacity. However, the contribution of surface mesothelial cells to the regulation of fibrin turnover and subsequent scar formation is not well understood. This thesis addresses the hypothesis that mesothelial cells actively participate in adhesion formation by promoting coagulation, inhibiting fibrinolysis and undergoing epithelial-mesenchymal transition (EMT) to upregulate extracellular matrix (ECM) deposition. Primary human omental cells, in addition to primary fibroblasts for comparison, were incorporated in an in vitro assay system aimed at identifying potential molecular biomarkers or therapeutic targets for the prevention of adhesion formation. We used a combination of exposure to the pro-fibrotic growth factor, TGF-β1, and culturing on a fibrin film or gel in vitro to mimic the post-injury environment during peritoneal repair. We identified an exaggerated TGF-β1-induced fibrogenic response by human pulmonary fibroblasts (HPFs) but not by a transformed mesothelial cell line, MeT-5A, suggesting primary mesothelial cells were preferable. Human omental cells (HOMCs) were isolated by protease digestion and characterised using a panel of mesothelial markers. TGF-β1 and fibrin substrates induced EMT in HOMCs from three patients however, expression of alpha-SMA and procollagens was highly variable. Furthermore, culturing cells on a fibrin gel enhanced the effects observed with TGF-β1 in HOMCs and promoted a hypercoagulable state (decreased tissue factor pathway inhibitor, TFPI) and reduced fibrinolysis (increased plasminogen activator inhibitor-1, PAI-1). Interestingly, HOMCs also expressed matrix cross-linking factor, tissue transglutaminase (TG2), in response to TGF-β1 when grown on a fibrin gel in a similar manner to HPFs suggesting they may be involved in the stabilisation of a newly formed adhesion. HPFs also promoted coagulation and inhibited fibrinolysis however the response appeared to be dependent on TGF-β1 rather than fibrin. In summary, this thesis indicates that fibrin is a key mediator in modulating HOMCs behaviour during the post-injury response and adhesion formation. High patient variation in the fibrogenic response suggests HOMCs undergo EMT and whilst the transitioned cells do not always contribute to excess collagen deposition, they may be involved in the stabilisation of newly deposited ECM. HOMCs potentially play a dual role during peritoneal remodeling by promoting coagulation and regulating fibrin persistence. TFPI may serve as a novel potential molecular biomarker or therapeutic target for the prevention of post-operative adhesions.
|Date of Award||31 Dec 2016|
- The University of Manchester
|Supervisor||Sarah Herrick (Supervisor) & Michael Sherratt (Supervisor)|