Role of the peritoneum and transforming growth factor β in the aetiology of endometriosis

Endometriosis is a benign inflammatory disorder, defined by the presence of endometrial \ntissue outside the uterus with lesions typically found on the pelvic peritoneum in close \nassociation with the peritoneal mesothelium. The prevalence of endometriosis is estimated at \n6-10% of women of reproductive age and it is associated with chronic pelvic pain, \ndysmenorrhoea, dyspareunia and infertility. Surgical excision can provide symptom relief, but \nsymptoms recur in up to 75% of surgical cases and available medical treatments have \nundesirable side effects. New treatments are limited due to our poor understanding of the \naetiology of endometriosis. To date, the majority of research has focused on changes within \nthe ectopic endometrial tissue to explain the development of endometriosis lesions, however, \nthere is increasing evidence that the peritoneal mesothelium plays an important role. \n \nAccording to Sampson’s theory of retrograde menstruation, ectopic endometrial cells must \nfirst have to attach to the surface of the peritoneum before undergoing invasion, proliferation, \nand neoangiogenesis. TGF-β1 is an inflammatory growth factor that regulates a variety of \ncellular functions including; cell adhesion, cell invasion and angiogenesis. Levels of TGF-β1 \nare increased in the peritoneal fluid of women with endometriosis compared to controls and \nresearch using a mouse model of endometriosis has demonstrated TGF-β1-null mice to \ndevelop smaller and fewer endometriosis lesions than their wild-type controls. Together these \nstudies suggest that TGF-β1 plays a major role in the development of peritoneal \nendometriosis lesions and that targeting this pathway may be of therapeutic potential. \nHowever the functional role that TGF-β1 plays in peritoneal endometriosis is still unclear. \nThe overall aim of this thesis was to determine if the peritoneal expression of TGF-β and its \ntarget genes are disrupted in women with endometriosis and whether this could contribute to \nthe development of endometriosis lesions. \n \nOur first aim was to determine if the peritoneum was a source of TGF-β expression and if \nreception and/or signalling were altered in women with endometriosis. We found that the \nperitoneal fluid of women with endometriosis contained increased concentrations of TGF-β1 \nand that peritoneal mesothelial cells adjacent to endometriosis lesions expressed significantly \nhigher levels of TGF-β1 mRNA. Analysis of TGF-β signalling targets within the peritoneum \nshowed that women with endometriosis express significantly higher levels of TGF-β targeted \ngenes associated with tumourigenesis processes including; EMT, invasion and angiogenesis. \nWe next asked if there are changes in the metabolic phenotype of endometriosis lesions and \nperitoneum in women with endometriosis, similar to the metabolic changes seen in tumour \ncells. Endometriosis lesions expressed markers of aerobic glycolysis, including HIF-1α, \nsuggesting that lesions may metabolise in a similar fashion to tumours. Furthermore, \nperitoneum adjacent to endometriosis lesions expressed significantly higher levels of markers \nof aerobic glycolysis, including HIF-1α, suggesting that the peritoneum may feed forward \nhigh-energy lactate, a by-product of glycolysis, to the endometriosis lesions. These \nobservations were supported by significantly increased lactate concentrations within the \nperitoneal fluid of women with endometriosis that positively correlated with levels of TGF- \nβ1. TGF-β1 was shown to increase expression of glycolysis markers and lactate expression in \nperitoneal mesothelial cells in-vitro, suggesting TGF-β may regulate this change. \n \nWe then determined if TGF-β1 was responsible for the change in peritoneal mesothelial cell \nmetabolism by signalling through the ID-HIF-1α pathway. ID proteins are transcription \nfactors, whose expression is regulated by the TGF-β superfamily. We found expression of \nID1 mRNA to be increased in the peritoneum of women with endometriosis and that TGF-β1 \nsignificantly increased ID1 but decreased ID2 expression in the peritoneal mesothelial cells \nin-vitro. ID1 siRNA knockdown decreased glycolysis initiator HIF-1α mRNA and ID2 \nsiRNA knockdown increased HIF-1α mRNA and lactate expression, suggesting TGF-β1 \nregulates mesothelial cell metabolism, at least in part, through the ID pathway. \nID transcription factors are also known to regulate VEGF-A expression, therefore we next \ndetermined if TGF-β1 induced ID1 and/or reduced ID2 expression in the peritoneum \npromoted VEGF-A mRNA and protein expression. VEGF-A, a cytokine essential for \nangiogenesis, was significantly increased in the peritoneal fluid of women with endometriosis \nand levels positively correlated with TGF-β1. TGF-β1 increased VEGF-A expression in-vitro \nand siRNA knockdown of ID1 decreased and siRNA knockdown of ID2 increased VEGF-A \nmRNA and protein expression in the peritoneal mesothelial cell. \n \nLastly we aimed to determine if TGF-β1 induces EMT in peritoneal mesothelial cells. The \nperitoneum of women with endometriosis expressed higher levels of EMT markers. Exposure \nof peritoneal mesothelial cells to TGF-β1 in-vitro induced EMT-like changes, including; \nchanges to cell morphology, gene expression and protein localisation. Peritoneal mesothelial \ncells were more migratory and invasive suggesting that TGF-β1 induced EMT may disrupt \nthe mesothelial cell monolayer allowing ectopic endometrial cells to invade the peritoneal \ntissue or for peritoneal mesothelial cells to migrate into endometriosis lesions. \nIn summary, the novel data presented in this thesis provides evidence that the pelvic \nperitoneum and in particular the peritoneal mesothelial cell may play a critical role in the \naetiology of peritoneal endometriosis. Expression of TGF-β1 and its transcriptional target \ngenes are dysregulated in the peritoneum of women with endometriosis. TGF-β regulated ID \nexpression may induce changes in cell metabolism and promote neoangiogenesis, prompting \nperitoneal endometriosis lesion development. Furthermore other TGF-β1 transcriptional \ntargets, such as those involved in EMT, are also altered in the peritoneum of women with \nendometriosis and may contribute the development and maintenance of lesion formation. \nThese results point to a central role for TGF-β1 expression and signalling in the aetiology of \nperitoneal endometriosis. Furthermore it is likely that changes within the expression profile \nand morphology of the peritoneal mesothelial cells contribute to peritoneal lesion formation. \nDrugs that target these pathways may provide new therapies for women with endometriosis.