Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Angiogenesis is maintained by a balance of pro-and antiangiogenic factors. Hence, concomitantly with the upregulation of proangiogenic factors, antiangiogenic proteins ADAMTS1 and regulator of calcineurin 1 (RCAN1) were upregulated by P CM treatment of HUVECs. They were subsequently shown to limit endothelial cell network formation and proliferation in response to P CM. Finally, the role of PGF2α in angiogenesis was investigated using two in vivo models. PGF2α treatment did not increase angiogenesis in a sponge matrigel mouse model. In a xenograft mouse model, PGF2α-FP signalling increased expression of angiogenic factors in human epithelial cells and mouse stroma but this did not enhance microvessel density. Taken together, this thesis had highlighted that PGF2α-FP receptor signalling stimulates expression of pro-and antiangiogenic factors that in turn regulate endothelial cell function. However, in vivo studies demonstrate that PGF2α-FP receptor interaction does not impact on the level of angiogenesis but may control other aspects of vascular function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563084 |
| Date | January 2010 |
| Creators | Keightley, Margaret Claire |
| Contributors | Sales, Kurt. ; Jabbour, Henry |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4809 |
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