Obstetrics and Gynaecology - Theses
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ItemCharacterisation of maternal pre-eclampsia susceptibility genes( 2015)Introduction: Pre-eclampsia (PE) has affected pregnant women throughout human history and remains a leading cause of maternal and fetal mortality and morbidity worldwide today. PE is clinically characterised by de novo hypertension and proteinuria developing after 20 weeks’ gestation that resolves with delivery of the fetus and placenta. Up to 8% of all pregnant women are afflicted with the disorder, which may necessitate premature delivery of the fetus in severe cases. The aetiology of the disorder remains unknown, although the placenta is widely accepted to be central to the pathogenesis of PE, as PE can occur in women with molar pregnancies, where there is no viable fetus. A family history of PE is a major risk factor, with heritability estimates of up to 54%. Genetic linkage and association studies in the Australian/New Zealand population previously identified susceptibility loci on chromosomes 2, 5 and 13 for PE. Bioinformatic analyses of these loci yielded the following candidate maternal PE susceptibility genes from various functional groups: ACVR1, ACVR1C, ACVR2A, INHA and INHBB from the activin/inhibin signalling group; ERAP1, ERAP2 and LNPEP from the M1 aminopeptidase family; and COL4A1 and COL4A2 from the connective tissue components group. The main aim of this thesis was to characterise the expression and determine the functional significance of these genes in the development of PE. Main findings: The bulk of the findings of this thesis has been published or is accepted for publication in multiple international peer-reviewed journals. The first Placenta paper in Chapter 3, demonstrated significantly increased mRNA expression of the candidate genes – ACVR1, INHBB, ERAP1, ERAP2, LNPEP, COL4A1 and COL4A2, in the pre-eclamptic maternal decidual tissue, which correlated with greater clinical severity of PE. The second Placenta paper in Chapter 4, examined the levels of arresten and canstatin fragments, which are derived from the COL4A1 and COL4A2 genes respectively, in the plasma from pre-eclamptic women and gestational age matched normotensive controls. Arresten levels were significantly increased as early as 16 weeks’ gestation in pre-eclamptic plasma. The subsequent two results chapters examined the functional roles of the activin A receptor, ACVR2A, in decidual stromal cells and endothelial cells. In Chapter 5, modelling the decreased expression observed in pre-eclamptic decidua, using an in vitro cell culture model, showed that decidual stromal cells had an impaired ability to decidualise and caused abnormal regulation of multiple extravillous trophoblast functions. Chapter 6, which forms the basis of the Pregnancy Hypertension paper, revealed that the higher baseline concentrations of activin A observed in PE resulted in vascular endothelial dysfunction, which was further exacerbated by a reduction in ACVR2A expression. Finally in Chapter 7, which was published in PLoS One, shared biological pathways of the susceptibility genes from the different functional group were identified through bioinformatics analyses. These shared pathways provide an insight as to the cumulative contribution of the different genes in the development of PE. Conclusions: The overall findings of the thesis provide evidence to support a causative, functional role of these maternal susceptibility genes in the pathogenesis of PE. The altered expression of genes, which are detectable before the onset of clinical disease, suggests the potential for the development of novel predictive biomarkers. Given how a single gene can affect the functions of multiple cell types present at the maternal-fetal interface, it is likely that the multiple gene alterations would have cumulative functional effects that ultimately influence the onset and severity of PE.
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ItemThe role of placental heparan sulphate proteoglycans in the pathogenesis of pre-eclampsia( 2015)Introduction: Uncomplicated pregnancies represent a hypercoaguable state. However, placental thrombosis is rare in these pregnancies which suggests that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia not only demonstrate an exaggerated increase in procoagulant activity which may contribute to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies, but there is evidence of substantial cell growth, differentiation and angiogenic functional defects. Proteoglycans are abundantly expressed within the placenta compared to other human tissues. Particularly of interest are heparan sulphate proteoglycans which have important anticoagulant, anti-inflammatory and angiogenic properties. Hypothesis: Altered abundance, structure or function of placental heparan sulphate proteoglycans contributes to the development of pre-eclampsia by: increasing placental thrombin generation, interfering with cellular growth, differentiation, disrupting normal angiogenesis and altering growth factor interactions. Aims : 1) To determine the mRNA expression, protein abundance and cellular localisation of placental heparan sulphate proteoglycans from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. 2) To investigate the functional consequence of reduced HSPGs in placental cells 3) To determine the differences in the abundance and structure of heparan sulphate proteoglycans and heparan sulphate GAGs from placentae obtained from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. Methods: 1) The mRNA, protein abundance and cellular localisation of placental heparan sulphate proteoglycans was determined using real-time PCR, western immunoblotting and immunofluorescence, respectively. 2) To investigate the functions of reduced heparan sulphate proteoglycans, a cell culture model using short interference RNA was used. Cellular growth will be assessed using the xCELLigence system, thrombin generation using the calibrated automated thrombogram system and angiogenesis will be determined using a matrigel based assay. Cellular differentiation and apoptosis will be determined using real-time PCR. Growth factor signalling will be determined using a real-time PCR growth factor array. 3) To isolate proteoglycans from placenta, anion exchange chromatography will be utilised. Enzymatic digestion will be used to isolate the glycosaminoglycans, and enzyme-linked immunosorbant assays will be undertaken to determine the abundance of PGs and GAGs. Results: The mRNA expression of heparan sulphate proteoglycans, Syndecan 1, Syndecan 2, Glypican 1 and Glypican 3 are significantly reduced in the placentae of women whose pregnancies are complicated by pre-eclampsia. A cell culture model was utilised to determine the functions of heparan sulphate proteoglycans in the placenta. Successful downregulation of heparan sulphate proteoglycans was achieved in the cell lines using short interference RNA treatment, and a number of functions were significantly altered as a result. The downregulation of Syndecan 1, Glypican 1 and Glypican 3 resulted in significant alterations in the downstream growth factor targets. Reduced Syndecan 2 expression resulted in a significant reduction in the thrombin generation potential of endothelial cells. Investigation into the abundance and structure of glycosaminoglycans within the placenta, demonstrated heparan sulphate glycosaminoglycans to be significantly reduced in pregnancies complicated with pre-eclampsia compared to gestation matched controls. Conclusion: The reduction in heparan sulphate proteoglycans expression observed in pregnancies complicated with pre-eclampsia may be responsible for the altered growth factor interaction commonly observed in preeclamptic pregnancies. This study has provided us with a greater understanding of the biological role of heparan sulphate proteoglycans within the human placenta and its potential implications in the pathogenesis of pre-eclampsia.
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ItemThe role of decidual mesenchymal stem cells in response to oxidative stress in normotensive and pre-eclamptic pregnancies( 2014)Preeclampsia (PE) is a serious clinical disorder of human pregnancy characterised by pregnancy-induced hypertension and proteinuria. If untreated, PE can lead to eclampsia, a convulsive life-threatening disorder. PE results from deficient placentation in early pregnancy which subsequently affects the mother, fetus, and placenta. Other than delivery of the fetus and placenta, cures for PE have not been forthcoming, which reflects the complexity of the disorder. The decidua is the maternal tissue between the placenta and the muscular uterine wall. It is the major site of pregnancy-associated uterine vascular remodelling of the spiral arterioles. PE is associated with abnormal vascular remodelling of the spiral arterioles. A consequence of abnormal remodelling is a reduced oxygen supply to the placenta and decidua. These hypoxic conditions lead to the production of reactive oxygen species and subsequently to oxidative stress. In PE, the maternal decidua is a major source of circulating reactive oxygen species that cause systemic vascular endothelium damage in the mother. Mesenchymal stem cells (MSC) can be isolated from both the placenta and decidua. The potential use of MSC in regenerative medicine has attracted considerable interest in the properties of MSC, but these investigations are limited to cell culture studies. The work in this thesis focuses on decidua-derived mesenchymal stem cells (DMSC). The role of DMSC in normal pregnancy and in PE is poorly understood. One aim of the work was to determine the anatomical location, or “niche”, of DMSC in the decidua. Another aim of the work was to determine the role DMSC play, if any, in response to oxidative stress. High resistance to oxidative stress is a common feature of MSC associated with increased cell survival and resistance to oxidative damage. Reactive aldehydes and lipid peroxides are destructive oxidative stress by-products, which are elevated in PE, and are detoxified by the aldehyde dehydrogenase (ALDH) family of enzymes. An aim of this thesis was to investigate whether an altered response to oxidative stress in DMSC contributes to the pathogenesis of PE. Informed consent was obtained for placental collection from PE-affected and normotensive patients. DMSC were isolated from the decidua basalis that remains attached to the placenta, using an enzymatic digestion-based method. Immunohistochemistry with MSC markers was performed to identify the DMSC niche. The flow-cytometry based “Aldefluor” assay was employed to quantify cells with high ALDH activity. The ALDH gene was inactivated by siRNA transfection in a DMSC-derived cell line. An xCELLigence cytotoxicity assay was performed to assess cell viability following H2O2-induced oxidative stress. Candidate ALDH activators were tested using the Aldefluor assay and real-time RT-PCR to examine changes in ALDH activity. Phenotypic characterisation showed DMSC fulfil the standard MSC criteria. DMSC were localised in the vascular niche in decidual vessels. In this study, ALDH expression was significantly decreased in preeclampsia-affected DMSC (PE-DMSC) compared with DMSC. ALDH gene knockdown by siRNA transfection was performed in DMSC to model the decreased ALDH expression observed in PE-DMSC. The ALDH-siRNA knockdown model showed that DMSC cell survival decreased in response to H2O2-induced oxidative stress. Therefore, ALDH expression in DMSC is required for cellular resistance to oxidative stress. Candidate ALDH activators were screened and two of the compounds were effective in up-regulating ALDH expression. This thesis provides proof-of-principle, in a cell culture model, for a novel therapeutic strategy that employs ALDH activators to restore resistance to oxidative stress in PE-DMSC and thereby potentially alleviates the symptoms of PE in the mother. In this study, DMSC were shown to be present in a vascular niche, and DMSC were removed during spiral arteriole remodelling in the decidua in normotensive pregnancies. In PE pregnancies however, shallow invasion and inadequate remodelling is expected to result in the presence of more unmodified spiral arteriole vessel walls, which contain PE-DMSC. Furthermore, this work showed PE-DMSC are abnormal, with decreased ALDH activity and reduced resistance to the high oxidative stress levels characteristic of PE. Thus, the work in this thesis suggests PE-DMSC are potentially novel and important contributors to the pathophysiology of PE.
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ItemAnti-angiogenic factors and pre-eclampsia: understanding disease pathophysiology for diagnostic and therapeutic translation( 2014)Pre-eclampsia is a condition that complicates 3-8% of all pregnancies, inflicting significant maternal and neonatal morbidity and mortality worldwide, causing over 63,000 maternal deaths annually. In the 21st century these statistics continue unabated due to a lack of progress in clinical developments to improve pre-eclampsia diagnosis and treatment. Excitingly, scientific knowledge of the pathophysiological processes underlying pre-eclamptic development has advanced significantly in recent years. We now understand pre-eclampsia to arise from a two-stage process; beginning with abnormal placental development leading to persisting placental hypoxia, with subsequent placental release of anti-angiogenic factors causing widespread endothelial dysfunction. This endothelial dysfunction produces the clinical features of disease, namely hypertension and proteinuria. Importantly, the discovery of the anti-angiogenic proteins, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), has presented new targets against which to develop potential therapeutic strategies to treat pre-eclampsia. The goal of such therapies would be to stabilise the maternal disease state to enable the pregnancy to progress to a gestation where delivery could occur with minimal risk to the infant. To address this goal, two aims provided the focus for the studies undertaken. Aim one sought to identify the molecular mechanisms underlying the development of pre-eclampsia with the goal of identifying new therapeutic targets, while aim two involved the development of antibodies targeting sFlt-1 and the exploration of their translational potential. This thesis details the studies performed to address these aims and their resulting findings in two parts, with each part addressing each of the aims respectively. In part one the final mechanisms of production of sFlt-1 and sEng from the placenta have been characterised for the first time. The identification that matrix metalloproteinase-14 cleaves membrane-bound endoglin on the placental surface, thereby releasing sEng into the maternal circulation, has identified a potentially new therapeutic target to prevent placental sEng release. Jumonji domain containing protein-6 has also been identified as regulating sFlt-1 production from placenta, significantly advancing our understanding of this pathway. Excitingly, jumonji domain containing protein-6 expression was also significantly reduced in pre-eclamptic placenta, being the first human condition linked to altered expression of this protein. Part two of this thesis saw the successful development of both polyclonal and monoclonal antibodies that target a placental specific sFlt-1 variant (sFlt-1 e15a) found predominately in humans. This variant could be the principle factor associated with pre-eclampsia. While exciting mRNA data exists in the literature, minimal work outlining the role of the protein exists. The development of these specific antibodies and their use in the creation of an enzyme linked immunosorbent assay has enabled for the first time the characterisation of this sFlt-1 variant in the maternal circulation of both normal and pre-eclamptic pregnancies. The translational potential for such antibodies as both diagnostic and therapeutic tools has been explored with encouraging preliminary findings. The significant findings resulting from this work not only advance pre-eclamptic scientific knowledge, but have also provided several new avenues for ongoing research. Improving outcomes for women and their babies afflicted with pre-eclampsia is now hopefully one step closer.
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ItemRole of trophoblast-derived proteins in the development of pre-eclampsia( 2011)Pre-eclampsia (PE) is a pregnancy-associated disorder that involves pregnancy-induced maternal hypertension and proteinuria. PE affects approximately 5-7% of pregnant women and despite extensive research the aetiology of this disorder remains unknown. Maternal endothelial cell (EC) dysfunction mediated by excess placenta-derived proteins in maternal plasma is a prominent component to the development of PE. Previous proteomic analysis, conducted by this laboratory, identified four candidate proteins that were increased in the syncytiotrophoblast microparticles (STBM) released from PE placentas compared to gestation matched controls (GMC). The candidate proteins were calreticulin, 14-3-3, Protein Disulfide Isomerase A3 (ERp57) and Valosin-containing protein (VCP). The investigations described in this study were undertaken on these candidate proteins. The overall aim of this study was to provide data from in vitro models that supports a role for trophoblast-derived proteins in the development of PE. The initial aim of this study was to measure the expression of the candidate proteins in the placenta by Western blot; comparing the expression of these proteins in the PE placenta versus GMC. The next aim was to measure the expression of candidate proteins in maternal plasma from normal and PE patients by Western blot. Western blots detected the expression of all four candidate proteins in the placenta, however, expression of the candidate proteins did not change between PE compared with the GMC. Western blots were also used to detect the presence of each protein in PE plasma compared with the GMC. The proteins calreticulin and 14-3-3 were both significantly increased with PE. ERp57 could not be detected in maternal plasma and there was no difference in the presence of VCP in PE maternal plasma compared to the GMC. Calreticulin was studied further. The aim was to measure the presence of calreticulin in plasma from women who at the time the sample was taken showed no clinical signs of PE, but who later develop PE compared to the GMC. Although the difference did not reach significance with the small number of samples available, this study observed an increase in maternal calreticulin concentrations at approximately 16 weeks gestation in women that subsequently develop PE compared with the GMC. The final aims of this study was to assess the effects of exogenous calreticulin at concentrations relevant to normotensive pregnancy (2µg/ml) and to PE (5µg/ml) on the human extravillous trophoblast cell line, HTR8/Svneo and human myometrial microvascular endothelial cells (utMVEC-Myo). Calreticulin, only at the concentration measured in PE plasma, inhibited HTR8/Svneo and stimulated utMVEC-Myo cell migration and reduced utMVEC-Myo cell numbers. Calreticulin at the lower concentration, 2µg/ml, had no effect on HTR8/Svneo or utMVEC-Myo cell function. This study has conducted further work on calreticulin during pregnancy and PE development and identified that the presence of 14-3-3 is increased in plasma with PE. Both these proteins, particularly calreticulin, are factors that may aid in identifying women at risk of developing PE and aid in increasing our knowledge on this pregnancy complication.