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Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 91-134) Also available in print.

Thesis (M.Sc.)--York University, 2005. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 83-93). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11803

Salmonella enterica species are intracellular bacteria causative agents of gastroenteritis and typhoid fever in humans. Pregnancy poses an increased risk of severe Salmonellosis in many mammalian species contributing to miscarriage and/or maternal illness. Previous studies indicated that Salmonella infection in pregnant mice caused rapid fetal and maternal death due to massive bacterial proliferation in the placenta. However, the susceptibility of human primary trophoblast cells (cTBCs) to Salmonella infection was not known. We hypothesized that human placental trophoblast cells are productively infected and provide a unique intracellular niche that permits uncontrolled Salmonella replication due to an ineffective maternal innate immune response to the virulent bacteria resulting in placental death. Firstly, we observed that S.Tm strains defective in the Salmonella pathogenicity island (SPI)-1 type III secretion system (TTSS) (S.Tm-ΔinvA) were unable to enter epithelial cells, but efficiently infected placental choriocarcinoma cell lines through scavenger receptor-mediated endocytosis. Next, we observed that S.Tm failed to grow vigorously in macrophages, but replicated rapidly within epithelial and placental trophoblast cells. Further examination of intracellular localization of S.Tm indicated that bacteria were arrested in early Rab5 expressing phagosomal vesicles within trophoblast cells, whereas phagosomal maturation progressed steadily in macrophages (with expression of lysosomal-associated membrane protein-1 (LAMP-1) and cathepsin D). Moreover, human primary cTBCs harboring S.Tm underwent rapid death of the cells. Infected cTBCs expressed phosphorylated-receptor-interacting serine/threonine-protein kinase (RIPK)-1 protein and phosphorylated-mixed lineage kinase domain-like (MLKL), suggesting induction of the necroptosis pathway of cell death. Furthermore, specific inhibition of necroptosis rescued S.Tm-induced death of cTBCs. Finally, S.Tm infected trophoblast cells produced interleukin (IL)-10, and signal transducer and activator of transcription (STAT)-3 signalling. This correlated to delayed phagosomal maturation which consequently facilitated intracellular pathogen proliferation. Overall, human trophoblast cells may act as reservoirs for S.Tm survival and may aid dissemination in the pregnant host.

Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR). Metabolomic profiling of placentas from such pregnancies has identified deranged sphingolipid metabolism as one of only a handful of pathways altered in PE/FGR. In other systems, the bioactive sphingolipid, sphingosine-1-phosphate (S1P) controls cell migration therefore this study aimed to determine its effect on extravillous trophoblast (EVT) function. S1P (50 nM–10 µM) attenuated the migration of the EVT cell lines, Swan-71 and SGHPL-4 (n = 6; p < 0.05) and also the outgrowth of trophoblast from explants of human first trimester placenta. Quantitative PCR and immunolocalisation studies demonstrated that both EVT cell lines and primary EVT express S1P receptors 1, 2 and 3 in similar abundance. Receptor inhibitors were used to reveal S1PR2 as the receptor responsible for mediating the inhibitory effect of S1P inhibitory effect; JTE-013 (100 nM) a specific S1PR2 inhibitor, abolished S1P- attenuated migration (n = 6; p < 0.05 versus S1P alone) whereas treatment with the S1PR1/3 inhibitor, FTY720 (100 nM; n = 6) had no effect on S1P activity. Ligand binding to S1PR2 can activate numerous intracellular signalling pathways via receptor association with the G proteins, Gα12/13, Gαq or Gαi; however, analysis of Swan-71 cell migration and actin cytoskeleton in the presence of S1P ± the Rho kinase inhibitor, Y-27632 (10 µM; n = 6) suggested preferential activation of Gα12/13. Nonetheless, S1P does activate Gαi in Swan-71 cells, as demonstrated by analysis of cAMP levels and phosphorylation of downstream signalling molecules; however attempts to shift the balance of intracellular pathway activation towards Gαi/Rac using siRNA-mediated knockdown of the Rac inhibitor ARGHGAP22 did not attenuate S1P inhibition of cellular motility, Subsequent experiments explored the possibility of preventing S1P’s actions by modulating EVT S1P receptor isoform expression using factors, including hormones and oxygen, previously reported to affect trophoblast migration or the expression of S1PR in other systems. Neither EGF nor low oxygen levels influenced S1PR expression however both IGF-II (10nM; p<0.05) and vitamin D (10nM; p<0.05) prevented the inhibitory effect of S1P on Swan-71 cell migration, the latter as a result of a significant reduction in S1PR2 expression (4-fold decrease; p<0.05).This study demonstrates that, although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2 / Gα12/13 to activate Rho and actin stress fibre formation and thereby acts as potent inhibitor of EVT migration. Strategies aimed at shifting the balance of receptor isoform expression, may provide a mechanism for improving impaired trophoblast migration in compromised pregnancies. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Thus these data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

ABSTRACT Preeclampsia (PE) is a one of the more common pregnancy complications that affects 5-8% of pregnancies worldwide and produces significant morbidity and mortality for mother and fetus. Shallow trophoblast invasion and insufficient maternal spiral artery remodeling early in gestation is believed to lead to a relatively hypoxic placenta with inflammatory and trophoblast endoplasmic reticulum (ER) stress. These stresses cause an imbalance in trophoblast expression of angiogenic/anti-angiogenic molecules with decreased placental growth factor (PGF) and increased soluble fms like tyrosine kinase-1 receptor (sFlt-1) production. The decrease in trophoblast PGF seems to be mediated at the transcriptional level while increased expression of sFlt-1 is mediated by alternative splicing. Two variants known to be elevated in PE are the sFlt-1i13 and sFlt-1e15a isoforms. Both share the first 13 exons of Flt-1. A read through into intron 13 and utilization of an alternative poly (A) signal sequence produces the sFlt-1i13 variants protein, while sFlt-1e15a results from alternative splicing of exon 14 to exon 15a, rather than exon 15, and utilization of an alternative poly (A) signal sequence. This angiogenic imbalance contributes to the clinical manifestations of PE later in pregnancy including maternal hypertension and proteinuria. Currently, there are no pharmacological options available for the prevention of PE and the only way to reverse PE symptoms is by delivery. The overall goal of my project was (1) to investigate potential therapeutic mechanisms that could be used to relieve the maternal symptoms of PE by correcting the angiogenic imbalance, and (2) to gain a better understanding of the alternative splicing mechanisms responsible for producing sFlt-1 gene expression in human trophoblast. PE shares some similar pathophysiology and risk factors with cardiovascular diseases. This has prompted use of statins as a potential therapy for PE. However, existing preclinical investigations for statin use has been mostly restricted to PE animal models without elucidating the cell types that respond to statin treatment. Therefore, we sought to determine the effect of statins on angiogenic gene expression in cells that are in direct contact with maternal blood during pregnancy and could contribute to PE: primary trophoblast and endothelial cells. Placental tissue and isolated cells were cultured under hypoxic stress (1% O2) as a model for the hypoxic environment noted in PE. We compared the effectiveness of two types of statins (hydrophilic vs hydrophobic) on angiogenic and anti-angiogenic gene expression from the human tissues and cells. Human placenta villus explants, umbilical vein endothelial cells (HUVECs), and cytotrophoblast were isolated from normal term placentae and cultured under low oxygen tension (1% O2) with serial concentrations of statins. Expression of proangiogenic genes (VEGF and PGF) and the prominent anti-angiogenic sFlt-1 isoforms (sFlt-1i13 & sFlt-1e15a) were analyzed. In villus explants, hypoxia (1% O2) tended to alter angiogenic gene expression in their predicted fashion, by increasing VEGF mRNA (hypoxia marker), decreasing PGF mRNA, and increasing both sFlt-1i13 and sFlt-1e15a mRNA expression. However, the changes in gene expression were quite variable and statistically not significant. Hypoxia significantly increased both sFlt-1i13 and sFlt-1e15a mRNA and protein expression in primary trophoblast but had limited effects on expression in HUVECs. Hypoxia significantly decreased PGF mRNA and protein expression in primary trophoblast, yet significantly increased PGF mRNA and protein expression in HUVECs. Concentrations of pravastatin or simvastatin used had limited effects on altering PGF mRNA and protein expression in any of the cell types. In primary trophoblast, lower concentrations of pravastatin (100/500 µg/ml) had no significant effects on sFlt-1i13 or sFlt-1e15a mRNA expression while higher concentrations (1000 µg/ml) significantly decreased sFlt-1i13 and tended to decrease sFlt-1e15a mRNA expression. Secreted sFlt-1 protein from trophoblast decreased with increasing concentrations of pravastatin. Similarly, simvastatin had limited effects and did not significantly decrease sFlt-1i13 or sFlt-1e15a expression in hypoxic primary trophoblast. Both pravastatin and simvastatin significantly down-regulated sFlt-1i13 and sFlt-1e15a mRNA expression and sFlt-1 protein production in HUVECs. To overcome the effects of statin treatments on sFlt-1 expression, primary HUVECs were treated with farnesyl pyrophosphate ammonium salt (FPP), an intermediate in the cholesterol synthesis pathway. FPP partially restored sFlt-1i13 and sFlt-1e15a mRNA expression. Our data support that the angiogenic imbalance seen in PE can be medicated by hypoxia, and that statin could be a promising medication to limit PE symptoms. The effect of statins may be more evident on endothelial cells than on trophoblast, and the reduction in sFlt-1 expression by statins seems to be partially mediated through the cholesterol synthesis pathway in endothelial cells. The antiangiogenic protein, sFlt-1, plays a central role in the pathophysiology of PE. Excessive amounts of the sFlt-1 receptor in maternal circulation leads to maternal endothelial cell dysfunction and subsequent clinical symptoms of PE. However, the mechanism governing sFlt-1 mRNA expression in trophoblast remains unclear. Jumonji C domain containing gene 6 (JMJD6) has been shown to be involved in splicing of sFlt-1i13 in endothelial cells, although with conflicting outcomes as to whether it increases or decreases alternative splicing of sFlt-1i13. It is unknown if JMJD6 functions to regulate splicing in human primary trophoblast. Therefore, we assessed whether JMJD6 expression is altered in primary trophoblast under hypoxia or ER stress and its ability to regulate alternative splicing of sFlt-1. Human cytotrophoblast were isolated from normal term placentae and were cultured in the presence or absence of ER stress inducer (tunicamycin) or at 1% O2 to simulate trophoblast stressors during PE. Expression of JMJD6, C/EBP homologous protein (CHOP), and sFlt-1 (sFlt-1i13, and sFlt-1e15a) variants were analyzed. Hypoxic stress significantly increases JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression. ER stress also tended to increase JMJD6, sFlt-1i13, and sFlt-1e15a mRNA expression in primary trophoblast. Collectively, our results show that low oxygen tension (1% O2) or ER stress increase JMJD6 mRNA expression which may contribute to increased sFlt-1i13 and sFlt-1e15a variant expression in primary trophoblast. Similarly, JMJD6 knock down with siRNA tends to slightly decrease sFlt-1i13 and sFlt-1e15a mRNA expression in primary trophoblast. JMJD6 overexpression in HTR-8 cells (choriocarcinoma) tended to increase sFlt-1i13 and sFlt-1e15a mRNA expression; however, results using HTR-8 were inconsistent due to extremely low expression of endogenous Flt-1 mRNA. To overcome this, a Flt-1 minigene plasmid was transfected into HTR-8 cell line. Under 1%O2 these cells increased expression of the sFlt-1i13 isoform. To more directly confirm effects of JMJD6 and hypoxia on sFlt-1 expression, HEK293 and JEG3 stable clones harboring the Flt-1 minigene were generated. Preliminary results from selected single colony isolates show that several stable clones express the Flt-1 minigene products. HEK293 and JEG3 stable clones harboring the Flt-1 minigene, HEK293-Flt1#5 and JEG3-Flt1#5 respectively, were cultured at 1%O2 for 48 or 72 hours. Hypoxic stress had no significant on altering sFlt-1 variant production or JMJD6 mRNA expression in HEK293-Flt1#5 cells. However, hypoxic JEG3-Flt1#5 cells significantly increased sFlt-1i13 isoform mRNA expression (˜6 fold) and mFlt-1 mRNA expression (2.5 Fold) and also increased JMJD6 mRNA expression (1.8 Fold). In summary, these data suggest a role for statins as a potential therapeutic approach for the prevention and treatment of PE by decreasing systemic sFlt-1 expression in endothelial cells. This effect seems most significant in endothelial cells. If substantiated by clinical studies, use of statins would offer an affordable and easily accessible therapy to lessen PE symptoms. Moreover, our preliminary data suggest a potential involvement of JMJD6 in splicing process of sFlt-1i13. Confirming of JMJD6 role in splicing of Flt-1 may provide therapeutic strategies to treat Flt-1 associated disorder.

People living near coal mines have raised concerns on how coal mining affects surrounding communities. Coal mining is a well-documented source of selenium inputs into the environment, and while there is considerable evidence demonstrating adverse effects of excess selenium on reproductive outcomes in fish, selenium toxicity in mammals is less understood. Studies in humans showed a correlation between high levels of selenium and increased adverse pregnancy outcomes, but the mechanisms behind this association are unclear. Importantly, many of the observed adverse pregnancy outcomes associated with high levels of selenium are linked to placental dysfunction. Mechanistically, supraphysiological concentrations of selenium have been shown to cause dysregulation of cortisol and induce ER stress. Balancing the amount of cortisol and ER stress during placental development is important, as a deficiency or surplus of either can cause aberrant placental development and/or placental dysfunction. Given that exposure to excess cortisol has been shown to induce ER stress, and ER stress has been shown to cause aberrant invasion and migration, which are important processes during placental development, the objective of my thesis is to test the hypothesis that excess selenium exposure impacts invasion and migration in first-trimester trophoblasts, and that these effects are mediated by the glucocorticoid and ER stress pathways. HTR-8/SVneo cells (human first-trimester trophoblasts) were exposed to environmentally relevant concentrations of sodium selenite (NaSe) for 24 or 48h. Cortisol was measured via ELISA, migration was measured via a wound-healing assay, and steady-state mRNA expression of genes involved in glucocorticoid homeostasis, ER stress, and invasion, migration, and angiogenesis were measured by qPCR. NaSe treatment caused increased cortisol and induced genes that are indicative of glucocorticoid receptor activation. NaSe also induced genes involved in ER stress as well as the regulation of invasion, migration and angiogenesis. NaSe also decreased migration as measured in the wound healing assay. When cells were co-treated with NaSe and either 1) metyrapone, an inhibitor of the enzyme responsible for synthesizing cortisol (CYP11B1), or 2) mifepristone, an antagonist of glucocorticoid receptor, the genes associated with increased cortisol did not decrease in the cells, suggesting that selenium may be activating the glucocorticoid pathway through alternate means. When the cells were co-treated with NaSe and ER stress inhibitor TUDCA, there was an attenuation of ER stress-related and invasion, migration and angiogenesis-related genes, as well as partial restoration of migration. Selenium treatment appears to have an impact on glucocorticoid activation, ER stress, and migration. While these results do not definitively identify the role that glucocorticoids play in the impact of selenium on migration, the results support the hypothesis that ER stress induced by selenium exposure partially affects migration in first-trimester trophoblasts cells. / Thesis / Master of Science (MSc)

Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes. / Master of Science

O biglicam é um proteoglicano é altamente expresso em células trofoblásticas de patologias placentárias com invasividade exacerbada. No entanto, as funções do biglicam no trofoblasto ainda não foram elucidadas. Sendo assim, verificamos a expressão e as funções de biglicam e seus receptores Toll-like (TLR)-2 e TLR-4 nas células trofoblásticas durante a gestação. As células do citotrofoblasto extraviloso (CTEV) foram positivas para todas as moléculas, menos para o biglicam em placentas a termo. Adição exógena de biglicam promoveu migração e invasão das células trofoblásticas. O biglicam estimulou a fosforilação de AKT nos sítios Thr308 e Ser473 nas células trofoblásticas. A migração e a invasão biglicam-dependentes e as fosforilações de AKT foram inibidas após a adição de anticorpos bloqueadores anti-TLR-2 e anti-TLR-4. O silenciamento gênico de AKT1 em células SGHPL-5 aboliu os efeitos do biglicam na motilidade. Em conclusão, o biglicam aumenta a motilidade de células trofoblásticas após sinalização por AKT através da ativação de TLR-2 e TLR-4. / Biglycan is a highly expressed proteoglycan in trophoblast cells from invasiveness-changed placental pathologies. However, biglycan functions in the trophoblast were not yet identified. Therefore, it was verified the expression and functions of biglycan and its receptors Toll-like (TLR)-2 and TLR-4 in trophoblast cells throughout pregnancy. The extravillous cytotrophoblast cells (EVT) were positive to all the molecules, although biglycan was negative in term placentas. Exogenous biglycan promoted migration and invasion of trophoblast cells. Biglycan stimulated AKT phosphorilation at Thr308 and Ser473 sites in trophoblast cells. The biglycan-dependent migration, invasion and AKT phosphorilation were inhibited upon addiction of anti-TLR-2 and anti-TLR-4 blocking antibodies. AKT1 genic silencing in SGHPL-5 cells abolished the motility effects. In conclusion, biglycan increases the motility of trophoblast cells after AKT signaliing throughout TLR-2 and TLR-4 activation.

O biglicam é um proteoglicano é altamente expresso em células trofoblásticas de patologias placentárias com invasividade exacerbada. No entanto, as funções do biglicam no trofoblasto ainda não foram elucidadas. Sendo assim, verificamos a expressão e as funções de biglicam e seus receptores Toll-like (TLR)-2 e TLR-4 nas células trofoblásticas durante a gestação. As células do citotrofoblasto extraviloso (CTEV) foram positivas para todas as moléculas, menos para o biglicam em placentas a termo. Adição exógena de biglicam promoveu migração e invasão das células trofoblásticas. O biglicam estimulou a fosforilação de AKT nos sítios Thr308 e Ser473 nas células trofoblásticas. A migração e a invasão biglicam-dependentes e as fosforilações de AKT foram inibidas após a adição de anticorpos bloqueadores anti-TLR-2 e anti-TLR-4. O silenciamento gênico de AKT1 em células SGHPL-5 aboliu os efeitos do biglicam na motilidade. Em conclusão, o biglicam aumenta a motilidade de células trofoblásticas após sinalização por AKT através da ativação de TLR-2 e TLR-4. / Biglycan is a highly expressed proteoglycan in trophoblast cells from invasiveness-changed placental pathologies. However, biglycan functions in the trophoblast were not yet identified. Therefore, it was verified the expression and functions of biglycan and its receptors Toll-like (TLR)-2 and TLR-4 in trophoblast cells throughout pregnancy. The extravillous cytotrophoblast cells (EVT) were positive to all the molecules, although biglycan was negative in term placentas. Exogenous biglycan promoted migration and invasion of trophoblast cells. Biglycan stimulated AKT phosphorilation at Thr308 and Ser473 sites in trophoblast cells. The biglycan-dependent migration, invasion and AKT phosphorilation were inhibited upon addiction of anti-TLR-2 and anti-TLR-4 blocking antibodies. AKT1 genic silencing in SGHPL-5 cells abolished the motility effects. In conclusion, biglycan increases the motility of trophoblast cells after AKT signaliing throughout TLR-2 and TLR-4 activation.