Dissertations / Theses on the topic 'Placental oxidative stress'

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietary selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.

Affecting 6-8% of all pregnancies, preeclampsia is the leading cause of maternal morbidity in the western world and is charactensed by hypertension, proteinuria, edema and platelet aggregation. Despite its prevalence and severity, no comprehensive theory or single factor has been suggested to explain the pathophysiology of this multi system disorder of pregnancy, with the only therapies being bed rest, pharmacological symptom management and if necessary early delivery. Oxidative stress plays an important role in the pathophysiology of preeclampsia, resulting from defective trophoblast invasion, reductions in placental perfusion and placental hypoxia/reoxygenation. The inability of endogenous antioxidant systems up regulated in normal pregnancy, to control increased levels of oxidative stress, is suggested as a possible factor in the feed forward generation of reactive oxygen species and placental oxidative stress. That in turn may stimulate increased syncytiotrophoblast apoptosis, endothelial cell activation and the maternal hyper immune response characteristic of preeclampsia. Analysis of the research literature revealed that previous evaluations of placental oxidation and antioxidant enzyme activity in preeclampsia were by no means comprehensive, and exhibited significant inter-study variations. It was the aim of this thesis to clarify the placental oxidative state and the endogenous antioxidant activity of glutathione peroxidase, thioredoxin reductase, thioredoxin and superoxide dismutase in human placentae in an attempt to determine if variations in antioxidant function were due to changes in gene expression or protein oxidation. The findings reported in this thesis indicate the presence of increased levels of oxidative stress in the preeclamptic placenta, associated with significant reductions in antioxidant enzyme capacity. Quantitative real-time PCR analysis of placental samples revealed that deceases in antioxidant capacity in the placenta are more likely to be related to the significant oxidative burden within the tissue rather than reductions in gene expression. A number of animal models exist to investigate components of preeclampsia pathophysiology, however the ability of these models to mimic the oxidative and antioxidant features of preeclampsia remains unclear. The exposure of pregnant rats to N(G)-nitro-L-arginine methyl ester is a widely used model of endothelial cell dysfunction during preeclampsia. It was the aim of this thesis to determine the biochemical characteristics of this model in an attempt to assess its effectiveness in mimicking oxidative changes in the preeclamptic placenta. Although this model is capable of producing a syndiome in rats similar to the disorder in terms of physiology, this is not manifest in terms of placental biochemistry. The importance of selenium in the synthesis of selenobased antioxidants such as glutathione peroxidase and thioredoxin reductase is well documented. Increasing demand for selenium by the developing fetus may be linked to reductions in selenium status during pregnancy. Considering preeclampsia is associated with significant reductions in selenium status it may be hypothesised that reductions in antioxidant function may be linked to selenium inadequacy. The modulation of dietaty selenium in pregnant rats was used to determine the importance of selenium during pregnancy and its effect on antioxidant function and placental oxidative stress. The results of this analysis revealed that selenium deficiency causes a pregnancy specific condition similar to preeclampsia. This condition was found to be associated with increased placental oxidative stress and significant reductions in the systemic activity of selenobased antioxidants that could be modified through selenium supplementation. In summary, data obtained in this thesis indicate that placental oxidative stress and reduced antioxidant enzyme activity play a significant role in the pathogenesis of preeclampsia. These studies support the hypothesis that antioxidant sufficiency is crucial in the maintenance of oxidative balance and that antioxidant dysfunction may result in damage to the placenta and the progression of the disease. These novel data further our understanding of the pathophysiology of preeclampsia and provide new insight into the pathogenesis of clinical complications exhibited in this condition, suggesting antioxidant therapy as a possible means for improving the health outcomes of both mother and baby.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

Affecting 5-7% of all pregnancies, pre-eclampsia is a dangerous complication of pregnancy which poses a serious health risk to both mother and fetus. It is a major cause of maternal and perinatal morbidity and is characterised by hypertension, proteinuria, edema and platelet aggregation. Despite the severity of the disease, no comprehensive theory has been determined to explain the pathogenesis of the disease, with delivery being the only curative treatment of this multisystem disorder of pregnancy. Oxidative stress plays a crucial role in the pathogenesis of pre-eclampsia, which results in placental injury causing a hypoxic environment and eventually levels of anti-oxidants are markedly decreased. This leads to the activation of cell death pathways such as apoptosis and necrosis. The findings reported in this thesis suggest that selenium supplementation could be a potential therapy for treating women who are at risk of developing pre-eclampsia. In this study, selenium supplementation was used to up-regulate the expression of endogenous anti-oxidants, Glutathione Peroxidase (GPx) and Thioredoxin-Reductase (Thx-Red). It has been well documented in the literature that pre-eclampsia is caused by oxidative stress and in an attempt to address this we investigated whether oxidative stress in trophoblast-like cells is protected by selenium supplementation. Trophoblast-like cells, BeWo, JEG-3 and Swan-71 cells were exposed to endogenous stressors such as Rotenone and Antimycin which are able to generate mitochondrial oxidative stress by inhibiting the electron transport chain.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Oxidative stress of the placenta is considered a key intermediary step in the pathogenesis of preeclampsia, but the cause for the stress remains unknown. Ischaemia-reperfusion injury, as a result of intermittent placental perfusion secondary to deficient trophoblast invasion of the endometrial arteries, is a possible mechanism. This thesis therefore tests whether hypoxia-reoxygenation (H/R) in vitro can induce placental oxidative stress, and cause increased apoptosis and production of tumour necrosis factor-α as seen in the preeclamptic placenta. The first aim was to examine the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species (ROS) was detected using a fluorescent marker when hypoxic villous samples were reoxygenated. The expression of oxidative stress markers including nitrotyrosine residues, 4-hydroxy-2-nonenal adducts, and inducible heat shock protein 72 was greatly increased in villous samples subjected to H/R compared to the controls maintained under constant hypoxia. Furthermore, preloading villous samples with ROS scavengers such as desferrioxamine and α-phenyl-N-tert-butylnitrone significantly reduced the levels of oxidative stress in H/R. Having demonstrated that in vitro H/R is capable of inducing oxidative stress in a reproducible and manipulable manner, investigations were next carried out to study the effects of resultant oxidative stress on apoptosis within the trophoblast. Compared to hypoxic and normoxic controls, there was a significant increase in the release of cytochrome c from mitochondria, activation of caspase , and cleavage of poly (ADP-ribose) polymerase in villous samples subjected to H/R. These events were associated with an increased number of syncytiotrophoblastic nuclei displaying apoptotic changes and increased lactate dehydrogenase release into the medium. The causal relationship between the generation of ROS and these apoptotic changes was revealed by the fact that pre-administration of desferrioxamine attenuated the insult.

Preeclampsia (PE) is the leading cause of poor maternal and perinatal outcomes in both developed and developing countries. Even though the condition is treatable in the developed world, mothers from developing countries still suffer dramatic events due to limited resources. There is the need to identify readily available measures in addition to existing biomarkers that can predict PE. Thus, this thesis evaluated suboptimal health status (SHS) and prospective levels of oxidative stress (OS) biomarkers and angiogenic growth mediators (AGMs) with placental anatomy and pathology in normotensive and preeclamptic births in a Ghanaian Population. This longitudinal nested case-control study was based on the Ghanaian Suboptimal Health Status Cohort Study (GHOACS) that recruited 593 normotensive pregnant women (NTN-PW) at baseline (10-20 weeks gestation) from the Komfo Anokye Teaching Hospital between June 2017 and May 2019. Suboptimal health status, a subjective health measure was evaluated using the Suboptimal Health Status Questionnaire-25 (SHSQ-25) at baseline, and participants were subsequently classified into suboptimal health status (SHS) and optimal health status (OHS). Baseline participants were followed at 21-31 weeks, 32-42 weeks of pregnancy and 48 hours-2 weeks postpartum. Of the 593, 488 pregnant mothers aged 18-45 years completed the study and 105 were lost to follow-up. Data on sociodemographic, clinical, obstetric and anthropometric characteristics were collected from participants in addition to urine and venous blood samples. The panel of objective biomarkers of AGMs (placental growth factor [PIGF], vascular endothelial growth factor-A [VEGF-A], soluble endoglin [sEng] and soluble VEGF receptor-1 also known as soluble fms-like tyrosine kinase [sFlt-1]) in addition to OS biomarkers (8-epiprostaglandinF2 alpha [8-epi-PGF2α], 8-hydroxy-2-deoxyguanosine [8-OHdG] and total antioxidant capacity [TAC]) were evaluated across the 4-time sampling periods (10-20, 21-31, 32-42 weeks, and 48 hours-2 weeks postpartum) using ELISA. Placenta tissues were collected after delivery and examined for macroscopical, histopathological and immunohistochemical analyses. Data were analysed using SPSS, GraphPad Prism, XLSTAT and R Core. There were significant associations between SHS and imbalances in AGMs and OS (Study I: cross-sectional). The incidence of PE was high among SHS pregnant women (30.7%) compared to OHS pregnant women (8.8%), and SHS was an independent risk indicator for PE (Study II: longitudinal nested case-control). Compared to using the biomarkers alone, combining biomarkers of AGMs and OS, particularly 8- OHdG/PlGF ratio measured at 21-31 weeks (mid-pregnancy) yielded the best area under the curve, sensitivity, specificity, positive and negative predicted values for predicting the likelihood of SHS and OHS pregnant women developing PE (Study III: longitudinal nested case-control). Placental abnormalities and imbalance in the expression of AGMs and OS were associated more with SHS pregnant mothers who developed PE, particularly early-onset PE than late-onset PE (Study IV: case-control). In contrast to normotensive pregnancy, longitudinal profiling of AGMs and OS showed different patterns across the 4-time sampling periods with significant imbalance among SHS more than OHS mothers who later developed PE. Unlike, the single biomarkers, combined AGMs and OS ratios measured at mid-pregnancy yielded more significant hazard ratios in association with PE development (Study V: longitudinal cohort). Thus, combined evaluation of SHS, biomarkers of AGMs and OS together with placental examination increased the understanding of the aetiology, diagnosis and prognosis of PE, and created a window of opportunity for developing potentially predictive, preventive and personalised medicine in both high-and low-resource maternal and child health settings.

Le tabagisme actif par la mère expose le fœtus en développement à des agents qui peuvent traverser la barrière placentaire et interférer avec les fonctions placentaires. Un large éventail de fonctions immunologiques, pourrait être compromises. Dans cette étude, nous avons évalué l'effet de l'extrait de la fumée de cigarette (CSE) sur les macrophages isolés à partir de placentas humains (pMφs), qui sont les principaux partenaires de l'immunité de fœto-maternelle innée. J’ai pu montrer que le CSE inhibe la formation des cellules géantes multinucléées (MGC). Cette propriété du CSE est spécifique aux macrophages car la fusion des macrophages dérivés des monocytes est inhibée lors de la formation de granulomes in vitro. J’ai également étudié l'absorption de particules et la production de cytokines par pMφs exposés au CSE. Le CSE a inhibé l'absorption des particules de zymosan, mais pas celle du zymosan opsonisé, ce qui suggère qu’il interfère avec les récepteurs phagocytaires et non phagocytaires. Le CSE augmente la libération de TNF et d'IL-33, et une diminue celle de l'IL-10, ce qui montre que l'équilibre entre les cytokines est affecté par le CSE. En outre, l’expression des métalloprotéinases telles que les MMP-1, MMP-10 et MMP-12, connues pour être impliquées dans le remodelage des tissus et la fusion des macrophages est dérégulée. Enfin, j’ai montré que la nicotine, l'un des principaux composés de tabac, n'a pas affecté les propriétés fonctionnelles des pMφs
Active smoking by the mother exposes the developing fetus to agents that can cross the placental barrier and interfere with placental functions. A wide range of immunological functions, including innate and adaptive immune responses, might be impaired. In this study, we assessed the effect of cigarette smoke extract (CSE) on macrophages isolated from human placentas (pMφs), which are major partners of innate feto-maternal immunity. I showed that CSE significantly inhibited the formation of multinucleated giant cells (MGCs). This property of CSE is specific to macrophages because the fusion of monocyte-derived macrophages is inhibited during the in vitro formation of granulomas. I also investigated particle uptake and cytokine production by pMφs exposed to CSE. CSE inhibited the uptake of zymosan, but not that of opsonized zymosan, suggesting that it interferes with phagocytic receptors, not with the phagocytic machinery of pMφs. CSE increased the release of Tumor Necrosis Factor and interleukin-33, and decreased that of interleukin-10, demonstrating that the balance between inflammatory and anti-inflammatory cytokines is affected by CSE. Furthermore, CSE enhanced the expression of metalloproteinase (MMPs) genes such as MMP-1, MMP-10 and MMP-12, known to be involved in tissue remodeling including macrophage fusion. Finally, I showed that nicotine, one of the major compounds of tobacco, did not affect the functional properties of pMφs

Contexte. Au cours de la pré-éclampsie (PE), le défaut d'invasion trophoblastique et de remodelage des artères utérines spiralées génère une mauvaise adaptation de la circulation utéro-placentaire associée à des phénomènes d'hypoxie/réoxygénation (H/R). Il en résulte un stress oxydant et un déséquilibre des facteurs angiogéniques/antiangiogéniques (diminution du VEGF et PIGF vs augmentation du sFlt1) responsables d'une placentation anormale, une dysfonction endothéliale et une inflammation systémique. Le dysfonctionnement de l'oxyde nitrique synthase endothéliale (eNOS) et la diminution de la biodisponibilité du NO, jouent un rôle critique dans la pathophysiologie de la PE. eNOS est la principale source de production du NO dans le placenta, et elle joue un rôle primordial dans l'homéostasie et la régulation du tonus vasculaire. Des données récentes indiquent que eNOS peut être modifiée au cours du stress oxydant, en particulier par S-glutathionylation, ce qui entraine son découplage avec une génération d'ion superoxyde et une réduction du NO. Objectif. Le but de ce travail a été d'étudier les conséquences du stress oxydant sur la eNOS placentaire, en particulier sa glutathionylation, et les modifications par les produits d'oxydation lipidique (LPO), en relation avec sa dysfonction observée au cours de la PE. Matériels et Méthodes. La modification de eNOS a été étudiée dans des tissus placentaires obtenus à partir de césariennes réalisées chez des patientes pré-éclamptiques (n=13), vs grossesses normales (n=9). Des études complémentaires sont réalisées sur des trophoblastes humains HTR-8/SVneo exposés à un stress oxydant induit par H/R, ou par exposition à des LPO. Résultats : Les études en immunofluorescence et microscopie confocale montrent une importante glutathionylation de eNOS dans les placentas de PE, sensible aux agents réducteurs (dithiotréitol), sans différence d'expression de eNOS totale entre PE et grossesses normales. La S-glutathionylation est confirmée par immunoprécipitation et Western Blot de la eNOS placentaire. L'exposition des trophoblastes HTR8 à des conditions d'H/R, génère une S-glutathionylation d'eNOS associée à une production réduite de NO, et une génération d'ion superoxyde. Le NO est nécessaire pour le potentiel invasif des trophoblastes, comme démontré par l'absence de migration des HTR8 après inhibition spécifique de eNOS par small ARN interference (siRNA), et rétablie après addition d'un donneur de NO, le NOC-18. Les trophoblastes exposés aux variations H/R, montrent des capacités de migration diminuées témoignant d'un potentiel invasif réduit, et rétabli par NOC-18. Dans la deuxième partie de ce travail, nous avons recherché la présence de LPO dans les placentas de PE, et nous avons émis l'hypothèse que eNOS puisse être une cible de ces agents. Nous montrons que les LPO tels que le 4-hydroxynonenal (4-HNE), et le 4-oxo-2-nonenal (ONE), s'accumulent dans les placentas de PE, en particulier sur la eNOS, alors qu'aucune modification n'est observée dans les placentas de grossesse normale. Les études en protéomique sur eNOS recombinante, montrent que ONE et 4-HNE modifient plusieurs épitopes (ONE-Lys, HNE-His, HNE-Cys). L'addition de 4-HNE ou de ONE aux HTR8, inhibe la production de NO et la migration des cellules, restaurée par l'addition de NOC-18. Conclusions et perspectives : Ces résultats montrent que la eNOS placentaire est une cible importante du stress oxydant au cours de la PE, avec des modifications par S-glutathionylation ou par formation d'adduits avec ONE ou 4-HNE, associées à une diminution de la production de NO. Ces modifications pourraient contribuer au dysfonctionnement de la eNOS placentaire observé au cours de la PE. En perspective, nous pourrons étudier les conséquences du stress oxydant et des LPO, sur le vieillissement accéléré du placenta, qui contribueraient à la physiopathologie de la PE
Context: During pre-eclampsia (PE), the defective trophoblastic invasion and remodeling of the uterine spiral arteries leads to poor adaptation of utero-placental circulation associated with hypoxia/reoxygenation phenomena. This induces oxidative stress and an imbalance between angiogenic/antiangiogenic factors (decrease in VEGF and PIGF vs. increase in sFlt1) responsible for abnormal placentation, endothelial dysfunction and systemic inflammation. Endothelial nitric oxide synthase dysfunction (eNOS) and decreased NO bioavailability play a critical role in the pathophysiology of PE. eNOS is the main source of placental NO production, and plays a key role in homeostasis and vascular tone regulation. Recent evidence indicates that eNOS may undergo glutathionylation in the vascular wall, and subsequent uncoupling in a prooxidant environment, this resulting in an increased generation of superoxide anion and a decreased production of NO. Objective: The purpose of this work was to study the consequences of oxidative stress on placental eNOS, in particular its glutathionylation and modification by lipid oxidation products (LPO), in relation to its dysfunction observed during PE. Materials and Methods: The modification of eNOS was studied in placental tissues obtained from preeclampsia-affected (n=13), vs normal pregnant women (n=9) and in HTR-8/SVneo human trophoblasts exposed to hypoxia/reoxygenation (H/R), or by exposure to LPO. Results: Immunofluorescence and confocal microscopy revealed a high glutathionylation of eNOS in PE placentas, reversed by dithiotreitol, which was confirmed by immunoprecipitation and western-blot experiments, with no difference in total eNOS expression between PE and normal pregnancy. Exposure of HTR8 trophoblasts to H/R conditions generates S-glutathionylation of eNOS associated with reduced NO production, and increased superoxide anion generation. NO is necessary for the invasive potential of trophoblasts, since trophoblasts exposed to H/R, or silenced for eNOS by small interfering RNAs (siRNA), showed a decreased migration capacity, which was restored by the NO donor, NOC-18. In the second part of this work, we investigated the presence of LPO in PE placentas, and hypothesized that eNOS could be a target of these agents. We show that LPO such as 4-hydroxynenal (4-HNE), and 4-oxo-2-nonenal (ONE), accumulate in PE placentas, particularly on eNOS, while no changes are observed in normal pregnancy placentas. Proteomics studies on recombinant eNOS show that ONE and 4-HNE modify several epitopes (ONE-Lys, HNE-His, HNE-Cys). The addition of 4-HNE or ONE to HTR8 inhibits NO production and cell migration, restored by the addition of NOC-18. Conclusions and perspectives: These results show that placental eNOS is an important target for oxidative stress during PE, with modifications by S-glutathionylation or adduct formation with ONE or 4-HNE, associated with a decrease in NO production. These changes could contribute to the dysfunction of placental eNOS observed during the PE. In perspective, we plan to study the consequences of oxidative stress and LPO on accelerated placental aging, which may contribute to the pathophysiology of PE, and beyond, of pathological pregnancies

Orientador: Leandro Gustavo de Oliveira
Resumo: Introdução: A gestação é uma condição fisiológica que pode apresentar maior suscetibilidade ao desequilíbrio entre fatores pró e antioxidativos, evoluindo assim com dano celular e resposta inflamatória. A autofagia é um processo que elimina organelas e proteínas danificadas do citoplasma, com potente mecanismo anti-inflamatório responsável pela manutenção da homeostase celular. A autofagia pode controlar a inflamação por meio da inibição da ativação do inflamassoma, complexo essencial para a liberação de citocinas pró-inflamatórias. Assim, alterações nesses processos podem relacionar-se com disfunções celulares e doenças sistêmicas. Objetivos: Este projeto teve como objetivo avaliar se a exposição de explantes placentários a diferentes concentrações de peróxido de hidrogênio (H2O2) é capaz de induzir autofagia e levar a ativação do inflamassoma NLRP3. Métodos: Explantes placentários de gestantes normais obtidos após o parto foram cultivados em diferentes concentrações de H2O2 por 4 e 24 h após a avaliação da viabilidade dos mesmos nesses períodos. As enzimas superóxido dismutase (SOD) e catalase foram avaliadas nos sobrenadantes das culturas após 4 h. As expressões gênicas de marcadores de autofagia (LC3-II, beclin-1 e p62), do inflamassoma (NLRP3 e caspase-1) e das citocinas IL-1β, IL-10 e TNF-α foram avaliadas por RT-qPCR. Os níveis de gonadotrofina coriônica (hCG), proteína de choque térmico 70 (Hsp70) e citocinas IL-1β, IL-10 e TNF-α foram determinados por ensaio imunoenz... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Submitted by Priscila Rezeck Nunes null (priscilarezeck@aluno.ibb.unesp.br) on 2017-03-08T14:58:45Z No. of bitstreams: 1 Versão final dissertação Priscila Rezeck Nunes.pdf: 4789833 bytes, checksum: 772d289b789606f7e85653d2c289a43a (MD5)
Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-13T14:38:45Z (GMT) No. of bitstreams: 1 nunes_pr_me_bot.pdf: 4789833 bytes, checksum: 772d289b789606f7e85653d2c289a43a (MD5)
Made available in DSpace on 2017-03-13T14:38:45Z (GMT). No. of bitstreams: 1 nunes_pr_me_bot.pdf: 4789833 bytes, checksum: 772d289b789606f7e85653d2c289a43a (MD5) Previous issue date: 2017-02-17
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Introdução: A gestação é uma condição fisiológica que pode apresentar maior suscetibilidade ao desequilíbrio entre fatores pró e antioxidativos, evoluindo assim com dano celular e resposta inflamatória. A autofagia é um processo que elimina organelas e proteínas danificadas do citoplasma, com potente mecanismo anti-inflamatório responsável pela manutenção da homeostase celular. A autofagia pode controlar a inflamação por meio da inibição da ativação do inflamassoma, complexo essencial para a liberação de citocinas pró-inflamatórias. Assim, alterações nesses processos podem relacionar-se com disfunções celulares e doenças sistêmicas. Objetivos: Este projeto teve como objetivo avaliar se a exposição de explantes placentários a diferentes concentrações de peróxido de hidrogênio (H2O2) é capaz de induzir autofagia e levar a ativação do inflamassoma NLRP3. Métodos: Explantes placentários de gestantes normais obtidos após o parto foram cultivados em diferentes concentrações de H2O2 por 4 e 24 h após a avaliação da viabilidade dos mesmos nesses períodos. As enzimas superóxido dismutase (SOD) e catalase foram avaliadas nos sobrenadantes das culturas após 4 h. As expressões gênicas de marcadores de autofagia (LC3-II, beclin-1 e p62), do inflamassoma (NLRP3 e caspase-1) e das citocinas IL-1β, IL-10 e TNF-α foram avaliadas por RT-qPCR. Os níveis de gonadotrofina coriônica (hCG), proteína de choque térmico 70 (Hsp70) e citocinas IL-1β, IL-10 e TNF-α foram determinados por ensaio imunoenzimático (ELISA) após 24 h de cultura. Resultados: Os níveis de LDH foram crescentes conforme o tempo de cultura, sendo que as culturas de 24 h apresentaram-se com viabilidade celular adequada para o estudo. Os níveis proteicos de catalase e Hsp70, bem como a expressão gênica de LC3-II, beclin-1 e p62 apresentaram níveis crescentes e relacionados às maiores concentrações de H2O2. As concentrações proteicas de SOD, hCG e TNF-α foram maiores nas culturas com 100 µM de H2O2. A expressão gênica de TNF-α, IL-1β, NLRP3 e caspase-1 foram elevadas em 1000 µM de H2O2. Além disso, a expressão proteica de IL-1β também foi maior nessa concentração. As concentrações gênicas e proteicas de IL-10 decresceram de acordo com o aumento da concentração de H2O2. Conclusões: Os resultados obtidos demonstraram que o H2O2 é capaz de induzir o estado de estresse oxidativo placentário, induzir autofagia, ativar o inflamassoma e assim aumentar a produção de citocinas inflamatórias.
Introduction: Pregnancy is a physiological condition characterized by increased susceptibility to oxidative stress, which can lead to cell damage and inflammatory response. Autophagy is a process that removes damaged organelles and proteins from the cytoplasm. It works as potent anti-inflammatory mechanism, responsible for maintaining cellular homeostasis. Autophagy can control inflammatory responses by regulating the activation of inflammasome, an essential complex for pro-inflammatory cytokine release. Objectives: The aim of this study was to evaluate whether placental explants exposure to hydrogen peroxide (H2O2) is able to induce autophagy and inflammasome activation. Methods: Placental explants achieved from normal pregnant women after delivery were cultured in different concentrations of H2O2 for 4 and 24 h after viability evaluation for these periods. Superoxide dismutase (SOD) and catalase were evaluated in culture supernatants after 4 h. Gene expressions of autophagy markers (LC3-II, beclin-1 and p62), inflammasome (NLRP3 and caspase-1) and IL-1β, IL-10 and TNF-α were assessed by RT-qPCR. Levels of chorionic gonadotrophin (hCG), heat shock protein (Hsp70) and IL-1β, IL-10 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) after 24 h of culture. Results: LDH levels were increased according to culture time, and the 24 h cultures presented adequate cell viability for the study. The protein levels of catalase and Hsp70, as well as the gene expression of LC3-II, beclin-1 and p62 presented increasing levels and related to the higher concentrations of H2O2. Protein concentrations of SOD, hCG and TNF-α were higher in cultures with 100 μM of H2O2. Gene expression of TNF-α, IL-1β, NLRP3 and caspase-1 were raised in 1000 μM of H2O2. In addition, IL-1β protein expression was also higher at this concentration. Gene and protein concentrations of IL-10 decreased as the H2O2 concentration increased. Conclusions: Our results demonstrate that H2O2 is able to induce a state of placental oxidative stress, induce autophagy, activate the inflammasome and then increase the production of inflammatory cytokines.
FAPESP: 2014/25611-5

Made available in DSpace on 2014-06-11T19:29:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T18:39:58Z : No. of bitstreams: 1 spada_apm_me_botfm.pdf: 214670 bytes, checksum: 608905a90c46918e2a91642487aaed16 (MD5)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Objetivo: avaliar o estresse oxidativo no sangue e na placenta de ratas com diabete de intensidade moderada. Métodos: O diabete foi induzido em ratas Wistar recém-nascidas (grupo diabete moderado) no dia do nascimento (dia 0) por streptozotocin (100 mg/kg, via subcutênea). As ratas do grupo nãodiabético (controle) receberam somente tampão citrato. Na vida adulta, as ratas (diabéticas e controle) foram submetidas ao acasalamento e o dia de diagnóstico positivo de prenhez foi considerado dia 0. A glicemia foi determinada nos dias 0, 7, 14 e 21 de prenhez. No 21º dia de prenhez, as ratas foram anestesiadas e dessangradas para determinação das atividades enzimáticas de superóxido dismutase (SOD), glutationa peroxidase (GSH-Px) e glutationa redutase (GSH-Rd) e das concentrações de grupos tiólicos (SH) e de espécies reativas ao ácido tiobarbitúrico (TBARS). Em seguida, as placentas foram retiradas e processadas para determinação das atividades de SOD e catalase e concentração de TBARS, gluationa reduzida e grupos tiólicos. Resultados: Ratas com diabete induzido no período neonatal (grupo diabético) apresentaram glicemia superior a 120mg/dl no dia 0 de prenhez e foi observada hiperglicemia no 14º dia de prenhez. A análise do estresse oxidativo em hemáceas lavadas mostrou que no grupo diabético houve aumento significativo na atividade da GSH-Px. No tecido placentário a atividade da catalase foi significativamente maior em ratas com diabete moderado. Conclusão: Frente às condições experimentais analisadas, o aumento dos biomarcadores do sistema antioxidante em ratas com diabete de intensidade moderada foram suficientes para conter o estresse oxidativo.
Objective: To evaluate the oxidative stress in blood sample and placental of female rats that received streptozotocin in the neonatal period. Methods: The diabetes was induced in female offspring (diabetic group) in the day of the birth (day 0) for streptozotocin (100 mg/kg, subcutaneous route). Female control rat (control group) received only citrate buffer. In the adult life, the female rats were submitted to the mating and the day the positive diagnosis, was considered day 0 of pregnancy. The glycemia was measured in the 0, 7, 14 and 21 of pregnancy. At day 21 of pregnancy, the female rats were anesthetized and died by decapitation for collection of the blood for determination of the enzymatic activity of the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) and of the concentrations of thiols group and thiobarbituric acid reactive substances (TBARS). Afterwards, placental were removed and processed for determinations of the enzymatic activity of the SOD and catalase and of the concentrations of the TBARS, glutathione reduced (GSH) and thiols group (SH). Results: Diabetic rats presented blood glucose concentration greater than 120 mg/dL in the day 0 of pregnancy and hyperglycemia in 14 º day of pregnancy. The analysis of the oxidative stress in maternal blood sample showed increased in GSH-Px activity. In placental tissue catalase activity of diabetic group is found to be increase in homogenate tissue in diabetic group. Conclusion: The hyperglycemia in diabetic rats increased antioxidant system biomarkers, however, these alterations were enough to control oxidative stress.

Resumo: Objetivo: avaliar o estresse oxidativo no sangue e na placenta de ratas com diabete de intensidade moderada. Métodos: O diabete foi induzido em ratas Wistar recém-nascidas (grupo diabete moderado) no dia do nascimento (dia 0) por streptozotocin (100 mg/kg, via subcutênea). As ratas do grupo nãodiabético (controle) receberam somente tampão citrato. Na vida adulta, as ratas (diabéticas e controle) foram submetidas ao acasalamento e o dia de diagnóstico positivo de prenhez foi considerado dia 0. A glicemia foi determinada nos dias 0, 7, 14 e 21 de prenhez. No 21º dia de prenhez, as ratas foram anestesiadas e dessangradas para determinação das atividades enzimáticas de superóxido dismutase (SOD), glutationa peroxidase (GSH-Px) e glutationa redutase (GSH-Rd) e das concentrações de grupos tiólicos (SH) e de espécies reativas ao ácido tiobarbitúrico (TBARS). Em seguida, as placentas foram retiradas e processadas para determinação das atividades de SOD e catalase e concentração de TBARS, gluationa reduzida e grupos tiólicos. Resultados: Ratas com diabete induzido no período neonatal (grupo diabético) apresentaram glicemia superior a 120mg/dl no dia 0 de prenhez e foi observada hiperglicemia no 14º dia de prenhez. A análise do estresse oxidativo em hemáceas lavadas mostrou que no grupo diabético houve aumento significativo na atividade da GSH-Px. No tecido placentário a atividade da catalase foi significativamente maior em ratas com diabete moderado. Conclusão: Frente às condições experimentais analisadas, o aumento dos biomarcadores do sistema antioxidante em ratas com diabete de intensidade moderada foram suficientes para conter o estresse oxidativo.
Abstract: Objective: To evaluate the oxidative stress in blood sample and placental of female rats that received streptozotocin in the neonatal period. Methods: The diabetes was induced in female offspring (diabetic group) in the day of the birth (day 0) for streptozotocin (100 mg/kg, subcutaneous route). Female control rat (control group) received only citrate buffer. In the adult life, the female rats were submitted to the mating and the day the positive diagnosis, was considered day 0 of pregnancy. The glycemia was measured in the 0, 7, 14 and 21 of pregnancy. At day 21 of pregnancy, the female rats were anesthetized and died by decapitation for collection of the blood for determination of the enzymatic activity of the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) and of the concentrations of thiols group and thiobarbituric acid reactive substances (TBARS). Afterwards, placental were removed and processed for determinations of the enzymatic activity of the SOD and catalase and of the concentrations of the TBARS, glutathione reduced (GSH) and thiols group (SH). Results: Diabetic rats presented blood glucose concentration greater than 120 mg/dL in the day 0 of pregnancy and hyperglycemia in 14 º day of pregnancy. The analysis of the oxidative stress in maternal blood sample showed increased in GSH-Px activity. In placental tissue catalase activity of diabetic group is found to be increase in homogenate tissue in diabetic group. Conclusion: The hyperglycemia in diabetic rats increased antioxidant system biomarkers, however, these alterations were enough to control oxidative stress.
Orientador: Débora Cristina Damasceno
Coorientador: Tiago Rodrigues
Mestre

Impaired placentation and oxidative stress are proposed to play major roles in the pathogenesis of pre-eclampsia (PE). It has recently been pointed out that PE might be more than one disease and may have several different pathogeneses. This thesis describes a new animal model for PE and examines the role of oxidative stress in early respective late onset PE.

The effects of Suramin injections on day 10 and 11 of pregnancy were investigated in normal and diabetic rats of two strains (U and H), with or without additional vitamin E treatment. Suramin caused placental dysfunction in both rat strains: foetal growth restriction, increased resorption rate, reduced placental blood flow, and decreased maternal blood volume in the placenta. In the U strain Suramin also caused maternal hypertension and reduced renal blood flow. Oxidative stress in the Suramin treated rats was indicated by increased levels of isoprostane 8-iso-PGF_2α in the placenta. Antioxidative treatment with vitamin E partly protected against the effects of Suramin. Streptozotocin-induced diabetes seemed to cause similar placental effects as Suramin, and in the diabetic rats the additional effects of Suramin were only moderate. In conclusion, Suramin-injected pregnant rats constitute a valid animal model for placental dysfunction (U and H rats) and PE (U rats).

Oxidative stress was estimated in women with early onset (≤ 32 weeks) or late onset (≥ 35 weeks) PE, in normotensive pregnant women of respective gestational length, and in healthy non-pregnant women. The ratio of PAI-1/PAI-2 was measured in serum, and the amount of isoprostane 8-iso-PGF_2α was measured in placenta, serum, and urine. The ratio of PAI-1/PAI-2 and placental isoprostane levels were higher in women with early onset PE compared with all other groups. Serum levels of isoprostane were similar between groups. Urinary levels of isoprostane were similar in all pregnant women, but lower in non-pregnant women. These data indicate that pregnancy increases general oxidative stress, and that early onset, but not late onset PE, causes increased oxidative stress also in placental tissue. The pathogeneses of early and late onset PE are, therefore, not likely to be identical.

Although not obvious at first sight, several parallels can be drawn between pregnancy andcancer. Many proliferative, invasive and immune tolerance mechanisms that supportnormal pregnancy are also exploited by malignancies to establish a nutrient supply andevade or edit the immune response of the host. The human placenta, of crucial importancefor pregnancy success, and its main cells, the trophoblast, share several features withmalignant cells such as high cell proliferation rate, lack of cell-contact inhibition andinvasiveness. Both in cancer and in pregnancy, the immune defense mechanisms,potentially threatening the survival of the tumor or the fetus, are progressively blunted oreven turned into tumor- or pregnancy-promoting players. Amongst immune mechanisms that are meant to protect the host from cancer and can be apotential threat to the fetus, the NKG2D receptor-ligand system stands out as the mostpowerful, stress-inducible “danger detector” system that comprises the activating NK cellreceptor NKG2D and its ligands, the MIC (MHC class I Chain-related proteins A and B)and ULBP (UL-16 Binding Proteins) families. It is the major cytotoxic mechanism in thebody promoting surveillance and homeostasis. In the present thesis we investigate theNKG2D receptor-ligand system in human early normal pregnancy and in theleukemia/lymphoma cell lines Jurkat and Raji and ask the questions “How is the NKG2Dreceptor-ligand system functioning in pregnancy and tumor? How is the danger of cytotoxicattack of the fetus avoided? Why is the immunosurveillance function compromised incancer patients?” We developed a method to isolate and culture villous trophoblast from early human normalplacenta and used it to study the NKG2D receptor-ligand system. We discovered that theNKG2D ligand families of molecules MICA/B and ULBP1-5 are constitutively expressedby the syncytiotrophoblast of the chorionic villi. Using immnunoelectron microscopy, westudied the expression of these molecules at the subcellular level and could show for thefirst time that they are preferably expressed on microvesicles in multivesicular bodies(MVB) of the late endosomal compartment and are secreted as exosomes. Exosomes arenanometer sized microvesicles of endosomal origin, produced and secreted by a great7variety of normal and tumor cells. The exosomes are packages of proteins and ribonucleicacids that function as “mail” or “messengers” between cells conveying different biologicalinformation. We isolated and studied exosomes from placental explant cultures. We foundthat they carry NKG2D ligands on their surface and are able to bind and down-regulate thecognate receptor on NK-, CD8+ and T cells. The down-regulation selectively causedimpairment of the cytotoxic response of the cells but did not affect their lytic ability asmeasured by perforin content and gene transcription. Thus, the NKG2D ligand-bearingexosomes suppress the cytotoxic activity of the cells in the vicinity of the placenta, leavingtheir cytolytic machinery intact, ready to function when the cognate receptor isrestored/recycled. These findings highlight the role of placental exosomes in the fetalmaternalimmune escape and support the view of placenta as an unique immunomodulatoryorgan. Next, we studied the expression and exosomal release of NKG2D ligands by tumor cellsusing the leukemia cell lines Jurkat and Raji as a tumor model. We found that NKG2Dligand-bearing exosomes with similar immunosuppressive properties as placental exosomesare constitutively secreted by the tumor cells, as a mechanism to blunt the cytotoxicresponse of the immune cells and thus protect themselves from cytotoxic attack by the host.Interestingly, we found that thermal- and oxidative stress up-regulates the exosomesecretion and the amount of exosome-secreted NKG2D ligands. Our results imply thattumor therapies that cause stress-induced damage, such as thermotherapy and stripping ofoxygen supply to the tumor, might have a previously unrecognized side effect causingenhanced exosome production and secretion, which in turn suppresses the natural antitumorimmune response and thus should be taken into account when designing an optimaltherapy of cancer patients. In conclusion, we describe a novel stress-inducible mechanism shared by placenta andtumors as an immune escape strategy. We found that placenta- and tumor-derived NKG2Dligand-bearing exosomes can suppress immune responses to promote the survival and wellbeing of the fetus or the tumor. Our work comprises an important contribution to theelucidation of the NKG2D ligand-receptor system and its mode of operation in the humanbody and opens new perspectives for designing novel therapies for infertility and cancer.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-25T12:46:12Z No. of bitstreams: 1 Natalie Emanuelle Ribeiro e Silva.pdf: 562491 bytes, checksum: e2fc3bb4739adc151a9f70d9d8e255e1 (MD5)
Made available in DSpace on 2016-07-25T12:46:12Z (GMT). No. of bitstreams: 1 Natalie Emanuelle Ribeiro e Silva.pdf: 562491 bytes, checksum: e2fc3bb4739adc151a9f70d9d8e255e1 (MD5) Previous issue date: 2013-02-26
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Intrauterine programmed cardiovascular disease may be linked to placental oxidative stress. It was investigated whether NaCl supplement, during pregnancy, affects placental oxidative stress and angiogenesis and imprints increased levels of oxidative stress in fetal liver. Dams were treated with 1.8% NaCl in drinking water from 20 days before and along pregnancy. Along pregnancy α-tocopherol, tempol or both were administered. Angiogenesis was evaluated throughout the expression of flt-1, the type 1 receptor for VEGF, in the villous bundles/labirinth. NaCl diminished flt-1 expression, as well as, reduced malonyldialdehide and augmented reduced gluthatione in placenta and fetal liver. α-Tocopherol led to an additional reduction in MDA levels in these organs, but diminished GSH. Otherwise, tempol increased MDA and diminished GSH in both placenta and fetal liver. No antioxidant changed the expression of flt-1. The reduced levels of MDA in placenta and fetal liver of dams under NaCl supplement might be due to reduced angiogenesis, that reduced oxygen supply, and also to the increased GSH levels. However the paradoxical pro-oxidant action of tempol indicates that the antioxidant reservoir in dams under sodium overload is limited. Furthermore, the parallel between the pattern of placental and fetal liver oxidative stress suggests that superoxide anions transferred from mothers may be important in programming cardiovascular diseases.
Doenças cardiovaculares programadas intrauterinamente podem está ligadas ao estresse oxidativo placentário. Neste trabalho, investigamos se a sobrecarga de NaCl, durante a gravidez, afeta o estresse oxidativo e angiogênese placentária, bem como se afeta os níveis de estresse oxidativo no fígado do feto. Mães foram tratadas com NaCl, 1,8%, na água de beber, 20 dias antes e ao longo da gravidez. α-Tocoferol, tempol ou ambos foram administrados ao longo da gravidez. A angiogênese foi avaliada por meio da expressão do flt-1, o receptor tipo 1 para o VEGF, nos feixes vilosos do labrinto. A suplementação com NaCl diminuiu a expressão de flt-1, bem como, reduziu os níveis de malonildialdeido e aumentou os níveis de glutationa reduzida na placenta e no fígado fetal. O α-tocoferol levou a uma redução adicional nos níveis de MDA nesses órgãos, mas diminuiu os níveis de GSH. De maneira diferente, o tempol aumentou os níveis de MDA e diminuiu os níveis de na placenta e no fígado fetal. O tratamento com os antioxidantes não alterou a expressão do flt-1. Os níveis reduzidos de MDA na placenta e no fígado do feto de mães submetidas a sobrecarga de NaCl podem ser devidos à angiogênese diminuida, o que reduziu o suprimento de oxigênio, ou aos os níveis aumentados de GSH. Entretanto, a ação paradoxal, pro-oxidante, do tempol indica que a reserva antioxidante nas mães submetidas a sobrecarga de sódio é limitada. Além disso, o paralelo entre o padrão de estresse oxidativo placentário e do fígado fetal sugere que ânions superóxidos transferidos a partir das mães podem ser importantes na programação das doenças cardiovaculares.

Research Doctorate - Doctor of Philosophy (PhD)
Stillbirth is a neglected public health problem affecting more than two million women and families globally each year with devastating and long-lasting psychosocial and financial impact. Rates of stillbirth, even in high-income countries with access to optimal obstetric care, have remained static in the past two decades. The causes of, or associations with, stillbirth that have been identified clinically include fetal factors such as genetic/structural abnormalities and growth restriction, maternal factors such as preeclampsia and infections and placental factors such as abruption and placenta previa. However, no specific cause has been established for the majority of stillbirths at term, and the rate of this category of death rises drammatically as gestation progresses beyond 38 weeks. Taking into account the functional definition of aging that is an increase in the risk of death with time, and the existence of placental pathologies in the unexplained stillbirth pregnancies resembling aging in other organs, we hypothesise that premature placental aging may be the primary factor in the aetiology of unexplained stillbirth. Premature aging may occur when cells experience increased oxidative stress that causes damage to cellular macromolecules, including DNA, RNA and lipids, and alters protein expression patterns, especially those that are crucial for cellular survival and function. Therefore, the primary aim of this thesis was to investigate evidence that the placenta from late-gestation shows biochemical signs of oxidative damage and aging that would also be present in placentas associated with stillbirths. A further aim was to investigate the pathways that mediate the oxidative damage and aging in the placenta in pathologic pregnancies. We have shown that placentas from both late-term and stillbirth pregnancies show biochemical signs of aging in the form of increased DNA and lipid oxidation. Also, the expression of aldehyde oxidase 1 (AOX1), which is known to be involved in reactive oxygen species (ROS) generation and oxidative stress, is increased in placental tissues obtained from both late-gestation and stillbirth pregnancies. We tested the association of AOX1 in stillbirth pregnancy as an RNA sequencing study performed in our laboratory identified a significant increase in AOX1 mRNA in late-term placentas compared to term healthy placentas (unpublished). The demonstration of G-protein coupled estrogen receptor 1 (GPER1), a cell surface estrogen receptor, localisation on the apical surface of the normal placental syncytiotrophoblast and its role in the reduction of ROS generation and oxidative damage indicate that this receptor may be a critical step in the pathway of placental ROS induced oxidative damage. Using a placental explant and a cell line culture model, we then tested the pathways that regulate placental oxidative damage and aging. Results presented in this thesis revealed that growth factor removal resulted in placental oxidative damage, with impaired mitochondrial function, decreased expression of sirtuins (proteins that control aging), alteration of nutrient sensing mammalianTORC1, and energy sensing AMP activated protein kinase pathways, all the changes are known to be associated with oxidative damage and aging in other tissues. Inhibition of AOX1 or stimulation of estrogen activation at GPER1 resulted in the blocking of all the changes observed after removal of growth factors. Together, these findings support the hypothesis that placental oxidation is regulated by estrogen activation at the GPER1 and inhibition of AOX1 leading to the inhibition of ROS generation and oxidative stress. Our study identifies potential biomarkers of oxidative damage and aging in stillbirth placentas that raise the possibility that these biomarkers of placental oxidative damage and aging may be released into the maternal blood where they may have diagnostic value in predicting the fetus at risk for stillbirth. Treatment targeting AOX1 and/or GPER1 may arrest the oxidative damage in the placenta in pregnancies identified at risk and may lead to novel therapeutic strategies for delaying placental aging, as well as preventing stillbirth and other age-related adverse pregnancy outcomes.

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation.
Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2013-07-05 14:37:05.15

The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

This thesis is comprised of seven chapters: a general thesis introduction (Chapter 1), one Published review of the literature (Chapter 2), one published research article (Chapter 3), one submitted research article (Chapters 4), one unpublished research chapter written in manuscript style (Chapters 5), a published systematic review (Chapter 6), and a final general discussion (Chapter 7). Chapter 2 is a published comprehensive review of the impact of maternal selenium and iodine on placental and child health (Habibi et al., Nutrients 2020). A few studies showed that selenium could increase antioxidant enzyme activity in placental cell lines. Additionally, population studies revealed that selenium and iodine deficiency separately were associated with a greater risk of pregnancy complications. However, there are no studies that have investigated the potential synergistic effect of iodine and selenium combined on placental health. To address this gap in the literature, the role of these two essential micronutrients during pregnancy and in relation to oxidative stress has been comprehensively reviewed. This review supports the hypothesis that selenium and iodine could potentially have a synergistic role in protecting against oxidative stress in the placenta. We went on to test this hypothesis in Chapters 3 and 4. Chapter 3 is a published original research study describing the effect of iodine and selenium on proliferation, viability, and oxidative stress in HTR-8/SVneo placental cells (Habibi et al., Biological Trace Element Research 2020). Our study showed that oxidative stress reduces HTR-8/SVneo cells viability and increases lipid peroxidation, which is oxidative damage to the cell membrane. Interestingly, selenium and iodine supplementation separately or together could protect cells against oxidative stress. A supraphysiological concentration of selenium caused some toxic effects in cells that were not exposed to oxidative stress. Interestingly, the same concentration in cells treated with oxidative stress was protective suggesting a higher demand of selenium in HTR-8/SVneo placental cells to combat oxidative stress. The combination of selenium and iodine provided a greater protection against oxidative stress to the cells compared to their individual supplementation suggesting a synergistic effect in HTR-8/SVneo placental cells. Chapter 4 is original research describing how selenium and iodine can protect first trimester human placenta against oxidative stress and what the effect of copper is in the placenta (submitted to Human Reproduction on 19th August 2020). For the first time, using laser ablation inductively coupled plasma-mass spectrometry, we showed the selenium and copper distribution in the treated human first trimester placenta explants. We investigated the potential mechanism of the effect of iodine, selenium and copper on first trimester human placenta. Oxidative stress increased DNA damage and apoptosis and supplementation with iodine or selenium was protective resulting in reduced DNA damage and apoptosis in first trimester placenta explants. Supplementation with a combination of iodine and selenium provided a greater reduction in oxidative damage to DNA molecules suggesting a synergistic effect against oxidative stress in the placenta. A high concentration of copper increased DNA damage and apoptosis in the placenta in the absence of induced oxidative stress. However, this was not seen when placenta tissue explants were exposed to oxidative stress suggesting a higher consumption of copper during oxidative stress. Chapter 5 is an unpublished original manuscript describing associations between buffy coat mitochondrial DNA content and maternal micronutrient status and pregnancy outcome (To be submitted to the International Journal of Molecular Sciences). Using 317 samples of participants of Screening for Pregnancy Endpoints (SCOPE) study in Adelaide we investigated the association between maternal mitochondrial DNA content at 15 ± 1 weeks’ gestation with micronutrient status and pregnancy outcomes. We found that higher inflammation, defined by increased C-reactive protein concentration, was associated with reduced mitochondrial DNA content. A lower mitochondrial DNA content at 15 ± 1 weeks’ gestation was associated with a greater risk of pregnancy complications. There was no association between mitochondrial DNA content and micronutrient status. Mitochondrial DNA content may reflect risk of pregnancy complications but it is not a strong predictor and it was not associated with maternal micronutrient status. Chapter 6 is a published systematic review on maternal diet and offspring telomere length (Habibi et al., Nutrition Reviews 2020). Maternal nutrition may be an important determinant of offspring telomere length that is a biomarker of ageing and chronic diseases. Therefore, we systematically reviewed all available literature about the association of maternal nutrition and offspring telomere length. We found little evidence on such an essential topic. However, there were seven studies on this topic and collectively they showed that higher maternal circulating folate and 25(OH)D3 and dietary caffeine intake, were associated with longer offspring telomere length, whereas dietary intake of carbohydrate, folate, n-3 PUFA, vitamin C or sodium, was not. This systematic review highlighted the necessity for further research in this area. Chapter 7 is the final chapter that presents a summary of my PhD work and general discussion of all findings of this project and suggests future research directions.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2020

Au cours de la grossesse, une perfusion placentaire adéquate est indispensable au bon développement du fœtus. Dans certaines maladies comme la prééclampsie, celle-ci est altérée, compromettant ainsi la vie du fœtus, mais aussi celle de sa mère. Le retrait du placenta mène à la disparition des symptômes de la prééclampsie, suggérant un rôle central de ce dernier dans la maladie. Le placenta étant dépourvu d’innervation autonome, le tonus vasculaire placentaire doit être sous le contrôle de facteurs humoraux et tissulaires. Les vaisseaux placentaires sont très réactifs aux prostanoïdes. Le rapport thromboxane A2 (TXA2)/prostacycline (PGI2) est fortement augmenté dans les placentas de grossesses avec prééclampsie. De plus, le taux d’isoprostane, marqueur du stress oxydatif, est accru dans les placentas de femmes avec prééclampsie. Finalement, la prééclampsie s’accompagne d’un stress oxydatif placentaire marqué. Les espèces réactives de l’oxygène sont connues d’une part, pour oxyder l’acide arachidonique (AA), formant ainsi des isoprostanes et d’autre part, pour augmenter la production de TXA2 dans différents tissus, suite à l’activation des cyclooxygénases (COXs). Nous proposons que : 1. les prostanoïdes sont parmi les molécules endogènes qui contrôlent le tonus vasculaire placentaire. 2. la maladie modifie la réponse aux isoprostanes dans les vaisseaux placentaires. 3. l’induction d’un stress oxydatif placentaire entraîne une réponse vasoactive par activation de la voie du métabolisme de l’AA. Nous avons tout d’abord montré, dans des placentas obtenus de grossesses normotensives, que l’U-46619, un mimétique de la TXA2, de même que l’isoprostane, 8-iso-prostaglandine E2 (8-isoPGE2), ont augmenté fortement la pression de perfusion dans les cotylédons perfusés in vitro et la tension dans les anneaux d’artères chorioniques suspendus dans des bains à organe isolé. En revanche, dans les artères chorioniques de placentas obtenus de grossesses avec prééclampsie, ces réponses étaient modifiées puisque la réponse maximale à l’U-46619 était augmentée et celle à la 8-isoPGE2 diminuée. D’autre part, nous avons montré que les réponses maximales aux deux prostanoïdes étaient augmentées dans les vaisseaux placentaires de grossesse normale ou avec prééclampsie issus d’une délivrance prématurée par rapport à ceux d’une délivrance à terme. Ceci suggère une évolution de la réactivité des artères placentaires au cours du 3e trimestre de grossesse. En outre, les vaisseaux placentaires ont répondu aux prostanoïdes de façon semblable qu’ils aient été issus d’un accouchement vaginal ou d’une césarienne élective. Ceci indique que les prostanoïdes placentaires n’interviennent pas dans le processus de délivrance. D’un autre côté, l’utilisation de bloqueurs spécifiques des récepteurs TP à la TXA2, le SQ29,548 et l’ICI192,605, et des récepteurs EP à la prostaglandine E2, l’AH6809, nous ont permis de mettre en évidence le fait que l’U-46619 et la 8-isoPGE2 pouvaient agir de façon non-sélective sur l’un ou l’autre des récepteurs. Ces résultats supportent donc nos 2 premières hypothèses : les prostanoïdes font partie des molécules endogènes qui peuvent contrôler le tonus vasculaire placentaire et la prééclampsie modifie la réponse aux isoprostanes dans les artères chorioniques d’une manière compatible avec l’augmentation de la production de ces substances qui elle, est probablement le résultat du stress oxydatif. En revanche, en ce qui concerne les substances capables de jouer la contrepartie vasodilatatrice, l’utilisation d’un inhibiteur des synthases de monoxyde d’azote, le L-NAME, et celle d’inhibiteurs des COXs, l’ibuprofène, l’indométacine et le N-2PIA, ne nous a pas permis de mettre en évidence un quelconque rôle du monoxyde d’azote ou des prostanoïdes vasodilatatrices à ce niveau. Finalement, nous avons montré que l’induction d’un stress oxydatif dans les cotylédons perfusés in vitro et les artères chorioniques entraînait une vasoconstriction marquée. Celle-ci semble résulter de l’action des prostanoïdes puisqu’un blocage des récepteurs TP ou des COXs diminuait significativement la réponse maximale au peroxyde d’hydrogène. Les prostanoïdes impliquées dans la réponse au stress oxydatif proviendraient essentiellement d’une activation des COXs puisque l’étude ne nous permet pas de conclure à une quelconque implication des isoprostanes dans cette réponse. Ces observations confirment donc notre hypothèse que, dans le placenta, le stress oxydatif possède des propriétés vasoactives par activation du métabolisme de l’AA. En résumé, les résultats obtenus dans les placentas de grossesses normotensives et avec prééclampsie suggèrent que les prostanoïdes sont des molécules d’importance dans la régulation du tonus vasculaire placentaire. Le fait que la prééclampsie modifie la réponse aux prostanoïdes pourrait expliquer pourquoi la perfusion placentaire est altérée chez ces patientes. En outre, il apparaît évident qu’il existe un lien étroit entre le stress oxydatif et la voie de synthèse des prostanoïdes placentaires. Cependant d’autres études sont nécessaires pour mieux comprendre la nature de ce lien, qui pourrait, d’une certaine façon, jouer un rôle important dans le développement de la prééclampsie.
Throughout pregnancy, appropriate placental perfusion is essential for the fœtus to grow properly. In disease such as preeclampsia, placental perfusion is impaired, compromising the fœtus and mother’s lives. Placenta delivery leads to a complete disappearance of the clinical symptoms of preeclampsia. This suggests that the placenta plays a central role in the disease. Placenta being devoid of autonomous innervation, placental vascular tone needs to be under the control of humoral and tissular factors; placental arteries are very reactive to prostanoids. The thromboxane A2 (TXA2)/prostacyclin (PGI2) ratio is increased in placenta from preeclamptic women. Furthermore, in placenta from preeclamptic pregnancies, isoprostane rate is increased, which is a marker of oxidative stress. Finally, preeclampsia is characterised by an important oxidative stress. Reactive oxygen species are known to form isoprostane through the oxidation of arachidonic acid (AA) and to increase TXA2 production in various tissues following an activation of the cyclooxygenases (COXs). We postulate that: 1. prostanoids are among the endogenous molecules that control placental vascular tone. 2. preeclampsia alters responses to isoprostanes in placental vessels. 3. induced placental oxidative stress leads to vasoactive responses through the activation of the AA metabolism. We first showed in placentas from normotensive pregnancies that the TXA2 mimetic U-46619 and the isoprostane 8-isoprostaglandin E2 (8-isoPGE2) markedly increased perfusion pressure in in vitro perfused cotyledons, as well as tension in isolated chorionic arteries. However, in placentas obtained from women with preeclampsia, those responses were altered in chorionic arteries. Indeed, maximal response to U-46619 was raised by preeclampsia, while the one to 8-isoPGE2 was decreased. We then showed that preterm delivery increased maximal responses to both prostanoids compared to term delivery. This observation suggests that placental arteries reactivity evolves along the 3rd trimester of pregnancy. Nevertheless, it appeared that delivery mode had no effect on vascular responses to prostanoids, suggesting that placental prostanoids are not involved in the delivery process. The use of specific blockers of the TXA2 TP receptors, SQ29,548 and ICI192,605, and of the prostaglandin E2 EP receptors, AH6809, revealed that U-46619 and 8-isoPGE2 could mediate their effects by acting on both receptors in a non-selective manner. Therefore, these results support our two first postulates: prostanoids could be the endogenous substances controlling the placental vascular tone and preeclampsia alters responses to isoprostanes in chorionic artery rings in a way compatible with the increased production of these substances possibly through the associated oxidative stress. Moreover, we were unable to identify any vasodilator substances capable of counteracting the effects of vasoconstrictors in the placental circulation. Indeed, blocker of nitric oxide synthases, L-NAME, as well as blockers of COXs, ibuprofen, indometacin and N-2PIA, did not reveal any effect of nitric oxide and vasodilator prostanoids at this level. Finally, we showed that induction of oxidative stress in in vitro perfused cotyledons and in isolated chorionic artery rings led to marked vasoconstriction. This would result from the action of prostanoids since a blockade of TP receptors or COXs significantly decreased maximal response to hydrogen peroxide. Prostanoids involved in this response would essentially come from COX activation. Indeed, the present results did not show any concrete involvement of isoprostane substances in the response to oxidative stress. Consequently, these observations confirm our hypothesis that, in the placenta, oxidative stress presents some vasoactive properties through the activation of the AA metabolism. In summary, results obtained in placentas from normotensive and preeclamptic pregnancies suggest that prostanoids are important in the regulation of the placental vascular tone. Furthermore, responses to prostanoids in chorionic arteries are altered by preeclampsia, which could explain why the placental perfusion is impaired in the disease. Moreover, it seems clear that there is a close relationship between oxidative stress and synthesis of placental prostanoids. However, more investigations are needed to better understand the nature of this relationship, which, in some way, could play an important role in the development of preeclampsia.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...