Abstract Abnormal placentation is associated with preeclampsia and placental insufficiency, both of which increase the risk for fetal growth restriction. So far the early recognition of the risk population for preeclampsia has been problematic. The first hypothesis of this study was that in preeclampsia, the maternal serum proteomic profile is different from that in uncomplicated pregnancies, and this difference is detectable already in early pregnancy. The findings of this study demonstrate that in clinical preeclampsia the maternal serum proteomic profile is different from that in uncomplicated pregnancies with increased levels of placental proteins and antiangiogenic factors in pregnancies with clinical preeclampsia. Furthermore, the early pregnancy maternal serum proteomic profile in women who later develop preeclampsia revealed a distinct and different pattern compared with the profile in clinical preeclampsia. In early pregnancy, the differentially expressed proteins belong to placental proteins, vascular and/or transport proteins and matrix and/or acute phase proteins, while angiogenic and antiangiogenic proteins were not significantly expressed in early pregnancy. Preeclampsia, placental insufficiency, fetal growth restriction and type-1 diabetes may have an impact on fetal cardiovascular hemodynamics. The second hypothesis in this thesis was that in placental insufficiency, abnormalities in fetal cardiovascular status correlate with biochemical markers of cardiac dysfunction and chronic hypoxia. In placental insufficiency, increases in fetal N-terminal pro-atrial (NT-proANP) and pro-B-type natriuretic peptide (NT-proBNP) and in fetal erythropoietin concentrations were related to increased pulsatility in the fetal umbilical artery and descending aorta. In addition, these fetuses demonstrated increased pulsatility in their systemic venous blood velocity waveforms. Thus, in placental insufficiency, biochemical markers of cardiac dysfunction and chronic hypoxia are associated with signs of increased fetal cardiac afterload and systemic venous pressure. Increased NT-proANP and NT-proBNP levels were also detected in fetuses of type-1 diabetic mothers with normal umbilical artery velocimetry. In these pregnancies, NT-proANP and NT-proBNP levels were related to poor maternal glycemic control during early pregnancy.

La prééclampsie est une maladie fréquente de la grossesse, caractérisée par l’apparition de novo d’une hypertension et d’une protéinurie à partir de la 20ème semaine d’aménorrhée. Ces symptômes s’aggravent au long de la grossesse, conduisant éventuellement à la mort maternelle en l’absence de prise en charge médicalisée. La thérapeutique définitive l’extraction du placenta, et donc du fœtus, ce qui induit une importante prématurité iatrogène. Les causes restent mal définies, mais il est bien admis que des anomalies au niveau de la mise en place du placenta sont au cœur de sa physiopathologie. Un défaut d’invasion trophoblastique des artères spiralées utérines semble être une constante de la maladie. Des données épidémiologiques démontrent qu’il existe une forte composante génétique dans la prééclampsie, et en 2005, un clonage positionnel dans des familles hollandaises, aboutit à l’identification de STOX1 comme le premier gène lié à cette maladie. STOX1 code un facteur de transcription intervenant dans le contrôle de la prolifération et de l’invasion des trophoblastes. Dans notre laboratoire, l’étude de STOX1 a été initiée par surexpression dans des cellules de choriocarcinome humain (modèle de trophoblastes) suivie d’une analyse transcriptomique. Celle-ci a révélé que les altérations d’expression génique observées suite à la surexpression de STOX1 étaient significativement corrélées à celles trouvées dans des placentas prééclamptiques. La création de souris transgéniques exprimant la version humaine de STOX1 sous le contrôle d’un promoteur ubiquitaire a alors été entreprise. Mes travaux de thèse ont principalement consisté à caractériser le phénotype de ces souris. Nous avons décidé de croiser des mâles transgéniques avec des souris sauvages afin de limiter l’expression du transgène à l’unité fœto-placentaire. Ces souris sauvages développent au cours de leur gestation une hypertension sévère, et une protéinurie. Elles constituent donc un nouveau modèle de prééclampsie. De plus, nous avons observé des anomalies que l’on trouve également chez les patientes : une fibrose rénale, une élévation des taux sériques de facteurs pro-angiogéniques (le récepteur soluble du VEGF et l’endogline soluble). Ces souris ont également des marqueurs d’hypertrophie cardiaque, attestant de l’impact sévère de l’hypertension. Pour mieux comprendre comment STOX1 peut induire ce syndrome, nous avons étudié son impact dans le modèle cellulaire surexprimant STOX1 et nous avons pu montré une altération de la gestion du stress oxydatif et de la fonction mitochondriale. En conclusion, nous avons obtenu et caractérisé un modèle de prééclampsie sévère, le seul existant montrant un phénotype hypertensif très marqué et très précoce. Ce modèle est un outil puissant pour découvrir de nouvelles voies impliquées dans la physiopathologie de la prééclampsie, pour rechercher de potentiels marqueurs diagnostiques précoces, tester des approches thérapeutiques innovantes et explorer les mécanismes responsables des conséquences à long terme de la prééclampsie. / Preeclampsia is a common disease of pregnancy characterized by de novo appearance of hypertension and proteinuria from the 20th week of gestation. These symptoms worsen during pregnancy, possibly leading to maternal death in the absence of medical management. The only final treatment is the extraction of placenta and so fetus, which is responsible to a significant iatrogenic prematurity. The causes remain unclear, but it is well accepted that abnormalities in the development of the placenta are at the heart of its pathophysiology. A lack of trophoblastic invasion of the uterine spiral arteries seems to be a constant of the disease. Epidemiological data show that there is a strong genetic component in preeclampsia, and in 2005, a positional cloning in Dutch families led to the identification of STOX1 as the first gene linked to the disease. STOX1 encode a transcription factor involved in the control of proliferation and invasion of trophoblasts. In our laboratory, the study of STOX1 was initiated by its overexpression in human choriocarcinoma cells (trophoblast model) followed by transcriptome analysis. This revealed that the gene expression changes observed after STOX1 overexpression were significantly correlated with those found in preeclamptic placentas. The creation of transgenic mice expressing the human version of STOX1 under the control of a ubiquitous promoter was then initiated. My work during this thesis consisted primarily to characterize the phenotype of these mice. We decided to cross transgenic males with wild-type mice to limit transgene expression in fetal-placental unit. These wild mice develop during gestation severe hypertension and proteinuria. They therefore constitute a new model of preeclampsia. In addition, we observed anomalies that are also found in patients: renal fibrosis, elevated serum levels of pro-angiogenic factors (soluble VEGF receptor and soluble endoglin). These mice also have markers of cardiac hypertrophy, attesting for the severe impact of hypertension. To better understand how STOX1 can induce the syndrome, we studied its impact on the cell model overexpressing STOX1 and we showed a change in the management of oxidative stress and mitochondrial function. In conclusion, we have obtained and characterized a model of severe preeclampsia, the only existing one showing a very strong and very early hypertensive phenotype. This model is a powerful tool for discovering new pathways involved in the pathophysiology of preeclampsia, for searching potential early diagnostic markers, for testing innovative therapeutic approaches and for exploring the mechanisms responsible for the long-term consequences of preeclampsia.

La prééclampsie est une maladie humaine qui affecte 3-8 % des grossesses dans le monde, cliniquement définie par l’apparition de novo d’une hypertension et d’une protéinurie. La cause initiale de la maladie semble être liée à un défaut de vascularisation placentaire, ce qui entraine des cycles d'hypoxie – ré-oxygénation, une ischémie placentaire et la libération de débris placentaires dans la circulation maternelle. Ces derniers sont responsables d'une activation endothéliale généralisée, exacerbée par un état pro-coagulant et pro-inflammatoire. Pour mieux caractériser la réponse des cellules endothéliales aux facteurs plasmatiques présent dans la circulation maternelle des femmes prééclamptiques , nous avons choisi une approche à l’échelle du génome entier pour évaluer le profil d'expression génique (grâce à des puces d’expression) de la lignée de cellules endothéliales humaines de la veine ombilicale (HUVEC) cultivée avec du plasma prééclamptique, comparée au profil de cellules cultivées avec du plasma humain provenant de grossesses normales. Cette étude nous a permis d'identifier différents gènes modulés dont celui codant la protéine de dimérisation Jun 2 (JDP2, diminué près de trois fois) qui pourrait être responsable d'une partie des modifications transcriptomiques trouvées. De façon intéressante en effet, inhiber JDP2 par une approche de siRNA régule significativement à la baisse (entre autres) l'expression du VEGF, imitant ainsi les effets du plasma prééclamptique sur les HUVEC. Dans la dernière partie de mon projet, nous nous sommes particulièrement concentrés sur l'impact de l’inhibition de JDP2 sur des gènes induits par l'hypoxie. La tension partielle basse en oxygène modifie l'expression génique par l'intermédiaire de la stabilisation du facteur de transcription HIF- 1a. En fait, dans un état hypoxique, HIF- 1a échappe à la dégradation par le protéasome, il forme alors des hétérodimères avec ARNT (HIF- 1ß) et induit l'expression de gènes ayant un élément de réponse à l’hypoxie (HRE) dans leur promoteur. L'induction de l'expression du VEGF dans des conditions d’hypoxie constitue un des premiers modèles de l’effet de l’hypoxie sur l’expression génique et c’est également un des mieux caractérisés. Afin d'évaluer le rôle de JDP2 sur l'expression du VEGF, et plus généralement sur des gènes cible de l’hypoxie, nous avons cultivé des cellules HUVEC dans des conditions de normoxie et d’hypoxie. Les mêmes conditions ont été utilisées en association avec la transfection de siRNA contre JDP2. En conclusion, dans des conditions d’hypoxie, l’inhibition de JDP2 a un impact négatif sur l'expression du VEGF. De plus, JDP2 semble être un médiateur essentiel de l'expression génique induite par l'hypoxie, car il est nécessaire à une activité complète de promoteur contenant des HRE (démontré dans des essais luciférase). / Preeclamspia is a unique human disorder which affects 3-8% of pregnancies worldwide, clinically defined as the new onset of hypertension and proteinuria. The root cause of the disease seems to be linked to a defect of placental vascularization, which enhances cycles of hypoxia –reoxygenantion, placental ischemia and the release of placental debris into maternal circulation. The latter ones are responsible for a widespread endothelial activation, exacerbated pro-coagulable and pro-inflammatory state. To best characterize the response of endothelial cells to the plasma factors present in maternal circulation of preeclamptic women, we chose a genome –wide approach in order to evaluate the gene expression profile of Human Umbilical Vein Endothelial Cells (HUVEC) line cultivated with preeclamptic plasma, compared to cells cultivated with human plasma coming from normal pregnancies. This study allows us to identify the gene Jun Dimerization Protein2 (JDP2) which could be responsible for part of transcriptomic modifications. Interestingly inhibiting JDP2 by the use of siRNA significantly down- regulates VEGF expression, thus mimicking the effects of preeclamptic plasma on HUVEC. In the last part of my project we focus specifically on the impact of JDP2 knock down on hypoxia- induced genes. Low oxygen tension modifies gene expression via the stabilization of the transcription factor HIF-1a. In fact under hypoxic condition, HIF-1a escapes from proteasomal degradation, it forms heterodimers with ARNT (HIF- 1ß) and induces the expression of genes having a Hypoxia Responsive Element (HRE) in their promoter. One of the first and best characterized models of the effect of hypoxia on gene expression is the induction of VEGF expression under hypoxic condition. In order to evaluate the contribution of JDP2 to VEGF expression, and more generally to hypoxia target genes, we cultivate HUVEC in normoxic and hypoxic condition. The same conditions were used in association with transfection of siRNA against JDP2. In conclusion, under hypoxic condition, JDP2 down- regulation has a negative impact on VEGF expression. Moreover, JDP2 seems to be an essential mediator of hypoxia –induced gene expression, since it is necessary for a full HRE promoter activity (demonstrated by Luciferase assays).

Preeclampsia and eclampsia are among the most common causes of maternal and fetal mortality and morbidity worldwide. There are no reliable means to predict eclampsia or cerebral edema in women with preeclampsia and knowledge of the brain involvement in preeclampsia is still limited. S100B and neuron specific enolase (NSE) are two cerebral biomarkers of glial- and neuronal origin respectively. They are used as predictors for neurological outcome after traumatic brain injuries and cardiac arrest but have not yet been investigated in preeclampsia. This thesis is based on one longitudinal cohort study of pregnant women (n=469, Paper I and III), one cross sectional study of women with preeclampsia and women with normal pregnancies (n=53 and 58 respectively, Paper II and IV) and one experimental animal study of eclampsia (Paper V). In Paper I and III, plasma concentrations of S100B and NSE were investigated throughout pregnancy in women developing preeclampsia (n=16) and in women with normal pregnancies (n=36) in a nested case control study. Plasma concentrations were increased in women developing preeclampsia in gestational week 33 and 37 for S100B and in gestational week 37 for NSE compared to women with normal pregnancies. In Paper II and IV, increased plasma concentrations of S100B and NSE were confirmed among women with preeclampsia compared to women with normal pregnancies. Furthermore, increased plasma concentrations of S100B correlated to visual disturbances among women with preeclampsia (Paper II) and plasma concentrations of S100B and NSE remained increased among women with preeclampsia one year after delivery (Paper IV). In Paper V, an experimental rat model of preeclampsia and eclampsia demonstrated increased serum concentrations of S100B after seizures in normal pregnancy (n=5) and a tendency towards increased plasma concentrations of S100B in preeclampsia (n=5) compared to normal pregnancy (n=5) without seizures. Furthermore, after seizures, animals with magnesium sulphate treatment demonstrated increased serum concentrations of S100B and NSE compared to no treatment. In conclusion; plasma concentrations of S100B and NSE are increased in preeclampsia during late pregnancy and postpartum and S100B correlates to visual disturbances in women with preeclampsia. The findings are partly confirmed in an animal model of eclampsia.

OBJETIVO: Analizar la relación que existe entre los estresores psicosociales y la presencia o ausencia de preeclampsia. MATERIAL Y MÉTODO: Estudio analítico de casos y controles. La muestra estuvo conformada por 120 puérperas, 60 casos con diagnóstico de preeclampsia durante la gestación pareados uno a uno con 60 puérperas de parto normal, según lugar de procedencia y religión. Se aplicó cuatro instrumentos: el Inventario de Ansiedad Rasgo Estado (IDARE), el Inventario de Depresión Rasgo Estado (IDERE), el APGAR familiar y la ficha de violencia para la identificación de estresores psicosociales (ansiedad, depresión, disfunción familiar y violencia). Para el análisis de los datos, además de los procedimientos descriptivos, se estimó Chi cuadrado (significativo p<0.05), la razón de momios con intervalos de confianza al 95% y la regresión logística bajo el método condicional pasos hacia atrás, aplicándose la prueba de Hosmer y Lemeshov (p>0.05) para determinar el modelo y la prueba R cuadrado de Cox y Snell para determinar el valor explicativo del modelo. RESULTADOS: La ansiedad (OR: 3.24; IC 95%: 1.24-8.49), la depresión (OR: 3.35; IC 95%: 1.12-9.99) y la violencia (OR: 2.41; IC 95%: 1.08-5.38) se asociaron significativamente con preeclampsia. No se encontró asociación entre disfunción familiar (OR: 1.65; IC 95%: 0.78-3.48) y preeclampsia. CONCLUSIÓN: Los estresores psicológicos (OR: 3.33; IC 95%: 1.42-7.82) y sociales (OR: 2.10; IC 95%: 1.01-4.36) se asociaron significativamente con preeclampsia. / Tesis

<p><a>Preeclampsia leads to increased risk of morbidity and mortality for both mother and fetus. Most previous studies have largely neglected mechanical compression of the left renal vein by the gravid uterus as a potential mechanism. In this study we first used a murine model to investigate the pathophysiology of left renal vein constriction. The results indicate that prolonged renal vein stenosis after 14 days can cause renal necrosis and an increase in blood pressure (BP) of roughly 30 mmHg. The second part of this study aimed to automate a diagnostic tool, known as the supine pressor test (SPT), to enable pregnant women to assess their preeclampsia development risk. A positive SPT has been previously defined as an increase of at least 20 mmHg in diastolic BP when switching between left lateral recumbent and supine positions. The results from this study established a baseline BP increase between the two body positions in non-pregnant female subjects and demonstrated the feasibility and utility of an automated SPT in pregnant women. Our results demonstrate that there is a baseline increase in BP of roughly 10-14 mmHg and that pregnant women can autonomously perform the SPT. Overall, this work in both rodents and humans suggests that 1) stenosis of the left renal vein in mice leads to elevation in BP and acute renal failure, 2) non-pregnant women experience a baseline increase in BP when they shift from left lateral recumbent to supine position, and 3) the SPT can be automated and used autonomously.</a></p> <br> <p> </p>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22640号 / 医博第4623号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 近藤 玄, 教授 斎藤 通紀, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22744号 / 医博第4662号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 竹内 理, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Preeclampsia (PE), intrauterine growth restriction (IUGR) and gestational diabetes (GDM) increase the risk of maternal and fetal morbidity and mortality. The roles of Advanced Glycation End-products (AGEs) are already well documented concerning inflammation, hypoxia and oxidative stress. AGEs bind to its receptor, Receptor for Advanced Glycation End-products (RAGE), and activate an inflammatory pathway. This pathway alters the efficacy of invasive trophoblast cells and in the placenta and can result in placental dysfunction. We hypothesized that the placental dysfunction found in PE, IUGR, and GDM resulted from an over activation of the RAGE-mediated inflammatory pathway. Using human placental samples, we found that RAGE protein expression via western blotting was increased in PE and decreased in IUGR while GDM remained similar to that of control placentas. We then wanted to determine the efficacy of RAGE activation to alter the invasive nature of invasive cytotrophoblasts cells. We found that the addition of AGEs to SW71 cells decreases invasion through the activation of JNK and ERK cellular signaling pathways. Altogether these findings suggest that RAGE activation in trophoblast cells seems result in insufficient placental pathogenesis causing PE, however the IUGR and GDM samples we obtained did not seem to have resulted from RAGE activation. We also found that RAGE activation can alter the ability of invasive trophoblasts to invade, thus limiting the ability of the placental cells to remodel the maternal spiral arteries. We believe that further research into specific triggers of IUGR (smoking-induced) and un-treated diabetes could result in RAGE stimulated placental insufficiency.

Determina los factores relacionados a la preeclampsia en adolescentes en el HSJL - 2018. Realiza un estudio, observacional, analítico y transversal. La población está constituida por las 424 pacientes adolescentes gestantes en el Hospital San Juan de Lurigancho - 2018. Encuentra que de las 424 pacientes gestantes adolescentes, se diagnosticó preeclampsia en 30 de ellas, siendo un 83.3% entre 17 a 19 años de edad, el 93,3% cuenta con estudios secundarios, el 76,7% reside en la zona urbana, el 80% son solteras, el 16,7% presentó un intervalo intergenésico mayor a 2 años, el 86,7% no presentó historial familiar de preeclampsia, el 36,3% es obesa. Del total de gestantes adolescentes, el 60% presentó chequeos prenatales mayor o igual a 4 y el 40% presentó menos de 4 chequeos prenatales, obteniéndose un valor de p< 0.05. Finalmente el 33.3% de gestantes adolescentes con preeclampsia presentaron obesidad, obteniéndose un valor de p< 0.05. Concluye que los chequeos prenatales insuficientes y la obesidad, presentaron una agrupación estadísticamente significativa en relación a la preeclampsia en adolescentes que fueron evaluadas en el HSJL durante el año 2018. / Tesis