The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term. Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue. The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta. No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.

In pregnancy, the maternal cardiovascular and placental circulation undergoes structural and functional changes to accommodate the growing fetus, but the mechanisms involved are not fully understood. Nitric oxide (NO) increases in normal pregnancy and lack of NO has been implicated in pregnancy related complications, preeclampsia and fetal growth restriction. Thus, the objective of the thesis was to determine if cardiovascular, uteroplacental and fetoplacental changes observed in human pregnancy also occur in mice and to assess the obligatory role of eNOS in mediating these changes. I showed that like humans, mice exhibit increases in maternal cardiac output, stroke volume, plasma volume, and uterine arterial blood flow, and a transient decrease in arterial pressure during pregnancy. Importantly, I showed that endothelial nitric oxide synthase (eNOS) plays an important role in promoting the progressive increase in maternal cardiac chamber dimensions and output and the enlargement of the aorta during pregnancy in mice. Another novel finding was that eNOS plays an important role in remodeling of the uterine and umbilical vasculatures during pregnancy. The remodeling of the uterine vasculatures, including the uterine and spiral arteries, were blunted in the eNOS KO mice with ko fetuses (KO(ko)) and this likely contributed to elevated vascular resistance and reduced perfusion of the uterine circulation during pregnancy. Impaired spiral artery remodeling may be caused by a deficiency in decidual uterine natural killer cells. Fetal placental vascularization was also impaired in eNOS KO(ko) mice, which likely increased vascular resistance and thereby reduced fetoplacental perfusion. Reduced vascularization may be due to decreased VEGF mRNA and protein expression in KO(ko) placentas. Decreased perfusion in both the uterine and umbilical circulations most likely contributed to elevated placental and fetal hypoxia in the eNOS KO(ko) mice. Interestingly, despite placental hypoxia, eNOS KO(ko) mice do not show the classical signs of preeclampsia including hypertension and proteinuria nor are maternal plasma sFlt1 levels elevated. Nevertheless, eNOS KO(ko) pups are growth restricted at term, and this is mainly due to the fetal genotype. These findings suggest that eNOS plays an essential role during pregnancy in remodeling of the maternal heart, aorta, and uterine and umbilical vasculatures thereby augmenting blood flow to the maternal and fetal sides of the placenta and thereby promoting fetal growth in mice.

In pregnancy, the maternal cardiovascular and placental circulation undergoes structural and functional changes to accommodate the growing fetus, but the mechanisms involved are not fully understood. Nitric oxide (NO) increases in normal pregnancy and lack of NO has been implicated in pregnancy related complications, preeclampsia and fetal growth restriction. Thus, the objective of the thesis was to determine if cardiovascular, uteroplacental and fetoplacental changes observed in human pregnancy also occur in mice and to assess the obligatory role of eNOS in mediating these changes. I showed that like humans, mice exhibit increases in maternal cardiac output, stroke volume, plasma volume, and uterine arterial blood flow, and a transient decrease in arterial pressure during pregnancy. Importantly, I showed that endothelial nitric oxide synthase (eNOS) plays an important role in promoting the progressive increase in maternal cardiac chamber dimensions and output and the enlargement of the aorta during pregnancy in mice. Another novel finding was that eNOS plays an important role in remodeling of the uterine and umbilical vasculatures during pregnancy. The remodeling of the uterine vasculatures, including the uterine and spiral arteries, were blunted in the eNOS KO mice with ko fetuses (KO(ko)) and this likely contributed to elevated vascular resistance and reduced perfusion of the uterine circulation during pregnancy. Impaired spiral artery remodeling may be caused by a deficiency in decidual uterine natural killer cells. Fetal placental vascularization was also impaired in eNOS KO(ko) mice, which likely increased vascular resistance and thereby reduced fetoplacental perfusion. Reduced vascularization may be due to decreased VEGF mRNA and protein expression in KO(ko) placentas. Decreased perfusion in both the uterine and umbilical circulations most likely contributed to elevated placental and fetal hypoxia in the eNOS KO(ko) mice. Interestingly, despite placental hypoxia, eNOS KO(ko) mice do not show the classical signs of preeclampsia including hypertension and proteinuria nor are maternal plasma sFlt1 levels elevated. Nevertheless, eNOS KO(ko) pups are growth restricted at term, and this is mainly due to the fetal genotype. These findings suggest that eNOS plays an essential role during pregnancy in remodeling of the maternal heart, aorta, and uterine and umbilical vasculatures thereby augmenting blood flow to the maternal and fetal sides of the placenta and thereby promoting fetal growth in mice.

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Introdução: O ambiente fetal é atualmente bem reconhecido pela sua importante contribuição à saúde e influência na predisposição a doenças crônicas como obesidade, hipertensão e diabetes mellitus tipo 2, ao longo da vida. Indivíduos nascidos com restrição de crescimento intrauterino (RCIU), especialmente do sexo feminino, em geral, apresentam preferência por uma dieta menos saudável, o que pode contribuir para o maior risco de doenças crônicas nessa população. A relação maternal de baixa qualidade na infância está associada a maior risco de desenvolver distúrbios psiquiátricos, como ansiedade e depressão, assim como obesidade na adolescência. Além disso, a sobreingestão alimentar emocional (definida como comer em resposta ao estímulo emocional ao invés de sentimentos de fome) está relacionada com o excesso alimentar, aumento do consumo de doces e gorduras e sobrepeso. É possível que crianças nascidas com RCIU sejam mais sensíveis a variações ambientais como o cuidado materno, e que a interação entre estes fatores seja capaz de modular o comportamento alimentar nestes indivíduos. Objetivo: Avaliar a interação entre a RCIU e o cuidado materno na sobreingestão emocional aos 4 anos de idade. Metodologia: Trata-se de uma análise preliminar de uma coorte canadense de nascimentos (Maternal Adversity, Vulnerability and Neurodevelopment – MAVAN). As avaliações do crescimento fetal foram baseadas na razão de peso ao nascer. Aos 48 meses de idade, foram aplicados o Questionário do Comportamento Alimentar Infantil (CEBQ) e filmada a interação mãe-criança durante uma tarefa estruturada (GESU), na qual as duplas são instruídas a reproduzir a imagem de uma casa, em conjunto, utilizando o brinquedo Etch-a-Sketch (Conhecido como Quadro Mágico, no Brasil). Utilizouse a escala “atmosphere” (ATM) da GESU, que avalia a atmosfera geral de toda a sessão por meio de uma escala que varia de 1, “muita discórdia e conflito, nenhuma expressão de sentimentos positivos” até 9 “Muito harmoniosa, agradável, pacífica, sem conflitos ou expressão de sentimento negativos”. Nessa fase, foram avaliadas 195 crianças. Um modelo de regressão linear, ajustado pelo IMC e separado por sexo, foi realizado para avaliar a correlação entre a RCIU e o escore de cuidado materno, aos 48 meses, no sobre-consumo emocional medido pelo CEBQ, considerando significativo um P <0,05. Resultados: Não havia diferenças entre RCIU e não RCIU na distribuição de sexos, educação e renda maternas, nem no escore “Atmosphere” do GESU. O modelo de regressão linear foi significativo para meninas (r2=0,204, p=0,012) com efeitos isolados do IMC (B=0,076, p=0,048) e da ATM (B=-0,229, p=0,019) na sobreingestão emocional. Além disso, vimos uma interação entre BWR e ATM (B=0,881, p=0,038), no qual meninas restritas e com pior escore ATM (pior qualidade de interação com a mãe) têm maior sobreingestão emocional aos 4 anos de idade. Nenhum efeito foi visto em meninos (r2 =0,012, p=0,331). Conclusão: A qualidade da interação mãe-filho parece ser um importante modulador da sobreingestão emocional em meninas, especialmente aquelas nascidas com baixo peso, aos 4 anos de idade. / Introduction: The fetal environment is nowadays well recognized for its important contribution to the long-term health and influence in the predisposition to chronic diseases such as obesity, hypertension and type 2 diabetes mellitus. Individuals born with intrauterine growth restriction (IUGR), especially females, demonstrate a general preference for a less healthy food choices during development, what could contribute for the increased risk for chronic disease in this population. Low maternal care in childhood is associated with increased risk for developing psychiatric disorders such as anxiety and depression, as well as obesity in adolescence. In addition, emotional overeating (defined as eating in response to the emotional stimulus rather than feelings of hunger) is related to excessive food consumption, an increased sweets and fats intake and overweight. It is possible that children born with intrauterine growth restriction are more sensitive to environmental changes such as maternal care, and the interaction between these factors are able to modulate feeding behavior in these individuals. Objective: Investigate the interaction between intrauterine growth restriction and maternal care on emotional overeating at 4 years old children. Methods: This is a preliminary analysis of a Canadian Birth Cohort (Maternal Adversity, Vulnerability and Neurodevelopment - MAVAN). The study sample included 195 children from the city of Montreal, Quebec, and Hamilton, Ontario. The IUGR assessment was based on fetal growth ratio of birth weight. At 48 months of age, mothers complete the Child Eating Behavior Questionnaire (CEBQ) and the mother-child interaction was filmed during a structured task (“Etch a sketch”) for posterior scoring of the maternal behavior, in which the pairs are instructed to reproduce a house image together using the Etch-a-Sketch toy. We used the scale "atmosphere" (ATM) of GESU, that characterizes the general atmosphere during the play activity through a scale ranging from 1, "a lot of disagreement and conflict, no expression of positive feelings" to 9 "Very harmonious, pleasant, peaceful, without conflict or negative feelings expressed". A linear regression model adjusted for BMI and split by sex was built to analyze the interaction between the ATM score and the birth weight status (IUGR or not) on the emotional overeating domain of the CEBQ. Results: There were no differences between IUGR and non-IUGR in the distribution of gender, maternal education and income, or the ATM score of GESU. The model was significant for girls (r2=0.204, p=0.012) with isolated effects of BMI (B=0.076, p=0.048) and ATM score (B=-0.229, p=0.019) on emotional overeating. Moreover, there was an interaction between IUGR and ATM (B=0.881, p=0.038), in which IUGR girls with worse ATM scores have increased emotional overeating at 4 years of age. No effects were seen in boys (r2=0.012, p=0.331). Conclusion: The quality of mother-child interaction seems to be important to prevent emotional overeating and overweight in girls, especially those born IUGR, at 4 years of age.

Introdução: O ambiente fetal é atualmente bem reconhecido pela sua importante contribuição à saúde e influência na predisposição a doenças crônicas como obesidade, hipertensão e diabetes mellitus tipo 2, ao longo da vida. Indivíduos nascidos com restrição de crescimento intrauterino (RCIU), especialmente do sexo feminino, em geral, apresentam preferência por uma dieta menos saudável, o que pode contribuir para o maior risco de doenças crônicas nessa população. A relação maternal de baixa qualidade na infância está associada a maior risco de desenvolver distúrbios psiquiátricos, como ansiedade e depressão, assim como obesidade na adolescência. Além disso, a sobreingestão alimentar emocional (definida como comer em resposta ao estímulo emocional ao invés de sentimentos de fome) está relacionada com o excesso alimentar, aumento do consumo de doces e gorduras e sobrepeso. É possível que crianças nascidas com RCIU sejam mais sensíveis a variações ambientais como o cuidado materno, e que a interação entre estes fatores seja capaz de modular o comportamento alimentar nestes indivíduos. Objetivo: Avaliar a interação entre a RCIU e o cuidado materno na sobreingestão emocional aos 4 anos de idade. Metodologia: Trata-se de uma análise preliminar de uma coorte canadense de nascimentos (Maternal Adversity, Vulnerability and Neurodevelopment – MAVAN). As avaliações do crescimento fetal foram baseadas na razão de peso ao nascer. Aos 48 meses de idade, foram aplicados o Questionário do Comportamento Alimentar Infantil (CEBQ) e filmada a interação mãe-criança durante uma tarefa estruturada (GESU), na qual as duplas são instruídas a reproduzir a imagem de uma casa, em conjunto, utilizando o brinquedo Etch-a-Sketch (Conhecido como Quadro Mágico, no Brasil). Utilizouse a escala “atmosphere” (ATM) da GESU, que avalia a atmosfera geral de toda a sessão por meio de uma escala que varia de 1, “muita discórdia e conflito, nenhuma expressão de sentimentos positivos” até 9 “Muito harmoniosa, agradável, pacífica, sem conflitos ou expressão de sentimento negativos”. Nessa fase, foram avaliadas 195 crianças. Um modelo de regressão linear, ajustado pelo IMC e separado por sexo, foi realizado para avaliar a correlação entre a RCIU e o escore de cuidado materno, aos 48 meses, no sobre-consumo emocional medido pelo CEBQ, considerando significativo um P <0,05. Resultados: Não havia diferenças entre RCIU e não RCIU na distribuição de sexos, educação e renda maternas, nem no escore “Atmosphere” do GESU. O modelo de regressão linear foi significativo para meninas (r2=0,204, p=0,012) com efeitos isolados do IMC (B=0,076, p=0,048) e da ATM (B=-0,229, p=0,019) na sobreingestão emocional. Além disso, vimos uma interação entre BWR e ATM (B=0,881, p=0,038), no qual meninas restritas e com pior escore ATM (pior qualidade de interação com a mãe) têm maior sobreingestão emocional aos 4 anos de idade. Nenhum efeito foi visto em meninos (r2 =0,012, p=0,331). Conclusão: A qualidade da interação mãe-filho parece ser um importante modulador da sobreingestão emocional em meninas, especialmente aquelas nascidas com baixo peso, aos 4 anos de idade. / Introduction: The fetal environment is nowadays well recognized for its important contribution to the long-term health and influence in the predisposition to chronic diseases such as obesity, hypertension and type 2 diabetes mellitus. Individuals born with intrauterine growth restriction (IUGR), especially females, demonstrate a general preference for a less healthy food choices during development, what could contribute for the increased risk for chronic disease in this population. Low maternal care in childhood is associated with increased risk for developing psychiatric disorders such as anxiety and depression, as well as obesity in adolescence. In addition, emotional overeating (defined as eating in response to the emotional stimulus rather than feelings of hunger) is related to excessive food consumption, an increased sweets and fats intake and overweight. It is possible that children born with intrauterine growth restriction are more sensitive to environmental changes such as maternal care, and the interaction between these factors are able to modulate feeding behavior in these individuals. Objective: Investigate the interaction between intrauterine growth restriction and maternal care on emotional overeating at 4 years old children. Methods: This is a preliminary analysis of a Canadian Birth Cohort (Maternal Adversity, Vulnerability and Neurodevelopment - MAVAN). The study sample included 195 children from the city of Montreal, Quebec, and Hamilton, Ontario. The IUGR assessment was based on fetal growth ratio of birth weight. At 48 months of age, mothers complete the Child Eating Behavior Questionnaire (CEBQ) and the mother-child interaction was filmed during a structured task (“Etch a sketch”) for posterior scoring of the maternal behavior, in which the pairs are instructed to reproduce a house image together using the Etch-a-Sketch toy. We used the scale "atmosphere" (ATM) of GESU, that characterizes the general atmosphere during the play activity through a scale ranging from 1, "a lot of disagreement and conflict, no expression of positive feelings" to 9 "Very harmonious, pleasant, peaceful, without conflict or negative feelings expressed". A linear regression model adjusted for BMI and split by sex was built to analyze the interaction between the ATM score and the birth weight status (IUGR or not) on the emotional overeating domain of the CEBQ. Results: There were no differences between IUGR and non-IUGR in the distribution of gender, maternal education and income, or the ATM score of GESU. The model was significant for girls (r2=0.204, p=0.012) with isolated effects of BMI (B=0.076, p=0.048) and ATM score (B=-0.229, p=0.019) on emotional overeating. Moreover, there was an interaction between IUGR and ATM (B=0.881, p=0.038), in which IUGR girls with worse ATM scores have increased emotional overeating at 4 years of age. No effects were seen in boys (r2=0.012, p=0.331). Conclusion: The quality of mother-child interaction seems to be important to prevent emotional overeating and overweight in girls, especially those born IUGR, at 4 years of age.

Introdução: O ambiente fetal é atualmente bem reconhecido pela sua importante contribuição à saúde e influência na predisposição a doenças crônicas como obesidade, hipertensão e diabetes mellitus tipo 2, ao longo da vida. Indivíduos nascidos com restrição de crescimento intrauterino (RCIU), especialmente do sexo feminino, em geral, apresentam preferência por uma dieta menos saudável, o que pode contribuir para o maior risco de doenças crônicas nessa população. A relação maternal de baixa qualidade na infância está associada a maior risco de desenvolver distúrbios psiquiátricos, como ansiedade e depressão, assim como obesidade na adolescência. Além disso, a sobreingestão alimentar emocional (definida como comer em resposta ao estímulo emocional ao invés de sentimentos de fome) está relacionada com o excesso alimentar, aumento do consumo de doces e gorduras e sobrepeso. É possível que crianças nascidas com RCIU sejam mais sensíveis a variações ambientais como o cuidado materno, e que a interação entre estes fatores seja capaz de modular o comportamento alimentar nestes indivíduos. Objetivo: Avaliar a interação entre a RCIU e o cuidado materno na sobreingestão emocional aos 4 anos de idade. Metodologia: Trata-se de uma análise preliminar de uma coorte canadense de nascimentos (Maternal Adversity, Vulnerability and Neurodevelopment – MAVAN). As avaliações do crescimento fetal foram baseadas na razão de peso ao nascer. Aos 48 meses de idade, foram aplicados o Questionário do Comportamento Alimentar Infantil (CEBQ) e filmada a interação mãe-criança durante uma tarefa estruturada (GESU), na qual as duplas são instruídas a reproduzir a imagem de uma casa, em conjunto, utilizando o brinquedo Etch-a-Sketch (Conhecido como Quadro Mágico, no Brasil). Utilizouse a escala “atmosphere” (ATM) da GESU, que avalia a atmosfera geral de toda a sessão por meio de uma escala que varia de 1, “muita discórdia e conflito, nenhuma expressão de sentimentos positivos” até 9 “Muito harmoniosa, agradável, pacífica, sem conflitos ou expressão de sentimento negativos”. Nessa fase, foram avaliadas 195 crianças. Um modelo de regressão linear, ajustado pelo IMC e separado por sexo, foi realizado para avaliar a correlação entre a RCIU e o escore de cuidado materno, aos 48 meses, no sobre-consumo emocional medido pelo CEBQ, considerando significativo um P <0,05. Resultados: Não havia diferenças entre RCIU e não RCIU na distribuição de sexos, educação e renda maternas, nem no escore “Atmosphere” do GESU. O modelo de regressão linear foi significativo para meninas (r2=0,204, p=0,012) com efeitos isolados do IMC (B=0,076, p=0,048) e da ATM (B=-0,229, p=0,019) na sobreingestão emocional. Além disso, vimos uma interação entre BWR e ATM (B=0,881, p=0,038), no qual meninas restritas e com pior escore ATM (pior qualidade de interação com a mãe) têm maior sobreingestão emocional aos 4 anos de idade. Nenhum efeito foi visto em meninos (r2 =0,012, p=0,331). Conclusão: A qualidade da interação mãe-filho parece ser um importante modulador da sobreingestão emocional em meninas, especialmente aquelas nascidas com baixo peso, aos 4 anos de idade. / Introduction: The fetal environment is nowadays well recognized for its important contribution to the long-term health and influence in the predisposition to chronic diseases such as obesity, hypertension and type 2 diabetes mellitus. Individuals born with intrauterine growth restriction (IUGR), especially females, demonstrate a general preference for a less healthy food choices during development, what could contribute for the increased risk for chronic disease in this population. Low maternal care in childhood is associated with increased risk for developing psychiatric disorders such as anxiety and depression, as well as obesity in adolescence. In addition, emotional overeating (defined as eating in response to the emotional stimulus rather than feelings of hunger) is related to excessive food consumption, an increased sweets and fats intake and overweight. It is possible that children born with intrauterine growth restriction are more sensitive to environmental changes such as maternal care, and the interaction between these factors are able to modulate feeding behavior in these individuals. Objective: Investigate the interaction between intrauterine growth restriction and maternal care on emotional overeating at 4 years old children. Methods: This is a preliminary analysis of a Canadian Birth Cohort (Maternal Adversity, Vulnerability and Neurodevelopment - MAVAN). The study sample included 195 children from the city of Montreal, Quebec, and Hamilton, Ontario. The IUGR assessment was based on fetal growth ratio of birth weight. At 48 months of age, mothers complete the Child Eating Behavior Questionnaire (CEBQ) and the mother-child interaction was filmed during a structured task (“Etch a sketch”) for posterior scoring of the maternal behavior, in which the pairs are instructed to reproduce a house image together using the Etch-a-Sketch toy. We used the scale "atmosphere" (ATM) of GESU, that characterizes the general atmosphere during the play activity through a scale ranging from 1, "a lot of disagreement and conflict, no expression of positive feelings" to 9 "Very harmonious, pleasant, peaceful, without conflict or negative feelings expressed". A linear regression model adjusted for BMI and split by sex was built to analyze the interaction between the ATM score and the birth weight status (IUGR or not) on the emotional overeating domain of the CEBQ. Results: There were no differences between IUGR and non-IUGR in the distribution of gender, maternal education and income, or the ATM score of GESU. The model was significant for girls (r2=0.204, p=0.012) with isolated effects of BMI (B=0.076, p=0.048) and ATM score (B=-0.229, p=0.019) on emotional overeating. Moreover, there was an interaction between IUGR and ATM (B=0.881, p=0.038), in which IUGR girls with worse ATM scores have increased emotional overeating at 4 years of age. No effects were seen in boys (r2=0.012, p=0.331). Conclusion: The quality of mother-child interaction seems to be important to prevent emotional overeating and overweight in girls, especially those born IUGR, at 4 years of age.

Durant ces dernières années, de nombreuses études épidémiologiques ont mis en évidence que les pathologies métaboliques (obésité, diabète) et cardiovasculaires pourraient, en partie, se déterminer dès la grossesse, via des perturbations de l’environnement intra-utérin. La notion de « programmation fœtale » implique qu’une altération durant la vie fœtale perturberait le développement du fœtus et le vulnérabiliserait au développement ultérieur de pathologies. Ainsi, un enfant qui naît avec un très faible poids de naissance (inférieur à 2,4 kg) ou à l’inverse avec un poids de naissance très élevé (supérieur à 4,0 kg) est statistiquement plus vulnérable au développement de ces maladies. Pour étudier ce phénomène et tenter d’en comprendre les mécanismes, nous avons utilisé des modèles expérimentaux (rat, souris) et évalué l’action et l’expression de deux substances vasodilatatrices : le Fasudil (un inhibiteur des Rho kinases) et l’hormone apeline. Les rates gestantes traitées par le L-NAME, un inhibiteur de la NO synthase (50 mg/jour) présentaient une hypertension artérielle et leurs nouveau-nés un retard de croissance intra-utérin (RCIU) de l’ordre de 20%. L’administration aux mères de Fasudil (10 mg/jour) permettait de restaurer une pression artérielle normale en fin de gestation et améliorait considérablement la croissance fœtale des animaux exposés au L-NAME. Cependant, alors que les animaux nés avec un RCIU (nouveau-nés L-NAME) ne présentaient que peu de perturbations métaboliques à l’âge adulte, les animaux exposés au Fasudil seul étaient rapidement en surpoids, présentaient une hyperglycémie à jeun et développaient des troubles du comportement alimentaire de type hyperphagique. D’autre part, par une étude menée chez des souris obèses et intolérantes au glucose après exposition à un régime hyperlipidique, nous avons démontré que l’expression génique de l’apeline est altérée dans plusieurs organes (foie, rein, tissu adipeux) bien que l’apelinémie des souris obèses reste inchangée. Des études en voie de finalisation sont menées afin de déterminer si le système apelinergique est modulé chez des souris gestantes obèses à la fois chez les compartiments maternels et fœtaux mais aussi dans le placenta. En conclusion, nous avons démontré que l’inhibition de la voie des Rho kinases en fin de gestation programme chez la descendance un surpoids, une hyperglycémie et à une altération de la prise alimentaire. Ayant démontré que le système apelinergique est altéré chez des souris femelles obèses et intolérantes au glucose, il nous reste à déterminer si ce système est aussi perturbé en condition de grossesse associée à l’obésité maternelle. / During the last decade, many epidemiological studies have shown that adult chronic metabolic (obesity, diabetes) and cardiovascular diseases may be determined, at least in part, during pregnancy through alterations of intrauterine environment. The “fetal programming” hypothesis implies that disturbances of the fetal development (intra uterine growth restriction – IUGR or macrosomia) increase the vulnerability to develop these pathologies in adulthood. To gain more insight into the mechanisms implicated in fetal programming, we used two experimental models of rodents (rat, mouse) and evaluated first the effect of an inhibition of the Rho-kinase pathway in utero on fetal growth and postnatal development in rats. In another study performed in mice, we aimed to assess the expression of apelin and its receptor APJ in obese and glucose intolerant mice fed with a high fat diet. Using data of this preliminary study, we speculated that this signaling system may be targeted during the pregnancy of obese mothers and could be implicated into the physiopathological consequences that may affect the fetoplacental unit. We demonstrated that pregnant rats treated by L-NAME, a NO synthase inhibitor (50 mg/day) were hypertensive and that their newborns presented a dramatic IUGR. Maternal treatment with the vasodilator Fasudil (10 mg/day) restored a normal maternal blood pressure and remarkably alleviated the fetal growth of L-NAME newborns. In adults, L-NAME male rats developed mild metabolic pathologies whereas rats exposed in utero to Fasudil presented an overweight, with hyperphagia and glucose intolerance. In obese and glucose intolerant mice fed with a high fat diet, we showed that apelin gene expression was altered in several organs (liver, kidney and adipose tissue) without any variation of apelin plasma concentration. Further studies are currently performed in our laboratory to unravel the expression of the apelin/APJ pathway in pregnant obese mice and their offsprings.

Le développement normal du placenta est la clé du succès de la grossesse. La croissance trophoblastique et vasculaire est une composante cruciale du développement placentaire. Un déficit dans ces processus conduit à des complications de la grossesse, telles que la Toxémie Gravidique (TG) ou le Retard de Croissance Intra-Utérin (RCIU). Récemment, un nouveau facteur angiogène spécifique des glandes endocrines vient d'être découvert : EG-VEGF pour « Endocrine Gland-derived Vascular Endothelial Growth Factor» ou PROK1 pour Prokineticin 1, facteur qui se lie à deux récepteurs couplés aux protéines G, PROKR1 et PROKR2. Dans des publications récentes du laboratoire, l'équipe a montré que I) EG-VEGF est fortement exprimé dans le placenta, II) son expression est majoritaire au 1er trimestre et III) les niveaux circulants d'EG-VEGF sont augmentés dans la TG, pathologie qui résulte de défauts vasculaires importants. L'ensemble de ces résultats convergeait vers un effet potentiel important d'EG-VEGF sur le placenta. Les objectifs de ma thèse ont été de caractériser (1) l'effet angiogène d'EG-VEGF sur les cellules endothéliales microvasculaires placentaires (HPEC), (2) d'étudier sa régulation par l'hCG (human Chorionic Gonadotropin), et (3) d'étudier son expression au 3ème trimestre dans les grossesses avec RCIU. J'ai montré que 1) EG-VEGF est un facteur angiogène important, et que ses effets angiogènes sont médiés par PROKR1, alors que c'est PROKR2 qui est impliqué dans ses effets sur la perméabilité, 2) l'hCG augmente la sécrétion d'EG-VEGF, ainsi que l'expression des PROKRs au 1er trimestre de la grossesse, et 3) EG-VEGF augmente la prolifération et la survie des trophoblastes, et est augmenté ainsi que ses récepteurs au 3ème trimestre dans les grossesses avec RCIU. L'ensemble de ces résultats montre qu'EG-VEGF est un nouveau facteur de croissance du placenta. Sa dérégulation dans la TG et le RCIU suggère qu'il pourrait servir de marqueur précoce prédictif pour ces pathologies et être utilisé comme cible thérapeutique potentielle dans l'avenir. / Growth of the placental villi is a key event in placental development during human pregnancy. Many growth factors have been shown to control this process; however their ubiquitous expression makes them less specific to this tissue. Recently, a new growth factor named EG-VEGF (Endocrine Gland-derived Vascular Endothelial Growth Factor), specific to endocrine glands including the placenta, has been identified. It mediates its biological activity via two G protein coupled receptors, Prokineticin Receptor 1 and 2 (PROKR1 and PROKR2). In recent work from our laboratory, we have shown that I) EG-VEGF is abundantly expressed in human placenta II) EG-VEGF levels are highest during the first trimester and III) EGVEGF circulating levels are increased in preeclampsia (PE). Altogether, our results suggested that EG-VEGF might be an important regulatory factor in the placenta. My aim was to investigate (1) EG-VEGF angiogenic effects on microvascular cells within the placenta (HPEC), (2) its hormonal regulation with hCG (human Chorionic Gonadotropin), and (3) its expression in IUGR (Intrauterine Growth Restriction) pregnancies in the third trimester. Our results show that 1) EG-VEGF increases angiogenic processes in HPEC cells via PROKR1, and also their permeability via PROKR2 2) hCG increases EG-VEGF secretion and PROKR expression in the first trimester placenta and 3) EG-VEGF increases both trophoblast proliferation and survival, and its expression is dysregulated in IUGR in the third trimester of pregnancy. Altogether, our results show that EG-VEGF is a new placental growth factor. Its dysregulation in PE and IUGR suggests that it can be considered as a potential diagnostic marker and a potential therapeutic target in future.

This thesis will be organized into two chapters discussing the placental expression of two proteins, pyruvate kinase M2 (PKM2) and heat shock protein 27 (HSP 27), in human placentas. Understanding the mechanisms of placental metabolism in healthy and diseased placentas helps us understand how placenta disorders occur and how we can treat these disorders. The goal is to investigate these proteins to gain an understanding of their roles in placental disorders and help decrease maternal and fetal mortality rates. Chapter one covers the background of pyruvate kinase M2 (PKM2) in cancer and embryonic tissues, and the expression of PKM2 in the human placenta. Cancer PKM2 has been studied extensively, but little is know about the role of placental PKM2. Expression of PKM2 is confirmed in normal human placenta samples and described in preeclamptic and intrauterine growth restriction (IUGR) affected human placentas. Proteins associated with elevated PKM2 in cancer are also associated with elevated PKM2 in human placentas. Comparing normal and diseased placenta samples helps understand the similarities between cancer PKM2 and placental PKM2. Understanding the mechanisms of placental metabolism and PKM2 expression in the human placenta will clarify how the placenta is affected by preeclampsia and IUGR and the role placental PKM2 plays in each of these diseases. Chapter two will cover a paper that I wrote on the expression of phosphorylated heat shock protein 27 (HSP27) in the human placenta. Heat shock proteins are involved in the stress response and help inhibit apoptosis. The object of the study was to look for correlations between p-HSP27 and apoptosis in human and ovine placenta samples. P-HSP27 was quantified in human placenta samples and in placenta sampled collected from ovine models. Pregnant control and hyperthermic sheep models were used to quantify expression of p-HSP27 across gestation. This study showed similarities between human IUGR and our ovine IUGR model, suggesting a link between decreased p-HSP27 and increased apoptosis in IUGR.