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Adult exposure to high fat diet (HFD) has been linked to increased risk of musculoskeletal, cardiovascular, and metabolic diseases; however, the contribution of gestational HFD to elevated oxidative stress (OS), perinatal cardiovascular, skeletal, and metabolic dysfunction as well as long-term effects on adult offspring are incompletely understood. Pathophysiologic mechanisms linking gestational HFD, OS, and insulin resistance to perinatal development and adult-onset chronic diseases are explored in the present study, and maternal antioxidant (quercetin) is offered as a potential preventive dietary supplement to reduce fetal and maternal sequelae of HFD. Female C57BL/6 mice were fed "cafeteria-style" HFD (including 32.1% saturated fat to mimic a typical fast food menu) with or without quercetin for one month prior to conception, and throughout gestation. HFD dams developed gestational diabetes with significantly increased placental OS and vasculopathy. Neonates were smaller at birth than age-matched controls, and surviving offspring developed type 2 diabetes, hypertension and osteoporosis during adulthood, despite having been fed healthy diet throughout their postnatal life. Additional measures of bone using three-dimensionally reconstructed computed tomographic image analysis (microCT) revealed microarchitectural changes of bone at birth, and at 6 and 12 months postnatally. Fetuses from HFD dams displayed diminished bone mineral density (BMD) and disrupted endochondral and intramembranous ossification with significantly shortened distal limb lengths, as compared to offspring of standard rodent chow dams. Skeletal malformation persisted into adulthood despite the fact that both control and HFD offspring were fed conventional rodent chow throughout postnatal life. The offspring gestationally exposed to HFD showed significant decreased femoral BMD at 6 months of age and dysregulation of distal femoral trabecular architecture at 12 months of age, indicating development of osteoporosis. We were able to reduce incidence of placental vasculopathy, fetal maldevelopment and adult-onset type 2 diabetes, hypertension and osteoporosis with concurrent maternal quercetin supplementation during pregnancy. Collectively, these data suggested that maternal HFD increases placental OS and vascular damage during pregnancy, which are associated with fetal malformation and elevated adult-onset multisystemic chronic diseases. Maternal quercetin supplementation must be further explored as a potential dietary intervention for improved placental integrity, fetal development and lifelong health. / Ph. D.

Throughout gestation, the blood vessel network of the placenta is formed sequentially by processes known as vasculogenesis and angiogenesis, which together meet the needs of the growing fetus. Normal placental angiogenesis is critical to support adequate fetal growth and assure the health of the offspring. Proper angiogenesis requires precise regulation of expression of agents that modulate this process; otherwise, pathologies of pregnancy such as preeclampsia may occur. The placenta is composed of different layers of tissue, including the lamellar (LZ), junctional, and glandular zones, each with a vascular morphology attuned to its function. We hypothesized that higher expression of pro-angiogenic factors is associated with increased morphological metrics in the LZ, the major vascularized zone. Thus, we aimed to characterize the major changes in morphology and vascular development in the placenta throughout pregnancy in cats, alongside a compressive analysis of the expression of major angiogenic factors and their receptors in the placenta, with an emphasis on the identification and interaction of different isoforms of the VEGF family. Microscopic analysis of tissue specimens from different stages of pregnancy revealed increased thickness of the LZ, especially during early to mid-gestation, at which time the tissue is composed of abundant materno-fetal interdigitations that appears rich in capillaries. VEGF proteins were detected in placental tissue in both fetal and maternal cells of the placenta, suggesting stimulatory interactions between different cell types to promote growth and angiogenesis. Gene expression analysis of placenta revealed upregulation of the pro-angiogenic factor VEGF-A in mid-pregnancy, followed by a steady decline toward term, consistent with morphologic changes in the LZ. In contrast, another pro-angiogenic factor, PlGF, showed a marked increase toward term; Flt-1, which acts as a receptor or reservoir for PLGF and VEGF A, was also upregulated at late pregnancy. Increased ratios of PLGF:VEGF-A may contribute to LZ proliferation in the last trimester. These findings are consistent with the creation of a proangiogenic placental state during gestation. Overall, we expect that this research will help elucidate mechanisms of placental vascularization, which can be applied to the design of improved strategies to treat vascular complications of pregnancy. Lastly, we applied the tools developed for placental studies to investigate pathologic angiogenesis in cutaneous squamous cell carcinoma (CSCC), a common skin cancer with major economic and medical impacts in humans and veterinary species. The creation of a new blood supply is essential for growth and metastasis of many tumor types. The goal of this study was to measure expression of variants of proteins that stimulate angiogenesis or transmit an angiogenic stimulus in feline CSCC. The results were mixed, with differences detected in expression of some regulatory agents and, for others, unexpectedly lower expression in CSSC compared to controls. Interestingly, the expression of VEGF-A relative to the protein that transmits its signal (KDR) was elevated in CSCC, suggestive of an altered signaling relationship. This finding supports our hypothesis and is consistent with human SCC studies. Our results encourage further studies on angiogenic factor variants in feline CSCC. / PHD

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成4年7月20日 淺田義正氏の博士論文として提出された

Introduction : La prééclampsie (PE) est un trouble hypertensif caractérisé par une tension artérielle au-dessus de 140/90 mmHg et touche 2 à 8% des grossesses globalement. À ce jour, la plupart des médicaments ne peuvent traiter ou prévenir la PE. La thrombospondine-1 (THBS1) est un facteur anti-angiogénique et un activateur principal du TGF-β, ce qui pourrait également être impliqué dans l'altération de l'angiogenèse dans les placentas prééclamptiques. Objectif : Notre objectif est de décrire l'expression de la THBS1 et de tester l’effet d’un inhibiteur, le peptide LSKL, sur la vascularisation placentaire dans les placentas d'un modèle murin de PE (in vivo) et sur la capacité angiogénique des cellules endothéliales placentaires humaines (in vitro). Méthodes : L'expression de THBS1 a été mesurée dans les placentas de souris par western blot et dans les placentas humains par immunohistochimie (IHC). Des cellules endothéliales placentaires (pECs) extraites de placentas humains et des souris enceintes hétérozygotes femelles surexprimant la rénine et l’angiotensinogène humaines (hR+A+) sont traitées avec LSKL, ou son peptide témoin SLLK (cellules : 30 μmol/L pendant24h ; animal 129 μmol/mL s.c. au jour de gestation 14,5). Des pEC ont été soumises à des conditions contrôle (8% d’oxygène) ou hypoxie (0,5% d’oxygène) ou thrombine (10 unités/mL) pendant 24 h. L’angiogenèse cellulaire a été évaluée sur matrigel, les protéines évaluées par western blot et la localisation de la THBS1 ainsi que la vascularisation placentaire par IHC. Résultats : Le traitement avec LSKL augmente le nombre de tubes formées dans les pECs exposées à la thrombine ou à l’hypoxie. L’inhibiteur de la voie canonique du TGF-β, ALK5/Smad2/3 (SB-505124, SB), n’a pas modifié la capacité de formation de tubes, contrairement à l’inhibiteur de la voie non canonique, TAK1/p38 ((5Z) -7-oxozeaenol, (OXO) qui a réduit la capacité angiogénique (p<0,05). Chez le modèle de souris de PE, LSKL a significativement amélioré la vascularisation placentaire, évaluée par le contenu des cellules endothéliales CD31+ marquées par IHC (p<0,05), qui s’est accompagnée d’une phosphorylation réduite de TAK1 et ERK dans ce tissu. Conclusion : Nos résultats indiquent une augmentation de l’expression de la THBS1 dans les vaisseaux et cellules endothéliales placentaires des grossesses atteintes de PE, ainsi qu’un effet pro-angiogénique placentaire du LSKL par inhibition de la vie non-canonique du TGF-β. Ces résultats contribueront à identifier de nouvelles cibles thérapeutiques capables d'améliorer la vascularisation placentaire dans PE. / Introduction: Pre-eclampsia (PE) is a hypertensive disorder characterized by blood pressure above 140/90 mmHg and affects 2% to 8% of pregnancies globally. To date, most drugs cannot treat or prevent PE. Thrombospondin-1 (THBS1) is an anti-angiogenic factor and a major activator of TGF-β, which may also be involved in impaired angiogenesis in pre-eclamptic placentas. Objective: Our aim is to describe the expression of THBS1 and to test the effect of its inhibitor, LSKL peptide, on placental vascularization in placentas from a mouse model of PE (in vivo) and on the angiogenic capacity of human placental endothelial cells (in vitro). Methods: THBS1 expression was measured in placentas of mouse by western blot and in human placentas by immunohistochemistry (IHC). Placental endothelial cells (pECs) extracted from hu-man placentas and pregnant female heterozygous mice overexpressing human renin and angio-tensinogen (hR+A+) were treated with LSKL or its control peptide SLLK (cells: 30 μmol/L for 24h; animals: 129 μmol/mL subcutaneously on gestation day 14.5). pECs were subjected to control conditions (8% oxygen) or hypoxia (0.5% oxygen) or thrombin (10 units/mL) for 24 hours. Cellular angiogenesis was assessed on Matrigel, proteins were evaluated by western blot, and the locali-zation of THBS1 as well as placental vascularization were examined by immunohistochemistry (IHC). Results: Treatment with LSKL increased the number of tubes formed in pECs exposed to thrombin or hypoxia. The inhibitor of the canonical TGF-β pathway, ALK5/Smad2/3 (SB-505124, SB), did not alter tube formation capacity, unlike the inhibitor of the non-canonical pathway, TAK1/p38 ((5Z)-7-oxozeaenol, (OXO)), which reduced angiogenic capacity. In the mouse model of PE, LSKL significantly improved placental vascularization, assessed by CD31+ endothelial cell content marked by IHC, which was accompanied by reduced phosphorylation of TAK1 and ERK in this tissue. Conclusion: Our results indicate an increase in THBS1 expression in the vessels and placental endothelial cells of pregnancies affected by PE, as well as a pro-angiogenic effect of LSKL through the inhibition of the non-canonical TGF-β pathway. These findings contribute to identifying new therapeutic targets capable of improving placental vascularization in PE.

CD146 est une glycoprotéine appartenant à la superfamille des immunoglobulines, exprimée de manière ubiquitaire sur l'endothélium, principalement au niveau des jonctions. Une forme soluble de CD146 (CD146s) est générée par clivage de la forme membranaire par un mécanisme dépendant des métalloprotéases. CD146 et CD146s interviennent dans le maintien de l'intégrité de l'endothélium, la transmigration endothéliale des monocytes et possèdent des propriétés angiogéniques. Les concentrations sériques de CD146s varient au cours de pathologies liées à une atteinte vasculaire. Notre travail a pour but d'étudier l'implication de CD146s dans deux contextes indépendants: la physiopathologie obstétricale et la sclérodermie systémique. La première partie de notre travail a consisté à analyser le rôle de la molécule au cours de la grossesse. Dans le placenta normal, l'expression de CD146 est restreinte aux trophoblastes possédant des capacités de migration et d'invasion, les trophoblastes extra-villeux (EVT). Ces EVT remodèlent les artères utérines spiralées afin d'établir une circulation foeto-maternelle. Au cours de ce processus, appelé pseudo-vasculogenèse, les EVT acquièrent un phénotype endothélial. De plus, l'expression placentaire de CD146 et les concentrations de CD146s varient au cours de grossesses pathologiques. L'hypothèse de notre travail est que CD146s régulerait les capacités invasives des EVT et interviendrait dans le développement placentaire. / CD146 is a member of the immunoglobulin superfamily. This is a membrane glycoprotein localized at the endothelial junction. CD146 also exists as a soluble form in the plasma (sCD146), generated by proteolysis of membrane CD146 through a mechanism involving metalloproteinases. CD146 and sCD146 are involved in endothelium integrity, monocyte transmigration and display angiogenic properties. CD146s level variations are observed in vascular pathologies. The aim of our work was to investigate the role of sCD146 in obstetrical physiopathology and in systemic sclerosis. In a first part, the involvement of our molecule was analyzed during pregnancy. In normal placenta, CD146 is expressed in intermediate and extra-villous trophoblasts (EVT), presenting migratory and invasive properties. EVT invade the spiral arteries and convert from an epithelial to an endothelial phenotype during the pseudo-vasculogenesis process. Moreover, CD146 placental expression and sCD146 levels are modified during pathologic pregnancies. We hypothesized that sCD146 could regulate EVT invasiveness and placental development. Using placental villous explants, we demonstrated that sCD146 inhibits EVT outgrowth. Consistently, we showed that sCD146 inhibits the ability of EVT cells (HTR8/SVneo) to migrate, invade and form tubes in Matrigel®. The involvement of sCD146 in human pregnancy was investigated by evaluation of sCD146 levels in 50 pregnant women. We observed physiological down-regulation of sCD146 throughout pregnancy. These results prompted us to investigate the effect of prolonged sCD146 administration in a rat model of pregnancy.

Quatre études épidémiologiques menées en Afrique ont montré que les enfants issus de mères ayant un placenta infecté par Plasmodium falciparum lors de l’accouchement font des infections palustres plus précocement que les autres enfants. Le fœtus serait sensibilisé in utero par les parasites infectants et développerait par la suite une tolérance aux infections palustres. Cette hypothèse nous conduit à supposer que i) la réponse anticorps spécifique de P. falciparum est différente chez les enfants en fonction du statut infectieux du placenta des mères à l’accouchement et ii) que cette sensibilité ne pourrait être induite que par les antigènes parasitaires porteurs des mêmes polymorphismes que ceux rencontrés in utero. Un troisième projet a consisté au développement d’une méthodologie permettant de distinguer les anticorps maternels de ceux néo-synthétisés par l’enfant dans le but de mesurer précisément l’acquisition de la réponse anticorps élaborée par l’enfant dès son plus jeune âge. Nous avons mis en place le suivi régulier et rapproché d’une cohorte de 620 nouveau-nés de la naissance à 18 mois au Bénin. Nous avons mesuré leurs réponses anticorps dirigées contre sept antigènes de P. falciparum candidats vaccins et constaté que le processus de maturation immunitaire commence à être mis en place à l’âge de 18 mois. L’infection palustre placentaire ne semble pas influer sur l’acquisition de la réponse anticorps spécifique jusqu’à 18 mois de vie. La méthodologie de distinction des anticorps maternels et néo-synthétisés a été validée. La caractérisation des polymorphismes des antigènes parasitaires présents à l’accouchement et pendant le suivi des enfants, mis en relation avec les données environnementales, a permis de valider en partie l’hypothèse de tolérance immunitaire. / Four epidemiological studies showed that infants born from mothers with Plasmodium falciparum placental malaria at delivery present a higher susceptibility to plasmodial infections than others. In connection with this observation, we hypothesized that i) the infants’ P. falciparum specific antibody responses are different according to presence or absence of placental malaria at delivery in their mothers and ii) susceptibility could only be induced by antigens that bring the same polymorphisms as those found in infected mothers. Another project consisted to develop a new methodology to distinguish maternal and neonatal antibodies in order to measure accurately neo-synthesized antibodies in the first months of life. A birth cohort of 620 newborns was established in an area endemic for malaria. Infants were followed-up until 18 months of age and their antibody responses specific for 7 P. falciparum antigens were quarterly measured. The emergence of the immune maturation process was observed in 18-months-infants. The acquisition of specific antibody responses was not impacted by placental malaria. The new methodological approach leading to distinguish maternal and neonatal antibodies was validated. The genetic characterization of the parasite antigen polymorphisms in mothers at delivery and their infants during the follow-up, in link to environmental data, led partially to the validation of the immune tolerance hypothesis.

Objetivo: Confeccionar curvas de normalidade do volume e dos índices de vascularização placentária segundo a idade gestacional (IG) e o peso fetal estimado (PFE). Métodos: Durante o período compreendido entre março e novembro de 2007 foi realizado estudo observacional transversal envolvendo 280 gestantes com idades gestacionais compreendidas entre 12 a 38 semanas. As gestantes foram submetidas à ultra-sonografia para avaliação do volume placentário tridimensional calculado pelo método VOCAL com quantificação da vascularização placentária por meio dos índices vasculares: índice de vascularização (IV), índice de vascularização e fluxo (IVF) e índice de fluxo (IF), usando-se o power Doppler. Os critérios de inclusão foram gestações únicas com idade gestacional confirmada à ultra-sonografia, sem doenças maternas e/ou malformações fetais. Foram derivadas equações matemáticas para as curvas do volume placentário e dos índices vasculares (IF, IV e IFV) por meio de modelo de regressão linear, assim como os percentis 10, 50 e 90 para volume placentário em relação à idade gestacional e o peso fetal estimado. Resultados: Foram incluídas no estudo 280 gestantes, das quais 14 (5%) foram excluídas por apresentarem intercorrências maternas, abortamento tardio e impossibilidade de obtenção dos dados no pósparto. Houve correlação significativa do volume placentário com a idade gestacional (r =0,572; p<0,001) e com o peso fetal estimado (r=0,505; p<0,001). O volume placentário médio variou de 83,0 cm3 para 12 semanas a 403,1 cm3 para 38 semanas. Os índices vasculares placentários não se correlacionaram com a idade gestacional, mantendo-se constante seus valores (IV vs. IG r=0,03, p=0,61; IF vs. IG r=0,03, p=0,58; VFI vs. IG r=0,06, p=0,27). Conclusão: Foram confeccionadas curvas de volume placentário segundo a idade gestacional e o peso fetal estimado, obtendo-se valores de referência. Diferentemente dos relatos prévios, os presentes resultados mostraram distribuição constante dos índices de vascularização em diferentes idades gestacionais. / Objectives: The purpose of this study was to construct normograms of placental volume and placental vascular indices in normal gestations according to gestational age and fetal weight by three dimensional ultrasound power Doppler. Methods: A study was performed with 280 normal pregnant women presenting 12 to 38 weeks of pregnancy, during the period between March and November 2007. Study patients were submitted to ultrasound examination and placental volume was obtained through VOCAL method. Placental perfusion was evaluated through three-dimensional power Doppler indices: (VI) vascularization index, (FI) flow index and (VFI) vascularization and flow index. The inclusion criteria were singleton pregnancies without known clinical complications or fetal abnormalities. Equations and regression coefficients for placental volume and vascular indices were calculated according to gestational age and fetal weight. The 10th, 50th, and 90th percentiles of volumes by gestational age and estimated fetal weight were calculated. Results: The sample studied consisted of 280 pregnant women, 14 (5%) of whom were excluded from the final analysis due to some presented problems, such as maternal clinical complications, late abortion or impossibility to obtain childbirth data. There was a statistically significant correlation between placental volumes, gestational age (r=0.572; p<0.001) and estimated fetal weight (r= 0.505; p< 0.001). Mean placental volume ranged from 83,0cm3 at 12 weeks to 403,1cm3 at 38. All placental vascular indices showed a constant distribution throughout gestational age. (VI vs. GA r=0, 03, p=0, 61; FI vs. GA r=0, 03, p=0, 58; VFI vs. GA r=0, 06, p=0,27). Conclusion: Normograms of placental volumes according to gestational age and estimated fetal weight were described, generating references values. Differently from previous reports, our results showed constant distributions of all 3D-power Doppler placental volumes according to gestational age.