Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, the principal angiogenic factor, vascular endothelial growth factor A (VEGFA), may influence follicle growth via regulation of angiogenesis. However, VEGFA may also influence follicular development through nonangiogenic mechanisms since its expression has been localized to nonvascular follicles and cells. Alternative mRNA splicing of 8 exons from the VEGFA gene results in the formation of different VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms are encoded by exon 8a while a sister set of isoforms with antiangiogenic properties are encoded by exon 8b. The antiangiogenic isoforms comprise the majority of VEGFA expressed in most tissues while expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at embryonic day 16. The antiangiogenic isoforms, Vegfa_165b and Vegfa_189b, were amplified and sequenced from rat ovaries and quantitative RT-PCR determined that Vegfa_165b mRNA was more abundant around embryonic day 18, but Vegfa_189b lacked a distinct pattern of abundance. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. Antiangiogenic VEGFA isoforms were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, postnatal day 3/4 rat ovaries were cultured with VEGFR-TKI, a tyrosine kinase inhibitor that blocks signaling through the VEGFA receptors, FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94%. In addition, treated ovaries had more primordial follicles, fewer early primary, transitional, and secondary follicles, and greater total follicle numbers compared with control ovaries. This suggests that VEGFA promotes follicle recruitment and early follicular development. These effects may be dependent upon increased ovarian vascularization or they may be mediated by nonvascular mechanisms.

The role of placental VEGFA in placental development and maternal pregnancy adaptations is unknown. In this thesis, one or both copies of Vegfa was knocked out from the placental spongiotrophoblast region using a mouse Cre-loxP system. Surprisingly, in pregnancies carrying 100% conceptuses with a single deletion of Vegfa from the spongiotrophoblast layer, maternal circulating VEGFA increased by 20% accompanied by a 15% decrease in arterial pressure while no impairment in embryo growth was found. In pregnancies carrying 50% conceptuses with both copies of Vegfa deleted from spongiotrophoblast, 17% of conceptuses had been reabsorbed by late gestation suggesting a function for spongiotrophoblast VEGFA in sustaining early pregnancy. In conclusion, spongiotrophoblast VEGFA affects maternal function during pregnancy but the exact mechanism remains to be defined.

The role of placental VEGFA in placental development and maternal pregnancy adaptations is unknown. In this thesis, one or both copies of Vegfa was knocked out from the placental spongiotrophoblast region using a mouse Cre-loxP system. Surprisingly, in pregnancies carrying 100% conceptuses with a single deletion of Vegfa from the spongiotrophoblast layer, maternal circulating VEGFA increased by 20% accompanied by a 15% decrease in arterial pressure while no impairment in embryo growth was found. In pregnancies carrying 50% conceptuses with both copies of Vegfa deleted from spongiotrophoblast, 17% of conceptuses had been reabsorbed by late gestation suggesting a function for spongiotrophoblast VEGFA in sustaining early pregnancy. In conclusion, spongiotrophoblast VEGFA affects maternal function during pregnancy but the exact mechanism remains to be defined.

No description available.

L'angiogenèse est un processus physiologique qui correspond à la formation de nouveaux vaisseaux sanguins à partir d'un réseau vasculaire préexistant et est régulée par l'équilibre entre les facteurs endogènes pro- et anti-angiogéniques. La rupture de cet équilibre est associée à de nombreuses pathologies dont l'ischémie, la rétinopathie ou encore la progression tumorale. Etant donné que les cellules endothéliales, principal type cellulaire composant les vaisseaux sanguins expriment les récepteurs à activité tyrosine kinase du facteur anticoagulant, la protéine S, Tyro3, Axl et Mer et produisent de la protéine S, l'objectif de ce travail est d'étudier le rôle, de la protéine S dans l'angiogenèse. Dans la première partie de ce travail, nous avons montré in vivo que la protéine S inhibe l'angiogenèse induite par les facteurs pro-angiogéniques (VEGFA et FGF2). Parallèlement, nous avons observé in vitro une inhibition par la protéine S de la prolifération et de la migration des cellules endothéliales induites par le VEGFA. Cet effet est corrélé à l'inhibition par la protéine S des voies de signalisation des MAP-Kinases et de la phosphatidylinositol 3-kinase (PIK3) induites par le VEGFA. Nous avons ensuite mis en évidence, par l'utilisation d'inhibiteurs pharmacologiques et de petits ARNs interférents, que la protéine S inhibe, via l'activation du récepteur Mer et le recrutement de la protéine phosphatase SHP2, l'activation du VEGFR2, le principal récepteur du VEGFA. Dans la deuxième partie, nous avons montré de manière intéressante que le rôle joué par la protéine S lors de l'angiogenèse est plus complexe, puisqu'elle est capable d'activer directement la voie de signalisatio / Angiogenesis is a physiological process that leads to new blood vessel formation and is regulated by a balance between pro-and anti-angiogenic endogenous factors. Disruption of this balance leads to many pathologies such as ischemia, retinopathies or tumor growth. Because endothelial cells, the main cellular type composing blood vessels, produce the anticoagulant factor, protein S and express its tyrosine kinase receptors Tyro3, Axl and Mer, we investigated the implication of protein S in angiogenesis. In the first part of this work, we demonstrated that protein S inhibits pro-angiogenic factors (VEGFA and FGF2)-induced angiogenesis in vivo. We also observed an inhibition of VEGFA-dependent endothelial cell proliferation and migration induced by protein S. These effects were correlated with protein S induced inhibition of VEGFA-dependent MAP-Kinases (Erk1, Erk2) and phosphatidylinositol 3-kinase (PIK3) activation. Furthermore, we demonstrated, using pharmacological inhibitors and small interfering RNAs, that protein S inhibits VEGFA-induced VEGFR-2 activation through Mer receptor activation and SHP2 protein phosphatase recruitment. In the second part, we demonstrated that, protein S on its own, is able to induce MAP-kinases pathway activation and endothelial cells proliferation. These cellular and molecular effects involved Mer receptor and SHP2 protein activation and required protein kinase SRC recruitment. Our results describe for the first time that protein S is an endogenous regulator for angiogenesis both in vitro and in vivo and may form the framework for the use of protein S as part of an anti-angiogenic treatment.

Made available in DSpace on 2016-12-23T13:49:02Z (GMT). No. of bitstreams: 1 Fabiano Honorato Pereira e Silva.pdf: 1723759 bytes, checksum: d36fad96c8f58abe13954947a41ba3ef (MD5) Previous issue date: 2013-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Obesity is a chronic and multifactorial disease resulting from a complex interaction of genetic and environmental risk. Despite efforts to develop actions to prevent and control, the prevalence of obesity continues to grow around the world. This disease is characterized by excess of body fat accumulation in adipose tissue deposits, subcutaneous (SAT) and visceral (VAT). Adipose tissue is highly vascularized and has intense angiogenic activity. For its expansion is required to occur the development of blood vessels from pre-existing vessels. The VEGFA gene is a important pro-angiogenic factors and acts promoting vasopermeability, migration and proliferation of endothelial cells. The objectives of this study were to observe the differences in the expression of VEGFA in SAT and VAT. The objectives of this study were to observe the differences in the expression of VEGFA in SAT and VAT. In addition, we also analyzed associations with clinical parameters and gene expression of adiponectin, a anti-inflammatory adipokine associated with protection against changes in glucose tolerance. In this study was performed, using the technique of quantitative PCR in real-time, the analysis of VEGFA gene expression in SAT and VAT of 33 female patients with morbid obesity (BMI ≥ 40). The sample was divided into two groups: a group with abnormal glucose tolerance (n = 9) and a group without these abnormalities (n = 19). In the total sample, the VEGFA was expressed similarly in both deposits. However, women with abnormal glucose tolerance had reduced expression of VEGFA in VAT. Moreover, in SAT the age was negatively correlated to this gene and in VAT the VEGFA gene was positively associated with overweight and negatively associated with total cholesterol and HDL. Furthermore, consistent positive correlations were observed between VEGFA and adiponectin, both SAT and in VAT. These results are important because they help to clarify the involvement and activity of VEGFA in adipose tissue / A obesidade é uma doença crônica e multifatorial que resulta de uma interação complexa entre fatores de risco genéticos e ambientais. Apesar de esforços para o desenvolvimento de medidas de prevenção e controle, a prevalência da obesidade continua crescendo em todo o mundo. Essa doença é caracterizada pelo excesso de peso e acúmulo de gordura corporal em depósitos de tecido adiposo subcutâneos (TAS) e viscerais (TAV). O tecido adiposo é altamente vascularizado e possui intensa atividade angiogênica. Para sua expansão é necessário que ocorra angiogênese, que é o desenvolvimento de vasos sanguíneos a partir de vasos pré-existentes. O gene VEGFA é um dos principais fatores pró-angiogênicos e atua promovendo a vasopermeabilidade, migração e proliferação de células endoteliais. Os objetivos desse trabalho foram verificar se há diferença na expressão de VEGFA em TAS e TAV e se a expressão desse gene está associada a parâmetros clínicos e à expressão do gene da adiponectina, uma adipocina anti-inflamatória associada à proteção contra alterações da tolerância à glicose. Foi realizada uma análise da expressão do gene VEGFA, por meio da técnica de PCR quantitativa em tempo real, em amostras de TAS e TAV de 33 pacientes do sexo feminino, com obesidade grau III (IMC ≥ 40), sendo que para 25 pacientes a análise pareada para TAS e TAV foi realizada. A amostra total foi dividida em dois grupos: um com alterações na tolerância à glicose (n= 9) e outro sem essas alterações (n=19). Na amostra total, o VEGFA foi expresso de forma semelhante nos dois depósitos. No entanto, mulheres com alterações na tolerância à glicose apresentaram expressão reduzida de VEGFA no TAV. Além disso, no TAS a idade foi correlacionada negativamente a este gene e no TAV o VEGFA foi positivamente associado ao excesso de peso e negativamente associado aos níveis de colesterol total e HDL. Também foram observadas correlações positivas consistentes entre VEGFA e adiponectina, tanto no TAS, quanto no TAV. Esses resultados são importantes, pois ajudam a esclarecer o envolvimento e a atividade do VEGFA no tecido adiposo

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the embryo that have great potential for regenerative therapies because of their ability to self-renew and differentiate into almost all cell types. However, this developmental potential is influenced by the local cellular microenvironment, including cell surface bound ligands. In this study, we synthesized an artificial stem cell niche wherein vascular endothelial growth factor A (VEGFA) was functionally immobilized in an agarose hydrogel. Immobilized VEGFA treatments were able to upregulate mesodermal markers, brachyury and VEGF receptor 2, by day 4 and were CD34+CD41+ by day seven. Subsequently, VEGFA immobilized treatments were able to generate colony forming cells by day fourteen. This work demonstrates our ability to use functionalized hydrogels to guide ESCs toward blood progenitor cells and serves as a useful tool to replicate aspects of the embryonic microenvironment.

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the embryo that have great potential for regenerative therapies because of their ability to self-renew and differentiate into almost all cell types. However, this developmental potential is influenced by the local cellular microenvironment, including cell surface bound ligands. In this study, we synthesized an artificial stem cell niche wherein vascular endothelial growth factor A (VEGFA) was functionally immobilized in an agarose hydrogel. Immobilized VEGFA treatments were able to upregulate mesodermal markers, brachyury and VEGF receptor 2, by day 4 and were CD34+CD41+ by day seven. Subsequently, VEGFA immobilized treatments were able to generate colony forming cells by day fourteen. This work demonstrates our ability to use functionalized hydrogels to guide ESCs toward blood progenitor cells and serves as a useful tool to replicate aspects of the embryonic microenvironment.

Dans les maladies chroniques du foie, l’angiogenèse et la fibrose sont étroitement liées. Nosprécédents travaux ont permis de montrer que les myofibroblastes portaux (MFP)contribuaient de façon importante à la fibrogenèse hépatique. L’objectif de ma thèse était dedéterminer si les MFP pouvaient aussi contribuer à l’angiogenèse hépatique. Nous avonsidentifié un nouveau marqueur spécifique des MFP, le collagène XV, grâce auquel nousavons pu mettre en évidence une prolifération des MFP dans des stades avancés de fibrose, àla fois dans des modèles animaux et chez les patients atteints d’hépatopathie chronique. Cetteprolifération des MFP était corrélée à celle des cellules endothéliales. Dans le foie cirrhotiquehumain, les MFP présentaient une distribution périvasculaire, entourant les capillaires àproximité de la réaction ductulaire. L’effet des MFP sur les cellules endothéliales a ensuite étéévalué par des tests d’angiogensèse in vitro et in vivo. Le milieu conditionné des MFPaugmentait la migration et la tubulogenèse des cellules endothéliales et stimulaitl’angiogenèse dans les implants de Matrigel chez la souris. En co-culture, les MFPdeveloppaient des jonctions intercellulaires avec les cellules endothéliales et augmentaient latubulogenèse. Nous avons montré que les MFP secrétaient des microparticules contenant duVEGF-A, capables d’activer VEGFR-2 dans les cellules endothéliales et de médier leur effetpro-angiogénique. Enfin, les cholangiocytes étaient capables d’accroître l’effet proangiogéniquedes MFP. En conclusion, les MFP jouent un rôle clef dans le remodelagevasculaire associé à la fibrose hépatique. / Liver angiogenesis and fibrogenesis are closely linked and most of studies have shown thatangiogenesis could worsen fibrosis in chronic liver diseases. Our previous works havedemonstrated that portal myofibroblasts (PMF) greatly contributed to liver fibrogenesis. Theaim of this present work was to determine if PMF could also contribute to liver angiogenesis.We identified collagene XV (col15a1) as a new specific marker for PMF. In vivo, weobserved PMF proliferation (measured by expression of col15a1) at advanced stages offibrosis both in liver from animals models ( CCl4 and BDL) and in livers from patients withchronic liver disease (primary biliary cirrhosis and non alcoolic fatty liver disease). PMFproliferation was correlated with endothelial proliferation. In human cirrhotic liver, PMF werelocated around vessels in fibrotic septa, in proximity to ductular reaction. PMF effects onendothelial cells were assessed in angiogenic tests in vitro and in vivo. PMF conditionedmedium enhanced migration and tubulogenesis of endothelial cells and stimulatedvascularization of matrigel plugs in mice. In coculture, PMF developed junctions withendothelial cells (demosomes and gap junctions) and enhanced endothelial tubulogenesis. Weshowed that PMF secreted VEGFA containing microparticles, able to activate VEGFR-2 inendothelial cells and to mediate their angiogenic function. Cholangiocytes could increasePMF angiogenic properties by stimulating VEGFA expression and microparticles secretion.In conclusion, PMF, studied with a new marker, col15a1, are key cells in hepatic vascularremodeling.

Les tumeurs des cellules de la granulosa (GCTs) sont des tumeurs avec un potentiel malin ayant tendance à récidiver, provoquant ainsi la mort dans 80% des cas de stade avancé consécutif à une rechute. Bien que les GCTs représentent 5% des tumeurs ovariennes, peu d’études ont évalué les protocoles de traitement adjuvant pour la maladie avancée ou récurrente. Notre but était d’évaluer l’efficacité de la voie de signalisation du facteur de croissance de l’endothélium vasculaire A (VEGFA) comme cible pour le traitement de la GCT utilisant le modèle murin transgénique Ptentm1Hwu/tm1Hwu; Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ (PCA) qui reproduit le stade avancé de la maladie humaine. Un anticorps anti-VEGFA a été administré une fois par semaine par voie intrapéritonéale (IP) à partir de 3 semaines d’âge. La thérapie anti-VEGFA a permis une réduction de la taille des tumeurs à 6 semaines d’âge (p<0.05) et une prolongation de la survie des animaux traités, lorsque comparé aux animaux contrôles. L’analyse des GCTs a montré une réduction significative de la prolifération cellulaire (p<0.05) et de la densité microvasculaire (p<0.01) mais aucune différence significative n’a été détectée dans l’apoptose cellulaire. p44/p42 MAPK, un effecteur de la signalisation pour le récepteur 2 de VEGFA (VEGFR2) associé à la prolifération cellulaire, était moins activé dans les tumeurs traitées (p<0.05). Par contre, l’activation d’AKT, un effecteur impliqué dans la survie cellulaire, était similaire d’un groupe à l’autre. Ces résultats suggèrent que l’anticorps anti-VEGFA réduit la prolifération cellulaire et la densité microvasculaire chez les souris PCA par inhibition de la voie de signalisation VEGFR2-MAPK, inhibant ainsi la croissance tumorale. En conclusion, l’efficacité de la thérapie anti- VEGFA mérite d’être évaluée en essais contrôlés randomisés pour le traitement des GCTs chez l’homme. / Ovarian granulosa cell tumors (GCTs) are potentially malignant tumors that have a tendency for late recurrence and cause death in 80% of women with advanced GCT due to recurrent disease. Although GCTs represent 5% of ovarian tumors in women, few studies have evaluated adjuvant treatment protocols for advanced or recurrent disease. Our goal was to determine the potential of targeting the vascular endothelial growth factor A (VEGFA) signaling pathway for the treatment of GCT. We used a genetically engineered mouse model, Ptentm1Hwu/tm1Hwu; Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ (PCA), which imitates the advanced human disease. A monoclonal anti-VEGFA antibody was administered by intra-peritoneal injection once a week beginning at 3 weeks of age. Anti-VEGFA therapy significantly decreased tumor weights by 6 weeks of age (p<0.05) and increased survival in treated animals in comparison to controls. Significant decreases in tumor cell proliferation (p<0.05) and microvessel density (p<0.01), but no significant difference in apoptosis was found in PCA tumors. p44/p42 MAPK, a VEGFA receptor 2 (VEGFR2) signaling effector associated with cell proliferation, was significantly less activated in anti-VEGFA-treated tumors (p<0.05). In contrast, AKT activation, a VEGFR2 signaling effector associated with cell survival was similar among all groups. These results suggest that anti-VEGFA therapy effectively reduces cell proliferation and microvessel density in PCA mice by inhibition of the VEGFR2-MAPK pathway, resulting in inhibition of GCT growth. We conclude that anti-VEGFA therapy merits further investigation in the form of controlled randomized trials for the treatment of human GCT.