Eine lokale Aktivierung des Komplementsystems im RPE ist ein pathogener Faktor der AMD. Neben der Wirkung von angiogenen Faktoren wie VEGF könnte eine Aktivierung des Blutgerinnungssystems im RPE dazu beitragen, dass sich aus einer trockenen eine feuchte AMD entwickelt. Dies könnte auf mehreren Ebenen geschehen: Gerinnungsfaktoren könnten die Expression der Komplementfaktoren und der angiogenen Faktoren regulieren sowie Wirkungen auf die Proliferation und Migration der RPE-Zellen besitzen. Eine Stimulierung der Proliferation und Migration der RPE-Zellen trägt zur Ausbildung von CNV-Membranen bei. Es ist aber bis jetzt nichts darüber bekannt, ob RPE-Zellen Faktoren des Blutgerinnungssystems exprimieren und ob z.B. Thrombin (als zentrale Protease des Blutgerinnungssystems) die Genexpression von Komplementfaktoren und von VEGF im RPE beeinflusst. Die Ziele der vorliegenden Dissertation waren daher: ● Nachweis der mRNA-Expression von Blutgerinnungs- und Komplementfaktoren im RPE; ● Nachweis der Wirkung von Thrombin auf die Expression von VEGF und von Komplementfaktoren, sowie auf die Proliferation und Migration der RPE-Zellen; und ● Nachweis der Wirkung der Komplementfaktoren C5a und C9 auf die Sekretion von VEGF und die Proliferation und Migration der RPE-Zellen.

La croissance d'une tumeur au-delà de quelques mm3 requiert la formation de son propre réseau vasculaire par angiogenèse ; la destruction de ces vaisseaux nourriciers pourrait conduire à une régression tumorale. L'angiogenèse est orchestrée par de nombreux facteurs de croissance, dont le VEGF (Vascular Endothelial Growth Factor). La thérapie photodynamique (PDT) est une modalité de traitement des petites tumeurs localisées, reposant sur l'action conjuguée d'un photosensibilisateur (PS), de la lumière et de l'oxygène. Outre des dommages cytotoxiques directs aux cellules tumorales, la PDT induit des dommages indirects, caractérisés par l'altération de la vascularisation tumorale et l'activation d'effecteurs immunitaires. <br />Un nouveau PS couplé à un heptapeptide (ATWLPPR) ciblant neuropiline-1, un co-récepteur du VEGF, a été synthétisé. Ce couplage ne modifie pas les propriétés photophysiques du PS.<br />Une étude in vitro sur cellules endothéliales de veine ombilicale humaine a montré une moindre cytotoxité à l'obscurité, une amélioration de l'incorporation intracellulaire et une meilleure activité photodynamique, suite au couplage du PS au peptide.<br />In vivo, chez des souris nude porteuses de gliomes malins humains, le PS conjugué s'accumule dans la tumeur à des taux supérieurs à ceux retrouvés dans la peau. En utilisant des conditions (dose de PS, fluence et irradiance lumineuses) optimisées par une approche de plan d'expériences, la PDT avec le PS couplé au peptide induit une réduction du flux sanguin pendant traitement, comparé au PS non couplé, une destruction des cellules endothéliales des vaisseaux sanguins, 24h après PDT, ainsi qu'un retard de croissance tumorale, statistiquement significatif comparé au PS non couplé.

Afin de mettre en évidence des signes génétiques et moléculaires d'irréversibilité de lésions prénéoplasiques bronchiques, pouvant améliorer le dépistage, la surveillance et la chimioprévention de patients fumeurs à risque, nous avons démontré la valeur prédictive de l'accumulation d'anomalies moléculaires dans les lésions prénéoplasiques et observé dans ces lésions une compétition sur leurs récepteurs communs les neuropilines 1 et 2 entre la Sémaphorine 3F, perdue précocement et dont le gène est un candidat suppresseur de tumeur proapoptotique, et VEGF, impliqué dans la progression et la migration tumorale, exprimé de façon croissante dans les lésions dysplasiques. Par ailleurs, la télomèrase, enzyme permettant le maintien de la taille des télomères et donc l'acquisition de l'immortalité, est ré exprimée précocement en réponse au raccourcissement télomérique et de façon croissante dans les dysplasies, parallèlement à l'inactivation des verrous du cycle cellulaire p16/Rb et p53.

Increased vascularization (angiogenesis) is a required adaptation for sustained tumor growth, and the primary mediator of de novo blood vessel formation is vascular endothelial growth factor (VEGF). The central transcriptional activator of VEGF is hypoxia inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of an inducible HIF-1alpha subunit and a constitutively expressed HIF-1beta subunit. In addition to HIF-1, it has recently been reported that signal transducer and activator of transcription 3 (Stat3) is required for VEGF production and angiogenesis. Although it is known that Stat3 is an important mediator of many of the oncogenic signaling pathways that regulate HIF-1alpha, it was not known if Stat3 regulates HIF-1alpha. To answer this important question, the effect of blocking Stat3 signaling on both HIF-1alpha and VEGF expression was examined. Treatment of cells with IL-6, a potent activator of Stat3, resulted in HIF-1alpha and VEGF induction during normoxia. By blocking protein synthesis with cycloheximide, it was determined that IL-6 induction of HIF-1alpha resulted from increased translation. When Stat3 was silenced with siRNA, both basal level expression and IL-6 induction of HIF-1alpha and VEGF were significantly reduced. Furthermore, it is likely that Stat3 is required for HIF-1alpha induction by a variety of growth signals, as both HIF-1alpha and VEGF expression resulting from EGF and heregulin were abolished when Stat3 signaling was blocked. Because we had observed that Stat3 was required for induction of HIF-1alpha by growth signals, we wanted to determine if Stat3 was also required for HIF-1a induction by hypoxia. When Stat3 was silenced and cells exposed to hypoxia, HIF-1a expression was again abolished. Furthermore, the hypoxic induction of VEGF and MMP-2 was also prevented.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Histidine-rich glycoprotein (HRG) is a heparin-binding plasma protein modulating immune, hemostatic and vascular functions. I have studied the antiangiogenic functions of HRG in vitro and in vivo in order to understand the molecular mechanisms of action of HRG as an angiogenesis inhibitor. Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is a central rate-limiting step of tumor development and thus a possible target for cancer therapeutics. Previous studies have shown that HRG has antiangiogenic functions in vivo and that the antiangiogenic effects are mediated via the proteolytically released His/Pro-rich domain of HRG. In this thesis we demonstrate that HRG can inhibit endothelial cell migration by interfering with focal adhesion and cytoskeletal turnover. Moreover we have identified the minimal active domain of HRG, a 35 amino acid peptide derived from the histidine- and proline-rich domain of HRG. Analyzing human tumor tissue samples, we have found that a His/Pro-rich fragment of HRG is bound to the vasculature from cancer patients but not to the vasculature from healthy individuals. The fragment is found in association with platelets, and we show that activated platelets can induce a functional microenvironment for the His/Pro-rich fragment. Cancer patients often display an increased coagulation and our data describe a new mechanism to confer specificity of an angiogenesis inhibitor for situations with enhanced platelet activation, as in the tumor. We have further studied the role of HRG in tumor growth by crossing HRG-deficient mice with a transgenic mouse model of pancreatic insulinoma. We show that mice lacking HRG display an elevated “angiogenic switch” and that the total tumor volume is larger in these mice than in wild type mice. HRG is also involved in regulation of platelet function and platelets can stimulate angiogenesis in various ways. We have depleted mice of platelets to study the possible connection between the function of HRG in angiogenesis and platelet regulation. Our data suggest an involvement of platelets in the antiangiogenic activities of HRG.

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.

La perméabilité vasculaire est une caractéristique cruciale de l’angiogenèse. Les acteurs principaux sont les cellules endothéliales qui la régulent en réponse à divers facteurs perméabilisant, tels que le « Vascular Endothelial Growth Factor » (VEGF). Dans le contexte pathologique du cancer, les cellules tumorales produisent de grandes quantités de VEGF qui stimulent la perméabilité, ce qui leur permet d’infiltrer le réseau vasculaire. Il est connu que la tyrosine kinase Src contrôle cette modulation de la perméabilité. Puisque notre laboratoire a préalablement démontré que la phosphatase de type récepteur (PTP) DEP-1 est impliquée dans l’activation de Src en réponse au VEGF, nous avons émis l'hypothèse que DEP-1 pourrait aussi jouer un rôle dans la perméabilité des cellules endothéliales. Grâce à des expériences de transfections d’ARN interférant, nous démontrons que DEP-1 est important pour la régulation de la phosphorylation de la VE-Cadhérine, un médiateur critique de la perméabilité. L’impact de DEP-1 sur la dissociation de jonctions intercellulaires est également démontré par microscopie à immunofluorescence de cellules endothéliales. DEP-1 est également nécessaire à l’augmentation de la perméabilité induite par VEGF in vitro. Deux résidus tyrosine retrouvés dans la queue carboxy-terminale de DEP-1 sont essentiels à l’activation de Src en réponse au VEGF. Suite à la transfection d’un plasmide encodant DEP-1 muté pour ces deux résidus, nous démontrons aussi leur implication dans la régulation de la perméabilité in vitro par DEP-1. Ces travaux permettent ainsi d’approfondir nos connaissances sur un nouveau régulateur potentiel de la perméabilité vasculaire. / Endothelial cell permeability is a crucial step of angiogenesis. The main actors behind permeability are endothelial cells who accomplish this in response to permeabilizing factors, most notably Vascular Endothelial Growth Factor (VEGF). In a pathological context, migrating tumor cells produce great quantities of VEGF that stimulate an increase of vascular permeability, which allows them to intravasate into the vasculature. Src has been shown to mediate this process. Our laboratory has previously shown that the protein tyrosine phosphatase DEP-1 is involved in the regulation of VEGF-dependant activation of Src. These data thus suggested that DEP-1 might play a role in endothelial cell permeability. Here, we show through siRNA experiments that DEP-1 is important for the regulatory phosphorylation of VE-Cadherin which is critical for the induction of permeability. The impact of DEP-1 on intercellular junction dissociation is also demonstrated through immunofluorescence microscopy of endothelial cells. We further show that DEP-1 is absolutely required for the VEGF-dependent increase of permeability as illustrated by in vitro permeability assay on siRNA-transfected endothelial cells. Finally, we show that tyrosine residues in DEP-1’s carboxy-terminal tail, which are crucial for mediating Src activity in response to VEGF, are implicated in VEGF-dependant increase in permeability by transfecting plasmids coding for DEP-1 mutants of these tyrosine residues. These findings shed light on a novel potential key regulator of in vivo permeability.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

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.