Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas. / Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells

L'hémangiome capillaire infantile (IH) est une tumeur vasculaire bénigne courante. Les formes sévères sont traitées depuis 2008 par propranolol bien que le mécanisme d’action reste à ce jour inexpliqué. La 1ère partie de ma thèse est une étude de tolérance chez les enfants traités par propranolol dans le cadre de l'ATU française. L'analyse de cette base de données prospective de 922 enfants a permis de confirmer la bonne tolérance du propranolol dans la population pédiatrique, et de cibler les situations à risque de complication. La 2e partie est une étude de la signalisation adrénergique à partir des tissus d'IH opérés. Tout d'abord, nous avons identifié par immunofluorescence les cellules exprimant les récepteurs béta-adrénergiques ADRB1, 2 et 3 : Nous avons observé une forte expression d'ADRB2 sur le mastocyte, et de manière plus modérée d'ADRB1 et 2 sur les vaisseaux. Ce profil d'expression était retrouvé quel que soit le degré d'involution de la tumeur, et était également observé sur des tumeurs vasculaires contrôles qui elles ne répondent pas au propranolol. Nous avons donc envisagé une activation des récepteurs ADRB propre aux IH prolifératifs. Cette hypothèse a été confirmée par la mise en évidence, de l'expression d'enzymes de synthèse des catécholamines et de marqueurs neuroendocrines sur les péricytes des IH prolifératifs, sur tissus inclus en paraffine et également péricytes d’IH mis en culture. La surexpression de HIF1-a sur ces cellules a conduit à tester l’effet du propranolol en hypoxie. Nous n’avons pas mis en évidence d'effet sur la prolifération cellulaire, mais par contre une inhibition de la sécrétion de VEGF induite par l’hypoxie. / Capillary infantile haemangioma (IH) is a common benign vascular tumour of infants. Severe forms are treated since 2008 by propranolol. However, its dramatic efficacy remains until now unexplained.The first part of my thesis is a safety study of children treated with propranolol as part of the French Compassionate Use Program. The prospective vigilance database, including 922 children, confirmed the safety of propranolol among the paediatric patients, and highlighted the circumstances at risk of complications.The second part is a study of adrenergic signaling in IH tissues. First, we identified by immunofluorescence the expression of beta-adrenergic receptors on IH cells. We observed a strong expression of ADRB2 on mast cells, and a moderate ADRB1 and 2 expression on vessels. This expression pattern was the same regardless of the degree of involution of the tumour, and has also been observed on tumour vascular controls which did not respond to propranolol. We therefore hypothesized that ADRB receptors may be activated in proliferative IH. We indeed detected the expression of catecholamine synthesis enzymes and of neuro-endocrine markers on pericytes in paraffin-embedded tissues, and also in IH pericytes in culture. We also detected HIF1-alpha overexpression and therefore explored propranolol effect during hypoxia. Propranolol had no effect on cell proliferation, but reduced hypoxia-induced secretion of VEGF-A.

Le carcinome à cellules rénales (RCC) exprimant le facteur inductible de l'hypoxie (HIF) en raison de l'inactivation du gène de von Hippel Lindau (vhl), représente un modèle d'hypoxie chronique. Le devenir des patients dépend du stade de dissémination des cellules tumorales. Par conséquent, déchiffrer les mécanismes de métastase est une préoccupation majeure. Le développement dépendant du VEGF-C (Vascular Endothelial Growth Factor C) d'un réseau lymphatique est en première ligne de propagation métastatique. Pour étudier le rôle de VEGFC dans la dissémination du RCC, nous avons étudié son expression dans l'hypoxie et nous avons invalidé son gène dans des lignées cellulaires humaines et murines. L'hypoxie régule négativement l'ARNm de VEGFC par une diminution de la transcription et de la stabilité de l'ARNm mais l'expression de la protéine VEGF-C est induite par l’hypoxie. Des capacités accrues de prolifération et de migration, et une meilleure expression des marqueurs mésenchymateux et des marqueurs souches caractérisent les cellules vegf-c -/-. Alors que les cellules vegfc -/- ne forment pas de tumeurs chez les souris immunodéficientes, elles développent des tumeurs agressives chez les souris immunocompétentes. La surexpression de VEGFC, est liée à une augmentation de la survie sans progression et globale chez les patients atteints de tumeurs non métastatiques alors qu'une diminution de la survie sans progression et globale est observée chez les patients métastatiques. Nos expériences décrivent une régulation subtile du VEGF-C par hypoxie et mettent en évidence son rôle bénéfique ou péjoratif. Par conséquent, le ciblage VEGF-C pour la thérapie doit être considéré avec prudence. / Hypoxic zones are common features of metastatic tumors. Renal cell carcinoma (RCC) expressing the Hypoxia Inducible Factor (HIF) because of inactivation of the von Hippel Lindau gene (vhl), represent models of chronic hypoxia. Their outcome depends on the extent of their dissemination at diagnosis. Therefore, deciphering the mechanisms of metastasis is a major concern. The Vascular Endothelial Growth Factor C (VEGFC)-dependent development of a lymphatic network is in front line of metastatic spreading. To address the role of VEGFC in RCC dissemination, we studied its expression in hypoxia and we invalidated its gene in human and mouse model cell lines of RCC. Hypoxia down-regulates VEGFC mRNA through a decrease in transcription and mRNA stability but concomitantly induced VEGFC protein expression. Increased proliferation and migration abilities, over-activation of the AKT signaling pathway and enhanced expression of mesenchymal and stem cell markers characterized vegfc-/- cells. Whereas vegfc-/- cells do not form tumors in immuno-deficient mice, they develop aggressive tumors in immuno-competent mice. Moreover, mouse RCC cells generate fast-growing tumors in mice invalidated for six1 or eya2, two major regulators of VEGFC expression. Lymphangiogenic markers overexpression including VEGFC is linked to increased disease-free and overall survival in patients with non-metastatic tumors whereas decreased progression-free and overall survival is observed for metastatic patients. Our experiments describe a subtle regulation of VEGFC by hypoxia and highlight its beneficial or pejorative role. Therefore, targeting VEGFC for therapy must be considered with caution.

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.

Les trois enzymes de la famille PRL (PRL-1,2,3), représentent un groupe de protéines tyrosine phosphatases intrigantes, impliquées dans un très grand nombre de maladies. Elles ont gagné beaucoup d'attention ces dernières années dans le contexte tumoral puisqu’elles sont très fréquemment associées à la formation de métastases, la prolifération cellulaire, l'invasion et la migration cellulaire. Bien que les propriétés oncogéniques des PRLs ne fassent plus de doute, ces phosphatases sont très peu caractérisées dans un contexte physiologique. Ainsi, aucun substrat biologique n’a été clairement identifié à ce jour et très peu de fonctions biologiques leurs sont associées, malgré un très haut degré de conservation inter-espèce suggérant un rôle critique de ces protéines dans les fonctions cellulaires. La caractérisation de la souris déficiente pour PRL-2 indique que cette phosphatase est probablement impliquée tout au long du développement. La phosphorylation des protéines est un phénomène important pour la régulation de l'angiogenèse en jouant des fonctions critiques et réversibles sur les voies de signalisation cellulaire. Cela nous a incités, dans le cadre de cette thèse, à étudier le rôle de PRL-2 dans la morphogenèse vasculaire en utilisant à la fois des modèles in vitro et un modèle de souris déficientes pour PRL-2. Dans le modèle murin de l'angiogenèse rétinienne, nous avons trouvé que la délétion de PRL-2 entraîne un retard dans la formation du plexus vasculaire avec une réduction de la vascularisation. De plus, on observe une angiogenèse et une ramification excessive au niveau du front vasculaire chez les souriceaux. En effet, le front de croissance des rétines de souris PRL-2 KO présente une densité vasculaire plus élevée associée à un bourgeonnement endothélial actif et multidirectionnel. Ces données présentent PRL-2 comme un composant potentiel du réseau de signalisation complexe qui orchestre la néo-angiogenèse. Nous avons ensuite examiné les conséquences de la perte de PRL-2 sur le comportement des cellules endothéliales in vitro. Nous avons observé des modifications de migration et d’invasion dans différents modèles. En particulier, l’absence de PRL-2 accroît le bourgeonnement de capillaires dans des tests d'angiogenèse in vitro, ce qui est en accord avec les données in vivo. En utilisant plusieurs siRNA, nous avons montré que la voie de signalisation de PRL-2 est liée à la signalisation VEGF/VEGFR. En effet, la stimulation des cellules endothéliales par le VEGF dépend de la présence ou de l'absence de PRL-2. En outre, une autre voie altérée par la régulation négative de PRL-2, est celle de Notch ainsi que celle du facteur de transcription Hey2. Ceci est cohérent avec les données in vivo où nous avons démontré un effet important sur la différenciation artérioveineuse. Ces données introduisent PRL-2 en tant que composant novateur du réseau de signalisation complexe qui orchestre l'angiogenèse développementale et, éventuellement, pathologique. / The three Phosphatase of Regenerative Liver (PRL-1, -2, -3) represent an intriguing group of protein tyrosine phosphatases that has been implicated in a number of diseases. They have gained much attention in the context of cancer. Indeed, they have been constantly associated with metastasis, cell proliferation, cell invasion and migration. To date, however, little is known about their physiological function and no biological substrates have been clearly identified. All three PRLs are highly conserved among mammals, underscoring the idea that they might have important roles in cellular functions. Characterization of the PRL-2 knockout mouse indicates that this phosphatase is likely involved throughout development. Protein phosphorylation is implicated in angiogenesis by playing critical and reversible functions in cell signaling pathways. This prompted us, in the frame of this thesis, to investigate the role of PRL-2 in vascular morphogenesis using both in vitro models and genetic loss-of-function mouse models. In the retinal angiogenesis mouse model, we found that PRL-2 deletion leads to delay in the formation of the retinal vascular plexus with a reduction of the advancing vasculature across the vitreal surface. Furthermore, excessive angiogenesis and branching at the leading edge in 6 day old pups is observed. Indeed, the growing front of PRL-2 KO mouse retinas showed a higher vascular density due to active, multidirectional hypersprouting of the vasculature. This data introduces PRL-2 as a potential component of the complex signaling network that orchestrates neo-angiogenesis. Based on these findings, we have examined whether the absence of PRL-2 can modify the behavior of endothelial cells in vitro. We showed an altered migration in various assays. In particular, sprouting in in vitro angiogenesis assays is altered, which is in agreement with the in vivo data. By using several siRNA, we showed that the signaling pathway of PRL-2 is dependent to VEGF/VEGFR signaling. Indeed, the stimulation of endothelial cells by VEGF is dependant of the presence or absence of PRL-2. Furthermore, other targets altered by PRL-2 downregulation, are Notch and the Hey2 transcription factor, which is consistent with in vivo data as we showed a strikingly effect on arteriovenous differentiation. Taken together, these data introduce PRL-2 as a novel component of the complex signaling network that orchestrates developmental and, possibly, pathological angiogenesis.

Das Ovarialkarzinom weist von allen gynäkologischen Tumorerkrankungen die höchste Sterblichkeit auf. Die Therapie umfasst eine radikale Operation sowie eine adjuvante, platin-basierte Chemotherapie. Darüber hinaus kommen seit einigen Jahren zielgerichtete Therapien wie der antiangiogenetische Antikörper Bevacizumab oder PARP-Inhibitoren bei Patientinnen mit einer homologen Rekombinationsdefizienz (HRD) zum Einsatz. Trotz dieser neuen Ansätze weisen vor allem Patientinnen mit einem fortgeschrittenen Ovarialkarzinom eine schlechte Prognose auf. Für Bevacizumab konnte zwar eine Verlängerung des progressionsfreien Überlebens nachgewiesen werden, es bleibt jedoch unklar, welche Patientinnen von einer Therapie mit Bevacizumab profitieren. Die Suche nach einem prädiktiven Biomarker für das Ansprechen auf Bevacizumab war Gegenstand verschiedener Studien. Auch wenn einige vielversprechende Ansätze publiziert wurden, konnte bislang keiner dieser Ansätze in der klinischen Praxis etabliert werden. In meiner Arbeit habe ich das molekulare Ziel von Bevacizumab, VEGF‑A, untersucht. VEGF‑A besteht aus verschiedenen Isoformen mit unterschiedlichen pro- und antiangiogenetischen Eigenschaften. Das Gleichgewicht zwischen pro- und antiangiogenetischen VEGF‑A-Isoformen scheint direkt mit der angiogenetischen Aktivität eines Tumor assoziiert zu sein. Daher war mein Ziel, die klinische Relevanz der Isoform VEGF‑A165b in Hinblick auf ihre prognostische Aussagekraft sowie ihr Potenzial als prädiktiver Marker für das Ansprechen auf Bevacizumab zu untersuchen. Die VEGF‑A165b-Expression wurde hierfür mittels Immunhistochemie in formalin-fixiertem, in Paraffin eingebettetem Gewebe von 413 Patientinnen untersucht, welches in Form eines Tissue Microarrays vorlag. Die Patientinnen waren Teil der deutschen Kohorte der internationalen, multizentrischen ICON7-Studie und wurden entweder mit der Standardchemotherapie oder mit der Standardchemotherapie und zusätzlich Bevacizumab behandelt. Die Ergebnisse der Immunhistochemie wurden mittels Lichtmikroskopie beurteilt. Mittels Kaplan-Meier- sowie Cox-Analysen wurde die Assoziation zwischen der VEGF‑A165b-Expressions und dem Ansprechen auf Bevacizumab untersucht. Die Kaplan-Meier- sowie die univariate Cox-Analyse zeigte keinen Unterschied im PFS sowie im Gesamtüberleben (OS) zwischen der Gruppe der Patientinnen mit einer niedrigen und der Gruppe der Patientinnen mit einer hohen VEGF‑A165b-Expression. Dies galt sowohl für die Gesamtkohorte als auch für die beiden Therapiegruppen bei getrennter Analyse. Somit zeigte sich die VEGF‑A165b-Expression als nicht prognostisch relevant in der untersuchten Kohorte. Anschließend erfolgte eine getrennte Analyse der Patientinnen mit einer niedrigen sowie der Patientinnen mit einer hohen VEGF‑A165b-Expression. Für jede der beiden Gruppen wurde das PFS und das OS zwischen den beiden Behandlungsarmen (Standardchemotherapie mit oder ohne Bevacizumab) verglichen. Für die Gruppe der Patientinnen mit einer hohen VEGF‑A165b-Expression konnte in der Kaplan-Meier- sowie der univariaten Cox-Analyse kein signifikanter Einfluss von Bevacizumab auf das PFS (HR: 0,759; 95%KI = 0,530 – 1,089; p = 0,134) oder das OS (HR: 0,898; 95%KI = 0,597 – 1,350; p = 0,606) nachgewiesen werden. Für die Gruppe der Patientinnen mit einer niedrigen VEGF‑A165b-Expression konnte in der univariaten Cox-Analyse eine signifikante Verbesserung sowohl des PFS (HR: 0,727; 95%KI = 0,538 – 0,984; p = 0,039) als auch des OS (HR: 0,662; 95%KI = 0,458 – 0,958; p = 0,029) unter Therapie mit Bevacizumab nachgewiesen werden. In der multivariaten Cox-Analyse erwies sich dieser Effekt sowohl für das PFS (HR: 0,610; 95%KI = 0,446 – 0,834; p = 0,002) als auch für das OS (HR: 0,527; 95%KI = 0,359 – 0,775; p = 0,001) als unabhängig von den etablierten prognostischen Faktoren. Damit zeigt meine Arbeit erstmals, dass die immunhistochemisch detektierte VEGF‑A165b-Expression prädiktiv ist für ein Ansprechen auf Bevacizumab bei Patientinnen mit einem Ovarialkarzinom. Die Ergebnisse stammen aus der retrospektiven Analyse eines umfangreichen Patientinnenkollektivs, welches Teil der internationalen ICON7-Studie war, und sind von hoher klinisch-translationaler Relevanz. Da der immunhistochemische Nachweis von VEGF‑A165b leicht in die pathologische Routinediagnostik zu integrieren ist, könnte dieser neue prädiktive Biomarker bei der Entscheidung helfen, ob eine Behandlung mit Bevacizumab für am Ovarialkarzinom erkrankte Patientinnen infrage kommt. Da der aktuelle Therapiestandard für HRD-positive Patientinnen eine Kombination von Bevacizumab mit einem PARP-Inhibitor vorsieht, ist eine prospektive Validierung der Ergebnisse in einem so behandelten Kollektiv notwendig, um die Relevanz der VEGF‑A165b-Expression als prädiktiver Marker für das Ansprechen auf diese Kombinationstherapie zu evaluieren. / Among female malignancies, ovarian cancer has the highest mortality rate. Therapy comprises radical tumor debulking, followed by adjuvant platinum-based chemotherapy. In recent years, targeted treatment approaches have been integrated into the standard treatment of ovarian cancer, such as the addition of the anti-angiogenic antibody bevacizumab or the addition of PARP inhibitors in patients with homologous repair deficiency (HRD). However, most patients with advanced ovarian cancer continue to face a poor prognosis. Although an improvement in progression-free survival (PFS) has been shown for bevacizumab, it remains hard to predict which patients can profit from an addition of bevacizumab to standard chemotherapy. Several study groups have been looking for a predictive marker for bevacizumab response in ovarian cancer patients. While promising approaches have been suggested, none of these have been implemented into clinical practice. In my thesis, I focussed on the molecular target of bevacizumab, VEGF‑A, which consists of several isoforms with pro- or antiangiogenic properties. It has been proposed that the balance of pro- and antiangiogenic VEGF‑A isoforms is directly linked to the angiogenic activity of a tumor. Therefore, the objective of my thesis was to investigate the clinical relevance of the VEGF‑A165b isoform in ovarian cancer patients with regard to i) its prognostic relevance and ii) its potential to predict response to bevacizumab. Expression of VEGF‑A165b was detected by immunohistochemistry using formalin-fixed paraffin-embedded tissue from 413 patients, arranged in a tissue microarray. The patients were participants in the German contribution to the ICON7 multicenter phase III trial and were each treated with standard platinum-based chemotherapy either with or without bevacizumab. Staining results were evaluated using optical microscopy. Kaplan-Meier analysis and Cox regression analysis were performed in order to explore the association between response to bevacizumab and VEGF‑A165b expression. Kaplan-Meier and univariate Cox regression analysis did not show a difference in PFS and overall survival (OS) between the VEGF‑A165b-low- and the VEGF‑A165b-high-expressing group. This was the case for the overall cohort as well as for both treatment arm groups separately. Thus, VEGF‑A165b expression in itself did not show a prognostic relevance in the study cohort. Subsequently, patients were stratified in a VEGF‑A165b-low- and a VEGF‑A165b-high-expressing group. For each group, PFS and OS were compared between the two treatment arms: standard platinum-based chemotherapy with or without bevacizumab respectively. For VEGF‑A165b-high-expressing patients, Kaplan-Meier and univariate Cox regression analyses did not show a significant effect of bevacizumab on PFS (HR: 0.759; 95%CI = 0.530 – 1.089; p = 0.134) or OS (HR: 0.898; 95%CI = 0.597 – 1.350; p = 0.606). However, for the VEGF‑A165b-low-expressing group, univariate Cox regression analysis showed a significant improvement in PFS (HR: 0.727; 95%CI = 0.538 – 0.984; p = 0.039) and OS (HR: 0.662; 95%CI = 0.458 – 0.958; p = 0.029) under bevacizumab. This effect was independent of established risk factors for both PFS (HR: 0.610; 95%CI = 0.446 – 0.834; p = 0.002) and OS (HR: 0.527; 95%CI = 0.359 – 0.775; p = 0.001). Therefore, the results of my thesis suggest for the first time that VEGF‑A165b protein expression as detected by immunohistochemisty is predictive for response to bevacizumab treatment in ovarian cancer patients. These findings were obtained from a retrospective analysis of a comprehensive patient cohort from an international clinical trial (ICON7) and are of high clinical-translational relevance. Since VEGF‑A165b detection is possible by standard immunohistochemistry, it can be envisioned that this novel predictive biomarker may guide bevacizumab related treatment decisions in ovarian cancer patients. Given the retrospective nature of my approach, a prospective validation of my results will be imperative to determine whether VEGF‑A165b expression also predicts response to combined treatment with beacizumab and PARP inhibitors, which is the new treatment standard for HRD-positive ovarian cancer patients.

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

Unusual DNA secondary structures, especially G-quadruplexes and i-motifs, play important roles in gene transcriptional regulation and have been identified as novel drug targets. In this dissertation, I explored their formation in the human VEGF and RET promoters and their roles in gene transcriptional regulation. VEGF is a key regulator of angiogenesis and is up-regulated in many types of tumors. A poly-guanine/poly-cytosine (polyG/polyC) tract in its proximal promoter (-85 to -50 base pairs relative to the transcription starting site) is essential for both basal and inducible VEGF expression. I demonstrated that the guanine-rich (G-rich) and cytosine-rich (C-rich) strands in the VEGF proximal promoter are able to form G-quadruplex and i-motif structures, respectively. The major G-quadruplex formed by the VEGF G-rich sequence is an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:4:1 arrangement of three double-chain-reversal loops (two single-base loops and one loop with four bases). The complementary C-rich sequence in the same region forms an intramolecular i-motif containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration (two double-base loops and one loop with three bases). The Gquadruplexes formed by the native VEGF G-rich and its derivative sequences were also confirmed by NMR. In addition, various transcription factors including Sp1, hnRNP K, CNBP and nucleolin, which recognize different DNA structural elements including single-stranded, double-stranded or G-quadruplex/i-motif DNA in the VEGF proximal promoter, have been confirmed by EMSA, siRNA and chromatin immunoprecipitation (ChIP) assay, suggesting that the DNA in the VEGF proximal promoter region is capable of undergoing transitions between those three structures. Based on my studies, I have proposed a model to describe how various transcription factors recognize different DNA structures in the VEGF proximal promoter to regulate transcription. In the proximal promoter of another important oncogene RET, I demonstrated that the guanine-rich strand forms an intramolecular parallel G-quadruplex containing three G-tetrads and a 1:3:1 arrangement of three double-chain-reversal loops. The complementary cytosine-rich strand forms an i-motif structure containing six semiprotonated cytosine-cytosine⁺ base pairs and a 2:3:2 loop configuration. Moreover, G-quadruplex-interactive compounds TMPyP4 and telomestatin were shown to further stabilize the RET G-quadruplex structure.

Vascular endothelial growth factor-B (VEGF-B), when initially discovered, was thought to be an angiogenic factor, due to its intimate sequence homology and receptor binding similarity to the prototype angiogenic factor, VEGF-A. Studies demonstrated VEGF-B, unlike VEGF-A, did not play a significant role in angiogenesis or vascular permeability and has become an active area of interest because of its role as a survival factor in pathological processes in a multitude of systems, including the brain. By characterization of important downstream targets of VEGF-B that regulate different cellular processes in the nervous system and cardiovascular system, it may be possible to develop more effective clinical interventions in diseases such as Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and ischemic heart disease, which all share mitochondrial dysfunction as part of the disease. Here we summarize what is currently known about VEGF-B function in pathological processes, compare probable mechanisms of action and elude to its potential as a homeostatic protective factor to increase mitochondrial function in the setting of neurological disease and cardiovascular disease.

The complex pathobiologic mechanisms of emphysema are not fully understood, leaving this deadly disease without effective pharmacotherapy for a cure. This project hypothesized that the sulfated dehydropolymer of caffeic acid (CDSO3) exhibits Fe2+ chelation-based hypoxia inducible factor-1a (HIF-1a) up-regulatory protective activities against in vitro emphysematous cell death and for in vivo reversal of emphysema induced with SU5416, a vascular endothelial growth factor blocker. Using in vitro chromogenic competitive inhibition assays, CDSO3 was shown to chelate Fe2+ (IC50 of 23 µM), but not Fe3+ ions. The trypan blue exclusion and lactate dehydrogenase assays were then employed to examine the cytoprotective activities of CDSO3 against inflammatory, oxidative, elastolytic, and apoptotic cell death using alveolar macrophages, epithelial and endothelial cells. CDSO3 at 10 µM produced significant protective activities against these emphysematous cell deaths by 50-154 %. These protective effects were opposed by the addition of the HIF-1a inhibitors, CAY10585 and echinomycin, and excess Fe2+, but not Fe3+, ions. Emphysema was then induced in rats following a subcutaneous injection of SU5416 at 20 mg/kg, after which CDSO3 at 60 µg/kg was administered to the lungs 3 times/week for two weeks. Treadmill exercise endurance (EE) was measured to assess the functional impairment, while lung tissues were removed for morphological assessments of alveolar airspace enlargement (MLI) and destruction (DI), as well as to measure protein levels using Western blot. SU5416 significantly impaired EE, MLI, and DI by 81 %, 47 %, and 5-fold, compared to the healthy animals, and these were significantly reversed by CDSO3 by 66, 74, and 87 %. CDSO3 treatment did not change the lung cytoplasmic expression of histone deacetylase 2 (HDAC2), HIF-1a, or a pro-apoptotic marker, BAX. However, induction with SU5416 significantly reduced VEGF expression by 52 % and increased cleaved caspase-3 expression by 1.5-fold, compared to the healthy animals, while CDSO3 normalized the expressions of both proteins in these emphysematous animals. However, when CDSO3 was pre-mixed with excess Fe2+, the reversal activities of CDSO3 were diminished. In conclusion, this study has demonstrated the Fe2+ chelation-based HIF-1a up-regulatory dependent in vitro and in vivo lung repairing efficacies for CDSO3 in emphysema.