Angiogenesis by either normal or neoplastic cells involves a delicate balance of both angiogenic and angiostatic regulators. In the ovary, normal physiological angiogenesis occurs around the developing follicle and corpus luteum in response to hormonal shifts. Interestingly, carcinomas arising from the ovary are usually highly vascularized and are commonly clinically observed to produce cyst fluids or ascites which contain both angiostatic and/or angiogenic regulators. However, in contrast to normal angiogenesis, angiogenesis associated with epithelial ovarian cancer usually produces aberrant vasculature that may promote neoplastic progression. Therefore, the ovary and ovarian cancers provide models to study the mechanisms governing the strict balance of angioregulators in both normal and tumor angiogenesis. While most studies to date have focused on angiogenic regulators for normal and aberrant angiogenesis, we investigated the potential for dysregulation of angiostatic regulators to contribute to the etiology of epithelial ovarian cancer. Therefore, in this study, we examined two angiostatic regulators, angiostatin and semaphorin 3F, in epithelial ovarian cancer. Angiostatin, a cleavage product of the circulating zymogen plasminogen, was isolated from serum and urine of mice bearing a Lewis lung carcinoma and in vivo studies have demonstrated its potent angiostatic properties. Thus, we investigated the potential prognostic/diagnostic advantage of aberrant angiostatin expression with epithelial ovarian cancer. We found that urinary angiostatin, compared to other angioregulators in plasma or urine, could serve as an effective biomarker for early detection of epithelial ovarian cancer, especially when used in combination with cancer antigen 125. Additionally, urinary angiostatin correlated with both recurrent disease as well as successful tumor ablation further supporting its potential as a disease biomarker. Alternative biological functions for the axon guidance molecule, semaphorin 3F, have been reported particularly in regard to angiogenesis, tumor progression and metastasis. However, the underlying mechanisms governing semaphorin 3F regulation and dysregulation remain unclear. Therefore, we first investigated the clinical relationship between semaphorin 3F expression and epithelial ovarian cancer progression. These immunohistological studies revealed that, similar to lung cancer, semaphorin 3F expression decreased with progression supporting a tumor suppressor-like role for semaphorin 3F. Additionally, we found that calcium, an essential cellular signaling molecule, could mediate transcriptional suppression of semaphorin 3F expression in a CREB-dependent manner. Lastly, given the antagonistic relationship between semaphorin 3F and vascular endothelial growth factor, we sought to determine whether semaphorin 3F and vascular endothelial growth factor promoted opposing effects on a common downstream target. In the course of these studies we determined that telomerase is a novel molecular target of semaphorin 3F in ovarian cancer cells such that semaphorin 3F suppresses telomerase activity while vascular endothelial growth factor promotes telomerase activity. In addition, we found that the inverse relationship between semaphorin 3F and telomerase was mediated through transcriptional inhibition of the hTERT promoter by semaphorin 3F. In conclusion, this research shows that dysregulation of the angiostatic regulators, angiostatin and semaphorin 3F, may contribute to the etiology of epithelial ovarian cancer. In the future, dysregulation of these and other angiostatic regulators may be exploited for therapeutic intervention or as biomarkers for early detection which would allow women more treatment choices and hopefully, reduce the mortality associated with this insidious disease.

Multiple Sclerosis (MS) is an inflammatory disease which causes areas of demyelination in the Central Nervous System (CNS) and affects only humans. Current therapies for MS are focused on anti-inflammatory treatment, which reduce the occurrence and clinical relapses of the disease. However, progressive disability of the disease is related to axonal degeneration. After demyelination, remyelination occurs, which helps repair the demyelinated lesions and protects axons from degeneration. However, this endogenous remyelination is inefficient, and currently there are no therapies available to enhance remyelination. The aim of this thesis was to first characterize a fast and reliable model to study CNS remyelination in vitro, and second to investigate the role of semaphorin 3a (Sema3A) and semaphorin 3f (Sema3F) signaling in CNS remyelination. Various in vivo models have been developed to investigate the pathology of multiple sclerosis, and can be used to test remyelination therapies. However, in vivo models are expensive, animal- and time- consuming. Until now, there has been no well-characterized and robust in vitro model for remyelination study. In this thesis, an ex vivo slice culture system with mouse brain and spinal cord was developed, and characterized by immunofluorescent microscopy and transmission electron microscopy, for CNS remyelination study. Automated (re)myelinating quantification by image pro plus software was developed and validated to provide a fast and reliable way for testing factors that change remyelination efficiency. Two such factors are Sema3A and 3F, which were initially identified as axon guidance cues during development. Sema3A (repulsive) and 3F (attractive) were proved to play a role in oligodendrocyte precursor cell (OPC) migration during development, and hypothesized to be important in remyelination. In this thesis, I investigated the effects and mechanisms for this by adding recombinant SEMA3A or SEMA3F or by knockdown their obligatory receptors Neuropilin (Nrp) 1 and 2, using lentivirus induced miRNAi. Slice culture and primary OPC culture were used to determine the effect on OPC survival, migration, proliferation, differentiation and myelination.

INTRODUCTION: Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive form of cancer with a high mortality rate, primarily due to lack of effective treatment options. Current therapeutic approaches are limited to surgical resection of the pancreas during early stages of the disease and to the use of non-specific chemotherapeutic drugs such as gemcitabine, neither of which has successfully improved the 5-year survival rate of PDAC. Both the lack of effective treatments and the high mortality of the disease call for the urgent need to develop new therapeutic options. OBJECTIVES: This thesis project focuses on an endogenous inhibitor of the neuropilin 2 receptor (NRP2) called semaphorin 3F (SEMA3F) and its use as a potential new drug in the fight against pancreatic cancer. By binding the transmembrane receptor neuropilin 2 (NRP2), SEMA3F can inhibit angiogenesis and cellular proliferation. Interestingly, given its role as a guidance molecule, it is also a potent mediator of cellular repulsion. All three of these effects will be analyzed in the context of this study. METHODS: Syngeneic pancreatic cancer cells were injected orthotopically in two separate groups of mice. One group involved the use of transgenic Nrp2-/- mice, and served as a way to analyze the absence of the receptor on the vasculature and how that affects the growth of the primary tumor and the formation of metastases in the liver. The other group received intravenous injections of SEMA3F-expressing and control adenovirus, and served to explore the effect of SEMA3F as a potential therapy against the growth of the primary tumor in the pancreas and distant metastases in the liver. RESULTS: We observed a decrease in pancreatic tumor and metastatic growth in the absence of Nrp2 in our transgenic mouse model compared to the WT control. Mice injected with SEMA3F-expressing adenovirus also showed a decrease in primary tumor growth as well as a reduction in the formation of metastases in the liver compared to the control. CONCLUSION: Nrp2 mediates angiogenesis in pancreatic cancer, which facilitates the growth of the primary tumor as well as the formation of metastases. Our results indicate that the anti-angiogenic, anti-proliferative and repulsive actions of SEMA3F could be used to develop an effective treatment option for PDACpancreatic ductal adenocarcinoma. / 2020-07-24T00:00:00Z

Edema or tissue swelling is exacerbated during inflammation due to increased leukocyte infiltration and vascular permeability, after which resolution returns the tissue to homeostasis. In acute inflammatory reactions, upregulated levels of vascular endothelial growth factor (VEGF) is shown to increase vascular permeability. Vascular endothelial cells (EC) form a selective barrier regulating the degree of microvascular exchange and permeability in normal physiological and pathological settings. Vascular EC express pro-permeability VEGF receptors and neuropilin co-receptors that can mediate both stimulatory and inhibitory signals. Secreted class 3 semaphorin-3F (SEMA3F) is a high affinity ligand for the NRP2 receptor and has been shown to be anti-angiogenic through its ability to inhibit cell migration and attachment. Importantly, SEMA3F has been shown to compete for binding with VEGF to the NRP2 receptor. However, the role, if any, of SEMA3F in inflammation has yet to be fully elucidated. We hypothesize that SEMA3F reduces edema by inhibiting vascular permeability thereby promoting a quickened resolution of inflammation. To generate inflammatory lesions, delayed-type hypersensitivity cutaneous reactions were induced on the ear skin of C57BL/6 mice through topical applications of oxazolone. Total ear thickness as a readout of tissue swelling was compared to baseline (Day 0). To determine the effects of depleting SEMA3F during inflammation, ear thickness was measured after SEMA3F antibody or control IgG intraperitoneal injection into Nrp2+/- mice. To assess the effects of increased systemic SEMA3F on edema, ear thickness was measured after intravenous delivery of SEMA3F adenovirus (Ad-3F) or control adenovirus into wild-type mice. We report that SEMA3F depletion via SEMA3F antibodies led to significantly prolonged edema compared to controls. Ad-3F treated mice exhibit lower levels of inflammatory edema compared to control. We demonstrate that the SEMA3F signaling cascade is a key mediator of fluid homeostasis in inflammation. Likely, SEMA3F serves as an anti-inflammatory mechanism preventing excessive edema. / 2020-02-20T00:00:00Z