Increased angiogenesis is an important characteristic of remodeling in asthmatic airways which stems from the imbalance between pro-angiogenic and anti-angiogenic factors. Surprisingly, the factors regulating this process in allergic asthma are poorly defined. The focus of this thesis is to investigate the effect of Semaphorin 3E (Sema3E) on angiogenesis events within the airways of murine model of allergic asthma. The role of Sema3E in asthma angiogenesis was tested in wild type and Sema3e-/- mice exposed to House Dust Mite (HDM) and monitored for changes in blood vessels number in the lungs. In addition, the potential of Sema3E, in reversing features of allergen inflammation, was tested in mouse model. In both cases immunohistochemistry and immunofluorescence staining of lung tissues used to assess the changes in the level of angiogenesis. Moreover, the expression of pro- and anti-angiogenic factors in total lung homogenate was assessed by ELISA and Real-Time PCR. The results showed that WT and Sema3e-/- mice both developed the HDM induced allergic asthma phenotype, but, the lung sections of HDM exposed Sema3e-/- mice had enhanced number of blood vessels compared to WT mice. The enhanced angiogenesis in Sema3-/- mice was coupled with increased level of angiogenesis driving factors VEGF and it receptor VEGFR-2. However, in WT mice the level of soluble VEGFR-1 secretion increased significantly which inhibited VEGF / VEGFR-2 binding. Besides, Sema3E treatment reduced the level of angiogenesis and inhibited HDM-induced secretion of VEGF and the expression of its receptor VEGFR-2 while increased the level of soluble VEGFR-1. Analyzing the ratio of VEGF / soluble VEGFR-1 revealed that in the presence of Sema3E in both models, soluble VEGFR-1 is the dominant factor which has an inhibitory role on angiogenic effect of VEGF. Taken together, this study provided the first evidence that Sema3E can modulate angiogenesis in allergic asthmatic airways. / February 2016

Asthma is a chronic condition characterized by variable airflow obstruction, bronchial hyper-responsiveness, airway inflammation and remodeling. In spite of tremendous advances, the regulatory mechanisms controlling these pathological features have not yet been completely addressed. From an immunological perspective, type 2 inflammation and eosinophilic infiltration are the most striking hallmarks of asthma. At physiological level, structural changes such as increase in smooth muscle mass take the center stage which is usually associated with clinical measures of asthma. There might be some regulatory mediators capable of tuning airway inflammation and remodeling under homeostatic conditions but abrogated in asthmatic conditions. Semaphorin 3E (Sema3E) is an axon guidance molecule that is ubiquitously expressed and plays diverse roles in structural and inflammatory cells such as regulation of cell migration, proliferation and angiogenesis. However, its role in clinical and experimental asthma remains unclear. In this thesis, I have set out to uncover the expression and function of Sema3E in allergic asthma. It is generally hypothesized that Sema3E is down-regulated in allergic asthma which orchestrates the function of inflammatory (dendritic cells and neutrophils) and structural (airway smooth muscle) cells. Replenishment of Sema3E, which is suppressed under asthmatic conditions, could confer protection against allergic asthma by modulation of cellular functions. I began by comparing the expression of Sema3E between allergic asthmatics and healthy subjects. A remarkable down-regulation of Sema3E under asthmatic patients was observed which was further confirmed in a mouse model of the disease. Decreased expression of Sema3E was specifically demonstrated on bronchial epithelial cells obtained from asthmatic patients at both mRNA and protein levels. To address the function of Sema3E in allergic asthma in vivo, I extended my studies to mouse models of the disease and demonstrated that Sema3e gene deletion results in exacerbated allergic asthma pathology induced by allergen exposure. To investigate the translational relevance of my findings, I performed treatment of an asthmatic mouse model with exogenous Sema3E in which its intranasal administration attenuated airway inflammation, remodeling and hyper-responsiveness. The mechanism underlying Sema3E’s role in pathogenesis of allergic asthma was extensively studied indicating a crucial role of this mediator in modulation of dendritic cells and neutrophils functions. Our data demonstrated that both dendritic cells and neutrophils express the Sema3E high affinity receptor, PlexinD1, which makes them responsive to Sema3E treatment. Then, I studied expression and function of PlexinD1 on human airway smooth muscle (ASM) cells. I found that PlexinD1 surface expression was reduced on ASM cells from asthmatic patients. Treatment of ASM cells with Sema3E inhibited their proliferation and migration as the characteristic feature of airway remodeling. Suppression of Rac1 GTPase activity and phosphorylation of Akt/PI3K and ERK/MAPK were found as signaling mechanisms underlying Sema3E’s inhibitory effects. Together, these findings show that Sema3E thereby appears as a novel regulatory mediator, upstream of pro-allergic events, suggestive of a new approach to attenuate allergic asthma deficits. / May 2016

Class III Semaphorins (SEMA3) comprise a family of chemokines that have been implicated as negative regulators of axonal guidance, angiogenesis and tumor progression. It has been demonstrated previously that one SEMA3, SEMA3F, may have therapeutic potential in the treatment of cancer. When transfected with SEMA3F, the highly metastatic human melanoma cell line A375SM was found to exhibit a highly-encapsulated, avascular phenotype with limited metastasis. Members of SEMA3 are regulated on many levels, including proteolytic processing. SEMA3F, like other SEMA3, is expressed as a 100 kD proprotein that is seen to be processed in vitro and in vivo to 95 and 65 kD isoforms. This has been largely attributed to furin-like endoproteases on the basis of furin inhibition studies. However, currently available small chemical or peptide inhibitors against the family of subtilisin/kexin-type proprotein convertases (PCSK), to which furin belongs, do not have good selectivity between PCSKs. Cleavage of SEMA3 to 65 kD have been shown to have differing effects. SEMA3A loses its ability to repel sympathetic ganglia and SEMA3E reverses its phenotype from chemorepulsant to chemoattractant for developing vasculature following cleavage. In order to further develop therapeutic strategies based on SEMA3F, it is therefore critical to better understand the proteolytic regulation of this molecule. In this study, it is shown that digest of purified SEMA3F with purified recombinant human furin does not result in proteolytic cleavage and suggested that the cleavage of SEMA3F to a 65 kD isoform may be mediated by other members of the PCSK family.

INTRODUCTION: Pancreatic cancer remains the fourth leading cause of cancer-related deaths with approximately 5% five-year survival and 3 months of median survival. The survival rate of pancreatic cancer has not improved substantially over the past 40 years. Therefore, a novel potential treatment for pancreatic cancer is urgently needed. Recently, a cell surface receptor, Neuropilin-2 (NRP-2), was found to competitively bind either stimulatory angiogenic ligands such as vascular endothelial growth factor-A (VEGFA) or inhibitory class 3 Semaphorin-3F (SEMA3F) ligands. Knowing that angiogenesis is necessary for pancreatic tumor growth, elucidating the role of NRP2 in angiogenesis may lead to curative treatment for pancreatic cancer. OBJECTIVES: Previously, NRP-2 has been shown to be expressed by human cells of pancreatic ductal adenocarcinoma (PDAC), one of the most lethal forms of pancreatic cancer. Additionally, knockdown of NRP-2 in vivo inhibited PDAC tumorigenesis. In our current study, we aimed to investigate the role of endothelial cell derived-Nrp-2 in PDAC-associated tumor angiogenesis. Furthermore, we studied the efficacy of SEMA3F as a potential inhibitory factor for pancreatic tumor growth. METHODS: To investigate the role of Nrp-2 in tumor-derived angiogenesis, we injected Panc0H7 cells, a C57BL/6 syngeneic mouse PDAC cell line, orthotopically into the pancreas of Nrp-2+/+, Nrp-2+/-, and Nrp-2 -/- mice and compared tumor growth and angiogenesis. We next injected control adenovirus (Ad-control) or SEMA3F adenovirus (Ad-3F), which actively encodes SEMA3F in vivo, followed by orthotopic injection of Panc0H7 cells into C57BL/6 mice three days later. We studied the efficacy of SEMA3F as a potential treatment for pancreatic cancer by comparing the tumor growth and tumor-associated angiogenesis of the two groups of adenovirus-treated mice. RESULTS: Our results showed that Panc0H7 tumors were significantly smaller in Nrp-2-deficient mice as compared to that of Nrp-2-intact mice. Furthermore, tumor microvessel density was significantly lower in Nrp-2-knockout mice compared to wild-type mice, while there was no difference in tumor weight or angiogenesis between wild-type and Nrp2 heterozygous mice. Our results also demonstrated that pancreatic tumors harvested from SEMA3F-treated mice were significantly smaller than the tumors from the control-treated mice. Furthermore, micrometastases were detected in the livers of mice treated with Ad-control but not in the Ad-3F group. CONCLUSIONS: Taken together, our results suggested that NRP2 might facilitate in vivo angiogenesis and tumor growth. Furthermore, SEMA3F could be a potential treatment to inhibit the growth and metastases of pancreatic tumors.

The generation of new lymphatic vessels through lymphangiogenesis has been implicated in many disease states. This process has some overlap with the better studied angiogenesis pathway, but is under distinct molecular control. Specifically, it has been shown that VEGFR-3 and neuropilin-2 are important mediators of lymphangiogenesis. A greater understanding of this process could lead to new therapies for cancer and lymphedemas. We investigated lymphatic vessel growth in a mouse model with a focus on the effects of neuropilin-2 knockout. First, we induced an immunogenic response via delayed-type hypersensitivity to examine lymphangiogenesis in the physiologic state. Our neuropilin-2 knockout mouse model displayed a decreased ability to resolve inflammation on exposure to an allergen. Next, we subcutaneously injected a highly invasive melanoma to examine lymphangiogenesis in the pathologic state. We noted significantly reduced tumor growth in our neuropilin-2 knockout. In addition, the neuropilin-2 knockout mice displayed reduced vessel area in comparison to their wild-type littermates, suggesting that inhibition of neuropilin-2 may prove a potent antitumor therapeutic strategy. These results highlight neuropilin-2's important role as a mediator of physiological and pathological angiogenesis and lymphangiogenesis.

Neuropilin-2 (NRP-2), expressed in capillaries and smooth muscle, is involved in a signaling axis featuring both stimulatory and inhibitory pathways. Ligand interaction with NRP-2 determines the effectual pathway: stimulatory VEGF, or inhibitory Semaphorin3. VEGF interaction causes NRP-2 localization with VEGFR-2, while Semaphorin3 (SEMA3) interaction causes NRP-2 to complex with Plexin A. VEGFR-2 interaction induces permeability and angiogenesis, making this an attractive target for anti-angiogenesis treatment. Plexin A interaction induces smooth muscle relaxation, offering a target for treatment of loss of bladder contractility. We aimed to further our understanding of the necessity of NRP-2 in the VEGFR-2-mediated induction of angiogenesis and permeability by performing assays in Nrp-2 knockout (KO) mice. We also tested an inhibitor against Semaphorin3/NRP-2 binding using U87MG cell, a human glioblastoma cell line. Angiogenesis assays were largely inconclusive, but suggested that NRP-2 does play a substantial role in contributing towards vessel growth. Permeability assays showed that NRP-2 plays a significant role in induction of permeability. The Semaphorin3F/NRP-2 inhibitor showed promise as a potential therapeutic, limiting the extent of Semaphorin3-based inhibition of VEGFR-2 pathway. / 2026-02-12T00:00:00Z

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.

Central nervous system (CNS) myelination is important for proper nervous system function in vertebrates. In demyelinating diseases such as multiple sclerosis, autoimmune-mediated myelin destruction results in neurological impairment; and although remyelination does occur spontaneously, it is poorly understood and insufficient in humans. Zebrafish (Danio rerio) are known to harbour tremendous regenerative capacity of various CNS tissues; however, there is presently only little knowledge of their myelin repair efficiency. An experimental model of myelin injury in zebrafish would permit study of the mechanisms involved in successful remyelination and could potentially guide the development of novel therapeutic agents for mammalian remyelination. This doctoral thesis describes the characterisation of the novel myelin protein Claudin k in zebrafish, demonstrates the establishment of adult zebrafish as an experimental model for CNS de- and remyelination and explores some mechanisms underlying myelin repair. A variety of myelin markers have previously been investigated in zebrafish, including myelin basic protein and myelin protein zero. However, the use of these is limited by either late developmental expression or presence in compact myelin only. Claudin k is a novel tight junction protein specific to zebrafish CNS and PNS, which can be observed early in development and throughout nervous system regeneration. Utilising specific antibodies and a novel transgenic zebrafish line, in which the claudin k promoter drives the expression of green fluorescent protein in myelinating cells, the studies herein characterise the expression of Claudin k, demonstrate the fidelity of the transgenic construct, and investigate the relationship of Claudin k with established myelin and CNS inflammation markers. Data demonstrate that Claudin k expression closely resembles expression patterns of the endogenous gene, and as such provides a key tool for examining CNS myelination in zebrafish. For the study of de- and remyelination in the zebrafish, the experiments herein describe the use of lysophosphatidylcholine (LPC), a detergent-like myelin toxin, which is used widely in rodent models to demyelinate axons. Its application to the adult zebrafish optic nerve induced focal demyelinating lesions, critically without detectable axonal injury, and permitted the study of time course and efficiency of remyelination. Myelin in the lesion area was reduced as detected by both immunohistochemistry and electron microscopy at 8 days post lesion (dpl), and return of the markers to control levels suggested regeneration by 28 dpl. In addition microglial activation was observed along the optic pathway, which also returned to levels compared to unlesioned control by 28 dpl. In young zebrafish (aged 4-6 months), the myelin thickness of remyelinated fibres showed no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. However, in old fish (aged 18+ months), remyelinated fibres presented with thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. While the zebrafish as an experimental system has tremendous benefits, such as potential for drug screens using the transparent larvae, capacity for transgenesis and live imaging, experimental models in zebrafish potentially bear several limitations, in particular their distant relationship to humans. To determine whether zebrafish remyelination involves homologous signalling mechanisms to mammals, demyelinated zebrafish optic nerves were treated with human recombinant Semaphorin 3A, an axonal guidance molecule which is well known to inhibit oligodendrocyte precursor cell (OPC) recruitment and remyelination in mammals. Results demonstrated fewer oligodendroglial cells at 14 dpl and less myelinated fibres at 28 dpl in the optic nerve lesion area compared to control treated animals, supporting the hypothesis that zebrafish remyelination may indeed respond to human signalling molecules. Taken together, the findings in this doctoral thesis suggest that this new experimental zebrafish-based model of CNS remyelination can be added to the suite of current models to better understand the remyelination process and that some signalling mechanisms observed in mammals around myelination and OPC recruitment are likely conserved in the zebrafish. In addition, it could potentially be used to discover novel therapeutic targets that promote myelination in injury.

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.

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