Ovarian Cancer is the 5th most common cause of cancer death in women and the second most common gynaecological cancer in the UK. Worldwide, around 152,000 women were estimated to have died from ovarian cancer in 2012. Survival rates for women with epithelial ovarian cancer have not significantly changed since platinum-based treatment was introduced over 30 years ago. This is particularly disconcerting considering the fact that there is a less than 5% five year survival rate for patients diagnosed with late stage high grade serous ovarian cancer. This thesis examines the role of BMP signalling in ovarian cancer using in vitro cancer cell models. It builds upon the initial published work by the Inman lab identifying autocrine BMP9 as a promoter of ovarian cancer cell proliferation. The findings of Chapters 3-5 provide strong evidence indicating BMP9 as a context specific modulator of ovarian cancer cell proliferation. This significantly builds upon on the sole pro-proliferative BMP9 growth response previously described. Responding cell lines were subjected to a microarray with and without BMP9 treatment In order to determine early BMP target genes which were subsequently transiently knocked down in order to determine their role in the aetiology of said growth phenotype. ID1 gene expression was found to significantly contribute to the BMP9 proproliferative phenotype. Moreover several other BMP genes identified significantly alter basal cell proliferation. It was subsequently determined that BMP9 implemented a cell growth phenotype by negating apoptosis. .Excitingly, preliminary evidence suggests a marked reduction in detectable levels of a recently described Bax isoform, Bax β that coincide with BMP9 addition and the resultant anti-apoptotic phenotype observed. This is very interesting as no prior evidence correlating the BMP family and Bax β currently exists. These findings provide an enhanced understanding of BMP9s contribution to ovarian cancer pathogenesis that may result in the development of effective and targeted therapeutic interventions upon further stratification of the contextuality of the BMP induced growth response.

There exists a dire need for improved therapeutics to achieve predictable and effective bone regeneration. Non-viral gene therapy is a safe method that can efficiently transfect target cells, therefore is a promising approach to overcoming the drawbacks of protein delivery of growth factors. The goal of this study was to employ cost-effective biomaterials to deliver genetic materials (DNA or RNA) in a controlled manner in order to address the high cost issues, safety concerns, and lower transfection efficiencies that exist with protein and gene therapeutic approaches. To achieve our goal, we set several aims: 1) To assess the bone regeneration capacity of polyethylenimine (PEI)-chemically modified ribonucleic acid (cmRNA) (encoding bone morphogenetic protein-2 (BMP-2)) activated matrices, compared to PEI-plasmid DNA (BMP-2)-activated matrices. 2) To explore the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. 3) To use collagen membranes as integral components of a guided bone regeneration protocol and to enhance the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or cmRNA encoding bone morphogenetic protein-9 (BMP-9). 4) To test whether the delivery of pDNA encoding BMP-2 (pBMP-2) and fibroblast growth factor-2 (pFGF-2) together can synergistically promote bone repair in a leporine model of diabetes mellitus, a condition that is known to be detrimental to union. 5) To investigated whether there is a synergistic effect on bone regeneration following delivery of pBMP-2 and pFGF-2, insulin and/or vitamin D. These investigations together provided new insights regarding the appropriate treatment methods for patients with fractures. Here we develop and test a non-viral gene delivery system for bone regeneration in challenging animal models utilizing a scaffold carrying PEI-nucleic acid complexes. We utilized three kinds of pDNA encoding either BMP-2, BMP-9 or FGF-2 as well as two kinds of cmRNA encoding either BMP-2 or BMP-9 formulated into PEI complexes. The fabricated nanoplexes were assessed for their size, charge, in vitro cytotoxicity, and capacity to transfect human bone marrow stromal cells (BMSCs). The in vivo functional potency of different nanoplexes embedded in scaffolds was evaluated using a calvarial bone defect model in rats, diaphyseal long bone radial defects in a diabetic rabbit model and intramuscular implantation in a diabetic rat. The results indicate that our non-viral gene delivery system induced migration and differentiation of resident cells to enhance bone regeneration. Together these findings suggest that scaffolds loaded with non-viral vectors harboring cmRNA or pDNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

The present work describes the preparation of hyaluronan derivatives and hydrogels with potential use in tissue engineering applications. A potentially injectable hydrogel consisting of hyaluronan and collagen was successfully used to grow neurons in vitro by encapsulation of neural stem and progenitor cells. Attempts were further made to establish a suitable modification strategy which could be used for the preparation of in vivo cross-linkable hyaluronan derivatives. The synthesis of a model substance consisting of a D-glucuronate derivative which could simplify the development of such a modification technique is described, although a new method to prepare hyaluronan derivatives was found without its use. The modification strategy involves the use of a triazine-reagent which enables the covalent attachment of hydrophilic and hydrophobic amines to hyaluronan carboxyl groups in a controlled fashion under mild conditions. Using triazine-activated amidation we synthesized an aldehyde-derivative of hyaluronan which was used to prepare gels by cross-linking with hydrazide-modified polyvinyl-alcohol. Gels were formed in less than 1 minute by mixing equal volumes of the polymer derivatives and they were subsequently used as a carrier for bone morphogenetic protein-2. An in vitro release study showed that approximately 88% of the growth factor is retained in the gel over a 4 week period. The ability to form new bone in vivo was further evaluated in an ectopic rat model by the injection of gels containing 30 µg BMP-2. Radiographic and histological examination 4 and 10 weeks after injection showed the formation of new bone without any signs of inflammation or foreign body response. Hydroxyapatite particles were further added to improve the mechanical properties of the gel, and a comparative study was conducted. This time the induced tissue consisted not only of bone, but also of interconnected cartilage and tendon, as confirmed by histology and immunohistochemistry.

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.

Complexed microspheres of poly(4-vinyl pyridine) (P4VN) and alginic acid were prepared by internal gelation method and subsequent freeze drying. The 4% and 10% microspheres were loaded with Bone Morphogenetic Protein-2 (BMP-2) and Bone Morphogenetic Protein-7 (BMP-7), respectively for in vitro studies and were entrapped in PLGA foams. Foams containing only 4%, BMP-2 microspheres, only 10%, BMP-7 microspheres and both populations were prepared. Control samples of each group were prepared with drug free microspheres. Bone marrow derived stem cells from rat femur and tibia isolated by a surgical operation, were seeded onto foams. Proliferation of cells on foams containing both microsphere populations was higher at all time points regardless of the presence of BMPs. This was attributed to different porosity characteristics. Proliferation was higher at all times in control samples in comparison to their positive samples for all groups, suggesting proliferation attenuation related enhancement in osteogenic activity due to BMP supply. Alkaline phosphatase (ALP) activities were lower at all time points for foams containing both microsphere populations regardless of BMP presence. This was attributed to different physical characteristics of foams confirmed by the inverse correlation between proliferation and osteogenic differentiation. Total and specific ALP activity results demonstrated the significant positive influence of all BMP containing types in enhancing osteogenic differentiation. Best results were obtained with co-administration of sequential delivery performing 4% and 10% microspheres loaded with BMP-2 and BMP-7, respectively.

Osteoarthritis (OA) is a disease characterized by the progressive degradation of articular cartilage. Current strategies for the disease are mainly towards relieving symptoms. This study was aimed to investigate the therapeutic potentials of Bone Morphogenetic Protein-9 (BMP-9), Raloxifene (Ral) and Pluronic F-68 (PLF-68) with a three-dimensional in vitro OA model. Articular chondrocytes isolated from rats were cultured in growth media and embedded in agarose to obtain agarose-chondrocyte discs. Dynamic hydrostatic mechanical stress was applied to discs. The discs were incubated with Aza-C for 48 hours for OA development. After its removal, chondrocytes were treated with different doses of BMP-9, Ral and PLF-68 for 10 days. The efficacies of treatments were evaluated by measuring cell number, glycosaminoglycan and collagen amount, and mechanical properties of the v discs. Measurements of these properties were performed with MTT, quantitative colorimetric assays, histochemical staining and mechanical tests, respectively. According to comparative results with healthy groups and controls (osteoarthritic chondrocytes without any treatment), it was found that BMP-9 had negative effect on osteoarthritic chondrocytes. On the other hand, Ral showed positive results related with matrix synthesis and mechanical properties especially at 5 &amp / #956 / M dose suggesting that it holds promise for the treatment of OA. The therapeutic effect of Ral on OA was documented for the first time in literature. The potential of PLF-68 for treatment of OA was also supported by this study considering its positive effects on cell number, collagen synthesis and mechanical properties. Yet, further investigations are also suggested for conclusive results on this agent.

In the United States alone, there are nearly 49,000 aortic valvular repairs or replacements each year, and this number is expected to rise. Unlike atherosclerosis, the molecular mechanisms contributing to this side-dependent disease development are limited, which contributes to the lack of therapeutic treatments. Once clinically manifested, options for treatment are limited to valvular replacement or repair. Therefore understanding the mechanobiology and cellular responses in aortic valves may provide important information for disease development and possible biomarkers or therapeutic treatments. Aortic valve disease occurs on one side of the valvular leaflet. The fibrosa side, which faces the aorta, is prone to disease development, while the ventricularis remains relatively unaffected. The hemodynamics is hypothesized to play a role in side dependent disease formation. The fibrosa endothelium is exposed to oscillatory flow while the ventricularis endothelium is exposed to a pulsatile unidirectional flow. Previous work by our group has shown that bone morphogenic protein-4 is a mechanosensitve inflammatory cytokine in the vasculature. In the following study, we proposed that mechanosensitive bone morphogenic proteins play a role in side specific aortic valve disease. Recently, the bone morphogenic proteins (BMPs) have been found in calcified human aortic valves. Furthermore, BMP-4 in vascular endothelial cells is increased by oscillatory shear stress. However, the role of the BMPs in aortic valve endothelial cells and their contribution to aortic valve calcification remains unstudied. Therefore, the overall objective of this dissertation was to investigate how disease and hemodynamics affects the BMP pathway and inflammation in human aortic valvular endothelial cells. By understanding how the bone morphogenic proteins are regulated and what roles they play in aortic valve disease, we will have better insight into endothelial cell regulation and contribution in aortic valve pathology. The central hypothesis of this project was that oscillatory flow conditions on the fibrosa side of the aortic valve stimulate endothelial cells to produce BMP-4, which then activates an inflammatory response leading to accumulation of inflammatory cells, calcification, and ultimately valve impairment. This hypothesis was tested through three specific aims using calcified human aortic valves, non-calcified human aortic valves, and side-specific human aortic valve endothelial cells. We first worked to establish the importance of the BMPs in the aortic valvular endothelium by looking at two populations of aortic valves: 1) calcified human aortic valves were obtained from patients undergoing valve replacement, and 2) non-calcified valves were obtained from recipient hearts of patients undergoing heart transplantation. Using immunohistochemical techniques, we examined the BMPs, BMP antagonists, and SMADs. Surprisingly, we identified that the ventricularis endothelium had higher BMP expression in both calcified and non-calcified human aortic valves. Furthermore, no disease-dependent BMP expression was detected. Next, we examined the BMP antagonists and found that there was robust BMP antagonist expression in the ventricularis endothelium and very low expression in the fibrosa endothelium. Finally, to determine if the BMP pathway was activated, we stained for the canonical BMP signaling molecule phosphorylated-SMAD 1/5/8 and found increased staining in the endothelium of calcified human aortic valves. Furthermore, a significant increase in SMAD 1/5/8 phosphorylation was seen in the endothelium of calcified fibrosa when compared to the non-calcified fibrosa. Finally, inhibitory SMAD 6 was significantly increased in the ventricularis endothelium of non-calcified human aortic valves. These findings suggest that preferential activation of BMP pathways, controlled by the balance between the BMPs and their inhibitors, play an important role in side-dependent calcification of human AVs. We next wanted to examine the role of shear stress in BMP regulation, but before doing so, we needed to examine the endothelial response to fluid shear stress to validate the phenotype of our isolated human aortic valve endothelial cells. KLF2 and eNOS expression in vascular endothelial cells has been shown to be increased by laminar flow and to have anti-inflammatory effects by decreasing VCAM-1 levels. Conversely, oscillatory shear stress has been shown to increase NF-kappa B translocation and increase ICAM-1 and E-selectin. We found laminar shear stress causes human aortic valve endothelial cells align parallel to flow and have robust increases of KLF2 and eNOS and decreases in VCAM-1 levels; however, laminar shear-treated cells had similar levels of NF-kappa B activation as oscillatory treated cells while ICAM-1 and E-selectin was not affected by shear stress. In contrast, oscillatory shear had higher levels of monocytes bound which may be due to eNOS's protective effects under laminar shear and robust VCAM-1 expression in oscillatory shear. These studies suggest differential regulation of human aortic valvular endothelial cells than published reports on human aortic endothelial cells which adds to the growing evidence that valvular endothelial cells are phenotypically different than vascular endothelial cells. After verifying the shear response of our endothelial cells, we next determined the shear response of the BMPs and BMP antagonists and described BMPs' effect on inflammation. Previously, BMP-4 has been shown in vitro and in vivo to be increased in endothelial cells exposed to oscillatory flow, while the closely-related BMP-2 has not been shown to be shear sensitive. In this study we have found that BMPs -2 and -4 are shear sensitive while BMP-6 is not. Furthermore, we have found that follistatin is decreased by laminar flow only in the ventricularis, while MGP1 is decreased in the fibrosa valvular endothelial cells under both oscillatory and laminar flow. Finally, incubation with noggin did not affect monocyte adhesion after shear, suggesting differential regulation of inflammation in human aortic valvular endothelial cells. By addressing the specific aims of this project, we have investigate disease- and side-dependent valvular endothelial BMP expression in vivo, shear regulation of valvular endothelial inflammation in vitro, and shear regulation of valvular endothelial BMP expression in vitro. Our results suggest that the BMP pathway is playing a role in side specific aortic valve disease development; however, regulation of the BMPs does not appear to be shear regulated in vivo. Other factors that may be affecting BMP production include including pulsatile pressures, bending stresses, cyclic stretch, and humeral stimuli present in the blood of the patients. However, in vitro we have found BMPs -2 and -4 to be shear-regulated in human aortic valvular endothelial cells. Shear-induced inflammation in human aortic valve endothelial cells seems to be VCAM-1-dependent, and BMP-independent. Finally, by identifying factors that are modulated in calcific- and shear-dependent processes, new targets for the early detection of aortic valve disease can be determined and new therapeutics to slow or stop the progression of aortic valve disease may be discovered.

Η χρόνια νεφρική ανεπάρκεια οφείλεται σε διάφορα αίτια βλάβης του σπειράματος και του διαμεσοσωληναριακού χώρου. Χαρακτηρίζεται ιστολογικά από την παρουσία σοβαρού βαθμού σπειραματικής σκλήρυνσης, ίνωσης του διάμεσου ιστού, ατροφίας των ουροφόρων σωληναρίων και υαλίνωσης των αρτηριδίων. Η αρχική βλάβη του σπειράματος που προκαλείται από διάφορα είδη ερεθισμάτων μπορεί να ακολουθήσει την οδό της αποκατάστασης ή να εξελιχθεί προς σκλήρυνση, διαδικασίες στις οποίες συμμετέχουν κυτταροκίνες και αυξητικοί παράγοντες που προέρχονται από ενδοθηλιακά, μεσαγγειακά, επιθηλιακά σωληναριακά κύτταρα, μονοκύτταρα και ινοβλάστες1 . Ο Transforming Growth Factor-β1 (TGF-β1) είναι ο κυριότερος αυξητικός παράγοντας που μέσω πολλαπλών μηχανισμών συμμετέχει στην ανάπτυξη σκληρυντικών αλλοιώσεων2. Προάγει την παραγωγή συστατικών της εξωκυττάριας θεμέλιας ουσίας και μειώνει την αποικοδόμησή τους, προκαλεί ενεργοποίηση των μυοϊνοβλαστών, δηλαδή κυττάρων με μεταναστευτικές ιδιότητες που παράγουν κολλαγόνο, συμμετέχει στη διαφοροποίηση των επιθηλιακών σωληναριακών κυττάρων προς ινοβλάστες και ευοδώνει την κυτταρική απόπτωση η οποία οδηγεί στην απογύμνωση του νεφρικού ιστού από τα φυσιολογικά του κύτταρα. Η χρήση αντισωμάτων κατά του TGF-β1 και αναστολέων του μετατρεπτικού ενζύμου της αγγειοτενσίνης σε διάφορα πειραματικά μοντέλα νεφρικής βλάβης έχει συμβάλλει στην κατανόηση μηχανισμών που συμμετέχουν στη διαδικασία εξέλιξής της. Σε πειραματικά μοντέλα έχει διαπιστωθεί ότι ο TGF–β ευθύνεται για την αυξημένη παρουσία μυοινοβλαστών, την εναπόθεση κολλαγόνου και την απώλεια του σωληναριακού επιθηλίου. Πιο πρόσφατα, ένα μέλος της υπερ-οικογένειας των ΤGF–β, η ΒΜΡ7, φάνηκε να εξουδετερώνει την ίνωση που προκαλείται μέσω του TGF–β. Οι δραστηριότητα των παραγόντων αυτών ελέγχεται από άλλες πρωτείνες οι οποίες μπορούν να αυξήσουν ή να καταστείλουν τη διέγερση των υποδοχέων των παραγόντων αυτών. Τα BMPs είναι ενδογενή μόρια που προστατεύουν το νεφρό από διάφορα είδη βλάβης όπως γενετικές βλάβες, ανοσολογικές αντιδράσεις, περιβαλλοντικοί παράγοντες, μεταβολικά αίτια και καταστάσεις οξείας ή χρόνιας νεφρικής βλάβης. / -

The present work addresses poor bone/implant integration and severe bone defects. In both conditions external stimuli is required for new bone to form. A multilayered functional implant coating, comprised of an inner layer of crystalline titanium dioxide (TiO2) and an outer layer of hydroxyapatite (HAP), loaded with bone morphogenetic protein-2 (BMP-2), was proposed as a tool for providing both improved initial bone formation and long-term osseointegration. The in vitro characterization of the implant coatings showed that TiO2 and HAP were more favorable for cell viability, cell morphology and initial cell differentiation, compared to native titanium oxide. Furthermore, significantly higher cell differentiation was observed on surfaces with BMP-2, indicating that a simple soaking process can be used for incorporating bioactive molecules. Moreover, the results suggest that there could be a direct interaction between BMP-2 and HAP, which prolongs the retention of the growth factor, improving its therapeutic effect. For treating severe bone defects a strategy involving BMP-2 delivery from hyaluronan hydrogels was explored. The hydrogels were prepared from two reactive polymers – an aldehyde-modified hyaluronan and a hydrazide-modified poly(vinyl alcohol). Upon mixing, the two components formed a chemically crosslinked hydrogel. In this work the mixing of the hydrogel components was optimized by rheological measurements. Furthermore, an appropriate buffer was selected for in vitro experiments by studying the swelling of hydrogels in PBS and in cell culture medium. A detection method, based on radioactive labeling of BMP-2 with 125I was used to monitor growth factor release both in vitro and in vivo. The results showed a biphasic release profile of BMP-2, where approximately 16 %  and 3 % of the growth factor remained inside the hydrogel after 4 weeks in vitro and in vivo, respectively. The initial fast release phase corresponded to the early ectopic bone formation observed 8 d after injection of the hydrogel formulation in the thigh muscle of rats. The hydrogel formulation could be improved by incorporation of HAP powder into the hydrogel formulation. Furthermore, bone formation could be increased by pre-incubation of the premixed hydrogel components inside the syringe prior to injection. Crushed hydrogels were also observed to induce more bone formation compared to solid hydrogels, when implanted subcutaneously in rats. This was thought to be due to increased surface area of the hydrogel, which allowed for improved cell infiltration.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.