A treatment strategy for cancer is the suppression of tumor growth by directing an immune response to the tumor vessels, which will destroy the tissue. In this thesis we describe the development of a vaccine that targets antigens expressed around angiogenic vasculature in most solid tumors. These antigens are alternative spliced extra domains of glycoproteins present in the extracellular matrix; e.g. the extra domain-B (ED-B) and extra domain-A (ED-A) of fibronectin and the C-domain of tenascin-C (TNCC). We show that it is possible to break self-tolerance and induce a strong antibody response against ED-B by vaccination. Furthermore, tumor growth was inhibited and the changes observed in the tumor tissue were consistent with an attack of the tumor vasculature by the immune system. For clinical development of therapeutic vaccines, targeting self-molecules like ED-B, a potent but non-toxic biodegradable adjuvant is required. The squalene-based Montanide ISA 720 (M720) in combination with CpG DNA fulfilled these requirements and induced an equally strong anti-self immune response as the preclinical golden standard Freund’s adjuvant. We have further characterized the immune response against ED-B generated with the adjuvant M720/GpG.  The ED-B vaccine also inhibited tumor growth in a therapeutic setting in a transgenic mouse model of pancreatic insulinoma in which tumorigenesis was already initiated. Furthermore, antibodies against ED-A and TNCC could be induced in mice and rabbits. We analyzed the expression of ED-A in breast tumors of transgenic MMTV-PyMT mice, a metastatic breast cancer model, with the aim to use this model to study the effect of an ED-A vaccine on metastasis. We also detected ED-B in canine mammary tumor tissue. Therefore vascular antigens might also represent potential therapeutic targets in dogs.  All together our preclinical data demonstrate that a vaccine targeting tumor blood vessels is a promising new approach for cancer treatment.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

Abstract The surface of multicellular organisms is covered with epithelial cells that provide a barrier to the external environment. As part of this barrier function, most epithelia produce apical extracellular matrices (aECMs). The generation of such chemical and physical barriers requires specialized deposition of macromolecules and is likely to involve a spatial and temporal coordination of biochemical activities at the apical surface. A challenging task is thus to characterize key proteins that underlie apical cell surface organization and correct aECM assembly. The Drosophila trachea provides an excellent system to study aECM formation, as they produce an ordered aECM, called the cuticle. The tracheal cuticle is unique by its presence of cuticular ridges, called taenidial folds, which prevent collapse of tracheal tubes while allowing them to expand and contract along their length. A gene called filzig encodes a transmembrane serine protease and is required for taenidial organization. The aim of this research was to further understand Filzig function through characterization of filzig mutants and Filzig protein expression. The results showed that Filzig is expressed in cuticle-producing epithelia as cuticle deposition begins. Moreover, Flz localized to the apical epithelial surface, as well as to the aECM. The apical Flz localization does not reflect the pattern of cuticle ridges, indicating that Flz-localization is not a determinant for taneidial patterning. Instead, Flz might act on extracellular targets that localize to the future taneidial folds. Alternatively, Filzig is involved in a cascade of self-organizing activity of cuticular components to form the regular taenidial folds.

Modular tissue engineering is a novel approach to creating scalable, self-assembling three-dimensional tissue constructs with inherent vascularisation. Under initial methods, the subcutaneous implantation of human umbilical vein endothelial cell (HUVEC)-covered collagen modules in immunocompromised mice resulted in significant host inflammation and limited HUVEC survival. Subsequently, a minimally-invasive injection technique was developed to minimize surgery-related inflammation, and cell death was attributed to extensive apoptosis within 72 hours of implantation. In confirmation of in vitro results, coating collagen modules with fibronectin (Fn) was shown in vivo to reduce short-term HUVEC apoptosis by nearly 40%, while increasing long-term HUVEC survival by 30% to 45%. Consequently, a 100% increase in the number of HUVEC-lined vessels was observed with Fn-coated modules, as compared to collagen-only modules, at 7 and 14 days post-implantation. Furthermore, vessels appeared to be perfused with host erythrocytes by day 7, and vessel maturation and stabilization was evident by day 14.

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.

Modular tissue engineering is a novel approach to creating scalable, self-assembling three-dimensional tissue constructs with inherent vascularisation. Under initial methods, the subcutaneous implantation of human umbilical vein endothelial cell (HUVEC)-covered collagen modules in immunocompromised mice resulted in significant host inflammation and limited HUVEC survival. Subsequently, a minimally-invasive injection technique was developed to minimize surgery-related inflammation, and cell death was attributed to extensive apoptosis within 72 hours of implantation. In confirmation of in vitro results, coating collagen modules with fibronectin (Fn) was shown in vivo to reduce short-term HUVEC apoptosis by nearly 40%, while increasing long-term HUVEC survival by 30% to 45%. Consequently, a 100% increase in the number of HUVEC-lined vessels was observed with Fn-coated modules, as compared to collagen-only modules, at 7 and 14 days post-implantation. Furthermore, vessels appeared to be perfused with host erythrocytes by day 7, and vessel maturation and stabilization was evident by day 14.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.

The conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.

Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.