Microbubble ultrasound (US) contrast agents have been used to monitor the progression of anti-angiogenic chemotherapies. However, US backscatter measurements used in contrast imaging are inherently variable, given the presence of many microbubbles of random position and size. A model was developed to investigate the influence of US scanner and microbubble characteristics on these variable measurements. The Coefficient of Variation was used to measure variability. It was found that an optimum excitation frequency exists that minimizes this variability. In the case of DefinityTM, a 2.25 MHz centre-frequency pulse yielded a less variable measurement than at 5 MHz. Conversely, decreasing microbubbble concentration was found to significantly increase variability. Evidence suggests that microbubbles are no longer Rayleigh scatterers at sufficient low concentrations. Post-processing was found to aid in reducing measurement variability by averaging samples where microbubble positions are uncorrelated. As well, reduction can be achieved by averaging about a region-of-interest of uniform perfusion.

Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.

Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.

Bartonella, a group of Gram negative facultative intracellular bacteria, are known to cause diseases, such as Cat Scratch Disease, Trench Fever and Carrion’s Disease, that involve angiogenesis during the infective cycle. B. bacilliformis, the etiological agent of Carrion’s Disease, causes a bi-phasic infection resulting in the formation of blood-filled angiogenic proliferative cutaneous nodules called verruga peruana. The work presented here was undertaken to characterize the mechanism by which these nodules are produced. Previous work in our laboratory suggested that the Bartonella henselae genome contains a homologue to the virB operon, a set of genes coding for a Type IV Secretion System (TFSS) that has been implicated in the pathogenesis of other α-2-proteobacteria. We identified virB operons in two additional Bartonella pathogens, B. quintana and B. clarridgeiae. No corresponding operon sequences were detected in B. bacilliformis DNA, however. This finding suggests that virB gene products are not required for verruga peruana formation. To continue our search for factors involved in B. bacilliformis-induced angiogenesis, we conducted a microarray analysis of differential gene expression in infected and uninfected endothelial cells. The results suggest similarities between later stage (36 hours) B. bacilliformis infection and that of HHV-8, the causative agent of Kaposi’s Sarcoma, particularly in relation to the host immune response. Finally, our research focused on the secreted factors that B. bacilliformis produces during its host infective cycle. Our data suggest that the B. bacilliformis homologue to the molecular chaperone GroEL not only induces angiogenesis in endothelial cells, but also protects endothelial cell tubule from the degradation seen when these cells are in the presence of live B. bacilliformis. In summary, the induction of verruga peruana nodules via B. bacilliformis may be the result of multiple factors over the course of persistent infection. Early infection may cause vascular damage, which induces VEGF and hypoxia factors. As infection persists, bacterial secretion of a unique GroEL may result in continued angiogenesis and the ensuing activation of immune cells, producing a localized environment of continual incomplete angiogenesis in areas of cutaneous infection.

Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

The mechanisms behind many important aspects of blood- and lymphatic vessel formation have yet not been elucidated in detail. The primary objectives of this thesis have therefore been to study the effects of hypoxia, platelet-derived growth factor (PDGF) and vascular endothelial growth factors (VEGFs) on vascular development and function. In conditions of low oxygen pressure, hypoxia, the survival of the organism is critically dependent on the ability to compensate for the reduced oxygen levels by promoting blood vessel growth and oxygen-independent energy production. Many direct effects of hypoxia in cells are attributed to the induction of a family of hypoxia-inducible transcription factors (HIFs) which control the expression of specific target genes. We found that capillary endothelial cells (ECs) respond to hypoxia with upregulation of genes involved in growth and remodeling of blood vessels. On the other hand, vein ECs responded to hypoxia with increased expression of genes involved in lymphatic vessel growth. Using differentiating embryonic stem (ES) cells, we have shown that hypoxia upregulates expression of VEGF receptor-3 (VEGFR-3) on blood vascular ECs. Furthermore, we have provided evidence for a critical role of VEGFR-3 in hypoxia-induced blood vessel development. Activation of PDGF receptor-β (PDGFR-β) on early vascular progenitors in differentiating ES cells or in mice induces blood vessel differentiation, while negatively influencing early hematopoiesis. PDGFR-β expression on vascular progenitors may therefore play a role in guiding differentiation of the vascular lineages. We have investigated the usefulness of differentiating ES cells as a model to study early lymphatic development. Administration of VEGF-C and VEGF-A induced formation of lymphatic vessel-like structures that seemed connected to the blood vasculature, supporting the general view that lymphatic ECs are derived from blood vascular ECs. In summary, this thesis has provided new insights in the contribution of different growth factors in hematopoietic, blood- and lymphendothelial development.

Angiogenesis, the growth of new blood vessels, is a complex process involving several cell types and molecular signals. Excessive vascular growth is a problem in tumors, and insufficient vascularization hampers the function of transplanted insulin-producing pancreatic islets. Understanding the mechanisms behind blood vessel growth generates increased means to control angiogenesis. In this thesis a model of pancreatic islet transplantation to muscle has been used to study the involvement of leukocytes in the development of new vasculature. Transplantation of isolated islets of Langerhans into mouse muscle promoted revascularization of the grafts to a level comparable to native islets in the pancreas. The complete and functional vascular restoration resulted in improved blood glucose control compared to the clinical standard implantation site, the liver. This proved muscle as a transplantation site to be a clinically relevant option for the treatment of type 1 diabetes. The rapid islet revascularization process was found to be dependent on a distinct subset of neutrophils characterized by high expression of the chemokine receptor CXCR4 and the enzyme matrix metalloproteinase 9 (MMP-9). These cells were recruited to recently transplanted and hypoxic grafts by islet-secreted vascular endothelial growth factor A (VEGF-A). Leukocyte migration and interactions in the engraftment area were monitored using a high-speed confocal microscope followed by software tracking. New software was developed to visualize migration statistics. This tool revealed areas around the islet graft where neutrophil gathering coincided with sites of angiogenesis. Macrophages in the engraftment area positioned themselves close to the newly formed vasculature and were shown to have a stabilizing effect on the vessels. When macrophages were removed, no pericytes were recruited to the forming vasculature. The perivascular macrophages also began to express a pericyte marker when in the graft, suggesting a close relationship between these cell types or macrophage plasticity. In conclusion, this thesis presents muscle as a proangiogenic transplantation site for pancreatic islets for the treatment of type 1 diabetes, where the revascularization of the grafts was dependent on the recruitment and actions of specialized immune cells.

Evidence from an abundant number of studies suggests that human female reproductive functions have become impaired over the past half century and that there might be a relationship between endocrine disrupting chemicals (EDCs) and reduced fertility. It is, however, not known by what mechanisms EDCs affect different reproductive functions such as endometrial receptivity, embryo implantation and placentation. The endometrium is continuously changing its morphological and functional properties, responding to cyclic changes of oestrogen and progesterone levels during the menstrual cycle. These changes include monthly preparation for embryo implantation through changed endometrial angiogenic activity and consequent changes in endometrial vasculature. Use of primary human endometrial endothelial cells (HEECs) in this work was evaluated as a possible screening tool for effects caused by EDCs on human endometrial vasculature and subsequently on various endometrial functions. In this study HEEC and endometrial stromal cells were isolated. HEECs were grown in monocultures, and together with stromal cells in co-cultures, and exposed to endocrine active substances. These were cadmium, which has oestrogenic properties, tamoxifen, with anti-oestrogenic effects, mifepristone, which is an anti-progestin, and bisphenol A, with oestrogenic properties. The effects were evaluated by using proliferation and viability assays, migration and tube formation assays, quantitative PCR (qPCR), immunohistochemistry and western blot. Cadmium affected the expression of angiogenesis-related genes, and caused different effects in HEECs cultured alone vs. HEECs co-cultured with stromal cells. Tamoxifen altered the expression of angiogenesis-related genes and reduced HEEC migration, thus having an anti-angiogenic effect. Mifepristone caused reduced formation of tubular structures in tube-formation assays involving HEECs co-cultured with stromal cells. Bisphenol A promoted tube formation in co-cultured HEECs which was related to changes in the expression of several angiogenesis-related genes as well as up-regulated expression of VEGF-D protein. In conclusion, we showed that EDCs have the ability to induce changes in endometrial angiogenic activity in vitro and may thus disturb normal endometrial functions related to fertility and pregnancy. HEECs grown in vitro may provide valuable information on the effects of EDCs on human endometrial functions. However, this model is not suitable as a large-scale screening tool.