Mathematical modeling of cancer is of significant interest due to its potential to aid in our understanding of the disease, including investigation into which factors are most important in the progression of cancer. With this knowledge and model different paths of treatment can be examined; (e.g. simulation of different treatment techniques followed by the more costly venture of testing on animal models). Significant work has been done in the field of cancer modeling with models ranging from the more broad systems, avascular tumor models, to smaller systems, models of angiogenic pathways. A preliminary model of a vascularized tumor has been developed; the model is based on fundamental principles of mechanics and will serve as the framework for a more detailed model in the future. The current model is a system of nonlinear partial differential equations (PDEs) separated into two basic sub-models, avascular and angiogenesis. The avascular sub-model is primarily based of Fickian diffusion of nutrients into the tumor. While the angiogenesis sub-model is based on the diffusion and chemotaxis of active sprout tips into the tumor. These two portions of the models allow the effects of microvessels on nutrient concentration within the tumor, as well as the effect of the tumor in driving angiogenesis, to be examined. The results of the model have been compared to experimental measurements of tumor growth over time in animal models, and have been found to be in good agreement with a correlation coefficient of (r2=0.98). / Master of Science

Peptide amphiphiles (PAs), peptides that self-assemble into hydrogels with a nanofibrous network, are interesting biomaterials due to their biocompatibility and biodegradability. Self-assembling peptide-based materials include a wide range of peptide motifs that form one-dimensional nanostructures in aqueous solution. Two different PAs are considered in this M.S. thesis work: lipidated peptides, and gas-releasing peptides (GRPs). These biomaterials have been developed to function as potential therapeutics that promote the growth of new blood vessels. The analyses conducted on the lipidated peptides, which were designed to include a peptide sequence that promotes angiogenesis, include cytotoxicity, viability, and tube formation assays. The GRPs were designed to release H2S, which is also capable of promoting angiogenesis. Several characteristic properties of the GRPs were analyzed, including morphology, mechanics, self-assembly, and gas release rates. Furthermore, cytotoxicity assays were conducted followed by the demonstration of gas uptake in endothelial cells. / Master of Science

The structural and functional abnormalities of the tumor vasculature generate regions of elevated interstitial fluid pressure and aberrant flow shear stress within the tumor microenvironment. While research has shown that the hydrodynamics of the tumor vasculature reduce transport and uptake of therapeutic agents, the underlying mechanisms by which fluid forces regulate vascular organization are not well known. Understanding the reciprocal interaction between tumor and endothelial cells to mediate angiogenesis, and the role of flow shear stress on this process, may offer insight into the design of improved therapeutic strategies to control vascularized tumors. Instrumental to this is the development of physiologically relevant models that enable tumor-endothelial co-culture under dynamic conditions. By integrating tissue-engineering strategies with cancer biology, micro-scale fluid mechanics, and optical flow diagnostics, the goal of this research was to develop a 3D in vitro microfluidic culture model to investigate tumor-endothelial cross talk under physiologically relevant flow shear stress. This objective was motivated by early findings demonstrating a contact-independent, paracrine-mediated mechanism by which endothelial cells enhance tumor-expressed angiogenic factors during 2D, static co-culture. The 3D tumor vascular model consists of a central microchannel embedded within a type I collagen hydrogel, through which a range of normal (4 dyn/cm^2), low (1 dyn/cm^2) and high (10 dyn/cm^2) microvascular wall shear stresses (WSS) were introduced. Endothelial cells lining the microchannel lumen form a confluent endothelium across which soluble growth factors are exchanged with tumor cells in the gel. Microscopic particle image velocimetry ("-PIV) was integrated within the model to enable noninvasive optical measurement of velocity profiles and quantification of WSS, which were then correlated with angiogenic potential. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, high WSS (10 dyn/cm^2) significantly down-regulates tumor-expressed angiogenic factors, suggesting flow shear stress-mediates endothelial cross talk with surrounding tumor cells. Collectively, this research demonstrates the utility of the 3D in vitro microfluidic culture model as a versatile platform for elucidating the role of tumor-relevant hydrodynamic stress on cellular response. / Ph. D.

No / Background: In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2. Experimental Design: Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity. Results: Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing 16 saccharide residues were investigated. Conclusions: Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

Yes / Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Inhibition of angiogenesis is an effective and low toxic therapeutic avenue for the treatment of cancer patients in addition to traditional interventions. Majority of current available angiogenesis inhibitors for cancer therapies are growth factor inhibitors and small molecule tyrosine kinase inhibitors. A number of endogenous proteins and/or proteolytic fragments of extracellular matrix proteins are shown to have the activity of inhibition of angiogenesis by directly targeting endothelial cells. Structural analyses have indicated that a common structure of anti-parallel β-sheet with a highly positively charged surface presents in many of those inhibitors. This common structural feature is critical for the maintenance of their anti-angiogenic function. With this structural information, we have designed and developed a new class of anti-angiogenic proteins by integrating the short anti-parallel β-sheet forming sequences of endogenous anti-angiogenic proteins into a stable host protein, the extracellular domain-1 of cluster of differentiation 2 molecule (CD2D1). 1D 1H NMR spectra analyses indicated that the designed anti-angiogenic protein (ref to as ProAgio) folded as a β-sheet structure similar to that of the parental protein, CD2D1. ProAgio inhibited the growth of human umbilical vein cells (HUVECs) without affecting the growth of epithelial cells, suggesting a specific effect to endothelial cells. ProAgio effectively reduced endothelial tubules formed by the co-culture of HUVECs and PC3 cells on matrix gel in vitro. The designed anti-angiogenic protein was further site-specifically PEGylated in order to improve PK/PD properties and reduce immunogenicity. Examinations with PC3 xenografts showed that both ProAgio and the PEGylated ProAgio dramatically inhibited tumor growth. Immunofluorescence staining analyses of the endothelial marker CD31 indicated dramatic decreases in tumor vessels in lengths and branching points. Histological and immunofluorescence staining analyses of tissue slices of major organs indicated that there were no pathological damages to the tissue structure or disruption of normal vessels associated with the treatment of our designed anti-angiogenic agent. Overall, our studies developed a novel anti-angiogenesis agent that may have great clinical potentials. Our concept of protein design can be extended to the development of other novel protein drugs.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 95 pages. Includes Vita. Includes bibliographical references.

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<p>The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its activity as a kinase. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1-mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.</p> / Dissertation

Angiogenesis is the growth of new blood vessels from existing vasculature; it requires the breakdown of existing blood vessel walls followed by the migration and proliferation of endothelial cells to form the new vessels. It is a complex process that is regulated by many pro- and anti-angiogenic factors and the roles of some of these factors are still unclear. Angiogenesis is a key feature of many pathological conditions including cancer, polycystic ovary syndrome and endometriosis so is an area of great research interest. There are several methods currently available for the study of angiogenesis, both in vitro and in vivo, and whilst all of these methods have enhanced understanding of angiogenesis, they also have limitations. The ovary is an excellent model for the study of angiogenesis as it undergoes intense vascular morphogenesis in a cyclical manner. The female reproductive system is unique as no other healthy adult tissue undergoes spontaneous angiogenesis. The tissues in the ovary undergo constant remodelling during both folliculogenesis and the formation and regression of the corpus luteum. Blood vessels are recruited from the ovarian stroma at the preantral stage to form vascular sheaths, in the thecal layer, which surround the developing follicle and supply nutrients, hormones and allow gaseous exchange. As follicular development progresses to the antral stage, when gonadotrophin-dependence is established, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Previous research into ovarian angiogenesis has focussed on the corpus luteum but the mechanisms of the regulation of angiogenesis during folliculogenesis need further elucidation. The work in this thesis aims to develop and utilise an in vitro angiogenesis assay using the culture of intact preantral and early antral follicles to provide a new approach to the study of follicular angiogenesis. During the course of this thesis this assay was utilised to investigate the effect of various factors on follicular angiogenesis and ovarian function. The role of the putative anti-angiogenic factor thrombospondin-1 (TSP-1) in the regulation of physiological angiogenesis was investigated using the in vitro angiogenesis assay developed during the course of this thesis and the role of TSP-1 in normal ovarian function was investigated using the culture of isolated granulosa cells. The results suggest that TSP-1 is able to inhibit angiogenesis and that it has an extravascular role in the ovary, in vitro. These findings were extended to an in vivo angiogenesis model where follicular angiogenesis was assessed by quantitative immunohistochemistry for bromodeoxyuridine and the endothelial cell marker CD31. The extravascular role for TSP-1 was also further investigated in vivo and was assessed by quantitative immunohistochemistry for activated caspase-3. The results confirmed the findings of the in vitro study, indicating that TSP-1 has antiangiogenic action and acts to clear non-dominant follicles from the ovary through the induction of atresia. Vascular endothelial growth factor (VEGF) is the main factor involved in stimulating angiogenesis and many advances have been made into elucidating the role, and the mechanisms of action, of VEGF on angiogenesis. Angiopoietin-1 (Ang-1) is considered to be one of the main factors acting in concert with VEGF to stabilise new blood vessels and its role in angiogenesis has been the subject of much discussion and controversy. This thesis investigates the effects of Ang-1 on follicular angiogenesis and development, using the in vitro angiogenesis assay, granulosa cell culture and RNA knockdown experiments. The results have shown that Ang-1 can induce follicular angiogenesis at high doses and that at low doses stimulates prosurvival pathways and inhibits apoptotic mediators. This thesis describes a novel in vitro culture system for the study of angiogenesis in ovarian follicles. Using this system the effects of various factors on follicular angiogenesis and on follicle development and survival have been investigated. Investigations into the mechanisms of action of these factors have also been performed. These studies have improved understanding of the regulation of follicular angiogenesis and have indicated extravascular roles for angiogenic factors in the ovary. Since angiogenesis is a key feature of many pathological conditions, the ability to manipulate angiogenesis and to investigate and quantify the effects of proor anti-angiogenic compounds may have important clinical implications.