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Physiologically relevant scaffold-based tissue engineered structures have been limited in scope and viability by the diffusion limits of oxygen and other nutrients and functions provided by native vasculature in vivo. This has prevented the maintenance of healthy cell populations in scaffolds that are more than 200痠 thick. Combining concepts from microfluidics with biomaterials engineering, this project set out to engineer a perfusable fibrin-based vascular network capable of physiologically relevant flow properties as well as diffusion that supports viable cell populations. To create this system, a small artery sized (1.5 mm wide) gelatin sacrificial structure was embedded inside of a block of robust fibrin gel (4.26% w/v fibrin) then melted and rinsed out to create a perfusable vascular network. Characterization consisted of morphometric and histological analyses for channel sizes compared to the sacrificial structures, particle tracking to observe flow properties, and fluorescent dextran diffusion to measure diffusivity into the fibrin scaffold. We found that channels derived from sacrificial structures maintain their size and shape inside of the gel. Flow properties of the fluid through the channels were found to be both laminar and within expected physiological rates compared to native vessels of similar sizes. Cells on the surface of the fibrin vascular device expressed fluorescent markers that were delivered through the vascular network and perfused through the fibrin scaffold. These findings suggest that a fibrin based vascular system may provide a platform creating a functional vascular layer and for developing tissue engineered systems of increased size and complexity.

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is important in post-injury scar formation but its role in scar-free regeneration remains relatively unexplored. This study investigates vascular regeneration during tail regeneration in the leopard gecko (Eublepharis macularius). It is hypothesized that blood vessel regeneration follows a conserved sequence of events similar to physiological angiogenesis. To test this hypothesis the onset and pattern of expression of common vascular and angiogenic proteins (von Willebrand factor, α-smooth muscle actin, vascular endothelial growth factor, thrombospondin-1 and cluster differentiation 36) was investigated. The effect of the anti-angiogenic peptide ABT-510 on tail regeneration was also explored by documenting changes in vascular morphology and histology of regenerate tails. Results show that the proteins of interest are expressed in a conserved sequence consistent with physiological angiogenesis. ABT-510 did not consistently prevent tail regeneration, but did have some small-scale effects. These results provide the basis for further investigations into the importance of angiogenesis during multi-tissue regeneration.

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The development of gene therapy for clinical use continues to face many hurdles. A major issue is the limitation of gene delivery technology. This body of work describes strategies for improving the selectivity and efficacy of gene delivery to vascular endothelium, with emphasis on delivery to pulmonary vasculature in vivo. Several important principles were established which continue to be of relevance to the field. The work progresses from vector development through to the use of new vector strategies in the application of novel gene delivery approaches in disease models. Work in gene therapy and vector development began in the Division of Human Gene Therapy, University of Alabama at Birmingham, under the mentorship of Prof David T Curiel and has continued through international collaborations and the establishment of my own laboratory in the Hanson Institute with affiliate links to the University of Adelaide. The work presented in this thesis consists entirely of published material, either as book chapters (three) or peer reviewed journal articles (twelve). The sequence of material progresses from a broad introduction to the field on Gene Therapy, more specific chapters dealing with pulmonary gene delivery including a detailed methodology chapter. The peer reviewed works contain an evolution of work dealing with the development of strategies to target adenoviral gene delivery vectors to the pulmonary vascular endothelium. This work encompasses the use of bi-specific conjugates, genetic modification of viral capsid (outer coat) proteins and the use of cell-specific promoters. The work progresses to a demonstration of the therapeutic gains achieved with the use of targeted over non-targeted vectors in animal models and culminates with a highly novel application of modulation of the bone morphogenetic protein pathway in pulmonary hypertension. A component of the work focuses on enhanced gene delivery to vein grafts exVIVO. There are many key original contributions encompassed within the work, including 1) first use of conjugate-based retargeting to vascular cells, 2) first demonstration that tropism modification could alter in vivo biodistribution of virus, 3) first demonstration of cell-specific retargeting of adenoviral vector after systemic vascular injection in vivo (a technique still unsurpassed in the field), 4) first demonstration of the in vivo selectivity gains achieved by combined cell-specific promoters with viral retargeting, 5) first demonstration of therapeutic gains achieved by targeting in a vascular context and 6) first demonstration that modulation of the BMPR2 pathway can have a therapeutic impact in pulmonary hypertension. Importantly, the targeting work I have developed has been adapted and used by others and laid a foundation for further vector improvements. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1349837 / Thesis (M.D.) -- University of Adelaide, School of Medicine, 2009

Title page, contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The development of gene therapy for clinical use continues to face many hurdles. A major issue is the limitation of gene delivery technology. This body of work describes strategies for improving the selectivity and efficacy of gene delivery to vascular endothelium, with emphasis on delivery to pulmonary vasculature in vivo. Several important principles were established which continue to be of relevance to the field. The work progresses from vector development through to the use of new vector strategies in the application of novel gene delivery approaches in disease models. Work in gene therapy and vector development began in the Division of Human Gene Therapy, University of Alabama at Birmingham, under the mentorship of Prof David T Curiel and has continued through international collaborations and the establishment of my own laboratory in the Hanson Institute with affiliate links to the University of Adelaide. The work presented in this thesis consists entirely of published material, either as book chapters (three) or peer reviewed journal articles (twelve). The sequence of material progresses from a broad introduction to the field on Gene Therapy, more specific chapters dealing with pulmonary gene delivery including a detailed methodology chapter. The peer reviewed works contain an evolution of work dealing with the development of strategies to target adenoviral gene delivery vectors to the pulmonary vascular endothelium. This work encompasses the use of bi-specific conjugates, genetic modification of viral capsid (outer coat) proteins and the use of cell-specific promoters. The work progresses to a demonstration of the therapeutic gains achieved with the use of targeted over non-targeted vectors in animal models and culminates with a highly novel application of modulation of the bone morphogenetic protein pathway in pulmonary hypertension. A component of the work focuses on enhanced gene delivery to vein grafts exVIVO. There are many key original contributions encompassed within the work, including 1) first use of conjugate-based retargeting to vascular cells, 2) first demonstration that tropism modification could alter in vivo biodistribution of virus, 3) first demonstration of cell-specific retargeting of adenoviral vector after systemic vascular injection in vivo (a technique still unsurpassed in the field), 4) first demonstration of the in vivo selectivity gains achieved by combined cell-specific promoters with viral retargeting, 5) first demonstration of therapeutic gains achieved by targeting in a vascular context and 6) first demonstration that modulation of the BMPR2 pathway can have a therapeutic impact in pulmonary hypertension. Importantly, the targeting work I have developed has been adapted and used by others and laid a foundation for further vector improvements. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1349837 / Thesis (M.D.) -- University of Adelaide, School of Medicine, 2009

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No / Endothelln-converting enzyme (ECE-1) is a critical enzyme in the production of the potent vasoconstrictor peptide endothelin (ET-1). It has previously been shown that the levels of both ET-1 and ECE-1 are raised in atherosclerosis, but the possible relevance of the isoforms of ECE-1 in these changes has not yet been investigated. The aim of this study was to examine the expression of the ECE-1a and ECE-1c isoforms in human atherosclerotic pathologies. Immunohistochemical analysis was carried out on sections from atherosclerotic and non-atherosclerotic vascular tissue using a combination of ECE-1 isoform-specific antibodies, anti-¿-actin antibodies to identify smooth muscle cells (SMC) and anti-CD68 antibodies to identify macrophages. ECE-1 isoform expression was also examined in cultured SMC and in macrophages isolated from human blood. Results indicated differences in isoform expression in atherosclerotic lesions, with distinct patterns of staining for ECE-1 a and ECE-1 c. ECE-1 c immunoreactivity was seen in macrophages, and also correlated with actin staining. ECE-1a was also localized to macrophages and SMC. Results of this study suggest that these local changes influence the expression patterns of the ECE-1 isoforms within individual cell types. Correlation of these isoform expression patterns with the stage of atherosclerosis could provide novel indicators of disease progression.

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