Argument: Hypertension is a main contributing risk factor to many cardiovascular diseases and may be the cause or the result of cardiovascular dysfunction. Black Africans, especially, suffer from hypertension because of lifestyle changes that occur during westernisation, which may lead to sympatho-adrenal hyperactivity. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are regulators of angiogenesis and are significantly up regulated during states of vascular dysfunction. Levels of angiogenic factors are unknown for African people and may not be the same as levels thus far reported for Caucasians. Aims: The aim of this study is firstly, to determine whether differences exist regarding the levels of VEGF-A and Ang-2 in urbanised compared to rural black Africans and secondly, to determine whether increased levels of VEGF-A and Ang-2 factors are related to hypertension in black Africans. Methodology: This is a sub study that is based upon the Prospective Urban and Rural Epidemiological (PURE) study. Apparently healthy, fasting African men and women (N=272, aged 35 to 50 years) from the North-West province of South Africa were selected by a medical doctor to participate in this study. Groups were stratified according to gender and urbanisation status based upon information derived from sociodemographic questionnaires. Cardiovascular parameters (Omron HEM-757), pulse wave velocity (PWV) (Compiler SP), plasma angiogenic factor levels (ELISA) and anthropometric measures were determined. An independent t-test and Pearson Chi-square test were used to compare urban and rural data, followed by an analysis of covariance (ANCOVA) while correcting for confounders (age, body mass index, physical activity and tobacco usage). ANCOVAs (corrected for confounders) were applied where hypertensive and normotensive groups were compared within the whole group and urbanised groups. Correlations, correcting for confounders, between cardiovascular variables and angiogenic factors were determined within the whole group and urbanised groups. Results and conclusion: Plasma VEGF-A values for all black Africans were very low while the ANG-2 levels were elevated compared to control values for Caucasians (normotensive and hypertensive) in literature. Urbanised men were more overweight and indicated a higher incidence of hypertension (42.47%) and elevated VEGF-A levels, but lower Ang-2 levels compared to rural men. Urbanised women were generally overweight, physically less active and smoked less, but indicated higher diastolic blood pressure (BP), VEGF-A levels and lower PWV compared with their rural counterparts. Ang-2 levels indicate a negative relationship to diastolic BP data in rural women. No relationships between hypertensive individuals and high angiogenic factor levels were uncovered. Conclusive evidence suggested that angiogenic factor levels were affected more by urbanisation than by the state of hypertension. If low levels of VEGF-2 occur, ANG-2 stimulation and properties may be altered, thereby switching ANG-2 from an anti-angiogenic to a pro-angiogenic molecule, inferring blood vessel destabilisation and vascular dysfunction, such as is observed in hypertensive urbanised men. Higher ANG-2 levels may result in Tie-2 receptor down regulation, hence causing VEGF-A levels to be lower. Further study is needed to ascertain this mechanism since Tie-2 receptor activity was not determined in this study. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2009.

Treatment of cardiac diseases involves transplantation of donor hearts, since the damaged heart has limited self-regeneration potential. An alternative treatment option has emerged as engineered cardiac tissues, grown in vitro by cultivation of cardiac cells on biomaterials, have comparable properties to native myocardium and can be implanted for cardiac repair. Major current limitations are a viable cell source and adequate vascularization to support cell survival. In this thesis, two proangiogenic biomaterials, a scaffold and a hydrogel, were developed to achieve vascularization in vitro and in vivo for cardiac repair. Scaffold patches are suitable for repairing congestive heart failure or congenital malformations, while injectable biomaterials allow minimally-invasive treatment post-myocardial infarction (MI). In the first aim, a collagen scaffold with covalently immobilized vascular endothelial growth factor (VEGF) was developed, and improved cell mobilization, survival and proliferation when used for free wall repair in adult rats. This increased angiogenesis, which aided in retaining the biomaterial size to allow tissue growth. In the second aim, a collagen-chitosan hydrogel with encapsulated thymosin β4 (Tβ4) was developed to 1) recruit cells from the heart epicardium for repair post-MI in vivo, and 2) guide capillary outgrowths from arteries and veins to form oriented capillary structure for in vitro cardiac tissue engineering. Results showed that the encapsulation of Tβ4 into collagen-chitosan hydrogels led to cell outgrowths from rat or mouse cardiac explants in vitro. A portion of the recruited cells were CD31-positive endothelial cells (ECs) that formed tubes. The hydrogel was injected in vivo to increase vascularization and number of cardiomyocytes within the infarct area post-MI, which improved left ventricular wall thickness. Tβ4-hydrogel also promoted the outgrowth of capillaries from vascular explants that followed the direction of the hydrogel-coated grooves of a micropatterned polydimethylsiloxane (PDMS) substrate. These capillary outgrowths eventually formed a vascular bed for engineering vascularized cardiac tissues. This thesis presents two bioinstructive biomaterials with sustained and localized delivery of angiogenic molecules to be used for in situ cardiac repair based on improved vascularization. The use of cell-free bioactive materials overcomes limitations of cell isolation and expansion as required for cell therapies or implantation of engineered tissues.

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

This manuscript presents an investigation and application of the medical radiographic technique of Dynamic Contrast-enhanced Computed Tomography with an emphasis on its application to the measurement of tissue perfusion using the techniques of CT Perfusion. CT Perfusion was used in association with Fluoro- Deoxy Glucose Positron Emission Tomography (FDG PET) to investigate altered blood flow due to the angiogenic effects of tumour in the clinical setting of medical imaging for cancer diagnosis and staging. CT perfusion, CT enhancement and Doppler ultrasound studies were compared in a series of patient studies performed for the assessment of metastatic liver disease. There was good correlation between all techniques for the arterial phase but not between Doppler measurements of the portal phase and any CT measurement. A new method was developed for quantifying CT perfusion and enhancement values, the Standardised Perfusion Value (SPV) and the Standardised Enhancement Value (SEV). The SPV was shown to correlate with FDG uptake in a series of 16 patient studies of lung nodules, an unexpected and potentially important finding that if confirmed in a larger study may provide an additional diagnostic role for CT in the assessment of lung nodules. Investigation of a commercially available package for the determination of CT Perfusion, CT Perfusion GE Medical Systems, was undertaken in a small series of brain studies for assessment of acute stroke. This data set showed the technique to positively identify patients with non-hemorrhagic stroke in the presence of a normal conventional CT, to select those cases where thrombolysis is appropriate, and to provide an indication for prognosis. An investigation of the accuracy and cost-effectiveness of FDG PET in solitary pulmonary nodules using Australian data was carried out. FDG PET was found to be accurate, cost saving and cost effective for the characterisation of indeterminate solitary pulmonary nodules in Australia. This work was expanded to include the impact of quantitative contrast enhancement CT (QECT) on the cost-effectiveness of FDG PET. The addition of QECT is a cost effective approach, however whether QECT is used alone or in combination with FDG PET will depend on local availability of PET, the cost of PET with respect to surgery and the prior probability of malignancy. A published review of CT perfusion, clinical applications and techniques, is included in the body of the work. Dynamic contrast-enhanced CT and FDG PET were used to investigate blood flow, expressed as SPV, and metabolic relationships in non-small cell lung cancers (NSCLC) of varying size and stage. A significant correlation between SPV and FDG uptake was only found for tumours smaller than 4.5 cm2. Blood flow-metabolic relationships are not consistent in NSCLC but depend on tumour size and stage. Dynamic contrast-enhanced CT as an adjunct to an FDG study undertaken using integrated PET-CT offers an efficient way to augment the assessment of tumour biology with possible future application as part of clinical care. In summary the work has developed a method for standardizing the results of dynamic contrast-enhanced CT and investigated its potential when applied with FDG PET to improve the diagnosis and staging of cancers.

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1365126 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Endometrial cancer is one of the most significant gynaecological malignancies that affect women from New Zealand and the rest of the world. One of the critical stages in the development of a tumour is the onset of hypoxia. The malignancy responds by having raised levels of Hypoxia Inducible Factor (HIF) that in turn induces increased production of Vascular Endothelial Growth Factor (VEGF). VEGF is a potent angiogenic factor that will mediate vascular supply of nutrients and oxygen to the developing tumour. The aim of this study was to investigate whether two compounds found in extracts of plant materials, Resveratrol (Resveratrol) and Epigallocatechin gallate (EGCG), altered the levels of VEGF in the supernatant of cultured endometrial cancer cells. Resveratrol is a phytoalexin that is found in many foods, such as grapes, nuts and berries, as well as in high concentrations in some red wines. 100 µM of resveratrol was added to cell cultures for 24 hours. VEGF levels in the supernatant were then analysed using ELISA. Resveratrol was found to have significant inhibitory effects in both primary endometrial cancer cell cultures and immortalised endometrial cancer cell cultures. Resveratrol was also shown to reverse the increase in VEGF caused by the hypoxia mimic cobalt chloride (CoCl₂). Epigallocatechin gallate (EGCG) is an antioxidant catechin extracted from green tea. The effect of EGCG was analysed using the same method as for resveratrol. 100 µM of EGCG was also shown to have a significant inhibitory effect on the level of VEGF in the supernatant of cultured endometrial cancer cells, as well as reducing the effect of CoCl₂. These results suggest that selected food compounds, resveratrol and EGCG, can reduce VEGF levels by inhibiting HIF. Further investigation This may have anti-tumour effects in women with endometrial cancer.

Doctor of Philosophy (PhD) / Asthma is an inflammatory disease characterised by tissue remodelling. A prominent feature of this remodelling is an increase in the number and size of the blood vessels- formed from pre-existing capillaries – angiogenesis (Siddiqui et al., 2007; Wilson, 2003). This is triggered by many different endogenous angiogenic stimulators such as vascular endothelial growth factor (VEGF), and inhibited by endogenous angiogenic inhibitors such as tumstatin. Tumstatin is the non-collagenous domain (NC1) of the collagen IV α3 chain which, when cleaved, inhibits endothelial cell proliferation and induces apoptosis. Experiments described in this thesis have for the first time demonstrated the absence of tumstatin in the airways of individuals with asthma and lymphangioleiomyomatosis (LAM) as well as the functional responses to tumstatin as an angiogenic inhibitor, both in vitro and in vivo, in the airway. Although tumstatin was absent from the airways of asthmatic and LAM individuals it was present in the airways of individuals with no airways disease, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. No significant difference was seen in the levels of the Goodpasture Binding Protein (GPBP), a phosphorylating protein responsible for the alternate folding of tumstatin, between asthmatic, LAM and individuals with no airways disease. The αvβ3 integrin, reported to be necessary for the activity of tumstatin, as well as the individual αv and β3 sub-units were shown to be equally expressed in the airways of all patient groups. Co-localisation of tumstatin, VEGF and the αvβ3 integrin was seen in the disease free airways, however, a different pattern of VEGF and the αvβ3 integrin expression was observed in asthmatic and LAM airways with minimal co-localisation. Tumstatin was detected in serum and bronchoalveolar lavage fluid (BAL-f) samples from asthmatics and individuals with no airway disease, however there was no significant difference in the level of expression between the two groups. It was demonstrated that the tumstatin detected in the serum and BAL-f samples from asthmatics and individuals with no airway disease was part of the whole collagen IV α3 chain and not in its free and potentially active form. The ability of recombinant tumstatin to inhibit tube formation and proliferation of primary pulmonary endothelial cells was demonstrated for the first time. Further, the functional response of tumstatin was demonstrated in vivo in a mouse model of allergic airway disease. Tumstatin inhibited angiogenesis in the airway and decreased airway hyperresponsiveness. Whether there is potential for tumstatin, or a derivative thereof, to be of therapeutic value in airways diseases in which angiogenesis is a component should be the subject of future studies.

Thesis (Ph. D.)--Michigan State University. Dept. of Biochemistry, 2007. / Title from PDF t.p. (viewed on Apr. 16, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 10, 2009). Includes bibliographical references.