The objective of this research is to investigate both mathematically and numerically the effects of vascular geometry upon the cellular dynamics in the endothelium and its consequence in the localisation of atherosclerosis. It is widely accepted that the formation of atherosclerotic plaques preferentially occurs at specific locations in the vasculature, such as arterial branches and bends. It has also been observed that, at the sites of plaque formation, the physiological functions of the vascular endothelium are impaired due to a defect in the production mechanisms of or diminished activities of endothelial nitric oxide (NO). From these observations, a correlation between the vascular geometry, which is effected via local haemodynamic forces, and local bioavailability of endothelial NO has been postulated. The research areas that have been involved in the investigation of atherosclerosis's localisation in the past, haemodynamics, medicine, calcium dynamics, NO kinetics and endothelial cell biology, have been studied individually, and there appears to be no integrated model to date that allows investigation of coupled haemodynamic and cellular mechanism applied in physiologically realistic model geometries. An integrated numerical model that includes these mechanisms will be developed in this research, which will lead to a further, more comprehensive understanding of the pathogenesis of atherosclerosis.

No description available.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

Subarachnoid hemorrhage (SAH) following cerebral aneurysm rupture is associated with substantial morbidity and mortality. The ability of SAH to induce vasospasm in large diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is unclear. Conversely, little is known regarding the impact of SAH on intracerebral (parenchymal) arterioles, which are critical for regulation of cerebral blood flow. To assess the function of parenchymal arterioles following SAH, measurements of diameter, intracellular Ca2+ ([Ca2+]i) and membrane potential were performed in intact arterioles from unoperated (control), sham-operated and SAH model rats. At physiological intravascular pressure, parenchymal arterioles from SAH animals exhibited significantly elevated [Ca2+]i and enhanced constriction compared with arterioles from control and sham-operated animals. Elevated [Ca2+]i and enhanced tone following SAH were observed in the absence of vascular endothelium and were abolished by the L-type voltage-dependent Ca2+ channel (VDCC) inhibitor nimodipine. Molecular assessment of the L-type VDCC CaV1.2 indicated unchanged mRNA and protein expression in arterioles from SAH animals. Increased CaV1.2 activity following SAH may also reflect enhanced pressure-induced membrane potential depolarization of arteriolar smooth muscle. Membrane potential measurements in arteriolar myocytes using intracellular microelectrodes revealed approximately 7 mV depolarization at 40 mmHg in myocytes from SAH animals. Further, when membrane potential was adjusted to similar values, arteriolar [Ca2+]i and tone were similar between groups. These results demonstrate that greater pressure-dependent membrane potential depolarization results in increased activity of CaV1.2 channels, elevated [Ca2+]i and enhanced constriction of parenchymal arterioles from SAH animals. Thus, impaired regulation of parenchymal arteriolar [Ca2+]i and diameter may restrict cerebral blood flow in SAH patients. Although nimodipine is used clinically to prevent delayed neurological deficits in SAH patients, the use of this drug has been limited by hypotension and treatment options remain inadequate. Therefore, our next objective was to explore strategies to selectively suppress CaV1.2 channels in the cerebral vasculature. To do so, we examined the physiological role of smooth muscle CaV1.2 splice variants containing the alternatively-spliced exon 9* in cerebral artery constriction. Using antisense oligonucleotides, we demonstrate that suppression of exon 9*-containing CaV1.2 splice variants results in substantially reduced cerebral artery constriction to elevated extracellular [K+]. In addition, no further reduction in constriction was observed following suppression of all Cav1.2 splice variants, suggesting that exon 9* splice variants are functionally dominant in cerebral artery constriction. In summary, results shown in this dissertation demonstrate that increased CaV1.2 activity following SAH results in enhanced constriction of parenchymal arterioles. Furthermore, evidence is provided supporting the concept that CaV1.2 splice variants with exon 9* are critical for cerebral artery constriction and may provide a novel target for the prevention of delayed ischemic deficits in SAH patients.

Endothelial dysfunction, or the inability of an artery to dilate sufficiently when subjected to excessive shear stress, serves both as a predictor of future cardiovascular events as well as an early indication of atherosclerosis. Several chronic disease states, including obesity, have been shown to alter endothelial function, which may be mediated through circulating pro- and anti-inflammatory adipokines. Still, the mechanisms by which obesity-related low-grade inflammation alters endothelial function are not fully elucidated. Acute and chronic endurance exercise training has previously been shown to be effective in improving endothelial function; however, chronic resistance exercise training is not universally regarded as beneficial to vascular functioning. Far fewer studies have examined the effect of acute resistance exercise on vascular function and adipokine release. To further understand the effects of resistance exercise training on vascular function, a meta-analysis was completed to examine the effects of resistance training on brachial artery flow mediated dilation (FMD), a common measure of endothelial function. The results of the meta-analysis indicate that resistance training has a small positive effect on FMD. Additionally, the effects of an acute bout of lower body resistance exercise on forearm blood flow (FBF) and two inflammatory cytokines were evaluated in obese (>30% body fat) and non-obese (≤30% body fat) subjects. It was hypothesized that the resistance exercise bout would increase FBF, that those changes would be greater in obese versus non-obese subjects, and that the changes in circulating cytokines (adiponectin and tumor necrosis factor-α) would be related to changes in FBF. The results indicate that FBF measures in obese and non-obese subjects react in a divergent pattern immediately following resistance exercise but return to baseline within 24 hours. These changes were not related to changes in adiponectin or TNF-α although changes in adiponectin were related to changes in TNF-α. In conclusion, resistance exercise training programs may have a small positive effect on vascular function which may reduce overall cardiovascular disease risk. Additionally, obese and non-obese subjects display differing patterns of vascular responses to an acute bout of resistance exercise, supporting the view that obesity, and its associated low-grade inflammatory response, may negatively alter vascular homeostasis.

Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts. Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (2–24 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection. Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 2–3 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10. The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.

Bibliography: leaves 300-326. / 326 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates an in vitro experimental system which was designed to assess functions of platelets and cultured endothelial cells when they were incubated either independently or in combination. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995

Thesis (M.Orth.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 137-147) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Bibliography: leaves 234-314. Examines the role of vascular endothelial cells in inflammation with particular reference to their participation in the immune response directed against a vascularised allograft (kidney)