published_or_final_version / Medicine / Master / Master of Medical Sciences

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

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medical Sciences / Master / Master of Medical Sciences

Thesis (Ph.D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves. Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2001.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

Abstract Adenoviral gene transfer is a valuable tool in molecular biology research. In order to be an efficient and safe vector, adenovirus structure and infection mechanism as well as molecular biology of the used transgene need to be well studied. The aim of this study was to evaluate the role of adenovirus as a gene transfer vector from several perspectives. Adenovirus uses receptor-mediated endocytosis in order to enter the target cell. The effect of Rab5 GTPase on adenovirus entry and gene transfer efficiency was examined first. Next, adenovirus was used as an investigatory tool in the cardiovascular research, focused on clarifying the role of adrenomedullin (AM) in heart and vascular remodeling. Finally, a model of adenoviral gene transfer into skin fibroblasts was used. The role of Rab5 GTPase in the adenovirus endocytosis was examined in HeLa cells using Cy3-labeled adenovirus, and gene transfer efficiency using β-galactosidase encoding adenovirus. Rab5 increased both adenovirus uptake and gene transfer, whereas dominant negative Rab5S34N decreased both endocytosis and gene transfer. The data indicate that Rab5 is needed in mediating the adenovirus uptake into the target cell. In the rat heart, adenovirus-mediated AM gene transfer transiently improved systolic function both in vivo and in vitro. AM caused activation of translocation of protein kinases C ε and δ, whereas phosphorylation of p38 mitogen activated protein kinase was decreased in the left ventricle. AM significantly attenuated the development of angiotensin II-induced cardiac hypertrophy. In rats with myocardial infarction, AM enhanced dilatation of left ventricle and thinning of anterior wall. The role of AM in neointima formation was evaluated in rat artery after endothelial injury. Intravascular AM gene transfer decreased neointimal growth and increased neointimal myofibroblasts apoptosis. These results show that AM regulates left ventricular systolic function and remodeling in the heart, and plays a role in pathological vascular remodeling. Adenovirus-mediated lysyl hydroxylase (LH) gene transfer into skin fibroblasts of type VI Ehlers-Danlos syndrome patient and rat skin increased functional LH production, elevated LH activity, and human LH mRNA production both in vitro and in vivo. LH gene replacement therapy may thus lead to possibilities to improve skin wound healing in Ehlers-Danlos syndrome patients.

Background: Adrenomedullin has been demonstrated to cause systemic vasodilation, and increased plasma adrenomedullin levels have been observed in cardiovascular disease states such as heart failure. While adrenomedullin receptors have been localized to the myocardium, the effects of adrenomedullin on human myocyte contractility remained unknown. Methods and Results: Left ventricular myocytes were isolated from myocardial biopsies of patients (n = 16) undergoing elective coronary artery bypass surgery with normal left ventricular ejection fractions (51 ± 1%). A total of 233 left ventricular myocytes were studied by videomicroscopy. Myocyte shortening velocity (μm/s) was measured at baseline and following the addition of either 3 nM, 30 nM, or 60 nM of adrenomedullin. The change in myocyte shortening velocity with increasing concentrations of adrenomedullin was computed. At all concentrations, adrenomedullin reduced myocyte shortening velocity from baseline values (P < 0.05). Next, the potential interaction of adrenomedullin with the β-adrenergic receptor system was examined using 25 nM isoproterenol. The β-adrenergic receptor-mediated increase in the myocyte shortening velocity was blunted with adrenomedullin (29 ± 7 vs 63 ± 13 μm/s, P < 0.05). Conclusions: These unique findings demonstrate that adrenomedullin reduced contractility in isolated human left ventricular myocytes and exhibited a negative interaction with the β-adrenergic receptor system. Past studies have shown that adrenomedullin induces nitric oxide synthesis and that nitric oxide can uncouple myocyte metabolism. Thus, while adrenomedullin causes systemic vasodilation, this peptide can also exert a negative contractile effect in human left ventricular myocytes.