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Theses, Masters


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Sucessfully submitted for the award of Postgraduate Diploma in Biomedical Science to the Technological University Dublin, 2009.


Each year cardiovascular disease causes over 4.3 million deaths in Europe and is the cause of nearly one in three deaths in the US. Nitric oxide (NO) is a toxic atmospheric gas which exists in tissues as a biological product of mammalian cells. It has been used to manage cardiovascular disease for over a century and to this day remains an important treatment option in cardiovascular medicine. NO is produced by many cells in the body including vascular endothelial cells. Because of its importance in vascular function, abnormal production of NO, which occurs in different disease states, can adversely affect blood flow and other vascular functions. NO binds to the haem moiety of the enzyme soluble guanylyl cyclase (sGC) which is found in vascular smooth muscle cells and most other cells of the body. NO diffuses from vascular endothelial cells into the vascular smooth muscle cells adjacent to the endothelium where it binds to and activates sGC. sGC is a heterodimer composed of two different subunits: α and β. The structure of sGC consists of a haem-containing regulatory domain, formed by N-terminal portions of both subunits, and a catalytic domain formed by the C-terminal half of both subunits. The N-terminal regions of α and β subunits are often referred to as the regulatory domain of the enzyme. Several isoforms of these subunits have been described: α1 and α2 and β1, β2 and β3. The heterodimer α1β1 is practically present in all tissues studied. In response to NO binding, sGC catalyses the conversion of guanosine-5′-triphosphate (GTP) to cGMP. cGMP is an intracellular messenger and acts on several cGMP-regulated receptor proteins, such as cGMP-stimulated protein kinases, cGMP-regulated phosphodiesterases (PDEs), and cGMP-gated ion channels which subsequently leads to vasorelaxation. Expression levels of sGC have been found altered in several models of cardiovascular disease however the mechanisms behind these alterations remain largely unknown. Several extracellular regulators such as inflammatory cytokines have been shown to affect sGC steady state levels. The regulation of sGC expression is unquestionably important to support cardiovascular function. The main objectives of this project were to study the effects of selected inflammatory cytokine stimulation on levels of sGC in human endothelial and smooth muscle cells. Primary human aortic vascular endothelial (HAOECs) and human aortic smooth muscle cells (HAOSMCs) were cultured and treated with the inflammatory cytokines GM-CSF and IL-4, to determine the effects they would have on sGC mRNA and protein expression. The mRNA level was determined using QPCR and the protein was analysed using Western blotting. QPCR results showed that cytokine treatment of HAOSMCs increase both of the sGC subunits. The opposite effect was seen in HAOECs, with a decrease in subunit mRNA expression being observed. Western blot analysis revealed that the level of sGC expression in primary cells is too low to be detected by this method, with very little antibody binding to either subunit. It has been suggested that alternative splicing of sGC may be an important mechanism in its regulation. A307838 is a recently discovered sGC splice form of the β1 subunit. Based on observations of the importance of sGC alternative splicing, a further objective of this project was to clone this splice form into an expression vector for future expression and testing in viral vectors. A307838 was PCR amplified as two separate fragments (1 and 2), which were transformed into separate vectors and ligated together in one plasmid vector. The ligation of fragment 1 and 2 was successful, however the orientation of fragment 1 was not correct by the time this study had finished. Work still continues on this splice form. The results from this study identify the effects of GM-CSF and IL-4 on sGC regulation in primary cells. Our findings point further to a role of cytokines in the regulation and activity of sGC. sGC regulation may be finely tuned, and this regulation may play an important part in the understanding of the development of many cardiovascular disorders. Considering the importance of sGC function, and reports concerning the functional significance of its splice forms, the study of A307838 should help us to gain further insights into the regulation of sGC.


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