Physiology - Theses
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ItemThe role of angiotensinogen in the brain renin-angiotensin system( 2011)The renin-angiotensin system (RAS) potently regulates blood pressure and fluid homeostasis. Athough originally described as circulating, humoral system, it is made more complex by the existence of several, potentially independent, tissue RAS. The brain RAS is the focus of the present PhD thesis and serendipitously, this year marks the 40th anniversary of its purported discovery by Fischer-Ferrario and Ganten. During this time, each component of the RAS has been identified within the brain, including enzymes of the extended RAS cascade such as angiotensin-converting enzyme type 2 and the mas receptor. The only known precursor of angiotensin peptides is angiotensinogen (AGT) and in the brain, it is produced predominantly by astrocytes. Physiological studies have shown that the predominant actions of angiotensin II (Ang II) in the brain involve cardiovascular regulation, consistent the systemic RAS. These studies have led to the hypothesis that the brain RAS functions and is regulated independently of the peripheral system. The first aim of this thesis was to determine whether Ang II modulates AGT production by astrocytes. Initial experiments were performed on primary astrocyte cultures from neonatal mice. Surprisingly, despite rigorous analyses, an Ang II binding site could not be detected in these cultures. Preliminary data from a transgenic mouse expressing a fluorescent protein via the angiotensin type 1A receptor (AT1AR) promoter did not reveal astrocytic expression of the receptor under basal conditions. Consistent with the absence of AT1ARs, AGT gene expression was not altered by incubation of cultured astrocytes with Ang II. However, when cultured astrocytes were transduced with AT1ARs they exhibited a profound decrease in AGT gene expression after 24 hours (91.4 % ± 1.8 %, P<0.01, n=4). To confirm this observation in vivo, recombinant adenoviral vectors were used to express the wild type or a constitutively active mutant (N111G) version of the AT1AR in rat astrocytes. A marked reduction in AGT immunoreactivity and gene expression was observed in astrocytes that expressed the constitutively active AT1AR, but not in astrocytes expressing the wild type receptor or control vector. Together, the studies described in Chapters two and three suggest a role for G-protein coupled receptor-mediated intracellular signalling pathways in the modulation of astrocytic AGT. Whilst all components of the RAS have been identified in the brain, their global distribution and subcellular location do not provide an intuitive model for Ang biosynthesis. In particular, it is not clear whether astrocytic AGT is required for local Ang II formation and activation of angiotensin receptors. Current methods are not adequate to resolve the relative importance and anatomy of the brain RAS in vivo without confounding influences by the systemic RAS. To address this issue, new technology incorporating novel RNA interference technology, was used to knockdown endogenous AGT gene expression specifically from astrocytes within a single brain region. Of the three microRNAs designed to knockdown AGT expression, the most efficient (miR930) was selected for use in vivo. A recombinant adenovirus was synthesised to encode miR930 and a reporter gene driven by a ubiquitous promoter. Substantial knockdown of AGT expression was observed seven days after microinjection of this construct into the rat brain nucleus of the solitary tract. However, AGT was also decreased following transduction with a negative-control microRNA produced in parallel with miR930, which has no homology to any known gene. This non-specific effect of the control microRNA was not observed in cultured cells transiently transfected with AGT. Therefore, it was reasoned that the recombinant adenoviral vector or foreign promoter were causing this non-specific AGT knockdown. Since the level of AGT knockdown induced by the control microRNA was as extensive as that of miR930, it was reasonable to conclude that a proportion of the knockdown achieved by this method was specific. Therefore, another vector was made to deliver the miR930 and its control in vivo. Recombinant lentiviruses were used, since they were considered less immunogenic. To direct expression in astrocytes, a Mokola lyssavirus protein coat was used and transgenes were driven by an astrocyte-specific promoter. Fourteen days after administration of this particular vector into the brain, robust AGT knockdown was observed. Unfortunately, the control microRNA also caused a robust decrease in AGT expression. Other astrocyte proteins were also affected by transduction with recombinant lentiviruses, somewhat consistent with the profile of activated astrocytes during an immune response. These non-specific effects could be attributed to a number of issues: including local mechanical perturbation; over-expression of transgenes; contaminants in the viral suspension; or local responses to viral infection. Further improvements in this technology and optimisation strategies will enable specific AGT knockdown in astrocytes in vivo and help resolve the organisation of this complex system in the brain.