Cardiovascular studies of AT2 receptor in various pathophysiological settings

The renin angiotensin system (RAS) plays an important role in blood pressure regulation and its importance in cardiovascular disease has been well researched. Hypertension is a major risk factor for the development of cardiovascular disease and the RAS is a chief regulator of arterial pressure. The main hormone of RAS, the octapeptide angiotensin II (Ang II), exerts its actions through stimulation of two major receptor subtypes, the angiotensin type-1 receptor (AT1R) and the angiotensin type-2 receptor (AT2R). The focus of current literature has been on the activation of the Ang II-AT1R axis and the unfavourable effects in cardiovascular pathology. However, shorter peptide fragments of Ang II are thought to have biological activity in their own right and elicit effects that oppose those mediated by Ang II by stimulating other RAS receptors such as AT2R. Given that hypertension is well established in aged populations, it is of particular interest to determine role of the AT2R in this setting. Additionally, it has been hypothesized that females have a greater vasodilator component of the RAS than males, which may contribute to sex differences in the rates of cardiovascular disease. Therefore, aspects of this thesis explored the function of AT2R stimulation with particular attention directed towards the influence of age and sex. In Chapter 2, HEK-293 cells stably transfected with either the AT1R or the AT2R were used to perform a systematic analysis of binding affinities of all the major Ang peptides. Additionally, the novel AT2R agonist, Compound 21, as well as the MasR agonist and antagonist, AVE0991 and A779 respectively, were tested for their ability to bind to the AT1R or the AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using [125I]- Sar1Ile8Ang II in AT1R transfected HEK-293 cells revealed only Ang II, Ang III and candesartan to have high affinity for AT1R. In AT2R transfected HEK-293 cells, blood pressure in adult normotensive rats via AT2R. However, in aged rats, the depressor effect of Ang (1-7) was maintained but also inhibited by either AT2R blockade or MasR blockade. At the same time, AT2R, MasR and ACE2 immunoreactivity was markedly elevated in aortic sections from aged animals. These results indicate that the Ang (1-7)-mediated depressor effect was preserved in aged animals and involved both AT2R and MasR, whereas Ang (1-7) effects were mediated exclusively via stimulation of AT2R in adult rats. Chapters 5 and 6 examined the effect of both chronic and acute treatment with the AT2R selective nonpeptide agonist, Compound 21. In Chapter 5, chronic treatment with Compound 21 failed to lower blood pressure in normotensive female mice or rats. However, in Chapter 6, the effect of acute administration of Compound 21 was examined in an hypertensive model in which β-5 Ile Ang II had already been shown to exert a vasodepressor effect (Chapter 3). In this case, against a background of AT1R blockade, Compound 21 reduced blood pressure in adult hypertensive rats, an effect that was reversed by AT2R blockade. Additionally, Compound 21 evoked vasorelaxation in both mouse and rat vessels via AT2R stimulation. The development of Compound 21 has resulted in a useful tool for further investigation of AT2R function but this thesis has also highlighted the inability of this compound to lower blood pressure under chronic conditions. In conclusion, this thesis provides evidence for the hypothesis that shorter Ang peptide fragments may act as endogenous AT2R ligands. Taken collectively, these results emphasise the importance of understanding the complex relationships that exist within the RAS. In particular, this thesis highlights that the AT2R axis as well as the Ang (1-7)/MasR axis (at least in aging) may act as counter-regulatory mechanisms to the Ang II/AT1R axis. Moreover, the development of additional AT2R ligands utilising relatively simple synthetic methods as shown in this thesis is warranted as these receptors are potential therapeutic targets for the treatment of hypertension and related cardiovascular remodelling.