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Vaso- and athero- protective effects of non-AT1 receptors in apolipoprotein-E deficient mice

thesis
posted on 16.01.2017, 23:28 by Tesanovic, Sonja
The renin angiotensin system (RAS) plays an important role in volume homeostasis and blood pressure regulation. It is also a major player in cardiovascular disease (CVD) development as the main hormone of the RAS, the octapeptide angiotensin (Ang) II, triggers oxidative stress in the vascular wall causing endothelial dysfunction, which is one of the key factors contributing to development of atherosclerosis. Over the years the RAS and its role in CVD has been well researched and recent evidence suggests that other peptide fragments of the RAS, such as the heptapeptide Ang (1-7), acting at non-AT1-receptor (AT1R) sites may counter-regulate the actions of Ang II, thus highlighting its potential therapeutic properties. Therefore, this thesis explores the effect of non-AT1R stimulation on endothelial dysfunction and atherosclerotic lesion development, using a well established apolipoprotein E-deficient (ApoE-/-) mouse model of atherosclerosis. Chapters 3, 4, 5 and 6 detail the effect of chronic infusion of Ang (1-7), AVE 0991 (an Ang (1-7) non-peptide mimetic), CGP42112 (an AT2R agonist) and perindopril (an ACE inhibitor) on endothelial dysfunction and atherosclerosis in ApoE-/- mice that were fed a high fat diet (HFD) - which is known to exacerbate lesion development in ApoE-/- mice - for 12 weeks prior to, and during, the chronic 4 week treatment period. Primary end points that were investigated included endothelium-dependent vasorelaxation to the endothelial-dependent vasodilator, acetylcholine (ACh), atherosclerotic lesion development, and plaque stability. Chapter 3 examined the effects of chronic Ang (1-7) treatment on endothelial dysfunction and atherosclerotic lesion development in ApoE-/- mice. Ang (1-7) treatment significantly improved endothelial function and inhibited atherosclerotic lesion development, measured as both fatty deposits and luminal encroachment. This enhanced vasorelaxation and inhibited lesion development appears to be mediated by the restored nitric oxide (NO) bioavailability given that chronic Ang (1-7) treatment significantly increased endothelial nitric oxide synthase (eNOS) expression and protein levels whilst concomitantly decreasing superoxide (O2.-) production. Interestingly, these effects appear to be mediated via both the Ang (1-7)/MasR and the AT2R, as both the MasR antagonist, A779, and the AT2R antagonist, PD123319, reversed the actions of Ang (1-7). Furthermore, both receptors were found to be upregulated in this ApoE-/- mouse model. In Chapter 4, the Ang (1-7)-mediated improvement in vascular function, decreased atherosclerotic lesion development and restored NO bioavailability was confirmed using the known Ang (1-7) non-peptide mimetic and MasR agonist, AVE0991. Similar to Ang (1-7), the AVE0991-mediated protective effects were reversed by both AT2R and MasR antagonists. Moreover, in Chapter 4, it was shown that AVE0991 not only inhibited atherosclerotic lesion development but also enhanced plaque stability assessed in the brachiocephalic artery. As noted for both Ang (1-7) and AVE0991, there is a complex interaction that involves both MasR and AT2R. Given the potential involvement of AT2R (Chapters 3 & 4), in Chapter 5, the effects of direct AT2R stimulation in ApoE-/- mice were examined utilizing the known AT2R agonist, CGP42112. Over the relatively narrow dose range tested (1, 5 and 10µg/kg/min) CGP42112 exhibited a vaso-protective effect which did not appear to be dose related. The results of this chapter demonstrate that direct AT2R stimulation mediates vaso- and athero-protection, an effect that was only attenuated with co-infusion of the AT2R antagonist, PD123319, and not by A779. Furthemore, CGP42112 significantly enhanced plaque stability, given that the collagen content was significantly increased and lipid content concomitantly decreased, thus showing that the AT2R is not only implicated in plaque development but also its cellular composition. Consistent with that observed in Chapters 3 and 4, PD123319 alone significantly impaired endothelial function when compared to vascular treatment. Moreover, PD123319 treatment alone further decreased plaque stability when compared to vehicle treatment, thus highlighting the importance of the functional AT2R in vascular function and plaque stability. Previous research has demonstrated that ACE inhibitors can lead to increased Ang (1-7) plasma levels, thus it has been hypothesized that Ang (1-7) could partially contribute to the beneficial effects seen with ACE inhibitors. Therefore, in the final experimental chapter, Chapter 6, the involvement of activation of either AT2R or MasR, presumably by Ang (1-7), in mediating the athero-protective effects of the ACE inhibitor, perindopril, were investigated. Indeed, it is shown that perindopril inhibited atherosclerotic lesion development and increased plaque stability (assessed by an increase in collagen and αSMC-actin content, with a concomitant decrease in lipid deposits within the brachiocephalic plaque). Perindopril also significantly decreased O2.- production and increased eNOS expression suggesting that improved NO bioavailability may contribute to the reduced atherosclerotic lesion development and stabilized plaque composition. Strikingly, the effects of perindopril, including blood pressure reduction, were reversed by either MasR or AT2R blockade, implicating a role of endogenous angiotensin peptides, such as Ang (1-7) acting at multiple sites, contributing to the effects of ACE inhibition. In conclusion, this thesis provides direct evidence that chronic stimulation of AT2R and MasR mediates vaso-protective and anti-atherosclerotic effects in the setting of atherosclerosis. Moreover, this thesis highlights the multifaceted interactions that occur within the RAS and highlights the importance of the Ang (1-7)/MasR/AT2R axis which may act as a counter-regulatory pathway against the pro-atherogenic ACE/Ang II/AT1R axis.

History

Principal supervisor

Robert Widdop

Year of Award

2010

Department, School or Centre

Pharmacology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences