Tracey Gaspari

Angiotensin AT2 receptors: cardiovascular hope or hype?

British Journal of Pharmacology (2003) 140, 809–824. doi:10.1038/sj.bjp.0705448

AT2 receptors: Functional relevance in cardiovascular disease

Abstract The renin angiotensin system (RAS) is intricately involved in normal cardiovascular homeostasis. Excessive stimulation by the octapeptide angiotensin II contributes to a range of cardiovascular pathologies and diseases via angiotensin type 1 receptor (AT1R) activation. On the other hand, tElsevier Inc.he angiotensin type 2 receptor (AT2R) is thought to counter-regulate AT1R function. In this review, we describe the enhanced expression and function of AT2R in various cardiovascular disease settings. In addition, we illustrate that the RAS consists of a family of angiotensin peptides that exert cardiovascular effects that are often distinct from those of Ang II. During cardiovascular disease, there is likely to be an increased functional importance of AT2R, stimulated by Ang II, or even shorter angiotensin peptide fragments, to limit AT1R-mediated overactivity and cardiovascular pathologies.

Angiotensin-(1–7) Acts as a Vasodepressor Agent Via Angiotensin II Type 2 Receptors in Conscious Rats

Given that angiotensin-(1–7) (Ang-[1–7]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(1–7) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of angiotensin II type 1 (AT1) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT1 receptor (AT1R) antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, Ang-(1–7) (5 pmol/min) alone, candesartan plus Ang-(1–7), and candesartan plus Ang-(1–7) and angiotensin II type 2 (AT2) receptor (AT2R) antagonist PD123319 (50 &mgr;g/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(1–7), or candesartan alone caused no significant alteration in MAP, whereas Ang-(1–7) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(1–7) during AT1R blockade was observed in SHR. Moreover, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response evoked by the Ang-(1–7)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(1–7) antagonist A-779 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE 140 (100 &mgr;g/kg IV) or the NO synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester (1 mg/kg IV) abolished the depressor effect of Ang-(1–7) in the presence of candesartan. Collectively, these results suggest that Ang-(1–7) evoked a depressor response during AT1R blockade via activation of AT2R, which involves the bradykinin–NO cascade.

Vasoprotective and Atheroprotective Effects of Angiotensin (1-7) in Apolipoprotein E–Deficient Mice

Objective—To evaluate the effectiveness of long-term angiotensin (Ang) (1-7) treatment to inhibit the progression of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Methods and Results—Ang (1-7) is a heptapeptide fragment that has been proposed to counterregulate the Ang II proatherogenic effects. The effect of long-term 4-week Ang (1-7) treatment on both inhibition of atherosclerotic lesion development and improvement of endothelial function was examined in apolipoprotein E−/− mice that had been fed an atherogenic high-fat (21%) diet for 16 weeks. Chronic Ang (1-7) treatment significantly improved endothelial function, an effect reversed with either angiotensin type 2 (AT2) or Mas receptor blockade. In these vessels, Ang (1-7) treatment significantly decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity when compared with vehicle treatment. These effects were blocked by both AT2 and Mas receptor antagonists. Lesion development, assessed as both fatty deposits (oil red O) and intima to media ratio, was also significantly decreased with Ang (1-7) treatment compared with respective controls. Cotreatment with either AT2 or Mas receptor antagonists reversed Ang (1-7)–mediated reduction in lesion development. Conclusion—Long-term Ang (1-7) treatment caused both vasoprotection, via improvement in endothelial function, and atheroprotection, with a reduction in lesion progression in a model of atherosclerosis. These effects appear to be mediated by the restoration of nitric oxide bioavailability and involve a complex interaction of both Mas and AT2 receptors.

A GLP-1 receptor agonist liraglutide inhibits endothelial cell dysfunction and vascular adhesion molecule expression in an ApoE-/- mouse model.

The glucagon like peptide-1 receptor (GLP-1R) agonist liraglutide attenuates induction of plasminogen activator inhibitor type-1 (PAI-1) and vascular adhesion molecule (VAM) expression in human vascular endothelial cells (hVECs) in vitro and may afford protection against endothelial cell dysfunction (ECD), an early abnormality in diabetic vascular disease. Our study aimed to establish the dependence of the in vitro effects of liraglutide on the GLP-1R and characterise its in vivo effects in a mouse model of ECD. In vitro studies utilised the human vascular endothelial cell line C11-STH and enzyme-linked immunosorbent assays (ELISA) for determination of PAI-1 and VAM expression. In vivo studies of vascular reactivity and immunohistochemical analysis were performed in the ApoE-/- mouse model. In vitro studies demonstrated GLP-1R-dependent liraglutide-mediated inhibition of stimulated PAI-1 and VAM expression. In vivo studies demonstrated significant improvement in endothelial function in liraglutide treated mice, a GLP-1R dependent effect. Liraglutide treatment also increased endothelial nitric oxide synthase (eNOS) and reduced intercellular adhesion molecule-1 (ICAM-1) expression in aortic endothelium, an effect again dependent on the GLP-1R. Together these studies identify in vivo protection, by the GLP-1R agonist liraglutide, against ECD and provide a potential molecular mechanism responsible for these effects.

The GLP-1 receptor agonist liraglutide inhibits progression of vascular disease via effects on atherogenesis, plaque stability and endothelial function in an ApoE−/− mouse model

Liraglutide, a once-daily glucagon-like peptide-1 receptor (GLP-1R) agonist, has been approved as a new treatment for type 2 diabetes and is the subject of a clinical trial programme to evaluate the effects on cardiovascular disease and safety. The current study aimed to determine the in vivo effect of liraglutide on progression of atherosclerotic vascular disease in the apolipoprotein E-deficient (ApoE−/−) mouse model and identify underlying mechanisms responsible. Liraglutide treatment inhibited progression of early onset, low-burden atherosclerotic disease in a partially GLP-1R-dependent manner in the ApoE−/− mouse model. In addition, liraglutide treatment inhibited progression of atherosclerotic plaque formation and enhanced plaque stability, again in a partially GLP-1R-dependent manner. No significant effect of liraglutide on progression of late onset, high-burden atherosclerotic disease was observed. In addition, no significant endothelial cell dysfunction was identified in ApoE−/− mice with early onset, low-burden atherosclerotic disease, although significant prevention of weight gain was observed in liraglutide-treated mice using this dietary protocol. Taken together, these results suggest a potential role for liraglutide in the prevention and stabilisation of atherosclerotic vascular disease together with possible protection against major cardiovascular events.

A Novel Histone Deacetylase Inhibitor Reduces Abdominal Aortic Aneurysm Formation in Angiotensin II-Infused Apolipoprotein E-Deficient Mice

Background/Aims: Aberrant expression of components of the matrix metalloproteinase (MMP) enzyme system is implicated in abdominal aortic aneurysm (AAA) formation. We aimed to investigate the influence of a novel histone deacetylase (HDAC) inhibitor (HDACi) metacept-1 (MCT-1), previously documented to reduce MMP expression, on HDAC activity and MMP expression in aortic smooth muscle cells and the in vivo incidence of AAAs. Methods: Western blot and gelatin zymography were used to determine HDAC activity and MMP-2 expression and activity in rat (rVSMCs) and human aortic vascular smooth muscle cells (hVSMCs) in vitro. In vivo AAAs were generated using apolipoprotein E-deficient mice infused with angiotensin (Ang) II. Immunohistochemistry detected MMP-2 and -9 expression in AAA tissue samples. Results: In vitro, MCT-1 inhibited HDAC activity in rVSMCs, and MMP-2 expression and proteolytic activity in hVSMCs.In vivo, Ang II treatment alone exhibited an AAA incidence of 84%. Doxycycline decreased the incidence of AAAs to 50%. Importantly, MCT-1 reduced AAA incidence to approximately 44%. MMP-2 and -9 immunoreactivity was reduced in MCT-1-treated aortic tissue. Conclusion: The novel HDACi MCT-1 inhibits MMP expressionand AAA incidence suggesting this compound may warrant further investigation in the context of AAA biology.

Direct AT2 receptor stimulation is athero-protective and stabilizes plaque in Apolipoprotein E-deficient mice

BACKGROUND The angiotensin II type 2 receptor (AT₂R) has been suggested to have an athero-protective role, however no studies have investigated the effect of direct stimulation of this receptor in atherosclerosis. Thus this study aimed to determine the effect of direct AT₂R stimulation in setting of atherosclerosis, using the known AT₂R agonist, CGP42112. METHODS AND RESULTS Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high fat (21%) diet for 16 weeks, with subcutaneous infusions of CGP42112 (1, 5 or 10 μg/kg/min) administered via osmotic mini-pumps in the final 4 weeks. CGP42112 treatment at all doses significantly improved endothelial function (p<0.001) when compared to acetylcholine mediated-vasorelaxation in aorta taken from vehicle-treated ApoE(-/-) mice. In aortic segments adjacent to those used for vascular reactivity studies, CGP42112 treatment at all doses concomitantly increased eNOS immunoreactivity and protein levels whilst superoxide (O2(-)) production was significantly (p<0.01) decreased compared to levels measured in aorta from vehicle-treated ApoE(-/-) mice. Moreover, CGP42112 (1 μg/kg/min) treatment significantly attenuated (p<0.05) atherosclerotic lesion progression (assessed as both lipid deposits and luminal encroachment in thoracic aorta and aortic arch) and significantly increased plaque stability in the brachiocephalic artery, a region normally prone to rupture. Both the vaso- and athero-protective effects of CGP42112 (1 μg/kg/min) were reversed with co-infusion of the AT2R antagonist, PD123319, but not the MasR antagonist, A779. CONCLUSION For the first time we have shown that direct stimulation of the AT₂R improves endothelial function, reduces atherosclerotic lesion progression and mediates plaque stability with these effects at least partly due to restoration of nitric oxide bioavailability.

Atrial Natriuretic Peptide Preserves Endothelial Function during Intimal Hyperplasia

Background: Atrial and C-type natriuretic peptides (ANP and CNP), acting through different receptors, have antiproliferative effects in vitro. Beneficial effects of CNP in vivo on early atherosclerosis have been described, but it is not known if ANP is antiproliferative in vivo. In the present study, the effects of chronic in vivo ANP were tested and compared with CNP on endothelial dysfunction and intimal thickening caused by peri-arterial collars. Methods: Non-occlusive collars were placed bilaterally around the common carotid arteries of rabbits. One collar was filled with saline vehicle. The contralateral collar was filled with ANP or CNP (1 or 10 µM, n = 5–7) with slow replacement of peptide via mini-pump (1 or 10 fmol/h). Results: After 7 days, endothelium-dependent vasorelaxation in saline-collared arteries was 33 ± 3% of maximum [averaged over 0.03–1 µM acetylcholine (ACh)] compared to 64 ± 2% in normal (uncollared) arteries (p < 0.05, n = 23). In vivo ANP restored the ACh relaxation to normal (e.g., 57 ± 6%, 1 µM ANP), similar to effects seen with CNP in vivo. Endothelium-independent vasorelaxation of collared-vessels was not altered by either peptide. Intimal hyperplasia induced by the collars was not prevented by peri-arterial natriuretic peptides. In additional rabbits (n = 6), CNP (100 pmol/h) given directly into the lumen of collared carotid arteries for 7 days reduced neointima formation by 16 ± 5% (p < 0.05), whereas ANP given intraluminally (100 pmol/h; n = 6) did not. Conclusions: The more potent actions of CNP on vascular smooth muscle cell migration and proliferation (established in vitro) may explain differences between the peptides on intimal hyperplasia in vivo. The major hallmark of atherosclerosis and restenosis, endothelial dysfunction, was prevented by chronic, peri-arterial administration of ANP or CNP.

Effect of tocopheryl phosphate on key biomarkers of inflammation: Implication in the reduction of atherosclerosis progression in a hypercholesterolaemic rabbit model.

1. Many studies have evaluated the effectiveness of α‐tocopherol (vitamin E) in the development and progression of cardiovascular diseases, with conflicting results reported on the protective effect of this anti‐oxidant.