Background-This study is the first to examine the effect of direct angiotensin II type 2 (AT 2 ) receptor stimulation on postinfarct cardiac function with the use of the novel nonpeptide AT 2 receptor agonist compound 21 (C21). Methods and Results-Myocardial infarction (MI) was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with C21 (0.01, 0.03, 0.3 mg/kg per day IP) was started 24 hours after MI and was continued until euthanasia (7 days after MI). Infarct size was assessed by magnetic resonance imaging, and hemodynamic measurements were performed via transthoracic Doppler echocardiography and intracardiac Millar catheter. Cardiac tissues were analyzed for inflammation and apoptosis markers with immunoblotting and real-time reverse transcription polymerase chain reaction. C21 significantly improved systolic and diastolic ventricular function. Scar size was smallest in the C21-treated rats. In regard to underlying mechanisms, C21 diminished MI-induced Fas-ligand and caspase-3 expression in the peri-infarct zone, indicating an antiapoptotic effect. Phosphorylation of the p44/42 and p38 mitogen-activated protein kinases, both involved in the regulation of cell survival, was strongly reduced after MI but almost completely rescued by C21 treatment. Furthermore, C21 decreased MI-induced serum monocyte chemoattractant protein-1 and myeloperoxidase as well as cardiac interleukin-6, interleukin-1␤, and interleukin-2 expression, suggesting an antiinflammatory effect. Conclusions-Direct

Abstract-Angiotensin II type 2 (AT 2 ) receptors can be regarded as an endogenous repair system, because the AT 2 receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT 2 receptor agonist, compound 21 (C21; dissociation constant for AT 2 receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: Ͼ10 000 nM). This study tested AT 2 receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor B. C21 dose-dependently (1 nM to 1 mol/L) reduced tumor necrosis factor-␣-induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT 2 receptor specificity was controlled for by inhibition with the AT 2 receptor antagonist PD123319 and by the absence of effects in AT 2 receptor-deficient cells. AT 2 receptor-coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor B, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-␣ in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT 2 receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT 2 receptor agonist C21. These data suggest that pharmacological AT 2 receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor B. (Hypertension. 2010;55:924-931.)

Studying the angiotensin type 2 receptor (AT 2 ) has been problematic in the past because a pharmacological tool for direct, specific in vitro and in vivo stimulation of the receptor has been lacking. Consequently, current knowledge about AT 2 receptor signalling and function had to be obtained by indirect approaches, like studying animals or cells with genetically altered AT 2 receptor expression levels, inhibitory experiments using specific AT 2 receptor antagonists, stimulation with angiotensin II under concomitant angiotensin II type 1 receptor blockade or stimulation with the peptide agonist CGP42112A, which has additional AT 2 receptor antagonistic properties. The recently developed non-peptide AT 2 receptor agonist Compound 21 now, for the first time, allows direct, selective and specific AT 2 receptor stimulation in vitro and in vivo. This new tool will certainly revolutionise AT 2 receptor research, enable many new insights into AT 2 receptor function and may also have the potential to become a future medical drug. This article reviews milestone findings about AT 2 receptor functional properties obtained by 'conventional' experimental approaches within the last 20 years. Moreover, it provides an overview of the first results obtained by direct AT 2 receptor stimulation with Compound 21, comprising effects on alkaline secretion, neurite outgrowth, blood pressure and post-infarct cardiac function.