Anti-fibrotic mechanisms of angiotensin AT2 -receptor stimulation.

The angiotensin AT2 -receptor is a main receptor of the protective arm of the renin-angiotensin system. Understanding of this unconventional G-protein coupled receptor has significantly advanced during the past decade, largely because of the availability of a selective non-peptide AT2 -receptor agonist, which allowed the conduct of a multitude of studies in animal disease models. This article reviews such preclinical studies that in their entirety provide strong evidence for an anti-fibrotic effect mediated by activation of the AT2 -receptor. Prevention of the development of fibrosis by AT2 -receptor stimulation has been demonstrated in lungs, heart, blood vessels, kidney, pancreas and skin. In lungs, AT2 -receptor stimulation was even able to reverse existing fibrosis. The article further discusses intracellular signalling mechanisms mediating the AT2 -receptor-coupled anti-fibrotic effect, including activation of phosphatases and subsequent interference with pro-fibrotic signalling pathways, induction of matrix-metalloproteinases and hetero-dimerization with the AT1 -receptor, the TGF-ßRII-receptor or the RXFP1-receptor for relaxin. Knowledge of the anti-fibrotic effects of the AT2 -receptor is of particular relevance because drugs targeting this receptor have entered clinical development for indications involving fibrotic diseases.

Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase.

The Angiotensin II type 2 receptor (AT2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT2R-mediated phosphorylation of eNOS at Ser1177, activation of phosphatases and dephosphorylation of eNOS at Tyr657 and Thr495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT2R-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; AT2R antagonist), l-NG-Nitroarginine methyl ester (l-NAME) (10 µM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na3VO4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser1177) or inhibitory eNOS residues (eNOS-Tyr657, eNOS-Thr495) was determined by Western blotting. Phosphorylation of Akt at Ser473 was measured to estimate Akt activity. AT2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, l-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT2R stimulation resulted in phosphorylation of eNOS-Ser1177 and dephosphorylation of eNOS-Tyr657 and eNOS-Thr495 Phosphorylation at eNOS-Ser1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr657, eNOS-Thr495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.