Enhancing the Interaction Between MAS and ETB Receptors is Vasoprotective

Abstract

Introduction The ET-1 system plays an important role in the pathophysiology of pulmonary hypertension (PH). We previously demonstrated that treatment of hypoxic mice (an experimental model of PH) with Ang(1-7) lead to amelioration of right ventricle hypertrophy and systolic pressure, vascular remodelling and hypercontractility to ET-1. The protective effects of Ang(1-7) were associated with an increase in ETBR expression. Peptide array analysis identified that the Ang(1-7) MAS receptor physically interacts with the ETBR. In this study, we evaluated the vascular effects of such interaction and whether enhancers of MAS/ETBR would modulate endothelial and vascular function. Methods Human aortic endothelial cells (HAEC) were used and stimulated with Ang(1-7) (0.1 μM) in the presence/absence of A779 (MAS antagonist, 1μM), BQ788 (ETBR antagonist, 1μM) and MAS/ETBR peptide disruptor (10μM). Protein expression and signalling activation was assessed by immunoblotting, NO production by DAF-FM fluorescence and vascular function by wire myography. Results In HAECs, ETBR expression was increased after stimulation with Ang(1-7) (Control=100% vs. 2h=147%±6.8, 4h=146.1%±5.5 and 24h=176%±21.9) in a MAS receptor dependent manner (+A779 - 2h= 88.2%±1.5, 4h= 84.3%±5.1 and 24h =86.7%±3.6) (p<0.05). Ang(1-7) also increased AKT (40%) and eNOS (35%) phosphorylation; followed by an increase in NO production (Control=100% vs. 10min= 242.5%±25.4) (p<0.05). The effects on endothelial cell signalling and No production were inhibited by MAS and ETBR antagonists, as well as, a MAS/ETBR interaction peptide disruptor. In a high through put screening of a 20K protein:protein interaction modulator library, we discovered 23 potential enhancers of the MAS/ETBR interaction, where we tested 4 in HAECs (Enh1-4: 10μM). From all enhancers, only enhancers 3 and 4 increased eNOS activation (Vehicle=100% vs. En3 10min= 202%±18.2, Enh4 10min= 194.7%±19.6) and NO production (Vehicle=100% vs. En3 10min= 121.2%±7.4, Enh4 10min= 123.6%±8.2). Mesenteric resistance arteries were pre-incubated with Enh3-4 for 30 minutes prior to acetylcholine (Ach) curves, to assess endothelium-dependent relaxation. Only Enh4 increased ACh-induced relaxation (Emax: 96.7±4.6 vs Emax control: 70.4±3.3), p<0.05. Conclusion Our data demonstrates that the MAS/ETBR interaction has protective effects in the endothelium. Enhancing MAS/ETBR crosstalk leads to NO production and improvement of endothelial function, demonstrating potential in the treatment of vascular dysfunction and associated diseases.