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.