Abstract 017: Ang-(1-7) Influences ET-1 Signaling Through MAS: ETBR Interactions: Implications in Pulmonary Hypertension

Abstract

ACE2 and Ang 1-7 have been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the important role of ET-1 in PH pathophysiology and endothelial dysfunction, we asked whether Ang 1-7 influences ET-1 signaling in endothelial cells and whether Ang 1-7 treatment influences the ET-1 system in PH. Human microvascular endothelial cells (HMEC) were stimulated with ET-1 in absence/presence of Ang 1-7 and showed that Ang 1-7 increased preproET-1 mRNA (250%), ET-1 release (125%) and ETBR protein (50%), p<0.05. ET-1 increases in e-selectin mRNA (400%), VCAM-1 protein (38%) and TNFα production (30%) were blocked by Ang 1-7, p<0.05. Pro-inflammatory effects were dependent on NO. Ang 1-7 increased NO production (257%) in a Mas and ETBR-dependent manner. Mas and ETBR interaction was observed by immunoprecipitation. To characterise physical interactions between Mas/ETBR, we utilised novel technology, employing a library of peptides scanning the MasR sequence, to define sites of ETBR binding. Substitution and truncation identified regions on MasR that confer specificity for ETBR binding. Peptide disruptors to prevent Mas/ETBR interaction were used for in vitro validation. We previously demonstrated in HMEC that Ang 1-7 stimulates Akt phosphorylation (180%), an effect inhibited by pre-incubation with peptide disruptors, p<0.05. To investigate pathophysiological significance of our findings, we investigated whether Ang 1-7 treatment ameliorates PH and whether this is associated with altered ET-1 status. Hypoxia was used to induce PH in mice: normoxic controls (NC), hypoxic PH (HP), normoxic (NA) and hypoxic PH (HA) treated with Ang 1-7 30μg/kg/day. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p<0.05) RVH (0.19 NC vs. 0.28 HP, p<0.01) and ET-1 levels (0.8 NC vs 2.4pg/ml HP, p<0.05) were increased and blocked by Ang 1-7. Hypercontractility and endothelial dysfunction in pulmonary arteries of HP mice was attenuated by Ang 1-7. These findings indicate that vasoprotective effects of Ang 1-7 may be mediated through MAS:ETBR dimerization. In vivo studies support a relationship between Ang 1-7/MAS and ET-1 systems. In conclusion we have identified a novel link between Ang 1-7 and ET-1 through physical interactions between MAS and ETBR.