Ang(1–7) has been shown to protect against pulmonary hypertension (PH). Mechanisms remain unclear. Considering the importance of ET-1 in PH pathophysiology and endothelial dysfunction, we questioned whether Ang(1–7) influences ET-1 signaling in endothelial cells and whether Ang(1–7) treatment influences the ET-1 system in PH.Design and Method:
Human endothelial cells (hEC) were stimulated with ET-1 in absence/presence of Ang(1–7). Mas and ETBR interaction was observed by immunoprecipitation. To characterize physical interactions, we utilized novel technology, employing a library of peptides spanning the MasR sequence, to define sites of ETBR binding. To investigate pathophysiological significance of our findings, we investigated whether Ang(1–7) treatment ameliorates PH. Hypoxia was used to induce PH in mice: normoxic (NV) and hypoxic vehicle (HV), normoxic (NA) and hypoxic PH (HA) treated with Ang(1–7) 30 μg/kg/day.Results:
Ang(1–7) increases ET-1 release (125%) and ETBR protein (50%). ET-1-induced increases in VCAM-1 protein (38%) and TNFα production (30%) were blocked by Ang(1–7). Pro-inflammatory effects were dependent on NO. Ang(1–7) increased NO production (257%) in a Mas and ETBR-dependent manner. Mutagenesis studies identified regions conferring specificity for ETBR binding. Peptide disruptors to prevent Mas/ETBR interaction were used for in vitro validation. We previously demonstrated in hEC that Ang(1–7) stimulates eNOS phosphorylation (180%), an effect inhibited by pre-incubation with peptide disruptors. In HP mice, RVSP (18.7 NV vs. 47.6mmHg HV, p < 0.05) RVH (0.19 NV vs. 0.28 HV, p < 0.01) and ET-1 levels (0.8 NV vs 2.4pg/ml HV, p < 0.05) were increased and blocked by Ang(1–7). Hypercontractility in pulmonary arteries of HV mice was attenuated by Ang(1–7).Conclusions:
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.