Abstract P094: Crosstalk Between Mas And ET _B R Elicits Protective Effects In Endothelial Cells And Vascular Function

Abstract

Mas and ET B receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET B R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET B R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10 -7 M) in the presence/absence of A779 (Mas receptor antagonist, 10 -5 M) and BQ788 (ET B R antagonist, 10 -5 M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H 2 O 2 )). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET B R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (p<0.05 vs ctl). High throughput screening of protein:protein interaction compounds in an in-house library identified 23 potential enhancers of the MAS/ET B R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10 -5 M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p<0.05) and H 2 O 2 production (54.7±11.1% vs ctl, p<0.05), while Enh3 increased H 2 O 2 generation (48.1±15.4% vs ctl, p<0.05) in ECs. Moreover, Enh4 increased Nrf2 (3.0 fold), Sod1 (2.0 fold) and Nqo1 (3.1 fold) mRNA expression (p<0.05 vs ctl). Enh3 and Enh4 increased NO production (Enh3: 21.2±7.4%; Enh4: 23.6±8.2% vs veh, p<0.05) in ECs. Acetylcholine (Ach) curves were performed to assess endothelium-dependent relaxation in the absence and presence of enhancers. Enh4 increased ACh-induced relaxation (Emax%: 96.7±4.6 vs ctl: 70.4±3.3, p<0.05), while other enhancers did not improve endothelial function. Taken together, increasing MAS/ET B R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET B R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.

Unraveling the crosstalk between renin‐angiotensin system receptors

“…78 Another RASrelated approach is the development of enhancers of MasR-ET B R heteromers for vasoprotection. 95 Another feature observed for heteromers with significant relevance for pharmacology is the phenomenon of cross-inhibition. This means that a specific antagonist for one receptor within the dimer also blocks signaling of the respective other receptor in the dimer.…”

“…Pharmacological approaches targeting heteromers which involve RAS receptors are, for instance, an interfering peptide for AT 1a R–SCTR heteromers, which when injected into the mouse lateral ventricle suppresses the water‐drinking behavior response after hyperosmotic shock associated with this heteromer 78 . Another RAS‐related approach is the development of enhancers of MasR–ET B R heteromers for vasoprotection 95 …”

“…MasR and ET B RMasR and ET B R receptors physically interact in endothelial cells and are involved in the increased NO generation and vasorelaxation induced by Ang-(1-7) 95. Thus, targeting MasR and ET B R heteromers may be a novel approach to enhance vascular function in individuals with cardiovascular disease.…”