Abstract 131: Phosphoproteomics Analysis Reveals That Angiotensin-(1-5) Inhibits MTOR Signalling Pathway In Endothelial Cells

Introduction: Our group demonstrated that Angiotensin-(1-5) [Ang-(1-5)] is a biologically active component of the protective arm of the Renin-Angiotensin System (RAS), displaying high efficacy towards the AT 2 R. However, signaling mechanisms for Ang-(1-5) in vitro are still unknown. Objective: To characterize Ang-(1-5) signaling signature in human aortic endothelial cells. Methods: Human aortic endothelial cells (HAEC) were stimulated with Ang-(1-5) (1μM) for 1, 3, 5 or 20 minutes. Proteins were isolated, digested and labeled with TMTpro TM 16-plex. Phosphorylation changes of proteins in response to Ang-(1-5) treatment were analyzed by mass spectrometry. Raw data were processed in Proteome Discoverer. General bioinformatic analysis was performed in R Studios and functional analysis was performed with DAVID and Cytoscape. Validation of phosphoproteomics data was performed by western blot in HAEC treated with Ang-(1-5) (1μM) for 1, 3, 5 or 20 minutes. Results: Treatment of HAEC with Ang-(1-5) significantly modified the phosphorylation status of 831 proteins at 1799 residues, majority of these being dephosphorylated (1079). Functional bioinformatic analysis revealed a cluster of proteins involved in cell cycle and cell division regulation. Upon further analysis, we observed that Ang-(1-5) led to dephosphorylation of residues of CLIP-170 (Ser 195 , Ser 200 , Ser 204 ), 4EBP1 (Ser 65 ), S6K1 (Ser 447 ) and ribosomal S6 protein (Ser 244 , Ser 247 ), suggesting an inhibitory effect on mTOR activity. Western blot analysis confirmed the inhibitory effect of Ang-(1-5) on mTOR signaling, as a significant dephosphorylation of Ser 65 -4EBP1 [% vs. CT = 69.9%] and Thr 389 -S6K1 [% vs. CT = 43.7%] was observed at all timepoints tested. These effects were AT 2 R-mediated, since PD123319 prevented dephosphorylation of Ser 65 -4EBP1 and Thr 389 -S6K1 driven by Ang-(1-5). Conclusion: This study provides an in-depth analysis of Ang-(1-5) signaling patterns in HAEC. The overall similarity of Ang-(1-5) and C21 intracellular signaling further supports that Ang-(1-5) is a hormone of the protective RAS. Since inhibition of mTOR in endothelial cells is known as an anti-senescence mechanism, further investigations are needed to confirm an anti-senescence effect of Ang-(1-5).