Identification of Phenotypic Modulation of Angiotensin-(1-7) in Thrombin-Stimulated Human Aortic Endothelial Cells from iTRAQ Quantitative Proteomics

Introduction:

The primary effector of renin-angiotensin system (RAS) system is angiotensin II. RAS activation causes many detrimental effects via AT1 receptor. Angiotensin-(1-7)/ angiotensin-converting enzyme 2/Mas axis is a newly identified counter-regulatory pathway against RAS system. Thrombin plays a critical role in coagulation and inflammation processes in vascular endothelium. Although RAS activation is associated with thrombotic complications, it is unknown whether angiotensin-(1-7) can modulate the pleotropic effects of thrombin. In this study, we investigate the proteomic changes of angiotensin-(1-7) effects on thrombin-stimulated human aortic endothelial cells (HAECs).

Materials and methods:

HAECs were pretreated with 10^-7M anigotenion-(1-7) for 1 h and stimulated with 2 units/mL thrombin for additional 5 h. Their proteomes were investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and MetaCore^TMsoftware.

Results:

A total of 653, 717 and 801 proteins were identified in triplicated iTRAQ analyses. MetaCore^TM pathway analysis identified that iTRAQ data showed the consistent pathway alterations (70%) in triplicated experiments. The same altered pathways included "Cytoskeleton remodeling_Cytoskeleton remodeling", "Cell adhesion_Integrin-mediated cell adhesion and migration", "Cell adhesion_Chemokines and adhesion" , "Cytoskeleton remodeling _ Regulation of actin cytoskeleton by Rho GTPases", "LRRK2 in neurons in Parkinson's disease", "Cytoskeleton remodeling_Fibronectin-binding integrins in cell motility", "Cytoskeleton remodeling _TGF, WNT and cytoskeletal remodeling" were among the top 10 statistically significant pathways. Additional experiments validated the phenotypes of angiotensin-(1-7) effects in thrombin-stimulated HAECs.

Conclusions:

Several regulatory pathways are altered by angiotensin-(1-7) in thrombin-stimulated HAECs, with cytoskeleton remodeling, cell adhesion and cell migration (motility) as the dominant altered phenotypes.