Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1–7)

Abstract

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1–7) and to determine potential intracellular signaling cascades.Human endothelial cells were stimulated with Ang-(1–7) for 1h, 3h, 6h, and 9h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies.The microarray identified 110 regulated proteins after 1h, 119 after 3h, 31 after 6h, and 86 after 9h Ang-(1–7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1–7) stimulation at 1h and 3h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1–7) treatment. The Ang-(1–7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1–7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.