Abstract 415: Identification of Intracellular Signaling Pathways in Human Endothelial Cells Stimulated by Angiotensin-(1-7)

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Abstract

Despite many publications regarding the physiological actions of Ang-(1-7) and its beneficial effects under pathophysiological circumstances, the intracellular signaling initiated by Ang-(1-7) remains still elusive. It was the aim of our study to identify proteins regulated by Ang-(1-7) and to determine potential intracellular signaling cascades.

Human Umbilical Vein Endothelial Cells (HUVEC) were stimulated either with 10-7M Ang-(1-7) or the solvent for 1h, 3h, 6h, and 9h. Effects of Ang-(1-7) on intracellular signaling were assessed via protein microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify potential intracellular Ang-(1-7) signaling pathways. Protein microarray data has been verified by Western blot and Real-Time PCR.

The microarray identified 107 regulated proteins after 1h, 139 after 3h, 33 after 6h, and 77 after 9h. Regulated proteins could be associated with high significance to metabolic pathways like “Molecular Mechanism of Cancer” and “Chronic Myeloid Leukemia” signaling, the latter fitting our recent findings that Ang-(1-7) stimulates hematopoetic progenitor cells. The regulation of identified proteins fitted into the signaling cascades in a time dependent manner. Western blot results for PIAS2 (early protein) were in accordance to the protein microarray data and displayed a decrease by almost 45% after Ang-(1-7) stimulation for 1h and 3h. Additionally, we were able to verify the protein microarray results for FBI-1/Pokemon (late protein) after stimulation for 9h, but not after 6h. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types like HDMEC and bEnd.3. Further experiments showed that PIAS2 protein was affected by Ang-(1-7) already after 30 min, whereby Real-Time PCR showed no influence of Ang-(1-7) on mRNA level.

Our experiments identified new proteins as the E3-type small ubiquitin-like modifier ligase PIAS2 and the proto-oncogene FBI-1/Pokemon in human endothelial cells, that are regulated in a time dependent manner by Ang-(1-7), and are mainly part of metabolic pathways related to cell death and cell survival, whereby PIAS2 is regulated through posttranslational modification after short-term stimulation with Ang-(1-7).

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