Abstract 698: Ets-1 is a Critical Regulator of p47phox and the Generation of Reactive Oxygen Species in Response to Angiotensin II
Background: We recently identified that the transcription factor Ets-1 is a critical downstream mediator of AngII-induced vascular inflammation. Gene targets of Ets-1 include the chemokine MCP-1 and the adhesion molecule VCAM-1. A major mechanism by which Ang II induces inflammation is through the generation of reactive oxygen species (ROS). In the current study, we tested the hypothesis that Ets-1 is a critical mediator of Ang II-induced ROS generation.
Methods and Results: Ang II-induced ROS production in HASMCs was markedly decreased by siRNA directed against Ets-1 as determined by DCF-DA assay (1028 ±;160 vs 1762 ±;202 arbitrary units, P < 0.05, n = 6). ROS release was also reduced in aortic segments obtained from Ets-1−/− mice after Ang II infusion, compared to littermate controls (Amplex Red Assay, Figure A⇓). We identified the NAD(P)H oxidase subunit p47phox as a downstream target gene of Ets-1. Ang II-induced p47phox expression in HASMCs was significantly blunted by siRNA directed against Ets-1 (Figure B⇓). Ang II-induced p47phox expression in the thoracic aorta was also markedly reduced in Ets-1−/− mice compared to wild type controls as determined by immunohistochemistry. Deletion and site-directed mutational analysis of human p47phox gene promoter identified an Ets-1 binding site at -45 upstream of transcription starting site that is critical for Ang II-induced p47phox gene expression, which was further validated by electrophoretic mobility shift assay and chromatin immunoprecipitation (Chip) analysis.
Conclusions: Ets-1 is a critical transcriptional mediator of Ang II-mediated ROS generation through the regulation of NAD(P)H oxidase subunits such as p47phox.