Abstract 13501: Antioxidant-1, a Novel Transcription Factor for NADPH Oxidase Organizer P47phox, Regulates Postnatal Neovascularization by Modulating Hydrogen Peroxide Production
Neovascularization is important for repair following ischemic injury and is dependent on angiogenesis and inflammation. Copper (Cu) plays an important role in regulating angiogenesis; however, its mechanism remains unknown. We previously showed that Cu chaperone protein antioxidant-1 (Atox1) functions as a Cu-dependent transcription factor and regulates Cu-induced cell growth. We thus investigated the role of Atox1 in postnatal neovascularization using mouse hindlimb ischemia model. Atox1 knockout (KO) mice exhibit a marked reduction in blood flow recovery at day 14, and infiltration of macrophage into ischemic muscles at day 3 (20% of WT) after ischemia. Atox1KO showed reduced adhesion molecules ICAM/VCAM mRNA levels in ischemic muscles and superoxide production (measured by DHE staining) in CD31+ endothelial cells (ECs). In cultured human ECs, Atox1 siRNA inhibited TNFα (100U/ml)-induced increase in ICAM/VCAM mRNA levels and H2O2 levels (80 and 95% inhibition, respectively). NFkB regulates ICAM/VCAM promoters via IkB degradation in a redox-sensitive manner; we examined the role of Atox1 in these responses. Chromatin immunoprecipitation assays showed that siAtox1 blocked p65 NFkB binding to the ICAM/VCAM promoters without affecting degradation of IkB. Mechanistically, HUVECs stimulated with TNFα increased mRNA levels of NADPH oxidase organizer p47phox (5 fold after 6 hours), which was abolished by siAtox1. Of note, Atox1 is shown to bind to DNA at Atox1 responsive elements (Atox1-RE) and the p47phox promoter contains 4 such cis elements. Overexpression of Atox1 increased p47phox promoter activity (2 fold) while overexpression of Atox1 mutants lacking the Cu binding or nuclear translocation sequence had no effect. Mutation and deletion of cis elements reduced p47phox promoter activity (60% of WT promoter) and lost TNFα induced increase. In conclusion, Atox1 binds to p47phox promoter at Atox1-RE in response to TNFα, thereby promoting endothelial cell H2O2 production that stabilizes p65 binding to DNA and adhesion molecule transcription. This promotes inflammatory cell recruitment and neovascularization. Thus, Atox1 is a potential therapeutic target for inflammation and angiogenesis-dependent ischemic cardiovascular diseases.
- © 2011 by American Heart Association, Inc.