OP-19 - Localized hydrogen peroxide metabolism and cardiovascular signal transduction

Abstract

The molecular events underlying the transition from “physiological” to “pathological” reactive oxygen species (ROS) in the vascular endothelium are poorly understood. We constructed differentially-targeted HyPer2 biosensors for H2O2 to identify the intracellular organelles and pathways responsible for dynamic regulation of endothelial H2O2 metabolism. We discovered that laminar (physiological) shear stress robustly increases H2O2 in the endothelial cell nucleus much more than in the cytosol. By contrast, oscillatory (pathological) shear increases H2O2 in the cytosol significantly more than in the nucleus. We generated H2O2 within specific subcellular locales in cultured endothelial cells using recombinant constructs expressing a D-amino acid oxidase (DAAO) that robustly generates H2O2 upon the addition of D-alanine to the cells. Using a series of differentially targeted DAAO constructs, we discovered H2O2 generation in distinct subcellular compartments differentially modulates endothelial transcription. Generation of H2O2 by DAAO in the endothelial cell nucleus enhanced transcription of Nrf2-modulated genes, whereas generation of H2O2 by DAAO targeted to the cytosol activated NF-kB-regulated genes. These findings have important implications for our understanding of flow-responsive genes in the normal blood vessel as well as in disease states associated with disordered blood flow and oxidative stress.