Abstract 13280: Localized Hydrogen Peroxide Production and Differential Activation of Redox-sensitive Transcription Factors Involved in Atherogenesis

Abstract

Reactive oxygen species modulate key physiological responses, yet ROS are also implicated in vascular disease states. At low concentrations, the stable ROS hydrogen peroxide (H 2 O 2 ) activates eNOS and enhances vascular barrier function. Yet at higher concentrations, vascular ROS lead to oxidative stress, increase permeability and promote atherosclerosis. The molecular events underlying the transition from “physiological” to “pathological” ROS in the endothelium are incompletely understood. Laminar shear stress activates “atheroprotective” redox-modulated transcription factors whereas turbulent/oscillatory flow activates “atherogenic” transcription factors that are sensitive to cellular redox state. In these studies, we constructed differentially-targeted HyPer biosensors for H 2 O 2 to identify the intracellular organelles and pathways responsible for dynamic mechanochemical regulation of endothelial H 2 O 2 metabolism. We discovered that laminar (physiological) shear stress increases H 2 O 2 in the endothelial cell nucleus much more than in the cytosol. By contrast, oscillatory (pathological) shear increases H 2 O 2 in the cytosol significantly more than in the nucleus. We next generated H 2 O 2 in specific subcellular locales in endothelial cells using recombinant constructs expressing a D-amino acid oxidase (DAAO) that robustly generates H 2 O 2 upon addition of D-alanine to the cells. By transfecting a series of differentially targeted DAAO constructs into endothelial cells, we discovered that generation of H 2 O 2 in distinct subcellular compartments differentially modulates transcriptional activation. Generation of H 2 O 2 by DAAO expressed in the endothelial cell nucleus led to enhanced transcription of Nrf2-modulated genes, whereas generation of H 2 O 2 by DAAO targeted to the cytosol instead activated NF-κB-regulated genes. This differential transcriptional response regulated by H 2 O 2 generated in distinct subcellular locales provides new insights into the roles of ROS and eNOS in mechanochemical coupling. Taken together, 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.