The proposed studies will use new biosensors and novel chemogenetic approaches to identify the molecular
mechanisms whereby reactive oxygen species (ROS) regulate nitric oxide (NO) signaling pathways in the
vascular endothelium. The proposed studies build on recent work in which we used chemogenetics to develop
a new animal model of cardiomyopathy caused by oxidative stress. Here we plan to expand this chemogenetic
approach to develop a new experimental program to study endothelial dysfunction and hypertension.
Many studies have implicated oxidative stress in endothelial dysfunction and hypertension, yet the underlying
molecular mechanisms remain incompletely understood. Low levels of the stable ROS hydrogen peroxide (H2O2)
modulate NO-dependent physiological responses, while higher ROS levels are associated with hypertension.
The proposed experiments exploit recent advances in chemogenetic and biosensor technologies to identify the
mechanisms underlying the transition from physiological H2O2 signaling to the development of hypertension and
other vascular disease states associated with pathological oxidative stress. We will pursue multispectral imaging
experiments that will simultaneously analyze H2O2, NO and Ca2+ using highly selective and sensitive HyPer7,
geNOp, and GECO biosensors. These studies will establish the mechanisms whereby purinergic P2Y2 receptors
modulate H2O2-, Ca2+-, and NO-dependent endothelial responses that control blood pressure.
Hemodynamic shear stress leads to eNOS activation and to increases in endothelial ROS that can promote
both physiological as well as pathophysiological responses. We found that physiological laminar shear stress
preferentially increases H2O2 in the endothelial cell nucleus, while pathological oscillatory shear stress increases
H2O2 more in the cell cytosol. We used a chemogenetic approach to generate H2O2 in endothelial cells, using
novel recombinant constructs expressing a yeast D-amino acid oxidase (DAAO) that robustly produces H2O2.
The recombinant yeast DAAO is quiescent since vascular cells contain L- but not D-amino acids. H2O2 can be
generated by adding D-alanine to cells expressing recombinant DAAO. Our studies showed that H2O2 generated
in the endothelial cell nucleus activates Nrf2-modulated transcripts, whereas generation of H2O2 in the cytosol
principally increases NF-kB-dependent transcripts. These differential transcriptional responses establish a
causal role for H2O2 and provide a strong connection between chemogenetic approaches and endothelial
pathophysiology. Studying in vitro, ex vivo, and in vivo models, we propose to extend these studies from cultured
human endothelial cells (Aim 1) to the investigation of arterial preparations and transgenic mice expressing
DAAO in the endothelium (Aim 2). This experimental program may lead to the development of a new
“chemogenetic” animal model of hypertension. These studies will use powerful new cell imaging approaches to
test the hypothesis that perturbations in intracellular H2O2 metabolism modulate endothelial responses both in
the normal vasculature and in hypertension, and in other vascular disease states caused by oxidative stress.