The endothelial isoform of nitric oxide synthase (eNOS) is a key determinant of vascular homeostasis and endothelial cell metabolism. eNOS catalysis involves several redox-active cofactors, and NO itself can interact with reactive oxygen species. The bioavailability of endothelium-derived NO is impaired in vascular disease states associated with, increased oxidative stress. We hypothesize that redox regulation ofthe eNOS pathway represents a critical determinant of NO- and ROS-dependent signaling and metabolic regulation in vascular endothelial cells. The proposed studies have important points of intersection with experimental plans proposed by other Projects in this Program. eNOS catalysis involves several redox-active cofactors; changes in intracellular redox state affect the concentrations of these key cofactors, and lead to alterations in eNOS-modulated responses. The formation of cellular NO adducts is influenced by reactive nitrogen and reactive oxygen species and by the cellular thiol redox state. Phosphorylation of eNOS by the AMP-activated protein kinase (AMPK) is influenced by reactive oxygen species, providing an important link between oxidative stress, eNOS signaling, and endothelial cell metabolism. Statins also activate AMPK and modulate endothelial ROS production. eNOSJs targeted to signal-transducing membrane microdomains termed caveolae, where the enzyme interacts with the scaffolding/signaling protein caveolin-1. We discovered that siRNA-mediated knockdown of caveolin leads to a striking increase in ROS production from endothelial cells, and found that caveolin-1-/- mice show dramatic increases in oxidative stress. eNOS-caveolin interactions are modulated by statins, yet the roles of statins in modulation of redox pathways involving eNOS remain incompletely understood. Experiments proposed in Specific Aim 1 will identify the mechanisms whereby altered biopterin and thiol metabolism affects eNOS post-translational modifications, subcellular targeting, and ROS generation. Experiments in Specific Aim 2 will determine the mechanisms whereby caveolin, statins, and reactive oxygen species modulate AMP-activated protein kinase (AMPK) signaling to eNOS.

Public Health Relevance

Cardiovascular drugs such as nitroglycerin are metabolized in the body to form nitric oxide (NO). NO is also synthesized in blood vessels by the enzyme eNOS (endothelial nitric oxide synthase). The function of eNOS is abnormal in cardiovascular diseases such as diabetes, which are characterized by oxidative stress. These studies explore how oxidative stress affects eNOS and lead to vascular disease and dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL048743-22
Application #
8644290
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
22
Fiscal Year
2014
Total Cost
$402,340
Indirect Cost
$176,940
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
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