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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
Project #
Application #
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Brigham and Women's Hospital
United States
Zip Code
Brown, Jonathan D; Lin, Charles Y; Duan, Qiong et al. (2014) NF-?B directs dynamic super enhancer formation in inflammation and atherogenesis. Mol Cell 56:219-31
Lee, Samuel; Min Kim, Soo; Dotimas, James et al. (2014) Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress. EMBO Mol Med 6:732-43
Taqueti, Viviany R; Di Carli, Marcelo F; Jerosch-Herold, Michael et al. (2014) Increased microvascularization and vessel permeability associate with active inflammation in human atheromata. Circ Cardiovasc Imaging 7:920-9
Zhao, Yuzheng; Yang, Yi; Loscalzo, Joseph (2014) Real-time assessment of the metabolic profile of living cells with genetically encoded NADH sensors. Methods Enzymol 542:349-67
Chatzizisis, Yiannis S; Blankstein, Ron; Libby, Peter (2014) Inflammation goes with the flow: implications for non-invasive identification of high-risk plaque. Atherosclerosis 234:476-8
Barroso, Madalena; Florindo, Cristina; Kalwa, Hermann et al. (2014) Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression. J Biol Chem 289:15350-62
Nallamshetty, Shriram; Le, Phuong T; Wang, Hong et al. (2014) Retinaldehyde dehydrogenase 1 deficiency inhibits PPAR?-mediated bone loss and marrow adiposity. Bone 67:281-91
Folco, Eduardo J; Sukhova, Galina K; Quillard, Thibaut et al. (2014) Moderate hypoxia potentiates interleukin-1? production in activated human macrophages. Circ Res 115:875-83
Kalwa, Hermann; Sartoretto, Juliano L; Martinelli, Roberta et al. (2014) Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium. Proc Natl Acad Sci U S A 111:3383-8
Shiroto, Takashi; Romero, Natalia; Sugiyama, Toru et al. (2014) Caveolin-1 is a critical determinant of autophagy, metabolic switching, and oxidative stress in vascular endothelium. PLoS One 9:e87871

Showing the most recent 10 out of 197 publications