The overall goal of these studies is to understand the endothelial nitric oxide synthase (eNOS) signaling system in health and disease. Regulation of endothelial NO production is a key determinant of blood pressure, platelet aggregation, and angiogenesis. The fundamental hypothesis of this research program is that eNOS post-translational modifications and protein-protein associations are essential determinants of NO-dependent signaling in the vascular wall. During the previous funding period, we investigated the relationships between eNOS subcellular targeting, acylation, and enzyme site-specific phosphorylation and dephosphorylation;identified novel kinase and phosphatase pathways involved in eNOS regulation;explored the inter-relationships of VEGF and sphingosine 1- phosphate (S1P) signaling pathways in eNOS regulation;developed and exploited siRNA-based methods to explore the role of caveolin in modulation of endothelial signal transduction;identified a key role for the small G protein Rac1 in control of kinase pathways that modulate eNOS;and discovered that eNOS undergoes reversible receptor-modulated S-nitrosylation. These discoveries set the stage for the proposed studies, which are organized into two Specific Aims.
Aim 1 proposes studies to explore the hypothesis that nitrosylation of eNOS inhibits its enzyme activity. We will examine the relationships between cellular redox state and eNOS nitrosylation in cultured endothelial cells, and will determine the mechanisms whereby eNOS phosphorylation and subcellular targeting influence eNOS nitrosylation and denitrosylation. The proposed studies will generate and characterize transgenic mice that express the nitrosylation-deficient eNOS mutant to test the hypothesis that eNOS nitrosylation serves to mitigate excessive NO generation in the vessel wall. Studies proposed in Aim 2 will identify the mechanisms of Rac1 activation by cell surface receptors in endothelial cells, and will determine the molecular basis for Rac1 inhibition by caveolin. These studies will explore the effects of HMG CoA reductase inhibitors (statins) on caveolin-Rac1 signaling in the endothelium, and will identify the roles of Rac1 in NADPH oxidase vs. PI3-kinase activation in responses to VEGF and S1P. Our studies of the cellular and molecular mechanisms that regulate eNOS may lead to the identification of new points for pharmacological intervention.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL046457-18
Application #
7758227
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Wood, Katherine
Project Start
1997-07-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
18
Fiscal Year
2010
Total Cost
$433,659
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Steinhorn, Benjamin; Sartoretto, Juliano L; Sorrentino, Andrea et al. (2017) Insulin-dependent metabolic and inotropic responses in the heart are modulated by hydrogen peroxide from NADPH-oxidase isoforms NOX2 and NOX4. Free Radic Biol Med 113:16-25
Kraus, Bettina J; Sartoretto, Juliano L; Polak, Pazit et al. (2015) Novel role for retinol-binding protein 4 in the regulation of blood pressure. FASEB J 29:3133-40
Tarrago, Lionel; Péterfi, Zalán; Lee, Byung Cheon et al. (2015) Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors. Nat Chem Biol 11:332-8
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
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Tatematsu, Satoru; Francis, Sanjeev A; Natarajan, Pradeep et al. (2013) Endothelial lipase is a critical determinant of high-density lipoprotein-stimulated sphingosine 1-phosphate-dependent signaling in vascular endothelium. Arterioscler Thromb Vasc Biol 33:1788-94
Michel, Thomas (2013) R is for arginine: metabolism of arginine takes off again, in new directions. Circulation 128:1400-4
Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine et al. (2013) Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling. Sci Signal 6:ra64
Sartoretto, Juliano L; Kalwa, Hermann; Romero, Natalia et al. (2013) In vivo imaging of nitric oxide and hydrogen peroxide in cardiac myocytes. Methods Enzymol 528:61-78

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