Abstract

Diminished production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a key role in the early pathogenesis of atherosclerosis. eNOS appears to be acylated and thereby targeted to cholesterol-enriched, plasmalemmal signal transduction domains called caveolae. Localization to caveolae is critical to eNOS function. The objectives of this proposal are to define the molecular mechanisms normally regulating eNOS trafficking to caveolae, and to determine if changes in cholesterol balance attenuate eNOS function by modifying eNOS trafficking to caveolae.
Aim 1 will test the hypothesis that eNOS is first targeted to the Golgi apparatus by myristoylation, and then targeted to caveolae by palmitoylation. eNOS trafficking will be assessed in COS-7 cells transfected with wild-type or mutant eNOS cDNAs by immunoprecipitation of biosynthetically-labelled eNOS in subcellular fractions, and by immunofluorescence and, immunoelectron microscopy.
Aim 2 will test the hypothesis that the integral membrane protein caveolin is necessary for eNOS trafficking to caveolae and for optimal eNOS function. Studies will be performed in permanently-transfected cell lines that are caveolin (+) or caveolin (-).
Aim 3 will employ endothelial cells to test the hypothesis that components of the eNOS activation system are colocalized in caveolae. Receptor-mediated eNOS stimulation will be evaluated in isolated caveolae, and the existence of an eNOS activation complex will be assessed by coprecipitation.
Aim 4 will test the hypothesis that eNOS trafficking to caveolae and eNOS function are attenuated when cholesterol content is abnormally low or high; experiments will be performed in transfected COS-7 cells, endothelial cells, and isolated plasma membranes. The studies planned will increase our basic knowledge of the regulation of endothelial NO production under normal conditions and during altered cholesterol balance, thereby revealing the pathogenetic link between hypercholesterolemia and endothelial dysfunction. In doing so, it is anticipated that the proposed work will lead to new preventative measures and treatment modalities for atherosclerosis that complement approaches aimed at cholesterol control.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058888-02
Application #
2735404
Study Section
Pathology A Study Section (PTHA)
Project Start
1997-07-15
Project End
2002-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of Texas Sw Medical Center Dallas
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390

  Publications

Lee, Wan Ru; Sacharidou, Anastasia; Behling-Kelly, Erica et al. (2015) PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle. PLoS One 10:e0124494
Saddar, Sonika; Carriere, Véronique; Lee, Wan-Ru et al. (2013) Scavenger receptor class B type I is a plasma membrane cholesterol sensor. Circ Res 112:140-51
Mineo, Chieko; Shaul, Philip W (2012) Novel biological functions of high-density lipoprotein cholesterol. Circ Res 111:1079-90
Mineo, Chieko; Shaul, Philip W (2012) Functions of scavenger receptor class B, type I in atherosclerosis. Curr Opin Lipidol 23:487-93
Mineo, Chieko; Shaul, Philip W (2011) PON-dering differences in HDL function in coronary artery disease. J Clin Invest 121:2545-8
Zhu, Weifei; Saddar, Sonika; Seetharam, Divya et al. (2008) The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity. Circ Res 102:480-7
Joshi, Mandar S; Mineo, Chieko; Shaul, Philip W et al. (2007) Biochemical consequences of the NOS3 Glu298Asp variation in human endothelium: altered caveolar localization and impaired response to shear. FASEB J 21:2655-63
Mineo, Chieko; Deguchi, Hiroshi; Griffin, John H et al. (2006) Endothelial and antithrombotic actions of HDL. Circ Res 98:1352-64
Seetharam, Divya; Mineo, Chieko; Gormley, Andrew K et al. (2006) High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I. Circ Res 98:63-72
Mineo, Chieko; Shaul, Philip W (2006) Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae. Cardiovasc Res 70:31-41

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