Endothelial dysfunction (ED) is a critical event in the pathophysiology of atherosclerosis and other cardiovascular diseases (CVD). The underlying causes of ED have not been fully established, although unbalanced production of nitric oxide (NO) and reactive oxygen production (ROS) are causally involved. Enzymatic production of NO is dependent on optimal basal tetrahydrobiopterin (BH4) levels in the endothelium. Increased availability of the cofactor by genetic manipulation or pharmacological supplementation has been shown to be beneficial, although the mechanisms controlling BH4 in the endothelium are poorly understood. The broad objectives of this renewal is designed to bridge the gap in knowledge and is based upon the hypothesis that altering BH4 metabolism by lipid peroxidation products and ROS has important consequences in normal NO/ROS fluxes and endothelial physiology favoring phenotypical changes associated with atherogenesis. The present proposal is built on three findings: (i) BH4-free endothelial nitric oxide synthase (eNOS) generates ~100 nmoles superoxide/min/mg protein which is inhibited by BH4 (micromolar range); (ii) BH4 depletion in endothelial cells increases superoxide production by calcium-dependent mechanisms; (iii) 4-hydroxy-2-nonenal (HNE) is a potent inhibitor of BH4 synthesis, depletes BH4 and NO, and increases superoxide production in endothelial cells. The evaluation of the consequences of increased superoxide production from eNOS is critical to the implications of NO and ROS oxidant signaling in disease. Specifically we will: 1) establish the mechanisms increasing uncoupled eNOS-dependent superoxide by HNE and peroxides; 2) investigate the influence of BH4 depletion by HNE and peroxides on increased mitochondrial-superoxide release and dysfunction; 3) elucidate the role of BH4 in limiting oxidative damage, mitochondrial dysfunction and changes in cell phenotype. In the execution of this proposal we will use established endothelial cell cultures and also cells isolated from GTPCH-transgenic mice to examine the influence of endogenous variation in BH4 in the endothelial responses which have been linked to protection, """"""""re-coupling"""""""" of eNOS and/or additional antioxidant activity. Generally this proposal is aimed at understanding these fundamental mechanisms that should provide the basis for developing new and improved strategies in the prevention and treatment of atherosclerosis and CVD. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL067244-06
Application #
7499544
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Goldman, Stephen
Project Start
2001-04-01
Project End
2011-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
6
Fiscal Year
2008
Total Cost
$262,674
Indirect Cost
Name
Medical College of Wisconsin
Department
Biophysics
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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Zielonka, Jacek; Srinivasan, Satish; Hardy, Micael et al. (2008) Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers. Free Radic Biol Med 44:835-46

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