A number of studies now implicate an increase in reactive oxygen species generation (ROS) in the pathogenesis of many cardiovascular diseases and this increase is believed to play a major role in the development of endothelial dysfunction. A major consequence of endothelial dysfunction is a decrease in bio-available nitric oxide (NO). The overall goals of our studies are to understand how reactive oxygen species (ROS) modulate endothelial nitric oxide synthase (eNOS) activity. Our recent studies have identified a novel reversible mechanism by which eNOS can be inhibited. We have found that hydrogen peroxide (H202) can induce a disruption of the eNOS dimer. However, recent published studies indicate that H202 can also be involved in activating the eNOS enzyme. In this proposal, we will test the hypothesis that the underlying mechanism for this dichotomous effect of H202 on eNOS activity is due to the endothelial cells ability to metabolize H202. In vivo, using primary cultures of pulmonary and aortic endothelial cells isolated from various gestational ages, we will determine whether there are developmental or vascular bed specific effects of H202 on eNOS activity. We will then follow up these studies by elucidating the mechanisms for the stimulatory and inhibitory effects of H202 on eNOS. Our recent studies indicate that the activation of eNOS by H202 appears to be linked to a c-Src mediated phosphorylation of HS90 and caveolin 1. The roles of these proteins in Enos activation by H202 will be elucidated using a COS-7 cell line stably expressing eNOS as a model system. We will then determine the effect of transient transfections with various wild type, dominant negative and dominant positive expression plasmids for caveolin 1, HSP90, and c-Src on H202-mediated eNOS activation. We will then determine the mechanism mediating the dimer disruption induced by H202. We will test the hypothesis that H202-mediated oxidation of tetrahydrobiopterin is involved in eNOS dimer disruption. We will investigate this in vivo using primary cultures of endothelial cells and in vitro using recombinant human eNOS protein purified from an E.coli expression system we have developed. The information garnered from the successful completion of these studies should prove to be significant in a number of physiological processes. Clinically, these studies may have important implications in interventions directed at pathophysiological disorders in which excess ROS production is though to be involved. These include pulmonary and systemic hypertension, diabetes, and atherosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL072123-01
Application #
6569431
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Goldman, Stephen
Project Start
2002-12-16
Project End
2003-09-08
Budget Start
2002-12-16
Budget End
2003-09-08
Support Year
1
Fiscal Year
2003
Total Cost
$301,245
Indirect Cost
Name
Northwestern University at Chicago
Department
Pediatrics
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Oishi, Peter E; Sharma, Shruti; Datar, Sanjeev A et al. (2013) Rosiglitazone preserves pulmonary vascular function in lambs with increased pulmonary blood flow. Pediatr Res 73:54-61
Kumar, Sanjiv; Oishi, Peter E; Rafikov, Ruslan et al. (2013) Tezosentan increases nitric oxide signaling via enhanced hydrogen peroxide generation in lambs with surgically induced acute increases in pulmonary blood flow. J Cell Biochem 114:435-447
Sharma, Shruti; Sun, Xutong; Rafikov, Ruslan et al. (2012) PPAR-? regulates carnitine homeostasis and mitochondrial function in a lamb model of increased pulmonary blood flow. PLoS One 7:e41555
Rafikov, Ruslan; Fonseca, Fabio V; Kumar, Sanjiv et al. (2011) eNOS activation and NO function: structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity. J Endocrinol 210:271-84
Sun, Xutong; Fratz, Sohrab; Sharma, Shruti et al. (2011) C-terminus of heat shock protein 70-interacting protein-dependent GTP cyclohydrolase I degradation in lambs with increased pulmonary blood flow. Am J Respir Cell Mol Biol 45:163-71
Black, Stephen M (2010) New insights into acute lung injury. Vascul Pharmacol 52:171-4
Wiseman, Dean A; Sharma, Shruti; Black, Stephen M (2010) Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator. Biometals 23:19-30
Fonseca, Fabio V; Ravi, Kandasamy; Wiseman, Dean et al. (2010) Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption. DNA Cell Biol 29:149-60
Hsu, Jong-Hau; Oishi, Peter; Wiseman, Dean A et al. (2010) Nitric oxide alterations following acute ductal constriction in the fetal lamb: a role for superoxide. Am J Physiol Lung Cell Mol Physiol 298:L880-7
Kumar, Sanjiv; Sud, Neetu; Fonseca, Fabio V et al. (2010) Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase C delta. Am J Physiol Lung Cell Mol Physiol 298:L105-16

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