Endothelial dysfunction is a key initiating step in the onset of multiple major diseases, including heart disease, arteriosclerosis, pulmonary hypertension, diabetes and acute lung injury. As such, an improved understanding of endothelial dysfunction could potentially affect tens of millions of people who suffer from these conditions, as well as avoid thousands of premature deaths. We believe the research plan laid out in this proposal could have significant impact on our knowledge regarding the role of zinc homeostasis in endothelial cell (EC) function. We have recently demonstrated that the disruption of cellular zinc (Zn2+) homeostasis plays a significant role in EC apoptosis following episodes of either acute oxidative or nitrosative stress, and that maintenance of Zn2+ homeostasis within the cell offers protection from these stresses. Data indicate that Zn2+ release, which occurs almost immediately following onset of acute stress, induces mitochondrial dysfunction and increased production of reactive oxygen species (ROS). However, additional preliminary evidence indicates that mitochondrial dysfunction is not due to the direct effect of Zn2+ on the mitochondria. This suggests that intermediate steps exist between Zn2+ release and the onset of mitochondrial dysfunction. We hypothesize that a mechanism involving Zn-mediated reduction of cellular antioxidant capacity leads to a shift in intracellular redox potential through blockade of glutathione recycling and the inhibition of the mitochondrial enzyme responsible for ATP export, adenine nucleotide translocator (ANT). To test this, we will characterize Zn-mediated effects on glutathione status and cycling as well as effects on mitochondrial function (NADH and ATP levels), and correlate these with effects on ANT activity. Secondly, we intend to characterize how mitochondrial dysfunction leads to induction of apoptosis. We have observed Bax translocation to mitochondria as a result of Zn-mediated stress. Interestingly, a recently identified anti-apoptotic factor, humanin, forms Zn-coordinated homodimers in its active form, and can bind Bax and other Bcl-2 pro-apoptotic factors, thus preventing cell death. Thus, using cultured EC, we will determine the role of mitochondrial ROS in blocking humanin activity, as well as evaluate the role of the cysteine residue at position 8 in this protective process. Thirdly, with a goal of identifying clinical therapeutic targets, we will extend our findings using a clinically relevant in vivo model. Preliminary data indicate that, in this mouse model of lipopolysaccharide-induced acute lung injury, Zn2+ homeostasis is disrupted and precedes an induction of cellular apoptosis. We will therefore determine if mitochondrial dysfunction secondary to a loss of GSH homeostasis is occurring and evaluate the roles of Zn2+, mitochondrial derived ROS, and humanin in this process. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32HL090198-01A1
Application #
7483953
Study Section
Special Emphasis Panel (ZRG1-F10-H (21))
Program Officer
Commarato, Michael
Project Start
2008-07-01
Project End
2009-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
1
Fiscal Year
2008
Total Cost
$52,898
Indirect Cost
Name
Georgia Regents University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
Wiseman, Dean A; Thurmond, Debbie C (2012) The good and bad effects of cysteine S-nitrosylation and tyrosine nitration upon insulin exocytosis: a balancing act. Curr Diabetes Rev 8:303-15
Wiseman, Dean A; Kalwat, Michael A; Thurmond, Debbie C (2011) Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis. J Biol Chem 286:16344-54
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
Sharma, Shruti; Kumar, Sanjiv; Wiseman, Dean A et al. (2010) Perinatal changes in superoxide generation in the ovine lung: Alterations associated with increased pulmonary blood flow. Vascul Pharmacol 53:38-52
Sud, Neetu; Wiseman, Dean A; Black, Stephen M (2010) Caveolin 1 is required for the activation of endothelial nitric oxide synthase in response to 17beta-estradiol. Mol Endocrinol 24:1637-49
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
Tian, Jing; Hou, Yali; Lu, Qing et al. (2010) A novel role for caveolin-1 in regulating endothelial nitric oxide synthase activation in response to H2O2 and shear stress. Free Radic Biol Med 49:159-70
Alieva, Irina B; Zemskov, Evgeny A; Kireev, Igor I et al. (2010) Microtubules growth rate alteration in human endothelial cells. J Biomed Biotechnol 2010:671536
Sharma, Shruti; Kumar, Sanjiv; Sud, Neetu et al. (2009) Alterations in lung arginine metabolism in lambs with pulmonary hypertension associated with increased pulmonary blood flow. Vascul Pharmacol 51:359-64

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