The proposed study will address a novel function of Copper (Cu)-transporting ATPase (ATP7A), a Cu transporter for extracellular SOD (ecSOD), in preventing endothelial dysfunction and eNOS uncoupling in diabetes. Oxidative stress and endothelial dysfunction contribute to pathogenesis of diabetes melitus (DM), characterized by impaired insulin-Akt signaling. Although role of oxidative stress in vascular dysfunction in DM has been extensively studied, little is known about the function of antioxidant enzymes in these pathological diseases. ecSOD is a Cu-containing enzyme synthesized and secreted by vascular smooth muscle cells (VSMCs), and anchored to endothelial surfaces and extracellular matrix. Because of its extracellular location, ecSOD can increase nitric oxide (NO) bioavailability released from the endothelium by preventing O2?-- mediated inactivation of NO and formation of peroxynitrite (ONOO-). Our laboratory previously demonstrated that full activation of vascular ecSOD requires Cu transporter ATP7A. However, the role of ecSOD and ATP7A in vascular dysfunction in DM is entirely unknown. Based on our preliminary studies, we will test the novel hypothesis that Akt-mediated phosphorylation of ATP7A is required for increasing ATP7A stability and translocation to the caveolae/lipid rafts where ecSOD obtains Cu to increase its activity. Thus, impaired insulin-Akt signaling in DM decreases ATP7A function and ecSOD activity in VSMCs, thereby promoting overproduction of O2"- and ONOO- in the extracellular space. This may facilitate the "out- side in signaling" leading to eNOS uncoupling and endothelial dysfunction in ECs.
Aim1 will determine the molecular mechanism by which function of ATP7A, a Cu-transporting ATPase for ecSOD, is impaired in cultured diabetic VSMC. We will test the hypothesis that insulin-Akt-mediated phosphorylation of ATP7A is required for ATP7A stabilization by inhibiting its ubiquitination/degradation as well as ATP7A translocation to caveolae/lipid rafts where ecSOD obtains Cu to promote ecSOD activity in VSMCs.
Aim 2 will determine the protective role of ATP7A, a Cu-transporting ATPase for ecSOD, in eNOS uncoupling and vascular dysfunction in murine and human diabetic vessels. We will examine if vessels from DM mice or those from biopsies obtained from patients with DM show decrease in ecSOD activity and ATP7A expression, thereby increasing O2"- level, ONOO- level, eNOS uncoupling, and endothelial dysfunction. We will use type1 and type2 DM mice models crossed with or without ecSOD KO, ATP7A mutant, or ATP7A overexpressing transgenic mice. We will also perform rescue experiments using adenoviral gene transfer of Akt phosphorylation sites mimetic ATP7A mutant in DM vessels ex vivo. X-ray fluorescence microscopy will be used to analyze Cu distribution in DM vessels and VSMCs. These studies should provide new insight into Cu transport system for ecSOD as a novel therapeutic strategy for treatment of oxidative stress-dependent cardiovascular diseases such as diabetes.

Public Health Relevance

Endothelial dysfunction, an early feature of diabetic vascular disease, is characterized by increased vascular oxidative stress and it plays an important role in a wide spectrum of cardiovascular diseases including atherosclerosis, which has an enormous impact on our health. The current studies will provide a new insight into a role of copper transporter for extracellular SOD, which protects endothelial function by scavenging superoxide anion in the vessel wall, as potential therapeutic targets for oxidative stress-dependent cardiovascular diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Gao, Yunling
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University of Illinois at Chicago
Internal Medicine/Medicine
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United States
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Urao, Norifumi; Ushio-Fukai, Masuko (2013) Redox regulation of stem/progenitor cells and bone marrow niche. Free Radic Biol Med 54:26-39
Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin et al. (2013) Copper transporter ATP7A protects against endothelial dysfunction in type 1 diabetic mice by regulating extracellular superoxide dismutase. Diabetes 62:3839-50
Kohno, Takashi; Urao, Norifumi; Ashino, Takashi et al. (2013) Novel role of copper transport protein antioxidant-1 in neointimal formation after vascular injury. Arterioscler Thromb Vasc Biol 33:805-13
Urao, Norifumi; Sudhahar, Varadarajan; Kim, Seok-Jo et al. (2013) Critical role of endothelial hydrogen peroxide in post-ischemic neovascularization. PLoS One 8:e57618
Mao, Mao; Sudhahar, Varadarajan; Ansenberger-Fricano, Kristine et al. (2012) Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway. Free Radic Biol Med 52:427-35
Urao, Norifumi; McKinney, Ronald D; Fukai, Tohru et al. (2012) NADPH oxidase 2 regulates bone marrow microenvironment following hindlimb ischemia: role in reparative mobilization of progenitor cells. Stem Cells 30:923-34
Fukai, Tohru; Ushio-Fukai, Masuko (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15:1583-606
Kaplan, Nihal; Urao, Norifumi; Furuta, Eiji et al. (2011) Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis. Free Radic Res 45:1124-35
Oshikawa, Jin; Urao, Norifumi; Kim, Ha Won et al. (2010) Extracellular SOD-derived H2O2 promotes VEGF signaling in caveolae/lipid rafts and post-ischemic angiogenesis in mice. PLoS One 5:e10189
Urao, Norifumi; Razvi, Masooma; Oshikawa, Jin et al. (2010) IQGAP1 is involved in post-ischemic neovascularization by regulating angiogenesis and macrophage infiltration. PLoS One 5:e13440

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