The central focus of this application is to address three hypotheses: 1) NAD(P)H oxidase 1 (NOX1) mediates eNOS uncoupling in diabetes;2) Folic acid (FA)-dependent restoration of eNOS cofactor tetrahydrobiopterin (H4B) salvage enzyme dihydrofolate reductase (DHFR) recouples eNOS in diabetes;3) Recoupling of eNOS impedes diabetic atherogenesis. Endothelial nitric oxide synthase (eNOS) is a major protector of vascular homeostasis by producing nitric oxide (NO?) that has potent anti-inflammatory and anti-atherosclerotic effects. Studies in the past decade have however established that eNOS can become uncoupled to produce superoxide (O2?-) rather than NO?, when its cofactor H4B was deficient, i.e. consequent to peroxynitrite mediated oxidation. This transformation may potentially sustain oxidant stress that has been implicated in diabetic etiology and acceleration of cardiovascular complications. Indeed, others and we have reported eNOS-derived, L-NAME-sensitive O2?- production from aortas of diabetic mice or rats. We have further demonstrated that diabetic uncoupling of eNOS is mediated by angiotensin II (Ang II), as Ang II signaling attenuators Candesartan or Captopril effectively recoupled eNOS to restore aortic H4B content and NO? production, while diminishing eNOS-derived O2?- production. Diabetic uncoupling of eNOS is also associated with a loss in H4B salvage enzyme dihydrofolate reductase (DHFR), which mediates Ang II uncoupling of eNOS in cultured endothelial cells. We have previously shown that Ang II uncouples eNOS via NOX-dependent H2O2 production and H2O2- dependent DHFR deficiency in cultured aortic endothelial cells. What remain to be elucidated is which specific NOX isoform lies upstream of uncoupled eNOS in diabetes (Aim 1), whether DHFR deficiency plays an important role in diabetic uncoupling of eNOS and whether folic acid (FA) can restore DHFR expression and activity to recouple eNOS (Aim 2). In preliminary experiments we found intriguing evidence that FA recoupled eNOS in cultured aortic endothelial cells and Ang II infused mice.
In specific aim 3 we will examine whether recoupling of eNOS is effective in impeding atherogenesis in diabetic mice. The overall hypothesis is that endothelial NOX1 is activated by hyperglycemia/diabetes in vivo, resulting in an initial production of ROS (Ang II-dependent), consequent DHFR deficiency, and uncoupling of eNOS, which in turn, exaggerates oxidant stress to accelerate diabetic atherogenesis.

Public Health Relevance

An increase in reactive oxygen species (ROS) production has been implicated in diabetic etiology and acceleration of cardiovascular complications. The proposed studies will delineate novel mechanisms underlying NAD(P)H oxidase-dependent dysfunctional regulation of the cardiovascular protective enzyme eNOS in diabetes, and the consequences of these molecular changes relevant to diabetic atherogenesis. Innovative findings from these studies may ultimately lead to novel therapeutic options for diabetic cardiovascular complications.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Special Emphasis Panel (ZRG1-VH-D (03))
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Charette, Marc F
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University of California Los Angeles
Schools of Medicine
Los Angeles
United States
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Zhang, Yixuan; Liu, Norika Mengchia; Wang, Yongchen et al. (2017) Endothelial cell calpain as a critical modulator of angiogenesis. Biochim Biophys Acta Mol Basis Dis 1863:1326-1335
Cai, Hua; Wang, Chen (2017) Surviving With Smog and Smoke: Precision Interventions? Chest 152:925-929
Cai, Hua; Wang, Chen (2017) Graphical review: The redox dark side of e-cigarettes; exposure to oxidants and public health concerns. Redox Biol 13:402-406
Zhang, Yixuan; Li, Qiang; Youn, Ji Youn et al. (2017) Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells: IMPLICATIONS IN VEGF-DEPENDENT ANGIOGENESIS AND DIABETIC WOUND HEALING. J Biol Chem 292:407-416
Siu, Kin Lung; Li, Qiang; Zhang, Yixuan et al. (2017) NOX isoforms in the development of abdominal aortic aneurysm. Redox Biol 11:118-125
Liu, Norika Mengchia; Siu, Kin Lung; Youn, Ji Youn et al. (2017) Attenuation of neointimal formation with netrin-1 and netrin-1 preconditioned endothelial progenitor cells. J Mol Med (Berl) 95:335-348
Zhang, Yixuan; Cai, Hua (2017) Editorial commentary: Endothelial-to-mesenchymal transition: When the good one goes bad. Trends Cardiovasc Med 27:394-396
Sun, Haipeng; Olson, Kristine C; Gao, Chen et al. (2016) Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure. Circulation 133:2038-49
Siu, Kin Lung; Gao, Ling; Cai, Hua (2016) Differential Roles of Protein Complexes NOX1-NOXO1 and NOX2-p47phox in Mediating Endothelial Redox Responses to Oscillatory and Unidirectional Laminar Shear Stress. J Biol Chem 291:8653-62
Wu, Jing; Saleh, Mohamed A; Kirabo, Annet et al. (2016) Immune activation caused by vascular oxidation promotes fibrosis and hypertension. J Clin Invest 126:50-67

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