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)
Research Project (R01)
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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
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Bouhidel, Jalaleddinne Omar; Wang, Ping; Siu, Kin Lung et al. (2015) Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy. Biochim Biophys Acta 1852:277-89
Siu, Kin Lung; Lotz, Christopher; Ping, Peipei et al. (2015) Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS. J Mol Cell Cardiol 78:174-85
Zhang, Yixuan; Shimizu, Hirohito; Siu, Kin Lung et al. (2014) NADPH oxidase 4 induces cardiac arrhythmic phenotype in zebrafish. J Biol Chem 289:23200-8
Siu, Kin Lung; Miao, Xiao Niu; Cai, Hua (2014) Recoupling of eNOS with folic acid prevents abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E null mice. PLoS One 9:e88899
Youn, Ji-Youn; Siu, Kin Lung; Lob, Heinrich E et al. (2014) Role of vascular oxidative stress in obesity and metabolic syndrome. Diabetes 63:2344-55
Bouhidel, Jalaleddinne Omar; Wang, Ping; Li, Qiang et al. (2014) Pharmacological postconditioning treatment of myocardial infarction with netrin-1. Front Biosci (Landmark Ed) 19:566-70
Youn, Ji-Youn; Zhang, Jun; Zhang, Yixuan et al. (2013) Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase. J Mol Cell Cardiol 62:72-9
Youn, J Y; Gao, L; Cai, H (2012) The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes. Diabetologia 55:2069-79
Youn, Ji Youn; Wang, Ting; Blair, John et al. (2012) Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension. Am J Physiol Heart Circ Physiol 302:H2243-9
Gao, Ling; Siu, Kin L; Chalupsky, Karel et al. (2012) Role of uncoupled endothelial nitric oxide synthase in abdominal aortic aneurysm formation: treatment with folic acid. Hypertension 59:158-66