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)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-VH-D (03))
Program Officer
Charette, Marc F
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Los Angeles
Schools of Medicine
Los Angeles
United States
Zip Code
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
Siu, Kin Lung; Li, Qiang; Zhang, Yixuan et al. (2016) NOX isoforms in the development of abdominal aortic aneurysm. Redox Biol 11:118-125
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
Zhang, Yixuan; Li, Qiang; Youn, Ji Youn et al. (2016) PTP1B in Calpain-dependent Feedback Regulation of VEGFR2 in Endothelial Cells: Implication in VEGF-dependent Angiogenesis and Diabetic Wound Healing. J Biol Chem :
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
Youn, Ji-Youn; Zhou, Jun; Cai, Hua (2015) Bone Morphogenic Protein 4 Mediates NOX1-Dependent eNOS Uncoupling, Endothelial Dysfunction, and COX2 Induction in Type 2 Diabetes Mellitus. Mol Endocrinol 29:1123-33
Li, Qiang; Wang, Ping; Ye, Keqiang et al. (2015) Central role of SIAH inhibition in DCC-dependent cardioprotection provoked by netrin-1/NO. Proc Natl Acad Sci U S A 112:899-904
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
Miao, Xiao Niu; Siu, Kin Lung; Cai, Hua (2015) Nifedipine attenuation of abdominal aortic aneurysm in hypertensive and non-hypertensive mice: Mechanisms and implications. J Mol Cell Cardiol 87:152-9
Ping, Peipei; Gustafsson, Ã…sa B; Bers, Don M et al. (2015) Harnessing the Power of Integrated Mitochondrial Biology and Physiology: A Special Report on the NHLBI Mitochondria in Heart Diseases Initiative. Circ Res 117:234-8

Showing the most recent 10 out of 29 publications