The central focus of the proposed studies is to investigate an innovative role of eNOS uncoupling in the formation of abdominal aortic aneurysm (AAA). Three hypotheses will be addressed: 1) Angiotensin II (Ang II) infusion of eNOS pre-uncoupled mice (hph-1) induces severe AAA;2) Ang II and uncoupled eNOS synergistically promotes AAA formation via NA(D)P)H oxidase (NOX)-dependent further uncoupling of eNOS, and consequent vascular remodeling highlighted by inflammation and MMP-mediated elastin and collagen degradation;3) Recoupling of eNOS is highly effective in preventing AAA formation in Ang II-infused hph-1 mice and apoE null mice. eNOS functions as a major protector of vascular homeostasis by producing nitric oxide (NO7), which has potent anti-inflammatory and anti-atherosclerotic effects. Studies in the past decade have however demonstrated that eNOS can become uncoupled to produce superoxide (O27-) rather than NO7, when its cofactor tetrahydrobiopterin (H4B) was deficient, i.e. consequent to peroxynitrite mediated oxidation 1-9. This transformation potently sustains oxidant stress. During the previous funding cycle we proposed to examine contribution to hypertension of uncoupled eNOS (Aim 4). A mouse model of eNOS uncoupling (hph-1) was employed. Ang II infusion (0.7 mg/kg/day, 14 days) of wild-type and hph-1 male mice resulted in similarly increased blood pressure (BP) up to day 10. To our surprise Ang II-infused hph-1 mice then started to have a decline in BP;some died suddenly. Ultrasound monitoring of abdominal aorta diameter in the survived mice and immediate dissection of the suddenly died mice revealed expansive or rupturing AAA. The total AAA incidence rate is 83.3%;and the mortality rate is 22.2%. During the previous funding cycle we have identified novel mechanisms responsible for Ang II uncoupling of eNOS, and new interventions effective in recoupling eNOS in vitro and in vivo (Aims 1-3). In this competitive renewal we will first characterize AAA formation in Ang II-infused hph-1 mice (Aim 1), and then delineate the underlying molecular mechanisms (Aim 2). Based on our preliminary data and anticipated results from Aims 1- 2, we will examine whether recoupling of eNOS is highly effective in preventing AAA formation in Ang II-infused hph-1 and apoE null mice (Aim 3). The overall hypothesis is that Ang II activation of NOX induces further H4B deficiency and eNOS uncoupling via endothelium-specific attenuation of the expression and activity of the H4B salvage enzyme dihydrofolate reductase (DHFR) in hph-1 mice, resulting in exaggerated NO7 deficiency, excessive reactive oxygen species (ROS) production and severe NO7/ROS imbalance (determined by H4B threshold, see alternative approaches discussion in the specific Aim 3), ROS-dependent inflammation, vascular remodeling and AAA formation. This pathway may prove similarly important in the Ang II-infused apoE null mice, where NOX could be activated by Ang II to induce further uncoupling of eNOS. It has been previously shown that eNOS uncoupling occurs in apoE null mice at baseline 3 10,11. Inhibition of the specific NOX isoform(s) responsible for Ang II further uncoupling of eNOS, and recoupling of eNOS by our newly established approaches of oral folic acid administration or DHFR overexpression 1,25, may then prove to have novel therapeutic potentials for AAA.

Public Health Relevance

Abdominal aortic aneurysm (AAA) is a prevalent and severe human disease, affecting approximately 2% of the population. Ruptured AAA is the 15th leading cause of death in US, accounting for more than 15,000 deaths each year. Due to lack of mechanistic insight into its etiology, therapeutic options have been limited to surgical correction. The current project aims to delineate molecular mechanisms of AAA never revealed before, and to assess innovative therapeutic options based on the new mechanistic findings.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Special Emphasis Panel (ZRG1-VH-D (02))
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Maric-Bilkan, Christine
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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
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