Regions of the circulation, such as the carotid bulb, the proximal coronary arteries and the distal aorta are exposed to disturbed, often oscillatory flow and are predisposed to development of atherosclerosis. There is currently no therapy to prevent lesion development at these sites. Tetrahydrobiopterin (BH4) is a critical cofactor for the nitric oxide synthase (NOS) enzymes and in its absence, the NOS enzymes become uncoupled, so that they produce superoxide (O2-) rather than NO. Our laboratory has discovered a new mechanism by which endothelial cells modulate BH4 levels in response to shear. We found that laminar shear stimulates BH4 levels by 30-fold, and increases the activity of GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme for BH4 production, by a similar extent. Shear stress dissociates GTPCH-1 from its feedback regulatory protein (GFRP), and this allows phosphorylation of GTPCH-1 on serine 81 (serine 72 in the mouse) by casein kinase alpha prime. In contrast, oscillatory shear stress does not dissociate GFRP and GTPCH-1, and does not cause GTPCH-1 phosphorylation, causing BH4 levels to be insufficient. This reduces NO production and increases O2" levels, and markedly enhances atherosclerotic lesion development in a mouse model with disturbed carotid flows. We therefore propose that GTPCH-1 phosphorylation represents a critical switch that alters endothelial cell phenotype, promotes oxidative injury, reduces NO production and predisposes to atherosclerosis. To test this hypothesis in vivo, we have successfully targeted knock-in of both an aspartate (to mimic phosphorylation) and an alanine (to prevent phosphorylation) in murine embryonic stem cells.
In aims 1, we will to study mice with the aspartate knock-in (KI[GCH/S72D] mice) to determine if maintaining GTPCH-1 activation prevents atherosclerosis and preserves NO function.
In aim 2, we will study mice in which GTPCH-1 cannot be activated by shear ( K I [GCH/S72A] mice), hypothesize that these animals will have enhanced atherosclerosis lesion. In the final aim, we plan to continue studies to discover new molecules that cause dissociation of GFRP and GTPCH-1. To accomplish this, we have developed a high-throughput screening assay and have already used this to screen 34,000 molecules. We have promising preliminary data to show that this approach will allow discovery of genes that can increase endothelial cell BH4 levels. We propose that agents discovered using this approach will provide of novel approach for prevention of atherosclerosis at sites of disturbed flow in vivo.

Public Health Relevance

While strategies such as lipid lowering and risk factor modification have proven effective in treatment of atherosclerosis, there are currently no specific therapies to prevent atherosclerosis in regions of the circulation with disturbed blood flow. In this project, we will test the hypothesis that a major cause of atherosclerosis at these sites is deficient tetrahydrobiopterin, and wilt attempt to discover new treatment strategies for prevention of atherosclerosis at these sites.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
Project #
Application #
Study Section
Special Emphasis Panel (ZGM1-PPBC-6)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
United States
Zip Code
Simpson, Scott A; Zaccagni, Hayden; Bichell, David P et al. (2014) Acetaminophen attenuates lipid peroxidation in children undergoing cardiopulmonary bypass. Pediatr Crit Care Med 15:503-10
Shonesy, Brian C; Bluett, Rebecca J; Ramikie, Teniel S et al. (2014) Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation. Cell Rep 9:1644-53
Wu, Jing; Thabet, Salim R; Kirabo, Annet et al. (2014) Inflammation and mechanical stretch promote aortic stiffening in hypertension through activation of p38 mitogen-activated protein kinase. Circ Res 114:616-25
Xu, Shu; Hermanson, Daniel J; Banerjee, Surajit et al. (2014) Oxicams bind in a novel mode to the cyclooxygenase active site via a two-water-mediated H-bonding Network. J Biol Chem 289:6799-808
Neau, David B; Bender, Gunes; Boeglin, William E et al. (2014) Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase. J Biol Chem 289:31905-13
Shuck, Sarah C; Wauchope, Orrette R; Rose, Kristie L et al. (2014) Protein modification by adenine propenal. Chem Res Toxicol 27:1732-42
Hermanson, Daniel J; Gamble-George, Joyonna C; Marnett, Lawrence J et al. (2014) Substrate-selective COX-2 inhibition as a novel strategy for therapeutic endocannabinoid augmentation. Trends Pharmacol Sci 35:358-67
Dikalov, Sergey I; Nazarewicz, Rafal R; Bikineyeva, Alfiya et al. (2014) Nox2-induced production of mitochondrial superoxide in angiotensin II-mediated endothelial oxidative stress and hypertension. Antioxid Redox Signal 20:281-94
Kirabo, Annet; Fontana, Vanessa; de Faria, Ana P C et al. (2014) DC isoketal-modified proteins activate T cells and promote hypertension. J Clin Invest 124:4642-56
Trott, Daniel W; Thabet, Salim R; Kirabo, Annet et al. (2014) Oligoclonal CD8+ T cells play a critical role in the development of hypertension. Hypertension 64:1108-15

Showing the most recent 10 out of 31 publications