Our long-term objective is to improve therapies for chronic progressive pulmonary hypertension (CPPH) in infants suffering from chronic cardiopulmonary disorders associated with persistent and episodic hypoxia. To do this, we developed a model of CPPH in newborn pigs. We have shown that after 3 days of chronic hypoxia, pulmonary hypertension develops and pulmonary vascular production of the vasodilator NO is intact. When hypoxic exposure is extended to 10 days, pulmonary hypertension worsens and is accompanied by reduced pulmonary vascular NO production. It follows that counteracting or restoring impairments in NO signaling could ameliorate CPPH. Our experimental design will test the hypothesis that treatment with oral L-citrulline, a precursor for L-arginine and NO, increases pulmonary vascular NO production and ameliorates the progressive development of chronic hypoxia-induced pulmonary hypertension.
The aims of this proposal are to: 1) evaluate the ability of and mechanisms by which L-citrulline increases pulmonary vascular NO production 2) evaluate the efficacy and safety of oral L-citrulline to ameliorate chronic hypoxia-induced pulmonary hypertension. Treatments started on the day of placement in hypoxia and continued throughout 3 or 10 days total hypoxic exposure will determine the ability to prevent pulmonary hypertension. Treatments started at the end of the 3rd day of hypoxia and continued for the subsequent 7 days of hypoxia will evaluate the ability to arrest or reverse the progression of pulmonary hypertension. As part of the first aim, studies will be performed to address current gaps in our knowledge about L-citrulline sources, availability, and processing. This will include studies to determine whether chronic hypoxia reduces plasma (extracellular) or intracellular levels of L- citrulline, diminishes the expression of neutral amino acid (L-citrulline) transporters, alters L-citrulline uptake, and/or impairs the amounts, activities or complexing of the enzymes and co-precursors (aspartate) needed for adequate intracellular generation/recycling of L-citrulline (e.g. diminished interaction of eNOS and the L- citrulline to L-arginine recycling enzymes, argininosuccinate and argininosuccinate lyase). As part of the second aim, we will perform pharmacokinetic studies of oral L-citrulline to optimize the therapeutic regimen. We will determine if optimized L-citrulline therapy improves the parameters of NO signaling that are perturbed with exposure to chronic hypoxia. These studies will provide invaluable information about offsetting and restoring impaired NO signaling pathways that can ultimately be translated into important clinical trials to treat infants with chronic cardiopulmonary conditions and CPPH due in part to hypoxia.

Public Health Relevance

Pulmonary hypertension is a well recognized complication of infants with chronic lung and heart disorders. Currently there are few good options for treating these infants. The goal of this project is to use a relevant animal model to help us understand why infants with lung and heart disorders associated with chronic or intermittent hypoxia develop progressive pulmonary hypertension, determine what happens in the lung blood vessels during disease development, and develop treatments for this disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CVRS-G (02))
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Lin, Sara
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Dikalova, Anna; Aschner, Judy L; Kaplowitz, Mark R et al. (2016) Tetrahydrobiopterin oral therapy recouples eNOS and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs. Am J Physiol Lung Cell Mol Physiol 311:L743-L753
Aschner, Judy L; Fike, Candice D (2015) Plasma asymmetric dimethylarginine in infants with bronchopulmonary dysplasia: a promising biomarker despite uncertain pathogenic significance. J Pediatr 166:222-4
Fike, Candice D; Dikalova, Anna; Kaplowitz, Mark R et al. (2015) Rescue Treatment with L-Citrulline Inhibits Hypoxia-Induced Pulmonary Hypertension in Newborn Pigs. Am J Respir Cell Mol Biol 53:255-64
Fike, Candice D; Summar, Marshall; Aschner, Judy L (2014) L-citrulline provides a novel strategy for treating chronic pulmonary hypertension in newborn infants. Acta Paediatr 103:1019-26
Dikalova, Anna; Fagiana, Angela; Aschner, Judy L et al. (2014) Sodium-coupled neutral amino acid transporter 1 (SNAT1) modulates L-citrulline transport and nitric oxide (NO) signaling in piglet pulmonary arterial endothelial cells. PLoS One 9:e85730
Fike, Candice D; Aschner, Judy L (2013) Spread the word, children are still not ""small adults"". Pulm Circ 3:3-4
Fike, Candice D; Aschner, Judy L; Kaplowitz, Mark R et al. (2013) Reactive oxygen species scavengers improve voltage-gated K(+) channel function in pulmonary arteries of newborn pigs with progressive hypoxia-induced pulmonary hypertension. Pulm Circ 3:551-63
Fike, Candice D; Dikalova, Anna; Slaughter, James C et al. (2013) Reactive oxygen species-reducing strategies improve pulmonary arterial responses to nitric oxide in piglets with chronic hypoxia-induced pulmonary hypertension. Antioxid Redox Signal 18:1727-38
Fike, Candice D; Aschner, Judy L (2013) Looking beyond PPHN: the unmet challenge of chronic progressive pulmonary hypertension in the newborn. Pulm Circ 3:454-66
Fike, Candice D; Sidoryk-Wegrzynowicz, Marta; Aschner, Michael et al. (2012) Prolonged hypoxia augments L-citrulline transport by system A in the newborn piglet pulmonary circulation. Cardiovasc Res 95:375-84