In premature infants, O2 toxicity and antioxidant deficiencies contribute to the development of bronchopulmonary dysplasia (BPD). Affecting up to 10,000 infants annually, BPD represents the impact of injury, including O2 toxicity, to the immature developing lung resulting in arrested lung development. Though clinicians have limited O2 exposure, BPD remains a significant cause of neonatal morbidity. Attempts to prevent BPD by therapeutic antioxidant administration have also failed. Thus, there exists a need for novel approaches to lessen the impact of O2 toxicity and promote normal lung development in premature infants. Recent studies suggest a potential for nuclear factor E2-related factor 2 (Nrf2) agonists to enhance endogenous antioxidant expression, preserve GSH levels, and prevent O2-mediated lung injury. Nrf2 significantly influences alveolarization and hyperoxic susceptibility in newborn mice. Thioredoxin reductase-1 (TrxR1) is best known for regenerating the active site of oxidized thioredoxin-1 (Trx1). A growing body of evidence suggests that TrxR1 inhibition may be a common feature of Nrf2 agonists. Aurothioglucose (ATG) and auranofin (AFN) potently inhibit TrxR1 and are used clinically to treat rheumatoid arthritis. Our previous studies in vivo demonstrated that ATG treatment increases Nrf2 activation, preserves lung GSH levels, and prevents hyperoxic lung injury in adult mice. We recently demonstrated in vitro that AFN treatment increases Nrf2-mediated antioxidant responses and increases GSH levels. The protective effects of ATG and AFN are lost upon GSH system disruption. Our novel preliminary data indicate that ATG lessens O2-mediated lung developmental deficits in newborn mice. Collectively, our data support a working model in which protection by TrxR1 inhibitors are mediated via Nrf2 and GSH-dependent mechanisms. The utility of TrxR1 inhibition to induce Nrf2 activation, enhance GSH levels, and prevent O2-mediated neonatal lung injury has not been tested. The objective of this application, therefore, is to utilize newborn transgenic animal, primary and immortalized lung epithelial culture systems to: 1) determine the impact of altered lung TrxR1 expression on Nrf2 activation, GSH levels and O2-mediated injury; 2) evaluate the safety and efficacy of TrxR1 inhibition to attenuate experimental O2-mediated neonatal lung injury; and 3) distinguish the contributions of Nrf2 and GSH toward these effects. Our central hypothesis is that TrxR1 inhibition will attenuate O2-mediated neonatal lung injury via Nrf2 and GSH-dependent mechanisms. To test this hypothesis, the following specific aims are proposed:
Specific Aim 1 will test the hypothesis that TrxR1 gene dosage alters Nrf2 activation, GSH levels, and O2-mediated neonatal lung injury. In this aim, TrxR1 expression will be genetically altered in vivo and in vitro. We will determine the effect of altered TrxR1 expression on Nrf2 activation, GSH levels, and O2-mediated injury in a BPD mouse model. TrxR1 expression will be altered in vivo using heterozygous and homozygous Club (Clara) cell-specific and alveolar type 2 (AT2) cell-specific conditional TrxR1 knockout mice. TrxR1 expression will be altered in vitro using primary cultured Club and AT2 cells from heterozygous and homozygous TrxR1 knockout mice. TrxR1 will be altered in murine transformed Club cells (mtCC) and AT2 cells (MLE-12) using TrxR1-specific siRNA.
Specific Aim 2 will test the hypothesis that pharmacologic TrxR1 inhibition attenuates O2-mediated neonatal lung injury via Nrf2 and GSH-dependent mechanisms.
This aim will use a BPD mouse model to evaluate the safety and efficacy of ATG to prevent O2-mediated lung injury. The contributions of Nrf2 and GSH will be determined using genetic and pharmacologic approaches. TrxR1 will be inhibited in newborn Nrf2+/+ and Nrf2-/- pups by ATG administration to either 1 d newborn pups or E19 dams. Buthionine sulfoximine (BSO) will be used to deplete GSH in the pups prior to hyperoxic exposure. In vitro, AFN-treated Nrf2+/+ and Nrf2-/- primary Club and AT2 cells and AFN-treated Nrf2-deficient mtCC and MLE-12 cells will be exposed to hyperoxia in the presence and absence of BSO. The studies outlined in this project, which will be straightforward given the expertise of the assembled research team, will determine the safety and efficacy of TrxR1 inhibition as a novel approach to attenuate O2-mediated neonatal lung injury and arrested lung development. Our findings will establish the rationale for future investigations of TrxR1 inhibitors to prevent BPD, a significant and costly cause of morbidity in preterm infants.

Public Health Relevance

Bronchopulmonary Dysplasia (BPD) is a lung disease that causes significant health problems in premature infants. BPD is caused by exposure to abnormally high oxygen levels (hyperoxia) and poor antioxidant defenses. This project focuses on using an innovative therapy, inhibition of thioredoxin reductase-1, to boost antioxidant defenses, protect against hyperoxia, and prevent BPD.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL119280-03
Application #
8842700
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Lin, Sara
Project Start
2015-05-01
Project End
2019-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Pediatrics
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Ali, Mehboob; Heyob, Kathryn; Tipple, Trent E et al. (2018) Alterations in VASP phosphorylation and profilin1 and cofilin1 expression in hyperoxic lung injury and BPD. Respir Res 19:229
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Li, Qian; Li, Rui; Wall, Stephanie B et al. (2018) Aurothioglucose does not improve alveolarization or elicit sustained Nrf2 activation in C57BL/6 models of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 314:L736-L742
Tindell, Rachael; Tipple, Trent (2018) Selenium: implications for outcomes in extremely preterm infants. J Perinatol 38:197-202
Tindell, Rachael; Wall, Stephanie B; Li, Qian et al. (2018) Selenium supplementation of lung epithelial cells enhances nuclear factor E2-related factor 2 (Nrf2) activation following thioredoxin reductase inhibition. Redox Biol 19:331-338
Liu, Taiming; Zhang, Meijuan; Terry, Michael H et al. (2018) Hemodynamic Effects of Glutathione-Liganded Binuclear Dinitrosyl Iron Complex: Evidence for Nitroxyl Generation and Modulation by Plasma Albumin. Mol Pharmacol 93:427-437
Durrani-Kolarik, Shaheen; Pool, Caylie A; Gray, Ashley et al. (2017) miR-29b supplementation decreases expression of matrix proteins and improves alveolarization in mice exposed to maternal inflammation and neonatal hyperoxia. Am J Physiol Lung Cell Mol Physiol 313:L339-L349
Li, Qian; Wall, Stephanie B; Ren, Changchun et al. (2016) Thioredoxin Reductase Inhibition Attenuates Neonatal Hyperoxic Lung Injury and Enhances Nuclear Factor E2-Related Factor 2 Activation. Am J Respir Cell Mol Biol 55:419-28

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