Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia
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Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPARγ) is a key regulator of Nox4 expression. Targeted depletion of PPARγ results in pulmonary hypertension phenotype whereas activation of PPARγ attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPARγ-signaling is disrupted during late gestation and H2O2 production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPARγ is decreased in nitrofen-induced CDH.
Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPARγ, Nox4 and Nox4-activation (p22phox).
There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPARγ levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22phox and decreased protein expression of PPARγ in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22phox accompanied by decreased perivascular PPARγ expression in lungs of nitrofen-exposed fetuses compared to controls.
To our knowledge, the present study is the first to report increased Nox4 production in the pulmonary vasculature of nitrofen-induced CDH. Down-regulation of the PPARγ-signaling pathway may lead to increased superoxide production, resulting in pulmonary vascular dysfunction and contributing to pulmonary hypertension in the nitrofen-induced CDH model. PPARγ-activation inhibiting Nox4 production may therefore represent a potential therapeutic approach for the treatment of pulmonary hypertension in CDH.
KeywordsCongenital diaphragmatic hernia Persistent pulmonary hypertension Nitrofen PPAR Nox
- 3.Dorfmuller P, Chaumais MC, Giannakouli M, Durand-Gasselin I, Raymond N, Fadel E, Mercier O, Charlotte F, Montani D, Simonneau G, Humbert M, Perros F (2011) Increased oxidative stress and severe arterial remodeling induced by permanent high-flow challenge in experimental pulmonary hypertension. Respir Res 12:119PubMedCrossRefGoogle Scholar
- 7.Mittal M, Roth M, Konig P, Hofmann S, Dony E, Goyal P, Selbitz AC, Schermuly RT, Ghofrani HA, Kwapiszewska G, Kummer W, Klepetko W, Hoda MA, Fink L, Hanze J, Seeger W, Grimminger F, Schmidt HH, Weissmann N (2007) Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature. Circ Res 101(3):258–267PubMedCrossRefGoogle Scholar
- 10.Barlier-Mur AM, Chailley-Heu B, Pinteur C, Henrion-Caude A, Delacourt C, Bourbon JR (2003) Maturational factors modulate transcription factors CCAAT/enhancer-binding proteins alpha, beta, delta, and peroxisome proliferator-activated receptor-gamma in fetal rat lung epithelial cells. Am J Respir Cell Mol Biol 29(5):620–626PubMedCrossRefGoogle Scholar
- 11.Ameshima S, Golpon H, Cool CD, Chan D, Vandivier RW, Gardai SJ, Wick M, Nemenoff RA, Geraci MW, Voelkel NF (2003) Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circ Res 92(10):1162–1169PubMedCrossRefGoogle Scholar
- 12.Hansmann G, de Jesus Perez VA, Alastalo TP, Alvira CM, Guignabert C, Bekker JM, Schellong S, Urashima T, Wang L, Morrell NW, Rabinovitch M (2008) An antiproliferative BMP-2/PPARgamma/apoE axis in human and murine SMCs and its role in pulmonary hypertension. J Clin Invest 118(5):1846–1857PubMedCrossRefGoogle Scholar
- 16.Chang YT, Ringman Uggla A, Osterholm C, Tran PK, Eklof AC, Lengquist M, Hedin U, Tran-Lundmark K, Frenckner B (2012) Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 302(11):L1159–L1166PubMedCrossRefGoogle Scholar
- 19.Luong C, Rey-Perra J, Vadivel A, Gilmour G, Sauve Y, Koonen D, Walker D, Todd KG, Gressens P, Kassiri Z, Nadeem K, Morgan B, Eaton F, Dyck JR, Archer SL, Thebaud B (2011) Antenatal sildenafil treatment attenuates pulmonary hypertension in experimental congenital diaphragmatic hernia. Circulation 123(19):2120–2131PubMedCrossRefGoogle Scholar
- 23.Li S, Tabar SS, Malec V, Eul BG, Klepetko W, Weissmann N, Grimminger F, Seeger W, Rose F, Hanze J (2008) NOX4 regulates ROS levels under normoxic and hypoxic conditions, triggers proliferation, and inhibits apoptosis in pulmonary artery adventitial fibroblasts. Antioxid Redox Signal 10(10):1687–1698PubMedCrossRefGoogle Scholar
- 24.Gosemann JH, Friedmacher F, Fujiwara N, Alvarez L, Corcionivoschi N, Puri P (2012) Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen induced congenital diaphragmatic hernia. Paper presented at the American Academy of Pediatrics, New OrleansGoogle Scholar