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Pediatric Surgery International

, Volume 29, Issue 1, pp 3–8 | Cite as

Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia

  • Jan-H Gosemann
  • Florian Friedmacher
  • Manuela Hunziker
  • Luis Alvarez
  • Nicolae Corcionivoschi
  • Prem Puri
Original Article

Abstract

Aim

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.

Methods

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).

Results

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.

Conclusion

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.

Keywords

Congenital diaphragmatic hernia Persistent pulmonary hypertension Nitrofen PPAR Nox 

References

  1. 1.
    Keijzer R, Puri P (2010) Congenital diaphragmatic hernia. Semin Pediatr Surg 19(3):180–185PubMedCrossRefGoogle Scholar
  2. 2.
    Teng RJ, Eis A, Bakhutashvili I, Arul N, Konduri GG (2009) Increased superoxide production contributes to the impaired angiogenesis of fetal pulmonary arteries with in utero pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 297(1):L184–L195PubMedCrossRefGoogle Scholar
  3. 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
  4. 4.
    Taira Y, Yamataka T, Miyazaki E, Puri P (1998) Adventitial changes in pulmonary vasculature in congenital diaphragmatic hernia complicated by pulmonary hypertension. J Pediatr Surg 33(2):382–387PubMedCrossRefGoogle Scholar
  5. 5.
    Lu X, Murphy TC, Nanes MS, Hart CM (2010) PPAR{gamma} regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-{kappa}B. Am J Physiol Lung Cell Mol Physiol 299(4):L559–L566PubMedCrossRefGoogle Scholar
  6. 6.
    Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK (2002) Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation 105(12):1429–1435PubMedCrossRefGoogle Scholar
  7. 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
  8. 8.
    Hwang J, Kleinhenz DJ, Lassegue B, Griendling KK, Dikalov S, Hart CM (2005) Peroxisome proliferator-activated receptor-gamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol 288(4):C899–C905PubMedCrossRefGoogle Scholar
  9. 9.
    Nisbet RE, Bland JM, Kleinhenz DJ, Mitchell PO, Walp ER, Sutliff RL, Hart CM (2010) Rosiglitazone attenuates chronic hypoxia-induced pulmonary hypertension in a mouse model. Am J Respir Cell Mol Biol 42(4):482–490PubMedCrossRefGoogle Scholar
  10. 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. 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. 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
  13. 13.
    Gosemann JH, Doi T, Kutasy B, Friedmacher F, Dingemann J, Puri P (2012) Alterations of peroxisome proliferator-activated receptor gamma and monocyte chemoattractant protein 1 gene expression in the nitrofen-induced hypoplastic lung. J Pediatr Surg 47(5):847–851PubMedCrossRefGoogle Scholar
  14. 14.
    Dingemann J, Doi T, Ruttenstock E, Puri P (2010) Abnormal platelet-derived growth factor signaling accounting for lung hypoplasia in experimental congenital diaphragmatic hernia. J Pediatr Surg 45(10):1989–1994PubMedCrossRefGoogle Scholar
  15. 15.
    Kling DE, Aidlen JT, Fisher JC, Kinane TB, Donahoe PK, Schnitzer JJ (2005) Nitrofen induces a redox-dependent apoptosis associated with increased p38 activity in P19 teratocarcinoma cells. Toxicol In Vitro 19(1):1–10PubMedCrossRefGoogle Scholar
  16. 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
  17. 17.
    Runo JR, Loyd JE (2003) Primary pulmonary hypertension. Lancet 361(9368):1533–1544PubMedCrossRefGoogle Scholar
  18. 18.
    Corbett HJ, Connell MG, Fernig DG, Losty PD, Jesudason EC (2012) ANG-1 TIE-2 and BMPR signalling defects are not seen in the nitrofen model of pulmonary hypertension and congenital diaphragmatic hernia. PLoS ONE 7(4):e35364PubMedCrossRefGoogle Scholar
  19. 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
  20. 20.
    Brennan LA, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, Black SM (2003) Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. Circ Res 92(6):683–691PubMedCrossRefGoogle Scholar
  21. 21.
    Corcionivoschi N, Alvarez LA, Sharp TH, Strengert M, Alemka A, Mantell J, Verkade P, Knaus UG, Bourke B (2012) Mucosal reactive oxygen species decrease virulence by disrupting campylobacter jejuni phosphotyrosine signaling. Cell Host Microbe 12(1):47–59PubMedCrossRefGoogle Scholar
  22. 22.
    Nisbet RE, Graves AS, Kleinhenz DJ, Rupnow HL, Reed AL, Fan TH, Mitchell PO, Sutliff RL, Hart CM (2009) The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice. Am J Respir Cell Mol Biol 40(5):601–609PubMedCrossRefGoogle Scholar
  23. 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. 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

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Jan-H Gosemann
    • 1
  • Florian Friedmacher
    • 1
  • Manuela Hunziker
    • 1
  • Luis Alvarez
    • 1
  • Nicolae Corcionivoschi
    • 1
  • Prem Puri
    • 1
    • 2
  1. 1.National Children’s Research CentreOur Lady’s Children’s HospitalDublin 12Ireland
  2. 2.School of Medicine and Medical Science and Conway Institute of Biomedical ResearchUniversity College DublinDublinIreland

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