Advertisement

NADPH Oxidases pp 487-496 | Cite as

NADPH Oxidases and Aging Models of Lung Fibrosis

  • Karen Bernard
  • Victor J. ThannickalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

There is a growing recognition that aging is a risk factor for fibrosis that affects a number of organ systems, including the lung. Despite this understanding, most studies of experimental fibrosis have been conducted in young mice that typically resolve injury-induced lung fibrosis over the course of several months. Our studies demonstrate that aged mouse models may recapitulate human disease by generating a more persistent fibrotic response to injury. This is, in part, due to an imbalance in the expression and activity of NADPH oxidase (NOX) enzymes, in particular the NOX4 isoform, and a related deficiency in antioxidant responses in pathogenic myofibroblasts. These pathogenic myofibroblasts acquire features of cellular senescence and become resistant to apoptosis. In this chapter, we present methods and procedures to apply the aging model of lung fibrosis in mice that will allow interrogation of myofibroblast functions and the expression and activity of NOX4 in cells. We provide recommendations for best laboratory practices to assess the severity and resolution of fibrosis in murine models of aging.

Key words

NADPH oxidase Fibrosis Myofibroblasts NOX4 Bleomycin injury Aging Oxidative stress 

Notes

Acknowledgments

This work was supported by grants from the National Institute of Health: P01 HL114470 and R01 AG046210 (to VJT).

References

  1. 1.
    Holmes B, Page AR, Good RA (1967) Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46(9):1422–1432CrossRefGoogle Scholar
  2. 2.
    Dinauer MC (1993) The respiratory burst oxidase and the molecular genetics of chronic granulomatous disease. Crit Rev Clin Lab Sci 30(4):329–369CrossRefGoogle Scholar
  3. 3.
    Pollock JD, Williams DA, Gifford MA, Li LL, Du X, Fisherman J, Orkin SH, Doerschuk CM, Dinauer MC (1995) Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9(2):202–209CrossRefGoogle Scholar
  4. 4.
    Klebanoff SJ (1968) Myeloperoxidase-halide-hydrogen peroxide antibacterial system. J Bacteriol 95(6):2131–2138PubMedPubMedCentralGoogle Scholar
  5. 5.
    Davies MJ, Hawkins CL, Pattison DI, Rees MD (2008) Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid Redox Signal 10(7):1199–1234CrossRefGoogle Scholar
  6. 6.
    Bedard K, Lardy B, Krause KH (2007) NOX family NADPH oxidases: not just in mammals. Biochimie 89(9):1107–1112CrossRefGoogle Scholar
  7. 7.
    Nisimoto Y, Diebold BA, Cosentino-Gomes D, Lambeth JD (2014) Nox4: a hydrogen peroxide-generating oxygen sensor. Biochemistry 53(31):5111–5120CrossRefGoogle Scholar
  8. 8.
    Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, Thannickal VJ (2009) NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 15(9):1077–1081CrossRefGoogle Scholar
  9. 9.
    Hecker L, Logsdon NJ, Kurundkar D, Kurundkar A, Bernard K, Hock T, Meldrum E, Sanders YY, Thannickal VJ (2014) Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance. Sci Transl Med 6(231):231ra247CrossRefGoogle Scholar
  10. 10.
    Ley B, Collard HR, King TE Jr (2011) Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 183(4):431–440CrossRefGoogle Scholar
  11. 11.
    Thannickal VJ, Lee DY, White ES, Cui Z, Larios JM, Chacon R, Horowitz JC, Day RM, Thomas PE (2003) Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J Biol Chem 278(14):12384–12389CrossRefGoogle Scholar
  12. 12.
    Huang X, Yang N, Fiore VF, Barker TH, Sun Y, Morris SW, Ding Q, Thannickal VJ, Zhou Y (2012) Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction. Am J Respir Cell Mol Biol 47(3):340–348CrossRefGoogle Scholar
  13. 13.
    Thannickal VJ, Zhou Y, Gaggar A, Duncan SR (2014) Fibrosis: ultimate and proximate causes. J Clin Invest 124(11):4673–4677CrossRefGoogle Scholar
  14. 14.
    Paik YH, Iwaisako K, Seki E, Inokuchi S, Schnabl B, Osterreicher CH, Kisseleva T, Brenner DA (2011) The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice. Hepatology 53(5):1730–1741CrossRefGoogle Scholar
  15. 15.
    Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schroder K, Brandes RP, Devaraj S, Torok NJ (2012) Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic Biol Med 53(2):289–296CrossRefGoogle Scholar
  16. 16.
    Sancho P, Mainez J, Crosas-Molist E, Roncero C, Fernandez-Rodriguez CM, Pinedo F, Huber H, Eferl R, Mikulits W, Fabregat I (2012) NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development. PLoS One 7(9):e45285CrossRefGoogle Scholar
  17. 17.
    Raghu G, Weycker D, Edelsberg J, Bradford WZ, Oster G (2006) Incidence and prevalence of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 174(7):810–816CrossRefGoogle Scholar
  18. 18.
    Fell CD, Martinez FJ, Liu LX, Murray S, Han MK, Kazerooni EA, Gross BH, Myers J, Travis WD, Colby TV, Toews GB, Flaherty KR (2010) Clinical predictors of a diagnosis of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 181(8):832–837CrossRefGoogle Scholar
  19. 19.
    Collard HR (2010) The age of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 181(8):771–772CrossRefGoogle Scholar
  20. 20.
    Thannickal VJ (2013) Mechanistic links between aging and lung fibrosis. Biogerontology 14(6):609–615CrossRefGoogle Scholar
  21. 21.
    Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153(6):1194–1217CrossRefGoogle Scholar
  22. 22.
    Mora AL, Bueno M, Rojas M (2017) Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis. J Clin Invest 127(2):405–414CrossRefGoogle Scholar
  23. 23.
    Schafer MJ, Haak AJ, Tschumperlin DJ, LeBrasseur NK (2018) Targeting senescent cells in fibrosis: pathology, paradox, and practical considerations. Curr Rheumatol Rep 20(1):3CrossRefGoogle Scholar
  24. 24.
    Bernard K, Logsdon NJ, Ravi S, Xie N, Persons BP, Rangarajan S, Zmijewski JW, Mitra K, Liu G, Darley-Usmar VM, Thannickal VJ (2015) Metabolic reprogramming is required for myofibroblast contractility and differentiation. J Biol Chem 290(42):25,427–25,438CrossRefGoogle Scholar
  25. 25.
    Bernard K, Logsdon NJ, Benavides GA, Sanders Y, Zhang J, Darley-Usmar VM, Thannickal VJ (2018) Glutaminolysis is required for transforming growth factor-beta1-induced myofibroblast differentiation and activation. J Biol Chem 293(4):1218–1228CrossRefGoogle Scholar
  26. 26.
    Jenkins RG, Moore BB, Chambers RC, Eickelberg O, Konigshoff M, Kolb M, Laurent GJ, Nanthakumar CB, Olman MA, Pardo A, Selman M, Sheppard D, Sime PJ, Tager AM, Tatler AL, Thannickal VJ, White ES, ATS Assembly on Respiratory Cell and Molecular Biology (2017) An official American thoracic society workshop report: use of animal models for the preclinical assessment of potential therapies for pulmonary fibrosis. Am J Respir Cell Mol Biol 56(5):667–679CrossRefGoogle Scholar
  27. 27.
    Thannickal VJ, Roman J (2010) Challenges in translating preclinical studies to effective drug therapies in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 181(6):532–533CrossRefGoogle Scholar
  28. 28.
    Srivastava AK, Khare P, Nagar HK, Raghuwanshi N, Srivastava R (2016) Hydroxyproline: a potential biochemical marker and its role in the pathogenesis of different diseases. Curr Protein Pept Sci 17(6):596–602CrossRefGoogle Scholar
  29. 29.
    Carnesecchi S, Deffert C, Donati Y, Basset O, Hinz B, Preynat-Seauve O, Guichard C, Arbiser JL, Banfi B, Pache JC, Barazzone-Argiroffo C, Krause KH (2011) A key role for NOX4 in epithelial cell death during development of lung fibrosis. Antioxid Redox Signal 15(3):607–619CrossRefGoogle Scholar
  30. 30.
    Gorin Y, Cavaglieri RC, Khazim K, Lee DY, Bruno F, Thakur S, Fanti P, Szyndralewiez C, Barnes JL, Block K, Abboud HE (2015) Targeting NADPH oxidase with a novel dual Nox1/Nox4 inhibitor attenuates renal pathology in type 1 diabetes. Am J Physiol Renal Physiol 308(11):F1276–F1287CrossRefGoogle Scholar
  31. 31.
    McGovern TK, Robichaud A, Fereydoonzad L, Schuessler TF, Martin JG (2013) Evaluation of respiratory system mechanics in mice using the forced oscillation technique. J Vis Exp (75):e50172Google Scholar
  32. 32.
    Gilhodes JC, Jule Y, Kreuz S, Stierstorfer B, Stiller D, Wollin L (2017) Quantification of pulmonary fibrosis in a bleomycin mouse model using automated histological image analysis. PLoS One 12(1):e0170561CrossRefGoogle Scholar
  33. 33.
    Ruscitti F, Ravanetti F, Essers J, Ridwan Y, Belenkov S, Vos W, Ferreira F, KleinJan A, van Heijningen P, Van Holsbeke C, Cacchioli A, Villetti G, Stellari FF (2017) Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice. Multidiscip Respir Med 12:8CrossRefGoogle Scholar
  34. 34.
    Waghray M, Cui Z, Horowitz JC, Subramanian IM, Martinez FJ, Toews GB, Thannickal VJ (2005) Hydrogen peroxide is a diffusible paracrine signal for the induction of epithelial cell death by activated myofibroblasts. FASEB J 19(7):854–856CrossRefGoogle Scholar
  35. 35.
    Bernard K, Logsdon NJ, Miguel V, Benavides GA, Zhang J, Carter AB, Darley-Usmar VM, Thannickal VJ (2017) NADPH oxidase 4 (Nox4) suppresses mitochondrial biogenesis and bioenergetics in lung fibroblasts via a nuclear factor erythroid-derived 2-like 2 (Nrf2)-dependent pathway. J Biol Chem 292(7):3029–3038CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Division of Pulmonary, Allergy and Critical Care Medicine, Department of MedicineUniversity of Alabama at BirminghamBirminghamUSA

Personalised recommendations