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.
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References
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):1422–1432
Dinauer MC (1993) The respiratory burst oxidase and the molecular genetics of chronic granulomatous disease. Crit Rev Clin Lab Sci 30(4):329–369
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):202–209
Klebanoff SJ (1968) Myeloperoxidase-halide-hydrogen peroxide antibacterial system. J Bacteriol 95(6):2131–2138
Davies MJ, Hawkins CL, Pattison DI, Rees MD (2008) Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid Redox Signal 10(7):1199–1234
Bedard K, Lardy B, Krause KH (2007) NOX family NADPH oxidases: not just in mammals. Biochimie 89(9):1107–1112
Nisimoto Y, Diebold BA, Cosentino-Gomes D, Lambeth JD (2014) Nox4: a hydrogen peroxide-generating oxygen sensor. Biochemistry 53(31):5111–5120
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):1077–1081
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):231ra247
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):431–440
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):12384–12389
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):340–348
Thannickal VJ, Zhou Y, Gaggar A, Duncan SR (2014) Fibrosis: ultimate and proximate causes. J Clin Invest 124(11):4673–4677
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):1730–1741
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):289–296
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):e45285
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):810–816
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):832–837
Collard HR (2010) The age of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 181(8):771–772
Thannickal VJ (2013) Mechanistic links between aging and lung fibrosis. Biogerontology 14(6):609–615
Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153(6):1194–1217
Mora AL, Bueno M, Rojas M (2017) Mitochondria in the spotlight of aging and idiopathic pulmonary fibrosis. J Clin Invest 127(2):405–414
Schafer MJ, Haak AJ, Tschumperlin DJ, LeBrasseur NK (2018) Targeting senescent cells in fibrosis: pathology, paradox, and practical considerations. Curr Rheumatol Rep 20(1):3
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):25,427–25,438
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):1218–1228
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):667–679
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):532–533
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):596–602
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):607–619
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–F1287
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):e50172
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):e0170561
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 12:8
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):854–856
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):3029–3038
Acknowledgments
This work was supported by grants from the National Institute of Health: P01 HL114470 and R01 AG046210 (to VJT).
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Bernard, K., Thannickal, V.J. (2019). NADPH Oxidases and Aging Models of Lung Fibrosis. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_29
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DOI: https://doi.org/10.1007/978-1-4939-9424-3_29
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