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International Ophthalmology

, Volume 38, Issue 5, pp 1895–1905 | Cite as

Comparative proteome analysis of the tear samples in patients with low-grade keratoconus

  • Fatih Yenihayat
  • Özgül Altıntaş
  • Murat Kasap
  • Gürler Akpınar
  • Nil Güzel
  • Onur Sinan Çelik
Original Paper
  • 150 Downloads

Abstract

Purpose

To elucidate the metabolic processes playing roles in the formation of keratoconus (KC).

Methods

Tears samples were collected using capillary glass tubes without stimulation and without prior anesthesia from 17 patients and 16 controls. Proteomic analysis by fluorescent 2D gel electrophoresis (DIGE) coupled with MALDI-TOF/TOF was performed. The identified proteins that were differentially regulated were subjected to Ingenuity Pathway Analysis (IPA). Corneal topography analyses with Sirius topography system (Costruzioni Strumenti Oftalmici, Florence, Italy) were performed on all participants. The steepest keratometry index was lower than 50 diopters in all keratoconus patients.

Results

DIGE analysis showed changes in abundance of nine proteins. Six of these proteins, namely serum albumin, Keratin Type II Cytoskeletal 1, IgG gamma chain-1, GAPDH, alpha-1 antitrypsin and ApoA-I, were down-regulated in the KC samples in comparison with the controls. In addition, we detected up-regulation of lysozyme C, keratin type I cytoskeletal 10 and lipocalin. The subsequent IPA predicted that NADH repair pathway is activated in the KC patients. This pathway involves generation of NADHX as a by-product via catalysis by GAPDH. NADHX is an inhibitor of several dehydrogenases and must be removed.

Conclusion

The involvement of NADHX repair pathway in KC should be investigated, since preliminary clues obtained in this study point to that direction. In particular, showing the presence of ATP-dependent NAD(P)H-hydrate dehydratase that eliminates NADHX would strengthen our findings and would be a major step toward understanding KC.

Keywords

Tear proteome Keratoconus DIGE MALDI-TOF/TOF 

Notes

Acknowledgements

This study was supported by Kocaeli University Scientific Research Project Coordination Unit (KOU-BAP: Project Number: 2014/052).

Compliance with ethical standards

Conflict of interest

Author Fatih Yenihayat declares that he has no conflict of interest. Author Özgül Altıntaş declares that she has no conflict of interest. Author Murat Kasap declares that he has no conflict of interest. Author Gürler Akpınar declares that he has no conflict of interest. Author Nil Güzel declares that she has no conflict of interest. Author Onur Sinan Çelik declares that he has no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional ethical committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Krachmer JH, Feder RS, Belin MW (1984) Keratoconus and related non inflammatory corneal thinning disorders. Surv Ophthalmol 28:293–322CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Rabinowitz YS (1998) Keratoconus. Surv Ophthalmol 42:297–319CrossRefPubMedCentralGoogle Scholar
  3. 3.
    Vazirani J, Basu S (2013) Keratoconus: current perspectives. Clin Ophthalmol 7:2019–2030. doi: 10.2147/OPTH.S50119 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lema I, Sobrino T, Durán JA et al (2009) Subclinical keratoconus and inflammatory molecules from tears. Br J Ophthalmol 93:820–824. doi: 10.1136/bjo.2008.144253 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kenney MC, Brown DJ (2003) The cascade hypothesis of keratoconus. Cont Lens Anterior Eye 26(3):139–146. doi: 10.1016/S1367-0484(03)00022-5 CrossRefGoogle Scholar
  6. 6.
    Kao WW, Vergnes JP, Ebert J et al (1982) Increased collagenase and gelatinase activities in keratoconus. Biochem Biophys Res Commun 107:929–936CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Lema I, Durán JA (2005) Inflammatory molecules in the tears of patients with keratoconus. Ophthalmology 112:654–659. doi: 10.1016/j.ophtha.2004.11.050 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Toprak I, Kucukatay V, Yildirim C et al (2014) Increased systemic oxidative stress in patients with keratoconus. Eye (Lond) 28:285–289. doi: 10.1038/eye.2013.262 CrossRefGoogle Scholar
  9. 9.
    Chwa M, Atilano SR, Hertzog D et al (2008) Hypersensitive response to oxidative stress in keratoconus corneal fibroblasts. Invest Ophthalmol Vis Sci 49:4361–4369. doi: 10.1167/iovs.08-1969 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Buddi R, Lin B, Atilano SR et al (2002) Evidence of oxidative stress in human corneal diseases. J Histochem Cytochem 50:341–351. doi: 10.1177/002215540205000306 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kenney MC, Chwa M, Atilano SR et al (2005) Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci 46:823–832. doi: 10.1167/iovs.04-0549 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Rabinowitz YS, Garbus J, McDonnell PJ (1990) Computer-assisted corneal topography in family members of patients with keratoconus. Arch Ophthalmol 108:365–371 (PMID: 2310336) CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Kenney MC, Chwa M, Escobar M, Brown D (1989) Altered gelatinolytic activity by keratoconus corneal cells. Biochem Biophys Res Commun 161:353–357CrossRefPubMedCentralGoogle Scholar
  14. 14.
    Kaldawy RM, Wagner J, Ching S, Seigel GM (2002) Evidence of apoptotic cell death in keratoconus. Cornea 21:206–209CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Sawaguchi S, Twining SS, Yue BY, Wilson PM, Sugar J, Chan SK (1990) Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res 50:549–554CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Chandler JW (1985) Immunology of the ocular surface. Int Ophthalmol Clin 25:13–23CrossRefPubMedCentralGoogle Scholar
  17. 17.
    Bonini S, Lambiase A, Sgrulletta R, Bonini S (2003) Allergic chronic inflammation of the ocular surface in vernal keratoconjunctivitis. Curr Opin Allergy Clin Immunol 3:381–387. doi: 10.1097/01.all.0000092610.76804.8a CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhou L, Sawaguchi S, Twining SS, Sugar J, Feder RS, Yue BY (1998) Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci 39:1117–1124PubMedPubMedCentralGoogle Scholar
  19. 19.
    Collier SA, Madigan MC, Penfold PL (2000) Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas. Curr Eye Res 21:662–668CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Galvis V, Sherwin T, Tello A, Merayo J, Barrera R, Acera A (2015) Keratoconus: an inflammatory disorder? Eye 29:843–859. doi: 10.1038/eye.2015.63 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Shoham A, Hadziahmetovic M, Dunaief JL, Mydlarski MB, Schipper HM (2008) Oxidative stress in diseases of the human cornea. Free Radic Biol Med 45:1047–1055. doi: 10.1016/j.freeradbiomed.2008.07.021 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chwa M, Atilano SR, Reddy V, Jordan N, Kim DW, Kenney MC (2006) Increased stress-induced generation of reactive oxygen species and apoptosis in human keratoconus fibroblasts. Invest Ophthalmol Vis Sci 47(5):1902–1910. doi: 10.1167/iovs.05-0828 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Arnal E, Peris-Martínez C, Menezo JL, Johnsen-Soriano S, Romero FJ (2011) Oxidative stress in keratoconus? Invest Ophthalmol Vis Sci 52:8592–8597. doi: 10.1167/iovs.11-7732 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wojcik KA, Kaminska A, Blasiak J, Szaflik J, Szaflik JP (2013) Oxidative stress in the pathogenesis of keratoconus and Fuchs endothelial corneal dystrophy. Int J Mol Sci 14:19294–19308. doi: 10.3390/ijms140919294 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Acera A, Vecino E, Rodriguez-Agirretxe I et al (2011) Changes in tear protein profile in keratoconus disease. Eye 25:1225–1233. doi: 10.1038/eye.2011 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lema I, Brea D, Rodríguez-González R, Díez-Feijoo E, Sobrino T (2010) Proteomic analysis of the tear film in patients with keratoconus. Mol Vis 16:2055–2061PubMedPubMedCentralGoogle Scholar
  27. 27.
    Zadnik K, Barr JT, Gordon MO, Edrington TB (1996) Biomicroscopic signs and disease severity in keratoconus. Collaborative longitudinal evaluation of keratoconus (CLEK) study group. Cornea 15:139–146CrossRefPubMedCentralGoogle Scholar
  28. 28.
    Acheson SA, Kirkman HN, Wolfenden R (1988) Equilibrium of 5,6 hydration of NADH and mechanism of ATP-dependent hydration. Biochemistry 27(19):7371–7375CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Marbaix AY, Noël G, Detroux AM, Vertommen D, Van Schaftingen E, Linster CL (2011) Extremely conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide repair. J Biol Chem 286(48):41246–41252. doi: 10.1074/jbc.C111.310847 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Colinas M, Shaw HV, Loubéry S, Kaufmann M, Moulin M, Fitzpatrick TB (2014) A pathway for repair of NAD(P)H in plants. J Biol Chem 289(21):14692–14706. doi: 10.1074/jbc.M114.556092 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Runström G, Mann A, Tighe B (2013) The fall and rise of tear albumin levels: a multifactorial phenomenon. Ocul Surf 11(3):165–180. doi: 10.1016/j.jtos.2013.03.001 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Balasubramanian SA, Pye DC, Willcox MD (2012) Levels of lactoferrin, secretory IgA and serum albumin in the tear film of people with keratoconus. Exp Eye Res 96(1):132–137. doi: 10.1016/j.exer.2011.12.010 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhou L, Beuerman RW, Chan CM et al (2009) Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics. J Proteome Res 8(11):4889–4905. doi: 10.1021/pr900686s CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Yamada M, Mochizuki H, Kawai M, Tsubota K, Bryce TJ (2005) Decreased tear lipocalin concentration in patients with meibomian gland dysfunction. Br J Ophthalmol 89(7):803–805. doi: 10.1136/bjo.2004.055822 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Breustedt DA, Korndörfer IP, Redl B, Skerra A (2005) The 1.8-A crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands. J Biol Chem 280(1):484–493. doi: 10.1074/jbc.M410466200 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wojnar P, Dirnhofer S, Ladurner P, Berger P, Redl B (2002) Human lipocalin-1, a physiological scavenger of lipophilic compounds, is produced by corticotrophs of the pituitary gland. J Histochem Cytochem 50(3):433–435. doi: 10.1177/002215540205000314 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Redl B (2000) Human tear lipocalin. Biochim Biophys Acta 1482(1–2):241–248CrossRefPubMedCentralGoogle Scholar
  38. 38.
    Glasson M, Stapleton F, Willcox M (2002) Lipid, lipase and lipocalin differences between tolerant and intolerant contact lens wearers. Curr Eye Res 25(4):227–235CrossRefPubMedCentralGoogle Scholar
  39. 39.
    Prause JU (1983) Serum albumin, serum antiproteases, and polymorphonuclear leucocyte neutral collagenolytic protease in the tear fluid of normal healthy persons. Acta Ophthalmol (Copenh) 61(2):261–271CrossRefGoogle Scholar
  40. 40.
    Sawaguchi S, Yue BYJT, Sugar J, Gilboy JE (1989) Lysosomal enzyme abnormalities in keratoconus. Arch Ophthalmol 107:1507–1510CrossRefPubMedCentralGoogle Scholar
  41. 41.
    Sawaguchi S, Twining SS, Yue BYJT et al (1994) a2-Macroglobulin levels in normal human and keratoconus corneas. Invest Ophthalmol Vis Sci 35:4008–4014PubMedPubMedCentralGoogle Scholar
  42. 42.
    Gupta AK, Sarin GS, Mathur MD, Ghosh B (1988) α1-Antitrypsin and serum albumin in tear fluids in acute adenovirus conjunctivitis. Br J Ophthalmol 72:390–393CrossRefPubMedCentralGoogle Scholar
  43. 43.
    Ananthi S, Venkatesh Prajna N, Lalitha P, Valarnila M, Dharmalingam K (2013) Pathogen induced changes in the protein profile of human tears from Fusarium keratitis patients. PLoS ONE 8(1):e53018. doi: 10.1371/journal.pone.0053018 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Barrett A, Gnehm D, Jones J, Trask BC (2014) α1-antitrypsin and C-reactive protein levels in tear fluid after continuous contact lens wear. Clin Exp Optom 97:66–71. doi: 10.1111/cxo.12093 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Mao J, Liu W, Wang Y (2014) Apolipoprotein A-I expression suppresses COX-2 expression by reducing reactive oxygen species in hepatocytes. Biochem Biophys Res Commun 454(3):359–363. doi: 10.1016/j.bbrc.2014.10.094 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Kawai S, Nakajima T, Hokari S, Komoda T, Kawai K (2002) Apolipoprotein A-I concentration in tears in diabetic retinopathy. Ann Clin Biochem 39(Pt 1):56–61. doi: 10.1258/0004563021901748 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Li B, Sheng M, Li J et al (2014) Tear proteomic analysis of Sjögren syndrome patients with dry eye syndrome by two-dimensional-nano-liquid chromatography coupled with tandem mass spectrometry. Sci Rep 4:5772. doi: 10.1038/srep05772 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Sen DK, Sarin GS (1979) Immunoglobulin concentrations in human tears in ocular diseases. Br J Ophthalmol 63(5):297–300CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of OphthalmologyKocaeli State HospitalKocaeliTurkey
  2. 2.Department of Ophthalmology, Medical SchoolAcıbadem UniversityIstanbulTurkey
  3. 3.DEKART Proteomics Laboratory, Medical SchoolKocaeli UniversityKocaeliTurkey
  4. 4.Department of Medical Biology, Medical SchoolKocaeli UniversityKocaeliTurkey
  5. 5.Department of OphthalmologyTunceli State HospitalTunceliTurkey

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