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Transforming Growth Factor: β3 Regulates Cell Metabolism in Corneal Keratocytes and Fibroblasts

  • D. Karamichos
  • J. M. Asara
  • J. D. Zieske
Chapter
Part of the Oxidative Stress in Applied Basic Research and Clinical Practice book series (OXISTRESS)

Abstract

Corneal keratocytes (HCKs) are specialized cells that reside in the corneal stroma, the largest layer of the human cornea. HCKs play a major role in corneal transparency and orchestrate corneal wound healing upon injury. Following an injury to the cornea HCKs adjacent to the wound undergo apoptosis and HCKs outside the wound area are activated, and they assume the role of remodeling the damaged stroma. Once activated the cells are termed fibroblasts (HCF). One of the key factors of this activation is thought to be transforming growth factor-beta (TGF-β). Five TGF-β (-β1,-β2,-β3,-β4, and -β5) have been identified and three (-β1,-β2, and -β3) are found in humans. We have recently identified TGF-β3 as a growth factor that can reduce or rescue corneal fibrosis in vitro. In this chapter, we present evidence that TGF-β3 plays a major role in regulating the metabolism of both HCKs and HCFs in vitro. We investigated both a conventional monolayer 2D system and a 3D self-assembled extracellular matrix (ECM) model. We targeted a total of 256 endogenous water-soluble metabolites by LC-MS/MS of which more than 60 were significantly regulated between different groups. These findings are expected to help achieve a better understanding of the specific and redundant functions of these metabolites.

Keywords

Metabolomics Keratocytes Fibroblasts Corneal stroma 

Abbreviations

ECM

Extracellular matrix

GNG

Glucogenesis

HCF

Human corneal fibroblasts

HCK

Human corneal keratocytes

TCA

Tricarboxylic acid cycle

TGF-β

Transforming growth factor-beta

Notes

Acknowledgments

This work was supported by National Institutes of Health Grant EY023568 (D.K) and EY020886 (DK and JDZ)

References

  1. 1.
    Patel SV, McLaren JW, Hodge DO, Bourne WM. Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci. 2001;42(2):333–9.PubMedGoogle Scholar
  2. 2.
    Reinstein DZ, Archer TJ, Gobbe M, Silverman RH, Coleman DJ. Stromal thickness in the normal cornea: three-dimensional display with artemis very high-frequency digital ultrasound. J Refract Surg. 2009;25(9):776–86.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Maurice DM. The structure and transparency of the cornea. J Physiol. 1957;136(2):263–86.PubMedCentralPubMedGoogle Scholar
  4. 4.
    Muller LJ, Pels E, Schurmans LR, Vrensen GF. A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma. Exp Eye Res. 2004;78(3):493–501.PubMedCrossRefGoogle Scholar
  5. 5.
    Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci. 1995;108(Pt 3):985–1002.PubMedGoogle Scholar
  6. 6.
    Karamichos D, Hutcheon AE, Zieske JD. Transforming growth factor-beta3 regulates assembly of a non-fibrotic matrix in a 3D corneal model. J Tissue Eng Regen Med. 2011;5(8):e228–38.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Karamichos D, Rich CB, Zareian R, Hutcheon AE, Ruberti JW, Trinkaus-Randall V, et al. TGF-beta3 stimulates stromal matrix assembly by human corneal keratocyte-like cells. Invest Ophthalmol Vis Sci. 2013;54(10):6612–9.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Dettmer K, Aronov PA, Hammock BD. Mass spectrometry-based metabolomics. Mass Spectrom Rev. 2007;26(1):51–78.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Rochfort S. Metabolomics reviewed: a new “omics” platform technology for systems biology and implications for natural products research. J Nat Prod. 2005;68(12):1813–20.PubMedCrossRefGoogle Scholar
  10. 10.
    Greiner JV, Kopp SJ, Glonek T. Phosphorus nuclear magnetic resonance and ocular metabolism. Surv Ophthalmol. 1985;30(3):189–202.PubMedCrossRefGoogle Scholar
  11. 11.
    Risa O, Saether O, Lofgren S, Soderberg PG, Krane J, Midelfart A. Metabolic changes in rat lens after in vivo exposure to ultraviolet irradiation: measurements by high resolution MAS 1H NMR spectroscopy. Invest Ophthalmol Vis Sci. 2004;45(6):1916–21.PubMedCrossRefGoogle Scholar
  12. 12.
    Fu H, Khan A, Coe D, Zaher S, Chai JG, Kropf P, et al. Arginine depletion as a mechanism for the immune privilege of corneal allografts. Eur J Immunol. 2011;41(10):2997–3005.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Klyce SD. Stromal lactate accumulation can account for corneal oedema osmotically following epithelial hypoxia in the rabbit. J Physiol. 1981;321:49–64.PubMedCentralPubMedGoogle Scholar
  14. 14.
    Nguyen TT, Bonanno JA. Lactate-H+ transport is a significant component of the in vivo corneal endothelial pump. Invest Ophthalmol Vis Sci. 2012;53(4):2020–9.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Guo X, Hutcheon AE, Melotti SA, Zieske JD, Trinkaus-Randall V, Ruberti JW. Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts. Invest Ophthalmol Vis Sci. 2007;48(9):4050–60.PubMedCrossRefGoogle Scholar
  16. 16.
    Karamichos D, Guo XQ, Hutcheon AE, Zieske JD. Human corneal fibrosis: an in vitro model. Invest Ophthalmol Vis Sci. 2010;51(3):1382–8.PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Webhofer C, Gormanns P, Reckow S, Lebar M, Maccarrone G, Ludwig T, et al. Proteomic and metabolomic profiling reveals time-dependent changes in hippocampal metabolism upon paroxetine treatment and biomarker candidates. J Psychiatr Res. 2013;47(3):289–98.PubMedCrossRefGoogle Scholar
  18. 18.
    Yuan M, Breitkopf SB, Yang X, Asara JM. A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc. 2012;7(5):872–81.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Xia J, Mandal R, Sinelnikov IV, Broadhurst D, Wishart DS. MetaboAnalyst 2.0—a comprehensive server for metabolomic data analysis. Nucleic Acids Res. 2012;40(Web Server issue):W127–33.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Donohoe DR, Garge N, Zhang X, Sun W, O’Connell TM, Bunger MK, et al. The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon. Cell Metab. 2011;13(5):517–26.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Tanaka H, Nishida T. Butyrate stimulates fibronectin synthesis in cultured rabbit cornea. J Cell Physiol. 1985;123(2):191–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Lee VH, Chien DS, Sasaki H. Ocular ketone reductase distribution and its role in the metabolism of ocularly applied levobunolol in the pigmented rabbit. J Pharmacol Exp Ther. 1988;246(3):871–8.PubMedGoogle Scholar
  23. 23.
    Tsai CP, Lin PY, Lee NC, Niu DM, Lee SM, Hsu WM. Corneal lesion as the initial manifestation of tyrosinemia type II. J Chin Med Assoc. 2006;69(6):286–8.PubMedCrossRefGoogle Scholar
  24. 24.
    Mahmoud SS. Corneal toxicity associated with ammonia exposure investigated by Fourier transform infrared spectroscopy. Biophys Rev Lett. 2009;04(4):331.CrossRefGoogle Scholar
  25. 25.
    Nelson DL, Cox MM. Lehninger principles of biochemistry. New York: Worth Publishers; 2000. p. 724.Google Scholar
  26. 26.
    Young JW. Gluconeogenesis in cattle: significance and methodology. J Dairy Sci. 1977;60:1–15.PubMedCrossRefGoogle Scholar
  27. 27.
    George SJ, Bernard AW, Albers RW, Stephen FK, Michael UD, editors. Basic neurochemistry. 6th ed. Philadelphia: Lippincott-Raven; 1999.Google Scholar
  28. 28.
    Lowenstein JM. Methods in enzymology, volume 13: citric acid cycle. Boston: Academic; 1969.Google Scholar
  29. 29.
    Krebs HA, Weitzman PDJ. Krebs’ citric acid cycle: half a century and still turning. London: Biochemical Society; 1987.Google Scholar
  30. 30.
    Lane N. Life ascending: the ten great inventions of evolution. New York: W. W. Norton & Co; 2009.Google Scholar
  31. 31.
    Monty K, Matthew PS, Matsudaira PT, Lodish HF, Darnell JE, Lawrence Z, et al. Molecular cell biology. 5th ed. San Francisco: W. H. Freeman; 2003. p. 973.Google Scholar
  32. 32.
    Lehninger AL, Nelson DL, Cox MM. Principles of biochemistry. 5th ed. New York, NY: W.H. Freeman and Company; 2008. p. 528.Google Scholar
  33. 33.
    Schutte E, Schulz I, Reim M. Lactate and pyruvate levels in the aqueous humor and in the cornea after cyclodiathermy. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1972;185(4):325–30.PubMedCrossRefGoogle Scholar
  34. 34.
    Lamers Y, O’Rourke B, Gilbert LR, Keeling C, Matthews DE, Stacpoole PW, et al. Vitamin B-6 restriction tends to reduce the red blood cell glutathione synthesis rate without affecting red blood cell or plasma glutathione concentrations in healthy men and women. Am J Clin Nutr. 2009;90(2):336–43.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Lima CP, Davis SR, Mackey AD, Scheer JB, Williamson J, Gregory 3rd JF. Vitamin B-6 deficiency suppresses the hepatic transsulfuration pathway but increases glutathione concentration in rats fed AIN-76A or AIN-93G diets. J Nutr. 2006;136(8):2141–7.PubMedGoogle Scholar
  36. 36.
    Hsu JM, Buddemeyer E, Chow BF. Role of pyridoxine in glutathione metabolism. Biochem J. 1964;90(1):60–4.PubMedCentralPubMedGoogle Scholar
  37. 37.
    Davis SR, Quinlivan EP, Stacpoole PW, Gregory 3rd JF. Plasma glutathione and cystathionine concentrations are elevated but cysteine flux is unchanged by dietary vitamin B-6 restriction in young men and women. J Nutr. 2006;136(2):373–8.PubMedGoogle Scholar
  38. 38.
    Robertson DG, Watkins PB, Reily MD. Metabolomics in toxicology: preclinical and clinical applications. Toxicol Sci. 2011;120 Suppl 1:S146–70.PubMedCrossRefGoogle Scholar
  39. 39.
    Chan AA, Hertsenberg AJ, Funderburgh ML, Mann MM, Du Y, Davoli KA, et al. Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype. PLoS One. 2013;8(2):e56831.PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Petroll WM, Lakshman N, Ma L. Experimental models for investigating intra-stromal migration of corneal keratocytes, fibroblasts and myofibroblasts. J Funct Biomater. 2012;3(1):183–98.PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Jester JV, Budge A, Fisher S, Huang J. Corneal keratocytes: phenotypic and species differences in abundant protein expression and in vitro light-scattering. Invest Ophthalmol Vis Sci. 2005;46(7):2369–78.PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Khaw PT, Shah P, Elkington AR. Injury to the eye. BMJ. 2004;328(7430):36–8.PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Netto MV, Mohan RR, Ambrosio Jr R, Hutcheon AE, Zieske JD, Wilson SE. Wound healing in the cornea: a review of refractive surgery complications and new prospects for therapy. Cornea. 2005;24(5):509–22.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of OphthalmologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Division of Signal Transduction and Mass Spectrometry Core, Beth Israel Deaconess Medical Center and the Department of MedicineHarvard Medical SchoolBostonUSA
  3. 3.Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of OphthalmologyHarvard Medical SchoolBostonUSA

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