June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Arginase, P4H and iNOS expression and the effect of iNOS inhibitor 1400W on urea, hydroxyproline and nitrite formation of keratoconus keratocytes
Author Affiliations & Notes
  • Tanja Stachon
    Department of Ophthalmology, Saarland University Medical Center, Homburg , Germany
  • Lorenz Latta
    Department of Ophthalmology, Saarland University Medical Center, Homburg , Germany
  • Achim Langenbucher
    Experimental Ophthalmology, Saarland University, Homburg, Germany
  • Berthold Seitz
    Department of Ophthalmology, Saarland University Medical Center, Homburg , Germany
  • Nóra Szentmáry
    Department of Ophthalmology, Saarland University Medical Center, Homburg , Germany
    Department of Ophthalmology, Semmelweis University, Budapest, Hungary
  • Footnotes
    Commercial Relationships   Tanja Stachon, None; Lorenz Latta, None; Achim Langenbucher, None; Berthold Seitz, None; Nóra Szentmáry, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3557. doi:
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      Tanja Stachon, Lorenz Latta, Achim Langenbucher, Berthold Seitz, Nóra Szentmáry; Arginase, P4H and iNOS expression and the effect of iNOS inhibitor 1400W on urea, hydroxyproline and nitrite formation of keratoconus keratocytes
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):3557.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Keratoconus (KC) is a stromal disease of the cornea, which leads to conical shape and loss of vision in later stages. Arginase is a cytoplasmatic enzyme that catalyzes the conversion of L-arginine to urea and ornithine, which serves as a precursor for the synthesis of proline and hydroxyproline (determinate parts of collagen strands of the corneal stroma). The competing enzyme for the substrate L-arginine is nitric-oxid synthase (iNOS). In a previous study we determined decreased arginase activity, urea and hydroxyproline concentration in KC keratocytes. The purpose of our present study was to evaluate arginase, prolyl-4-hydroxylase and iNOS expression in KC keratocytes and to determine the effect of the selective iNOS inhibitor 1400W on urea, hydroxyproline and nitrite (NO) formation of these cells.

Methods : Primary human KC (n=8) and normal (n=8) keratocytes were isolated by digestion in collagenase A from human corneal buttons, and cultured in DMEM/Ham’s F12 medium supplemented with 5% fetal calf serum. Arginase and iNOS were evaluated using qPCR and Western blot analysis. The amount of prolyl-4-hydroxylase was determined using flow cytometry. Following 10, 20 and 40µM iNOS inhibitor 1400W treatment, urea, hydroxyproline and NO concentration in KC keratocytes were assessed using colorimetric assays.

Results : KC keratocytes showed a 6-fold increased iNOS mRNA expression compared to normal cells, whereas Arginase expression remained unchained. Arginase could not be detected using Western blot analysis, and iNOS expression showed no differences in normal and KC keratocytes. The amount of prolyl-4-hydroxylase was decreased in KC keratocytes compared to controls (p=0.038). Using all of the above inhibitor concentrations, urea (p<0.01) and NO (p<0.007) concentrations were significantly decreased in KC keratocytes, but hydroxyproline concentration remained unchanged (p>0.09).

Conclusions : Our results indicate that the expression of the urea cycle enzymes is altered in KC keratocytes. The iNOS inhibitor 1400W decreases urea and NO formation without changes in hydroxyproline concentration in these cells. Therefore, its topical use does not seem to be a potential treatment option for keratoconus patients. Further investigations concerning the regulatory processes in collagen synthesis are needed to fully understand the pathogenesis of keratoconus.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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