May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Urokinase down–regulation by aprotinin after photorefractive keratectomy in rabbit corneal epithelial cells
Author Affiliations & Notes
  • A. Csutak
    Ophthalmology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • D.M. Silver
    Ophthalmology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
    Applied Physics Laboratory, Johns Hopkins University, Laurel, MD
  • T. Sperka
    Biochemistry & Molecular Biology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • J. Kadas
    Biochemistry & Molecular Biology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • G. Vereb
    Biophysics & Cell Biology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • A. Berta
    Ophthalmology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • J. Tőzsér
    Biochemistry & Molecular Biology,
    University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary
  • Footnotes
    Commercial Relationships  A. Csutak, None; D.M. Silver, None; T. Sperka, None; J. Kadas, None; G. Vereb, None; A. Berta, None; J. Tőzsér, None.
  • Footnotes
    Support  Hungarian Scientific Research Fund, OTKA Grant #T038351
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 1415. doi:
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      A. Csutak, D.M. Silver, T. Sperka, J. Kadas, G. Vereb, A. Berta, J. Tőzsér; Urokinase down–regulation by aprotinin after photorefractive keratectomy in rabbit corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1415.

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

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Abstract

Abstract: : Purpose: To investigate the effect of aprotinin, a serine protease inhibitor, on urokinase–type plasminogen activator (uPA) in rabbit corneal epithelial cells during wound healing after photorefractive keratectomy (PRK). Methods: Both eyes of 4 rabbits had PRK and were treated with antibiotic. One eye of each was treated additionally with aprotinin. The animals were sacrificed at 4 hours, 1, 3 and 5 days. A fifth rabbit served as control (one eye no PRK, other eye PRK but not treated) and was sacrificed 2 hours after PRK. Frozen sections of corneal tissues were prepared for in situ zymography to detect uPA activity. Reverse transcription (RT) and real–time quantitative PCR (QPCR) were used to determine uPA mRNA in corneal tissue samples. The animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Results: In situ zymography revealed that plasminogen–dependent casein hydrolysis was restricted to the leading edge of migrating epithelial cells in no–aprotinin and aprotinin treated corneal tissue after PRK. The hydrolysis was inhibited by a polyclonal anti–uPA antibody, amiloride, but not with polyclonal anti–tPA antibody. No plasminogen–independent lysis was observed. The control uPA mRNA was zero from the no–PRK eye and 0.0026 uPA/18S RNA from the PRK eye at 2 hours post–op. Relative to the 2–hour post–op uPA mRNA control, the no–aprotinin–treated uPA mRNA values were 58% at 4 hours and 21% at 1 day, while the aprotinin–treated values were 31% and 15%, respectively. No uPA mRNA was found at 3 or 5 days. Conclusions: In our previously published rabbit study, aprotinin treatment for a few days after PRK suppressed uPA activity in tears. The present zymography qualitatively shows uPA–specific activity in migrating epithelial cells even in the presence of aprotinin, while the RT–QPCR quantitatively indicates down–regulation of uPA by aprotinin at the mRNA level in these cells. Taken together, these experiments demonstrate that aprotinin reduces the normally available concentration of uPA from both tears and intracellular corneal sources. With this post–PRK aprotinin–induced uPA deficiency, the rabbit corneas eventually develop haze, as found in the previous study. This suggests that uPA needs to be available at levels exceeding some minimum threshold to support the uPA enzyme cascade responsible for promoting proper corneal wound healing without haze.

Keywords: wound healing • enzymes/enzyme inhibitors • cornea: basic science 
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