June 2013
Volume 54, Issue 15
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ARVO Annual Meeting Abstract  |   June 2013
Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride
Mary Feng; Julia Baryla; Hong Liu; Gordon Laurie; Robert McKown; Negin Ashki; Dinesh Bhayana; Cindy Hutnik
Author Affiliations & Notes
  • Mary Feng
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Julia Baryla
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Hong Liu
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Gordon Laurie
    Department of Cell Biology, University of Virginia, Charlottesville, VA
  • Robert McKown
    Department of Integrated Science and Technology, James Madison University, Harrisonburg, VA
  • Negin Ashki
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Dinesh Bhayana
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Cindy Hutnik
    Department of Ophthalmology, University of Western Ontario, London, ON, Canada
  • Footnotes
    Commercial Relationships Mary Feng, None; Julia Baryla, None; Hong Liu, None; Gordon Laurie, UVa Patent Foundation (F); Robert McKown, EyeRx Research, Inc. (I); Negin Ashki, None; Dinesh Bhayana, None; Cindy Hutnik, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 973. doi:
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      Mary Feng, Julia Baryla, Hong Liu, Gordon Laurie, Robert McKown, Negin Ashki, Dinesh Bhayana, Cindy Hutnik; Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride. Invest. Ophthalmol. Vis. Sci. 2013;54(15):973.

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Abstract
 
Purpose
 

Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the prosecretory and mitogenic properties of lacritin confer protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK.

 
Methods
 

Recombinant human lacritin and negative control fragment C-25 were cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established after exposure of subconfluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK.

 
Results
 

BAK reduced CRL-11515 cellular metabolic activity in a time and dose dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (P<0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 hours prior to BAK exposure significantly dampened levels of LC3-II (P<0.05) and promoted a 12% increase in cellular metabolic activity (P<0.01) when compared to BAK alone.

 
Conclusions
 

These results suggest lacritin protects cultured HCE cells stressed with BAK and it may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK.

 
BAK treatment of CRL-11515 cells increases autophagy. (A) CRL-11515 cells treated with BAK (0.001%, 0.004%) for 1 minute increased cellular lipidated LC3, known as ‘LC3-II‘ with increasing concentration of BAK (**P<0.01, *P<0.05; n=6). (B) Western blot of LC3-II with GAPDH as loading control.
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BAK treatment of CRL-11515 cells increases autophagy. (A) CRL-11515 cells treated with BAK (0.001%, 0.004%) for 1 minute increased cellular lipidated LC3, known as ‘LC3-II‘ with increasing concentration of BAK (**P<0.01, *P<0.05; n=6). (B) Western blot of LC3-II with GAPDH as loading control.
 
BAK treatment of CRL-11515 cells increases autophagy. (A) CRL-11515 cells treated with BAK (0.001%, 0.004%) for 1 minute increased cellular lipidated LC3, known as ‘LC3-II‘ with increasing concentration of BAK (**P<0.01, *P<0.05; n=6). (B) Western blot of LC3-II with GAPDH as loading control.
BAK treatment of CRL-11515 cells increases autophagy. (A) CRL-11515 cells treated with BAK (0.001%, 0.004%) for 1 minute increased cellular lipidated LC3, known as ‘LC3-II‘ with increasing concentration of BAK (**P<0.01, *P<0.05; n=6). (B) Western blot of LC3-II with GAPDH as loading control.
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Lacritin treatment of CRL-11515 cells rescues BAK induced autophagy. (A) LC3-II increased in CRL-11515 cells treated with BAK (0.004%) compared to control (**P<0.01 vs. control; n=5). L24h/BAK significantly reduced LC3-II compared to BAK treated cells (†P<0.05 vs. BAK; n=5). (B) Western Blot of LC3-II with GAPDH as loading control. BAK = BAK, 1 min. BAK+L = BAK and lacritin, 1 min. L24h/BAK = Pre-incubation with lacritin, 24 hours, followed by BAK treatment for 1 min.
View OriginalDownload Slide
 
Lacritin treatment of CRL-11515 cells rescues BAK induced autophagy. (A) LC3-II increased in CRL-11515 cells treated with BAK (0.004%) compared to control (**P<0.01 vs. control; n=5). L24h/BAK significantly reduced LC3-II compared to BAK treated cells (†P<0.05 vs. BAK; n=5). (B) Western Blot of LC3-II with GAPDH as loading control. BAK = BAK, 1 min. BAK+L = BAK and lacritin, 1 min. L24h/BAK = Pre-incubation with lacritin, 24 hours, followed by BAK treatment for 1 min.
 
Lacritin treatment of CRL-11515 cells rescues BAK induced autophagy. (A) LC3-II increased in CRL-11515 cells treated with BAK (0.004%) compared to control (**P<0.01 vs. control; n=5). L24h/BAK significantly reduced LC3-II compared to BAK treated cells (†P<0.05 vs. BAK; n=5). (B) Western Blot of LC3-II with GAPDH as loading control. BAK = BAK, 1 min. BAK+L = BAK and lacritin, 1 min. L24h/BAK = Pre-incubation with lacritin, 24 hours, followed by BAK treatment for 1 min.
Lacritin treatment of CRL-11515 cells rescues BAK induced autophagy. (A) LC3-II increased in CRL-11515 cells treated with BAK (0.004%) compared to control (**P<0.01 vs. control; n=5). L24h/BAK significantly reduced LC3-II compared to BAK treated cells (†P<0.05 vs. BAK; n=5). (B) Western Blot of LC3-II with GAPDH as loading control. BAK = BAK, 1 min. BAK+L = BAK and lacritin, 1 min. L24h/BAK = Pre-incubation with lacritin, 24 hours, followed by BAK treatment for 1 min.
View OriginalDownload Slide
 
Keywords: 482 cornea: epithelium • 426 apoptosis/cell death  
© 2013, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2024 Association for Research in Vision and Ophthalmology.
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