July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Targeting of Dynamin, an Essential Regulator of Endocytosis, Protects the Ocular Surface against Oxidative Stress
Author Affiliations & Notes
  • Andrew Webster
    Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Shravan K Chintala
    Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Joseph Barr
    Optometry, Ohio State University, Columbus, Ohio, United States
  • Noorjahan A Panjwani
    Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Pablo Argueso
    Schepens Eye Research Institute, Boston, Massachusetts, United States
  • Shinwu Jeong
    Roski Eye Institute, Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • M Elizabeth Fini
    Keck School of Medicine, University of Southern California, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Andrew Webster, Andrew Webster (P); Shravan Chintala, None; Joseph Barr, None; Noorjahan Panjwani, None; Pablo Argueso, None; Shinwu Jeong, Shinwu Jeong (P); M Elizabeth Fini, M Elizabeth Fini (P)
  • Footnotes
    Support  EY026479; Research for Preventing Blindness
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3855. doi:
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      Andrew Webster, Shravan K Chintala, Joseph Barr, Noorjahan A Panjwani, Pablo Argueso, Shinwu Jeong, M Elizabeth Fini; Targeting of Dynamin, an Essential Regulator of Endocytosis, Protects the Ocular Surface against Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3855.

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

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Abstract

Purpose : Accumulating evidence implicates oxidative stress in dry eye and other diseases of the ocular surface. Previously we showed (ARVO 2017) that oxidative stress causes corneal epithelial cell damage, as evidenced by uptake of vital dyes, and that co-treatment with endocytosis inhibitors is protective. Dynamin, the founding member of a GTPase family implicated in membrane remodeling, has a critical role in endocytic membrane fission events. Here we defined protective effects of the dynamin inhibitor, Dynasore, both in vitro and ex vivo.

Methods : Monolayer cultures of human corneal limbal epithelial (HCLE) cells, stratified cultures with mucosal differentiation, and enucleated mouse eyes were used. To create oxidative stress, t-butyl hydroperoxide (tBHP) was added to cultures at 10-50 mM for 2-3 h at 37°C. This was done in the presence or absence of Dynasore (40 uM). At the end of each experiment, one or more of the following cell damage assays were performed: 1) galectin-3 shedding from the glycocalyx, as determined by Western blotting of culture medium; 2) cytotoxicity, as determined by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) uptake; 3) vital dye (rose-bengal) uptake.

Results : Oxidative stress stimulated galectin-3 shedding by 3-fold (over untreated control) increased by tBHP, and that this release was reversed to 1.4-fold by dynasore. Both WST-1 and MTT assays indicated increased cell cytotoxicity by tBHP, but these events were blocked by dynasore, determined by WST-1 (p=0.003) and MTT (p=0.046) assays. Mouse corneas treated ex vivo with tBHP showed 7-fold increase in rose bengal uptake at the ocular surface (p=0.03); uptake was reduced to 3-fold by dynasore treatment (p=0.09). Dynasore alone had little effect on any of these parameters.

Conclusions : Dynasore protects the ocular surface against oxidative stress, suggesting it may have clinical value in treating dry eye and other forms of ocular surface disease.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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