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.