June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Unraveling the mechanism by which Dynasore protects the ocular surface
Author Affiliations & Notes
  • Rafael Martínez-Carrasco
    New England Eye Center, Tufts Medical Center, Boston, Massachusetts, United States
    Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Pablo Argueso
    Schepens of Mass. Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • M Elizabeth Fini
    New England Eye Center, Tufts Medical Center, Boston, Massachusetts, United States
    Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Rafael Martínez-Carrasco, None; Pablo Argueso, None; M Elizabeth Fini, University of Southern California (P)
  • Footnotes
    Support  NIH/NEI Grant R01EY026479. Massachusetts Lions Eye Research Fund. Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3265. doi:
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    • Get Citation

      Rafael Martínez-Carrasco, Pablo Argueso, M Elizabeth Fini; Unraveling the mechanism by which Dynasore protects the ocular surface. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3265.

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

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Abstract

Purpose : Corneal epithelium is the first barrier of the eye against external threats and protecting epithelial cells from damage is crucial to prevent ocular surface disease. In a recent study from our lab (PMID: 30303976), a new protective role for the dynamin inhibitor, dynasore, was proposed for corneal epithelial cells exposed to oxidative stress. Our purpose in this study was to unravel the mechanisms behind this effect

Methods : We used the oxidant tert-Butyl hydroperoxide (tBHP) (10 mM) to create oxidative stress in stratified cultures of immortalized human corneal limbal epithelial cells with mucosal differentiation (HCLE cells (PMID:12766048), together with dynasore (80 µM; Sigma) or chemical inhibitors of different processes such as Necrostatin-1 (Nec-1; necroptosis inhibitor) (300 µM; Sigma). We assessed barrier function (permeability to Rose Bengal and Dextran (70 kDa)) and cell membrane integrity (Trypan Blue). Calcein-AM/CoCl2 assay (Thermo) was used to determine mitochondrial permeability transition pore (mPTP) opening. We determined the expression levels of sXBP1 and CHOP by qPCR to evaluate ER stress and activation of the unfolding response. For Ca2+ experiments, a kit containing Fluo-4 (Thermo) was used

Results : Oxidative stress caused a significant loss of Calcein staining (P<0.05) and an increase in Trypan Blue uptake (P<0.001) that were abolished by treatment with either dynasore or Nec-1. Rose Bengal and Dextran uptake were also increased in stressed cells (P<0.01 and P<0.001 respectively) but also in Nec-1 treated cells (P<0.01 and P<0.001 respectively), while no increase was observed in presence of dynasore. While dynasore did not prevent the increase in sXBP1 expression caused by tBHP (P<0.001), the increase in CHOP gene expression (P<0.05) was reverted. Cytosolic Ca2+ was accumulated after oxidative stress induction, which was also inhibited by dynasore

Conclusions : Dynasore not only prevented the uptake of different dyes caused by oxidative stress, but also maintained membrane integrity and protected mitochondria. Dynasore inhibits a process of cell death mediated by RIPK1, as its inhibitor, Nec-1, also prevented membrane and mitochondrial damage. We propose that dynasore prevents cell death caused by ER stress by inhibiting cytosolic Ca2+ accumulation and, thus, the activation of PERK/CHOP pathway. Dynasore reveals as a promising new treatment for the ocular surface

This is a 2020 ARVO Annual Meeting abstract.

 

Proposed Dynasore protective mechanism

Proposed Dynasore protective mechanism

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