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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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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
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
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
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
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