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Hamid Alemi, Tomas Blanco, Rohan Bir Singh, Hayate Nakagawa, Zala Luznik, Jia Yin, Sunil Chauhan, Reza Dana; 1α,25-dihydroxy vitamin D3 has a cytoprotective effect against inflammatory stress-induced corneal endothelial cell death. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1462.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of corneal endothelial cell (CEnC) viability is the predominant cause of graft failure following corneal transplantations. The role of 1α,25-dihydroxy vitamin D3 (VitD) present in the aqueous humor on CEnC integrity is yet to be elucidated. In this study, we investigated the cytoprotective function of VitD on CEnC against inflammatory stress-induced cell death using an ex-vivo murine corneal model.
Murine cornea cups (n=5/group) were harvested from C57BL/6 mice and incubated in RPMI+10% BSA media in presence of IFN-γ (100 ng/mL) and TNF-α (100 ng/mL) to induce inflammatory stress in CEnC, with or without VitD (10-11 M, 10-9 M or 10-7 M) for 12 hours. The tight junction protein-1 was stained with FITC-conjugated anti ZO-1 antibody. The CEnC apoptosis was evaluated using the TUNEL assay. The images were acquired by using laser scanner confocal microscopy and subsequently analyzed using ImageJ. The frequency of CEnC apoptosis (TUNEL+ vs. ZO-1+) and density of ZO-1+ cells were compared in groups incubated with different concentrations of VitD.
The corneal cups incubated with IFN-γ and TNF-α had a significantly higher frequency of CEnC apoptosis compared to corneal cups that were not exposed to inflammatory stress (6.85±1.39% vs. 1.81±1.39%, p<0.001). CEnC apoptosis was significantly reduced following induction of inflammatory stress upon supplementation the media with 10-11 M VitD (3.75±0.96%, p=0.003), 10-9 M (2.69±0.95%, p<0.001) and 10-7 M (1.86±0.83%, p<0.001) of VitD, in comparison to the non-treated controls (6.85±1.39%).
Our results demonstrate that CEnC death induced by inflammatory stress is significantly reduced by VitD in a dose-dependent fashion. Notably, VitD treatment even at the lowest dose, which approximates its natural concentration in the aqueous humor, resulted in a significant reduction in CEnC death. These findings indicate a possible cytoprotective effect of VitD on corneal endothelial cells.
This is a 2020 ARVO Annual Meeting abstract.
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