Abstract
Purpose :
Congenital Hereditary Endothelial Dystrophy (CHED) occurs due to a mutation in SLC4A11 gene resulting in degeneration and dysfunction of endothelial cells leading to progressive opacity of cornea and loss of vision in infants. Depletion of SLC4A11 causes thickening of Descemets membrane and increases apoptosis in human corneal endothelial cells. Besides the genetic factor, oxidative stress also plays an important role in the pathogenesis of CHED, which causes degeneration of endothelial cells in corneal endothelial dystrophy. In India and Middle East countries, CHED accounts for 21% of all pediatric keratoplasty. Currently the only available treatment for CHED is by surgical intervention. But a large gap exists between the demand and supply of transplantable corneas. Hence, it is essential to study the role of SLC4A11 during oxidative stress for developing significant non-invasive therapeutic interventions to prevent or atleast delay corneal endothelial degeneration.
Methods :
We have cultured primary human corneal endothelial cells (HCEnC) from cadaveric corneas and checked of SLC4A11 expression by NRF2 inducers by immunocytochemistry and western blotting. The expression of SLC4A11 in corneal endothelial cells after knockdown of NRF2 by siRNA in presence or absence of a NRF2 inducer was analyzed by immunocytochemistry and quantitative PCR. To determine a possible link between SLC4A11 and NRF2, chromatin immuno-precipitation was done.
Results :
Elevated levels of SLC4A11 expression were seen in HCEnC that were treated with two different NRF2 inducer in comparison to the non-treated cells. Reduced SLC4A11 expression was observed in cells depleted of NRF2 indicating possible regulation of SLC4A11 by NRF2. NRF2 inducers caused increased expression of antioxidant genes and reduced the level of reactive oxygen species that were detected in cells depleted of SLC4A11. Bioinformatics analysis revealed the presence of putative NRF2 binding sites on the promoter of SLC4A11. Chromatin immuno-precipitation analysis further showed a positive binding of NRF2 on SLC4A11 promoter.
Conclusions :
Our study shows that NRF2 inducer can combat oxidative stress and enhance antioxidant signaling in cells depleted of SLC4A11 and might have a potential role as a therapeutic application to reduce the oxidative stress present in endothelial cells of CHED patients.
This is a 2020 ARVO Annual Meeting abstract.