Abstract
Purpose :
Our previous study found that in solute carrier family 4 member 11 (SLC4A11) knock out mice, lactate efflux from corneal endothelial cells was decreased, as the consequence, the corneal thickness increased from 98µm to 198µm on average. To explain the relevance of SLC4A11 expression to corneal endothelial pump function and monocarboxylate transporters (MCT1, 2, and 4), this study is testing the hypothesis that oxidative stress has a role in diminishing MCTs expression. A novel mitochondrial-targeted antioxidant (Visomitin) is tested for its potential to reverse SLC4A11 dysregulation.
Methods :
The SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCEC) was knocked down (KD) by siRNA transfection. SLC4A11 RNA and protein were measured by qPCR and immunoblotting. The expression of three monocarboxylate transporters, MCT1, 2 and 4, was examined in the same cell context. 24 hours post siRNA transfection, Visomitin was added and maintained for 15 hours. By incubating the cells (4x105 cells/0.5ml) in bicarbonate-rich ringer, lactate production from KD, treated with Visomitin and control cells were measured for initial efflux. The cellular stress caused by KD SLC4A11 was examined via MitoSOX-based assays to detect mitochondrial reactive oxygen species (ROS). Tert-butyl hydroperoxide (tBHP), an inducer of oxidative stress, was used as positive control.
Results :
In comparison to control, siRNA transfection caused SLC4A11 RNA and protein reduction of 81% (+ 5.5%) and 88% (+ 7.1) respectively. It also resulted in reduction of MCT1 (40% + 1.1%) and MCT2 (37% + 0.9%) expression. MCT4 expression did not change. In KD cells treated with Visomitin, the transcripts of MCT1 and MCT2 were increased to 73% (+1.0%) and 74% (+0.9%) of control. Lactate production was greatest in the control cells (51.71+0.35 nmol) > KD/Visomitin (46.42+0.77 nmol) > KD (40.18+0.39 nmol) over initial 30 minutes. In the measurement of ROS, MitoSOX fluorescence intensity in the cells was tBHP (1215+96) > KD (906+69) > KD/Visomitin (589+57) > Control (294+46).
Conclusions :
Knocking down SLC4A11 shows the effect on the expression of MCT1 and MCT2 in corneal endothelial cells, and lactate transport is consequently attenuated. SLC4A11 dysregulation causes oxidative stress which when attenuated by Visomitin reversed MCT expression and increased lactate efflex, indicating that MCT gene expression is sensitive to oxidative stress.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.