Abstract
Purpose :
Corneal fibrosis occurs as a response to corneal injury or trauma[DK1] as well as infection. It is known to be the third leading cause of blindness worldwide [DK2] with corneal transplantation as the main treatment for severe cases. Monocarboxylate transporters (MCTs) are a family of proton linked plasma membrane transporters that carry monocarboxylates like lactate and pyruvate across the plasma membrane. Our recent[DK3] studies have revealed the existence and modulation of MCTs in the human cornea in vitro. The aim of this study was to investigate the effects of small interfering RNAs (siRNA)-mediated silencing of monocarboxylate transporters (MCT2, and MCT4) in corneal fibrosis in vitro.
Methods :
Primary Human Corneal Fibroblasts (HCFs) were isolated and transfected with siRNAs using nucleofection (Lonza, 4D-Nucleofector X unit). The following groups were tested: controls, nucleofected MCT2 siRNA, and nucleofected MCT4 siRNA. Nucleofection was performed in antibiotic-free media containing nucleofector solution with 120nM MCT2 and MCT4 siRNA. Post-nucleofection cells were seeded in 2D 6-well plates containing cell culture media (Eagles Minimum Essential Media (EMEM), 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AA). After 72 hours in culture, the expression of MCTs and specific markers of corneal fibrosis were analyzed using qRT-PCR.
Results :
After 72 hours in culture, we observed 45% and 30% knockdown of MCT2 and MCT4 mRNA, respectively. Significant upregulation of collagen 3 (Col3) expression was seen with both siRNA MCT2 and siRNA MCT4. Significant downregulation of smooth muscle actin (SMA) in siRNA MCT2, but not with siRNA MCT4 was also observed. Collagen 1 (Col1) was significantly downregulated with both siRNA MCT2 and siRNA MCT4.
Conclusions :
Our study, revealed the effects of MCT2 and MCT4 in corneal fibrosis. The modulation of corneal fibrotic targets, after silencing MCT2 and MCT4, provide novel insights in the field and warrants further investigations in order to fully delineate the role of MCTs in human corneal wound healing process.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.