Abstract
Purpose :
Corneal fibrosis can lead to partial or complete vision loss. Currently, the only treatment for severe corneal fibrosis is corneal transplantation, with limitations including risks of rejection and donor shortages. Signaling sphingolipids, such as sphingosine 1-phosphate (S1P), have been associated with fibrosis in various tissues and organs, including the cornea. We previously reported that S1P signaling is tightly related to TGF-β and corneal fibrogenesis. This study aimed to investigate the effect of knocking down the major S1P-generating enzyme, Sphingosine Kinase 1 (SphK1), on the regulation of S1P, TGF-β, and fibrotic markers in human corneal cells in vitro.
Methods :
Healthy human corneal fibroblasts (HCFs) were isolated, transfected with SphK1 siRNA via nucleofection, and subcultured for 72 hours. HCFs were subjected to nucleofection in antibiotic-free media containing nucleofector solution and 120nM SphK1 siRNA. Post-nucleofection, wells were supplied with fresh media containing antibiotics and subcultured for 72 hours. Cultures without treatment/transfection served as controls. Using qPCR, the samples were examined for mRNA expression of SphK1, SphK2, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (SMA), Collagen 3 (Col3), and Collagen I (Col1).
Results :
Following siRNA concentration optimization, we observed approximately 40% knockdown of SphK1 mRNA expression via nucleofection with 120nM SphK1 siRNA after 72 hours. Upon examining mRNA expressions, we observed significant upregulation of Sphingosine Kinase 2 (SphK2) and Sphingosine-1-Phosphate Receptor 3 (S1PR3) following SphK1 knockdown. Additionally, we observed that SphK1 knockdown led to significant downregulation of TGF-β1 and SMA but had no effect on Col1 or Col3 expression.
Conclusions :
Utilizing a novel nucleofection knockdown method on HCFs in vitro, we have demonstrated the impact of SphK1 knockdown on SPLs and corneal fibrosis. Knocking down SphK1 and consequential activation of SphK2 and S1PR3 may indicate an effective way of reducing fibrosis via SMA and TGF-β1 inhibition without excessive ECM accumulation. Further investigations are necessary in order to fully uncover the potential of SphK1 knockdown as a novel therapy for corneal fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.