Abstract
Purpose :
Human corneal fibrosis can cause opacity, which can lead to partial or complete vision loss. The only current treatment for severe cases is corneal transplantation, which comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly linked to both transforming growth factor-beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to elucidate the role of SPLs and targeted TGF-β family members, their crosstalk, and downstream signaling in the context of corneal fibrosis in vitro.
Methods :
Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and cultured in EMEM+FBS+VitC (construct medium) for 4 weeks. The following treatments were prepared in construct medium: Control cells cultured in construct medium; 0.1ng/mL TGF-β1 (β1), 1μM sphingosine-1-phosphate (S1P), and 5μM SPHK I2 (I2). Five groups were tested: (1) Control; Rescue groups: (2) β1/S1P, (3) β1/I2 ; Prevention groups: (4) S1P/ β1, (5) I2/β1. Each treatment was administered for 2 weeks with one group and then switched to another for 2 weeks. Cultures without treatment served as controls. Using Western Blot analysis, 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members.
Results :
Our data revealed that β1/I2 rescue treatment caused significant downregulation in the expression of SMA, Collagen 3, TGF-βRI, TGF-βRII, SMAD4, pSMAD2, and S1PR3 compared to controls. I2/β1 prevention treatment leads to significant downregulation of SMA, Collagen 3, SMAD4, and pSMAD2 and significant upregulation of TGF-βRI. Interestingly, both β1/S1P and S1P/β1 groups lead to significant upregulation of Collagen 3 and activation of TGF-βRs and SMADs.
Conclusions :
We demonstrated that I2 treatment with β1 induced reversal and prevention of fibrosis regulated by inhibiting SphKs, TGF-β receptors, and the downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing I2 as a novel therapy for corneal fibrosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.