Abstract
Purpose :
Corneal fibrosis can cause the cornea to become opaque and can result in partial or complete vision loss for which the only current treatment is corneal transplantation. Recently sphingolipids (SPL) have been linked to fibrosis and found to be modulated by the transforming growth factor-β (TGF-β) pathway. The aim of this study is to delineate the actions of SPLs in corneal fibrosis and their signaling mechanism interactions with TGF-β.
Methods :
Healthy human corneal fibroblasts (HCFs) were cultured in EMEM with 10% FBS and 0.5mM 2-O-α-D-glucopyranosyl-L-ascorbic acid in 3D constructs and allowed to grow for 4 weeks in the presence of 0.01μM, 0.1μM, or 5μM sphingosine-1-phosphate (S1P), and 2μM, 5μM, or 10μM sphingosine kinase inhibitor 2 (SphKI2) . Cultures without any growth factors/ inhibitors served as Controls. After 4 weeks the constructs were examined for the expression of SPL specific pathway signaling that focused on sphingosine 1-phosphate receptor 3 (S1PR3) and sphingosine kinases 1 and 2 (SphK1/SphK2). Corneal fibrotic markers such as α-SMA and collagens I, III, and V were also examined using Western Blot analysis.
Results :
We observed that the 5μM concentration of S1P treatment led to significant downregulation (p= <0.0001) of SphK1, SphK2, S1PR3, α-SMA, collagen I, collagen V, and collagens ratio I/V. In terms of SPL – TGF-β signaling interactions our data showed significant upregulation of TGF-β1 and TGF-β3 upon treatment with 0.1µM S1P treatment and significant downregulation when treated with the 5µM concentration of S1P. In contrast, significant upregulation of S1PR3 was observed upon treatment with 5μM (p= 0.04) and 10μM (p= 0.02) concentrations of SphKI2, collagen V with 10μM concentration (p= 0.01), and collagens ratio I/III with 2μM concentration (p= 0.03). And significant downregulation of collagen III at concentrations of 2μM, 5μM, 10μM (p= 0.002, 0.03, and 0.02, respectively) and collagens I/V ratio at 10μM concentration (p= 0.005) were observed upon treatment with SphKI2.
Conclusions :
The S1P-mediated regulation of corneal fibrotic markers that we observed in our 3D in vitro tissue engineered model provided valuable information to understand the role of S1P signaling in corneal wound healing which would beof great importance in our effort to understand the molecular mechanisms of corneal scarring and wound healing process.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.