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Sarah E Nicholas, megan stiles, Nawajes A Mandal, Dimitrios Karamichos; Sphingolipids and TGF-β signaling in corneal fibrosis: the continuing mission. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4328. doi: https://doi.org/.
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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. Sphingolipids (SPL) have been linked to fibrosis and recently, we have shown that SPLs are tightly related to the corneal wound healing process. The aim of this study is to determine how SPL signaling and interplay with canonical TGF-β signaling pathway are able to modulate corneal fibrosis.
In vitro: Healthy human corneal fibroblasts (HCFs) were cultured on polycarbonate membranes and allowed to grow for 4 weeks stimulated with stable Vitamin C and in the presence of sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2). The constructs were examined for the expression of TGF-βRI and TGF-βRII, as well as other markers involved in corneal fibrosis. In vivo: Alkali burn was created in Sphk1-/- (KK) and wild type (WW) mice. Corneas were harvested at 1, 3, and 7 days post-injury and evaluated for cornea opacity and neovascularization. Both in vitro constructs and mice corneas were examined for the expression of fibrotic markers, sphingolipid-related markers, as well as TGF-β signaling pathway members.
In vitro: Using 3D constructs stimulated with S1P, we observed significant downregulation of TGF-βRII and cFN, significant upregulation of IL6, and upregulation of αSMA. Whereas SPHK I2 stimulation led to significant upregulation of cFN, significant downregulation of αSMA, and upregulation of sphingosine-1-phosphate receptor 3 (S1PR3). In vivo: In WW mice, TGF-βRII was significantly downregulated post-injury at days 1 and 3, returning to normal levels by day 7. TGF- βRII was significantly upregulated in KK mice on day 7 with no modulation at day 1 and 3. cFN was significantly upregulated in WW and KK mice at day 3 plateauing at day 7. We observed downregulation of IL6 in KK mice directly compared to WW mice. S1PR3 was significantly upregulated post-injury at day 1 and returned to near-normal levels by day 7. Lastly, following injury, we observed fibrosis in WW mice with reduced levels of fibrosis in KK mice.
Using complimentary in vitro and in vivo models we have made some key affirmations that have aided us in identifying the role of SPLs and TGF- β signaling pathways in corneal wound healing. Further studies are necessary to validate SPHK I2 as a potential therapeutic agent for corneal fibrosis.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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