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Sarah E Nicholas, Nawajes A Mandal, Dimitrios Karamichos; Sphingolipids and TGF-β: Seeking out novel treatments for corneal fibrosis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4681.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal fibrosis can lead to opacity and may ultimately cause partial or complete vision loss. Currently, the only treatment for severe corneal fibrosis is corneal transplantation. Sphingolipids (SPLs) are known to be associated with fibrosis; we have recently demonstrated that SPLs are tightly related to both, TGF-β signaling and corneal fibrogenesis. The aim of this study was to further elucidate the role of SPL and TGF-β signaling interactions in corneal fibrosis using both in vitro and in vivo models.
In vitro: Healthy human corneal fibroblasts (HCFs) were isolated and 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) and cultures without treatment served as controls. The constructs were examined for the expression of SMAD signaling members (pSMAD2, pSMAD3, SMAD4, and SMAD7), MAPK members (ERK 1/2, JNK, and p38), tyrosine kinases (FAK and Src), and a matricelluar protein (CTGF). In vivo: Alkali burn was created using 0.5N NaOH soaked filter paper (2 mm) applied to the cornea of anesthetized mice for 20 seconds in Sphk1-/- (KK) and wild type (WW) mice. Corneas were harvested at 7 and 14 days post-injury and evaluated for the expression of SMAD signaling members. Cell lysates were examined using Western Blot analysis.
In vitro: Using 3D constructs stimulated with or without S1P, we began unravelling the downstream signaling mechanism. In agreement with our in vivo data, there was significant downregulation in pSMAD2, SMAD4, and SMAD7, but not in pSMAD3 expression, in constructs stimulated with S1P. We also observed significant downregulation of ERK 1/2, p38, CTGF, FAK, and SRC but found no expression of JNK in S1P treated constructs. In vivo: In Sphk1-/- (KK) mice, SMAD7 early (7 days), pSMAD2 and SMAD4 at later wound healing time points (14 days) were upregulated when compared to WW, indicating their critical role in the formation of fibrosis.
Using in vitro and in vivo models we have identified key downstream interactions between SPLs and TGF- β signaling pathways. These findings have revealed even more connections, further explaining how S1P and TGF- β3 together are able to regulate corneal wound healing. Further studies are necessary to validate S1P as a potential therapeutic agent/ target for corneal fibrosis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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