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Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and 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 related to both, transforming growth factor beta (TGF-β) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-β and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-β1 (β1), 1 μM sphingosine-1-phosphate (S1P), and 5 μM Sphingosine kinase inhibitor 2 (I 2 ). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I 2 ; prevention groups; (4) S1P/β1; and (5) I 2 /β1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-β signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-β binding proteins (LTBPs), TGF-β receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I 2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I 2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I 2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-β receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I 2 as a novel therapy for corneal fibrosis.

The purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1−/−) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk−/− corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1−/− corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision. [ABSTRACT FROM AUTHOR]

Corneal haze brought on by fibrosis due to insult can lead to partial or complete vision loss. Currently, corneal transplantation is the gold standard for treating severe corneal fibrosis, which comes with the risk of rejection and the issue of donor tissue shortages. Sphingolipids (SPLs) are known to be associated with fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to Transforming Growth Factor β (TGF-β) signaling and corneal fibrogenesis. This study aimed to elucidate the interplay of SPLs, specifically sphingosine-1-phosphate (S1P) signaling, and its' interactions with TGF-β signaling through detailed analyses of the corresponding downstream signaling targets in the context of corneal fibrosis, in vitro. Healthy human corneal fibroblasts (HCFs) were isolated, plated on polycarbonate membranes, and stimulated with a stable Vitamin C derivative. The 3D constructs were treated with either 5 μM sphingosine-1-phosphate (S1P), 5 μM SPHK I 2 (I 2 ; inhibitor of sphingosine kinase 1, one of the two enzymes responsible for generating S1P in mammalian cells), 0.1 ng/mL TGF-β1, or 0.1 ng/mL TGF-β3. Cultures with control medium-only served as controls. All 3D constructs were examined for protein expression of fibrotic markers, SPLs, TGF-βs, and relevant downstream signaling pathways. This data revealed no significant changes in any LTBP (latent TGF-β binding proteins) expression when stimulated with S1P or I 2. However, LTBP1 was significantly upregulated via stimulation of TGF-β1 and TGF-β3, whereas LTBP2 was significantly upregulated only with TGF-β3 stimulation. Significant downregulation of TGF-β receptor II (TGF-βRII) following S1P stimulation but significant upregulation following I 2 stimulation was observed. Following TGF-β1, S1P, and I 2 stimulation, phospho-SMAD2 (pSMAD2) was significantly downregulated. Furthermore, I 2 stimulation led to significant downregulation of SMAD4. Adhesion/proliferation/transcription regulation targets, SRC, FAK, and pERK 1/2 were all significantly downregulated by exogenous S1P, whereas I 2 only significantly downregulated FAK. Exogenous TGF-β3 caused significant upregulation of AKT. Interestingly, both I 2 and TGF-β3 caused significant downregulation of JNK expression. Lastly, TGF-β1 led to significant upregulation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 3 (S1PR3), whereas TGF-β3 caused significant upregulation of only SphK1. Together with previously published work from our group and others, S1P inhibition exhibits great potential as an efficacious anti-fibrotic modality in human corneal stromal ECM. The current findings shed further light on a very complex and rather incompletely investigated mechanism, and cement the intricate crosstalk between SPLs and TGF-β in corneal fibrogenesis. Future studies will dictate the potential of utilizing SPLs/TGF-β signaling modulators as novel therapeutics in corneal fibrosis. • TGF-β3 modulates LTBP1 and LTBP2 expression, where TGF-β1 only modulates LTB1 expression. • TGF-βRII is downregulated by S1P but upregulated by I 2. • TGF-β3 and I 2 both downregulated JNK expression. [ABSTRACT FROM AUTHOR]