Amelioration of Fibrosis via S1P Inhibition Is Regulated by Inactivation of TGF-β and SPL Pathways in the Human Cornea

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 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) β1/S1P; (3) β1/I2; prevention groups; (4) S1P/β1; and (5) I2/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 I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 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 I2 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 I2 as a novel therapy for corneal fibrosis.


Introduction
Corneal fibrosis is a leading cause of blindness worldwide, affecting over 10 million people [1].Injury or trauma to the cornea can initiate resident keratocyte differentiation into myofibroblasts, causing high expression levels of α smooth muscle actin (αSMA) and Collagen III, which can cause irregular deposition of extracellular matrix (ECM) components [2,3].This disruption of the homeostatic corneal environment can cause cornea scarring, which can result in vision loss.While the corneal clarity is maintained by orchestrated signaling cascades, the specific mechanisms driving corneal fibrosis are very complex and still not well understood [1,[4][5][6].Unfortunately, for severe corneal scars, corneal transplantation is often the only viable option for those suffering from this condition.

Latent Transforming Growth Factor Beta Binding Proteins (LTBPs)
Latent TGF-β binding proteins-1 through -4 (LTBPs 1-4) are known activators of TGF-β and were investigated here for their protein expressions following treatment with The present study sought to understand the role of SPLs and TGF-β signaling pathway members, their cross-talks, and downstream targets in corneal fibrosis.Future studies will reveal whether S1P inhibition can be tailored as a novel therapy for the management of corneal fibrosis.

Transforming Growth Factor Beta Receptors (TGF-βRs)
TGF-β receptors I and II (TGF-βRI and TGF-βRII), which are activated via binding with active TGF-β, were investigated for their protein expressions for all groups tested.TGF-βRI expression was significantly upregulated by stimulation with β1-S1P compared

Effects of S1P and TGF-β Treatment Groups on HCF Cellular Migration
Cellular migration of HCFs was examined in response to stimulation with all groups tested over a period of 24 h.Following 12 h of stimulation with the I2-β1 group, we observed significantly increased wound closure compared with the controls (p = 0.0006) and S1P-β1 (p < 0.0001; Figure 7A).Similarly, the β1-I2 group caused significantly increased wound closure after 12 h compared to the controls (p = 0.0024) and β1-S1P (p < 0.0001) stimulation (Figure 7A).After 18 h, we observed significantly increased wound closure with I2-β1 compared to the controls (p = 0.0008) and β1-S1P (p < 0.0001) and in β1-I2 compared to the controls (p = 0.0008) and β1-S1P (p < 0.0001) stimulation (Figure 7A).After 24 h, all treatment groups reached 100% wound closure (Figure 7A).Representative cell migration images with all groups tested over the course of 24 h are shown in Figure 7B.

Effects of S1P and TGF-β Treatment Groups on HCF Cellular Migration
Cellular migration of HCFs was examined in response to stimulation with all groups tested over a period of 24 h.Following 12 h of stimulation with the I2-β1 group, we observed significantly increased wound closure compared with the controls (p = 0.0006) and S1P-β1 (p < 0.0001; Figure 7A).Similarly, the β1-I 2 group caused significantly increased wound closure after 12 h compared to the controls (p = 0.0024) and β1-S1P (p < 0.0001) stimulation (Figure 7A).After 18 h, we observed significantly increased wound closure with I 2 -β1 compared to the controls (p = 0.0008) and β1-S1P (p < 0.0001) and in β1 -I 2 compared to the controls (p = 0.0008) and β1-S1P (p < 0.0001) stimulation (Figure 7A).After 24 h, all treatment groups reached 100% wound closure (Figure 7A).Representative cell migration images with all groups tested over the course of 24 h are shown in Figure 7B.

Discussion
The mechanisms involving TGF-β and S1P in the cornea have largely remained a mystery due to the complexity of their signaling effects the lack of studies.Our group previously reported on the signaling expressions of SPLs, TGF-β members, canonical downstream SMAD, non-canonical downstream, and fibrotic markers in HCF 3D constructs treated with S1P, I2, TGF-β1, and TGF-3 [26].Our current study demonstrated the impact of I2-induced fibrotic prevention and rescue modulated by SPLs and TGF-β family member signaling in HCF 3D constructs.
Inactive TGF-β isoforms are secreted from cells and are activated in covalent association with LTBP molecules [30,66].Acosta et al., 2023, reported the presence of increased LTBP1 expression in murine corneal fibroblasts [16].Another recent study

Discussion
The mechanisms involving TGF-β and S1P in the cornea have largely remained a mystery due to the complexity of their signaling effects the lack of studies.Our group previously reported on the signaling expressions of SPLs, TGF-β members, canonical downstream SMAD, non-canonical downstream, and fibrotic markers in HCF 3D constructs treated with S1P, I 2 , TGF-β1, and TGF-3 [26].Our current study demonstrated the impact of I 2 -induced fibrotic prevention and rescue modulated by SPLs and TGF-β family member signaling in HCF 3D constructs.
TGF-β and S1P overlapping convergence and cell signaling effects have been well documented [62,95], and recent studies involving endometriosis [57], pulmonary fibrosis [58,61], EMT/asthma [59], and renal interstitial fibrosis [60] have reported their involvement in the development of the aforementioned disorders.Although TGF-β and S1P cross-talks have been rigorously investigated in various cells and tissues, their role in the cornea has been understudied and is not yet well understood.Herein, we observed the upregulation of TGF-βRI and II expressions following S1P-β1 and β1-S1P treatment groups but significant downregulation following I 2 -β1 and β1-I 2 group treatments, indicating that S1P prevention and rescue activated TGF-β receptors, whereas I 2 prevention and rescue treatments did not.
SMADs are major downstream signaling transducers for TGF-β receptors and have been previously documented for their role in corneal fibrosis [65,66].Recent studies have reported the impact of fibrosis in response to SMAD inhibition in the cornea.A reduction in corneal fibrosis was observed via the inhibition of SMAD2/3 in human [96,97] and mouse corneas [98].One study previously demonstrated that murine corneal fibrosis was regulated in part TGF-β1/SMAD2 activation [99].Nuwormegbe et al., 2021 [100], revealed that TGF-β1-induced fibrosis was suppressed via SMAD3 signal in the human cornea.This finding was substantiated by another group, which reported that SMAD3 overexpression enhanced TGF-β1-induced fibroblasts to myofibroblast differentiation in HCFs [101].Our current study demonstrated pSMAD2/3 and SMAD4 downregulation in response to I 2 -β1 treatment and upregulation following S1P-β1 treatment.Additionally, β1-I 2 treatment caused the downregulation of pSMAD2 and SMAD4, but β1-S1P led to the upregulation of SMAD4 only.
In the sphingosine rheostat, sphingosine kinase is known as the "fulcrum" due to its critical role in controlling the balance between S1P and ceramide levels [44,46].Many previous studies have implicated S1P as a fibrotic inducer in various cells and tissues [62].S1P is generated from ceramide, which is phosphorylated by sphingosine kinases, SphK1 and Sphk2, which can demonstrate oppositional effects.S1P produced by SphK1 in the cytosol can act as a second messenger or can be secreted to bind to S1P receptors and TGF-β receptors, whereas SphK2 resides in the cell nucleus where S1P is generated and regulates gene expression [55].Recently, SphK1 was found to influence S1P upregulation more than SphK2, and S1PR3 was linked to fibrotic manifestations in the lung [44].Wang et al., 2023, reported that abnormal S1P content in the circulation affected cardiovascular disorder pathogenesis and S1PR3 mediation of cell proliferation and vascular permeability [45].Furthermore, the S1PR3 antagonist was observed to improve graft viability in rat heart transplants [102].Another recent study revealed that S1P-induced epithelial endometriotic cell fibrosis was reliant on S1PR3 activation [103].Although investigations on S1P in the cornea have been under documented in the past, two recent studies have demonstrated the effects of SphK1/S1P in mouse corneas.Yasuda et al., 2021, revealed that TGF-β1-induced injury increased S1P via SphK1 upregulation modulated by S1PR3 and VEGF-A and angiogenesis [104].Wilkerson et al., 2022, reported that SphK1 knockout mice had reduced corneal neovascularization following injury [55].Previously, our group reported that TGF-β1 induced the upregulation of S1PR3 in HCF 3D in vitro constructs [26].In the current study, we observed the upregulation of S1PR3 following S1P-β1 and β1-S1P treatment groups but a significant downregulation following I 2 -β1 and β1-I 2 group treatments.
S1P is known to elicit cell and tissue-specific effects but is known largely as an inducer of fibrosis.A recent study observed that S1P stimulation enhanced retinal pigment epithelial cell migration, activated S1PR3, and stimulated αSMA transcription [105].Meanwhile, another study found that exogenous S1P treatment in human Müller glial cells led to the significant upregulation of α-SMA expression [106].Yang et al., 2024, found that an S1P agonist reduced cell migration compared to high glucose treatment in rat retinal Müller cells [107].Our current study found increased cell migration following stimulation with I 2 -β1 and β1-I 2 treatment groups, with decreased cell migration following stimulation with S1P-β1 and β1-S1P groups after 12 h and 18 h post-scratch.Moreover, we observed reduced fibrosis via αSMA and Collagen III expression regulated by I 2 -β1 and β1-I 2 treatments.
Our observations demonstrated that S1P complementation to TGF-β1-induced fibrosis led to the activation of SPL and TGF-β pathways, whereas I 2 treatment inhibited the pathways and resulted in reduced corneal fibrosis.Future investigations would explore the potential involvement of SMAD pathway inhibitors and S1P inhibition as a novel therapy for corneal fibrosis management.

Ethical Approval
Primary human corneal stromal fibroblasts (HCFs) were isolated from human cadaver corneas with no history of ocular or systemic disease and were de-identified prior to analysis.All cadaver corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA, USA).All studies herein were approved by the North Texas Regional Institutional Review Board (IRB # 2020-030) and adhered to the Declaration of Helsinki.

Human Corneal Fibroblast Cell Isolation and 3D In Vitro Model Cultures
HCFs were isolated from healthy donors by scraping away the epithelium and endothelium, cutting the stromal tissue into 2 × 2 mm pieces, and allowing them to adhere in T25 flasks.The corneal explants were cultured in complete media consisting of Eagle's Minimum Essential Medium (EMEM: ATCC; Manassas, VA, USA) with 10% fetal bovine serum (FBS: R&D Systems, Minneapolis, MN, USA) and 1% antibiotic-antimycotic (A.A.; Gibco, Life Technologies; Grand Island, NY, USA).HCFs were seeded onto polycarbonate transwell membranes in 6-well plates with 1 × 10 6 cells/well.The cells were incubated for 24 h hours to allow adherence to the membranes and thereafter were stimulated with 0.5 mM stable vitamin C (0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid [108], Sigma-Aldrich, St. Louis, MO, USA) in a complete medium containing the following treatments: 0.1 ng/mL TGF-β1 (β1), 1 µM sphingosine-1-phosphate (S1P), or 5 µM SPHK I 2 (I 2 ).The TGF-β1 treatment was administered for the first two weeks and was then switched to the S1P or I 2 treatment for the last two weeks.Additionally, the S1P or I 2 treatment was administered for the first two weeks and was then switched to the TGF-β1 treatment for the last two weeks.The treatment groups are abbreviated as follows: S1P-β1 (S1P prevention), β1-S1P (S1P rescue), I 2 -β1 (I 2 prevention), and β1-I 2 (I 2 rescue).Constructs with complete media and vitamin C only served as the controls.Fresh treatments were supplied every other day for a total of four weeks.A TGF-β1 stock solution was made at a concentration of 20 µg/mL by dissolving TGF-β1 powder (#240-B; R&D Systems; Minneapolis, MN, USA) in 1 mg/mL bovine serum albumin (BSA) and 4mM HCl.An S1P stock solution was prepared at a concentration of 125 µm by dissolving S1P powder (#860492P; Avanti Polar Lipids; Alabaster, AL, USA) in 4 mg/mL BSA in water at 37 • C inside a glass vessel.A stock solution of SPHK I 2 (#10009222; Cayman Chemicals; Ann Arbor, MI, USA) was made at a concentration of 5 mM by dissolving the powder in DMSO.Protein was extracted from the 3D constructs for Western blot analysis.

2D Scratch Assay-Cell Migration
HCFs were seeded at a density of 5 × 10 5 cells/well in 12-well plates in a control medium.Following 24 incubations, a scratch was administered through the confluent cell layer using a 10 µL pipette tip, and cell migration was observed at 0, 12, 18, and 24 h post-scratch.The TGF-β1 treatment was administered for the first 6 h and the S1P or I 2 treatment was administered for the last 6 h.Additionally, the S1P or I 2 treatment was administered for the first 6 h and the TGF-β1 treatment was administered for the last 6 h.An EXI-310 inverted microscope (Accu-Scope Inc.; Commack, NY, USA) was used to capture images of the wound closure progression.Cell migration was measured and quantified using ImageJ 1.53e software.All experimental conditions were repeated 5 times.

17 Figure 1 .
Figure 1.S1P and TGF-β signaling cross-talk: TGF-β receptor activation induces SMAD signaling cascades, resulting in the regulation of gene expression, including SphK regulation.Stimulation of SphK1 leads to the formation of S1P, which activates S1P receptors, inducing fibrotic cell responses.Created with Biorender.com.