Dataset Information

Unravelling the interplay of sphingolipids and TGF-? signaling in the human corneal stroma.

ABSTRACT:

Purpose

To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-?) in order to develop novel, non-invasive therapies.

Methods

Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis.

Results

Data showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5?M and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01?M, 0.1?M, and 5?M; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1?M and 5?M S1P and upregulated by 5?M and 10?M SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-? isoforms and S1P/SPHK I2 treatments and found that TGF-?1 and TGF-?3 were both significantly upregulated with the 0.1?M S1P but were significantly downregulated with the 5?M S1P concentration. When TGF-?1 was compared directly to TGF-?3 expression, we observed that TGF-?3 was significantly downregulated compared to TGF-?1 in the 5?M concentration of S1P. No changes were observed upon SPHK I2 treatment.

Conclusion

Our study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

SUBMITTER: Nicholas SE

PROVIDER: S-EPMC5555661 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma.

Nicholas Sarah E SE Rowsey Tyler G TG Priyadarsini Shrestha S Mandal Nawajes A NA Karamichos Dimitrios D

PloS one 20170814 8

<h4>Purpose</h4>To delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies.<h4>Methods</h4>Human corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis.<h4 ...[more]

PMID: 28806736

Similar Datasets

Project description:Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.