Abstract
Purpose :
Fibrosis occurs in corneas after injury or infection. Quiescent corneal stromal keratocytes (CSK) transit to the repair-type stromal fibroblasts (SF), which further differentiate into myofibroblasts (MyoF). The latter deposit excessive extracellular matrix (ECM) proteins in a disorganized manner, resulting in opacities that compromise the corneal transparency. TGFβ family contains key regulators of fibrosis, mechanistically acting through the canonical TGFβ/Smad and non-Smad pathways. TGFβ1 (Tβ1) mediates fibrosis whereas TGFβ3 (Tβ3) acts with anti-fibrotic effects and assists tissue regeneration. This study examined the effect of Tβ3:Tβ1 ratio on modulating the fibrosis-associated changes in cultured human CSK versus SF.
Methods :
Human CSK isolated from donor corneal stroma (n=3) were expanded using the ERI protocol. SF were generated from CSK cultures added with 2% fetal bovine serum for 2 passages. Both cell types were characterized by RNA sequencing and pathway analysis. Different ratios of recombinant human Tβ1 and Tβ3 were added to CSK and SF cultures, respectively, with fresh medium replenished every 24 hours, with reference to their half-lives determined by specific ELISA. Changes of cell size, cell stress response (myosin/F-actin intensity ratio) and the expression of fibrosis and inflammatory markers were examined by immunofluorescence and qPCR. The statistical significance was analyzed through one-way ANOVA using Sidak’s multiple comparisons test.
Results :
The cultivated human CSK and SF exhibited differential gene expression (>3700 genes in CSK versus >800 genes in SF with fold changes >2 or <0.5; P<0.05), affecting the enriched gene ontology terms, including GO:0005581~collagen trimer (P=4.41E-14) and GO:0007156~ cell adhesion (P=3.21E-8). In culture with varying Tβ3:Tβ1 ratios, the expression of fibronectin (FN) and tenascin C (TNC) was upregulated in CSK treated by both Tβ1 and Tβ3, and only in Tβ1-treated SF. By immunofluorescence, both Tβ1-treated CSK and SF showed an upregulated expression of αSMA, which was suppressed by the presence of Tβ3 (P <0.0001)
Conclusions :
Our work showed that corneal stromal cell fibrosis was modulated by Tβ1 and Tβ3 treatments. These results suggest a potential regulation of corneal fibrosis and wound healing through modulating the Tβ3:Tβ1 ratio.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.