TGFβ1 and TGFβ2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) beta 1 and TGF beta 2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGF beta 1 or TGF beta 2, with Image-J quantitation, and keratocan, vimentin, alphasmooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to 4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFfil and tear TGFP2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGF beta 1 and/or TGF beta 2 in the stroma in some corneas. TGF beta 1 and TGF beta 2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four 4.5D PRK corneas developed significant SMA myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in 4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGF beta 1 and TGF beta 2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

Automated summary

The study aimed to investigate the expression and localization of TGF beta 1 and TGF beta 2 in rabbit corneas with and without stromal fibrosis, as well as defective perlecan incorporation in the epithelial basement membrane. The findings revealed delayed regeneration of epithelial and EBM barriers in high injury corneas, leading to increased myofibroblast development and stromal fibrosis. Corneas with more severe injuries showed prolonged myofibroblast viability and triggered stromal scarring fibrosis due to delayed regeneration of epithelial and EBM barriers.