Abstract
Purpose :
The hallmark of keratoconus (KC) is biomechanical failure of the cornea, with fragmentation of Bowman’s layer (BL) contributing to reduced corneal strength. Proteomic studies of collagen I in the KC corneal epithelium have shown underexpression of this major structural component of the extracellular matrix. Collagen I spatially localizes to BL and is produced in part by corneal epithelium. Wnt signaling, which regulates extracellular matrix production, has been shown to be dysregulated in KC, and our transcriptomic study of progressive KC corneal epithelium demonstrates underexpression of WNT10A mRNA. However, it is unclear whether WNT10A regulates collagen I expression in the corneal epithelium.
Methods :
RNA-sequencing was performed on corneal epithelium samples of five progressive KC and five myopic control eyes. WNT10A underexpression was validated using TaqMan qPCR on 31 additional independent samples; protein level validation with Western blot analysis. Immunohistochemistry was performed on tissue microarrays containing cores from over 100 KC and control cases. Additionally, WNT10A was overexpressed in vitro in immortalized corneal epithelial cells.
Results :
WNT10A was underexpressed in KC corneal epithelium as compared to myopic controls (transcript ratio KC/control=0.59, p=0.02 per RNA-sequencing study; transcript ratio=0.66, p=0.03 per qPCR; protein ratio=0.07, p=0.06 per Western blot). Immunohistochemical analysis demonstrated WNT10A absence or marked decrease in BL of KC corneas (p<0.0001). Finally, WNT10A positively regulated COL1A1 expression in corneal epithelial cells (protein ratio=11, p=0.02).
Conclusions :
Though long suggested historically, our results provide for the first time a potential molecular mechanism supporting the hypothesis that alterations of the epithelium contribute to stromal pathogenesis in KC. Specifically, we find that WNT10A promotes COL1A1 expression, and hypothesize that when WNT10A levels are low, reduced deposition of collagen I in BL may compromise biomechanical strength and potentiate breaks typical of KC. These studies also suggest that the Wnt pathway represents a therapeutic target, as its manipulation in the corneal epithelium may induce collagen I production and increase tensile strength in KC.
This is a 2021 ARVO Annual Meeting abstract.