Collagen type XII is undetectable in keratoconus Bowman's layer

Abstract

Purpose: Corneal biomechanical failure is the hallmark of keratoconus (KC); however, the cause of this failure remains elusive. Collagen type XII (COL12A1), which localises to Bowman's layer (BL), is thought to function in stress-bearing areas, such as BL. Given the putative protective role of COL12A1 in biomechanical stability, this study aims to characterise COL12A1 expression in all corneal layers involved in KC.

Methods: TaqMan quantitative PCR was performed on 31 corneal epithelium samples of progressive KC and myopic control eyes. Tissue microarrays were constructed using full-thickness corneas from 61 KC cases during keratoplasty and 18 non-KC autopsy eyes and stained with an antibody specific to COL12A1. Additionally, COL12A1 was knocked out in vitro in immortalised HEK293 cells.

Results: COL12A1 expression was reduced at transcript levels in KC epithelium compared with controls (ratio: 0.58, p<0.03). Immunohistochemical studies demonstrated that COL12A1 protein expression in BL was undetectable, with reduced expression in KC epithelium, basement membrane and stroma.

Conclusions: The apparent absence of COL12A1 in KC BL, together with the functional importance that COL12A1 is thought to have in stress bearing areas, suggests that COL12A1 may play a role in the pathogenesis of KC. Further studies are necessary to investigate the mechanisms that lead to COL12A1 dysregulation in KC.

Keywords: Cornea; Experimental laboratory; Pathology.

Figure 1.

qPCR validation of under-expression of COL12A1 at transcript level in keratoconus epithelium compared to control (median and interquartile range displayed).

Figure 2.

Representative immunohistochemical staining of COL12A1 in control and keratoconus central corneas. Epithelium, basement membrane, Bowman’s layer, and anterior stroma are shown. Note the undetectable staining of COL12A1 in keratoconus Bowman’s layer and reduced staining in stroma (arrows aim at Bowman’s layer). Images taken at 40× magnification.

Figure 3.

COL12A1 expression in tissue microarrays of central corneas. (A) Relative intensity scores show that COL12A1 expression is undetectable in keratoconus Bowman’s layer and that its expression is also reduced in keratoconus basement membrane. (B) H-scores demonstrate reduced COL12A1 staining in keratoconus stroma and epithelium (median and interquartile range are displayed in both panels). *marks statistical significance

Figure 4.

(A) qPCR validation of COL12A1 knockdown in HEK293 cells in three biological repeats. Relative gene expression shows marked knockdown of COL12A1 in targeted HEK293 cells. (B) qPCR results demonstrate over-expression of COL3A1 in COL12A1 knockouts.

Figure 5.

(A) COL3A1 (band size 139 kDa) is over-expressed at the protein level in COL12A1 targeted HEK293 cells. (B) Densitometry results using GAPDH as a loading control.