Expression of SFRP Family Proteins in Human Keratoconus Corneas

Abstract

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.

Accumulation of sequence variants in genes of Wnt signaling and focal adhesion pathways in human corneas further explains their involvement in keratoconus

“…Interestingly, a significant increase of mRNA and protein expression of secreted frizzled-related protein 1 (SFRP1), which is a Wnt antagonist, has been detected in KTCN corneal epithelium and corneal buttons (Sutton et al, 2010;You et al, 2013a). In contrast, tear SFRP1 level has been significantly decreased in KTCN patients compared to control individuals (You et al, 2013b). The c.1238G>A variant in FZD2 was identified in the cornea of KC16 patient in combination with c.41C>T variant in VANGL1 (Wnt signaling) and c.2347G>A variant in COL4A4 (focal adhesion).…”

Expression of HGF and c-Met Proteins in Human Keratoconus Corneas

“…Immunolabelling of the thinned cone area of KC buttons was examined in each specimen but was not graded for comparison with controls because of the obviously altered morphology (only 1-2 epithelial layers present). The grading scale used was based on the intensity of the immunofluorescence (0 = no staining, 0.5 = very weak, 1 = weak, 2 = moderate, and 3 = strong) and the percentage (%) area immunolabelled (0 = 0%, 1 = 1% to 10%, 2 = 11% to 50%, and 3 > 50%) as described previously [ 19 ]. A final grade of 0 to 6 (intensity + % area immunolabelled) was then given for each specimen and this data is used to generate frequency histograms for HGF and c-Met immunostaining in KC and control specimens, respectively.…”

Peer Review #2 of "Accumulation of sequence variants in genes of Wnt signaling and focal adhesion pathways in human corneas further explains their involvement in keratoconus (v0.2)"

“…50 Interestingly, a significant increase of mRNA and protein expression of secreted frizzled-related protein 1 (SFRP1), which is a Wnt antagonist, has been detected in KTCN corneal epithelium and corneal buttons. 51,52 In contrast, tear SFRP1 level has been significantly decreased in KTCN patients compared to control individuals. 53 The c.1238G>A variant in FZD2 was identified in the cornea of KC16 patient in combination with c.41C>T variant in VANGL1 (Wnt signaling) and c.2347G>A variant in COL4A4 (focal adhesion).…”