Subepithelial corneal fibrosis partially due to epithelial-mesenchymal transition of ocular surface epithelium

Abstract

Purpose: To determine whether epithelial-mesenchymal transition is involved in the development of corneal subepithelial fibrosis (pannus).

Methods: Frozen samples of pannus tissue removed from human corneas with a diagnosis of total limbal stem cell deficiency were characterized by immunostaining for both epithelial and mesenchymal markers. We selected transformation-related protein 63 (p63) and pancytokeratin as epithelial markers and vimentin and α-smooth muscle actin (α-SMA) as mesenchymal markers. Immunostaining for β-catenin and E-cadherin was performed to determine wingless-Int (Wnt)-pathway activation. RT-PCR analysis was also performed on epithelial tissue obtained from pannus samples after dispase digestion.

Results: Immunohistochemistry revealed strong nuclear expression of p63 and weak intercellular expression of E-cadherin in epithelial basal cells of pannus tissue. Furthermore, translocation of β-catenin from intercellular junctions to the nucleus and cytoplasm was also observed. Double-positive cells for both p63 and α-SMA were observed in the subepithelial stroma of pannus tissue, which was supported by RT-PCR and cytospin analysis.

Conclusions: Epithelial-mesenchymal transition may be partially involved in the development of subepithelial corneal fibrosis due to total limbal stem cell deficiency.

Figure 1

Histopathology of pannus. Clinical appearance of patient (case 7) showed thin pannus covering whole cornea (A). Removed pannus in case 7 (B). Representative pannus photo of hematoxylin-eosin staining (C) showing irregular epithelial basal layer and hyper-epithelialization associated with increased subepithelial fibroblasts.

Figure 2

Immunostaining of pannus tissue against pancytokeratin (CK22). Epithelium of pannus in all cases stained positive for pancytokeratin (red), particularly in superior hyperepithelial layers. However, basal cells showed weak staining (A and B). C: pancytokeratin staining in normal cornea as a control. The scale bar indicates 50 μm.

Figure 3

Immnostaining of pannus tissue against vimentin and collagen IV. Compared to control (C, normal cornea), collagen IV expression (red) was irregular in pannus, and was also found in vessel walls (A and B). Vimentin(+) (green) stromal cells were uniformly observed in normal central cornea, but supra- and sub-epithelial activated vimentin(+) cells were observed in pannus. Cells double-positive for vimentin and p63 were observed (D, indicated by arrow). The scale bar indicates 50 μm.

Figure 4

Immunohistochemistry of E-cadherin and β-catenin. Epithelial basal cells in pannus showed weak staining for E-cadherin (green) and translocation of β-catenin (red) from intercellular junctions to cytoplasm. A: Pannus with double-staining for E-cadherin and β-catenin. B: Normal cornea. C: βcatenin, D: E-cadherin, E: E-cadherin and DAPI, in pannus epithelium was shown. A was a combination of C, D, and E. The scale bar indicates 50 μm.

Figure 5

Immunostaining of pannus tissue against p63 and α-SMA. Nuclear p63 (red) and cytoplasmic α- SMA (green) double-positive cells were observed in subepithelial location in pannus tissue (A and B). B was syntheses photo of p63 (C), α- SMA (D), and DAPI (E). Scale bar indicates 50 μm.

Figure 6

Immunostaining of cytospin of pannus tissue. Double-immunostaining for p63 (red) and α-SMA (green) was observed in individual cells (indicated by arrows) in cytospin of pannus (A, with DAPI staining; B, without DAPI staining).  Scale bar indicates 50 μm.

Figure 7

RT-PCR of pannus epithelium. α-SMA, MMP-2, and MMP-9 were all expressed in pannus epithelium, but not in limbal epithelium.  Weak Snail-1 expression was observed in pannus epithelium. E-cadherin and β-catenin were expressed in both  limbal epithelial cells and pannus.