Ultrastructural morphology and expression of proteoglycans, betaig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy

Abstract

Aims: To investigate the ultrastructural localisation of proteoglycans (PG), betaig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas.

Methods: Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins betaig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-betaig-h3, mouse anti-TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique.

Results: Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with betaig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling.

Conclusions: Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins betaig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as betaig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response.

Figure 1

Electron micrographs showing ultrastructural alteration in a bullous keratopathy cornea. (A) Electron lucent vacuole (V) in a degenerate basal epithelial cell (E) and vacuoles in the subepithelial region. (B) Subepithelial vacuole (V) between an epithelial cell (E) and Bowman's layer (B). (C) Thick basement membrane (BM) between epithelial cells (E) and Bowman's layer (B). (D) Basement membrane (BM) containing anchoring filaments (AC) above Bowman's layer (B). (E) Subepithelial pannus (P) containing microfilaments (MF) and rounded keratocytes (KR).

Figure 2

Light and electron micrograph of bullous keratopathy cornea. (A) Epithelial fold containing basement membrane material (BM). Keratocytes (KR) are arranged in a line above Bowman's layer (B) and the anterior stroma. (B) High magnification of the epithelial fold shown in (A). Keratocytes (KR) are present in the epithelial fold which contains basement membrane material (BM). (C) Part of an epithelial fold showing disrupted anchoring filaments (AC) in basement membrane material (BM). (D) Electron lucent lake (L) between the stroma (S) and Descemet's membrane (DM).

Figure 3

Electron micrographs of the posterior part of bullous keratopathy corneas. (A) Part of Descemet's membrane (DM) with a break (BR). (B) Collagen fibres (CF) and electron dense material in a break (BR) present in Descemet's membrane (DM). (C) Posterior collagenous layer (PCL) containing wide spacing collagen (LC) and microfilaments (MF). (D) Below the PCL, an endothelial cell (EN) containing melanin granules (ML). (E) A degenerate keratocyte (KR) containing melanin granules (ML). (F) Part of anterior stroma containing abnormal proteoglycans (PG).

Figure 4

Electron micrograph showing labelling of βig-h3 in BK corneas. (A) Control for immunoreaction. (B) Clumped labelling in epithelial cell (E). (C) Moderate clumped labelling in normal looking Bowman's layer (B). (D) Heavy labelling in basement membrane (BM) and around disrupted anchoring filaments. (E) Diffuse labelling on fibrotic pannus tissue (P) but not in the keratocyte (KR). (F) No labelling in the keratocyte but heavy labelling close to the keratocyte (KR) in the anterior stroma (S).

Figure 5

Electron micrograph showing labelling of βig-h3 in BK corneas. (A) Labelling on electron dense material present among collagen fibrils in the anterior stroma (S). (B) Labelling on fibrous (or fibrillar) material (FB) in the posterior stroma (S). (C) Labelling on fine fibrous (or fibrillar) material (FB) close to degenerate keratocyte (KR) but not in the keratocyte in the middle stroma (S). (D) Heavy labelling on banded Descemet's membrane (BDM). (E) Labelling on microfilaments (MF) in the PCL. (F) Labelling on electron dense material but not wide spacing collagen in the PCL.

Figure 6

Electron micrographs of BK corneas showing labelling of BC10, in (A) epithelium (E), (B) Basement membrane (BM) and around anchoring filaments (AC), (C) Bowman's layer (B), (D) stroma (S), (E) banded Descemet's membrane (BDM), (F) posterior collagenous layer (PCL).

Figure 7

Electron micrograph of BK corneas showing labelling of fibrillin-1-MAB2502, fibrillin-MAB1919, and fibronectin. (A) Fibrillin-1-MAB2502 labelling in epithelial cell (E), (B) diffuse labelling of fibrillin-1-MAB2502 on microfibrils (F) near a degenerate keratocyte (KR) in the pannus (P). (C) fibrillin-MAB1919 labelling on collagen fibrils in stroma (S), (D) Fibrillin-MAB1919 labelling on banded Descemet's membrane (BDM). (E) Labelling of fibronectin on the surface of the epithelium (E), and (F) in the stroma (S).