The involvement of transforming growth factor beta in excessive extracellular matrix production of corneal endothelial cells in Fuchs’ endothelial corneal dystrophy

Abstract

Purpose: Fuchs’ endothelial corneal dystrophy (FECD) is characterized by progressive loss of corneal endothelial cells (CECs), thickening of Descemet’s membrane and deposition of extracellular matrix (ECM) in the form of guttae. However, the molecular mechanism by which ECM is produced in FECD has yet to be elucidated. Here we demonstrate the involvement of epithelial-to-mesenchymal transition (EMT)-related genes in the excessive ECM production by CECs in FECD.

Methods: Human CECs (HCECs) derived from 3 FECD patients and HCECs derived from 3 normal donor corneas were cultured and then immortalized by SV40 and hTERT to produce iFECD and iHCEC cell lines. To elucidate ECM production, iFECD and iHCEC cells were cultured in Transwell^® Inserts, and ECM deposition was analyzed by hematoxylin-eosin staining. Type-I and type-IV collagen and fibronectin were evaluated by real-time polymerase chain reaction (PCR) and immunostaining. EMT-related gene (Snail1, Snail2, and ZEB1) levels were analyzed by real-time PCR. To evaluate the involvement of Snail1 and ZEB1 in the excessive ECM production, siRNA was used to knockdown those two genes. To evaluate the involvement of the transforming growth factor beta (TGF-β) signaling pathway, cells were treated with TGF-β or with SB431542 (an inhibitor of TGF-β type-I ALK receptors).

Results: ECM thickness was significantly increased in iFECD compared to iHCEC (6.65±0.82μm and 3.14±0.64μm, respectively) (p<0.01). PCR and immunostaining revealed increased production of type-I collagen, type-IV collagen, and fibronectin in iFECD compared to iHCEC. In iFECD, expression of Snail1 and ZEB1 was significantly increased (p<0.01), while Snail2 was not increased. TGF-β markedly increased the expression of Snail1 and ZEB1 associated with the increased production of ECM both in iFECD and iHCEC. On the other hand, siRNA of Snail1 and ZEB1 suppressed ECM production of iFECD (p<0.01). Moreover, SB431524 significantly suppressed the expression of Snail1 and ZEB1, as well as the production of ECM (p<0.01).

Conclusions: Our findings demonstrate that increased levels of EMT-related genes may induce excessive ECM production in FECD and that inhibition of the TGF-β signaling pathway may be a potent therapeutic target for the treatment of FECD.

Keywords: 480 cornea: basic science • 481 cornea: endothelium • 512 EMT (epithelial mesenchymal transition)