Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is a major cause of corneal transplantation. It is characterized by abnormal deposition of extracellular matrix (ECM), such as corneal guttae, accompanied by a loss of endothelial cells. Although recent studies have revealed several genomic factors, the molecular pathophysiology of FECD has not yet been revealed. In this study, we establish a cellular in vitro model by using immortalized corneal endothelial cells obtained from late-onset FECD and control patients and examined the involvement of epithelial mesenchymal transition (EMT) on excessive ECM production. We demonstrate that the EMT-inducing genes ZEB1 and SNAI1 were highly expressed in corneal endothelial cells in FECD and were involved in excessive production of ECM proteins, such as type I collagen and fibronectin through the transforming growth factor (TGF)-β signaling pathway. Furthermore, we found that SB431542, a specific inhibitor of TGF-β type I ALK receptors, suppressed the expression of ZEB1 and Snail1 followed by reduced production of ECM. These findings suggest that increased expression levels of ZEB1 and Snail1 in FECD cells were responsible for an increased responsiveness to TGF-β present in the aqueous humor and excessive production of ECM. In addition, these results suggest that the regulation of EMT-related genes by blocking the TGF-β signaling pathway may be a feasible therapeutic strategy for FECD.

PurposeLysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance to aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of the TM to LPA, however, is not completely understood. In this study, we explored the possible involvement of mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), transcriptional activation in extracellular matrix (ECM) production by LPA-induced contractile activity in human TM cells (HTM).MethodsThe responsiveness of genes encoding LPA receptors (LPARs), LPA hydrolyzing lipid phosphate phosphatases (LPPs), and the LPA-generating autotaxin (ATX) to cyclic mechanical stretch in HTM cells, was evaluated by RT-quantitative (q)PCR. The effects of LPA and LPA receptor antagonists on actomyosin contractile activity, activation of YAP/TAZ, and levels of connective tissue growth factor (CTGF), and Cyr61 and ECM proteins in HTM cells were determined by immunoblotting, mass spectrometry, and immunofluorescence analyses.ResultsCyclic mechanical stretch significantly increased the expression of several types of LPARs, LPP1, and ATX in HTM cells. LPA and LPA receptor–dependent contractile activity led to increases in both, the protein levels and activation of YAP/TAZ, and increased the levels of CTGF, Cyr61, α-smooth muscle actin (α–SMA), and ECM proteins in HTM cells.ConclusionsThe results of this study reveal that LPA and its receptors stimulate YAP/TAZ transcriptional activity in HTM cells by modulating cellular contractile tension, and augment expression of CTGF that in turn leads to increased production of ECM. Therefore, YAP/TAZ-induced increases in CTGF and ECM production could be an important molecular mechanism underlying LPA-induced resistance to AH outflow and ocular hypertension.

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This study investigated changes in collagen fibril architecture and the sulphation status of keratan sulphate (KS) glycosaminoglycan (GAG) epitopes from central to peripheral corneal regions. Freshly excised adult bovine corneal tissue was examined as a function of radial position from the centre of the cornea outwards. Corneal thickness, tissue hydration, hydroxyproline content, and the total amount of sulphated GAG were all measured. High and low-sulphated epitopes of keratan sulphate were studied by immunohistochemistry and quantified by ELISA. Chondroitin sulphate (CS) and dermatan sulphate (DS) distributions were observed by immunohistochemistry following specific enzyme digestions. Electron microscopy and X-ray fibre diffraction were used to ascertain collagen fibril architecture. The bovine cornea was 1021 ± 5.42 μm thick at its outer periphery, defined as 9–12 mm from the corneal centre, compared to 844 ± 8.10 μm at the centre. The outer periphery of the cornea was marginally, but not significantly, more hydrated than the centre (H = 4.3 vs. H = 3.7), and was more abundant in hydroxyproline (0.12 vs. 0.06 mg/mg dry weight of cornea). DMMB assays indicated no change in the total amount of sulphated GAG across the cornea. Immunohistochemistry revealed the presence of both high- and low-sulphated epitopes of KS, as well as DS, throughout the cornea, and CS only in the peripheral cornea before the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS remained constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also higher at mid-depth in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12 mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the distribution of low-sulphated KS.

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