Menadione Induces Endothelial Mesenchymal Transition in Human Corneal Endothelial Cells

Abstract

 

Purpose

 

Fuchs Endothelial Corneal Dystrophy (FECD) causes endothelial cell loss via oxidative stress and guttae formation is the main clinical hallmark of FECD. We hypothesize that oxidative stress-induced endothelial mesenchymal transition (EnMT) plays a role in the morphological changes and cell loss seen in FECD. The purpose of this study was to quantify endothelial cell stress response and EnMT changes in telomerase-immortalized human endothelial cells (HCEnC-21T) in response to oxidative stress.

 

Methods

 

HCEnC-21T were grown to confluence and exposed to 100 µM of menadione bisulfite (MN) for 1-5 hours in low glucose Dulbecco's Modified Eagle Medium (DMEM). Non-treated cells served as controls. Morphology was assessed by phase-contrast microscopy every hour during treatment. Rosette formation was quantified and adjusted to the cell number. Subcellular localization of EnMT markers (vimentin, E-cadherin, N-cadherin, and alpha-smooth muscle actin) was determined using immunofluorescence confocal microscopy.

 

Results

 

MN treatment resulted in endothelial cell rosette formation, where cells clustered around circular areas of distinct cell loss. There was a time-dependent increase in loss of cell hexagonality and development of fibroblast-like morphology. There was a linear rise in the number of rosettes formed in response to MN treatment, with significant increase at 5 hours (37±5.72) compared with 1 hour (6.0±0.82; p=0.01). MN treatment resulted in positive discrete cytoplasmic staining of vimentin and alpha-smooth muscle actin as compared to non-treated controls, which were negative for both antibodies. Staining of E-cadherin and N-cadherin was more intense in MN-treated samples as compared to controls.

 

Conclusions

 

MN treatment results in endothelial morphologic changes seen in FECD. There is an upregulation of EnMT markers during corneal endothelial cell rosette formation in response to MN treatment. These findings suggest that EnMT may play an important role in FECD pathogenesis.

 

 

Representative confocal images of non-treated (A) and treated (B) telomerase-immortalized human endothelial cells (HCEnC-21T). Discrete cytoplasmic vimentin staining (B) was noted in cells treated with 100uM of Menadione for 5 hours. Final magnification: 400X with 4 zoom.

 

Keywords: 481 cornea: endothelium • 480 cornea: basic science