April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Menadione Induces Endothelial Mesenchymal Transition in Human Corneal Endothelial Cells
Author Affiliations & Notes
  • Duna Raoof
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, MA
  • Kishore Reddy Katikireddy
    Schepens Eye Research Institute, Boston, MA
  • Thore Schmedt
    NBE Analytical, AbbVie Deutschland GmbH & Co. KG, Ludwigshafen, Germany
  • Ula Jurkunas
    Cornea, Massachusetts Eye and Ear Infirmary, Boston, MA
  • Footnotes
    Commercial Relationships Duna Raoof, None; Kishore Reddy Katikireddy, None; Thore Schmedt, None; Ula Jurkunas, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3584. doi:
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      Duna Raoof, Kishore Reddy Katikireddy, Thore Schmedt, Ula Jurkunas; Menadione Induces Endothelial Mesenchymal Transition in Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3584.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.
 
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  
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