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Veronique Beaulieu Leclerc, Olivier Roy, Stephanie Proulx; Effect of dexamethasone on the phenotype of cultured corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1141.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal endothelial cells (HCEC) tend to lose their endothelial morphology in culture, a phenomenon known as endothelial-to-mesenchymal transition (EndMT). The purpose of this study was to evaluate the ability of a synthetic glucorticoïd, dexamethasone (Dex), to block EndMT of cultured HCEC.
HCEC (n=4 populations, passage 2) with a mixed phenotype were passaged and grown in a proliferation medium containing Dex (1 µM). The medium was changed for a maturation medium still containing Dex after reaching confluency and the cells were cultured for another 7 days. Two other HCEC populations were cultured in the proliferation medium alone until confluency and Dex was added to the maturation medium for 7 days. Cultures without Dex were used as controls. Morphology was assessed by phase contrast microscopy. Immunofluorescence and western blots were performed (endothelial functionality markers ZO-1, Na+/K+ATPase and pan-cadherin; mesenchymal markers α-SMA and type I collagen) in order to analyze the phenotype.
Adding Dex during the proliferation phase of HCEC had no significant effect on the morphology of the cells. However, morphology was improved during the maturation phase. Immunofluorescence and western blot analysis showed that when Dex was used in both cultivation phases, it reduced the expression of mesenchymal markers, compared to the controls. Dex also improved the localisation of ZO-1 at cell junctions and increased Na+/K+ATPase expression. When Dex was only used in the maturation phase, the morphology greatly improved, from mixed/fibroblastic to endothelial. Expression of type I collagen was not significantly different (p>0.05) and expression of α-SMA was higher than the controls. Cells that went through the maturation phase in the presence of Dex had an increased expression of the endothelial markers ZO-1, Na+/K+ATPase and pan-cadherin as well as a better cytolocalisation when compared to the controls (without Dex) and compared to cells in the proliferation phase.
Addition of Dex to the culture media, in both culture phases, helps to acquire an endothelial phenotype. Results also demonstrate that the maturation of HCEC in the right conditions plays a role in the establishment of an endothelial phenotype and expression of function-related proteins.
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