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Peter W. Chen, Lauren Tien, Jessamee Mellon; Epigenetic Regulation of Corneal Endothelial Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5121.
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To determine if endothelial cell growth arrest is epigenetically regulated, and whether manipulation of epigenetic regulatory mechanisms can promote corneal endothelial cell proliferation.
Corneal endothelial sheets were harvested from BALB/c or C57BL/6 mice and either cultured in Shem’s Medium containing 10% FBS and immortalized with human papilloma virus E6/E7 genes or used after fresh isolation. Corneal endothelial cells treated with TGFβ for 24 hours, and harvested for RNA isolation. Real time PCR using gene-specific primers was used to quantitate expression of de novo methyltransferase genes. DNA and histone methylation was quantitated by fluorometric ELISA-based kits according to manufacturer’s instructions. Corneal endothelial cells were treated with the demethylating agents 5-Aza 2-deoxycytidine or Vorinostat for 24 hours, allowed to recover for 48 hours and assessed for proliferation in a conventional 3H thymidine release assay.
TGFβ-treated immortalized corneal endothelial cells and freshly isolated corneal endothelial cells upregulated de novo methyltransferases and exhibited an increase in global methylation. Proliferation of TGFβ-treated cells was suppressed compared to untreated controls. However, there was no difference in histone methylation between TGFβ - treated and untreated corneal endothelial cells. Corneal cell proliferation was significantly increased by treatment with Vorinostat (P< 0.002) but was not significantly increased by treatment with 5-Aza 2-Deoxycytidine.
Corneal endothelial cells can utilize de novo methylation to impart epigenetic gene regulation when exposed to TGFβ. Demethylation or inhibition of histone deacetylases promotes corneal endothelial cell proliferation which may play an important role in repopulating and restoring the endothelial cell layer in corneal diseases and in corneal transplantation.
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