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Ricardo F Frausto, Jaffer Kattan, Judy Chen, Benjamin Lin, Doug Chung, Anthony J Aldave; Effects of increased ZEB1 expression on proliferation and apoptosis of corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5301. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the role that ZEB1, discovered to underlie the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays on corneal endothelial cell proliferation and apoptosis.
A human corneal endothelial cell line (HCEnC-21T) and a cell line that lacks endogenous ZEB1 expression (Ishikawa) were transfected with either a vector overexpressing ZEB1 (pReceiver-MO2 ZEB1) or an empty vector (pCMV6-Entry), used as a control. Twenty-four hours after transfection, cells were seeded at either 10% (for proliferation) or 90% (for apoptosis) confluence. To measure cell proliferation, the number of cells (N) was enumerated at 3 hours (defined as N0) and compared to the number of cells at 24, 48 and 72 hours post-seeding (NT/N0). To measure cell apoptosis, cells were treated with 50mJ (HCEnC-21T) or 100mJ (Ishikawa) of UVC. Cells were lysed at 0, 2, 4, 6 and 8 hours after treatment (HCEnC-21T and Ishikawa). Apoptosis was monitored using phase-contrast microscopy, while whole-cell protein lysates were used for the detection of cleaved caspase3 (cCASP3) by Western blotting to assess the activation of the caspase-mediated pathways of apoptosis. Quantification of the protein bands was performed using ImageJ. Data were graphed and statistical analyses performed using GraphPad Prism 5 software.
Compared with cells transfected with the empty vector construct, HCEnC-21T cells overexpressing ZEB1 demonstrated a statistically significant (p<0.05) increase in cell proliferation at 48 and 72 hrs. In contrast, the proliferation of Ishikawa cells was not altered following ZEB1 overexpression. Cleaved CASP3 was generated in both HCEnC-21T and Ishikawa cells following UVC treatment, but no statistically significant difference in cCASP3 levels was observed between empty vector and ZEB1 transfected cells.
Increased ZEB1 expression results in increased proliferation but no effect on caspase3-mediated apoptosis of a human corneal endothelial cell line. While we observed increased corneal endothelial cell proliferation in response to increased ZEB1 expression, the significance of this finding to the pathogenesis of PPCD3, which is presumed to result from ZEB1 haploinsufficiency, remains to be determined in the context of reduced ZEB1 expression.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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