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Siddharth Mahajan, Daniel Thieme, Friedrich E Kruse, Thomas Armin Fuchsluger; Screening apoptosis markers in human corneal endothelium and epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Anti-apoptotic treatment of corneal endothelium is a promising approach to protect the endothelial monolayer in corneal grafts. The goal of the presented study was to detect an apoptosis marker equally suitable in cell suspension and tissue.
Apoptotic cleavage of Caspases (3, 7, 9), PARP and Lamin A or Lamin C in corneal epithelial cell suspension (HCE), corneal endothelial cell suspension (HCEC12), Descemet’s membrane explants and full thickness corneasProtein content and general applicability of these proteins were analyzed using western blot and quantified with ImageJ (AU=area units).
After staurosporine treatment Caspase3 cleavage is 12 fold higher in endothelial cells, 10 fold higher in epithelial cells while PARP cleavage was increased 4fold in both corneal cell suspensions but undetectable in DMEKs or tissue. Analog data were obtained for Caspase9 cleavage for endothelial cells (7fold), epithelial cells (4fold) and DMEKs (6fold) but failed to obtain reproducible results for full thickness corneas. With cleaved Lamin A we found a marker in all biological backgrounds which can be obtained in a quality making it possible to quantify. Lamin A itself is not changed due to staurosporine treatment in cells (control: 1495 +/- 90 AU vs 2.5µM STS 1400 +/-141 AU), in DMEKs (control: 7188 +/- 1031 AU vs 2m5µM STS 7143 +/- 408 AU) or whole tissues (control: 2578 +/- 273 AU vs 2.5µM STS 3026 +/- 113 AU). But cleaved Lamin A is induced 16 fold (control: 82 +/- 9 AU vs 2m5µM STS 1095 +/- 27AU) in HCEC12 cells, 13 fold in DMEK transplants (control: 203 +/- 126 AU vs 2.5µM STS 2826 +/- 789AU) and 12 fold in whole cornea grafts (control: 13.5 +/- 2 AU vs 2,5 µM STS 170 +/- 68 AU) due to staurosporine treatment.
Our project analyzes many different approved downstream targets of apoptosis pathways in cell lines, DMEK transplants and whole corneal grafts. Although we were able to detect most of the respective markers for apoptosis induction in cell lines or tissues, only Lamin A cleavage can be used as a marker in all biological backgrounds. With this data in hand we can now compare our studies on anti-apoptosis treatment for all our model backgrounds.
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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