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Viridiana Kocaba, Kishore Reddy Katikireddy, Marianne Price, Francis Price, Ula V Jurkunas; Corneal endothelial cell and guttae interaction in Fuchs endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3799.
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© ARVO (1962-2015); The Authors (2016-present)
Fuchs Endothelial Corneal Dystrophy (FECD) is characterized by a progressive corneal endothelial cell loss and formation of extracellular matrix deposits, called guttae. When designing endothelial cell therapy, the role of guttae on endothelial cell (EC) biogenesis is important. In this study, we examined the effect of guttae in Fuchs dystrophy on human corneal endothelial cell (HCEnC) monolayer formation.
2,000 HCEnCs, were seeded on normal (n=20) and Fuchs (n=50) Descemet’s membranes (N-DM & FECD-DM, respectively). Phase-contrast and live cell microscopy along with ImageJ were used to determine cell shape, behavior, morphology and migration patterns. Actin and ZO-1 staining were used to determine the % of coverage, number of cells, and number and size of guttae. EC surrounding guttae were isolated by laser capture dissection processed for isolation of RNA. Differential gene expression profiles of markers of endothelial to mesenchymal transition (EnMT) were analyzed and compared in function of guttae size (large guttae > 30μm; middle guttae 15-30μm; small guttae <15μm). Apoptosis was analyzed with TUNEL assay.
At day 7, HCEnC covered 97.7% [±8.5] of N-DM and 72.8% [±11] of FECD-DM, p=0.02. Mean number of EC was 2,083cell/mm2 (±153) on N-DM and 1,541cell/mm2 (±221), p=0.01. Actin and ZO-1 staining revealed 3 different growth patterns of cells encompassing guttae on FECD-DM. The 1st pattern with a mean guttae size of 10.5μm (±3.4) did not impede EC growth enabling coverage over guttae. In the 2nd pattern, a mean guttae size of 17μm (±4.3), enabled the coverage of only the cytoplasm but nuclei remained on the periphery of guttae. The 3rd pattern (>31.31μm ±5.2) completely abolished EC growth on guttae but surrounded them in a rosette pattern as seen in FECD specimens. EC surrounded large guttae showed an upregulation of EnMT compare to cells surrounded small guttae (respectively p=0.0007 & p=0.039). The % of apoptotic cells on FECD-DM was significantly higher compare to cells on N-DM (6.6% vs. 0.06%, p=0.04). Mean Guttae size surrounded by apoptotic cells was 108.45μm (±42.56).
These results suggest guttae may have a direct effect on the EC biogenesis by causing stress and apoptosis. It highlights the central role of guttae in determining endothelial cell growth, migration and coverage. This would suggest cell therapy procedures should be guided by guttae size.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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