Abstract
Purpose :
To investigate if corneal endothelial cells (CECs) in Fuchs endothelial corneal dystrophy (FECD), with or without Transcription Factor 4 (TCF4) repeat expansion, have increased cellular migration compared to controls.
Methods :
Descemet membrane and endothelium were collected from normal donor corneas (67 year old female), and FECD patients undergoing endothelial keratoplasty (54 year old female (54F), 74 year old female (74F)). Primary CECs were immortalized using the simian virus 40 T antigen followed by puromycin selection. Genetic analysis with triplet-repeat primed PCR from blood samples identified an expansion of the CTG repeat sequence in the TCF4 gene in the 54F (TCF4+) and not in the 74F (TCF4-). Live cell imaging was performed on normal and FECD CEC lines and ex-vivo specimens transfected with green fluorescent protein using lipid nanoparticles. Migration rates were determined as a function of cellular density using automated cell tracking (TrackMate) by analyzing 4 to 8 fields of view per sample. CEC lines and ex-vivo specimens were classified as FECD low cell density (n=3) or high cell density (n=3); or normal low cell density (n=3) or high cell density (n=3). Live imaging of scratch assays was performed on cell lines seeded at high cell density.
Results :
FECD cells with TCF4+ (1.96 +/-0.02 μm/min) and TCF4- (2.23 +/-0.02 μm/min) displayed increased mean speed compared to normal cells (1.57 +/-0.02 μm/min; p<0.001) under low cell density conditions. FECD cells with TCF4+ and TCF4- displayed increased mean speed compared to normal cells under high cell density conditions as detected by scratch assay (37.2 +/-1.1 % area vs 44.3 +/- 4.1% area vs 70.7 +/- 5.2% area; p<0.001). In ex-vivo specimens, FECD CECs in low cell density areas displayed increased cell migration compared to normal CECs (0.39 +/-0.005 μm/min vs 0.36 +/-0.005 μm/min; p<0.001) but no difference in mean speed was found in high cell density areas.
Conclusions :
CECs in low cell density conditions display increased cellular migration likely due to decreased contact inhibition. Furthermore, FECD cells have increased cellular migration under low cell density and high cell density conditions regardless of TCF4 repeat expansion status. FECD cells in ex-vivo specimens have increased cellular migration in low cell density areas, likely impacting cellular migration during Descemet stripping only.
This is a 2020 ARVO Annual Meeting abstract.