ABSTRACT Purpose Fuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy – a form of autophagy that degrades ferritin to release labile ferrous iron – and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. Methods Endothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS). Results Surgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. Conclusions Our results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

PURPOSE:The aims of this study were (1) to compare "front" and "rear" methods for loading Descemet membrane endothelial keratoplasty (DMEK) tissue into both micro-Jones and standard-Jones tubes and (2) to evaluate the efficacy of a cone-shaped glass funnel adapter designed to make loading DMEK tissue safer for corneal endothelial cells.METHODS:The corneal endothelium was stained with 0.06% trypan blue to confirm equivalence between mate corneas. The tissues were then processed using the Iowa Lions Eye Bank standard DMEK protocol. In comparison 1, one mate was loaded into the rear of a micro-Jones or standard-Jones tube and the other was loaded into the front of the same tube. In comparison 2, one mate was loaded into the front of the micro-Jones tube and the other was loaded through the cone-shaped funnel adapter into the rear. All tissues were ejected through the front of the modified Jones tubes and assessed for endothelial cell loss (ECL) with calcein AM staining, FIJI, and Trainable Weka Segmentation; scroll widths were measured digitally.RESULTS:There were no statistically significant differences in ECL between front and rear loading [micro (N = 6 pairs): front 15.74% vs. rear 17.95%; standard (N = 6 pairs): front 19.58% vs. rear 19.17%; all P > 0.05]. DMEK scrolls loaded with the funnel adapter exhibited lower ECL compared with scrolls loaded through the front [micro (N = 8 pairs): front 13.53% vs. loading funnel 2.40%; P < 0.001]. Loading with the adapter was not faster (front 6.66 seconds vs. loading funnel 5.52 seconds; P = 0.24).CONCLUSIONS:Using a cone-shaped DMEK loading funnel may reduce ECL sustained during preloading.