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PurposeWe sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM).MethodsA Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort.ResultsMice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls.ConclusionsThere are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

Fuchs endothelial corneal dystrophy (FECD), a progressive polygenic disease that causes degeneration of the corneal endothelium, is the leading indication for corneal transplantation in the U.S. Characteristically, loss of corneal endothelial cells and formation of degenerative extracellular “guttae” result in failure to maintain appropriate corneal hydration through active ion pumping to counter the passive leakage of aqueous humor. FECD pathogenesis is linked to oxidative stress and environmental exposure to ultraviolet A (UVA), and impaired endogenous response to oxidative stress is common to the disease across multiple genotypes. UV irradiation is also known to cause cellular damage by ferroptosis, a nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation. Although a possible role for ferroptosis in FECD has been postulated to account for the increased susceptibility to oxidative damage and lipid peroxidation, this has not been explored systematically. In this study, we investigated the roles of genetic background and UVA exposure in causing lipid membrane damage and endothelial cell degeneration in FECD. We first sought clinical evidence of iron-mediated lipid peroxidation in surgical samples obtained from FECD patients undergoing endothelial keratoplasty, and found increased levels of cytosolic ferrous iron (Fe2+) and evidence of lipid peroxidation were present in end-stage diseased tissues compared with healthy human donor corneas. Next, using immortalized (F35T) and primary cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found that alterations in gene and protein expression involved in ferroptosis were conserved in both cell culture and tissue models of FECD disease compared to controls, including elevated levels of the ferroptosis-specific marker transferrin receptor 1. F35T immortalized cells showed significantly higher basal lipid peroxidation than control HCEC-B4G12 cells, indicating higher susceptibility to ferroptosis attributable to genetic background. Then, we tested the impact of physiologically relevant doses of UVA irradiation on F35T cell cultures and found increased cytosolic Fe2+ iron levels indicating a role for ferroptosis in FECD disease progression. Finally, we tested the effects of inhibitory ferroptosis molecules. We found that F35T cells were more prone to RSL3 induced ferroptosis than healthy controls with deferoxamine chelation which indicated increased susceptibility in FECD cells, and cell death could be prevented after experimentally-induced ferroptosis using solubilized antioxidant ubiquinol indicating a role for anti-ferroptosis therapies. This investigation demonstrates that FECD genetic background and UVA exposure contribute to basal and incidental iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated oxidative damage and disease progression in FECD.