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Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet’s membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεβ) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

This study addressed limitations in calcein-AM-based endothelial viability assays, specifically focusing on pre-stripped DMEK grafts. Key challenges included the suboptimal calcein staining and the incompatibility of the viability assay with subsequent immunofluorescence (IF). Using human corneal grafts, we employed two strategies to optimize calcein staining. Firstly, we improved calcein staining in corneal endothelium by adjusting calcein-AM concentration and diluent, resulting in a threefold increase in fluorescence intensity with 4 µM calcein in Opti-MEM compared to the conventional 2 µM calcein in PBS. Secondly, introducing the trypan blue (TB) post-viability assay greatly reduced non-specific fluorescence, enhancing the contrast of calcein staining. This improvement significantly and importantly decreased both inter-operator’s variability and the time required for viability counting. For the subsequent double IF, an extensive wash is recommended on the fixed and permeabilized graft after the viability assay, which was carried out using Hoechst-Calcein (HC) labeling. The simple technical tips outlined in this study are not only effective for pre-stripped DMEK grafts but may also prove beneficial for other types of corneal grafts, such as PK and DSAEK.

Fuchs endothelial corneal dystrophy (FECD) is a bilateral, progressive corneal endothelial disease characterized by the formation of extracellular matrix (ECM) excrescences called guttae. This study integrated proteomic and transcriptomic analyses to elucidate the molecular composition and spatial organization of ECM proteins in the Descemet membrane (DM) of FECD patients. Through shotgun proteomics of FECD-derived DM specimens and RNA sequencing data from FECD ( n  = 10) and control ( n  = 7) corneal endothelial cells, we identified 19 significantly upregulated molecules in FECD, including 13 ECM-related proteins. Gene Ontology and Reactome analyses revealed ECM-related pathways as central to FECD pathology. Immunofluorescence analyses of flat-mounted and cross-sectional specimens from FECD patients undergoing Descemet membrane endothelial keratoplasty (DMEK) and controls demonstrated distinct spatial patterns for six ECM proteins. Fibronectin and collagen VI α1 were detected on the outer surfaces of guttae, matrilin-3 and biglycan localized around guttae, while LTBP2 and tenascin were strongly associated with the posterior fibrillar layer (PFL). The peripheral corneal regions predominantly exhibited scattered guttae, whereas the central region displayed buried guttae encapsulated by ECM deposition. This study comprehensively examined ECM protein expression patterns, revealing distinct spatial distributions across guttae, PFL, and surrounding DM regions. These findings suggest that clinical assessments should incorporate both guttae confluence and the presence of ECM-rich PFL to achieve a more comprehensive understanding of FECD progression, thereby informing more accurate staging and optimal surgical planning.

AimTo investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD).MethodsDescemet’s membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk).ResultsThe physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients.ConclusionsCCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.

The cornea, the anterior meniscus-shaped transparent and refractive structure of the eyeball, is the first mechanical barrier of the eye. Its functionality heavily relies on the health of its endothelium, its most posterior layer. The treatment of corneal endothelial cells (CECs) deficiency is allogeneic corneal graft using stored donor corneas. One of the main goals of eye banks is to maintain endothelial cell density (ECD) and endothelial barrier function, critical parameters influencing transplantation outcomes. Unlike in vivo , the stored cornea is not subjected to physiological mechanical stimuli, such as the hydrokinetic pressure of the aqueous humor and intraocular pressure (IOP). YAP (Yes-Associated Protein), a pivotal transcriptional coactivator, is recognized for its ability to sense diverse biomechanical cues and transduce them into specific biological signals, varying for each cell type and mechanical forces. The biomechanical cues that might regulate YAP in human corneal endothelium remain unidentified. Therefore, we investigated the expression and subcellular localization of YAP in the endothelium of corneas stored in organ culture (OC). Our findings demonstrated that CEC morphology, ECD and cell–cell interactions are distinctly and differentially associated with modifications in the expression, subcellular localization and phosphorylation of YAP. Notably, this phosphorylation occurs in the basal region of the primary cilium, which may play central cellular roles in sensing mechanical stimuli. The sustained recruitment of YAP in cellular junctions, nucleus, and cilium under long-term OC conditions strongly indicates its specific role in maintaining CEC homeostasis. Understanding these biophysical influences could aid in identifying molecules that promote homeostasis and enhance the functionality of CECs.