Purpose To review history of Descemet stripping only (DSO) in Fuchs endothelial corneal dystrophy, describe the results of a clinical trial of topical ripasudil after DSO (K-321-201 study), and discuss future directions. Methods A 1-year, phase 2, randomized, placebo-controlled multicenter clinical trial of two doses of K-321 (ripasudil) administered for 12 weeks after DSO surgery in Fuchs endothelial corneal dystrophy was performed. The primary endpoint, central corneal endothelial cell density (ECD) at 12 weeks after surgery, was determined by an independent reading center that was masked to study group assignment. Duration of corneal edema, need for medical or surgical rescue therapy, corneal thickness, and central ECD throughout the entire study period were also examined. Adverse events and exploratory endpoints were collected. Results Sixty-five subjects were enrolled (21 in the QID group, 22 in the BID, and in the placebo group). Over 95% of subjects completed the trial. The QID group had a higher central ECD 12 weeks after DSO than the placebo group (531 ± 312 cells/mm2 vs 228 ± 298 cells/mm2, P = .0065). Corneal edema cleared in 17/21 (81.0%) of the QID group at 12 weeks, compared with 2/22 (9.1%) of the placebo group (P < .0001). Rescue was required in 2/21 (9.5%) subjects in the QID group and 6/22 (27.3%) subjects in the placebo group (P = .0092). Adverse events were mild and did not lead to discontinuation of treatment. Conclusions Topical K-321 given QID improves DSO outcomes, as demonstrated by a higher ECD, more rapid resolution of corneal edema, and reduced failure rate. The medication was well-tolerated.

Purpose:Fuchs endothelial corneal dystrophy (FECD) involves pathological extracellular matrix (ECM) accumulation within Descemet's membrane, leading to guttae formation, endothelial dysfunction, and vision impairment. As current treatment is primarily surgical, we investigated whether Rho-associated protein kinase (ROCK) inhibition can reduce fibrotic ECM remodeling in FECD and related corneal endothelial diseases. Methods:Endothelial cell-Descemet membrane specimens from FECD, pseudophakous bullous keratopathy (PBK), pseudoexfoliation keratopathy (PEX-K), and normal donor corneas were treated ex vivo with ripasudil or comparator ROCK inhibitors (netarsudil, Y-27632). Complementary in vitro studies used human corneal endothelial cells. ROCK activity, ECM expression, matrix metalloproteinase (MMP) activity, and TGF-β/Smad signaling were analyzed using molecular and imaging techniques. Results:Relative to controls, FECD samples showed constitutive ROCK and TGF-β signaling activation. Ripasudil suppressed ROCK activity and downregulated fibrosis-associated ECM components (including collagens I/III, fibronectin, agrin, TGFBI, clusterin, and tenascin-C), while shifting the MMP-tissue inhibitor of metalloproteinases (TIMP) balance to enhance ECM turnover. These effects were accompanied by reduced ECM deposition and attenuation of TGF-β/Smad signaling, alongside broad transcriptional reprogramming related to fibrosis, inflammation, and cytoskeletal dynamics. Similar ECM-modulating effects were observed in PBK and PEX-K, with ripasudil showing the most pronounced effects among tested inhibitors. Conclusions:ROCK activation contributes to pathological ECM alterations in FECD, and its pharmacologic inhibition with ripasudil suppresses pro-fibrotic ECM production, promotes ECM-remodeling pathways, and supports restoration of ECM homeostasis. These findings suggest ROCK inhibition as a promising non-surgical therapeutic strategy for FECD and related corneal endothelial disorders, warranting further validation in preclinical studies.

PURPOSE:The aim of this study was to analyze the influence of donor characteristics and tissue preservation conditions on the scrolling behavior of grafts in Descemet membrane endothelial keratoplasty (DMEK). METHODS:This retrospective, single-center study included 3150 consecutive DMEK grafts after uneventful preparation for DMEK surgery. The grafts were prepared from either organ-cultured corneoscleral buttons (n = 1414) or corneoscleral buttons stored in Optisol-GS (n = 1736). The main donor characteristics were age, endothelial cell density, death-to-preservation time, preservation method, and storage duration. The thickness of Descemet membrane (DM) was analyzed in a subgroup of 74 grafts using transmission electron microscopy. Scrolling behavior was quantified as roll width using calipers (mm). RESULTS:Older donor grafts exhibited less pronounced scrolling, with a significant correlation between donor age and roll width (r = 0.561, P < 0.001, R2 = 0.308). Organ-cultured grafts exhibited larger roll widths (1.764 ± 0.65 mm) than Optisol-GS-stored grafts (1.516 ± 0.51 mm; P < 0.001). A significant correlation was found between DM thickness and roll width (Spearman ρ = 0.562, P < 0.001, R2 = 0.312), while DM thickness was also significantly correlated with donor age (r = 0.545, P < 0.001, R2 = 0.304). CONCLUSIONS:In this large cohort of DMEK grafts, donor age was confirmed as the most decisive factor influencing the scrolling behavior of DMEK grafts, with younger tissues demonstrating tighter scroll formation while the correlation gradually weakened in older donor groups. The DM thickness was confirmed as an independent contributor, as thicker membranes were associated with less tightly scrolled grafts.

Rho-associated kinase (ROCK) inhibitors have gained popularity as novel treatment options in the management of glaucoma and corneal endothelial disorders. Among the various ocular side effects, reticular corneal epithelial edema has been most frequently reported, mainly after treatment with netarsudil. To explain the potential mechanisms, we comparatively analyzed the effects of ripasudil and netarsudil on corneal endothelial and epithelial function in vitro. Primary human corneal endothelial and epithelial cells were incubated with netarsudil dihydrochloride and ripasudil hydrochloride dihydrate for up to 7 days. Gene and protein expression analyses were performed by real-time PCR and immunocytochemistry. Functional assays assessed the cell migration, proliferation, viability, Na+/K+-ATPase activity, transcellular electrical resistance, and FITC–dextran permeability. Reticular bullous corneal epithelial edema was observed in a patient following netarsudil 0.02%/latanoprost 0.005% ophthalmic solution (Roclanda®) for elevated intraocular pressure. In the subsequent laboratory analyses, both netarsudil and ripasudil were found to improve the corneal endothelial pump and barrier function, but they showed differential effects on corneal epithelial cells. Whereas ripasudil improved the epithelial barrier function by upregulating major components of the tight and adherens junctions and reducing paracellular permeability, netarsudil had no or even adverse effects on the epithelial barrier properties by downregulating the expression levels of cell-junction-associated genes. The expression changes normalized after discontinuation of ROCK inhibitors. The findings support the concept that ROCK inhibitors can act as a double-edged sword by having beneficial effects on corneal endothelial cells and adverse effects on epithelial cells.

PURPOSE:To determine the clinical and ultrastructural characteristics of secondary graft failure after Descemet membrane endothelial keratoplasty (DMEK). METHODS:Retrospective, single-center case series of eyes with failed DMEK grafts undergoing repeat DMEK between January 2014 and December 2023 at the Department of Ophthalmology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany. Potential risk factors for graft failure were correlated with the interval between the primary and repeat DMEK. Transmission electron microscopy (TEM) of excised Descemet membranes was used to detect ultrastructural alterations related to secondary graft failure after DMEK. RESULTS:Among 6268 DMEKs, 156 cases (2.5%) met study inclusion criteria of secondary graft failure after an initial DMEK. Mean interval to repeat DMEK was 40 ± 30 months (range 5-132 months). Approximately 44% (n = 68) of eyes undergoing repeat DMEK had required air injections after the first DMEK because of graft detachment, compared with 10% in the primary DMEK cohort (P < 0.001). Glaucoma was present in 33% (n = 51) of eyes with secondary graft failure compared with 8% of our primary DMEK cohort (P < 0.001). Among the 51 glaucoma cases, 61% had primary open-angle glaucoma, 31% pseudoexfoliation glaucoma, and 8% chronic angle-closure glaucoma.TEM revealed the formation of a posterior collagenous layer more frequently in glaucoma eyes (92.5%) than in nonglaucoma eyes (76.5%). Posterior collagenous layer was significantly thicker in eyes with glaucoma. CONCLUSIONS:Eyes with secondary graft failure exhibited a higher prevalence of glaucoma and rebubbling procedures after the primary DMEK. TEM findings suggest that pressure-independent mechanisms might contribute to graft failure in glaucomatous eyes.

Purpose:Pseudoexfoliation (PEX) glaucoma is caused by progressive accumulation of abnormal fibrillar aggregates in aqueous humor outflow tissues. The current lack of effective models for PEX research, replicating the key feature of PEX material production, represents a critical gap in understanding the molecular pathomechanisms and identifying specific therapeutic targets. We have developed an advanced in vitro model using stromal fibroblasts from iridectomy specimens of PEX glaucoma patients with an appropriate genetic background. Methods:Peripheral iridectomy specimens were obtained from patients with PEX glaucoma (n = 35) during routine trabeculectomy. Stromal fibroblasts were enriched from heterogenous cell cultures. Extracellular matrix formation was stimulated and modulated in two- and three-dimensional (3D) spheroid cultures by TGF-β1, Ficoll 400, all-trans-retinoic acid (ATRA), and curcumin. Cell cultures were analyzed by immunocytochemistry, transmission electron microscopy and qPCR. Immortalized cell lines (n = 4) were generated by SV40 large T-antigen transfection. Results:Fibroblasts carrying the high-risk haplotype of LOXL1 (lysyl oxidase-like 1) expressed PEX-relevant matrix components and assembled an elastic fibrillar network, particularly upon treatment with TGF-β1 and the macromolecular crowding agent Ficoll 400. In 3D spheroid cultures, the presence of typical fibrillar PEX aggregates could be demonstrated by electron microscopy. Matrix production could be effectively suppressed by ATRA and curcumin. Similar to primary cells, immortalized cell lines formed 3D spheroids and expressed PEX-relevant matrix components including typical fibrillar aggregates. Conclusions:This advanced in vitro model, which recapitulates the hallmark of PEX syndrome/glaucoma, may provide a useful, easy-to-handle platform for studying disease mechanisms, assessing the impact of genetic and external factors, and exploring effects of targeted therapies for PEX-associated fibrosis.

Purpose: To investigate transcriptomic differences between patients with Fuchs endothelial corneal dystrophy (FECD) with and without trinucleotide repeat (TNR) expansion in the transcription factor 4 gene, as the genetic basis for patients with FECD lacking TNR expansion remains unclear. Methods: Bulk RNA sequencing was performed on corneal endothelial cells from 17 subjects: 10 patients with FECD [4 with repeat expansion lengths <50 (RE-) and 6 with expansions of 50 or more (RE+)] and 7 controls. Differential gene expression analysis was conducted using DESeq2. Principal component analysis (PCA), correlation matrix analysis, and heatmap visualization were used to evaluate gene expression patterns. Gene Ontology enrichment analysis was performed on differentially expressed genes (DEGs). Results: Analysis identified 509 DEGs (281 upregulated, 228 downregulated) between RE- and controls, and 640 DEGs (292 upregulated, 348 downregulated) between RE+ and controls. PCA revealed distinct gene expression patterns in both RE- and RE+ groups compared with controls. Between RE- and RE+ groups, only 10 DEGs were identified: ALDH3A1, ENSG00000286252, RNU6-645P, SNORD113-7, RNU6-429P, SNORA16A, COCH, EGFL6, SEMA3E, and ENSG00000228463. PCA, correlation matrix analysis, and heatmap visualization demonstrated high similarity in overall gene expression patterns between RE- and RE+ groups. Conclusions: Contrary to expectations, gene expression profiles showed remarkable similarity between patients with FECD with and without TNR expansion. Further investigation of the identified DEGs may provide insights into the role of TNR expansion in FECD pathophysiology.

Fuchs endothelial corneal dystrophy (FECD) remains a leading cause of corneal blindness globally, with corneal transplantation being the primary treatment. FECD is characterized by the formation of guttae, extracellular matrix (ECM) deposits beneath the corneal endothelium, and progressive endothelial cell loss. These pathological changes cause visual deterioration through light scattering by guttae and corneal edema due to endothelial cell loss. However, limitations such as donor shortage and graft failure necessitate alternative therapeutic approaches. We employed computational drug screening using three platforms (L1000FWD, L1000CDS2, and SigCom LINCS) to identify compounds capable of normalizing FECD-associated differentially expressed genes (DEGs). Analysis of transcriptome data from FECD patients with TCF4trinucleotide repeat expansion identified 706 upregulated and 962 downregulated genes. The screening platforms identified 200, 35, and 76 compounds through L1000FWD, L1000CDS2, and SigCom LINCS, respectively, with five compounds commonly predicted across all platforms. Among these, LDN193189 and cercosporin were selected for further evaluation based on availability and lack of cytotoxicity. Both compounds significantly decreased the expression of ECM-related genes (FN1, MATN3, BGN, and LTBP2) in FECD cell models and suppressed TGF-β-induced fibronectin expression. Additionally, both compounds reduced aggresome formation to normal control levels, suggesting protection against endoplasmic reticulum stress-induced cell death. This study demonstrates the feasibility of computational drug screening for identifying therapeutic candidates for FECD, with LDN193189 and cercosporin showing promise in normalizing FECD-associated pathological changes.

Purpose: Fuchs endothelial corneal dystrophy (FECD) displays a higher incidence in females than in males, yet the underlying mechanism remains unclear. This study aimed to elucidate sex-dependent differential gene expressions in corneal endothelial cells (CECs) from healthy non-FECD individuals and from patients with FECD. Methods: RNA-Seq data from CECs of non-FECD subjects (3 males, 4 females) and FECD subjects (5 males, 5 females) were analyzed to identify differentially expressed genes (DEGs) between the sexes. We used heatmaps and principal component analysis for expression pattern visualization and Gene Ontology analysis for functional categorization of DEGs. Results: Among the non-FECD subjects, we identified 341 DEGs—143 upregulated and 198 downregulated—in females relative to males. For FECD subjects, 309 DEGs were discovered, with 215 upregulated and 94 downregulated in females compared with males. Heatmaps exhibited hierarchical clustering by sex, whereas principal component analysis delineated distinct male and female clusters in both non-FECD and FECD cohorts. Gene Ontology enrichment analysis linked the upregulated genes in non-FECD females to steroid hormone response, and downregulated ones to cyclin-dependent protein kinase activity. In females with FECD, upregulated genes were associated with immune responses and downregulated genes with peptide hormone binding. Conclusions: To our knowledge, these findings are the first to reveal distinct gene expression patterns in CECs between sexes. The observed variations suggest a potential genetic basis for the observed sex disparity in FECD prevalence. Further investigation is warranted to explore these associations and their implications for the pathogenesis of FECD.

Background:Rovunaptabin neutralises functional autoantibodies targeting G-Protein coupled receptors (GPCR-fAAbs), observed in patients with Post-COVID syndrome. As we hypothesise an improvement of PCS by rovunaptabin, the aim of reCOVer was to investigate safety, tolerability, and clinical effects of rovunaptabin in PCS patients. Methods:reCOVer is a prospective, exploratory, placebo-controlled, double-blind, randomised phase IIa clinical investigator initiated trial with 1350 mg rovunaptabin with additional cross-over at the Universitätsklinikum Erlangen, Germany. The trial was registered in EudraCT, 2022-001781-35. Screening was done between 21·11·2023 and 25·06·2024. Eligible participants (18-80 years) showed GPCR-fAAbs, at least 3/8 defined PCS symptoms persisting ≥3 months after COVID-19 and fatigue as major symptom. Participants were randomly assigned (1:1) to either receive rovunaptabin or placebo at day 0 (d0) and d48 with a follow-up of 28 days, respectively. Primary endpoint was the number of treatment emergent adverse events (TEAE) at d28 (co-primary endpoint: TEAE at d70); secondary endpoint focused on fatigue and quality of life. Findings:Thirty PCS patients were randomised and analysed. RCT analysis showed nine (rovunaptabin) and five TEAEs (placebo), yet without statistically significance (p = 0·1299; CI -14·80%; 63·02%); one serious adverse event, not related to treatment, was recorded. Rovunaptabin showed a neutralisation of GPCR-fAAb and a significant improvement of FACIT Fatigue Scale (effect size = 2·10, p = 0·0378), Bell score (effect size = 3·64, p = 0·0004), Fatigue Severity Scale (effect size = -2·66, p = 0·0088), and quality of life (4/8 items). Interpretation:As this proof-of-concept study showed effects on the patient-centred endpoint PCS and a good safety profil, subsequent studies are needed to confirm these results in a larger cohort. Funding:German Federal Ministry of Education and Research, German Research Foundation.

Purpose:Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disorder characterized by excessive extracellular matrix (ECM) accumulation and corneal endothelial cell death. CTG trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene represents the most significant genetic risk factor. This study aimed to elucidate the role of TCF4 in FECD pathogenesis through comprehensive proteomic analysis. Methods:Corneal endothelial cells isolated from patients with FECD harboring TCF4 trinucleotide repeat expansion were immortalized to establish an FECD cell model (iFECD). CRISPR/Cas9-mediated genome editing was employed to generate TCF4-knockout iFECD cells. Whole-cell proteome analysis was performed using liquid chromatography-mass spectrometry, followed by pathway enrichment analysis of differentially expressed proteins (DEPs). The effects of TCF4 deletion on TGF-β-mediated protein aggregation and cell death were evaluated using Western blot analysis, flow cytometry, and aggresome detection assays. Results:Proteomic analysis identified 88 DEPs among 6510 detected proteins. Pathway analysis revealed significant enrichment in ECM-associated pathways, oxidative stress responses, and cellular motility. TCF4 deletion attenuated TGF-β-induced cell death in iFECD cells. Concordantly, Western blot analysis demonstrated that TCF4 deletion suppressed TGF-β2-mediated cleavage of caspase-3 and poly (ADP-ribose) polymerase. Flow cytometric analysis of Annexin V-positive cells confirmed reduced apoptosis in TCF4-deleted cells following TGF-β2 treatment. Additionally, aggresome detection assays revealed that TCF4 deletion diminished TGF-β2-induced protein aggregation. Conclusions:This study demonstrates a crucial role for TCF4 in FECD pathogenesis, particularly in ECM regulation and protein aggregation-induced cell death.

PURPOSE:The objective of this study was to compare the capacity of anterior segment optical coherence tomography (AS-OCT) and Scheimpflug camera in detecting tomographic changes over 1 year in a cohort of patients with Fuchs endothelial corneal dystrophy. METHODS:We conducted a single-center prospective study which consisted of 2 identical visits spaced 1 year apart. At each visit, tomographic analyses were performed using Scheimpflug imaging (Pentacam HR, Oculus) and swept source AS-OCT (Casia SS-1000 Tomey). The resulting images were analyzed by 3 experienced independent observers. The main outcome was the agreement between the 2 devices for detecting subclinical corneal edema, as assessed using the interdevice reliability measured by the Cohen kappa coefficient. The criteria for subclinical edema were the presence of irregular isopachs, displacement of the thinnest point, and presence of posterior surface depression. RESULTS:We analyzed 129 eyes of 81 patients with good quality images for both devices. The overall agreement between the 2 devices to detect subclinical edema was good, but the performance was significantly different for 2 parameters (thinnest point displacement and posterior depression). The interdevice reliability was 0.84 for the loss of parallel isopachs, 0.55 for the displacement of the thinnest point, and 0.40 for the focal posterior corneal surface depression. CONCLUSIONS:Corneal tomographic features of Fuchs endothelial corneal dystrophy can be easily quantified in clinical routine and clinical research by AS-OCT and Scheimpflug imaging, but both are not fully interchangeable. A longitudinal follow-up should always be done with the same device. Neither appears to be more sensitive than the other for detecting minimal changes over 12 months.

Purpose: To analyze the feasibility and outcome of Descemet membrane endothelial keratoplasty (DMEK) for treatment of endothelial failure in primary angle closure suspect (PACS) eyes. Methods: Retrospective, single-center case series of eyes treated by DMEK for endothelial failure caused by PACS. Main study parameters were success rate of DMEK, best-corrected visual acuity, anterior chamber depth, central corneal thickness, and endothelial cell density. Mean follow-up time was 16 ± 13 months. Results: Ten eyes of 9 patients receiving DMEK for the treatment of corneal endothelial failure because of PACS were included. Except for 2 eyes that had undergone cataract surgery, none of the eyes had previous ocular surgery. DMEK combined with cataract surgery was performed in 5 eyes, DMEK alone with second-step cataract surgery in 2 eyes. The eyes with corneal edema after cataract surgery received DMEK only. DMEK surgery was successful in nine out of 10 eyes, 1 patient required repeat DMEK because of primary graft failure. In the group of phakic eyes, mean preoperative internal anterior chamber depth was 1.74 ± 0.18 mm. In eyes with corneal edema, central corneal thickness was 849 ± 205 μm before DMEK surgery, and 517 ± 24 μm at the final postoperative visit (P = 0.002). Conclusions: DMEK is a feasible option in eyes with endothelial failure because of primary angle closure. In case of advanced corneal edema, a second-step procedure (first DMEK, second cataract surgery) is a possible approach if visibility of the lens is too poor for simultaneous cataract surgery.