Descemet Endothelial Thickness Comparison Trial II (DETECT II): multicentre, outcome assessor-masked, placebo-controlled trial comparing Descemet membrane endothelial keratoplasty (DMEK) to Descemet stripping only (DSO) with adjunctive ripasudil for Fuchs dystrophy.

INTRODUCTION: It remains uncertain whether Descemet membrane endothelial keratoplasty (DMEK) or Descemet stripping only (DSO) yields better outcomes in patients with symptomatic Fuchs endothelial corneal dystrophy (FECD). This paper presents the protocol for the Descemet Endothelial Thickness Comparison Trial II (DETECT II), a multicentre, outcome-masked, randomised, placebo-controlled, clinical trial comparing DMEK to DSO with ripasudil (DSO-R) for this patient population. METHODS AND ANALYSIS: A total of 60 patients with endothelial dysfunction due to symptomatic FECD will be enrolled from seven participating sites in the USA. The patients will be randomly assigned in a 1:1 ratio to one of the following treatment groups: group 1-DMEK plus topical placebo and group 2-DSO plus topical ripasudil 0.4%. The enrolment period is 24 months. The primary outcome is best spectacle-corrected visual acuity at 12 months. Secondary outcomes include peripheral and central endothelial cell density, visual acuity, vision-related quality of life and Pentacam Scheimpflug tomography. Study outcomes will be analysed using mixed effects linear regression. Adverse events, including rebubble procedures, endothelial failure and graft rejection, will be documented and analysed using appropriate statistical methods. DETECT II aims to provide evidence on the comparative effectiveness of DMEK and DSO-R. The results of this trial will contribute to optimising the treatment of FECD, while also exploring the cost-effectiveness of these interventions. Dissemination of findings through peer-reviewed publications and national/international meetings will facilitate knowledge translation and guide clinical practice in the field of corneal transplantation. ETHICS AND DISSEMINATION: A data and safety monitoring committee has been empanelled by the National Eye Institute. All study protocols will be subject to review and approval by WCG IRB as the single IRB of record. This study will comply with the National Institute of Health (NIH) Data Sharing Policy and Policy on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. Data from the trial will be made available on reasonable request. TRIAL REGISTRATION NUMBER: NCT05275972.

[Pathogenesis of Fuchs endothelial corneal dystrophy, the fibrillar layer and individualized treatment]. / Pathogenese der Fuchs-Endotheldystrophie, die fibrilläre Schicht und individualisierte Therapie.

Fuchs endothelial corneal dystrophy (FECD) is a genetic and age-associated corneal disease characterized by an accelerated loss of corneal endothelial cells and an increased subendothelial deposition of extracellular matrix (ECM). Clinically, advanced disease leads to corneal edema with subsequent reduction in visual acuity. In the majority of patients with advanced FECD, a fibrillar layer (FL) appears on the posterior corneal surface. This FL is mostly localized in the inferotemporal corneal quadrant, marks areas with significantly reduced endothelial cell density and increased corneal thickness in the sense of edema and can be visualized and measured using Scheimpflug backscatter analysis due to increased backscatter. FECD is currently the most common indication for corneal transplantation worldwide, usually in the form of Descemet membrane endothelial keratoplasty (DMEK). New treatment approaches include variations of DMEK surgery such as hemi- or quarter DMEK with individualized and smaller grafts or Descemet membrane stripping only (DSO). In the future, clinical imaging of the FL as a particularly affected endothelial area could be important for FECD progression assessment and planning of surgical interventions. This article provides an overview of the current state of research on the clinical aspects, pathogenesis, fibrillar layer and individualized treatment of FECD.

Fuchs' Endothelial Corneal Dystrophy evaluation using a high-resolution wavefront sensor.

This study aims to evaluate the applicability of the high-resolution WaveFront Phase Imaging Sensor (WFPI) in eyes with Fuchs' Endothelial Corneal Dystrophy (FECD) through qualitative and quantitative analysis using a custom-designed Automatic Guttae Detection Method (AGDM). The ocular phase was measured using the t · eyede aberrometer and then was processed to obtain its High-Pass Filter Map (HPFM). The subjects were pathological and healthy patients from the Fundación Jiménez-Díaz Hospital (Madrid, Spain). The AGDM was developed and applied in pupils with 3 and 5 mm of diameter. A set of metrics were extracted and evaluated like the Root-Mean-Square error (RMS), Number of guttae, Guttae Area, and Area of Delaunay Triangulation (DT). Finally, a Support Vector Machine (SVM) model was trained to classify between pathological and healthy eyes. Quantitatively, the HPFM reveals a dark spots pattern according to the ophthalmologist's description of the slit-lamp examination of guttae distribution. There were significant statistical differences in all the metrics when FECD and Healthy groups were compared using the same pupil size; but comparing both pupil sizes for the same group there were significant differences in most of the variables. This sensor is a value tool to objectively diagnose and monitor this pathology through wavefront phase changes.

Hepatocyte Growth Factor Modulates Corneal Endothelial Wound Healing In Vitro.

In this study, we assessed the impact of hepatocyte growth factor (HGF) on corneal endothelial cells (CECs), finding that HGF concentrations of 100-250 ng/mL significantly increased CEC proliferation by 30%, migration by 32% and improved survival under oxidative stress by 28% compared to untreated controls (p < 0.05). The primary objective was to identify non-fibrotic pharmacological strategies to enhance corneal endothelial regeneration, addressing a critical need in conditions like Fuchs' endothelial dystrophy (FED), where donor tissue is scarce. To confirm the endothelial nature of the cultured CECs, Na+/K+-ATPase immunohistochemistry was performed. Proliferation rates were determined through BrdU incorporation assays, while cell migration was assessed via scratch assays. Cell viability was evaluated under normal and oxidative stress conditions using WST-1 assays. To ensure that HGF treatment did not trigger epithelial-mesenchymal transition, which could lead to undesirable fibrotic changes, α-SMA staining was conducted. These comprehensive methodologies provided robust data on the effects of HGF, confirming its potential as a therapeutic agent for corneal endothelial repair without inducing harmful EMT, as indicated by the absence of α-SMA expression. These findings suggest that HGF holds therapeutic promise for enhancing corneal endothelial repair, warranting further investigation in in vivo models to confirm its clinical applicability.

Rapid detection of guttae area using aniline blue staining in Fuchs endothelial corneal dystrophy mouse model.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial degeneration resulting in impaired visual acuity. Excessive deposition of extracellular matrix (guttae) on Descemet's membrane (DM) is the hallmark of FECD. We sought to detect the guttae area rapidly using aniline blue (AB) staining in FECD mouse model. FECD mouse model was established via ultraviolet A (UVA) exposure. Masson's trichrome staining was utilized to stain the corneal sections. AB staining was utilized to stain both whole cornea tissues and stripped Descemet's membrane-endothelium complex (DMEC) flat mounts, while immunofluorescence staining of collagen I was employed to stain guttae areas. In Masson's trichrome staining, corneal collagen fibrils were stained blue with AB. The DMEC flat mounts were stained into relative dark blue areas and relative light blue areas using 2% AB staining. The areas of dark blue could almost overlap with collagen I-positive areas, and have an acellular centre and a moderately distinct boundary line with the surrounding corneal endothelial cells. In conclusion, AB staining is a rapid and effective method for the evaluation of the guttae areas in the FECD mouse model.

Change in Visual Acuity of Patients With Fuchs Endothelial Corneal Dystrophy Over 1 Year.

PURPOSE: To determine whether the clinical and paraclinical course of Fuchs endothelial corneal dystrophy (FECD) over 1 year is related to the extent of triplet repetition in the transcription factor 4 (TCF4) gene. METHODS: A prospective study with a 1-year follow-up was conducted. A total of 104 patients (160 eyes) with FECD and an equivalent number of age- and sex-matched control subjects without FECD were included. At inclusion, the corneas were graded using the modified Krachmer grade (KG) and patients were genotyped for the number of trinucleotide repeats (TNRs) in the TCF4 gene by the short tandem repeat assay. Visual acuity, Scheimpflug tomographic features, and the Visual Function and Corneal Health Status using a visual disability instrument were measured on 2 visits at 1-year intervals. RESULTS: KGs ranged from 1 to 6, and 46% of eyes had grades 1 to 4. 71% of the patients harbored TNR expansion (>40) versus 13% in control subjects ( P < 0.001). Severity at inclusion was higher in the presence of TNR expansion when considering eyes independently (mean grade ±SD, 4.08 ± 1.42) without TNR expansion and 4.66 ± 1.27 with TNR expansion ( P = 0.024). In 1 year, the ETDRS score significantly decreased by -2.97 (95% confidence interval -4.69 to -1.26, P = 0.001) and the ETDRS score with glare by -4.25 (95% confidence interval -6.22 to -2.27, P < 10 -5 ). There was no relationship between the rate of decline and TNR expansion or KG. Central corneal thickness and Visual Function and Corneal Health Status scores did not significantly vary. CONCLUSIONS: It is possible to measure a subtle progression of FECD over a period as short as 1 year. We did not find a relationship between the presence of TNR expansion and the speed of deterioration over 1 year. This work should facilitate the design of future clinical trials on FECD.

Tissue-specific TCF4 triplet repeat instability revealed by optical genome mapping.

BACKGROUND: Fuchs endothelial corneal dystrophy (FECD) is the most common repeat-mediated disease in humans. It exclusively affects corneal endothelial cells (CECs), with ≤81% of cases associated with an intronic TCF4 triplet repeat (CTG18.1). Here, we utilise optical genome mapping (OGM) to investigate CTG18.1 tissue-specific instability to gain mechanistic insights. METHODS: We applied OGM to a diverse range of genomic DNAs (gDNAs) from patients with FECD and controls (n = 43); CECs, leukocytes and fibroblasts. A bioinformatics pipeline was developed to robustly interrogate CTG18.1-spanning DNA molecules. All results were compared with conventional polymerase chain reaction-based fragment analysis. FINDINGS: Analysis of bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin. However, clusters of CTG18.1 molecules, encompassing ∼1800-11,900 repeats, were exclusively detected in diseased CECs from expansion-positive cases. Additionally, both progenitor allele size and age were found to influence the level of leukocyte-specific CTG18.1 instability. INTERPRETATION: OGM is a powerful tool for analysing somatic instability of repeat loci and reveals here the extreme levels of CTG18.1 instability occurring within diseased CECs underpinning FECD pathophysiology, opening up new therapeutic avenues for FECD. Furthermore, these findings highlight the broader translational utility of FECD as a model for developing therapeutic strategies for rarer diseases similarly attributed to somatically unstable repeats. FUNDING: UK Research and Innovation, Moorfields Eye Charity, Fight for Sight, Medical Research Council, NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Grantová Agentura Ceské Republiky, Univerzita Karlova v Praze, the National Brain Appeal's Innovation Fund and Rosetrees Trust.

MitoQ relieves mitochondrial dysfunction in UVA and cigarette smoke-induced Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD), a degenerative corneal condition, is characterized by the droplet-like accumulation of the extracellular matrix, known as guttae and progressive loss of corneal endothelial cells ultimately leading to visual distortion and glare. FECD can be influenced by environmental stressors and genetic conditions. However, the role of mitochondrial dysfunction for advancing FECD pathogenesis is not yet fully studied. Therefore, in the present study we sought to determine whether a combination of environmental stressors (ultraviolet-A (UVA) light and cigarette smoke condensate (CSC)) can induce mitochondrial dysfunction leading to FECD. We also investigated if MitoQ, a water-soluble antioxidant, can target mitochondrial dysfunction induced by UVA and CSC in human corneal endothelial cells mitigating FECD pathogenesis. We modeled the FECD by increasing exogenous oxidative stress with CSC (0.2%), UVA (25J/cm2) and a combination of UVA + CSC and performed a temporal analysis of their cellular and mitochondrial effects on HCEnC-21T immortalized cells in vitro before and after MitoQ (0.05 µM) treatment. Interestingly, we observed that a combination of UVA + CSC exposure increased mitochondrial ROS and fragmentation leading to a lower mitochondrial membrane potential and increased levels of cytochrome c release leading to apoptosis and cell death. MitoQ intervention successfully mitigated these effects and restored cell viability. The UVA + CSC model could be used to study stress induced mitochondrial dysfunction. Additionally, MitoQ can serve as a viable antioxidant in attenuating mitochondrial dysfunction, underscoring its potential as a molecular-focused treatment approach to combat FECD pathogenesis.

Double-Cannula Maneuver for the Double-Roll Configuration of Donor Descemet Membrane Outside Recipient Anterior Chamber During Descemet Membrane Endothelial Keratoplasty.

PURPOSE: The purpose of this study was to present a new surgical technique to convert a single roll of Descemet membrane (DM) into a double roll using 2 cannulas in a balanced salt solution-filled Petri dish during DM endothelial keratoplasty. METHODS: A single DM roll stained with trypan blue was placed in a balanced salt solution-filled Petri dish. Two cannulas (28G) were introduced from opposite ends of the single roll, inserted into the roll, and slowly spread apart to change the single roll into a double roll. The DM was aspirated into the modified Jones tube and loaded, maintaining a double-roll configuration with endothelium-down orientation in a bevel-up position. The modified Jones tube with the bevel down was inserted into the recipient anterior chamber through the main wound. The modified Jones tube was rotated to the bevel-up orientation. After checking the graft orientation, the DM was inserted into the recipient anterior chamber. The double-roll DM was easily unfolded by tapping the center of the cornea using a cannula. A 28G cannula was inserted under the DM, and the anterior chamber was filled with air. RESULTS: Three months after surgery, the patient's corrected visual acuity in the right eye was 6/7.5 and the endothelial cell count was 1095/mm 2 . The corneal thickness was 533 µm, and the cornea was clear. CONCLUSIONS: The double-cannula maneuver mechanically changes the single roll of the donor DM to a double roll outside the recipient anterior chamber, making DM unfolding easier and minimizing the risk of upside-down apposition of the donor DM.

Neuropeptide alpha-Melanocyte stimulating hormone preserves corneal endothelial morphology in a murine model of Fuchs dystrophy.

Fuchs endothelial corneal dystrophy is a heterogenous disease with multifactorial etiology, and genetic, epigenetic, and exogenous factors contributing to its pathogenesis. DNA damage plays a significant role, with ultraviolet-A (UV-A) emerging as a key contributing factor. We investigate the potential application of neuropeptide α-melanocyte stimulating hormone (α-MSH) in mitigating oxidative stress induced endothelial damage. First, we examined the effects of α-MSH on a cultured human corneal endothelial cell line (HCEnC-21T) exposed to hydrogen peroxide (H2O2) induced oxidative DNA damage. We performed immunofluorescence and flow cytometry to assess DNA damage and cell death in the cultured cells. Additionally, we used an established mouse model that utilizes ultraviolet light to induce corneal endothelial cell damage resulting in decreased CEnC number, increased cell size variability, and decreased percentage of hexagonal cells. This endothelial decompensation leads to an increase in corneal thickness. Following UV-A exposure, the mice were systemically treated with α-MSH, either immediately after exposure (early treatment) or beginning two weeks post-exposure (delayed treatment). To evaluate treatment efficacy, we analyzed CEnC density and morphology using in vivo confocal microscopy, and central corneal thickness using anterior segment optical coherence tomography. Our findings demonstrated that α-MSH treatment effectively protects HCEnC-21T from free-radical induced oxidative DNA damage and subsequent cell death. In vivo, α-MSH treatment, mitigated the loss of CEnC density, deterioration of cell morphology and suppression of the resultant corneal swelling. These results underline the potential application of α-MSH as a therapeutic agent for mitigating corneal endothelial damage.

Exploring single-cell RNA sequencing as a decision-making tool in the clinical management of Fuchs' endothelial corneal dystrophy.

Single-cell RNA sequencing (scRNA-seq) has enabled the identification of novel gene signatures and cell heterogeneity in numerous tissues and diseases. Here we review the use of this technology for Fuchs' Endothelial Corneal Dystrophy (FECD). FECD is the most common indication for corneal endothelial transplantation worldwide. FECD is challenging to manage because it is genetically heterogenous, can be autosomal dominant or sporadic, and progress at different rates. Single-cell RNA sequencing has enabled the discovery of several FECD subtypes, each with associated gene signatures, and cell heterogeneity. Current FECD treatments are mainly surgical, with various Rho kinase (ROCK) inhibitors used to promote endothelial cell metabolism and proliferation following surgery. A range of emerging therapies for FECD including cell therapies, gene therapies, tissue engineered scaffolds, and pharmaceuticals are in preclinical and clinical trials. Unlike conventional disease management methods based on clinical presentations and family history, targeting FECD using scRNA-seq based precision-medicine has the potential to pinpoint the disease subtypes, mechanisms, stages, severities, and help clinicians in making the best decision for surgeries and the applications of therapeutics. In this review, we first discuss the feasibility and potential of using scRNA-seq in clinical diagnostics for FECD, highlight advances from the latest clinical treatments and emerging therapies for FECD, integrate scRNA-seq results and clinical notes from our FECD patients and discuss the potential of applying alternative therapies to manage these cases clinically.

Guttae Morphology After Cultured Corneal Endothelial Cell Transplant in Fuchs Endothelial Corneal Dystrophy.

Importance: Whether guttae in Fuchs endothelial corneal dystrophy (FECD) can be removed by polishing without Descemet stripping and whether postoperative maintenance of reduced guttae can be achieved through cultured corneal endothelial cell (CEC) transplant therapy are critical issues to be addressed. Objective: To investigate the decrease of guttae through polishing degenerated CECs and abnormal extracellular matrix (ECM) without Descemet stripping and to observe the behavior of guttae following cultured CEC transplant. Design, Setting, and Participants: This case series prospective observational study was conducted in a hospital outpatient clinic setting. Between December 2013 and January 2019, 22 eyes with corneal endothelial failure caused by FECD received cultured CEC transplant therapy at Kyoto Prefectural University Hospital. Of these, 15 eyes were consistently monitored at the same central corneal area during the preoperative phase, as well as in the early (within 1 year) and late (after 3 years) postoperative phases. The images from these phases were categorized into 3 groups: typical guttae, atypical guttae, and no guttae. Exposures: Cultured CEC transplant therapy. Main Outcomes: Proportion of guttae in the observable area was measured, comparing the early and late postoperative phases for each group. Results: The mean age of the patients at the time of surgery was 69 years (range, 49-79 years). All 15 eyes exhibited the presence of confluent guttae preoperatively (100%). Among these, 3 of 15 eyes belonged to male patients. The early postoperative phase of guttae morphologies was classified into 3 groups: 5 eyes with typical guttae, 7 with atypical guttae, and 3 with no guttae. The decrease in the number of these guttae was achieved by surgical procedures. The median percentage of guttae in the typical guttae, atypical guttae, and no guttae groups was 41.8%, 44.4%, and 16.2%, respectively, in the early phase, and 42.2%, 38.2%, and 18.8%, respectively, in the late phase. Conclusions and Relevance: The findings demonstrate that in some cases of FECD, guttae can be removed by scraping and polishing abnormal ECM and degenerated CECs, while preserving the Descemet membrane. Furthermore, cultured CEC transplant resulted in no increase in guttae for up to 3 years, providing insights into surgically eliminating guttae.

Open-angle glaucoma and Fuchs dystrophy.

A 62-year-old woman with a history of moderate myopia, long-standing open-angle glaucoma (OAG), and Fuchs dystrophy in both eyes was referred for consultative care. She had prior trabeculectomy in 1984 and 1992 in the left and right eyes, respectively. She is 3 months post-Descemet-stripping endothelial keratoplasty (DSEK) in the left eye, now referred with uncontrolled intraocular pressure (IOP) despite maximum tolerated medical therapy. Current medical therapy for IOP consists of acetazolamide 250 mg by mouth 2 times a day, brimonidine 2 times a day in the left eye, dorzolamide 2 times a day in the left eye, and timolol 2 times a day in the left eye. The patient has a history of presumed steroid response; however, her corneal surgeon has requested that the steroid be continued for the next several months because of the recent DSEK. The IOP in the left eye has ranged from the mid-20s to mid-30s since DSEK. The right eye has consistently had pressure in the low teens and below for many years without topical antihypertensive medications. Examination revealed stable visual acuity at 20/30 and 20/40 in the right and left eyes, respectively, IOP was 12 mm Hg in the right eye and 25 mm Hg in the left eye by Goldman applanation, irregular but reactive pupils without afferent defect, and full confrontational visual fields. Slitlamp examination showed superior low avascular bleb, moderate-to-severe guttae, and posterior chamber IOL in the right eye. The left eye showed superior low diffuse bleb, clear DSEK graft, quiet chamber, superonasal iridectomy, and posterior chamber IOL with an open posterior capsule. The conjunctiva was moderately scarred but a repeat trabeculectomy or Xen Gel stent (Abbvie) appeared possible. The angles were wide open in each eye. Fundus examination was normal aside from myopic, anomalous-appearing nerves with an approximate cup-to-disc ratio of 0.90 in both eyes. Humphrey visual field showed nonspecific changes on the right and moderate nasal defect on the left eye, stable to previous examinations dating back to 2018 (Figure 1JOURNAL/jcrs/04.03/02158034-202407000-00018/figure1/v/2024-07-10T174240Z/r/image-tiff and Figure 2JOURNAL/jcrs/04.03/02158034-202407000-00018/figure2/v/2024-07-10T174240Z/r/image-tiff). Optical coherence tomography (OCT) of the retinal nerve fiber layer (RNFL) revealed moderated thinning in both eyes that was also stable to prior examinations (Figure 3JOURNAL/jcrs/04.03/02158034-202407000-00018/figure3/v/2024-07-10T174240Z/r/image-tiff). Her axial length measured 25.23 and 26.34 mm in the right and left eyes, respectively. Central corneal thickness was 553 µm in the right eye and 563 µm in the left eye before her DSEK procedure. What would be your approach to management of this patient's left eye, addressing the following: Rationale for your procedure of choice? Would you over-rule the corneal surgeon and stop the steroid in an attempt to obviate the need for glaucoma surgery? Does the age of onset of glaucoma affect your surgical decision making? Note that patient age at the time of trabeculectomy was 22 years. Are some procedures better suited for patients after DSEK surgery?

Comparison of rebubbling rate between preloaded endothelium-in and preloaded no-touch endothelium-out Descemet membrane endothelial keratoplasty transplantation.

BACKGROUND: To compare the difference in rebubbling rates between patients undergoing Descemet membrane endothelial keratoplasty (DMEK) with endothelium-in using a standard IOL cartridge and those with endothelium-out DMEK utilizing a no-touch technique with borosilicate glass cartridge transplantation. METHODS: This retrospective study included all eyes that underwent preloaded endothelium-in or endothelium-out DMEK transplantation from June 2019 to December 2023 at the Hanusch Hospital, Vienna, Austria. All DMEKs were harvested, prepared and preloaded at the European Eye Bank of Venice, Italy. DMEK surgeries were done by one experienced surgeon and the procedure was completed by air tamponade of the anterior chamber. RESULTS: Overall, 32 eyes each of 31 endothelium-out patients and of 29 endothelium-in patients were included. 32 preloaded endothelium-in procedures were followed by 32 preloaded endothelium-out procedures. Rebubbling rate for endothelium-in was 15/32 (47%) and for endothelium-out was 7/25 (28%) (p = 0.035, Pearson's chi-squared test). Donor age was the most important variable for rebubbling in a random forest algorithm model (ROC: 0.69). CONCLUSIONS: Rebubbling rate in endothelium-out DMEK was less than two-thirds compared to endothelium-in DMEK favoring no-touch endothelium-out DMEK as the preferred technique of DMEK transplantation.

Enhanced Migration of Fuchs Corneal Endothelial Cells by Rho Kinase Inhibition: A Novel Ex Vivo Descemet's Stripping Only Model.

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

Dysregulation of the TCF4 Isoform in Corneal Endothelial Cells of Patients With Fuchs Endothelial Corneal Dystrophy.

Purpose: This study evaluated the dysregulation of TCF4 isoforms and differential exon usage (DEU) in corneal endothelial cells (CECs) of Fuchs endothelial corneal dystrophy (FECD) with or without trinucleotide repeat (TNR) expansion in the intron region of the TCF4 gene. Methods: Three RNA-Seq datasets of CECs (our own and two other previously published datasets) derived from non-FECD control and FECD subjects were analyzed to identify TCF4 isoforms and DEU events dysregulated in FECD by comparing control subjects to those with FECD with TNR expansion and FECD without TNR expansion. Results: Our RNA-Seq data demonstrated upregulation of three TCF4 isoforms and downregulation of two isoforms in FECD without TNR expansion compared to the controls. In FECD with TNR expansion, one isoform was upregulated and one isoform was downregulated compared to the control. Additional analysis using two other datasets identified that the TCF4-277 isoform was upregulated in common in all three datasets in FECD with TNR expansion, whereas no isoform was dysregulated in FECD without TNR expansion. DEU analysis showed that one exon (E174) upstream of the TNR, which only encompassed TCF4-277, was upregulated in common in all three datasets, whereas eight exons downstream of the TNR were downregulated in common in all three datasets in FECD with TNR expansion. Conclusions: This study identified TCF4-277 as a dysregulated isoform in FECD with TNR expansion, suggesting a potential contribution of TCF4-277 to FECD pathophysiology.

Associations Between Visual Functions and Severity Gradings, Corneal Scatter, or Higher-Order Aberrations in Fuchs Endothelial Corneal Dystrophy.

Purpose: The purpose of this study was to investigate the associations between visual function and severity grading, corneal scatter, or higher-order aberrations (HOAs) in patients with Fuchs endothelial corneal dystrophy (FECD). Methods: This observational case series study included 49 eyes of 27 patients with FECD and 10 eyes of 10 healthy individuals. We evaluated corrected distance visual acuity (CDVA) using Landolt-C and Early Treatment Diabetic Retinopathy Study charts and contrast sensitivity using the CSV-1000E chart and CSV-1000RN letter chart. We analyzed the associations between visual function and explanatory variables, including age, modified Krachmer grade, central corneal thickness (CCT), anterior segment optical coherence tomography (AS-OCT)-based grade, HOAs, intraocular straylight, and corneal densitometry. We additionally conducted receiver operating characteristic (ROC) analysis to identify the corneal densitometry thresholds for decreased visual function. Results: There were significant associations between visual function and the modified Krachmer grade, CCT, AS-OCT-based grade, HOAs, intraocular straylight, and corneal densitometry. A modified Krachmer grade ≥ 3 was identified as a threshold for decreased visual function. Multivariate analysis showed that corneal densitometry was significantly associated with all visual function parameters, and HOAs were significantly associated with CDVA but not with contrast sensitivity. ROC analysis revealed that corneal densitometry of the posterior layer at 0 to 2 mm ≥ 10 grayscale units (GSU), was identified as a threshold for decreased visual function. Conclusions: HOAs, forward and backward light scatter affected visual function, with backward light scatter being the most influential. In patients with FECD, modified Krachmer grade ≥ 3 and corneal densitometry ≥ 10 GSU were thresholds for visual disturbance.

Clinical characteristics of viral-associated Fuchs uveitis syndrome and Posner-Schlossman syndrome in a Chinese population.

PURPOSE: To identify the types of viral infection in aqueous humor (AqH) among patients diagnosed as Fuchs uveitis syndrome (FUS) or Posner-Schlossman syndrome (PSS) and investigate their relevance to clinical manifestations and visual outcome. METHODS: A total of 375 patients and 171 patients were diagnosed as FUS or PSS in our department. AqH and serum samples from 68 FUS patients and 16 PSS patients were obtained during eye surgery. The viral etiologies, clinical features, auxiliary tests and visual prognosis of patients with FUS or PSS who underwent AqH analysis were analysed and compared. RESULTS: Among 68 FUS patients, rubella virus (RV), cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella-zoster virus were identified in 17, 11, 1 and 1 patients, respectively. Seven patients with CMV and 1 with HSV were identified in 16 PSS patients. In both FUS and PSS groups, virus-associated eyes had higher proportion of secondary glaucoma and worse visual prognosis as compared with non-virus-associated eyes (all P < 0.05). In FUS group, specifically, CMV infection manifested as more obvious anterior segment inflammation and lower corneal endothelial cell density (CECD). RV infection showed a higher percentage of vitritis. In PSS group, CMV-associated PSS had a lower retinal nerve fiber layer thickness and CECD, worse visual prognosis as compared with non-virus-associated PSS (all P < 0.05). CONCLUSION: Our study identified 4 types of viral infection in FUS and 2 types of viral infection in PSS. Virus-associated patients are usually associated with more obvious clinical signs and poor visual prognosis.

Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.