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.

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.

Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Use of Topical Rho Kinase Inhibitors in the Treatment of Fuchs Dystrophy After Descemet Stripping Only.

PURPOSE: Fuchs corneal dystrophy (FD) is a common cause of endothelial keratoplasty. Recently, a series of FD cases treated with Descemet stripping only (DSO) demonstrated recovery of the central endothelium without transplantation of donor cells. Ripasudil, a rho kinase inhibitor, has been shown to promote corneal endothelial wound healing in animal models. This study prospectively evaluated the use of ripasudil in patients undergoing DSO for FD. METHODS: Enrolled patients underwent DSO with or without cataract surgery, performed by 1 surgeon. On the first postoperative day, patients were assigned to topical ripasudil 0.4% (Glanatec) 4 times a day for 2 months or no ripasudil and followed up monthly for the first 6 months and then at 9 and 12 months after surgery. Endothelial cell density (ECD) and pachymetry were evaluated at each postoperative visit. RESULTS: Eighteen patients were enrolled, including 8 women and 1 man in each group. Overall, patients who underwent DSO with ripasudil recovered vision more quickly (4.6 vs. 6.5 weeks, P < 0.01). In addition, the ripasudil group had a statistically significantly higher average ECD at 3, 6, and 12 months. The patients in the DSO observation group had a 10% decrease in peripheral ECD when comparing counts before surgery with counts 12 months after surgery (P < 0.05). In the DSO ripasudil group, there was no significant difference between peripheral ECD at preoperative baseline versus 12 months after surgery. CONCLUSIONS: DSO with topical rho kinase inhibitors may be an alternative treatment for patients with FD and a peripheral ECD greater than 1000 cells/mm.

Descemetorhexis Without Grafting for Fuchs Endothelial Dystrophy-Supplementation With Topical Ripasudil.

PURPOSE: To report the safety and efficacy of descemetorhexis without grafting as a primary intervention in Fuchs dystrophy, and the use of a ROCK inhibitor, ripasudil as a salvage agent in failing cases. METHODS: Twelve eyes of 11 patients underwent central descemetorhexis not exceeding 4 mm. All had Fuchs dystrophy-producing visual symptoms, requesting intervention. Exclusion criteria were a peripheral endothelial cell count <1000 and central edema. Corneal clearance and visual parameters were recorded monthly until corneal clearance was observed, then at intervals of 6 months. Cases failing to clear by month 2 were considered for salvage treatment. This consisted of treatment with 1 of 2 formulations of Rho-associated kinase inhibitor eye drops. Endothelial keratoplasty was planned as the final salvage procedure in unsuccessful cases. RESULTS: Nine of 12 eyes cleared spontaneously between 2 and 6 months. One eye failed to clear by month 5 and topical Y-27632 was administered, without success. Endothelial keratoplasty was performed. In 2 eyes, healing stalled at 3 and 2 months. In both cases, topical ripasudil administered 6 times a day for 2 weeks resulted in complete corneal clearance. In cases achieving corneal clearance, best spectacle corrected visual acuity improved from a mean of 0.26 to 0.125 (logMAR) with subjective improvement in quality of vision. CONCLUSIONS: In Fuchs dystrophy with visual degradation due to central guttae, descemetorhexis without grafting is a viable procedure for visual rehabilitation. Careful patient selection is required, but the advent of topical ripasudil as a salvage agent suggests that a broader application of the surgery may be possible. Further study into the use of this agent is now needed.

Selecting Fuchs patients for drug trials involving endothelial cell proliferation.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) might be managed by drug treatment before becoming severe enough to require surgery. For a clinical trial of such a drug, we hypothesize that selecting an adequate number of patients with FECD with only moderately compromised cell densities will be challenging. Thus, the purpose of the present study was to measure the prevalence of patients with FECD exhibiting moderately decreased corneal cell densities. METHODS: A retrospective data mining study (cross-sectional study) was performed on patient charts presenting at a large US northwestern academic health center by searching for diagnosis ICD-9 code 371.57 and Fuchs corneal dystrophies, including those with prior cataract surgeries and/or existing glaucoma. Patients with prior corneal transplants were excluded. Noncontact specular photomicroscopic data (Topcon 2000) were obtained from the central region whenever possible, and individual eyes were grouped according to cell density (cells/mm2): severe (<800), moderate (800-1,500), and mild (>1,500). RESULTS: The values for 98 eyes from 61 patients with FECD were as follows (mean ± SD): corneal thickness 573 ± 59 µm, cell size 627 ± 336 µm2/cell, coefficient of variation 23 ± 7, and density 1,883 ± 703 cells/mm2. The moderate subgroup with cell density values averaging 1,184 ± 212 (26) comprised 27% of the total FECD patient pool. CONCLUSIONS: Only approximately 1 out of 4 patients with FECD will show moderately compromised corneal cell densities. A moderate level of damage may be optimal for clinical trials for testing topical drugs on endothelial cell viability. Thus, investigators will need to initially screen a fourfold excess of all patients with FECD.

New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops.

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.

Rho-associated kinase inhibitor eye drop treatment as a possible medical treatment for Fuchs corneal dystrophy.

PURPOSE: To report a case of Fuchs corneal dystrophy that was successfully treated by Rho-associated kinase (ROCK) inhibitor eye drops, subsequent to transcorneal freezing of damaged corneal endothelial cells. METHODS: A 52-year-old Japanese man with a diagnosis of late-onset Fuchs corneal dystrophy was referred to our hospital as a candidate for keratoplasty. Best-corrected vision was 20/20 in the right eye and 20/63 in the left eye. Multiple guttae were observed in both eyes. The right cornea was clear, but the left showed severe central edema, with a central corneal thickness of 703 µm. We were unable to perform specular microscopy in the central cornea, but endothelial cells were observed in the midperiphery at a density of 757 cells per square millimeter. The patient was treated by a corneal endothelial denudation in the prepupillary region followed by the topical administration of a selective ROCK inhibitor, Y-27632, as eye drops for 1 week. Follow-up of 24 months is reported. RESULTS: Corneal clarity recovered and vision improved to 20/20 two weeks after the treatment. At 6 months, vision had improved to 20/16 and central corneal thickness measured was 568 µm, significantly lower than its pretreatment value. Endothelial function and vision have been well maintained up to the most recent observation, 24 months after the treatment. The average corneal endothelial density in the central and peripheral cornea was 1549.3 ± 89.7 and 705.0 ± 61.1 cells per square millimeter, respectively. CONCLUSIONS: The case highlights the possibility of medical treatments involving the use of ROCK inhibitor eye drops as an alternative to graft surgery for certain forms of corneal endothelial disease.