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The aim of this study was to evaluate which amino acid(s) among the 20 standard protein amino acids is indispensable for the survival of cultured human corneal endothelial cells (HCECs).HCECs were cultured in amino acid screening media that were missing 1 specific amino acid, and cell growth was evaluated. After this first selection, we conducted a further evaluation of cell growth in response to the addition of 4 amino acids (cysteine, methionine, valine, and arginine) to amino acid-free culture media. We then evaluated the antioxidant effect of cysteine compared with other antioxidants in terms of apoptosis of HCECs, rabbit corneal endothelial cell (CECs), monkey CECs, and ex vivo human donor corneas.Culture in an amino acid-free Dulbecco Modified Eagle Medium (DMEM) decreased the cell numbers to 11.0% when compared with culture in normal DMEM. Removal of cysteine, methionine, valine, or arginine from DMEM significantly suppressed cell numbers (27.7%, 61.4%, 75.5%, and 60.6%, respectively) (P0.01), whereas removal of other amino acids did not significantly decrease cell numbers. A lack of cysteine induced apoptosis, but addition of antioxidants reversed this. Removal of cysteine induced in vitro apoptosis in HCECs, rabbit CECs, monkey CECs, and ex vivo human donor corneas, whereas the presence of cysteine almost completely suppressed this apoptosis.Cysteine seems to be an indispensable amino acid for HCEC growth and survival. Its necessity might reflect a high requirement for antioxidants to protect HCECs from oxidative stress, as HCECs have high aerobic metabolic activity.

Corneal endothelial cells (CECs) show poor regenerative ability in humans, and noncompensatory damage of CECs causes irreversible corneal haziness in cases of bullous keratopathy. Although corneal transplantations provide considerable clinical benefits, allograft rejection, primary graft failure, and the shortage of donor corneas are problems that still need to be overcome. The establishment of new treatment therapies is the key to solving these problems, and we have attempted to establish a new clinical intervention for corneal endothelial dysfunction. We previously demonstrated that the inhibition of Rho/Rho kinase (ROCK) signaling by Y-27632, a specific ROCK inhibitor, promoted cell adhesion, inhibited apoptosis, and enhanced cell proliferation in cultured primate CECs. These results raise the possibility that the ROCK inhibitor might serve as a new tool for establishing an effective culture method for newly emerging cultivated CEC transplantation therapies. Moreover, because Y-27632 enhances cell proliferation in vitro, we hypothesized that the use of a ROCK inhibitor could be a new pharmacological intervention for the treatment of corneal endothelial dysfunction. We demonstrated that the topical instillation of a ROCK inhibitor promotes corneal endothelium wound healing in an animal model. This indicates that use of a ROCK inhibitor is a less invasive and novel therapy that should prove promising for the treatment of corneal endothelial dysfunction.

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.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.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.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.