Feasibility of cell-based therapy combined with descemetorhexis for treating Fuchs endothelial corneal dystrophy in rabbit model.

Corneal transparency is maintained by the corneal endothelium through its pump and barrier function. Severe corneal endothelial damage results in dysregulation of water flow and eventually causes corneal haziness and deterioration of visual function. In 2013, we initiated clinical research of cell-based therapy for treating corneal decompensation. In that study, we removed an 8-mm diameter section of damaged corneal endothelium without removing Descemet's membrane (the basement membrane of the corneal endothelium) and then injected cultured human corneal endothelial cells (CECs) into the anterior chamber. However, Descemet's membrane exhibits clinically abnormal structural features [i.e., multiple collagenous excrescences (guttae) and thickening] in patients with Fuchs endothelial corneal dystrophy (FECD) and the advanced cornea guttae adversely affects the quality of vision, even in patients without corneal edema. The turnover time of cornea guttae is also not certain. Therefore, we used a rabbit model to evaluate the feasibility of Descemet's membrane removal in the optical zone only, by performing a small 4-mm diameter descemetorhexis prior to CEC injection. We showed that the corneal endothelium is regenerated both on the corneal stroma (the area of Descemet's membrane removal) and on the intact peripheral Descemet's membrane, based on the expression of function-related markers and the restoration of corneal transparency. Recovery of the corneal transparency and central corneal thickness was delayed in areas of Descemet's membrane removal, but the cell density of the regenerated corneal endothelium and the thickness of the central corneal did not differ between the areas with and without residual Descemet's membrane at 14 days after CEC injection. Here, we demonstrate that removal of a pathological Descemet's membrane by a small descemetorhexis is a feasible procedure for use in combination with cell-based therapy. The current strategy might be beneficial for improving visual quality after CEC injection as a treatment for FECD.

Nonconcordant regulation of mitochondrial respiratory complexes in the kidneys of 5/6 nephrectomized mice.

Hyperglycemia induces nonconcordant regulation of renal mitochondrial respiratory complexes, increases oxidative stress, and causes diabetic nephropathy. Hypertension is a complication associated with diabetes and involves glomerular hyperfiltration, the effects of which on mitochondrial respiratory complexes are not well understood. To investigate the effect of glomerular hyperfiltration on renal mitochondrial respiratory complexes, we used the 5/6 nephrectomized BKS. Cg-Dock7m+/+Leprdb/J, Dock7m+/+Leprdb mice (db/m-5/6Nx mice) as a model for glomerular hyperfiltration. The BKS. Cg-Dock7m+/+Leprdb/J, +Leprdb/+Leprdb mice (db/db mice), a model for type 2 diabetes, was used as the positive control. We investigated the activities and protein levels of the mitochondrial complex, and the mitochondrial DNA and adenosine triphosphate content in the kidneys of these models. Blood chemistry and renal histopathological examination were performed for characterization of the disease. Both models showed expansion of the mesangial matrix of the glomeruli, which is indicative of glomerular hyperfiltration. The activities of complexes I and IV and the protein levels of complexes I and III were nonconcordant in db/m-5/6Nx mice. In conclusion, we demonstrated that nonconcordant regulation of mitochondrial complexes in db/m-5/6Nx mice involved with glomerular hyperfiltration. The progression and/or severity of nephropathy might be affected through a synergistic effect of mitochondrial dysfunction in hyperglycemia and glomerular hyperfiltration. J. Med. Invest. 64: 255-261, August, 2017.

Generation and Feasibility Assessment of a New Vehicle for Cell-Based Therapy for Treating Corneal Endothelial Dysfunction.

The corneal endothelium maintains corneal transparency by its pump and barrier functions; consequently, its decompensation due to any pathological reason causes severe vision loss due to corneal haziness. Corneal transplantation is the only therapeutic choice for treating corneal endothelial dysfunction, but associated problems, such as a shortages of donor corneas, the difficulty of the surgical procedure, and graft failure, still need to be resolved. Regenerative medicine is attractive to researchers as a means of providing innovative therapies for corneal endothelial dysfunction, as it now does for other diseases. We previously demonstrated the successful regeneration of corneal endothelium in animal models by injecting cultured corneal endothelial cells (CECs) in combination with a Rho kinase (ROCK) inhibitor. The purpose of the present study was to optimize the vehicle for clinical use in cell-based therapy. Our screening of cell culture media revealed that RELAR medium promoted CEC adhesion. We then modified RELAR medium by removing hormones, growth factors, and potentially toxic materials to generate a cell therapy vehicle (CTV) composed of amino acid, salts, glucose, and vitamins. Injection of CECs in CTV enabled efficient engraftment and regeneration of the corneal endothelium in the rabbit corneal endothelial dysfunction model, with restoration of a transparent cornea. The CECs retained >85% viability after a 24 hour preservation as a cell suspension in CTV at 4°C and maintained their potency to regenerate the corneal endothelium in vivo. The vehicle developed here is clinically applicable for cell-based therapy aimed at treating the corneal endothelium. Our strategy involves the generation of vehicle from a culture medium appropriate for a given cell type by removing materials that are not favorable for clinical use.