Citation: Okumura N, Koizumi N, Kay EP, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2013;54:243954: -250254: . DOI:10. 1167 PURPOSE. To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction.METHODS. As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mM of ROCK inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients.RESULTS. Slit-lamp microscopy revealed that both Y-27632-treated and -nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na þ / K þ -ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm 2 , as opposed to 1500 cells/mm 2 in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema. 2 However, several severe problems can arise associated with corneal transplantation, including allograft rejection, primary graft failure, and severe loss of cell density. To the best of our knowledge, no clinically practical medical therapy has been developed to effectively treat corneal endothelial dysfunction.As an alternative to corneal transplantation, regenerative medical procedures might be a plausible path of therapy for treating severe corneal endothelial dysfunction. Several research groups, including ours, have reported transplantations of cultivated CECs in an animal model to establish a new clinical intervention for corneal endothelial dysfunction. [3][4][5][6][7][8][9] We recently reported the use of cell therapy to successfully achieve the recovery of corneal transparency in both rabbit and primate corneal endothelial dysfunction models. 9 However, in cases of early-stage corneal endothelial dysfunction, in which stem cells or progenitor cells are still maintained in the tissue, drug therapy may provide a less-invasive or antiprogression treatment. Our group, as well as several other groups, reported that pharmaceutical agents such...

Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na+/K+-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

Our previous work demonstrated that both polymorphonuclear leukocytes (PMNs) and protein fractions released from PMNs induced de novo synthesis of fibroblast growth factor 2 (FGF-2), which in turn becomes the direct mediator of endothelial mesenchymal transformation observed in corneal endothelial cells (CECs). To identify the protein factor, we used ProteinChip Array technology. Protein fractions obtained from the conditioned medium released by PMNs were resolved by twodimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 17 kDa, were sequentially subjected to in-gel trypsin digestion and mass spectrometry. The 17-kDa peptide band was identified as interleukin-1␤ (IL-1␤). Biological activities of IL-1␤ were further determined; IL-1␤ altered the shape of CECs from polygonal to fibroblastic morphologies in a time-and dose-dependent manner, whereas neutralizing IL-1␤ antibody, neutralizing antibody to FGF-2, and LY294002 blocked the action of IL-1␤. IL-1␤ greatly increased the levels of FGF-2 mRNA in a time-and dose-dependent manner; IL-1␤ stimulated expression of all isoforms of FGF-2. IL-1␤ initially induced nuclear accumulation of FGF-2 and facilitated translocation of FGF-2 to plasma membrane and extracellular matrix. IL-1␤ activated phosphatidylinositol (PI) 3-kinase, the enzyme activity of which was greatly stimulated after a 5-min exposure to IL-1␤. This early and rapid activation of PI 3-kinase greatly enhanced FGF-2 production in CECs; pretreatment with LY294002 hampered the induction activity of IL-1␤. These observations suggest that IL-1␤ takes part in endothelial to mesenchymal transformation of CECs through its inductive potential on FGF-2 via the action of PI 3-kinase.

No abstract