Shinichiro Nakano

Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing.

PURPOSE To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect. METHODS The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated. RESULTS Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM. CONCLUSIONS These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.

Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction.

The corneal endothelium maintains corneal transparency; consequently, its dysfunction causes severe vision loss. Tissue engineering-based therapy, as an alternative to conventional donor corneal transplantation, is anticipated to provide a less invasive and more effective therapeutic modality. We conducted a preclinical study for cell-based therapy in a primate model and demonstrated regeneration of the corneal endothelium following injection of cultured monkey corneal endothelial cells (MCECs) or human CECs (HCECs), in combination with a Rho kinase (ROCK) inhibitor, Y-27632, into the anterior chamber. We also evaluated the safety and efficacy of Good Manufacturing Practice (GMP)-grade HCECs, similar to those planned for use as transplant material for human patients in a clinical trial, and we showed that the corneal endothelium was regenerated without adverse effect. We also showed that CEC engraftment is impaired by limited substrate adhesion, which is due to actomyosin contraction induced by dissociation-induced activation of ROCK/MLC signaling. Inclusion of a ROCK inhibitor improves efficiency of engraftment of CECs and enables cell-based therapy for treating corneal endothelial dysfunction as a clinically relevant therapy.

Effect of the Rho Kinase Inhibitor Y-27632 on Corneal Endothelial Wound Healing.

PURPOSE The purpose of this study was to investigate the feasibility of using Rho-associated kinase (ROCK) inhibitor eye drops for treating severe corneal endothelial damage due to surgical invasion. METHODS A rabbit corneal endothelial damage model was created by mechanically scraping half the area of the corneal endothelium of eighteen eyes of Japanese white rabbits. A selective ROCK inhibitor, Y-27632 (10 mM), was applied topically for 2 weeks, and then the anterior segment was evaluated by slitlamp microscopy. The corneal endothelium was evaluated by phalloidin staining and immunohistochemical analysis. We then conducted pilot clinical research and applied Y-27632 eye drops topically to three patients who exhibited severe corneal edema due to corneal endothelial damage. RESULTS In the corneal endothelial damage rabbit model, more Ki67-positive cells were detected in Y-27632-treated eyes than in control eyes. Five of six corneas became transparent in Y-27632-treated eyes, whereas zero of six corneas became transparent in the control eyes (P < 0.01). Actin fibers were distributed at the cell cortex in the eyes treated with Y-27632, whereas actin distribution was partially disrupted, and stress fibers were observed in control eyes. N-cadherin and Na+/K+-ATPase were expressed in almost all cells in Y-27632-treated eyes, but expression decreased in control eyes. Preliminary human cases confirmed that ROCK inhibitor eye drops were considerably effective for treatment of corneal edema associated with cataract surgery. CONCLUSIONS ROCK inhibitor may be developed as an eye drop for treating acute corneal endothelial damage to prevent progression of bullous keratopathy. (University Hospital Medical Information Network Clinical Trial Registry no. UMIN000003625; www.umin.ac.jp/ctr).

Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine.

Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Transiently Alters the Morphology of Corneal Endothelial Cells

PURPOSE Ripasudil (Glanatec), a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved in Japan in September 2014 for the treatment of glaucoma and ocular hypertension. The purpose of this study was to investigate the effect of ripasudil eye drops on corneal endothelial morphology, as ROCK signaling is known to modulate the actin cytoskeleton. METHODS Morphological changes in the corneal endothelium were evaluated in human subjects by specular and slit-lamp microscopy, following topical administration of ripasudil. We also used a rabbit model to evaluate the effect of ripasudil on clinical parameters of the corneal endothelium. Twenty-four hours after ripasudil application, corneal specimens were evaluated by phalloidin staining, immunohistochemical analysis, and electron microscopy. RESULTS Specular microscopy revealed morphological changes in human eyes, and slit-lamp microscopy showed guttae-like findings. The rabbit model showed morphological changes similar to those seen in human eyes after ripasudil administration. Electron microscopy demonstrated that these alterations are due to the formation of protrusions along the cell-cell borders, but this formation is transient. Expression of corneal endothelial function-related markers was not disrupted; corneal thickness and corneal volume were not changed; and no cell death was observed following ripasudil administration. CONCLUSIONS Ripasudil induces transient guttae-like findings in humans, most likely due to protrusion formation along intracellular borders caused by the reduction in actomyosin contractility of the corneal endothelial cells. No severe adverse effects were observed. Physicians should be aware that ROCK inhibitors can cause these guttae-like findings, to avoid misdiagnosing patients as having Fuchs endothelial corneal dystrophy. (www.umin.ac.jp/ctr number, UMIN000018340.).

Immune Cells on the Corneal Endothelium of an Allogeneic Corneal Transplantation Rabbit Model

Purpose Corneal endothelial cell density undergoes a progressive decrease for many years after transplantation, eventually threatening patients with late endothelial failure. The purpose of this study was to investigate the possibility of an immunologic response in successfully grafted corneal endothelium. Methods The corneal endothelium of patients who had undergone corneal transplantation was evaluated by specular microscopy. Rabbit models were subjected to penetrating keratoplasty (PK) with either syngeneic or allogeneic corneal transplants and Descemets stripping endothelial keratoplasty (DSEK) with allogeneic corneal transplants. The presence of immune cells and expression of proinflammatory cytokines were determined by immunostaining. The corneal endothelium and immune cells were also evaluated by scanning electron microscopy. Results Scanning slit contact specular microscopy of patients with no features of graft rejection revealed cell-like white dots on the grafted corneal endothelium. The corneal endothelium of the allogeneic PK and DSEK rabbit models displayed the presence of immune cells, including CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD68+ macrophages, and neutrophils, but these immune cells were rarely observed in the syngeneic PK model. These immune cells also produced proinflammatory cytokines. Notably, some of the corneal endothelial cells situated near these immune cells exhibited features of apoptosis. Conclusions T lymphocytes, B lymphocytes, macrophages, and neutrophils are present on the grafted corneal endothelium in both PK and DSEK allogeneic rabbit models. The potential involvement of immune cells as an underlying pathophysiology for late endothelial failure deserves further examination.

Truncated Contact Lenses For Peripheral Vitrectomy

In vitrectomy, excision of the peripheral vitreous is important to obtain better surgical outcomes as well as to prevent postoperative complications, such as retinal detachment and endophthalmitis.1–3 There are several methods to observe peripheral fundus during vitrectomy: the wide-angle viewing system, prismatic contact lenses, and scleral depression. Although the wide-angle viewing system allows for observation of wider area, image resolution and stereopsis are limited when compared with the contact lens system.4 The conventional prismatic lens system provides a clear view of the surgical field, but prismatic lenses with greater prism angle (30 or 45°) have significant amount of chromatic aberrations, resulting in blurring of the peripheral image.5 In addition, observation of the ora serrate is not possible with the prismatic lenses. Scleral depression method offers observation with good image resolution and stereopsis, but it can be painful for patients and technically challenging in sutureless microincision vitrectomy. We developed new truncated prismatic contact lenses for clear view of the peripheral fundus during vitrectomy. Because the lenses are very thin with smaller prism angles, they have less chromatic aberrations and distortion, allowing for clearer view of the peripheral fundus.

Effects of astigmatic defocus on binocular contrast sensitivity

Purpose To determine the effects of astigmatism on contrast sensitivity (CS). Methods Eighteen normal volunteers (30.5 ± 6.0 [mean ± SD] years) were recruited. After correcting each refractive error by spectacles, against-the-rule (ATR) or with-the-rule (WTR) astigmatism of +1.00, +2.00 and +3.00 D was intentionally produced in both eyes, and then binocular CS was measured. The cylindrical addition of different powers (+1.00–+3.00 D) was compensated with spherical lenses so that the spherical equivalent refraction became zero in each eye. Subsequently, the above cylindrical addition was monocularly induced, and binocular CS was measured again. The relation between CS and astigmatic power, axis, and monocular or binocular astigmatism was investigated. Results With binocular ATR and WTR astigmatism, increases in astigmatic power significantly correlated with decreases in the area under the log contrast sensitivity function (AULCSF). With monocular astigmatic defocus, astigmatic power addition did not affect AULCSF. With binocular astigmatic defocus of high-power (+2.00 and +3.00 D), ATR astigmatism deteriorated AULCSF more than WTR astigmatism. In a comparison between binocular and monocular astigmatic defocus, CS was significantly worse with binocular astigmatic defocus than with monocular astigmatic defocus at higher spatial frequencies regardless of astigmatic power. Conclusions Binocular astigmatic defocus deteriorates CS depending on the amount of astigmatic power. ATR astigmatism reduces CS more than WTR astigmatism dose. In addition, binocular astigmatic defocus affects CS more severely than monocular astigmatic defocus especially at high spatial frequencies.

Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Corneal Endothelial Cell Proliferation

Purpose We have performed clinical research on cell-based therapy for corneal endothelial decompensation since 2013. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for promoting proliferation of human corneal endothelial cells (HCECs). Methods HCECs were cultured in media supplemented with various low-molecular-weight compounds to screen for the effect of those compounds on cell proliferation. Activation of substrates of p38 MAPK and cell cycle regulatory proteins were evaluated by western blotting. Corneal endothelial wounds were created in a rabbit model, and p38 MAPK was applied in eye drop form, followed by evaluation of cell proliferation in the corneal endothelium by Ki67-immunostaining. Results HCECs cultured with SB203580 exhibited hexagonal morphology and similar size and morphology, whereas control HCECs cultured without inhibitor exhibited monolayer morphology and varied in size and morphology. Flow cytometry demonstrated that cell proliferation was significantly increased by SB203580. Western blotting showed activation of ATF2 and HSP27 (substrates of p38 MAPK), and upregulation of cyclin D and downregulation of p27 were induced by inhibiting p38 MAPK. In the rabbit model, promotion of wound healing of the corneal endothelium was associated with significant upregulation of Ki67-positive proliferating cells following topical administration of SB203580 when compared with untreated endothelium (50.9% and 36.1%, respectively). Conclusions Activation of p38 MAPK signaling due to culture stress might suppress the proliferation of HCECs, whereas a p38 MAPK inhibitor can counteract this activation and enable efficient in vitro HCEC expansion.

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal

Purpose To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. Methods A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. Results The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemets area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. Conclusions Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction.