Junji Hamuro

Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.

PURPOSE The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. METHODS MCECs of cynomolgus monkeys were cultured in a medium containing 10 microM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. RESULTS The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours. CONCLUSIONS The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

ROCK Inhibitor Converts Corneal Endothelial Cells into a Phenotype Capable of Regenerating In Vivo Endothelial Tissue

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na(+)/K(+)-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.

The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing

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(+)/K(+)-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. CONCLUSIONS These findings show that rock inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.).

Toll-like receptor 3 gene polymorphisms in Japanese patients with Stevens–Johnson syndrome

Background and aim: Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute-onset mucocutaneous diseases induced by infectious agents and/or inciting drugs. Given the association between the onset of SJS/TEN and infections, the possibility that there is an association between SJS/TEN and a disordered innate immune response was considered. The first line of defence against infection is comprised of evolutionarily conserved sets of molecules, the Toll–like receptors (TLRs). TLR3 recognises double-stranded RNA associated with viral infections. Methods: The Japanese single-nucleotide-polymorphism (JSNP) database reports 7 polymorphisms consisting of 7 SNPs in the human TLR3 gene; 3 of the 7 SNPs are coded in exon regions, (ie, 293248A/G, 293391A/G and 299698T/G), and the other 4 are coded in intron regions, (ie, 294440G/C, 294732C/T, 208036T/C and 298054C/T). These 7 SNPs were analysed in 57 Japanese patients with SJS/TEN with ocular surface complications and in 160 Japanese healthy controls. Results: SNP 299698T/G and the genotype patterns of 293248A/A and 299698T/T were strongly associated with SJS/TEN. Conclusion: The results suggest that polymorphisms in the TLR3 gene could be associated with SJS/TEN in the Japanese population.

Intrathoracic esophageal replacement by in situ tissue-engineered esophagus

OBJECTIVE This study aimed to evaluate in situ tissue-engineered esophagus in a canine model after experimental resection and replacement of a full circumferential defect of the intrathoracic esophagus. METHODS Two types of scaffolding were fabricated. In the KF(+) group (n = 6), oral keratinocytes and fibroblasts cultured on human amniotic membrane were sheeted on polyglycolic acid felt with smooth muscle tissue and were then rolled around tubes. In the KF(-) group (n = 6), the same procedure was followed, but the keratinocytes and fibroblasts were omitted. Both scaffolds were wrapped in omentum and implanted in the abdomen. In the KF(+) group, at 3 weeks after implantation, the scaffold developed into a tube with a well-differentiated lumen of stratified squamous cells surrounded by a thick smooth muscle-like tissue (in situ tissue-engineered esophagus). A part of the esophagus was resected and replaced by the graft in the same dogs. RESULTS In the KF(-) group, strictures developed after esophageal replacement, with almost complete obstruction within 2 to 3 weeks. In contrast, in the KF(+) group, the in situ tissue-engineered esophagus showed good distensibility and the dogs remained without feeding problems through 420 days. Esophageal peristalsis transferred food to the stomach, despite the absence of peristaltic activity in the in situ tissue-engineered esophagus itself. The thickness of the squamous epithelial layer and the smooth muscle layer of the in situ tissue-engineered esophagus were similar to that of the adjacent native esophagus. CONCLUSION The in situ tissue-engineered esophagus can successfully replace the intrathoracic esophagus, and this procedure may offer a promising surgical approach to esophageal diseases.

Enhancement of corneal endothelium wound healing by Rho-associated kinase (ROCK) inhibitor eye drops

Aim To demonstrate the efficacy of Rho-associated kinase (ROCK) inhibitor Y-27632 for corneal endothelial wound healing both in in vitro and in vivo models. Methods As an in vitro model, cultivated cynomolgus monkey corneal endothelial cells were scraped to create a linear defect. The wound distance was then determined during a 24-h culture in the presence or absence of 10 μM of Y-27632. As an in vivo model, central corneal endothelium of Japanese white rabbits was damaged by transcorneal freezing, then 10 mM of Y-27632 was applied topically six times daily for 48 h. The wound area of the corneal endothelium was evaluated after 48 h. Results The mean wound distance in the cultured corneal endothelial cells was significantly shorter in the Y-27632 group than in the control group. In the rabbit model, the mean wound area of the Y-27632 group was significantly smaller than that of the control group. Conclusion This study demonstrated that ROCK inhibitor Y-27632 promotes corneal endothelial wound healing both in in vitro and in vivo.

Essential roles of DC-derived IL-15 as a mediator of inflammatory responses in vivo

Interleukin (IL)-15 is expressed in a variety of inflammatory diseases. However, the contribution of dendritic cell (DC)–derived IL-15 to the development of diseases is uncertain. Using established models of Propionibacterium acnes (P. acnes)– and zymosan-induced liver inflammation, we observed granuloma formation in the livers of wild-type (WT) and RAG-2−/− mice but not in those of IL-15−/− mice. We demonstrate that this is likely caused by an impaired sequential induction of IL-12, IFN-γ, and chemokines necessary for monocyte migration. Likewise, lethal endotoxin shock was not induced in P. acnes– and zymosan-primed IL-15−/− mice or in WT mice treated with a new IL-15–neutralizing antibody. In both systems, proinflammatory cytokine production was impaired. Surprisingly, neither granuloma formation, lethal endotoxin shock, nor IL-15 production was induced in mice deficient for DCs, and adoptive transfer of WT but not IL-15−/− DCs restored the disease development in IL-15−/− mice. Collectively, these data indicate the importance of DC-derived IL-15 as a mediator of inflammatory responses in vivo.

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 &mgr;m. 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 &mgr;m, 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.

The Role of Interleukin-33 in Chronic Allergic Conjunctivitis

PURPOSE The authors discovered a genetic association between the ST2L gene and atopy. The ST2L gene encodes a membrane-bound functional marker for Th2 cells. Recently, a novel Th2 cytokine, interleukin-33 (IL-33), was discovered to be a specific ligand for ST2L. The authors investigated the role of IL-33 in chronic allergic conjunctivitis. METHODS Immunohistochemical analysis was carried out using giant papillae samples obtained from patients with atopic keratoconjunctivitis. The authors used proinflammatory stimuli to clarify IL-33 mRNA/protein-inducing signals with cultured human conjunctival epithelial cells, fibroblasts, human umbilical vascular endothelial cells, and mast cells. These cells were also used to examine the expression of ST2L (IL-33R). Finally, cultured mast cells were stimulated with recombinant IL-33 (rIL-33) to examine the downstream signals. RESULTS The authors found IL-33 protein expression in human vascular endothelial cells in the giant papillae and in the control conjunctivae. IL-33 expression was also observed in conjunctival epithelium of the giant papillae but not in the control conjunctivae. IL-1 beta stimulation upregulated IL-33 mRNA expression in conjunctival fibroblasts. The authors also confirmed mature IL-33 protein expression in ocular resident cells by Western blot analysis. Preferential ST2L expression was observed in human mast cells, and phosphorylation of p38 MAPK and IL-13 mRNA induction was observed in human cultured mast cells after rIL-33 stimulation. Phosphorylation of p38 MAPK was inhibited by soluble ST2 protein. CONCLUSIONS The IL-33-ST2 signaling cascade plays some roles in the pathophysiology of chronic allergic conjunctivitis through the activation of mast cells.

Association of Fas Ligand gene polymorphism with Stevens-Johnson syndrome.

Background: Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute severe blistering diseases of the skin and also two of the most devastating ocular surface diseases leading to corneal damage and loss of vision. The extreme rarity of cutaneous and ocular surface reactions to drug therapies led us to suspect individual susceptibility. SJS/TEN patients in the acute stage were reported to manifest increased serum levels of Fas Ligand (FasL). Thus, we performed SNP association analysis of the FasL gene. Methods: In 76 Japanese SJS/TEN patients with ocular surface complications and 160 Japanese healthy controls, we examined four SNPs of FasL reported in the Japanese Single Nucleotide Polymorphisms (JSNP) database by sequencing. Results: The SNP rs.3830150 A/G showed a significant strong inverse association with SJS/TEN. Analysis of the genotype pattern of SNPs rs.3830150 and rs.2639614 (rs.3830150 A/A–rs.2639614 G/G) also manifested a strong inverse association with SJS/TEN. Conclusion: FasL gene polymorphisms might be associated with SJS/TEN.