Suguru Nakagawa

Toxicity Evaluation of Antiglaucoma Drugs Using Stratified Human Cultivated Corneal Epithelial Sheets

PURPOSE To investigate the toxicity profiles of seven antiglaucoma topical eye drops and benzalkonium chloride (BAC) using stratified cultivated human corneal epithelial cell sheets (HCES) in a serum-free culture system. METHODS A range of prostaglandin analogies and preservatives, including BAC, sofZia (SZ), sodium benzoate (SB), and polyquaternium-1 (PQ) were tested. The barrier function and cell viability were examined by a carboxyfluorescein permeability assay and WST-1 assay. Histological evaluation of the HCES was also performed after application of each solution. RESULTS The carboxyfluorescein permeability assay had a higher sensitivity for the detection of toxicity of test solutions than the WST-1 assay or histological examination. Latanoprost BAC, latanoprost/timolol BAC, and 0.02% or higher concentration of BAC were the most toxic, followed by latanoprost SB, latanoprost preservative-free, BAC 0.002%, and travoprost/ latanoprost PQ. Travoprost SZ and tafluprost BAC (preserved with 0.001% BAC) was the least toxic in our experimental conditions. CONCLUSIONS The carboxyfluorescein permeability assay using HCES in a serum-free system was the most useful for the quantification of toxicity of ophthalmic solutions. Among the regimens examined, a BAC concentration of 0.001% or lower or non-BAC preservative sofZia was suggested to be the least toxic to the ocular surface.

Angiopoietin-like protein 2 is a potent hemangiogenic and lymphangiogenic factor in corneal inflammation

PURPOSE We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo. METHODS Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1β (IL-1β) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed. RESULTS Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1β were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas. CONCLUSIONS Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1β expression. Angptl2 may be a novel therapeutic target for preventing blindness.

Angiopoietin-Like 7 Is an Anti-Angiogenic Protein Required to Prevent Vascularization of the Cornea

Purpose We sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea. Methods Human umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and in vivo in mice. Results Areas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo, intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels. Conclusions ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.

Stromal bed quality and endothelial damage after femtosecond laser cuts into the deep corneal stroma

Aim To evaluate the stromal bed quality and endothelial damage after femtosecond laser (FSL) cuts into the deep corneal stroma. Methods Using a 150-kHz FSL, a lamellar cut was aimed at a depth of 100, 300, or 500 μm in porcine corneas. Stromal bed smoothness was graded from light microscopy and scanning electron microscopy images. Rabbit corneas were cut at remaining thicknesses of 70, 100 and 150 μm using the FSL. The effects of peeling off the corneal flap and the distance between laser spots (2 or 4 μm) were examined. Results The ratio of damaged cells in the group with a remaining depth of 70 μm was significantly larger than that in the groups with a remaining depth of 150 μm. The ratio of damaged cells in the group with a 4-μm spot separation and the flap peeled off was significantly larger than that in the group with a 4-μm spot separation and the flap not peeled off. Conclusions Corneal endothelial damage is likely to increase when the remaining depth is less than 70 μm, and peeling off the flap damages corneal endothelial cells when the remaining depth is less than 100 μm.

Frequency doubling technology for earlier detection of functional damage in standard automated perimetry-normal hemifield in glaucoma with low-to-normal pressure.

PurposeTo compare the longitudinal change of frequency doubling technology (FDT) perimetry and standard automated perimetry (SAP) results in SAP-normal hemifield in open-angle glaucoma (OAG) eyes with low-to-normal intraocular pressure (IOP). Materials and MethodsFDT perimetry with the N-30 full threshold protocol and SAP using the Humphrey Field Analyzer with the 30-2 Swedish Interactive Threshold Algorithm-standard protocol were periodically performed for at least 3 years in 39 eyes of 39 OAG patients with low-to-normal IOP and visual field damage confined to only one hemifield. The time courses of the mean of the threshold values of FDT and the mean of total deviations (TDs) in SAP-normal and SAP-abnormal fields were analyzed using a linear mixed model. ResultsThe average follow-up was 4.9 years and the average IOP during follow-up was 12.6 mm Hg with or without medication. The aging effect-corrected rate of change in the mean FDT threshold values was significantly negative (P=0.004) in the SAP-normal hemifield, whereas that of mean of TD values by SAP did not significantly differ. In the SAP-abnormal hemifield, the rate of change was significantly negative for both tests (P<0.001). The mean of the slope of the mean TD values in the SAP-abnormal hemifield was significantly more negative than that in the SAP-normal hemifield (P=0.011), whereas that of the mean FDT threshold values showed no significant difference between the 2 hemifields. ConclusionsFDT is useful for monitoring functional damage in the SAP-normal hemifield in OAG eyes with low-to-normal IOP.

Toxicity of Topical Antifungal Agents to Stratified Human Cultivated Corneal Epithelial Sheets

PURPOSE Prolonged use of topical antifungal agents may compromise corneal epithelial integrity. Here, we used an in vitro model of human stratified corneal epithelium to compare the ocular toxicity profiles of 4 different antifungal eye drops. METHODS Human corneal epithelial cell sheets were cultured in a serum-free medium containing 0.1% micafungin, 1% voriconazole, 5% pimaricin, 0.1% amphotericin B, or controls (saline or 5% glucose). Cell viability and barrier function were measured by WST-1 assay and carboxyfluorescein permeability assay, respectively. Cell migration was measured on a wound healing assay. RESULTS WST-1 assay and carboxyfluorescein permeability assay revealed that amphotericin B was the most toxic drug, followed by pimaricin, micafungin, and voriconazole. Cell migration on a wound healing assay was decreased in the following order, amphotericin B, pimaricin, micafungin, and voriconazole. CONCLUSIONS Topical micafungin and voriconazole appeared to be the least toxic to the corneal epithelium. Drug prescription should consider not only fungal species and susceptibility but also ocular toxicity and stage of treatment.

A high-salt diet enhances leukocyte adhesion in association with kidney injury in young dahl salt-sensitive rats

Salt-sensitive hypertension is associated with severe organ damage. Generating oxygen radicals is an integral component of salt-induced kidney damage, and activated leukocytes are important in oxygen radical biosynthesis. We hypothesized that a high-salt diet causes the upregulation of immune-related mechanisms, thereby contributing to the susceptibility of Dahl salt-sensitive rats to hypertensive kidney damage. For verifying the hypothesis, we investigated leukocytes adhering to retinal vessels when Dahl salt-sensitive rats were challenged with a high-salt (8% NaCl) diet using acridine orange fluoroscopy and a scanning laser ophthalmoscope. The high-salt diet increased leukocyte adhesion after 3 days and was associated with a significant increase in mRNA biosynthesis of monocyte chemotactic protein-1 and intercellular adhesion molecule-1 (ICAM-1) -related molecules in the kidney. Losartan treatment did not affect increased leukocyte adhesion during the early, pre-hypertensive phase of high salt loading; however, losartan attenuated the adhesion of leukocytes during the hypertensive stage. Moreover, the inhibition of leukocyte adhesion in the pre-hypertensive stage by anti-CD18 antibodies decreased tethering of leukocytes and was associated with the attenuation of functional and morphological kidney damage without affecting blood pressure elevation. In conclusion, a high-salt challenge rapidly increased leukocyte adhesion through the over-expression of ICAM-1. Increased leukocyte adhesion in the pre-hypertensive stage is responsible for subsequent kidney damage in Dahl salt-sensitive rats. Immune system involvement may be a key component that initiates kidney damage in a genetic model of salt-induced hypertension.

Deformation-based data reduction of Time-Varying Meshes for displaying on mobile terminals

In this paper, a drastical compression method for Time-Varying Meshes (TVMs) is presented aiming at displaying on mobile terminals, where the resources are quite limited. In addition to our previous work of the skeleton-based temporal compression for TVMs, we have implemented spatial compression using 3D model morphing and mesh simplification. In addition, we have developed an interface for skeleton adjustment and modified the skeleton-based motion analysis so that rotation motion can be represented properly. As a result, we have achieved a very high compression rate of 1/5,000 and the rendering speed has been enhanced by six times as compred to our previous method. The feasibility of the algorithm has been confirmed on an actual cell phone.

Oxidative stress induces ferroptotic cell death in retinal pigment epithelial cells

ABSTRACT The dysfunction and cell death of retinal pigment epithelial (RPE) cells are hallmarks of late‐stage dry (atrophic) age‐related macular degeneration (AMD), for which no effective therapy has yet been developed. Previous studies have indicated that iron accumulation is a source of excess free radical production in RPE, and age‐dependent iron accumulation in RPE is accelerated in patients with dry AMD. Although the pathogenic role of oxidative stress in RPE in the development of dry AMD is widely accepted, the mechanisms of oxidative stress‐induced RPE cell death remain elusive. Here, we show that ferroptotic cell death, a mode of regulated necrosis mediated by iron and lipid peroxidation, is implicated in oxidative stress‐induced RPE cell death in vitro. In ARPE‐19cells we observed that the ferroptosis inhibitors ferrostatin‐1 and deferoxamine (DFO) rescued tert‐butyl hydroperoxide (tBH)‐induced RPE cell death more effectively than inhibitors of apoptosis or necroptosis. tBH‐induced RPE cell death was accompanied by the three characteristics of ferroptotic cell death: lipid peroxidation, glutathione depletion, and ferrous iron accumulation, which were all significantly attenuated by ferrostatin‐1 and DFO. Exogenous iron overload enhanced tBH‐induced RPE cell death, but this effect was also attenuated by ferrostatin‐1 and DFO. Furthermore, mRNA levels of numerous genes known to regulate iron metabolism were observed to be influenced by oxidative stress. Taken together, our observations suggest that multiple modes of cell death are involved in oxidative stress‐induced RPE cell death, with ferroptosis playing a particularly important role. HIGHLIGHTSOxidative stress‐induced RPE cell death is rescued by ferroptosis inhibitors.Lipid peroxidation, glutathione depletion, and Fe2+ accumulation are observed.Iron overload enhances oxidative stress‐induced RPE cells death.Gene expressions of iron metabolism regulators are affected by oxidative stress in RPE cells.

Bilateral limbal stem cell deficiency with chromosomal translocation of 3p and 9p

We describe a case of bilateral limbal stem cell defi ciency (LSCD) with systemic abnormalities, including sensorineural deafness and pituitary dwarfi sm. The patient showed balanced translocation of 3p27 and 9p13, which to our knowledge has not been reported in the literature. We performed ocular surface reconstruction by autologous cultivated conjunctival epithelial cells (CjECs) transplantation on the amniotic membrane. To the best of our knowledge, this is also the fi rst report of cultivated CjECs transplantation for bilateral LSCD in humans.