Kei Takayama

Plasma-activated medium suppresses choroidal neovascularization in mice: a new therapeutic concept for age-related macular degeneration

Choroidal neovascularization (CNV) is the main pathogenesis of age-related macular degeneration (AMD), which leads to severe vision loss in many aged patients in most advanced country. CNV compromises vision via hemorrhage and retinal detachment on account of pathological neovascularization penetrating the retina. Plasma medicine represents the medical application of ionized gas “plasma” that is typically studied in the field of physical science. Here we examined the therapeutic ability of plasma-activated medium (PAM) to suppress CNV. The effect of PAM on vascularization was assessed on the basis of human retinal endothelial cell (HREC) tube formation. In mice, laser photocoagulation was performed to induce CNV (laser-CNV), followed by intravitreal injection of PAM. N-Acetylcysteine was used to examine the role of reactive oxygen species in PAM-induced CNV suppression. Fundus imaging, retinal histology examination, and electroretinography (ERG) were also performed to evaluate PAM-induced retinal toxicity. Interestingly, HREC tube formation and laser-CNV were both reduced by treatment with PAM. N-acetylcysteine only partly neutralized the PAM-induced reduction in laser-CNV. In addition, PAM injection had no effect on regular retinal vessels, nor did it show retinal toxicity in vivo. Our findings indicate the potential of PAM as a novel therapeutic agent for suppressing CNV.

Interleukin-18 Induces Retinal Pigment Epithelium Degeneration in Mice

PURPOSE To examine the effectiveness of interleukin-18 (IL-18) on choroidal neovascularization (CNV) and retinal pigment epithelium (RPE) in humans and mice. METHODS Serum IL-18 levels in patients with wet and dry AMD who were older than 50 years were measured and compared with those of age-matched controls. In mice, laser photocoagulation was performed in the retina to induce experimental CNV, and CNV volume was measured in eyes injected with recombinant IL-18 (rIL-18) and IL-18 neutralizing antibody (nIL-18Ab) compared with those injected with control. Tube formation assay was performed on human retinal endothelial cells (HREC) with rIL-18 administration in vitro. After subretinal injection of rIL-18, fundus change in the injected eyes was evaluated; active caspase-3 level was measured in the RPE/choroid complex, and tight junction integrity in RPE was visualized by zonula occludens-1 (ZO-1) staining. RESULTS Serum IL-18 levels in dry AMD patients were higher than those in control. Mouse rIL-18 or nIL-18Ab did not induce significant change in CNV volume compared with controls or change tube formation in HREC. Subretinal injection of rIL-18 induced retinal degeneration in the mice fundus; ZO-1 staining showed considerably disturbed RPE structure, and active caspase-3 expression was significantly higher after rIL-18 induction. CONCLUSIONS Interleukin-18 did not show a pro- or antiangiogenic effect on mouse laser-induced CNVs (laser-CNVs), whereas it directly induced RPE cell apoptosis in the mouse eye. Our results suggested that IL-18 is associated with dry AMD, but not with wet AMD.

Malondialdehyde induces autophagy dysfunction and VEGF secretion in the retinal pigment epithelium in age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries and is closely related to oxidative stress, which leads to lipid peroxidation. Malondialdehyde (MDA) is a major byproduct of polyunsaturated fatty acid (PUFA) peroxidation. Increased levels of MDA have been reported in eyes of AMD patients. However, little is known about the direct relationship between MDA and AMD. Here we show the biological importance of MDA in AMD pathogenesis. We first confirmed that MDA levels were significantly increased in eyes of AMD patients. In ARPE-19 cells, a human retinal pigment epithelial cell line, MDA treatment induced vascular endothelial growth factor (VEGF) expression alternation, cell junction disruption, and autophagy dysfunction that was also observed in eyes of AMD patients. The MDA-induced VEGF increase was inhibited by autophagy-lysosomal inhibitors. Intravitreal MDA injection in mice increased laser-induced choroidal neovascularization (laser-CNV) volumes. In a mouse model fed a high-linoleic acid diet for 3 months, we found a significant increase in MDA levels, autophagic activity, and laser-CNV volumes. Our study revealed an important role of MDA, which acts not only as a marker but also as a causative factor of AMD pathogenesis-related autophagy dysfunction. Furthermore, higher dietary intake of linoleic acid promoted CNV progression in mice with increased MDA levels.

Successful Treatment with Infliximab for Behçet Disease during Pregnancy

Purpose: To report a case of successful treatment with infliximab for Behçet disease (BD) during pregnancy. Design: A case report. Methods: A 30-year-old woman was diagnosed with BD at 12 weeks of pregnancy. Additional symptoms occurred, and infliximab at a dose of 5 mg/kg body weight was initiated at 18 weeks and repeated. Results: All symptoms improved, and a 3950-g infant was delivered uneventfully without any abnormality at 39 weeks. A few weeks after delivery, uveitis and systemic symptoms relapsed. Infliximab was reinitiated and all symptoms were resolved. Conclusions: Infliximab may be safe and effective for BD during pregnancy.

Phototoxicity of Indocyanine Green and Brilliant Blue G under Continuous Fluorescent Illumination on Cultured Human Retinal Pigment Epithelial Cells

PURPOSE We compared the phototoxicity of indocyanine green (ICG) and Brilliant Blue G (BBG) in cultured RPE cells under fluorescent lamp illumination imitating ambient light. METHODS Cultured human RPE line cells were stained with ICG or BBG solution at concentrations of clinical use, and cultured in a colorless medium for 24 hours in the dark or under illumination from a fluorescent lamp. After culture, cell morphology and TUNEL-positive apoptotic cells were observed. Cell viability and cell death rate were evaluated. Absorption spectral changes of BBG before and after incubation were measured. RESULTS ICG-stained cells cultured under illumination changed to an oval morphology with increased number of apoptotic cells, whereas ICG-stained cells cultured in the dark, and BBG-stained cells cultured under illumination and dark conditions maintained a flat morphology without increase in apoptotic cells. Cell viability decreased and cell death rate increased only in cells stained by ICG followed by culture under illumination. Staining cells with ICG at one-tenth concentration of clinical usage induced no cytotoxicity after culture under illumination. Approximately 30% of total BBG retained in the stained cells was released into the culture supernatant after incubation for 24 hours. The absorption spectrum of BBG did not change after fluorescent light irradiation. CONCLUSIONS Illumination with a fluorescent lamp caused cell death via apoptosis in ICG-exposed, but not in BBG-exposed cultured RPE cells. BBG may be a safer dye than ICG because of low light-induced cytotoxicity and rapid elution from stained cells.

Optical coherence tomography angiography in leber hereditary optic neuropathy.

treated using the same protocol and the difference in response may still reflect a variable reaction. It is possible that in unresponsive patients, switching to a higher frequency of aflibercept injections would result in a more favourable outcome (Arcinue et al. 2015). Whereas all our patients demonstrated a reduction in CRT following conversion, the visual function did not change accordingly, suggesting that in patients with continued visual deterioration despite anatomical stability, changing agents may be advised to achieve maximal therapeutic effect.

Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2 −/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2 −/− mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress.

Evaluation of microincision vitrectomy surgery using wide-viewing system for complications with ocular sarcoidosis.

AbstractWe evaluate the outcomes of microincision vitrectomy surgery (MIVS) using wide-viewing system for complications with ocular sarcoidosis resistance to medical treatment.Consecutive clinical records of 24 eyes (19 patients) with complications of ocular sarcoidosis underwent MIVS between April 2010 and December 2013 were retrospectively reviewed. MIVS and phacoemulsification were performed in 18 eyes and MIVS only in 6 eyes. Best-corrected visual acuity (BCVA), inflammation scores in the anterior segment and in the posterior segment, and central retinal thickness (CRT) of eyes with cystoid macular edema (CME) before surgery and after 1 week, 1, 3, 6, and 12 months were evaluated.LogMAR (log of the minimum angle of resolution) converted from BCVA was improved in 83.3% after 12 months and 66.7% showed improvement of more than 2 lines. The mean LogMAR was significantly improved from 1.14 ± 1.18 to 0.36 ± 0.79 in all eyes and 0.83 ± 0.86 to 0.23 ± 0.41 in eyes with MIVS and phacoemulsification, although no improvement was observed in eyes with MIVS only. Significant decrease of the mean anterior inflammation score was observed after 1 month in eyes with MIVS only and after 12 months in eyes with MIVS and phacoemulsification, and the mean posterior inflammation scores decreased after 1 week in all eyes. In eyes with preoperative CME, mean CRT was significantly decreased from 1 week after surgery. There was no case in which ocular inflammation was exacerbated by surgical stress.Improvement of visual acuity and resolution of ocular inflammation could be achieved by MIVS using wide-viewing system for complications of ocular sarcoidosis.

Role of Caveolin-1 for Blocking the Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy.

Purpose Proliferative vitreoretinopathy (PVR) is one of the most severe ocular diseases. Fibrotic changes in retinal cells are considered to be involved in the pathogenesis of PVR. Epithelial-mesenchymal transition (EMT) of RPE cells is one of the main concepts in the pathogenesis of fibrovascular membranes (FVMs) in PVR. In this study, we examined the expression of Caveolin-1 in human FVMs from patients with PVR. We also examined the role of Caveolin-1 in the pathogenesis of PVR. Methods Western blotting, real-time PCR, and immunohistochemistry were performed with human FVMs and mouse eyes with PVR. Cell migration assays were performed to evaluate the involvement of Caveolin-1 in EMT using primary human and mouse RPE cells. Results Caveolin-1 was expressed in human FVMs and upregulated in the mouse eyes with PVR. The alpha-smooth muscle actin (αSMA) expression and migration ability were increased in RPE cells with knockout or knockdown of Caveolin-1, whereas zonula occludens-1 (ZO-1) immunohistochemistry showed reduced morphology and expression of ZO-1. In addition, migration assays showed that Caveolin-1 reduction increased RPE cell migration abilities. Conclusions These results indicated that Caveolin-1 in RPE cells prevents PVR by blocking EMT.

Expression of Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells Induced by Amyloid-β Is Depressed by an Endoplasmic Reticulum Stress Inhibitor

Purpose: Amyloid-β (Aβ) is a 36- to 43-amino-acid peptide that is a constituent of drusen, and it has been demonstrated to upregulate vascular endothelial growth factor (VEGF) expression by retinal pigment epithelial (RPE) cells. This study aimed to determine whether 4-phenylbutyl phosphonylacetate (PBA), a known endoplasmic reticulum (ER) stress inhibitor, can reduce Aβ-induced expression of VEGF in RPE cells. Methods: Aβ was added to the medium of regularly cultured or polarized ARPE-19 cells, a human RPE cell line, with or without PBA. The levels of VEGF and ER stress markers, namely GRP78/Bip, cleaved caspases 4 and 12 and GADD153/C-EBP homologous protein, were determined by enzyme-linked immunoassay, immunocytochemistry and Western blotting. Results: Exposure of ARPE-19 cells to Aβ induced GRP78/Bip expression and activated caspases 4 and 12; however, their expression was decreased by simultaneous exposure to PBA. Aβ increased the expression of VEGF both in regularly cultured and polarized ARPE-19 cells, but it was suppressed by PBA. PBA did not cause RPE cell apoptosis. Conclusion: Aβ has been suggested to be involved in the development of age-related macular degeneration; therefore, our findings suggest that drugs that target ER stress should be considered for the treatment of age-related macular degeneration.