Megumi Honjo

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammatory responses and proangiogenic factors. It has been suggested that S1P upregulates choroidal neovascularization (CNV) and may be deeply involved in the pathogenesis of exudative age-related macular degeneration (AMD). Recent studies have suggested that apolipoprotein M (ApoM), a carrier protein for S1P, modulates the biological properties of S1P in the pathogenesis of atherosclerosis. However, the role of ApoM/S1P in AMD has not been explored. We investigated the effect of S1P on proangiogenic factors in human retinal pigment epithelium (RPE) cell lines in vitro. S1P promoted the expression of vascular endothelial growth factor in RPE cells. Hypoxia inducible factor-1α expression was also upregulated. These S1P-induced enhancements in growth factors and chemotactic cytokines in RPE cells were significantly inhibited by ApoM treatment. Additionally, in vivo experiments using a laser-induced CNV murine model demonstrated that intravitreal ApoM injection significantly reduced the progression of CNV formation. Although the detailed mechanisms remain to be elucidated, the present results provide a novel potential therapeutic target for AMD, and demonstrate a suppressive role for ApoM and S1P in the pathology of CNV progression.

Glaucoma is common and shows high prevalence in older adults. However, there are few studies on the structure/function relationship in older adults with glaucoma. This prospective, cross-sectional study (conducted between February and August 2014), enrolled 102 eyes of 102 subjects aged over 75 years, including 57 eyes with primary open angle glaucoma (POAG), 15 eyes with pseudoexfoliation glaucoma (PXG), and 30 healthy eyes. Multiple regression analysis was used to determine the correlation of circumpapillary retinal nerve fiber layer thickness (cpRNFLT) and macular parameters to mean deviation (MD) to and standard automated perimetry (SAP)-measured sensitivity, assessed with the 30–2 and 10–2 programs. In each 10–2 SAP test point, Spearman’s rank correlation coefficient was used to compare macular retinal nerve fiber layer thickness (mRNFLT), macular ganglion cell-inner plexiform layer thickness (GCIPLT), and mRNFL+GCIPL thickness (GCCT) with sensitivity after adjusting for retinal ganglion cell (RGC) displacement. In eyes with POAG and PXG, cpRNFLT was significantly correlated with 30–2 MD and 30–2 sensitivity. Multiple regression analysis revealed that the POAG had significantly lower cpRNFLT, mRNFLT, GCIPLT, and GCCT according to the severity of disease than control eyes after adjusting for sensitivity, age, sex, and axial length. The PXG eyes had significantly lower cpRNFLT, mRNFLT, and GCCT when compared with the early to moderate POAG eyes. GCCT was significantly correlated with 10–2 sensitivity, except in one juxtafoveal point, (r = 0.338–0.778) in the POAG eyes. The periphery of the central 10° area showed a good correlation between sensitivity and mRNFLT, while the central 5.8° showed a good correlation between sensitivity and GCIPLT. The correlation between structure and function was significant, and objective and quantitative method with OCT assessing glaucoma that does not require patient ability could be a possible parameter to assess diagnosis and progression in older patients with glaucoma.

Some of the cytokines and growth factors in the aqueous humor activate Rho, and the Rho/ROCK signal transduction participates in signaling pathways via rearrangement of actin cytoskeleton that leads to various cellular reactions. In a previous study, we demonstrated that a selective ROCK inhibitor significantly reduced intraocular pressure, the mechanism of which was attributed to improved outflow. ROCK inhibitors induced the actomyosin assembly, cell adhesive interactions, and the expression of extracellular matrix (ECM) proteins in cultured TM cells. The inhibition of Rho also has been implicated in pathological wound healing by regulating neovascularization, migration, and ECM in scar tissue. In this study, we investigated the role of ROCK inhibitor in regulating human Tenon fibroblast (HTF) activity and postoperative scar formation in a rabbit sclerostomy model. ROCK inhibitor showed decreased aSMA expression in HTF, and prevented enhanced contractility, assembly of actin stress fibers, and myofibroblastic transdifferentiation. In vivo sclerostomy studies showed that the bleb survival was significantly improved in ROCK inhibitor-treated eyes. In another study by us, ROCK inhibitor showed neuroprotective effects against rat retinal ischemia reperfusion injury. Collectively, ROCK inhibitors are thus a potential new strategy for developing medical therapy for glaucomatous dis-

Purpose. To investigate the role of Y-27632, a specific inhibitor of Rho-associated protein kinase (ROCK) in regulating human Tenon fibroblast (HTF) activities including proliferation, adhesion, contraction, migratory response, and myofibroblast transdifferentiation. Effects of Y-27632 on prevention of postoperative scar formation were also examined in a rabbit model of glaucoma filtration surgery. methods. After treatment of HTFs with Y-27632, cell toxicity, proliferation, migration, adhesion, and contraction were studied. The cytoskeleton and α-smooth muscle actin (α-SMA) expression were examined via immunohistochemistry. In vivo studies in Japanese white rabbits consisted of a full-thickness sclerostomy followed in the 7-day postoperative period by topical application of Y-27632. Intraocular pressure, morphologic changes in bleb features, and histology of surgical sites were evaluated. results. Y-27632 had no direct toxicity or significant effects on cell proliferation of HTF. The cell adhesion assay showed that Y-27632 promoted adhesiveness to both fibronectin and collagen type I. Use of Y-27632 significantly inhibited collagen gel contraction and α-SMA expression in HTFs. Y-27632 also increased HTF motility. In vivo, Y-27632 inhibited wound healing and fibroproliferation after filtration surgery and significantly improved surgical outcome compared with the vehicle. Histologic examination revealed that blebs in the Y-27632-treated group differed from those in the vehicle-treated group in that they lacked significant collagen deposition in the sclerostomy area. conclusions. Y-27632 had profound effects on activities of HTFs and was effective in preventing fibroproliferation and scar formation in a rabbit model of glaucoma surgery. A ROCK inhibitor may be an effective anti-scarring agent after glaucoma filtering surgery.

N-syndecan is a transmembrane heparan sulfate proteoglycan, that is highly expressed in neural tissues. In the current study, changes in N-syndecan expression during retinal development were examined. Localization of N-syndecan in developing rat retina was examined by immunohistochemistry and in situ hybridization. The amount of the core protein was evaluated by immunoblot analysis, using retinal homogenates at various developmental stages. In addition, mRNA expression was semiquantified by reverse transcription-polymerase chain reaction (RT-PCR). To understand better the localization of N-syndecan in retinal neuronal cells, we performed immunocytochemistry using retinal ganglion cells in culture. N-syndecan is highly expressed in nerve fiber-rich layers of the retina at early postnatal stages (between postnatal day [P]0 and P14). In contrast, immunoreactivity was faint during embryonic stages and late postnatal stages. In addition, in retinal flatmounted sections, N-syndecan immunoreactivity was observed on the axons of retinal ganglion cells. Intense signals were observed in the ganglion cell layer during in situ hybridization. Immunoblot analyses demonstrated that the amount of N-syndecan core protein reached a peak at approximately P14. The RT-PCR analyses using N-syndecan primers showed that an intense amplified band was observed in the cDNA derived from P14 retinas, whereas only faint bands were detected in the embryonic day (E)16 and P42 retinas. In retinal ganglion cells in culture, N-syndecan was located on the long, extended neurites. The data show that N-syndecan is transiently expressed, primarily in retinal neural fibers, during retinal development, indicating that it may be involved in formation of the retinal neural network.