Akiko Nishiwaki

Anti-angiogenic effects of the receptor tyrosine kinase inhibitor, pazopanib, on choroidal neovascularization in rats

Neovascularization in the eye is a major cause of irreversible vision loss. The present study was undertaken to determine mechanisms through which pazopanib, a drug that targets multiple receptor tyrosine kinases such as VEGF receptors, inhibits angiogenesis and experimental choroidal neovascularization (CNV). Pazopanib inhibited VEGF expression by retinal pigment epithelium (RPE) cells and choroidal endothelial cells (CEC), decreased VEGF-induced cellular migration in a dose-dependent manner and suppressed extracellular signal-regulated kinase (ERK)-1/-2 phosphorylation. To assess the impact of pazopanib in vivo, CNV was induced in rats by rupturing the Bruchs membrane by laser coagulation. These experiments demonstrated that twice-daily topical eye drop treatment significantly (P<0.001) decreased leakage from photocoagulated lesions by 89.5%. Furthermore, the thickness of the developed CNV lesions was significantly inhibited by 71.7% (P<0.001) in pazopanib-treated eyes, and immunoreactivity of VEGF was lower than in control eyes. Our data suggest that pazopanib is a promising inhibitor of angiogenesis leading to an effective inhibition of CNV development in vivo. This activity can be largely ascribed to the down-regulation of VEGF release in the retina as well as to impaired VEGF-induced signaling and chemotaxis. Using a convenient topical dosing regimen, pazopanib may prove useful for treating a variety of ocular neovascular diseases such as neovascular age-related macular degeneration.

Expression of glia maturation factor during retinal development in the rat

Glia maturation factor plays important roles in the development and growth of glia and neurons. We investigated the expression and localization of Glia maturation factor-beta (GMFB) and Glia maturation factor-gamma (GMFG) in the rat retina. By northern blot analysis, both GMFB and GMFG mRNAs were detected in retina as early as embryonic day (E) 18 and persisted until adult. The expression of GMFB mRNA was always much greater than that of GMFG mRNA. In situ hybridization showed that the GMFB mRNA signal was positive in the retina from E14 till adult. Immunostaining revealed that GMFB protein was present in the inner layer of retina at E14 and P1, and in Müller cells in adult. GMFG immunoreactivity was observed only in the inner limiting membrane from E14 to P1 rat retina, and was not detected in the adult retina. These results show that GMFs are synthesized and localized mainly in Müller cells in the rat retina, and suggest that they may contribute to the development and growth of glia and neurons.

Glycoxidized particles mimic lipofuscin accumulation in aging eyes: a new age-related macular degeneration model in rabbits

PurposeThe biogenesis of drusen, a hallmark of age-related macular degeneration (AMD), is still unclear. Lipofuscin, which extensively accumulates with age in RPE cells, is hardly soluble, derived in part from oxidation byproducts of the photoreceptor outer segments. The purpose of the current study is to develop a new AMD model in rabbits using glycoxidized particles as imitation lipofuscin, and determine whether accumulation of lipofuscin as insoluble material may play a role in drusen biogenesis and other pathogenesis of AMD.MethodsTo mimic the accumulation of insoluble lipofuscin, glycoxidized microspheres (glycox-MS) were made through a glycoxidation process with albumin and glycolaldehyde, α-hydroxy aldehyde. As a control, microspheres made with glutaraldehyde (cMS) and soluble glycoxidized (glycox-) albumin were prepared. Each material was implanted into the subretinal space in rabbits. The implanted area was assessed by funduscopy, fluorescein angiography, histology, and transmission electron microscopy (TEM).ResultsCompared with control microspheres, glycox-MS stagnated for a prolonged period in the cytoplasm of RPE cells. Eyes implanted with glycox-MS produced drusen-like deposits at a significantly higher frequency, when compared with the controls. Glycox-MS were observed at the margin of or beneath the drusen-like deposits in all cases. In some eyes with glycox-MS, late-onset sub-RPE choroidal neovascularization was observed, while control groups did not have these findings.ConclusionsThese results suggest that the accumulation of indigestible granules such as lipofuscin in RPE or subsequent depositions toward Bruch’s membrane may play a role in drusen biogenesis as a trigger of inflammation or via other mechanisms. This model of AMD may be useful to elucidate drusen biogenesis and pathogenesis of AMD.

Possible implications of acid-sensing ion channels in ischemia-induced retinal injury in rats

BackgroundRetinal ischemia in eyes with diabetic retinopathy and retinal vein occlusion leads to local tissue acidosis. Acid-sensing ion channels (ASICs) are expressed in photoreceptors and other neurons in the retina, and may play a role in acid-induced cell injury. The purpose of this study was to investigate the neuroprotective effects of amiloride, an ASIC blocker, on induced retinal ischemia in rats.MethodsTransient retinal ischemia was induced in male Long–Evans rats by the temporary ligation of the optic nerve. Just before the induction of ischemia, the experimental eyes underwent intravitreal injection of amiloride. On day 7, the retinal damage in eyes that underwent amiloride treatment (and in those that did not undergo the treatment) was evaluated by histology and electroretinogram (ERG).ResultsTransient retinal ischemia caused retinal degeneration with thinning of the inner layer of the retina. The blockage of ASICs with amiloride significantly prevented retinal degeneration. ERG demonstrated that the reduction in a- and b-wave amplitudes induced by the transient retinal ischemia was significantly prevented by the application of amiloride.ConclusionsThe present study suggests that ASICs might, at least in part, play a pathophysiological role in ischemia-induced neurodegeneration. Blockage of ASICs may have a potential neuroprotective effect in ocular ischemic diseases.

Three-Dimensional Spheroidal Culture Visualization of Membranogenesis of Bruch's Membrane and Basolateral Functions of the Retinal Pigment Epithelium

PURPOSE Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruchs membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE. METHODS Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide. RESULTS The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruchs membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruchs membrane. Western blotting showed expression of RPE differentiation markers and components of Bruchs membrane and RPE lipoproteins. CONCLUSIONS This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.

Pitavastatin Attenuates Leukocyte-Endothelial Interactions Induced by Ischemia-Reperfusion Injury in the Rat Retina

Purpose: Statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) have been shown to lower serum cholesterol levels in clinical use. Moreover, it has been reported that statins exert pleiotropic and beneficial effects on vascular endothelium. Therefore, we investigated the effects of pitavastatin, a new statin, on leukocyte accumulation during ischemia-reperfusion injury. Materials and Methods: Transient retinal ischemia was induced in Long-Evans rats for 60 min by temporal ligation of the optic nerve. Pitavastatin (0.12, 0.35, or 1.1 mg/kg) was administered 5 min prior to the induction of retinal ischemia. Leukocyte-endothelial interactions in the post-ischemic retina were evaluated in vivo with acridine orange digital fluorography. The number of rolling leukocytes, number of accumulated leukocytes, and diameters of the major retinal artery and vein were evaluated. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression in the retina was semiquantitatively studied using the RT-PCR method. Results: Pitavastatin-treated rats at doses of 0.35 and 1.1 mg/kg showed mild arterial narrowing (p < 0.01) and venous dilation (p < 0.01) compared with vehicle-treated (ischemic) rats. In rats treated with 0.35 mg/kg pitavastatin, the number of rolling leukocytes was significantly reduced by 35.5% (p < 0.01) 12 hr after reperfusion compared with that of vehicle-treated rats. With treatment at a dose of 0.35 mg/kg pitavastatin, the number of accumulated leukocytes was reduced to 68.7% (p < 0.01) 24 hr after reperfusion. Moreover, pitavastatin treatment significantly reduced ICAM-1 mRNA expression in the retina during ischemia-reperfusion injury. Conclusions: Pitavastatin effectively attenuated ischemia-induced leukocyte-endothelial interactions in the rat retina.

Evaluation of peripheral fundus autofluorescence in eyes with wet age-related macular degeneration

Purpose We aimed to evaluate the prevalence of abnormal peripheral fundus autofluorescence (FAF) in wet age-related macular degeneration (AMD) using wide-field imaging instrument. Patients and methods A retrospective, case-controlled study involving 66 eyes of 46 Japanese wet AMD patients and 32 eyes of 20 control patients was performed. Wide-field FAF images were obtained for typical AMD (37 eyes/28 patients), polypoidal choroidal vasculopathy (PCV) (22 eyes/20 patients), and retinal angiomatous proliferation (RAP) (seven eyes/four patients). Two masked ophthalmologists independently graded the images for mottled, granular, and nummular patterns. Main outcome measures were abnormal peripheral FAF frequencies and relative risks by disease subgroups and treatments. Results Abnormal peripheral FAF patterns were found in 51.5% of wet AMD eyes compared with 18.8% of control eyes (P<0.001). Mottled, granular, and nummular patterns were found in 45.5%, 31.8%, and 16.7%, respectively, of wet AMD eyes. Each disease subgroup (typical AMD, 54.1%; PCV, 36.4%; and RAP, 85.7%) showed significantly higher frequencies of peripheral FAF (P<0.001, P=0.03, and P<0.001, respectively) than control eyes (18.8%). There were no significant differences (P=0.76) between the frequencies in untreated and treated eyes. Conclusion Eyes of Japanese wet AMD patients had a higher abnormal FAF prevalence compared with control eyes. Among the three disease subtypes, abnormal patterns were least prevalent in PCV eyes.

In vitro drusen model: three-dimensional spheroid culture of retinal pigment epithelial cells

ABSTRACT Age-related macular degeneration (AMD) is a leading cause of blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids of human RPE cells constructed a well-differentiated monolayer of RPE with a Bruchs membrane. We determined that RPE spheroids exhibited drusen formation between the RPE and Bruchs membrane with expression of many drusen-associated proteins, such as amyloid β and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases, such as AMD. Summary: Drusen, a hallmark of age-related macular degeneration, are produced by three-dimensional spheroids of retinal pigment epithelial cells. This spheroid culture can be used as a new tool to investigate drusen biogenesis.