Shozo Sonoda

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

Genetic association of manganese superoxide dismutase with exudative age-related macular degeneration

PURPOSE To elucidate whether any polymorphic genes for xenobiotic-metabolizing and antioxidant enzymes are associated with the development of exudative age-related macular degeneration. METHODS A hospital-based case-control study was performed on a consecutive series of 102 Japanese patients with the exudative form of age-related macular degeneration who were recruited between 1993 and 1998 in the Kagoshima University Hospital. Controls were 200 systemically healthy individuals who had no senescent ocular disorders and were over 50 years of age. There was no evidence of age-related macular degeneration in the 200 controls. Genomic DNA from peripheral bloods was examined using polymerase chain reaction and defined for the genetic polymorphisms of cytochrome P-450 1A1, glutathione S-transferases, microsomal epoxide hydrolase, and manganese superoxide dismutase. RESULTS We found a significant association of manganese superoxide dismutase gene polymorphism, valine/alanine polymorphism at the targeting sequence of the enzyme, with age-related macular degeneration. The patients had an increased frequency of alanine allele and alanine/alanine genotype (odds ratio = 10.14, 95% confidence interval = 4.84 to 2.13; P =.0005 after Bonferroni correction). We also observed a weak association of microsomal epoxide hydrolase exon-3 polymorphism with age-related macular degeneration (odds ratio = 2.20, 95% confidence interval = 4. 02 to 1.20; P =.020 after Bonferroni correction). Cytochrome P-450 1A1, glutathione S-transferases, and microsomal epoxide hydrolase exon-4 were polymorphic, but their genotype frequency distributions did not show a statistically significant difference between the patients and controls. CONCLUSIONS The results suggest that manganese superoxide dismutase gene polymorphism is associated with exudative age-related macular degeneration. Microsomal epoxide hydrolase is another enzyme that may be associated with the disease. The exudative form of age-related macular degeneration may have genetic risk factors against oxidative stress and/or effects of xenobiotics. Further association studies in other polymorphic genes for xenobiotic-metabolizing enzymes are needed to elucidate the environmental-genetic interaction in the underlying cause of age-related macular degeneration.

Choroidal Structure in Normal Eyes and After Photodynamic Therapy Determined by Binarization of Optical Coherence Tomographic Images

PURPOSE To determine changes in choroidal structure by binarization of optical coherence tomographic (OCT) images. METHODS Choroidal images were recorded by enhanced depth imaging OCT. The subfoveal choroidal images were analyzed, and the luminal and interstitial areas were converted to binary images by the Niblack method. The interrater, intrarater, and intersession agreements of the binary images were determined for healthy eyes. In eyes with age-related macular degeneration (AMD), the binary images of the choroid before photodynamic therapy (PDT) were compared to those after PDT. The untreated fellow eyes were studied as controls. RESULTS In healthy eyes, the average ratio of the luminal to choroidal area was 65.4%. The interrater agreement rate was high, with intraclass correlation coefficient (ICC) 0.985 and 0.988 for the choroid and luminal areas, respectively. The intrarater ICC was 0.996 for the choroid and 0.997 for the luminal areas. The intersession ICC was 0.993 for the choroid and 0.984 for the luminal areas. In eyes with AMD, the subfoveal choroidal area, the luminal area, and the interstitial areas were thinner 6 months after PDT (all P < 0.01, Wilcoxon signed-rank sum test). The ratio of the luminal to choroidal area was significantly decreased to 62.8% (P < 0.01, Wilcoxon signed-rank sum test). The ratio for the fellow eyes was not significantly changed. CONCLUSIONS The Niblack binarization method can be used to analyze the luminal area of choroid in an OCT image with good repeatability and reproducibility. The change in the subfoveal choroidal area after PDT is due mainly to a decrease in the luminal areas.

Polarized Secretion of PEDF from Human Embryonic Stem Cell–Derived RPE Promotes Retinal Progenitor Cell Survival

PURPOSE Human embryonic stem cell-derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs. METHODS The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium-derived factor (PEDF) levels from conditioned media. RESULTS Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody. CONCLUSIONS hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD.

Cytochrome P450 1B1 gene mutations in Japanese patients with primary congenital glaucoma1

PURPOSE To report a novel missense mutation and DNA polymorphism of the CYP1B1gene in Japanese patients with primary congenital glaucoma. METHODS A series of 11 unrelated patients with primary congenital glaucoma was examined. Patients were followed in the Kagoshima University Hospital between 1979 and 1998. DNA was extracted from leukocytes of the patients, their families, and unrelated healthy individuals. Amplicons spanning the coding regions of the CYP1B1 gene were examined by direct sequencing and enzyme-restriction detection. RESULTS In the 11 unrelated patients, besides the previously reported insertional mutation (1620 ins G), a novel missense mutation was identified at codons 444 to replace arginine with glutamine (R444Q) in one patient. The novel missense mutation cosegregated in the relevant family as an autosomal recessive pattern and was not found in other patients or control individuals. In addition, five polymorphic sites were found at codons 48, 119, 330, 432, and 449. These polymorphic alleles did not cosegregate with the disease, and they were found in healthy individuals as well. CONCLUSIONS Approximately 20% of Japanese patients with primary congenital glaucoma may be affected by mutations in the CYP1B1 gene. Further studies are justified to explore whether a relationship exists between the phenotypic expressivity of the disease and the type of mutation.

Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

Extensive Chorioretinal Atrophy in Vogt–Koyanagi–Harada Disease

PURPOSE To report extensive chorioretinal atrophy during the long-term course of Vogt-Koyanagi-Harada (VKH) disease not treated properly in the initial phase. CASES Four patients with VKH disease were examined more than 10 years after onset of the disease. OBSERVATIONS They presented initially with classic features of VKH disease, except 1 patient who had developed bilateral, acute angle-closure glaucoma as the initial sign. Two patients received systemic corticosteroid therapy at the acute phase of the disease. During the follow-up of 13-34 years subsequent to onset, these patients had chronic recurrent anterior uveitis with apparently stable depigmented fundus. Eventually, they developed diffuse, extensive chorioretinal atrophy that resulted in severe visual loss. One patient had an unusual familial occurrence of the disease. CONCLUSIONS Failure to prescribe proper corticosteroid therapy in the initial phase of VKH disease may lead to chronic recurrent uveitis. Long-standing uveitic reactions may eventually result in severe visual loss due to extensive chorioretinal degeneration.

A novel PAX6 gene mutation (P118R) in a family with congenital nystagmus associated with a variant form of aniridia

Abstract Background: A variety of PAX6 gene mutations were identified in patients with aniridia and/or allied ocular dysgenesis such as keratopathy, Peters’ anomaly, foveal hypoplasia, and nystagmus. To scrutinize the etiology of a four-generation Japanese family with autosomal dominant nystagmus associated with anterior and posterior segment anomalies, the PAX6 gene was examined. Patients and methods: A Japanese family showed a variant aniridia phenotype in four successive generations. Affected individuals had congenital nystagmus, microcornea with shortened axial length, superficial peripheral corneal opacification with pannus formation, dislocated pupil, and foveal hypoplasia. Analysis of the PAX6 gene mutation was performed in affected and unaffected individuals. Results: A novel missense mutation in the PAX6 gene was found in all affected individuals examined, but neither in unaffected individuals nor in unrelated healthy individuals. This mutation predicted a proline to arginine change at codon 118 (P118R) in the paired domain of PAX6 protein. Conclusion: The reported family illustrates that mutations in the PAX6 gene, in particular missense mutations, may manifest atypical clinical expression or forme fruste of aniridia.

Two patients with severe corneal disease in KID syndrome

PURPOSE To report two independent Japanese patients with keratitis, ichthyosis, and deafness (KID) syndrome and severe corneal disorder. DESIGN Observational case reports. METHODS Clinical observation of a 5-year-old boy (Patient 1) and a 64-year-old man (Patient 2) with KID syndrome, presenting prominent corneal diseases. Molecular genetic assessment of the GJB2 gene encoding connexin-26 was performed. RESULTS Patient 1 had bilateral diffuse superficial punctuate keratopathy with severe corneal neovascularization. He had a missense mutation of the GJB2 gene. Patient 2 had bilateral corneal stromal keratitis and right corneal ulceration with rupture of the Descemet membrane. He did not have any pathologic mutation of the GJB2 gene. The area of palisades of Vogt was diminished and tear production reduced in both patients. Topical eye drops, and corticosteroid or antibiotics, respectively, relieved them effectively. CONCLUSION The impaired ocular surface regulating system might be a cause of corneal disease in KID syndrome and it can be treated by eye drops.

Retinal morphologic changes and concentrations of cytokines in eyes with diabetic macular edema.

Purpose: To determine the relationship between the retinal morphologic changes and concentrations of intravitreal cytokines in eyes with diabetic macular edema. Methods: A retrospective comparative study was performed. The preoperative optical coherence tomography images were evaluated to determine the presence of serous retinal detachments (SRDs), retinal cystic changes, and retinal swelling. The concentrations of vascular endothelial growth factor, interleukin (IL)-6, and IL-8 in vitreous samples collected during vitrectomy were determined. The correlations between optical coherence tomography parameters, other clinical factors, and the concentration of cytokines were calculated. Results: Fifty-two eyes (52 patients) were investigated. An SRD was found in 19 of the 52 eyes (36.5%). Multivariate regression analysis showed that IL-6 was the only factor significantly associated with the presence of an SRD (P = 0.001; odds ratio, 1.268; 95% confidence interval, 1.105–1.452). The other morphologic changes, such as retinal cystic changes and retinal swellings, were not significantly associated with the concentrations of intravitreal cytokines. Conclusion: The significant association of SRD with intravitreal IL-6 indicates that inflammation may play an important role in the development of SRD in diabetic macular edema.