Nobuyo Yanagihara

Hepatocyte growth factor stimulates proliferation and migration during wound healing of retinal pigment epithelial cells in vitro

PURPOSE A defect in retinal pigment epithelial (RPE) cells may cause dysfunction of the neural retina, so rapid recovery of differentiated RPE cells is required after RPE injury. We investigated the effect of hepatocyte growth factor (HGF) on wound healing in RPE cells. METHODS Confluent monolayers of bovine RPE cells were denuded, and the cells were allowed to recover in the presence or absence of HGF. The effect of HGF on RPE cell proliferation was evaluated by a 3-(4;5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetraz olium assay. In a migration assay, mitomycin C was used to inhibit proliferation, and the number of migrated cells was counted. The signaling pathways involved were examined using inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and protein kinase C pathways. RESULTS At 80 ng/mL, HGF stimulated the wound closure of RPE monolayers and rendered the restituted cells more epithelioid in shape. HGF at 10 ng/mL stimulated RPE cell migration the most, whereas 80 ng/mL of HGF inhibited migration, but stimulated proliferation the most. In particular, PI3 kinase and MAPK inhibitor inhibited PRE cell migration and proliferation, respectively. CONCLUSIONS HGF stimulated wound closure in cultured RPE cells, and rendered restituted cells epithelioid in shape. HGF may become a therapeutic candidate for RPE wound healing.

Progressive association of a “soluble” glycolytic enzyme with the detergent-insoluble cytoskeleton during in vitro morphogenesis of MDCK epithelial cells

In MDCK epithelial cells, cell contact at confluency initiates a protracted process of morphogenesis during which several proteins known to bind the cytoskeleton become progressively associated with the detergent-resistant cell fraction and distributed to their characteristic polarized domains. Using extraction protocols that identify this tight cytoskeletal linkage, here we show a similar but slower, time-dependent enrichment in the detergent resistant fraction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly abundant glycolytic enzyme that is traditionally considered soluble. Similar enrichment did not occur for two other glycolytic enzymes, phosphoglycerate mutase or lactate dehydrogenase. Insoluble GAPDH was not homogeneously distributed in the cytoplasm but rather displayed several discrete patterns that varied within and among MDCK cells. It also localized prominently to a few nuclei in the phenotypically heterogeneous cells of late confluency cultures. Disruptors of cytoskeletal filaments were relatively ineffective in the postconfluent epithelial monolayers, although use of disrupting agents implicated actin as the cytoplasmic filament that tethers insoluble GAPDH. Catalytic activity could be demonstrated in the insoluble fraction of GAPDH from postconfluent cultures, but only after release by mechanical disruption of insoluble extracts. Treatment of postconfluent cells with agents that deplete ATP diminished the fraction of cytoskeletally associated GAPDH, and levels of insoluble GAPDH were restored with ATP repletion, suggesting that ATP levels may regulate cytoskeletal linkage and thereby local enzyme activity. We conclude that the highly abundant and ubiquitous enzyme GAPDH becomes progressively enriched in detergent stable subcellular compartments during the process of epithelial morphogenesis. The process that produces GAPDH compartments is slow, suggesting that epithelial cells just at confluency, when they are typically analyzed, have not yet maximized the organizational state that can be attained in monolayer culture.

Long-term follow-up of severe central serous chorioretinopathy using indocyanine green angiography

Background: The severe types of central serous chorioretinopathy (CSC) have a chronic nature, suggesting that a pathological process persists subclinically. Indocyanine green(ICG) angiography recently revealed intrachoroidal dye leakage and its static nature in CSC. As the intrachoroidal dye leakage was suspected to be relevant to the disease process, the long-term persistence of intrachoroidal ICG leakage was examined in four patients of the severe types of CSC. Methods: ICG angiography was performed periodically over more than three years in three patients and two years in one patient. One patient had CSC with bullous retinal detachment, and the other three had chronic CSC or diffuse retinal pigment epitheliopathy. Results: Intrachoroidal ICG leakage persisted in all the patients. However, a change in location of persistent intrachoroidal leakage or disappearance of intrachoroidal leakage regardless of no progression of retinal pigment epithelial alteration was noted in one eye of two patients. Conclusions:Pathology causing intrachoroidal ICG leakage persisted subclinically for a long period. However, location and extent of the intrachoroidal leakage could change during along-term follow-up period.

Fundus autofluorescence in patients with pseudoxanthoma elasticum.

Purpose: To characterize changes in fundus autofluorescence in patientswith pseudoxanthoma elasticum (PXE). Fundus autofluorescence intrinsicallyderives from lipofuscin, and the degree of autofluorescence is thought to indicatethe degree of retinal pigment epithelium (RPE) metabolic activity. Methods:Twelve eyes of 6 patients (2 men, 4 women) with PXE were studied with aconfocal scanning laser ophthalmoscope. Patient age ranged from 42 to 62years. The autofluorescence of abnormal retinal areas was compared digitallywith that of neighboring, presumed healthy control areas. When the averagegray level of a fundus region was 2 SDs above or below the average graylevel of a control area, autofluorescence of the fundus region was consideredabnormal. Results: In all 12 eyes, some segments of the angioid streaksshowed decreased fundus autofluorescence, and other segments of the streaksshowed normal autofluorescence. Areas of peripapillary chorioretinal atrophyseen in 2 eyes and of disciform scarring seen in 3 eyes showed decreasedautofluorescence. Solitary or multiple drusen-like spots showed increasedautofluorescence in all 12 eyes. Conclusion: Atrophic and degenerativeRPE regions showed decreased fundus autofluorescence in areas of chorioretinalatrophy and in some segments of the angioid streaks. Some drusen-like spotsshowed increased autofluorescence. The characteristic changes in autofluorescencethat we observed in PXE patients suggest that the content of the drusen-like substancediffers from that of senile drusen and that the drusen-like lesions are similar to thesub-RPE deposits seen in macular dystrophy.

Effect of Retinoic Acid on Proliferation and Polyamine Metabolism in Cultured Bovine Retinal Pigment Epithelial Cells

Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 µM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.

Age-related scattered hypofluorescent spots on late-phase indocyanine green angiograms

Purpose: Scattered hypofluorescent spots may be seen on indocyanine green (ICG) angiograms of regions that do not show abnormalities when viewed with an ophthalmoscope. Hypofluorescent spots are found in several pathologic conditions, typically in inflammatory diseases. In this report, we describe hypofluorescent spots in normal fundi and show that such spots can be age-related.Methods: Video-fundus camera ICG angiograms of 115 eyes of 109 patients aged 12 to 85 years with normal fundi or with only age-related maculopathy were reviewed. The relation between age and scattered hypofluorescent spots, and between age-related maculopathy and spots was examined utilising regression analysis.Results: Scattered hypofluorescent spots were seen throughout the posterior pole in 24 eyes of 23 patients and in a portion of the posterior pole in 30 eyes of 29 patients. The hypofluorescent spots were noted between 26 and 37 minutes after dye injection. Patient age ranged from 51 to 80 years, and regression analysis showed that the frequency of hypofluorescent spots increased significantly with aging (p < 0.05). However, age-related maculopathy did not show a significant relation to the spots.Conclusion: Scattered hypofluorescent spots seen in the posterior pole during the late-phase of ICG angiograms can apparently be due to aging of the fundus.

Does ascorbic acid prevent retinopathy during interferon therapy in patients with chronic hepatitis C

Purpose. Ascorbic acid was administered to patients with chronic hepatitis C to elucidate the mechanism of onset of retinopathy during interferon (IFN) therapy, and its prevention. Methods. The subjects were 62 patients with chronic hepatitis C who had been admitted to our hospital. For the IFN therapy, 6 MIU of natural IFN-α or 10 MIU of recombinant human IFN-α 2b was administered every day for the first 2 weeks, followed by administration three times a week for 22 weeks. The patients were randomly assigned to a group receiving 600 mg/day of ascorbic acid or a group not receiving ascorbic acid (control group). The optic fundi were examined by ophthalmologists before the IFN therapy began and subsequently at weeks 2 and 4 and then every 4 weeks during the IFN therapy. Results. Retinopathy was found in 9 of the 31 patients (29%) in the ascorbic acid-treated group and in 11 of the 31 patients (35%) in the control group. The cumulative incidence of hemorrhage in the ascorbic acid-treated group was lower than that in the control group during the IFN therapy, but the difference between the two groups was not significant (P = 0.186). The cumulative incidence of cotton-wool spots in the ascorbic acid-treated group was almost same as that in the control group during the IFN therapy. The median platelet counts before the therapy was begun were 11.8 × 104/mm2 in the group with hemorrhage and 16.6 × 104/mm2 in the group without, and the lowest platelet counts during IFN therapy were 7.3 × 104/mm3 in the group with hemorrhage and 9.5 × 104/mm3 in the group without, indicating significantly lower values in the group with hemorrhage (P = 0.018 and P = 0.020, respectively). The lowest platelet counts during IFN therapy were 7.4 × 104/mm3 in the group with cotton-wool spots and 9.7 × 104/mm3 in the group without, indicating a significantly lower value in the group with cotton-wool spots (P = 0.036). Conclusions. Ascorbic acid was not considered to be useful for the prevention of the retinopathy associated with IFN therapy in patients with chronic hepatitis C.

Indocyanine Green Angiograms of Choroidal Nevi: Comparison Between Confocal and Nonconfocal Scanning Laser Ophthalmoscope and Fundus Video Camera

PURPOSE A fundus video camera and a nonconfocal scanning laser ophthalmoscope (SLO) detect direct light and indirect light, whereas a confocal SLO detects mostly direct light. Differences in confocal and nonconfocal SLO images and fundus video camera images are most likely due to their different optical systems. These differences were examined in indocyanine green (ICG) angiograms of a choroidal nevus. METHODS A confocal SLO, a nonconfocal SLO, and a high resolution digital fundus video camera were used to obtain ICG angiograms of pigmented choroidal nevi in 4 patients for 30 minutes following dye injection. RESULTS All the angiograms showed a hypofluorescent region in the nevus until 10-14 minutes after dye injection, except in 1 patient in whom no hypofluorescent region was seen in an early confocal-SLO angiogram. From 20 minutes to 30 minutes postinjection, the hypofluorescent regions were still visible in all fundus video camera angiograms and nonconfocal SLO angiograms but not in confocal SLO angiograms. CONCLUSIONS Early angiograms taken with the three angiography systems showed a similar appearance of the choroidal nevus. However, late ICG angiograms with a confocal SLO showed different images from those taken with a nonconfocal SLO or a fundus video camera. It is suggested that the angiography system and the aperture size of an SLO should be selected according to the aspect of the pigmented choroidal nevus that is of interest in late-phase ICG angiography.

Incising the Thick Retrolental Fibrovascular Tissue With a Hooked Sclerotome in Persistent Hyperplastic Primary Vitreous

A technique for incising thick retrolental fibrovascular tissue and extensive cyclitic membrane is reported in a case of anterior persistent hyperplastic primary vitreous. A membranectomy was performed in a 1-month-old post-lensectomy baby via a limbal approach. A sclerotome tip was hooked to cut through an extremely thick fibrovascular tissue by rotating the sclerotome by its grip. Sutherland microscissors (Grieshaber, Switzerland) and a vitrectomy cutter were used for further membranectomy. The baby was followed-up until age 18 months. A total of 3 membranectomy sessions were required because of rapid cyclitic membrane formation, severe centripetal retraction of the membrane on the ciliary processes, and posterior synechia. Thorough membranectomy and cutting the iris edge maintained a clear pupillary area during the 13-month postoperative period. Extremely thick retrolental fibrovascular tissue is a challenging condition that can be dealt with by delicate instrumentation.

A Simple, Safe Bimanual Technique for Subincisional Cortex Aspiration

We developed a bimanual manipulation technique to facilitate the removal of the subincisional lens cortex in small-incision phacoemulsification cataract surgery. A separate aspiration handpiece, not connected to an aspiration tube, is passed into the anterior chamber through a side-port corneal incision. Under irrigation with a standard infusion/aspiration (I/A) handpiece through a tunnel incision, the cortex is stripped off with the separate handpiece and removed with the I/A handpiece. In 227 eyes, subincisional cortex removal and subsequent capsule polishing was performed safely with the separate handpiece. Rupture of the posterior lens capsule occurred in 3 high-risk eyes.