Jung Il Moon

Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina

Heat shock proteins (HSPs) are highly conserved proteins playing a protective role under deleterious conditions caused by a wide variety of pathophysiological, including environmental stresses. Glutathione (GSH) is known to play a critical role in the cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH depletion induced cell death in the retina, but the mechanism(s) of cellular protection were not clear. Unregulated oxidative stress was induced by depletion of intracellular GSH by systematic administration of buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. After 0, 1, 4 and 7 days of BSO administration, we examined expression of both large and small HSP mRNAs (hsp90alpha, hsp90beta, hsp70, hsp60 and hsp25) in oxidative-stressed mouse retina. Of large HSPs, only hsp70 expression was significantly decreased from 1 day after BSO injection, whereas expression of other large hsps was not changed on day 1. Expression of hsp60 decreased on 4 days, whereas expression of hsp90 decreased on 7 days after BSO administration. Different from large HSPs, a small HSP, hsp25 increased its expression to a great extent from 1 day after BSO administration. Taken together, our results show that unregulated oxidative stress could induce differential expression of HSPs, which, in turn, may play distinct roles in the cellular defense. Targeting HSPs, therefore, may provide novel tools for treatment of retinal degenerative diseases such as glaucoma, retinopathy or age-related macular degeneration.

Glutathione depletion induces differential apoptosis in cells of mouse retina, in vivo

Oxidative stress affects numerous intracellular macromolecules, and may result in cell death unless precisely regulated. Unregulated oxidative stress can be controlled by various cellular defense mechanisms such as glutathione (GSH) which can critically counteract the damaging effects of oxidative stress in mammalian cells. We determined the effects of unregulated oxidative stress induced by GSH depletion on cells in mouse retina. Mice were intraperitoneally injected with buthionine sulphoximine (BSO) at 1.5 g/kg. After 0, 1, 4, and 7 days of BSO administration, retinas were excised and sections were subjected to GSH assay and terminal uridine deoxynucleotidyl nick end labeling (TUNEL) analysis. After 4 days of BSO administration, the number of TUNEL positive cells was significantly increased. However, after 7 days, TUNEL positive cells returned to the basal level. The retinal region most affected by the BSO treatment appeared to be the outer nuclear layer where the photoreceptor cells reside. Different from cells in other regions, retinal cells in the inner nuclear layer increased in their apoptosis even after the first day of BSO injection, and the increase was further potentiated after 4 days. Taken together, our studies suggested that GSH depletion may cause unregulated oxidative stress to the cells in the retina and indeed increased cell death in the retina. The cells in the inner nuclear layer seemed to be affected earlier than the cells in other layers of the retina. The GSH level in the retina may be a crucial therapeutic target in preventing blindness.

Antifibrotic effects of pirfenidone on Tenon’s fibroblasts in glaucomatous eyes: comparison with mitomycin C and 5-fluorouracil

PurposeThe purpose of this study was to evaluate the antifibrotic effects of pirfenidone (PFD) on primary cultured human Tenon’s fibroblasts (HTFs) from primary open-angle glaucoma (POAG) eyes, compared to mitomicin C (MMC) and 5-fluorouracil (5-FU).Materials and methodsSamples of human Tenon’s capsule were obtained during respective surgeries from three groups of patients: patients with cataract (CAT group), patients with POAG who underwent glaucoma filtration surgery (GFS) (POAG1 group), and patients with POAG who underwent GFS due to failed bleb of previous GFS (POAG2 group). Cell toxicity, cell migration, and the expression level of α-smooth muscle actin (α-SMA) protein were evaluated in primary cultured HTFs from the three patient groups after treatment (PFD, MMC, or 5-FU).ResultsOverall, cell viability after PFD treatment was higher compared to MMC treatment (82.3u2009±u20095.1xa0% vs 56.7u2009±u20093.8xa0%; pu2009=u20090.001) and comparable to 5-FU treatment (82.3u2009±u20095.1xa0% vs 85.7u2009±u200910.7xa0%, pu2009=u20090.214) at the same concentration (0.4xa0mg/ml). Both 0.3xa0mg/ml PFD and 0.1xa0mg/ml MMC inhibited cell migration compared to control (without treatment) cells (pu2009=u20090.014 and 0.005, respectively), while 0.2xa0mg/ml 5-FU showed the highest degree of cell migration among the three agents in the POAG1 group (PFD vs MMC vs 5-FU; 29.5u2009±u20092.1xa0% vs 34.5u2009±u20090.7xa0% vs 76.0u2009±u20098.5xa0%, PFD vs MMC; pu2009=u20091.000, PFD vs 5-FU; pu2009=u20090.008, MMC vs 5-FU; pu2009=u20090.011). PFD (0.1 or 0.3xa0mg/ml) and MMC (0.05 and 0.1xa0mg/ml) treatment significantly reduced the protein expression level of α-SMA in the POAG 1 group (all pu2009<u20090.05), and the α-SMA protein level following treatment with 0.3xa0mg/ml PFD was lower than that of 0.1xa0mg/ml MMC (pu2009=u20090.040).ConclusionPFD showed less cytotoxicity compared to MMC. PFD and MMC inhibited cell migration and reduced α-SMA protein expression levels, while 5-FU showed neither inhibition of cell migration nor reduction in α-SMA expression level. These findings indicate PFD as a potential adjunctive antifibrotic agent to prevent bleb failure during GFS.

The Effect of Additional Topical Cyclosporine or Vitamin A on the Ocular Surface during Antiglaucoma Medication Administration

Purpose: To investigate the effects of topical application of cyclosporine or vitamin A on the ocular surface during the concurrent administration of antiglaucoma drugs. Methods: Thirty rabbits were randomized into 5 groups. Group 1 was administered timolol, group 2 received travoprost, group 3 received a travoprost/timolol fixed combination solution, group 4 received timolol and travoprost, and group 5 received timolol, travoprost, and dorzolamide. Each group was divided into a subgroup that received only the antiglaucoma medication (subgroup A), a subgroup that received topical cyclosporine in addition to the antiglaucoma medication (subgroup B), and a subgroup that received topical vitamin A in addition to the antiglaucoma medication (subgroup C). Conjunctival impression cytology specimens were collected at baseline and at weeks 1, 3, and 6. Conjunctival biopsy specimens were collected at week 6. Results: The impression cytologic study results are as follows: statistically significant differences were found between groups 4A and 4B and between groups 4A and 4C at week 6 (p = 0.004, p = 0.006, respectively) and between groups 5A and 5B and between groups 5A and 5C at weeks 3 and 6 (p = 0.006, p = 0.008 at week 3, p = 0.003, p = 0.004 at week 6, respectively). No statistically significant differences were found between subgroup B and subgroup C in any of the groups at any of the times evaluated (p > 0.05). The conjunctival biopsy specimens from groups 1, 2, and 3 showed no distortion, but groups 4A and 5A showed distortion of the conjunctival epithelial structures. Groups 4B, 4C, 5B, and 5C showed less distortion of the conjunctival epithelial structures. Conclusion: Administration of cyclosporine or vitamin A may reduce the adverse ocular surface changes caused by long-term administration of antiglaucoma drugs.

Effects of antiglaucoma drugs on the ocular surface in rabbits: a fixed-combination drug versus two concomitant drugs.

PurposeWe investigated the effects of a fixed-combination antiglaucoma drug and compared it with two concomitant antiglaucoma drugs on the ocular surface.MethodsTwenty-four rabbits were randomized into four groups. Group 1 was administered timolol, group 2 travoprost, group 3 a travoprost/timolol fixed-combination solution, and group 4 timolol and travoprost. Conjunctival impression cytology specimens were collected at baseline and weeks 1, 3, and 6, and conjunctival biopsy specimens at week 6.ResultsThe impression cytology study results were as follows: No statistically significant differences among group 1–3 at any time (pxa0>xa00.05); a statistically significant difference between groups 3 and 4 at week 6 (pxa0=xa00.003); a statistically significant difference between baseline and group 4 at week 6 (pxa0=xa00.008). Conjunctival biopsy specimens of group 1–3 showed no distortion of the conjunctival epithelial structures, but group 4 showed decreased layers of epithelial cells with fewer periodic acid-Schiff (PAS) (+) goblet cells.ConclusionsA fixed-combination antiglaucoma drug is beneficial in reducing adverse ocular surface changes in long-term use. This is believed to be due to the smaller concentration of preservatives contained in the fixed-combination drug.

A Family with Axenfeld-Rieger Syndrome: Report of the Clinical and Genetic Findings

Purpose To describe clinical findings in a Korean family with Axenfeld-Rieger syndrome. Methods A retrospective review of clinical data about patients with diagnosed Axenfeld-Rieger syndrome. Five affected members of the family underwent a complete ophthalmologic examination. We screened the forkhead box C1 gene and the pituitary homeobox 2 gene in patients. Peripheral blood leukocytes and buccal mucosal epithelial cells were obtained from seven members of a family with Axenfeld-Rieger syndrome. DNA was extracted and amplified by polymerase chain reaction, followed by direct sequencing. Results The affected members showed iris hypoplasia, iridocorneal adhesions, posterior embryotoxon, and advanced glaucoma in three generation. None had systemic anomalies. Two mutations including c.1362_1364insCGG and c.1142_1144insGGC were identified in forkhead box C1 in four affected family members. Conclusions This study may help to understand clinical findings and prognosis for patients with Axenfeld-Rieger syndrome.

Effectiveness of the ICare rebound tonometer in patients with overestimated intraocular pressure due to tight orbit syndrome

AbstractPurposenTo evaluate the effectiveness of the ICare rebound tonometer in patients with overestimated intraocular pressure (IOP) due to tight orbit syndrome and to identify factors affecting the development of tight orbit syndrome in glaucoma patients.MethodsWe investigated 84 eyes in 84 glaucoma patients, of which 14 eyes were classified in the tight orbit syndrome group and 70 eyes in the control group. IOP was measured using the ICare tonometer and the Goldmann applanation tonometer (GAT). The demographic data, medical histories, ocular histories, and detailed ocular drug histories of the two groups were compared to identify factors contributing to the development of tight orbit syndrome.ResultsIn the tight orbit syndrome group, the ICare tonometer significantly underestimated the IOP by approximately 8.6xa0mmHg compared with the GAT. In the control group, the IOP readings of the GAT and the ICare tonometer did not differ significantly. Bland–Altman analysis showed that the mean difference between measurements taken using the GAT and those taken using the ICare tonometer was 2.5xa0± 6.3xa0mmHg. The difference between the GAT and ICare tonometer measurements was greater in the tight orbit syndrome group (8.6xa0±xa05.3xa0mmHg) than in the control group (1.3xa0±xa02.7xa0mmHg). Multivariate regression analysis revealed that only the use of prostaglandin analogs (PGAs) was associated with the development of tight orbit syndrome.ConclusionsThe ICare tonometer is a suitable alternative device for use in patients with tight orbit syndrome in whom the IOP may be overestimated with the GAT. The prolonged use of PGAs is significantly associated with the development of tight orbit syndrome.

Effects of AFP-172 on COX-2-induced angiogenic activities on human umbilical vein endothelial cells

PurposeTo investigate the angiogenic effect of the free acid of tafluprost (AFP-172) on human umbilical vascular endothelial cells (HUVECs).MethodsHUVECs cultured in the presence or absence of FP receptor antagonist (10xa0nM AL-8810) were exposed to escalating concentrations of 10−7, 10−6, 10−5, 10−4 and 10−3 M AFP-172 (the free acid of tafluprost). For cell proliferation assays, the numbers of cells were derived from a CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega) by Microplate reader (Bio-Rad, Benchmark). Endothelial cell migration was evaluated by a BD Biocoat™ Angiogenesis System using FluoroBlok ™ 24-well inserts (BD Biosciences, Bedford, MA). BioTek FLx800 fluorescence plate reader was used for quantitative measurement of fluorescently-labeled invasive vascular endothelial cells. Endothelial capillary-like tube formation was evaluated by BD Biocoat Angiogenesis System using Matrigel Matrix 96-well plate. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the gene expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and endothelial nitric oxide synthase (eNOS). COX-2 protein was detected by immunofluorescent staining and Western blot assay. Students t-test was used for statistical analysis.Results10−4 M AFP-172 treated cells stimulated the proliferation, migration and tube formation of HUVECs as compared to 10−5, 10−6, 10−7 M AFP-172 treated cells and control (Pu2009<u20090.01). RT-PCR showed that incubation of HUVECs with 10−4 M AFP-172 stimulated the expression of COX-2 mRNA (Pu2009<u20090.05). Western blot assay revealed that AFP-172 caused cells to increase in COX-2 protein at the concentrations of 10−4 M.Conclusions>AFP-172 showed the angiogenic effects on HUVECs at the concentrations of 10−4 M by inducing COX-2 protein.

Spectral domain optical coherence tomography cross-sectional image of optic nerve head during intraocular pressure elevation

AIMnTo analyze changes of the optic nerve head (ONH) and peripapillary region during intraocular pressure (IOP) elevation in patients using spectral domain optical coherence tomography (SD-OCT).nnnMETHODSnBoth an optic disc 200×200 cube scan and a high-definition 5-line raster scan were obtained from open angle glaucoma patients presented with monocular elevation of IOP (≥30 mm Hg) using SD-OCT. Additional baseline characteristics included age, gender, diagnosis, best-corrected visual acuity, refractive error, findings of slit lamp biomicroscopy, findings of dilated stereoscopic examination of the ONH and fundus, IOP, pachymetry findings, and the results of visual field.nnnRESULTSnThe 24 patients were selected and divided into two groups: group 1 patients had no history of IOP elevation or glaucoma (n=14), and group 2 patients did have history of IOP elevation or glaucoma (n=10). In each patient, the study eye with elevated IOP was classified into group H (high), and the fellow eye was classified into group L (low). The mean deviation (MD) differed significantly between groups H and L when all eyes were considered (P=0.047) and in group 2 (P=0.042), not in group 1 (P=0.893). Retinal nerve fiber layer (RNFL) average thickness (P=0.050), rim area (P=0.015), vertical cup/disc ratio (P=0.011), cup volume (P=0.028), inferior quadrant RNFL thickness (P=0.017), and clock-hour (1, 5, and 6) RNFL thicknesses (P=0.050, 0.012, and 0.018, respectively), cup depth (P=0.008), central prelaminar layer thickness (P=0.023), mid-inferior prelaminar layer thickness (P=0.023), and nasal retinal slope (P=0.034) were significantly different between the eyes with groups H and L.nnnCONCLUSIONnRNFL average thickness, rim area, vertical cup/disc ratio, cup volume, inferior quadrant RNFL thickness, and clock-hour (1, 5, and 6) RNFL thicknesses significantly changed during acute IOP elevation.