Jeong Hun Kim

Intravenously administered gold nanoparticles pass through the blood?retinal barrier depending on the particle size, and induce no retinal toxicity

The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 +/- 5%), endothelial cells (17 +/- 6%) and peri-endothelial glial cells (8 +/- 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.

The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation

The pathological angiogenesis in the retina is the major cause of vision loss at all ages. In particular, retinopathy of prematurity (ROP) is a leading cause of blindness in children. This study investigated whether gold nanoparticle (GNP) could inhibit retinal neovascularization in the animal model of ROP. Intravitreal injection of GNP significantly inhibited retinal neovascularization in the mouse model of ROP. In addition, GNP effectively suppressed VEGF-induced in vitro angiogenesis of retinal microvascular endothelial cells including proliferation, migration and capillary-like networks formation. GNP blocked VEGF-induced auto-phosphorylation of VEGFR-2 to inhibit consequently ERK 1/2 activation. GNP never affected on the cellular viability of retinal microvascular endothelial cells and induced no retinal toxicity. Our data suggest that GNP could be a potent inhibitor to retinal neovascularization without retinal toxicity. Furthermore, GNP could be extensively applied to variable vaso-proliferative retinopathies mediated by VEGF.

Recruitment of pericytes and astrocytes is closely related to the formation of tight junction in developing retinal vessels

During retinal development, retinal vascularization begins in the inner retinal layer and sprouts radially from the optic nerve to reach the periphery of the retina. Subsequently, retinal vessels sprout into the deep retinal layer to form three parallel of the nerve fiber layer and two plexiform layers. In this process, endothelial cells are closely related to astrocytes and pericytes with strict chronological order. Here, we provide that the recruitment of pericytes and astrocytes to vascular tube of endothelial cells is closely associated with the formation of tight junction in developing retinal vessels. At P4, endothelial cells of retinal vessels behind the invading front directly contact to pericytes, but not to foot processes of astrocytes, where ZO‐1 was already weakly immunoreactive along retinal endothelial cells. With the progression of retinal development, foot processes of astrocytes are gathered around retinal vessels and the maturation of tight junction in endothelial cells is clearly defined, which was temporally and spatially accordant to the expression of a tight junction protein, ZO‐1. In addition, tight junction could be formed with contact of pericytes to endothelial cells without the prominent ensheathment of astrocytic foot processes, which coincided with the appearance of a tight junction protein, ZO‐1. Therefore, these data demonstrate that the tight junction of endothelial cells in the blood–retina barrier could be developed by cellular interactions between pericytes, astrocytes, and endothelial cells. Moreover, ZO‐1 as well as occludin or claudin might demonstrate the tightness of blood–retina barrier in developing retina.

Protective effect of clusterin from oxidative stress-induced apoptosis in human retinal pigment epithelial cells.

PURPOSEnOxidative stress to retinal pigment epithelial (RPE) cells is thought to play a critical role in the pathogenesis of age-related macular degeneration (AMD). This study was conducted to investigate whether clusterin protects human RPE cells from ROS-induced apoptosis through a PI3K/Akt survival pathway.nnnMETHODSnThe preventive effect of clusterin on reactive oxygen species (ROS) production and RPE cell death induced by hydrogen peroxide was determined in ARPE-19 cells. The ability of clusterin to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and DAPI staining. Furthermore, the protective effect of clusterin via the PI3K/Akt pathway was determined by Western blot analysis.nnnRESULTSnClusterin prevented ARPE-19 cells from H(2)O(2)-induced cell death and ROS production. H(2)O(2)-induced oxidative stress increased caspase-3 activity, which was significantly inhibited by clusterin, as determined by the abrogation of apoptotic bodies. Interestingly, clusterin induced Akt phosphorylation in human RPE cells under oxidative stress, which contributed to cell viability in ARPE-19 cells. This cell survival by clusterin was blocked by a PI3K inhibitor.nnnCONCLUSIONSnClusterin may play a protective role in responding to the local redox environment of human RPE cells, which contributes to the cell survival via the PI3K/Akt pathway. Therefore, clusterin could be considered for the preventive approach to AMD.

Protective Effect of Clusterin on Blood–Retinal Barrier Breakdown in Diabetic Retinopathy

Purpose. To investigate whether clusterin attenuates blood-retinal barrier (BRB) breakdown in diabetic retinopathy. Methods. Mice with streptozotocin-induced diabetes and advanced glycation end product-treated human retinal microvascular endothelial cells (HRMECs) were used to determine the effect of clusterin on vascular permeability and tight junction protein expression, through perfusion of retinal vessels with FITC-bovine serum albumin, a [(3)H]sucrose permeability assay, a cell viability assay, Western blot analysis, immunocytochemistry, immunohistochemistry, and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling. Results. Up to 20 mug/mL of clusterin, which is 20 times the effective therapeutic concentration, did not affect the viability of the HRMECs. Moreover, it caused no toxicity in the retina. It effectively inhibited vascular endothelial growth factor-induced hyperpermeability in the HRMECs and the retinas. The antipermeability activity of clusterin was related to the restoration of tight junction proteins. Finally, it was shown to reduce leakage from the vessels in the diabetic retinas and to restore the expression of the tight junction proteins. Conclusions. The data suggest that clusterin, a well-known antipermeability factor naturally secreted by cells, may have therapeutic potential in the treatment of diabetic BRB breakdown.

Anti-angiogenic effect of luteolin on retinal neovascularization via blockade of reactive oxygen species production.

PURPOSEnOxidative stress-induced vascular endothelial growth factor (VEGF) is thought to play a critical role in the pathogenesis of retinopathy of prematurity (ROP). This study was performed to investigate the anti-angiogenic effect of luteolin against reactive oxygen species (ROS)-induced retinal neovascularization.nnnMETHODSnThe toxicity of luteolin was evaluated through modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human retinal microvascular endothelial cells (HRMECs) as well as TUNEL staining in the retina of C57BL/6J mice. After intravitreal injection of luteolin in the mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting. Anti-angiogenic activity of luteolin was evaluated by VEGF-induced migration and tube formation assay. The effect of luteolin on tertiary-butylhydroperoxide (t-BH)-induced ROS production was measured with 27-dichlorofluorescein diacetate. The effect of luteolin on t-BH-induced and hypoxia-induced VEGF transcription and expression were evaluated by RT-PCR and Western blot, respectively.nnnRESULTSnLuteolin never affected the viability of HRMECs up to 10 μM, where luteolin never induced any structural change in all retinal layers. Luteolin inhibited retinal neovascularization in the mouse model of ROP. Moreover, VEGF-induced migration and tube formation were significantly decreased by cotreatment of luteolin. Luteolin attenuated VEGF transcription via blockade of t-BH-induced ROS production. Luteolin suppressed hypoxia-induced VEGF expression via attenuating hypoxia inducible factor 1 α expression.nnnCONCLUSIONSnOur results suggest that luteolin could be a potent anti-angiogenic agent for retinal neovascularization, which is related to anti-oxidative activity to block ROS production and to subsequently suppress VEGF expression and the pro-angiogenic effect of VEGF.

Long-term visual outcomes of penetrating keratoplasty for Peters anomaly

BackgroundTo investigate the long-term results and visual outcomes of penetrating keratoplasty (PKP) in Peters anomaly.MethodsTwenty-three eyes from 22 patients with Peters anomaly who underwent PKP from 1998 to 2008 were reviewed retrospectively. Patients who were followed for more than 3xa0years after the first PKP were included in this study. The systemic and ophthalmic features of the recipients were assessed, and the various prognostic factors for graft survival were evaluated. Disease severity was determined according to other accompanying eye anomalies in mild or severe form. The final visual outcomes were presented with respect to graft clarity.ResultsAmong the 22 patients, 14 patients had unilateral disease, and eight patients had bilateral disease. Associated systemic anomalies were observed in six patients. The mean age at the first PKP was 42.4xa0months. Nineteen eyes (83xa0%) underwent PKP after 12xa0months of age. The graft failure rates at 1xa0year, 3xa0years, 5xa0years, and 10xa0years after PKP were 30xa0%, 39xa0%, 70xa0%, and 77xa0% respectively. Graft rejection within 1xa0month after PKP and severe disease were significant risk factors for graft failure. The mean final VAs in the clear-graft group and the failed-graft group were 1.883 logMAR and 2.767 logMAR (Pu2009<u20090.001).ConclusionThe results of delayed PKP in Peters anomaly were not inferior compared to the results of PKP performed at an earlier period in previous studies. If other congenital ophthalmic anomalies were present or graft rejection occurred within 1xa0month after PKP, the chance of graft failure was significantly increased.

The Clinical Outcomes of Proton Beam Radiation Therapy for Retinoblastomas That Were Resistant to Chemotherapy and Focal Treatment

Purpose To evaluate the clinical results of proton beam radiation therapy (PBRT) for treatment of retinoblastoma. Methods Children with retinoblastoma who were treated with chemotherapy and focal treatment such as brachytherapy and thermotherapy but showed no response or developed recurrences later received PBRT. The PBRT strategy was designed to concentrate the radiation energy to the retinoblastoma and spare the surrounding healthy tissue or organs. Results There were three patients who received PBRT. The first patient received PBRT because of an initial lack of tumor regression with chemotherapy and brachytherapy. This patient showed regression after PBRT. The second patient who developed recurrence of retinoblastoma as diffuse infiltrating subretinal seeding was taken PBRT. After complete regression, there was recurrence of tumor and the eye was enucleated. The third patient had unilateral extensively advanced retinoblastoma. Initial chemotherapy failed and tumor recurred. The tumor responded to PBRT and regressed significantly. However, the eye developed sudden multiple recurrences, so we had to perform enucleation. Conclusions PBRT for retinoblastoma was effective in cases of showing no response to other treatment modalities. However, it should be carefully applied when there was recurrence of diffuse infiltrating subretinal seeding or extensively advanced retinoblastoma initially.

Visual Prognosis of Retinoblastoma in the Posterior Pole Treated with Primary Chemotherapy Plus Local Treatments

Purpose To evaluate the visual outcomes of retinoblastoma in the posterior pole (RBPP) treated with chemotherapy plus local treatments and to address the prognostic factors that influence such outcomes. Methods The medical records of patients with RBPP diagnosed at the Department of Pediatric Ophthalmology, Seoul National University Childrens Hospital between August 1987 and September 2007 were reviewed retrospectively. Only those patients treated via primary chemotherapy plus local treatments were included. The presence of foveal involvement and tumors in the posterior pole before and after treatment, the type of regression pattern and the best corrected visual acuity (BCVA) of each patient were evaluated. Results A total of 13 eyes in 12 patients were included. The mean final BCVA for treated RBPP was 20/210 (range, hand motion to 20/16). However, eight eyes (61.5%) had an acuity of 20/200 or better and seven eyes (53.8%) had an acuity of 20/50 or better. The mean final BCVA was significantly better in cases with negative foveal involvement; however, four eyes (37.5%) with positive foveal involvement had an acuity of 20/200 or better. Tumors area in the posterior pole and the type of regression pattern were not significantly related to final BCVA. Conclusions Over one half of the studied RBPP patients had working vision. Although the eyes had RBPP with positive foveal involvement, about one-third of the patients had working vision. Vision preservation should be considered when deciding on RBPP treatment.

Antitumor activity of arsenic trioxide on retinoblastoma: cell differentiation and apoptosis depending on arsenic trioxide concentration.

PURPOSEnArsenic trioxide (ATO) targets multiple pathways in malignant cells, resulting in the promotion of differentiation or in the induction of apoptosis. The antitumor activity of ATO on retinoblastoma was investigated.nnnMETHODSnHuman retinoblastoma cells were incubated with various ATO concentrations. The antiproliferative effect of ATO was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the effect of ATO on cell-cycle progression was validated by flow cytometry. At a low concentration, the ATO-induced differentiation of retinoblastoma cells was evaluated by neurofilament expression and extracellular signal-regulated kinase (ERK)1/2 activation, which was confirmed by the inhibition of ERK1/2. At a high concentration, ATO-induced H(2)O(2) production was investigated with the cell-permeable fluorescent dye 27-dichlorofluorescein-diacetate, and the relationship of ATO-induced H(2)O(2) production with caspase-3-dependent apoptosis was validated by Western blot and 46-diamidino-2-phenolindole staining, which was confirmed by reactive oxygen species (ROS) inhibition. The effect of ATO on tumor formation was assessed with an orthotopic animal model of retinoblastoma.nnnRESULTSnThe antitumor activity of ATO in retinoblastoma was related to two main mechanisms, differentiation and apoptosis, which were determined by the level of ATO. At a low dose (<or= 1 microM), ATO induced the differentiation of retinoblastoma cells through ERK1/2 activation, whereas ROS generation by a high dose (>or= 2 microM) of ATO induced apoptosis in retinoblastoma cells. Moreover, ATO at low and high doses effectively inhibited tumor formation.nnnCONCLUSIONSnThese results suggest that ATO can be used as an effective alternative therapeutic for the treatment of retinoblastoma.