Induction of a proliferative response in the zebrafish retina by injection of extracellular vesicles.

Ongoing research using cell transplantation and viral-mediated gene therapy has been making progress to restore vision by retinal repair, but targeted delivery and complete cellular integration remain challenging. An alternative approach is to induce endogenous Müller glia (MG) to regenerate lost neurons and photoreceptors, as occurs spontaneously in teleost fish and amphibians. Extracellular vesicles (EVs) can transfer protein and RNA cargo between cells serving as a novel means of cell-cell communication. We conducted an in vivo screen in zebrafish to identify sources of EVs that could induce MG to dedifferentiate and generate proliferating progenitor cells after intravitreal injection into otherwise undamaged zebrafish eyes. Small EVs (sEVs) from C6 glioma cells were the most consistent at inducing MG-derived proliferating cells. Ascl1a expression increased after intravitreal injection of C6 sEVs and knockdown of ascl1a inhibited the induction of proliferation. Proteomic and RNAseq analyses of EV cargo content were performed to begin to identify key factors that might target EVs to MG and initiate retina regeneration.

Characterization of Vitreous and Aqueous Proteome in Humans With Proliferative Diabetic Retinopathy and Its Clinical Correlation.

AIMS: Proliferative diabetic retinopathy (PDR) is associated with microvascular complications that cause biochemical changes in the human retina and alter the proteome of vitreous humor and aqueous humor (AH). METHODS: Human vitreous humor and AH of PDR subjects were collected. Subjects who had surgery for epiretinal membrane or macular hole served as controls. Protein profiles were obtained and analyzed after running the samples on a liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: In vitreous humor, 16 unique proteins were noted in PDR patients, but not in controls. Those were associated mainly with coagulation, complement, and kallikrein-kinin systems. Under coagulation, fibrinogen and prothrombin proteins were more evident and may emphasize the importance of angiogenesis in the development of PDR. Vitreous proteins showed replicative presence in AH too. As for AH samples, we detected 10 proteins found in PDR patients, which were related to transport, coagulation, and inflammatory responses. CONCLUSIONS: We found 57 proteins in human vitreous and 39 proteins in AH. Identification of these proteins that are involved in various pathways will be helpful to understand diabetic retinopathy pathogenesis and to develop proteome as a biomarker for PDR.

Sirtuins Expression and Their Role in Retinal Diseases.

Sirtuins have received considerable attention since the discovery that silent information regulator 2 (Sir2) extends the lifespan of yeast. Sir2, a nicotinamide adenine dinucleotide- (NAD-) dependent histone deacetylase, serves as both a transcriptional effector and energy sensor. Oxidative stress and apoptosis are implicated in the pathogenesis of neurodegenerative eye diseases. Sirtuins confer protection against oxidative stress and retinal degeneration. In mammals, the sirtuin (SIRT) family consists of seven proteins (SIRT1-SIRT7). These vary in tissue specificity, subcellular localization, and enzymatic activity and targets. In this review, we present the current knowledge of the sirtuin family and discuss their structure, cellular location, and biological function with a primary focus on their role in different neuroophthalmic diseases including glaucoma, optic neuritis, and age-related macular degeneration. The potential role of certain therapeutic targets is also described.

Osmolarity and spectrophotometric property of brilliant blue green define the degree of toxicity on retinal pigment epithelial cells exposed to surgical endoilluminator.

OBJECTIVE: To evaluate the effect of varying concentrations of brilliant blue green (BBG) and their different biochemical characteristics on retinal pigment epithelial (RPE) cells under xenon light source illumination at varying distances to identify safe parameters for intraoperative use. METHODS: Human retinal RPE cells (ARPE-19) were exposed to two concentrations (0.25 and 0.50 mg/mL) of BBG and illuminated with a xenon surgical illuminator at varying distances (10 and 25 mm), intensity levels, and time intervals (1, 5, and 15 minutes). Additionally, the effect of osmolarity was examined by diluting BBG in different concentrations of glucose. Cytotoxicity of BBG and osmolarity effects on cell viability were evaluated using a WST-1 assay. Light absorption and emission characteristic of BBG in different solvents were measured using a plate reader at different wavelengths. Lastly, the activity of caspase-3 was also studied. RESULTS: Cell viability of ARPE-19 cells was 77.4%±12.7%, 78.7%±17.0%, and 65.0%±19.7% at 1, 5, and 15 minutes to exposure of high illumination xenon light at 10 mm (P<0.05) compared to controls. At both distances of illumination (10 and 25 mm), similar cell viabilities were seen between 1 and 5 minutes of exposure. However, there was a decline in viability when the illumination was carried out to 15 minutes in all groups (P<0.05). There was no significant reduction in cell viability in presence or absence of xenon light in different osmolar solutions concentrations of glucose (P>0.05). Maximal light absorption of BBG was noted between 540 and 680 nm. Activated caspase-3 level was not significant in both the concentrations of BBG (P>0.05). CONCLUSION: Our findings suggest that BBG at 0.25 mg/mL during vitreoretinal surgery is safe and not toxic to RPE cells up to 5 minutes under focal high illumination (10 mm) and up to 15 minutes under medium diffuse illumination (25 mm). BBG was safe to be mixed with isotonic glucose solution at the concentration range of 2.5%-10%, regardless of the illumination status.

Effect of distance and duration of illumination on retinal ganglion cells exposed to varying concentrations of brilliant blue green.

BACKGROUND: The objective of the study was to determine the safety parameters of using brilliant blue green (BBG) for chromovitrectomy by assessing the cytotoxicity of BBG on cultured retinal ganglion cells (RGCs) exposed to illumination. METHODS: RGCs were exposed to two concentrations of BBG (0.25 and 0.5 mg/mL) under metal halide illumination at varying distances (1 and 2.5 cm), intensities (990 and 2,000 Fc), and durations (1, 5 and 15 minutes). Cell viability was assessed using the WST-1 and CellTiter 96(®) AQueous One solution cell proliferation assays. RESULTS: Using the WST-1 assay, with high-intensity illumination, viability of RGCs ranged from 97.5±16.4% of controls with minimum BBG and light exposure (0.25 mg/mL BBG and illuminated for 1 minute at 2.5 cm distance) to 53.1±11.3% of controls with maximum BBG and light exposure (0.50 mg/mL and illuminated for 15 minutes at 1 cm distance; P < 0.01). With medium-intensity illumination, RGCs showed better viability, ranging from 95.1±7.2% of controls with minimum BBG and light exposure to 72.3±12.8% of controls with maximum BBG and light exposure. CellTiter 96(®) AQueous One assay showed similar results. CONCLUSION: RGCs seem to safely tolerate up to 5 minutes of exposure to 0.5 mg/mL BBG under diffuse medium-intensity illumination (990 Fc).

Deprivation of bFGF Promotes Spontaneous Differentiation of Human Embryonic Stem Cells into Retinal Pigment Epithelial Cells.

BACKGROUND: The retinal pigment epithelium (RPE), an important tissue monolayer of retina, sustains visual function and retinal homeostasis. In disease conditions such as Retinitis Pigmentosa (RP) and Age related Macular degeneration (AMD), the integrity and functional capacity of RPE monolayer is compromised. Human embryonic stem cells derived RPE (hESC-RPE) is ideal for cell based therapy because of their ability to morphologically and functionally mimic native fetal and adult RPE. However protocols for optimum culture of hESC-RPE are not well established. AIM: To describe a simplified protocol for differentiating human embryonic stem cells (hESC) into retinal pigment epithelial cells. METHODS: hESC (WA09-DL-11) cell lines were grown with standard stem cell culture protocol. After cell colonies were established, basic fibroblast growth factor (bFGF) was deprived (day 0). hESC colonies expressing pigmentation were characterized for expression of RPE65 and Zonular occludens--1 (ZO-1) with immunocytochemistry on days 0, 36, 42, 56 and 70 and western blot analysis on days 0, 40, 48, 53 and 63. In addition, morphological assessment was conducted on transformed cells longitudinally. RESULTS: Pigmented cells were noted 36 days after deprivation of bFGF from growth media. Immunofluorescence demonstrated progressive up regulation of RPE specific proteins (ZO-1 & RPE 65). Immunofluorescence of ZO-1 (in pixels) was (3.08 ± 0.31) on day 42, (5.33 ± 0.89, p = 0.0001) on day 56 and (4.87 ± 0.57, p = 0.0011) on day 70. Similarly expression of RPE 65 was (2.44 ± 0.31) on day 42, which continued to increase (4.23 ± 0.60, p = 0.0011) on day 56 and (5.59 ± 0.36, p < 0.0001) on day 70. Protein expression patterns using western blot confirmed the trends seen in immunofluorescence. Western blot analysis of ZO-1 expression (in optical density unit) was 272.57 ± 31.75 on day 40, 4212.20 ± 911.31 (p = 0.0004) on day 48, 5182.43 ± 1230.38 (p = 0.030) on day 53 and 5848.76 ± 241.04 (p < 0.0001) on day 63. Protein expression of RPE 65 was 1607.64 ± 247.76 on day 40, 2448.07 ± 152.66 on day 48 and (2341.15 ± 52.84) on day 63. hESC-RPE cells displayed a series of specific morphological changes (cytoplasmic, nuclear pigmentary and cell shape) over the course of time frame. By day 70, cells with hexagonal pattern, dark dense nucleus and uniform cytoplasm were noted in densely pigmented RPE colonies. CONCLUSION: bFGF deprivation leads to successful differentiation of hESC into RPE cells. Longitudinal transformative changes were confirmed with measurement of ZO-1 and RPE 65, specific markers of RPE.

Retinal thickness measurement obtained with spectral domain optical coherence tomography assisted optical biopsy accurately correlates with ex vivo histology.

BACKGROUND: This study determines 'correlation constants' between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice. METHODS: Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as 'correlation constants'. RESULTS: Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (± 1.09) and 219.58 µm (± 2.67), respectively. The mean 'correlation constant' for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations. CONCLUSION: This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

Growth factors/chemokines in diabetic vitreous and aqueous alter the function of bone marrow-derived progenitor (CD34⁺) cells in humans.

Ocular ischemic microenvironment plays a critical role in the progression of diabetic retinopathy (DR). In this study, we investigated the effect of vitreous and aqueous obtained from proliferative DR patients on the function of CD34⁺ cells derived from healthy humans. Human CD34⁺ cells were incubated with vitreous or aqueous of subjects with PDR. After incubation, cell migration of CD34⁺ was evaluated with CXCL12. Intracellular levels of nitric oxide (NO) were measured with DAF-FM. Tube formation assay was used to evaluate the effect of treated CD34⁺ cells on in vitro angiogenesis. Angiogenic protein array and mass spectrometry (MS) were performed to ascertain the factors secreted by healthy nondiabetic CD34⁺ cells exposed to diabetic vitreous or aqueous. PDR vitreous/aqueous reduced migration of CD34⁺ cells (672.45 ± 42.1/736.75 ± 101.7 AFU; P < 0.01) and attenuated intracellular NO levels (182 ± 1.4/184.5 ± 6.3 AFU, P = 0.002). Pretreatment with PDR vitreous suppressed tube formation of human retinal endothelial cells (64 ± 1.6 vs. 80 ± 2.5). CD34⁺ exposed to PDR vitreous resulted in the increased expression of CXCL4 and serpin F1, whereas CD34⁺ exposed to PDR aqueous showed increased expression of CXCL4, serpin F1, and endothelin-1 (ET-1). MS analysis of CD34⁺ (exposed to PDR vitreous) expressed J56 gene segment, isoform 2 of SPARC-related modular calcium-binding protein 2, isoform 1 of uncharacterized protein c1 orf167, integrin α-M, and 40s ribosomal protein s21. Exposure of healthy nondiabetic CD34⁺ cells to PDR vitreous and aqueous resulted in decreased migration, reduced generation of NO, and altered paracrine secretory function. Our results suggest that the contribution of CD34⁺ cells to the aberrant neovascularization observed in PDR is driven more by the proangiogenic effects of the retinal cells rather than the influence of the vitreous.

Aqueous interleukin-6 levels are superior to vascular endothelial growth factor in predicting therapeutic response to bevacizumab in age-related macular degeneration.

Objective. To prospectively evaluate the effect of intravitreal bevacizumab on aqueous levels of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in patients with exudative age-related macular degeneration (AMD) and correlate clinical outcomes with cytokine levels. Methods. 30 eyes of 30 patients with exudative AMD underwent intravitreal injection of bevacizumab three times at monthly intervals. The aqueous samples prior to the 1st injection (baseline) and 3rd injection were analyzed for VEGF and IL-6 levels. Subjects were subgrouped based upon change in the central subfield (CSF) macular thickness on SD-OCT at 8 weeks. Group 1 included patients (n = 14) with a decrease in CSF thickness greater than 10% from the baseline (improved group). Group 2 included patients (n = 16) who had a decrease in CSF thickness 10% or less (treatment-resistant). Results. In subgroup analysis, in both groups 1 and 2 patients, compared to aqueous VEGF, aqueous IL-6 levels showed a better correlation with CSF thickness on SD-OCT (r = 0.72 and 0.71, resp.). Conclusions. Aqueous IL-6 may be an important marker of treatment response or resistance in wet macular degeneration. Future therapeutic strategies may include targeted treatment against both VEGF and IL-6, in patients who do not respond to anti-VEGF treatment alone.

Assessment of the effect of distance and duration of illumination on retinal pigment epithelial cells exposed to varying doses of Brilliant Blue Green.

PURPOSE: To assess the cytotoxicity of varying concentrations of Brilliant Blue Green (BBG) on human retinal pigment epithelial (HRPE) cells exposed to metal halide surgical endoilluminator (SE) at varying distances of illumination. METHODS: HRPE (ARPE-19) were exposed to 2 concentrations (0.25 and 0.5 mg/mL) of BBG and illuminated with SE for 1, 5, and 15 min. Illumination (measured with light meter) was positioned at varying distances (1 and 2.5 cm) to mimic surgical distance of illumination. Cell viability (WST-1 assay) as well as structural changes in cells was quantified. RESULTS: At 1 cm distance of illumination, 0.25 mg/mL BBG decreased viability of HRPE to 89.6%±4.3%, 83.9%±10.9%, and 38.9%±5.1% of controls after 1, 5, and 15 min of exposure, respectively. Similarly, 0.5 mg/mL BBG at 1 cm distance of illumination reduced the viability of HRPE to 93.7%±2.8%, 59.6%±16%, and 34.7%±3.5% of controls. At the distance of 2.5 cm, HRPE showed improved viability; cells exposed to 0.25 mg/mL BBG maintained 98.85%±3.3%, 95.31%±7.12%, and 62.07%±3.0% of viability compared with controls after 1, 5, and 15 min of exposure. Morphometric evaluation of RPE cells showed increased width (swelling) of HRPE. CONCLUSION: BBG at its clinically used concentration (0.25 mg/mL) during vitreoretinal surgery is safe and not toxic to HRPE for up to 5 min under focal illumination (1 cm) and up to 15 min under diffuse illumination (2.5 cm) from the commonly used SE. RESULTS of our study are useful in establishing safety parameters for the use of BBG dye in vitreoretinal surgery.

Lutein protects retinal pigment epithelium from cytotoxic oxidative stress.

CONTEXT: Lutein (LUT) and zeaxanthin (ZEA) are currently under investigation in clinical trials as prophylactic nutritional agents for age-related macular degeneration (AMD). However, dose used in these trials is empirical and not been investigated in in vitro studies. OBJECTIVE: In this study, we investigated the dose-response effect of LUT and ZEA in protecting retinal pigment epithelium (RPE) from oxidative stress, a common underlying pathology in AMD. METHODS: Three thousand cultured human retinal pigment epithelial cells (ARPE-19) were plated in 72-well plate and after 24 h were exposed to increasing concentrations of hydrogen peroxide (H2O2). ARPE-19 cells were exposed to four different concentrations of LUT (0.5, 1, 2 and 4 µg/mL) and ZEA (0.1, 0.2, 0.4 and 0.8 µg/mL). After 24 h incubation, cells were subjected to oxidative stress induced with H2O2. Cultures containing saline solution and dichloromethane served as controls. Cell viability was assessed using the WST-1 assay. Pathophysiological pathways were evaluated by measuring caspase-3 levels as an indicator of apoptosis induction. Reactive oxygen species (ROS) levels were measured using dihydrorhodamine-123. RESULTS: Cell viability as a percentage of control was 81.3%, 81.1%, and 88.8% at 0.5, 1, and 2 µg/ml, respectively of LUT (p < 0.001). The maximum cytoprotective effect was seen with LUT at 2 µg/mL. ZEA did not show any cytoprotective effect at all concentrations used in the study. Caspase-3 showed a corresponding decrease in levels with LUT (1 and 2 µg/ml). Significant decrease in ROS levels were measured only with LUT at 4 µg/ml (p = 0.02). DISCUSSION AND CONCLUSIONS: Results from our study provide in vitro data to support the epidemiologic studies, which are currently underway to provide evidence that lutein may act as cofactor that modulates processes implicated in AMD pathogenesis.

Resveratrol inhibits proliferation of hypoxic choroidal vascular endothelial cells.

PURPOSE: Resveratrol, a polyphenolic phytoalexin present in red wine, has a protective role against tumor-induced angiogenesis. Exudative age-related macular degeneration is characterized by hypoxia-induced choroidal vascular endothelial cell (CVEC) proliferation. In this study, we evaluated the effect of resveratrol on hypoxic CVECs and the underlying signaling pathways involved. METHODS: CVECs (RF/6A) after induction of hypoxia with cobalt chloride (CoCl2, 200 µM) were exposed to increasing doses of resveratrol (2, 4, 6, 8, 10, and 12 µg/ml). Cell viability was measured with 4-[3-(4Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) colorimetric assay. The effect of resveratrol on hypoxia-induced vascular endothelial growth factor (VEGF) release was analyzed with enzyme-linked immunosorbent assay. The mechanistic pathway was further evaluated by analyzing phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) using immunoblot and cleaved caspase-3 with In-Cell enzyme-linked immunosorbent assay. RESULTS: Resveratrol inhibited hypoxic CVEC proliferation. Hypoxia-induced VEGF release (30.9±2.6 pg/ml) was inhibited in a dose-dependent fashion by 2, 4, 6, 8, 10, and 12 µg/ml resveratrol to 12.4±2.1, 11.0±1.9, 10.3±3.0, 7.5±1.9, 5.5±2.0, and 5.5±2.3 pg/ml, respectively. SAPK/JNK increased by 1.8-fold and 3.9-fold after treatment with 4 and 12 µg/ml resveratrol, respectively. Significant increase in caspase-3 levels was observed with 12 µg/ml resveratrol. CONCLUSIONS: Our study demonstrates that resveratrol suppresses hypoxic CVEC proliferation through activation of the SAPK/JNK pathway. Resveratrol, a nutritional supplement and inhibitor of CVECs, may be a useful adjunct to current anti-VEGF therapy in wet age-related macular degeneration.

Retinal Thickness Normative Data in Wild-Type Mice Using Customized Miniature SD-OCT.

OBJECTIVE: To report normative data for retinal thickness in wild-type C57BL/6 mouse utilizing a miniature SD-OCT system. METHODS: THIRTY ADULT MICE (RANGE: 3-5 months) were anesthetized and secured into the Bioptigen Spectral Domain Ophthalmic Imaging System. Right eye SD-OCT images were standardized by centralizing the optic nerve head (ONH) prior to image acquisition. Global and quadrant total retinal thickness (TRT) values were measured from retinal nerve fiber layer to retinal pigment epithelial layer. Posterior segment analyses also included the outer retinal layer (ORL) and inner retinal layer (IRL). Further sublayer analyses of four layers from the ORL and three layers comprising the IRL were also performed. RESULTS: The overall mean±SD global TRT in a C57BL/6 mouse model was 204.41±5.19 µm. Quadrant mean TRT values were 204.85±5.81 µm inferiorly, 204.97±6.71 µm nasally, 205.08±5.44 µm temporally, and 202.74±4.85 µm superiorly. Mean±SD thickness for ORL, and IRL were 126.37±10.01 µm, and 107.03±10.98 µm respectively. The mean±SD estimates for the four layers of the ORL were 18.23±2.73 µm, 26.04±4.21 µm, 63.8±6.23 µm, and 19.22±4.34 µm. Mean±SD values for the three IRL sublayers were 27.82±4.04 µm, 59.62±6.66 µm and 19.12±3.71 µm. CONCLUSION: This study established normative values for the total retinal thickness and sublayer thickness for the wild-type C57BL/6 mice. Moreover, it provides a standard of retinal morphology, in a commonly used animal model, for evaluating therapeutic interventions and retinal disease pathophysiology.

Differential sensitivity of choroidal endothelial, retinal ganglion, and retinal pigment epithelial cells in vitro to proton radiation.

PURPOSE: To evaluate the differential sensitivity of choroidal endothelial, retinal pigment epithelial, and retinal ganglion cells to escalating doses of proton beam radiation and to establish a safe dose range for the management of choroidal neovascularization associated with age-related macular degeneration (AMD). DESIGN: Laboratory investigation. METHODS: Proliferating simian choroidal endothelial cells (RF/6A), differentiated rat retinal ganglion cells (RGC-5), and serum-starved human retinal pigment epithelial cells (ARPE-19) were exposed to 2, 4, 8, and 12 cobalt gray equivalent of proton beam radiation and cell viability was quantified on day 9. Reactive oxygen species levels were analyzed. RESULTS: Significant decline of choroidal endothelial cell viability was noted as dose escalated from 4 to 8 cobalt gray equivalent with maximum effect observed at 12 cobalt gray equivalent. RGC-5 and ARPE-19 cell count decreased to 95% and 62.7% at 8 cobalt gray equivalent, respectively. Sub-analysis between 4 and 8 cobalt gray equivalent radiation revealed significant decrease in choroidal endothelial cell viability (43.1% at 7 cobalt gray equivalent and 32.3% at 8 cobalt gray equivalent of radiation). Correspondingly, RGC-5 and ARPE-19 cells did not show decrease in cell count or viability. Reactive oxygen species levels significantly increased in radiation-treated choroidal endothelial cells (8.3%-11.9%). CONCLUSIONS: At 6-8 cobalt gray equivalent proton beam radiation, retinal ganglion and retinal pigment epithelial cells are preserved while choroidal endothelial cells are completely inhibited. This dosage offers optimum therapeutic safety window for treatment using proton beam radiation for exudative AMD.

Modified protocol for in vivo imaging of wild-type mouse retina with customized miniature spectral domain optical coherence tomography (SD-OCT) device.

This protocol outlines and evaluates a modified scanning procedure for a customized spectral domain optical coherence tomography (SD-OCT) imaging apparatus within the wild-type C57Bl/6 mouse posterior segment. This modified protocol allows for the capture of a 50 degree field of view spanning 3 mm by 3 mm perimeter with the optic disc as the central point. By utilizing this scanning protocol a more reliable measurement of retinal thickness can be achieved outside the fluctuating region of the optic disc. This protocol, when applied to this high resolution device, enables non-invasive in vivo histological imaging and biometric assessment of the various layers of the rodent posterior segment within a 20 - 30 min procedural time-frame. This protocol could establish a standardized method for evaluating morphological changes, with this commercial SDOCT device, when assessing longitudinal disease pathophysiology and treatment response in mouse models for future vision science research.

Evaluation of sirtuin role in neuroprotection of retinal ganglion cells in hypoxia.

PURPOSE. Hypoxia-induced apoptosis is responsible for reduced retinal ganglion cell (RGC) viability in a variety of chronic ocular disorders. Sirtuin 1 (SIRT1) plays an important role in preserving cell viability during hypoxia. We investigated the role of SIRT1 in sustaining RGC viability in an in vitro model of hypoxia. METHODS. Staurosphorine-differentiated RGCs (RGC-5) received varying hypoxic concentrations (100-500 µM) of cobalt chloride (CoCl2) for 24 hours. Hypoxia-induced cell viability was assessed by WST-1 assay. The role of SIRT1 in promoting viability was determined indirectly via sirtinol (SIRT1 inhibitor). Hypoxia-induced apoptosis was evaluated by measuring stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) and caspase 3 activity. Vascular endothelial growth factor (VEGF) was measured to ascertain the influence of SIRT1. RESULTS. CoCl2 concentrations greater than 100 µM resulted in significantly reduced RGC viability (P=0.01). CoCl2 treatment increased SIRT1 levels significantly (P<0.01): 100 (6.5-fold), 200 (6-fold), 300 (3.5-fold), and 400 µM (4.5-fold). Phosphorylated SAPK/JNK increased 36-fold (200 µM CoCl2 concentration), then plateaued at the 300- (25-fold) and 400-µM (27.8-fold) CoCl2 concentrations (P<0.01). CoCl2 and sirtinol treatment increased Caspase 3 activity (P<0.05). VEGF release was significantly higher than control at the 100-µM CoCl2 concentrations (P<0.01). Sirtinol reduced RGC viability, SIRT1 levels, and VEGF release (P<0.01) while having greater effect on SAPK/JNK phosphorylation. CONCLUSIONS. SIRT1 significantly influences RGC viability. Sirtinol's effect reflects the interaction SIRT1 has with apoptotic signaling proteins. This investigation demonstrated SIRT1 importance in forestalling the effects of hypoxia-induced apoptosis.

Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor-2α.

PURPOSE: Hypoxia is a critical pathological factor in a variety of retinal diseases, including age-related macular degeneration. It upregulates angiogenic growth factors and promotes neovascularization. Hypoxia changes the cellular redox state and activates class III histone deacetylase sirtuin1 (SIRT1). Activated SIRT1 signals hypoxia inducible factor (HIF)-2α, which transactivates vascular endothelial growth factor (VEGF) and erythropoietin. In this study, we investigated the role of hypoxia induced SIRT1 in choroidal neovascularization in relation to age-related macular degeneration. METHODS: Choroidal endothelial cells (RF/6A) were maintained in a semiconfluent state and hypoxia was induced by exposing the cells to cobalt chloride for 24 h. Induction of hypoxia was confirmed by flow cytometric analysis and the levels of SIRT1 were noted in a hypoxic condition as well in the cells after blocking SIRT1 activity using sirtinol. The role of SIRT1 in the activation of HIF-2α and nuclear factor-κB (RelA/p65) during hypoxia in the presence or absence of SIRT1 was assessed using immunoblot analysis. VEGF levels were quantified using enzyme-linked immunosorbent assay. RESULTS: Hypoxic induction was confirmed using flow cytometric analysis, which showed cell cycle arrest starting at a 200 µM concentration of cobalt chloride. Hypoxic treatment (200 µM concentration of cobalt chloride) increased SIRT1 levels to 7.8%, which reduced to control level after its activity was inhibited (p<0.05). Activated SIRT1 mediates HIF-2α and nuclear factor-κB (RelA/p65) expression to 4.5 fold and fivefold, respectively, compared to control, and the levels were suppressed following sirtinol treatment (4.1% and 39.3% respectively; p=0.01). Hypoxic treatment increased VEGF levels by 94.9±19.6 pg/ml compared to control levels (25.58±3.58 pg/ml). These levels decreased to 10.29±0.2 pg/ml after blocking SIRT1 activity using sirtinol, compared to control (p<0.01). CONCLUSIONS: Our study results demonstrate that hypoxia mimetic cobalt chloride induces SIRT1 and augments HIF-2α, which activates and releases VEGF.

Evaluation of proton beam radiation sensitivity of proliferating choroidal endothelial and retinal ganglion cells with clonogenic assay.

CONTEXT: Proton beam therapy offers the advantage of precise delivery with limited damage to the healthy tissue and is being tested in the management of exudative age-related macular degeneration (AMD). However, the dosages tested are empirical and not based on preclinical studies. OBJECTIVE: In this study we evaluated the effects of varying doses of proton beam radiation on choroidal endothelial cells (CECs) and retinal ganglion cells (RGCs) using clonogenic assay to determine differential sensitivity. MATERIALS AND METHODS: Each cell type has different efficiency to replicate (plating efficiency (PE)). PE of CEC (RF/6A) and RGC (RGC-5) grown in culture flasks was determined by plating 250 cells each (without any treatment) and counting the number of colonies after 13 days. Radiation induced sensitivity was determined by exposing the semi-confluent RF/6A and RGC-5 cells to proton beam at the doses of 0 (control), 2, 4, 8 and 12 cobalt gray equivalent (CGE). The ability of the cells to repair and replicate to form colonies were analyzed 13 days after radiation with crystal violet stain and the survival ratio was calculated. The significance of survival was analyzed using ANOVA (Graphpad Instat.3). RESULTS: The PE of CEC and RGC was 12.96 ± 0.29% and 40.7 ± 1.48%, respectively. A survival ratio of CEC at 2, 4, 8 and 12 CGE proton radiation was 66.0 ± 8.6%, 44.3 ± 6.5%, 7.6 ± 0.3% and 1.14 ± 0.06% on exposure to 2, 4, 8 and 12 CGE proton radiation, respectively, p < 0.01). Survival ratio of RGC was 71.1 ± 22.4% (p = 0.05), 40.2 ± 7.9%, 8.89 ± 2.6% and 0.78 ± 0.31% at 2, 4, 8 and 12 CGE dosages (p < 0.001). DISCUSSION: CEC showed dose-dependent decrease in survival rate with values attaining significance at all radiation dosages. In contrast, RGC was comparatively radio resistant and were able to replicate at lower doses and sensitive at higher doses after proton beam radiation. CONCLUSION: Since CECs proliferate during neovascularization, this clonogenic assay is a useful assay to assess the sensitivity of CEC to radiation. This study identified that CEC were more sensitive to proton beam radiation than RGC at all doses. This may provide a therapeutic window for administration of proton beam radiation in the management of AMD.

A review: role of ultraviolet radiation in age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the western world. The retina is highly susceptible to photochemical damage from continuous exposure of light and oxygen. The cornea and the lens block a major portion of the ultraviolet (UV) radiation from reaching the retina (<295 nm). The relationship between UV light exposure and AMD is unclear, although short wavelength radiation and the blue light induce significant oxidative stress to the retinal pigment epithelium. Epidemiologic evidence indicates a trend toward association between severity of light exposure and AMD. In this review, we discuss type 1 and type 2 photochemical damage that occurs in response to UV exposure. We examine the impact of different doses of exposure to UV radiation and the subsequent production of oxidative stress in AMD. Local and systemic protective mechanisms of the retina including antioxidant enzymes and macular pigments are reviewed. This article provides a review of possible cellular and molecular effects of UV radiation exposure in AMD and potential therapies that may prevent blindness resulting from this disease.

Tumor necrosis factor-alpha (TNF-α) levels in aqueous humor of primary open angle glaucoma.

PURPOSE: Tumor necrosis factor alpha (TNF-α), a macrophage/monocyte derived pluripotent cytokine is associated with tissue ischemia, neuronal damage and remodeling. The physiological level of TNF-α in aqueous humor of normal and glaucomatous eyes is unknown. In this study, we evaluated the TNF-α levels in aqueous in patients with primary open angle glaucoma (POAG) and compared them to controls. METHODS: 50-100 µL of undiluted aqueous humor samples were obtained from eyes of 32 POAG patients who underwent cataract extraction, trabeculectomy or aqueous shunt implantation. Controls were obtained from 32 normal subjects who underwent routine cataract surgery. TNF-α levels were quantified using singleplex bead immunoassay analysis. RESULTS: The average TNF-α level in POAG samples was 2.72 ± 1.5 pg/mL (mean ± SD). The average TNF-α level in normal samples was 1.59 ± 0.46 pg/mL (mean ± SD). Significant increase of TNF-α levels in POAG samples was noted in comparison to normal samples (P < 0.001). CONCLUSION: TNF-α levels are elevated in aqueous in patients with POAG compared to normal subjects based on highly sensitive Luminex(®) bead immunoassay and may be a reliable biomarker in the progression of glaucoma.