18 - Ocular Photobiology, 1
Florida 3 08:00 - 10:00
|Chair(s): Beth Gaillard, Tad Sarna|
08:00 The accumulation of lipofuscin and A2E depend on retinal anatomy Z Ablonczy*, Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA
; J Herrmann, Allergan, Inc., Irivine, CA, USA; P Pallitto, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA; RR Drake, Department of Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA; Y Koutalos, Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA; RK Crouch, Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA; J Donello, Allergan, Inc., Irivine, CA, USA
Abstract: Both lipofuscin and its major component, A2E, has been implicated in retinal pigment epithelium (RPE) phototoxicity in vitro. In mice, lipofuscin and A2E exhibit a high spatial correlation increasing with the amount of A2E; however in humans, they do not co-localize. Therefore, we set out to determine the relationship of lipofuscin and A2E distributions in species with distinct retinal anatomies. RPEs from rabbits and macaques were mounted flat onto conductive slides and autofluorescence (AF) was imaged with a Maestro 2 hyperspectral imaging system. After coating with matrix (sinnapinic acid in 80% ethanol), the tissues were analyzed in a Bruker Autoflex II TOF-TOF mass spectrometer at a 25-250 μm spatial raster in the range of m/z 250-2500. The comparison of AF and mass spectrometric images revealed remarkable differences in lipofuscin, A2E, and lipid distributions between the species. Rabbits have a merangiotic retina. In rabbits, lipofuscin accumulated most in the middle of the visual streak, while A2E was highest in the far periphery and decreased toward the visual streak in a linear manner. Macaques have a fovea. In macaques, lipofuscin was markedly highest in the perimacula, while A2E was most abundant in the far periphery and decreased toward the perimacular area in a concentric fashion. In both species, lipofuscin broadly correlated with literature reports on vascular and photoreceptor density and A2E exhibited a distinctly inverse relationship. Taken together with our previous data on mice and humans, these results support the idea that lipofuscin accumulates in the RPE adjacent to intense retina metabolism while elevated levels of A2E represent retinal areas with a reduced metabolic demand. However, it is still unclear whether bisretinoids accumulate in the RPE in consequence of higher rates of formation or lower rates of breakdown in the periphery.
08:30 The effect of iron enrichment on expression of heme oxygenase-1 and ferritin in retinal pigment epithelium cells containing phagocytized melanosomes AK Pilat*, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
; AC Zadlo, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; TJ Sarna, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Abstract: ARPE-19 cells containing phagocytized photobleached melanosomes or untreated melanosomes were previously shown to exhibit differential ability to bind metal ions by the containing, which could affect the cells susceptibility to oxidative stress. Here we asked whether the protective mechanism involves differential expression of heme oxygenase-1 (HO-1) and ferritin. ARPE-19 cells on day 7-post plating were loaded by phagocytosis with untreated or photobleached melanosomes isolated from bovine RPE or black latex beads used as control particles. Heme oxgenase-1 and ferritin were quantified by western blot analysis after selected time post-phagocytosis. The stress was confirmed as sub-lethal by cell survival analysis using real-time quantification of propidium iodide fluorescence. Particle phagocytosis induced transient increased in HO-1 protein level and the increase was greater for melanosomes than for black latex beads. By the day 7 post-phagocytosis, HO-1 level were equivalent in cells containing melanosomes versus black latex beads. Iron enrichment increased HO-1 and ferritin in a dose-dependent manner. We did not observe any cytotoxic effect of iron enrichment in cells containing phagocytized untreated or photobleached melanosomes. Phagocytosis of photobleached melanosomes induced an increase in HO-1 and GPx protein level. The results indicate possible role for HO-1 and ferritin in RPE iron homeostasis. Our observation raises the possibility that cells containing pigment granules may differ in expression levels of other iron sensitive proteins aside from ferritin. Supported by grant for young researchers (the department statutory activity) K/DSC/003274 (AP).
09:00 The Effect of In Vitro Photoaging of Bovine RPE Melanosomes on Morphology, Reactivity and Photoprotective Efficiency of the Partially Photobleached Pigment Granules A Zadlo, Jagiellonian University
; G Szewczyk, Jagiellonian University; M Sarna, Jagiellonian University; A Pilat, Jagiellonian University; TJ Sarna*, Jagiellonian University
Abstract: Melanosomes in the human retinal pigment epithelium (RPE) undergo distinct age-related changes that could affect their biological functions. To analyze physicochemical nature of such changes, purified bovine RPE melanosomes were subjected to irradiation with intense visible light " an in vitro model for melanin photoaging. Melanin content, size and morphology of untreated and partially photobleached melanosomes were examined by electron paramagnetic resonance (EPR) spectroscopy and atomic force microscopy (AFM). The efficiency of control and photobleached melanosomes to inhibit photosensitized oxidation of lipids mediated by methylene blue (MB) was compared. The topography of melanin functional groups and their reactivity in the studied pigment granules were determined by monitoring their interaction with the nitroxide TEMPO choline, dysprosium (III) ions, and singlet oxygen, employed as molecular probes. Although the results show that the more extensively photobleached melanosomes, compared to untreated melanosomes, inhibited the photosensitzed oxidation of lipids less efficiently, weakly photobleached melanosomes were actually a better protective agent. The latter effect could be due to an increased exposure of melanin functional groups, responsible for the interaction with the tested reagents, while the reduced efficiency of more extensively photobleached melanosomes to inhibit photosensitized oxidation of lipids could be explained by partial degradation of the melanin active sites. AFM analysis of control and photobleached melanosomes confirmed this by showing that weakly photobleached melanosomes were mostly stripped of outer layer structures and exhibited exposed melanin nanoaggregates. An increased exposure of melanin functional groups dramatically affected the efficacy of photobleached melanosomes to interact with reagents as demonstrated by saturation recovery EPR employing dysprosium (III) as a powerful relaxing agent
09:30 Early Diagnosis of Diabetes Through The Eye DK Karumanchi*, Northern Illinois University
; ER Gaillard, Northern Illinois University; Devi Kalyan Karumanchi
Abstract: Diabetes mellitus is a metabolic disorder characterized by high blood sugar levels which give rise to complications in the eye, kidneys and the brain. Diabetes triggers the development of ocular diseases like diabetic retinopathy, glaucoma and cataracts which are the leading cause of blindness around the world. The most common method for the diagnosis of diabetes involves measuring the blood sugar levels in the body. One major disadvantage of this method is the fluctuating blood sugar levels which contribute to false negative results. This leads to delay in treatment, eventually causing permanent damage to the organs. Therefore, diagnosis of diabetes at an early stage is very crucial. One biomarker for diabetes related diseases is the formation of Advanced Glycation Endproducts (AGEs) that result from the Maillard reaction of proteins with glucose. α-crystallin in the ocular lens is a small heat shock protein with no protein turnover and hence acts as a record for post-translational modifications especially glycation which forms fluorescent AGEs. We have used steady state and time resolved fluorescence measurements to study the spectroscopic changes in alpha crystallin with increase in time of glycation and the intact lenses from diabetic and non-diabetic donors. Overall, this study was focused on developing a non-invasive diagnostic tool for early detection of diabetes mellitus.
09:45 Investigating the Cell Death Mechanisms of ARPE-19 Cells using Modified ARPE-Derived ECM to Model Aging and Disease ER Gaillard*, Northern Illinois University
; JC Tournear, Northern Illinois University
Abstract: Purpose: RPE cell death is a symptom of age related macular degeneration (AMD), but it is unclear what mechanisms of cell death are involved in this process. This study aims to modify ARPE-derived extra cellular matrix (ECM) in order to investigate the ARPE-19 cell death mechanism associated with various stresses modeling both age-related modifications and inflammation. Methods: A series of modifications were performed on ECM derived from ARPE-19 cells to model aging and inflammation. These modifications included non-enzymatic glycation, A2E, blue light irradiated A2E and nonenzymatic nitration. These modifications were done on ECM coating 24-well plates. Post modification, healthy ARPE19 cells were seeded onto the ECM and allowed to attach for 30 min. Unattached cells are removed and fixed for further study. Attached cells were allowed to grow prior to viability analysis. After 24 hours of growth, both unattached and attached cells were stained with Annexin-V and PI then subjected to analysis using flow cytometry to investigate apoptosis versus necrosis as mechanisms of cell death. Results: Cell viability is observed to decrease under all stresses when compared to unmodified ECM. Cells grown on blue light irradiated A2E modified ECM showed not only a loss in viability, but also a change in proliferation and cell morphology. There was evidence of both apoptosis and necrosis as mechanisms of cell death. When analyzing both the unattached and attached simultaneously, a further decrease in cell viability was observed suggesting that attachment to modified ECM is impaired. Conclusions: These data suggest that more than one mechanism of cell death is involved under aging and disease conditions. Both necrotic and apoptotic cells were observed supporting the idea that cells have difficulty in both cell attachment and proliferation. This study provides insight into the mechanisms of RPE cell death in AMD and can help to further understand the pathogenesis of the disease.