Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneause of a high resolution MRM approach to develop assays forbiologically important tear proteins.Methods: Human tear samples were collected from 1000 consentingpatients with no eye complaints (411 male, 589 female, average age55.5 years, SD 14.5 years) using the Schirmer tear test strips andpooled into a single global control sample. Quantification of proteinsis carried out by selecting “signature” peptides derived by trypsindigestion of the target protein. A 2-hour nanoLC-MS/MS run wasused to separate the tryptic peptides and perform quantitation ofhuman tear proteins in HR-MRM mode. Samples were analyzed intriplicate. Twenty-one high abundant proteins were further accessedfor signal reproducibility.Results: Fifty-three peptide assays that represent 51 high andintermediate abundant tear proteins were developed. All assaysshowed consistent retention time with a coefficient of variation (CV)of less than 2%. As for peak area, 17 out of the 21 assays showed areproducible peak area with CV less than 20%. Some wellcharacterized tear proteins, lacritin, mammaglobin-B, S100A4,S100A8, and prolactin inducible protein (PIP) showed the highestreproducibility of peak area, with CV less than 2%.Conclusions: These multiplexed MRM-based assays show greatpromise to be further developed for biomarker validation in humantear samples.Commercial Relationships: Lei Zhou, Singapore Eye ResearchInstitute (P); Louis Tong, None; Roger W. Beuerman, Allergan (F),SERI (P), Santen (R)Support: CG from NMRC, Singapore, Core facility funding fromSingHealth Foundation283 EndotheliumMonday, May 06, 2013 2:45 PM-4:30 PMTCC 304 Paper SessionProgram #/Board # Range: 2196-2202Organizing Section: CorneaProgram Number: 2196Presentation Time: 2:45 PM - 3:00 PMDecline in DJ-1 Leads to Decreased Nuclear Translocation ofNrf2 and Results in p53-mediated Apoptosis of Human CornealEndothelial CellsCailing Liu, Yuming Chen, Ula V. Jurkunas. Schepens/MassachusettsEye and Ear, Department of Ophthalmology, Harvard MedicalSchool, Boston, MA.Purpose: Dowregulation of DJ-1 and decreased nuclear localizationof nuclear factor erythroid-derived 2-like 2 (Nrf2) has been detectedin Fuchs endothelial corneal dystrophy endothelium. DJ-1 has beenreported to stabilize Nrf2, which binds to antioxidant responseelement in the nucleus to protect cells from apoptosis. In this study,we investigated the effect of DJ-1 downregulation on Nrf2 nucleartranslocation, Nrf2-associated protein regulation, and cornealendothelial cell susceptibility to oxidative stress.Methods: An immortalized normal human corneal endothelial cellline (HCECi) was transfectedwith 50 nM of siRNA specific to the human DJ-1 gene usingLipofectamine. Scrambled siRNA was used as control. Nrf2 and p53levels in nuclear and cytosolic extracts were evaluated by westernblotting. Nrf2-associated proteins such as Cul3 and Keap1 weredetected by immunoprecipitation (IP) with anti-Nrf2 antibody,followed by western blotting with target antibodies. Oxidative stresswas induced by exposing HCECi cells to a UVA broadband lampwith the fluence of 10 J/cm 2 . Cell pellets were harvested for westernblotting with anti-caspase 3 and p53 antibodies, while supernatantswere collected for a cell viability assay.Results: Despite similar levels of cytoplasmic Nrf2, nuclear Nrf2protein levels decreased by 2.2-fold (p=0.04) in DJ-1 siRNA-treatedHCECi cells as compared to scrambled siRNA-treated cells. IPstudies detected Nrf2 association with Cul3 and Keap1 with anincrease in Cul3-Nrf2 complex in DJ-1 siRNA-treated cells relativeto controls. UVA irradiation led to an 8.5% increase in cell death inDJ-1 siRNA-treated cells as compared to controls. Moreover,downregulation of DJ-1 led to a 16.4% increase in active caspase 3levels. This effect was augmented by UVA treatment, which led to a28.6% increase in caspase 3 as compared to controls. Downregulationof DJ-1 resulted in an increase in cytoplasmic p53 levels, while UVAirradiation resulted in a 17.7% increase in total p53 levels in DJ-1siRNA treated cells as compared to controls.Conclusions: Downregulation of DJ-1 impairs nuclear translocationof Nrf2 potentially by targeting Nrf2 for degradation through Cul3and Keap1 pathways. Decline in DJ-1 levels leads to heightened cellsusceptibility to UVA-induced apoptosis and activates p53-dependentpathway in corneal endothelium.Commercial Relationships: Cailing Liu, None; Yuming Chen,None; Ula V. Jurkunas, 61/482,769 (P), Altheos (C)Support: NIH/NEI Grant R01 EY20581 (UVJ), a Research toPrevent Blindness Award (UVJ)Program Number: 2197Presentation Time: 3:00 PM - 3:15 PMMicroRNA Analysis in Fuchs Endothelial Corneal DystrophyMario Matthaei 1, 2 , Jianfei Hu 1 , Laura Kallay 1 , Claus Cursiefen 2 ,Jiang Qian 1 , Albert S. Jun 1 . 1 Anterior Segment / Cornea, Wilmer EyeInst, Johns Hopkins Univ, Baltimore, MD; 2 Department ofOphthalmology, University of Cologne, Cologne, Germany.Purpose: MicroRNAs (miRNAs) are a class of endogenousnoncoding RNA which posttranscriptionally modulate geneexpression during development and disease. Our study investigatedthe differential miRNA expression pattern in human FuchsEndothelial Corneal Dystrophy (FECD) compared to normalendothelium.Methods: Total RNA was extracted from corneal endothelial cells ofFECD eyes (n=6) and age-matched normal autopsy globes (n=6). Theexpression of 754 well characterized miRNA sequences was analyzedin preamplified cDNA samples using OpenArray plate technology onthe QuantStudio 12K Flex system for high-throughput real-timequantification and individual targets were validated by Taqman qPCRassays.Results: We detected differential expression of at least 37microRNAs using global normalization and applying a fold-changeof 2 and p=0.05 as cut-off values. Of these 37 miRNAs, 32 showeddownregulation whereas 5 miRNAs were upregulated. MiRNA targetgenes were predicted and further analyzed by gene ontologyassessment.Conclusions: We present the first endothelial cell microRNA profilein FECD and normal endothelial samples and identify dysregulatedmiRNA expression in FECD.Commercial Relationships: Mario Matthaei, None; Jianfei Hu,None; Laura Kallay, None; Claus Cursiefen, Gene Signal (C),Alcon (R), Allergan (R), Bayer (R); Jiang Qian, None; Albert S.Jun, Johns Hopkins University (P)Support: Deutsche Forschungsgemeinschaft (DFG MA 5110/2-1 toM.M.), Richard Lindstrom/Eye Bank Association of AmericaResearch Grant (to M.M.), National Institutes of Health (EY019874to A.S.J.), Research to Prevent Blindness (to Wilmer Eye Institute)Program Number: 2198©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.