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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynovel biomarkers for AMD. We performed the whole proteomicprofiling of AH of patients, conditioned media (CM) from ARPE-19cells exposed to 400 µM paraquat 24 h and exosomes derived fromAH and CM by LC-MS/MS. Exosome pellets from AH and CM wereobtained with ExoQuickTM Exosome Precipitation Solution. Toconfirm exosome and exosomal proteins, transmission electronmicroscopy (TEM) and Western blot analysis for exosomal markerproteins (CD63 and Hsp70) were performed. Finally, liquidchromatography multiple reaction monitoring (LC-MRM) analysis ofAH of 15 treatment-naïve patients with neovascular AMD andcontrol subjects were performed to validate the target proteins.Results: TEM revealed that exosomes from the AH of patients andCM from ARPE-19 cells as round shaped membrane vesicles sized50-100 nm in diameter. CD63 and Hsp70 in exosomes from AH andCM were detected by Western blot analysis. Among 70 proteinsidentified both in AH and CM, six proteins including cathepsin D and26S proteasome non-ATPase were found in exosomes of AH. Sevenproteins including cytokeratin 8 and Hsp70 were found in exosomesof CM. Two proteins were detected in exosomes from both. In total,11 exosomal proteins were increased in AH of 15 patients withvarious levels of decrease after intravitreal injection of ranibizumabby LC-MRM.Conclusions: The present study has identified potential biomarkersand therapeutic target proteins of AMD and provided an effectiveapproach to identify AMD-associated proteins that are secreted byRPE cells in vivo. In addition, we provide new evidence that thesecretory mechanisms of these proteins might be closely related toexocytic activity as in the formation of drusens which are considereda risk factor for developing AMD.Commercial Relationships: Hyewon Chung, None; Ae Jin Choi,None; Eun Sook Lee, None; Gum-Yong Kang, None; SoyoungChoi, None; Hyunjung J. Lim, NoneSupport: National Research Foundation of Korea (NRF) funded bythe Ministry of Education, Science and Technology(2012M3A9B2028333)Program Number: 3655Presentation Time: 3:45 PM - 4:00 PMShort-term exposure to thrombin induces long lasting disruptionof barrier properties and proangiogenic signals in RPETami Livnat 1 , Omer Bialer 2 , Yael Nisgav 1 , Mor Dachbash 1 , RimaDardik 1, 3 , Dov Weinberger 2, 4 . 1 Laboratory of Eye Research,Felsenstein Medical Research Center, PetachTikva, Israel;2 Department of Ophthalmology, Rabin Medical Center, Petah Tiqwa,Israel; 3 The Israeli National Hemophilia Center and Institute ofThrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer,Israel; 4 Sackler School of Medicine, Tel Aviv University, Tel Aviv,Israel.Purpose: Thrombin is a multifunctional protease playing a centralrole in coagulation. Apart from its major function in vein and arteryocclusion, thrombin was found to play a significant role ininflammatory diseases. Thrombin exerts cellular effects through itsmembrane receptor (PAR). In vivo testing of thrombin is difficultsince its existence in plasma is short-term and transient. The retinalpigment epithelium (RPE) forms the outer blood retina barrier (BRB)and its integrity is essential for normal function of the retina. Retinalpathologies involving BRB disruption, such as diabetic retinopathy,inflammation, vascular occlusions and tumors may be accompaniedby elevation in thrombin levels.In the present work, we aimed to explore the impact of thrombin onthe integrity and function of the RPE blood barrier. We induced invitroa short-term exposure of RPE to pathological levels of thrombinand studied the immediate and long lasting changes in the RPEpermeability and angiogenic balance.Methods: ARPE-19 cells were grown for a month to achieve definitepolarity properties. Following a short (10 minutes) exposure tothrombin, the cells were washed and covered with new medium untilmeasurements were performed. Permeability was evaluated based onspectrophotometric monitoring of the leakage of labeled dextranmolecules. The expression of pro- and anti- angiogenic genes wasevaluated using real time PCR. Protein levels were measured byELISA or by FlowCytomix. MMPs activity was examined byzymography.Results: Short-term exposure to thrombin induced a long lasting (forhours) increase in RPE permeability to 10, 40 and 70 KD dextran.Decrease in PEDF mRNA and increase in VEGF and HGF mRNAexpression and protein levels were detected even 24 hours after the10 minute exposure to thrombin. MMPs 2 and 9 activities were alsoincreased hours after the short-term exposure to thrombin.Conclusions: Short-term exposure to thrombin inducesproangiogenic signals in RPE and disruption of barrier properties.The data indicate that changes in permeability, gene expression,proteins levels and activity persisted for hours after short-termexposure to thrombin. Based on our findings, we suggest thataccumulation of short-term exposures to thrombin over yearscontributes to the pathological processes involved in CNVdevelopment in the elderly.Commercial Relationships: Tami Livnat, None; Omer Bialer,None; Yael Nisgav, None; Mor Dachbash, None; Rima Dardik,None; Dov Weinberger, NoneProgram Number: 3656Presentation Time: 4:00 PM - 4:15 PMDICER1 is regulated via cAMP in an EPAC/Rap1-dependentmanner in human retinal pigment epithelial cells - implicationsfor age-related macular degenerationThomas P. Sauer, Sabine Kurth, Frank G. Holz, Tim U. Krohne.Ophthalmology, University of Bonn, Bonn, Germany.Purpose: DICER1 deficiency has recently been implicated in thepathogenesis of geographic atrophy (GA) due to age-related maculardegeneration (AMD). However, little is known about DICER1 generegulation in RPE cells. Cyclic adenosine monophosphate (cAMP)regulates DICER1 in human melanocytes. Therefore, we investigatedthe effects of cAMP on DICER1 gene expression in human RPE cellsMethods: The human RPE cell line ARPE-19 was treated withadenylate cyclase agonist forskolin, protein kinase A (PKA) inhibitorH89, Rap1 inhibitor GGTI-298, and Epac agonist 8-pCPT-2-0-MecAMP,both as single and combination treatments. Subsequently,cells were lysed and total mRNA was isolated. To assess DICER1gene expression, real-time quantitative PCR was carried out usingDICER1- specific primers: forward 5-CCCGGCTGAGAGAACTTACG-, reverse 5-CTGTAACTTCGACCAACACCTTTAAA-3Results: In forskolin-treated cells, increased cAMP levels resulted indownregulation of DICER1 (0.04 ± 0.036 relative gene expression).Inhibition of PKA did not prevent cAMP-induced downregulation(0.47 ± 0.41) whereas inhibition of Rap1 did (5.67 ± 5.23). Evenwithout forskolin treatment, activation of Epac downregulatedDICER1 (0.21 ± 0.21) whereas inhibition of Rap1 resulted in anupregulation (4.37 ± 3.52)Conclusions: cAMP-mediated downregulation of DICER1 in humanRPE cells is not solely explained by PKA action but involves theEpac/Rap1 pathway. As DICER1 has been demonstrated to preventAlu RNA-mediated damage in RPE, Rap1 represents a noveltherapeutic target for advanced dry AMD©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.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCommercial Relationships: Thomas P. Sauer, None; SabineKurth, None; Frank G. Holz, Acucela (C), Allergan (C), Genentech(F), Heidelberg Engineering (F), Zeiss (F), Novartis (F), Novartis(C), Optos (F), Merz (C), Bayer (F), Bayer (C), BoehringerIngelheim (C); Tim U. Krohne, Novartis Pharma GmbH,Nuremberg, Germany (F), Novartis Pharma GmbH, Nuremberg,Germany (C), Novartis Pharma GmbH, Nuremberg, Germany (R)Program Number: 3657Presentation Time: 4:15 PM - 4:30 PMUsing extremes to identify rare pathogenic variants in agerelated-maculardegenerationCodrut C. Paun 1, 2 , Dzenita Smailhodzic 1 , Camiel J. Boon 1 , Lies H.Hoefsloot 2 , Mohamed R. Daha 3 , Carel B. Hoyng 1 , Anneke I. DenHollander 1, 2 . 1 Ophthalmology, Radboud Univ Nijmegen Med Ctr,Nijmegen, Netherlands; 2 Human Genetics, Radboud UniversityNijmegen Medical Centre, Nijmegen, Netherlands; 3 Nephrology,Leiden University Medical Center, Leiden, Netherlands.Purpose: Age-related macular degeneration (AMD) has a stronggenetic component, and it has been estimated that 50% of theheritability of AMD is explained by common variants in thecomplement system. Despite the progress that has been made inidentifying common variants in AMD, few studies have assessed therole of rare variants in the pathogenesis of AMD. The purpose of thisstudy was to identify rare variants in AMD patients with high levelsof complement activation.Methods: Complement component C3 and the activation fragmentC3d were measured in serum samples of 197 AMD patients and 150unaffected age-matched controls. The C3d/C3 ratio was calculated asan indicator of C3 activation. DNA samples of patients with highC3d/C3 ratios (>3), were screened for mutations in the CFH gene bySanger sequencing. DNA samples of 8 patients with the highestC3d/C3 ratios (>4) were analyzed by exome sequencing.Results: AMD patients displayed an increased median C3d/C3 ratiocompared to controls (p3 were observed in37 AMD patients, but only in 4 control individuals. Sequenceanalysis of the CFH gene revealed a heterozygous frameshiftmutation (c.1769_1779del; p.Gly590fsX) in a patient with a C3d/C3ratio of 4.4. Exome sequencing in 8 patients with C3d/C3 ratios >4revealed rare variants in 5 patients in key components of thecomplement pathway.Conclusions: High levels of complement activation in AMD patientscan be caused by rare variants in components of the complementpathway, which have previously not been associated with AMD.High complement activation may therefore direct a genetic searchtoward complement gene abnormalities. This genotype-serotypecorrelation approach is able to identify new risk factors in AMD.Commercial Relationships: Codrut C. Paun, None; DzenitaSmailhodzic, None; Camiel J. Boon, None; Lies H. Hoefsloot,None; Mohamed R. Daha, None; Carel B. Hoyng, None; Anneke I.Den Hollander, NoneSupport: Foundation Fighting Blindness USA center grant C-GE-0811-0548-RAD04374 Lipids, Retinoids and Macular PigmentsTuesday, May 07, 2013 2:45 PM-4:30 PMExhibit Hall Poster SessionProgram #/Board # Range: 3756-3783/A0095-A0122Organizing Section: Biochemistry/Molecular BiologyProgram Number: 3756 Poster Board Number: A0095Presentation Time: 2:45 PM - 4:30 PMAnalysis of Retinal Gangliosides by Hydrophilic InteractionLiquid Chromatography - Electrospray Ionization Tandem MassSpectrometryOlivier Berdeaux 1, 2 , Elodie A. Masson 2 , Stéphanie Cabaret 1, 2 , AnneAthias 3 , Jean-Paul Pais De Barros 3 , Lionel Bretillon 2 . 1 ChemoSensPlatform, INRA, Dijon, France; 2 Eye & Nutrition Research Group,INRA, Dijon, France; 3 Lipidomic Analytical Platform, University ofBurgundy, Dijon, France.Purpose: Gangliosides (GG) are sialic acid-containingglycosphingolipids. A wide variety of GG have been described basedon differences in the oligosaccharidic chain, fatty acids and longchain sphingoid bases of the ceramide moiety. The GG profile isspecific to the organ, its differentiation and pathophysiological status.An efficient method of separation, identification and quantification istherefore crucial ifthe huge heterogeneity among these compounds isto be understood and changes highlighted. We report here a powerfulanalytical method based on hydrophilic interaction liquidchromatography-electrospray ionization tandem mass spectrometry(HILIC-ESI-MS/MS), further tested on rat retina. Indeed, GG areparticularly abundant in the central nervous system, including theretina. While their involvement in the development of the retina andtheir neuroprotective action have been demonstrated, their preciserole in the retina and its pathologies is still poorly understood.Methods: GG classes were separated under HILIC conditions. Usinga triple quadrupole MS instrument equipped with an ESI source,GM3 and GM2 molecular species were structurally characterized bycollision-induced dissociation (CID) of [M-H] - and GD3, GD1a,GD1b, GT1b and GQ1b by CID of [M-XH]X- in negative modes.The semi-quantitative analysis of the GG molecular species wasperformed in the negative mode by single reaction monitoring(SRM). An N-acetylneuraminic acid fragment at m/z 290 wasproduced in the collision cell from the GG molecular species andused in SRM for quantification.Results: This method enables the major and minor GG species to beeffectively separated in a highly heterogeneous GG mixture.Furthermore, it provides an accurate characterization of the widelyvariable ceramide moiety, whose role is not well understood. In ratretina, the main long chain bases are d18:1 and d20:1. Fatty acids aresaturated or monounsaturated, mainly 16:0, 18:0, 20:0 and 22:0. Thedistribution of the different ceramide species varies among the GGclasses, even though d18:1/18:0 and d18:1/20:0 (or d20:1/18:0)consistently appear to be the major ones.Conclusions: Applied to retinal samples, this glycolipidomicapproach offers an efficient, sensitive, straightforward, highlyaccurate and reliable tool to investigate the role of GG in the functionand pathologies of the retina.Commercial Relationships: Olivier Berdeaux, None; Elodie A.Masson, None; Stéphanie Cabaret, None; Anne Athias, None;Jean-Paul Pais De Barros, None; Lionel Bretillon, NoneProgram Number: 3757 Poster Board Number: A0096Presentation Time: 2:45 PM - 4:30 PMCirculating markers of retinal and optic nerve lipidsNiyazi Acar 1 , Olivier Berdeaux 1 , Zhiguo He 2 , Stéphanie Cabaret 1 ,Philippe Gain 2 , Gilles Thuret 2 , Catherine P. Garcher 3, 1 , Alain M.Bron 3, 1 , Lionel Bretillon 1 . 1 INRA, University of Burgundy, Eye &Nutrition Research Group, Dijon, France; 2 EA2521, University JeanMonnet, Laboratory "Biology, Imaging, and Engineering of CornealGrafts", Saint-Etienne, France; 3 University Hospital, Department ofOphthalmology, Dijon, France.Purpose: Blood lipids are frequently used as a surrogate of lipidcomposition of peripheral tissues. Even if it is well accepted, such arelationship has never been clearly demonstrated for the eye. The aim©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.
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