Biochemistry/Molecular Biology - ARVO

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.