Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologymethylcytosine (5mC) generated by the enzymatic action of TenEleven Translocation (TET) family members. The functional role of5hmC is largly unknown. However, studies in embryonic stem cellsand cancer tissues suggest that TET enzymes and 5hmC may beinvolved in gene regulation. Eye opening at postnatal week (pw) 2 isa key time point during mouse retinal development. Maturation ofretinal cells as well as formation of the retinal network is ongoingafter eye opening and is completed only one to two weeks later. Here,we analyzed the role of 5hmC during this phase of postnatal retinaldevelopment in the mouse.Methods: 5hmC was localized to specific retinal cell types usingimmunohistochemistry. Global 5hmC levels in retinal samples werequantified using ultra high pressure liquid chromatography-tandemmass spectrometry (UHPLC-MS/MS). 5hmC marks were mapped tothe retinal genome by hydroxymethylated DNA immunoprecipitation(hMeDIP) followed by next generation sequencing and weresubsequently correlated with retinal gene expression data obtainedfrom microarray experiments.Results: At pw 2 5hmC weakly localized to nuclei of cells within theganglion cell layer and the inner nuclear layer of the retina. Thelevels of 5hmC in these cells were increased at pw 3. In addition,5hmC was now detectable in retinal photoreceptors were it colocalizedwith histone marks of the euchromatin. Global levels of5hmC also increased from pw 2 to pw 3, whereas 5-methylcytosinewas unchanged. hMeDIP experiments revealed a developmentallyprogrammed acquisition of 5hmC during retinal maturation at generichregions and in genes containing activating histone marks.Conclusions: Our data suggest that 5hmC is dynamically regulatedduring postnatal retinal development and is capable to elevate geneexpression of retina-specific genes.Commercial Relationships: Stylianos Michalakis, None; ArshanPerera, None; Susanne Koch, None; Mirko Wagner, None; LukasWindhager, None; Kerstin Nagel-Wolfrum, None; Tim M. Strom,None; Ralf Zimmer, None; Thomas Carell, None; Martin Biel,NoneSupport: Deutsche Forschungsgemeinschaftbut also therapeutic targets and vehicles. EVs were previously shownto participate in angiogenesis, tissue remodeling and tissueregeneration. In this work we aimed at first time characterization ofprotein and RNA profiles of EVs isolated from human vitreoussamples.Methods: We analyzed EVs from vitreous fluid collected duringvitrectomy from patients with proliferative diabetic retinopathy andprimary rhegmatogenous retinal detachment. EVs were isolated usingdifferential centrifugation, and were visualized by transmissionelectron microscopy (TEM). Size histograms of EV preparationswere determined by a resistive pulse sensing approach (qNano).Cellular origin of EVs was determined by flow cytometry (FC).Protein and RNA profiles of EVs were analyzed by massspectrometry (MS) and bioanalyzer assay (Agilent).Results: TEM clearly shows various populations of EVs in thevitreous fluid (Figure 1). Peak EV size was around 150 nm indiameter. The presence of EVs in vitreous fluid was also confirmedusing FC based on annexin V binding. Most EVs in the vitreous fluidwere derived from platelets and endothelial cells. MS revealedclassical EV-associated proteins including actin, actin-bindingproteins (e.g. ankyrin), tubulin, clusterin, heat shock proteins andenzymes. However, we also identified eye-specific proteins in EVsincluding retinal dehydrogenase, retinol binding protein and lensspecific proteins (lensin, crystallin etc.). Most importantly RNAprofiling has revealed that miRNA molecules were present invitreous-fluid-derived EVs in very high amounts (Figure 2).Conclusions: In this work we successfully isolated and characterizedEVs from vitreous fluid, and demonstrated the presence of miRNAsin these structures. Demonstration of angiogenesis-inducing miRNAsin vitreous fluid EVs may lead to identification of new biomarkers ornovel therapeutic targets in eye disorders.325 Biochemical and Molecular Mechanisms of Diabetic andRetinal DiseaseTuesday, May 07, 2013 11:00 AM-12:45 PM6A Paper SessionProgram #/Board # Range: 3148-3153Organizing Section: Biochemistry/Molecular BiologyContributing Section(s): Biochemistry/Molecular BiologyFigure 1. EVs visualized by TEM. Magnification is 30,000xProgram Number: 3148Presentation Time: 11:00 AM - 11:15 AMAnalysis of extracellular vesicles in vitreous samplesBence Gyorgy 1, 2 , Zsuzsanna Récsán 2 , Ágnes Kittel 3 , KrisztinaPálóczi 1 , Lilla Turiák 4 , Károly Vékey 4 , Janos Nemeth 2 , Edit I. Buzas 1 ,Zoltán Zsolt Nagy 2 . 1 Department of Genetics, Cell- andImmunobiology, Semmelweis University, Budapest, Hungary;2 Department of Ophthalmology, Semmelweis University, Budapest,Hungary; 3 Institute of Experimental Medicine, Hungarian Academyof Sciences, Budapest, Hungary; 4 Chemical Research Center,Hungarian Academy of Sciences, Budapest, Hungary.Purpose: Extracellular vesicle (EV) secretion represents anevolutionally conserved feature of living cells. EVs are known totransfer protein and RNA cargos between cells placing EV analysisinto the mainstream of biomedical research. The assessment of EVsmay provide insight into the pathomechanism of various disorders.Furthermore, they may not only serve as potential novel biomarkers,Figure 2. RNA profile from EVs showing mostly small RNAmolecules.Commercial Relationships: Bence Gyorgy, None; ZsuzsannaRécsán, None; Ágnes Kittel, None; Krisztina Pálóczi, None; LillaTuriák, None; Károly Vékey, None; Janos Nemeth, None; Edit I.Buzas, None; Zoltán Zsolt Nagy, NoneSupport: This work was supported by OTKA K 73247, NK 84043and K77537, Kerpel-Fronius Ödön Fellowship, Baross Gábor (REG-KM-09-1-2009-0010) and FP7-PEOPLE-2011-ITN - PITN-GA-©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.