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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyindicated substantial inflammation compared to control eyes (OS).Cell counts from OS eyes were very low or undetectable andincreased to 2.1 x 106 cells/ml of AH in OD eyes. Protein from OSeyes averaged 3.2 mg/ml and increased to 32.9 mg/ml in OD eyes.VEGF and VEGF-C were below detection limits in OS eyes andincreased to 7.1 pg/ml and 75.5 pg/ml, respectively, in OD eyes.VEGF-D in OS eyes averaged 96.0 pg/ml and increased to 853 pg/mlin OD eyes.Conclusions: VEGF, VEGF-C, and VEGF-D levels increasedsignificantly in LPS treated eyes of NZW and are indicative of aninflammatory response. Ophthalmic exams, cell counts, and proteinincreases in LPS treated eyes support increased inflammation in thisrabbit model of uveitis. The significantly increased levels of VEGF-Cand VEGF-D in AH are new findings.Commercial Relationships: Douglas Decker, PharmOptima (I),PharmOptima (S); Jeffery E. Burr, PharmOptima LLC (E); KellyLester, PharmOptima (E); Vicki Zaworski, PharmOptima (E);Steven J. Weber, PharmOptima LLC (I), PharmOptima LLC (E)Program Number: 5011 Poster Board Number: A0140Presentation Time: 2:45 PM - 4:30 PMThe role of Cannabinoid receptors on light-inducedphotoreceptor degenerationTomoyo Imamura, Yuta Ohno, Kazuhiro Tsuruma, MasamitsuShimazawa, Hideaki Hara. Gifu Pharmaceutical University, Gifu,Japan.Purpose: Cannabinoid receptors are known as G protein-coupledreceptors and classified in CB1 receptor and CB2 receptor. CB1receptor is mainly localization in the presynaptic terminal of neurons,and regulates the release of neurotransmitters. In contrast, CB2receptors are expressed in the peripheral nervous system and organs,such as tonsils, spleen, these are involved in the regulation ofinflammation and immune response. It has been reported thatcannabinoid receptors are related to some neurodegenerativediseases, and expressed in retina. However, the role of cannabinoidreceptors in retina remains to be elucidated. Here, we investigated therole of cannabinoid receptors in light-induced photoreceptordegeneration, which is associated with retinitis pigmentosa and agerelatedmacular degeneration.Methods: An murine photoreceptor cell line, 661W, was irradiatedby white light at 2,500 lx. To examine the effect of CB1 and CB2receptors on light-induced retinal cell death, we used each receptorsagonist and antagonist, and evaluated cell viability by nuclearstaining with propidium iodine and Hoechst33342. In addition, 8weeks old male ddY mice were induced retinal damage by white lightirradiation at 8,000 lx illumination for 3 hours after the darkadaptation for 24 hours. To determine the expression of CB1 andCB2 receptor after light exposure, time-dependent changes ofexpression level and localization in retina and in 661W cells weremeasured by Western blotting and immunostaining.Results: CB1 and CB2 receptors existed in all layers of the retina.Furthermore, the expression level of CB1 receptor was increased bylight irradiation, whereas that of CB2 receptor was decreased. Inaddition, SR141617A, a CB1 receptor antagonist, showed asignificant protective effect. On the other hand, HU-308, a CB2receptor agonist, showed a significant protective effect, whereasSR144528, which is a CB2 antagonist, did not show a clear effect.Conclusions: CB1 receptor antagonist showed a significantprotective effect, whereas a CB2 receptor agonist showed asignificant protective effect. Furthermore, the expression level ofCB1 and CB2 receptor was changed in light retinal degenerationmodel. These results suggest that cannnabinoid receptors may beinvolved in the pathogenesis of retinal diseases such as retinitispigmentosa and age-related macular degeneration.Commercial Relationships: Tomoyo Imamura, None; Yuta Ohno,None; Kazuhiro Tsuruma, None; Masamitsu Shimazawa, None;Hideaki Hara, NoneProgram Number: 5012 Poster Board Number: A0141Presentation Time: 2:45 PM - 4:30 PMThe involvement of heparin-binding epidermal growth factor likegrowth factor on light-induced retinal degenerationYuki Inoue, Tomohiro Nakanishi, Astushi Oyagi, Yuta Ohno,Tomohiro Otsuka, Kazuhiro Tsuruma, Masamitsu Shimazawa,Hideaki Hara. Gifu Pharmaceutical University, Gifu, Japan.Purpose: Heparin-binding epidermal growth factor like growthfactor (HB-EGF) is a member of the EGF family of growth factor,and has been reported that it protects against various neuronal celldamages and expresses in the retina. Here, we investigated its role inlight-induced photoreceptor degeneration using 661W cells, atransformed mouse cone-cell line, and conditional Hb-egf knockout(KO) mice.Methods: 661W cells were used to investigate the effect of HB-EGFon light irradiation-induced cell-death using HB-EGF siRNA andHB-EGF treatments. Time-dependent changes in retinal HB-EGFwere measured using quantitative RT-PCR and Western blotting.Moreover, disruption of Hb-egf in the retinas from ventral forebrainspecific Hb-egf KO mice, was confirmed by LacZ staining and RT-PCR. Retinal damage was induced by exposure to whitefluorescentlight. Recombinant human HB-EGF was directly injectedinto vitreousbody in mice intravitreally. Electroretinogram (ERG)and histological analyses were performed.Results: The 661W cell death induced by light irradiation wasexacerbated by Hb-egf knockdown. Treatment with HB-EGFprotected against light-induced cell-death in 661W cells. Hb-egf andpro-HB-EGF levels were increased after light exposure in wild-type(WT) mice. LacZ-positive cells were observed, and Hb-egf deletionwas confirmed in the retinas of Hb-egf KO mice. Exposure to whitefluorescent light for 3 h reduced the a- and b-wave amplitudes of thedark-adapted ERG, and ONL thickness in Hb-egf KO mice versusWT mice. HB-EGF treatment improved both the a- and b-waveamplitudes, and the thickness of the ONL.Conclusions: These data suggest that HB-EGF plays a pivotal role inlight-induced retinal damage. It may become a potential therapeutictarget for age-related macular degeneration.Commercial Relationships: Yuki Inoue, None; TomohiroNakanishi, None; Astushi Oyagi, None; Yuta Ohno, None;Tomohiro Otsuka, None; Kazuhiro Tsuruma, None; MasamitsuShimazawa, None; Hideaki Hara, NoneSupport: None.Program Number: 5013 Poster Board Number: A0142Presentation Time: 2:45 PM - 4:30 PMComparison Study of EFEMP1 Gene Expression Levels inHuman ARPE-19 Cybrid Models Having Mitochondrial DNA ofHaplogroups H, J, K or LDanli Xing 1 , Deepika Malik 1 , Payam Falatoonzadeh 1 , Shari Atilano 1 ,Marilyn Chwa 1 , S Michal Jazwinski 2 , Miceli V. Michael 2 , Baruch D.Kuppermann 1 , Cristina M. Kenney 1 . 1 Ophthalmology, UC Irvine,Long Beach, CA; 2 Tulane Center for Aging, Tulane University, NewOrleans, LA.Purpose: EFEMP1 (Epidermal Growth Factor-containing fibulin-likeextracellular matrix protein) is a gene expressed in retinal pigmentepithelium (RPE) cells and is one of the high-risk genes associatedwith Age-related Macular Degeneration (AMD). EFEMP1 suppresses©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 BiologyVEGFA-induced angiogenesis, and mutations in this gene areassociated with drusen formation. Histology of AMD eyes shows thatEFEMP1 protein can be found between the RPE cell layer anddrusen. It has been suggested that misfolded EFEMP1 proteinaccumulates within RPE cells causing altered cellular function andinflammation. The aim of this study is to assess the baselineexpression of the EFEMP1 gene in a human ARPE-19 cybridcytoplasmic hybrid (cybrid) model where the cells have identicalnuclei, but contain mitochondrial DNA haplogroup H, J, K or L.Haplogroup H and J are of Northern-European descent, however, Jgenerally has more risk of developing AMD while H is protective.Haplogroup K, of Central-European descent, has some risk, andhaplogroup L, of African descent, has the least predisposition todeveloping AMD.Methods: Human ARPE-19 cells that were depleted of mitochondria(Rho0) were fused with platelets from individuals with known H, J,K or L haplogroups to create unique cybrid cell lines. RNA wasextracted from individual cybrids and cDNA was synthesized.EFEMP1 gene expression levels were measured using Q-PCR andanalyzed for changes in gene expression levels relative to cybrid H.Results: The expression levels of the EFEMP1 gene were found tobe significantly lower in Cybrids J, K and L when compared tocybrid H: cybrid J= 0.25 fold, p=0.0001; cybrid L= 0.4 fold, p=0.008;and cybrid K= 0.17 fold, p=0.003.Conclusions: This study demonstrates that human ARPE-19 cybridswith identical nuclei but variable mitochondrial DNA haplogroupshave differentially expressed levels of the EFEMP1 gene which isinvolved in VEGF-A induced angiogenesis. Cybrids J, K and Lshowed significantly lower levels of EFEMP1 gene expression ascompared to cybrid H in the following order H > L > J > K. Thisfinding may help us in further understanding the pathogenesis ofAMD with regards to differential risks amongst populationsCommercial Relationships: Danli Xing, None; Deepika Malik,None; Payam Falatoonzadeh, None; Shari Atilano, None; MarilynChwa, None; S Michal Jazwinski, None; Miceli V. Michael, None;Baruch D. Kuppermann, Alimera (C), Allegro (C), Allergan (C),Genentech (C), Glaukos (C), GSK (F), Novagali (C), Novartis (C),Ophthotech (C), Pfizer (C), Regeneron (C), Santen (C), SecondSight(C), Teva (C), ThromboGenics (C); Cristina M. Kenney, NoneSupport: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 5014 Poster Board Number: A0143Presentation Time: 2:45 PM - 4:30 PMInositol phosphatase INPP5E in primary cilia and eyedevelopmentNa Luo 1 , Michael Conwell 1 , Akhilesh Kumar 1 , Ryan M. Anderson 2 ,Yang Sun 1 . 1 Ophthalmology, Indiana University, Indianapolis, IN;2 Pediatrics/Endocrinology, Indiana University, Indianapolis, IN.Purpose: To investigate the function of inositol 5-phosphataseINPP5E in cilia development in the retina. Mutations of INPP5Ecause Joubert Syndrome, which is characterized by retinaldegeneration, renal cysts, polydactyly, and mental retardation.Previous studies have implicated primary cilia defects in JoubertSyndrome, but the function and mechanisms of INPP5E in ciliaformation and retina development are not well understood.Methods: Using antisense morpholino oligonucleotides, knockdownof INPP5E was performed in zebrafish and rescue experiments weredone using wild type and mutant human mRNA. INPP5E mRNAsmutations of R378C and R435Q were generated using QuikChangeIIand Ambion mMessage mMachine®. Immunofluorescence wasperformed to study the functional aspect of cilia in zebrafishembryos. mTOR inhibitors treatments were performed in zebrafishand cell lines to examine the role of INPP5E in cilia signaling.Results: In zebrafish INPP5E morphants, embryos showed a dosedependentphenotype of retinal degeneration, microphthalmia, andbody axis asymmetry. The Inpp5e morphant zebrafish exhibitedshortened and decreased cilia formation in the Kupffer’s vesicle andpronephric ducts as compared to control zebrafish. Epinephrinestimulatedmelanosome trafficking was delayed in the Inpp5ezebrafish morphants. The phenotypes were rescued by co-injection ofhuman wide type INPP5E but not R378C or R435Q hINPP5EmRNA. Rapamycin was found to slow the development of the retinalphenotype in the zebrafish morphants.Conclusions: Our data supports an important role of INPP5E inciliary development and maintenance and provides a novel model toevaluate small molecules in the function of retinal development.Commercial Relationships: Na Luo, None; Michael Conwell,None; Akhilesh Kumar, None; Ryan M. Anderson, None; YangSun, NIH (F), American Glaucoma Society (R), Knights TemplarEye Foundation (R), Reeves Foundation (R)Support: NIH-EY-K08-022058; American Glaucoma society;Knights Templar Eye foundationProgram Number: 5015 Poster Board Number: A0144Presentation Time: 2:45 PM - 4:30 PMHuman Complement Factor H (CFH) Transgene ExpressionRescues the Visual Function and Retina Abnormalities in Agedcfh-/- MiceJindong Ding 1 , Una L. Kelly 1 , Marybeth Groelle 1 , Catherine BowesRickman 1, 2 . 1 Ophthalmology, Duke University Medical Center,Durham, NC; 2 Cell Biology, Duke University Medical Center,Durham, NC.Purpose: Complement factor H (CFH) polymorphisms are thestrongest genetic risk factors associated with age-related maculardegeneration. We have previously generated bacterial artificialchromosome (BAC) transgenic mouse lines expressing the humanY402 or H402 CFH variants. We have also shown that human CFHprotein can replace mouse cfh in the mouse complement alternativepathway when crossed to the cfh-/- mice (CFH-Tg,cfh-/-). In thepresent work, we evaluated the ocular phenotype of the CFH-Tg,cfh-/- mice. Specifically, we examined whether the retinal degenerationin the cfh-/- mice can be rescued by the human CFH protein.Methods: Experiments were performed on two-year-old cfh-/-, CFH-Tg,cfh-/- and C57 mice. The retina visual function was measured byelectroretinogram (ERG). Eyes were fixed and embedded in resin.Semi-thin sections were examined to evaluate the gross morphologyof the retina. Approximately 60 transmission electron microscopicimages per retina were taken at regular intervals across the superiorinferiorplane to measure the thickness of sub-RPE deposit.Results: The b-wave amplitude of the ERG was significantly reducedin the cfh-/- mice, compared to the C57 mice. The b-wave amplitudeof the CFH-Tg,cfh-/- mice was partially recovered. Initial evaluationdid not detect any differences in the gross retina structure. Contraryto previous studies, we found that aged cfh-/- mice have considerablymore sub-RPE deposit than age-matched C57 mice. However, thethickness of these basal deposits were reduced in the CFH-Tg,cfh-/-mice.Conclusions: Human CFH protein can partially rescue the retinalERG deficit and reduce the amount of sub-RPE basal deposit causedby cfh gene deletion.©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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