Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPharmacology and Toxicology, Georgia Health Sciences University,Augusta, GA; 3 Department of Experimental Medicine and Pathology,University of Rome, La Sapienza, Italy; 4 Department of PetroleumChemistry, American University of Nigeria, Yola, Nigeria.Purpose: Enhanced expression and activity of TLR4 in the diabeticretina has been implicated in the pathogenesis of DiabeticRetinopathy (DR). Presently, it is not known how diabetes promotesTLR4 activity in retinal and peripheral blood cells. Here weinvestigated the role of thioredoxin-dependent peroxidases,peroxiredoxin 1 (Prx1) and 2 (Prx2), in modulating TLR4 signalingin the diabetic retina as well as in retinal cells in culture.Methods: Retinas of streptozotocin-induced diabetic rats (STZ-rats,4 weeks hyperglycemia) and non-diabetic rats (ND rats) as well aspost-mortem retinas of control and DR donors (obtained fromGeorgia Eye Bank), were examined. Interactions between Prxisoforms with thioredoxin-1 (Trx-1) were identified by coimmunoprecipitationand Western blotting in retinal tissues of theexperimental groups shown above. The expression and TLR4interaction of the oxidative stress-induced sulfonic forms of Prx(Prx1-SO3H and Prx2-SO3H) were also measured in retinal tissues,using specific antibodies. Retinal endothelial cells isolated frombovine eyes (BRECs) were transfected with siRNA against Trx-1 andanalyzed for the expression of Prx-SO3H and TLR4 association.Following stimulation of ECs with high glucose (HG, 25mM,48hours), TLR4 expression, interaction with Prx-SO3H, andsubcellular localization were analyzed by immunocytochemistry andco-immunoprecipitation.Results: Retinas of STZ-rats and donors with DR displayedaugmented protein levels of hyperoxized Prx isoforms, and increasedTLR4 association to both Prx-SO3H isoforms. HG stimulated thetranslocation of TLR4 to the plasma membrane as well as an increasein TLR4/Prx-SO3H interaction in ECs. Inhibition of Trx-1, achievedby transfection of the BREC with specific siRNAs, resulted inincreased expression of both isoforms of Prx-SO3H and promotedincreased TLR4/Prx-SO3H interactions. This effect was partiallyblunted with blockade of Trx-1.Conclusions: We have identified Prx-SO3H as a novel chaperone forTLR4 in the diabetic retina. This correlates with our findings that theanti-oxidant capacities of the Trx and Prx systems are compromisedin the diabetic milieu. This study highlights that inhibition of the Trxsystem leads to an accumulation of stable, oxidized Prx isoformswhich crosstalk with TLR4, a striking new pathway which maycontribute to the subclinical inflammation seen in DR.Commercial Relationships: Folami Lamoke, None; Sean Shaw,None; AnnaLisa Montemari, None; Wan Jin Jahng, None;Manuela Bartoli, NoneSupport: NIH/NEI NRSA F31EY022289Program Number: 1148 Poster Board Number: D0052Presentation Time: 1:00 PM - 2:45 PMMethylglyoxal Activates Chronic ER Stress Mediated EpigeneticLoss of Nrf2/Keap1 Gene Regulation in Diabetic CataractousLensesPalsamy Periyasamy 1 , Masahiko Ayaki 2 , Keshore R. Bidasee 3 ,Toshimichi Shinohara 1 . 1 Ophthalmology and Visual Sciences,University of Nebraska Medical Center, Omaha, NE;2 Ophthalmology, Mita Hospital, Tokyo, Japan; 3 Pharmacology andExperimental Neuroscience, University of Nebraska Medical Center,Omaha, NE.Purpose: Methylglyoxal (MGO) is a highly cytotoxic metaboliteproduced from glucose metabolism. Earlier studies indicate thatMGO is attributed to aging and diabetic complications throughformation of advanced glycation end products. But evidence forMGO-induced endoplasmic reticulum (ER) stress and epigenetic lossof Nrf2/Keap1 dependent antioxidant protection is emerging indiabetes. This study investigated the mechanism by which MGOinduced the ER stress and by what epigenetic mechanisms MGOinactivated the Nrf2/Keap1 dependent antioxidant protection in lensepithelial cells (LECs) during cataractogenesis.Methods: Human and Nrf2 knockout (KO) mouse LECs werecultured with MGO to study the ROS production and cell death.MGO was intraperitoneally administered to control and diabetic Nrf2KO mice. The expression profiles of markers of ER stress,Nrf2/Keap1 dependent antioxidant system and DNA methylation anddemethylation enzymes were analyzed by qPCR and Westernblotting. ER-Ca 2+ release was assessed by live cell imaging.Proteasomal degradation was studied by using an inhibitor, MG-132.Promoter DNA methylation status was sequenced by bisulfitegenomic DNA sequencing.Results: We found that MGO stimulates ROS production and celldeath in human and Nrf2 KO mouse LECs. MGO also reduced theER-Ca 2+ content of human LECs. The mRNA and proteinexpressions studies confirmed that MGO treatment significantlyactivates the ER stress-specific genes and suppresses the Nrf2/Keap1dependent antioxidant genes and DNA methyltransferases. Also, thelevels of glyoxalase-1 and DNA demethylation enzyme,methylcytosine dioxygenase were over-expressed by MGO. DNAsequencing results revealed significant demethylated DNA in theKeap1 promoter of diabetic cataractous lenses than clear lenses.Similarly, MGO treated human LECs resulted in significantdemethylation of Keap1 promoter and had a notable increase inKeap1 mRNA and protein.Conclusions: This study revealed a possible mechanistic rationale bywhich MGO induces ER stress mediated Nrf2/Keap1 dependentantioxidant system failure and altered expressions of DNAmethylation and demethylation enzymes. Also, epigeneticmodification of Keap1 promoter by MGO stimulates Keap1expression, which increases proteosomal degradation of Nrf2. So,redox-balance is altered towards lens oxidation and cataractformation.Commercial Relationships: Palsamy Periyasamy, None;Masahiko Ayaki, None; Keshore R. Bidasee, None; ToshimichiShinohara, NoneSupport: RPB, EY018172, HL085061Program Number: 1149 Poster Board Number: D0053Presentation Time: 1:00 PM - 2:45 PMTo determine changes in levels of cytokines in the anteriorchamber (AC) fluid of eyes in patients with diabetic macularedema (DME)whom have been treated with repeated injections ofranibizumab (RBZ)Yasir J. Sepah 1 , Mohamed A. Ibrahim 1 , Alyssa Morimoto 2 , AbeerAkhtar 1 , Mauricio Maia 2 , Kyu H. Hong 2 , Montserrat Carrasco-Triguero 2 , Diana V. Do 1 , Menno van Lookeren Campagne 2 , QuanDong Nguyen 1 . 1 Retinal Imaging Research and Reading Center,Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD;2 Bioanalytical Sciences, Genentech, Inc.,, San Francisco, CA.Purpose: To determine changes in levels of cytokines in aqueousfluid of eyes of patients with diabetic macular edema (DME) treatedwith serial injections of ranibizumab (RBZ).Methods: Aqueous levels of the pro-inflammatory cytokine IL6 andthe angiogenic chemokine IL8 were measured in serial samples from131 patients, collected at baseline (BL), months (M) 3, 6, 9 and 12from eyes with DME receiving treatment in the READ-3 Study.Patients received 6 monthly mandatory injections starting at BL.Thereafter, starting at M6, patients were retreated with RBZ if: 1)©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. 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