Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornealinked by translamellar keratocyte-keratocyte connections, but thisoccurs predominantly at the peripheral cornea. We propose a modelwhereby keratocytes form a single contiguous 3-D network, ratherthan a series of independent parallel networks, with a higher degreeof translamellar connectivity than previously appreciated.Commercial Relationships: Samuel D. Hanlon, None; Nancy C.Shenoi, None; Paul T. Harris, None; Paul T. Landry, None; Ali R.Behzad, None; Evelyn S. Brown, None; Margaret M. Gondo,None; Alan R. Burns, NoneSupport: NIH Grant EY017120, NIH Grant EY07551Program Number: 5239 Poster Board Number: C0158Presentation Time: 2:45 PM - 4:30 PMEffect of Serum Clot Activator on KeratocytesJi-Eun Lee 1 , Seung Uk Lee 2 , Jong Soo Lee 1 . 1 Ophthalmology, PusanNational University, Pusan, Republic of Korea; 2 Ophthalmology,Kosin University, Pusan, Republic of Korea.Purpose: To evaluate the effect of serum clot activator which couldbe used for making autologous serum eye drop on humankeratocytes.Methods: Cultured human corneal keratocytes were exposed to 10,20, and 30 % SiO2 for 24, 48, and 72 hours, MTT-based calorimetricassay was performed to determine the survival rate of keratocytes andlactate dehydrogenase (LDH) leakage assay to assess thecytotoxicity. Apoptotic response was evaluated with flow cytometricanalysis and fluorescence staining with Annexin V and propiodiumiodide. Cellular morphology was evaluated by inverted phasecontrastlight microscopy and electron microscopy.Results: The survival rate of human keratocytes and cytotoxicityshowed the concentration and time dependent response andsignificant response was found after treating with 30% SiO2 for 72hours. Apoptosis developed in flow cytometry and apoptotic cellswere demonstrated in fluorescent micrograph after treating with 30%SiO2 for 48 hours but cells were non viable after 72 hours. Humankeratocytes were more detached from the bottom of the dish anddamaged cells have degenerative changes like microvillidisappearance, vacuoles formation and chromatin of the nuclearremnant condensed along the nuclear periphery after treating with30% SiO2 for 72 hours.Conclusions: SiO2, the serum clot activator, showed the toxicity onhuman keratocytes at the concentration of 30% after 72 hourstreament. So blood should be extracted with tubes without clotactivator during making the autologous serum eye drop forpreventing the possible cytotoxicity on cornea.Commercial Relationships: Ji-Eun Lee, None; Seung Uk Lee,None; Jong Soo Lee, NoneProgram Number: 5240 Poster Board Number: C0159Presentation Time: 2:45 PM - 4:30 PMCo-cultures of Human Corneal Epithelium and Self-assembledKeratocyte and Fibroblast MatrixAudrey E. Hutcheon 1, 2 , Dimitrios Karamichos 1, 2 , Xiaoqing Q. Guo 1,2 , James D. Zieske 1, 2 . 1 Schepens Eye Research Institute/MEE,Boston, MA; 2 Department of Ophthalmology, Harvard MedicalSchool, Boston, MA.Purpose: During corneal wound repair the epithelium switches frominteracting with a mature matrix to a temporary matrix that ispartially assembled by wound-healing fibroblasts. The goal of thecurrent investigation was to develop and examine models that mimicthe interaction of epithelium interacting with a keratocyte-assembledmatrix versus a fibroblast-assembled matrix.Methods: Stromal cells were isolated from corneal explants byplacing tissue in either 1 or 10% FBS in DMEM. After culture andpassaging, the 1% (keratocyte-like) or 10% (fibroblast-like) cellswere plated in Transwell dishes with stabilized Vitamin C (VitC) ±0.1ng/ml of TGF-β3 and maintained in either 1 or 10% FBS inDMEM for 3 weeks. Immortalized human corneal-limbal epithelialcells (HCLE) were then placed atop the self-assembled matrix andcultured for 4 days; after which, the cultures were airlifted andmaintained for an additional 1 or 2 weeks. The co-cultures wereprocessed and examined by transmission electron microscopy (TEM)and indirect-immunofluorescence (IF) for morphology, extracellularmatrix components and fibrotic markers. Antibodies againstcollagens I, III, and V, thrombospondin-1 (TSP-1), cellularfibronectin (cFN), and smooth muscle actin (SMA) were examined.Results: Cells isolated with 1% serum but cultured without T3 didnot assemble a matrix. As observed by TEM, the morphology of thestromal cells isolated using 1% serum had a dendritic appearance,which is characteristic of keratocytes; whereas, the 10% serum cellswere spindle shaped, a fibroblastic characteristic. This distinctdifference in cell shape was also noted after IF with SMA, which wasfound to be present in both the keratocyte-like and fibroblast-likematrices. Collagens I, III, and V localization were similar in allcultures. Most interestingly, cFN and TSP-1 expression was greatlyreduced in the keratocyte-like co-cultures compared to the fibroblastlikeco-cultures.Conclusions: We have developed models that allow for thecomparison of the interaction of human corneal epithelial cells withkeratocyte-like and fibroblast-like self-assembled matrices. Thefibroblast-like matrix appears to stimulate wound-healing responses(as indicated by TSP-1 and cFN expression) to a greater extent thanthe keratocyte-like matrix.Commercial Relationships: Audrey E. Hutcheon, None; DimitriosKaramichos, None; Xiaoqing Q. Guo, None; James D. Zieske,NoneSupport: NIH Grants EY005665, EY020886, EY03790 (Core) andDOD W81XWH-11-1-0477Program Number: 5241 Poster Board Number: C0160Presentation Time: 2:45 PM - 4:30 PMRNAi Gene Silencing Of TGF-beta Signaling: A PowerfulApproach To Control Corneal FibrosisJason T. Rodier, Ajay Sharma, Ashish Tandon, Audra Stallard, RajivR. Mohan. Mason Eye Institute, University of Missouri-Columbia,Columbia, MO.Purpose: Transforming growth factor β (TGFβ) promotes keratocytetransdifferentiation to myofibroblast and cause corneal fibrosis invivo. Our siRNA studies demonstrated that TGFβ primarily usesSMAD signaling for this transformation in the cornea. The presentstudy tested the hypothesis that SMAD-2, SMAD-3 or SMAD-4 genesilencing is a novel approach to treat corneal scarring using an invitro model. We quantified the potency of SMAD gene silencing toabrogate TGFβ pathology and myofibroblast formation usingSMADs siRNA, and (2) examined whether RNA inference (RNAi) orshort-hairpin RNA (shRNA) is a better modality to achieve sustainedSMAD gene silencing in the cornea for gene therapy using an in vitromodel.Methods: : Human donor corneas were used to obtain human cornalefibroblasts (HCF). TGFβ1 (5ng/ml) was used to induce HCFtransformation myofibroblasts under serum-free conditions.Commercial pre-validated siRNA specific for SMADs were used.The shRNA and RNAi sequences specific for SMAD-2, SMAD-3and SMAD-4 were designed using RNAi software, and cloned intomammalian expression vectors to generated SMAD-shRNA/RNAivector constructs. Lipofectamine-2000 was used for transfection.Quantitative real-time PCR, western blotting and©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.