Corneal cell and molecular biology

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May 6, 2015 ... Stem Cell Biology, Aravind Medical Research Foundation, Madurai,. India. Purpose: .... high levels of α-SMA and were myofibroblasts (keratocan- lumican- ..... Forced Differentiation of Corneal Stromal Cell Spheroids Yields.

ARVO 2015 Annual Meeting Abstracts 470 Corneal cell and molecular biology Wednesday, May 06, 2015 3:45 PM–5:30 PM Exhibit Hall Poster Session Program #/Board # Range: 4896–4919/A0170–A0193 Organizing Section: Cornea Contributing Section(s): Genetics Program Number: 4896 Poster Board Number: A0170 Presentation Time: 3:45 PM–5:30 PM Wnt10b enhances human corneal endothelial cell proliferation through β-catenin and Rac1 activation JeongGoo Lee, J M. Heur. University of Southern California Eye Institute, Los Angeles, CA. Purpose: Wnt10b activates β-catenin dependent pathways for regulation of cellular functions that play crucial roles in wound healing including cell proliferation. Elucidation of Wnt10b signaling for induction of cell proliferation could help identify potential therapeutic targets for patients with vision loss due to endothelial dysfunction. However, Wnt10b signaling in corneal endothelial cells (CEC) has not been well characterized. Methods: Expression and/or activation of Wnt10b, LRP-6, β-catenin, Rac1, Tiam1 and Cyclin D1 were analyzed by immunoblotting. MTT assay was employed to measure cell proliferation rates. Activation of Rac1 and RhoA were determined by Rac-GTP pull-down assay and RhoA specific G-LISA assay, respectively. Results: Transient induction of Wnt10b by IL-1b-stimulation proceeds through both NF-kB and AP-1 pathway in human CECs. This leads to phosphorylation of LRP-6, resulting in activation of disheveled and subsequent nuclear translocation of Rac1 and β-catenin. Formation of nuclear β-catenin-Rac1 complex induced expression of Cyclin D1. Induction of cell proliferation by Wnt10b was completely blocked by NSC23766 (Rac1 inhibitor), but not by ML141 (Cdc42 inhibitor). Co-treatment of both Wnt10b and RhoA activator resulted in ~20% decrease of proliferation compared to Wnt10b treatment, suggesting that Rac1 plays key role for cell proliferation, whereas Cdc42 and RhoA have limited effect on cell proliferation induced by Wnt10b in human CECs. Conclusions: These findings suggest that Wnt10b induced by IL-1β through NF-kB and AP-1 in human CEC promotes cell proliferation through activation of Rac1 and β-catenin. Commercial Relationships: JeongGoo Lee, None; J M. Heur, None Support: RPB Career Development Award and NIH Grant EY021485 Program Number: 4897 Poster Board Number: A0171 Presentation Time: 3:45 PM–5:30 PM UVA Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3 Dependent Apoptosis in Corneal Endothelial Cells Cailing Liu1, Dijana Vojnovic1, Irene E. Kochevar2, Ula V. Jurkunas1. 1 Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA; 2Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA. Purpose: Previously, we reported that Nrf2 deficiency contributes to oxidant-antioxidant imbalance, causing apoptosis in Fuchs corneal endothelial dystrophy (FECD). To mimic the oxidative stress induced by sunlight, we utilized ultraviolet-A (UVA) irradiation to challenge human corneal endothelial cells (CEC). The aim of this project is to examine whether Nrf2-regulated antioxidant defense is activated in corneal endothelium by UVA light.

Methods: An immortalized human CEC (HCECi) was exposed to a UVA lamp to induce oxidative stress. Cells grown in complete medium were pretreated in OPTI-MEM medium 24 hrs prior to UVA treatment. Post UVA treatment with a dose of 2.5, 5, 10 or 25 J/ cm2, the cells were recovered in OPTI-MEM medium for 0, 3, 6, 18 or 24 hrs at 37 oC. Reactive Oxygen Species (ROS) were detected with carboxy-H2DCFDA. Cell viability was evaluated by Trypan Blue staining or by measuring the released lactate dehydrogenase in supernatants. The mRNA levels of Nrf2 target genes HO-1 and NQO1 were quantified by real-time PCR. The protein levels of phospho-p53 were examined by immunoblotting. Activated Caspase3 was examined by immunoblotting and by a fluorescence assay. At each recovery time, the non-UVA treatment was used as a control. Results: Exposure of HCECi cells to 5 J/cm2, 10 J/cm2 and 25 J/ cm2 UVA irradiation generated elevated levels of ROS production as compared to no UVA irradiation, but did not affect cell viability. At 6 hrs post irradiation, 5 J/cm2, 10 J/cm2 and 25 J/cm2 UVA irradiation led to a 1.5 to 1.6-fold increase in HO-1 mRNA as compared to controls. Similarly, 2.5 J/cm2 and 5 J/cm2 UVA irradiation caused a 1.3 to 1.5-fold increase in NQO-1 mRNA as compared to controls. Interestingly, 25 J/cm2 UVA treatment decreased NQO-1 mRNA levels at 6 hrs post irradiation. At 24 hrs post treatment, 5 J/cm2, 10 J/cm2 and 25 J/cm2 UVA exposure yielded a 1.3 to 1.8-fold increase in phospho- p53 and a 2.0 to 6.0-fold increase in activated Caspase3 levels as compared to no UVA treatment, resulting in 20-48% cell death. Conclusions: Irradiation of HCECi cell with UVA light resulted in ROS elevation and induction of Nrf2-regulated anti-oxidant defense followed by phospho-p53 and Caspase3 activation. The initial activation of antioxidant defense exerted a cytoprotective effect which was lost with increasing recovery times and doses of UVAinduced stress. Commercial Relationships: Cailing Liu, None; Dijana Vojnovic, None; Irene E. Kochevar, None; Ula V. Jurkunas, None Support: NIH/NEI Grant R01 EY20581 (UVJ) Program Number: 4898 Poster Board Number: A0172 Presentation Time: 3:45 PM–5:30 PM A Newly Discovered Transglutaminase-2-Paxillin Binding is Important for Adhesion Complex Regulation evelyn png1, 2, Kh Poon1, Louis Tong1, 2. 1Singapore Eye Research Institute, Singapore, Singapore; 2Department of Ophthalmology, Yong Loo Lin School of Medicine, Singapore, Singapore. Purpose: Cell adhesion and migration in ocular surface wound healing underpin diseases such as allergies and persistent epithelial defects. Transglutaminase (TG)-2 is a multifunctional protein which plays important role in cell adhesion and migration and can be found in the ocular surface. Previously we showed that reduction of TG-2 by RNA interference reduced cell adhesion, spreading and migration. Also, we found that interference with TG-2 was associated to reduce phosphorylation of paxillin at serine 178, which are important for cell adhesion and migration. We aimed to evaluate the possible interaction between TG-2 and paxillin. Methods: In this study, stable human corneal epithelial cell line expressing shRNA targeting TG-2 (shTG) and non-specific scrambled shRNA (control) were used. A combination of techniques such as co-immunoprecipitation, western blotting, in-vitro protein interaction assay based on non-label optic grating sensing, in-vitro autoradiographic kinase assay and immunohistochemistry were performed to evaluate the possible relationship between TG-2 and paxillin. Results: Paxillin co-immunoprecipitated with TG-2 in corneal epithelial cell lysates. Other adhesion proteins such as vinculin and

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ARVO 2015 Annual Meeting Abstracts focal adhesion kinase were also present in these paxillin containing complexes. In-vitro testing showed that TG-2 interacted significantly with recombinant paxillin in a dose dependent manner. As a positive control, recombinant beta-3 integrin also bind to immobilised paxillin. In our kinase assay, recombinant TG-2 was unable to phosphorylate purified paxillin. A library of 190 known serine/ threonine kinases was screened and after accounting for the autophosphorylation of the kinases, JNK1 was found to have the highest relative activity. Immunohistochemistry performed on migrating corneal epithelial cell sheet after scratch assay showed that paxillin in shTG cells were not localized in the advancing edge of the leading cell, unlike in control cells. Conclusions: TG-2 interacts directly with paxillin in adhesion complexes, but it does not directly phosphorylate paxillin. The relationship between TG-2 and paxillin could be used as a possible target in ocular wound healing. In addition, JNK1 may be the kinase that mediates the phosphorylation of paxillin. Commercial Relationships: evelyn png, None; Kh Poon, None; Louis Tong, None Support: NMRC/CSA/045/2012 Program Number: 4899 Poster Board Number: A0173 Presentation Time: 3:45 PM–5:30 PM miR-203 inhibits ΔNp63α dependent clonogenicity in corneal epithelial stem cells (CESCs) Jhansi Rani kasinathan, Veerappan Muthukkaruppan, Chidambaranathan Gowri Priya. Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Madurai, India. Purpose: One of the isoforms of the nuclear transcription factor p63 - ΔNp63α is expressed higher in CESCs in comparision to the differentiated cells. In keratinocytes, miR-203 has been reported to repress stemness by inhibiting ΔNp63α expression. This study aims to elucidate whether miR-203 has a similar influence in ΔNp63α isoform expression and in the maintanence of stemness in the nonkeratinized corneal epithelium. Methods: Limbal explant cultures were carried out using human globes (Arpitha et al., 2008). After 21 days of culture, the cells were trypsinized and 1.5 X 105cells were transfected with 20nm premiR-203, antago-miR-203 and scrambled sequence using HiPerfect transfection reagent (Qiagen) (Lena et al., 2008). After 48 hours of transfection, (i) 2000 cells were seeded in mitomycin treated 3T3 feeder layer and stained for rhodamine after culturing for 7 days to evaluate their colony forming efficiency (Arpitha et al., 2008). (ii) RNA extraction was performed using Qiagen RNeasy mini kit followed by semi quantitative RT-PCR to analyze the expression of ΔNp63α and β isoforms (Di Iorio et al., 2005) in the transfected cells. Results: Semi quantitative RT-PCR analysis revealed that premiR-203 represses ΔNp63α expression compared to control (transfected with scrambled sequence). In contrast, antago-miR-203 enhanced ΔNp63α expression. Similarly, colony forming efficiency of cells treated with antago-miR-203 was higher (2.2%) compared to control (1.4%) and was significantly reduced to 0.1% with premiR-203. Conclusions: miR-203 specifically inhibits the proliferative potential in CESCs by regulating ΔNp63α expression. Further studies are essential to understand the signaling pathways associated with this molecular regulation of stemness. Commercial Relationships: Jhansi Rani kasinathan, None; Veerappan Muthukkaruppan, None; Chidambaranathan Gowri Priya, None

Program Number: 4900 Poster Board Number: A0174 Presentation Time: 3:45 PM–5:30 PM Measurement of In Vivo Three-dimensional Corneal Cell Density and Size Using Two-photon Imaging in C57BL/6 Mice Hongmin Zhang, Siyu He, Susu Liu, Guoming Chen, Junjie Zhang, Shengtao Sun, Liya Wang. Henan Key Laboratory of Keratopathy, Henan Eye Institute Henan Eye Hospital, Zhengzhou, China. Purpose: To measure the cell size and cell density in five layers of the central cornea: the superficial epithelium, the basal epithelium, the anterior stroma, the posterior stroma, and the endothelium in the widely used inbred C57BL/6 mouse strain based on in vivo threedimensional (3D) Two-photon (2PH) imaging Methods: Corneas were scanned using a 2PH laser scanning uorescence microscope after staining with plasma membrane stain and Hoechst 33342. Cell density and Cell size measurements including cell surface area, cell volume, nuclear surface area, and nuclear volume were automatically quantified using the IMARIS software. The cell and nuclear surface-area-to-volume ratio (S:V ratio) and the cell nuclear-cytoplasmic (N:C) ratio were calculated. Results: The cell density was highest in the basal epithelium layer and lowest in the posterior stroma. The cell surface-area was highest in the anterior stroma, and the cell and nuclear volume was highest in the superficial epithelium. The cell surface-area, the cell and nuclear volume were both lowest in the basal epithelium. The cell and nuclear S:V ratio was highest in the basal epithelium and lowest in the superficial epithelium. The N:C ratio was highest in the basal epithelial cells and lowest in the posterior keratocytes. Conclusions: The present study is the first to quantify the cell density and size parameters in the five layers of the central cornea. These data provide important cell morphology features for the study of corneal physiology, pathology and disease in C57BL/6 mice. Commercial Relationships: Hongmin Zhang, None; Siyu He, None; Susu Liu, None; Guoming Chen, None; Junjie Zhang, None; Shengtao Sun, None; Liya Wang, None Program Number: 4901 Poster Board Number: A0175 Presentation Time: 3:45 PM–5:30 PM Galectin-3 and CD147 Colocalization in Sterile Corneal Ulcers Andrea Cruzat1, 2, Jerome Mauris2, Miguel Gonzalez1, 2, Marie-Claude Robert1, Kenneth Kenyon3, 2, James Chodosh1, Claes H. Dohlman1, Pablo Argueso2. 1Cornea / Ophthalmology, Harvard Medical Sch/ MEEI, Boston, MA; 2Schepens Eye Research Institute, Harvard Medical School, Boston, MA; 3New England Eye Center, Tufts University School of Medicine, Boston, MA. Purpose: Galectin-3 is a carbohydrate binding protein known to promote secretion of matrix metalloproteinases, a hallmark of ulceration, through interaction with the metalloproteinase inducer CD147. The aim of this study was to investigate the localization of galectin-3 and CD147 in patients with sterile corneal melting. Methods: Human tissue was collected from six patients with active corneal ulceration due to Boston keratoprosthesis implants, rheumatoid arthritis, and mucous membrane pemphigoid. Galectin-3 and CD147 were localized on human tissue cryosections by immunofluorescence microscopy. Biologic assessment of gelatinolytic activity was evaluated by in situ zymography. Results: Galectin-3 staining in the population study was predominant on apical cells of the stratified corneal epithelium, whereas discrete binding was observed on basal epithelial cells and keratocytes. Sporadic colocalization of CD147 and galectin-3 was observed at the epithelial-stromal junction and in corneal keratocytes, but not on the apical portion of the epithelium. Moreover, increased gelatinolytic activity was present within areas of galectin-3 and CD147 colocalization in the epithelial-stromal junction and keratocytes.

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ARVO 2015 Annual Meeting Abstracts Conclusions: Galectin-3 and CD147 colocalize in basal corneal epithelial cells and in corneal keratocytes within areas associated with increased gelatinolytic activity. Sustained up-regulation of these molecules may potentially lead to stromal matrix degradation and be clinically relevant to corneal ulceration. Commercial Relationships: Andrea Cruzat, None; Jerome Mauris, None; Miguel Gonzalez, None; Marie-Claude Robert, None; Kenneth Kenyon, None; James Chodosh, None; Claes H. Dohlman, None; Pablo Argueso, None Support: NIH/NEI EY024031 Program Number: 4902 Poster Board Number: A0176 Presentation Time: 3:45 PM–5:30 PM The epithelial basement membrane component perlecan is produced by stromal cells in vitro Abirami Santhanam1, Andre Torricelli2, Jiahui Wu1, Steven E. Wilson1. 1 Ophthalmology, Cleveland clinic foundation, Shaker heights, OH; 2 University of Sao Paulo, Sao Paulo, Brazil. Purpose: To investigate the production of the corneal epithelial basement membrane (BM) component perlecan by cultured stromal cells. Methods: Keratocytes were isolated from fresh rabbit corneal stroma treated with hyaluronidase and collagenase for 24 hours. Keratocytes were then grown in different serum conditions 1%, 5% and 10% FBS with or without the growth factors FGF-2 (40ng/ ml) and heparin sulfate (HS) (5ug/ml) or TGF-β (2ng/ml) for 60-72 hours. Different culture condition effects were analyzed by real time PCR, immunostaining and western blots for the cell specific markers keratocan, lumican and alpha-smooth muscle actin (SMA). Perlecan mRNA synthesis was analyzed by QPCR and protein production by western blotting, immunostaining and ELISA. Results: Keratocytes grown in serum-free medium expressed high levels of keratocan and lumican (keratocan+, lumican+ and SMA-) while those grown in 10% FBS with FGF-2 expressed low levels and were corneal fibroblasts (keratocan- lumican- and SMA-). As the serum concentration increased, the cultured cells became less keratocyte and more corneal fibroblast in phenotype. Analysis of α-SMA revealed that cells grown in 1% FBS with TGF-β expressed high levels of α-SMA and were myofibroblasts (keratocan- lumicanand SMA+). Keratocytes produced more perlecan protein than corneal fibroblasts or myofibroblasts (Fig.1). Conclusions: Keratocytes grown in vitro produce perlecan that likely contributes to epithelial basement membrane regeneration in vivo. Corneal fibroblasts and myofibroblasts also produce perlecan, albeit at lower levels than keratocytes.

Figure 1. Perlecan immunostaining under different culture conditions. Keratocytes produce more perlecan protein (red) compared to corneal fibroblasts and myofibroblasts. Blue is DAPI staining of cell nuclei. Magnification 400x. Commercial Relationships: Abirami Santhanam, None; Andre Torricelli, None; Jiahui Wu, None; Steven E. Wilson, None Support: Supported by EY10056 and Research to Prevent Blindness, New York, NY Program Number: 4903 Poster Board Number: A0177 Presentation Time: 3:45 PM–5:30 PM Elucidating the molecular basis of PPCD3: reduced ZEB1 protein levels do not affect corneal endothelial cell apoptosis Benjamin R. Lin, Ricardo F. Frausto, Judy L. Chen, Doug Chung, Anthony J. Aldave. Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, CA. Purpose: Posterior polymorphous corneal dystrophy 3 (PPCD3) is associated with a monoallelic deficiency secondary to nonsense and frameshift mutations in ZEB1. The purpose of this study was to investigate the impact of ZEB1 knockdown on the induction of apoptosis via doxorubicin hydrochloride (DOX) in a human corneal endothelial cell line. Methods: ZEB1 protein levels in a human corneal endothelial cell line (HCEnC-21T) were reduced by transfection with 4 nM ZEB1 siRNA, with scrambled siRNA used as a control. Forty eight hours after siRNA transfection, HCEnC-21T cells were treated with 2.5mM of DOX. Whole-cell protein lysates were prepared at 0, 3, 6, 9, and 12 hours after DOX treatment and subjected to SDS-PAGE. Apoptosis was monitored using phase-contrast microscopy. ZEB1 and cleaved caspase 3 (cCASP3), an apoptosis marker, were detected by immunoblotting. Densitometric analysis for ZEB1 and cCASP3 protein levels was performed using ImageJ software. Results: Forty-eight hours after transfection with ZEB1 siRNA, ZEB1 protein levels were reduced by 58% while no reduction was observed after transfection with scrambled siRNA. Phase-contrast microscopy demonstrated an increasing number of phase-positive cells and other features indicative of apoptosis in both ZEB1 siRNA and scrambled siRNA transfected cells as DOX treatment time

©2015, 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 permission to reproduce any abstract, contact the ARVO Office at [email protected]

ARVO 2015 Annual Meeting Abstracts increased. Transfection with ZEB1 siRNA did not significantly impact the induction or progression of apoptosis as measured by the appearance of cCASP3 protein 12 hours after DOX treatment. ZEB1 protein levels were inversely correlated with increasing cCASP3 protein levels. Twelve hours after DOX treatment, a significant decrease was observed in the levels of ZEB1 from levels 0-3 hours after DOX treatment in both ZEB1 siRNA and control siRNA transfected cells. Conclusions: A ZEB1 protein deficiency secondary to truncating mutations in ZEB1 is postulated as the cause of PPCD3. Reduction of ZEB1 protein levels in HCEnC-21T cells does not impact the induction or progression of apoptosis. However, an inverse relationship between ZEB1 levels and cCASP3 was observed, suggesting that ZEB1 levels are negatively regulated downstream of apoptotic stimuli. Although an initial reduction in ZEB1 levels does not have an impact on apoptosis, its reduction in response to apoptosis suggests that its role in this cellular process warrants further investigation. Commercial Relationships: Benjamin R. Lin, None; Ricardo F. Frausto, None; Judy L. Chen, None; Doug Chung, None; Anthony J. Aldave, None Support: NEI R01 EY022082, Research to Prevent Blindness Program Number: 4904 Poster Board Number: A0178 Presentation Time: 3:45 PM–5:30 PM ALDEHYDE DEHYDROGENASE 1A1 IN GUINEA PIG CORNEA: MOLECULAR FEATURES AND TISSUE LOCALIZATION Maria Fernanda F. Suarez1, Leandro Correa2, Evangelina Esposito2, Constanza Insfran1, Julio A. Urrets-Zavalia2, Horacio M. Serra1. 1 Department of Clinical Biochemistry, CIBICI CONICET, School of Chemical Sciences, National University of Cordoba, Cordoba, Argentina; 2Department of Ophthalmology, University Clinic Reina Fabiola and School of Medicine, Catholic University of Córdoba, Cordoba, Argentina. Purpose: Climatic droplet keratopathy (CDK) is a human degenerative corneal disease characterized by protein aggregation under the epithelium and caused by unfavorable environmental conditions (chronic exposure to ultraviolet radiation (UVR), constant erosions of the cornea) which induce oxidative stress. In an attempt to develop an experimental animal model for CDK, we previously studied the effect of ascorbate (antioxidant agent present in the cornea) in the cornea of healthy guinea pigs exposed to ultraviolet radiation (UVR) and erosions. Since aldehyde dehydrogenase 1A1 (ALDH1A1) also contribute to corneal preservation against oxidative stress, we decided to study the molecular aspects and localization of this enzyme in guinea pig cornea. Methods: Total corneal extract as well as epithelium, stroma and endothelium extracts from male guinea pigs were used. The expression of ALDH1A1 was studied by means of immunoblotting, and immunofluorescence assays. The existence of isoforms for the enzyme was investigated by isoelectric focusing. Results: ALDH1A1 from guinea pig corneas presents some unique characteristics compared with other mammals. This enzyme has a molecular weight of about 54 kDa, as another crystalline present in the cornea (ALDH3A1), previously characterized by our group in corneal epithelial cells of normal guinea pigs. Isoelectric focusing studies revealed the existence of a single isoenzyme for ALDH1A1 with a pI of approximately 9 as opposed to the two isoforms of the enzyme ALDH3A1. Immunofluorescence studies showed that ALDH1A1 enzyme as well as ALDH3A1 enzyme is expressed in epithelial cells, keratocytes and endothelial cells from the cornea.

Conclusions: In this work we identified and molecularly characterized for the first time, the enzyme ALDH1A1 in the different cellular compartments of normal guinea pig cornea. We also describe their tissue location and compare it to the location of the other crystalline (ALDH3A1). Commercial Relationships: Maria Fernanda F. Suarez, None; Leandro Correa, None; Evangelina Esposito, None; Constanza Insfran, None; Julio A. Urrets-Zavalia, None; Horacio M. Serra, None Support: SECYT-UNC, CONICET, FONCYT 2011-1846 Program Number: 4905 Poster Board Number: A0179 Presentation Time: 3:45 PM–5:30 PM Hyperosmotic shock activates RhoA and Rac1 in corneal endothelial cells Diana Santander-Garcia1, 2, Beatriz Marcos-Ramiro2, Susana Barroso2, Ignacio Jimenez-Alfaro3, 4, Jaime Millan2. 1Ophthalmology, Hospital Universitario Rey Juan Carlos, Móstoles, Spain; 2Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain; 3 Department of Ophthalmology, Fundación Jiménez Díaz, Madrid, Spain; 4Surgery Department, Universidad Autónoma, Madrid, Spain. Purpose: To study the involvement of Rho GTPase-mediated pathways in the corneal endothelial barrier response to hypertonic stress. Methods: Experiments were performed in human endothelial cell line HCEC B4G12 in vitro. After exposure to hypertonic stress, the activity of the Rho GTPases RhoA, Rac1 and Cdc42 was analyzed by pulldown assay. The activation of downstream effectors of Rho was detected by western blot. Electric Cell-Substrate Impedance Sensing (ECIS) of endothelial monolayers was measured to assess the influence of osmotic stress on barrier function. Endothelial barrier integrity was also analyzed by immunofluorescence and confocal microscopy. To determine the relative contribution of RhoA- and Rac1 mediated pathways to the corneal endothelial responses, we tested the effect of Y-27632, the inhibitor of the RhoA effector Rho kinase (ROCK), and of EHop-016, a new inhibitor of Rac1, on endothelial barrier disruption in response to hypertonic stress. Results: Exposure to NaCl (487 mM; 10 min) generated cell contraction, which was detected by a 60% decrease in transendothelial electrical resistance. NaCl induced the activation of both RhoA (p=0, 0441) and Rac1 (p= 0,0437), but not of Cdc42. Phosphorylation of myosin light chain and ezrin-radixin-moesin proteins, which are activated downstream RhoA was also induced 2 fold (p=0.08) and 1.67 fold (p=0.043), respectively. Incubation with the ROCK inhibitor Y-27632 (5 microM) attenuated resistance decrease upon NaCl exposure and delayed barrier recovery after stress withdrawal. In contrast, EHop-016 (1 microM) did not produce any change in the barrier response to stress. Conclusions: Both RhoA and Rac1 signalling are activated during the contraction of human corneal endothelial cells after exposure to hypertonicity. ROCK signalling inhibition by Y-27632 reduces the dynamics of barrier remodeling upon exposure and withdrawal of hyperosmotic stress. Commercial Relationships: Diana Santander-Garcia, None; Beatriz Marcos-Ramiro, None; Susana Barroso, None; Ignacio Jimenez-Alfaro, None; Jaime Millan, None

©2015, 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 permission to reproduce any abstract, contact the ARVO Office at [email protected]

ARVO 2015 Annual Meeting Abstracts Program Number: 4906 Poster Board Number: A0180 Presentation Time: 3:45 PM–5:30 PM Blockage of Smad-Signaling Pathway in Human Corneal Fibroblasts Xiaoqing Q. Guo, Jennifer A. Tran, Audrey E. Hutcheon, James D. Zieske. Department of Ophthalmology, Harvard Medical School, Schepens Eye Research Institute/MEE, Boston, MA. Purpose: Transforming growth factor beta (TGFβ) affects both corneal epithelial and stromal wound healing mechanisms by activating several signaling pathways. One of these being the Smad pathway, which, in addition to TGFβ, requires the presence of Smad Anchor for Receptor Activation (SARA) to become activated. Previous studies in human corneal epithelial cells (HCE) have shown that Trx-SARA, an inhibitor of SARA, can effectively inhibit the Smad pathway. In the current study, we investigated the use of Trx-SARA in primary human corneal fibroblasts (HCF) to discern the relationship between the Smad pathway and the activation of a known TGFβ-target protein, thrombospondin-1 (TSP-1), and HCF proliferation. Methods: Retroviruses (RTV) were made with either Trx-SARA or Trx-GA, a control plasmid. HCF ± Trx-SARA or -GA were serum starved and then grown with 2ng/ml TGFβ1 overnight. Samples with no TGFβ1-treatment served as controls. Samples were collected and then processed to examine the effects of Trx-SARA on TSP1 expression by western blotting (WB) or on Ki67 expression by indirect-immunofluorescence (IF). Results: Inhibition of Smad signaling reduced HCF proliferation by almost 50%; however, blocking Smad signaling had no effect on TSP-1 expression in HCF, which is in contrast to HCE data where TSP-1 expression was reduced by 90%. Conclusions: Trx-SARA is an effective inhibitor of the Smad pathway and a useful tool for studying TSP-1 activation. TSP1 response to TGFβ1 was not inhibited by Trx-SARA in HCF, suggesting that different signaling pathways are used to activate TSP-1 in HCF and HCE. Interestingly, Trx-SARA inhibited HCF proliferation indicating that proliferation is dependent on Smad signaling. Commercial Relationships: Xiaoqing Q. Guo, None; Jennifer A. Tran, None; Audrey E. Hutcheon, None; James D. Zieske, None Support: NIH Grant R01EY005665, NIH Grant P30EY03790 Program Number: 4907 Poster Board Number: A0181 Presentation Time: 3:45 PM–5:30 PM TSLP bridging innate and acquired immunity in corneal cell lines challenged by Aspergillus fumigates Xinyi Wu, Leyi Wang, Luping Wang, Xiaoxiao Ren. Ophthalmology, Qilu Hospital, Shandong University, Jinan, China. Purpose: We investigated the expression and function of thymic stromal lymphopoietin (TSLP) in telomerase-immortalized human corneal epithelial cells (THCEs) and telomerase-immortalized human stroma fibroblasts (THSFs) challenged by A. fumigatus hyphae and its relationship with Toll-like receptors (TLR). Methods: We stimulated THCEs and THSFs with TLR2 Ligand zymosan, TLR4 ligand lipopolysaccharide (LPS), human recombinant TSLP or A. fumigatus hyphae for various periods, with or without the inhibition of TLR2, TLR4 or TSLP using monoclonal antibody or small interfering RNA previously. The release and expression of TLR2, TLR4, TSLP, IL-4, IL-8, IL-13 and TNF-α were measured by means of ELISA, quantitative RT-PCR or western blot. Results: It was demonstrated that enhanced expression of TSLP, IL-4 and IL-13 was associated with the treatment of A. fumigatus hyphae in human corneal cell lines. Stimulation of THCEs and THSFs with TLR2 Ligand zymosan or TLR4 ligand lipopolysaccharide (LPS)

induced upregulated gene expression and release of TSLP; Inhibition of either TLR2 or TLR4 with monoclonal antibody impaired the A. fumigatus hyphae–induced TSLP and its downstream cytokines expression. The human recombinant TSLP augmented genetic and protein levels of TLR2 and TLR4 in human corneal cell lines; Small interfering RNA–mediated knockdown of TSLP hindered the expression of TLRs, IL-8 and TNF-α in AF-treated THCEs and THSFs. Conclusions: These results indicated that TSLP, as a critical factor in adaptive immunity, participated in the immune response of THCEs and THSFs challenge by Aspergillus fumigates, which is closely related to the function of TLRs, key players in innate immunity. At the same time, the innate immunity mediated by TLRs could be enhanced by TSLP in AF-treated THCEs and THSFs. Thus, TSLP may serve as a link between the innate and adaptive immune responses in corneal cell lines infected by Aspergillus fumigates. Commercial Relationships: Xinyi Wu, None; Leyi Wang, None; Luping Wang, None; Xiaoxiao Ren, None Support: NSFC Grant 81070707 Program Number: 4908 Poster Board Number: A0182 Presentation Time: 3:45 PM–5:30 PM CFSE fluorescence staining to detect daily cell movement in normal human corneas Chiara Bonzano1, Sara Olivari1, Barbara Canciani2, Marina Papadia1, Alessandro Bagnis1, Ranieri Cancedda2, Carlo E. Traverso1. 1DINOGMI, Eye Clinic University of Genoa, Genoa, Italy; 2Department of Oncology, Biology and Genetics, University of Genoa, Genoa, Italy. Purpose: We used our human limbal stem cell labelling method previously described to investigate epithelial cell movements and record cell migration daily for one week. Methods: Fresh normal human corneas from donors not suitable for transplantation were provided by the Melvin Jones Lions Eye Bank (Genoa, Italy) and used for the study. Carboxyfluorescein diacetate succinimidyl esters (CFSE) (Molecular Probes) was used to target corneal epithelial cells; fluorescence was digitally recorded for seven consecutive days (T1-T7), and the rate of cell movement was determined by tracing the different position of the CFSE-labelled limbal stem cells. A daily time-lapse sequence was digitally recorded. Cell movement was analyzed in frozen cross- sections by fluorescence microscopy. Results: Daily average CFSE-labelled limbal stem cell movement was 0.073 cm ± 0.01 (± SD) / day. Conclusions: CFSE staining allowed us to track corneal epithelial cells, from the limbal basal layer centripetally to the superficial epithelium and showed to be a reliable method able to observe and quantify the migration of epithelial cells on a daily basis. CFSElabelled cells moved towards the corneal surface in one week. Commercial Relationships: Chiara Bonzano, None; Sara Olivari, None; Barbara Canciani, None; Marina Papadia, None; Alessandro Bagnis, None; Ranieri Cancedda, None; Carlo E. Traverso, None Program Number: 4909 Poster Board Number: A0183 Presentation Time: 3:45 PM–5:30 PM Pigment epithelial derived factor (PEDF) and 44-mer and 34-mer PEDF peptides in the presence of docosahexaenoic acid (DHA) selectively increase neurotrophins and related genes in mouse epithelium Azucena H. Kakazu, Jorgelina M. Calandria, Jiucheng He, Haydee E. Bazan. Ophthalmology/Neuroscience Center, LSU Health Sciences Center, New Orleans, LA.

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ARVO 2015 Annual Meeting Abstracts Purpose: Previous studies in our laboratory have shown that PEDF and the 44-mer PEDF peptide (with neurotrophic activity), but not the 34-mer PEDF (with antiangiogenic activity), in association with DHA stimulates nerve regeneration. The mechanism of axonal regeneration is not completely understood but could involve the action of neurotrophins stimulated in the target tissue. Here we investigate the pattern of neurotrophin genes activated by PEDF and its peptides (44 mer and 34 mer) plus DHA in mouse corneal epithelium. Methods: C57BL/6 mouse eyes were incubated in DMEM/F12 alone or in the presence of 50ng/ml PEDF + 50nM DHA, 5ng 34-mer PEDF+DHA, or 5ng/ml 44-mer PEDF+DHA at 370C for 3h. Epithelia were collected and RNA was purified and reversetranscripted using iScript Reverse Transcription Supermix for RTqPCR (Bio-Rad). cDNA was amplified using SsoAdvanced SYBR Green Supermix (Bio-Rad); 10 ul of PCR mix was added to each well from the PrimePCR Custom Plate 384 wells for 62 genes (Bio-Rad). Data was collected and analyzed using CFX Manager 3.0 software. Values more than 2 fold increase were considered significant. Results: Nine out of 62 genes were found to increase after the different treatments. Treatment with PEDF+DHA and with the two PEDF peptides in the presence of DHA increased the expression of Fgf2 gene between 7 and 12 fold with respect to control. This gene encodes the synthesis of fibroblast growth factor-2 (FGF-2). The expression of the Ntf3 gene was increased 6 times when the corneas were incubated in PEDF+DHA, but was not stimulated in the presence of the PEDF derivatives. Expression of Npff and Ppy1 genes increased more than 2 fold in the presence of PEDF+DHA or the 44-mer PEDF+DHA. Conclusions: Several genes that have neurotrophic activities and have not been described previously were found in the corneal epithelium when incubated with PEDF, the 44-mer PEDF or the 34mer PEDF in the presence of DHA. Fgf2 expression increased in the three conditions. In the cornea, FGF-2 accelerated epithelial wound healing. Our previous studies show that PEDF+DHA treatment increases proliferation of epithelial cells and wound healing, and this function could be related to the entire PEDF molecule as well as the 34 mer peptide without neurotrophic properties. Commercial Relationships: Azucena H. Kakazu, None; Jorgelina M. Calandria, None; Jiucheng He, None; Haydee E. Bazan, None Support: NIH Grant EY019465 Program Number: 4910 Poster Board Number: A0184 Presentation Time: 3:45 PM–5:30 PM Modification of Supplemented Hormonal Epithelial Medium to Improve the Expansion of Human Limbal Epithelial Stem/ Progenitor Cells Sheyla Gonzalez, Hua Mei, Sophie X. Deng. Ophthalmology, Jules Stein Eye Institute UCLA, Los Angeles, CA. Purpose: To investigate supplemented hormonal epithelial medium (SHEM) in combination with embryonic stem cell medium (ESCM) for the expansion of human limbal epithelial stem/progenitor cells (LSCs). Methods: Limbal explants were cultured on denuded amniotic membrane in SHEM supplemented with 5% human serum, ESCM and a mixture of SHEM-ESCM (1:1). Single LSCs cultured on 3T3 feeder cells in SHEM were used as a control. Cell morphology, cell size, cell growth rate, outgrowth size and expression level of putative stem cell and stromal markers were analyzed. Results: Cell growth rate in the ESCM culture was the lowest compared to that in SHEM and SHEM-ESCM cultures (all p