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8. List of Contributors

Author

Abad, Juan-Carlos, primary, Abelson, Mark B, additional, Abramson, David H, additional, Acquadro, Martin A, additional, Adamis, Anthony P, additional, Adams, Wesley H, additional, Afshari, Natalie A, additional, Ai, Everett, additional, Aiello, Lloyd M, additional, Aiello, Lloyd P, additional, Akduman, Levent, additional, Albano, Marissa L, additional, Albert, Daniel M., additional, Alexandrou, Terry J, additional, Alfonso, Eduardo C, additional, Alié, Jorge L, additional, Alizadeh, Hassan, additional, Al Jadaan, Ibrahim A, additional, Al-Jastaneiah, Sabah, additional, Allen, Calliope E, additional, Allen, David, additional, Allen, Robert C, additional, Alm, Albert, additional, Al-Swailem, Samar, additional, Alt, Abigail K, additional, Altaweel, Michael M, additional, Anderson, Russell, additional, Andreoli, Christopher M, additional, Androudi, Sofia, additional, Ang, Leonard PK, additional, Anzaar, Fahd, additional, Apple, David J, additional, Arrigg, Claudia A, additional, Artal, Pablo, additional, Asbell, Penny, additional, Asdourian, George K, additional, Atebara, Neal, additional, Atmaca-Sonmez, Pelin, additional, Audo, Isabelle, additional, Auffarth, Gerd U, additional, Avery, Robin K, additional, Azar, Dimitri T, additional, Baker, Ann S, additional, Balles, Mark, additional, Barnes, Scott D, additional, Barnett, Donald M, additional, Barney, Neal P, additional, Barouch, Fina C, additional, Bartley, George B, additional, Barton, Jason JS, additional, Behlau, Irmgard, additional, Belda, Jose I, additional, Bennett, Jeffrey L, additional, Bennett, Timothy J, additional, Berdy, Gregg J, additional, Bernardino, Carlo Roberto, additional, Bernardino, Vitaliano, additional, Berson, Eliot L, additional, Bharadwaj, Amitabh, additional, Bhisitkul, Robert, additional, Bhui, Ravinder D, additional, Bilyk, Jurij, additional, Biousse, Valérie, additional, Bird, Alan C, additional, Blair, Norman Paul, additional, Blodi, Barbara A, additional, Blumenkranz, Mark S, additional, Boldt, H Culver, additional, Borchert, Mark S, additional, Borrillo, Luigi, additional, Borodic, Gary E, additional, Boruchoff, S Arthur, additional, Bose, Swaraj, additional, Boulton, Michael E, additional, Bowman, RW, additional, Bradley, Elizabeth A, additional, Brazitikos, Periklis D, additional, Breeze, Robert, additional, Bressler, Neil M, additional, Bressler, Susan B, additional, Brini, Alfred, additional, Budenz, Donald L, additional, Buffenn, Angela N, additional, Burk, Scott E, additional, Butrus, Salim, additional, Callanan, David, additional, Cameron, J Douglas, additional, Cantor, Louis B, additional, Cantore, William A, additional, Cantu-Dibildox, Jorge, additional, Casas, Victoria, additional, Casper, Miriam, additional, Casten, Robin J, additional, Catoira, Yara P, additional, Cavallerano, Jerry, additional, Chai, Samantha J, additional, Chalita, Maria R, additional, Chamberlain, Sherman M, additional, Chan, Audrey S, additional, Chan, Chi-Chao, additional, Chan, Paul, additional, Chapin, Matthew J, additional, Chapman, Karen L, additional, Chen, Eric, additional, Chen, Joe, additional, Chen, Julie A, additional, Chen, Teresa C, additional, Chen, Zhou, additional, Chévez-Barrios, Patricia, additional, Chew, Emily Y, additional, Chiang, Mark, additional, Chodosh, James, additional, Chong, Eva-Marie, additional, Chun, Denise, additional, Chylack, Leo T, additional, Ciardella, Antonio P, additional, Civan, Mortimer, additional, Clamen, Liane, additional, Clark, John I, additional, Cockerham, Glenn, additional, Cohen, Andre, additional, Cohen, Elisabeth J, additional, Colby, Kathryn A, additional, Coleman, Anne L, additional, Coleman, Hanna R, additional, Colin, Joseph, additional, Collier, J Michael, additional, Comer, Grant M, additional, Conlon, M Ronan, additional, Cooper, Kim E, additional, Corbett, James J, additional, Coma, Miguel C, additional, Cyrlin, Marshall N, additional, Dagi, Linda R, additional, Dahlgren, Matthew A, additional, Daley, Timothy J, additional, Da Mata, Andrea P, additional, Damato, Bertil, additional, D'Amico, Donald J, additional, Dana, Reza, additional, Danan-Husson, Aude, additional, Danesh-Meyer, Helen B, additional, Danis, Ronald P, additional, Darlington, Jason K, additional, Davidson, Stefanie L, additional, Davis, Janet L, additional, Davis, Elizabeth A, additional, de la Cruz, Jose J, additional, de la Garza, Adam G, additional, DeAngelis, Margaret M, additional, DeMartelaere, Sheri L, additional, Demer, Joseph L, additional, Dhaliwal, Avninder, additional, Dieckert, J Paul, additional, Do, Diana V, additional, Doane, Marshall G, additional, Dodds, Christopher, additional, Dohlman, Claes H, additional, Donati, Guy, additional, Donnenfeld, Eric D, additional, Drack, Arlene, additional, Dryja, Thaddeus P, additional, Dueker, David, additional, Duker, Jay S, additional, Dunbar, Jennifer A, additional, Dunn, James P, additional, Dupps, William J, additional, Durand, Marlene L, additional, Dutton, Jonathan J, additional, Eandi, Chiara M, additional, Edward, Deepak P, additional, Egan, Robert A, additional, Eichenbaum, David A, additional, Eklund, Susan E, additional, Engle, Elizabeth C, additional, Erickson, Kristine, additional, Esmaeli, Bita, additional, Fay, Aaron, additional, Feiner, Leonard, additional, Fekrat, Sharon, additional, Ferris, Frederick L, additional, Fine, Howard F, additional, Fletcher, Donald C, additional, Flikier, Paul, additional, Floyd, Richard P, additional, Flynn, Harry W, additional, Fong, Donald S, additional, Font, Ramon L, additional, Forbes, Brian JR, additional, Foroozan, Rod, additional, Foster, Bradley S, additional, Foster, C Stephen, additional, Foster, Jill A, additional, Foulks, Gary N, additional, Fountain, Tamara R, additional, Fox, Gregory M, additional, Freddo, Thomas F, additional, Freedman, Sharon F, additional, Freund, K Bailey, additional, Friberg, Thomas R, additional, Friedman, Alan H, additional, Friedman, David, additional, Friedman, Deborah I, additional, Friedman, Ephraim, additional, Fu, Arthur D, additional, Fulton, Anne B, additional, Galal, Ahmed, additional, Galetta, Steven, additional, Gallardo, Mark, additional, Gallie, Brenda, additional, Garner, Alec, additional, Garrity, James A, additional, Gatinel, Damien, additional, Gedde, Steven J, additional, Geist, Craig E, additional, Gerber, Steve, additional, Ghanem, Ramon C, additional, Gieser, Jon P, additional, Gilmore, Michael S, additional, Gimbel, Howard V, additional, Gipson, Ilene K, additional, Glover, Tyrone, additional, Goldberg, Robert A, additional, Goldenfeld, Mordechai, additional, Goldstein, Scott M, additional, Gomi, Cintia F, additional, Gong, Haiyan, additional, Gonzales, John A, additional, Goosey, John, additional, Gottlieb, Justin L, additional, Gould, Joshua, additional, Gragoudas, Evangelos S, additional, Granet, David B, additional, Greaney, Michael J, additional, Green, Daniel G, additional, Grehn, Franz, additional, Greiner, Jack V, additional, Greven, Craig M, additional, Griepentrog, Gregory J, additional, Groenewald, Carl, additional, Grosskreutz, Cynthia L, additional, Grover, Lori Latowski, additional, Gullapalli, Vamsi K, additional, Gulur, Padma, additional, Gunther, Jonathan, additional, Gupta, Manish, additional, Gupta, Mayank, additional, Guyer, David R, additional, Haivala, Darin R, additional, Haller, Julia A, additional, Halmagyi, GM, additional, Halperin, Lawrence S, additional, Hamdi, Islam M, additional, Hamilton, Steven R, additional, Hammersmith, Kristin M, additional, Han, Dennis P, additional, Hansen, Ronald M, additional, Harbour, J William, additional, Hariprasad, Seenu M, additional, Harissi-Dagher, Mona, additional, Hassan, Shirin E., additional, Hatton, Mark P, additional, Hawley, Pamela, additional, Hayashida, Yasutaka, additional, Heckenlively, John R, additional, Hedges, Thomas R, additional, Heggie, Alfred D, additional, Heher, Katrinka L, additional, Heier, Jeffrey S, additional, Hejtmancik, J Fielding, additional, Henderson, Bonnie A, additional, Hersh, Peter S, additional, Hidayat, Ahmed A, additional, Higginbotham, Eva Juliet, additional, Hirose, Tatsuo, additional, Ho, Allen C, additional, Ho, ThucAnh T, additional, Hogan, R Nick, additional, Holck, David E, additional, Holekamp, Nancy M, additional, Hovland, Peter G, additional, Hsu, Thomas C, additional, Hsu, William C, additional, Huang, Andrew JW, additional, Hughes, Mark S, additional, Hui, Jennifer, additional, Hunter, David G, additional, Huryn, Laryssa A, additional, Husain, Deeba, additional, Hyndiuk, Robert A, additional, Ip, Michael, additional, Jacobs, Brian J, additional, Jakobiec, Frederick A, additional, Jampol, Lee M, additional, Jensen, Harold G, additional, Ji, Fei, additional, Johnson, David L, additional, Johnson, Douglas H, additional, Johnson, Mark W, additional, Johnson, R Paul, additional, Johnson, Robert N, additional, Joos, Karen M, additional, Joyce, Nancy C, additional, Jumper, J Michael, additional, Jurkunas, Ula V., additional, Kahana, Alon, additional, Kahook, Malik Y, additional, Kanner, Elliott, additional, Kalwerisky, Kevin, additional, Kaplan, Henry J, additional, Karatza, Ekaterini C, additional, Kardon, Randy, additional, Katowitz, James A, additional, Katowitz, William R, additional, Kazlas, Melanie, additional, Keefe, Kelly S, additional, Kelley, Lara, additional, Kent, Charles J, additional, Kenyon, Kenneth R, additional, Khan, Bilal F, additional, Khan, Jemshed A, additional, Khan, Naheed W, additional, Khaw, Peng Tee, additional, Kherani, Femida, additional, Kim, Eva C, additional, Kim, Hee Joon, additional, Kim, Ivana K, additional, Kim, Jonathan W, additional, Kim, Rosa Y, additional, Kim, Stella K, additional, Kim, Tae-Im, additional, Klais, Christina M, additional, Klapper, Stephen R, additional, Klein, Barbara EK, additional, Kleinmann, Guy, additional, Klink, Thomas, additional, Klisovic, Dino D, additional, Klyce, Stephen D, additional, Kocaturk, Tolga, additional, Kohnen, Thomas, additional, Kojima, Takeshi, additional, Koller, Tobias, additional, Kostick, David A, additional, Kraut, Joel A, additional, Krishnan, Chandrasekharan, additional, Krueger, Ronald R, additional, Krug, Joseph H, additional, Krupsky, Sara, additional, Kuchtey, Rachel W, additional, Kurban, Ramsay S, additional, Kurz, Paul A, additional, Kuszak, JR, additional, Kwon, Young H, additional, Labbe, Thad A, additional, Lam, Deborah L, additional, Lamkin, Jeffrey C, additional, Lamping, Kathleen A, additional, Lane, Anne Marie, additional, Lane, Katherine A, additional, Lane, Keith J, additional, Lass, Jonathan H, additional, Lawrence, Mary G, additional, Lee, Andrew G, additional, Lee, Carol M, additional, Lee, Michael S, additional, Lee, Paul P, additional, Lee, William B, additional, Leibovitch, Igal, additional, Lemke, Bradley N, additional, Lemley, Craig A, additional, Leonardi, Andrea, additional, Lessell, Simmons, additional, Levin, Leonard A, additional, Levy-Clarke, Grace A, additional, Lew, Julie C, additional, Lewis, Craig, additional, Li, Wei, additional, Lim, Laurence S, additional, Lim, Lyndell L, additional, Lim, Wee-Kiak, additional, Liu, Grant T, additional, Loewenstein, John I, additional, Lott, McGregor N, additional, Lowry, Jonathan C, additional, Lyon, David B, additional, Lytle, Robert E, additional, MacCumber, Mathew, additional, Mackool, Bonnie T, additional, Madiwale, Nalini A, additional, Mah, Francis, additional, Mainster, Martin A, additional, Manning, Michael H, additional, Mansberger, Steven L, additional, Marc, Robert E, additional, Marchong, Mellone, additional, Marcus, Dennis M, additional, Mares, Julie A, additional, Marr, Brian P, additional, Martinez, Carlos E, additional, Massof, Robert W, additional, Matsumoto, Yukihiro, additional, Mattox, Cynthia, additional, Maus, Marlon, additional, McCabe, Cathleen M, additional, McCormick, Steven A, additional, McCrakken, Michael, additional, McCulley, James P, additional, McDermott, John A, additional, McDonald, H Richard, additional, McDonald, Marguerite B, additional, McDonnell, Peter J, additional, McGillivray, Robert, additional, McKeown, Craig A, additional, McLaughlin, James, additional, McMullen, W Wynn, additional, Melamed, Shlomo, additional, Meligonis, George, additional, Mendrinos, Efstratios, additional, Meyer, Dale R, additional, Meyerle, Catherine B, additional, Mieler, William F, additional, Migliori, Michael, additional, Mihm, Martin C, additional, Miller, Darlene, additional, Miller, David, additional, Miller, Joan W, additional, Miller, Neil R, additional, Mills, David M, additional, Mills, Monte D, additional, Milman, Tatyana, additional, Mogk, Lylas, additional, Mogk, Marja, additional, Monés, Jordi, additional, Montes-Micó, Robert, additional, Morse, Christie L, additional, Morton, Asa D, additional, Moskowitz, Anne, additional, Mukai, Shizuo, additional, Murphree, A Linn, additional, Murphy, Robert P, additional, Murray, Timothy G, additional, Murray, Philip I, additional, Nagao, Karina, additional, Neitz, Jay, additional, Neitz, Maureen, additional, Netland, Peter A, additional, Neufeld, Arthur H, additional, Newman, Nancy J, additional, Ng, Eugene WM, additional, Nguyen, Quan Dong, additional, Niederkorn, Jerry Y, additional, Noecker, Robert J, additional, Nussenblatt, Robert B, additional, O'Brien, Joan M, additional, O'Brien, Paul D, additional, O'Brien, Terrence P, additional, O'Day, Denis, additional, Olk, R Joseph, additional, Olsen, Karl R, additional, Onal, Sumru, additional, Ooi, Yen Hoong, additional, Opremcak, E Mitchel, additional, Ousler, George, additional, Ozment, Randall R, additional, Packer, Samuel, additional, Palmer, Millicent L, additional, Papaliodis, George N, additional, Park, DJ John, additional, Parke, David W, additional, Parsa, Cameron F, additional, Parsons, M Andrew, additional, Pasquale, Louis R, additional, Patel, Neha N, additional, Patel, Sayjal J, additional, Patrianakos, Thomas D, additional, Patrinely, James R, additional, Pavan-Langston, Deborah, additional, Peli, Eli, additional, Pepin, Susan M, additional, Perez, Victor L, additional, Pérez-Santonja, Juan J, additional, Perfect, John R, additional, Perry, Henry D, additional, Piatigorsky, Joram, additional, Pieramici, Dante, additional, Pierce, Eric A, additional, Pineda, Roberto, additional, Pless, Misha L, additional, Pomeranz, Howard D, additional, Pournaras, Constantin J, additional, Power, William, additional, Prakash, Manvi, additional, Prasad, Anita G, additional, Purvin, Valerie, additional, Quillen, David A, additional, Quinn, Graham E, additional, Rabena, Melvin D, additional, Rae, James L, additional, Raizman, Michael B, additional, Randazzo, Alessandro, additional, Rao, Narsing A, additional, Rapuano, Christopher J, additional, Reeves, Sherman W, additional, Regillo, Carl D, additional, Reichel, Elias, additional, Reinke, Martin H, additional, Rhee, Douglas, additional, Richter, Claudia U, additional, Rizzo, Joseph F, additional, Robb, Richard M, additional, Roden, Anja C, additional, Rodgers, I Rand, additional, Rodrigues, Merlyn M, additional, Ron, Yonina, additional, Rose, Geoffrey E, additional, Rosen, Emanuel S, additional, Rosenbaum, James T, additional, Rosenthal, Perry, additional, Rouse, Strutha C, additional, Rovner, Barry W, additional, Rozanowska, Malgorzata, additional, Rubin, Michael P, additional, Rubin, Peter AD, additional, Rumelt, Shimon, additional, Rustgi, Anil K, additional, Rutar, Tina, additional, Ruttum, Mark S, additional, Rutzen, Allan R, additional, Ryan, Edward T, additional, Sadun, Alfredo A, additional, Sahel, José-Alain, additional, Saligan, Leorey, additional, Salim, Sarwat, additional, Salmon, John F, additional, Salomão, Diva R, additional, Sami, David, additional, Sandberg, Michael A, additional, Sangwan, Virender S, additional, Saornil, Maria A, additional, Sassani, Joseph W, additional, Sayegh, Rony R, additional, Schachat, Andrew P, additional, Schell, Wiley A, additional, Schefler, Amy C, additional, Scheufele, Tina, additional, Schiedler, Vivian, additional, Schneider, Gretchen, additional, Schroeder, Alison, additional, Schuchard, Ronald A, additional, Schuman, Joel S, additional, Schwab, Ivan R, additional, Scott, Adrienne, additional, Scott, Ingrid U, additional, Sears, Marvin L, additional, Seddon, Johanna M, additional, Seiler, Theo, additional, Selkin, Robert P, additional, Semba, Richard D, additional, Serbanescu, Irina, additional, Sexton, Briar, additional, Shaarawy, Tarek M, additional, Shah, Peter, additional, Shapiro, Aron, additional, Sharma, Savitri, additional, Shein, Jean, additional, Shetlar, Debra J, additional, Shields, M Bruce, additional, Shields, Carol L, additional, Shields, Jerry A, additional, Shingleton, Bradford J, additional, Shore, John W, additional, Shuba, Lesya M, additional, Simon, Guy J Ben, additional, Simmons, Richard J, additional, Simpson, Michael, additional, Singh, Arun D, additional, Singh, Omah S, additional, Sisley, Karen, additional, Sit, Arthur J, additional, Smerdon, David, additional, Smiddy, William E, additional, Smith, Ronald E, additional, Smith, Terry J, additional, Snebold, Neal G, additional, Sobrin, Lucia, additional, Sorenson, John A, additional, Soukiasian, Sarkis H, additional, Spaeth, George L, additional, Spaide, Richard F, additional, Srivastava, Monika, additional, Srivastava, Sunil K, additional, Stangos, Alexandros N, additional, Starck, Tomy, additional, Stark, Walter J, additional, Stein, Joshua D, additional, Steinert, Roger F, additional, Strauss, Leon, additional, Streeten, Barbara W, additional, Streilein, J Wayne, additional, Strong, James D, additional, Sugino, Ilene K, additional, Suhler, Eric B, additional, Sullivan, Timothy J, additional, Sun, Jennifer K, additional, Sunness, Janet S, additional, Sutula, Francis C, additional, Syed, Nasreen A, additional, Ta, Christopher N, additional, Takei, Hidehiro, additional, Talamo, Jonathan H, additional, Tamesis, Richard R, additional, Tamhankar, Madhura, additional, Tarbet, Kristen J, additional, Tarver-Carr, Michelle, additional, Terry, Mark A, additional, Thomas, Joseph M, additional, Thompson, Vance, additional, Thorne, Jennifer E, additional, Thurtell, Matthew J, additional, Tingey, David P, additional, To, King W, additional, Tobaigy, Faisal M, additional, Tolentino, Michael J, additional, Tong, Melissa G, additional, Torkildsen, Gail, additional, Toth, Cynthia A, additional, Traboulsi, Elias I, additional, Trucksis, Michele, additional, Tsai, James C, additional, Tsai, Julie H, additional, Tse, David T, additional, Tseng, Scheffer CG, additional, Tu, Elmer Y, additional, Udell, Ira J, additional, Valenzuela, Alejandra A, additional, Van Gelder, Russell N, additional, Van Stavern, Gregory P, additional, Vander Veen, Deborah K, additional, Vavvas, Demetrios, additional, Verity, David H, additional, Vinciguerra, Paolo, additional, Vinger, Paul F, additional, Volpe, Nicholas J, additional, Wackernagel, Werner, additional, Wadhwa, Sonal Desai, additional, Wagoner, Michael D, additional, Waheed, Nadia K, additional, Walton, David S, additional, Wand, Martin, additional, Wang, Jie Jin, additional, Warden, Scott M, additional, Webb, Lennox, additional, Weber, David, additional, Wee, Daniel, additional, Westerfeld, Corey B, additional, Westfall, Christopher T, additional, Whitcup, Scott M, additional, White, Valerie A, additional, White, William L, additional, Wickens, Jason, additional, Wiggs, Janey L, additional, Wilensky, Jacob T, additional, Wilkinson, Charles P, additional, Williams, Patrick D, additional, Wilson, David J, additional, Wilson, M Roy, additional, Wilson, Steven E, additional, Winokur, Jules, additional, Wirostko, William J, additional, Wollstein, Gadi, additional, Wong, Albert Chak Ming, additional, Wong, Tien Y, additional, Woog, John J, additional, Wride, Michael, additional, Wu, Carolyn S, additional, WuDunn, Darrell, additional, Yang, Jean, additional, Yannuzzi, Lawrence A, additional, Yaremchuk, Michael J, additional, Yeatts, R Patrick, additional, Yee, Richard W, additional, Yeh, Steven, additional, Young, Lucy HY, additional, Yu, Jenny Y, additional, Yue, Beatrice YJT, additional, Zacks, Charles M, additional, Zagelbaum, Bruce M, additional, Zamani, Maryam, additional, Zarbin, Marco, additional, Zografos, Leonidas, additional, and Zoumalan, Christopher I, additional

Published
2008
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12. PGE2: a mediator of corneal endothelial wound repair in vitro

Author

Joyce, Nancy C. and Meklir, Barry

Subjects
Wound healing -- Research, Endothelium -- Cytology, Prostaglandins E -- Research, Biological sciences
Abstract

Studies on corneal endothelial monolayer reveal that prostaglandin E2 (PGE2) stimulates the cAMP pathway leading to greater single cell migrations during the corneal endothelial wound repair. The cycloxygenase inhibitor indomethacin lowers migratory response levels in wounded cultures. The mechanism of PGE2 action involves cytoskeletal changes, including actin modification and consequent enhancement of migratory movements.

Published
1994

14. Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium

Author

Joyce, Nancy C., Harris, Deshea L., Markov, Vladimir, Zhang, Zhe, and Saitta, Biagio

Subjects
Wound Healing, Gene Expression Profiling, Endothelium, Corneal, Membrane Proteins, Mesenchymal Stem Cells, Cadherins, Fetal Blood, Phosphoproteins, Models, Biological, eye diseases, Culture Media, Tissue Culture Techniques, fluids and secretions, Intercellular Junctions, Gene Expression Regulation, embryonic structures, Cell Adhesion, Zonula Occludens-1 Protein, Humans, Calcium, sense organs, Egtazic Acid, Biomarkers, Research Article, Oligonucleotide Array Sequence Analysis
Abstract

Purpose To test the feasibility of altering the phenotype of umbilical cord blood mesenchymal stem cells (UCB MSCs) toward that of human corneal endothelial cells (HCEC) and to determine whether UCB MSCs can “home” to sites of corneal endothelial cell injury using an ex vivo corneal wound model. Methods RNA was isolated and purified from UCB MSCs and HCECs. Baseline information regarding the relative gene expression of UCB MSCs and HCEC was obtained by microarray analysis. Quantitative real-time PCR (q-PCR) verified the microarray findings for a subset of genes. The ability of different culture media to direct UCB MSCs toward a more HCEC-like phenotype was tested in both tissue culture and ex vivo corneal endothelial wound models using three different media: MSC basal medium (MSCBM), a basal medium used to culture lens epithelial cells (LECBM), or lens epithelial cell-conditioned medium (LECCM). Morphology of the MSCs was observed by phase-contrast microscopy or by light microscopic observation of crystal violet-stained cells. Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy. Formation of cell-cell junctions was tested by treatment with the calcium chelator, EGTA. A second microarray analysis compared gene expression between UCB MSCs grown in LECBM and LECCM to identify changes induced by the lens epithelial cell-conditioned culture medium. The ability of UCB MSCs to “home” to areas of endothelial injury was determined using ZO1 immunolocalization patterns in ex vivo corneal endothelial wounds. Results Baseline microarray analysis provided information regarding relative gene expression in UCB MSCs and HCECs. MSCs attached to damaged, but not intact, corneal endothelium in ex vivo corneal wounds. The morphology of MSCs was consistently altered when cells were grown in the presence of LECCM. In tissue culture and in ex vivo corneal wounds, UCB MSC treated with LECCM were elongated and formed parallel sheets of closely apposed cells. In both tissue culture and ex vivo corneal endothelial wounds, ZO1 and N-cadherin localized mainly to the cytoplasm of UCB MSCs in the presence of MSCBM. However, both proteins localized to cell borders when UCB MSCs were grown in either LECBM or LECCM. This localization was lost when extracellular calcium levels were reduced by treatment with EGTA. A second microarray analysis showed that, when UCB MSCs were grown in LECCM instead of LECBM, the relative expression of a subset of genes markedly differed, suggestive of a more HCEC-like phenotype. Conclusions Results indicate that UCB MSCs are able to “home” to areas of injured corneal endothelium and that the phenotype of UCB MSCs can be altered toward that of HCEC-like cells. Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

Published
2012

15. Decreasing expression of the G1-phase inhibitors, p21Cip1 and p16INK4a, promotes division of corneal endothelial cells from older donors

Author

Joyce, Nancy C. and Harris, Deshea L.

Subjects
Adult, Cyclin-Dependent Kinase Inhibitor p21, Aging, Cell Survival, Cell Cycle, Endothelium, Corneal, G1 Phase, Down-Regulation, Endothelial Cells, Middle Aged, Electroporation, Humans, RNA, Small Interfering, Cell Division, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p16, Research Article, Aged
Abstract

Purpose The current studies were conducted to determine whether the cyclin-dependent kinase inhibitors, p21Cip1 (p21 cyclin-dependent kinase-interacting protein 1) and p16INK4a (p16 cyclin-dependent kinase inhibitor 1A), help mediate G1-phase inhibition in human corneal endothelial cells (HCEC) by testing the effect of siRNA (small interfering RNA)-mediated down-regulation of the expression of these inhibitors on cell cycle entry and proliferation in HCEC cultured from older donors. Methods HCEC were obtained from National Disease Research Interchange, Philadelphia, PA, and cultured according to published methods. Cells were electroporated in the presence of either a non-silencing siRNA control or p21+p16 siRNA. The efficiency of siRNA transfer was observed by fluorescence microscopy of Cy3-labeled control siRNA. Viability was determined by direct counting of cells before and after electroporation. The ability of p21+p16 siRNA to decrease the protein expression of p21Cip1 and p16INK4a was determined by semi-quantitative analysis of western blots. The effect of siRNA treatment on cell cycle progression and proliferation was determined 1, 5, and 11 days after electroporation by counting Ki67-positive cells and total DAPI-stained nuclei. Results siRNA was efficiently transferred to HCEC by the electroporation method. The average cell loss was 41.25% at 24 h following electroporation. Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment. This treatment significantly increased the average number of Ki67-positive cells over controls and increased the total number of cells in a time-dependent manner. Conclusions Both p21Cip1 and p16INK4a are involved in negative regulation of the cell cycle in HCEC and, thereby, provide an effective barrier to cell division. The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers. Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.

Published
2010

16. Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

Author

Harris, Deshea L. and Joyce, Nancy C.

Subjects
Male, Protein Tyrosine Phosphatase, Non-Receptor Type 1, Time Factors, Epidermal Growth Factor, Reverse Transcriptase Polymerase Chain Reaction, Blotting, Western, Cell Cycle, Endothelium, Corneal, Immunohistochemistry, Rats, ErbB Receptors, Rats, Sprague-Dawley, Cinnamates, Animals, Tissue Distribution, RNA, Messenger, Enzyme Inhibitors, Phosphorylation, Protein Tyrosine Phosphatases, Oligopeptides, hormones, hormone substitutes, and hormone antagonists, Cells, Cultured, Research Article
Abstract

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that EGFR is internalized in response to EGF stimulation suggests that it could interact with and be regulated by PTP1B. The ability of PTP1B inhibitor to sustain EGFR Tyr992 phosphorylation and increase the number of Ki67-positive cells indicates that PTP1B plays a role in the negative regulation of EGF-induced signaling and helps suppress cell cycle entry.

Published
2007

31. Staff Report to the President's Commission on the accident at Three Mile Island. Report of the public's right to information task force

Author

Rubin, David M., primary, Chaapel, Holly A., additional, Cunningham, Ann Marie, additional, Edison, Nadyne G., additional, Franklin, Mary Beth, additional, Friedman, Sharon M., additional, Hill, Wilma I., additional, Joyce, Nancy C., additional, Popkin, Roy S., additional, Sandman, Peter M., additional, Stephens, Mitchell, additional, Wells, Patricia E., additional, Beer, Ruth Anne, additional, Glassman, Ellen, additional, Hollister, Martha, additional, Kantrowitz, Jolie, additional, O'Grady, Donald, additional, Schwartz, Nora, additional, Stephens, Beth, additional, Thompson, Trisha, additional, Woerner, Ronald, additional, and Woznicki, Marilynn, additional

Published
1979
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37. Protein Tyrosine Phosphatase-1B (PTP1B) Helps Regulate EGF-induced Stimulation of S-phase Entry in Human Corneal Endothelial Cells

Author

Ishino, Yutaka, Zhu, Cheng, Harris, Deshea L., and Joyce, Nancy C.

Abstract

Purpose: Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods: Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results: PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions: Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B expression, but not EGFR expression, was elevated in HCEC from older donors, suggesting that the reduced proliferative activity of these cells in response to EGF is due, at least in part, to increased PTP1B activity. The fact that inhibition of PTP1B increased the relative number of cells entering S-phase strongly suggests that PTP1B helps negatively regulate EGF-stimulated cell cycle entry in HCEC. These results also suggest that it may be possible to increase the proliferative activity of HCEC, particularly in cells from older donors, by inhibiting the activity of this important protein tyrosine phosphatase.

Published
2008

38. Morphologic and biochemical evidence for a contractile cell network within the rat intestinal mucosa

Author

Joyce, Nancy C., Haire, Marcy F., and Palade, George E.

Abstract

Subepithelial and pericryptal fibroblastlike cells form a two-dimensional network immediately subjacent to the epithelial basal lamina in the small intestine and colon in several mammalian species. Stellate-shaped cells with similar, but not identical characteristics, form a three-dimensional network deep within the villar lamina propria. Electron microscopic studies indicate that these cells (a) contain a putative contractile apparatus, (b) are attached to each other and to apparently organized elements of the extracellular matrix by typical adhesive devices, and (c) form gap junctions with each other. Comparative in situ immunoperoxidase localization studies document the presence in these cells of four contraction-associated proteins (smooth muscle isotropomyosin, cyclic guanosine monophosphate-dependent protein kinase, both nonmuscle and smooth muscle isomyosin, and actin) in amounts generally greater than those found in connective tissue fibroblasts, but less than in smooth muscle cells. Taken together, these results strongly suggest a smooth muscle-like, contractile function for these cells and indicate that this cellular network may provide a supportive tonus for the epithelium, as well as provide the force needed for active movement of the villus, expulsion of crypt secretion products, and propulsion of absorption products in the lamina propria, the microvasculature, and lacteals of the intestinal villus.

Published
1987
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39. Activity of Minocycline Against Acinetobacter calcoaceticusVar. anitratus(Syn. Herellea vaginicola) and Serratia Marcescens

Author

Maderazo, Eufronio G., Quintiliani, Richard, Tilton, Richard C., Bartlett, Raymond, Joyce, Nancy C., and Andriole, Vincent T.

Abstract

The activity of minocycline and tetracycline against 23 isolates of Acinetobacter calcoaceticusvar. anitratus(syn. Herellea vaginicola) and 178 strains of Serratia marcescenswas determined by disk and microdilution methods. The results indicate that minocycline is highly active against this species of Acinetobacter, all but one strain being inhibited by 0.007 μg of the antibiotic per ml. Tetracycline was also active, though to a lesser degree, against A. calcoaceticus. Of the 178 strains of S. marcescenstested, only seven (3.9%) had a minimum inhibitory concentration of 2 μg or less of minocycline per ml. Tetracycline was less active than minocycline against S. marcescens; with 2 μg of tetracycline per ml, only 2 of 152 (1.3%) strains were inhibited. At concentrations of 8 and 16 μg of minocycline per ml, which can be achieved in the urine with the usual doses, 44.9 and 63.5% of S. marcescensstrains were inhibited, which implies its possible usefulness for the therapy of urinary tract infection due to this organism.

Published
1975
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44. EMDR Within a Phase Model of Trauma-Informed Treatment.

Author

Joyce, Nancy C.

Subjects
*EMDR (Eye-movement desensitization & reprocessing), *NONFICTION
Abstract

The article reviews the book "EMDR Within a Phase Model of Trauma-Informed Treatment," by Ricky Greenwald.

Published
2009

45. The culture of limbal stromal cells and corneal endothelial cells.

Author

Polisetti N and Joyce NC

Subjects
Cell Culture Techniques, Cells, Cultured, Corneal Stroma cytology, Endothelium, Corneal cytology, Humans, Limbus Corneae cytology, Endothelial Cells cytology, Stromal Cells cytology
Abstract

The cornea is the transparent front part of the eye and comprises three distinct cell layers. One of these cell layers is a self-renewing epithelium long believed to harbor a resident stem cell population. The location and characteristics of corneal epithelial stem cells have now been confirmed by several research groups, and these cells are currently applied therapeutically. The corneal stroma and endothelium are largely quiescent after infancy, and until recently they were not considered to undergo self-renewal or to maintain stem cells. This view was overturned during the last two decades. At present, cell populations with characteristics of adult stem cells are routinely isolated and characterized from the limbal stroma and the corneal -endothelium. This chapter describes methods for isolation and culture of limbal stromal cells and corneal endothelial cells.

Published
2013
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46. Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells.

Author

Ishino Y, Zhu C, Harris DL, and Joyce NC

Subjects
Adolescent, Aged, Aging metabolism, Blotting, Western, Bromodeoxyuridine metabolism, Cells, Cultured, Cornea enzymology, Cytoplasm metabolism, Endothelium, Corneal enzymology, ErbB Receptors metabolism, Humans, Immunohistochemistry, In Vitro Techniques, Middle Aged, Protein Tyrosine Phosphatase, Non-Receptor Type 1 metabolism, Tissue Distribution, Endothelium, Corneal cytology, Epidermal Growth Factor pharmacology, Protein Tyrosine Phosphatase, Non-Receptor Type 1 physiology, S Phase drug effects, S Phase physiology
Abstract

Purpose: Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation., Methods: Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation., Results: PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation., Conclusions: Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B expression, but not EGFR expression, was elevated in HCEC from older donors, suggesting that the reduced proliferative activity of these cells in response to EGF is due, at least in part, to increased PTP1B activity. The fact that inhibition of PTP1B increased the relative number of cells entering S-phase strongly suggests that PTP1B helps negatively regulate EGF-stimulated cell cycle entry in HCEC. These results also suggest that it may be possible to increase the proliferative activity of HCEC, particularly in cells from older donors, by inhibiting the activity of this important protein tyrosine phosphatase.

Published
2008

47. Differential protein expression in human corneal endothelial cells cultured from young and older donors.

Author

Zhu C, Rawe I, and Joyce NC

Subjects
Adolescent, Adult, Aged, Cells, Cultured, Electrophoresis, Gel, Two-Dimensional, Eye Proteins isolation & purification, Humans, Microscopy, Phase-Contrast, Middle Aged, Tissue Extracts, Endothelium, Corneal cytology, Endothelium, Corneal metabolism, Eye Proteins metabolism, Proteomics, Tissue Donors
Abstract

Purpose: To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors., Methods: Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry., Results: Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3-10 or pH 4-7 IPG strips. Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation., Conclusions: This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC, including differences that may affect proliferative capacity. Results indicate that HCEC from older donors exhibit reduced expression of proteins that support important cellular functions such as metabolism, antioxidant protection, protein folding, and protein degradation. These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.

Published
2008

48. Age and topographical comparison of telomere lengths in human corneal endothelial cells.

Author

Konomi K and Joyce NC

Subjects
Adolescent, Adult, Aged, Cell Separation, Child, Endothelial Cells cytology, Fluorescein-5-isothiocyanate metabolism, Humans, Peptide Nucleic Acids metabolism, Aging physiology, Endothelial Cells metabolism, Endothelium, Corneal cytology, Endothelium, Corneal metabolism, Telomere metabolism
Abstract

Purpose: Human corneal endothelium exhibits both age-related and topographical differences in relative proliferative capacity and in senescence characteristics. The purpose of these studies was to compare telomere lengths in human corneal endothelial cells (HCEC) from the central and peripheral areas of corneas from young and older donors to determine whether these changes may be due to replicative senescence or to stress-induced premature senescence., Methods: Pairs of corneas from five young (<30 years old) and six older donors (>65 years old) were separated into central and peripheral areas using a 9.5 mm diameter trephine to remove scleral tissue and a 6.0 mm diameter trephine to mark the central-peripheral boundary. One of the pair of corneas was cut into quarters and stained with a peptide nucleic acid (PNA)/fluorescein isothiocyanate (PNA/FITC) probe that specifically binds to telomere repeats. HCEC from the central (0-6.0 mm) and peripheral areas (6.0-9.5 mm) were isolated from the second cornea, mounted on slides by Cytospin, and stained with the PNA/FITC probe. Fluorescence confocal microscopy was used to obtain digital images. The average FITC intensity of nuclei was compared between the central and peripheral areas within and between the two age groups. Ccl185 and 1301 cells were analyzed as controls. Student's unpaired t-test was used to determine the statistical significance of the data., Results: Average FITC intensity from the central endothelium was 205.8+/-4.2 (younger) and 194.2+/-10.5 (older) and from the peripheral endothelium was 208.1+/-9.3 (younger) and 195.9+/-10.8 (older). Average intensity of single cells isolated from central endothelium was 113.9+/-31.1 (younger) and 107.9+/-26.1 (older) and from the periphery was 109.9+/-12.0 (younger) and 106.9+/-32.4 (older). Average FITC intensity of Ccl185 cells and 1301 cells was 50.5+/-5.0 and 206.9+/-19.4, respectively. Comparison of the results indicates no statistically significant difference between the central and peripheral areas within each group or between the young and older age group., Conclusions: Results indicate that the age-related and topographical reduction in relative proliferative capacity and senescence characteristics observed in HCEC are not due to replicative senescence caused by critically short telomeres but implicate stress-induced premature senescence as a cause of these clinically important changes.

Published
2007

49. p27kip1 Antisense-induced proliferative activity of rat corneal endothelial cells.

Author

Kikuchi M, Harris DL, Obara Y, Senoo T, and Joyce NC

Subjects
Animals, Blotting, Western, Bromodeoxyuridine metabolism, Cell Cycle Proteins metabolism, Cell Division drug effects, Cell Survival, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p27, Down-Regulation, Endothelium, Corneal metabolism, Gene Transfer Techniques, Immunohistochemistry, Ki-67 Antigen metabolism, Male, Oligonucleotides, Antisense genetics, Rats, Rats, Sprague-Dawley, Transfection, Tumor Suppressor Proteins metabolism, Cell Cycle Proteins genetics, Endothelium, Corneal cytology, Oligonucleotides, Antisense pharmacology, Tumor Suppressor Proteins genetics
Abstract

Purpose: To determine whether antisense downregulation of p27(kip1) will overcome G(1)-phase arrest and promote cell cycle progression in rat corneal endothelial cells (CECs)., Methods: Confluent cultures of rat CECs were incubated for 24 hours in the presence of p27(kip1) antisense (AS) oligonucleotides (oligoS) using nonliposomal lipid transfection. Control cultures were incubated under one of the following conditions: no oligos or lipid-containing buffer, lipid-containing buffer alone, or lipid-containing buffer plus missense (MS) p27(kip1) oligo. Viability was tested by a cell-viability assay after 0, 24, 48, and 72 hours. After postincubation for 0, 24, 48, or 72 hours, cultures were fixed and immunostained for p27(kip1), to test for downregulation, or for Ki67 or BrdU, to detect actively cycling cells. Western blot and immunocytochemistry (ICC) studies were conducted to determine the effect of p27(kip1) antisense treatment on the relative protein level and subcellular localization of several cell cycle proteins, including cyclin-D1, -E, -A, and -B1; CDK2 and -4; p21(cip1); and p15(INK4b). Proliferation was determined by direct counting of propidium iodide (PI) or 4',6'-diamino-2-phenylindole (DAPI)-stained cells., Results: Viability was not significantly affected by lipid-based oligo transfection for up to 48 hours, after which a decline was noted. The protein level of p27(kip1) was reduced after AS transfection in a time-dependent manner. Nuclear staining for p27(kip1) was greatly reduced in CECs incubated with AS oligo. No change in p27(kip1) levels was observed in controls at any time point tested. p27(kip1) AS oligo transfection increased cyclin-D1, -E, -A, and -B1 protein levels, and all cyclins were localized to the nucleus. No changes in protein level were observed for CDK2, CDK4, p21(cip1), or p15(INK4B). A time-dependent increase in the relative number of Ki67- and BrdU-positive cells was noted in CECs incubated with AS oligo. In contrast, no to few Ki67- or BrdU-positive cells were observed in CECs incubated with MS oligo or the buffer-treated control cells. The percentage increase in the number of cells transfected with AS oligo increased with time, compared with that of cells transfected with MS oligo., Conclusions: Treatment with p27(kip1) antisense oligonucleotides followed by postincubation in 10% FBS lowers endogenous p27(kip1) protein levels and promotes proliferation in confluent cultures of rat CECs.

Published
2004
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50. Effect of overexpressing the transcription factor E2F2 on cell cycle progression in rabbit corneal endothelial cells.

Author

Joyce NC, Harris DL, Mc Alister JC, Ali RR, and Larkin DF

Subjects
Animals, Cell Survival, Cyclin B metabolism, Cyclin B1, E2F2 Transcription Factor, Fluorescent Antibody Technique, Indirect, Green Fluorescent Proteins, Helix-Loop-Helix Motifs genetics, Humans, In Situ Nick-End Labeling, Ki-67 Antigen metabolism, Luminescent Proteins metabolism, Male, Plasmids, RNA, Messenger metabolism, Rabbits, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors metabolism, Transfection, Cell Cycle physiology, Endothelium, Corneal metabolism, Gene Expression physiology, Transcription Factors genetics
Abstract

Purpose: To test the hypothesis that overexpression of the transcription factor E2F2 promotes cell cycle progression in nonproliferating corneal endothelial cells., Methods: Ex vivo rabbit corneas were transfected with a lipid transfection reagent and either a control plasmid containing full-length cDNA for enhanced green fluorescent protein (pIRES2-EGFP) or a plasmid containing full-length cDNA for both E2F2 and EGFP (pIRES2-E2F2/EGFP). Transfection control experiments consisted of corneas incubated in buffer without transfection reagent or plasmid or incubated in reagent without plasmid. After transfection, corneas were incubated for various periods in 0.1% FBS (a concentration that maintains cell health, but does not promote proliferation). Immunocytochemical (ICC) localization tested for overexpression of E2F2 in transfected corneal endothelial cells and permitted calculation of transfection efficiency. Endothelial cell viability was tested in transfected ex vivo corneas and confluent cultures by using a cell-viability assay. Apoptosis was detected in confluent cultures by TUNEL assay. RT-PCR tested for mRNA expression of Ki67 (a marker of actively cycling cells) and cyclin B1 (a marker for the G2-phase of the cell cycle). Semiquantitative densitometric analysis compared the relative amounts of PCR reaction products., Results: ICC demonstrated the colocalization of E2F2 and EGFP in corneal endothelium with a transfection efficiency of 10% to 12%, using the pIRES2-based plasmid and transfection reagent. The cell-viability assay revealed very few dead cells in ex vivo corneal endothelium that overexpressed E2F2. Cell viability and TUNEL assays of confluent cultures revealed that approximately 27% of cells died in all cultures incubated with transfection reagent, but death appeared not to be due to apoptosis. No additional cell death was noted by either assay in cells that overexpressed E2F2. RT-PCR of endothelial samples obtained 48 hours after transfection showed the presence of higher levels of reaction product for Ki67 (a 5.1-fold increase) and cyclin B1 (a 2.3-fold increase) in cells that overexpressed E2F2 than in control samples., Conclusions: Overexpression of the transcription factor E2F2 in nonproliferating rabbit corneal endothelial cells induces cell cycle progression without inducing significant apoptosis.

Published
2004
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