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ARVO 2015 Annual Meeting Abstracts 435 Mechanisms of wound repair: Lessons learned from the eye Minisymposium Wednesday, May 06, 2015 11:00 AM–12:45 PM 605/607 Minisymposium Program #/Board # Range: 4386–4391 Organizing Section: Lens Contributing Section(s): Cornea, Immunology/Microbiology Program Number: 4386 Presentation Time: 11:00 AM–11:25 AM Site specific differences in the inflammatory response to injury Luisa A. DiPietro. University of Illinois at Chicago, Chicago, IL. Presentation Description: Problems with skin wound healing are a major health issue in the US, as more that 6 million persons develop serious problems with wound healing each year. The two most important problems experienced by patients are 1) non-healing wounds, and 2) wounds that heal with excessive scars. Interestingly, scars are rare in wounds of the oral cavity, suggesting that this tissue heals differently than skin. The comparison of the wound healing process in skin and oral mucosa has shown that oral mucosal wounds heal more quickly, with reduced inflammation, and with a more refined healing response. The study of the response of the oral mucosa to injury suggests therapeutics, including the modulation of inflammation, that might be employed to improve healing outcomes in other tissues. Commercial Relationships: Luisa A. DiPietro, None Support: NIH GM50875 Program Number: 4387 Presentation Time: 11:25 AM–11:45 AM Neutrophils Express Oncomodulin and Promote Optic Nerve Regeneration Larry Benowitz. 1Depts. of Neurosurgery and Ophthalmology, Harvard Medical School, Boston, MA; 2Neurosurgery, Neurobiology, Boston Children’s Hospital, Boston, MA. Presentation Description: Like most mature CNS pathways, the optic nerve cannot regenerate if injured, leaving victims of ischemic or traumatic nerve damage, glaucoma or other degenerative diseases with permanent visual losses. The induction of intraocular inflammation partially reverses this inability and enables retinal ganglion cells (RGCs) to regenerate axons part-way through the injured optic nerve. In cell culture, we found that cells of myeloid origin secrete a protein that enables mature RGCs to grow axons in the presence of two co-factors, mannose and elevated cAMP. We isolated the active protein by HPLC, SDS-PAGE, and cell culture bioassays, and identified it via mass spec as oncomodulin (Ocm), a small Ca++ -binding protein related to parvalbumin1,2. Ocm mRNA and protein increase dramatically in the eye within 12 hours of inducing intraocular inflammation, and gain- and loss- of function studies show that Ocm mediates most of the pro-regenerative effect of inflammation in vivo2-4. Combining intraocular inflammation with deletion of the pten gene enables some RGCs to regenerate axons from the eye to the brain in ~ 10 weeks, forming connections in anatomically appropriate target areas and restoring simple visual responses5. Flow cytometry, immunostaining, and quantitative PCR show that, whereas activated neutrophils and macrophages both express high levels of Ocm, neutrophils represent the more important biological source. The number of neutrophils greatly exceeds that of macrophages in the first several days, and immune-depletion of neutrophils nearly eliminates the effect of pro-inflammatory agents on axon regeneration despite the persistence of macrophages6. Injection of a peptide antagonist of Ocm or a neutralizing antibody similarly produce a near-complete loss of regeneration following nerve injury and pro-inflammatory stimulation3,6. These results show that inflammation enables mature RGCs to undergo extensive axon regeneration, and that this effect is mediated primarily by oncomodulin, an atypical growth factor produced by cells of myeloid origin. ________________________ 1 Y. Yin et al., J Neurosci 23, 2284 (2003); 2Y. Yin et al., Nat Neurosci 9, 843 (2006); 3Y. Yin et al., Proc Natl Acad Sci U S A 106, 19587 (2009); 4T. Kurimoto et al., J Neurosci 30, 15654 (2010); 5S. de Lima et al., Proc Natl Acad Sci U S A 109, 9149 (2012); 6T. Kurimoto et al., J Neurosci 33, 14816 (2013). Commercial Relationships: Larry Benowitz, Boston Children’s Hospital (P) Support: NIH/NEI EY 05690, NIH/IDDRC P30 HD018655, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, Kawasaki Medical School Alumni Association Fund for Foreign Study, Grant-in-Aid for Young Scientists B (23792021) Program Number: 4388 Presentation Time: 11:45 AM–12:00 PM Subbasal Nerves Fail to Project to the Mouse Corneal Apex After Corneal Debridement Mary Ann Stepp. Anatomy and Regenerative Biology Dept., George Washington University, Washington, DC. Presentation Description: Spontaneous erosions form within 2-4 weeks after debridement wounds to the mouse cornea but the exact causes of this pathology remain under investigation. Here we studied leukocyte recruitment and reinnervation of the corneal subbasal nerves after 1.5 mm corneal wounds to determine the mechanisms that lead to the development of epithelial defects in the adult Balb/c mouse. The mouse cornea has dense network of subbasal axons that enter the cornea at the limbus and run parallel to the basement membrane beneath the corneal epithelial basal cells towards the center of the cornea terminating in a swirling pattern termed the vortex at the corneal apex. A 1.5 mm trephine was placed on the ocular surface and an impression was made by rotating it. The impression serves as a guide to allow generation of a 1.5 mm wound; it penetrates the corneal epithelium and severs the subbasal nerves partially denervating the 1.5 mm area. When we wounded corneas by removing 1.5 mm of corneal epithelial tissue by either dulled blade or rotating burr, we found that subbasal axons were unable to target their projections all the way to the corneal apex and fully reinnervate the cornea. By contrast, after using the trephine alone and leaving the epithelium intact, the corneas all reinnervated completely with subbbasal axons targeting their projections to the corneal apex where they reformed a vortex. To allow cells to migrate to the corneal center after dulled blade and rotating burr wounds, the corneal epithelial cells at the periphery disassemble their hemidesmosomes. After migration is completed, they resynthesize a basement membrane and hemidesmosomes to allow firm adhesion; these events can take 2 weeks or longer. Trephine only injuries induce hemidesmosome reassembly in the epithelial cells at the site of trephine mark; the corneal cells at the apex, where the mechanical forces due to blinking are maximal, maintain their hemidesmosomes after trephine wounding. Defective axon targeting is seen after wounds that induce corneal epithelial cell migration but not after denervation alone without induction of cell migration. In addition to gaining insight into corneal erosions, this presentation will highlight the advantages to using the mouse cornea to gain insight into axonal regeneration in the peripheral nervous system. Commercial Relationships: Mary Ann Stepp, None Support: R21 23106 (MAS, APG), R01 21784 (MAS, ASM), RO1 8512 (MAS) ©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: 4389 Presentation Time: 12:00 PM–12:15 PM Regulation of cell motility by galectin-3 and MMP9 Pablo Argueso. Schepens Eye Research Institute, Harvard University, Boston, MA. Presentation Description: Epithelial cells require significant fluidity to change their shape and rearrange their position to assume a migratory phenotype during tissue repair—this involves the remodeling of tight junctions with neighboring cells and of the extracellular matrix. However, the dynamics that initiate these movements remain to be determined. Here, we reveal a hitherto unknown function of the carbohydrate-binding protein galectin-3 in destabilizing cell-cell junctions and cell-cell interactions by inducing matrix metalloproteinase expression. Further, we demonstrate a major role for the oligomeric form of galectin-3 in these processes by interacting with and clustering the matrix metalloproteinase inducer CD147 on the cell surface. Commercial Relationships: Pablo Argueso, None Support: NH Grant EY014847 Program Number: 4390 Presentation Time: 12:15 PM–12:30 PM Mechanisms of wound-healing in the lens: The role of repair cell subpopulations A. Sue Menko. Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA. Presentation Description: In epithelial tissues wound healing involves the collective movement of the injured epithelium directed by a population of mesenchymal leader cells at the wound edge. We have studied epithelial wound repair in the visual system using a lens mock cataract surgery model. Here we found that the leader cells that direct repair of this injured ocular epithelium can be traced to a vimentin-rich progenitor subpopulation of mesodermal origin resident to the lens. In the uninjured lens, these repair cell progenitors are located in niches amongst the cells of the epithelium. This finding was unexpected as prior to our studies the lens was thought to contain only two cell types, lens epithelial cells and their differentiated counterparts, the lens fiber cells. The fiber cell mass is removed during cataract surgery, leaving behind an injured epithelium, the denuded basement membrane of the posterior lens capsule, and the repair cell progenitors. This presentation will provide evidence of the presence of cells in the lens with reparative properties that are of both mesoderm and leukocyte lineages. These repair cells are present in embryonic and adult lens tissue. We will show how these repair cells become associated with the lens during development, and discuss the possibility that association of repair cells with the lens could be a dynamic process that continues in the adult. The mesenchymal repair cells of the lens were found to be rapid responders to injury, migrating immediately to the wound edge and extending lamellipodial processes along the denuded basement membrane of the lens capsule. These features are dependent on vimentin intermediate filament function, as is the repair process itself. We will discuss the function of repair cells in modulating an effective wound response of the epithelium. In addition, we will show evidence that these repair cells have high profibrotic potential, and share unique molecular features with mesenchymal repair cells in other tissue types that have similar ability to attain a myofibroblast phenotype. Evidence will be presented that the transition of repair cell to a myofibroblast is promoted through a mechanotransduction-signaling event. Commercial Relationships: A. Sue Menko, None Support: NIH Grant EY021784 Program Number: 4391 Presentation Time: 12:30 PM–12:45 PM Myofibroblast generation in cornea and lens: modulation of Smad signal by extracellular matrix Shizuya Saika. Dept of Ophthalmology, Wakayama Medical University, Wakayama, Japan. Presentation Description: Myofibroblast appears in the fibrotic tissues that are undergoing wound healing process. In the eye it is observed in corneal stroma, endothelium, crystalline lens or proliferative vitreoretinopathy tissue of origin of retinal pigment epithelium. Its phenotype is quite similar among myofibroblasts of each ocular tissue although the embryonic germinal origins differ from each other. Transforming growth factor β (TGFβ)/Smad signal plays a central role in the process of myofibroblast generation; keratocyte-myofibroblast conversion or epithelial-mesenchymal trantision (EMT), and is known to be further modulated by signals derived from binding of extracellular matrix (ECM) to cell surface receptors. In the current talk the mechanisms of modulation of TGFβ signal and EMT as well as keratocyte-myofibroblast conversion by matricellular proteins, i. e., osteopontin, tenscin-C and lumican will be reviewed. Although overall signals derived from these ECM components support Smad signal and positively modulate EMT or keratocyte-myofibroblast conversion, the detailed mechanisms of actions seem differ among those by each ECM component Commercial Relationships: Shizuya Saika, None Support: The Grant from the Ministry of Education, Science, Sports and Culture of Japan (C19592036) ©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].