Wound healing occurs without complications in most of us. Minor scrapes and cuts heal within a few days and are soon forgotten. Some people, however, do not heal well and either get infections which can be life threatening or scar excessively. An injury to the clear outer surface of the eye, the cornea, induces a complex series of events requiring new protein synthesis, cell migration, cell proliferation, and apoptosis. At a molecular level, the early wound response in the cornea is similar to that in the skin with a sheet of epithelial cells migrating into the wound area. My research has focused on developing a better understanding of how epithelial tissues in the cornea respond to injuries and identifying the proteins which mediate cell migration and cell proliferation. As we extend our understanding of how corneal tissues respond to injury, we are applying our knowledge to epidermal wound healing. Acknowledging that proper wound healing demands that epithelial cells interact with underlying stromal fibroblasts, we have recently begun to study the ways that epithelial cells interact with underlying fibroblasts in the cornea and skin. We have also studied how the cornea is formed during development with the idea that to understand how to fix something, it is important to know how it is put together. Those developmental studies lead to studies aimed at characterizing the adult corneal epithelial stem cells and the proteins they produce. Ultimately, we would like to help improve the quality of life for those suffering from complications caused by poor wound healing.
Specifically, we have been studying corneal and epidermal wound healing at the cellular and molecular level in the cornea in wild-type and various strains of knockout mice lacking genes encoding proteins suspected to be involved in wound healing. We perform experiments on anesthetized animals and remove the epithelial cell layers of the cornea leaving the underlying basement membrane intact. This type of debridement wound is analogous to a blister on the skin. In the clinic, ophthalmologists routinely remove the epithelium from the corneas of their patients prior to performing laser surgery to correct vision for myopia, in order to “clean up” a ragged edge to improve the quality of healing after trauma to the ocular surface, and during retinal surgery to improve their ability to image the retina through the cornea. Thus, our wound-healing model is clinically relevant to patients undergoing these procedures.
The ability of the cells of the wounded corneal epithelium to reduce their tight adhesion to the underlying basement membrane and begin migrating over the exposed basement membrane within hours after injury is mediated by adhesion molecules of the integrin family. Our lab was the first to demonstrate in a PNAS paper in 1990 that α6β4 integrin was an integral membrane component of the hemidesmosomes using the mouse cornea; it took another year for skin researchers to confirm that the same is true of the hemidesmosomes in skin. Since then, we have shown that expression of several members of the integrin family are upregulated in response to debridement wounding and induction of epithelial cell migration and that these changes require that the cornea be allowed to heal in vivo, since in vitro corneal organ cultures respond differently to wounding than do corneas wounded in vivo. We published in 2002 a study in Journal of Cell Science on wound healing in the cornea and skin in mice lacking a gene for the heparan sulfate proteoglycan syndecan-1. We are now moving on to discover the underlying mechanisms behind the delayed healing in cultured cells derived from the cornea of these mice using time-lapse studies of cell migration in both 2-D and 3-D cell culture systems. In 2014 I attended the Proteoglycan Gordon Research Conference and wrote a review article on syndecan-1 and it’s impact on wound healing. That paper is entitled “Syndecan-1 and It’s Expanded Contacts List” and was published in April 2015 in Advances in Wound Care. We have made important contributions to understanding the roles cell:substrate interaction plays in mediating cell migration and in maintaining adhesion of the adult stem-like cells within their niche as shown in a study appearing in Investigative Ophthalmology and Visual Sciences in 2004. In that study, we developed in vivo mouse models for the study of recurrent epithelial erosions and corneal epithelial stem cell deficiency. These models are now helping other labs design studies to develop treatments for these disabling conditions.
One of the integrins whose expression is altered during corneal wound healing is α9 integrin. In the course of working out the methods for quantitation of data from our whole mount staining of corneas for various integrins, we designed a study to assess the expression of α9 integrin during the maturation of the mouse eye from birth until 8 weeks of age. This study demonstrated the feasibility of evaluating the expression and localization of a protein over the entire flat-mounted mouse cornea assessing both regional (inferior, temporal, superior, and nasal) and left-right differences over time during development and in response to wounding. α9 integrin expression in adult unwounded mouse corneas was shown to be regionally restricted to a subpopulation of basal cells at the limbus. This study, which came out in July 2004 in Developmental Dynamics, looked at the developmental regulation of α9 integrin on the mouse ocular surface during corneal maturation and was appreciated by the journal editors and reviewers so much that an image from our work appeared on the cover of the journal. We followed that study up with data on bromodeoxyuridine (BrdU) positive label retaining cells (LRCs) in an article in the journal Stem Cells as well as with a review article in Experimental Eye Research both in 2006 where we clarify the role of α9 integrin in the maintenance of the corneal epithelial stem cells. We proved that α9 integrin, while not a surface marker for corneal epithelial stem cells, is a marker for early transit amplifying cells which arise as progeny from division of the stem cells themselves. By looking at the limbus at BrdU positive LRCs, integrins, and matrix molecules in the basement membrane zone such as laminins and collagen XVII, we are developing an understanding of the niche– the site where the stem cells are maintained and protected. It seems likely that integrins function to retain the stem cells at the niche but how do they do that? What is special about the niche? As we began formulating these questions, it became increasingly clear to us that we could make more progress in the future by using our corneal wound healing model to study how the niche is lost over time in our experimental model of corneal epithelial stem cell deficiency. Doing so has resulted in several additional publications in Stem Cells and J of Cell Science.
We also collaborated on a study appearing in Journal of Investigative Dermatology in 2008 of a tissue specific transgenic mouse that looses expression of α9 integrin in keratin 14 expressing cells beginning about 10 days of development. As expected from our published studies on α9 integrin in the limbal niche, the epithelial cells on the corneas of these mouse are defective and we look forward to being able to do additional studies on these mice to further define the roles of α9 integrin in the maintenance of the corneal epithelium.
One of our research goals is to extend our research findings from their obvious implications for the ocular surface to the skin. To this end, it was enormously beneficial to spend a sabbatical year in 2002 at the NIH National Cancer Institute with Stuart Yuspa, a skin carcinogenesis expert. Dr. Yuspa’s NCI lab which I worked in for a year and continue to visit weekly, as time permits, consists of a mixture of molecular biologists, cancer specialists, and dermatologists working towards understanding how skin cancers develop and progress. My knowledge of epithelial cell biology gained from years of work on the mouse cornea has allowed me to both contribute to their ongoing research efforts and to move quickly towards developing models for the study of epithelial wound healing in mice lacking the proteoglycan, syndecan-1. That work allows us to link skin and corneal wound healing defects through common molecular events that occur in cells derived from epidermal and corneal tissues derived from the syndecan-1 knockout mouse. We published a study in Journal of Cell Science in 2007 that shows that the epithelial cells in these mice actually have altered functions of several different classes of integrins on their surface and another study in Wound Repair and Regeneration in 2008 that describes the differences we see in the dermal fibroblasts from these mice and images from that study have also been selected to appear on the cover of the journal. In addition to a paper on the similarities and differences between migration and integrin function in corneal fibroblasts from wt and sdc-1 null mice that appeared in Experimental Cell Research in 2010. We also published a paper showing differences in skin cancer in sdc-1 null mice in the journal Molecular Carcinogenesis in collaboration with colleagues at the NCI in 2010 and one on delayed healing in CLIC-4 null mice in American Journal of Pathology in 2012.
Our work on corneal wound healing has been leading us into the role played by the immune system in corneal wound healing. We have a mouse model we use for these experiments that allows wounds to resolve and another model that permits spontaneous formation of recurrent erosions on the cornea. There are differences in the types of immune cells initially recruited into the cornea soon after the two different types of wounds. There are also differences in the way the nerves regrow into the cornea in wounds that resolve compared to those which develop erosions. By looking at the expression of mRNAs for molecules that inhibit nerve regrowth, we believe that we will be able to propose new treatments for recurrent erosions in the cornea that may apply to other conditions where reinnervation of sensory nerves is a problem as in diabetes and in the elderly. We have a paper that will come out soon in Investigative Ophthalmology and Visual Science that begins to describe the immune response to sterile injury in the mouse cornea. The development of animal models for the study of wound healing in the cornea has been a topic that we have a great deal of expertise in. We believe that sharing that knowledge with the vision research community will help those interested in doing corneal wound healing research develop better animal models. We recently spent a great deal of time writing a review article for Experimental Eye Research on this topic. Our co-authors included James D. Zieske at Harvard Medical School and the Mass. Eye and Ear Infirmary and Vickery Trinkaus-Randall from Boston University School of Medicine. We hope this article will have a positive impact on the field. I appreciated having the time to work on it after my term as chair of the NIH BVS study section was completed.
One way we have been able to contribute to the research community at GWU has been to work with faculty in the Department of Mechanical and Aerospace Engineering in the School of Engineering and Applied Science. We have been fortunate to collaborate with Dr. Michael Keidar on a project to better understand the impact of cold plasma atmospheric jets on biological tissues and to help develop biological applications for this new technology. The GWU Interdisciplinary Research Fund has been generous with their support for our projects. Dr. Keidar is an expert in the use of atmospheric plasma; in our lab, we understand cell biology and can assess the impact of treatment of cells and tissues with cold plasma on a cell’s viability and ability to migrate. We have collaborated on three papers in the Applied Physics Journal, Plasma Processes Polymers, and Plasma Medicine. I also co-mentored a talented Ph.D. student in Mechanical Engineering (Olga Volotskova) with Dr. Keidar. Olga graduated in August 2012 and is now a post-doctoral fellow at Stanford University in CA.
We have an NIH grant with colleagues at Thomas Jefferson University to look at the roles played by immune cells in mediating tissue repair responses in the lens and cornea. These studies are showing that immune cells are present in the developing and adult lens. The roles they play in cataract development and the ability of the lens to respond to injury is not known. These studies are the most basic of those we have been doing and resulted in an exciting paper recently published recently in Molecular Biology of the Cell that looks at the role of the intermediate filament protein vimentin in mediating the wound response of the lens to injury. The paper in Molecular Biology of the Cell was highlighted independently in 2 reviews that appeared in Current Opinions in Cell Biology [Leduc and Etienne-Manneville, Current Opinions in Cell Biology 32:102-112, 2015; Leube, et al., Current Opinions in Cell Biology 32:13-20, 2015] as an important finding that enhances our understanding of how the intermediate filaments are involved in regulating cell migration. We look forward to continuing these studies as well as those described above.