In response to trauma to the cornea, spontaneous recurrent erosions can develop after wounds have initially healed. Wounds that involve the peripheral cornea near the corneal epithelial stem cells can lead to a corneal epithelial stem cell deficiency (CESCD) that causes scratchy eyes and blood vessels to grow onto the cornea and interfere with vision. In the aging cornea, healing of wounds becomes slower due to diseases such as diabetes. Using various strains of normal and genetically modified mice and the culture of mouse epithelial cells, experimental model systems have been developed to study these conditions and to develop new treatments. Past research has lead to the hypothesis that expression of proteins called integrins is altered when healing is delayed and when recurrent erosions and/or CESCD are present. To better understand the causes and develop treatments for these conditions, the following Specific Aims are proposed: 1. To test in vivo in mice whether trauma-induced recurrent erosions are due to incomplete reassembly of adhesion complexes which, in turn, induces chronic activation of ADAM17 by: la. Evaluating adhesion complex reassembly using 3-D confocal imaging of whole mouse corneas and TEM after superficial keratectomy and debridement wounds, Ib. Determining the timing of ADAM-17 up-regulation and activation after corneal debridement wounding, and Ic. Reducing the frequency of erosions by inhibiting the activity of MMPs and ADAM17 using the small molecule inhibitor GM6001. 2. To test the hypothesis that CESCD is caused by the induction of proliferation of early transit amplfying (eTA) cells during reepithelialization, CESCD will be quantified in vivo by measuring K8+Muc5ac+ goblet cells on the cornea using whole mount confocal microscopy 4 wk after the following variations of the manual corneal debridement wound model: 2a. Inducing CESCD by increasing the size of the wounds above 1.5 mm and measuring eTA cell proliferation. 2b, Generating annular wounds leaving a central corneal epithelial cell island to reduce proliferation and migration of eTA and CESCs after injury, and 2c. Reducing CESCD by treating the nasal limbus with either mitomycin C or tryphostin AG1478 immediately after debridement wounds that normally induce CESCD. 3. To test whether altered integrin expression and EGFR-mediated signaling are responsible for the increase in keratinocyte migration that occurs in response to TGFpl and compare to corneas in organ culture by: 3a. Determining if migration and EGF receptor signaling differs between wt and syndl null cells in response to TGFpl and/or EGF, 3b. Inducing the syndl null phenotype in wt mouse skin cells and in a human corneal epithelial cell line by infecting cells with syndl directed siRNA-expressing lentivirus, 3c. Using debridement wounded wt and syndl null mouse corneas in organ culture to determine whether TGF(31 and EGF induce similar responses. Significant progress has been made in understanding how the cornea heals but improved treatment options need to be developed for patients with pathological healing due to trauma.
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