For the past 20 years, the long-range goal of our laboratory has focused on understanding the cellular and molecular mechanisms of cornea! wound healing. Importantly, our studies as well as others indicate that TGFB induced corneal myofibroblasts differentiation plays a critical role in wound healing and that myofibroblasts are responsible for the development of corneal haze and fibrosis after injury and refractive surgery. Recently, we have identified that: 1) the stroma contains a population of bone marrow-derived cells that exhibit dynamic searching activities and initiate corneal keratocyte activation upon stimulation, 2) myofibroblast differentiation and survival depend respectively on a p38 and PI3-kinase alternate TGFB signaling pathway, and 3) resolution of corneal haze after injury correlates with the disappearance of myofibroblast suggesting myofibroblast apoptosis. Based on these findings we propose the following Hypotheses: (1) Corneal injury results in activation of corneal macrophages that directly interact with keratocytes leading to TGFB mediated keratocyte activation and myofibroblasts differentiation. (2) Myofibroblast differentiation involves multiple TGFB signaling pathways including TAK1/p38/ATF2 and PI3-kinase/Akt that control myofibroblast differentiation and survival. And (3) Loss of growth factor signaling leads to down regulation of PI3-kinase/Akt survival pathway and the induction of myofibroblast apoptosis. Using these novel microscopic approaches involving 4-D live cell imaging and in vivo retroviral gene transfer we will test our hypotheses using the following Specific Aims: (1) Determine the role of corneal macrophages in myofibroblast differentiation by: (a) Characterizing the macrophage population in normal and injured mouse cornea, (b) Assessing the interaction between corneal macrophages and keratocytes. (c) Identifying the effects of macrophage stimulation on myofibroblast differentiation of cultured rabbit keratocytes. 2. Determine the role of alternative TGFp signaling pathways for myofibroblasts differentiation and survival by: (a) Measuring phosphorylation of TAK1, ATF2 and Akt during TGFp induced myofibroblast differentiation and IL-1a mediated myofibroblast apoptosis in cultured rabbit keratocytes. (b) Assess the effects of dominant negative interference and gene knockdown of ATF2 and Akt on myofibroblast differentiation and survival, (c) Characterize the effects of topically administered inhibitors of p38 (SB203580), PI3-kinase (LY234002) and Akt (AG1879) on in vivo myofibroblast differentiation and survival in rabbit wounds. 3. Determine the fate of corneal myofibroblasts after wound healing by: (a) Assessing the cell fate of EGFP transduced cells in normal and injured rabbit corneas, (b) Characterizing the effect of apoptosis inhibition on myofibroblast survival by transducing rabbit wound healing fibroblasts with baculovirus gene for inhibitor of apoptosis (cplAP). ? ? ? ?
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