Fibrosis, or scarring, is an evolutionarily developed method to rapidly close and repair a wound. Excessive deposition of extracellular matrix (ECM) and the generation of contractile fibroblasts (myofibroblasts) characterize fibrosis. In the cornea, severe wounds heal using fibrosis. However, in contrast to other tissues where function is maintained, fibrosis in the cornea leaves an opaque cornea that can impair vision. In the past funding period, we made the exciting observation that expression of integrin v6 in the epithelium after wounding appears to play a major role in directing the fibrotic response in the cornea. Integrin v6 binds to ECM, but more importantly, also has the ability to activate TGF-1 (T1) and -3 (T3). These isoforms of TGF- have been linked to stimulating (T1) and blunting (T3) fibrosis. Interestingly, our preliminary data indicate that a single application of T after wounding almost completely blunts the scarring response and that in an in vitro model, T3 may even reverse fibrosis. The hypothesis we propose to test is: Enhanced expression of v6 and subsequent activation of T1 is a crucial initiating step in wound healing and fibrosis. Activation of T3 later in the healing process dampens the progression towards fibrosis and scarring. We propose three specific aims to address the following questions. First, does v6 differentially activate T1 and T3 in wounds that resolve versus those that scar? Second, is v6 expression required for the stimulation of a fibrotic response? Third, does T3 promote regeneration in corneal wounds? In vivo wound healing models in mice will be used to examine these questions. However, the majority of experiments will make use of an innovative co-culture model that combines human corneal epithelial cells with human corneal fibroblasts that have been stimulated to secrete a stroma-like matrix. Specific components of fibrosis will be examined along with signaling components of fibrosis. Immunofluorescence microscopy, qRT-PCR, and protein assays will be the major analysis tools. In addition, specific inhibitors of v6 expression and function will be used. Scarring processes to be examined include: myofibroblast generation and apoptosis, TGF--signaling pathways and ECM deposition. Relevance to Public Health - Over 10 million people world-wide suffer from blinding due to corneal scarring. Currently, no therapies are available except for corneal transplantation. Our studies will also evaluate if manipulating v6 or if treating wounds with T3 can reduce corneal scarring in a human model.
Fibrosis, or scarring, is an evolutionarily developed method to rapidly close and repair a wound; however, in corneas, fibrosis leaves the cornea opaque and thus, non-functional. TGF-?1 (T1) has been linked to stimulating fibrosis and TGF-?3 (T3) to blunting it. Integrin ?v?6, which is known to activate both T1 and T3, is upregulated in the migrating epithelium that covers a corneal wound. Therefore, we propose to use a co-culture system consisting of human corneal epithelial cells and fibroblasts, as well as ?v?6-/- mice, to determine the mechanisms involved in ?v?6 activation of fibrosis, if integrin ?v?6 is required to stimulate fibrosis, and if T3 blunts the scarring response.
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