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|Title:||The Role of Cytoskeletal Signaling in Lens EMT|
|Abstract:||Cataract is an opacification of the ocular lens and is the leading cause of visual impairment and blindness worldwide. The only form of treatment is removal of lens cells through cataract surgery, however, this procedure can lead to complications, the most common of which is the development of a secondary form of cataract known as posterior capsule opacification (PCO). PCO is caused by lens epithelial cells not removed during cataract surgery, which have the capacity to migrate to the previously cell-free posterior capsule. Here, these remnant cells can undergo epithelial-mesenchymal transition (EMT) into myofibroblasts that impair vision through the promotion of a fibrotic environment involving excessive deposition of extracellular matrix (ECM) and capsule wrinkling. This process is mediated by surgery-induced activation of transforming growth factor (TGF)-β. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT, however, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin related transcription factor A (MRTF-A), an actin-binding protein, and MMP-9 may be important mediators of lens EMT. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFβ-induced cell-cell contact disassembly and ultimate α-smooth muscle actin (αSMA) expression, key transdifferentiation events. Using a rat lens epithelial explant model, my work demonstrated that Y-27632, a ROCK inhibitor, prevented TGFβ-induced nuclear accumulation of MRTF-A, E-cadherin/β-catenin complex disassembly, and expression of MMP-9 and αSMA. Furthermore, using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, my work further demonstrated that MRTF-A nuclear localization and activity is essential to the induction of αSMA expression. Lastly, using rat lens explants treated with recombinant human MMP-9 (rhMMP-9) and MMP-9 KO mouse lens explants I demonstrated a key role for MMP-9 in the proteolytic processing and regulation of membrane-associated E-cadherin. Overall, the findings outlined in this thesis strongly suggest that ROCK-mediated cytoskeletal signaling is indispensable to lens EMT, and involved in several key aspects of the TGF signaling pathway. The role of canonical TGF signaling through Smads has been widely studied in the lens, however, only recently has the academic lens community begun to appreciate the involvement of other signaling pathways. As a result, our understanding of how actin dynamics are involved in lens EMT is still at an early stage. The work in my thesis progresses our knowledge of the interplay between cytoskeletal signaling and its specific downstream mediators that result in a sustained EMT response. This work also has implications beyond lens EMT, impacting our understanding of the pathogenic processes common to a broad array of fibrotic diseases. Importantly, my findings have implications for the therapeutic targeting of ROCK-mediated signaling in the prevention of fibrosis of the lens, and other tissues.|
|Appears in Collections:||Open Access Dissertations and Theses|
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|Anna Korol PhD Thesis 2017 ABSOLUTE FINAL.pdf||25.63 MB||Adobe PDF||View/Open|
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