TGF-beta, matrix, and myofibroblasts in hepatic fibrosis

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DESCRIPTION (provided by applicant): This application is in response to NOT-OD-09-058 - Notice entitled NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of this competitive revision application for R01-DK058123 is to add an additional specific aim - directly related to the three existing aims and derived from data already generated - to investigate mechanical factors in bridging fibrosis. The goal of the parent grant is to develop a model of fibrosis that incorporates soluble, cellular, and mechanical factors. The original proposal concentrated on early fibrosis, in particular on early mechanical changes in the liver that resulted in a favorable environment for myofibroblastic differentiation. The three original specific aims were: 1) To determine the role of integrins in hepatic stellate cell (HSC) mechanosensing during myofibroblastic differentiation; 2) To develop a mechanical model of fibrosis that incorporated both HSC and portal fibroblast (PF) differentiation, with an emphasis on characterizing regional mechanical differences; and 3) To identify mediators of matrix stiffness in fibrosis, with a particular focus on the lysyl oxidase (LOX) family of collagen cross-linking enzymes. Our original model extended to the point where HSC and PF differentiated into fibrogenic myofibroblasts. We propose here to extend the model to encompass the architectural changes that occur in bridging fibrosis. We suggest that bridging fibrosis results mechanistically from the regional mechanics of the liver combined with the contractile properties of HSC and PF in a stiff environment. We propose the following hypothesis: that mechanical heterogeneity in early fibrosis combined with myofibroblast contractility can explain the development of bridging fibrosis. The new specific aim is to determine the contribution of mechanical factors (myofibroblast contractility and regional increases in liver stiffness) to the development of bridging fibrosis. The research plan includes 1) a detailed morphological analysis of fibrotic rat liver tissue for evidence of collagen fibril translocation and alignment; 2) an analysis of collagen fibril orientation using second harmonics and a comparison, using microindentation techniques, of the stiffness in portal and intervening regions in early fibrosis; and 3) use of ROCK1 mice in an in vivo fibrosis model to determine whether cell-mediated integrin adhesion and traction is required for bridging fibrosis to occur. If the hypothesis underlying this proposal is proven correct, it would have major implications for understanding and, potentially, treating fibrosis. PUBLIC HEALTH RELEVANCE: Late stage liver fibrosis and the end-stage of the disease, cirrhosis, are devastating complications of chronic liver injury. The mechanisms whereby fibrosis progresses, eventually becoming cirrhosis, are not well understood. This application proposes a new model to explain components of fibrosis progression, that, if supported by the experiments proposed here, would have significant implications for the understanding and treatment of fibrosis.