DESCRIPTION (provided by applicant): Fibrosis is pathobiological process common to many tissues and diseases which results in tissue remodeling and loss of function, often necessitating organ replacement or leading to end-stage disease. No therapies are currently available that successfully arrest or reverse fibrosis, and this represents a significant unmet clinical need. Fibrosis occurs predominantly in soft tissues (liver, lung, kidney, heart, skin) through fibroblast proliferation and deposition of extracellular matrix. Our recent work in the lung, and that of others in the liver, demonstrates that extracellular matrix stiffening is an early and prominent event in fibrosis. Critically, we and others have found that matrix stiffening from normal to fibroic levels supports fibroblast activation to a proliferative/matrix synthetic state, and the effects of matrix stiffness are independent of (and/or add to) the effects of TGF-beta, the dominant pro-fibrotic soluble factor. Increasing matrix stiffness thus creates a mechanobiological positive feedback loop that drives progressive fibrosis. We therefore believe fibroblast behaviors should be studied in physiologically relevant matrix stiffness conditions to identify new targets for potential therapeutic intervention relevant to fibrosis. To address this need, we have developed a cell culture platform to study fibroblast biology on matrices of stiffness matched to emerging fibrotic lesions in the lung. Importantly, our approach offers the first opportunity to study fibroblast phenotypic responses to molecular screening within a physiologically relevant mechanical environment compatible with a high throughput, discovery oriented approach. We propose here to screen a library of bioactive molecules and measure effects on key disease-relevant cellular phenotypes in a reference lung fibroblast cell line, and then test candidate molecules for their ability to alter fibrogenic activation of disease relevant primary fibroblasts from IPF and control lungs, all on matrices with stiffness matched to emerging fibrotic lesions. Success will be defined by identification of validated hits with broadly functional effects in down regulating fibrogenic activation of disease-related primary human lung fibroblasts. The identification of stiffness-specific therapies could provide new opportunities for targeted deactivation of fibroblasts and move the field toward new approaches for arresting or reversing progressive fibrosis.

PUBLIC HEALTH RELEVANCE: The prognosis for patients with pulmonary fibrosis remains overwhelmingly negative, thus new therapies and new directions for therapeutic development are sorely needed. The fibroblast and its transition to an activated proliferative, contractile and matrix synthetic state appears to be a key target for therapeutic development. The proposed studies will identify new anti-fibrosis candidates by measuring fibroblast responses to a library o known bioactive molecules on matrices with stiffness matched to emerging fibrotic lesions. __SpecificAimsTextDelimiter__ SPECIFIC AIMS Fibrosis is a pathobiological process common to many tissues and diseases which results in tissue remodeling and loss of function, often necessitating organ replacement or leading to end-stage disease. No therapies are currently available that successfully arrest or reverse fibrosis, and this represents a significant unmet clinical need. Fibrosis occurs predominantly in soft tissues (liver, lung, kidney, heart, skin) through fibroblast proliferation and deposition of extracellular matrix. Our recent work in the lung1, and that of others in the liver2, demonstrates that extracellular matrix stiffening is an early and prominent event in fibrosis. Critically, we and others have found that matrix stiffening from normal to fibrotic levels supports fibroblast activation to a proliferative/matrix synthetic state1,3-5, and the effects of matrix stiffness are independent of (and/or add to) the effects of TGF-beta1,3,6, the dominant pro-fibrotic soluble factor. Increasing matrix stiffness thus creates a mechanobiological positive feedback loop that drives progressive fibrosis1, while also changing fibroblast responses to molecular interventions7. We therefore believe fibroblast behaviors should be studied in physiologically relevant matrix stiffness conditions to identify new targets for potential therapeutic intervention relevant to fibrosis. To address this need, we have developed a cell culture platform to study fibroblast biology on matrices of stiffness matched to emerging fibrotic lesions in the lung7. Importantly, our approach offers the first opportunity to study fibroblast phenotypic responses to molecular screening within a physiologically relevant mechanical environment compatible with a high throughput, discovery oriented approach. We propose here to screen a library of bioactive molecules and measure effects on key disease-relevant cellular phenotypes in a reference lung fibroblast cell line, and then perform follow-on testing of hits identified in this screen using disease relevant primary fibroblasts from IPF and control lungs. Specifically, we propose to: Aim 1: Screen a library of bioactive molecules to discover compounds that tilt a reference fibroblast line toward maximal activation or quiescence on matrices with stiffness matched to early fibrotic lesions. Fibroblasts are acutely sensitive to their mechanical environment, but drug screening on matrices of controlled stiffness has not been possible until now. Using a reference lung fibroblast line (CCL-210) grown on collagen-coated synthetic matrices approximating the stiffness of early fibrotic lesions, we will search for candidate molecules capable of reverting fibroblasts to the quiescent state or pushing them toward maximal activation. We will measure proliferation, pro-collagen expression, F-actin content and cell morphology in a high-content image based screening approach. Candidate molecules will be ranked by multiplex effects on fibroblast activation, and high scoring hits will be validated in follow-on experiments and evaluated using secondary assays to measure candidate effects on additional fibrosis relevant behaviors (matrix synthesis and MMP activity, matrix contraction, production of pro- and anti-fibrotic soluble factors), allowing further prioritization of hits based on broad efficacy against pro-fibrotic behaviors. To further test the efficacy of candidate molecules, assays will be repeated on an alternative disease-relevant matrix coating (fibronectin), and in the presence of varying serum (0-10%) and TGF-beta (0.1-10ng/ml) concentrations. Aim 2: Test the efficacy of candidate molecules in disease relevant primary fibroblasts on matrices with stiffness matched to early fibrotic lesions. Fibroblasts within fibrotic lungs are phenotypically diverse, and likely vary in behavior and therapeutic responsiveness across individuals. Thus, candidate molecules should be tested against primary cells harvested directly from fibrotic lungs. Hence, we will test high priority hits identified in Aim 1 on primary low passage fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and control fibroblasts harvested from normal lung tissue. These experiments will be repeated on both collagen and fibronectin coated synthetic matrices, and in the presence of varying serum and TGF-beta concentrations to test the broad efficacy of candidate molecules for their anti-fibrogenic potency. Assays will then be repeated across a broad range of matrix stiffness conditions to test candidate molecule utility across a broad pathophysiologically relevant range of mechanical environments. Outcome and Impact: Success will be defined by identification of validated hits with broadly functional effects in down-regulating fibrogenic activation of IPF fibroblasts within physiologically relevant mechanical environments. The resulting insights could provide new opportunities for targeted deactivation of fibroblasts and move the field toward new approaches for arresting or reversing progressive fibrosis. Successful completion of this project would open multiple avenues for further advances, including: in vivo testing of identified candidates in fibrosis model systems, expansion of screening efforts using larger and more diverse molecular libraries, and molecular dissection of mechanisms by which identified candidate compounds control fibroblast activation.