Project Summary/Abstract Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease characterized by progressive scarring of the lungs, ultimately impeding the ability to breathe. Pathological recruitment of fibroblasts to sites of tissue injury and subsequent activation into scar-forming myofibroblasts are critical steps in the development and progression of pulmonary fibrosis. Accordingly, the identification of the molecular mediators directing fibroblast recruitment and myofibroblast activation, will not only further enhance our understanding of the pathogenesis of lung fibrosis, but also provide rational therapeutic targets for novel anti-fibrotic therapies. We and others have recently shown that increased matrix stiffness in fibrotic lungs promotes mechano-activation of fibroblasts. Further, matrix stiffness amplifies tissue fibrosis by locking stiffness-activated myofibroblasts on a mechanical positive feedback loop, by not fully understood mechanisms. We have recent evidence that matrix stiffness gradients produced in fibrotic lung tissues promote fibroblast recruitment to sites of tissue injury via durotaxis – the directed migration of cells from regions of lower to higher stiffness, which occurs independently of diffusible chemoattractants or substrate-bound haptotactic cues. As fibroblast “durotax” to regions of increased stiffness, the stiffness of these regions would drive the arriving fibroblasts to differentiate into myofibroblasts. Consequently, the central hypotheses of this proposal are that: (1) fibroblasts are recruited to sites of focal tissue injury via durotaxis, a mechanism in which cells migrate up stiffness gradients independently of chemotactic signals; and (2) that inhibition of fibroblast durotaxis has the potential to be a new therapeutic strategy for IPF. The studies proposed in this application are designed to visualize fibroblast durotaxis in vivo, to define molecular mechanisms of fibroblast durotaxis and to develop novel therapeutic strategies to inhibit durotaxis. Specifically, we propose: (1) To image fibroblast durotaxis ex vivo using multiphoton microscopy in precision cut lung slices and to investigate the therapeutic efficacy of targeting fibroblast durotaxis in vivo in a mouse model of lung fibrosis by inhibiting the mechanosensitive FAK/Paxillin pathway; (2) To define mechanisms by which the αvβ3/FAK/Paxillin pathway regulates matrix rigidity sensing and durotaxis in IPF fibroblasts. We will investigate both biochemical and biophysical regulators of αvβ3 integrin and their role in fibroblast durotaxis; and (3) To define mechanisms by which actin-microtubule crosstalk mediates fibroblast durotaxis. Specifically, we will investigate mechanisms by which α-TAT1-mediated microtubule acetylation controls dynamic recycling of αvβ3/FAK/Paxillin complexes in durotactic cells. We will also test the role of αTAT-1 in fibroblast durotaxis and pulmonary fibrosis in vivo in the bleomycin model of lung fibrosis, using fibroblasts-specific αTAT-1 KO mice generated by our laboratory. The experiments proposed in this application will provide new knowledge regarding the role of fibroblast durotaxis in the development and progression of lung fibrosis, and the potential efficacy of anti-durotactic therapy for the treatment of lung fibrosis.

Project Narrative Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease characterized by progressive scarring of the lungs. We have recent evidence that matrix stiffness gradients produced in fibrotic lungs promote progressive recruitment of fibroblasts to sites of active fibrosis via durotaxis – the directed migration of cells from regions of lower to higher stiffness. The studies proposed in this application are designed to visualize fibroblast durotaxis in vivo, to define the molecular mechanisms of fibroblast durotaxis and to develop novel therapeutic strategies to inhibit durotaxis in patients with IPF, an incurable and deadly disease.