Project Summary Interstitial lung diseases (ILD) are devastating disorders causing progressive scarring (i.e. fibrosis) of lung tissue, and result from reciprocal interactions of cellular abnormalities and extracellular matrix (ECM) dysregulation. Current ILD treatment primarily targets cellular signaling and remains unable to halt or reverse fibrogenesis. Despite being a key pathological hallmark of ILD, the ECM has rarely been directly targeted for therapeutic intervention. Fundamentally, the extracellular mechanism underlying lung fibrosis progression remains elusive. To bridge this gap, our objective is to develop an innovative approach for selective profiling of newly synthesized ECM (newsECM) along lung fibrogenesis. The proposed technology will selectively label newsECM produced over defined short time spans by incorporating chemoselective azido-tags via post- translational glycosylation. This will enable enrichment of newsECM free from the pre-existing ECM, and thereby enables sensitive proteomic detection of the dynamic ECM synthesis with unprecedented daily temporal resolution, irrespective of the abundant pre-existing ECM background. Our proposed approach has an inherent preference to ECM proteins, the majority of which are glycosylated. The proposed newsECM profiling will address a major technical barrier in conventional, non-selective mass spectrometry analysis, which suffers from limited sensitivity in detecting the dynamic new ECM deposition that is usually in low abundance. The proposed newsECM profiling technology is versatile and will be implemented in three lung fibrosis models, including in vivo mouse fibrosis models, an ex vivo donor lung perfusion model, and an in vitro synthetic fibrosis model. We intend to pursue the following specific aims. Aim 1 will track newsECM dynamics in vivo during the progression and resolution of lung fibrogenesis, which is expected to reveal pro- and anti-fibrotic ECM factors. Aim 2 will establish the correlation between the in vivo and ex vivo newsECM profiles using murine lungs as a model, and apply the resulting optimized newsECM profiling condition and algorithm to the ex vivo lung perfusion (EVLP) of donor human lungs bearing idiopathic pulmonary fibrosis (IPF), the most common form of ILD, to reveal human-specific pathogenic ECM mechanism. Finally, Aim 3 will establish a synthetic lung fibrosis model tissue-engineered combining fibroblast, epithelium, endothelium and macrophage within decellularized native lung ECM scaffold, and use it as an experimentally tractable system to further decode fibrogenic lung cell-ECM interaction. Furthermore, combining newsECM labeling and native ECM biomaterial engineering, we will offer a chemoselective platform for effective functional evaluation of candidate fibrosis-modulating ECM factors in a biomimetic, ECM-associated manner. In summary, this research will facilitate a paradigm shift in ILD treatment by promoting ECM-targeted therapeutic development and by enabling combined therapeutic interventions aiming at both cellular and extracellular targets to bring new hope for the impacted patients.

Project Narrative The proposed research will markedly advance our understanding of how extracellular matrix dysregulation leads to lung fibrosis (interstitial lung disease). It will promote extracellular targeted therapeutic development that is expected to complement and boost the efficacy of existing cell-centric therapies in treating lung fibrosis. Thus, knowledge derived from this research will bring new hope to patients suffering from this devastating, currently irreversible respiratory condition.