ABSTRACT Identifying and understanding the mechanisms whereby key hepatocyte-derived danger signals drive the fibrogenic response to liver injury remains a current challenge to make progress in this field of research. Our recent studies revealed that high-mobility group box-1 (HMGB1) is up-regulated, oxidized and secreted largely by damaged hepatocytes. Therefore, since hepatocyte-derived HMGB1 can communicate injury to neighboring cells, specific isoforms could target hepatic stellate cells, the key pro-fibrogenic cell type in the liver, to elicit scarring.PreliminaryresultssupportingthisapplicationshowthatinductionofHMGB1expressioncorrelates with fibrosis stage in patients and mice. Importantly, hepatocyte-derived HMGB1 undergoes unique post- translational modifications in response to oxidant stress. While fully reduced followed by oxidized or disulfide HMGB1 significantly rise at peak fibrosis; yet, sulfonate HMGB1 appears mostly during fibrosis resolution whereas the other isoforms decline. Hmgb1 ablation in hepatocytes (Hmgb1ΔHep) protects from fibrosis progression. Administration of oxidized HMGB1 enhances liver fibrosis more than reduced HMGB1. In contrast, injection of sulfonate HMGB1 lowers collagen-I and induces stellate cell apoptosis, critical for fibrosis resolution. Moreover, ablation of the HMGB1 receptor for advanced glycation end-products (Rage) in stellate cells blocks the HMGB1-mediated collagen-I increase in vitro and RAGE neutralization or ablation in stellate cells (RageΔHSC) prevents scarring in vivo. While we demonstrate that the oxidized HMGB1 isoforms are mostly produced by damaged hepatocytes, their specific contribution to fibrosis progression and/or resolution is still undefined. Our central hypothesis is that the redox-sensitive dynamic changes in hepatocyte-derived HMGB1 isoforms signal via RAGE-dependent mechanisms to drive the stellate cell pro-fibrogenic phenotype and fate, therefore contributing to the progression and/or resolution of liver fibrosis. In Aim 1, to determine the HMGB1 isoforms’ affinity and potential competition among them for RAGE, we will: 1) study the binding ability and kinetics of each HMGB1 isoform to RAGE with surface plasmon resonance; 2) identify the precise amino acid residues from each HMGB1 isoform that interact with RAGE by nuclear magnetic resonance spectroscopy; and 3) prove the ligand-receptor interaction using in vitro and in vivo approaches. In Aim 2, to identify how the HMGB1 isoforms signal via RAGE-dependent mechanisms to drive the stellate cell pro-fibrogenic phenotype and fate, we will use complementary in vitro systems and in vivo approaches. Fibrosis progression and resolutionwillbemonitoredbymagneticresonanceelastographyandconventionalparametersofliverinjury. Next, the signals transduced by each isoform in stellate cells via RAGE to up-regulate collagen-I and drive the cell fate will be identified with a proteomics approach followed by in vitro validation; then, the key s...