PROJECT SUMMARY / ABSTRACT Liver disease affects hundreds of millions of people worldwide. Liver transplantation remains the only treatment for end stage liver disease (ESLD); however, transplantation faces a major burden due to a shortage of liver donors. The liver is known for a remarkable regenerative ability through hepatocyte-driven regeneration, the proliferation of the main functional liver cell type, the hepatocyte. However, in ESLD, hepatocyte proliferation becomes exhausted. An alternative strategy to treat ESLD would be to promote liver regeneration through biliary epithelial cell (BEC)-driven regeneration as a novel mechanism that could be exploited as a therapeutic target. BEC-driven liver regeneration is a process by which BECs proliferate, transition to a liver progenitor cell (LPC) stage, a process defined as ductular reaction (DR), and differentiate into functional hepatocytes and restore liver function. BEC-driven regeneration has been experimentally demonstrated in mouse models in which hepatocyte proliferation was compromised and in our zebrafish models after near complete ablation of hepatocytes. Observation of DR in virtually all chronic and acute human liver diseases and of budding hepatocytes from BECs in human cirrhotic livers suggests that BEC-driven repair occurs in human, yet differentiation into hepatocytes is insufficient to alleviate the liver disease. Our goal is therefore to identify and test a druggable pathway that triggers BEC-to-hepatocyte conversion for the efficient therapeutic use of BECs to treat liver diseases. We propose that VEGFA promotes BEC-to-hepatocyte conversion and rescues liver function in complementary mouse and zebrafish liver injury models. Our preliminary data demonstrate that delivery of VEGFA in injured mouse livers via the non-integrative and safe nucleoside-modified mRNA complexed with lipid nanoparticles (mRNA-LNP) induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. Moreover, blocking VEGFR2, the main receptor for VEGFA, or downstream mediators PI3K/AKT abrogates BEC-driven liver regeneration in zebrafish, suggesting the key contribution of the VEGFR2/PI3K/AKT axis. Previous studies showed that VEGFA promotes liver repair in rodents by stimulating VEGFR2 on endothelial cells (ECs) that induce the secretion of hepatocyte mitogens and thus drives hepatocyte-driven repair. However, it has not been reported that VEGFA directly affects BECs for BEC-driven repair, which is suggested by our preliminary data showing VEGFR2 induction in a subset of BECs in injured mouse livers, an observation also reported by others in injured rats. Therefore, our findings combined with studies from others lead us to test 2 hypotheses using the complementary mouse and zebrafish liver injury models: (Aim 1) VEGFA delivery in vivo triggers BEC-to- hepatocyte conversion to replenish the lost cell mass and restore liver functions, and that (Aim 2) VEGFA acts directly on BECs and/or i...