Project Summary/Abstract Liver disease is the 10th leading cause of mortality annually. Although most of these deaths are related to cirrhosis from known causes, an estimated 10-30% of individuals have liver disease of unknown etiology. Whole exome sequencing (WES) can provide an actionable diagnosis in 10-30% of these patients. The Vilarinho Laboratory recently used WES to diagnose a cohort of patients with non-cirrhotic portal hypertension with loss of function mutations in the gene GIMAP5. GIMAP5 is a small GTPase that has previously been implicated in immune cell development but had never been associated with liver disease. Using a mouse model of Gimap5 loss of function, we determined that liver sinusoidal endothelial cells (LSECs) capillarize and lose their organotypic features in GIMAP5-mediated disease. In my preliminary work I performed pseudotime analysis on single cell RNA sequencing data to determine that LSECs dedifferentiate into capillarized endothelial cells. Endothelial capillarization is a pathogenic process that occurs in numerous liver disorders and involves loss of fenestrae and development of a basement membrane. Additionally, in order to better understand the organ wide dysfunction caused by endothelial capillarization, I performed confocal microscopy to evaluate zonation in hepatocytes. This demonstrated severe disruption of normal metabolic zonation of the liver. Collectively, these findings suggest that GIMAP5-mediated liver disease is an LSEC-intrinsic disease process that consequently effects hepatocyte zonation and regulates liver homeostasis. I hypothesize that Gimap5 is critical to maintaining LSEC identity and subsequently hepatocyte zonation and function. My first aim is to determine the role of Gimap5 in LSECs. I have created a novel mouse model, that uses a Cre-Lox system in order to knockout genes within LSECs in an inducible and selective manner. I will use this model to knockout Gimap5 within LSECs and then evaluate endothelial capillarization as well as organ-wide dysfunction. I will use a combination of flow cytometry, confocal and electron microscopy to visualize changes due to this dysfunction. My second aim is to determine the role of endothelial Gimap5 in maintaining hepatocyte zonation and function. It has been well recognized that endothelial derived Wnt signaling is necessary for the proper maintenance of hepatocyte metabolic zonation. Preliminary data shows that these Wnt signals are significantly reduced in Gimap5 loss of function endothelial cells. I will perform single cell RNA sequencing to investigate transcriptional alterations of hepatocytes in Gimap5 loss of function mice. I will also isolate mouse hepatocytes from Gimap5 loss of function mice to evaluate alterations in hepatocyte metabolic activity using functional assays. If successful, this proposal will identify the pathomechanisms behind GIMAP5- mediated liver disease and potentially elucidate a novel mechanism of endothelial capillariza...