The Overall Objectives of this proposal are to define the mechanism of liver sinusoidal endothelial cell (LSEC) endotheliopathy and its pathogenic role in portal hypertension and liver inflammation in metabolic dysfunction-associated steatohepatitis (MASH). MASH pathogenesis involves both lipotoxicity (toxic lipid-induced cellular stress) and sterile inflammatory responses. Lipotoxicity in LSEC triggers aberrant signaling, resulting in LSEC dysfunction and a proinflammatory phenotype, which we refer to as endotheliopathy. Emerging data implicate LSEC endotheliopathy in liver inflammation. Furthermore, LSEC dysfunction leading to subclinical portal hypertension in non-cirrhotic MASH patients has been recognized as a driver of liver fibrosis\o "Baffy, 2022 #36". However, the molecular mediators of LSEC endotheliopathy and functional consequences in MASH are largely unknown. We identified glycogen synthase kinase (GSK)3β as the top kinase in LSEC phosphoproteomics and focal adhesion and transendothelial migration (TEM) as the major pathways of multiomics integration on LSECs from mice with MASH. Our preliminary data indicate that during lipotoxicity: 1) GSK3β phosphorylates myosin light chain 2, generating contractile stress fibers and increasing cellular stiffness, thereby augmenting vascular resistance; 2) GSK3β increases the LSEC expression of the adhesion molecule intercellular adhesion molecule 1 (ICAM1) and enhances myeloid cell adhesion and TEM; 3) pharmacological GSK3 inhibition reduces liver inflammation and fibrosis, and endothelial cell-specific Gsk3β deletion reduces portal pressure in mice with diet-induced MASH. Based on these novel observations, we formulated the CENTRAL HYPOTHESIS that lipotoxicity-induced GSK3β activation in LSEC increases vascular resistance and mediates TEM of myeloid cells, thereby promoting portal hypertension and liver inflammation in MASH. The proposal will test this hypothesis and provide insights into two independent and integrated specific aims. First, we will study the in vitro mechanism and functional outcome of GSK3β-mediated LSEC cytoskeletal rearrangement and increased cellular stiffness. Using mice with diet induced MASH, and a novel GSK3 inhibitor, or mice with conditional deletion of Gsk3β in endothelial cells, we will define the LSEC vs hepatocyte specific role of GSK3β in MASH, and the potential for therapeutic targeting. Second, we will define the mechanism and functional outcome of GSK3β-mediated ICAM1 transcriptional upregulation and the therapeutic potential of ICAM1 pharmacological inhibition and endotjelial cell genetic repression in mouse models of MASH. We will then validate the human relevance of LSEC endotheliopathy and myeloid cells TEM in MASH by spatial transcriptomics.