The human liver scavenges extracellular matrix material, including hyaluronic acid, chondroitin sulfate, and dead red blood cells, from circulation, in addition to serving as a metabolic power house. This scavenging activity is performed by liver sinusoidal endothelial cells (LSECs) expressing the Stabilin-2 receptor. Upon activation by a subset of ligands, Stabilin-2 induces the MAP kinase and NF-kB pathways, by which all LSECs sense their environment and reciprocate using small molecules like nitric oxide. LSECs serve as the interface between the blood and hepatocytes and contain fenestrae (sieve plates) that regulate macromolecules perfusing through to the hepatocytes. Damage of LSECs often reduces the number and size of the fenestrae, affecting overall liver metabolism. Liver diseases, such as fatty liver, affect more than a third of the adult population, and these diseases directly affect the scavenger/sensing capacity of LSECs. Damaged LSECs lead to a build-up of extracellular matrix material in the blood that then negatively affects kidney function. The liver clears dead cells from blood, but it is also a site for metastatic cancer, both of which are intimately related to the health of LSECs. To more fully understand the clearance role of LSECs, the project leader proposes to examine them under shear stress conditions that mimic conditions in the hepatic sinusoid. This study will be the first time that the clearance activity of LSECs has been examined under variant flow to assess activity of Stabilin-2 as the major receptor. Questions to be addressed include: What effect does shear stress have on simple ligands or complex ligands like pericellular HA coated cells? How do LSECs respond to their ligands during shear stress, and what effect does fatty liver, a common ailment, have on LSEC Stabilin-2-mediated endocytosis? From his previous studies with Stabilin-2, the project leader hypothesizes that specific ligand binding to Stabilin-2 by circulating hyaluronan-rich cells is enhanced under shear stress, allowing them to cease circulation and proliferate in liver, and that expression levels of Stabilin-2 directly correlates with liver clearance function. The central hypothesis of the proposed research is that ligand-Stabilin-2 interactions, followed by endocytosis and activation of cell signaling, are influenced by shear stress at the sinusoids and hepatic metabolic status (e.g., fatty liver), which affects blood clearance and cancer cell metastasis to liver. The project leader will test this hypothesis using stable recombinant cells and primary LSECs from normal and disease models of rat livers. Purified LSECs from these livers will be evaluated under flow cell culture conditions to monitor ligand binding and signaling events propagated by Stabilin-2. To accomplish the project goal, the project leader will pursue three specific aims: 1) Determine Stabilin-2 binding of ligands under flow conditions, 2) Determine the effects of shear stress on cellular signaling in LSECs, and 3) Determine Stabilin-2 expression and endocytic activity in normal and fatty liver.
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