The overall goal of this research proposal is to define the molecular mechanisms by which biliary proteins interact with cholesterol vesicles in bile to promote cholesterol crystal nucleation, crystal growth and gallstone formation. Cholesterol gallstones account for 70% of gallbladder stones seen in the United States and result in over 500,000 cholecystectomies annually with an estimated health care cost in excess of $5 billion. Gallstone formation is multifactorial and biliary proteins play a critical role in the nucleation and growth of cholesterol monohydrate crystals. Gallbladder mucin the major component of the mucus gel in the gallbladder acts as the """"""""glue"""""""" which binds plates of cholesterol to form gallstones. Mucus hypersecretion may occur prior to gallstone formation resulting in an ideal environment for the growth of cholesterol crystals. Non-mucin proteins, such as biliary immunoglobulins and aminopeptidase N, also promote cholesterol crystal nucleation from supersaturated model and human biles. We hypothesize that these poorly described protein-lipid interactions are critical for cholesterol crystal nucleation and that nucleation and growth preferentially occur in the mucus gel layer of the gallbladder.
The specific aims of this proposal are to examine the effect of biliary proteins on the earliest step in nucleation, the fusion or lysis of cholesterol-phospholipid vesicles in model and human bile. This will be achieved by fluorescent assays of vesicle fusion or lysis and confirmation of morphological changes using biophysical techniques such as light scattering and electron microscopy. Comparison of these assays with nucleation time will determine if vesicle-protein interactions are indeed the earliest events in crystal formation and define the kinetics of nucleation. The effect of mucin gels on vesicle diffusion and motion will be studied and compared with synthetic gels to determine the unique properties of mucin on cholesterol solubility and nucleation. Comparison of vesicles to inert microspheres in mucin gels using dynamic and forced Raleigh light scattering will differentiate between effects of gel viscosity and vesicle fusion on movement through physiological gels.Finally, cholesterol crystal growth in mucin and synthetic gels will be examined by phase microscopy in a dynamic model to identify the role of gels on crystal growth. The cholesterol donors, in model and human bile, to crystal growth in gels will be identified. These studies will determine the unique interactions between bilary proteins and cholesterol vesicles in solution and in gels and define their contribution to crystal nucleation and gallstone growth perhaps leading to novel therapies for the treatment and prevention of this common disease.
