The long-term objective of this study is to elucidate how PAI-1 modulation affects fundamental aspects of human monocyte/macrophage adhesion and migration in vitro. We hypothesize that a complete loss of PAI- 1 activity in these cells will promote increased """"""""net"""""""" accumulation of resident macrophages within the vessel wall through enhancement of monocyte adhesion and transmigration and macrophage pericellular proteolysis mechanisms. This would increase the cellular substrate necessary for development of pathologic foam cells, which in turn would promote the development of atherosclerotic plaque. If demonstrated, this would be a novel and fundamental description of the role of PAI-1 in monocyte adhesion and migration.
Specific Aim 1 : Characterize the Effects of PAI-1 Modulation in Human Monocytes and Macrophages on Adhesion, Migration, and Pericellular Proteolysis using Functional Assays. Monocytes obtained from healthy volunteers will undergo in vitro PAI-1 gene silencing with siRNA, PAI-1 adenoviral driven over-expression, or will be left unmodified. These cells will subsequently placed in a calcein adhesion assay, a chemotaxis directed transwell migration chamber, and matrigel pericellular proteolysis assay.
Specific Aim 2 : Determine Substrate, Membrane Receptor, and Intracellular Signaling Dependent Mechanisms related to Adhesion and Migration resulting from PAI-1 Modulation in Human Monocytes. We will demonstrate vitronectin to aVB3 integrin dependent and independent adhesion as result of PAI-1 modulation using THP-1 cells, a monocyte cell line that is substrate dependent for adhesion. We will demonstrate the effect of PAI-1 modulation in macrophages on uPA/uPAR dependent pericellular proteolysis and uPAR receptor density. We will determine if there is an intracellular or biochemical relationship between Talin and PAI-1 by using immunocolocaliztion methods, fluorescence polarization binding assays, and fluorescence proteolytic assays.
Specific Aim 3 : Determine if HUVEC secreted PAI-1 or exogenously added PAI-1 can interfere with Monocyte Transendothelial Migration. We will test the ability of monocytes PAI-1 modulation to migrate through stimulated endothelial cells (HUVECs) purified PAI-1 in a transwell migration chamber. Coronary artery disease (plaque buildup in the heart arteries) is the fundamental cause of heart attacks and ischemic heart disease, which is the number one cause of death and hospitalization in our country. This research proposal attempts to define the role of a gene called PAI-1 within the cells that cause coronary artery disease called monocytes. This could serve the public health interest by spurring development of novel therapies for the prevention or treatment of atherosclerosis, which is what causes coronary artery disease. ? ? ?
