An early feature of the developing atherosclerotic lesion is accumulation of cholesterol in artery wall cells. This accumulation may be modulated by cellular pathways that transport cholesterol from intracellular storage compartments to the cell surface for excretion. The overall objective of this project is to characterize the structural and regulatory properties of cellular cholesterol trafficking pathways and to evaluate the role of these pathways in atherogenesis. We have shown that cholesterol trafficking pathways in cultured cells are regulated in response to changes in the growth state and cholesterol status of cells and by the interaction of high.density lipoprotein (HDL) and platelet-derived growth factor (PDGF) with cell-surface binding sites or receptors. HDL cellular interactions function to stimulate translocation to the cell surface and excretion of intracellular stores of excess cholesterol, whereas PDGF receptor binding appears to stimulate cholesterol translocation to the plasma membrane for use as a structural component of newly-synthesized membranes. We propose to use these different regulated conditions to characterize the properties of cellular trafficking pathways and to test the hypothesis that the functionally-diverse stimuli, HDL and PDGF, may modulate these pathways by common mechanisms. We will examine the intracellular compartments and structures involved in HDL- and PDGF- regulated cholesterol transport using cell fractionation techniques, fluorescent microscopy, and intracellular transport inhibitors. We also propose to characterize intracellular signals that modulate HDL-mediated cholesterol trafficking by assaying cells for activation of different phospholipases, protein kinases, and other signals associated with stimulation of cholesterol transport and excretion. Additional studies will identify' signalling pathways involved in PDGF-mediated cholesterol trafficking using signalling enzyme inhibitors, different isoforms of PDGF, and cell lines expressing different wild-type and mutant forms of PDGF receptors. Lastly, we will evaluate whether proteins involved in sterol trafficking are expressed in lesion and non-lesion areas of the artery wall. These studies will generate important information about the mechanisms of cholesterol trafficking in cells and provide insight into how the lipoprotein and cytokine environment of the artery wall may influence cellular cholesterol homeostasis and contribute to atherogenesis.
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