Effect of lysosomal cholesterol accumulation on lysosomal and vacuolar-ATPase activity

Abstract

This project is concerned with the lysosomal accumulation of lipid in the developing macrophage foam cell as occurs in the atherosclerotic lesion. Lysosomal lipid accumulation in atherosclerosis is a feature of advanced atherosclerotic lesions, but an under explored area of research. In this dissertation I explored the potential mechanisms behind the lysosomal accumulation of lipid. We found that, upon incubation of macrophages with physiological lipid particles, there was an increase in lysosome pH over time such that the number of lysosomes having an active pH decreased. This could be duplicated by pharmacologically sequestering free cholesterol within the lysosome. To analyze for the potential mechanism behind lysosomal inactivation a procedure for isolating lysosomes and monitoring their activation in vitro was established. The studies presented here are the first to monitoring how cholesterol affects the activation of the human macrophage vacuolar-ATPase, which is responsible for maintaining the acidic nature of the lysosome. These experiments determined that exogenously increasing the lysosomal membrane cholesterol inhibited the vacuolar-ATPase. Furthermore, data indicate that the entire vacuolar-ATPase complex is inhibited and not just the pumping of hydrogen ions into the lysosomal lumen. This inhibition of the vacuolar-ATPase does appear to be a reversible process if the membrane cholesterol levels are returned to normal. These data suggest that, if the cholesterol could be removed from the lesion, the lysosomes could potentially recover their v-ATPase activity and become reactivated. These studies should aid in the understanding of lysosome biology and provide a more comprehensive understanding of macrophage foam cell metabolism in the atherosclerotic lesion.