Novel mechanisms of macrophage foam cell formation. - Project Summary In the development of atherosclerotic plaques, macrophages infiltrate the intima of the aortic wall to digest modified lipoproteins. Over time, these macrophages become lipid laden foam cells. Most modified lipoproteins are aggregated and crosslinked to the extracellular matrix of the vessel wall, which prevents them from being phagocytosed by macrophages. We have described how macrophages digest aggregated LDL extracellularly using a process termed digestive exophagy. Macrophage first form a sealed, acidified extracellular compartment around aggregated LDL. Lysosomes are then secreted into this compartment, allowing lysosomal acid lipase to hydrolyze cholesteryl esters into free cholesterol. However, it remains unclear how this excess extracellular free cholesterol is transported into macrophages for lipid droplet formation. Moreover, recent cell-lineage analyses showed that most foam cells found in plaques are of smooth muscle cell origin and not macrophages. This discovery is intriguing because smooth muscle cells are not capable of digestive exophagy. My proposed research aims to elucidate the molecular mechanisms that can help address these two existing research questions, which would provide alternative mechanisms for foam cell formation. Based on my preliminary results, I hypothesize that the cholesterol transport protein, STARD4, can mediate the transport of free cholesterol from the plasma membrane of macrophages to the ER for lipid droplet biogenesis (Aim 1). In Aim 1.1, I propose to investigate changes in HDL metabolisms in STARD4 knockout macrophages with lipid mass spectrometry. In Aim 1.2, I will examine if the in vitro finding would translate to a delay in plaque formation in mouse models. Furthermore, I hypothesize that the lysosomal cholesterol transport proteins NPC1 and NPC2 can help transporting and inserting free cholesterol from the extracellular compartment to the plasma membrane of macrophages (Aim 2). Finally, I hypothesize that smooth muscle cells turn into foam cells by absorbing free cholesterol released by neighboring macrophages during digestive exophagy (Aim 3). My proposed studies will expand our understanding of the basic biology of Mϕ-agLDL interactions – a poorly understood and often overlooked process that underlies the development of atherosclerotic plaques. My pilot studies on STARD4 demonstrated that loss of STARD4 specifically impaired aggregated LDL-mediated foam cell formation and not acetylated LDL-mediated foam cell formation. Thus, the characterization of how extracellular free cholesterol released from digestive exophagy enters Mϕs and contributes to foam cell formation would open an avenue for novel therapeutic approaches. Designing or identifying inhibitors that slow down the incorporation of PM free cholesterol into lipid droplets could slow down foam cell formation in a manner that is independent of current treatments, and this may provide additive effect when used in combination with current treatments.
