The Function Of Apolipoprotein E And LDL Receptor-Related Protein1 In Atherogenesis

Macrophages play a critical role in atherogenesis. Macrophages avidly engorge modified LDL particles trapped in the subendothelial space in the artery intima where the efflux machinery cannot pump out a corresponding amount of cholesterol, eventually resulting in foam cell formation. As foam cell formation is an obligatory step for initiation of atherosclerosis, the consequent vessel wall local inflammation and cell apoptosis triggered by macrophage foam cells are the driving force for atherosclerosis progression and the plaque rupture. Many molecules participate in the macrophage cholesterol homeostasis maintenance, inflammatory response, apoptotic transformation and dead cell clearance. Among them, apolipoprotein E (apoE) and LDL receptor-related protein (LRP) are critical.A number of studies have proved that apoE has a multiple functions including the lipoprotein remnants clearance in the liver and neuronal plasticity in the brain. Macrophages contribute to less than 10% of plasma apoE, and in the unique microenvironment of the vessel wall, macrophage-derived apoE plays a crucial protective role in the pathology of atherosclerosis. In the compact intima space where exogenous cholesterol acceptors have limited availability, macrophage-derived apoE is uniquely positioned as a molecule in efficiently mediating cholesterol efflux from foam cells. Bone marrow transplantation studies have convincingly demonstrated that macrophage apoE deletion is the most efficient single macrophage gene deletion in promoting atherogenesis irrespective of the degree of hypercholesterolemia.LRP is both an endocytic and a signaling receptor through binding to over 30 distinct ligands and a large number of cytoplasmic adaptor proteins. The fact that the systemic LRP knockout in mice is lethal demonstrates an irreplaceable role of LRP in development and survival, also presents a challenge in studying its function in vivo through a systemic gene deletion approach. In recent years, through a tissue-specific knockout approach, LRP has been shown to protect the vessel wall from atherosclerosis at the levels of vascular smooth muscle cells (SMCs), hepatocytes and macrophages. In SMCs, LRP may inhibit PDGF-mediated signaling through incompletely understood mechanisms, thus suppressing SMC proliferation and migration. In hepatocytes, LRP not only mediates remnant clearance through apoE-LRP interaction, contributing significantly to plasma lipoprotein metabolism, but also has other anti-atherogenic roles independent of the remnant clearance. At the macrophage level, LRP was previously speculated to be pro-atherogenic since it functions in lipoprotein uptake. However, ours and another study demonstrated that macrophage LRP is anti-atherogenic, even though the mechanisms are largely unknown.ApoE is one of the most important ligands for LRP. In hepatocytes, apoE-LRP interaction mediates lipoprotein remnants clearance. In neurons, apoE-LRP mediated signaling modulates many neuron functions. In macrophages, LRP may be utilized as a receptor to mediate lipoprotein uptake via binding to apoE residing on the surface of the lipoprotein particles and thereafter to route the internalized cholesterol to a specific intracellular handling pathway; apoE-LRP may also mediate lipid-independent signaling pathway to regulate macrophage functioning. Recently, we demonstrated an intracellular direct physical interaction between apoE and LRP uniquely in macrophages which regulates macrophage apoE secretion, highlighting a close apoE-LRP relation in macrophages. In the current study, we use both in vivo atherosclerosis analysis and in vitro macrophage assays to investigate the impact of apoE-LRP interaction in macrophage function and atherogenesis.Previous studies demonstrated an atheroprotective role for macrophage LRP. We found macrophage LRP deletion promotes atherosclerosis, although those macrophages produce and secrete more apoE than wild type macrophages. Since macrophage apoE is a potent anti-atherogenic molecule, we propose that the failure of the increased expression of apoE in LRP-/- macrophages in reducing atherosclerosis may be due to the abrogation of apoE-LRP axis-mediated benefits. So in the current study, we aim at investigating the role of apoE-LRP interaction in macrophage function and atherosclerosis.Atherosclerosis analyses of bone marrow transplanted mice showed that ApoE-/-MФLRP-/- bone marrow recipient LDLR-/- mice developed 79% atherosclerosis lesion in promimal aorta compared to ApoE-/- bone marrow recipient LDLR-/- mice, even though they did not have difference in plasma lipoprotein levels. We also found that combined apoE and LRP deletion in macrophages increased the numbers of macrophage foam cell and apoptotic cell in atherosclerotic lesion by138% and 89.5%, respectively, compared to apoE single deletion. To eliminate the effects of systemic apoE from other tissues on macrophage in the recipient mice, we further directly compared the atherosclerosis lesion sizes and macrophage foam cell numbers in proximal aortas of apoE-/- mice and ApoE-/-MФLRP-/- mice. We found that in apoE-/- MΦLRP-/- mice, the average oil red O stained area was increased by 123%, and the MOMA-2 stained area was increased by 112%, compared to apoE-/- mice. These results demonstrate that macrophage LRP is athero-protective regardless of the absence or presence of macrophage or systemic apoE.In vitro, we collected peritoneal macrophages from mice of four different genotypes (WT, ApoE-/-, MФLRP-/- and apoE-/-MФLRP-/-), and performed experiments to study the cholesterol efflux, cell apoptosis and inflammatory response in order to study the correlation of macrophage apoE-LRP in macrophage functioning. We found that LRP deletion impaired macrophage cholesterol efflux regardless of the state of apoE expression. It demonstrates that LRP has an apoE-independent role in promoting cholesterol efflux from macrophages. These data also highlights the pivotal role of macrophage apoE in maintaining the cholesterol homeostasis in the absence of exogenous cholesterol acceptors. Macrophage apoptosis analysis suggests that macrophage LRP and apoE work both independently and inter-dependently in promoting macrophage survival. Cytokine measurement indicates that apoE and LRP correlatively modulate the inflammatory response to LPS stimulation.Taken together, our data confirmed the atheroprotective role of macrophage LRP in mice irrespective of the degree of hypercholesterolemia. Macrophage apoE and LRP work in both independent and interdependent fashions to modulate macrophage cholesterol homeostasis, apoptosis and inflammation, limiting atherosclerosis development.