Abstract-Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP Ϫ/Ϫ bone marrow into lethally irradiated female LDLR Ϫ/Ϫ recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of 125 I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-␣, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses. (Circ Res. 2007;100:670-677.) Key Words: low-density lipoprotein receptor-related protein Ⅲ atherosclerosis Ⅲ lipoproteins Ⅲ metalloproteinase Ⅲ macrophage F irst defined as a complex for removal of ␣ 2 -macroglobulin, 1,2 and then later identified as the lowdensity lipoprotein receptor (LDLR)-related protein (LRP), 3 LRP is a 600-kDa membrane receptor linked to numerous cellular functions and intracellular signaling events. 4 It has multiple extracellular ligands, including apolipoprotein E (apoE), lipoprotein lipase, plasma proteases (urokinase-type and tissue plasminogen activators), fibrinolytic factors (IXa and VIIIa), thrombospondin 1 and 2, and chaperone proteins receptor associated protein and heat shock protein-96 (reviewed elsewhere 5 ). The cytoplasmic tail of LRP binds to multiple intracellular adapter and scaffold proteins including disabled-1 and FE65. 6,7 LRP is present in numerous cell types, including macrophages and hepatocytes, and its systemic expression is essential for embryonic development. 8 A fundamental role for hepatic LRP in the clearance of plasma remnants has been demonstrated, as conditional hepatic LRP deletion results in increased plasma triglyceride and chylomicron levels, particularly in the absence of the LDLR. 9 Furthermore, decreased expression of hepatic LRP causes delayed chylomicron remnant clearance, supporting a protective effect of hepatic LRP on atherogenesis via reduced plasma lipoprotein burden. 10 Besides the effect on lipoprotein remnants, hepatic LRP may provide additional vascular protection by mediating the clearance of other proinflammatory ligands including matrix...

Objective The balance between apoptosis susceptibility and efferocytosis of macrophages is central to plaque remodeling and inflammation. LRP1 and its ligand, apoE, have been implicated in efferocytosis and apoptosis in some cell types. We investigated the involvement of the macrophage LRP1/apoE axis in controlling plaque apoptosis and efferocytosis. Method and Results LRP1-/- macrophages displayed nearly 2-fold more TUNEL positivity compared to WT cells in the presence of DMEM alone or with either LPS or oxidized LDL. The survival kinase, pAkt, was barely detectable in LRP1-/- cells, causing decreased pBad and increased cleaved caspase-3. Regardless of the apoptotic stimulation and degree of cell death, LRP1-/- macrophages displayed enhanced inflammation with increased IL-1β, IL-6, and TNFα expression. Efferocytosis of apoptotic macrophages was reduced by 60% in LRP1-/- versus WT macrophages despite increased apoE expression by both LRP1-/- phagocytes and WT apoptotic cells. Compared to WT macrophage lesions, LRP1-/- lesions had 5.7-fold more necrotic core with more dead cells not associated with macrophages. Conclusion Macrophage LRP1 deficiency increases cell death and inflammation by impairing pAkt activation and efferocytosis. Increased apoE expression in LRP1-/- macrophages suggests that the LRP1/apoE axis regulates the balance between apoptosis and efferocytosis thereby preventing necrotic core formation.

Low-density lipoprotein receptor-related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for IntroductionLow-density lipoprotein (LDL) receptor-related protein (LRP-1) is a member of the LDL receptor gene family, which includes type I transmembrane proteins that function in receptor-mediated endocytosis and cell signaling. 1 LRP-1 was first recognized as a receptor for apolipoprotein E and for the protease inhibitor, ␣ 2 -macroglobulin (␣ 2 M) 2,3 ; however, it is now clear that LRP-1 binds more than 40 different ligands, well beyond the more limited scope of the LDL receptor. 1 LRP-1 ligands include proteases, such as tissuetype plasminogen activator (tPA) and matrix metalloprotease-9, growth factors, extracellular matrix proteins, and foreign toxins. LRP-1 also mediates the endocytosis of plasma membrane proteins, including the urokinase receptor (uPAR), amyloid precursor protein, and tissue factor. 4 By regulating cell-surface uPAR, LRP-1 controls the activity of cell signaling factors downstream of uPAR, including extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and Rac1. 5,6 In mice, systemic LRP-1 gene deletion is embryonic lethal. 7 LRP-1 is synthesized as a 600-kDa single-chain precursor and processed by a furin-like protease into the mature 2-chain form. 8 The 85-kDa ␤-chain includes a small ectodomain, a single transmembrane domain, and the cytoplasmic tail. The 515-kDa ␣-chain is entirely extracellular but coupled to the ␤-chain through strong noncovalent forces. 8 The ␣-chain includes 4 clusters of cysteine-rich complementtype repeats (CRs). 1 The second and fourth clusters of CRs mediate most ligand-binding interactions, with the exception of the protease inhibitor, ␣ 2 M, which binds to the first and second CR clusters. 9 In addition to its role as a regulator of cell signaling downstream of uPAR, LRP-1 directly activates cell signaling upon binding of ligands, including tPA, activated ␣ 2 M, and apolipoprotein E. [10][11][12] The exact mechanism by which ligand binding to LRP-1 initiates cell signaling is unclear; however, the LRP-1 ␤-chain contains 2 NPxY motifs, the more C-terminal of which binds signaling adaptor proteins, such as Shc, c-Jun amino-terminal kinase (JNK)-interacting protein-1/2 (JIP-1/JIP-2), Dab-1, and FE65. [13][14][15] Adaptor protein binding to the LRP-1 ␤-chain is controlled by tyrosine phosphorylation, which occurs in response to ligand binding or the activity of nonreceptor tyrosine kinases. 11,13,16 LRP-1-JIP complex sequesters JNK and thereby prev...

Human apoE is a multifunctional and polymorphic protein synthesized and secreted by liver, brain, and tissue macrophages. Here we show that apoE isoforms and mutants expressed through lentiviral transduction display cell-specific differences in secretion efficiency. Whereas apoE3, apoE4, and a natural mutant of apoE4 (apoE-Cys 142 ) were efficiently secreted from macrophages, apoE2 and a non-natural apoE mutant (apoE-Cys 112 /Cys 142 ) were retained in the perinuclear region and only minimally secreted. The secretory block for apoE2 in macrophages was not affected by the ablation of LDLR (low density lipoprotein receptor), ABCA-1, or SR-BI (scavenger receptor class B type I) but was released in the absence of low density lipoprotein receptorrelated protein (LRP). In co-immunoprecipitation experiments, an anti-apoE antibody pulled down two times more LRP in apoE2-transduced macrophages than in apoE3-expressing macrophages. Non-reducing SDS-PAGE/Western blot analyses showed that macrophage apoE2 is mostly dimeric and multimeric, whereas apoE3 is predominantly monomeric. ApoE2 retention and multimer formation also occurred in human macrophages derived from the monocyte cell line THP-1. These results were specific for macrophages, as in transduced mouse primary hepatocytes: 1) ApoE2 was secreted as efficiently as apoE3 and apoE4; 2) all isoforms were exclusively in monomeric form; 3) there was no co-immunoprecipitation of apoE and LRP. A microsomal triglyceride transfer protein (MTP) inhibitor nearly deleted apoB100 secretion from hepatocytes without affecting apoE secretion. These data show that macrophages retain apoE2, a highly expressed protein carried by about 8% of the human population. Given the role of locally produced apoE in regulating cholesterol efflux, modulating inflammation, and controlling oxidative stress, this unique property of apoE2 may have important impacts on atherogenesis.ApoE, 2 a 299-residue exchangeable plasma apolipoprotein, is a major player in lipoprotein metabolism and cardiovascular disease (1). It also plays critical roles in many other important biological processes, including Alzheimer disease and cognitive function, immunoregulation and response to infectious agents, intracellular cholesterol trafficking and control of oxidation, and apoptosis (2). ApoE is synthesized and secreted primarily by the liver but also by brain, skin, and tissue macrophages throughout the body (1, 3). Although most lipoprotein-associated apoE is of hepatic origin, macrophage apoE is a major regulator of cholesterol entry and exit within the atherosclerotic plaque. The atheroprotective role of macrophage apoE has been well documented. Macrophage-derived apoE has been shown to protect against atherosclerosis, both early (4 -6) and late during plaque development (7,8), and even when expressed in amounts too low to affect plasma lipid levels (9). Moreover, C57BL/6 mice reconstituted with apoE Ϫ/Ϫ bone marrow develop 10-fold more atherosclerosis than control mice fed a butter fat diet without any differences ...