Macrophages in the Pathogenesis of Atherosclerosis

In atherosclerosis, the accumulation of apolipoprotein B-lipoproteins in the matrix beneath the endothelial cell layer of blood vessels leads to the recruitment of monocytes, the cells of the immune system that give rise to macrophages and dendritic cells. Macrophages derived from these recruited monocytes participate in a maladaptive, nonresolving inflammatory response that expands the subendothelial layer due to the accumulation of cells, lipid, and matrix. Some lesions subsequently form a necrotic core, triggering acute thrombotic vascular disease, including myocardial infarction, stroke, and sudden cardiac death. This Review discusses the central roles of macrophages in each of these stages of disease pathogenesis. In atherosclerosis, the accumulation of apolipoprotein B-lipoproteins in the matrix beneath the endothelial cell layer of blood vessels leads to the recruitment of monocytes, the cells of the immune system that give rise to macrophages and dendritic cells. Macrophages derived from these recruited monocytes participate in a maladaptive, nonresolving inflammatory response that expands the subendothelial layer due to the accumulation of cells, lipid, and matrix. Some lesions subsequently form a necrotic core, triggering acute thrombotic vascular disease, including myocardial infarction, stroke, and sudden cardiac death. This Review discusses the central roles of macrophages in each of these stages of disease pathogenesis. Atherosclerosis underlies the leading cause of death in industrialized societies and is likely soon to attain this status worldwide (Lloyd-Jones et al., 2010Lloyd-Jones D. Adams R.J. Brown T.M. Carnethon M. Dai S. De Simone G. Ferguson T.B. Ford E. Furie K. Gillespie C. et al.American Heart Association Statistics Committee and Stroke Statistics SubcommitteeExecutive summary: heart disease and stroke statistics—2010 update: a report from the American Heart Association.Circulation. 2010; 121: 948-954Crossref PubMed Scopus (459) Google Scholar). Atherosclerosis first involves a decades-long expansion of the arterial intima, a normally small area between the endothelium and the underlying smooth muscle cells of the media, with lipids, cells, and extracellular matrix (Figure 1). Although this process itself rarely leads to major symptoms due to preservation of the arterial lumen, a few of these lesions undergo necrotic breakdown, which precipitates acute, occlusive lumenal thrombosis and its consequences: myocardial infarction (“heart attack”), unstable angina (accelerating chest pain due to ongoing heart muscle ischemia), sudden cardiac death, and stroke (Virmani et al., 2002Virmani R. Burke A.P. Kolodgie F.D. Farb A. Vulnerable plaque: the pathology of unstable coronary lesions.J. Interv. Cardiol. 2002; 15: 439-446Crossref PubMed Google Scholar). This Review discusses the impact of monocyte-derived macrophages in both early atherogenesis and advanced plaque progression. Atherosclerosis is a focal disease process that occurs predominantly at sites of disturbed laminar flow, notably, arterial branch points and bifurcations. Careful morphological and functional studies of the earliest stages of atherogenesis in human and animal models indicate that the key initiating step is subendothelial accumulation of apolipoprotein B-containing lipoproteins (apoB-LPs; Figure 2) (Williams and Tabas, 1995Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar). ApoB-LPs are made by liver and intestinal cells and consist of a core of neutral lipids, notably cholesteryl fatty acyl esters and triglycerides, surrounded by a monolayer of phospholipid and proteins. Hepatic apoB-LPs are secreted as very low-density lipoproteins (VLDL), which are converted in the circulation to atherogenic low-density lipoprotein (LDL), and intestinal apoB-LPs are secreted as chylomicrons, which are converted by lipolysis into atherogenic particles called remnant lipoproteins. The key early inflammatory response to retained apoB-LPs, which may be enhanced by oxidative modification of the LPs, is activation of overlying endothelial cells in a manner that leads to recruitment of blood-borne monocytes (Glass and Witztum, 2001Glass C.K. Witztum J.L. Atherosclerosis. the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (1583) Google Scholar, Mestas and Ley, 2008Mestas J. Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis.Trends Cardiovasc. Med. 2008; 18: 228-232Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Activated endothelial cells secrete chemoattractants, or “chemokines,” that interact with cognate chemokine receptors on monocytes and promote directional migration (Figure 2 and Movie S1 available online). Importantly, prevention of monocyte entry by blocking chemokines or their receptors prevents or retards atherogenesis in mouse models of atherosclerosis (Mestas and Ley, 2008Mestas J. Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis.Trends Cardiovasc. Med. 2008; 18: 228-232Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). It should be noted that, although early apoB-LP retention precedes and triggers endothelial activation and monocyte entry, monocyte-derived macrophages in the lesion may subsequently secrete apoB-LP-binding proteoglycans (Williams and Tabas, 1995Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar). This mechanism likely plays an important role in the amplification of LP retention once lesions become established, which in turn can help to explain why the inflammation in atherosclerotic lesions fails to resolve (Tabas, 2010aTabas I. Macrophage death and defective inflammation resolution in atherosclerosis.Nat. Rev. Immunol. 2010; 10: 36-46Crossref PubMed Scopus (208) Google Scholar). Monocytes originate from bone marrow-derived progenitor cells, and this early stage of monocyte development may be regulated by cellular cholesterol content in a manner that can affect atherogenesis. Mice whose monocyte progenitor cells have defective cholesterol efflux due to deficiency of ABCA1 and ABCG1 transporters show an increase in circulating monocyte number (monocytosis) and increased atherosclerosis (Yvan-Charvet et al., 2010aYvan-Charvet L. Pagler T. Gautier E.L. Avagyan S. Siry R.L. Han S. Welch C.L. Wang N. Randolph G.J. Snoeck H.W. Tall A.R. ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation.Science. 2010; 328: 1689-1693Crossref PubMed Scopus (117) Google Scholar). Importantly, both the monocytosis and increased atherosclerosis are reversible by restoring cholesterol efflux from monocytes to high-density lipoprotein (HDL), and there are correlations in humans among high HDL, lower blood monocyte counts, and decreased risk for atherosclerosis (Coller, 2005Coller B.S. Leukocytosis and ischemic vascular disease morbidity and mortality: is it time to intervene?.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 658-670Crossref PubMed Scopus (129) Google Scholar). The role of monocyte subsets in atherogenesis has been a topic of great interest throughout the last decade. In mice, hypercholesterolemia is associated with an increase in an inflammatory monocyte subset referred to as Lychi, and these cells enter developing atherosclerotic lesions more readily than Lyclo monocytes. Data from other models of inflammation have suggested that Lychi monocytes participate in the acute response to inflammation, whereas Lyclo monocytes are associated with inflammation resolution (Arnold et al., 2007Arnold L. Henry A. Poron F. Baba-Amer Y. van Rooijen N. Plonquet A. Gherardi R.K. Chazaud B. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.J. Exp. Med. 2007; 204: 1057-1069Crossref PubMed Scopus (326) Google Scholar). However, this scenario may not be directly applicable to atherosclerosis because of the chronic nature of the inflammatory process, and maximal suppression of atherogenesis requires genetic manipulations that block entry of both subsets (Tacke et al., 2007Tacke F. Alvarez D. Kaplan T.J. Jakubzick C. Spanbroek R. Llodra J. Garin A. Liu J. Mack M. van Rooijen N. et al.Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.J. Clin. Invest. 2007; 117: 185-194Crossref PubMed Scopus (419) Google Scholar). Moreover, there are important differences in the characterization and roles of monocyte subsets in humans versus mice. Thus, the key issue as to whether monocyte subsets—and the macrophages and other cells (e.g., dendritic cells) derived from them—have different roles in atherogenesis or plaque progression remains unresolved. Following chemokinesis, monocytes become tethered and roll on endothelial cells overlying retained apoB-LPs, notably through the interaction of monocyte P-selectin glycoprotein ligand-1 (PSGL-1) with endothelial selectins (Mestas and Ley, 2008Mestas J. Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis.Trends Cardiovasc. Med. 2008; 18: 228-232Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Monocytes then become firmly adhered to lesional endothelial cells through the interaction of monocyte integrins with endothelial cell ligands, and recent evidence has implicated monocyte type I interferon signaling in this process (Goossens et al., 2010Goossens P. Gijbels M.J. Zernecke A. Eijgelaar W. Vergouwe M.N. van der Made I. Vanderlocht J. Beckers L. Buurman W.A. Daemen M.J. et al.Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.Cell Metab. 2010; 12: 142-153Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Immunohistochemical analysis of human lesions and molecular-genetic causation studies in gene-targeted mice suggest that the monocyte integrins VLA-4 (very late antigen-4) and LFA-1 (lymphocyte function-associated antigen 1) and their respective endothelial cell ligands, VCAM-1 (vascular cell adhesion molecule) and ICAM-1 (intercellular adhesion molecule-1), may be particularly important in the setting of early atherogenesis. Moreover, it should be noted that platelet aggregation on endothelium overlying atherosclerotic lesions may also promote monocyte-endothelial interactions by activating NF-κB signaling and expression of adhesion molecules and by depositing platelet-derived chemokines on activated endothelium (Mestas and Ley, 2008Mestas J. Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis.Trends Cardiovasc. Med. 2008; 18: 228-232Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Koenen et al., 2009Koenen R.R. von Hundelshausen P. Nesmelova I.V. Zernecke A. Liehn E.A. Sarabi A. Kramp B.K. Piccinini A.M. Paludan S.R. Kowalska M.A. et al.Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice.Nat. Med. 2009; 15: 97-103Crossref PubMed Scopus (157) Google Scholar). Finally, firm adhesion of monocytes is followed by their entry into the subendothelial space (diapedesis) (Kamei and Carman, 2010Kamei M. Carman C.V. New observations on the trafficking and diapedesis of monocytes.Curr. Opin. Hematol. 2010; 17: 43-52Crossref PubMed Scopus (29) Google Scholar). Although diapedesis has been studied extensively in other models of inflammation, work in atherogenesis is limited. In particular, molecules that have been implicated in diapedesis in vitro, such as tissue factor and PECAM-1, have not been shown to participate in diapedesis in the setting of atherogenesis per se (Goel et al., 2008Goel R. Schrank B.R. Arora S. Boylan B. Fleming B. Miura H. Newman P.J. Molthen R.C. Newman D.K. Site-specific effects of PECAM-1 on atherosclerosis in LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 1996-2002Crossref PubMed Scopus (33) Google Scholar, Harry et al., 2008Harry B.L. Sanders J.M. Feaver R.E. Lansey M. Deem T.L. Zarbock A. Bruce A.C. Pryor A.W. Gelfand B.D. Blackman B.R. et al.Endothelial cell PECAM-1 promotes atherosclerotic lesions in areas of disturbed flow in ApoE-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 2003-2008Crossref PubMed Scopus (38) Google Scholar). Part of this complexity may be due to the multiple functions of these molecules and the difficulty of assaying diapedesis in vivo. Driven by macrophage colony-stimulating factor (M-CSF) and probably other differentiation factors, the majority of monocytes in early atherosclerotic lesions become cells with macrophage- and/or dendritic cell-like features (Johnson and Newby, 2009Johnson J.L. Newby A.C. Macrophage heterogeneity in atherosclerotic plaques.Curr. Opin. Lipidol. 2009; 20: 370-378Crossref PubMed Scopus (53) Google Scholar, Paulson et al., 2010Paulson K.E. Zhu S.N. Chen M. Nurmohamed S. Jongstra-Bilen J. Cybulsky M.I. Resident intimal dendritic cells accumulate lipid and contribute to the initiation of atherosclerosis.Circ. Res. 2010; 106: 383-390Crossref PubMed Scopus (78) Google Scholar). There has been great interest in macrophage heterogeneity in atherosclerotic lesions, particularly regarding macrophages involved in proinflammatory processes (M1) versus those involved in resolution and repair (M2), but a clear picture has not yet emerged from these studies (Johnson and Newby, 2009Johnson J.L. Newby A.C. Macrophage heterogeneity in atherosclerotic plaques.Curr. Opin. Lipidol. 2009; 20: 370-378Crossref PubMed Scopus (53) Google Scholar). Much of the theory in this area has been driven by in vitro studies exploring gene/protein expression patterns and functional attributes of monocytes or macrophages subjected to various treatments, including growth/differentiation factors; cytokines derived from type 1 versus type 2 helper T cells; transcription factors, notably PPARs; and even atherogenic lipoproteins and lipids (Johnson and Newby, 2009Johnson J.L. Newby A.C. Macrophage heterogeneity in atherosclerotic plaques.Curr. Opin. Lipidol. 2009; 20: 370-378Crossref PubMed Scopus (53) Google Scholar, Kadl et al., 2010Kadl A. Meher A.K. Sharma P.R. Lee M.Y. Doran A.C. Johnstone S.R. Elliott M.R. Gruber F. Han J. Chen W. et al.Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2.Circ. Res. 2010; 107: 737-746Crossref PubMed Scopus (76) Google Scholar). However, the situation in the atherosclerotic subendothelium is almost certainly more complex, and there is a significant gap between the in vitro and in vivo observations. Future studies will need to focus on macrophage heterogeneity characterized by differential expression of specific molecules or molecular networks that have functional significance for atherogenesis rather than heterogeneity based simply on the aforementioned M1 versus M2 paradigm (Kadl et al., 2010Kadl A. Meher A.K. Sharma P.R. Lee M.Y. Doran A.C. Johnstone S.R. Elliott M.R. Gruber F. Han J. Chen W. et al.Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2.Circ. Res. 2010; 107: 737-746Crossref PubMed Scopus (76) Google Scholar). Moreover, as discussed below, any discussion of “macrophage heterogeneity” must take into account the role of dendritic-like cells and the plasticity of myeloid-derived cells. Even at very early stages of atherogenesis, many macrophages and dendritic-like cells have membrane-bound lipid droplets in the cytoplasm. These lipid-loaded cells are called “foam cells,” and their formation begins when phagocytes ingest and process apoB-LPs (Figure 2 and Movie S1). The mechanism of this uptake is a widely studied and hotly debated area. Early work suggested that uptake of oxidized LDL occurs via scavenger receptors, notably the type A scavenger receptor (SRA) and a member of the type B family, CD36 (Kunjathoor et al., 2002Kunjathoor V.V. Febbraio M. Podrez E.A. Moore K.J. Andersson L. Koehn S. Rhee J.S. Silverstein R. Hoff H.F. Freeman M.W. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages.J. Biol. Chem. 2002; 277: 49982-49988Crossref PubMed Scopus (350) Google Scholar). However, recent gene-targeting studies in apolipoprotein E (ApoE-deficient mice), a widely used model of atherosclerosis in which plasma lipoproteins are elevated due to absence of the lipoprotein-clearing protein ApoE, indicate that additional mechanisms of foam cell formation are also operational in atherosclerosis (Moore et al., 2005Moore K.J. Kunjathoor V.V. Koehn S.L. Manning J.J. Tseng A.A. Silver J.M. McKee M. Freeman M.W. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice.J. Clin. Invest. 2005; 115: 2192-2201Crossref PubMed Scopus (191) Google Scholar, Manning-Tobin et al., 2009Manning-Tobin J.J. Moore K.J. Seimon T.A. Bell S.A. Sharuk M. Alvarez-Leite J.I. de Winther M.P. Tabas I. Freeman M.W. Loss of SR-A and CD36 activity reduces atherosclerotic lesion complexity without abrogating foam cell formation in hyperlipidemic mice.Arterioscler. Thromb. Vasc. 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Further mechanistic and in vivo studies are needed to fully assess the relative importance of these processes, taking into account stage and location of lesions and the particular model being investigated. Once ingested, the cholesteryl esters of the LPs are hydrolyzed in late endosomes to cholesterol, often referred to as free cholesterol, and fatty acids (Maxfield and Tabas, 2005Maxfield F.R. Tabas I. Role of cholesterol and lipid organization in disease.Nature. 2005; 438: 612-621Crossref PubMed Scopus (411) Google Scholar). Late endosomal free cholesterol is trafficked to peripheral cellular sites by poorly understood mechanisms involving the late endosomal proteins NPC1 and NPC2 and the lipid lysobisphosphatidic acid. Recent work also points to a possible role of the ABC transporter protein ABCG1 in this process (Tarr and Edwards, 2008Tarr P.T. Edwards P.A. ABCG1 and ABCG4 are coexpressed in neurons and astrocytes of the CNS and regulate cholesterol homeostasis through SREBP-2.J. Lipid Res. 2008; 49: 169-182Crossref PubMed Scopus (62) Google Scholar). Delivery of free cholesterol to the endoplasmic reticulum (ER) has important roles in downregulating both LDL receptors and endogenous cholesterol synthesis by suppressing the sterol-regulatory element binding pathway (SREBP) (Brown and Goldstein, 1997Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (1816) Google Scholar). Moreover, the free cholesterol undergoes re-esterification to cholesteryl fatty acid esters (the “foam” of foam cells) by the ER enzyme acyl-CoA:cholesterol ester transferase (ACAT) (Brown et al., 1980Brown M.S. Ho Y.K. Goldstein J.L. The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters.J. Biol. Chem. 1980; 255: 9344-9352Abstract Full Text PDF PubMed Google Scholar). Defects in ACAT function or processes involved in cholesterol efflux from cells lead to free cholesterol-induced cytotoxicity and may promote macrophage death in advanced lesions, or “atheromata” (discussed below). Free cholesterol released from lysosomes and from rehydrolyzed cholesteryl ester droplets can also traffic to the plasma membrane and thus be available for efflux out of the cell (Tall et al., 2008Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Wang N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Rothblat and Phillips, 2010Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transport.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (95) Google Scholar). Cholesterol efflux is thought to be a major process involved in plaque regression when hypercholesterolemia is reversed. This expansive area has been covered in many reviews over the last decade, and here we will briefly summarize several key points. The mechanisms and exact route of cholesterol transport to the plasma membrane are not fully known, although Golgi-to-plasma membrane vesicular transport may be involved (Chang et al., 2006Chang T.Y. Chang C.C. Ohgami N. Yamauchi Y. Cholesterol sensing, trafficking, and esterification.Annu. Rev. Cell Dev. Biol. 2006; 22: 129-157Crossref PubMed Scopus (178) Google Scholar, Maxfield and Tabas, 2005Maxfield F.R. Tabas I. Role of cholesterol and lipid organization in disease.Nature. 2005; 438: 612-621Crossref PubMed Scopus (411) Google Scholar). Interestingly, trafficking of free cholesterol out of lysosomes may become defective in lesional macrophages, which would constitute a barrier to cholesterol efflux and lesion regression (Jerome, 2006Jerome W.G. Advanced atherosclerotic foam cell formation has features of an acquired lysosomal storage disorder.Rejuvenation Res. 2006; 9: 245-255Crossref PubMed Scopus (23) Google Scholar). Once at the plasma membrane, cholesterol is transferred to the outer leaflet, where it is removed from the cells by ABCA1- and ABCG1-mediated transport to apolipoprotein A1 and HDL, respectively, or by “passive diffusion” to cholesterol-poor HDL (Tall et al., 2008Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Wang N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Rothblat and Phillips, 2010Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transport.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (95) Google Scholar). As predicted, deletion of both ABCA1 and ABCG1 in macrophages enhances atherosclerosis in mice (Tall et al., 2008Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Wang N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Interestingly, deletion of ABCG1 alone in macrophages suppresses atherogenesis, which has been ascribed to either compensatory induction of ABCA1 and ApoE or to “beneficial” depletion of early lesional macrophages by oxysterol-induced apoptosis (Ranalletta et al., 2006Ranalletta M. Wang N. Han S. Yvan-Charvet L. Welch C. Tall A.R. Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1-/- bone marrow.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2308-2315Crossref PubMed Scopus (95) Google Scholar, Tarling et al., 2010Tarling E.J. Bojanic D.D. Tangirala R.K. Wang X. Lovgren-Sandblom A. Lusis A.J. Bjorkhem I. Edwards P.A. Impaired development of atherosclerosis in Abcg1-/- Apoe-/- mice: identification of specific oxysterols that both accumulate in Abcg1-/- Apoe-/- tissues and induce apoptosis.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1174-1180Crossref PubMed Scopus (19) Google Scholar). Extensive in vitro and in vivo work has focused on how sterol-regulated transcription factors, LXRα and LXRβ (LXR), induces ABCA1 and ABCG1 and, through this and other mechanisms, promote regression of foam cell lesions (Calkin and Tontonoz, 2010Calkin A.C. Tontonoz P. Liver x receptor signaling pathways and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1513-1518Crossref PubMed Scopus (73) Google Scholar). Evidence that ABC transporters, LXR, and other molecules carry out cholesterol efflux and “reverse cholesterol transport” in vivo has come from studies in which wild-type and gene-targeted mice have been injected with macrophages loaded with radiolabeled cholesterol, and then the appearance of label in blood and feces is followed (deGoma et al., 2008deGoma E.M. deGoma R.L. Rader D.J. Beyond high-density lipoprotein cholesterol levels evaluating high-density lipoprotein function as influenced by novel therapeutic approaches.J. Am. Coll. Cardiol. 2008; 51: 2199-2211Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). As a therapeutic strategy to promote lesion regression, investigators have attempted to enhance macrophage cholesterol efflux by increasing HDL or HDL-like particles or by increasing ABC transporters (Tall et al., 2008Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Wang N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Rothblat and Phillips, 2010Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transport.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (95) Google Scholar). Examples include cholesteryl ester transfer protein inhibitors, LXR activators, and HDL mimetics (see section below on therapeutic strategies). Though no drugs have yet been approved for this purpose, this set of strategies continues to be a major focus in cardiovascular drug discovery. A central question in atherogenesis is how the loading of macrophages with lipoprotein-derived cholesterol alters macrophage function, particularly with regard to specific proatherogenic processes. Although markedly excessive loading of free cholesterol in the face of defective cholesterol esterification can have profound effects on macrophages in advanced lesions (discussed below), the influence of cholesteryl ester storage is less certain because it is stored in membrane-bound neutral lipid droplets in the cytoplasm and is thus sequestered from cellular membranes (Maxfield and Tabas, 2005Maxfield F.R. Tabas I. Role of cholesterol and lipid organization in disease.Nature. 2005; 438: 612-621Crossref PubMed Scopus (411) Google Scholar). However, even with normal esterification, macrophages exposed to atherogenic lipoproteins may have some degree of enrichment of free cholesterol in the plasma membrane, which may enhance inflammatory signaling through clustering-mediated activation of signaling receptors due to the increased order parameter of cholesterol-enriched membranes (Yvan-Charvet et al., 2007Yvan-Charvet L. Ranalletta M. Wang N. Han S. Terasaka N. Li R. Welch C. Tall A.R. Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice.J. Clin. Invest. 2007; 117: 3900-3908Crossref PubMed Scopus (0) Google Scholar, Zhu et al., 2008Zhu X. Lee J.Y. Timmins J.M. Brown J.M. Boudyguina E. Mulya A. Gebre A.K. Willingham M.C. Hiltbold E.M. Mishra N. et al.Increased cellular free cholesterol in macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of macrophages.J. Biol. Chem. 2008; 283: 22930-22941Crossref PubMed Scopus (94) Google Scholar, Tang et al., 2009Tang C. Liu Y. Kessler P.S. Vaughan A.M. Oram J.F. The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor.J. Biol. Chem. 2009; 284: 32336-32343Crossref PubMed Scopus (80) Google Scholar). In addition, other proatherogenic lipids delivered to cells by lipoproteins, particularly modified lipoproteins, may have potent effects. As an example, oxysterols and oxidized phospholipids delivered through the uptake of oxidatively modified forms of lipoproteins can induce macrophage apoptosis (Seimon et al., 2010bSeimon T.A. Nadolski M.J. Liao X. Magallon J. Nguyen M. Feric N.T. Koschinsky M.L. Harkewicz R. Witztum J.L. Tsimikas S. et al.Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress.Cell Metab. 2010; 12: 467-482Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). As a new approach to the question of how lipoprotein loading affects macrophage biology, a recent study used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to identify proteins secreted by macrophages using an in vivo model of unloaded versus cholesterol-loaded macrophages (Becker et al., 2010Becker L. Gharib S.A. Irwin A.D. Wijsman E. Vaisar T. Oram J.F. Heinecke J.W. 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