Subcell Biochem. ; doi: /_8. Lipoprotein modification and macrophage uptake: role of pathologic cholesterol. It is thus important to understand the processes involved in, and outcome of, macrophage lipoprotein uptake. This review will cover lipoprotein uptake by the. Oxidized low-density lipoprotein (OxLDL) is a risk factor for Tissue-resident cells such as macrophages and mast cells release inflammatory.
Nuclear Factor erythroid-derived 2 -like 2; oxLDL: Tumour Necrosis Factor Introduction Macrophages are a major cell type in the atherosclerotic plaque which play a key role in plaque development, progression and ultimately, rupture. An early and ongoing process in plaque development is macrophage uptake of modified LDL a major carrier of cholesterol that has accumulated in the sub-endothelial space [ 12 ]. This ingestion of retained lipoprotein transforms the macrophages into foam cells [ 3 ] and an inflammatory response ensues.
However, this response is maladapted as the macrophage foam cells do not leave but are retained within the vessel wall [ 2 ].
The Bidirectional Relationship between Cholesterol and Macrophage Polarization
Foam cell accumulation, their apoptosis and subsequent necrosis leads to development of the necrotic core - a major contributor to plaque instability [ 45 ]. It is thus important to understand the processes involved in, and outcome of, macrophage lipoprotein uptake. This review will cover lipoprotein uptake by the different macrophage phenotypes identified in the plaque and, in addition, as cholesterol uptake or efflux can further modify macrophage function, the effect of LDL and HDL on macrophage polarization is also discussed.
Macrophage phenotypes in the atherosclerotic plaque While macrophage polarization suggests two extremes of phenotype, and indeed the terms M1 and M2 predominate in the literature, it is well appreciated that there is a spectrum of macrophage phenotypes that can be adopted with considerable plasticity between them . This is especially apparent in atherosclerosis where monocytes, and subsequently macrophages, are exposed to an array of factors throughout plaque initiation and progression and in the advanced plaque.
Furthermore, the advanced plaque is complex and heterogeneous in nature, often containing regions of intra-plaque hemorrhage IPH. Aside from these identified forms, a range of intermediate phenotypes may also be present. In addition, other monocyte-derived cells which share overlapping functions with macrophages are also present in the plaque, such as dendritic cells [ 2324 ] and fibrocytes [ 25 ]. However, oxLDL uptake itself can also alter macrophage phenotype.
Macrophage Foam Cell Formation with Native Low Density Lipoprotein
The numerous bioactive compounds present in LDL, and their modified forms, exert specific effects. Oxidized phospholipids, for example, promote formation of the Mox macrophage, which is distinct from the M1 and M2 macrophage forms [ 26 ].
Cholesterol is also present in the plaque in crystalline form.Cholesterol Metabolism, LDL, HDL and other Lipoproteins, Animation
Cholesterol crystals activate different and distinct pathways in the macrophages. It is noted, though, that M-CSF stimulation induces expression of a substantial portion of the M2 transcriptome [ 31 ].
Furthermore, M-CSF differentiated macrophages have also been called M0 or resting macrophages [ 32 ]. Indeed, macrophages lacking CD are evident in the plaque [ 16 ], though these could also be M1 macrophages. Furthermore, the loss of CD was accompanied by an inability of hemoglobin-haptoglobin Hb: Hp to induce hemoxygenase 1 HO-1 expression [ 16 ].
Transcriptome analysis and functional studies show that M4 macrophages are distinct from M-CSF differentiated as well as M1 and M2 polarized macrophages [ 36 ]. CD36 expression is lower on M4 macrophages than M-CSF differentiated macrophages and, accordingly, they have less intracellular cholesterol upon incubation with oxLDL [ 36 ]. The increased ABCG1 gene expression suggests that this may be through both reduced lipid uptake and increased efflux [ 36 ].
It is not clear whether M4 macrophages are predominantly pro- or anti-atherogenic [ 36 ]. However, as CXCL4 deficiency results in decreased atherosclerotic plaque burden [ 37 ], M4 macrophages may play a pro-atherosclerotic role [ 16 ].
The lipid handling capacities of the M1 and M2 macrophages have been examined in vitro and the presence of M1 and M2 foam cells examined in both mouse and human plaques. Although it is noted that monocytes in this later study were from obese subjects with diabetes. However, in healthy controls, ApoA1- and HDL3-cholesterol efflux was found to be lower in M2a compared to resting macrophages [ 18 ]. Furthermore, the level of expression of both the ABCA1 and ApoE genes was lower in M2a compared to M1 and resting macrophages [ 18 ], suggesting that lower cholesterol efflux does contribute to increased cholesterol accumulation in M2 macrophages.
Consistent with this increased lipid uptake, mannose receptor MR: In human plaques, although the presence of inflammatory macrophages has long been recognized, the first identification of M2 macrophages was by Bouhlel in [ 15 ]. In addition, both M1 and M2 markers were found in the cap, primarily on spindle shaped cells [ 19 ]. Whether M2 macrophages and their foam cell forms predominate in the early human atherosclerotic plaque is not clear, but with M-CSF promoting expression of M2 related genes [ 31 ], then M2 macrophages and subsequently M2 derived foam cells may arise early in plaque development.
Cholesterol uptake promotes ER stress which triggers the unfolded protein response [ 4244 ]. Since M2 IL derived foam cells are more sensitive to the unfolded protein response than other forms of macrophages [ 45 ], the lack of M2 macrophages in advanced human plaque may, in part, stem from increased cell death.
M1 macrophages are thought to be detrimental to plaque stability as aside from contributing to formation of the necrotic core, they contribute to thinning of the fibrous cap. M1 macrophages are found in the rupture-prone shoulder regions of the plaque [ 47 ] and there is an inverse relationship between the level of M1 CD86but not M2 CDwith carotid plaque cap thickness [ 19 ].
These findings are consistent with known role of M1 macrophages in tissue destruction [ 6 ]. This is consistent with their known role in tissue repair. Mhem macrophages Intraplaque hemorrhage is a common feature in the advanced plaque and its presence is associated with plaque progression [ 49 ]. Red blood cells have cholesterol enriched membranes which can result in increased cholesterol deposition and subsequent enlargement of the core [ 50 ].
Furthermore, RBC lysis releases hemoglobin. Heme and hemoglobin are strong oxidizers which could potentially increase lipid oxidation [ 51 ]. Hemoglobin is bound by haptoglobin to form the Hb: Hp complex [ 52 ] and macrophages scavenge this complex through CDthe Hb: Hp receptor [ 52 ]. This results in the release of IL, which forms an autocrine loop, stimulating further expression of CD [ 22 ].
Such IPH localized macrophages are known as Mhem macrophages [ 20 ]. In vitro studies show that Hb: Hp driven adoption of the Mhem phenotype is IL dependent [ 22 ] which suggests that the Mhem macrophages are related to the M2c form. While transcriptome analysis shows that Mhem are distinguishable from M2 and M1 macrophages by the expression of ATF [ 20 ], this comparison was made between IL-4 stimulated M2a but not IL stimulated macrophages.
However, exposure to Hb: Hp or heme [ 53 ] will no doubt trigger upregulation of specific genes which would allow distinction of these cells from M2c macrophages.
HO-1 breaks down hemoglobin to carbon monoxide, biliverdin which is rapidly converted to bilirubin and free iron [ 54 ] which is either used by the cell or bound by ferritin and exported out of the cell via the iron exporter ferroportin [ 21 ].
ATF-1 induced upregulation of HO-1 enables the safe handling of iron. As such, Mhem macrophages are resistant to lipid loading as they have lower expression of genes associated with lipid uptake, but higher expression of genes involved in reverse cholesterol transport [ 2021 ]. In the plaque, Mhem macrophages are found distant from the core and do not take up lipid [ 20 - 22 ].
The anti-inflammatory functions of the Mhem form are athero-protective. Non-HDL cholesterol levels capture all of the apoB containing lipoproteins in one number and are useful in assessing risk in the setting of hypertriglyceridemia.
Here, we also describe the current landscape of HDL metabolism. Furthermore, we describe many beneficial properties of HDL that antagonize atherosclerosis and how HDL dysfunction may promote cardiometabolic disease. The discovery by Virchow more than years ago that atheroma contained a yellow fatty substance, later identified as cholesterol by Windaus, suggested a role for lipids in the pathogenesis of atherosclerosis2.
Indeed, the goal of this chapter is to focus on the role of lipids and lipoproteins in the pathogenesis of atherosclerosis as well as their critical roles in risk assessment and as targets of therapy. The recognition that atherosclerosis is an inflammatory disease Figure 1 Initiation of the atherosclerotic lesion. The fatty streak phase of atherosclerosis begins with dysfunctional endothelial cells and the retention of apoB-containing lipoproteins LDL, VLDL, and apoE remnants in the subendothelial space.
Retained lipoproteins are modified oxidation, glycation, enzymaticwhich, along with other atherogenic factors, promotes activation of endothelial cells. Activated endothelial cells also promote the recruitment of other immune cells including dendritic cells, mast cells, regulatory T T-reg cells, and T helper 1 Th-1 cells. The monocytes differentiate into macrophages and express receptors that mediate the internalization of VLDL, apoE remnants, and modified LDL to become foam cells.
In addition, inflammatory signaling pathways are activated in macrophage foam cells leading to more cell recruitment and LDL modification. First, we provide brief description of the cellular and molecular events in the key stages of atherosclerosis.
The Role of Lipids and Lipoproteins in Atherosclerosis - Endotext - NCBI Bookshelf
Initiation and Fatty Streak Phase of Atherosclerotic Lesions The endothelial lining of arteries responds to mechanical and molecular stimuli to regulate tone,4 hemostasis,5 and inflammation6 throughout the circulation. Endothelial cell dysfunction is an initial step in atherosclerotic lesion formation and is more likely to occur at arterial curves and branches that are subjected to low shear stress and disturbed blood flow atherosclerosis prone areas 7, 8.
These mechanical stimuli activate signaling pathways leading to a dysfunctional endothelium lining that is barrier compromised, prothrombotic, and proinflammatory9. In atherosclerosis susceptible regions, the endothelial cells have cuboidal morphology, a thin glycocalyx layer, and a disordered alignment8, 10, In addition, these regions have increased endothelial cell senescence and apoptosis as evidenced by ER stress markers In contrast, less atherosclerosis prone endothelium is exposed to laminar shear stress causing activation of signaling pathways that maintain endothelial cell coaxial alignment, proliferation,13, 14 glycocalyx layer,15 and survival12, The increased nitric oxide NO production promotes endothelial cell migration and survival thereby maintaining an effective barrier In addition, the expression of superoxide dismutase SOD is increased to reduce cellular oxidative stress In atherosclerosis susceptible regions, reduced expression of eNOS and SOD leads to compromised endothelial barrier integrity Figure 1leading to increased accumulation and retention of subendothelial atherogenic apolipoprotein B apoB -containing lipoproteins low-density lipoproteins LDL and remnants of very low-density lipoproteins VLDL and chylomicrons 21, In addition, endothelial cell activation leads to increased production of reactive oxygen species25 that can cause oxidative modification of apoB-containing lipoproteins Besides mechanical stimuli, endothelial cell activation is increased by various molecular stimuli, including oxidized LDL, cytokines, advanced glycosylation end products, and pathogen-associated molecules In contrast, an atheroprotective function of HDL is to prevent endothelial activation and enhance NO production to maintain barrier integrity see details below Monocyte Recruitment and Foam Cell Formation Activation of endothelial cells causes a monocyte recruitment cascade involving rolling, adhesion, activation and transendothelial migration Figure 1.
Selectins, especially P-selectin, mediate the initial rolling interaction of monocytes with the endothelium Potent chemoattractant factors such as MCP-1 and IL-8 then induce migration of monocytes into the subendothelial space Ly6hi monocytes, versus Ly6lo, preferentially migrate into the subendothelial space to convert to proinflammatory macrophages in mice The enhanced migration of Ly6hi versus Ly6lo monocytes likely results from increased expression of functional P-selectin glycoprotein ligand In addition, the number of blood monocytes originating from the bone marrow and spleen, especially Ly6hi cells, increases in response to hypercholesterolemia Although macrophages are the main infiltrating cells, other cells contribute to development of lesions including dendritic cells41, 42 mast cells and T cells Figure 1 43, T cells regulate the proinflammatory phenotype of macrophages.
Antigen-specific activated T helper 1 Th-1 cells produce interferon IFN that converts macrophages to a proinflammatory M1 phenotype. During the initial fatty streak phase of atherosclerosis Figure 1the monocyte-derived macrophages internalize the retained apoB-containing lipoproteins, which are degraded in lysosomes, where excess free cholesterol is trafficked to the endoplasmic reticulum ER to be esterified by acyl CoA: Modification of apoB lipoproteins via oxidation and glycation enhances their uptake through a number of receptors not down-regulated by cholesterol including CD36, scavenger receptor A, and lectin-like receptor family see details below Figure 2 47, Enzyme-mediated aggregation of apoB lipoproteins enhances uptake via phagocytosis Figure 2 49, The LDL is endocytosed and trafficked to lysosomes, where the cholesteryl ester CE is hydrolyzed to free cholesterol FC by the acid lipase.
Cholesterol regulation of the LDLR prevents foam cell formation via this receptor in the setting of hypercholesterolemia. Uptake of native LDL by fluid phase pinocytosis may also contribute to foam cell formation.
Modifications of apoB containing lipoproteins induce significant cholesterol accumulation via a number of mechanisms. Enzyme-mediated aggregation of apoB lipoproteins enhances uptake via phagocytosis.
Cytoplasmic CE is cleared by two main pathways. In one pathway, removal of FC from the plasma membrane stimulates transport of FC that has been generated by neutral cholesterol esterase away from ACAT to the plasma membrane. Alternatively, cytoplasmic CE is packaged into autophagosomes, which are transported to fuse with lysosomes, where the CE is hydrolyzed by acid lipase and the resulting FC is then transported to the plasma membrane. The efflux of FC to lipid-poor apolipoproteins or HDL occurs by a number of mechanisms to reduce foam cell formation.
ApoE produces the most buoyant, FC-enriched particles.
ABCG1 may also play a role in the intracellular trafficking of cholesterol. Figure 2 51, Uptake of native LDL by fluid phase pinocytosis may also contribute to foam cell formation Figure 2 53, The triggering of macrophage inflammatory pathways is also a critical event in lesion development.
Oxidative stress, modified lipoproteins, and other lesion factors bioactive lipids, pattern recognition molecules, cytokines are capable of inducing inflammation via receptors55, 56, In addition, plasma membrane cholesterol in macrophage foam cells enhances signaling via inflammatory receptors62, Cytoplasmic CE is cleared by two major pathways.
Alternatively, cytoplasmic CE is packaged into autophagosomes, which are trafficked to lysosomes, where the CE is hydrolyzed by acid lipase73, 74, generating free cholesterol that is made available for efflux mainly via ABCA1 Figure 2 73, Furthermore, HDL and apoA-I protect against atherosclerosis by reducing inflammation via mechanisms independent of cholesterol efflux31, 75 see details below.
ApoE serves as the ligand for clearance of all of the apoB containing lipoproteins from the blood by the liver except for LDL. Gene knockout of apoE in mice results in hypercholesterolemia and spontaneous atherosclerotic lesion development 77, Hence, ApoE deficient mice have been used widely to study mechanisms of atherosclerotic lesion development.
Bone marrow transplantation studies were used to examine the role of macrophage apoE in lipoprotein metabolism. Interestingly, ApoE protects against atherosclerosis via several mechanisms. Expression of apoE by hematopoietic stem cells reduces monocyte proliferation and infiltration into the intima In addition, apoE on apoB lipoproteins reduces the lysosomal accumulation of cholesterol by enhancing the expression of acid lipase Importantly, secretion of apoE by macrophages stimulates efflux in the absence and presence of exogenous acceptors, including HDL and lipid-free apoA-I Figure 2 Recent studies demonstrated that macrophage apoE facilitates reverse cholesterol transport in vivo Endogenous apoE is required for efficient formation of the most buoyant, cholesterol-enriched particles by macrophages Figure 2 84, In addition to cholesterol efflux, macrophage apoE prevents inflammation and oxidative stress The local production of apoE is likely a critical atheroprotective mechanism considering that areas of atherosclerotic lesions have limited accessibility to plasma apoA-1 and HDL80, 81, Humans express three common apoE polymorphisms that predict CAD rates independently from plasma cholesterol levels ApoE3 C, R is the most common isoform and is functionally similar to mouse apoE.
Compared to apoE3 and apoE2 C, CapoE4 R, R are impaired in stimulating cholesterol efflux and in preventing inflammation and oxidation 97, Consistent with the compromised function of apoE4, human carriers exhibit increased risk of CAD compared to humans expressing apoE3 or apoE2 heterozygous, Figure 3 Progression of the atherosclerotic plaque.
Macrophage foam cell and endothelial cell inflammatory signaling continues to promote the recruitment of more monocytes and immune cells into the subendothelial space. Transition from a fatty streak to a fibrous fatty lesion occurs with the infiltration and proliferation of tunica media smooth muscle cells.
Smooth muscle cells are recruited to the luminal side of the lesion to proliferate and generate an extracellular matrix network to form a barrier between lesional prothrombotic factors and blood platelets and procoagulant factors.
A subset of smooth muscle cells express macrophage receptors and internalize lipoproteins to become foam cells. Fibrous fatty lesions are less likely to regress than fatty streaks. Progression to Advanced Atherosclerotic Lesions Fatty streaks do not result in clinical complications and can even undergo regression. However, once smooth muscle cells infiltrate, and the lesions become more advanced, regression is less likely to occur, Small populations of vascular smooth muscle cells VSMCs already present in the intima proliferate in response to growth factors produced by inflammatory macrophages In addition, macrophage-derived chemoattractants cause tunica media smooth muscle cells to migrate into the intima and proliferate Figure 3.
Critical smooth muscle cell chemoattractants and growth factors include PDGF isoforms, matrix metalloproteinases, fibroblast growth factors, and heparin-binding epidermal growth factor Figure 3 HDL prevents smooth muscle cell chemokine production and proliferation. The accumulating VSMCs produce a complex extracellular matrix composed of collagen, proteoglycans, and elastin to form a fibrous cap over a core comprised of foam cells Figure 4 A vital component of the fibrous cap is collagen, and macrophage-derived TGF- stimulates its production Figure 4 In addition, HDL maintains plaque stability by inhibiting degradation of the fibrous cap extracellular matrix through its anti-elastase activity This smooth muscle cell phenotype produces less -actin and expresses macrophage markers, including CD68 and Mac, While studies have shown that VSMCs express the VLDL receptor and various scavenger receptors, data showing that these cells robustly load with CE, similar to macrophages via these mechanisms is lacking.
As lesions advance, substantial extracellular lipid accumulates in the core, in part due to large CE-rich particles arising from dead macrophage foam cells, Regardless of the mechanisms of cholesterol enrichment, VSMCs compared to macrophages are inefficient at lysosomal processing and trafficking of cholesterol, and express much less ABCA, which all contribute to impaired cholesterol efflux However, macrophages in more advanced plaques also have reduced lysosome function and trapping of free and esterified cholesterol within their lysosomes contributes to the overall sterol accumulation in the lesion The reduced lysosome function appears multifactoral but includes direct and indirect inhibition of lysosomal acid lipase, the enzyme responsible for hydrolysis of cholesteryl esters in lysosomes, and a reduced capacity for transferring cholesterol from lysosomes In cell culture models of human macrophage foam cells, the inability to clear cholesterol from macrophages with compromised lysosome function continues even in the presence of compounds that stimulate efflux, Proteomic analysis of foam cells shows that changes in a number of lysosome proteases are related to macrophage sterol accumulation Thus, at least in the advanced stages of atherosclerosis, lysosome dysfunction contributes to the overall lesion severity.
As the intimal volume enlarges due to accumulating cells, there is vascular remodeling to lessen protrusion of the lesion into the lumen Figure 4thereby decreasing occlusion and the appearance of clinical symptoms for much of the life of the lesion Figure 4 Features of the stable fibrous plaque.