Phagocytosis in atherosclerosis: Molecular mechanisms and implications for plaque progression and stability

Macrophages play a key role in atherosclerotic plaque destabilization and rupture. In this light, selective removal of macrophages may be beneficial for plaque stability. However, macrophages are phagocytic cells and thus have an important additional role in scavenging of modified lipoproteins, unwanted or dead cells and cellular debris via phagocytosis. The concept of phagocytosis as well as the underlying mechanisms is well defined but the effect of phagocytosis in terms of plaque stability remains poorly understood. Recent findings point towards a complex role of macrophage phagocytosis in atherogenesis. Macrophages are necessary for removal of apoptotic cells from plaques, but exert strong proatherogenic properties upon phagocytosis of lipoproteins, erythrocytes and platelets. Apart from heterophagy, autophagocytosis better known as autophagy may occur in advanced atherosclerotic plaques. Several lines of evidence indicate that autophagy is initiated in plaque smooth muscle cells as a result of cellular distress. Since autophagy is well recognized as a survival mechanism, autophagic smooth muscle cells in the fibrous cap may reflect an important feature underlying plaque stability. All together, phagocytosis is a crucial process involved in atherogenesis that may significantly affect the stability of the atherosclerotic plaque.     

巨噬细胞移出动脉粥样硬化斑块调控机制

巨噬细胞对动脉粥样硬化斑块的发生发展及不稳定起着关键作用,减少斑块中巨噬细胞累积对逆转斑块起着尤为重要的作用。诱导巨噬细胞移出斑块是减少斑块中巨噬细胞数量的主要策略之一。文章以诱导巨噬细胞移出斑块的受体CCR7及抑制巨噬细胞移出斑块的受体UNC5b、PlexinD1及氧化应激为主线,介绍相关调控机制研究进展。     

Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium-diethyltriaminepentaacetic acid (DTPA) were tested in ApoE-/- and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE-/- mice compared with only 34% using untargeted micelles and no enhancement using gadolinium-DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture.     

Selective clearance of macrophages in atherosclerotic plaques by autophagy.

OBJECTIVES: The purpose of this study was to investigate whether stent-based delivery of an inhibitor of mammalian target of rapamycin (mTOR) can selectively clear macrophages in rabbit atherosclerotic plaques. BACKGROUND: Current pharmacologic approaches to stabilize atherosclerotic plaques have only partially reduced the incidence of acute coronary syndromes and sudden death. Macrophages play a pivotal role in plaque destabilization, whereas smooth muscle cells (SMC) promote plaque stability. METHODS: Stents eluting the mTOR inhibitor everolimus were implanted in atherosclerotic arteries of cholesterol-fed rabbits. In addition, in vitro experiments using explanted atherosclerotic segments and cultured macrophages as well as SMC were performed. RESULTS: Stents eluting everolimus led to a marked reduction in macrophage content without altering the amount of SMC compared with polymer control stents. In vitro studies showed that everolimus treatment induced inhibition of translation in both cultured macrophages and SMC. However, cell death occurred only in macrophages and was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. Everolimus-induced autophagy was mediated by mTOR inhibition, because cell viability was not affected using tacrolimus, an mTOR-independent everolimus analog. Moreover, mTOR gene silencing was associated with selective induction of macrophage cell death. Autophagic macrophage cell death was confirmed by transmission electron microscopy both in cultured cells and in atherosclerotic explants. CONCLUSIONS: Stent-based delivery of everolimus selectively cleared macrophages in rabbit atherosclerotic plaques by autophagy, an mTOR inhibition-dependent and novel mechanism to induce cell death in mammalian cells.     

Selective depletion of macrophages in atherosclerotic plaques via macrophage-specific initiation of cell death

Macrophages play a central role in atherosclerotic plaque destabilization, leading to acute coronary syndromes and sudden death. Removal of macrophages from plaques via pharmacological therapy may therefore represent a promising approach to stabilize vulnerable, rupture-prone lesions. In this review, we summarize the current therapeutic means to induce macrophage cell death in atherosclerotic plaques without affecting smooth muscle cell viability, and their potential pitfalls.     

Toll-like receptor 7 stimulation by imiquimod induces macrophage autophagy and inflammation in atherosclerotic plaques

Atherosclerotic plaques tend to rupture as a consequence of a weakened fibrous cap, particularly in the shoulder regions where most macrophages reside. Macrophages express Toll-like receptors to recognize pathogens and eliminate intracellular pathogens by inducing autophagy. Because Toll-like receptor 7 (TLR7) is thought to be expressed in macrophages but not in smooth muscle cells (SMCs), we investigated whether induction of macrophage autophagic death by TLR7 ligand imiquimod can affect the composition of atherosclerotic plaques in favor of their stability. Immunohistochemical staining of human carotid plaques as well as Western blotting of cultured macrophages and SMCs confirmed that TLR7 was expressed in macrophages, but not in SMCs. In vitro experiments showed that only TLR7 expressing cells underwent imiquimod-induced cell death, which was characterized by autophagosome formation. Imiquimod-treated macrophages activated nuclear factor-κB (NF-κB) and released pro-inflammatory cytokines and chemokines. This effect was inhibited by the glucocorticoid dexamethasone. Imiquimod-induced cytokine release was significantly decreased in autophagy-deficient macrophages because these cells died by necrosis at an accelerated pace. Local in vivo administration of imiquimod to established atherosclerotic lesions in rabbit carotid arteries induced macrophage autophagy without induction of cell death, and triggered cytokine production, upregulation of vascular adhesion molecule-1, infiltration of T-lymphocytes, accumulation of macrophages and enlargement of plaque area. Treatment with dexamethasone suppressed these pro-inflammatory effects in vivo. SMCs and endothelial cells in imiquimod-treated plaques were not affected. In conclusion, imiquimod induces macrophage autophagy in atherosclerotic plaques, but stimulates plaque progression through cytokine release and enhanced infiltration of inflammatory cells.     

Targeting of vulnerable plaque macrophages with polymer-based nanostructures

Macrophages are key cellular elements of atherosclerotic plaque pathogenesis and are a significant risk factor for plaque rupture. Current diagnostic techniques for the detection of plaque macrophages are often limited by insufficient sensitivity and selectivity and have not reached broad clinical practice until now. Supramolecular nanometer-sized structures such as conjugates, nanoparticles, micelles, or vesicles built from novel polymers promise to be useful in cell-specific delivery and may be of particular value for the detection and treatment of vulnerable plaque macrophages. Key properties of polymer-based nanostructures are high stability, improved biocompatibility, long circulation half-lives, defined biodegradation, targeting moieties, and triggerable controlled release. This review gives an insight into several promising research projects with polymer-based nanostructures for macrophage detection or treatment that might enter cardiologic practice in the near future.     

Niemann-Pick C heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis

Macrophage death in advanced atherosclerotic lesions leads to lesional necrosis and likely promotes plaque instability, a precursor of acute vascular events. Macrophages in advanced lesions accumulate large amounts of unesterified cholesterol, which is a potent inducer of macrophage apoptosis. We have shown recently that induction of apoptosis in cultured macrophages requires cholesterol trafficking to the endoplasmic reticulum (ER). Moreover, macrophages from mice with a heterozygous mutation in the cholesterol-trafficking protein Npc1 have a selective defect in cholesterol trafficking to the ER and are protected from cholesterol-induced apoptosis. The goal of the present study was to test the importance of intracellular cholesterol trafficking in atherosclerotic lesional macrophage death by comparing lesion morphology in Npc1+/+;Apoe-/- and Npc1+/-;Apoe-/- mice. Although advanced lesions in Npc1+/+;Apoe-/- mice had extensive acellular areas that were rich in unesterified cholesterol and macrophage debris, the lesions of Npc1+/-;Apoe-/- mice were substantially more cellular and less necrotic. Moreover, compared with Npc1+/-;Apoe-/- lesions, Npc1+/+;Apoe-/- lesions had a greater number of large, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)-positive areas surrounding necrotic areas, indicative of macrophage apoptosis. These differences were observed despite similar total lesion area and similar plasma lipid levels in the two groups of mice. These data provide in vivo evidence that intact intracellular cholesterol trafficking is important for macrophage apoptosis in advanced atherosclerotic lesions and that the ER-based model of cholesterol-induced cytotoxicity is physiologically relevant. Moreover, by showing that lesional necrosis can be diminished by a subtle defect in intracellular trafficking, these findings suggest therapeutic strategies to stabilize atherosclerotic plaques.     

巨噬细胞免疫代谢与动脉粥样硬化的研究进展

巨噬细胞在动脉粥样硬化斑块的起始、进展阶段直至斑块破裂中起核心作用,改变代谢途径是决定巨噬细胞功能和疾病进展的关键因素。本文阐述了动脉粥样硬化斑块微环境的核心因素如何影响巨噬细胞代谢变化以及代谢变化如何反过来改变巨噬细胞的免疫效应和组织修复功能,并探讨利用免疫代谢调节疾病中巨噬细胞反应的挑战和机遇。     

Free cholesterol-induced cytotoxicity a possible contributing factor to macrophage foam cell necrosis in advanced atherosclerotic lesions

A major characteristic of advanced atherosclerotic lesions is the necrotic, or lipid, core, which likely plays an important role in the clinical progression of these lesions. Recent data suggest that the necrotic core forms primarily as a consequence of macrophage foam cell necrosis. Lesional macrophages initially accumulate mostly cholesteryl esters, but macrophages in advanced lesions contain large amounts of unesterified, or free, cholesterol (FC). Although there are many theories as to why macrophage foam cells die in advanced lesions, the fact that a high FC:phospholipid (PL) ratio in cellular membranes can be toxic to cells suggests that FC-induced cytotoxicity may contribute to foam cell necrosis. The mechanism of FC cytotoxicity can be explained by disturbances in membrane protein function as a result of "stiffening" of the bilayer and by formation of intracellular FC crystals that can cause physical damage to cellular organelles. Macrophages appear to respond to FC loading by a fascinating adaptive response, namely the induction of PL biosynthesis, which initially keeps the cellular FC:PL ratio below toxic levels. Studies with cultured macrophages have demonstrated that a failure of this adaptive response leads to FC-induced foam cell cytotoxicity and necrosis, and thus a similar series of events in advanced atherosclerotic lesions could provide an explanation for the development of the necrotic core. (Trends Cardiovasc Med 1997;7: 256-263). ? 1997, Elsevier Science Inc.     

Decreased expression of insulin-like growth factor-1 and apoptosis of vascular smooth muscle cells in human atherosclerotic plaque

Insulin-like growth factor-1 (IGF-1) plays an important role in migration, cell cycle progression and survival of vascular smooth muscle cells (VSMC). We investigated the specific localization of IGF-1 and its receptor (IGF-1R) and their association with apoptosis and the expression of apoptosis-related proteins in early and advanced atherosclerotic lesions. Human atherosclerotic plaques (n=23) from patients undergoing aortic, carotid or femoral arterial surgery were studied. Immunohistochemistry and in situ hybridization revealed significantly higher expression of IGF-1 and IGF-1R in the media than in the intima of early atherosclerotic lesions (P<0.01). Medial VSMC positive for BAX, a proapoptotic protein of the B-cell CLL/lymphoma 2 (BCL2) family, showed colocalization of IGF-1. Apoptosis, as detected by DNA in situ terminal deoxynucleotidyl transferase end labeling (TUNEL), was not present in these early lesions. In advanced atherosclerotic plaques, the expression of IGF-1 and IGF-1R was significantly lower in the intimal regions with macrophage infiltration than in those without macrophage infiltration or than in the media (P<0.01). Furthermore, IGF-1 and IGF-1R immunoreactivity was markedly lower in intimal TUNEL-positive VSMC compared with intimal BAX-positive and medial VSMC (P<0.01). We conclude that IGF-1 and IGF-1R expression are reduced in the deep intima of early atherosclerotic lesions and in areas of advanced plaques with macrophage infiltration. Since IGF-1 is a potent survival factor for VSMC, poor expression of IGF-1 and IGF-1R in intimal regions with macrophage infiltration would likely contribute to triggering VSMC apoptosis potentially leading to plaque weakening, plaque rupture and acute coronary events.     

Mitochondrial uncoupling protein 2 mediates temperature heterogeneity in atherosclerotic plaques

AIMS: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue. METHODS AND RESULTS: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production (DeltaT = 0.13 +/- 0.04 degrees C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature. CONCLUSION: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.     

Human arterial wall cells secrete factors that are chemotactic for monocytes.

Macrophages and arterial smooth muscle cells comprise the cellular components of the atherosclerotic plaque. The vessel wall accumulation of macrophages occurs by a process of increased circulating monocyte migration into the vessel wall. In these studies it is demonstrated that human macrophages and arterial smooth muscle cells in culture secrete potent chemotactic factors for freshly isolated human monocytes. In contrast, human fibroblast-conditioned medium has no chemotactic activity. The effect of macrophage-conditioned medium is a function of macrophage differentiation and can be potentiated by macrophage activation. These results suggest that secretory products of human macrophages and arterial smooth muscle cells may be important stimuli for increased monocyte migration into the vessel wall in vivo.     

Proteolysis of the pericellular matrix: a novel element determining cell survival and death in the pathogenesis of plaque erosion and rupture

The 2 major general concepts about the cell biology of atherogenesis, growth of smooth muscle cells, and lipid accumulation in macrophages, ie, foam cell formation, have not been able to satisfactorily explain the genesis of acute coronary syndromes. Rather, the basic pathology behind the acute atherothrombotic events relates to erosion and rupture of unstable coronary plaques. At the cellular level, we now understand that a switch from cellular growth to cellular death, notably apoptosis, could be involved in turning at least some types of atherosclerotic plaques unstable. Because intimal cells require a proper matrix environment for normal function and survival, the vulnerability of an atherosclerotic plaque may critically depend on the integrity of the pericellular matrix of the plaque cells. In vitro studies have revealed that plaque-infiltrating inflammatory cells, such as macrophages, T-lymphocytes, and mast cells, by secreting a variety of proteases capable of degrading pericellular matrix components, induce death of endothelial cells and smooth muscle cells, and so provide a mechanistic explanation for inflammation-dependent plaque erosion and rupture. Thus, a novel link between inflammation and acute coronary syndromes is emerging. For a more explicit understanding of the role of proteases released by inflammatory cells in the conversion of a clinically silent plaque into a dangerous and potentially killing plaque, animal models of plaque erosion and rupture need to be established.     

Expression of matrix metalloproteinase 3 in experimental atherosclerotic plaques.

In atherosclerotic lesions, matrix metalloproteinases produced by foam cells (macrophages) are thought to increase plaque instability, promote plaque rupture, by degradating extracellular matrix. To investigate the relationship between the expression of these proteinases and the histologic appearance of atheromas, immunohistochemical analysis of matrix metalloproteinase 3 and cell-type markers was performed in atherosclerotic plaques induced in rabbit abdominal aortas by high-cholesterol diets and mechanical injury. In addition to an antibody against matrix metalloproteinase 3, RAM-11 and HHF-35 were used to detect macrophages and smooth muscle cells, respectively. Matrix metalloproteinase 3 was expressed diffusely within the plaques with a fibrofatty histologic pattern. In plaques with foam cell accumulation, matrix metalloproteinase 3 was seen in areas rich in foam cells and the smooth muscle cells near the lumen. In the plaques with fewer macrophages, the proteinase was expressed only in such smooth muscle cells. Matrix metalloproteinase 3 was expressed in the smooth muscle cells in plaques of all histologic types, and macrophages also expressed the metalloproteinase when present in significant numbers. These findings suggest that macrophage accumulation plays an important pathophysiologic role in causing the instability of atherosclerotic lesions by increasing the levels of matrix metalloproteinase 3.     

Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits.

Apoptotic macrophages are regularly found in atherosclerotic plaques indicating programmed cell death as one of their regulatory controls. The objective of this study was to characterize in more detail apoptotic macrophages in atherosclerotic lesions of humans and heritable hyperlipidemic (HHL) rabbits. Macrophages were immunohistochemically analyzed using antibodies directed against alphaMbeta2-integrins (CD11b/CD18), CD44, major histocompatibility complex (MHC) class I and II, inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), tumor necrosis factor alpha (TNFalpha), p53, c-jun/AP-1 and rabbit macrophages (RAM-11) and the TUNEL (TdT-mediated dUTP nick end labeling) technique. Colocalization studies of human atherosclerotic carotid and aortic tissue showed apoptotic plaque macrophages also being MnSOD-, alphaMbeta2-integrin-, CD44-, MHC class I- and II-, iNOS-, TNFalpha- and p53-immunoreactive. Similar results occurred in atherosclerotic aortas of HHL rabbits. Computer-assisted morphometric analyses revealed a positive correlation of the area density of MnSOD-immunoreactive macrophages with those of alphaMbeta2-integrin- and CD44-immunoreactive ones, but not with those of MHC class I- and II- as well as of RAM-11-immunoreactive macrophages. We conclude that apoptotic macrophages located in atherosclerotic vessel wall are activated, antigen-presenting, integrin-expressing and oxidatively stressed cells. Since all these processes have been demonstrated to cause apoptosis of macrophages in vitro, we propose their potency accelerates the susceptibility of the macrophages to programmed cell death in atherosclerotic lesions.     

Broad-spectrum chemokine inhibition reduces vascular macrophage accumulation and collagenolysis consistent with plaque stabilization in mice

BACKGROUND: A major determinant of the risk of myocardial infarction is the stability of the atherosclerotic plaque. Macrophage-rich plaques are more vulnerable to rupture, since macrophages excrete an excess of matrix-degrading enzymes over their inhibitors, reducing collagen content and thinning the fibrous cap. Several genetic studies have shown that disruption of signalling by the chemokine monocyte chemoattractant protein 1 reduced the lipid lesion area and macrophage accumulation in the vessel wall. METHODS: We have tested whether a similar reduction in macrophage accumulation could be achieved pharmacologically by treating apolipoprotein-E-deficient mice with the chemokine inhibitor NR58-3.14.3. RESULTS: Mice treated for various periods of time (from several days to 6 months) with NR58-3.14.3 (approximately 30 mg/kg/day) consistently had 30-40% fewer macrophages in vascular lesions, compared with mice treated with the inactive control NR58-3.14.4 or PBS vehicle. Similarly, cleaved collagen staining was lower in mice treated for up to 7 days, although this effect was not maintained when treatment time was extended to 12 weeks. The vascular lipid lesion area was unaffected by treatment, but total collagen I staining and smooth muscle cell number were both increased, suggesting that a shift to a more stable plaque phenotype had been achieved. CONCLUSIONS: Strategies, such as chemokine inhibition, to attenuate macrophage accumulation may therefore be useful to promote stabilization of atherosclerotic plaques.     

Proteasome inhibitor bortezomib promotes a rupture-prone plaque phenotype in ApoE-deficient mice

The ubiquitin–proteasome system is involved in the development and progression of atherosclerosis. The aim of this study was to investigate whether plaque composition is affected by proteasome function. In vitro, the potent and selective proteasome inhibitor bortezomib induced apoptosis in both cultured smooth muscle cells (SMCs) and activated macrophages. This effect was associated with increased expression of C/EBP homologous protein and cleavage of caspase-12, indicative of endoplasmic reticulum stress. The sensitivity to the proapoptotic effects of proteasome inhibition correlated with the protein synthesis rate. Proteasome inhibition in explanted atherosclerotic plaques of ApoE-deficient mice resulted in a significant decrease in SMCs and macrophages, indicating that both cell types in the atherosclerotic plaque were susceptible to the proapoptotic effects of proteasome inhibition. In vivo proteasome inhibition in ApoE-deficient mice did not affect plaque size or composition of early atherosclerotic plaques, but resulted in a significant decrease in collagen content as well as a significant enlargement of the necrotic core in advanced atherosclerotic plaques. In conclusion, our results indicate that an impaired proteasome function promotes features of a more rupture-prone plaque phenotype.     

Autophagy in atherosclerosis

Autophagy is a catabolic pathway for bulk destruction of long-lived proteins and organelles via lysosomes. Basal autophagy represents a reparative, life-sustaining process, but unrestrained autophagic activity promotes cell death. A growing body of evidence suggests that autophagy occurs in advanced atherosclerotic plaques. Vascular smooth muscle cells, macrophages, or endothelial cells treated in vitro with proatherogenic stimuli reveal certain features typical of autophagy, such as LC3 processing, formation of myelin figures, and extensive vacuolization. However, despite the increasing interest in autophagy, its role in atherosclerosis remains poorly understood. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading the damaged intracellular material. In this way, autophagy is antiapoptotic and contributes to cellular recovery in an adverse environment. Because atherosclerosis is an inflammatory disorder of the arterial intima, pharmacologic approaches have recently been developed to stabilize vulnerable, rupture-prone lesions through selective induction of macrophage autophagic death.