Molecular and cellular mechanisms of macrophage survival in atherosclerosis

Macrophages play a key role in the initiation and progression of atherosclerotic plaques. Although a significant number of macrophages undergoes cell death during plaque development as a result of atherogenic stressors, advanced plaques are characterized by a large macrophage content. Macrophage accumulation is mediated by continuous recruitment of monocytes, reduced emigration of macrophages and poor phagocytosis of dead cells which may trigger secondary necrosis and amplification of plaque inflammation. Moreover, an increasing body of evidence indicates that macrophages have developed several strategies to survive and to proliferate in the adverse environment of an advanced atherosclerotic plaque. Macrophages contain organic molecules or enzymes that provide enhanced antioxidant protection. In addition, synthesis of anti-apoptotic proteins is upregulated and several cellular protection mechanisms such as the unfolded protein response and autophagy are activated in macrophages to promote cellular survival. In this review, we discuss these macrophage survival mechanisms that allow growth and destabilization of advanced atherosclerotic plaques.     

不要低估血管平滑肌细胞在动脉粥样硬化斑块形成中的作用

血管内皮细胞、平滑肌细胞(SMC)和巨噬细胞共同参与动脉粥样硬化(As)斑块形成。近年研究表明,SMC来源的细胞占As斑块中细胞总数的70%以上。As斑块中的SMC通过自分泌细胞因子促进自身的增殖、迁移和炎症反应,通过旁分泌激活单核/巨噬细胞并将其募集到As损伤部位,同时通过其细胞膜表面表达的脂蛋白受体摄取脂质形成泡沫细胞。SMC在As斑块形成中扮演十分重要的角色,应进一步深化对SMC在As发生发展中的作用及作用机制的研究。     

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.     

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.     

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.     

肥大细胞在动脉粥样硬化斑块形成和破裂中的意义

肥大细胞,作为炎症细胞参与了人类动脉粥样硬化病变的早期和后期机制。体外研究表明,活化的即有免疫学活性的肥大细胞既可以水解低密度脂蛋白3使胆固醇在细胞内积累,又可以水解高密度脂蛋白使细胞内胆固醇流出减少,从而促进巨噬细胞和平滑肌细胞转成泡沫细胞;它能够激活基质金属蛋白酶、降解细胞外基质,还能抑制平滑肌细胞增生、诱导平滑肌细胞凋亡、抑制胶原合成和促进新血管形成而使斑块倾向破裂。以上这些作用使它在斑块形成和斑块破裂中起着重要作用。     

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.     

Apoptosis in atherosclerosis: beneficial or detrimental?

Several groups have demonstrated apoptotic cell death in atherosclerotic plaques. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and smooth muscle cells undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. Smooth muscle cell apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in early stages of atherosclerosis smooth muscle cells become susceptible to undergoing apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that smooth muscle cells may be killed by activated macrophages. The loss of the smooth muscle cells can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies are removed. Apoptotic cells that are not scavenged in the plaque activate thrombin which could further induce intraplaque thrombosis. It can be concluded that apoptosis in the primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreases after lipid lowering which could be important in our understanding of the cell biology of plaque stabilization.     

自噬在动脉粥样硬化中的作用及其调控

自噬是一种通过溶酶体系统降解长半衰期蛋白和细胞器的过程.大量证据提示,进展型动脉粥样硬化斑块中存在着自噬现象.在离体实验中,利用一些能导致动脉粥样硬化形成的刺激物处理血管平滑肌细胞、巨噬细胞和内皮细胞,这些细胞均呈现一些自噬的特点,如髓鞘样结构、细胞质中泛素化包涵体聚集以及空泡形成增多.然而,尽管对自噬的研究兴趣不断增加,但对其在动脉粥样硬化中的确切作用还不清楚.因此,了解自噬的潜在细胞和分子机制,将为研究动脉粥样硬化性疾病的机制和治疗提供一条新的思路.     

老年患者股动脉和颈动脉及冠状动脉粥样硬化斑块稳定性的比较

目的　探讨股动脉、颈动脉、冠状动脉粥样硬化斑块的稳定性。方法　收集我院老年尸体解剖病例 15例 ,将所有病例的两侧股动脉、两侧颈动脉、左冠状动脉前降支进行连续取材 ,常规病理检查 ,部分节段行α 平滑肌肌动蛋白、CD6 8、bax染色。结果　股动脉粥样硬化斑块中的平滑肌细胞、巨噬细胞数量与颈动脉相近。与冠状动脉比较 ,股动脉粥样硬化斑块中的平滑肌细胞相对多 ,巨噬细胞相对少 ;bax在巨噬细胞的表达多 ,在平滑肌细胞的表达少。结论　平滑肌细胞、巨噬细胞数量的不同导致了 3种动脉粥样硬化斑块不同的稳定性。股动脉中的粥样硬化斑块较冠状动脉更稳定。     

Macrophages and smooth muscle cells express lipoprotein lipase in human and rabbit atherosclerotic lesions.

Lipoprotein lipase (LPL; EC 3.1.1.34) may promote atherogenesis by producing remnant lipoproteins on the endothelial surface and by acting on lipoproteins in the artery wall. In vitro, smooth muscle cells and macrophages synthesize LPL, but in human carotid lesions only a few smooth muscle cells were reported to contain LPL protein. Endothelial cells do not synthesize LPL in vitro, but in normal arteries intense immunostaining for LPL is present on the endothelium. We used Northern blot analysis, in situ hybridization, and immunocytochemistry of human and rabbit arteries to determine cellular distribution and the site of the synthesis of LPL in atherosclerotic lesions. Northern blot analysis showed that LPL mRNA was detectable in macrophage-derived foam cells isolated from arterial lesions of "ballooned" cholesterol-fed rabbits. In situ hybridization studies of atherosclerotic lesions with an antisense riboprobe showed a strong hybridization signal for LPL mRNA in some, but not all, lesion macrophages, which were mostly located in the subendothelial and edge areas of the lesions. Also, some smooth muscle cells in lesion areas also expressed LPL mRNA. Immunocytochemistry of frozen sections of rabbit lesions with a monoclonal antibody to human milk LPL showed intense staining for LPL protein in macrophage-rich intimal lesions. The results suggest that lesion macrophages and macrophage-derived foam cells express LPL mRNA and protein. Some smooth muscle cells in the lesion areas also synthesize LPL. These data are consistent with an important role for LPL in atherogenesis.     

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.     

Role of PPARγ in macrophage biology and atherosclerosis

Macrophages carry out key functions by defending a host from microbial invaders and by clearing endogenous cellular debris. Molecules that are essential for the recognition, phagocytosis and clearance of pathogens also mediate the uptake and degradation of pathogenic lipoproteins. During atherogenesis, for example, scavenging trapped lipoproteins leads to the formation of foam cells and subsequently the activation of these lipid-laden macrophages. Although they are initially clinically silent, these fatty streaks evolve into complex inflammatory plaques that cause significant morbidity and mortality. Thus, interventions that decrease foam cell formation and reduce the inflammatory response of macrophages could become effective therapies for coronary artery disease. Thiazolidinediones (TZDs) might be developed as anti-atherogenic agents on the basis of their actions as ligands for peroxisome proliferator-activated receptor-gamma (PPARgamma).     

Localization of T lymphocytes and macrophages in fibrous and complicated human atherosclerotic plaques

The cellular composition of aortic atherosclerotic plaques was analyzed by immunocytochemistry using cell type-specific monoclonal antibodies. T lymphocytes and monocytes/macrophages were detected both in early, fibrous plaques, and in more advanced, complicated ones. Many smooth muscle cells in these plaques expressed the class II MHC antigen, HLA-DR. Since this antigen is inducible by T cell products, our findings suggest that T cell-smooth muscle interactions occur during atherogenesis.     

Macrophage migration inhibitory factor induces MMP-9 expression: implications for destabilization of human atherosclerotic plaques

Macrophage migration inhibitory factor (MIF) has been shown to participate in both experimental and human atherogenesis. Expression of MMP-9 has been shown to play a role in the instability of atherosclerotic plaque. Thus, we hypothesize that MIF may participate in the destabilization of atherosclerotic plaques by stimulating MMP-9 expression. This hypothesis was investigated by examining the expression of MIF and MMP-9 in human atherosclerotic plaques using two-color immunostaining and by determining the potential role of MIF in the induction of MMP-9 expression in vascular smooth muscle cells (VSMC) and macrophages in vitro. Two-color immunohistochemistry demonstrated that MIF was strongly upregulated by macrophages and VSMCs. This was associated with marked increase in MMP-9 expression in vulnerable atheromatous plaques, but not in the fibrous lesions. Upregulation of MIF and MMP-9 in vulnerable atheromatous plaques was associated with the weakening of fibrous caps. The role of MIF in MMP-9 expression was demonstrated by the ability of MIF to directly induce MMP-9 mRNA and protein expression in macrophages and in VSMCs in a dose and time-dependent manner, which was blocked by a neutralizing MIF antibody. In conclusion, MIF and MMP-9 are markedly upregulated in vulnerable atheromatous plaques. The ability of MIF to induce MMP-9 expression in VSMCs and macrophages suggests that MIF may play a role in the destabilization of human atherosclerotic plaques.     

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.     

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.     

Inhibition of intercellular communication in smooth muscle cells of humans and rats by low density lipoprotein, cigarette smoke condensate and TPA

Inhibition by tumor promoting chemicals of intercellular communication via gap junctions may be important in carcinogenesis. In order to investigate the possible role of gap junctional intercellular communication in atherogenesis, we examined the effect of known inhibitors of intercellular communication, 12-O-tetradecanoylphorbol-13-acetate (TPA) and cigarette smoke condensate (CSC), and low density lipoproteins (LDL) and high density lipoproteins (HDL) on cellular communication in smooth muscle cells of human and rat by the microinjection-dye transfer technique. When lucifer yellow CH solution is injected into a cell, the average numbers of human and rat smooth muscle cells that become fluorescent is about 22 and 6, respectively. The tumor promoter (TPA) almost completely blocked gapjunctional communication between smooth muscle cells at 100 ng/ml after 4 h exposure. LDL and CSC were able to inhibit intercellular communication in human and rat cells in a dose-dependent manner up to 60%. LDL-pretreatment of human smooth muscle cells did not affect inhibition of intercellular communication, which suggests that this effect is mainly non-receptor mediated. HDL did not influence junctional communication. The results indicate that inhibition of intercellular communication may also contribute to the pathogenesis of atherosclerotic lesions, such as plaques.     

Discoidin domain receptor 1 (ddr1) deletion decreases atherosclerosis by accelerating matrix accumulation and reducing inflammation in low-density lipoprotein receptor-deficient mice

Collagens are abundant within the atherosclerotic plaque, where they contribute to lesion volume and mechanical stability and influence cell signaling. The discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that binds to collagen, is expressed in blood vessels, but evidence for a functional role during atherogenesis is incomplete. In the present study, we generated Ddr1(+/+);Ldlr(-/-) and Ddr1(-/-);Ldlr(-/-) mice and fed them an atherogenic diet for 12 or 24 weeks. Targeted deletion of Ddr1 resulted in a 50% to 60% reduction in atherosclerotic lesion area in the descending aorta at both 12 and 24 weeks. Ddr1(-/-);Ldlr(-/-) plaques exhibited accelerated deposition of fibrillar collagen and elastin at 12 weeks compared with Ddr1(+/+);Ldlr(-/-) plaques. Expression analysis of laser microdissected lesions in vivo, and of Ddr1(-/-) smooth muscle cells in vitro, revealed increased mRNA levels for procollagen alpha1(I) and alpha1(III) and tropoelastin, suggesting an enhancement of matrix synthesis in the absence of DDR1. Furthermore, whereas plaque smooth muscle cell content was unchanged, Ddr1(-/-);Ldlr(-/-) plaques had a 49% decrease in macrophage content at 12 weeks, with a concomitant reduction of in situ gelatinolytic activity. Moreover, mRNA expression of both monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 was reduced in vivo, and Ddr1(-/-);Ldlr(-/-) macrophages demonstrated impaired matrix metalloproteinase expression in vitro. These data suggest novel roles for DDR1 in macrophage recruitment and invasion during atherogenesis. In conclusion, our data support a role for DDR1 in the regulation of both inflammation and fibrosis early in plaque development. Deletion of DDR1 attenuated atherogenesis and resulted in the formation of matrix-rich plaques.     

New oxidized LDL receptors and their functions in atherogenesis

Oxidized low density lipoprotein (Ox-LDL) appears to play key roles in atherosclerotic progression and plaque rupture. Biological effects of Ox-LDL on vascular cells may, at least in part, be mediated by cell surface receptors for Ox-LDL. Lectin-like oxidized LDL receptor (LOX)-1 and scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) are type II and I membrane glycoprtoeins, respectively, both of which can act as cell-surface endocytosis receptors for atherogenic oxidized LDL (Ox-LDL). LOX-1 expression can dynamically be induced by proinflammatory stimuli, and is detectable in cultured macrophages and activated vascular smooth muscle cells (VSMC), in addition to endothelial cells. LOX-1-dependent uptake of Ox-LDL induced apoptosis of cultured VSMC. In vivo, endothelial cells that cover early atherosclerotic lesions, and intimal macrophages and VSMC in advanced atherosclerotic plaques dominantly express LOX-1. LOX-1 expressed on the cellsurface can be cleaved, in part, and released as soluble molecules, suggesting the diagnostic significance of plasma soluble LOX-1 levels. SR-PSOX appeared to be identical to CXCL16, a novel membrane-anchored chemokine directed to CXCR6-positive lymphocytes, suggesting another role of SR-PSOX as T-cell chemoattractant. In contrast to LOX-1 expressed by a variety of cell types. SR-PSOX expression appeared relatively confined to macrophages in atherogenesis. Taken together, LOX-1 and SR-PSOX may play important roles in atherogenesis and athrosclerotic plaque rupture.