The role of chemokines in atherosclerosis: recent evidence from experimental models and population genetics

PURPOSE OF REVIEW: Atherosclerosis is an inflammatory disease process. This review discusses the recent genetic evidence from animal models and human populations that highlight the importance of chemokines in atherosclerosis. RECENT FINDINGS: CC-chemokine/CC-chemokine receptors (CCR), including CCR2/ MCP-1 (monocyte chemoattractant protein-1) and CCR5/RANTES (regulated on activation, normal T-cell expressed and secreted), have been shown in animal knockout and transgenic studies to have significant effects on atherosclerotic lesion size and macrophage recruitment. More recently fractalkine (CX3C1) and its receptor (CX3CR1) have emerged as another important pathway in atherosclerosis. For example, fractalkine is present in human atherosclerotic lesions and is able to stimulate platelet activation and adhesion. CX3CR1 is expressed on human aortic smooth muscle cells and CX3CR1/apolipoprotein E double knockout mice have significantly reduced atherosclerotic lesion size and macrophage recruitment. Human population genetic studies have tried to assess the importance of chemokines in human atherosclerosis. Currently, there is conflicting evidence regarding an association between polymorphisms in CCR2/MCP-1 and CCR5/RANTES and coronary artery disease. There is evidence, however, for an association between the fractalkine receptor polymorphism (CX3CR1-I249) and coronary artery disease in both human population and function studies. SUMMARY: Recent transgenic and gene knockout studies in murine models of atherosclerosis have highlighted the importance of chemokines and their receptors in atherosclerosis. Genetic evidence for a role of chemokines and their receptors in human population studies remains under investigation. Identifying chemokine polymorphisms could help to determine pathways that are important in atherosclerosis disease pathology and that may suggest novel therapeutic targets.     

Insulin-like growth factor II plays a central role in atherosclerosis in a mouse model.

Insulin-like growth factor II is a fetal promoter of cell proliferation that is involved in some forms of cancer and overgrowth syndromes in humans. Here, we provide two sources of genetic evidence for a novel, pivotal role of locally produced insulin-like growth factor II in the development of atherosclerosis. First, we show that homozygosity for a disrupted insulin-like growth factor II allele in mice lacking apolipoprotein E, a widely used animal model of atherosclerosis, results in aortic lesions that are approximately 80% smaller and contain approximately 50% less proliferating cells compared with mice lacking only apolipoprotein E. Second, targeted expression of an insulin-like growth factor II transgene in smooth muscle cells, but not the mere elevation of circulating levels of the peptide, causes per se aortic focal intimal thickenings. The insulin-like growth factor II transgenics presented here are the first viable mutant mice spontaneously developing intimal masses. These observations provide the first direct evidence for an atherogenic activity of insulin-like growth factor II in vivo.     

Functional role for toll-like receptors in atherosclerosis and arterial remodeling

PURPOSE OF REVIEW: Activation of inflammatory cascades is causally related to the development of atherosclerotic disease. Toll-like receptors are innate immune receptors that recognize pathogen-associated molecular patterns. In this review the pathways by which toll-like receptors might play a role in the development and progression of atherosclerosis will be discussed according to recent literature. RECENT FINDINGS: Toll-like receptors are expressed in atherosclerotic tissue. Next to pathogens, endogenous toll-like receptor ligands have been linked with the development of arterial occlusive disease. In mouse models of hyperlipidemia, a potential role for the toll-like receptor pathway has been suggested in hypercholesterolemia-induced atherosclerosis. Recent in-vitro studies revealed a mechanism by which toll-like receptor ligation results in a strong inhibition of cholesterol efflux from macrophages. In addition, oxidized lipoproteins interact with toll-like receptors. Furthermore, activation of the apoptotic cascade, which is important during atherogenesis, enhances the toll-like receptor pathway resulting in upregulation of proinflammatory cytokines. Human epidemiologic studies have linked TLR4 polymorphism with atherosclerosis. However, data on the association between atherosclerosis progression and TLR4 polymorphisms are conflicting. Next to plaque growth, arterial remodeling is an important determinant of luminal narrowing in atherosclerosis. Recently, a possible role for TLR4 signaling in arterial remodeling has been revealed in mouse models. SUMMARY: A clarification of the molecule [corrected] mechanisms by which the toll-like receptor signaling cascade influences atherosclerosis might [corrected] lead to novel strategies to intervene in the development of this life-threatening disease.     

Experimental atherosclerosis: a historical overview

Almost one-hundred years ago the first evidence of experimental atherosclerosis was reported. Over the past century, significant advances have been made in the development of animal models of human coronary artery disease. In this minireview, induction of atherosclerotic lesions in several animal models including rodents (mice, rabbits, rats, hamsters, guinea pigs), avian (pigeons, chickens, quail), swine, carnivora (dogs, cats), and non-human primates is discussed. The limitations and advantages of the animal models of atherosclerosis have been summarized. The transgenic/knockout animal models have greatly enhanced our understanding of atherosclerosis. Compared to wild-type counterparts, the knockout/transgenic animals develop atherogenesis faster without a need for a highly atherogenic diet. Although almost all investigations support a causal role for increased plasma cholesterol levels in the development of atherosclerotic vascular disease, an increasing body of evidence indicates serious invqlvement of other factors including oxidative stress, inflammation, infection and other emerging risk factors.     

The role of complement in atherosclerosis

PURPOSE OF REVIEW: Although it has long been recognized that atherosclerotic lesions show evidence of complement activation, the functional roles of the complement system in atherogenesis are not yet fully resolved. This article highlights recent publications on the complement system in the atherosclerosis field. RECENT FINDINGS: There have been a number of recent papers reporting on the association of complement proteins and complement regulators with high density lipoproteins, complement activation by enzymatically-modified LDL, signalling pathways downstream of C3a and C5a receptors and membrane C5b-9 assembly, and the prevention of C5b-9 assembly on endothelial cells via upregulation of CD59 expression in response to arterial laminar flow. C1q has been found to play a protective role in early lesion formation in LDL receptor deficient mice, and Crry-Ig and soluble C1 inhibitor have both been shown to have therapeutic effects in models of vascular injury in ApoE deficient mice. The possibility that the Y402H Factor H polymophism influences atherosclerosis has been supported in a recent paper showing increased risk in white hypertensive individuals. SUMMARY: The articles that have emerged over the last year highlight the relevance of the complement system to the atherosclerosis field.     

The role of transforming growth factor beta in atherosclerosis: novel insights and future perspectives

PURPOSE OF REVIEW: Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Transforming growth factors beta 1, 2 and 3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system, and have been suggested to play a role in the pathogenesis of atherosclerosis. RECENT FINDINGS: In the present review, we discuss recent developments in the role of transforming growth factor beta in the regulation of the immuno-inflammatory balance that modulates atherosclerosis. Such studies strongly suggest that the inhibition of endogenous transforming growth factor beta signalling favours the development of atherosclerotic lesions with an increased inflammatory component (T cells and macrophages) and decreased collagen content, features that are characteristic of unstable atherosclerotic plaques. SUMMARY: Transforming growth factor beta is identified as a critical modulator of the immuno-inflammatory balance in atherosclerosis, and a crucial plaque-stabilizing factor. Future studies should aim at defining the precise molecular mechanisms responsible for this protective effect, and developing immunomodulatory strategies based on the promotion of transforming growth factor beta activity (T regulatory T helper type 3 cells) to limit disease complications.     

Iron in arterial plaque: modifiable risk factor for atherosclerosis

It has been proposed that iron depletion protects against cardiovascular disease. There is increasing evidence that one mechanism for this protection may involve a reduction in iron levels within atherosclerotic plaque. Large increases in iron concentration are seen in human atherosclerotic lesions in comparison to levels in healthy arterial tissue. In animal models, depletion of lesion iron levels in vivo by phlebotomy, systemic iron chelation treatment or dietary iron restriction reduces lesion size and/or increases plaque stability. A number of factors associated with increased arterial disease or increased cardiovascular events is also associated with increased plaque iron. In rats, infusion of angiotensin II increases ferritin levels and arterial thickness which are reversed by treatment with the iron chelator deferoxamine. In humans, a polymorphism for haptoglobin associated with increased cardiovascular disease is also characterized by increased lesional iron. Heme oxygenase 1 (HO1) is an important component of the system for mobilization of iron from macrophages. Human HO1 promoter polymorphisms causing weaker upregulation of the enzyme are associated with increased cardiovascular disease and increased serum ferritin. Increased cardiovascular disease associated with inflammation may be in part caused by elevated hepcidin levels that promote retention of iron within plaque macrophages. Defective retention of iron within arterial macrophages in genetic hemochromatosis may explain why there is little evidence of increased atherosclerosis in this disorder despite systemic iron overload. The reviewed findings support the concept that arterial plaque iron is a modifiable risk factor for atherogenesis.     

Apoptotic cell death in atherosclerosis

PURPOSE OF REVIEW: Apoptosis is a critical regulator of homeostasis in many tissues, including the vasculature. Apoptosis in atherosclerotic lesions is triggered by inflammatory processes, both via cell-cell contact and by cytokines and oxidized lipids. Apoptosis of vascular smooth muscle cells, endothelial cells and macrophages may promote plaque growth and pro-coagulation and may induce rupture, the major consequence of atherosclerosis in humans. RECENT FINDINGS: Studies over the past year have clearly demonstrated the significance of cell death in atherosclerosis. Some of the key cellular, cytokine and molecular regulators that contribute to the apoptosis of cells within the atherosclerotic lesion have been identified and their mechanism of action elucidated. Other studies have shed some light on the identity of cells whose loss by apoptosis contributes to plaque instability. SUMMARY: The identification of which cell types undergo apoptosis within the atherosclerotic lesion, the extracellular factors that impinge on these cells, and the intracellular mechanisms that govern their demise have begun to be elucidated. This information is critical in the design of further in-vivo experiments such as the exploitation of animal models, and ultimately, in applying this knowledge to clinical practice.     

Urotensin II and atherosclerosis

Urotensin II, through its interaction with its UT receptor, is a potent vasoactive peptide in humans and in several animal models. Recent studies have demonstrated elevated plasma U-II levels in patients with atherosclerosis and coronary artery disease. U-II is expressed in endothelial cells, smooth muscle cells and infiltrating macrophages of atherosclerotic human coronary arteries. UT receptor expression is up-regulated by inflammatory stimuli. Activation of UT receptor by U-II stimulates endothelial and smooth muscle cell proliferation and monocytes chemotaxis. Therefore, in addition to its primary vasoactive effect, these observations suggest a role of U-II and UT receptor in the initiation and/or progression of atherosclerosis.     

Iron in arterial plaque: A modifiable risk factor for atherosclerosis

It has been proposed that iron depletion protects against cardiovascular disease. There is increasing evidence that one mechanism for this protection may involve a reduction in iron levels within atherosclerotic plaque. Large increases in iron concentration are seen in human atherosclerotic lesions in comparison to levels in healthy arterial tissue. In animal models, depletion of lesion iron levels in vivo by phlebotomy, systemic iron chelation treatment or dietary iron restriction reduces lesion size and/or increases plaque stability. A number of factors associated with increased arterial disease or increased cardiovascular events is also associated with increased plaque iron. In rats, infusion of angiotensin II increases ferritin levels and arterial thickness which are reversed by treatment with the iron chelator deferoxamine. In humans, a polymorphism for haptoglobin associated with increased cardiovascular disease is also characterized by increased lesional iron. Heme oxygenase 1 (HO1) is an important component of the system for mobilization of iron from macrophages. Human HO1 promoter polymorphisms causing weaker upregulation of the enzyme are associated with increased cardiovascular disease and increased serum ferritin. Increased cardiovascular disease associated with inflammation may be in part caused by elevated hepcidin levels that promote retention of iron within plaque macrophages. Defective retention of iron within arterial macrophages in genetic hemochromatosis may explain why there is little evidence of increased atherosclerosis in this disorder despite systemic iron overload. The reviewed findings support the concept that arterial plaque iron is a modifiable risk factor for atherogenesis.     

The multiple actions of angiotensin II in atherosclerosis

Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system, has been implied in the pathogenesis of atherosclerosis on various levels. There is abundant experimental evidence that pharmacological antagonism of Ang II formation by angiotensin converting enzyme inhibition or blockade of the cellular effects of Ang II by angiotensin type 1 receptor blockade inhibits formation and progression of atherosclerotic lesions. Angiotensin promotes generation of oxidative stress in the vasculature, which appears to be a key mediator of Ang II-induced endothelial dysfunction, endothelial cell apoptosis, and lipoprotein peroxidation. Ang II also induces cellular adhesion molecules, chemotactic and proinflammatory cytokines, all of which participate in the induction of an inflammatory response in the vessel wall. In addition, Ang II triggers responses in vascular smooth muscle cells that lead to proliferation, migration, and a phenotypic modulation resulting in production of growth factors and extracellular matrix. While all of these effects contribute to neointima formation and development of atherosclerotic lesions, Ang II may also be involved in acute complications of atherosclerosis by promoting plaque rupture and a hyperthrombotic state. Accordingly, Ang II appears to have a central role in the pathophysiology of atherosclerosis.     

Igf-I and longevity

Recent animal studies have demonstrated evidence of the involvement of insulin and insulin-like growth factor (IGF)-I signalling in the control of ageing and longevity. Disruption of insulin/IGF-I signalling pathways significantly extends lifespan in several animal models. Similarities among these signalling pathways in animals and humans raise the possibility that modifications in the IGF-I signalling system could also extend lifespan in humans. However, in contrast to the findings in animal studies, reduced IGF-I activity in humans is not associated with longevity. In humans, low IGF-I activity is even associated with an increased risk of developing cardiovascular disease and diabetes. High IGF-I activity in humans is associated with an increased risk of developing cancer. In addition, genetic predisposition and lifestyle play a major role in determining age-associated disease. For each individual there is probably a specific optimal 'setpoint' for the insulin/growth hormone/IGF-I axis which co-determines survival.     

Anti-inflammatory drugs and atherosclerosis

PURPOSE OF REVIEW: Inflammation contributes to the formation and progression of atherosclerosis and the therapeutic potential of some anti-inflammatory drugs has been evaluated for possible antiatherosclerotic effects. This review will briefly describe the mechanisms underlying the inflammation-atherosclerosis connection, the effect of various anti-inflammatory therapies on atherosclerotic disease and a sampling of the potential targets and agents under evaluation. RECENT FINDINGS: Some agents with anti-inflammatory properties appear to have beneficial effects on atherosclerosis or subsequent risk for cardiovascular events, while others have been disappointing. The anti-inflammatory actions of statins have been linked retrospectively with their favorable effects on atherosclerosis progression and clinical outcomes. The cardiovascular safety of COX-2 inhibitors is being assessed prospectively in patients with atherosclerosis. Potential new therapeutic agents targeting other inflammatory mechanisms and oxidative stress are being evaluated in animal models and clinical trials. SUMMARY: Due to the contributory inflammatory pathways in atherosclerosis, the properties of existing and novel anti-inflammatory agents are being carefully and actively evaluated in cardiovascular disease. Advances in our understanding of both atherosclerosis and the inflammatory contributors may play an important role in future strategies to decrease the incidence of atherosclerotic cardiovascular disease.     

Genetic and genomic insights into the molecular basis of atherosclerosis

Atherosclerosis is a complex disease involving genetic and environmental risk factors, acting on their own or in synergy. Within the general population, polymorphisms within genes in lipid metabolism, inflammation, and thrombogenesis are probably responsible for the wide range of susceptibility to myocardial infarction, a fatal consequence of atherosclerosis. Genetic linkage studies have been carried out in both humans and mouse models to identify these polymorphisms. Approximately 40 quantitative trait loci for atherosclerotic disease have been found in humans, and approximately 30 in mice. Recently, genome-wide association studies have been used to identify atherosclerosis-susceptibility polymorphisms. Although discovering new atherosclerosis genes through these approaches remains challenging, the pace at which these polymorphisms are being found is accelerating due to rapidly improving bioinformatics resources and biotechnologies. The outcome of these efforts will not only unveil the molecular basis of atherosclerosis but also facilitate the discovery of drug targets and individualized medication against the disease.     

Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis

Spontaneous atherosclerosis is largely an occlusive disease of medium-size arteries whose progression in a hyperlipidemic environment reflects chronic interactions among injury stimuli to the vessel wall and "responses to injury" by vascular tissue and certain blood components. Development of vessel lesions in animal models of spontaneous atherosclerosis and (at least in principle) in man largely reflects responses of three major cell types (vascular endothelial cells, vascular smooth muscle cells, monocytes-macrophages) as well as the content and distribution of lipids among various lipoprotein subclasses and the increased atherogenicity of modified (e.g., oxidized) lipoproteins. The severe clinical complications associated with spontaneous atherosclerosis, along with its rather common incidence in man, have focused attention on the prevention and therapy of this vascular disease state. Some pharmacological studies in animal models of spontaneous atherosclerosis and some retrospective epidemiological studies in man suggest that vitamin E, the principal (if not sole) lipid-soluble chain-breaking tissue antioxidant, might have therapeutic benefit as an antiatherosclerotic agent. This suggestion gains support from a variety of compelling in vitro evidence demonstrating direct influences of vitamin E on cells and lipoproteins likely involved in the pathogenesis of spontaneous atherosclerosis. Biochemical and cellular data indicate that the potential antiatherogenic activity of vitamin E could reflect its activities as a regulator of endothelial, smooth muscle, or monocyte-macrophage function, an inhibitor of endothelial membrane lipid peroxidation, a modulator of plasma lipid levels and lipid distribution among circulating lipoproteins, and a preventor of lipoprotein oxidative modification. On the other hand, there is a comparative lack of conclusive evidence from animal models regarding: (a) the importance to atherogenesis of vascular and cellular processes modulated by vitamin E; (b) the influence of vitamin E on these processes in vivo and, consequently, on the initiation/progression of spontaneous atherosclerosis. Therefore, pharmacologic investigation of vitamin E (and synthetic, vitamin E-like antioxidants) in nutritional and hyperlipidemic animal models of spontaneous atherosclerosis is required to establish whether any atherosclerotic impact is associated with vitamin E and, if so, what the mechanistic basis of the therapeutic benefit is. Such a line of experimental inquiry should also increase our understanding of the pathogenesis of atherosclerotic vessel disease per se.     

Role of complement activation in atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is characterized by a strong inflammatory component. One factor contributing to inflammation in the arterial intima is the complement system. Here we summarize recent progress in the field of complement research on atherogenesis. RECENT FINDINGS: The complement system is activated in human atherosclerotic lesions and is actively regulated by the local synthesis of complement components and of complement regulatory proteins. Potential triggers of complement activation in the arterial intima include immunocomplexes, C-reactive protein, modified lipoproteins, apoptotic cells, and cholesterol crystals. Complement activation releases anaphylatoxins, and anaphylatoxin receptors have been identified in human atherosclerotic lesions. However, experiments on genetically engineered mice with severe hyperlipidemia have been unable to show a major role for complement in experimental atherogenesis. SUMMARY: In humans there is extensive circumstantial evidence for a role of complement in atherosclerosis, which is somewhat contradictory to recent modest or negative findings in atherosclerosis-prone genetically engineered hyperlipidemic mice.     

Nuclear receptors and inflammatory diseases

It is well known that the steroid hormone glucocorticoid and its nuclear receptor regulate the inflammatory process, a crucial component in the pathophysiological process related to human diseases that include atherosclerosis, obesity and type II diabetes, inflammatory bowel disease, Alzheimer's disease, multiple sclerosis, and liver tumors. Growing evidence demonstrates that orphan and adopted orphan nuclear receptors, such as peroxisome proliferator-activated receptors, liver x receptors, the farnesoid x receptor, NR4As, retinoid x receptors, and the pregnane x receptor, regulate the inflammatory and metabolic profiles in a ligand-dependent or -independent manner in human and animal models. This review summarizes the regulatory roles of these nuclear receptors in the inflammatory process and the underlying mechanisms.     

Extracellular vesicle signalling in atherosclerosis

Atherosclerosis is a major cardiovascular disease and in 2016, the World Health Organisation (WHO) estimated 17.5 million global deaths, corresponding to 31% of all global deaths, were driven by inflammation and deposition of lipids into the arterial wall. This leads to the development of plaques which narrow the vessel lumen, particularly in the coronary and carotid arteries. Atherosclerotic plaques can become unstable and rupture, leading to myocardial infarction or stroke. Extracellular vesicles (EVs) are a heterogeneous population of vesicles secreted from cells with a wide range of biological functions. EVs participate in cell-cell communication and signalling via transport of cargo including enzymes, DNA, RNA and microRNA in both physiological and patholophysiological settings. EVs are present in atherosclerotic plaques and have been implicated in cellular signalling processes in atherosclerosis development, including immune responses, inflammation, cell proliferation and migration, cell death and vascular remodeling during progression of the disease. In this review, we summarise the current knowledge regarding EV signalling in atherosclerosis progression and the potential of utilising EV signatures as biomarkers of disease.     

Atherosclerotic lesion-specific copper delivery suppresses atherosclerosis in high-cholesterol-fed rabbits

Dietary cholesterol supplements cause hypercholesterolemia and atherosclerosis along with a reduction of copper concentrations in the atherosclerotic wall in animal models. This study was to determine if target-specific copper delivery to the copper-deficient atherosclerotic wall can block the pathogenesis of atherosclerosis. Male New Zealand white rabbits, 10-weeks-old and averaged 2.0 kg, were fed a diet containing 1% (w/w) cholesterol or the same diet without cholesterol as control. Twelve weeks after the feeding, the animals were injected with copper-albumin microbubbles and subjected to ultrasound sonication specifically directed at the atherosclerotic lesions (Cu-MB-US) for target-specific copper delivery, twice a week for four weeks. This regiment was repeated 3 times with a gap of two weeks in between. Two weeks after the last treatment, the animals were harvested for analyses of serum and aortic pathological changes. Compared to controls, rabbits fed cholesterol-rich diet developed atherosclerotic lesion with a reduction in copper concentrations in the lesion tissue. Cu-MB-US treatment significantly increased copper concentrations in the lesion, and reduced the size of the lesion. Furthermore, copper repletion reduced the number of apoptotic cells as well as the content of cholesterol and phospholipids in the atherosclerotic lesion without a disturbance of the stability of the lesion. The results thus demonstrate that target-specific copper supplementation suppresses the progression of atherosclerosis at least in part through preventing endothelial cell death, thus reducing lipid infiltration in the atherosclerotic lesion.