www.medscape.com Authors and Disclosures Rupinder K Kanwar1, Rajneesh Chaudhary2, Takuya Tsuzuki2 & Jagat R Kanwar*1 Nanomedicine, Laboratory of Immunology & Molecular Biomedical Research (LIMBR), Center for Biotechnology & Interdisciplinary Biosciences, Institute for Frontier Materials (IFM), Deakin University, Waurn Ponds, Victoria 3217, Australia 2 Nanomaterials, Institute for Frontier Materials (IFM), Deakin University, Waurn Ponds, Victoria 3217, Australia 1 Financial & competing interests disclosure This work was supported by the Department of Innovation, Industry, Science, Research and Commonwealth of Australia, AISRF grant (BF030016) and BioDeakin, Deakin University. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. *Author for correspondence Tel.: +61 352 271 148 Fax: +61 352 273 402 jagat.kanwar@deakin.edu.au From Nanomedicine Emerging Engineered Magnetic Nanoparticulate Probes for Molecular MRI of Atherosclerosis How Far Have We Come? Rupinder K Kanwar; Rajneesh Chaudhary; Takuya Tsuzuki; Jagat R Kanwar Posted: 07/10/2012; Nanomedicine. 2012;7(6):899-916. © 2012 Future Medicine Ltd. Abstract and Introduction Abstract Atherosclerosis is a chronic, progressive, immunoinflammatory disease of the large and medium-sized arteries, and a major cause of cardiovascular diseases. Atherosclerosis often progresses silently for decades until the occurrence of a major catastrophic clinical event such as myocardial infarction, cardiac arrest and stroke. The main challenge in the diagnosis and management of atherosclerosis is to develop a safe, noninvasive technique that is accurate and reproducible, which can detect the biologically active high-risk vulnerable plaques (with ongoing active inflammation, angiogenesis and apoptosis) before the occurrence of an acute clinical event. This article reviews the events involved in the pathogenesis of atherosclerosis in light of recently advanced understanding of the molecular pathogenesis of the disease. Next, we elaborate on the interesting developments in molecular MRI, by describing the recently engineered magnetic nanoparticulate probes targeting clinically promising molecular and cellular players/processes, involved in early atherosclerotic lesion formation to plaque rupture and erosion. Introduction With the evolved understanding of the pathogenesis of atherosclerosis, it is now recognized as a chronic, progressive, immunoinflammatory disease of medium-sized and large arteries that originates with lesion formation at the arterial wall.[1,2] Currently affecting millions of people worldwide, atherosclerosis is the underlying cause of the majority of cardiovascular diseases (CVDs). Atherosclerosis progresses symptomatically silent for decades until the occurrence of an often fatal acute clinical event such as myocardial infarction, cardiac arrest and stroke. Prevention of blood flow through the coronary artery as a result of an atheromatous process leads to myocardial infarction, and ischemic stroke occurs due to plaque rupture in the carotid arteries. Atherosclerotic plaque rupture in coronary arteries, the most severe complication of the disease, causes approximately 70% of acute coronary events, such as fatal acute myocardial infarction and/or sudden coronary death.[3] Plaque erosion is the culprit for the remaining deaths due to an acute coronary event.[4] Critical limb ischemia, angina pectoris, hypertension, abdominal aortic aneurysms and renal impairment are some of the other complications of atherosclerosis. Through its acute clinical manifestations, atherosclerosis threatens to become a leading cause of death worldwide by 2020. Within the next decade, CVDs are predicted to be the major cause of death globally, because of the rapidly increasing prevalence in eastern Europe and developing countries, and also because of the rising incidence of obesity and diabetes worldwide.[2,5] The commonly associated risk factors for atherosclerosis include dyslipidemia, insulin resistance/diabetes and hypertension (particularly mediated by the renin–angiotensin–aldosterone system).[6] The earlier optimistic hope that by treating hypercholesterolemia and hypertension, coronary artery disease could be eliminated by the end of the 20th century has needed revision. [2] More recently, in the global etiology of atherosclerosis the role of underlying anthropological stimuli such as diet, physical activity, smoking and socioeconomics has emerged in laying the foundations of atherogenesis.[7] A healthy diet accompanied by regular exercise and management of known risk factors, with available methods, has accomplished an important role in the prevention of atherosclerosis. However, for many individuals these measures are not enough.[8] There is still a vital need to revisit the causes of CVDs and reflect on devising new strategies for prediction/diagnosis, treatment and prevention. In recent past, the quest to identify biologically high-risk 'vulnerable' plaques (e.g., heightened plaque inflammation, angiogenesis or apoptosis) has led to significant developments in the field of molecular imaging.[9] Among new generation imaging modalities, noninvasive, nonionizing, safe high-resolution molecular MRI has, in particular, recently become a promising valuable imaging modality for the functional characterization of atherosclerosis processes at the cellular and molecular levels with tremendous clinical interest.[10] With the improved understanding of the pathophysiology of atherosclerosis, a number of different cellular and molecular targets, such as intraplaque macrophages, their scavenger functions and receptors, oxidized low-density lipoproteins (oxLDLs), cell adhesion molecules (CAMs), apoptosis, neovascularization, activated platelets and fibrin, have recently been studied to validate a range of nanoparticulate contrast agents developed for molecular MRI of atherosclerosis. We have reviewed in detail the recently developed magnetic MRI nanoparticulate probes targeting macrophages, their phagocytic activities, surface receptors and molecular products such as neutrophil gelatinase-associated lipocalin.[10] In this article, in order to highlight the potential of molecular MRI to characterize the processes involved in lesion initiation and plaque progression, we first briefly describe the events involved in the pathogenesis of atherosclerosis. The applications of the exponentially growing field of nanotechnology in driving recent development for targeted molecular MRI of CAMs, apoptosis, neovascularization, activated platelets and fibrin, are also reviewed. Pathogenesis of Atherosclerosis The formation of atherosclerotic lesions is a multifactorial, complex immunoinflammatory process that starts at lesion-prone areas with a dysfunctional endothelium, in medium-sized and large arteries.[1,2,11] As a result of decades of long immunoinflammatory signaling, atherosclerotic lesions progress mainly through three stages: fatty streak, fibrous plaque (fibroatheroma) and a complex thrombosis-prone plaque. Figures 1 & 2 (see Figure 3 for key) show the schematic representation of the atherosclerosis process. The arterial wall comprises of three layers. The innermost arterial layer, tunica intima at the luminal side, comprises a monolayer of endothelial cells (ECs) with a basement membrane. The middle and the thickest layer of the wall, tunica media, contains concentric vascular smooth muscle cells (SMCs), elastin fibers and an extracellular matrix (ECM). Internal elastic lamina composed of elastin protein polymer separates the intima and media. The outermost layer, tunica adventitia, consists of loose connective tissues with intermixed SMCs, fibroblasts and fat cells separated from the media by an external elastic lamina.[12–14] The tunica adventitia of most elastic and large muscular arteries, including aorta, coronary, carotids and femoral, contains a number of small blood vessels termed vasa vasorum. The endothelial monolayer is a vital organ of the circulatory system. Its health is essential to normal vascular physiology and its dysfunction – a systemic disorder – is a crucial factor in the pathogenesis of atherosclerosis and its complications.[11] Figure 1. Formation of atherosclerotic lesions at the region of arterial bifurcation (refer to page 903 for figure key). The earliest appearance and subsequent progression of atherosclerotic lesions are associated with regions of arterial bifurcations, branch points and major curves. The figure shows the arterial bifurcation region with high atherosclerosis susceptibility showing altered endothelial structure, function and expression. The uniform laminar hemodynamic blood flow, changes into a complex disturbed nonlaminar flow at the regions of arterial branching with flow separation and recirculation. The endothelial cells in this region show enhanced permeability to low-density lipoproteins and other biomacromolecules. (A) Low-density lipoprotein permeation, entry and accumulation. (B) Macrophage rolling, activation, adhesion and transmigration. (C) Lymphocyte entry. (D) Smooth muscle cell migration. (E) Atherosclerotic lesion with smooth muscle cell fibrous capping and calcification (fibroatheroma). Figure 2. Atherosclerotic disease progression from lesion initiation to plaque rupture (refer to page 903 for figure key). (A) Due to endothelial dysfunction and increased permeability, low-density lipoprotein accumulation and oxidation occurs. This is followed by overlying endothelial cell activation with upregulated expression of heat shock proteins, proinflammatory cytokines, cellular adhesion molecules (VCAM-1, ICAM-1, P-selectin, PECAM-1), chemokines leading to recruitment of monocytes and, to a lesser extent, lymphocytes. The monocytes differentiate and proliferate into macrophages, ingest the oxidized low-density lipoprotein forming foam cells which accumulate within the subintimal region and form fatty streaks. (B) The accumulated foam cells undergo apoptosis leading to accumulation of cell debris within the intimal region. This is followed by the proliferation and migration of smooth muscle cells leading to the formation of a smooth muscle cell fibrous cap over the necrotic core. The continuous inflammation leads to lesion advancement, calcification below the necrotic core, release of matrix degrading enzymes and finally lesion rupture releasing the thrombus into the blood compartment. Figure 3. Atherosclerotic disease progression from lesion initiation to plaque rupture. Key corresponding to figures 1 & 2 on pages 901 & 902. Endothelial Dysfunction There is strong evidence of a role for endothelial dysfunction as a marker of atherosclerotic risk and it also seems to play a fundamental role in the pathogenesis of acute coronary syndromes, such as unstable angina and acute myocardial infarction.[15] Endothelial dysfunction is characterized by a decrease in the bioavailability of vasodilators, in particular nitric oxide (NO), and an increase in the endothelial contracting factors.[16] Impairment in endothelial vasodilation leads to platelet activation.[17] The expression of P-selectin – a platelet activation marker and CAM – is known to be inhibited by NO. NO also suppresses the conformational change during activation of the glycoprotein IIb/IIIa receptor on platelet surfaces, which is required for fibrinogen binding.[18] NO inhibits migration of monocytes, proliferation of SMCs, activation and aggregation of platelets, and adhesion.[19,20] Thus, owing to its reduced anti-inflammatory potential, due to the decreased NO production and increased oxidative process, a dysfunctional endothelium may further contribute to plaque destabilization.[16,19,20] To summarize, endothelial-derived NO plays a critical role for maintaining an anti-atherogenic environment in the arterial wall. Atherosclerotic lesion formation starts to take place under an intact but leaky, activated and dysfunctional endothelium at the lesion-prone sites. Later, ECs may die out and the advanced lesions contain de-endothelialized (denuded) areas, with or without platelets adhering to the exposed subendothelial tissue.[21] Furthermore, as reviewed earlier, the reduction in anticoagulatory potential of endothelium and an increase in the production of procoagulatory agents, such as tissue factor (TF) and plasminogen activator inhibitor, are the characteristics of endothelial dysfunction resulting in a thrombogenic vascular environment.[16] Taken together, a dysfunctional endothelium leads to increased permeation of biomacromolecules such as lipoproteins,[22] increased expression of chemotactic cytokines and CAMs resulting in enhanced leukocyte recruitment, intimal accumulation of foam cells, decreased regeneration of ECs, and altered hemostatic/fibrinolytic balance, favoring thrombin generation, and platelet and fibrin deposition. Endothelial dysfunction also plays a role in plaque destabilization, contributes to enhanced plaque vulnerability, and may trigger plaque rupture by favoring thrombus formation. Endothelial Upregulation (Activation) Lesion initiation begins when the arterial endothelium becomes dysfunctional, stressed and activated by a range of risk factors. The established atherosclerosis risk factors, such as high blood pressure, hypercholesterolemia levels, infection, hemodynamic forces, oxLDL – a biochemically modified LDL cholesterol – dietary factors, toxins and chemical insults, can activate the endothelial stress response and induce heat shock proteins (HSPs).[1,23] Under normal circumstances, HSPs, also known as stress proteins, are a family of highly conserved ubiquitous proteins that act as molecular chaperones and aid cells in coping with stressful environments.[24] There is substantial evidence from human and animal experimental studies that mammalian HSPs 60 and 70 are present in the atherosclerotic lesions and produced by cells of the arterial wall to protect against any local damage.[1,25,26] Anti-HSP60 and antiHSP70 antibodies are associated with established human atherosclerosis, predictive of mortality, and mediate endothelial cytotoxicity and activation.[26–30] In the multifactorial and complex pathogenesis of atherosclerosis, the involvement of inflammatory and immunological mechanisms is widely supported, where the immune response acts as a double-edged sword during the development and progression of atherosclerosis.[31] Among these mechanisms is an autoimmune reaction against HSPs. [23,32,33] HSP60 and HSP70, because of their inducibility, strong immunogenicity and high interspecies homology, have been associated with triggering autoimmune disease.[1,26] HSPs have been implicated as antigenic targets for the activation of lesional T cells in atherosclerotic plaques.[25] Recently, the possible role of HSP90 as a candidate autoantigen in carotid atherosclerosis has also been reported. [34] Whether these HSPs will become therapeutic targets remains to be established. This may be due to the ambiguity regarding their multifaceted role in atherosclerosis. In this regard, HSP60 is proposed to act as an autoantigen triggering both cell- and antibody-mediated immune responses, while HSP70 is proposed to be a cytoprotector.[26] We have shown earlier that HSP60 and HSP70 are disease markers and can be used to stage the progression of atherosclerosis.[1] By employing 3–69-week-old apoE-deficient (apoE–/–; apoE knockout) mice, the most widely used robust mouse model of human atherosclerosis, we found that HSPs 60 and 70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis. This further suggests that few cells escape the toxic environment of the atherosclerotic plaque. The aortas of 3–69-week-old normocholesterolemic apoE+/+ mice did not express HSPs. Our study provided the first evidence that expression of HSP60 and HSP70 is strongly upregulated very early at lesionprone sites in the aortas of young apoE–/– knockout mice and then dramatically downregulated in the chronic lesions of aged mice. At the lesion-prone sites of 3-week-old apoE–/– mice, both mammalian HSPs detected were newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia. Although HSP expression often correlated with oil red O-detectable lipid deposits on aortic valves, the apparently lipid-free aortic wall adjacent to the valve commissures also expressed HSPs in these 3-week-old mice. This could be attributed to mechanical stresses from turbulent blood flow patterns at this site, where the lipid is also first deposited. The local hemodynamic forces that act at different arterial geometries with disturbed flow patterns, affecting the endothelium lining, play an important role in the localization of atherosclerotic lesions.[35–37] As shown in Figure 1 (see Figure 3 for key), the earliest appearance and subsequent progression of atherosclerotic lesions are associated with regions of arterial bifurcations, branch points and major curves. A number of in vitro and in vivo studies have focused on the effect of hemodynamic flow on the structure and function of the endothelial monolayer.[16,38–40] The ECs at the undisturbed laminar flow regions are ellipsoidal in shape and are axially aligned in the direction of flow. ECs at the disturbed flow regions show a disrupted orderly patterned alignment leading to increased macromolecular permeability and lipoprotein accumulation.[12] The first stage of atherosclerosis progression is the passive diffusion, accumulation and retention of the LDLs through the endothelial junction into the intimal layer due to hemodynamic flow stressinduced EC dysfunction at the arterial branching or curvature.[16,35] Circulating plasma molecules and lipoprotein particles, depending on their size and concentration, permeate through the ECs and accumulate in the intima. Particles such as apoB100 bind to proteoglycans, through ionic interactions of the ECM.[22,41] The potentially trapped LDLs are modified to oxLDL – a cytotoxic, proinflammatory and chemotatic molecule.[21,42] Although mechanisms for the atherogenic modification of LDL are not known, they could include oxidative modifications mediated by myeloperoxidase, 15-lipoxygenase and/or NO synthase, or by reactive oxygen species such as HOCl, phenoxyl radical intermediates or peroxynitrite generated in the intima during inflammation.[8,43] The process of lipoprotein oxidation leads to the inhibition of NO production which is anti-atherogenic and a vasodilator.[19,42] The endothelium in the flow-disturbed regions has also been shown to express CAMs and HSPs,[1,37] leading to monocyte adhesion and recruitment. The decreased bioavailability of CAM inhibitory and anti-atherogenic, NO at the dysfunctional endothelium, is therefore considered a contributory factor leading to leukocyte recruitment.[19,40,44] The atherogenic and proinflammatory oxLDL particles are known to stimulate overlying ECs to produce HSPs, CAMs, chemokines and cytokines.[43,45,46] In summary, endothelium activation occurs as a result of the deposition of LDL and oxLDL in the intima, and also by the hemodynamic stress turbulent blood flow at arterial branching points. The activation of the endothelium leads to upregulated expression of CAMs, chemokines and proinflammatory cytokines on ECs, and thereby promotes leukocyte recruitment. Endothelial stress and activation is also associated with the shedding of CAMs, HSPs and cytokines into the circulation,[13] and these molecules are therefore useful markers of endothelial activation/dysfunction.[26,29] Leukocyte Recruitment Monocytes and T lymphocytes (to a lesser extent) are recruited from the circulation, and their transendothelial migration is one of the earliest cellular responses in atherosclerosis. The human lesions are not found to be rich with T cells; a macrophage/T-cell ratio of between approximately 4:1 and 10:1 has been reported.[43] Leukocyte recruitment is predominantly mediated by CAMs, which are expressed on the endothelial surface of arteries and on circulating leukocytes in response to an inflammatory stimulus. Basic and clinical research provides several lines of evidence that support a crucial role of CAMs in the development of atherosclerosis and plaque instability. Chemokines such as CCL2, CCL5 (also known as RANTES), CXCL10 (also known as IP10) and fractalkine (CX3CL1) act in synergy with CAMs to guide monocytes, dendritic cells and T lymphocytes into the intima. [47] Mouse studies reveal that chemokine CCL2, also known as MCP-1, and its receptor, CCR2, play a key role in the initiation of atherosclerosis by attracting circulating monocytes and also T lymphocytes to the activated endothelia.[21,48,49] During leukocyte recruitment, CAMs act in a programmed, sequential manner to form what has been has been termed as the leukocyte–endothelial cascade.[50] The slowing and rolling (tethering) of circulating leukocytes is mediated by selectins (P, E and L) and their ligands (mainly P-selectin ligand). Immunoglobulin superfamily members, such as ICAM-1 and VCAM-1 expressed on endothelia, and their interactions with activated leukocyte integrins such as LFA-1, Mac1 and VLA-4/α4β1, induce tight adhesion of the leukocytes to the luminal surface of the endothelium. PECAM-1 and junctional adhesion molecule-A are involved in the extravasation of cells from the blood compartment into the vessel and underlying tissue (diapedesis).[51,52] Proinflammatory cytokines such as IL-8, IL-1 and TNF-α also aid the leukocyte recruitment as they are known to induce CAM expression.[53] The focal expression of VCAM-1 and ICAM-1 was shown to correspond to the subsequent position at which the fatty streak forms and this focal pattern indicates that hemodynamic forces play an important role in the localization of atherosclerotic lesions.[37,54] Intimal retention of lipoproteins and dominant recruitment of monocytes in the vessel wall comprises the first stage of atherosclerosis. This stage may diminish upon resolution of inflammation or progress to advanced plaques. Thus, the perseverance of this cellular recruitment process seems to underlie disease progression. Among other immune cells recruited during lesion progression are B lymphocytes and plasma cells, although they are rarely found in the intimal lesions and appear abundantly in adventitia next to advanced intimal disease.[21] Activated mast cells may also be found both in plaques and adventitia, particularly in culprit lesions causing acute ischemic events. The mast cell attractant CCL11 (also known as eotaxin) guides their recruitment.[21,31] Foam Cell Formation & Macrophage Differentiation The presence of foam cells (lipid-loaded macrophages) is a characteristic of both early (fatty streaks) and late atherosclerotic lesions. The innate immune responses, mounted in the defense of the stressed and activated endothelium, are important players in the initiation of atherosclerosis and involve internalizing (scavenger) as well as signaling pattern-recognition receptors, as reviewed earlier.[43] Cytokines, such as macrophage colony-stimulating factor secreted by ECs, play an important role in the differentiation and proliferation of recruited monocytes into intimal macrophages, which show different phenotypes including inflammatory and foam cells. A panel of classic and newer cytokines regulating the cellular processes controlling foam cell formation has been reviewed recently.[53] Ingestion of lipoproteins (native LDL and oxLDL) retained in the intima by macrophages involves their interaction with scavenger receptors such as CD36, CD68, SR-PSOX (CXCL16), LOX1, SR-AI/II and SR-B1 expressed on the macrophages.[10] The disruption of the normal homeostatic mechanism governing cholesterol transport and storage within macrophages results in the formation of foam cells containing cholesteryl esters. The anti-atherogenic high-density lipoproteins play an important role in the removal of excess cholesterol from peripheral tissues and inhibition of lipoprotein oxidation.[55] Foam cell formation lays the foundation of fatty streaks, indicating early stage disease, induces a vicious circle of defective inflammation and provides a focal point for atherosclerotic plaque development. The process of resolution of inflammation is impaired in atherosclerosis.[56] By comparison to the native LDL receptor, macrophage scavenger receptors are not downregulated by intracellular cholesterol accumulation. Therefore, the continuous presence of atherogenic lipoproteins in the intima results in their internalization by macrophages till death.[21] Plaque Progression The cycle of formation and death of foamy macrophages in the intima drives the plaque progression. Death of foam cells by apoptosis and necrosis leads to the formation of a soft, destabilizing lipid-rich necrotic core within the intima. Macrophage differentiation and foam cell formation help to bridge the innate and adaptive response to atherosclerosis, and recruited T lymphocytes join macrophages in the intima and direct adaptive immune responses. Data from both animal and human studies reveal that recruited T lymphocytes and macrophages are localized in the shoulder regions in particular, where the lesion grows and immune activation is an ongoing process in atherosclerotic lesions. [43] Several different subtypes of T lymphocytes, such as proinflammatory T helper cells/CD4+, Tregs and CD8+ T cells can be found in the atherosclerotic lesions. Atherosclerosis is driven by the Th1 response, and the role of Th2 immune responses in atherosclerosis remains unclear.[43] T lymphocytes produce proinflammatory cytokines such as IFN-γ and CD40 ligand (CD40L, CD154), which contribute to the amplification of local inflammation leading to plaque progression.[12] Cytokines present in atherosclerotic lesions promote a Th1 response, inducing activated T lymphocytes to differentiate into Th1 effector cells.[12] A signature cytokine of Th1, IFN-γ, causes a range of effects, such as induction of other proinflammatory cytokines, enhanced activation of macrophages and ECs, higher expression of MHC class II, less collagen fiber formation, increased protease and chemokine secretion and upregulation of CAM expression.[57] T lymphocytes, resident macrophages of the plaque as well as ECs, secrete cytokines and growth factors that mediate proliferation and migration of SMCs from the tunica media into the intima, which then synthesize a SMC-derived ECM and form a fibrous cap on the lipid core. This leads to the maturation of fatty streaks into more advanced fibrofatty lesions. The fibrous cap encloses a lipid-rich necrotic core composed of oxLDL, cholesterol, and necrotic and apoptotic cells that failed to survive due to the unavailability of nutrients. SMC migration and proliferation is a highly regulated process controlled by various mediators. The costimulatory interactions between CD40 (on macrophages and ECs) and its ligand CD40L (on Thelper cells) play an important role in the process.[19] CD40 activation results in release of the chemokine IL-8 and other proinflammatory cytokines, such as IL-1 and IFN-γ.[53] The subsequent downstream effects of CD40 signaling and cytokine release result in a phenotypic change in SMCs. The quiescent and 'contractile' phenotype of medial SMCs changes to an active 'synthetic' and migratory one. Thus, this change makes SMCs capable of migrating (into the intima) and proliferating in order to mediate repair. Various mediators regulating the migratory and proliferative activities of SMCs have been reviewed recently.[58] These include: growth promoters such as PDGF, FGF, endothelin-1, thrombin and IL-1; inhibitors such as heparin sulfates, NO and TGF-β. Matrix metalloproteinase (MMP)-9 and other proteinases also participate in the process of SMC migration as they degrade the ECM, catalyze the process to remove the basement membrane around SMCs and facilitate contacts with the interstitial matrix.[58] Owing to the repairing and protective capabilities of SMCs, their migration, proliferation and synthetic activities are considered beneficial, although they may contribute to luminal narrowing in the artery. By formation of a fibrous cap, the lipid core is isolated from circulating blood and the collagen-rich matrix products of SMCs confer stability to plaques. The continuous formation of a fibrous cap results in stable complex lesions (fibroatheroma) with an enlarged necrotic core. The stability of the plaque is dependent on a balance between ECM synthesis and its breakdown as a result of ongoing immunoinflammation. In addition, evidence has been reviewed that Treg cells, which help in controlling or dampening the immune responses, may play a beneficial role and their cytokine products, TGF-β and IL-10, have shown intense atheroprotective effects in mouse models. Genetically modified mice with reduced Treg lymphocytes demonstrated enhanced lesion inflammation and accelerated atherosclerosis.[43,59] On the other hand, angiogenesis (plaque neovascularization) is another contributor to the growth of atherosclerotic plaques. In the absence of atherosclerosis, the nourishment of blood vessels is accomplished by adventitial vasa vasorum or oxygen diffusion from the vessel lumen. As the plaque progresses, the intimal thickness increases and it exceeds the normal oxygen diffusion threshold. Vasa vasorum proliferation in the inner layers then becomes the major source for nutrients to the vessel wall. Angiogenesis, the predominant form of neovascularization in atherosclerosis, is largely related to hypoxia, although hypoxia-independent pathways mediated by inflammation and Toll-like receptors have also been described.[60] Angiogenesis contributes to lesion progression in many ways. It serves as another portal for leukocyte entry to rupture-prone regions of the plaque, including the cap and shoulders.[61] Furthermore, these fragile neovessels can lead to focal intraplaque hemorrhage thereby generating intraplaque thrombin-mediated activation of ECs, monocytes/macrophages, SMC, and platelets. As reviewed earlier, the response to thrombin by these cells results in increased inflammation through the involvement of a broad array of inflammatory mediators that favor the thrombotic complications of atherosclerosis.[57] Another common feature of plaque progression is the focal calcification in atherosclerotic plaques which increases with age. Clinical evidence suggests that the pattern of plaque calcification differs among plaques and is also responsible for different clinical manifestations. The culprit lesions responsible for acute coronary syndromes, such as unstable angina and acute myocardial infarction, generally have less calcification than plaques responsible for stable angina.[21,62] As atherosclerotic plaques further develop, the macrophages, ECs and SMCs die by apoptosis or necrosis. Apoptosis (programmed cell death) contributes dramatically to the high TF activity and thrombogenicity of the lipid-rich core. Apoptotic cells shed membrane apoptotic microparticles rich in an anionic phospholipid, phosphatidylserine, that is redistributed on the surface of cells during apoptosis. These microparticles, produced in considerable amounts within the lipid core, carry almost all TF activity, and as reviewed earlier,[63] are responsible for the procoagulant activity of the plaque. Moreover, luminal EC apoptosis may be responsible for the thrombus formation on eroded plaques without rupture. In biologically active complex plaques with ongoing inflammation, death of foam cells and loss of SMCs may lead to damaging consequences, such as the formation of a destabilizing lipidrich avascular, hypocellular core, and a fragile and rupture-prone fibrous cap. Plaque Rupture & Atherothrombosis The term 'plaque rupture' is proposed for "a plaque with deep injury with a real defect or gap in the fibrous cap that had separated its lipid-rich atheromatous core from the flowing blood, thereby exposing the thrombogenic core of the plaque".[64] This is the most common cause of coronary artery thrombosis. As described above, the ongoing inflammation due to the intense adaptive immune reactions can interfere with the integrity of the interstitial collagen of the fibrous cap. The intraplaque immunoinflammatory signaling stimulates the destruction of existing collagen fibers and inhibits the synthesis of new collagen. IFN-γ inhibits the collagen synthesis by SMCs. CD40L interactions with CD40, as well as IL-1 secreted by T cells lead further to the release of interstitial collagenases, including MMP-1, -8 and -13 from activated macrophages.[57,65] The shoulder regions of plaques, as well as areas of foam cell accumulation, express MMP-9 and the experimental data provide support for an important role of MMP-9 during several stages of atherosclerosis. Human plaque analysis has revealed that MMP-9 is catalytically active and may thus be a contributing player to the dysregulation of ECM proteins that leads to plaque rupture, during atherothrombosis.[57,66] It has also been suggested that locally overexpressed MMP-9 promotes intravascular thrombus formation through the increased TF expression and TF-mediated activation of the coagulation cascade.[67] Thus, when a fibrous cap ruptures, the thrombus causes most acute clinical complications of atherosclerosis. During the pathogenesis of atherosclerosis, multiple sites of endothelial denudation and plaque rupture develop and heal.[21] The endothelial denudation and plaque rupture allow the flowing blood to make contact with the highly thrombogenic material in the lipid core or the subendothelial tissue, leading to platelet adherence and fibrin strand formation. In the pathogenesis of arterial thrombosis, platelet aggregation causes the initial flow obstruction but fibrin formation is required for the ensuing stabilization of the platelet-rich plug/thrombosis. Both platelets and fibrin may accumulate over eroded and ruptured plaques and the magnitude of this thrombotic response depends on the thrombogenic stimulus, as plaque rupture appears much more thrombogenic than plaque erosion.[21] Nanoparticulate Probes for Molecular MRI of CAMs, Apoptosis, Angiogenesis & Thrombosis In order to meet one of the major challenges (i.e., to develop a noninvasive method to detect 'vulnerable' high-risk atherosclerotic plaques prior to thrombosis or plaque rupture/erosion), cardiovascular imaging has evolved impressively in recent years. Nonionizing, high-resolution MRI is presently considered as one of the most promising noninvasive modalities for imaging vessel wall anatomy and atherosclerotic plaque composition (the discrimination of lipid core, fibrosis, intraplaque hemorrhage deposits and calcification). Conventional MRI has an inherent low sensitivity issue with regards to the application of this technique for target-specific imaging of the atherosclerotic plaque. Although the lesion/plaque progression-related biomarkers such as CAMs, macrophages and their scavenger receptors, MMPs, ECM proteins, αvβ3 integrin, oxLDL and fibrin are upregulated in the diseased tissue; they are often present at relatively low levels (10−9–10−13 M/g tissue).[68,69] Molecular MRI thus relies on the development of new-generation contrast agents. It has recently been shown to play an important role in investigating molecular and cellular targets associated with atherosclerotic plaque development. We have recently reviewed various magnetic contrast nanoplatforms such as superparamagnetic iron oxide nanoparticles (SPION), liposomes, micelles and nanoemulsions that have been prepared by conjugating different targeting ligands, such as monoclonal antibodies, antibody fragments, peptidomimetics, small peptides and recombinant proteins, to nanoparticles.[10] Both T1 contrast agents containing either paramagnetic gadolinium (Gd3+) ion complexes or SPIONbased T2 contrast agents have been validated with success, at the preclinical and clinical levels, for molecular MRI of atherosclerosis. Since our earlier article was focused on macrophage targeting, here we highlight the emerging MRI probes targeting other clinically promising molecular and cellular players/processes, involved in early atherosclerotic lesion formation to plaque rupture and erosion. CAMs CAMs are key players in the recruitment of leukocytes during the immunoinflammatory pathogenesis of atherosclerosis. Therefore, CAMs have been considered as one of the interesting targets for the development of nanoparticulate-based probes for molecular MRI. Due to its strict temporal and spatial expression/regulation, VCAM-1 has received the most attention as an ideal target for imaging and therapy of atherosclerosis. Different VCAM-1 targeting cross-linked iron oxide (CLIO)-based nanoparticulate magnetic resonance (MR) contrast agents have been engineered in the recent past. By employing a phage display approach, Kelly et al. identified a peptide sequence containing a VHSPNKK motif that mediates cell internalization via VCAM-1 in murine endothelium under physiological conditions.[70] The peptide sequence showed very high affinity to VCAM-1-expressing ECs, low affinity to macrophages and inhibited leukocyte–endothelial interactions, in vitro. Using the peptide sequence, a novel VCAM-1-targeting magnetofluorescent imaging agent (VNP) with CLIO for optical and MR (dual) imaging was synthesized. In vivo, VNP successfully identified VCAM-1expressing ECs in a murine TNF-α-induced inflammatory model and colocalized with VCAM-1expressing cells in atherosclerotic lesions of cholesterol-fed apoE–/– mice. The same team later developed another novel, second-generation VCAM-1-targeted agent with enhanced affinity and sufficient sensitivity.[71] By employing a linear peptide affinity ligand VHPKQHR, homologous to VLA-4 (a known ligand for VCAM-1), they developed a multivalent agent (VINP-28), detectable by MRI and optical imaging. VINP-28 showed a 20-times higher affinity than the earlier agent VNP, and allowed noninvasive imaging of VCAM-1-expressing ECs and macrophages in atherosclerosis. Atheromata of atorvastatin-treated apoE–/–mice showed reduced VINP-28 deposition and VCAM-1 expression. In another interesting study, a model of adoptive human EC (HUVEC) transfer (by implanting in matrigel to athymic nude mice) was used to test the E-selectin-specific MRI agent, consisting of CLIO nanoparticles conjugated to antihuman endothelial selectin, E-selectin antibody (CLIO-F[ab']2).[72] Highresolution MR images were obtained before and after the administration of CLIO-F(ab')2, which showed specific hypointensity only if treated with proinflammatory cytokine IL-1β. A three-times larger CLIO-induced MR signal decrease on T2* images was measured in HUVEC implants in response to IL1β treatment. The study observations further revealed that HUVEC-containing neovessels were perfused and that IL-1β inducible E-selectin expression in these vessels was detectable with noninvasive imaging by using targeted nanoparticles. P-selectin (CD62P) is overexpressed on pathologically activated endothelium surfaces and activated platelets during atherosclerosis initiation, progression, rupture and thrombosis.[73] More recently, polyethylene glycol and dextran-coated iron oxide nanoparticles, named versatile ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, coupled to an antihuman P-selectin antibody (VH10), were developed and validated preclinically in apoE–/– mouse as a bimodal magnetofluorescent agent, for MRI and optical imaging of inducible P-selectin expression in human activated platelets involved in early stages of atherosclerosis.[74] Iron oxide nanoparticles typically in the size range of a few micrometers, known as microparticles of iron oxide (MPIOs), have also been developed and tested as contrast agents for molecular MRI. MPIOs deliver a high payload of iron resulting in stronger MR contrast and represent a novel tool for molecular MRI.[75] A novel dual CAM targeting strategy has been employed for targeted imaging of VCAM-1 and P-selectin in apoE–/– mice aorta using MPIOs (4.5 µm) conjugated with monoclonal antibodies against VCAM-1 (VCAM-MPIO) and/or P-selectin (P-selectinMPIO).[76] The study showed that dual-ligand MPIOs can be used for functional MRI of these CAMs in mouse atherosclerosis. The strategy employed in this study exploited the opportunity of endovascular accessibility with the use of MPIOs (of similar size to circulating red blood cells) that could convey a substantial contrast payload larger than the one carried by USPIO. As reviewed recently, [77] iron oxide nanoparticle-based contrast agents while effective for most applications, are not suitable for application in molecular endothelial imaging, due to their small size, where the delivery of sufficient contrast volume is challenging on the 'planar' target that is on the surface of the blood vessel wall. The dual ligand-targeted MPIO approach, targeting VCAM-1 and P-selectin, appears promising for in vivo molecular MRI of CAMs (expressed in vascular inflammation, as well as on activated platelet thrombosis), in atherosclerosis as well as in a diverse range of vascular disease models, such as acute vascular inflammation, thrombosis, ischemia–reperfusion injury and ischemic stroke. To overcome the issue of nonbiodegradable MPIOs, recently biodegradable multimeric (mMPIOs) have been developed for targeting VCAM-1.[75] These novel mMPIOs are degraded by macrophages in culture. Biodistribution studies following intravenous injection show a faster clearance rate from the body in comparison with equivalently sized monomeric MPIOs. VCAM-mMPIOs specifically bind to IL1 activated endovascular endothelium in vivo in a brain inflammation murine model. The findings suggest that these mMPIOs may constitute a promising nanoplatform for molecular MRI and should be tested in atherosclerotic models. A slow-clearance blood-pool paramagnetic agent (CMD-A2-GdDOTA [1,4,7,10-tetraazacyclododecane-N,N',N',N'tetraacetic acid]:P717) chemically modified to create a functionalized product (F-P717; hydrodynamic radius 5.7 nm) was synthesized as a new macromolecular imaging agent for noninvasive in vivo molecular MRI and evaluated in an apoE–/– mouse model.[78] In this study, a biomimetic approach was employed by grafting key chemical groups involved in the interaction between P-selectin and its endogenous ligand (PSGL-1) onto a paramagnetic macromolecular gadolinium agent (carboxymethyl-dextran DOTA-Gd) for dual fluorescence and MRI detection. F-P717 significantly enhanced the MRI signal in the abdominal aortic wall of apoE–/– mice, with a greater than 50% signal-to-noise ratio increase between 10 and 30 min, but not in control mice. The MRI data were correlated to histopathology and immunofluorescence in mouse aortic tissue and colocalized F-P717 with the inflammatory area revealed by P-selectin labeling. Apoptosis Apoptosis of intraplaque cells and ECs is linked to plaque destabilization, vulnerability, deendothelization and erosion. Since annexin V has a high binding affinity for phosphotidylserine residues translocated to the outer leaflet of the plasma membrane in apoptotic cells, a nanoplatform was developed using annexin V– to potentially deliver superparamagnetic contrast agents for MRI to sites containing apoptotic cells, such as high-grade atherosclerotic lesions.[79] Biochemically derivatized (annexin V) SPIONs were tested in two rabbit models of human atherosclerosis and myocardial infarction, namely Watanabe heritable hyperlipidemic rabbit and Watanabe heritable hyperlipidemic myocardial infarction, respectively. The latter rabbit model has plaque morphology that resembles the human vulnerable plaques and myocardial infarctions. Postinjection vascular targeting by annexin VSPIONs was atheroma-specific and signal hypointensity was seen in atheromatous lesions, but not in the healthy arteries in a rabbit. Another study demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel CLIO nanoparticles (annexin-CLIOCy5.5; a bimodal [magnetic and near-infrared fluorescence] nanoparticle).[80] This nanoparticulate agent validated in a left anterior descending coronary artery ligation model may have utility in apoptosis detection in atherosclerotic plaques. Plaque Angiogenesis During angiogenesis, targeting the specific proteins expressed excessively in angiogenic blood vessels is the key point in developing anti-angiogenic therapeutics and theranostics. Although angiogenesis research and diagnostics were originally the domain of cancer nanomedicine,[81] recently the potential of angiogenesis targeting in atherosclerosis instability and inflammation has emerged. The presence of the neovessels has been associated with plaque inflammation and instability. Plaque neovessels can be targeted by using αvβ3 integrin biomarkers as the activated ECs express these surface integrins that are not expressed by resting ECs of blood vessels in nondiseased tissues. Two strategies have been employed to image plaque angiogenesis: one approach relies on targeting αvβ3 integrin biomarkers; the second is to directly measure the effect of increased blood in the adventitia due to angiogenesis by applying a dynamic contrast-enhanced MRI method.[82] Winter et al. prepared paramagnetic Gd-diethylenetriaminepentacetate-bis-oleate-containing nanoemulsions of perfluorocarbon, functionalized to detect plaque angiogenesis by coupling with a Arg-Gly-Asp mimetic.[83] These paramagnetic nanoparticles noninvasively targeted the αvβ3 integrin expression in the aortic wall of hyperlipidemic rabbits during early atherosclerosis. Specific detection of the neovasculature within 2 h postinjection was performed by routine MRI at a clinically relevant field strength (1.5 T). The same team later showed that a single application of αvβ3 integrin-targeted paramagnetic nanoemulsion for site-specific delivery of fumagillin (an endothelium-selective antiangiogenic compound) is enough to inhibit angiogenesis in a rabbit model.[84] In their next follow-up study, it was demonstrated that these nanoparticles decreased aortic angiogenesis for 3 weeks after treatment and their anti-angiogenic effect was acute, but this effect was prolonged when given in combination with oral atorvastatin.[85] These findings indicate that this theranostic nanomedicine-based approach could translate into a clinically relevant strategy to evaluate prolonged anti-angiogenic treatment and atherosclerotic plaque stability. Thrombosis (Platelet Activation/Aggregation & Fibrin Targeting) Thrombi can be found at the later stages of atherosclerosis, and plaque rupture or erosion is regarded as the precipitating event for thrombus formation. Platelets not only play a role in the development of atherosclerosis, but are the key to thrombus formation. Thrombi also contain highly abundant fibrin protein. Therefore, noninvasive imaging of activated platelets and fibrin is of great clinical interest. Von Zur Muhlen et al. evaluated the ability of a MRI contrast agent consisting of MPIOs (1-µm size) and a single-chain antibody that selectively binds to ligand-induced binding sites (LIBS) on the activated platelet glycoprotein IIb/IIIa (also known as integrin αIIbβ3).[86] The study was carried out in vivo using a mouse model of ferric chloride-induced carotid artery thrombosis. With LIBS-MPIO, activated platelets can be detected by in vivo MRI with excellent contrast properties and the agent also allowed monitoring of thrombolytic (urokinase) therapy. Furthermore, through ex vivo MRI and histological observations of the surface of symptomatic human carotid plaques, LIBS-MPIOs binding was confirmed to areas of platelet adhesion/aggregation but not for control MPIOs. In their later study, the same group tested the capability of a LIBS-MPIO contrast agent to target activated platelets deposited on injured vessel walls in a mouse model of endothelial injury.[87] The agent being able to convey a substantial contrast payload conferred functional specificity to image-activated platelets adhering to the vessel wall. Fibrin is a highly expressed and clinically relevant target of atherosclerosis. Fibrin deposition is not only one of the earliest signs of plaque rupture or erosion, it also accounts for (along with intraplaque hemorrhage) a considerable part of the core of growing lesions.[88] Fibrin is one of the key elements in thrombus formation following plaque rupture. Flacke and coworkers tested a fibrin-specific paramagnetic MR contrast agent that could allow clinically enhanced, sensitive detection and quantification of occult microthrombi within the intimal surface of atherosclerotic vessels in symptomatic patients and provide direct evidence to support acute therapeutic intervention. [89] The contrast agent was formulated using liquid perfluorocarbons as the core. A lipid-encapsulated liquid perfluorocarbon nanoemulsion conjugated to antifibrin F(ab)' fragments on the outer lipid membrane surface was synthesized with high avidity and a prolonged systemic half-life. It could carry high Gddiethylenetriaminepentaacetate payloads for high detection sensitivity. The study demonstrated the enhanced sensitive detection of clots both in vitro and in vivo. Human fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium levels (0, 2.5 and 20 mol% Gd) and 1.5 T. Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. The nanoparticulate paramagnetic contrast agent described above was shown previously to detect human fibrin clots in vitro with sizes from 0.5 to 7.0 mm with high-resolution MRI at 4.7 T.[90] In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The results of the study suggest that molecular imaging with fibrintargeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin. In the light of recent safety concerns in some patients with renal disease or following liver transplant, leading to US FDA restrictions over the use of gadolinium-based contrast agents, Lanza's team has recently extended their fibrin targeting work to use manganese-based MR as T1 imaging probes.[91] They are the first to report synthesis and MR characterization of fibrin-specific 'soft' type manganese oxide nanocolloids and manganese oleate nanocolloids incorporating divalent manganese. [91] These nanocolliods were constrained to the vasculature by size (>120 nm) to avoid extravasation into nontarget, nonclearance tissues within the arterial wall or beyond. They also synthesized manganese(III)-labeled nanobialys (porphyrin encapsulated in an inverted micelle) as a potential theranostic agent.[92] All these nanoparticulate agents were targeted to fibrin via a fibrin-specific monoclonal antibody using classic biotin–avidin interactions. In both studies, utility of these contrast agents in MR-targeted imaging was supported by their high particulate r1 relaxivities (at 1.5 and 3.0 T) and the strong MR contrast enhancement of T1-weighted MR images, when targeted to in vitro clots. The efficacies of these contrast agents in vivo have yet to be investigated. Senpan et al. have prepared a T1-weighted colloidal iron oxide nanoparticle platform (CION), which was achieved by entrapping oleate-coated magnetite particles within a cross-linked phospholipid nanoemulsion.[93] The developed CIONs with fibrin-specific functional groups caused signal enhancement in T1-weighted ex vivo MRI of fibrin in ruptured atherosclerotic plaques from human carotid endarterectomy specimens without blooming artifacts. Fumagillin was also incorporated into these CIONs in an in vitro demonstration of their potential as theranostic agents. It is important to note that use of iron oxide nanoparticles as T2 imaging contrast agents typically results in negative contrast effects, markedly delayed imaging (they can only be imaged 24 h or more, after systemic administration due to persistent blood pool interference) and is associated with prominent dipole blooming artifacts. This study shows an interesting attempt to tailor the magnetic properties of iron oxide nanoparticles to decrease the r2/r1 ratio, which thereby enables T1-weighted (positive contrast) MRI. McCarthy and coworkers have synthesized novel efficient multimodal nanoagents targeted to fibrin and activated factor XIII (FXIIIa), the two different constituents of thrombi.[94] FXIIIa is responsible for the cross-linking of fibrin α- and γ-chains to stabilize thrombi and increase fibrinolytic resistance. As its activity diminishes over time, it is considered a hallmark of biologically acute thrombi. The nanoagents are targeted by conjugating peptide-targeted ligands to the surface of fluorescently labeled CLIO nanoparticles and functionalized with a fibrin-targeting peptide (GPRPPGGSKGC), and α2-antiplasmin (α2AP)-based peptide (GNQEQVSPLTLLKC) to target FXIIIa by both MRI and optical imaging modalities. The incorporation of two spectrally distinct fluorescent dyes, namely VT680 and Cy7, enabled simultaneous fluorescence imaging of FXIIIa and fibrin via multichannel fluorescence imaging approaches. Through in vitro near-infrared fluorescence and MRI, nanoagents were shown to bind with high affinities to clots. In vivo studies in a FeCl3-induced thrombosis mouse model and ex vivo analysis demonstrated further that both nanoagents readily accumulated within vascular thrombi preferentially over analogously synthesized control agents. Conclusion Atherosclerosis is recognized as a chronic progressive immunoinflammatory disease, and the underlying cause of the majority of CVDs. It is often present symptomatically silent for decades until the occurrence of a major catastrophic clinical event such as myocardial infarction, cardiac arrest and stroke. Because of its acute clinical manifestations, atherosclerosis threatens to be a leading cause of death worldwide in the future.[5] Although the pathogenesis of atherosclerosis is multifactorial and complex, inflammation and immune reactions are known to present from early lesion formation to plaque rupture. As a result of the recent evolution of cardiovascular imaging/diagnostics, it is now well established that plaque composition, not the degree of luminal narrowing, determines plaque vulnerability. High resolution, nonionizing MRI has emerged in particular, as the most promising modality for studying atherosclerosis in humans in vivo, and most importantly allows for the characterization of plaque composition. The growing body of evidence primarily from preclinical, and to some extent, from clinical studies shows that molecular MRI of atherosclerosis with emerging nanoparticulate-based probes is already playing an important role in basic science investigations and in drug development for targeting atherosclerosis. In order to overcome the inherent low sensitivity issue of conventional MRI, the targeted nanoparticulate probes (containing either paramagnetic gadolinium or SPIONs) have been developed for molecular MRI: to carry high-contrast payload for their detection with high sensitivity; to be biocompatible and; to bind strongly and specifically to the target. The experimental evidence cited in this review shows that molecular MRI nanoparticulate probes targeting the clinically promising molecular and cellular players of atherosclerotic plaque, other than macrophages,[10] are still in the stage of preclinical testing and under development. To summarize (Table 1), SPION based T2 contrast agents USPIO, CLIO, MPIO and mMPIO have been mainly employed as promising molecular probes, to target CAMs and thrombi constituents. Dual ligand (VCAM-1 and P-selectin)-targeting MPIOs and biodegradable VCAM-1-targeting mMPIOs appear to constitute another promising nanoplatform-based approach to convey large contrast payloads for molecular imaging on the endothelial surface of arterial vessels. Recent development of novel efficient multimodal nanoagents targeting two different constituents of thrombi (fibrin and FXIIIa) can be considered as an important step leading towards the diagnosis of biologically acute thrombi in clinics. Among the other new avenues being explored, anti-angiogenic paramagnetic perflourocarbonbased Arg-Gly-Asp functionalized nanoemulsions targeting αvβ3 integrin on neovessels have been investigated as site-specific drug delivery vehicles (nanotheranostic agents). Interestingly, in order to overcome the issues encountered with iron oxide-based negative contrast T2 mechanism-based imaging, the development of T1-weighted CION nanoemulsions and manganese-based probes appear to not only be important molecular MRI strategies targeting fibrin, but also present their potential as promising theranostic agents. These probes need further follow-up investigations to determine their clinical benefit. Similarly, bimodal (magnetofluorescent) annexin-CLIO-Cy5.5 designed to visualize cardiomyocyte apoptosis need further validation for their potential to target apoptosis in atherosclerotic plaques, as this process plays a dramatic role in high TF activity, thrombogenicity of the lipid-rich core, plaque destabilization and erosion. Table 1. Summary of nanoparticulate contrast agents targeting cell adhesion molecules, apoptosis, plaque angiogenesis and thrombosis (platelet activation/aggregation and fibrin targeting). Nanoparticle contrast agents Molecular/cellular targets Models Imaging modalities Ref. TNF-α induced CL57BL/6 MR and [70] Cell adhesion molecule targeting VNP (CLIO-based VCAM-1 mice, ApoE–/– mice nanoparticle contrast agent) fluorescence imaging VINP-28 (CLIO-based nanoparticle contrast agent) VCAM-1 MR and ApoE–/– mice; atrovastatinfluorescence response monitoring imaging [71] CLIO-F(ab')2 †; CLIO-based contrast agent E-selectin Athymic nu/nu mice; HUVEC implants MRI [72] USPIO-based contrast agent P-selectin ApoE–/– mice MR and fluorescence imaging [74] Multimeric MPIO-based contrast agent (biodegradable) VCAM-1 Murine brain inflammation MRI model [75] MPIO-based contrast agent VCAM-1 and Pselectin (dual ligand) ApoE–/– mice MRI [76] CarboxymethyldextranDOTA-Gd-based contrast agent P-selectin ApoE–/– mice MR and fluorescence imaging [78] Annexin V-SPION Phosphatidylserine residues (apoptotic cells) WHHL/WHHLMI rabbit strains MRI [79] Annexin-CLIO-Cy5.5 Phosphatidylserine residues (apoptotic cardiomocytes) Left anterior descending coronary artery ligation CL57BL/6 mice MR and NIRF imaging [80] αvβ3-integrin Hyperlipidemic New Zealand white rabbits MRI [83] αvβ3-integrin Hyperlipidemic New Zealand white rabbits (fumagillin-response monitoring) MRI [84] αvβ3-integrin Hyperlipidemic New Zealand white rabbits (combined therapeutic response monitoring atrovastatin and fumagillin) MRI [85] Apoptosis targeting Plaque angiogenesis targeting RGD-coupled Gd-DTPABOA-based nanoemulsion RGD-coupled, fumagillinloaded Gd-DTPA-BOAbased nanothernostic agent RGD-coupled, fumigillinloaded Gd-DTPA-BOAbased nanothernostic agent Thrombosis targeting (platelet activation/aggregation & fibrin targeting) MPIO-based contrast agent Activated platelet GPIIb/IIIa (αIIbβ3integrin) Ferric chloride-induced carotid artery mouse model MRI [87] Gd-DTPA-loaded perfluorocarbon nanoemulsion Fibrin Dog (open circulation) MRI [89] Collidal iron oxide nanoparticle Fibrin Human carotid endarterectomy specimens (ex vivo) MRI [93] Manganese nanocolloids Fibrin In vitro clot binding MRI [91] Manganese nanobialys Fibrin In vitro clot binding MRI [92] Fluorescent CLIO-based nanoagents Fibrin and FXIIIa In vitro and in vivo (FeCl3MR and NIRF induced thrombosis; imaging mouse model) [94] †CLIO conjugated to antihuman E-selectin antibody fragment. BOA: Bis-oleate; CLIO: Cross-linked iron oxide; DOTA: 1,4,7,10-tetraazacyclododecaneN,N',N',N'tetraacetic acid; DTPA: Diethylenetriaminepentacetate; FXIIIa: Activated factor XIII; Gd: Gadolinium; GP: Glycoprotein; HUVEC: Human umbilical vein endothelial cell; MPIO: Microparticles of iron oxide; MR: Magnetic resonance; NIRF: Near-infrared fluorescence; RGD: Arg-Gly-Asp; SPION: Superparamagnetic iron oxide nanoparticle; USPIO: Ultrasmall superparamagnetic iron oxide; VNP: VCAM-1-targeting magnetofluorescent imaging agent; WHHL: Watanabe heritable hyperlipidemic rabbit; WHHLMI: Watanabe heritable hyperlipidemic myocardial infarction. Adapted from [10]. Future Perspective Being a nonionizing imaging modality, the widespread future use of molecular MRI for clinical benefits holds much promise in diagnosis, to risk stratify patients and to allow long-term and serial monitoring of therapy response. As reviewed earlier,[10] the findings of the ATHEROMA trial follow-up with noninvasive USPIO (Sinerem®, Guerbet, Paris, France)-MRI has proven of value for monitoring biological readout of therapy by quantifying carotid artery inflammation (passively targeting macrophages). Importantly, Sinerem does not yet have FDA approval and is still investigational. Prior to the widespread future applications of these probes for clinical diagnostics and therapy monitoring, a number of steps and issues involved in the clinical product development need to be taken care of.[10] Several key issues about toxicity, in vivo stability/pharmacokinetics, as well as regulatory and economic hurdles, will have to be clearly addressed. The systemic nature of atherosclerosis disease involving large to medium-size arteries further requires the focus on different arterial structures and poses another challenge for molecular MRI. There is presently no single ideal approach for molecular MR probes. The selection of nanoparticle size and paramagnetic versus superparamagnetic MR probes will, therefore, mainly depend on the molecular target and the application. Both large and small-sized iron oxide nanoparticles have advantages and disadvantages. Small nanoparticles can deeply penetrate into tissue and are advantageous for extravascular biomarker targeting. Targeted iron oxide nanoparticles hold great potential, but to date, none have progressed to clinical trials. Detection of negative contrast with iron oxide nanoparticles is frequently considered as a challenge for molecular MRI of atherosclerosis than for the other pathologies such as cancer. The small size of arteries, signal from the adjacent vessel, breathing movements, beating of the heart and flow effects continue to pose challenges. Large nanoparticles, generally with a higher payload, improve the contrast and detectability. In future, with the development of a biodegradable version of the LIBS-MPIO agent to target activated platelets, there is a valuable clinical prospect to identify unstable atherosclerotic plaques with MRI. New cellular and molecular targets of atherosclerotic lesions/plaques should be explored and identified. As the pathophysiology of atherosclerosis is better understood, nanoparticulate probes with improved MRI and drug delivery potential can be developed. With ongoing advances in targeted MR nanoparticlulate probe development, the improved ability to visualize the amount or quantify the levels of atherosclerotic plaque inflammation with targeted probes will provide an insight into atherosclerosis progression and will help in assessing the probable risk of a clinical manifestation in the patient. Sidebar Executive Summary Atherosclerosis, as a result of plaque rupture, threatens to become a leading cause of death worldwide by 2020 through its acute clinical manifestations, such as myocardial infarction and stroke. In the multifactorial and complex pathogenesis of atherosclerosis, lesion formation begins when arterial endothelium becomes dysfunctional, stressed and activated by range of risk factors. Recruitment of monocytes and, to a lesser extent, T lymphocytes, is one of the earliest cellular responses in atherosclerotic lesion formation. Expression of cell adhesion molecules along with chemokines and proinflammatory cytokines by an activated endothelium plays a key role in this process. Lesions progress from early fatty streaks to advanced complex plaques. Endothelial cells, monocyte-derived macrophages, T lymphocytes and smooth muscle cells are the major players in the pathogenesis of atherosclerosis. Heightened inflammation, angiogenesis and apoptosis contribute to plaque destabilization and enhanced plaque vulnerability/rupture. In the recent past, in order to identify high-risk vulnerable plaques prior to thrombosis or rupture and erosion, cardiovascular imaging has evolved impressively. In particular, high-resolution, nonionizing MRI has emerged as one of the most promising noninvasive safe modalities for imaging vessel wall anatomy and atherosclerotic plaque composition. With the development of new generation-targeted nanoparticulate magnetic resonance contrast agents, molecular MRI has widened the scope of conventional MRI for functional characterization of the atherosclerosis process at the cellular and molecular levels, with remarkable clinical interest. Superparamagnetic and paramagnetic nanoparticulate probes with T 1 and T 2 contrast mechanisms, respectively, have been synthesized to target the clinically promising molecular and cellular players of atherosclerotic plaque. Preclinical and clinical data indicate that molecular MRI of atherosclerosis with emerging nanoparticulate-based probes is already playing an important role in basic science investigations and in drug development for targeting atherosclerosis. The probes targeting cell adhesion molecules (VCAM-1, P- and E-selectins), plaque angiogenesis, thrombi and apoptosis are still at the stage of preclinical testing and under development. A number of steps and issues involved in clinical product development need to be resolved of before their clinical benefits can be obtained. As the pathogenesis of atherosclerosis is better understood, nanoparticulate probes with improved targeting and drug delivery potential can be developed. In future, through multidisciplinary collaborations, the wide spread use of molecular MRI for clinical benefits holds much promise in diagnosis, to risk stratify patients and to allow longterm and serial monitoring of drug response. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Kanwar RK, Kanwar JR, Wang D, Ormrod DJ, Krissansen GW. Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in apoE-deficient mice. Arterioscler. Thromb. Vasc. Biol.21(12),1991–1997 (2001). •• The apoE-deficient mouse has become the most important and well-known animal model for the study of atherosclerosis. This study provides the first evidence that heat shock proteins 60 and 70 are disease markers that can be used to stage the progression of atherosclerosis, and suggests that the apoE-deficient mouse will be a useful model in which such a study could be undertaken. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med.352(16),1685–1695 (2005). •• Highlights the role of inflammation in the pathogenesis of atherosclerotic coronary artery disease. It describes the evidence that immune mechanisms interact with metabolic risk factors to initiate, propagate and activate lesions in the arterial tree. Naghavi M, Libby P, Falk E et al. From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I. Circulation108(14),1664–1672 (2003). Burke AP, Farb A, Malcom GT, Liang YH, Smialek J, Virmani R. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N. Engl. J. Med.336(18),1276–1282 (1997). Murray CJL, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet349(9064),1498–1504 (1997). Libby P, Aikawa M. Stabilization of atherosclerotic plaques: new mechanisms and clinical targets. Nat. Med.8(11),1257–1262 (2002). Lamon BD, Hajjar DP. Inflammation at the molecular interface of atherogenesis: an anthropological journey. Am. J. Pathol.173(5),1253–1264 (2008). Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell104(4),503–516 (2001). Osborn EA, Jaffer FA. The year in molecular imaging. JACC Cardiovasc. Imaging3(11),1181– 1195 (2010). Kanwar RK, Chaudhary R, Tsuzuki T, Kanwar JR. Emerging engineered magnetic nanoparticulate probes for targeted magnetic resonance imaging of atherosclerotic plaque macrophages. Nanomedicine (Lond.)7(5),735–749(2012). •• The first in a series of two reviews describing an update on recently engineered magnetic nanoparticulate probes targeting macrophages in atherosclerosis. Gimbrone MA, Topper JN, Nagel T, Anderson KR, Garcia-Cardeña G. Endothelial dysfunction, hemodynamic forces, and atherogenesisa. Ann. NY Acad. Sci.902(1),230–240 (2000). Douglas G, Channon KM. The pathogenesis of atherosclerosis. Medicine38(8),397–402 (2010). Andrew CN. An overview of the vascular response to injury: a tribute to the late Russell Ross. Toxicol. Lett.112–113(0),519–529 (2000). Lusis AJ. Atherosclerosis. Nature407(6801),233–241 (2000). Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation104(3),365–372 (2001). Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler. Thromb. Vasc. Biol.23(2),168–175 (2003). Diodati JG, Dakak N, Gilligan DM, Quyyumi AA. Effect of atherosclerosis on endotheliumdependent inhibition of platelet activation in humans. Circulation98(1),17–24 (1998). Anderson TJ. Assessment and treatment of endothelial dysfunction in humans. J. Am. Coll. Cardiol.34(3),631–638 (1999). 19. Kanwar JR, Kanwar RK, Burrow H, Baratchi S. Recent advances on the roles of NO in cancer and chronic inflammatory disorders. Curr. Med. Chem.16(19),2373–2394 (2009). • Comprehensive review on the role of nitric oxide in cancer, myocardial pathophysiology, CNS pathologies, inflammation, asthma, chronic liver diseases, inflammatory bowel disease and arthritis. 20. De Caterina R, Libby P, Peng HB et al. Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J. Clin. Invest.96(1),60–68 (1995). 21. Erling F. Pathogenesis of atherosclerosis. J. Am. Coll. Cardiol.47(Suppl. 8),C7–C12 (2006). 22. Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation116(16),1832–1844 (2007). 23. Xu Q. Role of heat shock proteins in atherosclerosis. Arterioscler. Thromb. Vasc. Biol.22(10),1547–1559 (2002). 24. Morimoto RI, Kline MP, Bimston DN, Cotto JJ. The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem.32,17–29 (1997). 25. Kleindienst R, Xu Q, Willeit J, Waldenberger FR, Weimann S, Wick G. Immunology of atherosclerosis. Demonstration of heat shock protein 60 expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in human atherosclerotic lesions. Am. J. Pathol.142(6),1927–1937 (1993). 26. Bielecka-Dabrowa A, Barylski M, Mikhailidis DP, Rysz J, Banach M. HSP 70 and atherosclerosis – protector or activator? Expert Opin. Ther. Targets13(3),307–317 (2009). 27. Schett G, Xu Q, Amberger A et al. Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity. J. Clin. Invest.96(6),2569–2577 (1995). 28. Pockley AG, Wu R, Lemne C, Kiessling R, De Faire U, Frostegard J. Circulating heat shock protein 60 is associated with early cardiovascular disease. Hypertension36(2),303–307 (2000). 29. Halcox JP, Deanfield J, Shamaei-Tousi A et al. Circulating human heat shock protein 60 in the blood of healthy teenagers: a novel determinant of endothelial dysfunction and early vascular injury? Arterioscler. Thromb. Vasc. Biol.25(11),e141–142 (2005). 30. Herz I, Rosso R, Roth A, Keren G, George J. Serum levels of anti-heat shock protein 70 antibodies in patients with stable and unstable angina pectoris. Acute Card. Care8(1),46–50 (2006). 31. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat. Rev. Immunol.6(7),508–519 (2006). •• Elegantly describes the role of immune mechanisms in the development and activation of atherosclerotic plaques. 32. Mandal K, Jahangiri M, Xu Q. Autoimmunity to heat shock proteins in atherosclerosis. Autoimmun. Rev.3(2),31–37 (2004). 33. Afek A, George J, Gilburd B et al. Immunization of low-density lipoprotein receptor deficient (LDL-RD) mice with heat shock protein 65 (HSP-65) promotes early atherosclerosis. J. Autoimmun.14(2),115–121 (2000). 34. Rigano R, Profumo E, Buttari B et al. Heat shock proteins and autoimmunity in patients with carotid atherosclerosis. Ann. NY Acad. Sci.1107,1–10 (2007). 35. Langille B, O'Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science231(4736),405–407 (1986). 36. Joris I, Zand T, Majno G. Hydrodynamic injury of the endothelium in acute aortic stenosis. Am. J. Pathol.106(3),394–408 (1982). 37. Walpola PL, Gotlieb AI, Cybulsky MI, Langille BL. Expression of ICAM-1 and VCAM-1 and monocyte adherence in arteries exposed to altered shear stress. Arterioscler. Thromb. Vasc. Biol.15(1),2–10 (1995). 38. Zand T, Hoffman AH, Savilonis BJ et al. Lipid deposition in rat aortas with intraluminal hemispherical plug stenosis: a morphological and biophysical study. Am. J. Pathol.155(1),85– 92 (1999). 39. Vallance P, Chan N. Endothelial function and nitric oxide: clinical relevance. Heart85(3),342– 350 (2001). 40. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH. Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling: molecular, cellular, and vascular behavior. J. Am. Coll. Cardiol.49(25),2379–2393 (2007). 41. Borén J, Olin K, Lee I, Chait A, Wight TN, Innerarity TL. Identification of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely affects proteoglycan interaction without affecting LDL receptor binding. J. Clin. Invest.101(12),2658– 2664 (1998). 42. Knowles JW, Reddick RL, Jennette JC, Shesely EG, Smithies O, Maeda N. Enhanced atherosclerosis and kidney dysfunction in eNOS(–/–) apoE(–/–) mice are ameliorated by enalapril treatment. J. Clin. Invest.105(4),451–458 (2000). 43. Hansson GK, Hermansson A. The immune system in atherosclerosis. Nat. Immunol.12(3),204–212 (2011). 44. Auffray C, Fogg D, Garfa M et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science317(5838),666–670 (2007). 45. Kol A, Bourcier T, Lichtman AH, Libby P. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J. Clin. Invest.103(4),571–577 (1999). 46. Ellins E, Shamaei-Tousi A, Steptoe A et al. The relationship between carotid stiffness and circulating levels of heat shock protein 60 in middle-aged men and women. J. Hypertens.26(12),2389–2392 (2008). 47. Zernecke A, Shagdarsuren E, Weber C. Chemokines in atherosclerosis: an update. Arterioscler. Thromb. Vasc. Biol.28(11),1897–1908 (2008). 48. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis. Nature394(6696),894–897 (1998). 49. Gu L, Okada Y, Clinton SK et al. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol. Cell.2(2),275–281 (1998). 50. Watanabe T, Fan J. Atherosclerosis and inflammation mononuclear cell recruitment and adhesion molecules with reference to the implication of ICAM-1/LFA-1 pathway in atherogenesis. Int. J. Cardiol.66(Suppl. 1),S45–S53; discussion S55 (1998). 51. Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis170(2),191–203 (2003). 52. Zernecke A, Liehn EA, Fraemohs L et al. Importance of junctional adhesion molecule-A for neointimal lesion formation and infiltration in atherosclerosis-prone mice. Arterioscler. Thromb. Vasc. Biol.26(2),e10–e13 (2006). 53. McLaren JE, Michael DR, Ashlin TG, Ramji DP. Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy. Prog. Lipid Res.50(4),331–347 (2011). 54. Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R. Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler. Thromb. Vasc. Biol.18(5),842–851 (1998). 55. Kontush A, Chapman MJ. Functionally defective high-density lipoprotein: a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol. Rev.58(3),342–374 (2006). 56. Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis. Nat. Rev. Immunol.10(1),36–46 (2010). 57. Packard RR, Libby P. Inflammation in atherosclerosis: from vascular biology to biomarker discovery and risk prediction. Clin. Chem.54(1),24–38 (2008). 58. Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med. Indones.39(2),86–93 (2007). 59. Mallat Z, Taleb S, Ait-Oufella H, Tedgui A. The role of adaptive T cell immunity in atherosclerosis. J. Lipid Res.50(Suppl.),S364–S369 (2009). 60. Moreno PR, Purushothaman K-R, Sirol M, Levy AP, Fuster V. Neovascularization in human atherosclerosis. Circulation113(18),2245–2252 (2006). 61. De Boer OJ, Van Der Wal AC, Teeling P, Becker AE. Leucocyte recruitment in rupture prone regions of lipid-rich plaques: a prominent role for neovascularization? Cardiovasc. Res.41(2),443–449 (1999). 62. Ehara S, Kobayashi Y, Yoshiyama M et al. Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction. Circulation110(22),3424–3429 (2004). 63. Tedgui A, Mallat Z. Apoptosis as a determinant of atherothrombosis. Thromb. Haemost.86(1),420–426 (2001). 64. Schaar JA, Muller JE, Falk E et al. Terminology for high-risk and vulnerable coronary artery plaques. Eur. Heart J.25(12),1077–1082 (2004). 65. Sukhova GK, Schonbeck U, Rabkin E et al. Evidence for increased collagenolysis by interstitial collagenases 1 and -3 in vulnerable human atheromatous plaques. Circulation99(19),2503–2509 (1999). 66. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J. Clin. Invest.94,2493–2503 (1994). 67. Morishige K, Shimokawa H, Matsumoto Y et al. Overexpression of matrix metalloproteinase-9 promotes intravascular thrombus formation in porcine coronary arteries in vivo. Cardiovasc. Res.57(2),572–585 (2003). 68. Lipinski Mj, Fuster V, Fisher Ea, Fayad Za. Technology insight: targeting of biological molecules for evaluation of high-risk atherosclerotic plaques with magnetic resonance imaging. Nat. Clin. Pract. Cardiovasc. Med.1,48–55 (2004). 69. Chen W, Cormode DP, Fayad ZA, Mulder WJM. Nanoparticles as magnetic resonance imaging contrast agents for vascular and cardiac diseases. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol.3(2),146–161 (2011). • Focuses on recent work that used nanoparticles for molecular MRI of vascular and cardiac diseases. 70. Kelly KA, Allport JR, Tsourkas A, Shinde-Patil VR, Josephson L, Weissleder R. Detection of vascular adhesion molecule-1 expression using a novel multimodal nanoparticle. Circ. Res.96(3),327–336 (2005). •• Shows for the first time that endothelial targets (adhesion molecules) can be visualized by in vivo MRI without monoclonal antibodies. The study approach has far-reaching implications in 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. nanodiagnostics, and the developed multimodal nanoparticle technology could be applied to other targets. Nahrendorf M, Jaffer FA, Kelly KA et al. Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis. Circulation114(14),1504– 1511 (2006). Kang HW, Torres D, Wald L, Weissleder R, Bogdanov AA. Targeted imaging of human endothelial-specific marker in a model of adoptive cell transfer. Lab. Invest.86(6),599–609 (2006). Woollard KJ, Chin-Dusting J. Therapeutic targeting of P-selectin in atherosclerosis. Inflamm. Allergy Drug Targets6(1),69–74 (2007). Jacobin-Valat M-J, Deramchia K, Mornet S et al. MRI of inducible P-selectin expression in human activated platelets involved in the early stages of atherosclerosis. NMR Biomed.24(4),413–424 (2011). Perez-Balderas F, Davis BG, Vankasteren SI. New biodegradable multimeric MPIO contrast agent shows rapid in vitro and in vivo degradation and high sensitivity contrast. Proc. Intl. Soc. Mag. Reson. Med.19,1689 (2011). McAteer MA, Schneider JE, Ali ZA et al. Magnetic resonance imaging of endothelial adhesion molecules in mouse atherosclerosis using dual-targeted microparticles of iron oxide. Arterioscler. Thromb. Vasc. Biol.28(1),77–83 (2008). McAteer MA, Akhtar AM, Von Zur Muhlen C, Choudhury RP. An approach to molecular imaging of atherosclerosis, thrombosis, and vascular inflammation using microparticles of iron oxide. Atherosclerosis209(1),18–27 (2010). Alsaid H, De Souza G, Bourdillon M-C et al. Biomimetic MRI contrast agent for imaging of inflammation in atherosclerotic plaque of apoE–/– mice: a pilot study. Invest. Radiol.44(3),151– 158. (2009). Smith B, Heverhagen J, Knopp M et al. Localization to atherosclerotic plaque and biodistribution of biochemically derivatized superparamagnetic iron oxide nanoparticles (SPIONs) contrast particles for magnetic resonance imaging (MRI). Biomed. Microdevices9(5),719–727 (2007). Sosnovik DE, Schellenberger EA, Nahrendorf M et al. Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle. Magn. Reson. Med.54(3),718–724 (2005). Kanwar JR, Mahidhara G, Kanwar RK. Antiangiogenic therapy using nanotechnological-based delivery system. Drug Discov. Today16(5–6),188–202 (2011). Kerwin WS, O'Brien KD, Ferguson MS, Polissar N, Hatsukami TS, Yuan C. Inflammation in carotid atherosclerotic plaque: a dynamic contrast-enhanced MR imaging study. Radiology241(2),459–468 (2006). Winter PM, Morawski AM, Caruthers SD et al. Molecular imaging of angiogenesis in earlystage atherosclerosis with αvβ3-integrin-targeted nanoparticles. Circulation108(18),2270–2274 (2003). Winter PM, Neubauer AM, Caruthers SD et al. Endothelial αvβ3 integrin–targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler. Thromb. Vasc. Biol.26(9),2103–2109 (2006). Winter PM, Caruthers SD, Zhang H, Williams TA, Wickline SA, Lanza GM. Antiangiogenic synergism of integrin-targeted fumagillin nanoparticles and atorvastatin in atherosclerosis. JACC Cardiovasc. Imaging1(5),624–634 (2008). 86. Von Zur Muhlen C, Von Elverfeldt D, Moeller JA et al. Magnetic resonance imaging contrast agent targeted toward activated platelets allows in vivo detection of thrombosis and monitoring of thrombolysis. Circulation118(3),258–267 (2008). 87. Von Zur Muhlen C, Peter K, Ali ZA et al. Visualization of activated platelets by targeted magnetic resonance imaging utilizing conformation-specific antibodies against glycoprotein IIb/IIIa. J. Vasc. Res.46(1),6–14 (2009). •• By employing microparticles of iron oxide targeting ligand-induced binding sites on the activated conformation of glycoprotein IIb/IIIa on platelets, the study conducted in mice opens an opportunity in the future to specifically image activated platelets involved in acute atherothrombosis with MRI. This study represents the first molecular imaging of a conformational change in a surface receptor. 88. Wickline SA, Neubauer AM, Winter PM, Caruthers SD, Lanza GM. Molecular imaging and therapy of atherosclerosis with targeted nanoparticles. J. Magn. Reson. Imaging25(4),667– 680 (2007). 89. Flacke S, Fischer S, Scott MJ et al. Novel MRI contrast agent for molecular imaging of fibrin. Circulation104(11),1280–1285 (2001). 90. Yu X, Song S-K, Chen J et al. High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent. Magn. Reson. Med.44(6),867–872 (2000). 91. Pan D, Senpan A, Caruthers SD et al. Sensitive and efficient detection of thrombus with fibrinspecific manganese nanocolloids. Chem. Commun. (22),3234–3236 (2009). 92. Pan D, Caruthers SD, Hu G et al. Ligand-directed nanobialys as theranostic agent for drug delivery and manganese-based magnetic resonance imaging of vascular targets. J. Am. Chem. Soc.130(29),9186–9187 (2008). 93. Senpan A, Caruthers SD, Rhee I et al. Conquering the dark side: colloidal iron oxide nanoparticles. ACS Nano3(12),3917–3926 (2009). •• A promising report on T1-weighted theranostic colloidal iron oxide nanoparticle platform technology with a potential to overcome the temporal and spatial imaging challenges associated with current iron oxide nanoparticle T2 imaging agents, and target unstable ruptured plaques or treating atherosclerotic angiogenesis. 94. McCarthy JR, Patel P, Botnaru I, Haghayeghi P, Weissleder R, Jaffer FA. Multimodal nanoagents for the detection of intravascular thrombi. Bioconj. Chem.20(6),1251–1255 (2009). •• Demonstrates the synthesis of two novel multimodal nanoagents for near-infrared fluorescence and magnetic resonance imaging, targeted to two different key molecules in thrombi, fibrin and FXIIIa. Papers of special note have been highlighted as: • of interest •• of considerable interest Nanomedicine. 2012;7(6):899-916. © 2012 Future Medicine Ltd.