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
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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.
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Papers of special note have been highlighted as:
• of interest
•• of considerable interest
Nanomedicine. 2012;7(6):899-916. © 2012 Future Medicine Ltd.