Advances in clinical chemistry vol 50

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CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.

SARIKA ARORA (65), Department of Biochemistry, Lady Hardinge Medical
College & Associated Hospitals, New Delhi, India
C.H. BRIDTS (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
THOMAS M. CONNOLLY (1), Discovery Translational Medicine, Wyeth
Research, Collegeville, Pennsylvania, USA
P.K. DABLA (65), Department of Biochemistry, Lady Hardinge Medical
College & Associated Hospitals, New Delhi, India
L.S. DE CLERCK (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
K.J. DE KNOP (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
JORIS R. DELANGHE (23), Department of Clinical Chemistry, Ghent University,
Gent, Belgium
D.G. EBO (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
MASSIMO FRANCHINI (47), Servizio di Immuno-ematologia e Medicina
Trasfusionale, Dipartimento di Patologia e Medicina di Laboratorio, Azienda
Ospedaliero-Universitaria di Parma, Parma, Italy
M.M. HAGENDORENS (87), Department of Paediatrics, Faculty of Medicine,
University of Antwerp, Antwerpen, Belgium

ix


x

CONTRIBUTORS

ALAN R. HIPKISS (123), School of Clinical and Experimental Medicine, College
of Medical and Dental Sciences, The University of Birmingham, Edgbaston,
Birmingham, United Kingdom
ISHMAEL KASVOSVE (23), Department of Chemical Pathology, University of
Zimbabwe College of Health Sciences, Harare, Zimbabwe
JASBIR KAUR (103), Department of Ocular Biochemistry, Dr. Rajendra Prasad
Centre for Ophthalmic Sciences, All India Institute of Medical Sciences,
New Delhi, India
GIUSEPPE LIPPI (47), Laboratorio di Analisi Chimico-Cliniche, Dipartimento di
Patologia e Medicina di Laboratorio, Azienda Ospedaliero-Universitaria di
Parma, Parma, Italy
GWINYAI MASUKUME (23), Department of Chemical Pathology, University of
Zimbabwe College of Health Sciences, Harare, Zimbabwe
MEDHA RAJAPPA (103), Department of Ocular Biochemistry, Dr. Rajendra
Prasad Centre for Ophthalmic Sciences, All India Institute of Medical
Sciences, New Delhi, India
PARUL SAXENA (103), Department of Ocular Biochemistry, Dr. Rajendra
Prasad Centre for Ophthalmic Sciences, All India Institute of Medical
Sciences, New Delhi, India
BHAWNA SINGH (65), Department of Biochemistry, G.B. Pant Hospital,
New Delhi, India
MARIJN M. SPEECKAERT (23), Department of Clinical Chemistry, Ghent

University, Gent, Belgium
REINHART SPEECKAERT (23), Department of Clinical Chemistry, Ghent
University, Gent, Belgium
W.J. STEVENS (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
GIOVANNI TARGHER (47), Sezione di Endocrinologia, Dipartimento di Scienze
Biomediche e Chirurgiche, Universita` di Verona, Verona, Italy


CONTRIBUTORS

xi

RICHA VAISHYA (65), Department of Biochemistry, G.B. Pant Hospital,
New Delhi, India
M.M. VERWEIJ (87), Department of Immunology‐Allergology‐Rheumatology,
Faculty of Medicine, University of Antwerp, Antwerpen, Belgium
XINKANG WANG (1), Discovery Translational Medicine, Wyeth Research,
Collegeville, Pennsylvania, USA
J.M. WALSHE (151), Department of Neurology, The Middlesex Hospital,
London, United Kingdom


PREFACE
I am pleased to present volume fifty of Advances in Clinical Chemistry
series for the year 2010.
In this first volume for the new decade, an array of interesting topics is
presented. This volume leads off with an interesting review on the identification of potential biomarkers in vulnerable atheromatous plaques. Rupture of
these plaques is associated with a host of coronary artery syndromes including myocardial infarction and stroke. The second review explores the unique
relationship of haptoglobin polymorphism and its functionally distinct phenotypes in vaccination, as well as susceptibility or resistance to common

infection. The role of bilirubin as a physiological antioxidant is presented
in the next chapter in support of its reported protective role in prevention of
cardiovascular morbidity and mortality. The oxidation theme is continued in
the next chapter as the role of NAD(P)H oxidase is investigated as the major
source of superoxide in vascular cells and myocytes. The importance of this
key enzyme in the pathophysiology of coronary artery disease is elucidated.
The next chapter deals with the application of microarray technology in the
component-resolved diagnosis of IgE-mediated allergies. An excellent chapter
on pathology of vision loss, specifically ocular disease and the biochemical
mechanisms, involved with angiogenesis. The identification and elucidation of
these unique markers may potentially facilitate early diagnosis or treatment
options. A comprehensive review on mitochondrial dysfunction and protein
alteration is next presented. The identification of these new biomarkers of both
diagnostic and prognostic significance will increase in importance as the
world’s population ages. This volume concludes with an interesting review
on monitoring copper in Wilson’s disease.
I extend my appreciation to each contributor of volume fifty and thank
colleagues who contributed to the peer review process. I also extend thanks to
my Elsevier editorial liaison, Gayathri Venkatasamy, for dedicated support.
I sincerely hope the first volume of the new decade will be enjoyed by our
readership. As always, comments and suggestions for future review articles
for the Advances in Clinical Chemistry series are always appreciated.
In keeping with the tradition of the series, I would like to dedicate volume
fifty to my mother Florence.
GREGORY S. MAKOWSKI
xiii


ADVANCES IN CLINICAL CHEMISTRY, VOL. 50

BIOMARKERS OF VULNERABLE ATHEROMATOUS
PLAQUES: TRANSLATIONAL MEDICINE PERSPECTIVES
Xinkang Wang1 and Thomas M. Connolly
Discovery Translational Medicine, Wyeth Research,
Collegeville, Pennsylvania, USA

1.
2.
3.
4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Background on Atherosclerosis and Vulnerable Plaques . . . . . . . . . . . . . . . . . . . . . . . . .
Concept of Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Imaging Biomarkers for Vulnerable Plaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Computed Tomography (CT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Positron Emission Tomography (PET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Magnetic Resonance Imaging (MRI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Optical Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Circulating Biomarkers for Vulnerable Plaques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Nonspecific Inflammatory Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Inflammatory Cytokines/Chemokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Adhesion Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Matrix Metalloproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Other Inflammatory Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6. Redox Biomarkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. FDA Perspectives of Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
2
4
4
4
6
7
8
8
9
9
10
11
12
12
13
13
14
15

1. Abstract
In cardiovascular disease rupture of a vulnerable atherosclerotic plaque is
the major causative factor of acute coronary syndromes, myocardial
infarction and stroke, and can ultimately lead to death. Identification of
biomarkers that could accurately predict the risk of plaque rupture would
1

Corresponding author: Xinkang Wang, e-mail:
1

0065-2423/10 $35.00
DOI: 10.1016/S0065-2423(10)50001-5

Copyright 2010, Elsevier Inc.
All rights reserved.


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WANG AND CONNOLLY

be a significant advance in guiding treatment of patients with this disease.
The use of these biomarkers would also facilitate the development of new
drugs to treat cardiovascular disease, particularly those that act through
novel mechanisms. There is currently a lack of specific biomarkers for
vulnerable plaque, and thus, it is an area of intense research including the
concepts of live detection versus retrospective characterization, molecular
imaging, and biochemical biomarker discovery. This review will focus on
recent advances on both imaging and circulating molecular biomarkers in
atherosclerosis. The use of combinations of different imaging modalities
(such as molecular, functional, and anatomical) and modalities with circulating/biochemical markers is the current trend and will likely provide the most
useful information for the assessment of the vulnerability of atherosclerotic
plaques.

2. Background on Atherosclerosis and Vulnerable Plaques
Atherosclerosis is a disease of medium and large arteries and involves
endothelial dysfunction, inflammation, and the buildup of lipid deposits,
fibrous tissue, and cellular debris to form a large and growing mass termed
a ‘‘plaque.’’ As the atheromatous plaque grows, the vessel may undergo

a remodeling to enlarge its dimension. Significant stenosis may occur only
after the plaque makes up a significant portion of the intima and then the
reduced lumen makes it more difficult for blood to flow through the artery
with a resulting reduced oxygen supply to the target organ. Furthermore,
erosion or rupture of an unstable plaque exposes the blood to thrombogenic
stimuli which can lead to thrombus formation and complete occlusion
and ensuing cardiovascular (CV) event. The formation of a plaque in an
artery, or atherosclerotic vascular disease, is a major health problem with
over 200 million cases worldwide. It is the causative factor that leads
to coronary artery disease (CAD; myocardial infarction and angina),
peripheral vascular diseases (PAD; critical limb ischemia and intermittent
claudication), and cerebral vascular disease (CVD; ischemic strokes).
Coronary heart disease (CHD) represents the number one and stroke is the
number three cause of death in the United States and most Western
countries. It is estimated that annually 785,000 individuals in the United
States suffer a first heart attack and an estimated 935,000 had a total attack
in 2006 [1].
Atherosclerosis is a complex and progressive disease. The progression of
the disease and composition of the plaque are influenced by inflammatory
cells and the mediators they secrete, and can be further affected by a metabolic condition such as diabetes (glucose), homocysteine, smoking status,


BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

3

and coagulation mediators. The complex interaction among inflammatory
cells, vascular cells, various lipoproteins/particles, and diverse local and
circulating mediators mark the vast complexity of the atherosclerosis
process. While the most severe and ultimate fate of atherosclerosis may be

vessel occlusion (mostly by atherothrombosis), the rate of atherosclerosis
progression is difficult to predict and varies among individuals. Early
atherosclerosis is thought to be initiated with the initial infiltration of inflammatory cells through the compromised endothelium and their progression to
become subendothelial macrophages, which accumulate cholesterol to form
foam cells and subsequently form fatty streak [2, 3]. Over time an intermediate lesion of atherosclerosis develops when smooth muscle cells migrate
into the subendothelium, proliferate, and lay down extracellular matrix to
form the fibrous cap. With the persistence of various risk factors, such
as high levels of LDL, inflammation, shear stress, and other oxidative stresses, the lesion grows. Multiple cellular components including macrophages,
T cells and smooth muscle cells, and various mediators produced by these
cells continuously drive the progression and remodeling of the atherosclerotic lesion [2, 3]. The stability of the atheroma may be weakened due to the
digestion of the fibrous cap by proteases including matrix metalloproteinases
(MMPs), which can ultimately lead to plaque erosion, rupture, and possible
occlusion of the vessel by thrombosis [4].
It is noted that less than 70% patients with acute coronary syndrome
(ACS) had significant stenotic plaques (as defined by angiography) [5, 6]
and that many acute myocardial infarctions (AMI) occur due to occlusion of
coronary arteries without significant structural stenosis [7]. Thus, plaque
dimensions (in particular stenotic phenotype) and clinical outcome are not
always closely related. Histopathological studies of postmortem specimens
suggest distinct composition and characteristics for high-risk/vulnerable plaques that could serve for diagnosis prior to their rupture, including a thin
fibrous cap, a large lipid-rich core, and increased macrophage activity.
Recent advances have provided a better understanding of the molecular/
biochemical mechanisms of atherosclerosis development and in imaging
technologies that provide better insights on plaques prone to rupture,
prone to erosion, or with calcified nodules (additional factor of plaque
vulnerability) [8]. The plaque prone to erosion is often rich in proteoglycans,
though in most cases it lacks a distinguishing structure such as a lipid pool or
necrotic core. In plaques that have lipid-rich cores, the fibrous cap is usually
thick and rich in smooth muscle cells [8, 9]. The plaque with a calcified nodule
often protrudes into the lumen and is associated with loss and/or dysfunction

of endothelial cells over a calcified nodule and thus the loss of fibrous cap,
which makes the plaque at high risk or vulnerable [8, 9].


4

WANG AND CONNOLLY

3. Concept of Biomarkers
An NIH working group has defined a biomarker as ‘‘a characteristic that is
objectively measured and evaluated as an indicator of normal biological
processes, pathogenic processes, or pharmacological responses to a therapeutic intervention’’ [10]. A biomarker may be measured in blood, urine, or a
tissue, or may be a recording of a process. Key criteria are that the biological
variables measured be done quantitatively and with acceptable reproducibility, sensitivity, and specificity. Biomarkers can be used for disease diagnosis,
therapeutic target validation, target engagement, pharmacokinetics and
pharmacodynamics relationship (as both drug efficacy and safety parameters), and patient selection and stratification [11].

4. Imaging Biomarkers for Vulnerable Plaques
Multiple diagnostic imaging modalities have been developed and applied
to detect atherosclerotic plaques. Table 1 summarizes key features of each
modality, including advantages and limitations of their applications in atherosclerosis [12–27]. While earlier methods provided anatomical information, the field is currently shifting toward imaging techniques that provide
information on plaque and vessel composition as well. The imaging modalities are summarized as invasive and noninvasive categories, of which the
invasive methodologies include angiography, intravascular ultrasound
(IVUS), angioscopy, optical coherence tomography (OCT), and noninvasive
methodologies include ultrasound, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and optical imaging [24, 25,
27, 28]. This review will focus on noninvasive imaging methodologies and
their applications in atherosclerosis. In addition, molecular imaging has been
rapidly advancing and there is great interest and potential for its application
in the molecular and functional aspects of atherosclerosis such as inflammation, protease activity, and angiogenesis. Some potential applications of
molecular imaging in atherosclerosis are also reviewed/highlighted in the

following sections along with each imaging modalities.

4.1. ULTRASOUND
Surface ultrasound has been successfully used to noninvasively assess
plaques in the carotid artery because of its high sensitivity and the proximity
of this artery to the body surface, thus allowing for excellent penetration


TABLE 1
ADVANTAGES AND DISADVANTAGES OF VARIOUS IMAGING MODALITIES FOR VULNERABLE PLAQUE ASSESSMENT
Modality
Invasive
Angiography
Angioscopy
IVUS
OCT
Intravascular MRI

Noninvasive
Ultrasound

CT
PET/SPECT
MRI
Optical imaging

Advantages

Standard for stenotic lesions/luminal diameter
Clinical experience

Excellent visual of lipid component in plaques and lumen surface
Direct imaging of vessel wall and plaques
Excellent for vessel wall penetration
High resolution
High resolution and morphological characterization of plaques

Excellent for wall and plaques
Reproducible
Quick
Clinical trial experience
Calcified plaque detection
Molecular imaging
High sensitivity
Anatomic and functional characterization of plaques
High resolution
Molecular imaging
Versatile, high sensitivity

Limitations

References

Provides only lumen dimensions

[12]

Poor penetration
Poor for lipids
Noncoronary assessment
Clinical applications to be developed

[13]
[14]
[15]

Invasive
Time-consuming
Potential heat buildup inside vessel wall

[16, 17]

Poor for lipids
Technically demanding
Noncoronary assessment

[18, 19]

Lack of clinical experience for plaques
Radiation exposure
Low resolution
Lack of clinical experience for plaques
Lack of clinical experience for plaques

[20, 21]

[24, 25]

Lack of clinical experience

[26, 27]

[22, 23]

IVUS, intravascular ultrasound; OCT, optical coherence tomography; MRI, magnetic resonance imaging; CT, computed tomography; PET,
positron emission tomography; SPECT, single photon emission computed tomography.


6

WANG AND CONNOLLY

through the tissues. Measurements of carotid wall thickness and quantitative
analysis of plaque are usually taken at B-mode and evaluated as carotid
intima–media thickness (CIMT) [29, 30], and is the most common use of this
methodology. On a more experimental nature, ultrasound has been used to
identify intraplaque hemorrhage and lipids (as hypoechoic heterogeneous
plaque) versus mostly fibrous (as hyperechoic homogenous plaque) [31, 32].
A variety of studies have shown that there is a correlation between CIMT
and CV risk factors [33–35]. Ultrasound technology is reproducible and
suitable for large, multicenter trials; low cost; and quick. CIMT imaging
has been frequently used in clinical trials as a surrogate end point for
determining the effectiveness of interventions that lower risk factors for
atherosclerosis, such as the following: the Kuopio Ischaemic Heart Disease
Risk Factor (KIDH) Study [36], Atherosclerosis Risk in Community (ARIC)
Study [37], the effect of aggressive versus conventional lipid lowering on
atherosclerosis progression in familial hypercholesterolemia (ASAP) [38],
Arterial Biology for the Investigation of the Treatment Effects of Reducing
Cholesterol 2, (ARBITER 2) [39], Measuring Effects on Intima–Media
Thickness: An Evaluation of Rosuvastatin (METEOR) [40], and Ezetimibe
and Simvastatin in Hypercholesterolemia Enhances Atherosclerosis Regression study (ENHANCE) [41]. The resolution of ultrasound for lipids and

plaque composition is limited, however, unless contrast agents are applied. It
is also technically challenging, especially to assess plaques in the coronary
artery. Other limitations are illustrated in a recent study which showed that
ultrasound measurement may underestimate the vessel wall thickness and
plaques compared to MRI [42], and that CIMT represents mainly hypertensive medial hypertrophy, which is more predictive of stroke than of myocardial infarction (MI) [28–30]. In addition to CIMT, carotid artery plaque
ulceration can be reliably detected by three-dimensional ultrasound [43].
Likewise, microemboli can be detected by transcranial Doppler in patients at
higher risk of stroke [44, 45]. In a new emerging application, ultrasound-based
molecular imaging of atherosclerotic plaques and CV disease with contrastenhanced ultrasonography, which relies on the detection of the acoustic signal
produced by microbubble or nanoparticle agents that are targeted to the specific
molecules at the sites of disease [46], has also been developed.

4.2. COMPUTED TOMOGRAPHY (CT)
CT provides high sensitivity to noninvasively detected calcified plaques
due to its substantially higher density over noncalcified tissues. CT is reliable
and widely used in the clinic, especially with the replacement by helical CT of


BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

7

electron bean CT for the detection of calcified coronary artery plaques.
The disadvantages of CT include radiation exposure and its inability to
differentiate the compositional changes in the noncalcified plaque areas.
One study performed by multislice detector CT showed that statin therapy
led to a significant reduction of noncalcified plaque burden that was not
reflected in calcium scoring or total plaque burden [47], suggesting the
potential to monitor medical treatment in patients with coronary atherosclerosis. Recent advances in CT are aided by contrast enhancement (CT angiography or CTA) and the use of multidetector row CT (MDCT) with
submillimeter collimation and retrospective ECG gating, that permit

high-resolution imaging of coronary artery stenosis and atherosclerotic plaques. However, the recent study of CTA using MDCT failed to reliably
identify the functional significance of coronary lesions in patients with stable
angina and atypical chest pain, suggesting that at this time a diagnostic
strategy relying on CTA alone should not be used for making revascularization decisions [48].

4.3. POSITRON EMISSION TOMOGRAPHY (PET)
PET is the study of human physiology by electronic detection of positronemitting radiopharmaceuticals. The simultaneous detection of these photons
(two high-energy photons emitted in opposite directions) is the basis of PET
imaging [49]. PET provides a measure of metabolic and functional activity of
living tissue based on the retention of positron-emitting tracers. Current
approaches in PET imaging for atherosclerosis use 18F-fluorodeoxyglucose
(FDG), as a radiolabeled tracer, which is taken up by metabolically active
cells and has been frequently used in cancer diagnosis. Two studies in
particular support the use of this ligand for atherosclerosis as a noninvasive
measure of carotid plaque inflammation. Rudd et al. reported a greater
uptake in symptomatic carotids versus asymptomatic vessels and that
this uptake was located near macrophages in endarterectomy samples [50].
In another report, FDG uptake was shown to correlate with the CD68
(macrophage) count in histological examination carotid endarterectomy
samples from patients [22]. PET is very sensitive, noninvasive, and provides
molecular and functional imaging of plaques. Further development of PET
could allow detection of the molecular and cellular events in atherosclerotic
plaques by the development of imaging probes that target MMPs [51] or
annexin A5 (99mTc), a marker of ongoing apoptosis [52]. Limitations of
PET are its poor resolution, which also requires then a coregistration
with CT or MRI, and the requirement of specific radioactive tracers


8

WANG AND CONNOLLY

that usually require a reactor/cyclotron, which are limited in scope of applications. The clinical application of PET in atherosclerosis is still in its
exploratory phase.

4.4. MAGNETIC RESONANCE IMAGING (MRI)
MRI has several advantages. It is noninvasive, has high resolution, and
provides a quantitative characterization of a full range of pathologic features
that could represent plaque rupture, including a lipid core and fibrous cap,
calcification, intima/media/adventitia dimensions as well as intraplaque hemorrhage and acute thrombosis [24]. Therefore, MRI has shown great promise
to study atherosclerosis in the carotid and coronary arteries, as demonstrated
in several clinical studies such as a longitudinal MRI study of atherosclerotic
patients in response to statin treatment [53] and a case–control subgroup
from the Familial Atherosclerosis Treatment Study (FATS) [54]. Exploration of MRI applications is still evolving, especially with the development of
various contrast agents for the assessment of cellular and molecular components of atherosclerosis progression and plaque rupture [24, 28]. For example, the use of ultrasmall particles of iron oxide (USPIO) allows the detection
of macrophage-rich atheroma by MRI [55, 56]. The development of contrast
agents that target specific molecular and cellular components of high-risk
plaques such as macrophage scavenger receptor (for macrophages) [57] and
endothelial adhesion molecules (for vascular inflammation) [58] will provide
excellent tools of MRI biomarkers to monitor atherosclerotic plaque vulnerability. However, the clinical applications of MRI for atherosclerosis burden
assessment and detection of vulnerable plaque remain to be further explored.

4.5. OPTICAL IMAGING
Optical imaging, in particular, near-infrared fluorescence (NIRF) imaging
(excitation 650–900 nm), provides a new and highly versatile platform for
noninvasive in vivo molecular imaging [27]. Optical imaging is extremely
sensitive (picomolar range), utilizes a variety of target platforms (peptide,
protein, antibody, nanoparticles, etc.), thereby providing further versatility,
and is flexible on the detection system needed and range of detection. Optical
imaging provides the ability to visualize atheroma inflammation, calcification, and angiogenesis [27]. For example, the application of a fluorescent

VINP-28 for VCAM-1 internalizing nanoparticle-28 was demonstrated to be
a sensitive method of optical imaging to noninvasively detect atherosclerotic
plaques in apo-E deficient mice [26].


9

BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

5. Circulating Biomarkers for Vulnerable Plaques
Circulating biomarkers that are involved in inflammation, endothelial
damage, or hemostasis are recognized as potential biomarkers for CV risk
and plaque vulnerability. For many of these molecules there is a mixture of
data that is both supportive, and not, for their linkage to CVD and their role
as biomarkers. Table 2 summarizes representatives of those peripheral blood
biomarkers [59–81].
5.1. NONSPECIFIC INFLAMMATORY BIOMARKERS
C-reactive protein (CRP), a member of the pentraxin family, is the most
extensively studied inflammatory and CHD biomarker [82]. Large clinical
studies provided positive associations between CIMT and plasma levels of
CRP [83] and suggested that CRP may have utility in identifying those at
high risk of atherosclerotic complications [84]. Previous studies comparing
patients with low and high CRP levels following statin therapy raise the
TABLE 2
BIOMARKERS ASSOCIATED WITH VULNERABLE PLAQUE IN PERIPHERAL BLOOD
Marker
CRP
TNF-a
IL-6
IL-18

CD40L
sICAM-1
sVCAM-1
sE-selectin
MMP-9
PAPP-A
Lp-PLA2
sRAGE
PAI-1
Adiponectin
Oxidized LDL

Detection method

References

Latex-particle-enhanced immunoassay
ELISA
ELISA
ELISA
ELISA; flow cytometry
ELISA
ELISA
ELISA
Zymography
ELISA
ELISA
ELISA
ELISA
ELISA

Bioanalysis; ELISA

[59, 60]
[61, 62]
[63, 64]
[65]
[66, 67]
[68]
[68]
[68]
[63, 69]
[70, 71]
[72, 73]
[74]
[75]
[76]
[77–80]

All of these markers have been studied in patients with coronary artery disease. While the
method for measuring CRP is robust, the other assays are for research purpose only and many
have very high coefficients of variation [81]. CRP, C-reactive protein; TNF, tumor necrosis
factor; IL, interleukin; MMP, matrix metalloproteinase; sICAM, soluble intercellular adhesion
molecule; sVCAM, soluble vascular cellular adhesion molecule; PAPP-A, pregnancy-associated
plasma protein-A; Lp-PLA2, lipoprotein-associated phospholipase A2; sRAGE, soluble receptor
for advanced glycation end products; PAI, tissue plasminogen activator; ELISA, enzyme-linked
immunosorbent assay.


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provocative possibility that CRP may serve as a CAD risk marker and
therapeutic intervention [85]. The recent finding in the Jupiter trial, where
patients with normal range LDL-c levels, but elevated CRP, showed a
significant drop in CRP and CV benefit with statin treatment [86] has ignited
significant debate on the value of monitoring CRP levels and the use of this
biomarker in atherosclerotic disease. However, many studies have been
inconclusive regarding a causal role of CRP in CHD [87]. In particular, a
recent genome-wide association study of CRP gene argues against a causal
association of CRP with CHD [88]. In addition, CRP is not a specific marker
of atherosclerosis, as it is derived from multiple sources throughout the body
and it is elevated in many disease states. The role of CRP as a predictor of
CAD over other known causative and modifiable factors such as LDL and
oxidized LDL remains to be vigorously validated [82, 89, 90].
5.2. INFLAMMATORY CYTOKINES/CHEMOKINES
The vulnerable plaque contains an extensive inflammatory milieu driven
by a complex set of inflammatory mediators. These mediators could be
monitored in the systemic circulation and thereby may provide insight on
the plaque inflammatory state which includes among others, interleukin-18
(IL-18), IL-6, CD40L, and chemokines.
IL-18 is a pleiotropic proinflammatory cytokine. Increased expression of
IL-18 was observed in human atherosclerotic lesions, and especially in those
prone to rupture by assessing endarterectomy specimens [91]. Elevated serum
concentration of IL-18 was shown to be a strong independent predictor of
CV death in patients with CAD [65], and could lead to accelerated vulnerability of atherosclerotic plaques. The prognostic value of plasma IL-18 levels
as a biomarker for atherothrombotic events remains to be further demonstrated. The Prospective Epidemiological Study of Myocardial Infarction
(PRIME) study showed the association of IL-18 levels with CHD risk [92]
but the Monitoring of Trends and Determinants in Cardiovascular Disease/
Kooperative Gesunheitsforschung in der Region Augsburg (MONICA/

KORA) study had no statistically significant association [93].
IL-6 is a key proinflammatory cytokine associated with the development of
atherosclerosis. The elevated levels of IL-6 expression were observed at the
site of coronary plaque rupture [94]. Various clinical and epidemiological
studies suggest predictive value of plasma IL-6 levels for CV events [95].
Yamagami et al. [64] demonstrated that patients with higher serum IL-6
levels, together with elevated CRP, have a lower echogenicity of their carotid
plaques. This finding supports the link between increased inflammation and
potential risk of vulnerable plaques. The MONICA/KORA study also
demonstrated the association of the elevated plasma concentrations of


BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

11

CRP and IL-6 with the increased CHD risk [93]. The reality of using IL-6 as a
biomarker of vulnerable plaques remains to be further explored.
CD40L is a pleiotropic immunomodulator expressed together with its
receptor (CD40) by cells known to actively contribute to atherosclerosis,
including endothelial cells, smooth muscle cells, monocytes/macrophages,
T-cells, and platelets. Its ligation on the surface of these cells triggers the
expression of other inflammatory and proatherogenic mediators [96]. Elevated plasma concentrations of soluble CD40L (sCD40L) have been observed in
patients with ACS in the CAPTURE study [66]. A similar association was
observed between sCD40L and CV risk in the FRISC study [97] that demonstrated the benefit from a single nucleotide polymorphism in the CD40L gene
that regulates the sCD40L plasma concentrations and shows a correlation
between elevated sCD40L levels and a prothrombotic state. On the other
hand, the Dallas Heart Study suggested that sCD40L was not associated with
most atherosclerosis risk factors or with subclinical atherosclerosis, casting
doubt on its utility in screening for high-risk patients [67].

Chemokines are inflammatory cytokines characterized by their ability to
cause directed migration of leukocytes into inflamed tissue, including atherosclerotic plaques. Elevated expression of chemokines, IL-8, neutrophil-activating peptide-2, interferon-g-inducible protein 10, monocyte chemoattractant
protein-1, and leukotactin-1 in atherosclerotic lesions has been demonstrated
[98]. Combined measurements of multiple chemokines are under consideration
as possibly representative of a ‘‘signature of disease’’ that could serve as an
accurate method to assess for the presence of atherosclerotic disease [98].

5.3. ADHESION MOLECULES
Adhesion molecules play an important role in the development of atherosclerosis. Previous studies demonstrated that the levels of soluble adhesion
molecules, such as soluble vascular cellular adhesion molecule-1 (sVCAM-1),
soluble intercellular adhesion molecule-1 (sICAM-1), and soluble E-selectin
(sE-selectin), were associated with increased risk of future death from CV
causes among patients with CAD [68]. Guray et al. [99] evaluated various
soluble adhesion molecules in patients with various clinical presentations of
coronary atherosclerosis, and compared them to those with angiographically
documented normal coronary arteries, and demonstrated that serum
sVCAM-1, sE-selectin, and sP-selectin levels were useful for predicting coronary plaque destabilization. These results suggest the presence of a more
severe and extensive chronic inflammation in the coronary circulation of
these patients. While the clinical application of soluble adhesion molecules
as a useful biomarker of vulnerable plaque remains to be demonstrated, as


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WANG AND CONNOLLY

discussed in the previous sections, molecular imaging targeting specific endothelial adhesion molecules may provide insights into atherosclerosis
development.
5.4. MATRIX METALLOPROTEINASES
MMPs are a family of zinc- and calcium-dependent endopeptidases that

play a key role in the regulation of extracellular matrix formation and
stability with a strong influence on arterial wall remodeling. The expression
and activity of MMPs are associated with atherosclerotic lesions with advanced CHD. In experimental models of atherosclerosis, degradation of the
matrix surrounding smooth muscle cells also promotes smooth muscle cell
migration [100], whereas higher expression levels of MMP-9 was shown to be
associated with destabilization of the plaque [101]. Clinical studies showed an
association of MMP-9 serum concentrations with CAD [102, 103] and with
unstable carotid plaques [68]. While the clinical value of measuring MMPs as
biomarkers remains to be established, these evidences support for an important role of MMPs in plaque destabilization and rupture. In addition, the
MMP activity could also be monitored by molecular imaging in vivo [104].
5.5. OTHER INFLAMMATORY MARKERS
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a 50-kDa calciuminsensitive lipase produced predominantly by macrophages and lymphocytes. This enzyme resides mainly in LDL particles in plasma. The plasma
levels and activity of Lp-PLA2 are significantly elevated in patients with
CAD and ischemic stroke risk [72, 105]. The enzyme activity of Lp-PLA2 is
implicated in the formation of inflamed, rupture-prone plaque [106]. Although Lp-PLA2 has been reported to exhibit both pro- and anti-inflammatory activities, its primary role appears to be proatherogenic. In this context,
Lp-PLA2 hydrolyzes oxidized phospholipids such as those within oxidized
LDL, generating proinflammatory moieties lysophosphatidylcholine and
oxidized fatty acids [104]. Lp-PLA2 was shown to be expressed within the
necrotic core and in macrophages, notably apoptotic macrophages, surrounding vulnerable and ruptured plaques in patients who suffered from
sudden coronary death [73]. Inhibition of Lp-PLA2 was demonstrated to
reduce complex coronary atherosclerotic plaque development in swine [107]
and in patients [108].
The receptor for advanced glycation end products (RAGE) is a cell-bound
receptor of the immunoglobulin superfamily, which can be activated by
a number of proinflammatory ligands, including advanced glycoxidation
end products, S100/calgranulins, high mobility group box 1, and amyloid


BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

13

b-peptide. RAGE can also act as a counter-receptor for the leukocyte integrin MAC-1. The soluble form of RAGE (sRAGE) lacks the transmembrane
domain and circulates in plasma to function as a decoy to neutralize the
ligands. Therefore, the high plasma levels of sRAGE are associated with a
reduced risk of CAD and other inflammation-related diseases [74]. Studies in
diabetic patients demonstrated that simvastatin inhibited plaque RAGE
expression and contributed to plaque stabilization [109]. A recent study
shows that sRAGE is inversely associated with coronary atherosclerosis
[110]. The feasibility of sRAGE as a biomarker of VP remains to be further
validated.
5.6. REDOX BIOMARKERS
Oxygen and other free radicals are excessively produced in an inflammation zone. Oxidized LDL plays a central role in atherosclerosis development.
It is associated with a number of pathophysiological events, including the
injury of ECs, leukocyte recruitment, foam cell formation, and transition of
plaques from stable to vulnerable and unstable by regulating the expression
and activity of MMPs. Elevated plasma levels of oxidized LDL were
observed in patients with MI [77] and shown to be the strongest predictor
of CHD events compared with a conventional lipoprotein profile and other
traditional risk factors for CHD [78]. Further studies are warranted to use
oxidized LDL as a biomarker to assess various stages of atherosclerosis and
for VP.

6. FDA Perspectives of Biomarkers
In order for a drug to be approved by the FDA for marketing in the United
States, it must be shown to be effective. The meaning of effectiveness is
defined in the Food, Drug, and Cosmetic Act ‘‘as the drug must meet
substantial evidence of effectiveness,’’ which means ‘‘. . .evidence consisting
of adequate and well-controlled investigations, including clinical investigations, . . .that the drug has the effect it purports or is represented to have
under the condition of use prescribed . . .in the labeling or proposed labeling

thereof ’’ [111]. In the case of atherosclerosis and CV disease, outcomes, for
example, MI, stroke, and death, are the ultimate measure of effectiveness.
Since 1990 the FDA has considered an elevated LDL cholesterol (LDL-c) as
a risk factor for CV disease and that lowering LDL-c reduces this risk [112].
LDL-c levels have served as an acceptable target of pharmacological treatment, that is, a surrogate endpoint. In order for this surrogate biomarker
to be used in the approval process, a series of criteria need to be met.


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These include the following: an epidemiological basis, supportive evidence in
animal models, a demonstration that the effect of treatment on the biomarker is consistent across multiple drug classes, and that the change in biomarker
parallels that of the disease and impacts disease outcome. Thus, in support of
LDL-c as an acceptable biomarker, lower total-c levels are associated with a
reduced CHD mortality rate [113]; the work of Brown and Goldstein demonstrated in a human disease, familial hypercholesterolemia, a linkage with
LDL receptor defects and cholesterol levels [114]; a variety of animal models
demonstrate a linkage between LDL-c and atherosclerosis [115]; and clinical
trials that demonstrate benefits of LDL-c reduction, by multiple mechanisms
including diet, niacin and most rigorously, statin treatment [116, 117].
No LDL-c lowering drug has ever been approved based on results from an
imaging study or from a CV outcomes trial. All outcome studies have been
conducted postmarketing and were not part of the approval process. Considerable discussion on the use of LDL-c as a surrogate endpoint to approve
drug marketing has recently arisen with the findings of the ENHANCE study
[41] in which a statin–ezetimibe combination (two drugs that each lower
LDL-c via distinct mechanisms, Vytorin), when compared to statin alone,
failed to show a greater decrease in CIMT despite a greater reduction in
LDL-c and CRP. While many have written about the interpretation of this
result and lessons learned [118] and some have advocated for the need of

outcomes data earlier in the process when a drug lowers LDL-c via a novel
mechanism of action, the FDA has not changed its position on the benefits of
lowering LDL-c [119]. Based on currently available data, they do not recommend any change in patients taking the statin–ezetimibe combination or
ezetimibe alone and indicate patients should talk to their doctor or other
health care professional if they have any questions about these drugs or the
ENHANCE trial. Clearly, this trial and the resulting high-profile discussions
that it has spawned are leading to a thorough evaluation of the LDL-c
biomarker policy and could lead to new guidance on the need for and timing
of outcome studies relative to a demonstration of LDL-c lowering activity.

7. Conclusion
The current concept of the VP or high-risk plaque has been defined mainly
based on histological specimens obtained from autopsies. Enhanced diagnostic methods using both imaging and circulating biomarkers carry the
hope for more accurate definition of the VPs and better selection and identification of high-risk patients. While various imaging technologies have been
used to evaluate VP in either preclinical models or in clinic, each has its
strengths and weaknesses. The measurement of IMT by ultrasound is one of


BIOMARKERS OF VULNERABLE ATHEROMATOUS PLAQUES

15

the most frequently used methodologies and has been used in a number of
clinical studies. A drawback of this technology is that it cannot be used to
image coronary arteries. CT angiography can provide a unique assessment of
high-risk calcified plaques, but a recent report showed that it did not predict
the functional significance of coronary lesions in patients with stable angina
and atypical chest pain [48]. MRI may represent the most versatile imaging
modality for the vessel wall, lumen, plaque volume, and composition of
plaques along with anatomic and functional capabilities, especially when

used with contrast agents. Optical and multimodality molecular imaging of
atherosclerosis offers new opportunities to study atherosclerotic plaque development and may provide a new and translatable strategy to evaluate
plaque vulnerability. While the latest imaging technologies are very
promising, more technical development and validation are needed to warrant
their clinical application to assess VP/high-risk plaques.
Likewise, while a number of circulating biomarkers have shown their great
promise for their tight association with VP/high-risk plaques, none can be
used as a surrogate biomarker. Validation is required to use these circulating
molecules as a useful biomarker of VP, including CRP, the best characterized
inflammatory biomarker that has been shown to be useful in aid of patient
stratification where high risk requires intensified medical intervention [84, 85].
To date, none of circulatory biomarkers and imaging biomarkers, neither
in initially healthy subjects nor in patients with stable and unstable CAD, has
been shown to add incremental value over traditional risk factors in global
risk assessment in the prediction of CHD. The lack of useful biomarkers for
accurate prediction of VP calls for more vigorous validation of the biomarker
candidates. It is important that the validated biomarkers could provide not
only qualitative assessment of VP but also quantitative measurement.
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