ADVANCES IN CLINICAL CHEMISTRY
VOLUME 54


This page intentionally left blank


Advances in
CLINICAL
CHEMISTRY
Edited by
GREGORY S. MAKOWSKI
Clinical Laboratory Partners
Newington, CT
Hartford Hospital
Hartford, CT

VOLUME 54

AMSTERDAM • BOSTON • HEIDELBERG • LONDON
NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Academic Press is an imprint of Elsevier


Academic Press is an imprint of Elsevier
32 Jamestown Road, London NW1 7BY, UK
225 Wyman Street, Waltham, MA 02451, USA
525 B Street, Suite 1900, San Diego, CA 92101-4495, USA
Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands

This book is printed on acid-free paper. ϱ
Copyright ß 2011, Elsevier Inc. All rights reserved
No part of this publication may be reproduced, stored in a retrieval system
or transmitted in any form or by any means electronic, mechanical, photocopying,
recording or otherwise without the prior written permission of the publisher
Permissions may be sought directly from Elsevier’s Science & Technology Rights
Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333;
email: Alternatively you can submit your request online by
visiting the Elsevier web site at and selecting
Obtaining permission to use Elsevier material
Notice
No responsibility is assumed by the publisher for any injury and/or damage to persons
or property as a matter of products liability, negligence or otherwise, or from any use
or operation of any methods, products, instructions or ideas contained in the material
herein. Because of rapid advances in the medical sciences, in particular, independent
verification of diagnoses and drug dosages should be made
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
ISBN: 978-0-12-387025-4
ISSN: 0065-2423
For information on all Academic Press publications
visit our website at www.elsevierdirect.com
Printed and bound in USA
11 12 13 14
10 9 8 7 6

5 4 3 2


1


CONTENTS
CONTRIBUTORS

................................................................................

ix

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Heat-shock Proteins in Cardiovascular Disease
JULIO MADRIGAL-MATUTE, JOSE LUIS MARTIN-VENTURA,
LUIS MIGUEL BLANCO-COLIO, JESUS EGIDO,
JEAN-BAPTISTE MICHEL, AND OLIVIER MEILHAC
1.
2.
3.
4.
5.
6.
7.

Abstract ... ...................................................................................
Introduction .................................................................................
Atherogenesis and Possible Stimuli of Inducible HSPs .................................
HSPs/Anti-HSPs as Biomarkers of Atherothrombosis .................................

Molecular Mechanisms: Bystanders or Actors? . ........................................
HSP as Therapeutic Targets in CVD/Atherothrombosis ...............................
Conclusions ..................................................................................
Acknowledgments...........................................................................
References. ...................................................................................

3
3
4
8
15
25
28
28
29

Polyamines in Cancer
EDWIN A. PAZ, JENARO GARCIA-HUIDOBRO, AND NATALIA A. IGNATENKO
1.
2.
3.
4.
5.
6.
7.

Abstract ... ...................................................................................
Introduction .................................................................................
Overview of Polyamine Regulation .......................................................
Deregulation of Polyamines in Cancer ...................................................

Genetic Variability in ODC Affecting Carcinogenesis..................................
EIF5A and Cancer..........................................................................
Chemoprevention Strategies Within Polyamine Pathway ..............................
Acknowledgments...........................................................................
References. ...................................................................................

v

46
46
47
50
54
56
60
63
63


vi

CONTENTS

Acquired Hemophilia A
MASSIMO FRANCHINI, AND GIUSEPPE LIPPI
1.
2.
3.
4.
5.


Abstract.......................................................................................
Introduction..................................................................................
Pathogenesis..................................................................................
Laboratory Diagnosis.......................................................................
Conclusions ..................................................................................
Acknowledgments ...........................................................................
References ....................................................................................

71
72
72
73
78
79
79

Hypobetalipoproteinemia: Genetics, Biochemistry,
and Clinical Spectrum
PATRIZIA TARUGI, AND MAURIZIO AVERNA
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.


Abstract.......................................................................................
Introduction..................................................................................
Pathways of apoB-Containing Lipoproteins Production . ..............................
Dominant Forms of Primary HBL ........................................................
Recessive Forms of Primary HBL .........................................................
Primary Orphan FHBL.....................................................................
Spectrum of Clinical Manifestations in Primary HBL ..................................
Main Clinical Issues of FHBL .............................................................
Secondary Hypobetalipoproteinemias ....................................................
Conclusions ..................................................................................
Addendum....................................................................................
Acknowledgment ............................................................................
References ....................................................................................

82
83
83
87
91
92
92
94
96
97
99
101
101

Sm Peptides in Differentiation of Autoimmune Diseases

MICHAEL MAHLER
1.
2.
3.
4.
5.
6.
7.
8.

Abstract.......................................................................................
Introduction..................................................................................
Systemic Lupus Erythematosus ............................................................
Mixed Connective Tissue Disease .........................................................
Biochemical Aspects of the Sm Antigen ..................................................
Characteristics of Anti-Sm Antibodies....................................................
Detection of Anti-Sm Antibodies..........................................................
Clinical Association of Anti-Sm Antibodies.. ............................................

109
110
110
112
112
113
114
118


CONTENTS

9.
10.
11.
12.
13.

Meta-Analysis of Anti-Sm Antibodies....................................................
Genesis of Anti-Sm Antibodies............................................................
(Sm) Peptides as Antigens..................................................................
Summary and Conclusion..................................................................
Take Home Messages ......................................................................
References. ...................................................................................

vii
118
119
119
122
122
122

Aromatase Activity and Bone Loss
LUIGI GENNARI, DANIELA MERLOTTI, AND RANUCCIO NUTI
1.
2.
3.
4.
5.
6.
7.

8.
9.

Abstract ... ...................................................................................
Introduction .................................................................................
Aromatase and Sources of Estrogen Production ........................................
The Aromatase Gene and Its Tissue-Specific Regulation ..............................
Aromatase Deficiency and the Bone ......................................................
Skeletal Consequences of Aromatase Excess ............................................
Threshold Estradiol Hypothesis for Skeletal Sufficiency ...............................
Variability in the Level of Aromatase Activity: Effects on Bone Metabolism .......
Summary and Conclusions.................................................................
References. ...................................................................................

129
130
131
133
134
145
146
148
153
154

Biochemistry of Adolescent Idiopathic Scoliosis
GIOVANNI LOMBARDI, MARIE-YVONNE AKOUME, ALESSANDRA COLOMBINI,
ALAIN MOREAU, AND GIUSEPPE BANFI
1.
2.

3.
4.
5.
6.
7.
8.

Abstract ... ...................................................................................
Introduction .................................................................................
Bone Biochemical Parameters .............................................................
Hormones . ...................................................................................
Trace Elements ..............................................................................
Hematological Parameters—Platelets.....................................................
Melatonin . ...................................................................................
Conclusions ..................................................................................
References. ...................................................................................

166
166
168
168
171
171
172
178
179

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183



This page intentionally left blank


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

MARIE-YVONNE AKOUME (165), Viscogliosi Laboratory in Molecular Genetics
of Musculoskeletal Diseases, Sainte-Justine University Hospital Research
Center; and Department of Biochemistry, Faculty of Medicine, Universite´ de
Montre´al, Montre´al, Quebec, Canada
MAURIZIO AVERNA (81), Department of Clinical Medicine and Emerging
Diseases, University of Palermo, Palermo, Italy
GIUSEPPE BANFI (165), IRCCS Istituto Ortopedico Galeazzi, Milano, Italy
LUIS MIGUEL BLANCO-COLIO (1), Vascular Research Lab, IIS, Fundacio´n
Jime´nez Dı´az, Auto´noma University, Av. Reyes Cato´licos 2, Madrid, Spain
ALESSANDRA COLOMBINI (165), IRCCS Istituto Ortopedico Galeazzi, Milano,
Italy
JESUS EGIDO (1), Vascular Research Lab, IIS, Fundacio´n Jime´nez Dı´az,
Auto´noma University, Av. Reyes Cato´licos 2, Madrid, Spain
MASSIMO FRANCHINI (71), Department of Pathology and Laboratory Medicine,
Immunohematology and Transfusion Center, University Hospital of Parma,
Parma, Italy
JENARO GARCIA-HUIDOBRO (45), Biochemistry and Molecular and Cellular
Biology Graduate Program, University of Arizona, Tucson, Arizona, USA
LUIGI GENNARI (129), Department of Internal Medicine, Endocrine-Metabolic
Sciences and Biochemistry, University of Siena, Siena, Italy
NATALIA A. IGNATENKO (45), Department of Cell Biology and Anatomy,
Arizona Cancer Center, Tucson, Arizona, USA


ix


x

CONTRIBUTORS

GIUSEPPE LIPPI (71), Clinical Chemistry Laboratory, Department of Pathology
and Laboratory Medicine, University Hospital of Parma, Parma, Italy
GIOVANNI LOMBARDI (165), IRCCS Istituto Ortopedico Galeazzi, Milano,
Italy
JULIO MADRIGAL-MATUTE (1), Vascular Research Lab, IIS, Fundacio´n
Jime´nez Dı´az, Auto´noma University, Av. Reyes Cato´licos 2, Madrid, Spain
MICHAEL MAHLER (109), INOVA Diagnostics Inc., San Diego, California,
USA
JOSE LUIS MARTIN-VENTURA (1), Vascular Research Lab, IIS, Fundacio´n
Jime´nez Dı´az, Auto´noma University, Av. Reyes Cato´licos 2, Madrid, Spain
OLIVIER MEILHAC (1), Inserm U698, Hemostasis, Bio-engineering and
Cardiovascular Remodeling, Hospital Bichat, Paris, France
DANIELA MERLOTTI (129), Department of Internal Medicine,
Endocrine-Metabolic Sciences and Biochemistry, University of Siena, Siena,
Italy
JEAN-BAPTISTE MICHEL (1), Inserm U698, Hemostasis, Bio-engineering and
Cardiovascular Remodeling, Hospital Bichat, Paris, France
ALAIN MOREAU (165), Viscogliosi Laboratory in Molecular Genetics of
Musculoskeletal Diseases, Sainte-Justine University Hospital Research
Center; Department of Biochemistry, Faculty of Medicine; and Department
of Stomatology, Faculty of Dentistry, Universite´ de Montre´al, Montre´al,
Quebec, Canada

RANUCCIO NUTI (129), Department of Internal Medicine, Endocrine-Metabolic
Sciences and Biochemistry, University of Siena, Siena, Italy
EDWIN A. PAZ (45), Cancer Biology Interdisciplinary Graduate Program,
Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA
PATRIZIA TARUGI (81), Department of Biomedical Sciences, University of
Modena and Reggio Emilia, Modena, Italy


PREFACE
I am pleased to present Volume 54 of Advances in Clinical Chemistry series
for 2011.
In the second volume for this year, a number of topics for clinical laboratories are reviewed. The first review explores the potential role of heat shock
proteins in cardiovascular disease including atherogenesis and atherothrombotic complications. Their role as biomarkers, mediators, and therapeutic
agents is discussed. The second chapter summarizes the biochemical mechanisms of polyamine regulation by tumor suppressor genes and oncogenes
during tumorigenesis. The role of autoantibodies in acquired hemophilia
A is discussed in the third chapter with a focus on pathogenesis, diagnosis,
and epidemiology. The fourth chapter investigates hypobetalipoproteinemias as a heterogenous group of disorders. The biochemistry, genetics, and
clinical spectrum of this disease are discussed. The fifth chapter explores
autoimmune disease associated with the generation of antibodies to small
nuclear ribonucleoproteins in systemic lupus erythematosus. The role of
aromatase in sex steroid hormone generation and their importance in acquisition and maintenance of bone mass in both males and females are elucidated in the sixth chapter. The volume concludes with the seventh chapter,
which discusses the biochemical, hormonal, and hematologic factors associated with development of adolescent idiopathic scoliosis. The potential role
of melatonin signaling dysfunction is explored as a pathologic mechanism in
disease onset and progression.
I thank each contributor of Volume 54 and those colleagues who contributed to the peer-review process. I extend my appreciation to my Elsevier
liaison, Gayathri Venkatasamy, for continued editorial support.
I hope the second volume for 2011 will be enjoyed and used. As always,
your comments and suggestions for clinical laboratory topics of interest for
the Advances in Clinical Chemistry series are always appreciated.
In keeping with the tradition of the series, I would like to dedicate Volume

54 to Uncle Rich.
GREGORY S. MAKOWSKI

xi


This page intentionally left blank


ADVANCES IN CLINICAL CHEMISTRY, VOL. 54

HEAT-SHOCK PROTEINS IN CARDIOVASCULAR DISEASE
Julio Madrigal-Matute,* Jose Luis Martin-Ventura,*
Luis Miguel Blanco-Colio,* Jesus Egido,*
Jean-Baptiste Michel,† and Olivier Meilhac†,1
´ n Jime
´ nez Dı´az,
*Vascular Research Lab, IIS, Fundacio
´ noma University, Av. Reyes Cato
´ licos 2, Madrid, Spain
Auto
†
Inserm U698, Hemostasis, Bio-engineering and Cardiovascular
Remodeling, Hospital Bichat, Paris, France

1.
2.
3.
4.


Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Atherogenesis and Possible Stimuli of Inducible HSPs . . . . . . . . . . . . . . . . . . . . . . . . . . .
HSPs/Anti-HSPs as Biomarkers of Atherothrombosis . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Antigenic Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Indirect Detection via Anti-HSP Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Molecular Mechanisms: Bystanders or Actors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Intracellular Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Extracellular Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. HSP as Therapeutic Targets in CVD/Atherothrombosis . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. HSP Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Immune Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3
3
4
8
9
12
15
15
21
25
25
27
28
29


Abbreviations
17-AAG
17-DMAG
acLDL
ACS
AIF
1

17-allylamino-17-demethoxygeldanamycin
17-desmethoxy-17-N,
N-dimethylaminoethylaminogeldanamycin
acetylated LDL
acute coronary syndrome
apoptosis inducing factor

Corresponding author: Olivier Meilhac, e-mail:
1

0065-2423/11 $35.00
DOI: 10.1016/B978-0-12-387025-4.00001-7

Copyright 2011, Elsevier Inc.
All rights reserved.


2
AMI
Ang-II
APAF1
ApoEÀ/À

BAECs
BMP
CAD
CD
CHD
CRP
CVD
eEF2 kinase
eNOS
ERK
Foxp3
GSH
HDF
Hip
HIV
HO-1
Hop
HOPE study
HSE
HSF
HSP
HSR
HUVECs
Ig
IL
kDA
LDL-RÀ/À
LDLs
LPS
MAPK

MCP1
MGP
MI
Mn-SOD
NF-B
NO
NOS

MADRIGAL-MATUTE ET AL.

acute myocardial infarction
angiotensin-II
apoptosis protease activating factor 1
apolipoprotein E knock out
bovine aortic endothelial cells
bone morphogenetic protein
coronary artery disease
cluster of differentiation
coronary heart disease
C-reactive protein
cardiovascular disease
eukaryotic elongation factor-2 kinase
endothelial NOS
extracellular signal-regulated kinase
forkhead box P3
glutathione
human diploid fibroblasts
HSP70-interacting protein
human immunodeficiency virus
heme oxygenase-1

HSP70–HSP90 organizing protein
the heart outcomes prevention evaluation study
heat-shock element
heat-shock factor
heat-shock protein
heat-shock response
human umbilical vein endothelial cells
immunoglobulin
interleukin
kilodalton
LDL receptor knock out
low-density lipoproteins
lipopolysaccharide
mitogen-activated protein kinases
monocyte chemoattractant protein 1
matrix Gla protein
myocardial infarction
manganese superoxide dismutase
nuclear factor kappa B
nitric oxide
NO synthases


HEAT-SHOCK PROTEINS IN CARDIOVASCULAR DISEASE

oxLDL
PAMPs
ROS
SA
SAPK/JNK

siRNA
SMCs
SR
TGF-b
Th2 cytokine
TLR
TNF-a
Tregs
VEGF
VSMCs
YC1

3

oxidized LDL
pathogen-associated molecular patterns
reactive oxygen species
stable angina
stress-activated protein kinase/c-Jun N-terminal kinase
small interference RNA
smooth muscle cells
scavenger receptor
transfoming growth factor beta
T-helper type 2 cytokine
toll-like receptor
tumor necrosis factor alpha
T regulatory cells
vascular endothelial growth factor
vascular SMCs
3-(50 -hydroxymethyl-20 -furyl)-1-benzyl-indazol


1. Abstract
Heat-shock proteins (HSPs) belong to a group of highly conserved families
of proteins expressed by all cells and organisms and their expression may be
constitutive or inducible. They are generally considered as protective molecules against different types of stress and have numerous intracellular functions. Secretion or release of HSPs has also been described, and potential roles
for extracellular HSPs reported. HSP expression is modulated by different
stimuli involved in all steps of atherogenesis including oxidative stress, proteolytic aggression, or inflammation. Also, antibodies to HSPs may be used to
monitor the response to different types of stress able to induce changes in HSP
levels. In the present review, we will focus on the potential implication of HSPs
in atherogenesis and discuss the limitations to the use of HSPs and anti-HSPs
as biomarkers of atherothrombosis. HSPs could also be considered as potential therapeutic targets to reinforce vascular defenses and delay or avoid
clinical complications associated with atherothrombosis.

2. Introduction
Heat-shock proteins (HSPs) belong to a group of highly conserved families
of proteins expressed by all cells and organisms from bacteria to humans in
response to a variety of different stress stimuli, including heavy metals, inflammatory cytokines, amino acid analogues, oxidative stress, or ischemia [1].


4

MADRIGAL-MATUTE ET AL.

The name of ‘‘stress proteins’’ would be more appropriate than ‘‘HSPs’’
but for historical reasons, due to the discovery of genes of the HSP family in
salivary gland cells of Drosophila subsequent to heat shock [2,3], this name is
still in use today. HSP expression may be constitutive or inducible. HSPs are
generally considered as protective molecules against different types of stress.
They have numerous intracellular functions including roles as molecular
chaperones, promoting correct protein folding of newly synthesized or denatured proteins [4], inhibitors of apoptosis [5], or maintainers of cellular

integrity by stabilization of the cytoskeleton [6]. Secretion or release of
HSPs has also been described, and potential roles for extracellular HSPs
reported. The compartmentalization of HSPs and their role as markers or
actors in atherosclerosis will be discussed in this chapter. Several other
reviews deal with HSPs and cardiovascular disease [7] including cardiac
protection [8] or neuroprotection [9]. In the present review, we will focus
on the potential implication of HSPs in atherogenesis and atherothrombotic
complications; we will discuss whether they may be considered as biomarkers, whether they participate in the etiology of vascular complications, as
well as their potential use as therapeutic agents.
HSPs are classified according to their molecular weight, ranging from 10 to
110 kDa. However, a new nomenclature has been recently proposed [10]. The
correspondences of the principal HSPs that we will discuss here are presented
in Table 1, but the old nomenclature will be used throughout this review.
Table 1 also summarizes the cardiovascular origin of the different HSPs,
their potential inducers, their reported functions, and whether their circulating levels (both antigens and antibodies directed against HSPs) are associated
with cardiovascular disease.

3. Atherogenesis and Possible Stimuli of Inducible HSPs
Several elements participating in atherogenesis have a strong impact on
HSP expression and their posttranslational modifications, such as phosphorylation. We will summarize the different steps of atherogenesis leading to
atherothrombotic complications and clinical manifestations with a particular
emphasis on molecular events reported to induce HSP expression (Fig. 1).
The formation of atheroma starts during childhood by the accumulation
of phagocytic cells in the intimal layer of the arterial wall. The intima is
constituted by the endothelial layer and subjacent extracellular matrix,
separated by the internal elastic lamina from the tunica media, principally
composed of smooth muscle cells (SMCs), elastic, and collagen fibers in
association with glycoproteins and proteoglycans. The intima represents a
very limited space in healthy arteries where accumulation of phagocytic cells,



TABLE 1
HSPS: NEW NOMENCLATURE, CELL EXPRESSION, INDUCING FACTORS, INTRA/EXTRACELLULAR FUNCTIONS AND USE OF HSPS AS CIRCULATING BIOMARKERS
Circulating biomarker
New
nomenclature

Cardiovascular
expression

Induced by

Intracellular function

Extracellular function

Antigen

Antibody
– " carotid atherosclerosis
[30]
– Associated with severity
of CAD [25,31]
– # MI compared to
CHD [32]
– Predicitive of 5-year
mortality in carotid
atherosclerosis [30]
– " higher risk of new CV
event [33]

– Predicted coronary risk
[34]
– Associated with infection
[35] and CVD [36–40]
– # CAD [56]
– not related with
prevalence of CAD [53]
and high risk of ACS [30]

HSP60

HSPD [11]

– Ubiquitously
expressed [11]

– Heat shock [12]
– miR-1/miR-206 [13]
– proinflammatory
cytokines [12]
– Hemodynamic factors
[14]

– Cell survival [15]
– Apoptosis [16]
– Protein trafficking
[17,18]
– Peptide hormone
signaling [19]
– Proliferation [20]


– Proinflammatory [21]
– Immunogen [22]
– Proapoptotic [16]

– " in carotid
atherosclerosis [23]
– Associated with IMT in
borderline
hypertension [24]
– Associated with
severity of CAD [25,26]
– " infection, stress,
myocardial necrosis
[27–29]

HSP70

HSPA [11]

– Smooth muscle
cells [41]
– Cardiac myocytes
[42]
– Monocytes/
Macrophages [41]

–
–
–

–
–
–

–
–
–
–

– Proinflammatory [49]
– Proliferation and
calcification [50]
– Immunogen [51]

– " levels associated with
decreased IMT in
hypertensive
patients [52]
– " levels associated with
low CAD risk [53]
– # carotid
atherosclerosis [54]
– Inversely correlated
with neutrophil
activation [54]
– " ACS [55]

Heat shock [12]
Mechanical stress [43]
Hyperlipidemia [44]

oxLDL [45]
HSP90 inhibitors [41]
Other
pharmacological
compounds [46]

Antiinflammatory [41]
Antiapoptotic [47]
Antioxidant [48]
Antiproliferative[46]

(continues)


TABLE 1 (Continued)
Circulating biomarker
New
nomenclature
HSP27

HSPB1 [11]

HSP90

HSPC [11]

"
#

Increased

Decreased

Cardiovascular
expression
– Smooth muscle
cells [57]
– Endothelial cells
[58]
– Cardiac myocytes
[59,60]
– Monocytes/
Macrophages [61]
– Neutrophils [62]
– Monocytes/
macrophages [41]
– Smooth muscle cells
[41]
– Endothelial cells
[72]

Induced by

Intracellular function

Extracellular function

– Chemical stressors [58]
– Heat shock [63]
– Hyperlipidemia [44]


–
–
–
–
–
–

Actin stabilization [64]
Muscle contraction [64]
Cell migration [64]
Cell survival [64]
Antioxidant [65]
Antiinflammatory [66]

–
–
–
–

Anti-inflammatory [61]
Antiapoptotic [67]
Antioxidant [62]
Proapoptotic [62]

– Heat shock [73]
– Hyperlipidemia [44]

–
–
–

–

Antioxidant [74]
Antiapoptotic [75]
Pro-angiogenic [76]
Proinflammatory [77]

– Prooxidant [78]
– Proinflammatory [79]

Antigen

Antibody

– # atherosclerosis [68]
– " ACS [69]

– " MI in patients with
ACS relative to
unstable angina [70]
– " acute chest pain [71]

– " atherosclerosis [80]

– " atherosclerosis [81]


HEAT-SHOCK PROTEINS IN CARDIOVASCULAR DISEASE

7


Blood-derived cells
Platelets
Monocyte
Cytokines

HSP expression

Lymphocyte
Macrophage

Proteases

Neutrophil

Inflammatory,
proteolytic, and
oxidative stress

ROS, MPO

Erythrocyte

Hemoglobin, iron

Hb

oxLDL

LDL,


Complement
fibrinolytic system

Plasma components
FIG. 1. HSP expression may be modulated by different types of stresses linked to atherogenesis [82] such as proteolytic aggression (e.g., HSP27 expression is increased in response to plasmin
in human vascular smooth muscle cells, [57]), stimulation by cytokines [83], or oxidative stress. In
particular, oxidized LDLs are reported to induce HSP expression [84]. Also, erythrophagocytosis
was shown to induce the synthesis of different HSPs in human monocytes/macrophages [85].
Injection of lysed blood was reported to induce HSP70 expression in the brain [86], suggesting
that free hemoglobin is able to trigger HSP expression. In response, HSPs may protect vascular
cells against different types of aggression within the atherothrombotic plaque.

named foam cells due to their vacuolated aspect, has been detected in human
fetal arteries, in particular in cases of maternal hypercholesterolemia [87].
Hypercholesterolemia is the major risk factor for the development of atheromatous disease. In particular, high circulating levels of low-density lipoproteins (LDLs) lead to their intimal deposition and the subsequent formation
of foam cells due to nonregulated uptake of modified LDLs. The accumulation of foam cells produces fatty streaks observable in ‘‘en face’’ preparations
of arterial samples. In humans, mutations of the LDL receptor (familial
hypercholesterolemia) lead to a strong increase in plasma LDL concentration, thus favoring the development of atheromatous plaques and associated
complications around the 3rd decade of life (myocardial infarction, stroke,
etc.). Animal models deficient or mutated for the LDL receptor are commonly used as models of atherosclerosis (LDL-R knock-out mice, Watanabe
heritable hyperlipidemic rabbits). LDLs, and in particular modified LDLs,
have been reported to participate in all steps of atherogenesis. The major


8

MADRIGAL-MATUTE ET AL.

modification of LDL shown to display atherogenic effects is their oxidation.

LDLs and oxidized LDLs, as well as oxidative stress in general, are known to
induce HSP expression in different cell types present in the pathological
arterial wall (Fig. 1), which may constitute a response to injury.
Evolution of the fatty streaks toward fibroatheroma involves proliferation
of SMCs within the intima that form the fibrous cap surrounding the foam
cells and accumulated extracellular lipids (lipid core), characterized by a
switch of the SMCs from a contractile to a secretory phenotype. HSPs
could play a role in this step by interacting with the cytoskeletal proteins,
such as actin, and thereby modifying SMC migration/proliferation [88].
Fibroatheromatous plaques evolve toward more complicated lesions that
are very heterogeneous but often characterized by the presence of sclerotic
material (calcifications) and the formation of a necrotic, lipidic, and hemocruoric core composed of cell debris, inflammatory, and blood cells (leukocytes, platelets, and red blood cells). HSPs may participate in processes
associated with the evolution toward complicated plaques, such as calcification [50,89]. The presence of blood within the plaque was recently reported to
be the major determinant of the clinical outcome in patients with carotid
artery disease [90]. It reflects local plaque hemorrhage and is associated with
increased intraplaque neovessels. Blood brings into the plaque both oxidative
and proteolytic activities, which are the main driving forces of plaque vulnerability toward rupture, via fragilization of the fibrous cap and by inducing
apoptosis of different vascular cells including SMCs. Many HSPs are induced in response to oxidative stress and proteolytic injury (Fig. 1); they may
therefore constitute sensitive markers of these processes but also a response
for restraining noxious insults potentially favoring plaque rupture and leading to clinical complications. These points will be discussed in detail in the
present review.

4. HSPs/Anti-HSPs as Biomarkers of Atherothrombosis
Our definition of a biomarker is a marker reflecting or integrating one or
several biological activities. Such markers may be any detectable and quantifiable molecules including proteins, peptides, lipids, nucleic acids, etc. This
notion is of major importance when considering HSPs as potential biomarkers of cardiovascular disease. Biomarkers are not specific of a disease but
rather reflect a biological activity associated with this pathology, at one time
point. We will discuss the studies reporting differences in HSP expression in
patients with atherosclerosis versus healthy subjects, directly by antigenic
methods such as ELISA or Western blots in plasma or tissues, or indirectly

by assessment of circulating antibodies raised against HSPs.


HEAT-SHOCK PROTEINS IN CARDIOVASCULAR DISEASE

9

4.1. ANTIGENIC DETECTION
4.1.1. HSP60
Different studies have analyzed the levels of circulating HSPs. Among
them, levels of HSP60 are increased in patients with carotid atherosclerosis,
suggesting its potential role as a diagnostic biomarker [23]. In patients with
borderline hypertension, serum HSP60 levels were associated with intima–
media thickness, a surrogate marker of atherosclerosis [24].
In addition, prospective data have confirmed an association between high
levels of sHSP60 and early carotid atherosclerosis [91]. Similarly, another
study has undertaken a prospective analysis of the association of HSP60 with
the severity of CAD, reporting that HSP60 levels were significantly correlated with both the extent index and stenoses [25]. These data have been
recently confirmed in a large case–control study, suggesting that the combination of HSP60 and anti-HSP60 antibody levels may predict this risk [26].
Potential explanations for the high HSP60 levels observed in atherosclerotic
patients may be responses to infection, stress, or myocardial necrosis [27–29].
In complement to the clinical observation of increased HSP antigens in
patients with atherothrombosis, different authors have analyzed the presence
of HSPs in atherosclerotic plaques. In initial studies, increased HSP65
expression and the presence of HSP65-specific T-cells both in experimental
and human atherosclerotic lesions were reported [14,92,93]. In subsequent
studies, chlamydial HSP60 was colocalized with human HSP60 in plaque
macrophages in human atherosclerotic lesions [94].
4.1.2. HSP70
An inverse relation between HSP70 and atherosclerosis has been reported

by several groups. Whereas HSP70 is detectable in serum of nondiseased
individuals [95], low serum HSP70 levels have been suggested to predict the
development of atherosclerosis [52–54]. In hypertensive patients, increased
concentrations of circulating HSP70 correlated with decreased intima/media
thickness [52]. In another study by Zhu et al., high serum levels of HSP70
were found to be associated with a low risk of coronary artery disease [53].
We have reported that plasma HSP70 concentrations were decreased in
patients with carotid atherosclerosis relative to control healthy subjects
[54]. Interestingly, circulating levels of neutrophil activation markers (myeloperoxidase, matrix metalloprotease 9/lipocalin complexes, and elastase)
were inversely correlated with those of HSP70, suggesting the proteolytic
degradation of this HSP under atherothrombotic conditions.
Under acute conditions, Zhang et al. recently reported that HSP70 was
increased in patients with acute coronary syndrome (ACS) relative to ageand sex-matched healthy controls [55]. HSP70 levels were associated with


10

MADRIGAL-MATUTE ET AL.

increased risk and severity of ACS. Interestingly, these authors monitored
HSP70 levels at the time of admission, 2, 3, and 7 days after acute myocardial
infarction (AMI). They report that HSP70 plasma concentration decreased
rapidly after the onset of AMI. It is likely that following ischemia, the
myocardial necrotic area releases large amounts of HSP70, as described in
response to heat shock where HSP70 was abundant in small blood vessels
found between the ventricular cardiomyocytes [96].
Berberian et al. first reported HSP70 expression in normal human aortas
and carotid atherosclerotic plaques [97]. In atherosclerotic tissue, the necrotic
core and its underlying media contained significantly more HSP70 staining
than did fibrotic areas [47]. Accumulation of HSP70 in VSMCs adjacent

to the necrotic core was suggested to be insufficient to protect them against
the noxious stimuli of the plaque. We have recently quantified HSP70 immunostaining in 60 human atherosclerotic plaques and showed an increased
expression of HSP70 in the shoulder region of the plaque compared to the
fibrous area, probably reflecting increased stress of this vulnerable region due
to blood flow. Interestingly, when atherosclerotic plaques were classified
according to the cap thickness, we observed that HSP70 expression is lower
in plaques with thin caps (< 165 m), suggesting that HSP70 plays an important role in the stability of advanced human atherosclerotic plaques [41].
4.1.3. HSP27
Plasma levels of HSP27 were shown to be decreased in atherosclerosis
following a proteomic comparison between conditioned medium obtained
from human carotid samples and healthy mammary endarteries [68]. At this
time, HSP27 was described as an intracellular protein ubiquitously expressed
by many cell types, including vascular cells. A noncandidate proteomicbased approach allowed us to discover HSP27 as a potential marker of
nondiseased vascular wall. The decreased solubilization of HSP27 under
atherothrombotic conditions was attributed, at least in part, to proteolytic
activities such as that of plasmin present in culprit plaques and able to digest
the soluble HSP27, potentially reducing its circulating levels [57].
In a prospective study including 255 female health care professionals
devoid of cardiovascular disease at the time of plasma sampling, we were
unable to show any association between baseline HSP27 plasma level and
incidence of cardiovascular events (myocardial infarction, ischemic stroke,
or cardiovascular death) during a follow-up period of up to 5.9 years [98].
These results may be explained by the apparently healthy state of the subjects
at study initiation. Therefore, the results may not be applicable to other
populations, such as those with advanced atherosclerosis or ACS.


HEAT-SHOCK PROTEINS IN CARDIOVASCULAR DISEASE

11


Following a global proteomic approach on homogenized carotid samples,
Park et al. [99] have also identified HSP27 as a protein which is overexpressed
in the nearby normal-appearing area compared with the plaque core area.
These authors showed that HSP27 plasma levels were increased in 27 patients
with ACS relative to patients with stable angina (SA), patients with coronary
risk factors, or healthy subjects. They concluded that increased HSP27
plasma levels may reflect the presence of vulnerable plaques. However,
since blood was sampled within 24 h of the onset of ACS, it cannot be
ruled out that the increase in HSP27 levels is secondary to myocardial
ischemia or necrosis, as previously suggested for HSP70 [69].
By immunohistochemistry, we found that both human atherosclerotic
plaques and mammary arteries expressed HSP27 protein [68]. Interestingly,
HSP27 expression, which was mainly present in the cap and media colocalizing with alpha-actin-positive VSMCs, was inversely correlated with markers
of apoptosis [57].

4.1.4. HSP90
In a recent paper, Businaro et al. have shown increased HSP90 serum
levels in patients with atherosclerosis. HSP90 was overexpressed in plaques
from patients with atherosclerosis, potentially contributing to plaque instability by inducing an immune response [81]. In agreement, we have shown an
increased expression of HSP90 in the vulnerable region of human atherosclerotic plaques. Moreover, atherosclerotic plaques with thin caps (< 165 m)
displayed higher total HSP90 levels, suggesting that HSP90 correlates
with events leading to the instability of advanced human atherosclerotic
plaques [41].
As mentioned above, extensive research has been undertaken on circulating HSPs, reported to be either positively (HSP60) or negatively (HSP70)
correlated with the presence and progression of atherosclerosis. HSP27 has
been known for a long time for its antiapoptotic, antioxidant and thus
antiatherogenic functions at a cellular level (discussed in more detail in the
Section 4). However, further studies are needed to clarify the potential role of
circulating HSP27 as a cardiovascular biomarker. More recently, HSP90 has

also been associated with increased atherosclerosis. Only a few studies have
addressed the predictive value of circulating HSPs in large patient cohorts.
There is thus a need for such studies in the future. In relation to HSP
expression in atherosclerotic plaques, it seems that whereas HSP70 and
HSP27 are associated with features of plaque stability, HSP60 and HSP90
display the opposite pattern.


12

MADRIGAL-MATUTE ET AL.

4.2. INDIRECT DETECTION VIA ANTI-HSP ANTIBODIES
Whereas HSP levels in plasma or serum may reflect transient variations in
their secretion or release, detection of antibodies directed against HSPs could
represent a more stable marker of a pathological state. Since HSPs are
basically intracellular proteins, their presence in the extracellular compartment may trigger an immune response and lead to the production of
anti-HSP antibodies. HSPs are highly conserved proteins that are also
good immunogens.

4.2.1. Anti-HSP60/65 Antibodies
HSP65 is one of the most highly conserved proteins: 97% homology
among prokaryotes and more than 70% homology between prokaryotic
and human HSP65 [100]. Heat-shock proteins can promote, as well as
regulate, autoimmunity. Therefore, antimicrobial HSP65 antibodies may
cross-react with self-HSP65 [101]. It is thus difficult to clearly establish
which antigen was originally responsible for the production of anti-HSP60/
65 antibodies (microbial or self-source).
Several studies have suggested an association between antibodies directed
against HSP60/65 (anti-HSP60/65) and atherothrombosis. In their earliest

study, Xu et al. reported increased levels of serum antibodies against HSP65
in patients with carotid atherosclerosis [30]. In a subsequent study from the
same group, HSP65 antibody titres were also increased in plasma of CAD
patients whereas no correlation to established cardiovascular risk factors was
observed. In contrast, HSP65 antibody levels were found to be significantly
lower in AMI, compared to coronary heart disease (CHD) [32]. Following
this study, Zhu et al. observed that anti-human HSP60 was also associated
with the presence and severity of CAD [31]. In a recent study, anti-HSP60
was independently associated with CAD risk, and a combination of high
anti-HSP60, hypertension, and diabetes was shown to be particularly detrimental for CAD risk [102]. The first study testing the potential prognostic
value of HSP antibody levels showed that HSP65 antibody levels were
predictive of 5-year mortality in patients with carotid atherosclerosis [30].
This initial observation was later confirmed in the HOPE study. Among
patients with previous CV events or at high risk of such events, high serum
concentration of antibodies to HSP65 was linked to a higher risk of developing new CV events during a mean follow-up of 4.5 years. This risk was even
higher when combined with high levels of fibrinogen [33].
In another study, the authors observed that high IgA-class anti-HSP60
antibody levels predicted coronary risk, although the effect was modest
without simultaneous occurrence of other classical risk factors [34].