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ADVANCES IN CLINICAL CHEMISTRY VOLUME 54

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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. ⬁ 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: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, 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


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 Montreál, Montreál, 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, Fundacioń Jimeńez Dıáz, Autońoma University, Av. Reyes Cato´licos 2, Madrid, Spain ALESSANDRA COLOMBINI (165), IRCCS Istituto Ortopedico Galeazzi, Milano, Italy JESUS EGIDO (1), Vascular Research Lab, IIS, Fundacioń Jimeńez Dıáz, Autońoma 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, Fundacioń Jimeńez Dıáz, Autońoma 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, Fundacioń Jimeńez Dıáz, Autońoma 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 Montreál, Montreál, 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

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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 Dıáz, *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: [email protected] 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 ApoE/ 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-R/ 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


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]


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


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.


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

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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.


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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.

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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].


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Among potential explanations for the increased levels of antibodies to HSPs observed in plasma, infections might play an important role. Mayr et al. observed that anti-HSP65 antibody titres correlated with human IgA to Chlamydia pneumoniae and with IgG to Helicobacter Pylori [35]. In subsequent studies, high levels of antibodies to human HSP60 and C. pneumoniae were observed in coronary atherosclerosis, showing that their simultaneous presence substantially increased the risk for disease development [36]. Further, Heltai et al. demonstrated associations of high levels of antihHSP60 and anti-C. pneumoniae antibodies with AMI and of the level of anti-HSP65 antibodies with SA [37]. In addition, serum levels of anti-human HSP60 IgG antibody and anti-chlamydial IgM antibody, but not IgG or IgA, were significantly higher in ACS patients than in stable ischemic heart disease patients or controls [38]. Finally, antibodies to mycobacterial HSP65 are associated with elevated levels of coronary calcification and also correlated with H. pylori infection [39]. In relation to the potential prognostic value of HSP60 antibodies commented above, it was observed that a high level of HSP60 IgA could be considered as a risk factor for coronary events, especially when it was present together with C. pneumoniae infection and inflammation [40]. 4.2.2. Anti-HSP70 Antibodies In accordance with studies suggesting that increased levels of circulating HSP70 are correlated with a low risk of coronary artery disease, a publication by Hertz et al. reports that levels of antibodies directed against HSP70 are decreased in patients with CAD (SA and unstable angina) versus control subjects [56]. In contrast, a previous study by Zhu et al. did not find any association between anti-HSP70 IgG seropositivity and the prevalence of CAD despite decreased serum HSP70 levels in these patients [53]. More recently, Zhang et al. [30] reported that lower anti-HSP70 antibody levels are independently associated with a higher risk of ACS. To date, the association between anti-HSP70 levels and coronary artery disease is still unsettled and deserves further investigation. 4.2.3. Anti-HSP27 Antibodies Antibody titres to HSP27 were reported to be elevated during the first 12 h following myocardial infarction in patients with ACS relative to patients with unstable angina [70]. These authors observed that anti-HSP27 antibody concentrations rapidly decrease during the 12–24 h period following MI. Shams et al. also reported increased anti-HSP27 titers in acute conditions, when patients where admitted to hospital with acute chest pain, as compared to patients without any history of CVD [71].

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4.2.4. Anti-HSP90 Antibodies Businaro et al. have recently shown increased HSP90 antibodies in serum from patients with atherosclerosis, implicating HSP90 as a possible autoantigen in the pathogenesis of carotid atherosclerosis [81]. 4.2.5. Limitations to the Use of HSPs and Anti-HSPs as Biomarkers of Atherothrombosis Since expression of inducible HSPs is dependent on a variety of stimuli, their levels may be modulated in different pathological states and even in physiological circumstances such as physical exercise [103,104]. For example, anti-HSP70 antibodies are increased in asthma [105], during HIV infection [106] or in patients with type II diabetes [107]. Also, increased titers of antiHSP27 antibodies have been reported in women with ovarian cancer [108]. Detection of antibodies to bacterial HSPs, such as mHSP65, is not specific of an atherothrombotic state but rather reflects the presence of bacteria that may be independent of CVD. Although the implication of bacteria in atherogenesis has been suggested, further studies are needed to establish a causal link between infection and atherosclerosis [109]. Similarly, circulating HSPs may reflect a secretion by virtually all cell types. In spite of the direct access of arterial wall cells to the blood compartment, the release (or lack of release) of HSPs from focal atherothrombotic lesions may not have sufficient impact on their plasma concentrations to explain the differences observed between patients and subjects free of CVD. Therefore, plasma concentrations of HSPs may, as is the case for C-reactive protein (CRP), reflect a general state of stress or inflammation, not directly linked to atherothrombotic plaque evolution or vulnerability. For example, the source of circulating HSP27 is still under debate since some authors could not detect it in cultured VSMCs whereas it is expressed by the medial layer in human artery samples [110]. However, incubation of human arteries devoid of atherosclerosis leads to a release of HSP27 in the conditioned medium, without trace of necrosis [68]. Macrophages can also be a source of HSP27; in vitro, human macrophages stimulated by estrogen secreted HSP27 via the exosomal pathway [111]. Since most HSPs are inducible, their expression and secretion may be rapidly modulated by an acute event. The above-mentioned work of Zhang et al. is a good example of the transient expression of HSPs [55]. These authors reported that plasma levels of HSP70 may predict risk of ACS which appears contradictory with all studies showing an inverse relation between high circulating levels of HSP70 and increased risk of atherothrombosis. Interpretation of the results should therefore take into account the time of blood sampling. It is likely that the expression of most HSPs,


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including HSP70 and HSP27, is stimulated under acute conditions such as myocardial infarction. HSP27 is a protein particularly easily detectable and identifiable by proteomic approaches. This protein is reported to be differentially expressed in many pathological situations. In fact, differential proteomics allowed identification of HSP27 as a potential marker of neuroblastoma [112], lymph node metastasis [113], chemotherapy response in patients with esophageal adenocarcinoma [114]. More than 120 publications are retrieved by a PubMed search when proteomics is combined with HSP27. Many pathological situations may module HSP expression and secretion. Caution must therefore be exercised before using HSPs as diagnostic or prognostic markers of any given disease.

5. Molecular Mechanisms: Bystanders or Actors? In addition to their well-described chaperoning and antiapoptotic functions, HSPs play different roles depending upon their cellular location. The hypothesis of the Heat-Shock Paradox [1] is based on the idea that extracellular and intracellular HSPs exert different functions. While intracellular HSPs have been reported to downregulate inflammation [115–119], extracellular HSPs have been suggested, for the most part, to be proinflammatory by triggering an immune response [120,121]. This hypothesis may not apply to all HSPs as in the case of HSP27; its atheroprotective role has been shown in both intracellular and extracellular compartments [111]. In physiological conditions, HSPs play their main role of molecular chaperones promoting the correct folding of proteins. In pathological conditions, increased HSP levels may represent a response to modulate inflammation. 5.1. INTRACELLULAR EFFECTS Heat-shock response (HSR) is triggered by a variety of stress conditions that interfere with correct protein folding, leading to accumulation of misfolded or aggregated proteins. HSR is mediated by HSF1, a transcription factor which binds to heat-shock elements (HSE), present in the promoter region of a wide range of target genes, including HSPs [122]. Under normal conditions, HSP70 [80] and HSP90 [123] remain bound to monomeric HSF1 and some other cochaperones (i.e., HSP70–HSP90 Organizing Protein, Hop [124]; HSP70-Interacting Protein, Hip [125]; members of the HSP40/DnaJ HSPs family [126] or p23 [127]) in the cytoplasmic compartment. Under stress conditions, HSF1 is released, translocates to the nucleus, trimerizes and activates the synthesis of HSPs [128–130]. In fact, a negative feedback

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mechanism modulates the stress response, since augmented levels of HSPs are able to sequester the free cytosolic HSF1 and therefore impede its translocation to the nucleus and the subsequent HSP synthesis. The complex interactions between the chaperones, cochaperones, and their client proteins decide the fate of a misfolded protein: either a new folding attempt or ubiquitination and subsequent degradation toward the proteasome pathway. However, in extreme oxidative conditions, ubiquitination can be bypassed [131]. Oxidative stress, inflammation, and apoptosis, among other processes, are involved in the initiation, development, and rupture of atherosclerotic plaques. Implication of HSPs in such events is gaining attention, and a number of studies are coming to light. 5.1.1. HSP60 Contradictory findings about the relationship of HSP60 with oxidative stress have been reported, although papers on this subject are scarce. Lee et al. raised this issue using normal human diploid fibroblasts (HDF) and found that sensitiveness to oxidative stress observed in young HDF cells was dependent upon HSP60 translocation from the mitochondria to the cytosol and subsequent massive activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) occurred [132]. In contrast, the use of a specific siRNA for HSP60 augmented resistance to oxidative stress [133]. As endothelial cells are the primary barrier in atherogenesis, HSP60 levels in endothelial cells were analyzed under stress conditions. HSP60 was expressed in the cytoplasm and on the surface of endothelial cells stressed by high temperature or TNF-a, and these cells were susceptible to complement-dependent lysis by HSP60-specific antibody [134]. Due to its association with infection, several studies analyzed the potential contribution of HSP60 in relation to bacteria/viruses in atherogenesis. Among them, C. pneumoniae was able to induce VSMC proliferation via HSP60 [20]. Also, during cytomegalovirus infection, antibodies against the virus can be generated, potentially cross-reacting with human HSP60 and leading to apoptosis of nonstressed endothelial cells [135]. Taking all these data into account, although the exact intracellular function of HSP60 is not clear, it may be considered as a potential mediator of oxidative stress and inflammation. 5.1.2. HSP70 5.1.2.1. In vitro. HSP70 has been suggested to exert antioxidative effects in cells exposed to H2O2 at the mitochondrial level [136], protecting cells by preserving levels of glutathione (GSH) [137]. Since endothelial damage after exposure to oxygen free radicals is considered to be important in the first steps of atherogenesis, HSP70 upregulation could be protective in these early stages of atherosclerosis. H2O2-induced oxidative stress in HUVECs was


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significantly decreased after mild heat shock, which increased HSP70 mRNA and protein levels, providing delayed protection (up to 20 h) after preconditioning [48]. The protective role of HSP70 against inflammation has been previously reviewed [138]. Indeed, HSP70 involvement in the protection against inflammation in endothelial cells has been suggested [139], and we recently showed that HSP70 upregulation decreased inflammatory markers in macrophages and in VSMCs [41]. In addition, it was shown that an inducer of HSP70 (YC1: 3-(50 -hydroxymethyl-20 -furyl)-1-benzyl-indazol) could effectively prevent VSMC proliferation induced by oxLDL [46]. The relation between HSP70 and apoptosis has been described in detail. APAF1 (apoptosis protease activating factor 1) binds to HSP70 and HSP90, thereby inhibiting the apoptotic signaling pathway [140–143]. HSP70 also inhibits AIF (apoptosis inducing factor) release from mitochondria [144,145]. In relation with CVD, inhibition of HSP70 expression has been shown to stimulate apoptosis and intimal hyperplasia in vein segments ex vivo by upregulating manganese superoxide dismutase (Mn-SOD) activity, an enzyme that protects mitochondria from injury in myocardial ischemia–reperfusion [146]. In vitro experiments performed by Wang et al. demonstrated that increased levels of HSP72, induced either by heat shock or by a nonheat-shock pretreatment, protected human endothelial cells against neutrophil-induced necrosis [147]. A similar study was undertaken in primary cultures of porcine endothelial cells, showing that protection against lipopolysaccharide arsenite-induced apoptosis was not only due to HSP70 upregulation but also to augmented levels of the inhibitor B alpha and decreased NF-B binding activity [148]. Experiments performed in cultured endothelial cells with transient overexpression of HSP70 suggested that HSP70 could be the main factor responsible for the HSR-mediated protection against LPS-induced apoptosis [149]. Similarly, Bernardini et al. showed that the synergistic action of HSP70, HSP32, and VEGF mediated protection against LPS-induced apoptosis in aortic endothelial cells [150]. In conclusion, in vitro studies highlight antioxidant, anti-inflammatory, and antiapoptotic properties of intracellular HSP70. 5.1.2.2. In vivo. HSP70 has been already shown to exert anti-inflammatory functions [151], by inhibiting leukocyte adhesion and recruitment [152] or by decreasing NF-B activation and the number of activated macrophages in a model of brain inflammation of mice overexpressing HSP70 [119]. Other approaches, such as the induction of HSPs by low dose alcohol consumption, have been proposed. The authors suggested that the cardioprotective effect showed in rats was mediated by increased HSP levels, namely HSP70 and HSP32 [153]. We have shown that treatment of ApoE/ mice with 17-AAG/17-DMAG upregulated HSP70 expression in the aortic arch, which was associated with

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attenuated inflammation and a significant reduction in plaque size and lipid content [41]. Recently, in a mouse model of ischemia and oxidative aggression induced by severe heat stress (42 C) for 1 h, HSP72-overexpressing mice displayed higher levels of antioxidant enzymes (glutathione peroxidase and glutathione reductase) [154]. A similar approach was used in a rat model of heat-stroke circulatory shock, in which rats were heat-shocked for 1 h at 43 C. HSP72 expression was assessed 16 and 96 h following this 1 hour preconditioning and showed that HSP72 expression in the striatum peaked at 16 h, paralleled by reduced oxidative stress markers, whereas at 96 h, HSP72 expression was similar to that of basal levels [155]. In vitro and in vivo studies in the field of CVD confirm the well-described cytoprotective role of intracellular HSP70. In atherothrombosis, HSP70 could act as an intracellular shield in various cells, inhibiting different processes involved in the formation, development, and rupture of the atheromatous plaque. 5.1.3. HSP27 5.1.3.1. In vitro. Reactive Oxygen Species (ROS) represent the main trigger of protein misfolding, causing an increase in HSP expression and other protective responses (i.e., antioxidant response) [65]. Perturbations in cytoskeletal structure are one of the major consequences of extensive oxidative stress [156]. Small HSPs (i.e., HSP27) as well as other HSPs, such as HSP90 or the HSP70 families, protect intermediate filaments and microfilaments, thus preventing damage of the centrosome [6,157]. Aggregated and native LDL are both able to induce HSP27 dephosphorylation, leading to its subcellular reorganization to the tip of actin stress fibers and focal adhesion structures [110]. HSP27 was shown long ago to be involved in F-actin assembly [158] and expressed by normal medial SMCs [159]. A possible role of HSP27 in the protection against chronic inflammatory response has been suggested, due to the decreased levels of HSP27 in complicated atherosclerotic plaques [68] or unstable plaques [160]. Using two different VSMC lines, Chen et al. found that HSF-1 silencing by small interfering RNA (siRNA) decreased HSP27 levels. Moreover, the inflammatory response to angiotensin-II (Ang-II) was exacerbated in HSF-1 siRNAtransfected cells, suggesting a role for HSF-1 and HSP27 in the modulation of the inflammatory response [161]. Accordingly, Voegeli et al. later showed in VSMCs that siRNA-targeting HSP27 increased the phosphorylation of the p65 subunit of NF-B induced by Ang-II [66]. In endothelial cells, inhibition of HSP27 phosphorylation via interference with VEGF-induced p38-MAPK signaling led to decreased actin polymerization and cell migration, indicating a potential role of HSP27 and its phosphorylation state in angiogenesis or neovascularization [162]. Recently, contrasting with this


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anti-inflammatory role, it has been reported that HSP27 may participate in switching transient high activation of NF-B into a chronic sustained activation in endothelial cells [163]. Augmented levels of HSP27 are also associated with higher levels of glutathione, which protect the cell against oxidative stress [65]. The wellknown cardioprotective effect of resveratrol (an antioxidant molecule) was further studied by Wang et al. who found that inhibition of human aortic SMC proliferation by resveratrol was accompanied by a significant increase in HSP27 levels [164]. Another widely investigated intracellular effect of HSP27 is its antiapoptotic properties. HSP27 has been shown to inhibit the release of mitochondrial cytochrome c [165,166] and to inactivate cytochrome c by direct binding [167,168]. HSP27 has also been shown to enhance resistance to apoptosis in many other tissues [169–171]. VSMC disappearance is involved in the weakening of the fibrous cap [172], and this loss may come from disruption of extracellular survival signals by proteases, which degrade extracellular matrix components [173,174]. Anoikis, an apoptotic process subsequent to detachment, may contribute to plaque instability in atherosclerosis originating from the loss of extracellular matrix [174]. We showed that siRNA-mediated silencing of HSP27 in VSMCs treated with plasmin led to cell detachment accompanied by apoptotic features [57]. By modulating VSMC apoptosis, HSP27 could favor plaque stability. 5.1.3.2. In vivo. In vivo studies in a model of vascular remodeling induced by surgical injury to the rat carotid artery showed that when carotid levels of HSP27 peaked at 14 days, activation of NF-B started to decrease, suggesting a possible role of HSP27 in modulating inflammation [175]. Using a mouse model overexpressing HSP27 cross-bred with ApoE/ mice fed with a high fat diet, Rayner et al. [111] have reported a reduced progression of atheromatous lesions associated with increased HSP27 in serum, particularly in female mice. These authors attribute this atheroprotective effect to a possible competition of HSP27 for the uptake of atherogenic lipids (i.e., modified LDL) via the Scavenger Receptor A, demonstrated in vitro in macrophages. In addition, they reported that macrophages overexpressing HSP27 displayed reduced cell adhesion and migration, properties that may participate in their atheroprotective role. Recently, the same group showed that extracellular release of HSP27 involved exosomes and confirmed that atheroprotection provided by HSP27 was estrogen-dependent [176]. Thus, the intracellular effects of HSP27 have been extensively studied and include cytoskeletal stablization and protection against oxidative stress, inflammation and apoptosis, supporting its beneficial role in atherosclerosis and CV-related diseases.

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5.1.4. HSP90 5.1.4.1. In vitro. Two main functions of HSP90 are related to oxidative stress; its association with nitric oxide synthases (NOS) [177] and its protection of proteasomes from oxidative insults [178]. NOS synthesize nitric oxide, which has been shown to play a protective role against oxidative stress. In macrophages and VSMCs [179–181], NO inhibits LDL oxidation, a welldescribed proatherogenic factor involved in endothelial dysfunction and foam cell formation [182]. HSP90 enhances eNOS activity [177] and upregulates NO synthesis, thereby inhibiting oxidation of LDL. Further, stimulation of bovine aortic endothelial cells (BAECs) with native and minimally oxidized LDL impaired the calcium-dependent association HSP90/eNOS [74]. This vascular protective feature has been extensively described in vitro, such as, for example, in porcine coronary artery endothelial cells [183]. In BAECs, stimulation with vascular endothelial growth factor (VEGF) promoted the association of Akt and eNOS with HSP90 which represents a scaffold favoring eNOS phosphorylation and subsequent activation by Akt [184]. HSP90 inhibitors (herbamycin or geldanamycin) were also shown to decrease estradiol-dependent eNOS activation in HUVECs [185]. In addition, inhibition of HSP90 with 17-AAG was associated with decreased endothelial migration and angiogenesis. The role of HSP90 in angiogenesis was suggested to be due to its interaction with Akt and eNOS [76]. In addition, HSP90 has been shown to protect the cells against oxidation. Aging and oxidative stress increase the levels of oxidized proteins inside the cells; the proteasome is then in charge of the clearance of damaged proteins that cannot be refolded. HSP90 may act as a shield for the 20S proteasome or multicatalytic proteinase [178,186,187], but to our knowledge there is no published data related to HSP90 and the proteasome in the field of vascular research. However, HSP90 was shown to mediate the phosphorylation of ERK1/2, promoting its nuclear translocation, and thus increasing rat VSMC proliferation in response to oxidative stress [188]. Whether VSMC proliferation is good or bad in atherogenesis is still a matter of debate: whereas it can be considered as a healing process in response to various noxious stimuli favoring plaque stability, it can also participate in arterial wall thickening and stenosis. HSP90 could also modulate monocyte proinflammatory response. It has been recently reported that mactinin, an inducer of monocyte maturation and present in vivo at sites of monocytic activation, associates with HSP90. Mactinin was found to promote production of a number of proinflammatory and chemotactic cytokines for monocytes via the inhibition of HSP90 activity [77]. In contrast, HSP90 has been shown to activate the formation of


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bradykinin on endothelial cells [72] via activation of the prekallikrein– kininogen complex [189]. Bradykinin is a member of the kinin family which is a family of proinflammatory peptides involved in CVD such as atherosclerosis [190]. HSP90 may promote macrophage survival upon stimulation with oxLDL by binding to eukaryotic elongation factor-2 kinase (eEF2 kinase). Indeed, dissociation of this complex by the HSP90 inhibitor, geldanamycin, decreased the viability of macrophages [191]. Antiapoptotic effects of HSP90 were also reported by Lin et al. who observed that increased formation of HSP90/eNOS complexes by adiponectin protected HUVECs from Ang-IIinduced apoptosis [75]. The same group found that apoptosis of HUVECs induced by high glucose, a feature of type II diabetes which is associated with a poor cardiovascular outcome, could be prevented by increased HSP90/ NOS complex formation and recruitment of activated Akt [192]. Similar data regarding the antiapoptotic effects of HSP90 in association with Akt/eNOS and other client proteins in endothelial cells [193–196] and in other cell types involved in the atherosclerotic lesion have been described [197]. 5.1.4.2. In vivo. Confirmation in vivo that HSP90 is a modulator of eNOS activity was shown in a model of newborn piglet pulmonary circulation [198] and in rats subjected to exercise. In the physically trained group, eNOS activity was significantly higher, as was the eNOS/HSP90 association, which could explain in part the dynamic changes in redox status following chronic exercise [199]. The largest amount of literature related to HSP90 and inflammation in CVD has arisen from the use of HSP90 inhibitors because, as mentioned before, using the HSP90 inhibitors derived from geldanamycin led to the upregulation of HSP70 whose anti-inflammatory functions have been widely demonstrated. Bucci et al. described a not very well-documented role of NO in acute inflammation. In a model of inflammatory response to carrageenan in eNOS-deficient mice, the authors suggested that eNOS and its interaction with HSP90 are key factors in the modulation of the vascular inflammatory response [200]. In conclusion, HSP90 is a novel player in atherosclerosis, displaying antiapoptotic functions and a potential protective role against oxidative stress via the stimulation of eNOS activity. Its role in promoting inflammation needs to be clarified. 5.2. EXTRACELLULAR EFFECTS Based on the idea of the heat-shock paradox [1], extracellular HSPs are viewed as a trigger for the immune response due to their potent immunogenic role and their potential function as intercellular signals. Extracellular HSPs

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and their involvement in vascular diseases are gaining attention due to their importance in processes, such as inflammation, related to chronic autoimmune disease. HSPs exit the cells to the extracellular milieu by two different mechanisms; a passive release, which usually follows prior cell damage, and active release, which involves exosomes [176,201,202] or lysosomes-like vesicles [111]. In addition, infections may represent a nonendogenous source of HSPs in the extracellular compartment as mentioned above [35–37,39,40]. 5.2.1. HSP60 5.2.1.1. In vitro. To delineate the potential mechanisms whereby HSP60 is involved in atherogenesis, in vitro experiments were performed. Human serum anti-HSP65 antibodies act as autoantibodies reacting with HSP60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity [203]. In addition to endothelial cells, macrophages expressing HSP60 can be lysed by autoantibodies against HSP65/60. Since macrophage death contributes to enlargement of the necrotic core, this effect can contribute to atherosclerotic plaque instability [204]. Further studies analyzed the potential effect of HSP60 on the mechanisms associated with atherothrombosis. Chlamydial HSP60 induced expression in vascular cells of matrix metalloproteases [205], key proteases promoting plaque rupture. In agreement, HSP60 was able to induce proinflammatory cytokines [21], possibly via the CD14 receptor [206], although it could be dependent on the cell type since in adult rat cardiomyocytes, extracellular HSP60 enhanced apoptosis via TLR-4 and completely independently of TLR-2 and CD14 [16]. 5.2.1.2. In vivo. To address the role of HSP60/HSP65 in atherosclerosis, in vivo studies were performed. In the earliest study, normocholesterolemic rabbits were immunized with different antigens with/without adjuvant. Atherosclerotic lesions in the intima of the aortic arch were found to have developed only in those animals immunized with antigenic preparations containing HSP65, either as part of the whole mycobacteria or purified recombinant HSP65, in spite of their normal serum cholesterol levels. Further, combined immunization with HSP65-containing material and a cholesterol-rich diet promoted the development of significantly more severe atherosclerosis [207]. C. pneumoniae infection combined with a cholesterol-rich diet induced the development of autoantibodies against mHSP60 and this was associated with the enhanced development of lipid lesions [22]. In agreement, anti-HSP60 autoantibodies isolated from blood of patients with CHD and injected into the tail vein of apolipoprotein E-deficient mice induced the formation of atherosclerotic lesions. Further, administration of a specific mouse


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monoclonal antibody to HSP60 effectively induced atherogenesis in apolipoprotein E-deficient mice [208]. Interestingly, high levels of HSP60 autoantibodies are considered to be an important prothrombotic factor that may impact cardiovascular disease [209]. Overall, extracellular HSP60 has been shown to exert a clear proatherogenic function. It has a close relationship with infection, probably due to the high homology between human and microbial HSP60/65, triggering the immune response and thus favoring a chronic state of inflammation. 5.2.2. HSP70 5.2.2.1. In vitro. Several studies have shown that HSPs, including HSP70 [210], are present in the extracellular milieu and that HSP70 can be exchanged between cells [211]. Johnson et al. reported that exogenous HSP70 was not internalized but remained associated with the cell surface of serumdeprived arterial SMCs, protecting the cells against noxious stimuli [212]. Also, HSP70 supplementation was shown to limit cytotoxic effects induced by serum starvation in aortic SMCs [47]. Oxidized LDLs were shown to induce HSP70 secretion by macrophages, which in turn stimulated IL-1 beta and IL-12 expression by naı¨ve macrophages. HSP70 could therefore have a paracrine effect. OxLDL also stimulated HSP70 production by SMCs and nonconfluent proliferating endothelial cells [45,213]. In human monocytes, the dual character of HSP70 was shown to be due to its extracellular proinflammatory function via activation of NF-B signaling and inducing proinflammatory cytokines TNF-a, IL-1b, and IL-6 [49]. Extracellular HSP70 was shown to induce proliferation of endothelial cells and tube formation in vitro and also promoted calcification of VSMCs. HSP70 may bind and activate Matrix Gla Protein (MGP), an inhibitor of Bone Morphogenetic Protein-4 (BMP-4). The authors hypothesized that HSP70 could be a key factor in unbalancing the positive feedback between BMP inhibitors and BMP signaling [50]. 5.2.2.2. In vivo. In a rat model of balloon-injury, immunization with HSP70 accelerated the intimal thickening. Increased release of HSP70 within the damaged artery may produce an anti-HSP70 response and contribute to a proinflammatory state, thus suggesting that HSP70 may be a potent immunogen and explaining the accelerated neointimal formation. Also, T-cells could migrate to HSP70-rich areas and promote SMC migration and leukocyte chemoattraction via cytokine production [51]. HSP70 release into the extracellular millieu results from various stress conditions. Extracellular HSP70 seems to trigger an immune response leading to increased levels of inflammation, proliferation, and calcification. However, some studies have provided information about the antiapoptotic

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role of HSP70. Therefore, further studies addressing the extracellular roles of HSP70 are neccesary to better understand its participation in atherothrombotic processes.

5.2.3. HSP27 Extracellular HSP27 has been suggested to play an anti-inflammatory role in atherosclerosis [111]. For example, HSP27 has been shown to induce IL-10 production by human monocytes and is thus proposed to represent an antiinflammatory stimulus [61]. Exogenous addition of recombinant HSP27 was able to reduce spontaneous apoptosis of human neutrophils isolated from peripheral blood, without any modification of cytokine secretion (TNF-a, IL-10, IL-12) by these cells [67]. In contrast, HSP70 was reported to accelerate neutrophil apoptosis, but also reduced oxidative stress and stimulated anti-inflammatory cytokine production [62]. Rayner et al. suggested the development of therapies based on supplementation by exogenous HSP27 which could interact with SR-A, leading to reduced cholesterol uptake into the vessel. They conclude suggesting that ‘‘Studies testing these innovative formulations of recombinant HSP27 are underway both pre- and postinduction of atherosclerosis.’’ [111].

5.2.4. HSP90 A possible role for HSP90 in modulating the extracellular activation of ERK1/2 in response to oxidative stress was proposed by Liao et al. in rat VSMCs [78]. Extracellular HSP90 has been recently implicated in the induction of the proatherogenic cytokine IL-8 via TLR-4 in human aortic SMCs, in addition to upregulation of IL-6, activation of ERK and p38 MAPK [79]. Also, the proinflammatory activation of the kinin-forming cascade by HSP90 in HUVECs may be extracellular in pathologic circumstances in which HSP90 is highly expressed or secreted [72]. Hence, extracellular HSP90, whose role in CVD has not been fully described, appears to be proatherogenic. It is important to note that an intense debate exists concerning whether experimental conditions have been carefully followed using recombinant HSPs as an in vitro model for extracellular HSPs [214]. LPS contamination, other PAMPs (Pathogen-associated molecular patterns) or molecules bound to and/or chaperoned by HSPs might be responsible for some of their immunogenic responses. Thus, caution should be exercised when analyzing data coming from the use of recombinant HSPs.


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6. HSP as Therapeutic Targets in CVD/Atherothrombosis Since several HSPs are considered to be protective in various stress conditions, modulation of their expression has been considered as a potential therapeutic strategy in cardiovascular disease. Other potentially deleterious HSPs have been the target of immunization. It was not until the late 1980s that HSPs were examined as potential restorative molecules in cardiovascular diseases [215–217]. 6.1. HSP INDUCTION 6.1.1. Thermal Preconditioning Thermal preconditioning was used in a rat model of atherosclerosis in which induction of HSPs (including HSP72 in the media) reduced not only the neointimal thickening but also the inflammatory infiltration (MCP1) and oxidative stress (p22-phox) [218]. Moreover, it has been found that moderate heating inhibited in-stent restenosis and neointimal hyperplasia in an atherosclerotic model in rabbits, and this beneficial effect has been associated with HSP70 overexpression [219]. These studies showed the importance of global HSP upregulation as a potential therapeutic strategy in CVD. Indeed, thermal therapy has been shown to improve vascular endothelial function in CVD patients [220,221]. This approach has the advantages of simplicity and feasibility. However, this strategy could potentially enhance both pro- and antiatherosclerogenic HSPs, as well as other beneficial or harmful mechanisms, which should be evaluated in detail. In any case, thermal therapy may only produce acute overexpression of certain HSPs, and its impact on a chronic disease such as atherosclerosis may thus be rather limited. 6.1.2. Gene Therapy/Recombinant Proteins New approaches to overexpress HSPs such as gene therapy were carried out in mice by Rayner et al. [111]. Interestingly, mice overexpressing HSP27 (ApoE/ HSP27o/e) showed significantly decreased lesion size relative to ApoE/ mice with normal levels of HSP27. It should be noted that female littermates displayed smaller atherosclerotic lesions than males, accompanied with a 10-fold higher HSP27 concentration in serum. The release of HSP27 was suggested to be mediated by acetylated LDL (acLDL) and estrogens, which could explain the differences found between female and male littermates. This was accompanied by a marked reduction in the secretion of the proinflammatory cytokine IL-1b and an increase in the extracellular levels of the anti-inflammatory cytokine IL-10. Moreover, HSP27 acts competitively by binding to scavenger receptor A, inhibiting thereby the

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engulfment of acLDL by macrophages, a step required to acquire the foam cell phenotype. In a model of rat heart transplantation, HSP70 gene overexpression caused an improvement in ventricular and endothelial function [222] and in cardioprotection against ischemia–reperfusion injury [223]. Recombinant HSPs have also been used as therapeutic compounds in a model of mice exposed to cigarette smoke [224]. Matsumoto et al. injected HSP70 intravenously into mice previously exposed to cigarette smoke, known to induce intimal thickening after arterial wall damage [225,226]. HSP70-injected mice showed a decreased intimal thickening compared to controls (saline-injected mice); the authors hypothesized that prevention of intimal thickening could be mediated by the inhibition of ERK activation. Overall, these data highlight the beneficial effects of specific upregulation of atheroprotective HSPs limiting atherosclerotic lesion development and intimal thickening associated with endothelial protection. However, suppression of proatherogenic HSP by other approaches, such as siRNAs, could also be used. In any case, to translate this approach into a clinical scenario, several parameters should be evaluated such as the safety of gene therapy, and the bio-distribution and stability of recombinant proteins or siRNAs. 6.1.3. Pharmacological Compounds These promising results obtained by thermal preconditioning and gene therapy have led to the development of pharmacological compounds affecting HSP expression for treatment of different diseases, among them atherothrombosis, because of the hazards of submitting patients to a heat shock and the difficulties of using approaches such as gene therapy in humans. In 2003, Connolly et al. found that stimulation by Herbamycin A resulting in the production of HSP27, but not of HSP70, was responsible for decreasing intimal hyperplasia in a rat carotid ballon injury model [227]. Another pharmacological compound called YC-1, an inducer of HSP70 [46], inhibited neointima formation in a model of balloon-injured rat carotid artery [228], thus suggesting a possible therapeutic use in treatment of vascular diseases. In an ApoE/ mouse model fed a high cholesterol diet, HSP70 upregulation by using low doses of HSP90 inhibitors (17-AAG/17-DMAG) decreased inflammatory markers and also reduced aortic atherosclerotic plaque size as well as its lipid content [41]. This approach using HSP90 inhibitors has been largely used in other fields such as cancer, where it is now entering clinical trials [229–233]. Finally, statins, the most widely administered pharmacological compounds used in the treatment of CVD around the world, have shown among their numerous pleiotropic effects, the ability to increase the expression of HSPs, such as HSP90, HSP70, HSP32 (HO-1), and to activate the HSP70 promoter via the HSE, thus activating heat-shock gene transcription [234].


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These latest positive data for the treatment of atherosclerosis in animal models raise the question of their use in the treatment of other CVD [235]. Thus, specific upregulation of atheroprotective HSP by pharmacological compounds could be a promising approach for the treatment of atherothrombosis. However, one important issue that should be considered is the dosage used since some of these compounds have proapoptotic actions that could have deleterious effects in terms of plaque stability. Data emanating from clinical trials using these pharmacological compounds should be examined in detail to highlight the pros and cons of their use in humans. For example, the dosage and toxicity of 17-AAG gained from phase I clinical trials in cancer patients should facilitate the evaluation of HSP90 inhibitors in non-neoplasic disorders, such as cardiovascular diseases.

6.2. IMMUNE THERAPY As stated earlier in this review, extracellular HSPs are likely to influence atherosclerosis by triggering an immune response associated with a proinflammatory state. HSP60, due to its homology with bacterial HSP65, has given rise to a number of studies in which the immune response initiated by HSP60/ HSP65 was investigated. HSP60 is now considered to be a major autoantigen in atherosclerosis [236,237]. Several approaches based on HSP60/65 immunization have been attempted in experimental models of atherosclerosis. In 2002, Harats et al. [238] tested the effect of inducing mucosal tolerance to HSP60 in an atherosclerosis model accelerated by Mycobacterium tuberculosis. LDL receptor-deficient mice (LDL-R/) were given HSP65 orally, and two different models of accelerated atherosclerosis were tested, i.e. a high fat diet or immunization with heat killed M. tuberculosis. In both proatherosclerotic scenarios, oral administration of HSP65 limited plaque formation. A possible explanation could be a cellular response to HSP65 leading to increased Th2 cytokine IL-4 in HSP65 immunized mice, which was previously described to possess a protective role in atherosclerosis [239]. A very similar paper by Maron et al. reported a decrease in atherosclerotic plaque formation in the aortic arch of LDL-R/ mice having a received nasal mucosal administration of HSP65. They hypothesized that this could be related to higher levels of IL-10 and to a greater Th2 type humoral (IgG1) response in animals which received nasal administration [240]. A third study along the same line showed that oral tolerance to a small immunogenic peptide of HSP60 or to that of full-length HSP60 both reduced the plaque size by 80%. They proposed that an increase in the number of CD4þ CD25þ Foxp3þ Tregs cells could participate in the augmented production of TGF-b and IL-10 and finally contribute to decrease inflammation in the atherosclerotic area [241]. The specificity of this response is

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shown by the fact that oral administration of a highly conserved sequence of HSP70 could not reduce atherosclerosis. In 2009 Xiong et al. showed, in wild-type rabbits fed with a high cholesterol diet, that nasal immunization with HSP65 could attenuate atherosclerosis and reduce lipid levels [242]. Similar attempts have been made in the field of other chronic inflammatory diseases such as rheumatoid arthritis [243]. Immunization against proatherogenic HSPs could open new therapeutic avenues in cardiovascular disease although similar concerns to those commented above for gene therapy should be taken into account. The extracellular roles of the different HSPs should be further studied in order to conceive new strategies aiming at modulating HSP circulating levels (supplementation or increased clearance depending on their beneficial or deleterious role in atherogenesis). 7. Conclusions HSP expression is modulated by different stimuli involved in all steps of atherogenesis including oxidative stress, apoptosis, proteolytic aggression, or inflammation. This could be reflected by changes in HSP protein levels in the extracellular compartment, potentially impacting their plasma levels. 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 field of cardiovascular disease, although several HSPs have been suggested to be markers of the pathology, caution must be exercised since their expression may be transient, in response to an acute event, such as myocardial infarction for example, or reflecting a chronic state of inflammation or oxidative stress rather than a specific risk of future cardiovascular event. The biological roles of intracellular HSPs in atherogenesis are potentially very important since most of them protect the cells of the vasculature against various noxious stimuli. The emerging role of extracellular HSPs appears to be linked to CVD. As a consequence, therapeutic efforts for treating CVD such as atherosclerosis should take into account modulations in the recognition, binding, and internalization of HSPs. HSPs could then be considered as potential therapeutic targets to reinforce vascular defenses and delay or avoid clinical complications associated with atherothrombosis. ACKNOWLEDGMENTS The authors’ work have been supported by the Leducq Foundation, the Spanish Ministerio de Ciencia y Tecnologıá (SAF 2010-21852), CAM (S2006/GEN-0247), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035; RD06/0014/0008), and European Network (HEALTH F2-2008-200647) and EUS2008-03565. The authors would like to thank Dr. Mary Osborne-Pellegrin for editing this chapter.


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POLYAMINES IN CANCER Edwin A. Paz,* Jenaro Garcia-Huidobro,† and Natalia A. Ignatenko‡,1 *Cancer Biology Interdisciplinary Graduate Program, Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA † Biochemistry and Molecular and Cellular Biology Graduate Program, University of Arizona, Tucson, Arizona, USA ‡ Department of Cell Biology and Anatomy, Arizona Cancer Center, Tucson, Arizona, USA

1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of Polyamine Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Deregulation of Polyamines in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Deregulation of ODC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Deregulation of Other Polyamine Metabolic Genes . . . . . . . . . . . . . . . . . . . . . . . . 5. Genetic Variability in ODC Affecting Carcinogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. EIF5A and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Chemoprevention Strategies Within Polyamine Pathway. . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Suppression of ODC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Induction of SAT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Evaluation of Antitumorigenic Properties of DFMO and NSAIDS in Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Combination Chemoprevention Strategies in Humans. . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46 46 47 50 50 53 54 56 60 60 60 62 62 63

Abbreviations COX DAX DFMO FAP 1

cyclooxygenase diamine exporter a-difluoromethylornithine familial adenomatous polyposis

Corresponding author: Natalia A. Ignatenko, e-mail: [email protected] 45

0065-2423/11 $35.00 DOI: 10.1016/B978-0-12-387025-4.00002-9


46 NSAID ODC SAT1

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nonsteroidal anti-inflammatory drug ornithine decarboxylase spermidine/spermine N1-acetyltransferase

1. Abstract Polyamines are organic cations shown to control gene expression at the transcriptional, posttranscriptional, and translational levels. Multiple cellular oncogenic pathways are involved in regulation of transcription and translation of polyamine-metabolizing enzymes. As a consequence of genetic alterations, expression levels and activities of polyamine-metabolizing enzymes change rapidly during tumorigenesis resulting in high levels of polyamines in many human epithelial tumors. This review summarizes the mechanisms of polyamine regulation by canonical tumor suppressor genes and oncogenes, as well as the role of eukaryotic initiation factor 5A (EIF5A) in cancer. The importance of research utilizing pharmaceutical inhibitors and cancer chemopreventive strategies targeting the polyamine pathway is also discussed.

2. Introduction The natural polyamines (putrescine, spermidine, and spermine) are ubiquitous low molecular weight aliphatic amines that play multifunctional roles in cell growth, differentiation, and survival. Polyamines are unique because of their flexible polycationic nature that allows them to bind electrostatically to negatively charged macromolecules including nucleic acids, acidic proteins, and membranes [1]. Polyamines regulate important cellular processes, including cell proliferation and viability. Genetic evidence indicates that polyamines are required for optimal growth of bacteria [2] and are essential for aerobic growth in yeast [3]. The cellular functions of polyamines also include intestinal mucosal maturation and cell migration [4,5]. Polyamines have been shown to influence transcription, RNA stabilization, and translational frameshifting [6,7]. Complex regulation controls intracellular polyamine pool sizes through combined actions of de novo synthesis, retroconversion, degradation, efflux, and uptake of polyamines. A significant number of reviews have summarized the functions and regulation of polyamines and their potential role in cancer disease [8–14]. The present review is focused on the regulation of polyaminemetabolizing enzymes during neoplastic transformation, evaluation of agents targeting the polyamine pathway to prevent or reverse neoplastic growth, and polyamine-mediated functions of the EIF5A in cells.

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3. Overview of Polyamine Regulation Polyamines are synthesized through the action of the enzyme ornithine decarboxylase (ODC). ODC is the first enzyme in polyamine biosynthesis which catalyzes the formation of putrescine from ornithine. Putrescine is subsequently converted into spermidine through the actions of S-adenosylmethionine decarboxylase 1 (AMD1) and spermidine synthase. Spermidine is then converted into spermine by AMD1 and spermine synthase. Important catabolic effectors maintain polyamine homeostasis as well. Spermidine/ spermine N1-acetyltransferase (SAT1) adds terminal acetyl groups to spermidine and spermine that subsequently promotes the export of acetylated polyamines. Spermine oxidase (SMO) converts spermine to spermidine. Acetylpolyamine oxidase (PAOX) also aids in polyamine homeostasis by converting acetylated spermidine and spermine back to putrescine and spermidine, respectively (Fig. 1). Multiple studies demonstrated that ODC is essential metabolic effector required for normal development in mammals. ODC gene knockout is lethal in murine embryos 3.5 days after fertilization [15]. ODC protein levels in cells are regulated by an ODC inhibitory protein called ornithine decarboxylase antizyme (OAZ) [16,17]. The regulation by antizyme involves the þ 1 frameshift of the translating ribosomes on the antizyme mRNA upon accumulation of polyamines, resulting in increased translation of the antizyme protein [7,16]. Full-length functional antizyme acts as a noncompetitive inhibitor of ODC and enhances ubiquitin-independent ODC degradation by the 26S proteasome [16]. In addition, antizyme increases polyamine efflux and suppresses polyamine uptake, thus decreasing the intracellular polyamine pool [18,19]. Antizyme is regulated by the antizyme inhibitor (AZIN1) which is a homolog of ODC but lacks the decarboxylation activity [20]. AZIN1 sequesters the intracellular pool of antizyme which causes an increase in levels of ODC protein, ODC activity, and polyamine levels. ODC is also regulated through transcriptional activation in response to hormones, growth factors, and tumor promoters via the response elements in the ODC gene promoter. Response elements in the ODC gene include cAMP response element, CAAT, and LSF sequences, activator proteins 1 and 2 (AP1 and AP2) sites, specificity protein (SP1) binding sites, and a TATA box (reviewed in Ref. [21]). Of special interest, the ODC gene promoter contains three Enhancer (E)-boxes (CACGTG) that are binding sites for the MYC, MAX, MAD, and MNT transcription factors [22]. AMD1 converts putrescine into the higher polyamines by producing the aminopropyl donor decarboxylated S-adenozylmethionine (dcSAM). The activity of AMD1 is highly regulated at the level of transcription, translation, and protein turnover [23]. The AMD1 gene contains a number

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48

Ornithine Antizyme DFMO

Degradation

ODC

Putrescine AMD1, spermidine synthase

NH3+

SAT1, PAOX

NH3+

Spermidine +

AMD1, spermine synthase

NH2+

H3N

NH3+

SAT1, PAOX, SMO

Spermine +

H 3N

NH2+ NH2+

NH3+

FIG. 1. Polyamine structure and metabolism. Enzymes are shown in purple squares, except for ODC shown in blue oval (ODC, ornithine decarboxylase; AMD1, S-adenosylmethionine decarboxylase 1; SAT1, spermidine/spermine N1-acetyltransferase; SMO, spermine oxidase; PAOX, acetylpolyamine oxidase). DFMO, a-difluoromethylornithine, an irreversible inhibitor of ODC.

of binding sites for different transcription factors including a spermidine response element. Putrescine can activate mammalian AMD1 by enhancing the production of the processed form of the enzyme and improving its catalytic activity [23]. Protein ubiquitination has also been shown to regulate AMD1 turnover [24,25]. Mammalian spermidine and spermine synthase are regulated by the availability of putrescine or spermidine and dcSAM. The recent studies by Forshell et al. [26] described the induction of the spermidine synthase gene transcription by the c-MYC oncogene in a murine model of B-cell lymphoma. The putative c-MYC binding site is associated with canonic E-boxes located upstream and downstream of exon 1 in the spermidine synthase gene. A key polyamine catabolic enzyme SAT1 is highly inducible in response to a variety of stimuli including elevated polyamine levels, synthetic


49

polyamine analogs, toxins, hormones, cytokines, heat-shock, ischemia– reperfusion injuries, and other stresses (reviewed in Ref. [27]). The regulation of SAT1 occurs via an activation of transcription, mRNA processing, mRNA translation, and protein turnover [27–30]. The SAT1 gene contains the binding sites for common regulators of gene expression such as SP1 and AP1, CCAAT/enhancer-binding protein b (C/EBPb), cAMPresponse-element-binding protein (CREB), nuclear factor kB (NF-kB), and peroxisome proliferator-activated receptors (PPARs) transcription factors. SAT1 also contains a polyamine-response element (PRE) which binds polyamines and polyamine-responsive transcription factors [31]. The posttranscriptional regulation of SAT1 involves regulation of mRNA stability, alternative splicing of SAT1 mRNA, translational regulation of SAT1 synthesis, and stabilization of SAT1 protein, which are reviewed elsewhere [32]. Translational regulation through upstream open reading frames (uORFs) plays a significant role in polyamine metabolism. The uORFs precede main coding sequences in mRNA structures of the polyamine metabolic genes ODC, OAZ, AZIN1, AMD1, spermine synthase, and SAT1, and influence the efficiency of polyamine-responsive regulation of their translation [33]. Polyamine transport mechanisms mediate intracellular polyamine homeostasis. Polyamines are imported into the cells from extracellular sources such as luminal bacteria and diet [34]. Belting et al. [35] have shown that the polyamine transport system involves endocytic pathways with cell surface heparin sulfate proteoglycans as a possible vehicle for polyamine uptake. The more recent study by Roy et al. [36] indicates that polyamine uptake occurs via a dynamin-dependent and a clathrinindependent endocytic uptake, and that a plasma membrane lipid raft protein caveolin-1 is a potential regulator of the polyamine pathway. The significance of caveolae-dependent endocytosis in polyamine uptake has also been studied in vivo using genetically engineered mice [37]. This study has shown that putrescine uptake in the intestinal tissue is regulated by a nitric oxide (NOS2)-dependent mechanism; when the intracellular polyamine content is low, uptake is regulated by a glycosylated heavy chain of the cationic amino acid transporter SLC3A2. Polyamines are exported out of mammalian cells in the acetylated form by a diamine exporter (DAX) [38]. Recently, Uemura et al. [39] described the molecular mechanism of polyamine export as a polyamine/arginine exchange reaction which involves the amino acid transporter SLC3A2. In this study, the polyamine catabolic enzyme SAT1 was found to be colocalized with SLC3A2, indicating that this efflux system facilitates excretion of acetylated polyamines. The tight regulation imposed on intracellular polyamine levels underscores the importance of these molecules for optimal cellular growth.

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4. Deregulation of Polyamines in Cancer Cancer is a major human health problem worldwide and is the second leading cause of death in the United States [40]. Over the past 40 years, significant progress has been made in understanding the molecular basis of cancer. It has been established that cancer is a neoplastic condition resulting from genetic changes that control the proliferation, maturation, metastatic behavior, and senescence of cells. These genetic changes are diverse in nature and can involve loss or gain of gene function. Tumorigenesis is a multistep process involving the acquisition of chromosomal abnormalities and development of mutations in different genes, which provide cancer cells selective growth advantages and cooperativity that leads to dysregulation of cell growth at multiple levels [41]. During tumorigenesis, cancers evolve mechanisms to deregulate polyamine metabolism. Canonical oncogenes and tumor suppressors have been shown to affect polyamine metabolism in transformed cells. Therefore, it is not surprising that deregulation of polyamine levels appear to play an essential role during tumorigenesis. The pleiotropic effects observed by alterations of the polyamines might be explained by their ability to regulate specific aspects of gene expression and protein translation.

4.1. DEREGULATION OF ODC 4.1.1. Role of ODC in Neoplastic Transformation ODC, a key polyamine biosynthesis enzyme, plays a major role in the process of carcinogenesis. An association between high levels of polyamines and cancer was first reported in the late 1960s by Russell and Snyder [42], who measured high levels of ODC activity in regenerated rat liver and in several human cancers. ODC deregulation occurs in response to a variety of oncogenic stimuli, including cancer promoters 12-O-tetradecanoylphorbol13-acetate and asbestos [43,44]. ODC is also regulated by androgens and ODC gene overexpression has been observed in human prostate cancer [45]. Numerous studies have documented changes in ODC regulation during carcinogenesis at the level of transcription, translation, and protein degradation (reviewed in Refs. [14,46]). The causative role of ODC in carcinogenesis has been observed upon overexpression of ODC by transfection in vitro and in vivo in transgenic mice (reviewed in Refs. [47,48]). Overexpression of the intracellular noncompetitive ODC inhibitor antizyme in transgenic mice has been shown to reduce carcinogenesis [49–51]. These genetic studies corroborate epidemiological studies that have documented elevated ODC expression and activity during colon tumorigenesis [52,53].


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4.1.2. Role of APC and c-MYC in ODC Induction Major oncogenic pathways are involved in regulation of ODC transcription and translation. We, and others, have shown that the ODC gene is regulated by the WNT signaling pathway, one of the major cascade governing epithelial development [54,55]. The adenomatous polyposis coli (APC) tumor suppression gene is a component of the WNT cascade, and is mutated or lost in the germ line of individuals with familial adenomatous polyposis (FAP), a heritable form of colon cancer [56,57]. The APC gene is also mutated in almost 90% of human colon cancers and 30% of melanoma skin cancers. We have shown that ODC gene expression and polyamine contents are elevated in the intestinal tissue, and a specific inhibitor of ODC suppresses intestinal carcinogenesis in the ApcMin/þ mouse, an animal model for FAP [58]. In this model, mutated APC led to a decrease in antizyme and SAT1 expression, indicating that APC controls the polyamine metabolic pathway in the colon. APC mutation is an early event in colon carcinogenesis, and is, therefore, considered to be an initiating event. In carcinogenic tissue, loss of APC and dysregulation of the WNT pathway leads to increased expression of the c-MYC oncogene. APC mutation prevents GSK-3b phosphorylation of b-catenin which would normally lead to its proteosomal degradation. Mutated APC leads to stabilization and accumulation of b-catenin in the nucleus where it forms a complex with TCF/LEF (T-cell factor/lymphoidenhancing factor) transcription factor [59,60]. The heterodimer complex binds to the specific regions in the promoter of c-MYC and other growthrelated genes and alters gene expression profiles [54,59]. c-MYC is a transcription factor that is required for the proliferation of normal cells, and its over expression can lead to uncontrolled growth and cancer [61]. During APC-dependent carcinogenesis, c-MYC activation affects transcription of ODC by binding to E-boxes or MYC-binding regions in the promoter region of the gene. In vivo studies using mouse model with a conditional deletion of c-MYC in the intestinal and colonic mucosa have shown that c-MYC plays an important role in the development of intestinal tumors with activating mutations in the APC gene via increased proliferation and suppressed apoptosis (Fig. 2) [62]. Both c-MYC and ODC present an attractive target for pharmacological inhibition. Transient c-MYC inactivation can be an effective therapy for certain cancers as demonstrated in the transgenic mouse model of osteogenic sarcoma [63]. Selective transcriptional silencing of the c-MYC promoter has been achieved using a porphyrin analog that binds to the G-quadruplex DNA structure within the c-MYC promoter [64,65].

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52 APCmut

c-MYC β-Catenin TCF ODC

DFMO

Polyamines

Proliferation

Apoptosis

Neoplasia

FIG. 2. APC-mediated effects on polyamine biosynthesis and tumorigenesis. APC mutation (APCmut) leads to proliferation and inhibition of apoptosis via upregulation of ornithine decarboxylase (ODC) activity. An irreversible inhibitor of ODC a-difluoromethylornithine (DFMO) is shown in red.

4.1.3. Role of Mutant K-RAS in ODC Induction Activating mutations of K-RAS oncogene have been observed in approximately 30% of human colon tumors [66]. Constitutive RAS activation by the substitution of amino acid residues at various positions is frequently found in human invasive cancers [66]. Oncogenic RAS influences different cellular processes via signaling pathways involving the Akt proto-oncogene, the serine/threonine protein kinase Raf-1, and the Rho small GTPase family [67]. The RAS gene family, consisting of H-RAS, K-RAS, and N-RAS genes, can also increase reactive oxygen species (ROS) levels via the RAS-mitogenactivated protein kinase kinase (MAPKK) and mitogen-activated protein kinase (MAPK) pathways [68]. Transformation of NIH 3T3 cells with the human H-RAS oncogene causes significant increase in ODC expression and polyamine contents [69]. Mutant K-RAS oncogene also increases cellular polyamine levels by increasing ODC enzyme activity [70]. Detailed analysis


53

of the RAS pathway using RAS partial-loss-of-function mutants and inhibitors showed that the regulation of ODC transcription occurs via Raf/Mek/ Erk pathway, while the PI3-kinase pathway mediates ODC translation [71]. 4.2. DEREGULATION OF OTHER POLYAMINE METABOLIC GENES 4.2.1. Deregulation of SAT1 During Tumorigenesis SAT1 is a key polyamine catabolic enzyme and an important regulatory step in maintaining polyamine content. In vitro experiments and genetic manipulations with SAT1 expression in experimental animal models confirmed SAT1 functions to facilitate polyamine efflux from cells [72,73]. Because tumorigenesis is associated with increased polyamine biosynthesis and polyamine levels, SAT1 expression and enzyme activity are regulated in response to these alterations via an increase in transcription, translation, and suppression of protein turnover (reviewed in Ref. [32]). Elevated SAT1 enzyme activity results in a sustained increase in acetylated polyamine levels that facilitates their excretion and degradation via the SAT1/PAOX pathway. Cancer can develop mechanisms to prevent the induction of SAT1 in order to maintain high polyamine levels. Particularly, it has been shown that the RAS pathway is implicated in SAT1 regulation via decreased expression of PPARg, a member of the nuclear hormone receptor family and an important regulator of cell proliferation and differentiation [74]. Specifically, an activated K-RAS suppressed SAT1 expression by a mechanism involving the PPARg response element 2 (PPRE-2) located at þ48 bp relative to the transcription start site of the SAT1 gene. SAT1 expression can be restored by transient expression of the PPARg protein, by treatment with the PPARg ligand ciglitazone or with the MEK1/2 inhibitor PD98059, suggesting that MAPKs are involved in the regulation of SAT1 expression by PPARg [75]. RAS is therefore a negative regulator of SAT1 that results in high polyamine levels. SAT1 expression can also influence tumorigenesis. Studies performed using genetically engineered mouse models showed that overexpression of the mouse Sat1 gene is associated with increased intestinal tumorigenesis and the knockout of Sat1 reversed this progression [76]. 4.2.2. Tumorigenesis and Polyamine Transport Experimental and clinical studies provide evidence that the polyamine transport system is hampered during neoplastic transformation. The study by Nilsson et al. [77] shows that polyamine uptake plays a significant role in the progression to lymphomagenesis. In the intestinal tract, it has been shown that

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depletion of exogenous polyamines which are normally obtained from either microbial flora activity or food, using antibiotics or polyamine-deficient diet, resulted in a compensatory increase in polyamine uptake [78,79]. Bachrach and Seiler have shown that various oncogenes can increase polyamine uptake as well [80]. The upregulation of polyamine uptake in cells expressing an activated K-RAS occurs via SRC-dependent caveolin-1 phosphorylation at tyrosine residue 14 and modulation of the urokinase plasminogen activated receptor [36,81]. Mutant K-RAS oncogene also acts to suppress the polyamine export system via negative regulation of SLC3A2 protein [39]. Roy et al. [36] provided evidence for the negative regulation of polyamine uptake by caveolin-1, which can function as a tumor suppressor [82].

5. Genetic Variability in ODC Affecting Carcinogenesis The ODC gene has three E-boxes in the region from 400 to þ 400 bp relative to the start of transcription. The heterodimer transcriptional activator c-MYC/MAX or transcriptional repressor MAD1/MAX binds to these E-box elements to regulate the transcription level of ODC gene in proliferative cells [21,22]. ODC promoter activity is influenced by cooperative interactions involving these neighboring E-boxes. A single nucleotide polymorphism (SNP) exists in intron 1 of the human ODC gene between two E-boxes with a G/A variation located 316 nucleotides downstream of the transcriptional start site [83,84]. Since ODC expression has been linked to cancer development, individuals with specific sets of SNPs may exhibit an increased predisposition for colon polyp development. According to the model of colon tumorigenesis proposed by Fearon and Vogelstein in 1990, colorectal cancer develops as a multistep process that involves the progression from normal mucosa to small and large adenomas leading to invasive cancer and metastasis [41,85,86]. Removal of adenomas is associated with a lower risk of colorectal cancer; however, recurrence is common [87,88]. It is also recognized that not all colon polyps will progress to invasive cancer [89,90]. The relationship between the ODC polymorphism and the risk of adenoma recurrence has been assessed in participants of a large randomized, double-blind wheat bran fiber colon cancer; prevention trial at the Arizona Cancer Center in Tucson, Arizona [90,91]. A substantial and statistically significant effect of the ODC polymorphism on risk of adenoma recurrence has been found on this trial in aspirin users with a strong correlation between recurrent polyp size and risk of colon cancer. This suggests a modification to the accepted model of the step-wide molecular process involved in the original polyp-carcinoma sequence. The actual model proposed by Martinez et al. [90,91] predicts that carcinogenesis

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resulting from certain initiating events (e.g., chemical carcinogens and genetic rick factor), and the responses to chemoprevention strategies are influenced by genetic variability among individuals (Fig. 3). Specifically, the þ 316 nucleotides SNP in the ODC promoter displayed functional consequences for E-box activation (e.g., c-MYC) and repression (e.g., MAD1), and association with recurrence of colon polyps. This association, between the genetic variability affecting ODC expression and the risk of colorectal adenoma recurrence, has since been confirmed in a number of studies, particularly for aspirin users [92–94]. A positive [95] and negative [96] association has been shown between sporadic colorectal cancer and genetic variability in ODC gene. The risk of adenoma recurrence was found lower in AA homozygous individuals who reported taking aspirin [91]. Barry et al. [93] in the Aspirin/ Folate Polyp Prevention Study found no association between ODC genotype and colorectal cancer recurrence in individuals randomly assigned to placebo

Epithelium Genetic and epigenetic changes (APC loss or mutate)

Genetic variability (SNP)

Small adenomas

Large adenomas

ODC 316 SNP A-allele

G-allele

Recurrence (cancer risk stratification)

No recurrence

Small adenomas

Large adenomas

Cancer

FIG. 3. Association between genetic variability and G316A ODC SNP. Genetic and epigenetic changes are associated with cancer progression where the final outcome is influenced by the original polyp sizes and genetic variability of G316A ODC SNP.

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or aspirin treatment (81 or 325 mg daily). This study also found that ODC genotype modified the effect of aspirin on adenoma risk. Although aspirin treatment had no protective effect among subjects with a GG genotype, it was associated with statistically significant reduced risks of any adenoma among subjects with at least one A allele. Hubner et al. [94] in a United Kingdom Colorectal Adenoma Prevention trial reported that the rare 316AA homozygotes had reduced colorectal adenoma recurrence risk, with an additional lower recurrence risk if aspirin was administered. At the same time, a recent study from Hughes et al. reported no correlation between ODC 316 SNP with colorectal adenomas in the Czech Republic case control series with no data on aspirin [96]. A population-based study of 400 of stages I–III colorectal cancer cases from the California Irvine Gene-Environment Study of Familial Colorectal Cancer reported that the specific outcome of colorectal cancer patients is dependent on the ODC 316 SNP genotype with the higher colorectal cancer-specific risk of death for individuals with ODC GA/ AA genotypes [95]. In summary, the ODC A-allele may be protective for colon adenoma recurrence and detrimental for survival after colon cancer diagnosis. However, it is unlikely to play a major role in susceptibility to colorectal cancer development.

6. EIF5A and Cancer Polyamine metabolism has been linked to protein synthesis through the unique posttranslational modification of the universal translation factor EIF5A. EIF5A is an essential gene that encodes a protein that is approximately 17 kDa in size and is conserved among eukaryotes and archaebacteria [97]. EIF5A is the only known protein that is modified at a specific lysine residue to yield hypusine. Although EIF5A has been extensively studied, its biological role has not been fully elucidated. Early studies using a methionylpuromycin assay suggested a translation initiation role for EIF5A, as it promoted first peptide formation [98]. However, subsequent reports have argued against a general translation initiation role due to the finding demonstrating the depletion of EIF5A in yeast only causing a minor decrease in protein synthesis [99]. Although EIF5A is still under intense investigation, its growth altering potential has provided yet another link between polyamines, eukaryotic translation, and cancer. Hypusination of EIF5A requires the polyamine biosynthetic pathway. It is mediated by two enzymes: deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). EIF5A hypusination is initiated by DHS transferring an aminobutyl moiety from the polyamine spermidine onto a specific EIF5A


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lysine residue. Deoxyhypusine is then hydroxylated by DOHH to yield hypusine. The importance of EIF5A hypusination is reflected through findings showing that mutating the lysine residue required for the posttranslational modification leads to growth defects in Saccharomyces cerevisae [100]. The importance of spermidine levels for EIF5A modification has also been highlighted through a study of yeast polyamine auxotrophs that were grown in low levels of spermidine. It was shown that yeast polyamine auxotrophs use a large proportion of the limited supply of spermidine for hypusination, emphasizing its importance for supporting growth [101]. Studies have also shown the vital importance of both DHS and DOHH for cell proliferation and are reviewed elsewhere [102]. The essential nature of hypusination in EIF5A reflects its importance for normal cell growth and in order to fully understand its effects, the identification of more functional binding partners may increase our understanding of its function during tumorigenesis. A recent study conducted by Lebska et al. demonstrated that maize EIF5A (ZmEIF5A) is phosphorylated at Serine 2 (Ser2) by casein kinase 2 (CK2) resulting in nuclear shuttling [103]. A proteomic approach in which CK2 binding partners were identified through mass spectrometry resulted in identification of ZmEIF5A as a novel potential substrate. Through an elegant approach, a thrombin cleavage assay was used to confirm that Ser2 of maize EIF5A is indeed phosphorylated. Phosphorylation of ZmEIF5A ultimately resulted in nuclear translocation. Confocal microscopy confirmed that replacing Ser2 with aspartic acid increased the ratio of nuclear ZmEIF5A. Although mammalian cells may not possess the Ser2 phosphorylation site, the possibility that the temporal–spatial control of EIF5A during growth is regulated by posttranslational modifications may provide insight about its biological role. One such report suggested that hypusinated EIF5A localizes mainly in the cytoplasm [104]. Although EIF5A’s role during translation has been established, further studies focusing on the nuclear presence of EIF5A could provide novel information regarding its oncogenic potential. EIF5A is also a translation factor that shares structural homology to elongation factor P (EF-P) of eubacteria. It has been suggested that EIF5A stabilizes tRNAi Met for peptidyl transferase center (PTC) positioning in the ribosome [105]. A recent report by Lee et al. [106] demonstrated a crystal structure of Thermus thermophilus EF-P bound to the 70S ribosome which promoted the alignment of charged initiator tRNA to the P site of the ribosome. EF-P was shown to bind between the P site and the E site, and appears to encourage conformational changes of a mature ribosome. The specific location of EF-P is thought to promote first peptide bond formation by properly positioning the tRNA molecule in the P site through noncovalent interactions to the tRNA backbone. Interestingly, a hypusine homology model was rendered that demonstrated the potential of a hypothetical

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hypusinated EF-P extending the hypusine chain near the PTC where the catalytic mechanism for peptide bond formation resides. This model suggests that hypusination might facilitate first peptide bond formation by promoting a thermodynamically favorable microenvironment in the PTC for protein synthesis. A biochemical model where hypusination is demonstrated to affect the activation entropy for peptide bond formation in an 80S PTC would strengthen the T. thermophilus hypusine homology model hypothesis. The findings that EIF5A is structurally similar to EF-P provide evidence for a direct role of EIF5A in elongation. GST pulldown assays have previously demonstrated that GST-EIF5A copurifies with eEF2, the 60S ribosomal protein P0, and a small ribosomal protein S5 [107], and demonstrated that EIF5A associates mostly with translationally active ribosomes. A recent paper published by Saini et al. [108] provided further evidence that EIF5A is a universally conserved elongation factor. An EIF5A degron mutant (tif51a-td) was constructed and grown in both permissive and nonpermissive temperatures to determine the effects of this translation factor on growth, protein production, and polysome profiles. Growth of the degron mutant at the nonpermissive temperature reduced the growth rate of the tif51a-td yeast strain, highlighting the essential requirement of EIF5A. Depletion of EIF5A also resulted in diminished levels of [35S]methionine protein incorporation. Sucrose gradient analysis further showed that the tif51a-td strain did not appear to have translation initiation defects, but that the retention of polysomes and slower ribosomal run-off suggested EIF5A plays a role during the elongation step of translation [108]. This finding was further corroborated by a report that mammalian EIF5A promotes translation elongation and stress granule formation [109]. In humans, there are two isoforms of EIF5A that share considerable sequence homology of approximately 84% at the amino acid level. Although the EIF5A1 isoform is constitutively expressed, EIF5A2 appears to be restricted to specific tissues. Using a human multiple-tissue expression array, it was demonstrated that EIF5A2 is expressed most highly in testis, adult neuronal tissues, and the colon adenocarcinoma cell line SW480 [110]. Consistent with this finding, other cancer cell lines, including ovarian cell line UACC-1598, displayed higher levels of EIF5A2 expression [111]. Multiple studies have now shown that EIF5A2 and polyamine-metabolizing enzymes are overexpressed in many human cancer cell models (Fig. 4). EIF5A2 is located near chromosome 3q26, which is a site of multiple amplification events in many solid tumors. Overexpressed EIF5A2 in NIH3T3 cells has been shown to increase colony formation in soft agar, while hydroxyurea treatment of the ovarian cell line UACC 1598 resulted in reduction of both EIF5A2 copy number and cell growth rate [112]. In an in vivo RNAi screen to identify tumor suppressors in hepatocellular

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Arginine

Ornithine

APCmut ODC

ODC RNA

ODC Putrescine

MYC

SAM

EIF5A2

dcSAM

EIF5A2 RNA

EIF5A2 (lysine 50)

AMD1

AMD1 RNA

AMD1

SRM

SRM RNA

SRM Spermidine

EIF5A2 (hypusine 50)

Oncogenic potential

FIG. 4. Role of eukaryotic translation initiation factor 5A2 (EIF5A2) in tumorigenesis and its relationship to polyamine biochemical pathway. APC and c-MYC target genes in the polyamine pathway to increase the intracellular polyamine levels. Spermidine, which is the substrate to the posttranslational modification of EIF5A2, converts a lysine residue to the novel amino acid hypusine in the EIF5A protein. Two unique chromosomal loci encode EIF5A1 and EIF5A2 isoforms.

carcinomas, exportin 4 (XPO4) was shown to genetically interact with EIF5A2 [113]. By utilizing a subcutaneous tumor growth assay, it was shown that EIF5A2 expression resulted in tumor formation of p53/;Myc liver progenitor cells. An MTT assay further demonstrated that knockdown of EIF5A2 led to decreased growth of a human hepatoma cell line with an XPO4 deletion (SK-Hep1). Furthermore, a colony formation assay of SKHep1 cells verified that EIF5A2 knockdown resulted in inhibition of proliferation. Previous reports indicated that XPO4 mediates export of EIF5A1 and Smad3 from the nucleus [113–115]. In fact, Zender et al. also demonstrated that XPO4 deletion resulted in EIF5A1 and EIF5A2 nuclear accumulation. Interestingly, EIF5A1 expression did not appear to induce higher subcutaneous tumor growth in mice. Although both translation factors appear to share large sequence homology, there is a biologically significant amount of difference leading to contrasting phenotypes. Future studies

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characterizing the role of EIF5A during cancer progression could provide novel information that could be exploited for novel drugs targeting EIF5A.

7. Chemoprevention Strategies Within Polyamine Pathway 7.1. SUPPRESSION OF ODC a-Difluoromethylornithine (DFMO) is the most widely used inhibitor of polyamine metabolism. DFMO was developed over three decades ago and is now used in both experimental studies [11] and clinical applications, including cancer prevention [116] and hair removal [117]. Racemic DFMO was shown to be an enzyme-activated, irreversible inhibitor of ODC [118]. Biochemical mechanisms for inactivation of ODC by DFMO have been proposed [119] and genetic studies have corroborated features of this model [120]. Upon treatment with DFMO, there are significant cytostatic effects on eukaryotic cells. The ability of DFMO-treated cells to regain proliferation upon addition of exogenous putrescine suggests a clear functional role of polyamines during cellular growth and proliferation. In vivo, DFMO treatment inhibited dietary arginine-induced colon carcinogenesis and reduced adenoma dysplasia grade in ApcMin/þ mice [121]. DFMO has been evaluated as a cancer therapeutic agent, but the results were generally not encouraging [122]. More recent experimental studies suggest that the failure of DFMO as a single agent, in either the cancer prevention or treatment setting, is due to compensatory mechanisms affecting polyamine transport and catabolism [8]. The experimental evidence suggests that DFMO can selectively reduce viability of colon cancer cells expressing mutant K-RAS oncogene [123]. This finding was corroborated by Lan et al. [124], who reported reduction of the growth of chemically induced skin tumors in mice by DFMO. DFMO also caused regression of existing spontaneous tumors in transgenic mice over expressing both ODC and mutant H-RAS in keratinocytes. DFMO was also found to be effective in suppressing the mutant K-RAS-mediated colon tumorigenesis in vivo since it reversed the changes in experimental cell migration and cell–cell communication genes [70]. 7.2. INDUCTION OF SAT1 Several structurally diverse nonsteroidal anti-inflammatory drugs (NSAIDs) act to promote transcription of the SAT1 by both COX-dependent and COX-independent mechanisms. Laboratory work demonstrated that sulindac sulfone [125] and aspirin [126] induce SAT1 transcription. The sulfone derivative of sulindac, which recognizes a unique DNA sequence

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in the SAT1 promoter, activates SAT1 gene to induce its transcription. The mechanism of activation involves the induction of PPARg protein and its binding to one of the peroxisome proliferators-activated receptors response elements (PPREs) in the SAT1 gene, namely PPRE-2. PPRE-2 is located þ 48 bp relative to the transcription start site; PPRE-1, which is located 323 bp relative to the start site, is not required for the induction of SAT1. Aspirin, which is structurally unrelated to sulindac, induces SAT1 transcription via a distinct mechanism involving NF-kΒ sites in the SAT1 promoter [126]. Studies in animal models confirm the in vitro findings and indicate that sulindac can suppress ODC enzyme activity in addition to activating SAT1 gene expression [127]. Effects on polyamine metabolism are a component of the COX-independent mechanisms by which some NSAIDS inhibit colon carcinogenesis, as depicted in Fig. 5. SAT1 expression is also induced by some synthetic polyamine analogs. Polyamine analogs are compounds that can interfere with polyamine functions in cells causing inhibition of tumor growth (for review, see [9]). Three classes of synthetic polyamine analogs have been developed: symmetrically substituted, asymmetrically substituted, and conformationally restricted analogs. These classes demonstrate multiple biological activities and improved targeting

APCmut K-RASmut

DFMO

ODC

Polyamines

Invasion

SAT1

Sulindac

Proliferation

Neoplasia

FIG. 5. Combination chemoprevention strategies targeting polyamine metabolism in cancer. Mutant APC (APCmut) and activated K-RAS (K-RAS mut) lead to upregulation of ODC and suppression of SAT1, respectively, which increases the polyamine concentration. Combined action of an irreversible ODC inhibitor DFMO and nonsteroidal anti-inflammatory drug sulindac will prevent polyamine-dependent cell proliferation and invasion and suppress neoplasia.

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abilities with small changes in molecular structure [9]. Mechanisms by which polyamine analogs exert their intracellular effects include the induction of catabolism and the displacement of natural polyamines from functional sites related to the transcriptional regulation of genes [128]. Polyamine analogs with the N-terminal alkyl group, such as symmetrically substituted analog BENSpm, cause significant and prolong activation of SAT1 enzyme activity by binding to the active site of the SAT1 protein at the polyamine-binding region causing its accumulation [29]. Other analogs, such as PG-11047, which belong to a class of conformationally restricted compounds, increase the SAT1 transcript level and deplete cellular spermidine leading to total polyamine depletion in colon cancer cells [129]. Since the synthetic polyamine analogs have antitumor activity, they are currently under clinical evaluation, alone and in combination with established drugs, such as DFMO, for the targeted depletion of polyamine levels and suppression of tumor growth. 7.3. EVALUATION OF ANTITUMORIGENIC PROPERTIES OF DFMO AND NSAIDS IN ANIMAL MODELS The molecular targets for antitumor activity of NSAIDs sulindac and celecoxib within the polyamine pathway have been evaluated using the experimental animal models including ApcMin/þ mice. Specifically, sulindac increased steady state RNA levels and enzymatic activity of the polyamine catabolic enzyme SAT1 [127]. Sulindac also decreased the activity of the biosynthetic enzyme ODC, but not AMD1. The effectiveness of sulindac to suppress intestinal carcinogenesis was partially abrogated by dietary putrescine [127]. Since high concentrations of putrescine can be found in certain dietary components, it may be advantageous to restrict dietary putrescine consumption in patients undergoing treatment with sulindac. The combination of DFMO with sulindac, a nonselective inhibitor of both COX-1 and COX-2, or celecoxib, a selective COX-2 inhibitor, was additive in suppressing tumorigenesis in ApcMin/þ mice [130]. At the same time, the combination of DFMO with sulindac was more effective in reducing the intestinal polyamine contents and incidence of high-grade intestinal adenomas than combination of DFMO with celecoxib [130]. 7.4. COMBINATION CHEMOPREVENTION STRATEGIES IN HUMANS The described experimental studies set a stage for the clinical trials of DFMO and sulindac. In 2000, the University of California-Irvine and the University of Arizona started a collaboration to conduct a prospective, randomized clinical trial of 150 mg sulindac, combined with 500 mg DFMO, daily for 3 years versus placebo [11,131]. The phase III clinical


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trial assessed the toxicity of this combination. The low nontoxic dose of DFMO used in this study was sufficient to suppress ODC in human rectal mucosa [122,132]. The major endpoint of the trial was colon polyp recurrence. This randomized trial showed markedly reduced recurrence of all adenomas (70% decrease), advanced adenomas (92% decrease), and recurrence of more than one adenoma (95% decrease) [116]. Putrescine and spermidine levels of rectal mucosal biopsies were also markedly reduced in the active intervention arm after both 12 and 36 months of treatment; PGE2 levels were unaffected. All toxicities, including clinical, audiologic, and cardiovascular affects, were not significantly different between the treatment and placebo groups. Larger and longer term studies will evaluate the absolute risk of this treatment [133]. The polyamine transport system presents another chemoprevention target for lowering the polyamine contents in cancer cells. A group of lipophilic polyamine analogs has been recently developed that inhibit the cellular polyamine uptake system and significantly increase the effectiveness of polyamine depletion when combined with DFMO [134]. Overall, polyamine metabolism offers an attractive direction for future advances in treatment of cancer risk factors. ACKNOWLEDGMENTS Authors would like to express their gratitude to Dr. Eugene W. Gerner, Department of Cell Biology and Anatomy, Arizona Cancer Center, for providing the helpful comments to this chapter and Rhiannon McGuire, MBA, Arizona Cancer Center, for her assistance in editing this chapter. Authors apologize to the contributors to this field, whose work is not cited due to space constraints.

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ACQUIRED HEMOPHILIA A Massimo Franchini*,1 and Giuseppe Lippi† *Department of Pathology and Laboratory Medicine, Immunohematology and Transfusion Center, University Hospital of Parma, Parma, Italy † Clinical Chemistry Laboratory, Department of Pathology and Laboratory Medicine, University Hospital of Parma, Parma, Italy

1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Laboratory Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Epidemiology and Clinical Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Treatment of Bleeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Inhibitor Eradication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 71 72 73 73 75 75 77 78 79

1. Abstract Acquired hemophilia A is a rare but often life-threatening hemorrhagic disorder characterized by an autoantibody directed against coagulation factor VIII. Fifty per cent of cases are idiopathic whereas the remaining 50% are associated with pregnancy, autoimmune conditions, malignancies and drugs. In this review the actual knowledge on diagnostic and therapeutic aspects of this disease will be summarized.

1

Corresponding author: Massimo Franchini, e-mail: [email protected] 71

0065-2423/11 $35.00 DOI: 10.1016/B978-0-12-387025-4.00003-0


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2. Introduction Acquired hemophilia A (AHA) is a rare disorder caused by circulating autoantibodies against factor VIII (FVIII) that inhibit the coagulation cascade. The majority of bleeding episodes in AHA are subcutaneous, but bleeds affecting gastrointestinal, lung, intracranial, and retroperitoneal sites, among others, also occur, and these are associated with increased mortality. This chapter will review the pathogenesis, diagnosis, epidemiology, and treatment of AHA.

3. Pathogenesis AHA is a rare disorder with an incidence of between 1.3 and 1.5 cases per million population annually [1,2]. AHA results in a defect in the coagulation cascade caused by autoantibodies against FVIII. These autoantibodies are oligoclonal and are generally of the IgG4 subclass, although IgG1 and IgG2 antibodies are also observed [3]. There are a number of differences between the FVIII autoantibodies in AHA and the alloantibodies that some individuals with hemophilia develop following treatment with recombinant or plasma-derived FVIII. Autoantibodies to FVIII predominantly target the A2, A3, and C2 domains of the molecule, and the majority of individuals (62%, 13/21) only have antibodies targeting one of these domains [4]. In contrast, 85% (29/34) of individuals with alloantibodies have antibodies that target multiple domains of the FVIII molecule [4]. Antibodies against FVIII are characterized as showing either type 1 (> 98% inhibition of FVIII and a linear relationship between the log of residual FVIII activity and antibody concentration) or type 2 kinetics (incomplete inactivation of FVIII at maximum available antibody concentrations and nonlinear kinetics) [5]. It has been reported that alloantibodies and autoantibodies exhibit type 1 and type 2 kinetics, respectively [6]. However, the clinical picture may be more complex, as type 1 kinetics were demonstrated in 5/7 (71%) of patients with AHA and an underlying pathology (post-pregnancy, drug-related, diabetes, hypertension, gastric ulcer, bullous pemphigoid and idiopathic thrombocytopenic purpura), while type 2 antibody kinetics were observed in 9/9 (100%) of individuals with AHA and no associated pathology [3]. The A2, A3, and C2 domains of FVIII that are targeted by autoantibodies are functionally important. The C2 domain contains the binding site for von Willebrand factor (VWF), which is vital for maintaining circulating levels of FVIII in the plasma [7]. Following vascular injury, FXa or thrombin induces the dissociation of FVIII and VWF. This dissociation allows FVIIIa to bind FIXa via the A2 and A3 domains of FVIII [8]. In addition, phospholipid

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binding sites on the C2 domain of FVIII are involved in directing the complex to the membrane surface of activated platelets [8]. The membranebound complex of FVIII and FIXa then acts to induce thrombin formation via the proteolytic activation of FX [7]. There are at least two potential mechanisms by which autoantibodies can inhibit FVIII activity. Catalytic/hydrolyzing IgG targeting FVIII is observed in around 50% of individuals with AHA [9]. Catalytic antibodies appear to have a functional effect, as changes in the levels of IgG-mediated hydrolysis over time were associated with changes in inhibitory activity in 9/12 individuals with AHA [9,10]. A second mechanism may involve direct inhibition of functional activity, such as antibodies that bind to the C2 region and interfere with phospholipid binding [11,12]. Antibodies inhibiting FIXa binding have also been described [13].

4. Laboratory Diagnosis Cases of unexplained bleeding with an isolated prolonged activated partial thromboplastin time (aPTT) suggest a diagnosis of AHA [14]. An aPTT mixing test, in which equal volumes of test and normal plasma are mixed, enables factor deficiency to be distinguished from the presence of inhibitors. FVIII inhibitors are time- and temperature-dependent, so prolongation should occur after incubation (1–2 h) at 37 C [14]. Other potential reasons for a prolonged aPTT, such as heparin contamination and lupus anticoagulants, should be ruled out before confirming the presence of FVIII inhibitors (Fig. 1) [15]. AHA is then confirmed by demonstrating low FVIII activity and raised inhibitor titer [1,14]. The inhibitor titer is measured by the Bethesda test, which assays the ability of the test versus control plasma to reduce a known amount of FVIII activity. The Nijmegen modification of this test reflects changes to the buffer in the test sample and the use of immunodepleted factor VIII deficient plasma in the control sample to increase discrimination between positive and negative samples [16]. The Bethesda assay classifies inhibitors as high >5 BU/ mL and low 65 years [1,17]. Thus, the median age of affected individuals was 72 years (range 13–19) in interim analysis of 319 individuals from the European Acquired Hemophilia registry (EACH2) [18] and 78 years (range 2–98) in a UK surveillance study of 154 patients [1]. These data do not appear to support a sex association, with between 43% and 53% of cases occurring in men

74

FRANCHINI AND LIPPI Sudden onset of bleeding episode Negative personal and family hemorrhagic history Confirmed prolonged APTT with normal PT

Exclude presence of heparin

Mixing study patient plasma/normal plasma Incubation 2 h at 37 °C

APTT mix normal

APTT mix prolonged Exclude lupus anticoaguant

Intrinsic pathway clotting factors deficiency

FVIII reduction FVIII inhibitor titration

Diagnosis of AHA

FIG. 1. Laboratory diagnosis of AHA.

[1,18]. There are a number of cases in younger females (often aged between 20 and 30 years), which reflects the development of postpartum inhibitors [19]. AHA is extremely rare in children, with only one case reported in the UK during a 2-year surveillance study [1]. Over half of all cases of AHA are idiopathic (Table 1) [1,18]. Underlying clinical conditions observed in the remaining individuals include autoimmune disorders, solid tumors, lymphoproliferative disorders, and pregnancy (Table 1). In addition, cases of AHA associated with penicillin, sulfonamides, chloramphenicol, the anticonvulsant diphenylhydantoin, and the immunomodulatory agents fludarabine and interferon-a have been reported [20]. Although AHA and congenital hemophilia A both result in deficiency in FVIII, bleed pathology is quite different. In AHA, the majority of bleeds (80%) are subcutaneous, while intramuscular (45%) or gastrointestinal (approximately 25%) bleeds are also common [1]. Joint bleeds were less common (approximately 2–8%) [1,18]. In contrast, joints are the most common site of bleeding episodes in individuals with congenital hemophilia [21]. Fatal bleeds were observed in 13/143 (9.1%) individuals with AHA [1]. Gastrointestinal and lung bleeds were associated with early deaths (within the first week of presentation), whereas intracranial and retroperitoneal bleeds resulted in later deaths (between 2 and 21 weeks) [1].


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TABLE 1 AHA AND UNDERLYING CLINICAL CONDITIONS [1,18]

Underlying diagnosis, % (n) None Pregnancy Autoimmune disorder Hematological malignancy History of malignancy Active malignancy Monoclonal gammopathy Other (drug association, infection) Dermatologic

EACH2 (N ¼ 319)

UK surveillance study (N ¼ 150)

55.0 (176) 8.5 (27) 7.2 (23) 2.2 (7) 3.1 (10) 4.4 (14) 2.5 (8) 19.4 (62) Not reported

63.3 (95) 2.0 (3) 16.6 (25) Not reported Not reported 14.6 (22) Not reported Not reported 3.3 (5)

4.2. TREATMENT AHA is a rare disorder with few comparative trials to guide treatment. There have been a number of published treatment guidelines, including a recent consensus guideline published in 2009, which are largely in agreement with treatment options [14,15,22–24]. Because AHA is a rare disorder and is associated with bleeding episodes in individuals who may not have a history of bleeding, initial presentation is often to non-specialist clinicians. Therefore, clinicians seeing a patient with an acute bleed and an unexplained prolonged aPTT test, particularly in elderly individuals or postpartum women, should consider AHA as a diagnosis and ensure that their patient receives specialist care at a hemophilia center [14]. The choice of treatment in AHA is dependent on the site of bleeding and the underlying disorder, although the two main goals are to control any bleeds and eradicate inhibitor antibodies to reduce the risk of further bleeds [14,15,23]. Treatment of the underlying disease may be appropriate [15,23], as there have been reports of inhibitors disappearing following successful treatment of malignancies [25,26].

4.3. TREATMENT OF BLEEDS Subcutaneous bleeds may not need immediate hemostatic treatment but should be closely observed; however, retroperitoneal, retropharyngeal, muscle, intracranial, gastrointestinal, pulmonary, or postoperative bleeds, as well as severe hematuria or bleeds from multiple sites, should be treated [14].

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Clinicians should take into account the risk and benefits of hemostatic treatment, particularly as many individuals with AHA are elderly with risk factors for thromboembolic events. 4.3.1. Bypassing Agents Recombinant-activated FVII (rFVIIa) and activated prothrombin complex concentrates (aPCCs) are recommended as first-line treatment for severe bleeds [14,15,23]. They are effective in 80–90% of bleeds; however, there are no comparative trials of rFVIIa and aPPC to demonstrate superior efficacy for either product [14,15,23]. rFVIIa is a recombinant protein that is manufactured to exclude the risk of human pathogen transmission, whereas aPCC is a plasma-derived product. The potential for human pathogen transmission with aPPC, although small, has led to suggestions that rFVIIa may be more appropriate in certain populations (e.g., postpartum women) [14]. EACH2 registry data suggest that rFVIIa is the more commonly used of the bypassing agents (Table 2) [27]. If one bypassing agent fails, individuals should be switched to the alternative agent [14]. Therapy with both rFVIIa and aPPC in alternating sequence has been reported in a limited number of individuals with congenital hemophilia, but no case reports exist in AHA [14]. Therefore, if this approach is used, it should be limited to limb-or life-threatening bleeds [14]. Thromboembolic complications with bypassing agents appear to be more common in AHA than in congenital hemophilia, reflecting the risk factors present in the generally older AHA population [14]. 4.3.2. Other Options Other hemostatic treatments such as human plasma-derived or recombinant FVIII or desmopressin may be used in individuals with low-titer inhibitors and minor bleeding episodes if bypassing agents are unavailable [14].

TABLE 2 HEMOSTATIC TREATMENT USED IN THE EACH2 REGISTRY [26] Hemostatic treatment rFVIIa aPCC Human FVIII Desmopressin Multiple therapies Not specified

Bleed treatment, n (%) (N ¼ 294) 143 (49) 72 (24) 35 (12) 8 (3) 13 (4) 23 (8)


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If a bleed is refractory to first-line treatment, the removal of the inhibitor antibody by plasmapheresis or immunoadsorption may be appropriate [14].

4.4. INHIBITOR ERADICATION Two first-line options for inhibitor eradication are a corticosteroid or a corticosteroid plus cyclophosphamide (Table 3) [14]. If no response is observed after 4–6 weeks, rituximab alone or with a corticosteroid is a second-line option [14]. Some experts recommend a more conservative approach to inhibitor eradication in some cases, for example postpartum AHA, in which inhibitors may resolve spontaneously [28]. However, other guidelines favor inhibitor eradication in all cases because of the continuing risk of fatal bleeds as long as inhibitor antibodies remain and the inability of clinical factors such as the patient’s presenting characteristics or inhibitor titer to predict bleed risk [14,23]. If inhibitor eradication is attempted in women of child-bearing age, cytotoxic agents should be avoided due to the potential risk of infertility [14,15]. Immunosuppressive therapy for inhibitor eradication can be associated with serious side effects such as neutropenia-related infections and sepsis, particularly in elderly individuals. Sepsis was reported in 37/112 (33%) of AHA patients with non-bleeding-related morbidity and caused death in 11% of cases [1]. In a smaller case series of 17 elderly patients aged > 76 years, 13 died, of which bleeding caused three deaths (23%) and infection related to immunosuppressive treatment caused five deaths (38%) [29]. Another case series including 13 patients aged 70 years or older demonstrated better outcomes (overall 31% mortality) [30]. However, treatment toxicity (infections

TABLE 3 AHA TREATMENT RECOMMENDATIONS Anti-hemorrhagic treatment – First line – Alternative treatmenta – Second line Inhibitor eradication – First line – Second line – Alternative treatment – Not useful

rFVIIa, aPCC hFVIII, DDAVP Immunoadsorption or plasmapheresis Corticosteroids cyclophosphamide Rituximab corticosteroids Azathioprine, vincristine, mycophenolate, cyclosporine Intravenous immunoglobulin

rFVIIa, recombinant activated factor VII; aPCC, activated prothrombin complex concentrates; hFVIII, human plasma-derived FVIII; DDAVP, desmopressin. a Low-titer inhibitors and minor bleeding.

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and hematological toxicity) was observed in 5/7 individuals on classical immunosuppression and 2/6 individuals receiving rituximab [30]. Rituximab is a monoclonal antibody that targets the CD20 antigen expressed on the cell surface of B cells [31]. Rituximab treatment depletes peripheral B cells and was initially developed for the treatment of CD20þ non-Hodgkin’s lymphomas [31], but has also been used in other disorders associated with autoantibodies [32]. Two literature reviews have reported efficacy rates of 80–90% in AHA, with no reported cases of opportunistic infection [32,33]. Data from the EACH2 registry did not show an advantage for rituximab (mostly administered with other agents) over steroids alone or steroids plus cytotoxic agents (complete response in 63%, 60%, and 84% of cases, respectively) [34]. Adverse events were seen in a higher proportion of individuals treated with steroids and cytotoxic agents (39%) than those treated with rituximab (26%) or steroids alone (25%) [34]. Although initial data are promising, they reflect uncontrolled studies and case reports in individuals also receiving other agents for inhibitor eradication, so rituximab is currently recommended as second-line therapy until further efficacy and long-term safety data become available [14]. Successful cases of inhibitor eradication after failure of first line-therapy have been reported with the use of cyclosporine, azathioprine, vincristine, or mycophenolate. Finally, current evidence does not support the use of intravenous immunoglobulin for inhibitor eradication in AHA, either as a single agent or in combination with steroids and cytotoxic agents [14,15].

5. Conclusions AHA is a rare bleeding disorder that can lead to fatal bleeds. The disease is characterized by autoantibodies against FVIII that inhibit the coagulation cascade by at least two potential mechanisms: catalysis of FVIII and functional blockade of FVIII interactions. Treatment needs to address both the control of acute bleeds and eradication of the inhibitor autoantibody to reduce the risk of further bleeding. Bypassing agents offer a clear first-line treatment choice for acute bleeds. Of the two bypassing agents, rFVIIIa excludes the risk of any transfer of human viral pathogens, which is a small risk in plasma-derived products. Inhibitor eradication with corticosteroids with or without cytotoxic agents is associated with high rates of morbidity and mortality due to neutropenia and infections. Rituximab is a promising agent for inhibitor eradication, with the potential for less treatment-related adverse side effects than classical immunosuppressive treatments; however, further studies are required to confirm initial efficacy and safety results.


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ACKNOWLEDGMENTS The authors take full responsibility for this chapter but are grateful to Mike Lappin, PhD, of Watermeadow Medical (supported by Novo Nordisk Farmaceutici S.p.A.) for writing assistance.

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[14] A. Huth-Ku¨hne, F. Baudo, P. Collins, J. Ingerslev, C.M. Kessler, H. Le´vesque, et al., International recommendations on the diagnosis and treatment of patients with acquired hemophilia A, Haematologica 94 (2009) 566–575. [15] M. Franchini, G. Lippi, Acquired factor VIII inhibitors, Blood 112 (2008) 250–255. [16] B. Verbruggen, I. Novakova, H. Wessels, J. Boezeman, M. van den Berg, E. MauserBunschoten, The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved specificity and reliability, Thromb. Haemost. 73 (1995) 247–251. [17] F. Baudo, F. de Cataldo, Acquired hemophilia: a critical bleeding syndrome, Haematologica 89 (2004) 96–100. [18] H. Levesque, P. Collins, F. Baudo, A. Huth-Kuhne, P. Knoebl, P. Marco, et al., Acquired haemophilia: descriptive data of the European acquired haemophila registry (EACH 2), J. Thromb. Haemost. 7 (Suppl. 2) (2009) PP-WE-604 (abstract). [19] M. Franchini, Postpartum acquired factor VIII inhibitors, Am. J. Hematol. 81 (2006) 768–773. [20] M. Franchini, F. Capra, N. Nicolini, D. Veneri, F. Manzato, F. Baudo, et al., Drug-induced anti-factor VIII antibodies: a systematic review, Med. Sci. Monit. 13 (2007) RA55–RA61. [21] N.W. Jansen, G. Roosendaal, F.P. Lafeber, Understanding haemophilic arthropathy: an exploration of current open issues, Br. J. Haematol. 143 (2008) 632–640. [22] M. Franchini, G. Targher, M. Montagnana, G. Lippi, Laboratory, clinical and therapeutic aspects of acquired hemophilia A, Clin. Chim. Acta 395 (2008) 14–18. [23] P.W. Collins, Treatment of acquired hemophilia A, J. Thromb. Haemost. 5 (2007) 893–900. [24] F. Baudo, T. Caimi, F. de Cataldo, Diagnosis and treatment of acquired hemophilia, Haemophilia 16 (2010) 102–106. [25] S. Ichikawa, K. Kohata, Y. Okitsu, M. Suzuki, S. Nakajima, M.F. Yamada, et al., Acquired hemophilia A with sigmoid colon cancer: successful treatment with rituximab followed by sigmoidectomy, Int. J. Hematol. 90 (2009) 33–36. [26] M. Shurafa, S. Raman, I. Wollner, Disappearance of factor VIII antibody after removal of primary colon adenocarcinoma, Am. J. Hematol. 50 (1995) 149–150. [27] F. Baudo, P. Collins, A. Huth-Ku¨hne, P. Knoebl, H. Le´vesque, M. Pascual, et al., Hemostatic therapy in acquired haemophilia: data from the European Acquired Haemophilia (EACH2) Registry, J. Thromb. Haemost. 7 (Suppl. 2) (2009) OC-WE-058 (abstract). [28] P.M. Mannucci, F. Peyvandi, Autoimmune hemophilia at rescue, Haematologica 94 (2009) 459–461. [29] O. Lambotte, J. Dautremer, B. Guillet, T. Boutekedjiret, M. Dreyfus, R. Kotb, et al., Acquired hemophilia in older people: a poor prognosis despite intensive care, J. Am. Geriatr. Soc. 55 (2007) 1682–1685. [30] S. Girault, K. Ly, A. Jaccard, V. Loustaud, P. Turlure, A. Julia, et al., Prognosis of acquired hemophilia in older people, J. Am. Geriatr. Soc. 56 (2008) 956–958. [31] D.G. Maloney, A.J. Grillo-Lo´pez, C.A. White, D. Bodkin, R.J. Schilder, J.A. Neidhart, et al., IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma, Blood 90 (1997) 2188–2195. [32] M. Franchini, Rituximab in the treatment of adult acquired hemophilia A: a systematic review, Crit. Rev. Oncol. Hematol. 63 (2007) 47–52. [33] W.R. Sperr, K. Lechner, I. Pabinger, Rituximab for the treatment of acquired antibodies to factor VIII, Haematologica 92 (2007) 66–71. [34] P. Collins, F. Baudo, A. Huth-Ku¨hne, P. Knobl, H. Levesque, P. Marco, et al., First line immunosuppressive treatment for acquired factor VIII antibodies: results of the European Acquired Haemophilia Registry (EACH2), J. Thromb. Haemost. 7 (Suppl. 2) (2009) PP-MO-609 (abstract).


HYPOBETALIPOPROTEINEMIA: GENETICS, BIOCHEMISTRY, AND CLINICAL SPECTRUM Patrizia Tarugi*,1 and Maurizio Averna† *Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy † Department of Clinical Medicine and Emerging Diseases, University of Palermo, Palermo, Italy

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathways of apoB-Containing Lipoproteins Production . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Assembly and Secretion of apoB-Containing Lipoproteins . . . . . . . . . . . . . . . . 3.2. Control of the Secretion of apoB-Containing Lipoproteins . . . . . . . . . . . . . . . . 3.3. Intracellular Degradation of apoB and Secretion of apoB-Containing Lipoproteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Clearance of apoB-Containing Lipoproteins via LDL-Receptor . . . . . . . . . . . 4. Dominant Forms of Primary HBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. FHBL Due to Defective Secretion of apoB-Containing Lipoproteins . . . . . . 4.2. FHBL Due to Increased Liver Uptake of apoB-Containing Lipoproteins via LDL-Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Prevalence of Primary FHBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Recessive Forms of Primary HBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Abetalipoproteinemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Chylomicron Retention Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Primary Orphan FHBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Spectrum of Clinical Manifestations in Primary HBL. . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Main Clinical Issues of FHBL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. FHBL and Fatty Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. FHBL and Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. FHBL and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Secondary Hypobetalipoproteinemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Patrizia Tarugi, e-mail: [email protected] 81

0065-2423/11 $35.00 DOI: 10.1016/B978-0-12-387025-4.00004-2


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Abbreviations ABL apoB CVD CM CMRD COP CHD ER ERAD FHBL HBL LDL-C LDL-R MTP PERPP PCSK9 TC TG

abetalipoproteinemia apolipoprotein B cardiovascular disease chylomicron chylomicron retention disease coat protein coronary heart disease endoplasmic reticulum endoplasmic reticulum associated degradation familial hypobetalipoproteinemia hypobetalipoproteinemia LDL-cholesterol LDL-receptor microsomal triglyceride transfer protein post-ER presecretory proteolysis proprotein convertase subtilisin/kexin type 9 total cholesterol triglyceride

1. Abstract Hypobetalipoproteinemias (HBL) represent a heterogeneous group of disorders characterized by reduced plasma levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5th percentile of the distribution in the population. HBL are defined as primary or secondary according to the underlying causes. Primary monogenic HBL are caused by mutations in several known genes (APOB, PCSK9, MTP, SARA2) or mutations in genes not yet identified. Familial hypobetalipoproteinemia (FHBL) is the most frequent monogenic form of HBL with a dominant mode of inheritance. It may be due to loss-of-function mutations in APOB or, less frequently, in PCSK9 genes. The rare recessive forms of primary monogenic HBL are represented by abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) due to mutations in MTP and SARA2 genes, respectively. The clinical phenotype of heterozygous FHBL is usually mild, being frequently characterized by fatty liver. The clinical phenotype of homozygous FHBL, ABL, and CMRD is usually severe being characterized by intestinal lipid malabsorption and fat-soluble vitamin deficiency. Secondary HBL are due to several nongenetic factors such as diet, drugs, and disease-related conditions. The aim of this review is to discuss the biochemistry, genetics, and clinical spectrum of HBL and to provide a clinical and laboratory diagnostic algorithm.

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2. Introduction HBL include a heterogeneous group of disorders characterized by reduced plasma levels of TC, LDL-C, and apoB below the 5th percentile of the distribution in the population. LDL, previously known as beta-lipoprotein, plays a key role in the transport of hydrophobic neutral lipids (mainly cholesterol) throughout the body. Since apoB is the main structural protein component of LDL, this lipoprotein class belongs to apoB-containing lipoproteins which also include plasma very low density lipoproteins (VLDL) and chylomicrons (CM) [1]. HBL may be caused by mutations in several known genes or mutations in unidentified genes (primary monogenic HBL) and by several nongenetic factors such as diet, drugs, and disease-related conditions (secondary HBL). In primary HBL, the biochemical phenotype segregates as an autosomal dominant or a recessive trait. The diagnosis of dominant HBL is driven by the multigenerational presence of related individuals carrying the biochemical HBL trait (low LDL-C and apoB plasma levels). The diagnosis of recessive HBL is primed by the presence, early in life, of the biochemical HBL trait associated with a generally severe clinical phenotype in offsprings of normolipidemic parents. Dominant HBL includes FHBL (OMIM 107730), recessive HBL includes ABL (OMIM 200100) and CMRD (OMIM 246700)—also called Anderson’s disease [1,2]. The spectrum of the clinical features in primary HBL ranges from absence or paucity of symptoms to a very severe syndrome (e.g., Section 7). In general, the genetic or acquired mechanisms that may reduce LDL-C and apoB plasma levels and cause primary or secondary HBL, respectively, must alter the production, assembly, secretion, or catabolism of apoB-containing lipoproteins. This chapter critically reviews the current knowledge on the pathophysiology, molecular genetics, and clinical features of HBL, and describes a diagnostic approach that may be helpful in a clinical setting.

3. Pathways of apoB-Containing Lipoproteins Production 3.1. ASSEMBLY AND SECRETION OF APOB-CONTAINING LIPOPROTEINS Liver and intestine are the main site of production of apoB-containing lipoproteins. apoB is expressed primarily in liver and intestine and plays a central role in the transport and metabolism of plasma cholesterol and triglycerides (TG) [1]. In human plasma, apoB occurs in two forms: apoB100 and apoB-48, which are encoded by the same gene (APOB) located on

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chromosome 2. apoB-100 (the full-length translation product of apoB mRNA) is a large monomeric protein of 4536 amino acids that is synthesized in the liver. apoB-100 is an essential component of liver-derived VLDLs, IDLs, and LDLs, where it serves as ligand for the LDL-receptor (LDL-R). apoB-48, a peptide consisting of the N-terminal 2152 amino acids of apoB100 (corresponding to 48% of apoB-100), is synthesized by the intestine and is essential for CM production, the lipoproteins that transport dietary lipids. apoB-48 results from a posttranscriptional modification of apoB mRNA (mRNA editing), which converts a glutamine codon at position 2153 into a stop codon [1]. apoB is essential for the production of apoB-containing lipoproteins. Naturally occurring mutations in APOB gene that result in structurally abnormal apoBs (e.g., apoB truncated at the C-terminal end) are associated with a reduction of plasma levels of apoB-containing lipoproteins [1–3] (e.g., Section 4.1.1). The assembly of VLDL in the liver and CM in the intestine occurs cotranslationally (during the synthesis of apoB): while the C-terminal end of apoB is still being synthesized, the N-terminal portion is translocated across the endoplasmic reticulum (ER) and is assembled as small lipoprotein particles for correct targeting to the pathways of lipoprotein secretion. The apoB is targeted to the ER via the signal sequence (spanning amino acids 1–27); its translocation occurs through the translocon, a proteinaceus channel in the ER membrane [4,5]. 3.2. CONTROL OF THE SECRETION OF APOB-CONTAINING LIPOPROTEINS In view of the essential role of apoB in the assembly and secretion of apoBcontaining lipoproteins, apoB levels are regulated at multiple levels. Several factors influence the translocation process of newly synthesized apoB. Most relevant is the availability of lipids at the site of apoB synthesis in the ER, which appears to dictate the amount of apoB secreted. In addition, the process of translocation is affected by the characteristics of apoB itself, including length, signal peptide polymorphism, and apoB folding to attain lipid-binding capability, which regulate its ability to assemble into lipoproteins [6]. Successful transport and correct conformation of apoB may lead to its final secretion as a lipoprotein constituent. In the case of lipid shortage, nascent apoB translocation into the ER lumen is inefficient and domains of apoB are exposed to the cytosol, where newly synthesized apoB undergoes rapid intracellular degradation (e.g., Section 3.3) [5]. The N-terminal assembly of lipids onto apoB during translocation requires the microsomal triglyceride transfer protein (MTP), an 894 amino acid protein located in the ER lumen that is a component of a protein complex involved in the early stages of apoB lipidation in liver and intestine [7]. MTP has been shown to bind to


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the first 1000 amino acids of apoB which form a domain capable of initiating nascent lipoprotein assembly (i.e., capable of recruiting lipids and facilitating the conversion of apoB into a buoyant lipoprotein particle) [8–12]. This ‘‘lipoprotein initiating’’ domain of apoB contains the regions spanning amino acid residues 1–264 and 512–721 or 270–570 involved in the binding with MTP [7,13]. The physical interaction between apoB and MTP is important for the initiation of translocation of the nascent apoB chain and for the cotranslational addition of lipids to this chain [4,5,7]. Via these mechanisms, MTP is believed to avoid improper folding and premature degradation of apoB. The crucial role of MTP in the assembly and secretion of apoBcontaining lipoproteins is substantiated by the observation that mutations in the MTP gene, which abolish MTP activity, are the cause of ABL, a severe recessive disorder in which VLDL and CM are not secreted (e.g., Section 5.1) [7,14]. Pharmacological inhibition of MTP in cultured cells and in vivo results in a dose-dependent inhibition of the secretion of apoB [15,16]. Recent evidence suggests that, in addition to MTP, other molecules with lipid transferase activity in the ER, chaperones, and modifying enzymes (such as PDI, BiP, Grp94, chaperon-like lectins, and cyclophilin) in different subcellular compartments may be involved in the lipoprotein formation [17]. The addition of lipids to apoB is believed to occur in two steps. In the first step, a small amount of lipids is added to apoB during its translation and translocation into the ER lumen. This initial lipidation prevents apoB degradation and leads to the formation of a partially lipidated small lipoprotein particle. In the second step, after apoB translation is completed, the bulk of neutral lipids is added to the primordial lipoprotein particle to form a mature particle [18,19]. The maturation of VLDLs and CMs, which starts in the ER, is completed in the Golgi [20]. Mature VLDLs and CMs present in the lumen of the smooth ER are transported from the ER via specialized vesicles to the Golgi apparatus for secretion [21]. The transport through the secretory pathway is mediated by the coat protein (COP) machinery. The COPII complex functions in ER-derived vesicle formation for anterograde transport. One of the subunits of the COPII complex (Sar1-GTPase) has been found to be critical for the vesicular transport of apoB-containing lipoproteins in rat hepatoma cells [22]. COPII associates with apoB-containing lipoprotein particles and forms ER-derived vesicles that initiate their intracellular transport to the Golgi apparatus before release into the circulation [22]. In humans there are two Sar1 proteins, designated Sar1a and Sar1b (encoded by Sar1-ADP-ribosylation GTPase, SARA1 and SARA2 genes, respectively), which differ by 20 amino acid residues. The role of Sar1b in vesicular transport of apoB-containing lipoproteins was demonstrated by the observation that mutations in the SARA2 gene are the cause of CMRD [23]. Whether the Sar1a isoform plays a distinct role in the transport of

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apoB-containing lipoproteins (e.g., is specifically required for VLDL secretion by the liver) is an appealing hypothesis that remains to be tested. 3.3. INTRACELLULAR DEGRADATION OF APOB AND SECRETION OF APOB-CONTAINING LIPOPROTEINS In contrast to most secreted proteins, the amount of apoB-containing lipoproteins excreted by the cells is mostly regulated by apoB degradation along the ER/Golgi secretory pathway. Targeting of apoB for degradation occurs when: (i) lipid availability is reduced, (ii) MTP activity is low, (iii) apoB is misfolded, or (iv) under cellular stress conditions. Two intracellular apoB degradation mechanisms have been described: (i) ER associated degradation (ERAD), catalyzed by the ubiquitin–proteasome system, occurs cotranslationally via retrotranslocation when misfolded apoB is present in the cell or lipid supply/availability is reduced; (ii) post-ER presecretory proteolysis (PERPP), achieved by autophagy, which delivers oxidized, aggregated apoB and partially assembled apoB lipoproteins to lysosomes for destruction [24,25]. Thus, intracellular degradation of apoB reduces the assembly and secretion of apoB-containing lipoproteins. Another mechanism involved in the control of the secretion of apoBcontaining lipoproteins is the LDL-R-mediated degradation. LDL-R promotes intracellular degradation of apoB-100 resulting in decreased secretion of VLDL by the liver. LDL-R knockout mice secrete apoB at a higher rate than control mice; this increase is prevented by overexpression of the LDL-R [26]. An in vivo turnover study in humans demonstrated that complete deficiency of the LDL-R is associated with an increased apoB production rate [27]. In addition, some naturally occurring mutations in LDL-R have been identified which cause retention of both the mutant LDL-R and apoB within the ER [28]. Taken together, these observations suggest that the LDLR facilitates the retention of apoB in the ER and its presecretory degradation and possibly also the reuptake of newly secreted apoB-containing lipoproteins on the cell surface. 3.4. CLEARANCE OF APOB-CONTAINING LIPOPROTEINS VIA LDL-RECEPTOR Most of the LDL particles are cleared from plasma by the liver via the LDL-R. LDL-R-mediated uptake and degradation of LDL is an important determinant of the concentration of LDL in the plasma, as clearly demonstrated by some monogenic disorders (autosomal dominant hypercholesterolemias) affecting LDL-R activity/number or the capacity of LDL to bind LDL-R. These disorders may result from: (i) loss-of-function mutations of LDL-R gene (reducing the number of LDL-Rs or their function),


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(ii) mutations in APOB gene (reducing the affinity of apoB for the LDL-R), or (iii) gain-of-function mutations of PCSK9 gene (resulting in a reduced number of LDL-Rs on the cell surface) [29]. The role of PCSK9 gene in the regulation of LDL-R number (and the regulation of plasma LDL level) has received great attention in recent years. PCSK9 gene, located on chromosome 1, encodes a 692 amino acid protein designated proprotein convertase subtilisin/kexin type 9 (PCSK9). This protein is mainly expressed in the liver where it is produced in the ER as a precursor protein which undergoes cotranslational autocatalytic cleavage in the Golgi to create the processed protease that is secreted. Cell culture and animal models have established that the LDL-R is one of the main downstream targets of PCSK9. Secreted mature PCSK9 binds the LDL-Rs on the surface of hepatocytes and promotes their internalization and degradation in a post-ER complex [30,31]. Thus, PCSK9 exerts its effect on the posttranslational regulation of LDL-R by controlling the LDL-R degradation and the number of receptors available on the cell surface. The mechanism by which PCSK9 reduces the number of LDL-R is only partially known. There is evidence that the secreted form of PCSK9 binds directly to the first epidermal growth factor-like repeat of the extracellular domain of the LDL-R. PCSK9 binding to this site is required for LDL-R degradation [32]. The ability of PCSK9 to promote LDL-R degradation, however, is independent of its catalytic activity [33]. PCSK9 would function as a chaperone molecule that prevents LDL-R recycling to the plasma membrane from endosomes and/or targets LDL-R to the lysosome for degradation [33].

4. Dominant Forms of Primary HBL FHBL is the main form of dominant primary HBL. FHBL is a genetically heterogeneous disorder which may be due to defective secretion or increased catabolism of apoB-containing lipoproteins. The majority of FHBL patients are heterozygotes; homozygous and compound heterozygous FHBL are very rare. 4.1. FHBL DUE TO DEFECTIVE SECRETION OF APOB-CONTAINING LIPOPROTEINS The main candidate gene in subjects with FHBL with a dominant inheritance is the APOB gene. Approximately 50% of FHBL heterozygotes are carriers of pathogenic mutations in the APOB gene (APOB-related FHBL) [34]. A large number of APOB mutations have been reported to be the cause of FHBL and novel mutations are continually being identified in FHBL subjects [2,3,34,35].

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4.1.1. APOB Gene Mutations Producing Truncated APOBs Most APOB gene mutations cause the formation of premature termination codons in the apoB mRNA. The translation of these mRNAs leads to the formation of truncated apoBs of various size which, to a variable extent, lose the capacity to form plasma lipoproteins in liver and/or intestine and to export lipids from these organs [2,3,34–37]. Truncated apoBs may or may not be detectable in plasma according to their size. Truncated apoBs longer than apoB-29/30 (i.e., with a size corresponding to 29–30% of that of apoB-100, according to a centile nomenclature) are detectable in plasma (by immunoblot with an anti-apoB antibody), as they are secreted into the plasma as constituents of plasma lipoproteins. The detection of a truncated apoB in plasma suggests the presence of a mutation located in a genomic region spanning from exon 26 to exon 29 of APOB gene. Truncated apoBs shorter than apoB-29/30, due to mutations located in the first 25 exons of APOB gene, are not detectable in plasma, as they are not secreted [3,34,35]. These short truncated apoBs account for 30% of all APOB mutations reported so far in FHBL [34]. Heterozygous FHBL subjects carrying truncated apoBs have a reduced production of apoB-containing lipoproteins in liver and, in some cases, in the intestine, which prevents the formation of VLDL and CM, respectively [38,39]. The production rate (in liver and intestine) of truncated apoBs, as compared with the corresponding wild-type forms of apoB (apoB48 and apoB-100), is greatly reduced for two main reasons: (i) the reduced lipid-binding capacity of structurally abnormal apoBs (notably short truncations) makes them prone to a rapid intracellular degradation, (ii) the presence of premature stop codons in apoB mRNAs due to frameshift or nonsense mutations may induce a rapid mRNA degradation (nonsense-mediated mRNA decay). In addition, FHBL carriers of long apoB truncations, which are secreted into the plasma, may have an increased removal of truncated apoB-containing lipoproteins by the liver (via the LDL-R) [38] or by the kidney via megalin receptor [40]. The plasma levels of LDL-C and apoB in FHBL heterozygotes carrying truncated apoBs are less than 30% of the values found in age- and gendermatched controls [3,34]. This reduction, which is far below the expected 50% values, is due not only to the extremely low levels of the secreted truncated forms of apoB but also to the reduced production rate of apoB-100 encoded by the normal allele which is 70–75% lower than that found in normal subjects [41,42]. The lower production of normal apoB-100 is the result of a reduced synthesis combined with an increased intracellular degradation. Very few FHBL homozygotes and compound heterozygotes have been reported. In APOB-related FHBL homozygotes or compound heterozygotes carrying apoB truncations, LDL-C and apoB are either not detectable


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(if both mutant alleles encode truncated apoB shorter than apoB-29/30) or are 10–20% of the control values (if both mutant alleles or at least one mutant allele encode(s) truncated apoBs longer than apoB-29/30) [34,43]. 4.1.2. APOB Gene Mutations Producing Amino Acid Substitutions in apoB Few nonconservative amino acid substitutions in apoB have been reported to be the cause of FHBL [44,45]. Two mutations, R463W and L343V, were found to cosegregate with FHBL in two large Libanese kindred. These mutations involve the N-terminal beta-alpha1 domain of apoB which contains sequence elements shown to be important for the proper folding of apoB [13,46], for the physical interaction between apoB and MTP (the chaperone molecule required for apoB lipidation) [8,10] and for lipid recruitment during lipoprotein assembly and secretion [9]. These missense mutations are associated with a decreased secretion of the mutant apoBs that are retained in the ER because of an increased binding to MTP [44,45]. Other carriers of R463W have been recently identified in Italian, Dutch, and Spanish FHBL subjects, suggesting that R463W may be a recurrent mutation in the population [34,37]. Recently, other missense mutations have been reported to be the cause of FHBL. Five missense APOB mutations located within the N-terminal 1000 amino acids of apoB, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of FHBL in the Italian population. Among these mutations, the A31P substitution in apoB completely blocked apoB-48 secretion when expressed in rat hepatoma cells [47]. In contrast to the two missense mutations L343V and R463W, the A31P mutant did not lead to ER retention as the aberrantly folded protein is degraded intracellularly by proteasomes and autophagosome/lysosome pathway [47]. Plasma levels of LDL-C and apoB in heterozygous carriers of the APOB missense mutations are comparable with those present in FHBL due to apoB truncations. The two homozygotes for the missense mutation R463W described so far have barely detectable apoB-containing lipoproteins in plasma [44] as observed in patients homozygotes/compound heterozygotes for short truncated apoBs [43,48]. 4.2. FHBL DUE TO INCREASED LIVER UPTAKE OF APOB-CONTAINING LIPOPROTEINS VIA LDL-RECEPTOR The large number of FHBL subjects in whom no APOB gene mutations were found suggests that other genes are involved in the pathogenesis of FHBL [3]. In the past few years, loss-of-function mutations in PCSK9 gene emerged as possible cause of FHBL (PCSK9-related FHBL).

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Studies in mice had shown that the inactivation of PCSK9 gene [49] was associated with a marked reduction of plasma cholesterol and LDL-C. This effect was due to an increased plasma LDL clearance, secondary to an increased number of LDL-Rs in the liver. This observation suggested that loss-of-function mutations of PCSK9 in humans would increase the receptormediated uptake and catabolism of plasma LDL, possibly resulting in reduced plasma level of LDL and a lipid phenotype similar to that found in APOB-related FHBL (e.g., Section 4.1). This hypothesis was confirmed when inactivating mutations in human PCSK9 (resulting in truncated proteins) were discovered in different populations (African-American, African, Caucasian, and Japanese populations) [50–54]. Heterozygous subjects carrying the loss-of-function mutations causing PCSK9 truncations (C679X, Y142X, A68fsL82X, and W428X) were found to have a reduction of plasma LDL-C levels ranging from 30% to 70% [50–54]. One homozygote for C679X and one compound heterozygote (Y142X/R97) were found to have an 80% reduction of LDL-C with respect to control values [51,52,55]. Population studies have demonstrated that some amino acid substitutions of PCSK9 (p.R46L, p.G106R, p.N157K, p.G236S, p.R237W, p.L253F, p. A443T, and p.S462P) are associated with a significant but variable reduction (ranging from 4% to 30%) in plasma levels of LDL-C [56–59]. All these variants were found to be more frequent in hypocholesterolemic subjects; however, only some of them were demonstrated to be loss-of-function mutations in vitro. Taken together, these observations strongly suggest that in humans, loss-of-function mutations of PCSK9 cause an FHBL phenotype by increasing the hepatic uptake of LDL. In subjects with PCSK9-related FHBL, the magnitude of reduction of plasma LDL-C and apoB appears to be less striking than that observed in subjects with APOB-related FHBL in whom the reduction usually ranges from 60% to 80% with respect to control values [34]. PCSK9-related FHBL homozygotes or compound heterozygotes have a less severe reduction of LDL-C and apoB with respect to APOB-related FHBL homozygotes/compound heterozygotes [43,48,51,52]. Plasma levels of LDL-C and apoB show large interindividual variability in FHBL heterozygotes, regardless of the gene involved (APOB or PCSK9) [34,50,60] that may be due to environmental factors (notably the diet) or to genetic factors affecting secretion and catabolism of apoB-containing lipoproteins. For example, it was shown that apoE genotype accounts for 15–60% of this variation in APOB-related FHBL heterozygotes carrying truncated apoBs [61]. It is also possible that factors affecting intestinal cholesterol absorption (i.e., variations in NPC1L1 gene) play a role in regulating apoB and LDL-C concentration in plasma of FHBL subjects [62].


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4.3. PREVALENCE OF PRIMARY FHBL Epidemiological data on the prevalence of primary FHBL are scarce. The population frequency of heterozygous APOB-related FHBL has been estimated to be 1 in 3000 [63]. Heterozygous PCSK9-related FHBL seems to be rare among Caucasians, while in African-Americans this disorder due to the two nonsense mutations (Y142X and C679X) has a prevalence of 2–2.6% [50]. The prevalence of homozygous and compound heterozygous APOBand PCSK9-related FHBL is exceedingly rare [2,59]. 5. Recessive Forms of Primary HBL Two very rare primary monogenic HBLs, usually diagnosed in infancy, segregate as autosomal recessive traits: ABL and CMRD. These disorders are characterized by the complete absence of apoB-containing lipoproteins (ABL) or by a selective absence of apoB-48-containing lipoproteins (CMRD) due to a defective assembly and secretion of these lipoproteins by liver and/or intestine [1]. 5.1. ABETALIPOPROTEINEMIA ABL is an ‘‘exceedingly rare’’ disorder that occurs in less than one in 1 million individuals. The plasma lipid profile of ABL patients is characterized by extremely low plasma levels of TC, VLDL, and LDL and an almost complete absence of apoB-100 and apoB-48. ABL-obligate heterozygotes have normal plasma lipids; in some cases, however, a mild reduction of TC and LDL-C has been reported [14]. ABL is due to mutations in the MTP gene which is required for the assembly and secretion of apoB-containing lipoproteins in both liver and intestine (e.g., Section 3.2). A variety of mutations in this gene, located on chromosome 4, have been described; most of them result in truncated proteins devoid of function [14,48,64–68]. Some MTP missense mutations have also been reported, which affect either the apoB-binding ability of MTP or its interaction with other components of the protein complex; they are associated with a milder form of the disease [65,66]. It is conceivable that the severity of ABL phenotype is related to the residual activity of MTP and the capacity to form VLDL and CM. The absence of MTP activity leads to the accumulation of large lipid droplets in the cytoplasm in hepatocytes and enterocytes. 5.2. CHYLOMICRON RETENTION DISEASE CMRD is a very rare recessive disorder characterized by the selective absence of apoB-48 in plasma, low plasma cholesterol, and fat-soluble vitamins. apoB-48-containing lipoproteins are not secreted into the plasma,

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neither fasting nor postprandially [1]. In CMRD the plasma levels of LDL-C and apoB are 25–40% of the control levels [69]. Affected subjects have an inability to export dietary lipids as CMs, leading to a marked accumulation of CM-like particles in membrane-bound compartments of enterocytes, which contain large cytosolic lipid droplets. CMRD is due to mutations in the SARA2 gene belonging to the Sar1ADP-ribosylation factor family of small GTPases. The SARA2 gene, located on chromosome 5, encodes the Sar1b protein, a single polypeptide of 198 amino acids [23,70] that is involved in the control of the intracellular trafficking of CMs in COPII-coated vesicles. CMs are selectively recruited by the COPII machinery for transport through the cellular secretory pathway [21,22]. Thus, CMRD may arise as a result of defects in the transport of CMs through the secretory pathway. Up to now, 14 mutations in the SARA2 gene have been identified in patients with the clinical diagnosis of CMRD-Anderson disease [23,71–74]. Missense mutations of SARA2 represent the most common cause of CMRD; these mutations were found in the Sar1b b-sheets region and are predicted to perturb the geometry of the guanosine diphosphate and guanosine triphosphate binding site of Sar1b [70]. Only two frameshift mutations have been reported so far [72,74].

6. Primary Orphan FHBL In a significant number of FHBL kindred, the search for causative mutations in the candidate genes is often inconclusive. Orphan FHBL kindred in which the HBL phenotype segregates as dominant trait have been studied by linkage analysis in order to identify the causative genes. Susceptibility loci have been identified in chromosomes 13q, 3p21.1-22, and 10 but no candidate genes have emerged so far [75–77].

7. Spectrum of Clinical Manifestations in Primary HBL Monogenic hypobetalipoproteinemias are characterized by a wide spectrum of clinical features. The clinical phenotypes of primary HBL due to defective secretion of apoB-containing lipoproteins (APOB-related FHBL, ABL, and CMRD) are highly variable, their severity being directly related to the degree of the impairment of the production of apoB-100 and/or apoB-48 [3]. The clinical phenotype is more severe in the primary recessive HBLs: in ABL and in CMRD the impaired formation/secretion of apoB-48 explains the severe


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intestinal lipid malabsorption and the related disorders (such as the growth retardation and the neurological manifestations caused by vitamin E deficiency) [78,79]. ABL displays a cluster of severe clinical manifestations such as malabsorption of dietary fat and fat-soluble vitamins, steatorrhea, ‘‘failure to thrive’’ and acanthocytosis that are present in infancy and childhood. Later in life, because of the deficiency of fat-soluble vitamins, coagulopathy, posterior column neuropathy and myopathy, anemia, spinocerebellar ataxia, and retinitis pigmentosa develop [78]. Few reports have documented a possible association of ABL with ileal adenocarcinoma [80] and metastatic spinal cord glioblastoma [81] and fatty liver was reported in some cases [82,83]. In CMRD, the clinical phenotype is strictly related to the impaired capacity to form CMs after a fat-containing meal. This leads to the development of steatorrhea, growth retardation, malnutrition, and an accumulation of lipid droplets within the enterocyte [84,85]. No neurological symptoms have been reported. However, the description of variable clinical phenotypes and a weak genotype–phenotype correlation in CMRD suggest that this disease might represent a more complex trait rather than a simple autosomal recessive disorder [69,74,79]. FHBL heterozygotes are generally asymptomatic but most of them develop fatty liver and sometimes a mild intestinal malabsorption [34,35]. FHBL homozygotes or compound heterozygotes have fatty liver and may suffer from severe fat malabsorption [43,48]. In homozygotes or compound heterozygotes of APOB-related FHBL carrying truncated apoBs, the severity of the clinical phenotype depends on the capacity of the truncated apoBs to bind lipids and to be secreted as lipoprotein particles by liver and intestine. apoBs shorter than apoB-29/30 are degraded intracellularly, mimicking an ABL-like condition [43,48]. The presence in these individuals of at least one mutant APOB allele producing truncations longer than apoB-48 may allow the secretion of small amounts of apoB-containing lipoproteins (specifically CMs), which contribute to mitigate the clinical phenotype [34,43]. In homozygotes/compound heterozygotes for long truncations (truncations longer than apoB-48), the clinical phenotype is usually mild, being characterized only by fatty liver [34]. Heterozygous APOB-related FHBL carrying short truncations (i.e., truncated apoBs shorter than apoB-48) usually have fatty liver and mild intestinal lipid malabsorption [34,86]; carriers of long truncations are generally asymptomatic but they tend to develop fatty liver [34]. Carriers of apoB missense mutations may be asymptomatic or manifest mild fatty liver disease and sometimes a mild fat malabsorption [44,45,47,87]. A common characteristic of the severe forms of primary HBL is fat-soluble vitamin deficiency, including vitamin E, secondary to fat malabsorption.

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Before the introduction of high-dose vitamin (A, D, K, and E) therapy, the majority of ABL patients did not survive past the third decade of life. The long-term vitamin treatment in ABL patients [78] was able to arrest the progression of the neurological complications, especially when initiated before the 16th month of life [88]. The current standard treatment for ABL, which can be extended to CMRD and homozygous FHBL, is based on a low fat diet eventually supplemented with medium-chain triglyceride and the administration of fat-soluble vitamins [79]. The rare cases of PCSK9-related FHBL homozygotes, despite a substantial reduction of plasma LDL-C (0.36–0.41 mmol/l), appear to be in good health since they are free of gastrointestinal symptoms and detectable increase in hepatic TG [51,52]. FHBL heterozygotes carrying PCSK9 mutations are completely asymptomatic in contrast with APOB-related FHBL. This difference is probably due to the biochemical pathways underlying PCSK9- and APOB-related FHBL. Increased hepatic catabolism of LDL present in PCSK9-related FHBL does not result in lipid accumulation within the hepatocytes or entherocytes, while such an accumulation is a key feature in the disorders characterized by defective assembly/secretion of apoB-containing lipoproteins (APOB-related FHBL, ABL, and CMRD).

8. Main Clinical Issues of FHBL 8.1. FHBL AND FATTY LIVER DISEASE In APOB-related FHBL the presence of fatty liver has been documented by abdominal ultrasound examination, magnetic resonance, or liver biopsy [89–93]. In FHBL heterozygous carriers of truncated forms of apoB, the mean liver TG content is approximately fivefold that of controls with a great interindividual variability despite similar apoB truncations and similar measures of obesity and glucose tolerance; this could indicate that other modifier genes than APOB may affect the magnitude of hepatic TG accumulation [94]. Histologically fatty liver of FHBL is similar to nonalcoholic fatty liver disease, which is highly prevalent in the general population, often being associated with obesity, dyslipidemia, hypertension, insulin resistance, type 2 diabetes mellitus; all these features, when clustered, characterize the metabolic syndrome [95,96]. In contrast to metabolic syndrome, in FHBL subjects the increased hepatic fat content is neither associated with glucose intolerance nor with insulin resistance [97]. The pathophysiology of fatty liver in APOB-related FHBL has been elucidated in animal models: apoB truncation-inducing mutations lead to the production of VLDL particles smaller than normal, with reduced


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capacities to bind TG; in addition apoB-100 produced by the normal allele is also reduced. The two combined defects of the VLDL export system produce the accumulation of TG in hepatocytes [98,99]. Fatty liver is also present in FHBL heterozygotes carrying missense mutations of APOB; these mutant apoBs are poorly lipidated and secreted as lipoprotein particles, causing an impaired export of hepatic TG [25]. The long-term outcome of fatty liver in FHBL is still unknown; anecdotal reports have documented an association between fatty liver and steatohepatitis, liver cirrhosis and hepatocarcinoma in FHBL patients [86,89–91,100]. Interestingly, fatty liver is not present in other forms of primary HBL such as PCSK9-related FHBL [51] and the orphan FHBL (linked to chromosomes 3 and 10 loci) [76,77]. 8.2. FHBL AND CARDIOVASCULAR DISEASE Elevated LDL-C is a major risk factor for cardiovascular disease (CVD). A recent meta-analysis of 61 prospective studies demonstrated that higher TC levels were associated with higher risks of coronary heart disease (CHD) and that there is no lower threshold. The association is log-linear suggesting that greater LDL-C absolute reductions should be associated with greater proportional reductions in risk of CHD [101]. In view of the low plasma LDL-C levels, it is reasonable to assume that primary HBL subjects: (i) might be naturally protected against CVD owing to reduced life-time exposure to atherogenic apoB-containing lipoproteins and (ii) represent a unique setting to verify the effects of a lifelong exposure to very low levels of LDL-C on the development of cardiovascular atherosclerosis-related diseases. To date no prospective studies have tested this hypothesis mainly because of the relatively small size of existing FHBL case series. Recently 14 APOB-related FHBL subjects have been clinically evaluated and found to be completely free from CVD [37]. A study designed to assess the noninvasive surrogate markers of CVD in FHBL showed that heterozygous APOB-related FHBL subjects (no. 41) were found to have a significant decrease in arterial stiffness as compared to control subjects, despite an increased prevalence of traditional cardiovascular risk factors [102]. Convincing evidence of the protection of lifelong reduction of plasma LDL-C has emerged with the observation that individuals with PCSK9-related FHBL have a marked reduction of CHD risk. Cohen et al. [103] examined the effect of PCSK9 loss-of-function mutations associated with reduced plasma levels of LDL-C on the incidence of CHD in a large population (Atherosclerosis Risk in Communities Study, ARIC). During a 15-year follow-up period among the carriers of the C679X mutation and the R46L variants, there was an 88% and a 47% reduction in the risk of CHD,

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respectively. These findings represent a strong ‘‘proof of concept’’ in favor of earlier initiation of lifestyle or, when appropriate, of drug interventions to lower LDL-C levels. 8.3. FHBL AND CANCER The association between low plasma cholesterol concentrations and increased risk of cancer has been reported in several studies and it has been explained as an example of ‘‘reverse causality’’ (subclinical cancer could reduce cholesterol levels) [104–106]. To test whether long-term low plasma cholesterol levels may increase cancer risk, cancer incidence has been evaluated in the ARIC study, among PCSK9-related FHBL [107]. There was no evidence that cholesterol-lowering variants of PCSK9 were associated with increased risk of total cancer.

9. Secondary Hypobetalipoproteinemias Environmental factors and several diseases may contribute to reduce the LDL-C and apoB plasma levels. Vegetarians who do not eat meat, fish, diary, or eggs have mean plasma TC and LDL-C levels lower than expected values of the control population. TC and LDL-C average plasma levels are 3.10–3.36 and 1.55–1.81 mmol/l, respectively, which are very close to the 5th percentile of the population distribution [108,109]. In patients with chronic parenchymal liver disease, lipid and lipoprotein plasma levels tend to be reduced [110]. The TC and LDL-C decrease parallels, the impairment of the hepatic synthesis due to the progressive liver failure [111]. Among cirrhotic patients classified according to the MELD score which determines the residual liver function, TC ranges between 3.88 and 2.58 mmol/l and LDL-C between 2.07 and 1.29 mmol/l [112]. Conditions like chronic pancreatitis (adults) and cystic fibrosis (children) are characterized by intestinal fat malabsorption which is associated with HBL. In these common diseases, exocrine pancreatic insufficiency causes steatorrhea when pancreas function is below 5% of normal function. Patients with chronic pancreatitis suffer from malabsorption and nutritional deficiencies. In advanced disease, TC and LDL-C are generally low, 3.10–4.14 and 1.81–2.33 mmol/l, respectively [113]. In cystic fibrosis, the presence of pancreatic insufficiency is associated with lower levels of TC (2.58–2.32 mmol/l) and LDL-C (1.29–1.81 mmol/l) [114]. In end-stage renal disease patients on hemodialysis, the LDL-C distribution curve is shifted to the left [115]; malnutrition and inflammation are


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believed responsible for the hypocholesterolemia associated with this condition [116]. Hyperthyroidism exhibits an enhanced excretion of cholesterol, a reduced output of hepatic VLDL and an increased turnover of LDL resulting in a decrease of plasma TC and LDL-C [117,118]. Low plasma levels of TC have been frequently described in a variety of hematologic disorders characterized by the presence of anemia [119]. The existence of HBL has been reported in all phenotypes of beta-thalassemia with TC and LDL-C levels in homo- and heterozygotes about 50% lower compared with healthy controls [120,121]. The mechanisms underlying the hypocholesterolemia observed in beta-thalassemia patients are unknown, and several explanations have been proposed: anemia, liver dysfunction, increased cholesterol consumption by the bone marrow, hyper-stimulation of the LDL-R by inflammatory cytokines, or an overactive reticuloendothelial system [122]. Hypocholesterolemia has also been documented in Sickle cell disease patients and it recognizes the same putative mechanisms as beta-thalassemia [123,124]. Hypocholesterolemia has been associated with cancer; there is no cause– effect relationship but the so-called unsuspected sickness phenomenon [125,126]: subclinical disease may lower cholesterol levels by a LDL-R hyperactivity in tumor cells [127].

10. Conclusions The plasma levels of LDL-C and apoB below the 5th percentile of the reference population’s values represent the biochemical landmark which drives the clinical diagnosis of monogenic hypobetalipoproteinemias. The magnitude of plasma LDL-C and apoB reduction is affected by: (i) the mode of transmission, dominant (APOB- or PCSK9-related FHBL) versus recessive (ABL and CMRD) and (ii) the heterozygous or homozygous state in the case of FHBL (Table 1). The first step in the differential diagnosis of HBL is the exclusion of the secondary forms of HBL that are associated with a variety of clinical conditions. If the clinical diagnosis suggests primary HBL, the study of the transmission of HBL trait in the family of the index case provides a guide for the molecular diagnosis. A mild clinical phenotype (moderate reduction of TC, LDL-C, and apoB possibly associated with fatty liver), when combined with the presence of the HBL trait in family members, strongly suggests the clinical diagnosis of heterozygous FHBL. A diagnosis of possible FHBL should also be considered in a subject with a mild phenotype whenever the mode of transmission of HBL trait is undefined (i.e., it cannot be ascertained

TABLE 1 MONOGENIC PRIMARY HYPOBETALIPOPROTEINEMIAS

Primary HBL

Mode of inheritance

Candidate gene

apoB or LDL plasma levels versus controls

APOB-related FHBL

Codominant

APOB

25)

P < 0.05 ANOVA 1 0.95 0.9 0.85 0.8 SS

S/L

L/L S/S CYP19A1 genotype

S/L

L/L

FIG. 7. Lumbar BMD values according to CYP19A1 (TTTA)n repeat genotype in subjects divided by BMI in (A) normal (BMI 25) and (B) overweight or obese groups (BMI > 25). Subjects were grouped according to short (S; TTTA 9) and long (L; TTTA > 9) repeats number (adapted from Ref. [138]).

Given the importance of estrogen in bone accrual, it is likely that deleterious CYP19A1 polymorphisms exert even a greater role in young individuals. Despite an early study in 140 middle-aged Finnish men that revealed an association between the number of TTTA repeat sequences and height and BMI but not with BMD [149], a larger analysis confirmed that CYP19A1 polymorphisms significantly affect the attainment of peak bone mass [150]. In a well-characterized cohort of 1068 men at the age of peak bone mass (18.9 0.6 years), both the TTTA repeat variation and a silent G/A polymorphism at Val80 of the CYP19A1 gene were predictors of areal BMD of the radius, lumbar spine, total body, and cortical bone size (cortical crosssectional area and thickness) of both the radius and tibia. To date, the molecular mechanisms through which the different CYP19A1 variants affect aromatase activity and bone metabolism remain in great part unknown. In a preliminary study in elderly men, higher in vitro aromatase efficiency and greater estrogen production were observed in fibroblasts from subjects with a high TTTA repeat sequence genotype in comparison to fibroblasts from a low TTTA repeat sequence genotype [138]. However, due to its location in intron 4 of the CYP19A1 gene, it is unlikely that this polymorphism directly affects aromatase activity. It is more likely that the different TTTA alleles are in linkage disequilibrium with other functional variants in the gene or with a nearby gene. Indeed, a different study described a strong degree of linkage disequilibrium between the (TTTA)n repeat polymorphism and the C/T substitution in exon 10, just 19 bp downstream of the termination site of translation [135]. In that study, the T allele was associated

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with a higher number of TTTA repeat sequences and showed a high activity phenotype, with increased aromatase activity, increased aromatase mRNA levels, and with a switch in promoter usage from adipose tissue promoter to the more active ovary promoter. Different studies also evidenced a functional role of other polymorphisms located within the complex promoter region of the CYP19A1 gene. In particular, a C/G polymorphisms in promoter I.2 (rs1062033) was shown to influence gene transcription by interacting with CEBPb [151], a transcription factor acting either as a stimulator or inhibitor of aromatase expression in different tissues [152,153]. In fact, in experiments of transient transfections of osteoblastic cell lines with luciferase reporters, inserts with the rs1062033 region stimulated the expression of the reporter gene, in an allele-specific way. The expression of the reporter gene was significantly higher in constructs bearing the G allele (which was also associated with higher BMD in population studies) than in those with the C allele. In the same model, cotransfecting with CEBPb increased luciferase expression by the aromatase constructs, especially in those with the G allele. Furthermore, evidence for differential allelic expression was found in bone tissue samples, again indicating the G allele as the more overexpressed. Although these studies, in the aggregate, provide data to argue for the importance of polymorphisms in CYP19A1 as determinants of estrogen production and bone strength, larger and more definitive studies are needed before any firm conclusions can be drawn. Finally, recent evidence also suggested that CpG methylation represents an important epigenetic mechanism for regulating CYP19A1 expression and that different methylation patterns may be responsible for the observed interindividual variation in promoter-driven expression of aromatase, at least in skin fibroblasts [154]. In fact, unmethylated constructs showed consistently higher promoter activity than methylated constructs. 8.2. ACQUIRED VARIATION IN AROMATASE ACTIVITY Besides genetic or epigenetic considerations, several additional mechanisms have been proposed in which aromatase activity could be modulated under certain circumstances in different tissues. It is known, for example, that aromatase is a marker of the undifferentiated adipose mesenchymal cell phenotype and that on a per cell basis, it is more highly expressed in these cells than in mature adipocytes. Thus, factors that stimulate adipocyte differentiation such as ligands of the PPARg receptor (i.e., troglitazone) could also lead to downregulation of aromatase gene and a reduction in aromatase activity. Of course, if there are more adipocytes, there could be more aromatase activity even with reduced production of estrogen per fat cell. In vitro studies support this hypothesis [155–157]. Similarly, phthalates, ubiquitous

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environmental toxins found in plasticizers, have been reported to activate the PPARg and PPARa pathways and to decrease aromatase activity, mRNA and protein levels in ovarian granulosa cells [158]. The clinical relevance of these environmental modulators on global aromatase activity and estrogen production in man remains unknown. Of interest, the activation of PPARa pathway by fenofibrate in female mice significantly reduced aromatase mRNA and activity, resulting in decreased femoral BMD and uterine size [159]. Several other contaminants may affect aromatase activity and estrogen production. In particular, glyphosate-based herbicides are toxic and endocrine disruptors in human cell lines that are widely used across the world. Their residues are frequent pollutants in the environment and are spread on most eaten transgenic plants, modified to tolerate high levels of these compounds in their cells. While up to 400 ppm of their residues are accepted in some feed, recent experimental studies demonstrated that aromatase transcription and activity were disrupted with subagricultural doses and with residues from 10 ppm [160]. In addition, cycloxygenase (COX) inhibitors, by reducing PGE2 production, may inhibit aromatase activity, at least in breast cancer cells, and in some studies showed strong chemopreventive activity against mammary carcinogenesis [161,162]. However, PGE2 appears also involved in the regulation of bone turnover [163], and its inhibition by the combination of relative COX-2 selective nonsteroidal anti-inflammatory drugs and aspirin was associated with high and not low BMD at multiple skeletal sites both in men and women [164]. A recent study showed that phytochemicals such as procyanidin B dimers contained in red wine and grape seeds inhibit aromatase activity in vitro and suppress aromatase-mediated breast tumor formation in vivo [165]. It has been estimated that daily consumption of 125 ml of red wine would provide adequate amounts of procyanidin B dimers to suppress in situ aromatase in an average postmenopausal woman. Similarly, myosmine, a minor tobacco alkaloid widely occurring in food products of plant and animal origin, inhibits the conversion of testosterone to estradiol by human aromatase with potential implications for sex hormone homoeostasis [166]. Another important and well-recognized modulator of aromatase efficiency in bone cells is vitamin D that has been shown to stimulate glucocorticoidinduced aromatase activity in cultured osteoblasts [167]. The magnitude of this effect varies largely among individuals, depending on the level of vitamin D receptor [168]. Of interest, vitamin D receptor knock-out mice showed reduced aromatase activity with respect to WT animals [169]. Finally, aromatase efficiency may be influenced by pathological conditions. It is known that increased androgen aromatization can be caused by hepatocellular carcinoma [170], adrenocortical tumors [171], and testicular


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tumors [172,173]. In these neoplastic conditions, inappropriate amounts of aromatase enzyme are expressed and estrogen levels are increased. Elevated plasma estradiol concentrations have also been described in men with liver cirrhosis together with decreased plasma testosterone [173,174]. In these patients, the metabolic clearance rate of estrogens seems to be unaltered, suggesting that the observed hyperestrogenism could be caused solely by an increase in androgen aromatization. Much less is known about a possible negative influence of pathological conditions on aromatase activity. In a preliminary study on elderly men, significant differences in estradiol levels in relation to Helicobacter pylori infection were observed, independently from circulating testosterone levels [175]. Levels of estradiol in infected CagApositive patients were significantly lower than in infected CagA-negative patients and this variation was associated with differences in bone turnover. The mechanism underlying this association is unknown and deserves further investigations. Indeed, aromatase activity and production of estradiol were recently demonstrated in gastric parietal cells [176]. Recent observations also suggested that diabetes negatively affects expression levels of aromatase both in ovary and testis [177,178]. However, the effects of this condition on major extragonadal sites of aromatase activity including bone remains to be determined. Moreover, experimental studies also evidenced that metformin, an oral antidiabetic agent, inhibits aromatase expression in both granulosa luteal cells and breast adipose cells while insulin stimulates aromatase mRNA expression in different cell lines [179,180]. Since a recent study evidenced that metformin-induced inhibition of aromatase expression occurs via downregulation of promoter II, I.3, and 1.4 [180], its potential negative effects on estrogen production and skeletal health should be investigated.

9. Summary and Conclusions Aromatase, the enzyme responsible for the transformation of androgens into estrogens, has a complex, tissue-specific regulation. The regulation of the level and activity of this enzyme determines the concentrations of estrogens that have endocrine, paracrine, and autocrine effects on several tissues including bone. Importantly, extraglandular aromatization of circulating androgen precursors is the major source of estrogen not only in men but also in women after the menopause. Several lines of clinical and experimental evidence now clearly indicate that aromatase activity and estrogen production are necessary for longitudinal bone growth, attainment of peak bone mass, the pubertal growth spurt, epiphyseal closure, and normal bone remodeling in young individuals. Moreover, with aging, individual differences in aromatase activity may significantly affect bone loss and fracture risk in both genders.

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BIOCHEMISTRY OF ADOLESCENT IDIOPATHIC SCOLIOSIS Giovanni Lombardi,* Marie-Yvonne Akoume,†,‡ Alessandra Colombini,* Alain Moreau,†,‡,} and Giuseppe Banfi*,1 *IRCCS Istituto Ortopedico Galeazzi, Milano, Italy † Viscogliosi Laboratory in Molecular Genetics of Musculoskeletal Diseases, Sainte-Justine University Hospital ´ al, Quebec, Canada Research Center, Montre ‡ Department of Biochemistry, Faculty of Medicine, ´ de Montre ´ al, Montre ´ al, Quebec, Canada Universite } Department of Stomatology, Faculty of Dentistry, ´ de Montre ´ al, Montre ´ al, Quebec, Canada Universite

1. 2. 3. 4. 5. 6. 7.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone Biochemical Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hematological Parameters—Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Melatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Melatonin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Interaction with Calcium Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Role of Melatonin in Pathogenesis of Scoliosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Giuseppe Banfi, e-mail: [email protected] 165

0065-2423/11 $35.00 DOI: 10.1016/B978-0-12-387025-4.00007-8


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1. Abstract This chapter reviews the biochemical, hormonal, and hematological factors in the onset and development of adolescent idiopathic scoliosis (AIS), an orthopedic entity of unknown etiology. Briefly, AIS is defined as a lateral curvature of the spine combined with vertebral rotation that occurs in patients of 10 years of age or older until bone maturity (18–20 years of age). AIS is predominant in females. If untreated, the curvature could evolve with negative long-term prognosis including psychosocial impact, back pain, pulmonary compromise, cor pulmonale, and even death due to respiratory failure. Causes of the disease have been postulated to involve genetics, abnormal muscle, connective tissue and bone structures, and neuroendocrine disorders. Psychological pathways have also been studied. Little data, however, have been collected on bone turnover in these patients. Some studies demonstrated decreased bone mineral density which may be suggestive of increased osteoblast activity. Other studies suggested a correlation to abnormal platelet morphology. Alterations in the spinal muscle contractile function may be responsible for spinal curvature. Measurement of trace elements in serum revealed impaired zinc and selenium metabolism, probably secondary to hormonal deregulation. Subsequent endocrine studies suggested a role for leptin and growth hormone in AIS. Recently, a neuroendocrine hypothesis has been proposed. This theory involves a unique melatonin-signaling dysfunction and opens new frontiers in the elucidation of the pathologic mechanisms for onset and progression of this disease.

2. Introduction Scoliosis is a three-dimensional deformity of the spine where lateral curvature may be combined with vertebral rotation. Throughout the eighteenth and nineteenth centuries, scoliosis was believed to be caused by postural positioning of the body. In addition, idiopathic scoliosis has historically been attributed to a wide variety of conditions ranging from poor posture to poor nutrition [1]. The Scoliosis Research Society (SRS) defined AIS as occurring in patients of 10 years of age or older, up until bone maturity at 18–20 years of age. This condition is distinct from the infantile, under 3 years of age, and juvenile, 3– 10 years of age spinal disorders. AIS is defined by a curvature of at least 10 as measured using the Cobb technique and vertebral rotation on a standing

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longitudinal radiograph of the spine combined with asymmetry on forward bending. The Cobb angle is defined as the angle formed by the intersection point of the perpendiculars of the parallel lines to the upper and lower plate of the curve-limiting vertebrae. The estimated worldwide prevalence of the disease is 0.5–3.0% with an incidence of 1–3% in adolescents, predominantly females [2,3]. As such, AIS represents an important pathologic entity with high clinical management costs and significant lifestyle psychosocial impact. Approximately 15–20% of spinal curvatures are attributable to developmental/genetic causes, neuromuscular, or metabolic disorders [4]. The remaining cases are deemed idiopathic. Although a small percentage of these cases are attributed to familial causes, the majority (85%) of these remain sporadic [5]. Spinal curvature is frequently recognized during a growth spurt, but varies in vertebral location and clinical impact. For example, thoracic curvatures are more prone to progression than lumbar or thoracolumbar scoliosis. Early onset of curvature is associated with poor prognosis [6]. If untreated, the curvature evolves with negative long-term prognosis, including psychosocial effects, back pain, pulmonary compromise, cor pulmonale, and even death due to respiratory failure [7]. Curve progression is the most important factor in the natural history of AIS. The risk of curve progression appears to predict spinal growth potential. Patients with skeletal immaturity and major curvature are at high risk for progression and warrant immediate treatment [3]. Although the clinical manifestations of scoliosis have been well described, the etiology of AIS, the most common form of scoliosis, continues to elude investigators. Various hypotheses have been postulated for defining the source of the disease. Genetic, neuroendocrine, muscle, connective tissue, and bone structure abnormalities have been suggested. Psychological pathways have also been studied and proposed as possible causes of scoliosis. Biochemical variations have been described and could be linked to the pathogenesis and evolution of this disease. The biochemistry of this disease is particularly important for evaluating the mechanisms of signaling dysfunction which appears to have a crucial role in clinical manifestation and for the development of potential diagnostic and prognostic tools. To date, diagnosis remains based on radiographic evaluation of the vertebral column. The lack of good prognostic indicators typically leads to therapies based on physician experience or diagnostic symptoms. This review summarizes biochemical, hormonal, and hematological parameters in AIS.

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3. Bone Biochemical Parameters Surprisingly, bone biochemical parameters have not been well studied in scoliosis. A study published 30 years ago investigated a variety of serum analytes in this disease [8]. This report found that calcium, phosphate, alkaline phosphatase (ALP), calcitonin, and parathyroid hormone (PTH) were not significantly different in scoliotic patients. A recent study compared a wide cohort of Asiatic AIS females (11–16 years of age) with 300 age-matched healthy females [9]. This study evaluated bone formation by measurement of serum bone-specific ALP and bone resorption by measurement of urine deoxypyridinoline. No differences were reported in the group of 12-years-old children. In AIS children aged 13 years and older, a significant difference was found for bone-specific ALP. The median concentration of bone-specific ALP was substantially increased in the 13-years group in AIS subjects versus healthy females (158 vs. 112 U/L). Bone-specific ALP was also increased in AIS in the 14-years (110 vs. 84 U/L) and 15-to-16-years groups (80 vs. 61.2 U/L) versus healthy females. Bone-specific ALP values were lower in absolute values as expected with decreased growth rate. Interestingly, urine deoxypyridinoline was significantly different only in the 15- to 16-years groups with higher median values in healthy females (13.5 nmol/ mmol creatinine) versus AIS subjects (9.4 nmol/mmol creatinine). A strong inverse correlation was found between bone-specific ALP and bone mass in AIS females. This appeared to be due to calcium intake and lower bone mass as determined by dual energy radiograph absorptiometry. Bone formation seems to be stimulated in AIS patients, despite low bone mass density. Bone resorption is not modified at all until 15 years of age [9].

4. Hormones Scoliotic adolescents, especially females, are usually taller than nonaffected age-matched subjects. Although higher secretion of growth hormone (hGH) has been described in scoliotic females, provocative tests were inconclusive [10]. Fasting hGH morning levels and 24-h testing regimes were, however, more indicative of association between this hormone and AIS [8,11]. Interpretation of these data was based on altered sensitivity of the hGH release mechanism in scoliotic females [10]. A large study investigated five different polymorphisms of the hGH receptor in 510 scoliotic females and 363 controls [12]. The hGH receptor did not appear to be a predisposing or disease-modifier gene in AIS. Bioassay measurements have found that insulin-like growth factors are not changed in scoliotic patients [13].


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An association between melatonin and hGH in scoliosis has been suggested [14], but appears limited to sporadic cases involving hGH therapy [15]. Sex hormones, namely estrogens, have a role in bone formation and promote increased ALP activity, collagen synthesis, and calcium deposition in the extracellular matrix. It has been demonstrated that estrogens are involved in the melatonin-signaling pathway in osteoblasts and exacerbate melatonin-signaling defects in AIS osteoblasts. Increased cAMP concentration, observed in a few scoliotic cases, was reduced in the presence of 17b-estradiol. This finding suggests a partial rescue of the signaling dysfunction that exists in this functional subgroup of scoliotic patients [16]. Although plasma estrogen concentration was initially found to be decreased in scoliotic females [17], this observation was not confirmed in subsequent studies [18,19]. A study of 174 scoliotic females revealed that mean estradiol levels were lower (50 pg/mL) versus age-matched (13–19 years) healthy controls (90 pg/mL). The same finding was also described for progesterone [20]. The influence of estrogens on AIS development is not concentration dependent, but appears to be associated with activity on their respective target cells [21]. The role of estrogens in AIS has been assumed due to the apparent association of age at menarche and scoliosis development. Late age of menarche, associated with null estrogen concentration, is associated with increased AIS prevalence [22]. Interestingly, the geographic latitude also matters, because sunlight exposure influences melatonin secretion, which can modify the sexual maturation and favor AIS development [23]. For example, low estrogen level predisposes young ballet dancers to scoliosis [22]. Estrogens appear to have a key role in regulating or triggering the melatonin-signaling dysfunction in AIS patients and activity of bone cells during bone remodeling. It is of particular interest that estrogens suppress cytokine IL-6, which induces osteoclast proliferation and bone resorption; a polymorphism of the IL-6 gene is associated with high risk of scoliosis [21,24]. Estrogen signaling is mediated by two receptors, ERa and ERb. These receptors belong to the nuclear receptor hormone superfamily which is widely expressed and present in osteoblasts. The CC genotype of the exon ØK of ERb is overrepresented in AIS patients [25]. It is also linked to curvature severity, suggesting that circulating estrogen concentration may impact different signaling pathways. In another study, four polymorphisms in exons encoding for the steroid-binding domain were identified [20]. Estradiol showed higher values with respect to peak height velocity in 24 females with AIS. The average peak was 10.5 1.8 cm/year at age 11.7 1 years. However, interindividual variability was high; very few high estradiol (> 100 pg/mL) values were obtained before the peak, but low values after the peak were nondiscriminatory. Other hormones

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such as dehydroepiandrosterone (DHEA) and osteocalcin were nondiscriminatory, whereas insulin growth factor 1 (IGF-1) was discriminatory, increasing 6 months before and continuing to do so until 12 months after the peak [26]. Only two contradictory studies reported androgen levels in scoliotic females. Testosterone concentration in 27 AIS patients was found to be higher than those in seven age-matched controls, whereas estradiol was not significantly different [19]. Interestingly, testosterone values in 174 AIS females aged 13–19 years were significantly lower than those measured in 104 healthy controls [20]. It should be noted that the controls demonstrated a high average testosterone value (4 ng/mL) for females. A recent hypothesis involves hormones that regulate energy balance, particularly leptin. Hypothalamic hypersensitivity to circulating leptin could induce an asymmetry as an adverse response through the sympathetic nervous system and the hGH–insulin axis, which worsens the axial abnormality during bone growth [27]. A comparison between a group of 120 scoliotic Chinese females and 80 nonscoliotic individuals showed a marked decrease in serum leptin in morning fasted subjects and during the follicular phase of menstrual cycle, independent from age and menstrual status. Leptin concentration was associated with body mass and height, but not with curvature magnitude, and was inversely correlated with age at menarche. This finding is of interest because leptin is a neurohormone that regulates satiety and displays high circulating levels in obese subjects due to increased adipose tissue mass. Leptin binds membrane receptors having a single transmembrane domain that belong to type I cytokine receptors exhibiting tyrosine kinase activity. There are two leptin receptor forms: the long one (OB-Rb) expressed in skeletal muscles and the short form (OB-Ra). Canpolat et al. [28] demonstrated that exogenous administration of melatonin in rats led to decreased circulating leptin. This finding is further strengthened by the fact that circulating leptin is increased in pinealectomized rats. As such, the melatonin deficiency hypothesis proposed by Machida and others cannot explain why circulating leptin is lower in AIS. However, Alonso-Vale et al. [29,30] demonstrated that increased cytosolic cAMP strongly inhibited leptin synthesis, which can be prevented by melatoninsignaling activity via interaction with MT1 receptors. This result provides insight into the role of melatonin-signaling dysfunction and could explain the diminution of circulating levels typically observed in AIS. Reduced leptin concentration could influence lower bone mass typically seen in AIS females [31]. The role of leptin could also be important in the genesis of scoliosis due to its critical role in bone formation and remodeling [32].


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5. Trace Elements Some research on the etiology of AIS has focused on trace elements. In 1980, a study described increased copper concentration in hair obtained from scoliotic patients [33]. High levels of copper in hair were also found in a subsequent study [34]. Interestingly, this report also found high hair zinc concentration, but low selenium concentration. Additional studies reported decreased blood selenium concentration in AIS, but no significant difference in zinc and copper concentrations [35]. In a separate study, zinc concentration in hair and serum in AIS subjects was similar to controls, but the back muscles of surgically treated scoliotic subjects were found to contain decreased zinc [36]. It is noteworthy that pinealectomized chickens show decreased serum zinc concentration versus control animals. It is likely that melatonin affects zinc metabolism, regulating mineral absorption in the gut [37]. The possible link between trace elements and axial skeleton disease is likely due to their seminal role in connective tissue metabolism. For example, copper is a coenzyme of lysyl oxidase, an enzyme involved in the synthesis and maturation of collagen. Variation in trace element concentrations could be linked to endocrine dysfunction, for example, increased hair copper and zinc concentration in hGH deficiency. Finally, trace elements are crucial to ensure optimal antioxidant enzyme function including superoxide dismutase and glutathione peroxidase. Both are regulated by melatonin.

6. Hematological Parameters—Platelets Because platelets and muscle have a similar actin:myosin contractile apparatus, they may share common abnormalities in AIS. In fact, a number of platelet abnormalities have been described including increased intracellular calcium and phosphorus and abnormal peptide structure of myosin chains [38], decreased activity of intracellular contractile proteins [39], decreased platelet aggregation [40], and increased intracellular dense bodies [41]. Poor aggregation and impairment of the contractile properties suggested a calcium transport defect related to membrane and contractile protein metabolism in scoliotic subjects [41]. Researchers have clearly demonstrated defective platelet maturation in AIS. Altered expression level of different Ca2þ ATPases (PMCA and SERCA) has been demonstrated in AIS patients [42]. Interestingly, AIS patients with low SERCA3a and high PMCA4b levels exhibited a right thoracic curvature, while a second group expressing high SERCA3a and PMCA4b levels exhibited a thoracolumbar curvature. Although these

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reports strongly associate defective megakaryocyte differentiation in AIS patients, they appear contradictory with a previous study that reported no morphologic or functional platelet abnormalities [43]. Irrespective of these findings, it is unlikely that these functional platelet characteristics can be used clinically for diagnostic or prognostic purposes in AIS. Calmodulin, a calcium-binding receptor protein, is a regulator of various enzymatic systems and intracellular calcium function [44]. It regulates the contractile apparatus of platelets and muscles via interaction of ionized calcium with actin and myosin. Platelet calmodulin concentration is increased in AIS. Increased calmodulin appears associated with disease progression and ultimately to therapeutic intervention via brace stabilization or vertebral fusion. Conversely, the calmodulin levels in stable nonprogressive and control groups were very similar [45]. Although platelet calmodulin levels were higher in those individuals with curvatures exceeding 30 and those with double major curves, this difference was not statistically significant. A recent report, however, did not find differences in platelet calmodulin concentration in 20 scoliotic adolescents. Analysis of intracellular calmodulin concentration in paravertebral muscles did not show differences between patients and controls, whereas comparison between left and right sides in AIS patients displayed higher levels on the convex side [46]. With respect to hematologic parameters, increased hemoglobin (Hgb) concentration was found in 23.2% of patients with early onset scoliosis (n ¼ 138). However, high Hgb concentration and hematocrit were secondary to hypoxemia due to thoracic deformation [47].

7. Melatonin 7.1. BACKGROUND Most biochemical evidence in AIS is linked to melatonin metabolism and its influence on bone formation and resorption. Melatonin (N-acetyl-5-methoxytryptamine) is produced by the pineal gland. Tryptophan is first converted by tryptophan hydroxylase to 5-hydroxytryptophan, which is decarboxylated to serotonin. The synthesis of melatonin is catalyzed by two enzymes (arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase) located in the pineal gland. It is known that the synthesis and release of melatonin are both stimulated by darkness and inhibited by light. The sympathetic nervous system transmits light exposure information from the retinal photoreceptors to the pineal gland. When darkness occurs, the release of norepinephrine from retinal receptors


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upregulates pinealocyte a1 and b1 adrenergic receptors. Some diseases interrupt the typical circadian rhythm, inducing low melatonin concentration during daylight. These include Turner and Klinefelter syndromes, Cushing’s disease, psoriasis, and sarcoidosis [48].

7.2. MELATONIN RECEPTORS There are two types of melatonin receptors: ML1 (high affinity) and ML2 (low affinity). ML1 is a Gi-protein-coupled receptor that inhibits adenylate cyclase activity, through GTPase activity. The high density of ML1 receptors in the hypothalamus (suprachiasmatic nucleus) is responsible for the chronobiology of the nervous system [48]. There are two subtypes of ML1 receptor: MT1 and MT2. The roles of both these types of receptors are illustrated in Fig. 1. Melatonin in normal osteoblasts binds to MT2 receptors inhibiting cAMP production by adenylyl cyclases via coupling to G inhibitory (Gi) proteins. A common melatonin-signaling defect in all AIS patients allows their functional classification into three groups or endophenotypes. Interestingly, the same group showed that, in a minority of AIS patients (functional group 1), when melatonin binds to its receptor, it activates cAMP production through Gs protein activation, as a consequence of an affinity switch in G-protein subtypes. Exposure to 17b-estradiol decreases the coupling of Gs protein with MT2 receptor. Receptor phosphorylation enhances the coupling with Gi proteins. Thus, the control of the phosphorylation of the Gi a subunit is of importance in the regulation of biologic processes and could represent candidate molecules in AIS pathogenesis. The modulation of Gi proteins is of crucial importance. Among kinases and phosphatases which modulate this protein, the protein kinase C d (PKCd), a serine–threonine kinase, has a role in desensitization of the MT2 receptor. It induces the blocking of inhibition of adenylyl cyclase causing increased cAMP in the cell [49,50]. Phosphorylation of the serine residues of Gi a subunits at their N-terminus is well known to block formation of heterotrimers with Gb and Gg subunits, thus preventing inhibition of adenylyl cyclase activity in the presence of melatonin. The translocation of PKCd within the cell membrane involves specific interactions with the receptor for activated protein C kinase 1 (RACK1) which acts as a scaffold protein binding only the activated form of PKCd. Different patterns of interaction between melatonin, receptors, and regulator proteins were found in osteoblasts derived from scoliotic subjects. However, the signaling dysfunction could be considered a real pathogenic mechanism in the development of scoliotic symptoms.

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174 A

Mel

MT1

AC PI2P Gqα

Giβγ Giα

ATP

cAMP

PLC

PKA DAG + IP3

[Ca2+]i

CREB

P-CREB

B Mel

MT2

AC PI2P Gqα

Giβγ Giα

PLC

ATP

cAMP

GC

GTP

cGMP

PKA

DAG + IP3

PKC

[Ca2+]i

CREB

P-CREB

Protein kinase cascades FIG. 1. Melatonin-signaling cascade through ML1 receptor subtypes (MT1 and MT2). (A) The signaling cascade through MT1. Melatonin binding to MT1 receptor inhibits cAMP production by adenylyl cyclase (AC) through Gia protein activation, thus inhibiting the protein kinase A (PKA) and phospho-CREB signaling; moreover, it activates phospholipase C, through the Gibg subunits and the Gqa activation, which in turn induces the intracellular calcium concentration increase. (B) The signaling cascade through MT2. The binding of melatonin to this receptor subtype, other than the induction of the signaling reported for MT1, is also able to inhibit the activity of guanilate cyclase (GC) and thus reduce the cGMP cellular content.


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7.3. INTERACTION WITH CALCIUM METABOLISM Melatonin biosynthesis is regulated by acetyltransferase expression and posttranslational control mechanisms via the changes in intracellular concentration of cAMP/calmodulin/Ca2þ following adrenergic stimulation [51]. Calmodulin is also a melatonin-binding protein of considerable regulatory significance. Its affinity to melatonin is sufficient for mediating effects at elevated physiological concentrations, and this binding is responsible for inhibition of calmodulin action. In particular, this interaction is specific to calcium-activated calmodulin and results in the inhibition of the CaM (calmodulin) kinase II. Moreover, melatonin binding to membrane receptors induces the activation of the bg complex of G proteins that stimulate PLCb and thus induces the activation of PKCa that in turn catalyzes production of phosphorylate calmodulin, perpetuating its inhibition. These interactions are important in inducing cytoskeleton rearrangements [51,52]. As reported above, calmodulin is a calcium-binding receptor protein that regulates cAMP-based enzyme systems, and thereby the contractile properties of muscle cells via cell membrane regulation of Ca2þ transport [53]. As such, melatonin may modulate diurnally many cellular functions involving calcium transport [52]. Melatonin modulates a specific cellular function through the kinetics of its binding to calmodulin [54]. Since calmodulin and melatonin exert a reciprocal antagonism in various tissues, and probably on skeletal muscle as well, it is reasonable to assume that the melatonin and calmodulin interplay could contribute to modulating paraspinal muscle tone and activity in AIS [48]. Because pineal deficiency modulates calcium-activated calmodulin, the spinal cord contractile proteins may be affected and neural cells may fail to grow in response to stretch. In addition, scavengers may not satisfactorily ‘‘mop-up’’ free radicals produced by stretch, causing cellular damage and inadequate cord growth. It has been proposed that asynchronous growth between the spinal cord and vertebrae (bone growth) could be part of the pathologic mechanism leading to scoliosis [55]. It has been demonstrated that administration of calmodulin antagonists, such as tamoxifen and trifluoperozine, mimicks the inhibitory effects of melatonin and can stop progression of this disease in mice [53]. Experiments conducted on bone-derived cell lines have proven that melatonin can increase expression of bone sialoprotein as well as several other essential bone marker proteins, including ALP, osteocalcin, and type I collagen. Melatonin stimulated both osteoblast differentiation and mineralization. In ovariectomized rats (a model of postmenopausal osteoporosis),

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the administration of physiologic melatonin doses with adequate estrogen or pharmacologic melatonin doses is required to increase bone mineral content and/or bone mineral density [56]. 7.4. ROLE OF MELATONIN IN PATHOGENESIS OF SCOLIOSIS The role of melatonin in scoliosis was first identified by experiments in chickens. Pinealectomy, performed in chickens shortly after they hatch, induced scoliosis [57–59]. Chickens, as bipedal animals, developed spinal disease with anatomic features similar to human AIS. It has been hypothesized that melatonin deficiency interferes with the normal symmetrical growth of the proprioceptive system involving the paraspinal muscles and the spine [60]. An asymmetric expression in bilateral paravertebral muscles of melatonin receptor MT2 mRNA in scoliotic patients was actually demonstrated, but it may merely be a secondary effect caused by forces exerted on the abnormally curved spine [61]. The administration of melatonin in pinealectomized chickens prevented scoliosis onset and development [60]. The same group also reported a significant decrease of nocturnal serum melatonin concentration in adolescents characterized by progressive scoliosis, whereas in patients with a stable deformity the concentration was not significantly different from controls [54]. Although experimental spinal disease was also reproduced in pinealectomized rats, it should be mentioned that scoliosis only developed in bipedal rats with surgically removed forelimbs and tails [62]. Similar findings were reported in a strain of mice (C57BL/6) where the gene controlling the melatonin pathway is naturally knocked out [63]. Melatonin deficiency secondary to pinealectomy alone does not produce scoliosis if the quadruped condition is maintained: the postural mechanism is thus crucial for inducing vertebral column abnormalities [1]. Although low melatonin concentration in severe progressive human scoliosis was corroborated by experimental animal studies [54], additional studies did not unequivocally confirm melatonin deficiency in AIS. For example, a study was performed using morning and evening urine samples collected from adolescent scoliotic females [64]. This report showed no difference in melatonin concentration (measured by high pressure liquid chromatography) versus controls [64]. Another study found that serum melatonin concentrations during the day (2 p.m.) and at night (2 a.m.) were not significantly different between a group of seven AIS patients and seven age-matched controls [14]. The same authors also did not confirm the previously demonstrated melatonin preventive effect on scoliotic development in pinealectomized chickens. In their experiments, the intraperitoneal injection of melatonin had no effect on scoliosis genesis or on disease progression [65].


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Melatonin is rapidly metabolized in the liver by hydroxylation. The principal catabolite in urine is 6-sulfatoxymelatonin, whose concentration can reflect the serum melatonin levels. The evaluation of its excretion in urine from AIS patients did not show differences during the entire 24-h collection period (or in diurnal and nocturnal collections) versus age- and gendermatched controls [66]. Similar results were reported by another team that compared serum melatonin and urine 6-sulfatoxymelatonin [67]. No differences in melatonin concentration were observed in homogenates of paravertebral muscles obtained from scoliotic and nonscoliotic adolescents or in comparison between sides (convex and concave) in AIS patients [46]. It should be noted that absolute melatonin concentration may be less important than its secretion rhythm, a property that could greatly influence cell metabolism through receptor occupancy and regulation. The role of melatonin in AIS remained uncertain due to the questionable relevance of avian studies versus humans. These issues were compounded by experimental data obtained from primates. For example, a 2-years study on 18 pinealectomized monkeys failed to induce scoliosis [68]. Positron emission tomography (PET) was used to evaluate F-18 fluorodeoxyglucose metabolism in the pineal gland versus the cerebellar area. No metabolic differences were found in AIS versus control subjects [69]. Urine 6-sulfatoxymelatonin concentration was also similar in the two groups. Biochemical and metabolic data did not support the hypothesis of absolute melatonin deficiency as a cause of AIS. Increased incidence of AIS was not found in children after surgical or radio-therapeutic pinealectomy due to cancers, despite melatonin deficiency. Interestingly, scoliotic patients typically suffer from sleep difficulty and disturbance. Transient deficiencies of melatonin synthesis or perturbation in its signal transduction could explain the discrepancies found between these reports. Administration of melatonin in AIS patients with a low concentration of endogenous melatonin was studied in a group of 40 subjects (28 with stable scoliosis and 12 with progressive scoliosis). Melatonin was measured by a radioimmunoassay from samples drawn every 3 h for 24 h. Environmental illumination was held constant from 7 a.m. to 9 p.m. Integrated melatonin concentration in 25 control subjects was 368 pg/mL (standard error 28.5) within a 24-h period: 183 (49.8) pg/mL for the nocturnal period (from midnight to 6 a.m.) and 10 (2.5) pg/mL for daytime (from 9 a.m. to 6 p.m.). The circadian rhythm was preserved in patients as well. Twenty two patients had melatonin concentrations similar to those of controls whilst 18 patients had lower concentrations. The administration of melatonin to patients did not alter the biologic rhythm, and prevented, in mild cases (curvature

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